Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.     

Attachment of glycosaminoglycans to collagenous matrices modulates the tissue response in rats

Biocompatibility and tissue regenerating capacity are essential characteristics in the design of collagenous biomaterials for tissue engineering. Attachment of glycosaminoglycans (GAGs) to collagen may add to these characteristics by creating an appropriate micro-environment. In this study, porous type I collagen matrices were crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide, in the presence and absence of chondroitin sulfate and heparan sulfate. The tissue response to these matrices was evaluated after subcutaneous implantation in rats. Biocompatibility of the matrices was established by the induction of a transitional inflammatory response, and the generation of new host tissue. Non-crosslinked collagen was gradually resorbed and replaced by collagenous connective tissue. By contrast, crosslinked matrices, with and without GAGs. retained their scaffold integrity during implantation, and supported the interstitial deposition and organization of extracellular matrix. In addition, crosslinking decreased tissue reactions at late time intervals. No calcification in any of the implants was observed. The presence of GAGs preserved porous lamellar matrix structures. Heparan sulfate in particular promoted angiogenesis at weeks 2 and 4, predominantly at the matrix periphery. The almost complete absence of macrophages and giant cells associated with collagen-GAG matrices, after 10 weeks implantation, indicated a reduced foreign body reaction. It is concluded that attachment of GAGs to collagen matrices modulates the tissue response. The potential of these biocompatible scaffolds for tissue engineering is increased by preserving porous matrix integrity. promoting angiogenesis and reducing foreign body reactions.     

Biomechanical properties of carbodiimide crosslinked collagen: influence of the formation of ester crosslinks

There is a growing interest in the use of collagen matrices for tissue engineering. To prevent rapid degradation and to improve their mechanical properties, collagen matrices have been modified using different crosslinking agents. Among the different agents used, water soluble carbodiimides (such as N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide, EDC) in combination with N-hydroxysuccinimide (NHS) are attractive systems, because no additional chemical entities are incorporated in the matrix. EDC/NHS crosslinking leads to amide bond formation between activated carboxyl groups and amine groups. Recently, we proposed that in addition to amide bond formation, ester links are also formed between activated carboxyl groups and hydroxyl groups. This was based on observations we made after development of a new method to quantify concentrations of carboxyl groups of collagen materials before and after crosslinking. The current study is directed to the influence of ester bond crosslinks formed after crosslinking of collagen with EDC/NHS on its physical-chemical and biomechanical properties. Reconstituted dermal bovine collagen patches (RDBC) were used as model material and were crosslinked with EDC/NHS. In one RDBC group, collagen amine groups were blocked with propionaldehyde prior to crosslinking, while in the other group unprocessed RDBC was crosslinked without additional matrix modifications. It was shown that after activation of collagen carboxyl groups with EDC and NHS, amide crosslinks as well as ester crosslinks with collagen hydroxyl groups were formed. It was furthermore demonstrated that the ester crosslinks of EDC/NHS-crosslinked RDBC could be removed by mild hydrolysis affording collagen matrices with improved mechanical properties.     

First steps towards tissue engineering of small-diameter blood vessels: preparation of flat scaffolds of collagen and elastin by means of freeze drying

Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.     

Glycosaminoglycan-targeted fixation for improved bioprosthetic heart valve stabilization

Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO(4)) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG-degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue.     

Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves

Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.     

The modification of scaffold material in building artificial dermis

Type X collagen is principal extracellular matrix (ECM) in natural dermis. To prepare artificial dermis, collagen is traditional, and most superior biomaterial. But beside collagen, the dermis also contains many other ECM. Among them, glycosaminoglycan (GAG) is another important substance. To imitate the natural dermis, and modificate the scaffold materials, two types of scaffolds were prepared: one is traditional type X collagen spongy scaffold, the other is collagen-chondroitin sulfate (CS) spongy scaffold. Collagen was blended with CS, one kind of GAG, and cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Dermis fibroblast was isolated from neonate prepuce, and dermis fibroblasts were cultured on the scaffolds. The physical and chemical properties of the scaffolds were tested, including SEM, DSC, H&E staining, immunohistochemical staining and CS content analysis and so on. The results indicated that EDC is an effective and non-cytotoxic cross-link reagent, and attaching CS into collagen scaffold could improve the stability and histocompatibility of scaffold.     

Rat Sertoli-spermatogenic cell co-cultures established on collagen-glycosaminoglycan crosslinked copolymers

Summary The role of permeable substrates on the proliferation and differentiation of rat spermatogenic cells in co-culture with Sertoli cells was evaluated. Co-cultures were prepared on substrate discs consisting of a thin, stable analog of extracellular matrix attached to a polyester mesh to facilitate handling. Substrate discs were used alone or mounted within a polysulfone reusable holder. Substrates included collagen type I alone or crosslinked with the glycosaminoglycans (GAG) chondroitin-6-sulfate (8%) or heparin (5%). Chemical crosslinking immobilizes collagen and GAG components, preserves the native triple-helical configuration of collagen type I, and renders the copolymer more resistant to degradation by cellular enzymes. Cell attachment and growth properties of Sertoli and spermatogenic cells on these substrates were compared with another permeable substrate (HATF, a mixture of cellulose esters) uncoated or coated with Matrigel (extracellular matrix material derived from EHS tumors) and with the more conventional nonpermeable glass or plastic substrates. We have found that Sertoli-spermatogenic cell co-cultures prepared from pubertal rats readily attach to collagen or collagen-GAG substrate discs as well as to HATF. However, the optical transparency of collagen or collagen-GAG, as compared to the opaque HATF substrate, facilitates monitoring cell attachment and growth by phase contrast microscopy. Substrate discs processed for light and electron microscopy using standard procedures demonstrate the organization of a structurally polarized epithelial layer with basally located Sertoli cells and spermatogenic cells associated by lateral and apical Sertoli cell surfaces. The permeable nature of the collagen-GAG substrate, its optical transparency, and the formation of an electrical-resistant, polarized Sertoli-spermatogenic cell epithelial layer opens new in vitro experimental possibilities for testing agents that may favor or disrupt the spermatogenic process.     

A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications

In order to develop efficient design strategies for a tissue-engineered heart valve, in vivo and in vitro models of valvular structure and cellular function require extensive characterisation. Collagen and glycosaminoglycans (GAGs) provide unique functional characteristics to the heart valve structure. In the current study, type I collagen-GAG hydrogels were investigated as biomaterials for the creation of mitral valve tissue. Porcine mitral valve interstitial cells (VICs) and endothelial cells (VECs) were isolated and co-cultured for 4 weeks in hydrogel constructs composed of type I collagen. The metabolic activity and tissue organisation of mitral valve tissue constructs was evaluated in the presence and absence of chondroitin sulphate (CS) GAG, and comparisons were made with normal mitral valve tissue. Both collagen and collagen-CS mitral valve constructs contracted to form tissue-like structures in vitro. Biochemical assay demonstrated that over 75% of CS was retained within collagen-CS constructs. Morphological examination demonstrated enhanced VEC surface coverage in collagen-CS constructs compared to collagen constructs. Ultrastructural analysis revealed basement membrane synthesis and cell junction formation by construct VECs, with an increased matrix porosity observed in collagen-CS constructs. Immunohistochemical analyses demonstrated enhanced extracellular matrix production in collagen-CS constructs, including expression of elastin and laminin by VICs. Both native valve and collagen-CS construct VECs also expressed the vasoactive molecule, eNOS, which was absent from collagen construct VECs. The present study demonstrates that collagen gels can be used as matrices for the in vitro synthesis of tissue structures resembling mitral valve tissue. Addition of CS resulting in a more porous model was shown to positively influence the bioactivity of seeded valve cells and tissue remodelling. Collagen-GAG matrices may hold promise for a potential use in heart valve tissue engineering and improved understanding of heart valve biology.     

Influence of chondroitin sulfate and hyaluronic acid on structure, mechanical properties, and glioma invasion of collagen I gels

To mimic the extracellular matrix surrounding high grade gliomas, composite matrices composed of either acid-solubilized (AS) or pepsin-treated (PT) collagen and the glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are prepared and characterized. The structure and mechanical properties of collagen/CS and collagen/HA gels are studied via confocal reflectance microscopy (CRM) and rheology. CRM reveals that CS induces fibril bundling and increased mesh size in AS collagen but not PT collagen networks. The presence of CS also induces more substantial changes in the storage and loss moduli of AS gels than of PT gels, in accordance with expectation based on network structural parameters. The presence of HA significantly reduces mesh size in AS collagen but has a smaller effect on PT collagen networks. However, both AS and PT collagen network viscoelasticity is strongly affected by the presence of HA. The effects of CS and HA on glioma invasion is then studied in collagen/GAG matrices with network structure both similar to (PT collagen-based gels) and disparate from (AS collagen-based gels) those of the corresponding pure collagen matrices. It is shown that CS inhibits and HA has no significant effect on glioma invasion in 1.0?mg/ml collagen matrices over 3 days. The inhibitory effect of CS on glioma invasion is more apparent in AS than in PT collagen gels, suggesting invasive behavior in these environments is affected by both biochemical and network morphological changes induced by GAGs. This study is among the few efforts to differentiate structural, mechanical and biochemical effects of changes to matrix composition on cell motility in 3D.     

Enhanced biological stability of collagen with incorporation of PAMAM dendrimer

The crosslinking methods of collagen using glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) are frequently performed in biomedical applications, but both methods still have their own disadvantages, including the GTA cytotoxicity and low degree of EDC/NHS crosslinking. In this study, we incorporated polyamidoamine (PAMAM) dendrimer with surface amine groups into the two aforementioned crosslinking methods to improve the biostability and structural integrity of collagen. Fifty micromolar of dendrimer concentration was found to have negligible in vitro cytotoxicity and was used for EDC and GTA crosslinking of collagen. The collagenase digestion assay showed that the collagen scaffolds crosslinked in the presence of PAMAM exhibited a higher denature temperature and higher resistance against collagenase digestion compared with their counterparts without dendrimer. Cell proliferation with human conjunctival fibroblasts showed that the incorporation of PAMAM in EDC crosslinking significantly increased the proliferation. All the crosslinked scaffolds also exhibited higher structural stability than the noncrosslinked scaffold. Crosslinking with EDC and PAMAM together yielded substantially higher proliferation and may be a suitable collagen scaffold for biomedical applications.     

Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress-strain characteristics were determined to evaluate the physico-chemical properties of equine- and bovine-collagen-based scaffolds. Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and time (p=0.0001) on degradation of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced swelling ratio compared to bovine collagen samples (p< 0.0001). The amino acid composition of equine collagen revealed a higher amount of hydroxylysine and lysine. Shrinkage temperatures of non-crosslinked samples showed a significant difference between equine (60 degrees C) and bovine collagen (57 degrees C). Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and matrix condition (p=0.0001) on rupture strength measured by stress-strain analysis. The ultrastructure, the crosslinking density and the strain at rupture between collagen matrices of both species showed no significant differences. For tissue engineering purposes, the higher enzymatic stability, the higher form stability, as well as the lower risk of transmissible disease make the case for considering equine-based collagen. This study also indicates that results obtained for scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-chemical properties.     

Characterization of Myxoid Substance of Human Aortic Sarcoma

An autopsy case of aortic sarcoma which was located in the intima of the abdominal aorta and characterized by abundant extracellular myxoid substances was examined. Histological, immunohistochemical and ultrastructural studies revealed no characteristics suggestive of endothelial, leiomyogenic or histiocytic differentiation. Histochemical and biochemical analyses revealed that the myxoid substances were glycosaminoglycans (GAGs) consisting of hyaluronic acid (85% of the total GAG) and chondroitin sulfate proteoglycan (15% of the total GAG). Neither heparan sulfate proteoglycan nor dermatan sulfate proteoglycan was detected. In the aorta, GAGs are synthesized mainly by arterial smooth muscle cells and endothelial cells. Among these cells, the arterial smooth muscle cell is the principal cell type that synthesizes aortic GAGs, predominantly chondroitin sulfate proteoglycan, whereas the endothelial cell synthesizes small amounts of GAGs, predominantly heparan sulfate proteoglycan. Therefore, the results of this study suggest that the tumor cells of the present case have a property similar to arterial smooth muscle cells in terms of GAG synthesis, and that the tumor originated most probably from arterial smooth muscle cells.     

Characterization of myxoid substance of human aortic sarcoma

An autopsy case of aortic sarcoma which was located in the intima of the abdominal aorta and characterized by abundant extracellular myxoid substances was examined. Histological, immunohistochemical and ultrastructural studies revealed no characteristics suggestive of endothelial, leiomyogenic or histiocytic differentiation. Histochemical and biochemical analyses revealed that the myxoid substances were glycosaminoglycans (GAGs) consisting of hyaluronic acid (85% of the total GAG) and chondroitin sulfate proteoglycan (15% of the total GAG). Neither heparan sulfate proteoglycan nor dermatan sulfate proteoglycan was detected. In the aorta, GAGs are synthesized mainly by arterial smooth muscle cells and endothelial cells. Among these cells, the arterial smooth muscle cell is the principal cell type that synthesizes aortic GAGs, predominantly chondroitin sulfate proteoglycan, whereas the endothelial cell synthesizes small amounts of GAGs, predominantly heparan sulfate proteoglycan. Therefore, the results of this study suggest that the tumor cells of the present case have a property similar to arterial smooth muscle cells in terms of GAG synthesis, and that the tumor originated most probably from arterial smooth muscle cells.     

Neomycin binding preserves extracellular matrix in bioprosthetic heart valves during in vitro cyclic fatigue and storage

Bioprosthetic heart valve (BHV) cusps have a complex architecture consisting of an anisotropic arrangement of collagen, glycosaminoglycans (GAGs) and elastin. Glutaraldehyde (GLUT) is used as a fixative for all clinical BHV implants; however, it only stabilizes the collagen component of the tissue, and other components such as GAGs and elastin are lost from the tissue during processing, storage or after implantation. We have shown previously that the effectiveness of the chemical crosslinking can be increased by incorporating neomycin trisulfate, a hyaluronidase inhibitor, to prevent the enzyme-mediated GAG degradation. In the present study, we optimized carbodiimide-based GAG-targeted chemistry to incorporate neomycin into BHV cusps prior to conventional GLUT crosslinking. This crosslinking leads to enhanced preservation of GAGs during in vitro cyclic fatigue and storage. The neomycin group showed greater GAG retention after both 10 and 50 million accelerated fatigue cycles and after 1 year of storage in GLUT solution. Thus, additional binding of neomycin to the cusps prior to standard GLUT crosslinking could enhance tissue stability and thus heart valve durability.     

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood-vessel development in the chick chorioallantoic membrane

This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.     

Growth promoting substrates for human dermal fibroblasts provided by artificial extracellular matrices composed of collagen I and sulfated glycosaminoglycans

The application of native extracellular matrix (ECM) components is a promising approach for biomaterial design. Here, we investigated artificial ECM (aECM) consisting of collagen I (coll) and the glycosaminoglycans (GAGs) hyaluronan (HA) or chondroitin sulfate (CS). Additionally, GAGs were chemically modified by the introduction of sulfate groups to obtain low-sulfated and high-sulfated GAG derivatives. Sulfate groups are expected to bind and concentrate growth factors and improve their bioactivity. In this study we analyzed the effect of aECM on initial adhesion, proliferation, ECM synthesis and differentiation of human dermal fibroblasts (dFb) within 8-48 h. We show that initial adhesion and cell proliferation of dFb progressively increased in a sulfate dependent manner. In contrast, synthesis of ECM components coll and HA was decreased on high-sulfated aECM coll/HA3.0 and coll/CS3.1. Furthermore, the matrix metallo-proteinase-1 (MMP-1) was down-regulated on coll/HA3.0 and coll/CS3.1 on mRNA and protein level. The fibroblast differentiation marker α-smooth muscle actin (αSMA) is not affected by aECM on mRNA level. Artificial ECM consisting of coll and high-sulfated GAGs proves to be a suitable biomaterial for dFb adhesion and proliferation that induces a "proliferative phenotype" of dFb found in the early stages of cutaneous wound healing.     

Fabrication of chondroitin sulfate-chitosan composite artificial extracellular matrix for stabilization of fibroblast growth factor

The development of a novel, three-dimensional, macroporous artificial extracellular matrix (AECM) based on chondroitin sulfate (ChS)-chitosan (Chito) combination is reported. The composite AECM composed of ChS-Chito conjugated network was prepared by a homogenizing interpolyelectrolyte complex/covalent conjugation technique through co-crosslinked with N,N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS). In contrast to EDC/NHS, two different reagents, calcium ion and glutaraldehyde, were used to react with ChS or Chito for the preparation of ChS-Chito composites containing crosslinked ChS or Chito network in the matrix. The stability and in vitro enzymatic degradability of the glutaraldehyde-, EDC/NHS-, and Ca2+ -crosslinked ChS-Chito composite AECMs were all investigated in this study. The results showed that crosslinking improved the stability of prepared ChS-Chito AECMs in physiological buffer solution (PBS) and provided superior protective effect against the enzymatic hydrolysis of ChS, compared with their non-crosslinked counterpart. Because ChS was a heparin-like glycosaminoglycan (GAG), the ChS-Chito composite AECMs appeared to promote binding efficiency for basic fibroblast growth factor (bFGF). The bFGF releasing from the ChS-Chito composite AECMs retained its biological activity as examined by the in vitro proliferation of human fibroblast, depending on the crosslinking mode for the preparation of these composite AECMs. Histological assay showed that the EDC/NHS-crosslinked ChS-Chito composite AECM, after incorporated with bFGF, was biodegradable and could result in a significantly enhanced vascularization effect and tissue penetration. These results suggest that the ChS-Chito composite AECMs fabricated in this study may be a promising approach for tissue-engineering application.     

Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.