Fibrin: A Versatile Scaffold for Tissue Engineering Applications

TISSUE ENGINEERING: Part B Volume 14, Number 2, 2008 # Mary Ann Liebert, Inc. DOI: 10.1089/ten.teb.2007.0435 Fibrin: A Versatile Scaffold for Tissue Engineering Applications TAMER A.E. AHMED, M.Sc., EMMA V. DARE, B.Sc., and MAX HINCKE, Ph.D. ABSTRACT Tissue engineering combines cell and molecular biology with materials and mechanical engineering to replace damaged or diseased organs and tissues. Fibrin is a critical blood component responsible for hemostasis, which has been used extensively as a biopolymer scaffold in tissue engineering. In this review we summarize the latest developments in organ and tissue regeneration using fibrin as the scaffold material. Commercially available fibrinogen and thrombin are combined to form a fibrin hydrogel. The incorporation of bioactive peptides and growth factors via a heparin-binding delivery system improves the functionality of fibrin as a scaffold. New technologies such as inkjet printing and magnetically influenced self-assembly can alter the geometry of the fibrin structure into appropriate and predictable forms. Fibrin can be prepared from autologous plasma, and is available as glue or as engineered microbeads. Fibrin alone or in combination with other materials has been used as a biological scaffold for stem or primary cells to regenerate adipose tissue, bone, cardiac tissue, cartilage, liver, nervous tissue, ocular tissue, skin, tendons, and ligaments. Thus, fibrin is a versatile biopolymer, which shows a great potential in tissue regeneration and wound healing. INTRODUCTION T due to aging or pathological conditions is a major human health problem. Tissue engineering, a new field in biomedical sciences, combines cellular and molecular biology on one hand and material and mechanical engineering on the other to provide an alternative to organ and tissue transplants that face a limited supply of donor organs.1,2 Current tissue engineering techniques utilize any combination of three critical components: a cellular component, biocompatible and mechanically stable carrier vehicle/matrix scaffold, and a bioactive component. The cellular component should consist of healthy, viable cells that are accessible, manipulable, and nonimmunogenic. The carrier component has a dual function, acting as both a delivery vehicle and a matrix scaffold. The bioactive component should promote differentiation and maturation of the cellular component.3–6 Three approaches have been established to regenerate tissues by tissue engineering. The first approach is to deliver a bioactive factor/scaffold combination that induces migration, proliferation, and differentiation of cells from the surrounding tissue.7 The second approach is to transplant cells microHE LOSS OR DAMAGE OF ORGANS AND TISSUES encapsulated within a semipermeable membrane with subsequent partial restoration of tissue functions.8 The third approach, which is most widely used now, aims to transplant cells into a three-dimensional supporting biodegradable matrix, which becomes capable of replacing the functions of the pathologically altered tissues.9,10 Tissue engineering generally requires an artificial extracellular matrix (ECM) (scaffold) in which the cells can proliferate and differentiate with subsequent new tissue generation. The scaffold must be biodegradable and allow reasonable cell adhesion. It should also provide sufficient mechanical support to withstand in vivo forces.11 A broad array of synthetic polymers (polyglycolic acids (PGA), polylactic acid (PLA), copolymers of glycolic and lactic acids, polyurethanes, polyhydroxybutyrate (PH4B), polyanhydrides and polyortho esters), and natural polymers (collagen, fibrin, glycosaminoglycans, and chitosan) have been used as scaffolds in tissue engineering.2,7,12,13 Hydrogels are a class of biomaterials that have great scaffolding potential in many tissue engineering applications due to their high tissue-like water content, high biocompatibility in general, mechanical properties that parallel the properties of soft tissues, efficient transport of nutrients and waste, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada. 199 AHMED ET AL. 200 powerful ability to uniformly encapsulate cells, and ability to be injected as a liquid that gels in situ.13–15 Hydrogels, which are based on water-soluble components, are either chemically or physically cross-linked; depending on their chemistry, they can be either degradable or nondegradable.14 Chemically cross-linked hydrogels can be polymerized using a chemical initiator16 or photoinitiator. The photoinitiated hydrogels use initiators that are inactive until they are exposed to light of a particular wavelength.17 In comparison to the most widely used scaffolds, fibrin gels combine some important advantages such as high seeding efficiency and uniform cell distribution.18 In addition, fibrin has adhesion capabilities.11 Further, it can be produced from the patient’s own blood and used as an autologous scaffold without the potential risk of foreign body reaction or infection.2 Fibrin is a biopolymer of the monomer fibrinogen. The fibrinogen molecule is composed of two sets of three polypeptide chains named Aa, Bb, and g, which are joined together by six disulfide bridges.19 Fibrin is formed after thrombin-mediated cleavage of fibrinopeptide A from the Aa chains and fibrinopeptide B from the Bb chains,20 with subsequent conformational changes and exposure of polymerization sites. This generates the fibrin monomer that has a great tendency to self-associate and form insoluble fibrin. Further, the blood coagulation factor XIIIa is a transglutaminase that rapidly cross-links g chains in the fibrin polymer21 by introducing intermolecular x-(g-glutamyl) lysine covalent bonds between the lysine of one g-chain and glutamine of the other.15 This covalent cross-linking produces a stable fibrin network that is resistant to protease degradation.22 This effect can be reinforced by introducing chemical cross-linker such as genipin.23 Fibrin and fibrinogen have critical roles in blood clotting, fibrinolysis, cellular and matrix interactions, the inflammatory response, wound healing, and neoplasia.20 Fibrin has been used clinically as a hemostatic agent in cardiac, liver, and spleen surgery. In addition, it can be used in surgery for patients with hemophilia. Further, fibrin is useful as a sealant in a variety of clinical applications, including procedures such as colonic anastomosis as well as in seroma prevention following soft tissue dissection. Moreover, it has been used to reduce suture vascular and intestinal anastomosis, to promote fistula closure, and in laparoscopic/endoscopic procedures.24 A number of allogeneic fibrin sealants such as TisseelÒ, EvicelTM, and CrossealTM have been approved by the Food and Drug Administration (FDA) for clinical use as hemostatic agents. However, this review will be restricted to discussion of the manipulation of fibrin for tissue engineering applications. The most widely used forms of fibrin scaffolds are fibrin hydrogels, fibrin glue, and fibrin microbeads (FMBs). FIBRIN HYDROGEL Fibrin hydrogels are constructed from commercially purified allogeneic fibrinogen and purified thrombin.25 Fibrin hydrogels have been used widely in the last decade in a variety of tissue engineering applications, and these include tissue engineering of adipose,26 cardiaovascular,2,27–30 ocular,31–33 muscle,34–37 liver,38,39 skin,40,41 cartilage,42–47 and bone tissues.48,49 In addition, fibrin hydrogels have applications for promoting angiogenesis50–52 and enhancing neurite extension.22,53,54 The fibrin hydrogel as a potential scaffold has three major disadvantages: the shrinkage of the gel that happens during the formation of flat sheets, low mechanical stiffness, and its rapid degradation before the proper formation of tissueengineered structures.2,28 Gel shrinkage can be prevented by incorporating a fixing agent such as poly-L-lysine into the fibrin gel during the culturing period.2 In order to improve the low mechanical stiffness for some tissue engineering applications, fibrin hydrogels can be combined with other scaffold materials to obtain constructs with desired mechanical strength. Examples of materials used for preparing composite scaffolds are polyurethane,55 polycaprolactone-based polyurethane,56 polycaprolactone,57 b-tricalciumphosphate (bTCP),58 b-tricalciumphosphate/ploycaprolactone (b-TCP/ PCL),59 and polyethylene glycol.60 Most commercial preparations of fibrinogen usually contain other plasma proteins such as fibronectin, growth factors, enzymes, enzyme inhibitors, and proenzymes.61 In addition, serum plasminogen is usually a component of the in vitro culture medium.2 Further, plasminogen and matrix metalloproteinases may be secreted from the encapsulated cells inside the fibrin hydrogel.25 These are all factors that contribute to the rapid degradation of the fibrin hydrogel, which is usually observed. The stability of the fibrin hydrogel can be prolonged using a number of strategies. One approach is to concomitantly optimize pH and the concentrations of fibrinogen and calcium ion (Ca2þ).42 Another strategy is to use FMBs, a highly cross-linked, dense, and denatured threedimensional fibrin matrix.62,63 Alternatively, fibrin can be modified with a molecule such as polyethylene glycol, which renders the fibrin structure more stable.64 The fourth strategy is to reduce the cell density.46 And finally protease inhibitors that are specific for plasmin and matrix metalloproteinases are often added to the in vitro culturing media,2,25 or alternatively, the plasmin inhibitor, aprotinin, can be immobilized into the fibrin hydrogel.65 An allied strategy is to modify the fibrin hydrogel to introduce additional characteristics that enhance its use in tissue engineering applications. This approach is described in more detail in the following section. Incorporation of biologically active peptides into fibrin hydrogels The usefulness of the fibrin hydrogels in many tissue engineering applications can be extended further by incorporating the biological activity of other proteins, such as those derived from the ECM proteins—fibronectin, vitronectin, laminin, and collagen. The peptide domains responsible for the biological activity of these proteins (bioactive domains) can be synthesized for covalent cross-linking to the fibrin FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING hydrogel through a transglutaminase-catalyzed reaction.22 The bioactive domain peptide is coupled to a transglutaminase substrate sequence (NQEQVSP) to generate a bifunctional peptide or bi-domain peptide. A variety of bioactive adhesion domains have been synthesized to incorporate the transglutaminase substrate sequence: these include IKVAV, YIGSR or RNIAEIIKDI derived from laminin, DGEA derived from collagen, and RGD derived from many ECM proteins (Fig. 1).22,66 By extension of this concept, a heparinbinding domain can be synthesized with addition of the transglutaminase substrate sequence for subsequent crosslinking to fibrin. The heparin-binding domain is derived from antithrombin III (K(bA)FAKLAARLYRKA), modified antithrombin III (R(bA)FARLAARLYRRA), neural cell adhesion molecule (KHKGRDVILKKDVR), or platelet factor-4 (YKKIIKKL). The heparin-binding domain binds heparin, which is then available to bind a wide variety of heparin-binding growth factors (HBGFs). This constitutes the heparin-binding delivery system, which enables sequestered growth factors to be slowly released by a cell-mediated degradation of the reservoir, such that release occurs primarily in response to cellular activity instead of by simple diffusion (Fig. 1). The release of growth factors is regulated by heparinase and plasmin enzyme activities.53,67 Heparin binds a wide variety of growth factors, which are selected to provide suitable inductive cues, including acidic and basic fibroblast growth factor (aFGF, bFGF), transforming growth factor-b1,b2 (TGF-b1,b2), neurotrophin-3 (NT-3), brainderived neurotrophic factor, and nerve growth factor (NGF). 201 Although some growth factors such as NGF bind heparin with lower affinity than bFGF, NGF delivered from heparinbinding delivery system is more effective than soluble NGF in promoting neurite extension of dorsal root ganglia (DRGs).53,67–70 Fibrin hydrogel inkjet printing Inkjet printing technology has been used recently in tissue engineering, as it offers a practical and efficient tool to dependably handle and dispense biological and/or material elements in a tunable manner to generate cellular and tissue structures, or even organ analogs.71,72 Proteins,73,74 growth factors,75 and even whole viable cells71,76 can be deposited with the inkjet printing method mainly with the aim of constructing cell patterns. In this technique, fibrin is used as a printable hydrogel to build three-dimensional constructs. Printing of thrombin onto fibrinogen leads to geometricspecific cross-linking and enables the rapid construction of three-dimensional fibrin scaffolds with specific structures and forms.71 Magnetically guided self-assembled fibrin hydrogel Fibrin gels with defined architecture on the nanometer scale can be fabricated using magnetic forces. Active thrombin is chemically cross-linked to the surface of magnetic microbeads that are positioned in a defined twodimensional array by a magnetic field. The geometric FIG. 1. Cartoon depicting incorporation of active biological peptides into fibrin gel. AHMED ET AL. 202 FIG. 2. Magnetically guided self-assembly of fibrin hydrogel using active thrombin that is chemically cross-linked to the surface of magnetic microbeads. patterns guide the self-assembly of fibrin fibrils through localized catalytic cleavage of soluble fibrinogen substrate (Fig. 2).77 FIBRIN GLUE Fibrin glue is clearly distinguished from fibrin hydrogels that are prepared from purified fibrinogen and thrombin. Fibrin glue, also referred to as fibrin sealant or fibrin tissue adhesive, has been widely used as a helper tool in many surgical fields within the last three decades because of its capability to promote hemostasis and tissue bonding.78 Further, fibrin glue accelerates wound healing, reduces blood loss, and protects against bacterial infection.79 Fibrin glue is usually obtained after the fractionation of pooled plasma and consists primarily of two components—enriched fibrinogen (also known as cryoprecipitate) and thrombin.78 The cryoprecipitate often contains factor XIIIa and fibronectin in addition to fibrinogen, while the thrombin component sometimes contains CaCl2 and antifibrinolytic drugs.80 Fibrin glue can be prepared either from allogeneic pooled plasma such as commercially available Tissucol/ TisseelÒ, BeriplastÒ, and QuixilTM, or from autologous plasma that is collected from the patient prior to surgery and processed using devices such as CryosealÒ-FS or VivostatÒ.79,81,82 Although commercially produced fibrin glue is available in standardized quality, autologous fibrin glue has two major advantages: the possibility of viral transmission and prion infection is greatly reduced, and the cost is lower.81–85 The total cost of 10 mL autologous fibrin glue obtained by cryoprecipitation, including the material cost, was estimated in 2005 to be around $50, while 1 mL of commercial fibrin glue costs approximately $200.86 Fibrinogen is the major component of fibrin glue, and several techniques have been developed for its isolation. The first technique is the cryoprecipitation of fresh-frozen plasma or stored plasma. After freezing at 808C for at least 12 h, then thawing for several hours at 48C, plasma is centrifuged and the supernatant is discarded. The remaining yellow precipitate of fibrinogen is resuspended in small volume of the plasma supernatant.87,88 The second technique depends on the Cell Saver equipment, which is a centrifugal blood concentrator that concentrates the blood by removing plasma from whole blood.89,90 The physical separation of cellular components by the centrifugal field is based on the differences in density and particle size.91 The third technique is chemical precipitation of fibrinogen using agents such as ethanol, polyethylene glycol, or ammonium sulfate.92 The last technique is the chromatographic method in which plasma is passed through a column containing ligand immobilized to a matrix such as agarose.93 The fibrinogen in the plasma binds to the ligand with high affinity and then is selectively eluted from the column. Many ligands have high affinity for fibrinogen such as heparin,94 protamine,95 insolubilized fibrin, and fibrin monomer.96 Cryoprecipitation, chemical precipitation, and chromatographic separation can be conducted in the laboratory. Fibrin glue for tissue engineering applications has two functions: it serves as a delivery vehicle and as a scaffolding matrix.4–6 Fibrin glue in combination with an appropriate cell source has been used in a variety of tissue engineering applications, including maxillofacial bone,97,98 periodontal bone,99 bone,100–103 ear cartilage,104–106 cartilage,107–109 cornea,110 heart,111 blood vessel,112 tendon, and ligament regeneration.113–115 In addition, fibrin glue has been used to promote the healing of severe burns and chronic wounds.116–118 Further, fibrin glue is widely used to affix a variety of tissue-engineered constructs at the implantation site due to its adhesive properties.119–123 The two components of fibrin glue can be mixed and processed in different ways.80,104,114,124 The spray application is more powerful in the treatment of extensive wounds, where the two components are injected separately into a continuous compressed air jet (Fig. 3).124 The spray system delivers an even and fine layer of rapidly polymerized fibrin onto the wound surface.125 The co-application of fibrin glue with cells using the spray system has many advantages. It creates a thin and homogenous film of fibrin glue, coating extensive surfaces with a small amount of fibrin glue and localizing the proliferating cells to the wound.124,126 Fibrin glue can be modified to enhance its mechanical strength by incorporating other polymers such as gelatin, hyaluronic acid, and chondroitin-6-sulfate.127 Further, heparin can be incorporated to potentiate the effects of HBGFs FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING 203 FIG. 3. Spray applicator of fibrin glue. The two components of the fibrin glue at room temperature are injected separately into continuous compressed air jet to form a thin even layer of cross-linked fibrin. on cell proliferation and migration. Heparin binds HBGFs and sequesters them inside the fibrin glue with subsequent slow release of growth factors. Binding of HBGFs to heparin protects them from thrombin-induced degradation.128–131 FIBRIN MICROBEADS FMBs are small spherical dense beads with a diameter ranging from 50 to 250 microns that consist of highly condensed and cross-linked fibrin. FMBs are produced from plasma fibrinogen obtained by fractionation.132 The fibrinogen is mixed with thrombin, and the activated fibrin is immediately and quickly stirred in a heated oil emulsion (corn oil: isooctane, 1:1; 758C) to yield spherical droplets that are further cross-linked into solid beads (Fig. 4).62,133 Fibrinogen denatures above 508C due to the instability of the D-domain, whereas factor XIIIa is much more stable and can cross-link proteins at higher temperatures. Denatured fibrinogen in the FMB is greatly haptotactic to mesenchymal-type cells, such as endothelial cells, smooth muscle cells, and fibroblasts.133 FMBs have been used widely to isolate and grow mesenchymal stem cells from both bone marrow and blood.62,63,132,134 Kidney gene and cell therapy has been tested in vitro using FMBs as a threedimensional platform, since a variety of transduced renal cells grow and differentiate in this material.134 Further, FMBs in combination with the appropriate cell source can be used in bone regeneration and wound healing.132,135 TISSUE ENGINEERING APPLICATIONS OF FIBRIN Adipose tissue Many synthetic and natural materials have been evaluated clinically for reconstruction of missing soft tissue due to mastectomy or lumpectomy in plastic and reconstructive surgery, but the results are frequently not satisfactory. For examples, silicone may elicit immune reactions in the recipient tissue, and collagen scaffolds shrink in vivo. Autologous adipose tissue has been used as a replacement; however, this strategy failed to maintain a stable volume.136 In the tissue engineering approach to regenerate adipose tissue, fibrin hydrogels have been evaluated using commercial plasminogenfree fibrinogen, factor XIIIa, and thrombin to encapsulate preadipocytes, which are a feasible cell source for adipose tissue regeneration.26,136 Although, scaffolds such as poly (lactic-co-glycolic acid) and collagen have been used in combination with preadipocytes to regenerate adipose tissue in rodent models, the volume of the regenerated tissue failed to be maintained over a long implantation period due to a rapid and unpredictable resorption rate.137–139 Fat tissue is a highly vascularized tissue; therefore, the formation of an efficient microcirculatory network in a cell–fibrin graft should allow the in vivo engineering of persistent three-dimensional implants. In support of this concept, highly vascularized fibrin matrix containing preadipocytes cells in a chorioallantoic membrane experimental model was volume stable.140 Basic AHMED ET AL. 204 FIG. 4. Formation of fibrin microbeads (FMBs). Fibrinogen, in conjugation with factor XIIIa, is mixed with thrombin, and the activated fibrin is immediately and quickly stirred in heated oil emulsion (758C) to yield spherical denatured droplets that are further cross-linked into solid beads by heat-stable transglutaminase. FGF was found to enhance both adipogenesis and neovascularization of the newly formed tissue in fibrin–preadipocyte constructs cultured in adipogenic media prior to implantation into athymic mice.26 This finding suggests that the heparinbinding delivery system approach could yield superior results. Another approach is to mechanically stabilize the space, for example, using a dome-shaped support structure for suitable regeneration of volume-stable adipose tissue. Preadipocytes mixed with fibrin matrix (space-filling matrix) were injected under such a dome structure fabricated from PGA and poly(L-lactic acid) and implanted subcutaneously in athymic mice. The volume of the newly formed adipose tissues was maintained for at least 6 weeks. This study demonstrated that volume-stable adipose tissues can be engineered in vivo using mechanical support structures, and it offers the potential for augmentation of adipose tissues with volume conservation.136 In another strategy to address the problem, fibrin–preadipocyte constructs were implanted into a pre-prepared capsule in the recipient rats. The capsule was created by implanting a silicone block into the recipient tissue, which induced capsule formation prior to the implantation of fibrin–adipocyte constructs. Volume retention was demonstrated in implanted areas up to 1 year after implantation. Using a prefabricated capsule pouch as the recipient site and fibrin glue as a transport vehicle seems to be a reliable and safe source of material for adipose tissue augmentation.139 Bone tissue In the field of orthopedics, tissue engineering has been used in attempts to treat a variety of bone defects that are a result of traumatic injury, as well as those that arise during bone development. Fibrin has been used alone or in combination as a scaffold for bone regeneration. In promising animal studies, rabbit autologous fibrin beads were used to treat defects created in the proximal tibial physis of New Zealand white rabbits.141 The fibrin beads were created by combining fibrin with alginate. After gel formation the alginate was removed and the construct was seeded with perichondrial cells and implanted into the defect. After 12 weeks it was found that the bones with untreated tibial defects were shorter and more deformed than those treated with the fibrin bead implant. This represents a possible future treatment for partial arrest of physeal growth. Plateletrich fibrin glue has also been used as a scaffold for bone tissue engineering.101 Platelet-rich fibrin gels have a high concentration of platelets that release growth factors and bioactive proteins to initiate and accelerate tissue repair and regeneration.142 When the platelet-rich fibrin glue was mixed with bone marrow mesenchymal stem cells and bone morphogenic protein-2 and injected subcutaneously into nude mice, nodules containing bone tissue formed after 12 weeks.101 Platelet-rich plasma mixed with fibrin glue and mesenchymal stem cells has been implanted into mandible FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING bone defects along with dental implants to augment bone volume in dogs.99 Much greater osseointegration was observed with the fibrin glue implant than in controls without any graft material. The results of these studies indicate that platelet-rich fibrin glue may be a suitable scaffold to enhance healing and integration of implants with bone. Fibrin glue has also been combined with other materials, such as hyaluronic acid and hydroxyapatite, for improved mechanical, adhesive, and biological properties of cellular scaffolds.143,144 The combination of calcium phosphate granules with fibrin glue leads to a porous, mineralized ultrastructure, which may provide favorable osteogenic properties for rapid healing of bone defects. The combination of these materials is predicted to improve mechanical properties compared to the ceramic alone, and enhances angiogenesis, cell attachment, and proliferation.145 Another approach, autologous bone fragments seeded into fibrin glue and mixed with btricalcium phosphate, efficiently regenerated cranial bone in situ in dogs.146 Cardiac tissue Congestive heart failure can be treated by several therapeutic strategies; however, ischemic or scarred myocardium resulting from congestive heart failure can lead to abnormal heart function.147 Cardiac tissue engineering may provide an alternative treatment to replace the damaged tissue. Fibrin has been used as a scaffold for the regeneration of myocardial tissue and heart valves. In one approach, a neonatal cardiac rat myocytes were suspended within a fibrin gel and implanted into silicone tubing. The construct was surgically placed around the femoral artery and vein of adult rats. After 3 weeks in vivo, the tissue within the silicone chamber resembled normal myocardial tissue.27 Tissue-engineered heart valves have many advantages over mechanical and bioprosthetic valves such as the ability to adapt to a growing patient, better durability, and low thrombogenicity.148 Williams et al.149 encapsulated human dermal fibroblasts or porcine valve interstitial cells into adherent fibrin gel disks. After 5 weeks of in vitro culture both cell types were able to remodel fibrin and deposit collagen and elastin. Mechanical stimulation of moulded fibrin gel scaffolds seeded with arterial cells was shown to enhance deposition of ECM proteins, including type I and III collagen. This study indicated that dynamic conditioning of autologous fibrin-based scaffolds may be necessary for tissue engineering of heart valves.150 Fibrin as a cell carrier has also been used in combination with polycaprolactone for aortic valve regeneration. Fibrin with knitted polycaprolactone provided good load-bearing properties and permitted cellular ingrowth. Both materials were found to be biocompatible and durable.57 Therefore, fibrin scaffolds show promise for this application, since the complex valve shape can be cast from fibrin using an injection moulding technique.30,151 205 Cartilage tissues Fibrin has successfully been used as a scaffold and adhesive to improve healing and regeneration of fibrocartilage, elastic cartilage, craniofacial cartilage, and articular cartilage. For fibrocartilage regeneration, mesenchymal stem cells have been seeded into fibrin glue and implanted into meniscal defects in rats. Meniscal healing in the avascular zone was observed after 8 weeks.152 It has also been shown that incorporation of a fibrin clot derived from whole blood into a meniscus repair site resulted in a reduction in failure rate from 61% to 8%.153,154 Regeneration of elastic cartilage by tissue engineering using fibrin has also been investigated. Ruszymah et al.155 seeded pediatric auricular chondrocytes into fibrin and implanted the constructs into nude mice. It was found that the tissue-engineered cartilage resembled native elastic cartilage. In combination with polytetrafluoroethylene as an artificial perichondrium, fibrin gel–seeded auricular chondrocytes regenerated flexible tissue that returned to its original shape without fracture after rigorous torsion.156 Fibrin glue has also been used along with autologous concha cartilage to reconstruct the middle ear canal wall in the chinchilla.157 Tissue engineering approaches to articular cartilage repair offer potentially important advantages over modern metallic and plastic joint prostheses.158 Fibrin gel has been widely investigated for tissue engineering of cartilage,42–47,159 often in combination with other biomaterials to incorporate the advantages of both biomaterials.160,161 Fibrin glue as scaffolding material has been used for the treatment of deep chondral defects in human.162,163 Genipin is a natural product that can be used to cross-link fibrin hydrogels. It has been shown to enhance the scaffold mechanical properties, and also induces chondrogenic differentiation of human primary articular chondrocytes.23 Genipin is significantly less toxic than other chemical cross-linkers such as glutaraldehyde.164 In other approaches, fibrin hydrogels can be mixed with a PGA mesh to form a solid cell delivery device that is easily manipulated in vitro.165 Fibrin gels mixed with hyaluronic acid provide a favorable environment for chondrocytes to maintain stable phenotype and to synthesize cartilage ECM in nude mice.161 Further, fibrin hydrogels with collagen allow stable graft fixation in a chicken model of cartilage repair.166 Similarly, fibrin glue can be mixed with alginate to allow initial cell proliferation and subsequent preservation of differentiated cells to establish a stable matrix structure in vitro and in rabbit.160,167,168 Further, fibrin glue has been combined with a polycaprolactone-based polyurethane scaffold to establish a long-term construct with a stable shape after implantation in mice.56 Moreover, when a tri-copolymer of gelatin, hyaluronic acid, and chondroitin6-sulfate is combined with fibrin glue, it promotes ECM secretion and inhibits ECM degradation in vitro.127 Chondrocytes have been used widely as a productive cue after encapsulation in fibrin during articular cartilage tissue AHMED ET AL. 206 engineering.42–44,47,56,127,160,161,165,166,168–173 However, many other cell lines are also used for cartilage tissue engineering, including periosteal cells,167 bone marrow stromal cells,174 adipose tissue–derived mesenchymal stem cells,175 and bone marrow–derived mononuclear cells.176 When added to the culture media, members of TGF-b family serve as inductive cues for the fibrin glue–encapsulated cells.159,167 However, other factors can alternatively be used, such as insulin-like growth factor I (IGF-I)171 and FGF-2.177 Bone morphogenic protein-2 and IGF-I genes have been transferred to mesenchymal cells through adenovirus-mediated transfection for use with fibrin glue for cartilage restoration in experimental partial thickness cartilage lesions.178 Liver tissue Fibrin matrix supports three-dimensional cell organization and new tissue formation in vitro; in addition, it is a robust carrier for hepatocytes transplantation. Liver transplantation can be orthotopic where liver cells are transplanted into the rats spleen or portal vein,38 or heterotopic where hepatocytes are transplanted either underneath the kidney capsules179 or intraperitoneally.180 Fibrin hydrogel has been used as a scaffold for orthotopic extravascular transplantation of hepatocytes, where fibrin–hepatocyte constructs were fabricated by mixing commercial fibrinogen with thrombin.38 This study showed that the injection of fibrin gel–immobilized hepatocytes into the rat liver parenchyma is technically feasible without significant safety problems or harmful side effects. Further, the transplanted cells are actively integrated into the recipient liver parenchyma. The fibrin matrix is resorbed by the donor cells, and its degradation products do not affect hepatocyte survival and differentiation.38 Microencapsulation of hepatocytes in combination with a fibrinogen– collagen mixture, by extrusion into a terpolymer solution, and submerging the resultant microcapsules into a thrombin solution, induced the formation of an insoluble fibrin network inside the microcapsules. The formation of a fibrin network inside the microcapsules not only increased the rates of urea and albumin synthesis by hepatocytes but also enhanced the mechanical strength in the interior of the microcapsules. This modification enabled microcapsules to withstand the large compressive and shear forces found in bioreactors and resulted in uniform perfusion.39,181 Nervous tissue Tissue-engineered neural structures may present an alternative strategy to treat serious clinical conditions of the nervous system, such as brain and spinal cord injuries and neurodegenerative diseases, in which functional neural cells are often lost or degenerate within the nervous system.71,182 Fibrin-based scaffolds have been used to treat both central and peripheral nerve injury in rat and chicken models.67,68 Fibrin gels within nerve guide tubes implanted in rat sciatic nerve injury models facilitate axonal regeneration and cell migration in short-gap nerve injuries.183 The regenerated axons and associated glia invaded and grew inside the fibrin. In the absence of fibrin, no nerve generation occurred. Neurite penetration in fibrin occurs by plasmin-mediated fibrinolysis localized to the neurite tip, since addition of plasmin inhibitors such as aprotinin decrease neurite extension dramatically in embryonic chicken DRGs.184 However, when murine embryonic stem cells are used as a cell source, aprotinin is required to optimize fibrin conditions for nerve regeneration.185 It has been shown that fibrin hydrogel alone does not allow proper nerve regeneration and fibrin must be covalently modified to incorporate exogenous heparin-binding domains, a class of adhesion domains that bind heparin and other sulfated glycosaminoglycans and promote nerve regeneration. Four different heparin-binding peptides, derived from antithrombin III, modified antithrombin III, neural cell adhesion molecule, and platelet factor-4 were covalently bound to fibrin separately through a bi-domain peptide. These peptides were found to enhance the degree of neurite extension from embryonic chicken DRGs.54 In a similar study, fibrin hydrogels modified through a bi-domain peptide with four laminin-derived oligopeptides—RGD, IKVAV, YIGSR, and RNIAEIIKDI—in combination resulted in an induction of neurite extension from DRGs in the same model.66 A third group examined the feasibility of using a fibrin-based drug delivery system, which was designed to provide controlled release of neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), brain-derived neurotrophin factor, and bNGF in vitro and in vivo. Therapy for nerve injury was successful using a heparin-based delivery system, where a linker peptide contained a factor XIIIa substrate was covalently linked to fibrin during polymerization. The linker captured heparin within the fibrin hydrogel, and the immobilized heparin bound NT-3 and bNGF, preventing their loss from the scaffold by diffusion. The result of these studies demonstrated that the incorporation of a delivery system providing controlled release of growth factors across short nerve gaps led to enhanced central and peripheral nerve regeneration in chicken and rat models, and represents a feasible method to enhance nerve generation in DRGs.53,67–69 Fibrin glue has also been used to bridge gaps between injured spinal cord segments and to secure dorsal root stumps after dorsal column injury in a rat model.186,187 Recently, fibrin was used as a printable hydrogel to build a three-dimensional neural structure. Complex cellular patterns and structures were created by automated and direct inkjet printing of rat primary embryonic hippocampal and cortical neurons onto fibrin. The printed fibrin-based neural constructs provide suitable cell or tissue sources for clinical treatments of serious neural injuries and degenerative diseases.71 Ocular tissue Tissue engineering of the cornea could overcome the disadvantages of corneal transplantation such as immune re- FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING 207 jection and a shortage of donor supply.188 Fibrin-based matrices have been used extensively for the treatments of many corneal diseases, including corneal perforation and total limbal cell deficiency.189,190 Transplantation of fibrin glue– cultured limbal stem cell constructs permanently restored corneal integrity in patients with total limbal deficiency.190 In addition, fibrin glue in combination with amniotic membrane transplant allowed total reepithelialization and subsequent repair of the corneal perforation associated with loss of the stroma in human.189 Encapsulation of corneal epithelial stem cells into an autologous fibrin gel rich in fibronectin produces a flexible and easily manipulated material in vitro, which is a potential source of a bioengineered ocular surface. It should be pointed out that aprotinin (plasmin inhibitor) should be added to the conditioned medium to prevent fibrin degradation.33 Fibrin glue is an effective scaffold for creating carrierfree transplantable corneal epithelial sheets in rabbit model. Carrier-free sheets are more differentiated than amniotic membranes and retain similar levels of colony-forming progenitor cells.110 However, fibrin has been combined with agarose to obtain better biomechanical properties, and has been used to create a full-thickness corneal model in rabbit, in which the three corneal cell types (endothelial, stromal, and epithelial) were layered sequentially within the fibrin– agarose scaffold.31 Fibrin has also been combined with collagen to create an in vitro model of the cornea with adjacent sclera. Fibrin cross-linked with poly(N-isopropylacryl amide)-co-acrylic acid coacryloxysuccinimide (PNiPAAmco-AAc) together with laminin and NGF was used for constructing sclera, and collagen plus chondrotin 6-sulfate cross-linked with glutaraldehyde was used for constructing the cornea. This approach led to complete innervation and vascularization.32 Skin tissue In the past few years, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements.191 Cultured keratinocytes have been used widely for the treatment of a variety of skin defects such as acute partial or full-thickness burns and chronic wounds such us ulcers in human.117 However, keratinocytes alone do not attach properly to the wound, and the presence of fibroblasts is essential for keratinocytes to maintain their function in mice.41 Clinically, fibrin glue is a common carrier and matrix vehicle for keratinocytes. Cultured keratinocyte suspensions in fibrin glue are used for the reepithelialization of deep, partial, and full-thickness wounds in both animal models and in humans.192,193 In addition, fibrin glue has been shown to improve the skin graft success rate, especially when incorporated into difficult grafting sites or sites associated with unavoidable movement. Further, when fibrin is associated with fibronectin, it supports keratinocyte and fibroblast growth, both in vivo and in vitro. Constructs of fibrin glue and keratinocytes plus fibroblasts provide similar advantages to those demonstrated with conventional skin grafts.118 Similarly, fibrin hydrogels seeded with keratinocytes and fibroblasts resulted in proper epidermal structure formation and enhanced migration of vascular endothelial cells in comparison to collagen in a mice model.41 Moreover, the fibrin gel increased the consistency of the wound healing response, reduced the time for keratinocyte activation, and promoted more complete reepithelialization in skin equivalent incisional wounds.194 Implantation in pig excisional wounds of fibroblasts encapsulated into FMBs resulted in high fibroblast proliferation, more angiogenesis, and TABLE 1. TISSUE ENGINEERING APPLICATIONS OF FIBRIN SCAFFOLDS Types of fibrin scaffold (reference numbers) Engineered tissue Adipose tissue Bone Cardiac tissue Cartilage Liver Fibrin hydrogel Fibrin glue Fibrin microbeads 26,136 139,140 – 48,49,58–60,141,174 97–103,121,143–146,177 62,63,135 2,27–30,57,149–151 111 – 9,23,25,42–47,156,159, 161,165,166,170,171, 173–175 38,39 55,56,104–109,121,127,152,155, 157,160,162,163,167–169,172, 176–178 – – – Muscle tissue 34–37 – – Nervous tissue 22,53,54,66–69, 71,184,185 31–33 186,187 – 110,189,190 – 40,41,194,197 116–118,125,126,191–193,195 132 – 113–115 – 12,18,50–52,128,130 129 – Ocular tissue Skin Tendons and ligaments Vascular tissue AHMED ET AL. 208 granulation tissue formation, especially when plateletderived growth factor-BB (PDGF-BB) was included in the FMBs.132 A more effective technique was the co-application to wounds of fibrin glue with either keratinocytes or mesenchymal stem cells using a fibrin glue spray system with a double-barreled syringe. This resulted in enhancement of the healing and subsequently accelerated repair processes in acute and chronic cutaneous wounds of animal models and human patients.125,126,195 On the contrary, poor physical properties such as low mechanical strength and rapid degradation rates are the major disadvantages of fibrin-based cell cultures. To overcome these disadvantages, synthetic polymeric membrane such as polycaprolactone, which is characterized by its biodegradability, biocompatibility, high tensile strength, slow degradation kinetics, and good barrier properties, can be coated with fibrin glue, which then serves as a biomimetic surface for the adherence and proliferation of keratinocytes in vitro.196 Although protease inhibitors such as aprotinin are usually added to the fibrin component to delay its degradation rate, aprotinin decreases the regeneration of granulation tissue and was not particularly beneficial to the wound healing process in rat.197 Tendons and ligaments Tendon and ligament tears are amongst the most common orthopaedic injuries. However, incomplete regeneration of the tissue often occurs during healing.198 Hankemeier et al.113 combined human bone marrow stem cells (BMSCs) with liquid fibrin glue and injected the mixture into patellar tendon defects of immunodeficient rats. An advantage of using fibrin as a matrix is that it can be injected as a liquid for in situ gelation. Injection of the fibrin/cell material leads to formation of tissue that is histologically more mature; however, biomechanical measurements will be important for determining the functionality of fibrin for this application. Similarly, fibrin glue has been used as a carrier for BMSCs and injected into transected rabbit Achilles tendons along with repair by suturing.114 It was found that fibrin/ BMSC therapy following primary repair led to an improvement of histological and biomechanical parameters at only the early stages of healing. Table 1 summarizes the diverse tissue engineering applications of fibrin scaffolds. CONCLUSIONS In this review we have summarized many important characteristics that make fibrin a robust hydrogel scaffold for tissue engineering. Fibrin can be manipulated as gel, glue, or microbeads to increase its utility in tissue engineering applications. In addition, it can be modified to incorporate several biologically active peptides to increase the local concentrations of growth factors and mimic the natural milieu surrounding the cells. The incorporation of these active peptides facilitates adhesion of the cells to the scaffold, promotes migration of the cells inside the gel, and enhances cell–cell interaction. Further, fibrin gels can be fabricated with defined architecture on the nanometer scale under the influence of magnetic forces. Fibrin can also be utilized as a printable hydrogel to establish specific cell patterns in the three-dimensional construct using inkjet-printing techniques. Fibrin as an ideal scaffold has a significant disadvantage: the gradual disintegration of the gel with subsequent loss of shape and volume before the proper formation of tissue-engineered constructs. This disadvantage can be overcome by optimizing the concentrations of fibrinogen, calcium ion (Ca2þ), and pH, by using a lower cell density or by adding specific protease inhibitors. Stability can also be enhanced by using highly denatured densely cross-linked FMBs or by combining fibrin with an artificial supporting polymer. Fibrin gel shrinkage and its low mechanical stiffness represent other disadvantages of fibrin scaffolds in some tissue engineering applications, which can be controlled by cross-linking or by combining fibrin with other artificial scaffolding material. Due to its numerous advantages, fibrin is used extensively to support threedimensional scaffolds in tissue engineering for adipose, bone, cardiac, cartilage, liver, nervous, ocular, skin, tendon, and ligament tissues. ACKNOWLEDGMENTS This research was supported by Ontario Centres of Excellence, EMK Network grant to M.H. and M.G. T.A.E.A. is a recipient of a Ph.D. scholarship from the Mission Department of the Egyptian Government, and expresses gratitude to Dr. M. El Demellawy for inspirational mentorship. The generous assistance of Dr. M. Griffith and Dr. G. Dervin is acknowledged. REFERENCES 1. Risbud, M. Tissue engineering: implication in the treatment of organ and tissues defects (review). Biogerontology 2, 117, 2001. 2. Jockenhoevel, S., Zund, G., Hoerstrup, S.P., Chalabi, K., Sachweh, J.S., Demircan, L., Messmer, B.J., and Turina, M. Fibrin gel—advantages of a new scaffold in cardiovascular tissue engineering. Eur. J. Cardiothorac. Surg. 19, 424, 2001. 3. Song, L., Baksh, D., and Tuan, R.S. Mesenchymal stem cellbased cartilage tissue engineering: cells, scaffold and biology. Cytotherapy 6, 596, 2004. 4. Leo, A.J., and Grande, D.A. Mesenchymal stem cells in tissue engineering. Cells Tissues Organs 183, 112, 2006. 5. Gruber, H.E., Leslie, K., Ingram, J., Norton, H.J., and Hanley, E.N. Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers. Spine J. 4, 44, 2004. FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING 6. Ringe, J., Kaps, C., Burmester, G.R., and Sittinger, M. Stem cells for regenerative medicine: advances in the engineering of tissues and organs. Naturwissenschaften 89, 338, 2002. 7. Whang, K., Tsai, D.C., Nam, E.K., Aitken, M., Sprague, S.M., Patel, P.K., and Healy, K.E. Ectopic bone formation via rhBMP-2 delivery from porous bioabsorbable polymer scaffolds. J. Biomed. Mater. Res. 42, 491, 1998. 8. Lanza, R.P., Jackson, R., Sullivan, A., Ringeling, J., McGrath, C., Kuhtreiber, W., and Chick, W.L. Xenotransplantation of cells using biodegradable microcapsules. Transplantation 67, 1105, 1999. 9. Hunter, C.J., Mouw, J.K., and Levenston, M.E. Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness. Osteoarthritis Cartilage 12, 117, 2004. 10. Risbud, M.V., and Sittinger, M. Tissue engineering advances in in vitro cartilage generation (review). Trends Biotechnol. 20, 351, 2002. 11. Rosso, F., Marino, G., Giordano, A., Barbarisi, M., Parmeggiani, D., and Barbarisi, A. Smart materials as scaffolds for tissue engineering (review). J. Cell Physiol. 203, 465, 2005. 12. Cummings, C.L., Gawlitta, D., Nerem, R.M., and Stegemann, J.P. Properties of engineered vascular constructs made from collagen, fibrin, and collagen-fibrin mixtures. Biomaterials 25, 3699, 2004. 13. Williams, C.G., Kim, T.K., Taboas, A., Malik, A., Manson, P., and Elisseeff, J. In vitro chondrogenesis of bone marrowderived mesenchymal stem cells in photopolymerizing hydrogel. Tissue Eng. 9, 679, 2003. 14. Lavik, E., and Langer, R. Tissue engineering: current state and perspectives (mini-review). Appl. Microbiol. Biotechnol. 65, 1, 2004. 15. Ramaswamy, S., Wang, D.A., Fishbein, K.W., Elisseeff, J.H., and Spencer, R.G. An analysis of the integration between articular cartilage and nondegradable hydrogel using magnetic resonance imaging. J. Biomed. Mater. Res. B. Appl. Biomater. 77, 144, 2006. 16. Stammen, J.A., Williams, S., Ku, D.N., and Guldberg, R.E. Mechanical properties of a novel PVA hydrogel in shear and unconfined compression. Biomaterials 22, 799, 2001. 17. Nguyen, K.T., and West, J.L. Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 23, 4307, 2002. 18. Swartz, D.D., Russel, J.A., and Andreadis, S.T. Engineering of fibrin-based functional and implantable small-diameter blood vessels. Am. J. Physiol. Heart. Circ. Physiol. 288, H1451, 2005. 19. Mosesson, M.W. Fibrinogen and fibrin structure and functions (review). J. Thromb. Haemost. 3, 1894, 2005. 20. Mosesson, M.W., Siebenlist, K.R., and Meh, D.A. The structure and biological features of fibrinogen and fibrin. Ann. N.Y. Acad. Sci. 936, 11, 2001. 21. Horan, J.T., and Francis, C.W. Fibrin degradation products, fibrin monomer and soluble fibrin in disseminated intravascular coagulation. Semin. Thromb. Hemost. 27, 657, 2001. 22. Schense, J.C., and Hubbell, J.A. Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug. Chem. 10, 75, 1999. 23. Dare, E.V., Griffith, M., Poitras, P., Kaupp, J.A., Waldman, S.D., Carlsson, D.J., Dervin, G., Mayoux, C., and Hincke, 209 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. M.T. Genipin cross-linked fibrin hydrogels for in vitro human articular cartilage tissue engineered regeneration (submitted). Spotnitz, W.D., and Prabhu, R. Fibrin sealant tissue adhesive— review and update. J. Long-Term Eff. Med. Implants 15, 245, 2005. Ahmed, T.A., Griffith, M., and Hincke, M. Characterization and inhibition of fibrin hydrogel-degrading enzymes during development of tissue engineering scaffolds. Tissue Eng. 13, 1469, 2007. Cho, S.W., Kim, I., Kim, S.H., Rhie, J.W, Choi, C.Y., and Kim, B.S. Enhancement of adipose tissue formation by implantation of adipogenic-differentiated preadipocytes. Biochem. Biophys. Res. Commun. 345, 588, 2006. Birla, R.K., Borschel, G.H., Dennis, R.G., and Brown, D.L. Myocardial engineering in vivo: formation and characterization of contractile, vascularized three-dimensional cardiac tissue. Tissue Eng. 11, 803, 2005. Mol, A., van Lieshout, M.I., Dam-de Veen, C.G., Neuenschwander, S., Hoerstrup, S.P., Baaijens, F.P., and Bouten, C.V. Fibrin as a cell carrier in cardiovascular tissue engineering applications. Biomaterials 26, 3113, 2005. Long, J.L., and Tranquillo, R.T. Elastic fiber production in cardiovascular tissue-equivalents. Matrix Biol. 22, 339, 2003. Ye, Q., Zund, G., Benedikt, P., Jochenhoevel, S., Hoerstrup, S.P., Sakyama, S., Hubbell, J.A., and Turina, M. Fibrin gel as a three-dimensional matrix in cardiovascular tissue engineering. Eur. J. Cardiothorac. Surg. 17, 587, 2000. Alaminos, M., Del Carmen Sanchez-Quevedo, M., MunozAvila, J.I., Serrano, D., Medialdea, S., Carreras, I., and Campos, A. Construction of a complete rabbit cornea substitute using fibrin-agarose scaffold. Invest. Ophthalmol. Vis. Sci. 47, 3311, 2006. Suuronen, E.J., Muzakare, L., Doillon, C.J., Kapila, V., Li, F., Ruel, M., and Griffith, M. Promotion of angiogenesis in tissue engineering: developing multicellular matrices with multiple capacities. Int. J. Artif. Organs 29, 1148, 2006. Han, B., Schwab, I.R., Madsen, T.K., and Isseroff, R.R. A fibrin-based bioengineered ocular surface with human corneal epithelial stem cells. Cornea 21, 505, 2002. Nieponice, A., Maul, T.M., Cumer, J.M., Soletti, L., and Vorp, D.A. Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cell within a three-dimensional fibrin matrix. J. Biomed. Mater. Res. A. 81, 523, 2007. Rowe, S.L., Lee, S., and Stegmann, J.P. Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels. Acta Biomater. 3, 59, 2007. Huang, Y.C., Dennis, R.G., Larkin, L., and Baar, K. Rapid formation of functional muscle in vitro using fibrin gels. J. Appl. Physiol. 98, 706, 2005. Hecker, L., Baar, K., Dennis, R.G., and Bitar, K.N. Development of a three-dimensional physiological model of the internal and sphincter bioengineered in vitro from isolated smooth muscle cells. Am. J. Physiol. Gastrointest. Liver. Physiol. 289, G188, 2005. Bruns, H., Kneser, U., Holzhuter, S., Roth, B., Kluth, J., Kaufmann, P.M., Kluth, D., and Fiegel, H.C. Injectable liver: a novel approach using fibrin as a matrix for culture AHMED ET AL. 210 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. and intrahepatic transplantation of hepatocytes. Tissue Eng. 11, 1718, 2005. Sun, T., Chan, M.L., Quek, C.H., and Yu, H. Improving mechanical stability and density distribution of hepatocytes microcapsules by fibrin clot and gold nanoparticles. J. Biotechnol. 111, 169, 2004. Balestrini, J.L., and Billiar, K.L. Equibiaxial cyclic stretch stimulates fibroblasts to rapidly remodel fibrin. J. Biochem. 39, 2983, 2006. Hojo, M., Inokuchi, S., Kidokoro, M., Fukuyama, N., Tanaka, E., Tsuji, C., Miyasaka, M., Tanino, R., and Nakazawa, H. Induction of vascular endothelial growth factor by a fibrin as dermal substrate for cultured skin substitutes. Plast. Reconstr. Surg. 111, 1638, 2003. Eyrich, D., Brandle, F., Appel, B., Wiese, H., Maier, G., Wenzel, M., Staudenmaier, R., Goepferich, A., and Blunk, T. Long-term stable fibrin gels for cartilage engineering. Biomaterials 28, 55, 2007. Mesa, J.M., Zaporojan, V., Weinand, C., Johnson, T.S., Bonassar, L., Randolph, M.A., Yaremchuk, M.J., and Butler, P.E. Tissue engineering cartilage with aged articular chondrocytes in vivo. Plast. Reconstr. Surg. 118, 41 (discussion 50–53), 2006. Peretti, G.M., Xu, J.W., Bonassar, L.J., Kirchhoff, C.H., Yaremchuk, M.J., and Randolph, M.A. Review of injectable cartilage engineering using fibrin gel in mice and swine models. Tissue Eng. 12, 1151, 2006. Connelly, J.T., Vanderploeg, E.J., and Levenston, M.E. The influence of cyclic tension amplitude on chondrocyte matrix synthesis: experimental and finite element analyses. Biorheology 41, 377, 2004. Passaretti, D., Silverman, R.P., Huang, W., Kirchhoff, C.H., Ashiku, S., Randolph, M.A., and Yaremchuk, M.J. Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer. Tissue Eng. 7, 805, 2001. Johnson, T.S., Xu, J.W., Zaporojan, V.V., Mesa, J.M., Weinand, C., Randolph, M.A., Bonassar, L.J., Winograd, J.M., and Yaremchuk, M.J. Integrative repair of cartilage with articular and nonarticular chondrocytes. Tissue Eng. 10, 1308, 2004. Chung, Y.I., Ahn, K.M., Jeon, S.H., Lee, S.Y., Lee, J.H., and Tae, G. Enhanced bone regeneration with BMP-2 loaded functional nanoparticles-hydrogel complex. J. Control. Release 121, 91, 2007. Weinand, C., Pomerantseva, I., Neville, C.M., Gupta, R., Weinberg, E., Madisch, I., Shapiro, F., Abukawa, H., Troulis, M.J., and Vacanti, J.P. Hydrogel-b-TCP scaffolds and stem cells for tissue engineering bone. Bone 38, 555, 2006. Liu, J.Y., Swartz, D.D., Peng, H.F., Gugino, S.F., Russell, J.A., and Andreadis, S.T. Functional tissue-engineered blood vessels from bone marrow progenitor cells. Cardiovasc. Res. 75, 618, 2007. Dietrich, F., and Lelkes, P.I. Fine-tuning of a threedimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels. Anginonesis 9, 111, 2006. Urech, L., Bitterman, A.G., Hubbell, J.A. and Hall, H. Mechanical properties, proteolytic degradability and biological modifications affect angiogenic process extension into native and modified fibrin matrices in vitro. Biomaterials 26, 1369, 2005. 53. Sakiyama-Elbert, S.E., and Hubbell, J.A. Development of fibrin derivatives for controlled release of heparin-binding growth factors. J. Control. Release 65, 389, 2000. 54. Sakiyama, S.E., Schense, J.C., and Hubbell, J.A. Incorporation of heparin-binding peptide into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. FASEB J. 13, 2214, 1999. 55. Lee, C.R., Grad, S., Gorna, K., Gogoleswski, S., Goessl, A., and Alini, M. Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis. Tissue Eng. 11, 1562, 2005. 56. Eyrich, D., Wiese, H., Maier, G., Skodacek, D., Appel, B., Sarhan, H., Tessmar, J., Staudenmaier, R., Wenzel, M.M., Goepferich, A., and Blunk, T. In vitro and in vivo cartilage engineering using a combination of chondrocytes-seeded long-term stable fibrin gels and polycaprolactone-based polyurethane scaffolds. Tissue Eng. 13, 2207, 2007. 57. Van Lieshout, M., Peters, G., Rutten, M., and Baaijens, F. A knitted, fibrin-covered polycaprolactone scaffold for tissue engineering of the aortic valve. Tissue Eng. 12, 481, 2006. 58. Weinand, C., Gupta, R., Huang, A.Y., Weinberg, E., Madisch, I., Qudsi, R.A., Neville, C.M., Pomerntseva, I., and Vacanti, J.P. Comparison of hydrogels in the in vivo formation of tissue-engineered bone using mesenchymal stem cells and beta-tricalcium phosphate. Tissue Eng. 13, 757, 2007. 59. Weinand, C., Gupta, R., Weinberg, E., Madisch, I., Jupiter, J.B., and Vacanti, J.P. Human shaped thumb bone tissue engineered by hydrogel-beta-tricalcium/poly-epsiloncarpolactone scaffolds and magnetically sorted stem cells. Ann. Plast. Surg. 59, 46, 2007. 60. Peled, E., Boss, J., Bejar, J., Zinman, C., and Seliktar, D. A novel poly (ethylene glycol)-fibrinogen hydrogel for tibial segmental defect repair in rat model. J. Biomed. Mater. Res. A. 80, 874, 2007. 61. Clark, R.A. Fibrin is a many splendored thing. J. Invest. Dermatol. 121, xxi, 2003. 62. Rivkin, R., Ben-Ari, A., Kassis, I., Zangi, L., Gaberman, E., Levdansky, L., Marx, G., and Gorodetsky, R. High-yield isolation, expansion, and differentiation of murine bone marrow-derived mesenchymal stem cells using fibrin microbeads (FMB). Cloning Stem Cells 9, 157, 2007. 63. Kassis, I., Zangi, L., Rivkin, R., Levdansky, L., Samuel, S., Marx, G., and Gorodetsky, R. Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads. Bone Marrow Transplant. 37, 967, 2006. 64. Dikovsky, D., Bianco-Peled, H., and Seliktar, D. The effect of structural alterations of PEG-fibrinogen hydrogel scaffolds on 3d-cellular morphology and cellular migration. Biomaterials 27, 1496, 2006. 65. Smith, J.D., Chen, A., Ernst, L.A., Waggoner, A.S., and Campbell, P.G. Immobilization of aprotinin to fibrinogen as a novel method for controlling degradation of fibrin gels. Bioconjug. Chem. 18, 695, 2007. 66. Schense, J.C., Bloch, J., Aebischer, P., and Hubbell, J.A. Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension. Nat. Biotechnol. 18, 415, 2000. 67. Lee, A.C., Yu, V.M., Lowe, J.B., Brenner, M.J., Hunter, D.A., Mackinnon, S.E., and Sakiyama-Elbert, S.E. Con- FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. trolled release of nerve growth factor enhances sciatic nerve regeneration. Exp. Neurol. 184, 295, 2003. Taylor, S.J., McDonald, J.W., and Sakiyama-Elbert, S.E. Controlled release of neurotrophin-3 from fibrin gels for spinal cord injury. J. Control. Release 98, 281, 2004. Sakiyama-Elbert, S.E., and Hubbell, J.A. Controlled release of nerve growth factor from a heparin-containing fibrin-based cell ingrowth matrix. J. Control. Release 69, 149, 2000. Lyon, M., Rushton, G., and Gallagher, J.T. The interaction of the transforming growth factor-bs with heparin/heparn sulfate is isoforms-specific. J. Biol. Chem. 272, 18000, 1997. Xu, T., Gregory, C.A., Molnar, P., Cui, X., Jolata, S., Bhaduri, S.B., and Boland, T. Viability and electrophysiology of neural cell structures generated by the inkjet printing method. Biomaterials 27, 3580, 2006. Mironov, V., Boland, T., Trusk, T., Forgacs, G., and Markwald, R.R. Organ printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol. 21, 157, 2003. Roth, E.A., Xu, T., Das, M., Gregory, C., Hickman, J.J., and Boland, T. Inkjet printing for high-throughput cell patterning. Biomaterials 25, 3707, 2004. Sanjana, N.E., and Fuller, S.B. A fast flexible ink-jet printing method for patterning dissociated neurons in culture. J. Neurosci. Methods 136, 151, 2004. Watanabe, K., Miyazaki, T., and Matsuda, R. Growth factor array fabrication using a color ink jet printer. Zool. Sci. 20, 429, 2003. Xu, T., Jin, J., Gregory, C., Hickman, J.J., and Boland, T. Inkjet printing of viable mammalian cells. Biomaterials 26, 93, 2005. Alsberg, E., Feinstein, E., Joy, M.P., Prentiss, M., and Ingber, D.E. Magnetically guided self-assembly of fibrin matrices with ordered nano-scale structure for tissue engineering. Tissue Eng. 12, 3247, 2006. Marx, G. Evolution of fibrin glue applicators. Transfus. Med. Rev. 17, 287, 2003. Buchta, C., Dettke, M., Funovics, P.T., Hocker, P., Knobl, P., Macher, M., Quehenberger, P., Treitl, C., and Worel, N. Fibrin sealants produced by the cryoseal FS system: product chemistry, material properties and possible preparation in the autologous preoperative setting. Vox San. 86, 257, 2004. Carless, P.A., Anthony, D.M., and Henry, D.A. Systematic review of the use of fibrin sealant to minimize perioperative allogeneic blood transfusion. Br. J. Surg. 89, 695, 2002. Buchta, C., Hedrich, H.S., Macher, M., Hocker, P., and Redl, H. Biochemical characterization of autologous fibrin sealants produced by Cryoseal and Vivostat in comparison to homologous fibrin sealants produced Tissucol/Tisseel. Biomaterials 26, 6233, 2005. Jackson, M.R. Fibrin sealants in surgical practice: an overview. Am. J. Surg. 182S, 1, 2001. Evatt, B., Austin, H., Leon, G., Ruiz-Saez, A., and de Bosch, N. Hemophilia treatment. Predicting the long-term risk of HIV exposure by cryoprecipitate. Haemophilia 6, 128, 2000. Kawamura, M., Sawafuji, M., Watanabe, M., Horinouchi, H., and Kobayashi, K. Frequency of transmission of human parvovirus B 19 infection in fibrin sealant used during thoracic surgery. Ann. Thorac. Surg. 73, 1098, 2002. 211 85. Hunter, N., and Houston, F. Can prion diseases be transmitted between individuals via blood transfusion: evidence from sheep experiments. Dev. Biol. (Basel) 108, 93, 2002. 86. Nakamura, H., Matsuyama, Y., Yoshihara, H., Sakai, Y., Katayama, Y., Nakashima, S., Takamatsu, J., and Ishiguro, N. The effect of autologous fibrin tissue adhesive on postoperative cerebrospinal fluid leak in spinal cord surgery: a randomized controlled trial. Spine 30, E347, 2005. 87. Dresdale, A., Rose, E.A., Jeevanandam, V., Reemtsma, K., Bowman, F.O., and Malm, J.R. Preparation of fibrin glue from single-donor fresh-frozen plasma. Surgery 97, 750, 1985. 88. Spotnitz, W.D., Mintz, P.D., Avery, N., Bithell, T.C., Kaul, S., and Nolan, S.P. Fibrin glue from stored human plasma. An inexpensive and efficient method for local blood bank preparation. Am. Surg. 53, 460, 1987. 89. Nakamura, Y., Masuda, M., Toshima, Y., Asou, T., Oe, M., Kinoshita, K., Kawachi, Y., Tanaka, J., and Tokunaga, K. Comparative study of cell saver and ultrafiltration nontransfusion in cardiac surgery. Ann. Thorac. Surg. 49, 973, 1990. 90. Sirieix, D., Chemla, E., Castier, Y., Massonnet-Castel, S., Fabiani, J.N., and Baron, J.F. Comparative study of different biological glues in an experimental model of surgical bleeding in anesthetized rats: platelet-rich and -poor plasmabased glue with and without aprotinin versus commercial fibrinogen-based glue. Ann. Vasc. Surg. 12, 311, 1998. 91. Koster, A., Sanger, S., Knörig, F.J., Kuppe, H., Hetzer, R., and Loebe, M. Autologous plasma and platelet sequestration at the beginning of cardiopulmonary bypass: a pilot investigation in five patients undergoing extended vascular surgery in deep hypothermia. ASAIO J. 48, 106, 2002. 92. Silver, F.H., Wang, M.C., and Pins, G.D. Preparation of fibrin glue: a study of chemical and physical methods. J. Appl. Biomater. 6, 175, 1995. 93. Burnouf, T., and Radosevich, M. Affinity chromatography in the industrial purification of plasma proteins for therapeutic use. J. Biochem. Biophys. Methods 49, 575, 2001. 94. Mohri, H., and Ohkubo, T. Fibrinogen binds to heparin: the relationship of the binding of other adhesive proteins to heparin. Arch. Biochem. Biophys. 303, 27, 1993. 95. Dempfle, C.E., and Heene, D.L. Purification of human plasma fibrinogen by chromatography on protamine-agarose. Thromb. Res. 46, 19, 1987. 96. Matthias, F.R., Hocke, G., and Lasch, H.G. Isolation of fibrinogen from plasma by affinity chromatography on insolubilized fibrinogen (FG-ag) and insolubilized fibrinmonomer (FM-ag). Thromb. Res. 7, 861, 1975. 97. Follmar, K.E., Prichard, H.L., DeCroos, F.C., Wang, H.T., Levin, L.S., Klitzman, B., Olbrich, K.C., and Erdmann, D. Combined bone allograft and adipose-derived stem cells autograft in a rabbit model. Ann. Plast. Surg. 58, 561, 2007. 98. Groger, A., Klaring, S., Merten, H.A., Holste, J., Keps, C., and Sittinger, M. Tissue engineering of bone for mandibular augmentation in immunocompetent minipigs: preliminary study. Scand. J. Plast. Reconstr. Surg. Hand. Surg. 37, 129, 2003. 99. Ito, K., Yamada, Y., Naiki, T., and Ueda, M. Simultaneous implant placement and bone regeneration around dental implants using tissue-engineered bone with fibrin glue, AHMED ET AL. 212 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. mesenchymal stem cells and platelet-rich plasma. Clin. Oral. Implants Res. 17, 579, 2006. Lechner, S., and Huss, R. Bone engineering: combining smart biomaterials and application of stem cells. Artif. Organs 30, 770, 2006. Zhu, S.J., Choi, B.H., Jung, J.H., Lee, S.H., Huh, J.Y., You, T.M., Lee, H.J., and Li, J. A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue. Oral. Surg. Oral. Med. Oral. Pathol. Oral. Radiol. Endod. 102, 175, 2006. Zhu, S.J., Choi, B.H., Huh, J.Y., Jung, J.H., Kim, B.Y., Lee, H.J., and Li, J. A comparative qualitative histological analysis of tissue-engineered bone using bone marrow mesenchymal stem cells, alveolar bone cells, and periosteal cells. Oral. Surg. Oral. Med. Oral. Pathol. Oral. Radiol. Endod. 101, 164, 2006. Lee, O.K., Coathup, M.J., Goodship, A.E., and Blunn, G.W. Use of mesenchymal stem cells to facilitate bone regeneration in normal and chemotherapy-treated rats. Tissue Eng. 11, 1727, 2005. Neumeister, M.W., Wu, T., and Chambers, C. Vascularized tissue-engineered ears. Plast. Reconstr. Surg. 117, 116, 2006. Westreich, R., Kaufman, M., Gannon, P., and Lawson, W. Validating the subcutaneous model of injectable autologous cartilage using a fibrin glue scaffold. Laryngoscope 114, 2154, 2004. Neovius, E.B., and Kratz, G. Tissue engineering by cocultivating human elastic chondrocytes and keratinocytes. Tissue Eng. 9, 365, 2003. Chang, J., Rasamny, J.J., and Park, S.S. Injectable tissue engineered cartilage using a fibrin sealant. Arch. Facial. Plast. Surg. 9, 161, 2007. Dragoo, J.L., Carlson, G., McCormick, F., Khan-Farooqi, H., Zhu, M., Zuk, P.A., and Benhaim, P. Healing fullthickness cartilage defects using adipose-derived stem cells. Tissue Eng. 13, 1615, 2007. Meinhart, J., Fussenegger, M., and Hobling, W. Stabilization of fibrin-chondrocyte constructs for cartilage reconstruction. Ann. Plast. Surg. 42, 673, 1999. Higa, K., Shimmura, S., Kato, N., Kawakita, T., Miyashita, H., Itabashi, Y., Fukuda, K., Shimazaki, J., and Tsubota, K. Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier. Invest. Ophthalmol. Vis. Sci. 48, 597, 2007. Kofidis, T., Lenz, A., Boublik, J., Akhyari, P., Wachsmann, B., Muller-Stahl, K., Hofmann, M., and Haverich, A. Pulsatile perfusion and cardiomyocytes viability in a solid three-dimensional matrix. Biomaterials 24, 5009, 2003. Arkudas, A., Tjiawi, J., Bleiziffer, O., Grabinger, L., Polykandriotis, E., Beier, J.P., Sturzel, M., Horch, R.E., and Kneser, U. Fibrin gel-immobilized VEGF and bFGF efficiently stimulate angiogenesis in the AV loop model. Mol. Med. 13, 480, 2007. Hankemeier, S., van Griensven, M., Ezechieli, M., Barkhausen, T., Austin, M., Jagodzinski, M., Meller, R., Bosch, U., Krettek, C., and Zeichen, J. Tissue engineering of tendons and ligaments by human bone marrow stromal cells in a liquid fibrin matrix in immunodeficient rats: results of 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. a histologic study. Arch. Orthop. Trauma. Surg. 127, 815, 2007. Chong, A.K., Ang, A.D., Goh, J.C., Hui, J.H., Lim, A.Y., Lee, E.H., and Lim, B.H. Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit Achilles tendon model. J. Bone Joint. Surg. Am. 89, 74, 2007. Soon, M.Y., Hassan, A., Hui, J.H., Goh, J.C., and Lee, E.H. An analysis of soft tissue allograft anterior cruciate ligament reconstruction in a rabbit model: a short-term study of the use of mesenchymal stem cells to enhance tendon osteointegration. Am. J. Sports Med. 35, 962, 2007. Johnsen, S., Ermuth, T., Tanczos, E., Bannasch, H., Horch, R.E., Zschocke, I., Peschen, M., Schopf, E., Vanscheidt, W., and Augustin, M. Treatment of therapy-refractive ulcera cruris of various origins with autologous keratinocytes in fibrin sealant. Vasa 34, 25, 2005. Kopp, J., Jeschke, M.G., Bach, A.D., Kneser, U., and Horch, R.E. Applied tissue engineering in the closure of severe burns and chronic wounds using cultures human autologous keratinocytes in a natural fibrin matrix. Cell. Tissue. Bank. 5, 89, 2004. Currie, L.J., Sharpe, J.R., and Martin, R. The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review. Plast. Reconstr. Surg. 108, 1713, 2001. Marlovits, S., Striessnig, G., Kutscha-Lissberg, F., Resinger, C., Aldrian, S.M., Vecsei, V., and Trattnig, S. Early postoperative adherence of matrix-induced autologous chondrocyte implantation for the treatment of full-thickness cartilage defects of the femoral condyle. Knee Surg. Sports Traumatol. Arthrosc. 13, 451, 2005. Kanemaru, S., Nakamura, T., Omori, K., Magrufov, A., Yamashita, M., and Ito, J. Regeneration of mastoid air cells in clinical applications by in situ tissue engineering. Laryngoscope 115, 253, 2005. Oshima, Y., Watanabe, N., Matsuda, K., Takai, S., Kawata, M., and Kubo, T. Behavior of transplanted bone marrow derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation. J. Histochem. Cytochem. 53, 207, 2005. Oshima, Y., Watanabe, N., Matsuda, K., Takai, S., Kawata, M., and Kubo T. Fate of transplanted bone-marrow-derived mesenchymal cells during osteochondral repair using transgenic rats to stimulate autologous transplantation. Osteoarthritis Cartilage 12, 811, 2004. Ronga, M., Grassi, F.A., and Bulgheroni, P. Arthroscopic autologous chondrocyte implantation for the treatment of a chondral defects in the tibial plateau of the knee. Arthroscopy 20, 79, 2004. Neuman, A.R., Weinberg, A., Benmeir, P., Eldad, A., and Wexler, M.R. Spray application of fibrin glue by a simple device. Plast. Reconstr. Surg. 88, 922, 1991. Falanga, V., Iwamoto, S., Chartier, M., Yufit, T., Butmarc, J., Kouttab, N., Shrayer, D., and Carson, P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng. 13, 1299, 2007. Grant, I., Warwick, K., Marshall, J., Green, C., and Martin, R. The co-application of sprayed cultured autologous kera- FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. tinocytes and autologous fibrin sealant in a porcine wound model. Br. J. Plast. Surg. 55, 219, 2002. Chou, C.H., Cheng, W.T., Kuo, T.F., Sun, J.S., Lin, F.H., and Tsai, J.C. Fibrin glue with gelatin/hyaluronic acid/ chondroitin-6-sulfate tri-copolymer for articular cartilage tissue engineering: the results of real-time polymerase chain reaction. J. Biomed. Mater. Res. A. 82, 757, 2007. Uriel, S., Brey, E.M., and Greisler, H.P. Sustained low levels of fibroblast growth factor-1 promote persistent microvascular network formation. Am. J. Surg. 192, 604, 2006. Erzurum, V.Z., Bian, J.F., Husak, V.A., Ellinger, J., Xue, L., Burgess, W.H., and Greisler, H.P. R136K fibroblast growth factor-1 mutant induces heparin-independent migration of endothelial cells through fibrin glue. J. Vasc. Surg. 37, 1075, 2003. Shireman, P.K., Hampton, B., Burgess, W.H., and Greisler, H.P. Modulation of vascular cell growth kinetics by local cytokine delivery from fibrin glue suspensions. J. Vasc. Surg. 29, 852, 1999. Greisler, H.P., Gosselin, C., Ren, D., Kang, S.S., and Kim, D.U. Biointeractive polymers and tissue engineered blood vessels. Biomaterials 17, 329, 1996. Gorodetsky, R., Clark, R.A., An, J., Gailit, J., Levdansky, L., Vexler, A., Berman, E., and Marx, G. Fibrin microbeads (FMB) as a biodegradable carriers for culturing cells and for accelerating wound healing. J. Invest. Dermatol. 112, 866, 1999. Marx, G., Mou, X., Hotovely-Salomon, A., Levdansky, L., Gaberman, E., Belenky, D., and Gorodetsky, R. Heat denaturation of fibrinogen to develop a biomedical matrix. J. Biomed. Mater. Res. B. Appl. Biomater. 84, 49, 2008. Shimony, N., Gorodetsky, R., Marx, G., Gal, D., Rivkin, R., Ben-Ari, A., Landsman, A., and Haviv, Y.S. Fibrin microbeads (FMB) as a 3D platform for kidney gene and cell therapy. Kidney Int. 69, 625, 2006. Gurevich, O., Vexler, A., Marx, G., Prigozhina, T., Levdansky, L., Slavin, S., Shimeliovich, I., and Gorodetsky, R. Fibrin microbeads for isolating and growing bone marrowderived progenitor cells capable of forming bone tissue. Tissue Eng. 8, 661, 2002. Cho, S.W., Kim, S.S., Rhie, J.W., Cho, H.M., Choi, C.Y., and Kim, B.S. Engineering of volume-stable adipose tissue. Biomaterials 26, 3577, 2005. Patrick, C.W., Zheng, B., Johnston, C., and Reece, J.P. Long-term implanation of preadipocytes-seeded PLGA scaffolds. Tissue Eng. 8, 283, 2002. von Heimburg, D., Zachariah, S., Heschel, I., Kuhling, H., Schoof, H., Hafemann, B., and Pallua, N. Human preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo. Biomaterials 22, 429, 2001. Wechselberger, G., Russell, R.C., Neumeister, M.W., Schoeller, T., Piza-katzer, H., and Rainer, C. Successful transplantation of three tissue-engineered cell types using capsule induction techniques and fibrin glue as a delivery vehicle. Plast. Reconstr. Surg. 110, 123, 2002. Borges, J., Mueller, M.C., Padron, N.T., Tegtmeier, F., Lang, E.M., and Stark, G.B. Engineered adipose tissue supplied by functional microvessels. Tissue Eng. 9, 1263, 2003. 213 141. Yoo, W.J., Choi, I.H., Chung, C.Y., Cho, T.-J., Kim, I.-O., and Kim, C.J. Implantation of perichondrium-derived chondrocytes in physeal defects of rabbit tibiae. Acta. Orthop. 76, 628, 2005. 142. Altmeppen, J., Hansen, E., Bonnländer, G.L., Horch, R.E., and Jeschke, M.G. Composition and characteristics of an autologous thrombocyte gel. J. Surg. Res. 117, 202, 2004. 143. Wittkampf, A.R. Fibrin glue as cement for HA-granules. J. Craniomaxillofac. Surg. 17, 179, 1989. 144. Nakamura, K., Koshino, T., and Saito, T. Osteogenic response of the rabbit femur to a hydroxyapatite thermal decomposition product-fibrin glue mixture. Biomaterials 19, 1901, 1998. 145. Le Nihouannen, D., Le Guehennec, L., Rouillon, T., Pilet, P., Bilban, M., Layrolle, P., and Daculsi, G. Microarchitecture of calcium granules and fibrin glue composites for bone tissue engineering. Biomaterials 27, 2716, 2006. 146. Umeda, H., Kanemaru, S.-I., Yamashita, M., Kishimoto, M., Tamura, Y., Nakamura, T., Omori, K., Hirano, S., and Ito, J. Bone regeneration of canine skull using bone marrowderived stromal cells and b-tricalcium phosphate. Laryngoscope 117, 997, 2007. 147. Stevenson, L.W., Warner, S.L., Steimyle, A.E., Fonarow, G.C., Hamilton, M.A., Moriguchi, J.D., Kobashigawa, J.A., Tissisch, J.H., Drinkwater, D.C., and Laks, H. The impending crisis awaiting cardiac transplantation: modeling a solution based on selection. Circulation 89, 450, 1994. 148. Schoen, F.J. Future directions in tissue heart valves: impact of recent insights from biology and pathology. J. Heart Valve Dis. 8, 350, 1999. 149. Williams, C., Johnson, S.L., Robinson, P.S., and Tranquillo, R.T. Cell sourcing and culture conditions for fibrin-based valve constructs. Tissue Eng. 12, 1489, 2006. 150. Flanagan, T.C., Cornelissen, C., Koch, S., Tschoeke, B., Sachweh, J.S., Schmitz-Rode, T., and Jockenhoevel, S. The in vitro development of autologous fibrin-based tissueengineered heart valves through optimised dynamic conditioning. Biomaterials 28, 3388, 2007. 151. Jockenhoevel, S., Chalabi, K., Sachweh, J.S., Groesdonk, H.V., Demircan, L., Grossman, M., Zund, G., and Messmer, B.J. Tissue engineering: complete autologous valve conduit– a new moulding technique. Thorac. Cardiovasc. Surg. 49, 287, 2001. 152. Izuta, Y., Ochi, M., Adachi, N., Deie, M., Yamasaki, T., and Shinomiya, R. Meniscal repair using bone marrow-derived mesenchymal stem cells: experimental study using green fluorescent protein transgenic rats. Knee 12, 217, 2005. 153. Henning, C.E., Lynch, M.A., Yearout, K.M., Vequist, S.W., Stallbaumer, R.J., and Decker, K.A. Arthroscopic meniscal repair using an exogenous fibrin clot. Clin. Orthop. Relat. Res. 252, 64, 1990. 154. Swenson, T.M. The use of exogenous fibrin clot to supplement meniscal surgery techniques. Orthopedics 30, 718, 2007. 155. Ruszymah, B.H., Lokman, B.S., Asma, A., Munirah, S., Chua, K., Mazlyzam, A.L., Isa, M.R., Fuzina, N.H., and Aminuddin, B.S. Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage. Int. J. Pediatr. Otorhinolaryngol. 71, 1225, 2007. 214 156. Jian-Wei, X., Randolph, M.A., Peretti, G.M., Nazzal, J.A., Roses, R.E., Morse, K.R., and Yaremchuk, M.J. Producing a flexible tissue-engineered cartilage framework using expanded polytetrafluoroethylene membrane as a pseudoperichondrium. Plast. Reconstr. Surg. 116, 577, 2005. 157. Siedentop, K.H., O’Grady, K., Bhattacharyya, T.K., and Shah, A. Fibrin tissue adhesive and autologous concha cartilage for reconstruction of the posterior-superior canal wall of the chinchilla middle ear. Otol. Neurotol. 25, 220, 2004. 158. Kim, H.W., and Han, C.D. An overview of cartilage tissue engineering. Yonsei Med. J. 41, 766, 2000. 159. Dare, E.V., Vascotto, S.G., Carlsson, D., Hincke, M.T., and Griffith, M. Differentiation of fibrin gel encapsulated chondrogenic cell line. Int. J. Artif. Organs 30, 619, 2007. 160. Perka, C., Spitzer, R.S., Lindenhayn, K., Sittinger, M., and Schultz, O. Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants. J. Biomed. Mater. Res. 49, 305, 2000. 161. Park, S.H., Park, S.R., Chung, S.I., Pai, K.S., and Min, B.H. Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation. Artif. Organs 29, 838, 2005. 162. Handl, M., Trc, T., Hanus, M., Stastný, E., Fricová-Poulová, M., Neuwirth, J., Adler, J., Havranová, D., and Varga, F. Therapy of severe chondral defects of the patella by autologous chondrocyte implantation. Acta. Chir. Orthop. Traumatol. Cech. 73, 373, 2006. 163. Visna, P., Pasa, L., Hart, R., Kocis, J., Cizmár, I., and Adler, J. Treatment of deep chondral defects of the knee using autologous chondrocytes cultured on a support-results after one year. Acta. Chir. Orthop. Traumatol. Cech. 70, 356, 2003. 164. Bedran-Russo, A.K., Pereira, P.N., Duarte, W.R., Drummond, J.L., and Yamauchi, M. Application of crosslinkers to dentin collagen enhances the ultimate tensile strength. J. Biomed. Mater. Res. B. Appl. Biomater. 80, 268, 2007. 165. Ameer, G.A., Mahmood, T.A., and Langer, R. A biodegradable composite scaffold for cell transplantation. J. Orthop. Res. 20, 16, 2002. 166. Perka, C., Schultz, O., Lindenhayn, K., Spitzer, R.S., Muschik, M., Sittinger, M., and Burmester, G.R. Joint cartilage repair with transplantation of embryonic chondrocytes embedded in collagen-fibrin matrices. Clin. Exp. Rheumatol. 18, 13, 2000. 167. Perka, C., Schultz, O., Spitzer, R.S., and Lindenhayn, K. The influence of transforming growth factor beta1 on mesenchymal cell repair of full-thickness cartilage defects. Biomed. Mater. Res. 52, 543, 2000. 168. Almqvist, K.F., Wang, L., Wang, J., Baeten, D., Cornelissen, M., Verdonk, R., Veys, E.M., and Verbruggen, G. Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks. Ann. Rheum. Dis. 60, 781, 2001. 169. Sims, C.D., Butler, P.E., Cao, Y.L., Casanova, R., Randolph, M.A., Black, A., Vacanti, C.A., and Yaremchuk, M.J. Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes. Plast. Reconstr. Surg. 101, 1580, 1998. 170. Silverman, R.P., Passaretti, D.M., Huang, W.M., Randolph, M.A., and Yaremchuk, M.J. Injectable tissue-engineered cartilage using a fibrin glue polymer. Plast. Reconstr. Surg. 103, 1809, 1999. AHMED ET AL. 171. Fortier, L.A., Mohammed, H.O., Lust, G., and Nixon, A.J. Insulin-like growth factor-I enhances cell-based repair of articular cartilage. J. Bone. Joint. Surg. Br. 84, 276, 2002. 172. Malicev, E., Radosavljevic, D., and Velikonja, N.K. Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds. Biotechnol. Bioeng. 96, 364, 2007. 173. Peretti, G.M., Campo-Ruiz, V., Gonzalez, S., Randolph, M.A., Wei, X.J., Morse, K.R., Roses, R.E., and Yaremchuk, M.J. Tissue engineered cartilage integration to live and devitalized cartilage: a study by reflectance mode confocal microscopy and standard histology. Connect. Tissue. Res. 47, 190, 2006. 174. Xu, X.L., Lou, J., Tang, T., Ng, K.W., Zhang, J., Yu, C., and Dai, K. Evaluation of different scaffolds for BMP-2 genetic orthopedic tissue engineering. J. Biomed. Mater. Res. B. Appl. Biomater. 75, 289, 2005. 175. Im, G.I., Shin, Y.W., and Lee, K.B. Do adipose tissuederived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells?. Osteoarthritis Cartilage 13, 845, 2005. 176. Chang, F., Ishii, T., Yanai, T., Mishima, H., Akaogi, H., Ogawa, T., and Ochiai, N. Repair of large full-thickness articular cartilage defects by transplantation of autologous uncultured bone-marrow-derived mononuclear cells. J. Orthop. Res. 26, 18, 2008. 177. Dragoo, J.L., Samimi, B., Zhu, M., Hame, S.L., Thomas, B.J., Lieberman, J.R., Hedrick, M.H., and Benhaim, P. Tissueengineered cartilage and bone using stem cells from human infrapatellar fat pads. J. Bone Joint Surg. Br. 85, 740, 2003. 178. Gelse, K., von der Mark, K., Aigner, T., Park, J., and Schneider, H. Articular cartilage repair by gene therapy using growth factor-producing mesenchymal cells. Arthritis Rheum. 48, 430, 2003. 179. Xiangdong, W., Ar’Rajab, A., Ahren, B., Andersson, R., and Bengmark, S. The effect of pancreatic islets on transplanted hepatocytes in the treatment of acute liver failure in rats. Res. Exp. Med. (Berl.) 191, 429, 1991. 180. Demetriou, A.A., Whiting, J.F., Feldman, D., Levenson, S.M., Chowdhury, N.R., Moscioni, A.D., Kram, M., and Chowdhury, J.R. Replacement of liver function in rats by transplantation of microcarrier attached hepatocytes. Science 233, 1190, 1986. 181. Chia, S.M., Leong, K.W., Li, J., Xu, X., Zeng, K., Er, P.N., Gao, S., and Yu, H. Hepatocyte encapsulation for enhanced cellular functions. Tissue Eng. 6, 481, 2000. 182. Taylor, J.P., Hardy, J., and Fischbeck, K.H. Toxic proteins in neurodegenerative disease. Science 296, 1991, 2002. 183. Williams, L.R., Longo, F.M., Powell, H.C., Lundborg, G., and Varon, S. Spatial-temporal progress of peripheral nerve regeneration within a silicone chamber: parameters for a bioassay. J. Comp. Neurol. 218, 460, 1983. 184. Herbert, C.B., Bittner, G.D., and Hubbell, J.A. Effects of fibrinolysis on neurite growth from dorsal root ganglia cultured in two and three-dimensional fibrin gels. J. Comp. Neurol. 365, 380, 1996. 185. Willerth, S.M., Arendas, K.J., Gottlieb, D.I., and SakiyamaElbert, S.E. Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells. Biomaterials 27, 5990, 2006. FIBRIN SCAFFOLDS FOR TISSUE ENGINEERING 186. Cheng, H., Cao, Y., and Olson, L. Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function. Science 273, 510, 1996. 187. Iwaya, K., Mizoi, K., Tessler, A., and Itoh, Y. Neurotrophic agents in fibrin glue mediate adult dorsal root regeneration into spinal cord. Neurosurgery 44, 589 (discussion 595), 1999. 188. Nishida, K. Tissue engineering of the cornea. Cornea 22, S28, 2003. 189. Duchesne, B., Tahi, H., and Galand, A. Use of human fibrin glue and amniotic membrane transplant in corneal perforation. Cornea 20, 230, 2001. 190. Rama, P., Bonini, S., Lambiase, A., Golisano, O., Paterna, P., de Luca, M., and Pellegrini, G. Autologous fibrincultured limbal stem cells permanently restore the corneal surface of patients with total limbal cell deficiency. Transplantation 72, 1478, 2001. 191. Horch, R.E., Kopp, J., Kneser, U., Beier, J., and Bach, A.D. Tissue engineering of cultured skin substitutes. J. Cell. Mol. Med. 9, 592, 2005. 192. Hafemann, B., Hettich, R., Ensslen, S., Kowol, B., Zuhlke, A., Ebert, R., Konigs, M., and Kirkpatrick, C.J. Treatment of skin defects using suspension of in vitro cultured keratinocytes. Burns 20, 168, 1994. 193. Kaiser, H.W., Stark, G.B., Kopp, J., Balcerkiewicz, A., Spilker, G., and Kreysel, H.W. Cultured autologous keratinocytes in fibrin glue suspension, exclusively and combined with STS-allograft (preliminary clinical and histological report of a new technique). Burns 20, 23, 1994. 215 194. Geer, D.J., Swartz, D.D., and Andreadis, S.T. Fibrin promotes in a three-dimensional in vitro model of wound regeneration. Tissue Eng. 8, 787, 2002. 195. Siedler, S., and Schuller-Petrovic, S. Wound healing enhancement in leg ulcers: a case report. Cell Tissue Bank 3, 25, 2002. 196. Khor, H.L., Ng, K.W., Htay, A.S., Schanz, J.T., Teoh, S.H., and Hutmacher, D.W. Preliminary study of a polycaprotone membrane utilized as epidermal substrate. J. Mater. Sci. Mater. Med. 14, 113, 2003. 197. Marx, G., and Mou, X. Characterizing fibrin glue performance as modulated by heparin, aprotinin, and factor XIII. J. Lab. Clin. Med. 140, 152, 2002. 198. Provenzano, P.P., Hurschler, C., and Vanderby, R., Jr. Microstructural morphology in the transition region between scar and intact residual segments of a healing rat medial collateral ligament. Connect. Tissue Res. 42, 123, 2001. Address reprint requests to: Max Hincke, Ph.D. Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa 451 Smyth Road Ottawa Ontario K1H 8M5 Canada E-mail: [email protected]