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2011

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citation for published version (APA) Weijers, E. M. (2011). matrices for tissue engineering: Naturally occurring variants alter cellular characteristics.

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Download date: 26. Sep. 2021

Review: Fibrin matrices for tissue engineering

Ester M. Weijers Moniek P.M. de Maat Victor W.M. van Hinsbergh Pieter Koolwijk

Submitted

26 Review: Fibrin matrices for tissue engineering

Abstract

A challenge in tissue engineering is making a scaffold that combines and cells that optimally restores, maintains and/or enhances tissue and organ functions in the patient. One promising scaffold material is fibrin, which provides a good environment for cell migration and proliferation. It serves as a reservoir of growth factors and its degradation synergies with tissue formation. Fibrin forms a natural biodegradable, biocompatible scaffold with high cell binding and signaling capacity. Moreover, autologous fibrin can be used to avoid the potential risk of foreign body reactions or infections. Together these properties inspired a broad field of research to use fibrin for tissue engineering applications. In this review the natural properties of fibrin(ogen) are given and various tissue engineering approaches using fibrin are highlighted.

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INTRODUCTION

Tissue engineering is an interdisciplinary field in biomedical engineering that aims at generating new biological tissues for replacing diseased or injured tissues using scaffolds and/or cells 1. During tissue engineering the healing capacity of the patient is increased and guided, thereby damaged tissues can be repaired and their natural functions can be restored. There are three principal therapeutic strategies for treating diseased or injured tissues in patients; (I) implantation of freshly isolated or cultured cells, (II) implantation of tissues assembled in vitro , from cells and scaffolds; and (III) in situ tissue regeneration 2. As one can appreciate, the approach to tissue engineering is varied and plentiful, where selecting the right for the scaffold is as important as choosing the right cell type 1. The ideal scaffold should have tissue-like mechanical properties, display immunologic integrity, and support , migration and differentiation. The provisional scaffold preferentially disappears through specific degradation, while new tissue is formed 3. Scaffolds can be made of natural materials, of synthetic materials or combinations of the two (hybrid scaffolds). Fibrin is one of the natural materials that has high potential for use in tissue engineering, and it can be isolated from the patients blood and used as an autologous scaffold, without the potential risk of a foreign body reaction or infections 4,5 . In vivo , fibrin(ogen) plays an important role in hemostasis, , and angiogenesis 6-8. Upon contact with fibrin, cells will gradually replace the fibrin scaffold by a mature tissue-specific . Taken together, these properties make fibrin an interesting and widely used protein for tissue engineered scaffolds.

Scope of review This review is intended to provide a focused survey on the use of the natural scaffold fibrin in various approaches in tissue engineering, e.g. in wound sealing, skin tissue engineering, vascular tissue engineering, heart valve replacement and stem cell research. Moreover, fibrin can be used as delivery tool for biomolecules, drugs and cells. Here, the natural properties of fibrin(ogen) are summarized and various tissue engineering approaches using fibrin are highlighted.

FIBRIN Fibrinogen Fibrinogen is an acute-phase protein, occurring at 2 to 4 g/L in human blood. During trauma, inflammation or upon treatment with interleukin-6 and glucocortocoids the fibrinogen concentration increases 9. Fibrinogen is a symmetrical glycoprotein of 340 kDa, composed of two sets of three polypeptide chains that are linked together by 29 disulfide bonds. Three separate genes on chromosome 4q22-23 encode for the fibrinogen A α-, B β- and γ-chains, with the B β- chain as rate limiting and regulatory gene 10 . Plasma fibrinogen shows a high degree of heterogeneity in healthy individuals, wherein the fibrinogen variants can have altered structural and functional characteristics 11 . Fibrinogen is primarily synthesized in the liver, however lung epithelial cells can produce small proportions 12,13 . Electron microscopy studies demonstrated a trinodular structure with a length of approximately 45 nm 6. In the central E-domain the amino-

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termini of the two A α-, two B β- and two γ-chains come together (Figure 1) 14 . Two α-helical coiled- coil rods are spanning from the E-domain and end in two distal D-domains. The D-domains contain the carboxy-termini of the B β- and γ-chains, whereas the carboxy-termini of the A α-chains extent and fold back towards the center of the fibrinogen molecule 15,16 .

Polymerization Fibrin is a natural polymer, which is formed by the enzymatic polymerization of fibrinogen. Soluble fibrinogen is converted to fibrin monomers by -catalyzed removal of fibrinopeptides A and B on the amino-termini of the A α- and B β-chains. The release of fibrinopeptides exposes new amino-termini, which initiate the polymerization process to form a fibrin matrix 6,17 . Soluble monomers bind to each other via ‘knob-hole’ binding and form insoluble fibrin fibers that also branch, which then results in a fibrin matrix. The fibrin matrix is stabilized by the factor XIIIa (FXIIIa), which cross-links two adjacent γ-chains or adjacent α- and γ-chains 18,19 . The cross-links within the fibrin matrix will result in a higher stability of the matrix and lower susceptibility to degradation via .

Figure 1. Fibrinogen structure. (A) Overview of the A α-, B β- and γ-chains of fibrinogen, wherein the central E-domain contains the amino-termini of all six chains, the two D-domains contain carboxy-termini of the B β- and γ-chains. (B) Schematic representation of the structural polypeptide arrangement and three nodules is given. The carboxy-termini of the A α-chains fold back towards the center of the molecule (This image was originally published in Blood by H.C.F Côté et al. Blood . 1998;92;7:2195-2212 14 . © the American Society of Hematology)

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Fibrin structure and properties The structure of the fibrin matrix influences its biological function. Variation in the fibrin structure is described by fiber thickness, fiber length, number of branch-points and porosity 20 . Alterations in the fibrinogen and thrombin concentration, the ionic strength, the pH and fibrinogen or FXIII polymorphisms can influence the fibrin structure 6,21-23 . Increasing the thrombin concentration during polymerization will lead to a more dense structure with thinner fibers 24 . Furthermore, various naturally occurring fibrinogen variants have been shown to influence the fibrin structure 11,25. The fibrinogen variant Nieuwegein, with partially truncated A α-chains, was shown to form a tighter fibrin network than normal fibrinogen (Figure 2A,B) 26 . Also the naturally occurring fibrin variant γA-γ′ showed a finer and more branched network than γA-γA fibrinogen (Figure 2C,D) 27 . Fibrin can directly bind growth factors, e.g. vascular endothelial (VEGF) -2 (FGF-2), -derived growth factor (PDGF) and transforming growth factor-β (TGF-β)28-30 . In addition, since thrombin, factor XIII and can bind to the fibrin matrix, the proteins binding to these molecules can indirectly bind to fibrin, for example heparin- binding growth factors 7. Finally, proteolytical enzymes, such as plasminogen, tissue-type plasminogen activator (tPA) and α2-antiplasmin can bind directly to fibrin, and thereby control its degradation 7,31 .

Figure 2. Scanning electron microscopy (SEM) of the fibrin network formed from normal and naturally occurring fibrinogen variants. SEM pictures of normal plasma (A) and plasma Nieuwegein (B) clotted with 1 U/ml thrombin; and fibrin γA-γA (C) and γA-γ′ (D) clotted with 0.1 U/ml thrombin. (This research was originally published in Blood . A. Collen et al. Blood . 2001;97;4:973-980 26 and K.R. Siebenlist et al. Blood . 2005;106;8;2730-2736 27 © The American Society of Hematology).

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Hemostasis and wound healing During the last step of the cascade a complex fibrin matrix is formed 8. A stable thrombus is formed by the interaction of an activated platelet clot with fibrin, this interaction is mediated by integrin αIIb β3 (GPIIb-IIIa) on 7. In time, the biochemical properties of the fibrin clot change and a more stable plug is formed. Next to its role in hemostasis, fibrin is an important protein during wound healing. Here, fibrin provides a temporary extracellular matrix that facilitates cell migration, invasion and proliferation and is a reservoir for growth factors and proteases. The extracellular matrix (ECM) plays an instructive role for cellular activities and cells possess receptors on their surface that respond to extracellular signals 3. The important role of fibrinogen in wound healing is indicated by the delayed tissue repair that is seen in patients with defects in clot formation and fibrin dissolution 32 . Moreover, dysfibrinogenemia results in impaired wound healing 33 .

Fibrinolysis In vivo , fibrinolysis is a protective mechanism, removing thrombi from the circulation and thereby restoring blood flow. During wound healing, fibrin will be gradually degraded and replaced by mature extracellular matrix, wherein the proteolytic activity of membrane-type matrix metalloproteinases (MT1-MMP) 34,35 and plasmin 36,37 locally degrades the fibrin matrix (Figure 3). Conversion of the inactive zymogen plasminogen into the active protease plasmin is mediated via receptor-bound urokinase-type plasminogen activator (uPA) and secreted tissue-type plasminogen activator (tPA) 38,39 . Several inhibitors of fibrinolysis are known, among which aprotinin, α2-antiplasmin, tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1 (PAI-1) 35,37,40,41 . Towards the tissue engineering approach, controllable fibrin degradation or fibrinolysis is of great interest. In vivo , fibrin is degraded in the order of days to weeks, and the rate of fibrinolysis is influenced by the fibrin structure, its cross-linking and the incorporation of protease inhibitors 5,40,42,43 . Inhibition of plasmin by the addition the protease inhibitor aprotinin, was shown to inhibit fast degradation of the fibrin matrix, and thereby provides a more controllable degradation 5,44 . Long-term stability and mechanical integrity may be essential for cells that require ample time and stiffness to remodel the matrix 45 . Fibrin matrices are gradually replaced by mature that is produced by invading cells, e.g. fibroblasts, endothelial progenitor cells and smooth muscle cells 32,46,47 .

Angiogenesis In vivo , the fibrin matrix also provides a good and temporary environment for the formation of new vascular structures from existing vessels (angiogenesis). During wound healing and tumor growth, endothelial cells from adjacent vessels can locally degrade the fibrin matrix. This proteolytic event depends on the action of membrane-type matrix metalloproteinases, in particular MT1-MMP, and/or uPA and plasmin activities (Figure 3) 35,36,39 . After local degradation, the endothelial cells migrate into the matrix, proliferate and form elongated structures. This is followed by vessel stabilization and reconstitution of the basement membrane 39 . Fibrin provides specific

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adhesion sites for receptors expressed on angiogenic endothelial cells, such as the RGD- 48,49 dependent integrins αVβ3, αVβ5 and α5β1 . In addition, fibrin is a reservoir for storage and release of growth factors, such as VEGF and FGF-2, which initiate and guide angiogenesis 50 . The structure of fibrinogen was shown to be an important determinant of the formation of neovascular structures 21,26 . We previously demonstrated that heterogeneity variants of fibrinogen influence angiogenesis, high molecular weight (HMW) fibrinogen facilitated the in vitro tube formation and in vivo vessel formation better than low molecular weight (LMW) fibrinogen 25 . Furthermore, fibrin 51 fragments can influence angiogenesis. Alphastatin (A α1-24 ) was shown as anti-angiogenic agent , as is true for fragment E, which represents the plasmin cleavage product of the central E-domain 52 of fibrinogen . The fibrin fragments B β15-42 , D-dimers and γC-domains were able to increase vascular permeability and thereby extravasation of fluid and molecules 53-56 .

Figure 3. Fibrinolysis by MT1-MMP and plasmin. The proteolytic activities of membrane-type 1 matrix metalloproteinase and plasmin can remodel and degrade fibrin. A schematic overview of the activation, regulation and action of both pathways is given. Membrane-bound molecules are indicated in boxes attached to the endothelial cell. Abbreviations: plasminogen activator inhibitor (PAI), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR), membrane-type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of matrix metalloproteinase (TIMP).

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TISSUE ENGINEERING

The ideal scaffold for tissue engineering should be biocompatible and biodegradable, and provide sufficient and adequate interactions with various cell types. The tissue engineered scaffold should not initiate an immunological or ‘foreign body’ response in the patient. Moreover, it should provide temporary mechanical support that can withstand in vivo forces and maintain space for tissue development. The mechanical support should be maintained until the engineered tissue has sufficient mechanical integrity by itself. The engineered scaffold should be a provisional tissue, which facilitates in situ regeneration, thereby helping the body to heal.

Natural, synthetic and hybrid materials There are three categories of scaffold materials in tissue engineering: natural (biological) materials, synthetic materials and hybrid (semi-synthetic) materials 1,57 . Naturally occurring materials have physiological activities, such as cell adhesion, mechanical properties and biodegradability 1,3 . Moreover, cells can adhere and interact with them via integrins, followed by activation of signal transduction pathways. Natural materials can be degraded and replaced upon the generation of new tissues. Difficulties with natural materials are to obtain them with a continuous supply and immunological integrity, and to fully understand the cell-scaffold interactions. Well-investigated natural scaffolds are chitosan, , collagen, fibrin and combinations hereof 3,30,58 . In addition, decellularized tissues can be used as natural scaffolds in tissue engineering 58 . Notwithstanding, synthetic scaffolds can be relatively easy manipulated and the risk of viral infection is absent. They can be reproducibly manufactured on a large scale, with a variety of specific properties 3. Synthetic polymers, e.g. (PEG), polyurethane (PUR), polyglycolic acid (PGA), polylactic adid (PLA) and polyhydroxybutyrate (PH4B), provide initial mechanical support and their degradation can be controlled 5. However, unlike natural scaffolds the synthetic scaffolds do not contain biologically functional molecules and the scaffolds are usually not or only slowly degraded. Hybrid materials combine natural and synthetic materials, by the incorporation of functional molecules into a synthetic backbone. Thereby the specific activity of natural materials is combined with the advantages of synthetic materials3. This approach is especially of interest for engineering tissues that require high mechanical strain, for example in bone tissues 59 .

Fibrin scaffolds The natural scaffold fibrin plays important roles in hemostasis and wound healing, as described above. In addition, fibrin facilitates a good environment for migration and proliferation of cells, it serves as a reservoir of growth factors, and its degradation synergies with new tissue formation 30 . Fibrin can be obtained autologously or commercially. As hemostatic sealant it can be directly applied on the wound area, where high fibrin concentrations seal the wound and prevent further blood loss. The polymerization of fibrin is initiated immediately after thrombin addition, thereby 3D-shape molding is relatively easy. It is also possible to use fibrin as an injectable scaffold. Together, these properties are of great interest for the development of tissue engineering applications. However, one should appreciate the possible tissue response on fibrin fragments, since fibrin degradation products are known to induce an acute phase response. During

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degradation, fibrin fragments such as, fragment B β15-42 , γC-domains, alphastatin, fragment-E and D-dimers, might appear in the circulation or act locally 51-56 . Moreover, modifying fibrin can also alter its structure and functional properties, as was shown after incorporation of the chemotherapeutic agent cisplatin 60 .

Advantages of fibrin scaffolds Fibrin is known to stimulate regenerative and remodeling responses in various cell types 58 . These responses are important, since the induction of e.g. elastin and collagen synthesis, will lead to improvement of the mechanical strength of the scaffold. Collagen synthesis, organization and fibril alignment of various cell types is enhanced in fibrin scaffolds 32,61 , and fibroblasts were shown to produce collagen in response to TGF-β in a fibrin scaffold 47,62 . Moreover, smooth muscle cells synthesize more collagen when entrapped in fibrin as opposed to collagen type I scaffolds 63 . Furthermore, fibrin plays an important role in angiogenesis. This is advantageous for tissue engineering, since one of the major problems in engineered tissues is the limited blood supply. During wound healing the fibrin matrix naturally provides a suitable environment for angiogenesis and thereby recovery of the blood supply 9,41 . Changing the fibrin structure can result in altered angiogenesis, as was shown with the more porous, naturally occurring HMW-fibrinogen, which enhances blood vessel formation 25,37 . Addition of pro-angiogenic growth factors can stimulate the rate of angiogenesis 39,50 , whereby a controlled and slow release results in functional well-organized vascular structures 64 . This is in contrast to high local VEGF concentrations that induce the formation of supernumerary, malformed vessels65 . Other important factors controlling angiogenesis are delta like ligand 4 (DLL4) and hypoxia inducible factor 1 α (HIF-1α)66,67 . In addition, fibrin is also used in stem cell research. Due to its cell adhesive properties, biocompatibility and biodegradation fibrin can be used in the isolation and the delivery of stem cells. The isolation of rat bone marrow-derived mesenchymal stem cells was 3 to 4-fold increased when cross-linked fibrin microbeads were used, compared to conventional plastic adhesion 68 . Stem cells can be delivered to a site of interest, by incorporating them in fibrin during polymerization 5,69 . Human mesenchymal stem cell delivery was optimal using a fibrin matrix composed of high fibrinogen and thrombin concentrations70 . Murine embryonic stem cells growing in embryoid bodies differentiate optimal toward neural lineages inside relatively low fibrinogen and thrombin concentrations 71 . The importance of optimization not only counts for stem cells, it is important for each cell type of personal interest.

Fibrin combined with synthetic materials Hybrid materials regulate the physical and biological cell signaling abilities of the scaffold and these materials have high mechanical strength. Combinations of biodegradable polyethylene glycol (PEG) and fibrinogen (PEGylated fibrin) can be used for smooth muscle, , cardiac, endothelial 72 and embryonic stem cell culturing 1. As a provisional scaffold, PEGylated fibrinogen functions as a bio-mimetic fibrin clot regulating the cell invasion, and maintains its stability in vivo for several months 1,72 . PEGylated fibrin was used for mesenchymal stem cell delivery onto injured myocardium and facilitated the regeneration of infarcted myocardium 73 . Moreover, PEGylated fibrinogen, was shown to be highly efficient in promoting bone defect healing 74 . In cartilage tissue

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engineering poly(lactic-co -glycolic acid) synthetic sponges embedded with fibrin increased the loading efficiency of bone marrow-derived mesenchymal stem cells. Together with a TGF-β1 containing DNA-vector this resulted in well-healing cartilage defects 75 .

Composite scaffolds containing fibrin As fibrin scaffolds provide a relatively low mechanical strength and fast degradation, these problems can be overcome by combining different natural scaffolds, also called composite scaffolds. A well-known composite scaffold is the combination of fibrin and fibronectin. When these mixed proteins are used as coating of polymeric surfaces, degradation of fibrin occurred together with deposition of elastin, collage type I and IV 62 . Composite scaffolds made of fibrin, fibronectin, gelatin and growth factors were shown to maintain a steady cell proliferation, prolonged cell survival and prevented dedifferentation of endothelial cells in vitro 76 . Moreover, unlike gelatin-coated polystyrene, these composite coatings did not increase the generation of thrombogenic molecules 76 .

Applications of fibrin in tissue engineering Wound sealing Fibrin sealants mimic the final stages of natural coagulation, forming a stable fibrin clot that enhances wound closure 9,77 . The FDA approved two commercial fibrin sealants Tisseel VH (Baxter Immuno, Vienna, Austria) and Hemaseel APR (Haemacure Corporation, Sarasota, USA). These fibrin sealants are non-autologous and consist of high concentrations fibrinogen and often contain additional components, including factor XIII, aprotinin and tranexamic acid 77 . Autologously obtained fibrin sealants are under investigation 78 . Fibrin sealants have been found useful in clinical settings for hemostatic and surgical sealing applications. Successful applications of fibrin sealants in cardiovascular, gastrointestinal, pneumothoracic and neurosurgery were shown 79 . For further details, the reader is directed reviews from Amrani et al. 33 and Spotnitz et al. 77 .

Skin tissue engineering Tissue engineered skin replacements might result in a successful application for full-thickness burns and chronic wounds 80 . For this application, keratinocytes are often encapsulated within fibrin scaffolds 81 . The fibrin-encapsulated keratinocytes showed increased production of PDGF- BB and TGF-β182 . Combining various skin cell types leads to a self-organization within the scaffold, wherein the architecture of the natural dermis is mimicked. Co-cultures of keratinocytes and dermal fibroblasts, showed migration of fibroblasts into the fibrin scaffold in 2-3 days, whereas keratinocytes did not 83 . Moreover, fibrin facilitated a good re-epithelialization of skin equivalents 84 . Although further investigation is necessary, fibrin scaffolds might provide a suitable approach in skin tissue engineering.

Vascular tissue engineering Synthetic materials have been successfully applied in many large-vessel reconstructions. However, small-caliber vascular conduits show high rates of thrombosis 85,86 . In tissue engineering of vascular grafts, the inside of a vascular construct should be non-thrombotic and ultimately have

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a well-orientated inner endothelial cell layer. Fibrin has been identified as the most non- thrombogenic adhesive protein with respect to platelet consumption and activation 69,87 . Tissue engineered vascular grafts of synthetic materials (Dacron ® or polytetrafluoroethylene (PTFE)) coated with fibrin might be a possibility for the future 58,69,88 . Fibrin holds opportunities, due to its controllable degradation and the stimulation of extracellular matrix remodeling. Fibrin was shown as natural scaffold for endothelialization, and enhanced endothelial cell attachment and resistance to endothelial cell loss by shear stress in fibrin coated PTFE vascular grafts 89 . Moreover, endothelial cells on fibrin scaffolds tend to orientate towards the direction of flow, without significant cell loss 90 . Reviews on this topic have been published by Stegemann et al. 58 and Shaikh et al. 69 .

Heart valve replacement Heart valves fully composed of fibrin are currently being developed, fibrin and vascular cells together with bioreactor culturing, almost mimics the natural aortic heart valves. However, the valve closing properties are not sufficient due to tissue shrinkage 91 . Dynamic straining and pulsating flow during bioreactor culturing increased extracellular matrix formation, and optimal mechanical and dynamic strain were obtained 92 . The tissue engineered human heart valve leaflets as described by Mol et al., which are mainly composed of degradable non-woven polyglycolic acid (PGA) meshes, coated with poly-4-hydroxybutyrate (P4HB), do not have this shrinkage problem. Cell seeding was performed using fibrin as myofibroblast cell carrier, thereby a non-porous tissue structure was obtained and cells were stimulated to produce collagen and elastin 46 .

Biomolecule and drug delivery Injection or systemic delivery of biomolecules or drugs often leads to rapid clearance 41,64 . Therefore, the spatio-temporal controlled delivery of these molecules within fibrin scaffolds is a progressing field of research. Fibrin scaffolds, sheets, microparticles and fibrin-coated drug particles have been used as delivery systems 30,93 . Growth factors (e.g. VEGF and TGF-β1) can naturally bind to the fibrin scaffold and subsequent degradation of the scaffold can release these growth factors 5,94,95 . Weakly binding proteins can be released simply by diffusion. Moreover, incorporation of heparin within the fibrin scaffold protects growth factors 93 and binds additional proteins, such as neurotropin-3 and 30,96 . A gradual release of growth factors by tissue specific degradation was shown by TGF-β1 incorporation in fibrin, in which approximately 50% of the amount TGF-β1 was released after 10 94 days . Immobilization of VEGF 165 and FGF-2 in fibrin, using the arteriovenous loop model in rats, resulted in a dose-dependent increase of angiogenesis 95 . Moreover, the angiogenesis or endothelialization can be stimulated by FXIIIa dependent incorporation of α2-antiplasmin- 41 VEGF 121 . Naturally, nerve generation takes place in fibrin 96 . Incorporation of bi-domain peptides, composed of -1 or N-cadherin domains with a FXIII domain, resulted in vivo in 85% more and 75% longer myelinated axons 97 . In vitro , an enhancement of neurite extension was also seen

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using PC12 cells in fibrin scaffolds with these bi-domain peptides; here the cross-link density of fibrin played an important role 98 . Another promising field of research is drug release from fibrin scaffolds. The chemotherapeutic drugs, carboplatin, embedded in fibrin remained cytotoxic on retinoblastoma cells 60 . However, by creating a drug-releasing fibrin scaffold, the possible influence of the drugs on fibrin polymerization and structure should be taken into account.

CONCLUSIONS

In this review an overview of the fibrin structure, its biological functions and the applications for tissue engineering has been given. Fibrin naturally forms hemostatic plugs and provisional matrices facilitating tissue repair. Furthermore, it can be used for numerous tissue engineering applications. Interestingly, angiogenesis is well facilitated by fibrin, and the formation of mature extracellular matrix is stimulated. The biodegradability of fibrin is an advantage for the release of biomolecules, drugs and cells. However, addition of these molecules to fibrin scaffolds can alter their structure and functional properties. The fast degradation and low mechanical stiffness of fibrin might be a disadvantage in some applications. Furthermore, one should take account of the possible tissue response of fibrin fragments, which may accumulate locally or appear in the circulation.

ACKNOWLEDGEMENTS

This research was supported by the Dutch Program for Tissue Engineering (DPTE) and The Netherlands Initiative for Regenerative Medicine (NIRM).

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39 Chapter 2

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