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Tissue Eng. Author manuscript; available in PMC 2010 February 12.
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Tissue Eng. 2006 December ; 12(12): 3307. doi:10.1089/ten.2006.12.3307.
Engineering Complex Tissues
ANTONIOS G. MIKOS1, SUSAN W. HERRING2, PANNEE OCHAREON2, JENNIFER

ELISSEEFF3, HELEN H. LU4, RITA KANDEL5, FREDERICK J. SCHOEN6, MEHMET TONER7,
DAVID MOONEY8, ANTHONY ATALA9, MARK E. VAN DYKE9, DAVID KAPLAN10, and
GORDANA VUNJAK-NOVAKOVIC11
1Department of Bioengineering, Rice University, Houston, Texas 2 Department of Orthodontics,

University of Washington, Seattle, Washington 3 Department of Biomedical Engineering, Johns
Hopkins University, Baltimore, Maryland 4 Department of Biomedical Engineering and College of
Dental Medicine, Columbia University, New York 5 Department of Pathology and Laboratory
Medicine, University of Toronto, Toronto, Ontario 6 Department of Pathology, Brigham and Women’s
Hospital and Harvard Medical School, Boston, Massachusetts 7 Massachusetts General Hospital,
Harvard Medical School and Shriners Hospital for Children, Boston, Massachusetts 8 Division of
Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 9 The Wake
Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston
Salem, North Carolina 10 Department of Biomedical Engineering, Tufts University, Medford,
Massachusetts 11 Department of Biomedical Engineering, Columbia University, New York
Abstract
This article summarizes the views expressed at the third session of the workshop “Tissue Engineering
—The Next Generation,” which was devoted to the engineering of complex tissue structures.
Antonios Mikos described the engineering of complex oral and craniofacial tissues as a “guided
interplay” between biomaterial scaffolds, growth factors, and local cell populations toward the
restoration of the original architecture and function of complex tissues. Susan Herring, reviewing
osteogenesis and vasculogenesis, explained that the vascular arrangement precedes and dictates the
architecture of the new bone, and proposed that engineering of osseous tissues might benefit from
preconstruction of an appropriate vasculature. Jennifer Elisseeff explored the formation of complex
tissue structures based on the example of stratified cartilage engineered using stem cells and
hydrogels. Helen Lu discussed engineering of tissue interfaces, a problem critical for biological
fixation of tendons and ligaments, and the development of a new generation of fixation devices. Rita
Kandel discussed the challenges related to the re-creation of the cartilage-bone interface, in the
context of tissue engineered joint repair. Frederick Schoen emphasized, in the context of heart valve
engineering, the need for including the requirements derived from “adult biology” of tissue
remodeling and establishing reliable early predictors of success or failure of tissue engineered
implants. Mehmet Toner presented a review of biopreservation techniques and stressed that a new
breakthrough in this field may be necessary to meet all the needs of tissue engineering. David Mooney
described systems providing temporal and spatial regulation of growth factor availability, which may
find utility in virtually all tissue engineering and regeneration applications, including directed in
vitro and in vivo vascularization of tissues. Anthony Atala offered a clinician’s perspective for
functional tissue regeneration, and discussed new biomaterials that can be used to develop new
regenerative technologies.
Address reprint requests to: Gordana Vunjak-Novakovic, Department of Biomedical Engineering, Columbia University, 363G
Engineering Terrace, Mail Code 8904, 1210 Amsterdam Ave., New York, NY 10027, [email protected].
MIKOS et al. Page 2
INTRODUCTION
Engineering complex tissues is perhaps the most ambitious goal of all tissue engineers.
Engineering of functional vascular networks, interfaces, structural hierarchy, and complex

functional features is emerging as an unparalleled scientific and technical challenge for the
next generation of tissue engineers. This translates into the need to establish compositional
gradients or subcompartments, temporal changes, and the use of cells to drive tissue and organ
morphogenesis. An additional level of complexity is imposed by the orientation toward a
“custom designed,” individualized approach to defect repair that takes into account multiple
additional requirements. New generations of interactive biomaterial scaffolds and bioreactors
are now being developed to emulate features found in vivo and to bring this ambitious goal
closer to reality. The fields of “adult biology,” associated with tissue remodeling; transplant
surgery; and immunology are also essential for supporting the efforts of tissue engineers in this
direction. To noninvasively monitor the maturation and remodeling of such grafts by molecular
imaging techniques, new sets of biomarkers may need to be developed. Efforts are under way
to develop new modalities and to improve the existing ones for preservation of component
cells and engineered tissues, in order to make tissue grafts available to patients in a timely
fashion.
ENGINEERING OF COMPLEX ORAL AND CRANIOFACIAL TISSUES

Antonios Mikos from Rice University discussed tissue engineering approaches to dental, oral,
and craniofacial rehabilitation for millions of people, as well as significant challenges that need
to be addressed before successful clinical applications can be realized. The current paradigm
that recognizes the complex interplay between biomaterial scaffolds, growth factors, and local
cell populations during healing has been consistently utilized to design constructs that attempt
to promote these interactions to restore the original architecture and function of complex
tissues. Nevertheless, issues related to each component of this paradigm reflect the fact that
“ideal” tissue engineering techniques are yet to be developed for the regeneration of complex
composite structures such as alveolar bone, periodontal ligament, and teeth.
Part of the difficulty in designing constructs exposed to the oral environment relates to a unique
set of interactions involved in the regenerative process in contrast to other parts of the body
(Table 1). Augmented healing of alveolar extraction sites, where bone regeneration materials
implanted into a tooth extraction socket encounter an environment very different from that of
healing long-bone fractures, provides an excellent example. Interactions between the implanted
material and the oral flora occur within an essentially “contaminated” wound-healing site, and
a progenitor cell population responsible for bone healing is believed to be derived from
remnants of the periodontal ligament, rather than from periosteum or endosteum. Further,
reconstitution of a healed extraction socket involves the coordinated regeneration of both bone
and epithelium. Thus, tissue engineering approaches to augmented healing of alveolar sockets
have attempted to recapitulate the temporal and spatial relationships of growth factors and cells
with the healing environment of the oral cavity, and constructs should be designed with these
considerations in mind.
Biomaterial scaffolds
Biomaterials and growth factor delivery systems used for alveolar bone engineering have
several fundamental shortcomings. From an anatomical standpoint, an ideal scaffold for bone
regeneration should be injectable so that it conforms to the irregularly shaped defects produced
by tooth extraction. Next, it should undergo an increase in mechanical stiffness, to mimic the
mechanical properties of bone, in order to sustain the physiological loading conditions imposed
by mastication. The scaffold material should also degrade at a rate complementary to bone
formation so as to preserve the morphology of the alveolar ridges—an important consideration

MIKOS et al. Page 3
for the prosthetic rehabilitation of the patient. If the material degrades too quickly, the resultant
defect will compromise denture construction or dental implant placement. A slower rate of
degradation will reduce tissue in-growth and regeneration.
Although moldable polymeric materials exist, which upon cross-linking exhibit compressive
and tensile strengths close to that of bone, these materials do not degrade on a timescale suitable
for optimal bone healing. In addition, these materials are hydrophobic, which renders them
unsuitable for cell encapsulation. Conversely, numerous synthetic and biocompatible materials
capable of forming hydrogels are available. These degrade quickly and promote tissue in-
growth, but their weak mechanical properties require rigid fixation devices to stabilize
mechanically unstable bone defects. Thus, the challenge is to create an injectable biomaterial
that has mechanical properties similar to bone, yet degrades quickly enough to support tissue
in-growth, and allows for cell encapsulation.
Aside from the mechanical properties of a scaffold, the interactions between cells and the
biomaterial surface also need to be addressed. While design improvements to biomimetic
scaffolds have allowed an increase in cell adhesion, the adhesion of specific cell populations
by altering scaffold properties has yet to be perfected. Surface properties are especially
important in periodontal ligament regeneration, where successful formation of the
periodontium and surrounding alveolar bone also requires the prevention of epithelial down-
growth into the periodontal defect site. An ideal periodontal regeneration material should
promote the attachment and in-growth of both osteogenic cells and periodontal ligament
fibroblasts, while excluding epithelial cells from the periodontal defect.
Growth factor delivery

Another important area in tissue engineering is growth factor delivery. Numerous methods
exist for controlled delivery of bioactive growth factors in bone and periodontal regeneration
applications. To ensure sufficient bioavailability to the regenerating tissues, growth factors
need to be loaded into scaffolds in concentrations that far exceed those normally seen in the
body. Gene therapy might be able to address these issues using a different approach. Successful
transfection of cells within the healing environment would up-regulate growth factor
production and raise the concentration to required levels. However, viral gene delivery vehicles
run the risk of becoming pathogenic and are limited in the size of genetic material they can
carry, while nonviral gene delivery vehicles tend to be inefficient at gene transfection and are
cytotoxic at high enough concentrations. Thus, from a cost perspective, more efficient growth
factor delivery systems are needed, especially if such constructs are to be used successfully in
clinical practice.
An equally important challenge in growth factor delivery for the engineering of complex tissues
is control over the kinetics of release. Much research has focused on the characterization of
key growth factors within the healing cascade and their temporal and spatial expression within
complex tissue defects. However, the ability to direct the release of numerous growth factors
on multiple timescales will determine the success of a tissue engineering scaffold in
recapitulating the complex wound-healing environment seen in oral and craniofacial tissues.
Scale-up
Issues of scale-up present additional challenges. Tissue engineering strategies that prove to be
successful in small experimental defects may fail in larger human applications where nutrient
diffusion into the center of an extensive cell-based construct may be limited during the initial
stages of healing before any appreciable angiogenesis takes place. Investigators must also
consider the possibility of differences in physiology between animal and human subjects, and

MIKOS et al. Page 4
whether or not potentially promising results obtained in animal models can be reproduced in
the clinical setting.
Animal models—With this consideration in mind, improvements in tissue engineering

strategies require the development of effective in vivo models for the assessment of implanted
constructs. While both cranial cavitational defect models and long-bone segmental defect
models are commonly employed to test bone regeneration materials in small rodents, they fail
to re-create the complex wound-healing environment found in the oral cavity. A model needs
to be developed that can create an easily reproducible critical-size defect, which communicates
with the oral cavity. The animal chosen must be large enough to make surgery practical, and
reasonably inexpensive to permit studies with sufficient statistical power. In addition, the
ability to quantify both the angiogenic and osteogenic potential throughout the volume of the
surgical defect is essential.
Future directions and potential for dental and craniofacial tissue engineering
While advances in tissue engineering have created a variety of approaches toward both alveolar
bone and periodontal ligament regeneration, an ideal material has yet to be developed.
Additional clinical challenges that must be addressed include the ability to control the
morphology of bone formed by an implanted biomaterial. For instance, the alveolar bone
present in the posterior maxilla tends to have a larger proportion of cancellous bone when
compared to the anterior mandible, which has implications for the primary stability of titanium
implants placed in that location. There currently exists no synthetic biomaterial capable of
controlling the architecture of bones generated de novo within a bony defect. Another challenge
is to create dental implant systems for tooth replacement that can regenerate a periodontal
ligament attachment to the surrounding alveolar bone and form a tight gingival seal between
the implant surface and the oral mucosa. Finally, one of the most difficult challenges is to
regenerate an entire tooth. Although some attempts have been made in this direction, they have
been rarely successful. The ability to regenerate a complete tooth and its periodontal ligament
within an extraction socket would be an extraordinary achievement, and would surely
revolutionize the practice of dentistry.
Dr. Mikos concluded that continuing developments in the field of tissue engineering would
undoubtedly lead to improvements in biomaterial scaffolds for cell-based therapeutics and the
delivery of bioactive molecules such as growth factors and DNA. He also emphasized that the
success of tissue engineering strategies would rely on the ability of the implanted construct to
control the proliferation and differentiation of specific cell populations within the wound-
healing site, using materials that are both cost effective and easy to manipulate in the clinical
setting. In this way, tissue regeneration can be optimized in the complex wound-healing
environment of the oral cavity.
OSTEOGENESIS AND THE VASCULATURE: SHOULD THE SCAFFOLD COME

FIRST?
Susan W. Herring from the University of Washington explained that the vascular arrangement
of the overlying periosteum precedes and dictates the architecture of the new bone, and
proposed that engineering of osseous tissue might benefit from preconstruction of an
appropriate vasculature and scaffolds with pore size and surface architecture conducive to the
growth of vessels.1,2 She emphasized that bone is completely dependent on its vasculature.
Vessels provide nutrients and minerals to osteogenic sites, provide access for osteoclasts and
other cells, and, possibly through their accompanying pericytes, supply a source of
mesenchymal osteoprogenitors. Beyond the requirements of homeostasis, however, there is a
morphogenetic connection. Vascular cells have dramatic inductive osteogenic effects,3 and, at

MIKOS et al. Page 5
the same time, processes that stimulate osteogenesis induce angiogenic factors.4 The
association between membranous ossification and the preexisting vascular pattern in the
connective tissue matrix is so anatomically close that one early worker referred to the process
as “angiogenic ossification”5 (translated by Inoue et al.6) and another described the bone as
“modeling itself along the course of the artery.”7 Arnold Caplan, a founding father of modern
tissue engineering, described the vasculature as the “orientor of osteogenesis” in vivo,8 and his
illustrations make clear that it is the vessels of the periosteum that provide the (negative) model
for newly apposed layers of bone.
If it is generally true that the normal developmental sequence is for mineralized matrix to be
patterned by a pre-existing vasculature, then the present practice violates the precept that tissue
engineering works best if it mimics ontogeny. Manufacturing the matrix first may not be the
most efficacious way to produce a well-perfused graft. Instead, the vascular network should
be prepared first, from which living, remodeled woven bone could be produced naturally.
To establish that normal skeletal growth proceeds from vasculature to bone, rather than the
reverse, Herring and colleagues assessed the role played by the periosteal vasculature in
organizing the newly deposited matrix of growing craniofacial bones in a pig model. Piglets,
2–6 weeks old (n = 5), were anesthetized, injected intravenously with calcein (12.5 mg/kg;
Sigma Aldrich, St. Louis, MO), and then perfused with heparinized saline followed by a
silicone rubber–based vascular fill (Microfil®; Flow Tech, Carver, MA). The head was frozen
overnight, and the zygomatic arch was cut into blocks and dehydrated and infiltrated with
plastic embedding media. Cured specimens were thick-sectioned (50–1000 μm) and examined
under epifluorescent illumination to compare the calcein-labeled matrix (mineralized in the
last 3 hours of life) to the periosteal vasculature. Additional material included periosteal whole
mounts from animals injected with Microfil® and histological sections of decalcified arches
from piglets that had received injections of bromodeoxyuridine 3 hours before sacrifice.9
Different portions of the periosteum of the zygomatic arch were strikingly different in
osteogenic activity. The medial surface was almost devoid of calcein label. Histological
observations confirmed that the medial surface was not appositional, and was indeed being
actively resorbed. The lateral surface showed extensive deposition of calcein, but the pattern
was different for the two bones that comprise the arch, reflecting their different structures.10
The more anterior zygomatic bone (Fig. 1) showed a continuous surface layer of calcein,
approximately 100 μm thick, whereas the more posterior temporal bone (Fig. 2) exhibited
calcein extending the tips of bony spicules by approximately 250 μm.
As has been described for the periosteum of limb bones,11 the nutrient vessels of craniofacial
periosteum were different from those of the bone-marrow spaces. Relatively large-caliber
vessels were found within the plane of the periosteum, parallel to the bone surface. Beyond
this general relationship, vascular pattern varied with region.
Despite its resorptive character, the medial periosteum was well vascularized with very large
vessels close to the bone surface. Histological sections suggested that these vessels were
composites of fusing smaller vessels and that they were continuous with the marrow circulation
in the resorbing medial bone. In contrast, the largest vessels of the appositional lateral
periosteum were more peripheral in the fibrous layer at some distance from the bone surface.
These large vessels, which stemmed from nearby muscles and ligaments, formed an
anastomosis in the plane of the periosteum. Closer to the bone surface, small-caliber vessels
formed another planar network, interconnected with the large, superficial one. Only very small
vessels penetrated the bone surface, and these frequently showed signs of angiogenesis, such
as sprouts and endothelial tubes.

MIKOS et al. Page 6
Both the zygomatic and the temporal bone showed an intimate association between newly
mineralized matrix and vascular architecture. In thick sections, labeled matrix could even be
visualized as tubes around blood vessels (Fig. 3). However, only for the zygomatic bone was
the vascular architecture that of the periosteum. Here the accreting matrix surrounded the deep
periosteal network, becoming the negative image of the vasculature (Fig. 1). On the temporal
bone, the mineralizing, elongating spicules were nested between radiating vessels that were
perpendicular to the periosteal plane. Rather than coming from the periosteum, these vessels
were clearly continuations of the intraosseous vasculature (Fig. 2).
In growing craniofacial bones, the relationship between new matrix and vascular architecture
was close enough to justify the idea that vascular pattern dictates where mineralized matrix is
laid down. However, the significant vasculature is not necessarily that of the periosteum. In
the slower-growing zygomatic bone, the deep periosteal network did appear to be engulfed by
mineralizing matrix and thus patterned the woven bone into its negative image. But in the
faster-growing temporal bone, the appearance was that of radially oriented intraosseous vessels
co-opting and transforming the deep periosteal network.
Susan Herring concluded that from a tissue engineering point of view, the important feature is
that, in both cases, bone formed around the vessels. Osteogenesis can be organized either by
internal vessels, as in the temporal bone, or by external vessels, as in the zygomatic bone. Thus,
it may not be necessary, or even advisable, to engineer three-dimensional porous scaffolds for
bone; rather, a vasculature should be constructed. Further, the vascular network need not be
three-dimensional if it is osteogenic. A two-dimensional periosteum creates a three-
dimensional bone. A two-dimensional vascular graft might be especially suitable for the
membrane bones of the skull, which lack cartilage predecessors and are entirely formed and
shaped by their periostea.
ORGANIZATION AND INTEGRATION OF ENGINEERED CARTILAGE

Jennifer Elisseeff from Johns Hopkins University discussed stem cell research in the context
of musculoskeletal tissues and in connection with novel engineering tools that are being
developed to improve tissue engineering techniques and facilitate their eventual clinical
translation. She suggested that stem cells and novel enabling technologies are critical for future
tissue engineering research.
The discovery of stem cells and the development of novel engineering technologies have
infused energy into the field of tissue engineering and stimulated new advances that will further
understanding of the tissue development process and promote clinical translation. Stem cells
address a major challenge in tissue engineering—procurement of adequate cell numbers to
place on a scaffold.13 The cell is the building block of the developing tissue, proliferating and
producing the desired tissue-specific extracellular matrix (ECM). Cells isolated from tissue
biopsies oftentimes have limited expansion capabilities, and instead readily differentiate and
secrete the tissue-specific proteins. The problems arise from the fact that during expansion
these differentiated cells often change phenotype and lose their capability to form tissue.14 In
contrast, stem cells have the capacity to proliferate in an undifferentiated state, providing a
potentially large source of cells.15 The subsequent challenge, though, is to differentiate the
stem cells during the tissue engineering process. In the case of adult stem cells, in particular,
bone marrow–derived mesenchymal stem cells (MSCs), many of the signals (e.g., growth
factors) required for tissue-specific differentiation have been elucidated.16 In the case of
embryonic stem cells (ESCs), manipulating or controlling differentiation remains a significant
challenge.

MIKOS et al. Page 7
Stem cells
Stem cells are characterized by their ability to proliferate significantly in an undifferentiated
state and, under appropriate conditions, to differentiate along one or more lineages. The
discovery of these cells has stimulated significant excitement in the field as large cell numbers
are required to seed the three-dimensional scaffolds, particularly if the scaffolds are the size
of clinically relevant defects.17
Adult stem cells can be isolated from a number of tissues, most notably, the bone marrow.18
Researchers continue to identify other sources of adult stem cells, including fat, cardiac, neural,
limbus, bone, and muscle tissues. While there is still some debate over identity and
classification, adult stem cells can be characterized by surface markers and their functional
differentiation. The ESCs can be isolated from the inner cell mass of the blastocyst and the
primordial gonadal ridge of the fetus19,20 These cells are cultured on fibroblast feeder layers
to maintain them in an undifferentiated state, for potentially very long periods of time. New
feeder-free stem-cell culture systems are under development by a number of groups.
The proliferative capacity of the ESCs is intriguing for researchers in regenerative medicine
who continually battle the challenge of having enough cells to combine with biomaterials to
build new tissues. Differentiation of ESCs is usually initiated by formation of embryoid bodies
(EBs), clusters of cells differentiating into all three germ layer lineages.21 A number of
researchers are selecting specific cell types or groups of cells from EBs to apply to tissue repair.
22 While these proliferation and differentiation characteristics lead one to assume unlimited
capacity for ESCs to make any tissue in the body, we are now just touching the surface on how
to control these cells and the unique requirements that we must elucidate to induce tissue
development. A number of problems remain to be solved before stem cell research can be
translated into clinical practice.
Tissue engineering of cartilage provides one important venue to evaluate a number of cell
types: fully differentiated chondrocytes from cartilage, adult stem cells, and ESCs.23–28 Figure
4 pictures cartilage engineered from chondrocytes isolated from cartilage tissue, bone marrow–
derived stem cells, and cells derived from EBs made from human and mouse ESCs. While the
base hydrogel scaffold was similar in all cases, embryonic cells required unique conditions for
differentiation in each case.
Technologies: Understanding biology and engineering organized tissues

Biomaterials, along with cells, are a critical component of tissue engineering. The biomaterial
scaffold provides a three-dimensional environment for cells and the developing tissue.
Researchers are aiming to design scaffolds that can promote a desired cell behavior, including
proliferation and differentiation.29 This aim is challenging, since many of the signals required
for stem cells, particularly ESCs, are unknown. This has led engineers to work with biologists
to help discover more about the biology of stem cells and how they behave in complex tissue
engineering scaffold systems.
Like many other tissues in the body, cartilage is structurally organized and contains a
superficial, a middle, and a deep layer. The tissues in each of these layers are distinct with
respect to matrix composition and organization and subsequent mechanical properties. The
superficial layer contains more collagen than the other layers, and the fibers are aligned parallel
to the joint surface. The deep layer of cartilage contains more proteoglycans and the collagen
fibers are aligned perpendicular to the cartilage joint surface. The cells in the deep layer are
larger compared to the superficial cells, which are smaller and flattened. The combination of
these two layers and the intermediate third layer in between is what provides cartilage its unique
mechanical properties and overall functionality as a smooth, lubricating surface that absorbs

MIKOS et al. Page 8
and transmits mechanical forces. Therefore, from a tissue engineering perspective, rebuilding
the structure of even a seemingly simple tissue such as cartilage is critical to building more
functional tissues. The need for organized structures applies to practically all tissues in the
body, and these technologies may be applied in a similar manner to other tissue types.
Hydrogels can be used as scaffolds to suspend chondrocytes (and stem cells) in a three-
dimensional environment for engineering cartilage.30 Hydrogels are cross-linked polymer
matrices capable of absorbing large amounts of water. Cells can be suspended in the liquid
polymer solution before cross-linking. Light triggers the cross-linking process, and this enables
a high degree of control over cross-linking—only when the solution is exposed to light does
gelation occur. This technology was developed primarily to ease implantation, giving
physicians control over gel scaffold formation in vivo.31
The light-based process is also useful for creating multilayered hydrogel structures. For
example, a bilayered hydrogel can be created in a mold by placing one layer of partially gelled
liquid polymer solution that forms a semi-solid, and then adding a second layer of liquid
polymer solution to the first. The whole construct is then fully cross-linked to form a bilayered
hydrogel. This technique can be extended to create more layers in the hydrogel. Different cell
types can be encapsulated in each layer, without migration. Moreover, the interface of the
hydrogel layers can be manipulated such that there is either a “clean” interface with no cell
mixing, or a “fuzzy” interface with some degree of contact and mixing of the cells. Cells labeled
with dyes encapsulated in a bilayered gel are pictured in Figure 5.
While the multilayered hydrogels are useful for creating tissues that resemble the normal
architecture of tissue, they also provide a venue to study how cells “talk” to each other.
Developmental biologists have for many years known the importance of cell organization,
boundaries, and interfaces between cells in regulating tissue development. This concept can
be applied to tissue engineering for enhancement of both tissue -formation rate and quality.
In both developmental biology and tissue engineering, little is known about how the cells in
the different layers of cartilage “talk” to each other to regulate tissue composition. Biologists
have determined a number of signals in limb development that may also be important in the
multilayered hydrogel system. The application of multilayered, organized hydrogel scaffolds
applies to the new realm of understanding how stem cells talk to other cells to regulate their
own differentiation and also behavior of other cells. When chondrocytes isolated from the deep
layer of cartilage were cocultured next to cells from the superficial layer, they decreased their
proliferation rate and produced more ECM compared to when they were cultured alone. While
we do not know all of the signals that were transmitted between the two cells, we can further
investigate the mechanisms for the observed differences in cell function. Most important, it is
clear that engineering biomaterial scaffolds to create organized tissue structures will be critical
for functional tissue engineering and for understanding mechanisms of cell communication at
the mm- and even the cm-length scales.
The hydrogel systems have also been used to study the behavior of mouse and human ESCs
and human embryonic germ cells (Fig. 4). While we learn a great deal from working with fully
differentiated cells and adult stem cells, the cell contact and matrix requirements for the above
mentioned cells are all different. In all cases, the three-dimensional environment played an
important role in differentiating the stem cells and even affected the cell response to growth
factors.25 Significant research still needs to be done to further understand the differences
between these cell types.
There has been much interest in and debate over the role of stem cells in repair. It is clear that
stem cells play a positive role in tissue repair, but questions arise as to whether these cells
differentiate and help form the repair tissue or whether they simply secrete the appropriate

MIKOS et al. Page 9
cytokines that stimulate host cells to form new tissue and also scar formation. Tissue
engineering systems can help in answering some of these questions. For example, MSCs were
co-cultured in hydrogels adjacent to chondrocytes. Previous investigations by Gerstenfeld et
al. have demonstrated that chondrocytes cultured in monolayers stimulate the differentiation
of MSCs toward an osteogenic phenotype.32 In similar coculture studies in a three-dimensional
hydrogel environment, the MSCs stimulated the chondrocytes to proliferate and to produce
more matrices. This effect was not observed with osteoblasts or fibroblasts, suggesting that
MSCs promote new cartilage development. Coculture studies have thus been crucial in utilizing
ESCs for cartilage tissue engineering applications.
Jennifer Elisseeff summarized that there are a number of exciting technologies in tissue
engineering that currently, and will in the future, have a significant impact on the field of tissue
engineering. Stem cells and enabling technologies will keep tissue engineers busy, as they
elucidate the scaffold environments for more powerful cell types (stem cells) and design
systems to enable translation of these technologies.
INTERFACE TISSUE ENGINEERING FOR SOFT TISSUE-TO-BONE

INTEGRATION
Helen Lu from Columbia University discussed engineering of a functional soft tissue–bone
interface, a problem critical for biological fixation of tendons and ligaments. She stressed that
interface tissue engineering will likely be instrumental in the development of a new generation
of fixation devices that can expedite the translation of orthopedic tissue engineering to the
clinical setting. Using the anterior cruciate ligament (ACL)-to-bone insertion site as a model
system, she explored the creation of the tissue interface by coculturing cells on a scaffold with
a biomimetic gradient of structural and functional properties.
Application of tissue engineering methods12,33 to musculoskeletal tissue regeneration has led

to tremendous advances, whereby bone,34–37 cartilage,38–43 and ligament-like44–48 tissues
have been engineered in vitro and in vivo. Recently, the emphasis in the field has shifted from
tissue formation to tissue function.49 In addition to engineering tissues with physiological
mechanical properties, a significant effort was invested into the biological integration of
engineered grafts.
Musculoskeletal function requires soft tissue to integrate with subchondral bone and function
in unison to facilitate joint motion. The insertion of ligaments or tendons into bone is usually
achieved through fibrocartilage interface.50–56 By design, the controlled matrix heterogeneity
minimizes the formation of stress concentrations, effectively transferring complex loads
between two distinct types of tissues.51,57 Current soft-tissue reconstruction methods do not
result in adequate graft integration, and the lack of interface can compromise graft function
and long-term outcome. Lu proposed that the regeneration of the soft tissue-to-bone interface
is a prerequisite for achieving biological fixation of soft-tissue grafts, and is essential for
functional orthopedic tissue engineering. An important long-term goal is to devise tissue
engineering strategies for regenerating the soft tissue-to-bone interface, and to apply these
strategies to design integrative fixation devices capable of promoting the biological fixation.
To this end, elucidating the structure-function relationships and the mechanisms of interface
regeneration will be important for the design of integrative fixation devices.
Design considerations
The ACL is the primary knee-joint stabilizer and the most frequently injured knee ligament,
58 with over 100,000 ACL reconstruction procedures performed annually in the United States.
59 Autologous hamstring tendon-based grafts are increasingly utilized due to donor-site

MIKOS et al. Page 10
morbidity associated with bone-patellar tendon-bone grafts.60 The soft tissue–based grafts can
restore the physiological range of motion and joint function through mechanical fixation;
however, biological fixation is not achieved as disorganized scar tissue forms within the bone
tunnels and the native insertion site is lost due to surgery.
The ACL connects to bone through a characteristic fibrocartilage interface, with controlled
spatial variation in cell type and matrix composition.50,51,53–56,61 The ligament-to-bone
interface consists of three distinct tissue regions: ligament, fibrocartilage, and bone (Fig. 6).
The ligament proper is composed of fibroblasts embedded in type I and type III collagens. The
fibrocartilage region is further divided into nonmineralized and mineralized zones. The
nonmineralized fibrocartilage matrix consists of ovoid chondrocytes, and collagen types I and
II within the proteoglycan-rich matrix. The mineralized fibrocartilage zone contains
hypertrophic chondrocytes surrounded by a mineralized matrix.54 Type X collagen, a marker
for hypertrophic chondrocytes, is detected only within this region.61 The last zone is the
subchondral bone, within which osteoblasts, osteocytes, and osteoclasts are embedded in a type
I collagen matrix.
The specific organization and controlled heterogeneity of the interface are important for
minimizing stress concentrations and facilitating the transfer of complex loads between soft
and hard tissues.51,62 This multitissue insertion site is lost following ACL reconstruction
surgery, and the graft is attached to bone via mechanical fixation using screws or pins. Without
a biological interface, the graft-bone junction has limited mechanical stability, and the lack of
integration is the primary cause of graft failure.63–65 To date, functional integration of soft-
tissue grafts with bone remains a problem, and advances are hindered by the lack of integrative
graft solutions and sufficient understanding of mechanisms that govern interface regeneration.
Functional integration of soft tissue to bone may be achieved through the regeneration of the
fibrocartilage interface, which in turn may require multiple types of cells, a multiphasic
scaffold, and the development of controlled matrix heterogeneity mimicking that of the native
insertion. Lu and colleagues have focused their efforts on three areas: (i) elucidating the
mechanism of interface regeneration using models of homotypic and heterotypic cell-cell
interactions, (ii) characterizing the structure-function relationship at the insertion site and
identify interface-relevant design parameters, and (iii) designing biomimetic scaffolds capable
of supporting multitissue regeneration.
Cell-cell interactions and mechanism of graft integration—It is well established that

tendon-to-bone healing following ACL reconstruction does not lead to the reestablishment of
the native insertion; rather, a fibrovascular or fibrocartilaginous tissue layer is formed within
the bone tunnel.66–69 This neo-tissue is located within the bone tunnel, whereas,
physiologically, the insertion is situated outside of subchondral bone (Fig. 6). The formation
of a fibrovascular tissue, instead of a mineralized matrix, within the bone tunnel results in a
weak link at the graft-to-bone fixation site.64 To promote biological fixation, it is thus
physiologically more relevant to develop a fibrocartilaginous zone outside of the bone tunnel.
The formation of a fibrocartilage layer where the tendon graft directly contacts the bone is
highly significant, and has led to the hypothesis that the interactions between cells derived from
tendon (e.g., fibroblasts) and bone (e.g., osteoblasts) play a role in interface or fibrocartilage
regeneration. Fujioka et al.70 reported that by suturing the Achilles tendon to its original
attachment site, cellular organization resembling that of the native insertion was observed over
time. In vivo cell-tracking studies have revealed that the tendon graft is populated by host cells
within 1 week of implantation.71 While the source and nature of these host cells are not known,
cell types other than osteoblasts and fibroblasts may be involved in fibrocartilage formation.
Based on these observations, it is thought that osteoblast-fibroblast interactions mediate

interface regeneration through two mechanisms: (i) phenotypic changes or transdifferentiation

of osteoblasts, fibroblasts, or both, and (ii) the recruitment of progenitor stem cells or MSCs
to the graft-bone interface, where they differentiate into fibrochondrocytes and form
fibrocartilage.
One of the coculture systems focused on the interaction of fibroblasts and osteoblasts,72 with
the goal of emulating the in vivo condition where the tendon is in direct contact with bone tissue
following ACL reconstruction. In this model, osteoblasts and fibroblasts were first seeded on
opposite sides of a tissue-culture well separated by a hydrogel divider preformed in the center
of the well (Fig. 7A). Once the cells reached confluence, the divider was removed, allowing
the osteoblasts and fibroblasts to migrate and interact within the interface region. In another
model system, an osteoblast monolayer was cultured atop a chondrocyte micromass73 (Fig.
7B). It is likely that osteoblast-fibroblast interactions facilitate the recruitment and
differentiation of stem cells or progenitor cells into chondrocytes or fibrochondrocytes. A
triculture model was used to evaluate the effects of fibroblast-osteoblast interactions on
chondrocytes.74,75 These studies demonstrated the utility of in vitro coculture systems for
investigating the mechanisms of interface regeneration.
Structure-function relationships at the ACL-bone interface—The first steps in

functional tissue engineering are the determination of the material properties of the tissue to
be replaced, the measurement of in vivo strains, and the calculation of stresses found in the
native tissue.49 Therefore, in order to successfully engineer the soft tissue-to-bone interface,
the structural and material properties of the insertion site must be characterized. Identification
of these properties will be critical for biomimetic scaffold design, and will serve as benchmark
criteria for judging the success of engineered interfaces.
Current knowledge of mechanical properties of the insertion site is based on theoretical

analyses,55,76 as direct measurement of these properties is difficult due to the complexity and
the relatively small-length scale of the interface (100 μm to 1 mm).50,56,62 Applying the novel
functional imaging method of ultrasound elastography, the strain distribution at the ACL-to-
bone interface could be determined.77 Elastography analyses of bovine tibiofemoral joints
loaded in tension revealed that the displacement across the insertion was the highest at the ACL
and decreased toward the bone (Fig. 8A). These regional differences suggest an increase in
tissue stiffness from ligament to bone. These results agree with finite-element analysis of the
medial collateral ligament, predicting that the maximum principal compressive stress occur
near the distal edge of the femoral insertion.76
Based on Wolff’s Law,78 a structure-function relationship likely exists at the insertion site, and
the observed interface organization and matrix composition are directly related to the nature
of the strain experienced in the region. Fibrocartilage is often found in regions subjected to
compressive stresses.79 Combining microscopic mechanical testing with optimized digital
image correlation methods, the region-dependent changes in compressive mechanical
properties of the fibrocartilage interface were quantified.80 Displacement under applied load
was imaged using epifluorescence microscopy, and digital image analysis was performed to
determine the mechanical properties. The incremental displacement decreased gradually from
the ligament, to the nonmineralized to the mineralized fibrocartilage, and then to bone,
implying an increase in tissue stiffness across these regions.
Analysis of these individual regions demonstrated decreased strain and a significantly higher
elastic modulus for the mineralized fibrocartilage compared to the nonmineralized
fibrocartilage region.80 Surface characterization of the insertion site (Fig. 8B) revealed a
corresponding increase in calcium and phosphate content progressing from ligament to
interface and then to bone.81 Benjamin et al. suggested that the amount of calcified tissue at

the insertion correlates to the force transmitted across the calcified zone.82 Thus, the increase
in elastic modulus is likely due to the mineral phase of the fibrocartilage interface.
Scaffold design and testing—A biomimetic scaffold is essential for providing an optimal
environment for fibrocartilage formation. In addition to supporting the growth and
differentiation of relevant cell types, the scaffold for interface tissue engineering must direct
heterotypic and homotypic cell-cell interactions to promote multitissue formation and the
maintenance of controlled matrix heterogeneity. Consequently, the scaffold should exhibit a
gradient of properties mimicking those of the native insertion zone. The interface scaffold must
be degradable and must exhibit mechanical properties comparable to those of the ligament
insertion site. It should also be adaptable to current ACL reconstruction methods or should be
preincorporated into the design of replacement grafts in order to facilitate in vivo graft
integration. A triphasic scaffold (Fig. 9A) with one region (Phase A) intended for soft tissue,
Phase B intended for fibrocartilage region, and Phase C designed for bone formation is an
example of a biomimetic scaffold for engineering a tissue interface. Through the interaction
of osteoblasts, chondrocytes, and fibroblasts on this triphasic scaffold with a gradient of
material properties, it is anticipated that a fibrocartilage-like interface may be formed in the
intermediate region, Phase B, under appropriate physical and chemical stimulation.
The interactions of osteoblasts and fibroblasts on the triphasic scaffold and the feasibility of
this scaffold for interface tissue engineering have been evaluated in vitro83 and in vivo.84
Specifically, fibroblasts and osteoblasts were seeded onto Phase A (polymer fiber mesh without
Ca-P) and Phase C (polymer-ceramic composite with Ca-P85), respectively, whereas Phase B
(polymer with lower amount of Ca-P than Phase C) was left unseeded. The migration of both
cell types into Phase B was monitored over time, and it was observed that fibroblasts (Calcein
AM, green) and osteoblasts (CM-DiI cell tracer, red) were localized primarily at the opposite
ends of the scaffold after initial seeding (Fig. 9B-i), with very few cells found in Phase B. After
4 weeks of culture, the fibroblasts and osteoblasts proliferated within their respective phases
and both cell types migrated into Phase B (Fig. 9B-ii). The controlled cell distribution resulted
in elaboration of matrix specific to each cell type on the relevant phase of the scaffold. A
mineralized matrix was detected in Phase C only, with extensive type I collagen matrix
deposition observed on both Phase A and Phase B.
When the triphasic scaffold cultured with osteoblasts and fibroblasts was evaluated in a
subcutaneous athymic rat model, abundant tissue formation was found (Fig. 9C). The
production of ECM compensated for the decrease in mechanical properties of the
biodegradable scaffold and, more important, controlled matrix heterogeneity in vivo.
Optimization studies are currently under way with the ultimate goal of developing integrative
fixation devices for use in soft-tissue reconstruction procedures.
Helen Lu summarized that the integrative graft solutions would be a significant component of
functional tissue engineering.86,87 Building upon the foundation of tissue engineering
methodologies developed in the past two decades, innovative approaches have been created to
address the challenges of biological fixation of soft-tissue grafts. The success of interface tissue
engineering will likely depend on a thorough understanding of the structure-function
relationships existing at the native insertion site, and the elucidation of the mechanisms
governing interface regeneration. The successful regeneration of the soft tissue-to-bone
interface will augment graft function and enhance translation potential of engineered grafts.
TISSUE ENGINEERING OF ARTICULAR JOINT SURFACE INTERFACES

Rita Kandel from the University of Toronto discussed the need for, and the potential and
challenges related to, the recreation of the cartilage-bone interface, in the context of tissue

engineered joint repair. Resurfacing the articulating surfaces of synovial joints with synthetic
prostheses is still a treatment of choice for end-stage diseases. She argued that though primary
joint replacements implanted since the late 1980s have shown reasonable success, failure rates
of up to 40% after 10 years have been reported depending on the type of implant.90–93 It has
been speculated that all joint-replacement implants made of synthetic materials will need
replacement if the patient lives long enough. Recent efforts have focused on developing new
treatment methods that will result in biological repair and preclude the need for non-degradable
synthetic materials. One of these approaches entails the use of tissue engineering methods to
regenerate articular cartilage either in vitro or in vivo.89,94
General requirements
The key to successful bioengineering of articular joints will be to generate implants that will
address the contrasting needs of the two different tissues, cartilage and bone, as well as the
cartilage-bone interface. Bone comprises well-organized trabeculae, consisting of mineralized,
mostly type I collagen matrices. In contrast, the ECM of articular cartilage is composed
predominately of water and type II collagen fibers admixed with large aggregating
proteoglycans. The deep aspect (adjacent to bone) of the cartilage is mineralized and this zone
anchors the hyaline cartilage to the underlying subchondral bone. In addition to this interfacing
function, the calcified zone is involved in the transmission of forces across the joint.95,96 The
stiffness of the calcified cartilage layer is greater than that of the nonmineralized hyaline
cartilage but is an order of magnitude less than that of bone, most likely due to the arrangement
of collagen fibers.97 It has been hypothesized that this stepwise transition in stiffness is
important for the proper functioning of cartilage and distribution of forces.97 It is well known
that repair of joint defects with fibrocartilage, which does not have the organization and
composition of hyaline articular cartilage, will degrade over time as it does not have sufficient
load-bearing capability.88,98 As the goal of cartilage tissue engineering is to generate articular
cartilage that will function similar to the native tissue, it is likely that regeneration of cartilage
with both of these zones will be required.
Biphasic tissue constructs

A variety of methods have been proposed to repair osteochondral defects. One way to
bioengineer these articulating surfaces is to generate biphasic constructs composed of cartilage
tissue overlying and integrated with a substrate that serves as the bone interfacing component
(osteochondral-type biphasic constructs).99–114 Similar to scaffolds, substrates are porous and
usually biodegradable.115,116 However, in contrast to scaffolds, the cells or tissue are placed
on the top surface and not seeded throughout the substrate. As a result, the regenerated cartilage
is anchored to the intended articulation surface of the substrate. The substrate both supports
cartilage formation and facilitates fixation after implantation by in-growth of bone into the
pores. Important characteristics of this substrate, if it is used to support cartilage formation in

vitro prior to implantation, are (i) it is fully porous, or chondrocytes will not form cartilage,
and (ii) the size and organization of the pores allow fluid flow but will not permit (or severely
restrict) cell infiltration into the full thickness of the substrate. Substrates utilized for these
types of constructs have been made from a variety of natural and synthetic materials, but as
the substrates are meant to be bone interfacing they must have sufficient strength for weight
bearing.99–114
A porous ceramic substrate composed of calcium polyphosphate (CPP), which has mechanical
properties approximating cancellous bone, has been developed.117 To generate the biphasic
construct, articular chondrocytes are placed on the articulation surface of the CPP substrate.
As the cartilage forms in vitro, the developing tissue fills and thereby integrates with the top
portion of the substrate (Fig. 10). The cartilage tissue contains type II collagen and large

proteoglycans.118 The CPP is biodegradable, and as the biomaterial is made of calcium and
phosphate the breakdown products do not incite an inflammatory reaction.119
Potential for cartilage repair

This type of biphasic construct, in which the cartilage tissue is formed prior to implantation,
has the potential to repair focal cartilage defects. As the cartilage is already formed, lateral
integration to the adjacent host cartilage is possible immediately upon implant insertion. As
the substrate (CPP) is porous, bone can grow into the pores after implantation, resulting in
secure implant fixation, and because it is biodegradable the CPP will ultimately be replaced
by bone. This approach allows generation of cartilage with a calcified zone.120–123 By using
chondrocytes isolated specifically from the deep zone of articular cartilage, it is possible to
generate cartilage with a mineralized zone adjacent to the bone substitute (Fig. 10). The mineral
that forms under these conditions is hydroxyapatite, similar in composition and size to that
present in the native calcified cartilage zone.122 The presence of a mineralized interface
improved the compressive properties of the tissue as the equilibrium modulus increased
approximately eightfold and the equilibrium stress increased twofold compared to cartilage
without a mineralized zone. The interfacial shear strength (measures strength of attachment of
the in vitro-formed cartilage to the substrate) also increased as the energy to failure increased
twofold (unpublished data). As most tissue engineering methods to date generate cartilage with
only a fraction of the mechanical properties of the in vivo tissue, the above approach
demonstrates the importance of bioengineering cartilage with an organization that mimics the
native cartilage.124
Rita Kandel summarized that the most important challenge is identifying a cell source that will
provide sufficient numbers of cells capable of forming hyaline cartilage large enough to repair
clinically relevant defects, and still having the capability to form cartilage with an upper non-
mineralized zone and a deep mineralized zone. Articular chondrocytes are phenotypically
unstable when grown in monolayer culture, and the cells can dedifferentiate to fibrocartilage-
type cells with even one passage.125 So, if the approach is to expand the cell number of
differentiated chondrocytes in culture, it will be necessary to identify cell culture conditions
that will allow for cell proliferation while still maintaining chondrocyte phenotype.126
Alternatively, one can design materials with appropriate characteristics so that when the
passaged cells are placed on the material it would induce the cells to redifferentiate toward
articular chondrocytes. Nanomaterials or “smart” materials may be more suitable for this
approach.127,128
Another approach currently being explored is the use of mesenchymal progenitor cells (MPCs).
Most of the work focusing on this cell type and its role in tissue engineering is preliminary, in
part because of our limited understanding of the biology of these cells and because the markers
used to separate out cells that can differentiate to chondrocytes from a mixed cell population
have not been well defined. However, MPCs after expansion and seeding into a scaffold are
able to differentiate toward chondrocytes under the appropriate culture conditions, but whether
they can generate tissue with the two zones is not known.129,130 The biphasic (osteochondral-
type) constructs can be created in a shape individualized for the defect, the soft tissue–hard
tissue interface can be re-created prior to implantation, and they should be relatively easy to
implant if designed appropriately.
HEART VALVE TISSUE ENGINEERING AND REGENERATION RESEARCH

The immense need for cardiovascular tissue engineering and regeneration has generated
considerable interest and investigation in this field.131–135 Frederick Schoen from Harvard
Medical School stressed that the engineering of tissue requires an understanding of the
relationships of structure to function in normal and pathological tissues (including mechanisms

of embryological development, and functional tissue biomechanics and other structure-

function correlations) and the ability to control cell and tissue responses to injury, physical
stimuli, and biomaterial surfaces.
Dr. Schoen described an approach to tissue engineering of heart valves based on concepts and
data derived from valve anatomy, physiology, development, remodeling, response to injury,
and substitution. He stressed that surgical repair or replacement of a diseased heart valve is a
common procedure (over 85,000 cases per year in the United States and 285,000 worldwide),
and the tissue of the heart valves cannot regenerate spontaneously. Although valve surgery
generally leads to enhanced survival and quality of life, presently available valve substitutes
provide imperfect functional restitution and have potential complications.136,137 Moreover, in
pediatric applications, where although physiologically corrective procedures can be
successfully performed, repairs of congenital deformities require very small valve sizes, and
subsequent (and repetitive) operations may be needed to accommodate growth of the patient,
even in cases where typical prosthesis-associated complications have been prevented. The goal
of heart valve tissue engineering is to overcome the limitations of contemporary valve
substitution methods by creating or regenerating a living valve replacement that functions well
hemodynamically, repairs ongoing tissue damage, and has long-term durability and growth
potential similar to the natural heart valves.138–141
Functional structure of heart valves: Role of valvular ECM and cells

The aortic valve (the most extensively studied, most frequently diseased, and most widely
transplanted valve) best illustrates the essential relationships of structure to function. The aortic
valve cusps open to form an obstruction-free orifice during ventricular systole and close rapidly
and completely in diastole. Although the pressure differential across the closed valve imposes
a large load on the cusps, regurgitation and cuspal prolapse are prevented by substantial
coaptation of the cusps, which is enabled by a microscopically inhomogeneous architecture
with three well-defined cellular tissue layers.142,143 Nearest to the outflow surface is the
fibrosa, composed predominantly of circumferentially aligned, densely packed collagen fibers,
largely arranged parallel to the cuspal free edge. The fibrosa provides strength and stiffness,
and minimizes sagging of the cusp centers. The central layer of the aortic valve cusp,
spongiosa, is composed of loosely arranged collagen and abundant glycosaminoglycans. This
layer accommodates the dynamic shape changes of the cusp during the cardiac cycle, lubricates
relative movement between ventricularis and fibrosa layers, and absorbs shock during closure.
The thin layer near the inflow surface, ventricularis, is rich in radially aligned elastic fibers,
and this enables the cusps to recoil and have minimal surface area when the valve is open, but
stretch in response to back-pressure of blood in the closed phase. The valves are lined by a
confluent layer of valvular endothelial cells (VEC); deep to the surface are valvular interstitial
cells (VIC).
Studies of normal, pathological, and substitute valves have demonstrated that the principal
determinant of valve durability is the valvular ECM, whose quantity and quality depend on
viability and function of VIC.144 The VIC comprise a population of resident cells of diverse
and dynamic phenotypes, largely along a spectrum of fibroblast-like to myofibroblast-like.
145–148 The VIC synthesize the valvular ECM molecules and express matrix degrading
enzymes such as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) that mediate
ECM remodeling and repair.149
In normal valves, VIC are fibroblast-like (i.e., immunoreactive to vimentin but not to smooth
muscle actin [SMA, a marker of myofibroblast function150,151], SMemb, or MMP-13). Indeed,
only 2–5% of VIC of normal intact valves express SMA.148 In contrast, previous in vitro studies
using isolated cells cultured from heart valves demonstrated that 56–78% of cells are SMA
positive.142 The higher percentage of myofibroblasts observed when VIC are removed from

the intact valve (i.e., and placed in culture) suggests that removal from the normal tissue
environment stimulates VIC growth. Moreover, treatment of isolated VIC with transforming
growth factor (TGF) strongly activates VIC to the myofibroblast phenotype.152
Cardiac valve development, maturation in utero, and postnatal changes

During normal development of the heart, the valve cusps or leaflets originate from
mesenchymal outgrowths known as endocardial cushions.153,154 Some endothelial cells in the
cushion-forming area that initially line the internal cardiac surface undergo endothelial to
mesenchymal “trans-differentiation” and migrate from the luminal surface to the
subendocardial ECM.155,156 These cells remodel the cushions into leaflets and cusps, in a
process also stimulated by TGF.157
The mechanisms of developmental maturation of heart valves were studied by analyzing human
semilunar valves obtained from fetuses at 14–19 and 20–39 weeks of gestation and neonates
24 hours to 30 days old, and comparing these valves with those of normal adults.158 Fetal VIC
showed a high proliferative index, an activated/myofibroblast-like phenotype (indicated by
SMA expression) and MMP-collagenases, indicating active matrix remodeling. In contrast,
cell turnover and VIC activation were low in adult valves. A trilaminar architecture, evident
by 36 weeks of gestation, was rudimentary compared to that of adult valves. Moreover, collagen
content increased during valve maturation but not subsequently, although collagen fiber
alignment was higher in adult than fetal valves.
Valve adaptation and repair

The VIC become activated and mediate functional biomechanical adaptation of valves when
exposed to an altered mechanical environment.148,159 Although the specific regulatory
mechanisms are unknown, this activation is potentially reversible, and when equilibrium is
achieved, the cells return to a quiescent state.148 It is thought that VIC phenotype is regulated
predominantly by the wall stresses in the valve leaflets, analogous to remodeling in cardiac
hypertrophy and vascular walls.160–162 Mechanical and chemical stimulation may be
synergistic.163
A unique opportunity to study cellular phenotypic changes and ECM remodeling of valves in
response to abruptly altered mechanical loading in vivo is provided by clinical replacement of
a diseased aortic valve by transplantation of the patient’s own pulmonary valve into the aortic
position (Ross procedure) done in some pediatric and young adult patients with aortic valve
disease.164 Analysis of the structure and interstitial cell phenotypes in clinical pulmonary-to-
aortic valve transplants early (2–10 weeks) and late (3–6 years) postoperatively showed near-
normal cuspal structure and collagen architecture, and viable VIC.165 In early autograft cusp
explants, VIC resembled myofibroblasts (19% had positive SMA) and had strong expression
of MMP-13, indicative of active ECM remodeling. Subsequently, there was late normalization
of VIC phenotypes toward those of normal aortic valve, with only 6% of VIC in late explants
and 5% in normal aortic valves expressing SMA.
Physiological wound healing in mitral valves is also associated with phenotypic modulation
of VIC from fibroblasts to myofibroblasts.166 Moreover, VIC in mitral valve leaflets from
patients with myxomatous mitral valve degeneration also express features of myofibroblasts,
suggesting that this disease comprises a nonequilibrium state, consistent with the progressive
course of this condition in many patients.167
The data summarized above on the dynamic VIC phenotypes that occur in several
circumstances suggest the following general mechanistic paradigm by which cardiac valvular
tissue is dynamically and reversibly responsive to environmental conditions, particularly

mechanical loading: (i) Under equilibrium conditions, the majority of VIC are quiescent
fibroblast-like cells, (ii) Altered mechanical loading (as in valve development, adaptation to
changes in the local mechanical environment and in pathologic states) stimulates VIC to
become activated and mediate connective tissue remodeling, and (iii) When equilibrium is
restored, the cells return to the quiescent state.
Studies of contemporary bioprosthetic heart valve substitutes

Structural dysfunction is the major cause of failure of the most widely used bioprostheses
(flexible-stent-mounted, glutaraldehyde-preserved porcine aortic valves and bovine pericardial
valves). Within 15 years following implantation, 30–50% of porcine aortic valves implanted
as either mitral or aortic valve replacements require replacement because of primary tissue
failure. Cuspal mineralization and noncalcific damage to the cuspal structural matrix are the
major responsible pathologic processes.168,169 Calcification is markedly accelerated in
younger patients. The mechanism of valve calcification involves the reaction of calcium
derived from plasma with organic phosphorous residing in the cross-linked, nonviable cells of
the preserved valve.170,171 Alterations in tissue structure and chemistry of bioprosthetic valves
that occur during valve fabrication and manufacture can contribute to the characteristic failure
modes as follows: (i) The microstructure of the cusps is fixed in a static geometry characteristic
of one phase of the cardiac cycle, inducing abnormal tissue stresses during function, (ii)
Damage to endothelial coverage allows penetration of plasma and inflammatory cells into the
cusps, (iii) Bioprosthetic valve VIC are nonviable due to the chemical pretreatments; without
viable cells, synthesis of collagen or other ECM components cannot occur, and structural
damage can accumulate.
Conceptual approach to heart valve tissue engineering

The rationale and design criteria for tissue engineered heart valves (TEHV) derive from five
key concepts of functionally adaptive valvular remodeling or regeneration established over the
past decade from studies in many laboratories of normal, developing, and diseased heart valves;
bioprosthetic valves; and other tissue valve substitutes: (i) The highly specialized arrangement
of collagen and other ECM components of heart valves (particularly elastin and proteoglycans)
enable physiological function and extended durability, (ii) The quality of valvular ECM
depends on the ability of VIC to modulate their phenotype and function and thereby mediate
valvular adaptation to different environments, (iii) Structural deterioration of native and
substitute valves is mediated by chemical and mechanical damage to collagen, (iv) Cell
viability in nearly all current bioprosthetic tissue valve substitutes is compromised or
completely eliminated during processing; thus, ECM damage that occurs following
implantation of current bioprosthetic valves cannot be repaired, (v) The long-term success of
a tissue engineered (living) valve replacement will therefore depend on the ability of its cellular
components to assume normal function with the capacity to repair structural injury, remodel
ECM, and potentially grow.
In the general paradigm of tissue engineering,12 cells are seeded on a synthetic polymer or
natural material that serves as a scaffold, and then a tissue is matured in vitro (bioreactor) until
proliferating cells produce a sufficient amount and quality of ECM. The tissue thus formed is
called a construct. In the second step, the construct is implanted in the appropriate anatomic
location, where further remodeling in vivo may occur to recapitulate normal functional organ
or tissue architecture. Alternatively, cardiovascular tissue engineering approaches can omit a
step or steps in the general paradigm by taking advantage of specific properties of initial
components (cell or scaffold) and endogenous cells. For example, in the cell-seeded scaffold
model, a confluent endothelial coverage could be placed on a cardiovascular prosthesis at the
time of implantation.172,173 The cell transplant model used in experimental and early clinical
trials of cell-based therapy for myocardial tissue repair uses cells injected at a specific tissue

site, in the hope that these cells will differentiate and proliferate, and/or stimulate repair by
functional tissue.174 More recent approaches to valve and vascular graft engineering have
utilized scaffolds designed to attract endogenous cells to repopulate and remodel a
decellularized matrix.175,176 Irrespective of the methodology, the key processes occurring

during the in vitro and in vivo phases of tissue formation and maturation are (i) cell adhesion,
proliferation, sorting and differentiation; (ii) ECM production and organization; (iii)
degradation of the scaffold; and (iv) remodeling and potential growth of the tissue. Key targets
for characterizing tissue engineered constructs include tissue composition, cellular gene
expression and phenotype, ECM, key effectors of tissue remodeling, and tissue quality.
Engineered heart valves constructed from biodegradable polymer scaffolds

Composite trileaflet valve-wall constructs fabricated from poly-4-hydroxybutyrate-coated
polyglycolic acid scaffolds seeded with autologous bovine endothelial and carotid artery
medial cells were cultured in vitro up to 14 days in a bioreactor that mimicked valve cyclical
motions by providing pulsed flow of cell culture medium at physiological pressure.148,177 The
ECM produced in this construct was predominantly proteoglycan, and collagen accumulation
(detected by picrosirius red staining observed under polarized light) was limited to a few weak
birefringence fibrils. Elastin was not detected histologically. Immunohistochemical staining
revealed that cells in the construct were activated myofibroblasts, as determined by strong
expression of SMA (microfilaments) and MMP-13 coexpression.
The constructs were implanted as pulmonary valves in lambs. Valves functioning for and
explanted at 4–20 weeks demonstrated the dynamic changes of VIC phenotype and ECM in
tissue engineered valve explants toward layered architecture and cellular configuration of
native valves. Some cells in explants at 4–8 weeks showed weak staining for SMA, but all cells
throughout the cusp stained strongly for MMP-13. Explants at 16–20 weeks had layered
leaflets, with fibroblast-like cells, having undetectable SMA; some cells were still positive for
MMP-13. Leaflets were partially covered with cells that stained positive for von Willebrand
factor, which is characteristic of endothelial cells. Movat stain demonstrated collagen in the
fibrous layer on the aortic (outflow) side, proteoglycans in the central loose layer, and elastin
near the ventricular (inflow) side. The structure resembled that of native valve. These studies
showed that an engineered tissue valve could be implanted into and function in the pulmonary
artery, and that the remodeling of these valves recapitulated features of tissue development,
response to injury and wound healing.
Harnessing the reparative potential of circulating endogenous cells

There is now accumulating evidence that circulating endogenous cells with the potential of
differentiating into vascular cells can be recruited in vivo from the bone marrow to sites of
biomaterials (and potentially unseeded scaffolds) or natural cardiovascular tissues. Endothelial

progenitor cells (EPCs) promote endothelial regeneration in dog models by covering implanted
Dacron grafts178 and in human models by covering implanted ventricular assist devices.179
Coating a nonseeded scaffold with appropriate cell-signaling molecules to encourage EPC
adhesion is an exciting opportunity. Recent evidence suggests that bone marrow could be a
source of progenitor cells contributing to smooth muscle-like cells in the vasculature, including
heart valves.180,181
Inflammatory cells may also play a role in mediating remodeling of a scaffold implanted in
vivo without prior cell seeding. In one experiment on pigs, each of the four pigs used had one
pulmonary valve leaflet excised and replaced with a leaflet constructed from porcine small
intestinal submucosa (SIS).182 Histology indicated that the implanted matrix was progressively
resorbed and replaced by fibrous connective tissue that had features of adult valve.
Alternatively, a biodegradable graft containing collagen microsponge was fabricated and

tested, with and without preseeding.183 In both cases (SIS and collagen microsponge) there
was no thrombus formation, the scaffold was absorbed, and there was endothelialization,
parallel smooth-muscle cell alignment, elastin, and collagen fibers. These results suggested
that the patch promoted in situ cellularization and regeneration of autologous tissue. However,
an important limitation of this study was that the implanted patches were small; cellularization
of a large patch may be less efficient.
Nevertheless, clinical applications of decellularized heart valves have been largely

unsuccessful. Simon et al. used SynerGraft decellularized porcine heart valves as homograft
valve replacements in the right ventricular outflow tract during Ross procedures in children.
184 Many valves failed, and postimplant examination of failed valves revealed incomplete
decellularization, lack of cell repopulation, lack of endothelialization with severe
inflammation, fibrous sheath formation inside and outside the graft, foreign body reaction,
calcification, and severe degeneration of leaflets and wall. Histological examination of
decellularized porcine Syner-Graft valves (Cryolife Kennesaw, GA) implanted without in
vitro preseeding for 6 months in sheep suggested only minimal growth of host cells on intact
leaflets and showed a lack of calcification.185
Challenges and future directions in clinical translation

Heart valve tissue engineering has exciting potential but many challenges and questions
remain. Early studies focused on the design of individual valve leaflets.186 As the field of tissue
engineering evolves, attempts have been made to transition from the design of separate
structures to the design of complete valved conduits.177 Importantly, the remodeling of a single
cusp attached to a native arterial wall may be different from those in a valved conduit.
There is a need to develop guidelines characterizing the safety, efficacy, and quality of tissue
engineered products (be they heart valves, other cardiovascular implants, or prostheses in other
clinical applications) before they can be implanted in humans. Challenges in tissue
characterization for heart valve tissue engineering are summarized in Figure 11. The major
research goals are to understand mechanisms, define animal models, develop biomarkers,
develop assays/tools, and define surrogate and true end points. The major clinical goals are to
characterize and assure control of quality of tissue constructs, accommodate patient-to-patient
heterogeneity in tissue remodeling, and predict outcomes as early as possible.
Demonstration of long-term efficacy (implantability, functionality, long-term performance)

and safety (biocompatibility, durability, modes of failure, ease of monitoring) of these valves
in humans will be a particular challenge. Since current heart valve replacements serve their
recipients predictably and moderately well in most patient populations (except children),
acceptance of tissue engineering by the surgical community may be slow. Indeed, a leading
cardiologist suggested that surgeons should consider the use of a tissue engineered valve in a
patient (beyond appropriately controlled clinical research) only when the 15-year lifetime of
conventional valve substitutes can be conclusively and predictably demonstrated.187 This will
be a substantial challenge since results from available animal models of bioengineered tissue
may not necessarily translate directly to humans, and the most suitable animal model for testing
TEHV has not yet been determined.
A key consideration is that in vivo behavior of TEHV may display considerable variability
among recipients, owing to intrinsic variation in inflammation and remodeling potential among
individuals.188–191 Simply stated, some patients might inadequately remodel their tissue
engineered valves, and the consequences could be disastrous. This issue is conceptually
analogous to the emerging area of pharmacogenomics,192 which shows that individual patient
characteristics (for example, genetic mutations or polymorphisms) have a profound influence
on drug metabolism outcome in some patients. Indeed, it has been shown that individuals with

genetic defects in coagulation proteins may be unusually susceptible to thrombosis of prosthetic

heart valves.193
To understand, monitor, and potentially control patient variability in wound healing and tissue
remodeling in vivo, tissue biomarkers measurable by chemical or molecular assay or molecular
imaging that predict success and failure of an engineered tissue must be identified. These
biomarkers may be employed in preclinical in vitro and in vivo experiments and clinical studies
using molecular tissue imaging,194,195 to track the presence, migration, proliferation, and
function of bone marrow–derived endogenous progenitor cells and cells used to preseed
scaffolds. Imaging of magnetically labeled MSCs injected into porcine myocardium has been
performed in vivo,196 a technique that can potentially be expanded to study magnetically
labeled EPCs and MPCs seeded on a scaffold and implanted into an animal or human model.
In vivo molecular imaging has been used to demonstrate key enzymatic and cellular events in
atherosclerosis and thrombosis.197,198 Molecular imaging can probe polymorphisms of ECM
gene expression in vivo in models of wound healing199 and cardiovascular disease.200,201
These studies can potentially be translated to perform real-time in vivo characterization of
scaffold matrices (either preseeded or with the potential of attracting endogenous cells)
implanted in animal models. Moreover, relevant biomarkers might be measured distant from
the tissues in which they are generated; for example, researchers are currently working to
identify serum-specific biomarkers of ECM remodeling in diseases, such as MMPs in acute
coronary syndromes,202 and urine-based biomarkers in early cancer progression.203
Frederick Schoen concluded that the assessment of bioactive and engineered implants will
require a broadened scope of the concept of biocompatibility, and that the approaches employed
in implant retrieval and evaluation will necessitate the identification of tissue characteristics
(biomarkers) that will be predictive of (surrogates for) success and failure. Although
considerable validation of these concepts and enabling technological advances will be
necessary, a most exciting possibility is that such biomarkers may be used to noninvasively
image or monitor the maturation or remodeling of engineered devices in individual patients
through bioassay or molecular imaging technology.
BIOPRESERVATION FOR TISSUE ENGINEERING

Mehmet Toner from Harvard Medical School addressed the critical dependence of tissue
engineering on the availability of large numbers of cells for growth and culture, and the related
need for cryopreservation. As the field expands, and the need for cells continues to grow,
preservation and storage of living cells for transport or later use will become increasingly
important. He reminded the audience that the field of cryopreservation has been active for more
than 50 years,204 with the primary goal to move toward the preservation of whole organs. This
goal has proven extremely difficult to achieve and failure to make significant progress has led
to disappointment, but, recently, with the emergence of tissue engineering and cell-based
therapies, cryopreservation research has been reinvigorated.
Cryopreservation and tissue engineering

From a tissue engineering perspective, the role of cryopreservation is not necessarily to
preserve whole organs, but to preserve the cells necessary to create tissue engineered products
as well as engineered tissues (Fig. 12). Cryopreservation research has led to the successful
preservation of a large number of cell types. Successful freezing protocols for most cells rely
on a combination of controlled freezing rates and the addition of cryoprotectant chemicals.
Somewhat surprisingly, however, there are many cells types that have not been successfully
preserved despite many years of effort. Illustratively, red blood cells have been successfully
preserved and used clinically for 50 years, while there are still no clinically viable techniques
for the long-term preservation of platelets and granulocytes. Cryopreservation techniques that

have been successful for one cell type have proven to be difficult to adapt to other cells types.
These problems have been compounded as the field has attempted to move into the preservation
of tissues or whole organs. In order to support the growth of tissue engineering, the
cryopreservation field, or the broader field of biopreservation, must create methods for the
long-term preservation of any cell type, and ultimately of complex tissues.
Cryoprotectants—The accepted paradigm for successful cryopreservation is to avoid ice

formation inside the cell during cooling. This is most easily accomplished using slow-freezing
techniques. In slow-freezing protocols the cells are cooled at a controlled rate and ice is usually
formed, or nucleated, outside the cells at a specific temperature. The formation of ice causes
an increase in the osmolality of the extracellular solution because ice rejects solutes as it forms,
and the cells shrink. As the cooling continues the cells continue to shrink, and because there
is very little water inside the cells, ice does not form within the cellular interior. The intracellular
solution eventually either forms a eutectic solid or enters into a glass phase. Unfortunately, this
massive cellular shrinkage and the increase in extracellular electrolyte concentration may lead
to cell death.205–207 To prevent this, cryoprotectants, usually glycerol or dimethyl sulfoxide
(DMSO), are added. These chemicals enter the cell, thereby reducing cellular shrinkage, and
somewhat dilute the electrolytes. In general, however, if cells are frozen slowly they die, even
with the addition of cryoprotectants.
In order to prevent cell death, the cells must be allowed to dehydrate, thereby reducing the
amount of intracellular water, and then they must be frozen quickly to avoid excess shrinkage
and damage from high electrolyte concentrations. Most successful cryopreservation protocols
use a period of slow cooling and the introduction of cryoprotectants to induce some cellular
shrinkage, followed by fairly rapid cooling to liquid nitrogen temperatures. If the cooling is
too fast, so that the cells do not shrink, then ice forms inside the cells and they die. If the cooling
is too slow, then the cells shrink too much and die from dehydration and electrolyte build-up.
If it is just right, the cells may survive.208 The three sources of cellular damage—dehydration,
intracellular ice formation, and cryoprotectant toxicity—are all independent. There is no
guarantee that for any particular cell type a cooling rate exists that will lead to high levels of
viability. A cell that is very sensitive to dehydration effects may have low viability at cooling
rates that extend right up to the point where ice starts to form inside the cells, which can kill
them. Some cells are very sensitive to cryoprotectants and cannot tolerate even low
concentrations of glycerol or DMSO.
Cooling rates—The rate at which cells must be cooled in order to induce some, but not too
much, shrinkage is highly dependent on cell type.209 For highly permeable cells such as
erythrocytes, the optimal cooling rate is more than 1000°C/min, but for relatively impermeable
stem cells the optimum is about 1°C/min. Determination of optimum cooling rates has been a
highly fruitful area of research, both empirically and theoretically.210 The original concept for
defining an optimum was proposed by Mazur211 as the fastest cooling rate possible that would
not promote intracellular ice formation. He proposed that intracellular ice would not form if
the cell had less than 10% of its initial water when it entered the ice nucleation zone of
temperature. These values were refined to 5% of the initial water content when the cell reached
−30°C.212 Based on this definition and the water transport equations originally developed, a
theory for determining optimum cooling rates for cell survival was developed.211,212 This
enabled prediction of optimal cooling rates that agreed very well with experimental values for
a wide variety of cells, based solely on knowledge of the cell membrane permeability and the
cell’s initial surface area to volume ratio. However, the fact that the optimum value is cell-type
dependent means that there is no one preservation protocol that will work for all cells. Also,
as we noted earlier, for many cells there is no protocol of this type that will work at all.209

Vitrification—Another method for avoiding ice formation inside the cell is to vitrify the entire
sample, that is, have the entire sample enter the glass phase with no ice inside or outside the
cells. There are two methods for achieving vitrification. The first method is to freeze the sample
very fast. If water is frozen fast enough, 1,000,000°C/s, it will form a glass phase even without
the addition of cryoprotectants.213 For single cells and small samples this has been done
successfully,209,214 but scaling techniques of this type for large samples or even large numbers
of small samples may be impossible. The second, more widely used, technique involves the
use of high concentrations of cryoprotectant chemicals so that ice will not form no matter how
slowly the sample is cooled. For instance, solutions containing 41–50% of propanediol will
form a glass phase when cooled at any rate without forming ice.215 Since ice is not formed
anywhere in the sample, the cells do not shrink when they are cooled. They may shrink if
cryoprotectants that do not enter the cell are used, and they usually are, but this shrinkage is
controlled by the amount of chemicals added. Vitrification protocols, therefore, do not depend
on cooling rate—which eliminates two of the three sources of damage in traditional freezing
protocols. This also enables preservation of large tissue samples, at least in theory. Cooling
large tissues at a uniform rate is impossible unless very slow cooling rates are used, but for
vitrification protocols, very slow cooling rates can be used and uniformity of cooling is not so
important. For these reasons, vitrification protocols are regarded by many as most promising
for the successful preservation of whole organs.216
The problem with vitrification protocols is that they require very high concentrations of
cryoprotectants, much higher than the concentrations used in freezing protocols. The mere
addition and removal of the chemicals can kill the cells due to osmotic stress, and the chemicals
are toxic.215,217,218 It seems clear that many cell types will never be successfully vitrified
unless new, nontoxic cryoprotectants can be found, and it is not clear that this will even happen.
Vitrification may ultimately be effective for the storage of certain organs whose cells can
tolerate the required chemicals, but it is unlikely to be the best method for preserving the many
different cell types the field of tissue engineering will require.
Challenges ahead
Research continues along the traditional lines of vitrification and freezing protocols that
attempt to prevent intracellular ice formation, but new research is moving in slightly different
directions. Recent work has shown that ice formation is strongly enhanced by cell-cell
interactions.219,220 This suggests that efforts to prevent intracellular ice in complex tissues
may be even more difficult than expected, since ice formation in even one cell of the tissue
could trigger intracellular ice formation in surrounding cells or throughout the system. On the
other hand, work by Acker and coworkers has shown that ice that forms inside a cell due to
interaction with a neighboring cell is not lethal.221,222 This complements some earlier studies
by Rall,223 which showed that the formation of intracellular ice is not always a lethal event.
These studies suggest that future cryopreservation research might aim at controlling the damage
caused by the formation of intracellular ice rather than its prevention. If this damage is
prevented then faster cooling rates could be achieved with lower concentrations of
cryoprotectants.
Other research is moving away from cryopreservation. Within the last 5 years there has been
significant research on the various forms of anhydrobiotic preservation—preserving cells in a
dried state. In 1992 Goodrich et al. reported the successful recovery of functional erythrocytes
after freeze drying.224 Using trehalose and sucrose to stabilize membranes and to promote glass
formation, researchers have been able to preserve a variety of cells using air drying and freeze
drying techniques.225–227 There are two potentially significant advantages of drying
techniques as a form of biopreservation for tissue engineering. The first is ease of storage and
transport. Samples preserved in a dry state can be stored at or near room temperature. The

convenience of off-the-shelf availability for the cells used in tissue engineering and for tissue
engineered products is significant. In contrast, cryopreserved cells must be maintained at liquid
nitrogen temperatures, which makes storage expensive and transport difficult. The second
advantage of drying techniques is that the major additives used to promote survival of dried
cells are sugars, usually trehalose or sucrose, which are nontoxic. This means that many cell
types tolerate the addition of the sugars and that the sugars do not need to be removed before
cell culture and clinical use. Drying techniques, therefore, may be applicable to cell types that
do not tolerate traditional cryoprotectants.
Mehmet Toner concluded that the field of tissue engineering has allowed the biopreservation
field to refocus. The long-held goal of whole-organ preservation remains important, but of new
importance is the need to preserve cells of all types (including stem cells), even in small
numbers, and engineered tissues. New techniques such as dried preservation, largely untested
for most cell types, may not be applicable to whole organs, but may provide substantial benefits
to tissue engineering. Techniques that are widely applicable to many important cell types do
not yet exist, and it is not even clear which class of techniques will likely provide answers in
the future. The traditional techniques of controlled freezing and vitrification have not yet
yielded the desired results. A new breakthrough in biopreservation may be necessary to meet
the full needs of the tissue engineering field.
TEMPORALLY AND SPATIALLY REGULATING GROWTH FACTOR

AVAILABILITY
David Mooney from Harvard University discussed the process of angiogenesis as a universally
relevant and useful model to study the relation of growth factor delivery to tissue regeneration,
and stressed the need for control over the spatial and temporal availability of individual or
combinations of factors. He reviewed the process of angiogenesis and what has been learned
from past efforts to promote angiogenesis with growth factor delivery, polymeric sustained
delivery systems, and certain key issues to address in future studies, as well as the potentially
broad importance of this topic for tissue engineering.
Polymeric systems allow one to regulate the tissue concentration, duration of signaling, and
simultaneous or sequential availability of multiple factors. These levels of control enable the
creation of networks of new blood vessels with controlled maturity and increased functionality
in rodent models. However, the utility of these systems must be evaluated in larger animals
and humans in the future, and, importantly, a quantitative understanding of growth factor
signaling and its dependence on the host environment will likely be required to exploit these
systems therapeutically. Systems that can provide desirable spatial and temporal signaling of
growth factors could find utility in virtually all tissue engineering and regeneration
applications.
Morphogens and growth factors regulate developmental, disease, healing, and regeneration
processes. The spatial and temporal availability of these molecules is integral to their ability
to regulate these processes and control the pattern of tissue formation. One type of tissue
formation in which this interplay has been extensively investigated is new blood vessel
formation (neovascularization). The cardiovascular system is the first organ system to develop
and function in the embryo,228 pointing to its central importance in development. While the
role of the vascular system in the transport of nutrients and waste products has long been
appreciated, its importance in regulating the availability of circulating growth factors and stem
cells has become clear only recently.229 Neovascularization results from the processes of
vasculogenesis, angiogenesis, and arteriogenesis.229,230 Angiogenesis is the major process by
which vessels are formed in the adult, and serves as an excellent model for examining the
importance of controlling growth factor availability in tissue engineering. The engineering of

any tissue of appreciable size (e.g., smallest dimension greater than a few hundred microns),
with the exception of cartilage, will require angiogenesis to allow cell survival and tissue
function.231 Cell transplantation approaches to promoting neovascularization will not be
specifically reviewed, and the interested reader is referred to a review of that topic.232
Angiogenesis and lessons from past studies

Angiogenesis occurs either via the sprouting of new vessels from the sides and ends of existing
vessels or via the longitudinal division of a vessel.233 Vascular endothelial growth factor
(VEGF) initiates activation, migration, and proliferation of endothelial cells to form immature
new vessels, and these immature vessels are ultimately stabilized through recruitment of mural
cells (smooth muscle cells and pericytes).229 Fibroblast growth factor (FGF) and angio-
poietin-2 (Ang-2) act with VEGF to initiate angiogenesis, while platelet derived growth factor
(PDGF), TGF-β, and angiopoietin-1 (Ang-1) act during the later stages to mediate maturation
of the new vessels by recruiting and promoting interactions with mural cells.234 The VEGF
appears to be a particularly important factor, as a 50% reduction in its expression results in
embryonic lethality.235 While the VEGF family contains many related proteins and isoforms
of these proteins, all references to VEGF in this review will refer to VEGF165, the most
commonly used isoform of VEGF-A.
The large number of patients suffering from ischemic diseases (e.g., coronary artery disease),
in concert with the availability of recombinant angiogenic growth factors, has led to significant
efforts to promote angiogenesis in a therapeutic context. In spite of promising early animal

studies and small-scale clinical studies of therapeutic angiogenesis, large clinical trials with
delivery of recombinant growth factors have not demonstrated as significant an effect.236 In
these approaches, single proteins (e.g., VEGF) have been delivered to drive angiogenesis as
either a bolus injection(s) into the site of disease or via systemic administration. This strategy
is limited due to the inherent instability of these proteins in vivo (e.g., the half life of VEGF is
90 min following intravenous injection,237) and the low targeting to the tissue of interest (e.g.,
single intravenous or intracoronary administration of growth factor leads to less than 0.1%
retention in the tissue of interest after 24 hours238). The premature removal of VEGF likely
leads to apoptosis of endothelial cells and regression of the newly formed vessels.239 These
issues have led investigators to deliver large, supraphysiological quantities of these factors in
the clinical trials, but this approach may be constrained by possible side effects at distant sites
(e.g., expansion of atherogenic plaques, growth of tumors)236 and a still too rapid decrease in
local factor concentration at the site of interest. Altogether, these results indicate that the
duration of growth factor availability, and spatial segregation of signaling to the desired tissue
will likely be crucial to the success of growth factor delivery to promote angiogenesis.
Another potential limitation of many efforts to promote angiogenesis may be a requirement to

provide multiple growth factors, in an appropriate sequence, to promote the formation of a

mature and stable vascular network. While VEGF is crucial in the initial stages, PDGF-BB
promotes the maturation of blood vessels by recruiting smooth muscle progenitors to the
endothelial lining of nascent vasculature. Arrival of these cells to the immature vessels is
hypothesized to eliminate the requirement for VEGF.234 The interaction of the endothelial cells
with the pericytes/smooth muscle progenitors then leads to activation of TGF-β, which induces
smooth muscle/pericyte differentiation and matrix deposition.234 Angiopoietin-2 has been
demonstrated to augment vascularization, presumably by destabilizing the existing vasculature
and making it more responsive to initiators such as VEGF.234 Angiopoietin-1, which binds to
the same cell surface receptor (Tie-2) as Ang-2, appears to play an important role in recruiting
and maintaining the endothelial support cells.234

Polymeric systems to control temporal and spatial growth factor availability

Polymeric vehicles allow for sustained and localized delivery of growth factors and may
provide an ideal means to regulate the local availability of single or multiple growth factors
over desired time frames to promote therapeutic angiogenesis in the context of ischemic disease
or as a component of a tissue engineering strategy. A variety of polymers and their varying
physical forms have been developed to allow for localized and sustained delivery of various
bioactive macromolecules.240 Both injectable systems that are amenable to minimally invasive
delivery,241,242 and implantable, porous scaffolds243–246 that may effectively serve as patches
or vehicles for the combined delivery of angiogenic factors and cells required to engineer
various tissues types (e.g., bone forming or neural cells) have been developed for angiogenic
factors.
Providing a multiweek sustained release of VEGF from polymeric systems, in contrast to bolus
delivery, leads to a significant increase in the density of blood vessels formed at the site of the
polymer implantation,244 enhances perfusion of ischemic limbs,245 and prevents necrosis of
these limbs (Chen and Mooney, unpublished data). Importantly, these effects can be achieved
with total VEGF doses of orders of magnitude lower than what is typically used for bolus
delivery, and the sustained VEGF presence in the tissue of interest is accompanied by little to
no systemic exposure to the growth factor.246
An alternate approach to potentially sustaining the presentation of angiogenic molecules within

a tissue of interest is to utilize localized gene therapy in order to promote a local production of
the factor by the cells resident in the tissue.230 The approaches to accomplish this goal to date,
while they have led to very promising initial results, may ultimately be limited in their success
due to the short-term expression of the plasmid DNA or adenoviral vectors used in these efforts.
One approach to extend, in a controllable manner, the expression of the plasmid encoding the
factor(s) of interest is to immobilize the plasmid in a polymer system that enables its sustained
release. Incorporation of plasmid DNA into poly (lactide-co-glycolide) scaffolds can lead to a
significant increase in the duration of expression,247 and condensation of the plasmid prior to
polymer encapsulation can significantly increase the level and duration of expression in vivo.
248
Regardless of whether one promotes angiogenesis via direct delivery of a protein, or via indirect
gene therapy approaches, it may be necessary to provide multiple factors in specific
combinations or sequences to drive the formation of mature and fully functional vessel
networks. A combined delivery of multiple factors important in the initiation of angiogenesis
(e.g., VEGF and Ang-2) can increase the density of new blood vessels in an additive manner
(Ennet and Mooney, unpublished data), but does not enhance the maturity of the vessel
networks. A simultaneous delivery of factors involved in angiogenesis initiation and
maturation decreases the formation of blood vessels,249 likely due to the conflicting roles of
the early versus later acting factors. However, providing sequential delivery of factors acting
in the different stages of angiogenesis can maintain the vessel-forming activity of the early
factors, while also promoting the subsequent maturation of these vessel networks.249
While the main focus in the development of polymeric systems for delivery of angiogenic
molecules is the chemistry of the polymer, physical aspects of the system may also be critical
to its performance. External mechanical stresses and strains imposed on the system may alter
the kinetics of factor release,250 and even the intrinsic mechanical properties of the polymer
system can alter the responsiveness of the surrounding cells to the therapeutic molecule (e.g.,
plasmid DNA uptake and expression251).

Future directions and potential for tissue engineering

Therapeutic angiogenesis clinical trials have, together with a large number of more basic
studies, identified a number of key issues for the use of angiogenic molecules. These issues
will likely be broadly applicable to the use of growth factors or morphogens in tissue
engineering and regeneration. Little function typically results from providing
supraphysiological concentrations of single factors for poorly controlled time frames and with
little regulation over the distance of signaling, even if biological processes are significantly
influenced. Instead, sophisticated delivery systems that provide the necessary concentrations,
factor gradients, and sequential availability of multiple factors are more likely to initiate and
drive the processes to a desired end point, and result in highly functional new tissues.
Polymeric systems offer great promise in enabling control over growth factor signaling, and
considerable success with this approach has been achieved in small animal models. The
function of these systems must, however, be tested in larger animals and humans to assess their
utility. Critically, it will be necessary to understand the quantitative biology of the systems of
interest, in addition to the qualitative understanding that is the norm, in order to appropriately
design these systems for specific applications. For example, the specific duration of signaling,
and the concentration and specific gradient of the factor(s) in the tissue of interest required to
both activate and guide tissue formation are typically unknown, and are likely to vary with the
age and disease state of the individual. A quantitative understanding of these issues will likely
require the development of novel in vitro and in vivo model systems, and better mathematical
models of the biological processes.
David Mooney concluded that polymeric systems that can drive therapeutic angiogenesis could
find broad utility in tissue engineering and regeneration. In addition to the obvious utility in
treating ischemic diseases,236 VEGF signaling and angiogenesis are now more broadly
appreciated as helpful in regulating many regenerative processes. Bone formation is dependent
on VEGF signaling,252 and the sustained delivery of VEGF from polymers can enhance bone
regeneration.242,253 Nerve and blood vessel wiring is dependent on common mechanisms,
254 and VEGF signaling may be beneficial to slow or reverse various neural diseases.255 More
broadly, angiogenesis is crucial to the survival of transplanted cells. Appropriate delivery of
angiogenic molecules has been demonstrated to increase the engraftment of various cell types,
including osteoprogenitors256 and hepatocytes.257 Stem cell populations may be dependent on
vascular niches,204 and systems that appropriately stimulate angiogenesis may reestablish
niches crucial to regeneration and maintenance of tissues.
THE ROLE OF BIOMATERIALS IN DEVELOPING REGENERATIVE

TECHNOLOGIES
Anthony Atala from Wake Forest University emphasized that the clinical application of tissue
engineering has been elusive, owing to the difficulties in expanding cells outside the body, the
challenge of providing adequate vascular support to growing constructs, and the nonavailability
of biomaterials capable of controlling cell fate. He stressed that, in nature, there are three pillars
of functional regeneration: soluble factors (autocrine and paracrine mechanisms), insoluble
factors (ECM), and physical forces (pressure, flow, and shear). For years investigators have
sought to use the marriage of biology, materials science, and bioengineering to master the
development of tissues either in vitro or in vivo, but our grip on these three mediating factors
is remarkably loose. In particular, the complex interactions between adult stem and progenitor
cells and the ECM, and the mechanisms that lead to functional tissue regeneration are poorly
defined. Dr. Atala discussed how regenerative cells function to restore injured tissue, and how
these mechanisms are being exploited to guide the development of better biomaterials
technologies.

Clinical translation
The term tissue engineering began appearing in the medical research literature as a distinct
derivative of its predecessor, cell therapy, in the late 1980s. Although the proceedings from
one of the first tissue engineering conferences were published soon thereafter,258 the potential
of tissue engineering to provide tissues and organs to millions of patients suffering from trauma,
congenital defects, and chronic diseases has not yet been fully recognized. Unfortunately, few
notable successes of clinical translation have been achieved since that time.
People often point to the first tissue engineered product approved for clinical use by the Food
and Drug Administration, Dermagraft™, as one of the few examples. Despite the obvious
technical and regulatory achievement, Dermagraft has been a commercial disappointment and
was sold by the developer, Advanced Tissue Sciences (ATS), to Smith & Nephew for only
$12 million in 2002, as part of ATS’s bankruptcy liquidation. In October 2005, Smith &
Nephew announced that it would exit Dermagraft and related products from the market. The
ATS and Dermagraft experience has shown that strong science does not necessarily translate
into strong sales. Knowing this, how do we as scientists bring tissue engineering technology
from the bench to the bedside?
Interactive biomaterials
In its simplest form, tissue engineering is the combination of biomaterials and cells into a
construct that will eventually become the regenerated tissue. In early years, the biomaterial
scaffolds were degradable synthetic polymers and naturally derived materials, primarily
collagen, that had been used for decades in medical device applications. The view at the time
was that if a proper temporary architecture was provided to the cells, functional tissue would
form. It was soon determined that cellular interaction with the insoluble environment was as
important as it was complex. In an effort to move the science forward, many investigators
began to consider the merits of combining the benefits of various types of materials.
Specifically, cell binding was considered to be of great importance to the maturation of a
construct into functional tissue.
It is well known that cellular recognition is facilitated by the binding of cell surface integrins
to specific amino acid motifs of the ECM.259,260 The predominant ECM proteins are collagen
and fibronectin, both of which have been extensively studied with regard to cell binding.261
Fibronectin contains several regions that support attachment by a wide variety of cell types.
262 Mould et al. showed that in addition to the widely known Arginine-Glycine-Aspartic Acid
(RGD) motif, the “X”-Aspartic Acid-”Y” motif on fibronectin is also recognized by the integrin
a4b1, where X represents Glycine, Leucine, or Glutamic Acid, and Y represents Serine or
Valine.263 Remarkably, it was not until well after the discovery of these important cellular
interactions that published reports of the efforts of biomaterial scientists to employ synthetic
schemes to incorporate protein binding motifs into synthetic polymers first began to appear in
the literature.264–267
One explanation for this apparent lag in the development of interactive biomaterials is the
necessity to employ materials with a proven history of clinical use. Another is that those most
knowledgeable about the interactions between growing tissue and its ECM are typically
developmental biologists, a group not heavily involved during the nascent stages of the tissue
engineering field. Nevertheless, the concept that a combination of natural and synthetic
polymers alone will yield successful results in our quest to recapitulate developmental biology
is unrealistic. The native ECM is simply too complex to be mimicked accurately.268–271 This
complexity has been shown to be highly cell specific, although most studies fail to elucidate
specific mechanisms, particularly with respect to the role ECM plays in recruiting and
controlling the differentiation of stem and progenitor cells.272,273 How then do we overcome

this circumstance? Do we continue down the current path, looking to the immediate past for
solutions to our future, or do we undertake yet another renaissance in biomaterials
development? It is apparent that recent advances in stem cell biology compel the biomaterials
research community to reexamine its course.
Cell sources
It is widely accepted that almost every tissue in the body, including brain,274 liver,275
circulating blood,276 and heart,277 as well as skin278 and fat,279 contains some type of stem
cell or progenitor cell. Preclinical application of these cell sources is beginning to take shape,
and as techniques are developed for the isolation, purification, and controlled differentiation
of these cells into mature, functional tissues, successful patient treatment paradigms can be
envisioned. However, while many differentiated cell types grow well in culture after being
isolated from native tissue, stem and progenitor cell characteristics can easily be lost and
progeny diverted from their desired terminal fate when cultured under inadequate conditions,
that is, without a supportive coating or feeder layer.
Methods to ameliorate this dilemma have been developed in certain cell types and are in routine
use at the basic and applied research levels. For example, coating tissue culture plastic with
natural biomaterials such as collagen or fibronectin is somewhat effective, and the use of three-
dimensional gels such as collagen or Matrigel™ has been successful. However, there is no
evidence to suggest that these are optimal biomaterial systems for inducing cell differentiation.
On the contrary, recent studies have shown that slight variations in the composition of ECM
surrogates can have a dramatic impact on cell growth and differentiation,280 but have yet to be
developed for many cell types. Moreover, several authors have pointed out that ECM
composition is more tissue specific than currently appreciated.281–283 Others have shown
experimentally that ECM is able to mediate a regenerative process by attracting stem cells,
279,284 but that these environments are not necessarily optimal for efficient differentiation and
functional tissue development. Given that even subtle changes in ECM composition can have
a dramatic effect on cellular differentiation,280,285 it is reasonable to assume that a multitude
of possibilities exist and we have merely scratched the surface.
Anthony Atala summarized that the application of developmental and stem cell biology to
biomaterials development offers an astounding number of possibilities, yet our understanding
of this interplay in the context of tissue engineering remains incomplete. Any textbook on the
composition of ECM will almost universally contain the same basic chapters: collagens,
fibronectin, elastin, laminin, and perhaps another section on proteoglycans. A recent literature
search of the key words “extracellular matrix” and “biomaterial” yielded 1,432 hits. If one adds
the term “biglycan,” a critical ECM proteoglycan in bone formation, the number of hits drops
to only three. This begs the question, are collagen, fibronectin, laminin, and elastin the most
widely studied ECM-related biomaterials because they are the most important, or because they
are the most easily studied?
Most approaches to biomaterials development for tissue engineering fail to take into account
the tremendous complexity of the ECM. This is understandable given that most analytical tools
for studying the composition of the ECM are solution-based techniques, with the exception of
immunostaining methods. Also, given the fact that complete digestion of the ECM without
significantly disrupting the integrity of the constituent molecules is extraordinarily difficult, it
is not surprising that biomaterials development has fallen so far behind advances in cell and
molecular biology. In order to overcome these inherent challenges, efforts need to be focused
on the application of existing tools in ways that have never been tried and the development of
new tools that will allow investigators to completely reverse engineer the ECM. Only through
a complete understanding of the compositional gradients, temporal changes, and remodeling

of even the most minute constituents of the ECM will we be able to use the solid cellular
environment to drive tissue and organ morphogenesis.
Acknowledgments
Antonios Mikos thanks Simon Young and Mark Wong for help with the preparation of this article, and the National
Institutes of Health (NIH) for funding support (R01 DE15164). Susan Herring thanks Patricia Emry and Frank Starr
for help with the procedures, and the National Institute of Dental and Craniofacial Research (NIDCR) for funding
support (PHS award DE08513). Helen Lu thanks Kristen Moffat for help with the preparation of this article, and the
Whitaker Foundation, the Wallace H. Coulter Foundation, the NIH (NIAMS AR052402), and the National Science
Foundation (GK-12 Graduate Fellowship) for funding support. Rita Kandel thanks M. Grynpas, M. Hurtig, and R.
Pilliar for help with the preparation of this article, and the Canadian Institutes of Health Research, the Natural Sciences
and Engineering Research Council, and the Arthritis Society for funding support. Mehmet Toner thanks Alex Fowler
for help with the preparation of this article. David Mooney thanks NIDCR and the NHLBI for funding support.
References
1. Ripamonti U. Soluble, insoluble and geometric signals sculpt the architecture of mineralized tissues.
J Cell Mol Med 2004;8:169. [PubMed: 15256065]
2. Yoshikawa H, Myoui A. Bone tissue engineering with porous hydroxyapatite ceramics. J Artif Organs
2005;8:131. [PubMed: 16235028]
3. Villanueva JE, Nimni ME. Promotion of calvarial cell osteogenesis by endothelial cells. J Bone Miner
Res 1990;5:733. [PubMed: 2396500]
4. Carvalho RS, Einhorn TA, Lehmann W, Edgar C, Al-Yamani A, Apazidis A, Pacicca DM, Clemens
TL, Gerstenfeld LC. The role of angiogenesis in a murine tibial model of distraction osteogenesis. J
Artif Organs 2004;34:849.
5. Krompecher S. Die Entwicklung der Knochenzellen und die Bildung der Knochengrundsubstanz bei
der Knorpelig and bindegewebig vorgebildeten sowie der primären reinen Knochenbildung. Anat Anz
1934;78:34.
6. Inoue N, Rafiee B, Toda I, Tamada Y, Suwa F, Aro HT, Chao EYS. Vascular and trabecular
microstructures in the early stage of cortical defect repair. J Biomech 2001;34:S81.
7. Lacroix, P. Organization of Bones. Philadelphia: Blakiston; 1951.
8. Caplan AI. Mesenchymal stem cells. J Orthop Res 1990;9:641. [PubMed: 1870029]
9. Ochareon, P. Ph.D. thesis. Department of Oral Biology, University of Washington; Seattle, WA: 2004.
Craniofacial periosteal cell capacities.
10. Rafferty KL, Herring SW, Artese F. Three-dimensional loading and growth of the zygomatic arch. J
Exp Biol 2000;203:2093. [PubMed: 10862722]
11. Simpson AHRW. The blood supply of the periosteum. J Anat 1985;140:697. [PubMed: 4077705]
12. Langer R, Vacanti J. Tissue engineering. Science 1993;260:920. [PubMed: 8493529]
13. Mooney DJ, Mikos AG. Growing new organs. Sci Am 1999;280:60. [PubMed: 10201117]
14. Schnabel M, Marlovits S, Eckhoff G, Fichtel I, Gotzen L, Vecsei V, Schlegel J. Dedifferentiation-

associated changes in morphology and gene expression in primary human articular chondrocytes in
cell culture. Osteoarthritis Cartilage 2002;10:62. [PubMed: 11795984]
15. Andrews PW, Benvenisty N. Starting from building blocks: tissue engineering using stem cells. Curr
Opin Biotechnol 2005;16:485.
16. Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis
Res Ther 2003;5:32. [PubMed: 12716446]
17. Elisseeff JH. Embryonic stem cells: potential for more impact. Trends Biotechnol 2004;22:155.
[PubMed: 15104108]
18. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti
DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science
1999;284:143. [PubMed: 10102814]
19. Gearhart J. New potential for human embryonic stem cells [comment]. Science 1998;282:1061.
[PubMed: 9841453]

20. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM.
Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145. [PubMed:
9804556]
21. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N.

Differentiation of human embryonic stem cells into embryoid bodies compromising the three
embryonic germ layers. Mol Med 2000;6:88. [PubMed: 10859025]
22. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable
neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129. [PubMed:
11731781]
23. Elisseeff J, Puleo C, Yang F, Sharma B. Advances in skeletal tissue engineering with hydrogels.
Orthod Craniofac Res 2005;8:150. [PubMed: 16022717]
24. Elisseeff J. Injectable cartilage tissue engineering. Expert Opin Biol Ther 2004;4:1849. [PubMed:
15571448]
25. Hwang NS, Kim MS, Sampattavanich S, Baek JH, Zhang Z, Elisseeff J. Effects of three dimensional
culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells. Stem
Cells 2006;15:295.
26. Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal
differentiation of cells derived from human embryonic germ cells. Stem Cells 2005;23:113.
[PubMed: 15625128]
27. Williams CG, Kim TK, Taboas A, Malik A, Manson P, Elisseeff J. In vitro chondrogenesis of bone
marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel. Tissue Eng 2003;9:679.
[PubMed: 13678446]
28. Elisseeff J, McIntosh W, Anseth K, Riley S, Ragan P, Langer R. Photoencapsulation of chondrocytes
in poly(ethylene oxide)-based semi-interpenetrating networks. J Biomed Mater Res 2000;51:164.
[PubMed: 10825215]
29. Hubbell JA. Bioactive biomaterials. Curr Opin Biotechnol 1999;10:123. [PubMed: 10209141]
30. Sharma B, Elisseeff JH. Engineering structurally organized cartilage and bone tissues. Ann Biomed
Eng 2004;32:148. [PubMed: 14964730]
31. Elisseeff J, Anseth K, Sims D, McIntosh W, Randolph M, Langer R. Transdermal
photopolymerization for minimally invasive implantation. Proc Natl Acad Sci USA 1999;96:3104.
[PubMed: 10077644]
32. Gerstenfeld LC, Cruceta J, Shea CM, Sampath K, Barnes GL, Einhorn TA. Chondrocytes provide
morphogenic signals that selectively induce osteogenic differentiation of mesenchymal stem cells. J
Bone Miner Res 2002;17:221. [PubMed: 11811552]
33. Skalak, R.; Fox, CF., editors. Tissue engineering. Granlibakken, Lake Tahoe, California. February
26–29, 1988; UCLA Symposia on Molecular and Cellular Biology; New York, NY: Liss; 1988.
Proceedings of a workshop
34. Crane GM, Ishaug SL, Mikos AG. Bone tissue engineering. Nat Med 1995;1:1322. [PubMed:
7489417]
35. Yaszemski MJ, Payne RG, Hayes WC, Langer R, Mikos AG. Evolution of bone transplantation:
molecular, cellular and tissue strategies to engineer human bone. Biomaterials 1996;17:175.
[PubMed: 8624394]
36. Laurencin CT, Ambrosio AA, Borden M, Cooper JA. Annual Review of Biomedical Engineering
1999;1:19.
37. Leach JK, Mooney DJ. Bone engineering by controlled delivery of osteoinductive molecules and
cells. Expert Opin Biol Ther 2004;4:1015. [PubMed: 15268670]
38. Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG, Langer R. Neocartilage formation in
vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res
1993;27:11. [PubMed: 8380593]
39. Mauck RL, Soltz MA, Wang CC, Wong DD, Chao PH, Valhmu WB, Hung CT, Ateshian GA.
Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded
agarose gels. J Biomech Eng 2000;122:252. [PubMed: 10923293]

40. Riley SL, Dutt S, De La Torre R, Chen AC, Sah RL, Ratcliffe A. Formulation of PEG-based hydrogels
affects tissue-engineered cartilage construct characteristics. J Mater Sci Mater Med 2001;12:983.
[PubMed: 15348352]
41. Caterson EJ, Li WJ, Nesti LJ, Albert T, Danielson K, Tuan RS. Polymer/alginate amalgam for
cartilage-tissue engineering. Ann N Y Acad Sci 2002;961:134. [PubMed: 12081882]
42. Almarza AJ, Athanasiou KA. Design characteristics for the tissue engineering of cartilaginous tissues.
Ann Biomed Eng 2004;32:2. [PubMed: 14964717]
43. Alhadlaq A, Elisseeff JH, Hong L, Williams CG, Caplan AI, Sharma B, Kopher RA, Tomkoria S,
Lennon DP, Lopez A, Mao JJ. Adult stem cell driven genesis of human-shaped articular condyle.
Ann Biomed Eng 2004;32:911. [PubMed: 15298429]
44. Jackson DW, Heinrich JT, Simon TM. Biologic and synthetic implants to replace the anterior cruciate
ligament. Arthroscopy 1994;10:442. [PubMed: 7945642]
45. Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of fibroblast-seeded ligament analogs
for ACL reconstruction. J Biomed Mater Res 1995;29:1363. [PubMed: 8582904]
46. Woo SL, Hildebrand K, Watanabe N, Fenwick JA, Papageorgiou CD, Wang JH. Tissue engineering
of ligament and tendon healing. Clin Orthop Relat Res 1999;367:S312. [PubMed: 10546655]
47. Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC, Kaplan DL. Silk matrix for tissue
engineered anterior cruciate ligaments. Biomaterials 2002;23:4131. [PubMed: 12182315]
48. Lu HH, Cooper JA Jr, Manuel S, Freeman JW, Attawia MA, Ko FK, Laurencin CT. Anterior cruciate
ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies.
Biomaterials 2005;26:4805. [PubMed: 15763260]
49. Butler DL, Goldstein SA, Guilak F. Functional tissue engineering: the role of biomechanics. J
Biomech Eng 2000;122:570. [PubMed: 11192376]
50. Cooper RR, Misol S. Tendon and ligament insertion. a light and electron microscopic study. J Bone
Joint Surg Am 1970;52:1. [PubMed: 4189231]
51. Benjamin M, Evans EJ, Copp L. The histology of tendon attachments to bone in man. J Anat
1986;149:89. [PubMed: 3693113]
52. Niyibizi C, Visconti CS, Kavalkovich K, Woo SL. Collagens in an adult bovine medial collateral
ligament: immunofluorescence localization by confocal microscopy reveals that type XIV collagen
predominates at the ligament-bone junction. Matrix Biol 1995;14:743. [PubMed: 8785589]
53. Wei X, Messner K. The postnatal development of the insertions of the medial collateral ligament in
the rat knee. Anat Embryol (Berl) 1996;193:53. [PubMed: 8838496]
54. Petersen W, Tillmann B. Structure and vascularization of the cruciate ligaments of the human knee
joint. Anat Embryol (Berl) 1999;200:325. [PubMed: 10463347]
55. Thomopoulos S, Williams GR, Gimbel JA, Favata M, Soslowsky LJ. Variations of biomechanical,
structural, and compositional properties along the tendon to bone insertion site. J Orthop Res
2003;21:413. [PubMed: 12706013]
56. Wang IE, Mitroo S, Chen FH, Lu HH, Doty SB. Age-dependent changes in matrix composition and
organization at the ligament-to-bone insertion. J Orthop Res 2006;24:1745. [PubMed: 16779829]
57. Woo SL, Gomez MA, Seguchi Y, Endo CM, Akeson WH. Measurement of mechanical properties of
ligament substance from a bone-ligament-bone preparation. J Orthop Res 1983;1:22. [PubMed:
6679572]
58. Johnson RJ. The anterior cruciate: a dilemma in sports medicine. Int J Sports Med 1982;3:71.
[PubMed: 7049974]
59. American Academy of Orthopaedic Surgeons. Arthoplasty and Total Joint Replacement Procedures:
United States, 1990 to 1997. 1997.
60. Beynnon BD, Johnson RJ, Fleming BC, Kannus P, Kaplan M, Samani J, Renstrom P. Anterior cruciate
ligament replacement: comparison of bone-patellar tendon-bone grafts with two-strand hamstring
grafts. A prospective, randomized study. J Bone Joint Surg Am 2002;84-A:1503. [PubMed:
12208905]
61. Niyibizi C, Sagarrigo VC, Gibson G, Kavalkovich K. Identification and immunolocalization of type
X collagen at the ligament-bone interface. Biochem Biophys Res Commun 1996;222:584. [PubMed:
8670248]

62. Woo SL, Buckwalter JA. AAOS/NIH/ORS workshop. Injury and repair of the musculoskeletal soft
tissues. Savannah, GA, June 18–20, 1987. J Orthop Res 1988;6:907. [PubMed: 3171771]
63. Friedman MJ, Sherman OH, Fox JM, Del Pizzo W, Snyder SJ, Ferkel RJ. Autogeneic anterior cruciate
ligament (ACL) anterior reconstruction of the knee: a review. Clin Orthop 1985;186:9. [PubMed:
3888475]
64. Kurosaka M, Yoshiya S, Andrish JT. A biomechanical comparison of different surgical techniques
of graft fixation in anterior cruciate ligament reconstruction. Am J Sports Med 1987;15:225.
[PubMed: 3303979]
65. Robertson DB, Daniel DM, Biden E. Soft tissue fixation to bone. Am J Sports Med 1986;14:398.
[PubMed: 3535550]
66. Rodeo SA, Arnoczky SP, Torzilli PA, Hidaka C, Warren RF. Tendon-healing in a bone tunnel: a
biomechanical and histological study in the dog. J Bone Joint Surg Am 1993;75:1795. [PubMed:
8258550]
67. Grana WA, Egle DM, Mahnken R, Goodhart CW. An analysis of autograft fixation after anterior
cruciate ligament reconstruction in a rabbit model. Am J Sports Med 1994;22:344. [PubMed:
8037275]
68. Blickenstaff KR, Grana WA, Egle D. Analysis of a semitendinosus autograft in a rabbit model. Am
J Sports Med 1997;25:554. [PubMed: 9240991]
69. Weiler A, Hoffmann RF, Bail HJ, Rehm O, Sudkamp NP. Tendon healing in a bone tunnel. Part II:
Histologic analysis after biodegradable interference fit fixation in a model of anterior cruciate
ligament reconstruction in sheep. Arthroscopy 2002;18:124. [PubMed: 11830805]
70. Fujioka H, Thakur R, Wang GJ, Mizuno K, Balian G, Hurwitz SR. Comparison of surgically attached
and non-attached repair of the rat Achilles tendon-bone interface. Cellular organization and type X
collagen expression. Connect Tissue Res 1998;37:205. [PubMed: 9862222]
71. Kobayashi M, Watanabe N, Oshima Y, Kajikawa Y, Kawata M, Kubo T. The fate of host and graft
cells in early healing of bone tunnel after tendon graft. Am J Sports Med 2005;33:1892. [PubMed:
16157856]
72. Wang IE, Shan JM, Chen FH, Lu HH. Effects of osteoblast and fibroblast interactions on cell growth
and differentiation. Biochem Biophys Res Commun. 2006 Submitted.
73. Jiang J, Nicoll SB, Lu HH. Co-culture of osteoblasts and chondrocytes modulates cellular
differentiation in vitro. Biochem Biophys Res Commun 2005;338:762. [PubMed: 16259947]
74. Wang IE, Lu HH. Tri-culture of bovine anterior cruciate ligament fibroblasts, osteoblasts and
chondrocytes with application in interface tissue engineering. Transactions of the Orthopaedic
Research Society. 2005
75. Shan JM, Wang IE, Lu HH. Effects of conditioned media on osteoblast and ligament fibroblast growth
and differentiation. Annual Meeting of the Biomedical Engineering Society. 2004
76. Matyas JR, Anton MG, Shrive NG, Frank CB. Stress governs tissue phenotype at the femoral insertion
of the rabbit MCL. J Biomech 1995;28:147. [PubMed: 7896857]
77. Spalazzi J, Gallina J, Fung-Kee-Fung S, Konofagou E, Lu H. Elastographic imaging of strain
distribution in the anterior cruciate ligament and at the ligament-bone insertions. J Orthop Res
2006;24:2001. [PubMed: 16900541]
78. Wolff, J. Das Gesetz der Transformation der Knochen. Hirschwald Verlag; Berlin: 1892.
79. Vogel KG, Koob TJ. Structural specialization in tendons under compression. Int Rev Cytol
1989;115:267. [PubMed: 2663761]
80. Moffat, KL.; Chahine, NO.; Hung, CT.; Ateshian, GA.; Lu, HH. Characterization of the mechanical
properties of the ACL-bone insertion. Proceedings of the ASME Summer Bioengineering
Conference; 2005.
81. Spalazzi JP, Costa KD, Doty SB, Lu HH. Characterization of the mechanical properties, structure,
and composition of the anterior cruciate ligament-bone insertion site. Transactions of the Orthopaedic
Research Society. 2004
82. Benjamin M, Evans EJ, Rao RD, Findlay JA, Pemberton DJ. Quantitative differences in the histology
of the attachment zones of the meniscal horns in the knee joint of man. J Anat 1991;177:127.
[PubMed: 1769887]

83. Spalazzi JP, Doty SB, Moffat KL, Levine WN, Lu HH. Development of controlled matrix
heterogeneity on a triphasic scaffold for orthopedic interface tissue engineering. Tissue Eng
2006;12:3497. [PubMed: 17518686]
84. Spalazzi JP, Dagher E, Doty SB, Rodeo SA, Lu HH. In vivo evaluation of a triphasic composite
scaffold for ACL-bone integration. Transactions of the International Symposium on Ligaments and
Tendons. 2006
85. Lu HH, El Amin SF, Scott KD, Laurencin CT. Three-dimensional, bioactive, biodegradable, polymer-
bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis
and mineralization of human osteoblast-like cells in vitro. J Biomed Mater Res 2003;64A:465.
86. Spalazzi JP, Dionisio KL, Jiang J, Lu HH. Osteoblast and chondrocyte interactions during coculture
on scaffolds. IEEE Eng Med Biol Mag 2003;22:27. [PubMed: 14699933]
87. Lu HH, Jiang J, Tang A, Hung CT, Guo XE. Development of controlled heterogeneity on a polymer-
ceramic hydrogel scaffold for osteochondral repair. Bioceramics 2005;17:607.
88. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current
status and prospects. Osteoarthritis Cartilage 2002;10:432. [PubMed: 12056848]
89. Redman SN, Oldfield SF, Archer CW. Current strategies for articular cartilage repair. Eur Cell Mater
2005;9:23. [PubMed: 15830323]
90. Berry DJ, Harmsen WS, Ilstrup D, Lewallen DG, Cabanela ME. Survivorship of uncemented
proximally porous-coated femoral components. Clin Orthop Relat Res 1995;319:168. [PubMed:
7554627]
91. Jacobsson SA, Djerf K, Wahlstrom O. Twenty-year results of McKee-Farrar versus Charnley
prosthesis. Clin Orthop Relat Res 1996;329:S60. [PubMed: 8769323]

92. Soderman P, Malchau H, Herberts P, Zugner R, Regner H, Garellick G. Outcome after total hip
arthroplasty: Part II: Disease-specific follow-up and the Swedish National Total Hip Arthroplasty
Register. Acta Orthop Scand 2001;72:113. [PubMed: 11372940]
93. Berend KR, Lombardi AV Jr, Mallory TH, Adams JB, Russell JH, Groseth KL. The long-term
outcome of 755 consecutive constrained acetabular components in total hip arthroplasty examining
the successes and failures. J Arthroplasty 2005;20:93. [PubMed: 16214009]
94. Kinner B, Capito RM, Spector M. Regeneration of articular cartilage. Adv Biochem Eng Biotechnol
2005;94:91. [PubMed: 15915870]
95. Oegema, TR., Jr; Thompson, RC, Jr. The zone of calcified cartilage: its role in osteoarthritis. In:
Kuettner, K., editor. Articular Cartilage and Osteoarthritis. New York: Raven Press; 1992. p. 319-331.
96. Burr DB. Anatomy and physiology of the mineralized tissues: role in the pathogenesis of
osteoarthrosis. Osteoarthritis Cartilage 2004;12(Suppl A):S20. [PubMed: 14698637]
97. Mente PL, Lewis JL. Elastic modulus of calcified cartilage is an order of magnitude less than that of
subchondral bone. J Orthop Res 1994;12:637. [PubMed: 7931780]
98. Smith GD, Knutsen G, Richardson JB. A clinical review of cartilage repair techniques. J Bone Joint
Surg Br 2005;87:445. [PubMed: 15795189]
99. Niederauer GG, Slivka MA, Leatherbury NC, Korvick DL, Harroff HH, Ehler WC, Dunn CJ,
Kieswetter K. Evaluation of multiphase implants for repair of focal osteochondral defects in goats.
100. Mahmoudifar N, Doran PM. Tissue engineering of human cartilage and osteochondral composites
using recirculation bioreactors. Biomaterials 2005;26:7012. [PubMed: 16039710]
101. Schaefer D, Martin I, Shastri P, Padera RF, Langer R, Freed LE, Vunjak-Novakovic G. In vitro
generation of osteochondral composites. Biomaterials 2000;21:2599. [PubMed: 11071609]
102. Sherwood JK, Riley SL, Palazzolo R, Brown SC, Monkhouse DC, Coates M, Griffith LG, Landeen
LK, Ratcliffe A. A three-dimensional osteochondral composite scaffold for articular cartilage repair.
103. Alhadlaq A, Mao JJ. Tissue-engineered osteochondral constructs in the shape of an articular condyle.
J Bone Joint Surg Am 2005;87:936. [PubMed: 15866954]
104. Schaefer D, Martin I, Jundt G, Seidel J, Heberer M, Grodzinsky A, Bergin I, Vunjak-Novakovic G,
Freed LE. Tissue-engineered composites for the repair of large osteochondral defects. Arthritis
Rheum 2002;46:2524. [PubMed: 12355501]

105. Tanaka T, Komaki H, Chazono M, Fujii K. Use of a biphasic graft constructed with chondrocytes
overlying a beta-tricalcium phosphate block in the treatment of rabbit osteochondral defects. Tissue
Eng 2005;11:331. [PubMed: 15738686]
106. Gao J, Dennis JE, Solchaga LA, Goldberg VM, Caplan AI. Repair of osteochondral defect with
tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan
sponge. Tissue Eng 2002;8:827. [PubMed: 12459061]
107. van Susante JL, Buma P, Homminga GN, van den Berg WB, Veth RP. Chondrocyte-seeded
hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat. Biomaterials
1998;19:2367. [PubMed: 9884051]
108. Guo X, Wang C, Duan C, Descamps M, Zhao Q, Dong L, Lu S, Anselme K, Lu J, Song YQ. Repair
of osteochondral defects with autologous chondrocytes seeded onto bioceramic scaffold in sheep.
Tissue Eng 2004;10:1830. [PubMed: 15684691]
109. Schek RM, Taboas JM, Segvich SJ, Hollister SJ, Krebsbach PH. Engineered osteochondral grafts
using biphasic composite solid free-form fabricated scaffolds. Tissue Eng 2004;10:1376. [PubMed:
15588398]
110. Chang CH, Lin FH, Lin CC, Chou CH, Liu HC. Cartilage tissue engineering on the surface of a
novel gelatin-calcium-phosphate biphasic scaffold in a double-chamber bioreactor. J Biomed Mater
Res B Appl Biomater 2004;71:313. [PubMed: 15386400]
111. Wang X, Grogan SP, Rieser F, Winkelmann V, Maquet V, Berge ML, Mainil-Varlet P. Tissue
engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study.
112. Gao J, Dennis JE, Solchaga LA, Awadallah AS, Goldberg VM, Caplan AI. Tissue-engineered
fabrication of an osteochondral composite graft using rat bone marrow-derived mesenchymal stem
cells. Tissue Eng 2001;7:363. [PubMed: 11506726]
113. Hung CT, Lima EG, Mauck RL, Taki E, LeRoux MA, Lu HH, Stark RG, Guo XE, Ateshian GA.
Anatomically shaped osteochondral constructs for articular cartilage repair. J Biomech
2003;36:1853. [PubMed: 14614939]
114. Kreklau B, Sittinger M, Mensing MB, Voigt C, Berger G, Burmester GR, Rahmanzadeh R, Gross
U. Tissue engineering of biphasic joint cartilage transplants. Biomaterials 1999;20:1743. [PubMed:
10503975]
115. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529.
[PubMed: 11071603]
116. Frenkel SR, Di Cesare PE. Scaffolds for articular cartilage repair. Ann Biomed Eng 2004;32:26.
[PubMed: 14964719]
117. Pilliar RM, Filiaggi MJ, Wells JD, Grynpas MD, Kandel RA. Porous calcium polyphosphate
scaffolds for bone substitute applications—in vitro characterization. Biomaterials 2001;22:963.
[PubMed: 11311015]
118. Waldman SD, Grynpas M, Pilliar RM, Kandel RA. Characterization of cartilaginous tissue formed
on calcium polyphosphate substrates in vitro. J Biomed Mater Res 2002;62:323. [PubMed:
12209917]
119. Grynpas MD, Pilliar RM, Kandel RA, Renlund R, Filiaggi M, Dumitriu M. Porous calcium
polyphosphate scaffolds for bone substitute applications in vivo studies. Biomaterials
2002;23:2063. [PubMed: 11996048]
120. Kandel RA, Boyle J, Gibson G, Cruz T, Speagle M. In vitro formation of mineralized cartilaginous
tissue by articular chondrocytes. In Vitro Cell Dev Biol Anim 1997;33:174. [PubMed: 9112125]
121. Yu H, Grynpas M, Kandel RA. Composition of cartilaginous tissue with mineralized and non-
mineralized zones formed in vitro. Biomaterials 1997;18:1425. [PubMed: 9375844]
122. Kandel R, Hurtig M, Grynpas M. Characterization of the mineral in calcified articular cartilaginous
tissue formed in vitro. Tissue Eng 1999;5:25. [PubMed: 10207187]
123. Allan KS, Pilliar RM, Wang J, Grynpas MD, Kandel RA. Formation of biphasic constructs
containing cartilage with a calcified zone interface. Tissue Eng. In press.
124. Waldman SD, Spiteri CG, Grynpas MD, Pilliar RM, Hong J, Kandel RA. Effect of biomechanical
conditioning on cartilaginous tissue formation in vitro. J Bone Joint Surg Am 2003;85-A(Suppl 2):
101. [PubMed: 12721351]

125. Cancedda R, Dozin B, Giannoni P, Quarto R. Tissue engineering and cell therapy of cartilage and
bone. Matrix Biol 2003;22:81. [PubMed: 12714045]
126. Masuda K, Sah RL, Hejna MJ, Thonar EJ. A novel two-step method for the formation of tissue-
engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC)

method. J Orthop Res 2003;21:139. [PubMed: 12507591]
127. Li WJ, Tuli R, Okafor C, Derfoul A, Danielson KG, Hall DJ, Tuan RS. A three-dimensional
nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells.
128. Rosso F, Marino G, Giordano A, Barbarisi M, Parmeggiani D, Barbarisi A. Smart materials as
scaffolds for tissue engineering. J Cell Physiol 2005;203:465. [PubMed: 15744740]
129. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological
characterization. Int J Biochem Cell Biol 2004;36:568. [PubMed: 15010324]
130. Tuli R, Nandi S, Li WJ, Tuli S, Huang X, Manner PA, Laquerriere P, Noth U, Hall DJ, Tuan RS.
Human mesenchymal progenitor cell-based tissue engineering of a single-unit osteochondral
construct. Tissue Eng 2004;10:1169. [PubMed: 15363173]
131. Nugent HM, Edelman ER. Tissue engineering therapy for cardiovascular disease. Circ Res
2003;92:1068. [PubMed: 12775655]
132. Echenhagen T, Zimmermann WH. Engineering myocardial tissue. Circ Res 2005;97:1220.
[PubMed: 16339494]
133. Rabkin E, Schoen FJ. Cardiovascular tissue engineering. Cardiovasc Pathol 2002;11:305. [PubMed:
12459430]
134. Leor J, Cohen S. Myocardial tissue engineering: creating a muscle patch for a wounded heart. Ann
N Y Acad Sci USA 2004;1015:312.
135. Isenberg BC, Williams C, Tranquillo RT. Small-diameter artificial arteries engineered in vitro. Circ
Res 2006;98:25. [PubMed: 16397155]
136. Hammermeister K, Sethi GK, Henderson WG, Grover FL, Oprian C, Rahimtoola SH. Outcomes 15
years after valve replacement with a mechanical versus a bioprosthetic valve: final report of the
Veterans Affairs Randomized Trial. J Am Coll Cardiol 2000;36:1152. [PubMed: 11028464]
137. Schoen, FJ. Pathology of heart valve substitution with mechanical and tissue prosthesis. In: Silver,
MD.; Gotlieb, AI.; Schoen, FJ., editors. Cardiovascular Pathology. 3. Philadelphia: Saunders; 2001.
138. Breuer CK, Mettler BA, Anthony T, et al. Application of tissue-engineering principles toward the
development of a semilunar heart valve substitute. Tissue Eng 2004;10:1725. [PubMed: 15684681]
139. Rabkin-Aikawa E, Mayer JE Jr, Schoen FJ. Heart valve regeneration. Adv Biochem Eng Biotechnol
2005;94:141. [PubMed: 15915872]
140. Vesely I. Heart valve tissue engineering. Circ Res 2005;97:743. [PubMed: 16224074]
141. Mendelson K, Schoen FJ. Heart valve tissue engineering: concepts, approaches, progress, and
challenges. Ann Biomed Eng 2006;34:1799. [PubMed: 17053986]
142. Schoen FJ. Aortic valve structure-functional correlations: role of elastic fibers no longer a stretch
of the imagination. J Heart Valve Dis 1997;6:1. [PubMed: 9044068]
143. Scott M, Vesely I. Aortic valve cusp microstructure: the role of elastin. Ann Thorac Surg
1995;60:5391.
144. Schoen FJ. Future directions in tissue heart valves: impact of recent insights from biology and
pathology. J Heart Valve Dis 1999;8:350. [PubMed: 10461233]
145. Della Rocca F, Sartore S, Guidolin D, Bertiplaglia B, Gerosa G, Casarotto D, Pauletto P. Cell
composition of the human pulmonary valve: a comparative study with the aortic valve—The
VESALIO project. Ann Thorac Surg 2000;70:1594. [PubMed: 11093493]
146. Taylor PM, Allen SP, Yacoub MH. Phenotypic and functional characterization of interstitial cells
from human heart valves, pericardium and skin. J Heart Valve Dis 2000;9:150. [PubMed:
10678389]
147. Taylor PM, Batten P, Brand NJ, Thomas PS, Yacoub MH. The cardiac valve interstitial cell. Int J
Biochem Cell Biol 2003;35:113. [PubMed: 12479860]

148. Rabkin E, Hoerstrup SP, Aikawa M, Mayer JE Jr, Schoen FJ. Evolution of cell phenotype and
extracellular matrix in tissue-engineered heart valves during in vitro maturation and in vivo
remodeling. J Heart Valve Dis 2002;11:308. [PubMed: 12056720]
149. Newby AC. Dual role of matrix metalloproteinases (Matrixins) in intimal thickening and
atherosclerotic plaque rupture. Physiol Rev 2005;85:1. [PubMed: 15618476]
150. Schurch W, Seemayer TA, Gabbiani G. The myofibroblast: a quarter century after its discovery.
Am J Surg Pathol 1998;22:141. [PubMed: 9500214]
151. Schmitt-Graff A, Desmouliere A, Gabbiani G. Heterogeneity of myofibroblast phenotypic features:
an example of fibroblastic cell plasticity. Virchows Arch 1994;425:3. [PubMed: 7921410]
152. Walker GA, Masters KS, Shah DN, Anseth KS, Leinwand LA. Valvular myofibroblast activation
by transforming growth factor-beta: implications for pathological extracellular matrix remodeling
in heart valve disease. Circ Res 2004;95:253. [PubMed: 15217906]
153. Mjaatvedt, CH.; Yamamura, H.; Wessels, A.; Ramsdell, A.; Turner, D.; Markwald, RR. Mechanisms
of segmentation, septation, and remodeling of the tubular heart: endocardial cushion fate and cardiac
looping. In: Harvey, RP.; Rosenthal, N., editors. Heart Development. San Diego: Academic Press;
1999.
154. Schroeder JA, Jackson LF, Lee DC, Camenisch TD. Form and function of developing heart valves:
coordination by extracellular matrix and growth factor signaling. J Mol Med 2003;81:392.
[PubMed: 12827270]
155. Eisenberg LM, Markwald RR. Molecular regulation of atrioventricular valvuloseptal
morphogenesis. Circ Res 1995;77:1. [PubMed: 7788867]
156. Armstrong EJ, Bischoff J. Heart valve development: endothelial cell signaling and differentiation.
Circ Res 2004;95:459. [PubMed: 15345668]
157. Parenya G, Vineberg S, Kaushal S, Roth SJ, Rabkin E, Schoen FJ, Bischoff J. Aortic valve
endothelial cells undergo TGF-beta-mediated transdifferentiation to a mesenchymal phenotype.
Am J Pathol 2001;159:1335. [PubMed: 11583961]
158. Aikawa E, Whittaker P, Farber M, et al. Human semilunar cardiac valve remodeling by activated
cells from fetus to adult. Implications for postnatal adaptation, valve pathology and tissue
engineering. Circulation 2006;113:1344. [PubMed: 16534030]
159. Merryman WD, Youn I, Lukoff HD, Krueger PM, Guilak F, Hopkins RA, Sacks MS. Correlation
between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen
biosynthesis. Am J Physiol Heart Circ Physiol 2006;290:H224. [PubMed: 16126816]
160. Grossman W, Jones D, McLaurin LP. Wall stress and patterns of hypertrophy in the human left
ventricle. J Clin Invest 1975;56:56. [PubMed: 124746]
161. Berenji K, Drazner MH, Rothermel BA, Hill JA. Does load-induced ventricular hypertrophy
progress to systolic heart failure? Am J Physiol Heart Circ Physiol 2005;289:H8. [PubMed:
15961379]
162. Pries AR, Reglin B, Secomb TW. Remodeling of blood vessels: responses of diameter and wall
thickness to hemodynamic and metabolic stimuli. Hypertension 2005;46:725. [PubMed: 16172421]
163. Merryman, WD.; Lukoff, HD.; Hopkins, RA.; Sacks, MS. Aortic valve interstitial cell phenotype
and biosynthesis: synergistic effects of cyclic tension and TGF-β1. Proceedings of BIO2006, ASME
Summer Bioengineering Conference; 2006.
164. Kouchoukos NT, Davila-Roman VG, Spray TL, Murphy SF, Perrillo JB. Replacement of the aortic
root with a pulmonary autograft in children and young adults with aortic-valve disease. N Engl J
Med 1994;330:1. [PubMed: 8259138]
165. Rabkin-Aikawa E, Aikawa M, Farber M, Kratz JR, Garcia-Cardena G, Kouchoukos NT, Mitchell
MB, Jonas RA, Schoen FJ. Clinical pulmonary autograft valves: pathologic evidence of adaptive
remodeling in the aortic site. J Thorac Cardiovasc Surg 2004;128:552. [PubMed: 15457156]
166. Tamura K, Jones M, Yamada I, Ferrans VJ. Wound healing in the mitral valve. J Heart Valve Dis
2000;9:53. [PubMed: 10678376]
167. Rabkin E, Aikawa M, Stone JR, Fukumoto Y, Libby P, Schoen FJ. Activated interstitial
myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart
valves. Circulation 2001;104:2525. [PubMed: 11714645]

168. Schoen FJ, Levy RJ. Calcification of tissue heart valve substitutes: progress toward understanding
and prevention. Ann Thorac Surg 2005;79:1072. [PubMed: 15734452]
169. Sacks MS, Schoen FJ. Collagen fiber disruption occurs independent of calcification in clinically
explanted bioprosthetic heart valves. J Biomed Mater Res 2002;62:359. [PubMed: 12209921]
170. Schoen FJ, Tsao JW, Levy RJ. Calcification of bovine pericardium used in cardiac valve
bioprostheses: implications for the mechanisms of bioprosthetic tissue mineralization. Am J Pathol
1986;123:134. [PubMed: 2421577]
171. Schoen FJ, Levy RJ, Nelson AC, Bernhard WF, Nashef A, Hawley M. Onset and progression of
experimental bioprosthetic heart valve calcification. Lab Invest 1985;52:523. [PubMed: 3990244]
172. Meinhart JG, Deutsch M, Fischlein T, Howanietz N, Froschl A, Zilla P. Clinical autologous in
vitro endothelialization of 153 infrainguinal ePTFE grafts. Ann Thorac Surg 2001;71:S327.
[PubMed: 11388216]
173. Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW, Rabkin E, Moran AM,
Schoen FJ, Atala A, Soker S, Bischoff J, Mayer JE Jr. Functional small-diameter neovessels created
using endothelial progenitor cells expanded ex vivo. Nat Med 2001;7:1035. [PubMed: 11533707]
174. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac
repair. J Clin Invest 2005;115:572. [PubMed: 15765139]
175. Steinhoff G, Stock U, Karim N, Mertsching H, Timke A, Meliss RR, Pethig K, Haverich A, Bader
A. Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo
restoration of valve tissue. Circulation 2000;102:III50. [PubMed: 11082362]
176. O’Brien MF, Goldstein S, Walsh S, Black KS, Elkins R, Clarke D. The SynerGraft valve: a new
acellular (nonglutaraldehyde-fixed) tissue heart valve for autologous recellularization first

experimental studies before clinical implantation. Semin Thorac Cardiovasc Surg 1999;11:194.
177. Hoerstrup SP, Sodian R, Daebritz S, Wang J, Bacha EA, Martin DP, Moran AM, Guleserian KJ,
Sperling JS, Kaushal S, Vacanti JP, Schoen FJ, Mayer JE Jr. Functional living trileaflet heart valves
grown in vitro. Circulation 2000;102:III44. [PubMed: 11082361]
178. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR,
Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial
cells. Blood 1998;92:362. [PubMed: 9657732]
179. Rafii S, Oz MC, Seldomridge JA, Ferris B, Asch AS, Nachman RL, Shapiro F, Rose EA, Levin HR.
Characterization of hematopoietic cells arising on the textured surface of left ventricular assist
devices. Ann Thorac Surg 1995;60:1627. [PubMed: 8787455]
180. Deb A, Wang SH, Skelding K, Miller D, Simper D, Caplice N. Bone marrow-derived myofibroblasts
are present in adult human heart valves. J Heart Valve Dis 2005;14:674. [PubMed: 16245507]
181. Visconti RP, Ebihara Y, LaRue AC, et al. An in vivo analysis of hematopoietic stem cell potential:
hematopoietic origin of cardiac valve interstitial cells. Circ Res 2006;98:690. [PubMed: 16456103]
182. Matheny RG, Hutchison ML, Dryden PE, Hiles MD, Shaar CJ. Porcine small intestine submucosa
as a pulmonary valve leaflet substitute. J Heart Valve Dis 2000;9:769. [PubMed: 11128782]
183. Iwai S, Sawa Y, Ichikawa H, Taketani S, Uchimura E, Chen G, Hara M, Miyake J, Matsuda H.
Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular

prosthesis. J Thorac Cardiovasc Surg 2004;128:472. [PubMed: 15354111]
184. Simon P, Kasimir MT, Seebacher G, Weigel G, Ullrich R, Salzer-Muhar U, Rieder E, Wolner E.
Early failure of the tissue engineered porcine heart valve SY-NERGRAFT in pediatric patients. Eur
J Cardiothorac Surg 2003;23:1002. [PubMed: 12829079]
185. Elkins RC, Dawson PE, Goldstein S, Walsh SP, Black KS. Decellularized human valve allografts.
Ann Thorac Surg 2001;71:S428. [PubMed: 11388241]
186. Zund G, Breuer CK, Shinoka T, Ma PX, Langer R, Mayer JE, Vacanti JP. The in vitro construction
of a tissue engineered bioprosthetic heart valve. Eur J Cardiothorac Surg 1997;11:493. [PubMed:
9105814]
187. Rahimtoola SH. The next generation of prosthetic heart valves needs a proven track record of patient
outcomes at > or = 15 to 20 years. J Am Coll Cardiol 2003;42:1720. [PubMed: 14642677]
188. Staros EB. Innate immunity: new approaches to understanding its clinical significance. Am J Clin
Pathol 2005;123:305. [PubMed: 15842058]

189. McBrearty BA, Clark LD, Zhang XM, Blankenhorn EP, Heber-Katz E. Genetic analysis of a
mammalian wound-healing trait. Proc Natl Acad Sci USA 1998;95:11792. [PubMed: 9751744]
190. Ye S. Polymorphism in matrix metalloproteinase gene promoters: implication in regulation of gene
expression and susceptibility of various diseases. Matrix Biol 2000;19:623. [PubMed: 11102751]
191. Jones GT, Phillips VL, Harris EL, Rossaak JI, van Rij AM. Functional matrix metalloproteinases-9
polymorphism (C-1562T) associated with abdominal aortic aneurysm. J Vasc Surg 2003;38:1363.
[PubMed: 14681642]
192. Weinshilboum R. Inheritance and drug response. N Engl J Med 2003;348:529. [PubMed: 12571261]
193. Gencbay M, Turan F, Degertekin M, Eksi N, Mutlu B, Unalp A. High prevalence of hypercoagulable
states in patients with recurrent thrombosis of mechanical heart valves. J Heart Valve Dis
1998;7:601. [PubMed: 9870192]
194. Jaffer FA, Weissleder R. Seeing within: molecular imaging of the cardiovascular system. Circ Res
2004;94:433. [PubMed: 15001542]
195. Jaffer FA, Weissleder R. Molecular imaging in the clinical arena. J Am Med Assoc 2005;293:855.
196. Hill JM, Dick AJ, Raman VK, Thompson RB, Yu ZX, Hinds KA, Pessanha BS, Guttman MA,
Varney TR, Martin BJ, Dunbar CE, McVeigh ER, Lederman RJ. Serial cardiac magnetic resonance
imaging of injected mesenchymal stem cells. Circulation 2003;108:1009. [PubMed: 12912822]
197. Jaffer FA, Tung CH, Gerszten RE, Weissleder R. In vivo imaging of thrombin activity in
experimental thrombi with thrombin-sensitive near-infrared molecular probe. Arterioscler Thromb
Vasc Biol 2002;22:1929. [PubMed: 12426227]
198. Chen J, Tung CH, Mahmood U, Ntziachristos V, Gyurko R, Fishman MC, Huang PL, Weissleder
R. In vivo imaging of proteolytic activity in atherosclerosis. Circulation 2002;105:2766. [PubMed:

12057992]
199. Blom IE, van Dijk AJ, de Weger RA, Tilanus MG, Goldschmeding R. Identification of human ccn2
(connective tissue growth factor) promoter polymorphisms. Mol Pathol 2001;54:192. [PubMed:
11376134]
200. Ogata T, Shibamura H, Tromp G, Sinha M, Goddard KA, Sakalihasan N, Limet R, MacKean GL,
Arthur C, Sueda T, Land S, Kuivaniemi H. Genetic analysis of polymorphisms in biologically
relevant candidate genes in patients with abdominal aortic aneurysms. J Vasc Surg 2005;41:1036.
[PubMed: 15944607]
201. Boecker W, Bernecker OY, Wu JC, Zhu X, Sawa T, Grazette L, Rosenzweig A, del Monte F, Schmidt
U, Hajjar RJ. Cardiac-specific gene expression facilitated by an enhanced myosin light chain
promoter. Mol Imaging 2004;3:69. [PubMed: 15296671]
202. Fukuda D, Shimada K, Tanaka A, Kusuyama T, Yamashita H, Ehara S, Nakamura Y, Kawarabayashi
T, Iida H, Yoshiyama M, Yoshikawa J. Comparison of levels of serum matrix metalloproteinase-9
in patients with acute myocardial infarction versus unstable angina pectoris versus stable angina
pectoris. Am J Cardiol 2006;97:175. [PubMed: 16442358]
203. Fernandez CA, Yan L, Louis G, Yang J, Kutok JL, Moses MA. The matrix metalloproteinase-9/
neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present
in the urine of breast cancer patients. Clin Cancer Res 2005;11:5390. [PubMed: 16061852]
204. Meryman, H. Foreword. In: Fuller, BJ.; Lane, N.; Benson, EE., editors. Life in the Frozen State.
Boca Raton, FL: CRC Press; 2004.
205. Lovelock JE. The mechanism of the protective action of glycerol against haemolysis by freezing
and thawing. Biochim Biophys Acta 1953;11:28. [PubMed: 13066452]
206. Mazur P, Cole KW. Roles of unfrozen fraction, salt concentration, and changes in cell volume in
the survival of frozen human erythrocytes. Cryobiology 1989;26:1. [PubMed: 2924590]
207. Pegg DE, Jacobsen IA, Diaper MP, Foreman J. The effect of cooling and warming rate on the packing
effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol. Cryobiology
1984;21:491. [PubMed: 6499496]
208. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984;247:125.
209. Fowler AJ, Toner M. Prevention of hemolysis in rapidly frozen erythrocytes by using a laser pulse.
Ann N Y Acad Sci 1998;858:245. [PubMed: 9917823]

210. Karlsson JOM, Eroglu A, Toth TL, Cravalho EG, Toner M. Fertilization and development of mouse
oocytes cryopreserved using a theoretically optimized protocol. Hum Reprod 1996;11:1296.
[PubMed: 8671443]
211. Mazur P. Equlibrium, quasi-equilibrium, and non-equilibrium freezing of mammalian embryos. Cell
Biophys 1990;17:53. [PubMed: 1704816]
212. Thirumala S, Devireddy RV. A simplified procedure to determine the optimal rate of freezing
biological systems. J Biomech Eng 2005;127:295. [PubMed: 15971707]
213. Johari GP, Hallbrucker A, Mayer E. Two calorimetrically distinct states of liquid water below 150
Kelvin. Science 1996;273:90. [PubMed: 8688057]
214. Goetz A, Goetz SS. Vitrification and crystallization of organic cells at low temperatures. J Appl
Phys 1938;9:718.
215. Taylor, MJ. Vitrification in tissue preservation: new developments. In: Fuller, BJ.; Lane, N.; Benson,
EE., editors. Life in the Frozen State. Boca Raton, FL: CRC Press; 2004. p. 603-641.
216. Kaiser J. New prospects for putting organs on ice. Science 2002;295:1015. [PubMed: 11834818]
217. Fahy GM, Suja EA. Cryopreservation of the mammalian kidney. Cryobiology 1997;35:114.
[PubMed: 9299103]
218. Song YC, Pegg DE, Hunt CJ. Cryopreservation of the common carotid artery of the rabbit:
optimization of dimethyl sulfoxide concentration and cooling rate. Cryobiology 1995;32:405.
[PubMed: 7587281]
219. Acker JP, Larese A, Yang H, Petrenko A, McGann LE. Intracellular ice formation is affected by
cell interactions. Cryobiology 1999;38:363. [PubMed: 10413578]
220. Irimia D, Karlsson JOM. Kinetics and mechanism of intercellular ice propagation in a micropatterned
tissue construct. Biophys J 2002;82:1858. [PubMed: 11916845]
221. Acker JP, McGann LE. Cell-cell contact affects membrane integrity after intracellular freezing.
Cryobiology 2000;40:54. [PubMed: 10679150]
222. Acker JP, McGann LE. Protective effect of intracellular ice during freezing. Cryobiology
2003;46:197. [PubMed: 12686211]
223. Rall WF, Reid DS, Farrant J. Innocuous biological freezing during warming. Nature 1980;286:511.
[PubMed: 7402331]
224. Goodrich RP, Sowenimo-Cokr SO, Zerez CR, Tanaka KR. Preservation of metabolic activity in
lyophilized human erythrocytes. Proc Natl Acad Sci USA 1992;89(3):967. [PubMed: 1736313]
225. Bhowmick S, Zhu L, McGinnis L, Lawitts J, Nath BD, Toner M, Biggers J. Desiccation tolerance
of spermatozoa dried at ambient temperature: production of fetal mice. Biol Reprod 2003;68:1779.
[PubMed: 12606475]
226. Acker JP, Fowler A, Lauman B, Cheley S, Toner M. Survival of desiccated mammalian cells:
beneficial effects of isotonic media. Cell Preserv Technol 2002;1:129.
227. Crowe, JH.; Crowe, LM.; Tablin, F.; Wolkers, W.; Oliver, AE.; Tsvetkova, NM. Stabilization of
cells during freeze-drying: the trehalose myth. In: Fuller, BJ.; Lane, N.; Benson, EE., editors. Life
in the Frozen State. Boca Raton, FL: CRC Press; 2004. p. 581-601.
228. Hamilton, WJ.; Boyd, JD.; Mossman, HW. Human Embryology. Baltimore: William and Wilkins;
1962.
229. Coultras L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development.
Nature 2005;438:937. [PubMed: 16355211]
230. Ennett AB, Mooney DJ. Tissue engineering strategies for in vivo neovascularization. Expert Opin
Biol Ther 2002;2:805. [PubMed: 12517260]
231. Colton CK. Implantable biohybrid artificial organs. Cell Transplant 1995;4:41.
232. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization
and regeneration. Nat Med 2003;9:702. [PubMed: 12778169]
233. Conway EM, Collen D, Carmeliet P. Molecularmechanisms of blood vessel growth. Cardiovasc Res
2001;49:507. [PubMed: 11166264]
234. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth
factors and blood vessel formation. Nature 2000;407:242. [PubMed: 11001067]

235. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck

A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal
blood vessel development and lethality in embryos lacking a single VEGF allele. Nature
1996;380:435. [PubMed: 8602241]

236. Simons M. Angiogenesis: where do we stand now? Circulation 2005;111:1556. [PubMed:
15795364]
237. Lazarous DF, Shou M, Scheinowitz M, Hodge E, Thirumurti V, Kitsiou AN, Stiber JA, Lobo AD,
Hunsberger S, Guetta E, Epstein SE, Unger EF. Comparative effects of basic fibroblast growth
factor and vascular endothelial growth factor on coronary collateral development and the arterial
response to injury. Circulation 1996;94:1074. [PubMed: 8790049]
238. Laham RJ, Rezaee M, Post M, et al. Intracoronary and intravenous administration of basic fibroblast
growth factor: myocardial and tissue distribution. Drug Metab Dispos 1999;27:821. [PubMed:
10383927]
239. Nor JE, Christensen J, Mooney DJ, Polverini PJ. Vascular endothelial growth factor (VEGF)-
mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2
expression. Am J Pathol 1999;154:375. [PubMed: 10027396]
240. Langer R. Drug delivery and targeting. Nature 1998;392:5. [PubMed: 9579855]
241. Zisch AH, Lutolf MP, Hubbell JA. Biopolymeric delivery matrices for angiogenic growth factors.
Cardiovasc Pathol 2003;12:295. [PubMed: 14630296]
242. Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland
JL, Daugherty A, van Bruggen N, Redmond HP, Carano RA, Filvaroff EH. Vascular endothelial
growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad
Sci USA 2002;99:9656. [PubMed: 12118119]
243. Sheridan ME, Shea LD, Peters MC, Mooney DJ. Bioabsorbable polymer scaffolds for tissue
engineering capable of sustained growth factor delivery. J Control Rel 2000;64:91.
244. Peters MC, Polverini PJ, Mooney DJ. Engineering vascular networks in porous polymer matrices.
J Biomed Mater Res 2002;60:668. [PubMed: 11948526]
245. Sun Q, Chen RR, Shen Y, Mooney DJ, Rajagopalan S, Grossman PM. Sustained vascular endothelial
growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb. Pharm Res
2005;22:1110. [PubMed: 16028011]
246. Ennett AB, Kaigler D, Mooney DJ. Temporally regulated delivery of VEGF in vitro and in vivo. J
Biomed Mater Res 2006;79:176.
247. Shea LD, Smiley E, Bonadio J, Mooney DJ. DNA delivery from polymer matrices for tissue
engineering. Nat Biotechnol 1999;17:551. [PubMed: 10385318]
248. Huang YC, Riddle K, Rice KG, Mooney DJ. Long-term in vivo gene expression via delivery of PEI-
DNA condensates from porous polymer scaffolds. Hum Gene Ther 2005;16:609. [PubMed:
15916485]
249. Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric systems for dual growth factor
delivery. Nat Biotechnol 2001;19:1029. [PubMed: 11689847]
250. Lee KY, Peters MC, Anderson KW, Mooney DJ. Controlled growth factor release from synthetic
extracellular matrices. Nature 2000;408:998. [PubMed: 11140690]
251. Kong HJ, Riddle K, Matsumoto T, Leach K, Mooney DJ. Non-viral gene delivery regulated by
stiffness of cell adhesion substrates. Nat Mater 2005;4:460. [PubMed: 15895097]
252. Gerber H, et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis
during endochondral bone formation. Nat Med 1999;5:623. [PubMed: 10371499]
253. Murphy WL, Simmons CA, Kaigler D, Mooney DJ. Bone regeneration via a mineral substrate and
induced angiogenesis. J Dental Res 2004;83:204.
254. Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438:932. [PubMed: 16355210]
255. Greenberg DA, Jin K. From angiogenesis to neuropathology. Nature 2005;438:954. [PubMed:
16355213]
256. Huang YC, Kaigler D, Rice KG, Krebsbach PH, Mooney DJ. Combined angiogenic and osteogenic
factor delivery enhances bone marrow stromal-cell driven bone regeneration. J Bone Miner Res
2005;20:848. [PubMed: 15824858]

257. Smith MK, Peters MC, Richardson TP, Garbern JC, Mooney DJ. Locally enhanced angiogenesis
promotes transplanted cell survival. Tissue Eng 2004;10:63. [PubMed: 15009931]
258. UCLA Symposia on Molecular and Cellular Biology. Tissue Eng 1990;(Suppl 14E):227.
259. Buck CA, Horwitz AF. Cell surface receptors for extracellular matrix molecules. Ann Rev Cell Biol
1987;3:179. [PubMed: 2825736]
260. Akiyama SK. Integrins in cell adhesion and signaling. Hum Cell 1996;3:181. [PubMed: 9183647]
261. McDonald, JA.; Mecham, RP., editors. Receptors for extracellular matrix. San Diego, CA: Academic
Press; 1991.
262. Pierschbacher MD, Ruoslahti E, Sundelin J, Lind P, Peterson PA. The cell attachment domain of
fibronectin: determination of the primary structure. J Biol Chem 1982;16:9593. [PubMed: 7050098]
263. Mould AP, Komoriya A, Yamada KM, Humphries MJ. The CS5 peptide is a second site in the IIICS
region of fibronectin recognized by the integrin a4b1. J Biol Chem 1991;266(6):3579. [PubMed:
1750929]
264. Massia SP, Hubbell JA. Human endothelial cell interactions with surface-coupled adhesion peptides
on a nonadhesive glass substrate and two polymeric biomaterials. J Biomed Mater Res 1991;25(2):
223. [PubMed: 1829082]
265. Lin HB, Garcia-Echeverria C, Asakura S, Sun W, Mosher DF, Cooper SL. Endothelial cell adhesion
on polyurethanes containing covalently attached RGD-peptides. Biomaterials 1992;13(13):905.
[PubMed: 1477259]
266. Pierschbacher MD, Polarek JW, Craig WS, Tschopp JF, Sipes NJ, Harper JR. Manipulation of
cellular interactions with biomaterials toward a therapeutic outcome: a perspective. J Cell Biochem
1994;56(2):150. [PubMed: 7829572]

267. Glass J, Blevitt J, Dickerson K, Pierschbacher M, Craig WS. Cell attachment and motility on
materials modified by surface-active RGD-containing peptides. Ann N Y Acad Sci 1994;745:177.
[PubMed: 7832507]
268. Bashkin P, Doctrow S, Klagsbrun M, Svahn CM, Folkman J, Vlodavsky I. Basic fibroblast growth
factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like
molecules. Biochemistry 1989;28:1737. [PubMed: 2541764]
269. Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB
J 1994;8:823. [PubMed: 8070631]
270. Streuli C. Extracellular matrix remodeling and cellular differentiation. Curr Opin Cell Bio
1999;11:634. [PubMed: 10508658]
271. Kleinman HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis. Curr Opin
Biotechnol 2003;14:526. [PubMed: 14580584]
272. Baharvand H, Azarnia M, Parivar K, Ashtiani SK. The effect of extracellular matrix on embryonic
stem cell-derived cardiomyocytes. J Mol Cell Cardio 2005;38:495.
273. Philp D, Chen SS, Fitzgerald W, Orenstein J, Margolis L, Kleinman HK. Complex extracellular
matrices promote tissue-specific stem cell differentiation. Stem Cells 2005;23:288. [PubMed:
15671151]
274. Gage FH. Mammalian neural stem cells. Science 2000;287(5457):1433. [PubMed: 10688783]
275. Zhang Y, Bai XF, Huang CX. Hepatic stem cells: existence and origin. World J Gastroenterol 2003;9
(2):201. [PubMed: 12532431]
276. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman
G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275
(5302):964. [PubMed: 9020076]
277. Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A, Colucci WS, Liao R. CD31- but not
CD31 + cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res
2005;97(1):52. [PubMed: 15947249]
278. Bartsch G, Yoo JJ, De Coppi P, Siddiqui MM, Schuch G, Pohl HG, Fuhr J, Perin L, Soker S, Atala
A. Propagation, expansion, and multilineage differentiation of human somatic stem cells from
dermal progenitors. Stem Cells Dev 2005;14(3):337. [PubMed: 15969629]
279. De Ugarte DA, Ashjian PH, Elbarbary A, Hedrick MH. Future of fat as raw material for tissue
regeneration. Ann Plast Surg 2003;50(2):215. [PubMed: 12567065]

280. Flaim CJ, Chien S, Bhatia S. An extracellular matrix microarray for probing cellular differentiation.
Nat Meth 2005;2(2):119.
281. Bottaro DP, Liebmann-Vinson A, Heidaran MA. Molecular signaling in bioengineered tissue
microenvironments. Ann N Y Acad Sci 2002;961:143. [PubMed: 12081884]

282. Rosso F, Giordano A, Barbarisi M, Barbarisi A. From cell-ECM interactions to tissue engineering.
J Cell Physiol 2004;199(2):174. [PubMed: 15039999]
283. Badylak SF. Regenerative medicine and developmental biology: the role of the extracellular matrix.
Anat Rec B New Anat 2005;287(1):36. [PubMed: 16308858]
284. Li F, Li W, Johnson S, Ingram D, Yoder M, Badylak S. Low-molecular weight peptides derived
from extracellular matrix as chemoattractants for primary endothelial cells. Endothelium 2004;11
(3–4):199. [PubMed: 15370297]
285. Davis ME, Motion JPM, Narmoneva DA, Takahashi T, Hakuno D, Kamm RD, Zhang S, Lee RT.
Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for
endothelial cells. Circulation 2005;111:442. [PubMed: 15687132]

FIG. 1.
Lateral surface of the zygomatic bone, coronal section, epifluorescent illumination. Light color
is the 3-hour calcein label in a continuous layer of newly mineralized matrix. Vascular fill (red)
is seen in the periosteal vessels and in cross-sectioned vessels within the calcein-labeled layer
(arrows). Color images available online at www.liebertpub.com/ten.

FIG. 2.
Lateral surface of the temporal bone, coronal section, epifluorescent illumination. The 3-hour
calcein label is discontinuous and forms the tips of bony spicules. Vascular fill (red) shows
that the radially arranged vessels of this layer are continuous with intraosseous vessels (arrows)
and differ in orientation from the periosteal vessels. Color images available online at
www.liebertpub.com/ten.

FIG. 3.
Lateral portion of the zygomatic bone, parasagittal section. An intraosseous vessel near the
surface can be seen encased in a bony tube (arrow). Color images available online at

FIG. 4.
Cartilage has been engineered using a number of different cell types. Primary bovine
chondrocytes (A), caprine MSCs (B), mouse EBs (C), and human EBs (D) demonstrate
Safranin-O positive proteoglycan production in polyethylene glycol (PEG) gels with the
exception of human EBs, which did not undergo chondrogenesis. Human embryonic stem (ES)
cells were aggregated into EBs and either encapsulated directly (2A) or disaggregated and
cultured for five passages (1) before encapsulation (2B). The hEBs that were encapsulated into
PEG gels did not stain positive for Safranin-O (D). Cells derived from the hEBs (1)
encapsulated in PEG gels also did not stain positive for Safranin-O when cultured in
chondrogenic medium with TGF-β1 (E). Incorporation of the adhesion peptide sequence
YRGDS into the PEG gels promoted homogenous differentiation and formation of cartilage-
like tissue from hES-derived cells (F), confirming the unique requirements for human ES cell
differentiation. Reproduced with permission from Annals of Biomedical Engineering. Color
images available online at www.liebertpub.com/ten.

FIG. 5.
Cells labeled with colored dyes encapsulated in a bilayered hydrogel. Organized tissue can be
created or interactions of coculture environments can be studied. Reproduced with permission
from Annals of Biomedical Engineering. Color images available online at

FIG. 6.
Anatomy and matrix organization of the ligament-to-bone insertion site. (A) The anterior
cruciate ligament (ACL) connects to the femur and tibia through two insertion sites (posterior
view). (B) The multitissue organization of the tibial insertion, transiting from the ACL to
fibrocartilage (FC) region, and then to the bone region (modified Goldner’s Masson Trichrome,
bar = 500 μm).49 (C) The fibrocartilage interface is further divided into the nonmineralized
fibrocartilage (NFC) and mineralized fibrocartilage (MFC) zones (von Kossa, bar = 200 μm).
Color images available online at www.liebertpub.com/ten.

FIG. 7.
Coculture models to evaluate interaction of interface-relevant cells. (A-i) In vitro coculture
model of fibroblasts (Fb) and osteoblasts (Ob) permit heterotypic and homotypic cell-cell
interactions.88 (A-ii) Fibroblast (CFDA-SE, green) and osteoblast (CM-DiI, orange-red)
distribution at day 7, bar = 100 μm. (B) In vitro coculture model of chondrocytes (Ch) and
osteoblasts (Ob), established by forming an osteoblast monolayer on top of the chondrocyte
micromass. Glycosaminoglycan distribution was restricted to the chondrocytes micromass
during coculture (day 10, Alcian blue, bar = 100 μm). Color images available online at

FIG. 8.
Structure-function relationship at the ligament-to-bone insertion. (A-i) Elastographic analysis
of the ACL-to-bone insertion (TI) under applied uniaxial tension.97 Displacement map
calculated from ultrasound radio frequency data (Increase in magnitude in mm: blue to red,
bar = 5 mm). A region-dependent decrease in displacement is a result of increasing tissue
stiffness from the ligament to fibrocartilage region and to the bone region. (A-ii)
Microcompression testing of the ACL-to-bone insertion also revealed region-specific increase
in tissue stiffness from the nonmineralized (NFC) to mineralized fibrocartilage (MFC) and to
bone.102 In the displacement curve, slope of the curve in each region represents the strain, with
a less steeper slope for MFC, indicating decreased strain compared to the NFC zone. (B) The
increase in tissue stiffness across the interface may be related to the higher calcium phosphate
distribution from the NFC to MFC, and to bone (i von Kossa and ii Elemental analysis of the
MFC revealed presence of Ca and P at the insertion104). Color images available online at

FIG. 9.
Biomimetic multiphasic scaffold for interface tissue engineering: Design, in vitro and in vivo
testing. (A) Triphasic scaffold modeled after the three regions of the interface112: Phase A for
soft tissue, Phase B for the fibrocartilage region, and Phase C for bone. Phase A consists of
knitted degradable polymer mesh, Phase B of sintered degradable polymer microspheres, and
Phase C of osteointegrative polymer-ceramic composite microspheres.11 (B) In vitro coculture
of fibroblasts and osteoblasts on the triphasic scaffold resulted in phase-specific cell
distribution and controlled matrix heterogeneity.112 Fibroblasts (Calcein AM, green) were
localized in Phase A and osteoblasts (CM-DiI, red) were found in Phase C at day 1 (i) and day
28 (ii). Both osteoblasts and fibroblasts migrated into Phase B by day 28. (C) In vivo evalution
of the triphasic scaffold cocutlured with fibroblasts and osteoblasts revealed abundant tissue
infiltration and matrix production at 4 weeks postimplantation.113 (modified Goldner’s
MassonTrichrome, bar = 200 μm). Color images available online at

FIG. 10.
(A) Line drawing of biphasic construct. (B) Histological appearance of biphasic construct at
8 weeks of culture. The cartilage is integrated with the upper aspect of substrate and has a
nonmineralized zone and a mineralized zone (arrowhead) adjacent to the substrate (von Kossa
and toluidine blue, ×50 original magnification). Color images available online at

FIG. 11.
Paradigm for translating research in heart valve tissue engineering from the laboratory to the
clinic. Biomarkers for cell and tissue characterization in conjunction with structural, chemical,
and molecular information obtained via in vitro and in vivo models are necessary for
understanding key biological processes in tissue engineering and regenerative medicine. These
concepts and data can be used to predict and measure patient success and failure. Data from
clinical experience further informs the development of appropriate biomarkers, which may
result in reassessment of the appropriate characterization parameters. Reproduced and
modified with permission from Mendelson et al.141 Color images available online at

FIG. 12.
Long-term storage of cells is critical for the successful applications of tissue engineered
products. Cell and tissue banks are needed at various steps in the development of tissue
engineered products. Reprinted with permission from Acker, J.P., Chen, T., Fowler, A., and
Toner, M. Engineering dessication tolerance in mammalian cells: tools and techniques. In:
Fuller, B.J., Lane, N., and Benson, E.E., eds. Life in the Frozen State. Boca Raton, FL: CRC
Press, 2004. Color images available online at www.liebertpub.com/ten.

Table 1
Key Aspects of Tissue Engineering of Oral and Craniofacial Tissues
Tissue Engineering Aspect Challenge
Wound healing environment • Regeneration of lost tissues in essentially “contaminated” conditions.
Scaffold design requirements • Injectability.

• Ability to encapsulate cells.
• Ability to harden to a state which mimics the mechanical properties
of bone.
• Degradation at a rate fast enough to allow the in-growth of
surrounding tissue.
Cell-surface interactions • Ability to selectively promote the adhesion of specific cell

populations while excluding the invasion of others.
Growth-factor delivery • Increased efficiency by loading physiological amounts of growth

factors into a scaffold allowing for improved cost-effectiveness.
• Spatial and temporal control over the release of multiple growth
factors using multiple kinetic rates.
Assessment • Development of clinically-relevant animal models for in vivo

qualitative and quantitative assessment of implanted materials.
Scale-up • Ability to use tissue engineered constructs in large defects where

diffusional limitations may limit the viability of encapsulated cells.
• Ability to determine the translatability of results seen with in vivo
animal models in the clinical setting.
Specific clinical issues which • Control over the morphology of regenerated bone.
remain unaddressed
• Periodontal ligament anchorage and effective gingival seal around
titanium dental implants.
• Regeneration of entire teeth with supporting structures in extraction
sockets.

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Tissue Eng. 2006 December ; 12(12): 3307. doi:10.1089/ten.2006.12.3307.

Engineering Complex Tissues

ANTONIOS G. MIKOS1, SUSAN W. HERRING2, PANNEE OCHAREON2, JENNIFER

1Department of Bioengineering, Rice University, Houston, Texas 2 Department of Orthodontics,

Engineering of functional vascular networks, interfaces, structural hierarchy, and complex

ENGINEERING OF COMPLEX ORAL AND CRANIOFACIAL TISSUES

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Growth factor delivery

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Animal models—With this consideration in mind, improvements in tissue engineering

environment of the oral cavity.

OSTEOGENESIS AND THE VASCULATURE: SHOULD THE SCAFFOLD COME

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

ORGANIZATION AND INTEGRATION OF ENGINEERED CARTILAGE

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Technologies: Understanding biology and engineering organized tissues

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

with dyes encapsulated in a bilayered gel are pictured in Figure 5.

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

INTERFACE TISSUE ENGINEERING FOR SOFT TISSUE-TO-BONE

Application of tissue engineering methods12,33 to musculoskeletal tissue regeneration has led

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

tunnels and the native insertion site is lost due to surgery.

Cell-cell interactions and mechanism of graft integration—It is well established that

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

interface regeneration through two mechanisms: (i) phenotypic changes or transdifferentiation

Structure-function relationships at the ACL-bone interface—The first steps in

Current knowledge of mechanical properties of the insertion site is based on theoretical

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

TISSUE ENGINEERING OF ARTICULAR JOINT SURFACE INTERFACES

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Biphasic tissue constructs

pores. Important characteristics of this substrate, if it is used to support cartilage formation in

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Potential for cartilage repair

HEART VALVE TISSUE ENGINEERING AND REGENERATION RESEARCH

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

of embryological development, and functional tissue biomechanics and other structure-

Functional structure of heart valves: Role of valvular ECM and cells

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Cardiac valve development, maturation in utero, and postnatal changes

Valve adaptation and repair

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Studies of contemporary bioprosthetic heart valve substitutes

Conceptual approach to heart valve tissue engineering

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

decellularized matrix.175,176 Irrespective of the methodology, the key processes occurring

Engineered heart valves constructed from biodegradable polymer scaffolds

Harnessing the reparative potential of circulating endogenous cells

biomaterials (and potentially unseeded scaffolds) or natural cardiovascular tissues. Endothelial

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

Nevertheless, clinical applications of decellularized heart valves have been largely

Challenges and future directions in clinical translation

Demonstration of long-term efficacy (implantability, functionality, long-term performance)

Tissue Eng. Author manuscript; available in PMC 2010 February 12.

genetic defects in coagulation proteins may be unusually susceptible to thrombosis of prosthetic

BIOPRESERVATION FOR TISSUE ENGINEERING