SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. GENERAL OVERVIEW
  4. EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING
  5. NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING
  6. CONCLUSIONS
  7. LITERATURE CITED

In this review, we focused our attention on the more important natural extracellular matrix (ECM) molecules (collagen and fibrin), employed as cellular scaffolds for tissue engineering and on a class of semi-synthetic materials made from the fusion of specific oligopeptide sequences, showing biological activities, with synthetic materials. In particular, these new “intelligent” scaffolds may contain oligopeptide cleaving sequences specific for matrix metalloproteinases (MMPs), integrin binding domains, growth factors, anti-thrombin sequences, plasmin degradation sites, and morphogenetic proteins. The aim was to confer to these new “intelligent” semi-synthetic biomaterials, the advantages offered by both the synthetic materials (processability, mechanical strength) and by the natural materials (specific cell recognition, cellular invasion, and the ability to supply differentiation/proliferation signals). Due to their characteristics, these semi-synthetic biomaterials represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration. © 2005 Wiley-Liss, Inc.


GENERAL OVERVIEW

  1. Top of page
  2. Abstract
  3. GENERAL OVERVIEW
  4. EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING
  5. NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING
  6. CONCLUSIONS
  7. LITERATURE CITED

Tissue engineering is an emerging interdisciplinary field in biomedical engineering and aims at regenerating new biological tissue for replacing diseased or devastated tissues by using cells (Patrick et al., 1998). Formation of new biological tissues by tissue engineering can be achieved both in vitro and in vivo. Tissue engineering generally requires an artificial extracellular matrix (ECM) for tissue regeneration, because cell proliferation and differentiation, resulting in tissue regeneration, would be difficult unless such a matrix is provided that functions as a cell scaffold. Since this artificial ECM should disappear through absorption into the body when the new tissue is regenerated, materials for the matrix should be prepared from biodegradable polymers. This requirement, as well as adequate cell adhesion onto the matrix surface, make biological materials attractive in tissue engineering.

Unlike ECM that represent the result of a millenary natural evolution, artificial biomaterials do not have such a complex structure and chemical composition. In consequence of this, the information content of man-made devices is very low and the quantity of signals they can transmit to the cells is scarce. Bearing these basic considerations in mind our efforts in trying in vitro tissue reconstruction must be driven toward the exact knowledge of cell function on one hand, and, on the other hand, toward the knowledge of interactions and signals that cells must receive from the environment to behave as in natural tissues. We know that ECM plays an instructive role for cellular activities and that cells possess on their surface receptors to respond to the extracellular signals. As soon as ligand-receptor interaction is established, the biochemical machinery involved in the control of gene expression starts.

EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING

  1. Top of page
  2. Abstract
  3. GENERAL OVERVIEW
  4. EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING
  5. NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING
  6. CONCLUSIONS
  7. LITERATURE CITED

Tissue engineering typically employs exogenous three-dimensional ECMs to engineer new natural tissues from isolated cells. One approach to designing exogenous ECMs for tissue engineering is to mimic the functions of the ECM molecules naturally found in tissues (Fouser et al., 1991).

The synthetic ECM should provide temporary mechanical support sufficient to withstand in vivo forces and maintain a potential space for tissue development. This mechanical support by the synthetic ECM should be maintained until the engineered tissue has sufficient mechanical integrity to support itself. The cells comprising the engineered tissue must express appropriate genes to maintain the tissue-specific function of the engineered tissue. The function of seeded cell is strongly dependent on the specific cell-surface receptor (e.g., integrins) used by the cell to interact with the material, on interactions with surrounding cells (Parsons-Wingerter and Saltzman, 1993), and on the presence of soluble growth factors (Deuel, 1997). These factors can be controlled by incorporating or integrating a variety of signals, such as cell-adhesion peptides (Hubbell, 1995) and growth factors (Mooney et al., 1996), into the synthetic ECM, or by subjecting it to mechanical stimuli (Banes, 1993).

The exogenous ECMs for tissue engineering can be fabricated from two classes of biomaterials: naturally derived materials and synthetic materials. Naturally occurring materials are composed of polypeptides, polysaccharides, nucleic acids, hydroxyapatites, or their composites. Biological materials have some remarkable advantages over synthetics, that is, their excellent physiological activities such as selective cell adhesion (e.g., collagen and fibrin), mechanical properties similar to natural tissues (e.g., animal heart valves and blood vessels), and biodegradability (e.g., gelatine and chitin). However, as with synthetics, biological materials have several deficiencies including risk of viral infection, antigenicity, unstable material supply, and deterioration, which accompanies long-term implantation. In addition, naturally derived materials offer limited versatility in designing an exogenous ECM with specific properties (e.g., porosity, mechanical strength). Synthetic materials, by contrast, can be manufactured reproducibly on a large scale, and can also be processed into an exogenous ECM in which the macrostructure, mechanical properties, and degradation time can be readily controlled and manipulated. Exogenous ECMs fabricated by biodegradable polymers will eventually erode in the body, avoiding a chronic foreign-body response.

The greatest disadvantage of synthetic materials, however, is the lack of cell-recognition signals. Toward this end, efforts are being made to incorporate cell-adhesion peptide into biomaterials, which normally exhibit few cellular interactions. Mechanical signals conveyed to cells via their adhesion to the matrix also clearly regulate the development of various tissues and the gene expression of many cell types in culture.

The concept of combining synthetic materials with cell-recognition sites of naturally derived biomaterials is very attractive. These hybrid materials could possess the favorable properties of synthetic materials, including widely varying mechanical and degradative properties, reproducible large-scale production, and good processability, as well as the specific biological activity of naturally derived materials. This last property may be needed to engineer complex tissue with multiple cell types organized in specific patterns. Several cell-adhesion ligands with highly specific recognition could potentially be displayed spatially in a desirable pattern to induce specific cell-organization schemes.

Naturally derived biomaterials for tissue engineering

Cells adhere and interact with their extracellular environment via integrins, and their ability to activate associated downstream signaling pathways depends on the character of adhesion complexes formed between cells and their ECM.

The main naturally derived biomaterials used as scaffolding for tissue engineering are of varying chemical nature. They comprise polypeptides, polysaccharides, polyesters, and inorganic materials. A mammalian body has different kinds of polypeptides including plasma, structural, and functional proteins. The majority of proteins used as biomaterials originate from blood plasmas and structural skeletons. Functional proteins such as enzymes, cell growth factors, and interleukins are also used, but are mostly incorporated into biomaterials as ingredients. Here we have focused our attention on the most frequently used natural scaffolds for tissue engineering, such as fibronectin and collagen.

Fibronectin

Fibronectin is a multifunctional component of the ECM. Intracellular signaling induced by cell adhesion on fibronectin plays a critical role in cytoskeletal organization, cell cycle progression, and cell survival (Hynes, 1990; Frisch and Ruoslahti, 1997). Cells assemble fibronectin into a fibrillar form that accumulates at their apical surface. Fibronectin matrix formation is initiated by fibronectin binding to cell surface receptors, followed by assembly and reorganization of the cell surface–associated fibronectin into fibrils (McDonald, 1988; Mosher et al., 1992; Mosher, 1993). The α5β1 integrin is the major receptor responsible for fibronectin matrix assembly (Ruoslahti, 1991; Wu et al., 1993). The αvβ3 integrin can also direct matrix assembly (Wennerberg et al., 1996; Wu et al., 1996), which may account for fibronectin matrix formation in α5 integrin null mice (Yang et al., 1993). A number of other integrins bind to fibronectin, but do not initiate fibril formation (Busk et al., 1992; Zhang et al., 1993; Wu et al., 1995). The first type III repeat of the fibronectin molecule is important in promoting matrix assembly (Morla and Ruoslahti, 1992; Aguirre et al., 1994; Hocking et al., 1994). Small fragments derived from this III1 module induce fibronectin polymerization at moderate concentrations, but inhibit it at high concentrations (Morla and Ruoslahti, 1992; Morla et al., 1994). Interaction of integrins with the actin cytoskeleton is also essential for matrix assembly (Hynes, 1990).

Bourdoulous et al. studied the role of the fibronectin matrix in cytoskeletal regulation by disassembling the fibronectin matrix with a 76-amino acid fragment derived from the first type III repeat of fibronectin (Morla and Ruoslahti, 1992; Morla et al., 1994), or by preventing matrix assembly with integrin antibodies. Interestingly, they found that removal of fibronectin matrix without altering the cell-substrate adhesion of the cells decreased the basal activity of ERK while slightly potentiating ERK response to growth factors. This effect and the activation of P38 MAPK and suppression of JNK in III1-C-treated cells showed that the effects of matrix removal on MAPKs are quite different from those of loss of substrate adhesion. In agreement with this, depleting fibronectin matrix had little effect on cell survival, while inhibiting cell proliferation. Thus, signals from fibronectin matrix seem to control cell proliferation, whereas cell substrate adhesion provides a survival signal. These signals can be modulated separately by removing the matrix or by allowing cells to attach to a substrate in the absence of matrix.

In summary, these results revealed a specialized role for cell surface fibronectin matrix in cytoskeletal organization, growth factor responses, and cell cycle control that cannot be substituted for by cell adhesion to a substrate.

Furthermore, it is known that α5β1 and αvβ3 integrins are central to regulating downstream events, including cell survival and cell-cycle progression. In contrast to previous findings that αvβ3 integrins promote angiogenesis (Bourdoulous et al., 1998), recent evidence argues that αvβ3 integrins may act as negative regulators of proangiogenic integrins such as α5β1. This suggests that fibronectin is critical for scaffold vascularization because it is the only mammalian adhesion protein that binds and activates α5β1 integrins. Cells are furthermore capable of stretching fibronectin matrices such that the protein partially unfolds, and recent computational simulations provide structural models of how mechanical stretching affects fibronectin function. Vogel and Baneyx (2003) recently proposed a model, whereby excessive tension generated by cells in contact with biomaterials may in fact render fibronectin fibrils non-angiogenic and may potentially inhibit vascularization. The model could explain why current biomaterials fail to vascularize, independent of their surface chemistries and textures.

In addition, other factors seem to modulate fibronectin fibril structure and thereby affect angiogenesis. Hall et al. (2001) showed that the molecular properties of fibrin-based matrices, such as fibrillar structure and covalent modifications with adhesion domains, influence the angiogenic behavior of human umbilical vein endothelial cells (HUVECs) in vitro. The fibrillar structure of fibrin-based matrices was influenced by pH but not by covalent incorporation of exogenous adhesion domains. Native fibrin-based matrices polymerized at pH 10 formed organized and longitudinally oriented fibrin fibrils, which provided a good angiogenic substrate for endothelial cells. Furthermore, upon covalent incorporation of the model ligand L1Ig6, which binds to the integrin most prominently expressed on the surface of angiogenic endothelial cells, alpha(v)beta3, these matrices became angiogenesis-promoting when polymerized at physiological pH. Most important, L1Ig6-modified matrices were very specific in inducing the angiogenic phenotype of HUVECs, whereas control cells did not differentiate on these matrices. These results indicate that artificial ECMs can influence cell behavior in two ways. One way is based on the three-dimensional fibril structure of the matrix molecules themselves, and the other provides specific binding sites for direct cell-matrix interactions that lead to the activation of second-messenger cascades, thus promoting angiogenic differentiation.

Moreover, starting from the observation that currently used biodegradable scaffolds in cardiovascular tissue engineering show toxic degradation and inflammatory reactions and are potentially immunogenic, Ye et al. (2000) proposed the use of a three-dimensional fibrin gel scaffold for vessel tissue engineering. In their experiments, human aortic tissue was harvested from the ascending aorta in the operation room and worked up to pure human myofibroblast cultures. These human myofibroblast cultures were suspended in fibrinogen solution and seeded into 6-well culture plates for cell development for 4 weeks and supplemented with different concentrations of aprotinin. Hydroxyproline assay and histological studies were performed to evaluate the tissue development in these fibrin gel structures. The light microscopy and the transmission electron microscopy studies for tissue development based on the three-dimensional fibrin gel structures showed homogenous cell growth and confluent collagen production. No toxic degradation or inflammatory reactions could be detected. Furthermore, fibrin gel myofibroblast structures dissolved within 2 days in medium without aprotinin, but medium supplemented with higher concentration of aprotinin retained the three-dimensional structure and had a higher collagen content and a better tissue development. They concluded that a three-dimensional fibrin gel structure could serve as a useful scaffold for tissue engineering with controlled degradation, excellent seeding effects, and good tissue development.

Collagen

Collagens are ubiquitous proteins responsible for maintaining the structural integrity of vertebrates and many other organisms (Myllyharju and Kivirikko, 2001). More than 20 genetically distinct collagens have been identified (Hulmes, 1992, 2002; Kadler et al., 1996; Ottani et al., 2001). In tissues that have to resist shear, tensile, or pressure forces, such as tendons, bone, cartilage, and skin, collagen is arranged in fibrils, with a characteristic 67 nm axial periodicity, which provides the tensile strength. Only collagen types I, II, III, V, and XI self-assemble into fibrils. The fibrils are composed of collagen molecules, which consist of a triple helix of approximately 300 nm in length and 1.5 nm in diameter. Collagen fibril formation is an extracellular process, which occurs through the cleavage of terminal procollagen peptides by specific procollagen metalloproteinases.

Some collagens form networks (types IV, VIII, and X), a typical example of which is the basement membrane, mostly made of collagen IV. Other collagens associate with fibril surfaces (types VI, IX, XII, and XIV). Yet other collagens are transmembranous proteins (types XIII and XVIII) or form periodic beaded structures (type VI).

Type I collagen occurs throughout the body, except in cartilage. It is the principal collagen in the dermis, fasciae, and tendons and is a major component of mature scar tissue. Type II collagen occurs in cartilage, the developing cornea, and in the vitreous body of the eye. Type III collagen dominates in the wall of blood vessels and hollow intestinal organs and co-polymerizes with type I collagen. Types V and XI collagen are minor components and occur predominantly co-polymerized with collagen I (type V) and collagen II (type XI).

Collagens are mostly synthesized by the cells comprising the ECM: fibroblasts, myofibroblasts, osteoblasts, and chondrocytes. Some collagens are also synthesized by adjacent parenchymal or covering (epithelial, endothelial, and mesothelial) cells. A typical example is type IV collagen, which is synthesized in a cooperative effort between the stromal cell and the parenchymal/covering cell.

Collagen is the most abundant protein in animals and because of its high mechanical strength and good resistance to degradation, it has been utilized in a wide range of products in industry (Ulrich et al., 1992), while its low antigenicity has resulted in its widespread use in medicine (Ramshaw et al., 2000). Collagen products can be purified from fibers, from molecules reconstituted as fibers, or from specific recombinant polypeptides with preferred properties. A feature common to all these biomaterials is the need for stable chemical cross-linking to control the mechanical properties and the residence time in the body, and to some extent the immunogenicity of the device. This can be achieved by a number of different cross-linking agents that react with specific amino acid residues on the collagen molecule imparting individual biochemical, thermal, and mechanical characteristics to the biomaterial (Silver et al., 1995).

For these reasons, collagen is currently used for tissue engineering. For example, Hudon et al. (2003) produced a new model of endothelialized reconstructed dermis that promotes the spontaneous formation of a human capillary-like network. The endothelialized dermis was prepared by co-culturing two human cell types, dermal fibroblasts and umbilical vein endothelial cells, in a collagen sponge biomaterial. Thereafter, they strove to study, quantitatively and qualitatively, the influence of angiogenic and angiostatic drugs on capillary-like tube (CLT) formation in vitro in the model. The visualization by confocal microscopy of the tubes present in the model showed that the endothelial structures were not cord-like but rather CLTs with well-defined lumina. Moreover, these tubes were organized in a complex network of branching structures. When angiogenic factors (vascular endothelial growth factor (VEGF) 10 ng/ml or basic fibroblast growth factor 10 ng/ml) were added to the model, 1.8 and 1.4 times more capillaries were observed, respectively, whereas the addition of progesterone (10 μg/ml) reduced by 2.4 times the number of tubes compared with the control. These results suggest that this model is a highly efficient assay for the screening of potentially angiogenic and angiostatic compounds.

Another example of collagen as scaffold for dermal tissue engineering came from recent studies by Guerret et al. (2003). They studied Apligraf, a bioengineered living skin, composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the ECM components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. They observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, that is, type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is, thus, very useful for studying long-term tissue remodeling of bioengineered living tissues.

Jux et al. (2003) used intestinal collagen layer (ICL), a highly purified (acellular) bioengineered type-I collagen derived from porcine submucosa, as septal occluder to replace a commercial occluder (CardioSeal) in percutaneous transcatheter closure of interventionally created atrial septal defects in lambs. A complete pathomorphological follow-up investigation including histology was carried out after 2, 4, and 12 weeks. Standard CardioSEAL implants served as a control group. After 2 weeks in vivo the devices were already covered completely by neo-endothelium. Compared with the conventional synthetic scaffold, ICL devices showed a quicker endothelialization, decreased thrombogenicity, and superior biocompatibility with no significant cellular infiltration observed in the histology of explants with ICL fabrics. After 3 months in vivo the collagen layer remained mechanically intact, but began to show the first histological signs of mild disintegration, gradual reabsorption, and remodeling. In conclusion, short-term results from preliminary in vivo experiments using a bioengineered collagen matrix as the occluder tissue scaffold showed excellent biocompatibility. This resulted in superior overall results: quicker endothelialization, decreased thrombogenicity, and decreased immunological host response. Another recent clinical application of collagen was provided by Sculean et al. (2003). They compared, clinically, the treatment of deep intrabony defects with a combination of a bovine-derived xenograft (BDX) and a bioresorbable collagen membrane to access flap surgery.

Twenty-eight patients suffering from chronic periodontitis, each of whom displayed one intrabony defect, were randomly treated with BDX  + collagen membrane (test) or with access flap surgery (control). Soft tissue measurements were made at baseline and at 1 year following therapy. At 1 year after therapy, the test group showed a reduction in mean probing depth (PD) from 9.2 ± 1.3 to 3.9 ± 0.7 mm and a change in mean clinical attachment level (CAL) from 10.2 ± 1.5 to 6.2 ± 0.5 mm. In the control group, the mean PD was reduced from 9.0 ± 1.2 to 5.2 ± 1.8 mm and the mean CAL changed from 10.5 ± 1.5 to 8.4 ± 2.1 mm. The test treatment resulted in statistically higher PD reductions and CAL gains than the control one. In the test group all sites (100%) gained at least 3 mm of CAL. In the control group no CAL gain occurred in four sites (29%), whereas at six sites (43%) the CAL gain was 2 mm. A CAL gain of 3 mm or more was measured in four defects (29%). From these studies, it can be concluded that at 1 year after surgery both therapies resulted in significant PD reductions and CAL gains, and treatment with BDX + collagen membrane resulted in significantly higher CAL gains than treatment with access flap surgery.

While these examples offer encouraging applications of collagen in tissue engineering, limits in its use arise from its low mechanical strength and a fast biodegradation when implanted in the human body. Thus, more research is needed to improve mechanical strength and to enhance the time of permanence in the human body.

NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING

  1. Top of page
  2. Abstract
  3. GENERAL OVERVIEW
  4. EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING
  5. NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING
  6. CONCLUSIONS
  7. LITERATURE CITED

Advances in the field of tissue engineering are connected to the performance of biomaterials that help in guiding tissue formation or regeneration. Design principles in biomaterials have typically been influenced by the function of the ECM. Because the ECM has been shown to play a key role in signal transduction (Howe et al., 1998; Schwartz and Baron, 1999; Streuli, 1999), the development of materials that can specifically and molecularly interact with cells has become an emerging area of research (Hubbell, 1999; Griffith, 2002). The regulation of cell behavior through receptor-mediated adhesion [e.g., by functionalizing materials with integrin-binding oligopeptides (Ruoslahti, 1996; Hubbell, 2000) and by growth factors (Whitaker et al., 2001) has thus been targeted.

Many researchers have been focusing on the development of synthetic materials that are targeted to assist tissue regeneration (Mann et al., 2001; Pratt and Hubbell, 2001; Halstenberg et al., 2002). Placed at the site of a defect, such materials should actively and temporarily participate in the regeneration process by providing a platform on which cell-triggered remodeling could occur. Consequently, these matrices must display some key characteristics of the provisional ECM.

For example, one of the critical initial functions of the fibrin-rich network that fills tissue defects after trauma, almost regardless of the injury site, lies in its ability to foster the invasion of inflammatory cells. These cells then initiate the remodeling process by partially degrading the matrix and by secreting molecular signals for attraction and differentiation of other cell types such as fibroblasts that build up new ECM (Clark, 1996). Because the provisional ECM often presents itself in such situations as a biophysical barrier to these cells, invasion and remodeling depend on the action of cell-secreted proteases enabling cell migration by clearing a path (Werb, 1997; Murphy and Gavrilovic, 1999). Thereby, matrix metalloproteinases (MMPs) have been implicated as key players (Woessner and Nagase, 2000; Sternlicht and Werb, 2001). Secreted as inactive proenzymes and activated near the cell surface or expressed at the surface in activated form as membrane-anchored MMPs (MT-MMP), these enzymes can cleave virtually all constituents of the ECM at specific sites. Besides their role in migration, they affect other cell functions such as proliferation and apoptosis (Vu and Werb, 2000).

Lutolf et al. (2003a) developed synthetic materials that can assist tissue regeneration by mimicking the MMP-mediated invasion of the natural provisional matrix. This was accomplished by cross-linking linear oligopeptide substrates for MMPs into 3D networks on reaction with multiarm endfunctionalized poly(ethylene glycol) (PEG) macromers (Lutolf and Hubbell, 2003) together with integrin-binding domains (here, RGDSP) attached in a pendant fashion to hydrogels. In this way, cells were anticipated to be able to migrate within these networks by MMP- and integrin-dependent mechanisms.

In a successive study, Lutolf et al. (2003b) used the same PEG-based hydrogels to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical-sized defects in rat crania. They showed that these defects were completely infiltrated by cells and were remodeled into bony tissue within 5 weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration. In the example of a critical size defect model of rat calvaria bone, the authors showed that material characteristics can be exploited in conducting bone regeneration. Primary human fibroblasts were shown to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network cross-linking density.

To function effectively as a cell-ingrowth matrix in vivo, the degradation behavior by MMPs and the cell invasion behavior of these artificial provisional matrices, respectively, must be well understood and optimized. In these two studies, the authors examined how the molecular composition of the material (variables: adhesiveness, sensitivity to MMPs, and network architecture) influenced its 3D cellular invasion. By using both in vitro and in vivo assays, they demonstrate that the proteolytic degradation of these hydrogel networks can be rationally controlled on multiple levels.

Degradation of gels was engineered starting from a characterization of the degradation kinetics (k(cat) and K(m)) of synthetic MMP substrates in the soluble form and after cross-linking into a 3D hydrogel network.

In conclusion, these semi-synthetic hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring ECM-derived materials such as fibrin or collagen, which require difficult purification procedures and carry the risks of immunogenicity and disease transmission.

Halstenberg et al. (2002) synthesized a protein-graft-PEG hydrogel, starting from an artificial protein, created by recombinant DNA methods, and modified by grafting of PEG diacrylate. The artificial protein contained repeating amino acid sequences based on fibrinogen and anti-thrombin III, comprising an RGD integrin-binding motif, two plasmin degradation sites, and a heparin-binding site. Adhesion studies showed that the artificial protein had specific integrin-binding capability based on the RGD motif contained in its fibrinogen-based sequence. Furthermore, heparin bound strongly to the protein's anti-thrombin III-based region. Protein-graft-PEG hydrogels were plasmin degradable, had Young's moduli up to 3.5 kPa, and supported three-dimensional outgrowth of human fibroblasts. Cell attachment in three dimensions resulted from specific cell-surface integrin binding to the material's RGD sequence. Hydrogel penetration by cells involved serine-protease mediated matrix degradation in temporal and spatial synchrony with cellular outgrowth.

Due to their characteristics, protein-graft-PEG hydrogels represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration. Zisch et al. (2003) synthesized a new class of bioactive synthetic hydrogel matrices based on PEG and synthetic peptides that exploits the activity of vascular endothelial growth factor alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking MMP substrate peptides. By using a Michael-type addition reaction, they incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. GENERAL OVERVIEW
  4. EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING
  5. NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING
  6. CONCLUSIONS
  7. LITERATURE CITED

In the first part of this review, we focused our attention on the more important natural ECM molecules (collagen and fibrin) employed as scaffold for tissue engineering. Their biological properties make these biomaterials currently employed in clinic applications for the regeneration of same damaged tissues and make them still interesting in the research area of tissue engineering. But the low mechanical properties, the risk of viral infection, the antigenicity, the unstability of materials, and deterioration, which accompanies long-term implantation limiting the clinical applications of these natural biomaterials.

In the second part, we focused our attention on the development of a new semi-synthetic biomaterials for tissue engineering, that represent a new attractive challenge for the industrial and institutional research in this field.

In particular, the ability to supply molecular signals able to guide the cellular response on these new semi-synthetic biomaterials represent a tremendous innovation in the area of tissue engineering.

For example, the insertion of metalloproteinases oligopeptide sequences and integrin-binding peptide in a PEG followed by physical entrappment of recombinant human bone morphogenetic protein-2 confer to this hydrogel the ability to be colonized by cells in function of their invasivness. Moreover, while cells adhere and colonised the semi-synthetic matrix througt the action of metalloproteinases enzymes, at the same time, the enzymatic degradation of polymeric scaffold release bone morphgenetic protein-2 that induce new bone formation (Fig. 1).

thumbnail image

Figure 1. Sequencial diagram of modified PEG hydrogel-cells interactions and new bone formation. Part A: cells adhesion on modified PEG hydrogel via integrin receptors and matrix metallo-proteinases (MMPs) production. Part B: MMPs remodeling of PEG modified hydrogel with bone morphogenetic protein-2 (BMP-2) local release and new bone formation.

Download figure to PowerPoint

Validity of the experimental approach of in vitro et in vivo tissue engineering of soft tissues is exaustively demonstrates by these works (Halstenberg et al., 2002; Zisch et al., 2003; Lutolf et al., 2003a,b).

Future research aiming to the design and synthesis of composite materials made from the fusion of these kind of semisynthetic “intelligent” biomaterials with other biomaterials able to confer high mechanical characteristics to the final composite, will represent a valid strategy to approach in vivo et in vitro engineering of hard tissues (i.e., bone tissue).

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. GENERAL OVERVIEW
  4. EXTRACELLULAR MATRIX FOR TISSUE ENGINEERING
  5. NEW SEMI-SYNTHETIC SMART MATERIALS FOR TISSUE ENGINEERING
  6. CONCLUSIONS
  7. LITERATURE CITED