Tollemar2016 PDF

Genes & Diseases
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Tollemar V et al
Stem cells, growth factors and scaffolds in craniofacial regenerative medicine
Running Title: Approaches to craniofacial defect repair
Viktor Tollemar1,2,3, Zach J. Collier1,2, Maryam K. Mohammed1,2, Michael J. Lee2,
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Guillermo A. Ameer4,5, Russell R. Reid3
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The University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA;
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Department of Orthopedic Surgery and Rehabilitation Medicine, The University of
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Chicago Medical Center, Chicago, IL 60637, USA;
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Laboratory of Craniofacial Biology and Development, Section of Plastic and
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Reconstructive Surgery, Department of Surgery, The University of Chicago Medicine,
Chicago, IL 60637, USA
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Department of Surgery, Feinberg School of Medicine, Chicago, IL 60611, USA;
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Biomedical Engineering Department, Northwestern University, Evanston, IL 60208
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Correspondence:
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Russell R. Reid, MD, PhD

Department of Surgery, Section of Plastic and Reconstructive Surgery
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The University of Chicago Medicine

5841 South Maryland Avenue
Chicago, IL 60637
Tel: 773-702-6302
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Fax: 773-702-1634
Email: [email protected]
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Abstract
Current reconstructive approaches to large craniofacial skeletal defects are often

complicated and challenging. Critical-sized defects are unable to heal via natural
regenerative processes and require surgical intervention, traditionally involving
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autologous bone (mainly in the form of nonvascularized grafts) or alloplasts. Autologous
bone grafts remain the gold standard of care in spite of the associated risk of donor site
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morbidity. Tissue engineering approaches represent a promising alternative that would
serve to facilitate bone regeneration even in large craniofacial skeletal defects. This
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strategy has been tested in a myriad of iterations by utilizing a variety of
osteoconductive scaffold materials, osteoblastic stem cells, as well as osteoinductive
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growth factors and small molecules. One of the major challenges facing tissue
engineers is creating a scaffold fulfilling the properties necessary for controlled bone
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regeneration. These properties include osteoconduction, osetoinduction,
biocompatibility, biodegradability, vascularization, and progenitor cell retention. This
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review will provide an overview of how optimization of the aforementioned scaffold

parameters facilitates bone regenerative capabilities as well as a discussion of common
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osteoconductive scaffold materials.

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Keywords: Scaffolds; regenerative medicine; bone regeneration; tissue engineering;

osteogenesis; craniofacial defects
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Introduction
Large craniofacial skeletal defects secondary to trauma, congenital condition, or cancer

resection pose serious challenges to reconstructive surgeons. Extensive defects which
prevent spontaneous re-ossification are termed ‘critical-sized’ and often require complex
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reconstructive approaches (Fig.1A).1 Repair of these defects has traditionally required
autologous bone grafts from a variety of sources, including cranium, tibia, rib, and iliac
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crest (Fig.1B).2,3 These procedures, although they have seen success clinically and are
currently the gold standard of care, necessitate a second surgical site with a significant
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risk of morbidity. In particular, undesirable sequelae at the donor site include infection,
bleeding, pain, swelling, unanticipated fractures, and injury to adjacent critical
structures.4–6 Additionally, autologous bone graft procedures have been complicated by
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unpredictable graft resorption rates, limited supply of autologous bone, and rapid bone
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remodeling in young children.2,3,7
Alternatives in the alloplast category, including demineralized bone matrix, bone
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ceramics, porous polyethylene implants, and various other polymers, have seen
variable success. However, they generally carry a greater risk of infection than
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autologous bone grafts and are more likely to fail over time.8–12 Permanent methods of
rigid fixation utilizing metals or metal alloys suffer similar limitations in addition to
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integrating poorly with the surrounding tissue.13 Because craniofacial reconstructive

surgeries are often performed on children (Fig. 1) who require repair capable of
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accommodating natural growth and development, permanent rigid fixation is not the
most favorable alternative.
Biocompatible implants that augment natural bone-regenerative capabilities
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currently represent the most promising and versatile approach to repairing critical-sized
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craniofacial defects.14 This tissue engineering-based strategy generally involves three

key elements: osteoconductive scaffolding, stem cells, and growth factors (Fig.2).
These three elements allow osteoblastic and endothelial progenitor cell differentiation,
bone formation, and integration with surrounding bone tissue even in large defects.15
Osteoblastic stem cells within an osteoconductive scaffold provide the possibility of a
tailored three-dimensional space for bone growth. Osteoblastic differentiation can be
induced by a variety of osteoinductive growth factors both in vivo and in vitro.16 Finally,
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efficacious bone regeneration requires integration with surrounding tissue, including

vascularization, fusion of the implant with autologous bone without fibrous tissue at the
bone-implant interface, and eventual complete replacement of the scaffold with new
bone.17–19
The goal of achieving these prerequisites has challenged tissue engineers to
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choose the optimum combination of cell types, scaffold properties, and growth factors.
The process is inherently complex and multidisciplinary due to requisite collaboration
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between molecular biology, materials science, surgery, and mechanical engineering.20
This review will explore current progress toward achieving reliable repair of craniofacial
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defects using osteoconductive scaffold and osteogenic stem cell-based tissue
engineering.
Stem Cells used for Bone Regeneration

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Irrespective of craniofacial bone defect size or complexity, healing is fundamentally
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dependent on the presence of osteogenic and vasculogenic precursor cells in

surrounding tissues.21 These precursors migrate to the injury site and differentiate into
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osteoblasts and endothelial cells, promoting bone formation and vascularization.22 In

recent years, clinical reports have suggested that stem cell supplementation may work
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synergistically with this natural progenitor cell migration and differentiation to produce
the best results in healing critical-sized bone defects.22–31
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Several stem cell types have been used both in vitro and in vivo to produce new
bone (Fig. 3). Bone marrow-derived mesenchymal stromal cells (BMSCs) are
increasingly being applied to craniofacial defect repair, and several studies have
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substantiated their effectiveness as osteoblastic precursors in critical-sized defect

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reconstruction.32–34 A recent phase I/II clinical trial determined that CD90+ osteoblastic
BMSCs and neovascularization-inducing CD14+ monocytes and macrophages seeded
onto a β-tricalcium phosphate (β-TCP) scaffold provided a viable treatment for patients
with severe maxillary bone deficiency.35,36 When compared with scaffold alone, the
progenitor cell-seeded scaffold treatment showed a higher proportion of regenerated
viable, highly vascularized, and mineralized bone in addition to a lower proportion of
residual β-TCP particles four months postoperatively.35 Mesenchymal stem cells derived
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from umbilical cord blood have also been used successfully, in conjunction with poly-
lactic co-glycolic acid (PLGA) implants, to heal critical-sized alveolar cleft defects in a
swine model. Investigators reported no inflammation and better bone quality than
autologous bone graft from the iliac crest by CT volumetric and histological analysis.37
However, despite its success, the use of BMSCs is limited by finite supply and the
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morbidity associated with procurement procedures.38
Adipose-derived stem cells (ADSCs) represent a promising alternative to BMSCs
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in that they are more plentiful, less painful to harvest, and easily expandable.39 ADSCs
have showed similar osteogenicity to BMSCs, with certain subpopulations
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demonstrating enhanced tendency toward osteoblast differentiation and others
successfully induced through gene therapy.34,40 The necessity for invasive procedures
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during harvesting still constrains ease of access to ADSCs and the scope of their
clinical significance.
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Urine-derived stem cells (USCs), which can be obtained from voided urine and
require no invasive procedures, have recently garnered a great deal of attention in the
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bone tissue engineering community as a promising, but still poorly studied, alternative
stem cell source. Research regarding USCs is still in its infancy, but recent studies by
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Guan et al. have demonstrated their applicability to bone regeneration.38,41–43 USCs are
biologically similar to ADSCs and are capable of osteogenic differentiation in vitro.43
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Furthermore, USCs have successfully differentiated into osteoblasts via calcium silicate
ion induction of the Wnt/β-catenin signaling pathway.38 They have also been shown to
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be compatible with both calcium sulfate/PLGA composite and β-TCP scaffolds.38,42

Neovascularization is a critical component of bone tissue engineering, and can
be facilitated by incorporation of endothelial progenitor cells (EPCs) in scaffold design.
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EPCs have been shown to enable neovascularization in response to ischemia.44,45 This

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ischemic response is seen in the context of critical-sized craniofacial defects, and EPCs
have been used in combination with MSCs and a thermoresponsive porous nano-
calcium sulfate/alginate scaffold to repair calvarial defects in rats.45 EPCs are also
compatible with β-TCP scaffolds, in which they have been shown to contribute directly
to neovasculogenesis through endothelial cell differentiation and recruitment of
additional host EPCs. Exogenous EPCs have also been shown to release pro-
angiogenic factors such as vascular endothelial growth factor (VEGF).46
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Osteoinductive Factors
A critical component of osteoblastic progenitor cell differentiation and subsequent
bone formation are osteoinductive growth factors (Table 1). Many growth factors are
known to enhance bone regeneration, including transforming growth factor β (TGF-β),
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fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and platelet
derived growth factor (PDGF).47–50 Several bone morphogenic proteins (BMPs),
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members of the TGF-β family, have been used clinically to induce bone regeneration in
critical-sized craniofacial defects as well as alveolar ridge and sinus augumentation.51–53
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They bind receptors on multiple stem cell types and induce osteoblastic differentiation
through the Smad protein signaling pathway.14 BMPs, particularly BMP-2 and BMP-7,
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have been studied extensively in bone healing and produce superior fusion rates with
fewer complications than autologous bone grafts.54–65 Infuse® Bone Graft (Medtronic
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and Wyeth) and Osigraft® (Stryker Biotech) are two FDA-approved collagen-based
scaffolds containing recombinant BMP-2 and BMP-7, respectively. The clinical success
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of these products demonstrates the importance of growth factors in osteogenesis and

underscores the potential of growth factor-infused scaffolds.
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Other osteoinductive BMPs include BMP-4, 6, and 9, and previous evidence

suggests that BMP-9, a relatively poorly characterized growth factor, is the most potent
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BMP in promoting in vitro and in vivo osteogenic differentiation of mesenchymal stem

cells.66–75 Despite such auspicious results, relatively high dose requirements, cases of
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ectopic bone formation, and paradoxical increase in bone resorption – particularly

observed with BMP-2 – have tarnished some of BMPs’ initial promise.76–79 Efforts are
ongoing to combine synergistic growth factors and carrier molecules to lower the
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necessary BMP dose and control its release.80,81

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Growth factor incorporation into scaffolds may be accomplished in a number of

ways, each of which confers unique properties. Soaking a scaffold in growth factor-
containing solution results in a loose association with the structural material and,
therefore, facilitates quick release of the desired stimulatory molecules. Conversely,
growth factors may be incorporated into and even covalently linked to the scaffold
microstructure for extended release. Cells modified to express and secrete
osteoinductive growth factors may also be seeded in the scaffold, achieving a similar
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effect.82 The necessary cell modifications typically involve gene therapy accomplished
either by viral or nonviral transduction. Viral transduction is the most effective means of
gene transfer and is generally carried out using retroviruses, adenoviruses, or adeno-
associated viruses.83,84 Gene transfer can also be accomplished via direct uptake of
gene-containing plasmids from solution or as a conjugate with a nucleus-bound
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biomolecule.84
Issues with growth factor-enriched scaffolds are generally associated with
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mismatched release profiles – the release of growth factor is often dictated by passive
diffusion or degradation rate, and does not appropriately parallel the rate of bone
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regeneration and healing.82 It has been shown that covalent linkage of the growth factor
to the scaffold may slow and improve its release profile to more closely approximate
cellular demands.85 For example, covalently incorporated VEGF in a fibrin scaffold
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results in a more tightly controlled release and, subsequently, a more organized
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vascularization in comparison to scaffold with unlinked VEGF.86 One risk inherent in
covalently incorporated growth factors is altering established mechanical,
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osteoconductive, or other properties of the scaffold material. Despite this, it has been
used in animal models to successfully repair mandibular, zygomatic, and calvarial bone
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defects.14,87
As a supplement to BMPs or other osteoinductive growth factor proteins, small
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molecules that help induce osteoblast differentiation have been used. Small molecules
are generally more cost-effective, easier to synthesize and handle, and diffuse rapidly.88
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Statins, as well as several immunosuppressants, are small molecules that have

demonstrated capability to induce osteoblastic differentiation and bone formation.89–92
Phenamil, an irreversible amiloride analogue, is another small molecule that has been
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shown to induce osteogenesis in dental pulp cells and BMSCs through robust activation
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of the BMP signaling pathway.93–96 Most recently, phenamil has demonstrated

synergistic effects with BMP-2 by inducing osteogenic differentiation of ADSCs in
calvarial defect repair.97
Characteristics of an Optimal Scaffold
Osteoconduction
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In designing scaffolds for bone regeneration, there are several key properties that tissue
engineers consider. First is the capacity to deliver exogenous osteoblastic and epithelial
progenitor cells to the defect site and/or to facilitate recruitment of host progenitor cells
that aid in bone generation and tissue integration. Osteoconduction refers to the ability
of the scaffold to not only act as a carrier for these progenitor cells but also to provide a
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viable template for bone growth.17 Osteoconductive materials that provide a supportive
microenvironment in which exogenous and endogenous progenitor cells can
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differentiate and produce vascularized bone are a key part of scaffold design.
Natural fracture healing is characterized by the formation of a cartilaginous
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callus, which undergoes mineralization, resorption, and replacement by new bone.98 It is
this role of the cartilaginous callus as an osteoconductive template that current scaffolds
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seek to emulate. However, whereas physiologic bone healing is limited to small defects,
scaffolds enhance these processes to bridge large segmental defects.99 Collagen and
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hydroxyapatite, the primary organic and mineral components of bone, respectively, are
prototype osteoconductive materials and will be discussed later in this review.100
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The concept of mimicking native bone ECM, which serves as a collagenous

framework for osteoblasts and a reservoir for growth factors, has played a significant
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role in scaffold design.101 Interplay between the scaffold and progenitor cells should
closely mimic natural cell surface receptor and ECM interactions.18 These interactions
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are critical in bone regeneration processes such as osteoblast adhesion, proliferation,

migration, differentiation, and matrix deposition.18 The importance of biophysical
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cell/scaffold interactions on cell function has been underscored by studies

demonstrating significant differences in cell adhesion and differentiation behavior with
changes in scaffold elasticity and surface microstructure.102,103
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Osteoinduction
In smaller fractures, natural regenerative healing occurs via recruitment of
mesenchymal stem cells from adjacent tissues and bone marrow to the site of injury,
where they are induced to differentiate into osteoblasts and deposit new bone to bridge
the fracture.98,104 Differentiation of these migratory progenitor cells is accomplished via
mechanical, biochemical, and biophysical factors in a process called osteoinduction.104
Osteoinductive scaffold designs seek to emulate this natural phenomenon through
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biochemical structure, progenitor cell adhesion properties, and delivery of growth

factors.44,105,106
Biocompatibility
Biocompatibility is an essential attribute of any scaffold implant, and in order to be
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clinically successful, it must not elicit a damaging inflammatory response. In the context
of biodegradable scaffolds, the most common way for unwanted inflammatory
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processes to occur is by production of reactive oxygen species (ROS). Accumulation of
degradation products may generate toxic levels of ROS.107–111 Approaches to
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minimizing the inflammatory response include incorporation of biomimicking materials
as well as conjugate antioxidants in the scaffold itself.112–115 Utilizing scaffolds that can
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be delivered through minimally invasive techniques, such as injectable hydrogels or
thermoresponsive scaffolds, is also an important tactic to reduce inflammation.116,117
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Biodegradability
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Osteoconductive scaffolds should act only as a temporary framework for bone

regeneration.18 Temporality is critically important, as the ideal scaffold is not meant to
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be a permanent prosthetic, but rather a provisional support for osteoblastic

differentiation, bone regeneration, and vascularization until fully functional tissue has
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replaced the scaffold and the defect is healed.18 Full resorption of the original scaffold is
necessary for uninterrupted bone remodeling and physiologic responses to mechanical
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stimuli.19 Unmatched rates of scaffold material resorption and bone formation may result
in incomplete bone regeneration or obstructed remodeling and tissue integration.118–120
Therefore, degradability of the scaffold into biocompatible byproducts is an essential
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property that is governed by scaffold chemical composition, micro- and macrostructure,

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and numerous host factors.19,121 Clinical factors affecting bone regeneration and
scaffold degradation rates, including patient co-morbidities and defect anatomy, must be
considered in selecting graft substitutes for repairing craniofacial defects.19
Vascularization
An extensive variety of scaffolds and stem cell therapy approaches to healing
craniofacial defects have been proposed and tested, but successful treatment ultimately
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depends on integration with surrounding tissue. That success hinges on two key factors
– the ability to recruit local osteoblastic and endothelial progenitor cells to the site of
injury and the existence of functioning vasculature near the defect.13,45 Vasculogenesis,
or formation of new blood vessels through differentiation of recruited endothelial
progenitor cells (EPCs), is a normal response to traumatic injury and is largely mediated
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by vascular endothelial growth factor (VEGF).16,122 Downstream effects of VEGF
culminate in proliferation of circulating EPCs, which initiate vasculogenesis at the defect
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site.16 Vasculogenesis and angiogenesis, collectively known as neovascularization, are
necessary prerequisites for osteogenesis, and it has been shown that bone
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regenerative capabilities are directly linked to circulating EPC levels.122
However, effective delivery of these EPCs is complicated by the vascular
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deficiency that often exists in the context of critical-sized craniofacial and other bone
defects.45 In order to promote vascularization despite these challenges, scaffolds can be
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enriched with both growth factors and endothelial progenitor cells. Several strategies
have been attempted, including direct integration of neovasculogenic growth factors and
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cytokines, incorporating cells capable of secreting these growth factors, featuring

adhesion proteins conducive to endothelial cell attachment and blood vessel formation,
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and seeding with endothelial progenitor cells.46,123–127 Multipotent bone marrow stromal
cells enriched for mesenchymal and endothelial phenotypes have also demonstrated
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capacity for highly vascularized bone generation in mandibular defect repair.128

The importance of vascular supply in bone reconstruction is well
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recognized.129,130 Osteoprogenitor cells associate with endothelial cells, which supply

not only oxygen and nutrients but also growth factors necessary for osteoblastic
differentiation.131 For this reason, neovascularization is an essential step in promoting
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sustained bone regeneration. Accommodating for endothelial progenitor cell invasion

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and attachment, delivery of pro-angiogenic factors, and blood vessel formation

necessitates a porous scaffold structure.132 It is thought that 150 to 500 µm is a
sufficient pore diameter to support neovascularization and blood vessel
invasion.133`However, porosity often relates inversely with material strength. The idea
that reduced porosity and higher density confers greater mechanical strength while
increased porosity facilitates growth factor delivery, cell migration, and vascularization
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has been a key principle of scaffold design.18,134,135 As a result, the ideal scaffold strikes
a balance between the two competing properties.18
Head and neck cancer treatments involving bone resection and radiation therapy
also pose a significant challenge for reconstructive surgeons due to the debilitating
nature of radiation toxicity on bone regeneration.13,136 Radiation therapy severely
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complicates bone development, remodeling, and fracture healing secondary to
progenitor cell loss and compromised vasculature.137–140 These complicating factors
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require a combination of neovasculogenic progenitor cells and growth factors to ensure
proper vascularization.141,142
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Biomaterials for Osteoconductive Scaffold Construction
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Although autologous bone grafts remain the gold standard for repairing critical-sized
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craniofacial defects, their use is cost-prohibitive, requires a second surgical site, is
associated with significant donor site morbidity, and is limited by the finite supply of
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autologous bone.3,4,143 The use of biocompatible scaffolds in healing these defects may
provide a more cost-effective and less complicated alternative to autologous bone
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grafts.121 Scaffolds provide an osteoconductive and osteoinductive extracellular matrix

analog to facilitate cellular migration, proliferation, adhesion, differentiation, and
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generation of new bone.105,121 A variety of materials for this purpose have been studied,
including ceramics, natural and synthetic polymers, various composite materials, silicon-
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based bioglass, and metals (Table 2).13,121,144
Demineralized bone matrix

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Demineralized bone matrix (DBM) is produced by acid extraction of allogenic bone, a

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process that removes the inorganic mineral component of bone and leaves a type I
collagen framework.145 Demineralization also exposes osteoinductive growth factors,
including BMPs, making DBM more osteoinductive than complete bone grafts. DBM is
currently available as powder, granules, gel, putty, and paste, but an intrinsic limitation
of all DBM types is poor mechanical strength and porosity.146 A recent retrospective
study investigating craniofacial defect reconstruction outcomes using bone cement,
autologous bone grafts, and DBM revealed the highest rate of residual defect using
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DBM.147 Because of such findings, DBM alone is not considered a promising scaffold
material. However, recent efforts using poly(lactic acid) (PLA)/DBM composite scaffolds
for bone engineering have proven to be more effective.145
Ceramics
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Some of the most promising initial scaffolds closely mimic the chemistry and structure of
native extracellular matrix in bone.13 Foremost among these are calcium phosphate
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ceramics, including hydroxyapatite (HA), β-TCP, and biphasic calcium phosphate.13
Due to their biocompatibility, safety, reliability, availability, ease of sterilization, and long
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shelf life, calcium phosphate scaffolds have considerable promise as an alternative to
bone grafts.148,149
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Hydroxyapatite bioceramics confer a high degree of osteoconductivity but are
brittle and resorbed at a rate much slower than desired, often taking several years. This
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is in contrast to tricalcium phosphate (TCP) scaffolds, which have been reported to fully
resorb within 12 weeks.18,150 By altering calcium-to-phosphate ratios, internal pore
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architecture, and other parameters of these TCP scaffolds, engineers have been able to
control resorption rates and improve osteogenicity.4–6 Furthermore, HA-TCP composite
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scaffolds have demonstrated both osteoconductivity and favorable resorption

rates.151,152 Similarly, it has been shown that HA/collagen composite implants are
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characterized by improved stiffness and osteointegration in comparison to collagen

alone in critical-sized rat calvarial defects.153 An injectable collagen/calcium phosphate
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hydrogel has also exhibited efficient umbilical cord-derived mesenchymal stem cell
(UCMSC) seeding and ability to support osteoblastic differentiation and osteogenesis.154
Although conferring essential osteoconductive, porous, and resorption properties,
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ceramic scaffolds are relatively brittle and do not have the strength optimally desired. To
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that end, more recent experiments have found that incorporating hydroxyapatite
nanoparticles into more structurally competent polymer scaffolds has resulted in a more
favorable combination of strength, protein loading, cell adhesion and migration, and
osteogenic properties.155 In addition, a scaffold comprised of calcium phosphate
ceramic tiles set within a titanium framework has recently been described in the context
of complex craniofacial defect repair.3
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Calcium carbonate is another potential ceramic material for osteoconductive

scaffold fabrication. It has better natural biodegradation properties than calcium
phosphate, and may prove useful in pediatric craniofacial reconstruction, where highly
active skeletal remodeling necessitates rapid scaffold resorption.14,156 As of yet, this
material has most significantly been used to repair burr holes from hematoma-related
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neurosurgery cases.156 Two studies have tested alveolar bone regenerative capabilities
of calcium carbonate scaffolds and concluded that its mechanism of supporting bone
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growth is primarily through space-provision rather than previously hypothesized
osteoconductive properties.157,158 Since then, little research has been done to further
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characterize bone tissue engineering applications for calcium carbonate.
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Polymers
Natural and synthetic polymers are often used as scaffold materials for bone tissue
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engineering because of a well-balanced combination of properties, including
biodegradability, biocompatibility, porosity, and ease of handling.159–161 Naturally-
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derived materials, such as collagen and fibrin proteins, or chitin-derived chitosan

polysaccharide, are also an option for bone tissue engineering.117,162 Such materials
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may confer greater cell adhesion and functional support properties than synthetic
materials, but in most cases, this is offset by several disadvantages. Natural polymers
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often offer less control over mechanical properties, sometimes exhibit immunogenicity,
and frequently exist in finite supply; therefore, they are difficult and expensive to obtain.
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Synthetic polymers, however, do not suffer from these shortcomings and have been a
more important source of biomaterials for osteoconductive scaffold construction.162
Synthetic polymers can be produced on a large scale using reproducible and
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tunable methods, providing fine control over mechanical and physical properties. They
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have a well-documented history of clinical application in craniofacial bone

reconstruction, especially in children.163 Synthetic polymers like poly(lactic acid) (PLA),
poly(glycolic acid) (PGA), and various iterations of combined poly(lactic-co-glycolic acid)
(PLGA) have been used for a range of clinical applications, including critical-sized
craniofacial defect repair.37,164
PLA is an FDA-approved synthetic biomaterial that has several properties
conducive to bone tissue engineering, including controllable biodegradation rate,
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biocompatibility, and good mechanical strength.165 It has been applied clinically to

fabrication of resorbable sutures, as a drug delivery scaffold, and as resorbable bone
fixation devices in fracture healing. However, its application as a scaffold biomaterial for
craniofacial bone regeneration is limited by poor osteoinductive properties.145 PGA is
another FDA-approved synthetic biomaterial with a variety of tissue engineering
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applications, including regeneration of cartilage, bone, tendon, muscle, and skin.166–168
Despite such adaptability, its mechanical properties are not ideal for the precision bone
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reconstruction necessary for craniofacial defect repair because of its softness and
inability to maintain shape. PGA and PLA alone are not suitable bone tissue
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engineering scaffold materials, but their respective softness and low osteoinductivity
have been partially addressed by combining them to form a PLGA composite
scaffold.169 PLGA has been shown to have a controllable degradation rate (through
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varying composition of its constituent homopolymers) in addition to supporting
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osteoblast attachment, growth, and differentiation both in vitro and in vivo.162,170–173
Nevertheless, PLGA’s mechanical properties and osteoconductivity are suboptimal for
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bone tissue engineering, and it is most often used as part of a composite material with
ceramics, bioglass, or other more osteoconductive materials.173,174
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Poly(propylene fumarate) (PPF) is a synthetic, unsaturated, linear polyester

polymer that is biodegradable, biocompatible, osteoconductive, injectable, and
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sufficiently strong for craniofacial bone tissue engineering.175–185 It generally requires a

small monomer accelerating agent, such as N-vinylpyrrolidone, in order to crosslink as
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an injectable polymer.186 A two-phase PPF cement incorporating cross-linked

microparticles to increase strength and lower setting temperature has been developed.
This PPF-based system has improved injectability, setting temperature, and setting time
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over clinically available polymethyl methacrylate (PMMA) bone cement and is believed
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to be suitable for application in craniofacial bone regeneration.175 PPF has also been
used as a co-polymer with polycaprolactone (PCL) as a scaffold for osteoblastic
differentiation and maturation in vitro.187 PCL is a non-aromatic polyester that is highly
flexible and has a controllable biodegradation rate owed to alterable substituent
molecular weight.188–190 Similarly, the PPF-PCL co-polymer setting time, setting
temperature, mechanical strength, and other physical properties can be tuned through
variation of substituent molecular weight as well as relative proportion of PPF and
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PCL.186,187 PPF-PCL’s chemical structure also allows for HA incorporation, which aids
osteoblast progenitor cell adhesion and proliferation.187
Polyamide (PA) is a synthetic polymeric collagen analog that provides excellent
strength as well as biocompatibility. Those properties have made PA a promising
partner for HA or other bioceramics in osteoconductive composite scaffolds. As a BMP-
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7-transduced MSC-laden composite with HA nanoparticles, PA has been successfully
used to repair mandibular defects in rabbits.191
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Metals
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Currently, metals such as titanium are used clinically in craniofacial reconstruction.
However, as inert alloplasts, they do not integrate with surrounding tissue and do not
stimulate new bone formation.13 Metals that degrade in a physiological setting have
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been proposed in order to solve this problem and promote more long-term success.
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Biodegradable metals, such as magnesium alloys, have generally been shown to
possess mechanical properties mimicking that of natural bone while retaining the critical
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ability to resorb over time.164,192 Mg-rare earth element compounds, Mg-Ca, pure Fe,
Fe-Mn alloys, and Fe foam have all been tested as osteoconductive scaffold materials
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for bone tissue engineering.193–204 In particular, Mg and its alloys have been shown to
support osteoblastic differentiation of progenitor cells and are degraded in vivo to Mg
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hydroxide and hydrogen gas.195 Given the importance of porosity for progenitor cell
migration and neovascularization, porous Mg scaffolds have been investigated and can
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be fabricated with preserved mechanical properties.164,205,206 Their strength, ductility,

biodegradability, and osteoconductive properties make Mg alloys, and potentially other
metals, possible alternatives to polymer or ceramic scaffolds.164
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Incorporating metal nanoparticles into polymer scaffold materials has also been
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an ongoing effort to produce higher strength composite scaffolds that retain their
osteoinductivity and osteoconductivity.144,155,207 Addition of other trace impurities, such
as zinc oxide, iron, and silicon dioxide, has been shown to confer a greater degree of
control in degradation rates, density, mechanical strength, and biocompatibility.105 The
addition of zinc and silicon has boosted both expression of type I collagen and
extracellular signaling promoting angiogenesis as well as osteoblast differentiation.208,209
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Bioglass
There are two major groups of glass-based osteogenic scaffolds: glass-ceramic and
glass-polymer porous composites.144 It has been demonstrated that silicon found in
glass enhances angiogenesis as well as gene expression regulating osteogenesis and
growth factor production in osteoblasts.13 Several studies have confirmed that silicate-
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based scaffolds are capable of stimulating osteogenesis.210–212 Accordingly, silicon has
been successfully incorporated into bioceramics in order to augment bioactivity and
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osteostimulatory effects.211,213–216
For example, silicon/HA scaffolds have also shown increased bone ingrowth over
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HA alone, but these hybrids are limited by low mechanical load strength.13 Alternatives
include calcium silicate (CS)-containing scaffolds, which are able to stimulate
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osteogenic differentiation of several adult stem cell lines, including BMSCs, and have
pro-angiogenic properties.38,215,217–221 Importantly, these scaffolds are able to have
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these effects without the addition of exogenous growth factors.217,218 Osteogenic and
angiogenic growth factors have previously been utilized in bone tissue engineering, but
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the prospect of a single scaffold capable of inducing both osteogenesis and

angiogenesis without exogenous growth factors has exciting implications.215,222,223
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Silicate bioglass as well as some ceramic scaffolds have been shown to posses this
dual-inductive attribute.211,215,221
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As previously discussed, composite scaffolds combining materials with different

desirable properties are a step toward the ideal. Silicate composite scaffolds have been
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tested, and varying the relative proportion of each component affords some degree of
control over mechanical properties, hydrophobicity, and degradation.217,218,224,225
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Injectable biomaterials
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Injectable biomaterials provide two major advantages over traditional solid scaffolds;
they can be delivered through minimally invasive means, and they spontaneously mold
to the shape of even the most complicated defects. This has important implications for
reducing inflammatory side effects and subsequent scar formation stemming from
invasive surgery and imprecise scaffold fit. Injectable biomaterials have been tested in
the context of tissue engineering and may be appropriate for facilitating osteogenesis in
craniofacial defects.117,226,227 In particular, thermoresponsive biomaterials have been
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shown to predictably undergo liquid-to-solid phase change at appropriate physiological

temperatures and may be a potent delivery mechanism for osteogenic growth factors
and progenitor cells.228–233
N-isopropylacrylamide (NIPAA) is a particularly well studied thermoresponsive
biomaterial, but it is limited by issues including toxicity, nondegradability, and
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hydrophobicity-driven syneresis with subsequent release of compounds or lysis of cells
entrapped within the scaffold.234–237 Many of these limitations may be overcome with
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incorporation of poly(polyethylene glycol citrate) acrylate (PPCac) to form a
poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) polymer.116 This
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material not only preserves the thermoresponsive properties of NIPAA but also
possesses higher protein loading efficiency, supports three-dimensional cell
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proliferation, retains viable cells for at least 72 days, and has intrinsic antioxidant
properties.116,238,239
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Hydrogels comprise another important class of osteoconductive scaffolds that
can be delivered through noninvasive means.13,240,241 They are water-absorbing
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matrices composed of cross-linked hydrophilic polymers that are well suited to

harboring growth factors and viable stem cells.241,242 As a result, hydrogels are ideal for
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stem cell and biofactor delivery that promote bone tissue regeneration.240–242 For
example, a composite hydrogel incorporating BMP-2 and synergistic chitosan
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(deacetylated chitin) has demonstrated controlled release of BMP-2 with minimal burst
phase and shows remarkable bone regenerative capability.81
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Other injectable scaffolds include hydroxyapatite or calcium sulfate pastes, but

are complicated by syneresis and contraction, as well as brittleness following
setting.243,244 Using a combination of these and other materials in injectable composites
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helps overcome many of the individual materials’ limitations and enhances

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osteoconductivity.245,246 For example, PLGA microspheres coated with HA form a

colloidal gel that can be seeded with osteoblastic progenitor cells and successfully
support osteogenesis in vivo.247,248 Furthermore, PLGA-HA microsphere gel is an
effective delivery vehicle for the anti-osteoporotic drug alendronate, demonstrating a
sustained drug release profile and minimal burst phase.249 If this can be replicated with
osteoinductive small molecules or growth factors, it would greatly enhance the
osteogenic potential of PLGA-HA as a biomaterial for bone tissue regeneration. Another
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composite microgel scaffold composed of chitin, polycaprolactone, and HA has been

investigated with ADSCs and has produced promising results for application in bone
tissue engineering.117 As with other composite scaffolds, relative proportions of each
component can be tuned to provide optimal degradation rate, viscoelastic and
mechanical properties, cell adhesion properties, and osteoconductivity.117,250,251
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Osteoinductive molecular structure
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In addition to the composition of the scaffold, the molecular structure is also a design
priority for optimizing osteoconductive and osteoinductive properties. It has been
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suggested that an optimal approach for bone regeneration should closely mimic that of
natural healing, and the design of an osteoinductive scaffold should reflect the basic
multicellular unit of corticocancellous bone.252 This basic structure consists of a long
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cylindrical unit in line with the bone’s long axis and is composed of osteoclasts on the
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leading end and osteoblasts laying down new bone on the lagging end. Designing
scaffolds to initiate this bone remodeling step without the need to first deposit a
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temporary bone matrix is a novel idea pursued by some investigators.252 This strategy
would utilize osteoinductive geometric cues within the scaffold to initiate bone formation
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without the need for exogenous osteogenic molecular signals.252,253

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Conclusions and Future Directions

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Thorough understanding of the physiology and molecular pathways involved in bone

formation and remodeling is a prerequisite for making advances in craniofacial bone
tissue engineering. Innovations in material science and molecular biology have allowed
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tissue engineers to augment physiologic bone healing and make bone regeneration via
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scaffold/stem cell therapy a clinical possibility. Combining biomaterials, often with

competing properties, to fabricate optimized scaffolds for use in craniofacial skeletal
regeneration is representative of current research trends and the most promising
strategy for tissue engineers and craniofacial surgeons. New advances unlocking the
osteogenic potential of several stem cell types, as well as the discovery of more readily
available stem cell sources (e.g. urine-derived stem cells), are also providing exciting
prospects for craniofacial bone regeneration.
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Despite such advances in tissue engineering, craniofacial bone reconstruction is

often complicated by scarring, osteomyelitis, osteonecrosis, or previous radiation
damage. The combination of stem cells, growth factors, small molecules, and scaffold
materials used in reparative bone tissue engineering will largely be guided by these and
other complicating factors. Still, relatively little research explores the behavior of tissue
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engineering approaches in the context of extensive medical comorbidities or
compromised wound healing capability. Craniofacial skeletal repair via tissue
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engineering remains the most promising alternative to autologous bone grafts, and
numerous modalities involving a variety of stem cells, growth factors, and
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osteoconductive scaffold materials have been tested and met with success in animal
models. In the future, strategies and materials must be refined to achieve more reliable
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outcomes and to address the various challenges posed by real clinical scenarios in
which craniofacial reconstruction is appropriate.
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Acknowledgments
The reported work was funded in part by the Chicago Biomedical Consortium with
support from the Searle Funds at The Chicago Community Trust (RRR, GA), and a
NIH/NIDCK Career Development Award (#1K08 DE020140-01; RRR). VT was a
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recipient of the Pritzker Summer Research Fellowship funded through a NIH T-35
training grant (NIDDK). ZC was a recipient of the Pritzker Research Fellowship. MKM
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was a recipient of Howard Hughes Medical Institute Medical Research Fellowship.
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Conflict of Interest
The authors declare no conflict of interest.
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Figure Legends
Fig.1 Case example of a pediatric craniofacial defect. A) Depicted is a large

craniofacial skeletal defect resulting from resorption of an autogenous bone graft
following emergency craniectomy and delayed replacement of the bone. B)
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Reconstruction was accomplished through a second autograft involving full-thickness
resection of large portions of the frontal and right parietal bones. The donor site was
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repaired using demineralized bone matrix and particulate bone graft. The use of these
CT images follows the guidelines of the University of Chicago Institutional Review
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Board.
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Fig.2 Tissue engineering paradigm for craniofacial defect repair. Illustration
depicting ideal modality for craniofacial defect repair. The strategy involves growth
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factor-induced osteoblastic differentiation and bone formation within an osteoconductive
and biodegrdadable scaffold.
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Fig.3 Osteoblastic stem cell sources. The potential sources of mesenchymal stem
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cells (MSCs) that can be used for bone tissue engineering and regeneration. The
recently described urine-derived stem cells (USCs) may represent one of the most
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promising and convenient sources of MSCs for tissue engineering and regenerative
medicine.
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Table 1. Osteoinductive growth factors. Growth factors that can be used in bone
tissue engineering and their general contribution to osteogenesis.
Table 2. Biomaterials for bone tissue engineering. Commonly used biomaterials for
bone regeneration in craniofacial defect repair.
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Osteoblastic
Growth Factor Osteoblast Proliferation Neovasculogenesis
Differentiation
TGF-B promoting promoting
FGF promoting
VEGF promoting/inducing
PDGF promoting* promoting promoting
BMP-2 inducing promoting early; inhibiting late
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*Only PDGF-AA has been shown to promote osteoblastic differentiation in MSCs
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Osteoconductive biomaterials for scaffold construction

Allogenic bone derivative Demineralized bone matrix (DBM)
Ceramics Hydroxyapatite (HA)
Tricalcium phosphate (TCP)
biphasic calcium phosphate
Calcium carbonate
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Polymers poly(lactic acid) (PLA)
poly(glycolic acid) (PGA)
poly(lactic-co-glycolic acid) (PLGA)
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Poly(propylene fumarate) (PPF)
Polycaprolactone (PCL)
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Polyamide (PA)
Chitosan
Metals Titanium
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Magnesium Alloy
Zinc (doping)
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Bioglass Silicon
Calcium-silicate (CS)
Thermoresponsive N-isopropylacrylamide (NIPAA)
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poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN)

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Genes & Diseases

Stem cells, growth factors and scaffolds in craniofacial regenerative medicine

Running Title: Approaches to craniofacial defect repair

Viktor Tollemar1,2,3, Zach J. Collier1,2, Maryam K. Mohammed1,2, Michael J. Lee2,

Russell R. Reid, MD, PhD

The University of Chicago Medicine

Current reconstructive approaches to large craniofacial skeletal defects are often

review will provide an overview of how optimization of the aforementioned scaffold

osteoconductive scaffold materials.

Keywords: Scaffolds; regenerative medicine; bone regeneration; tissue engineering;

Large craniofacial skeletal defects secondary to trauma, congenital condition, or cancer

integrating poorly with the surrounding tissue.13 Because craniofacial reconstructive

craniofacial defects.14 This tissue engineering-based strategy generally involves three

efficacious bone regeneration requires integration with surrounding tissue, including

Stem Cells used for Bone Regeneration

dependent on the presence of osteogenic and vasculogenic precursor cells in

osteoblasts and endothelial cells, promoting bone formation and vascularization.22 In

substantiated their effectiveness as osteoblastic precursors in critical-sized defect

be compatible with both calcium sulfate/PLGA composite and β-TCP scaffolds.38,42

EPCs have been shown to enable neovascularization in response to ischemia.44,45 This

of these products demonstrates the importance of growth factors in osteogenesis and

Other osteoinductive BMPs include BMP-4, 6, and 9, and previous evidence

BMP in promoting in vitro and in vivo osteogenic differentiation of mesenchymal stem

ectopic bone formation, and paradoxical increase in bone resorption – particularly

necessary BMP dose and control its release.80,81

Growth factor incorporation into scaffolds may be accomplished in a number of

Statins, as well as several immunosuppressants, are small molecules that have

of the BMP signaling pathway.93–96 Most recently, phenamil has demonstrated

Characteristics of an Optimal Scaffold

The concept of mimicking native bone ECM, which serves as a collagenous

are critical in bone regeneration processes such as osteoblast adhesion, proliferation,

cell/scaffold interactions on cell function has been underscored by studies

biochemical structure, progenitor cell adhesion properties, and delivery of growth

Osteoconductive scaffolds should act only as a temporary framework for bone

be a permanent prosthetic, but rather a provisional support for osteoblastic

property that is governed by scaffold chemical composition, micro- and macrostructure,

cytokines, incorporating cells capable of secreting these growth factors, featuring

capacity for highly vascularized bone generation in mandibular defect repair.128

recognized.129,130 Osteoprogenitor cells associate with endothelial cells, which supply

sustained bone regeneration. Accommodating for endothelial progenitor cell invasion

and attachment, delivery of pro-angiogenic factors, and blood vessel formation

grafts.121 Scaffolds provide an osteoconductive and osteoinductive extracellular matrix

based bioglass, and metals (Table 2).13,121,144

Demineralized bone matrix

Demineralized bone matrix (DBM) is produced by acid extraction of allogenic bone, a

scaffolds have demonstrated both osteoconductivity and favorable resorption

characterized by improved stiffness and osteointegration in comparison to collagen

Calcium carbonate is another potential ceramic material for osteoconductive

derived materials, such as collagen and fibrin proteins, or chitin-derived chitosan

have a well-documented history of clinical application in craniofacial bone

biocompatibility, and good mechanical strength.165 It has been applied clinically to

Poly(propylene fumarate) (PPF) is a synthetic, unsaturated, linear polyester

sufficiently strong for craniofacial bone tissue engineering.175–185 It generally requires a

an injectable polymer.186 A two-phase PPF cement incorporating cross-linked

be fabricated with preserved mechanical properties.164,205,206 Their strength, ductility,

the prospect of a single scaffold capable of inducing both osteogenesis and

As previously discussed, composite scaffolds combining materials with different

shown to predictably undergo liquid-to-solid phase change at appropriate physiological

matrices composed of cross-linked hydrophilic polymers that are well suited to