Development of tailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking, and ultrastructural aspects

The many biocharacteristics of glycosaminoglycans (GAGs) make them valuable molecules to be incorporated in collagenous biomaterials. To prepare tailor-made collagen-GAG matrices with a well-defined biodegradability and (bioavailable) GAG content, the crosslinking conditions have to be controlled. Additionally, the ultrastructural location of GAGs in engineered substrates should resemble that of the application site. Using chondroitin sulfate (CS) as a model GAG, these aspects were evaluated. The methodology was then applied for other GAGs. CS was covalently attached to collagen using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A maximum of about 155 mg CS/g matrix could be immobilized. CS incorporation and bioavailability, as evaluated by interaction with specific antibodies and glycosidases, was dependent on the molar ratio EDC:carboxylic groups of CS. The denaturation temperature could be modulated from 61 to 85 degrees C. The general applicability of EDC/NHS for immobilizing GAGs was demonstrated with dermatan sulfate, heparin, and heparan sulfate. These matrices revealed comparable physico-chemical characteristics, biodegradabilities, and preserved bioavailable GAG moieties. At the ultrastructural level, GAGs appeared as discrete, electron-dense filaments, each filament representing a single GAG molecule. Distribution was independent of GAG type. They were observed throughout the matrix fibers and at the outer sites, and located, either parallel or orthogonally, at the periphery of individual collagen fibrils. Compositional and ultrastructural similarity between matrices and tissue structures like cartilage and basement membranes can be realized after attachment of specific GAG types. It is concluded that EDC/NHS is generally applicable for attachment of GAGs to collagen. Modulation of crosslinking conditions provides matrices with well-defined GAG contents, and biodegradabilities. Ultrastructural similarities between artificially engineered scaffolds and their possible application site may favor the use of specific collagen-GAG matrices in tissue engineering.     

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.     

Crosslinking of collagen with dendrimers

Polypropyleneimine octaamine dendrimers were studied as an alternative means of generating highly crosslinked collagen. Crosslinking was effected by using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC). The multifunctional dendrimers were introduced as novel crosslinkers after the activation of the carboxylic acid groups of glutamic and aspartic acid residues in collagen. The conventional crosslinker glutaraldehyde was used as a control. EDC, itself an alternative crosslinker, which forms zero-length crosslinks by directly covalently binding collagen molecules, as well as a low molecular weight diamine and a low molecular weight triamine, were also studied. All of the resultant gels were freeze-dried to obtain sponges for characterization. Water uptake of the gels decreased from 90% to 60% after dendrimer crosslinking compared with EDC crosslinking. DSC results showed an increase of denaturation temperature of collagen after crosslinking with the various methods. The generation 2 and 3 dendrimer-crosslinked collagen samples had the highest denaturation temperature, at up to 90 degrees C compared with 50 degrees C in the uncrosslinked collagen control. The dendrimer-crosslinked collagen also showed unique thermal characteristics, with multiple denaturation temperature peaks in contrast to the single peak noted with the other crosslinked collagens. This is thought to be due to the heterogeneous nature of dendrimer crosslinking. Collagenase results revealed that the dendrimer-crosslinked collagen had a comparative resistance to proteolysis to glutaraldehyde-crosslinked collagen. Measurement of activated carboxylic acid groups before and after crosslinking indicated that 40-70% of the activated carboxylic acid was consumed during crosslinking with dendrimers. The results suggest that dendrimer crosslinking of collagen produces stable gels. The presence of a large number of excess amine groups in the dendrimers may also be useful for subsequent modification with biologically relevant groups.     

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.     

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.     

Collagen–hyaluronic acid scaffolds for adipose tissue engineering

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen–hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity ～85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen–HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen–HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen–HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen–HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen–HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.     

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.     

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.     

Novel collagen scaffolds prepared by using unnatural D-amino acids assisted EDC/NHS crosslinking

This work discusses the preparation and characterization of novel collagen scaffolds by using unnatural D-amino acids (Coll-D-AAs)-assisted 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxyl succinimide(NHS)-initiated crosslinking. The mechanical strength, hydrothermal and structural stability, resistance to biodegradation and the biocompatibility of Coll-D-AAs matrices were investigated. The results from Thermo mechanical analysis, Differential scanning calorimetric analysis and Thermo gravimetric analysis of the Coll-D-AAs matrices indicate a significant increase in the tensile strength (TS, 180?±?3), % elongation (% E, 80?±?9), elastic modulus (E, 170?±?4) denaturation temperature (T _d, 108?±?4) and a significant decrease in decomposition rate (T _g, 64?±?6). Scanning electron microscopic and Atomic force microscopic analyses revealed a well-ordered with properly oriented and well-aligned structure of the Coll-D-AAs matrices. FT-IR results suggest that the incorporation of D-AAs favours the molecular stability of collagen matrix. The D-AAs stabilizing the collagen matrices against degradation by collagenase would have been brought about by protecting the active sites in collagen. The Coll-D-AAs matrices have good biocompatibility when compared with native collagen matrix. Molecular docking studies also indicate better understanding of bonding pattern of collagen with D-AAs. These Coll-D-AAs matrices have been produced in high mechanical strength, thermally and biologically stable, and highly biocompatible forms that can be further manipulated into the functional matrix suitable in designing scaffolds for tissue engineering and regenerative medical applications.     

Swelling behavior of hyaluronic acid and type II collagen hydrogels prepared by using conventional crosslinking and subsequent additional polymer interactions

Polymer-polymer interactions, such as hydrophobic and electrostatic interactions, were introduced into covalently crosslinked composite matrices consisting of an anionic polysaccharide, hyaluronic acid (HyA) and a cationic polypeptide, type II collagen. The matrices were covalently crosslinked using a water soluble carbodiimide (WSC; 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride) and were immersed at different temperatures in excess water or 0.4 M NaCl aqueous solution, i.e. the optimal salt concentration to suppress polyion complex formation. Hydrophobic interaction was introduced in the matrices when the temperature was increased. Matrices with polymer-polymer interactions could be obtained when immersed in water at 37°C without any collagen denaturation. The molar ratios of [OH]/[COOH] and [NH2]/[COOH], and WSC concentration in precursor solutions of HyA-collagen composite matrices influenced the electrostatic interactions. The hydrophobic and electrostatic interactions in the matrix were maintained in water, even at low temperatures, while in salt solutions at elevated temperatures, these interactions were nullified.     

Swelling behavior of hyaluronic acid and type II collagen hydrogels prepared by using conventional crosslinking and subsequent additional polymer interactions

Polymer-polymer interactions, such as hydrophobic and electrostatic interactions, were introduced into covalently crosslinked composite matrices consisting of an anionic polysaccharide, hyaluronic acid (HyA) and a cationic polypeptide, type II collagen. The matrices were covalently crosslinked using a water soluble carbodiimide (WSC; 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride) and were immersed at different temperatures in excess water or 0.4 M NaCl aqueous solution, i.e. the optimal salt concentration to suppress polyion complex formation. Hydrophobic interaction was introduced in the matrices when the temperature was increased. Matrices with polymer-polymer interactions could be obtained when immersed in water at 37 degrees C without any collagen denaturation. The molar ratios of [OH]/[COOH] and [NH2]/[COOH], and WSC concentration in precursor solutions of HyA-collagen composite matrices influenced the electrostatic interactions. The hydrophobic and electrostatic interactions in the matrix were maintained in water, even at low temperatures, while in salt solutions at elevated temperatures, these interactions were nullified.     

Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: towards annulus fibrosus tissue engineering

A novel design of silk-based scaffold is developed using a custom-made winding machine, with fiber alignment resembling the anatomical criss-cross lamellar fibrous orientation features of the annulus fibrosus of the intervertebral disc. Crosslinking of silk fibroin fibers with chondroitin sulphate (CS) was introduced to impart superior biological functionality. The scaffolds, with or without CS, instructed alignment of expanded human chondrocytes and of the deposited extracellular matrix while supporting their chondrogenic redifferentiation. The presence of CS crosslinking could not induce statistically significant changes in the measured collagen or glycosaminoglycan content, but resulted in an increased construct stiffness. By offering the combined effect of cell/matrix alignment and chondrogenic support, the silk fibroin scaffolds developed with precise fiber orientation in lamellar form represent a suitable substrate for tissue engineering of the annulus fibrosus part of the intervertebral disc.     

Behavior of fibroblasts on a porous hyaluronic acid incorporated collagen matrix

A hyaluronic acid (HA) incorporated porous collagen matrix was fabricated at -70 degrees C by lyophilization. The HA incorporated collagen matrix showed increased pore size in comparison with collagen matrix. Biodegradability and mechanical properties of matrices were controllable by varying the ultraviolet (UV) irradiation time for cross-linking collagen molecules. Addition of HA to collagen matrix did not effect ultimate tensile stress after UV irradiation. HA incorporated collagen matrices demonstrated a higher resistance against the collagenase degradation than collagen matrix. In an in vitro investigation of cellular behavior using dermal fibroblasts on the porous matrix, HA incorporated collagen matrix induced increased dermal fibroblast migration and proliferation in comparison with collagen matrix. These results suggest that the HA incorporated collagen porous matrix assumes to enhance dermal fibroblast adaptation and regenerative potential.     

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.     

Preparation and evaluation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds for tissue engineering

Extracellular matrix components are valuable building blocks for the preparation of biomaterials involved in tissue engineering, especially if their biological, chemical and physical characteristics can be controlled. In this study, isolated type I collagen fibrils, elastin fibres and chondroitin sulphate (CS) were used for the preparation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds. A total of 12 different scaffolds were prepared with four different ratios of collagen and elastin (1:9, 1:1, 9:1 and 1:0), with and without chemical crosslinking, and with and without CS. Collagen was essential to fabricate coherent, porous scaffolds. Electron microscopy showed that collagen and elastin physically interacted with each other and that elastin fibres were enveloped by collagen. By carbodiimide-crosslinking, amine groups were coupled to carboxylic groups and CS could be incorporated. More CS could be bound to collagen scaffolds (10%) than to collagen-elastin scaffolds (2.4-8.5% depending on the ratio). The attachment of CS increased the water-binding capacity to up to 65%. Scaffolds with a higher collagen content had a higher tensile strength whereas addition of elastin increased elasticity. Scaffolds were cytocompatible as was established using human myoblast and fibroblast culture systems. It is concluded that molecularly-defined composite scaffolds can be composed from individual, purified, extracellular matrix components. Data are important in the design and application of tailor-made biomaterials for tissue engineering.     

Bone tissue engineering on patterned collagen films: an in vitro study

This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane. Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27 microm, groove depth: 12 microm, ridge width: 2 microm) and macropatterned (groove width: 250 microm, groove depth: 250 microm, ridge width: 100 microm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation. Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used. This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.     

Influence of different crosslinking treatments on the physical properties of collagen membranes

The physical properties of collagen-based biomaterials are profoundly influenced by the method and extent of crosslinking. In this study, the influence of various crosslinking treatments on the physical properties of reconstituted collagen membranes was assessed. Five crosslinking agents viz., GTA, DMS, DTBP, a combination of DMS and GTA and acyl azide method were used to stabilize collagen matrices. Crosslinking density, swelling ratio, thermo-mechanical properties, stress-strain characteristics and resistance to collagenase digestion were determined to evaluate the physical properties of crosslinked matrices. GTA treatment induced the maximum number of crosslinks (13) while DMS treatment induced the minimum (7). Of the two diimidoesters (DMS and DTBP), DTBP was a more effective crosslinking agent due to the presence of disulphide bonds in the DTBP crosslinks. T(s) for DTBP and DMS crosslinked collagen were 80 degrees C and 70 degrees C, and their HIT values were 5.4 and 2.85MN/m(2), respectively. Low concentration of GTA (0.01%) increased the crosslinking density of an already crosslinked matrix (DMS treated matrix) from 7 to 12. Lowest fracture energy was observed for the acyl azide treated matrix (0.61MJ/m(3)) while the highest was observed for the GTA treated matrix (1.97MJ/m(3)). The tensile strength of GTA treated matrix was maximum (12.4MPa) and that of acyl azide treated matrix was minimum (7.2MPa). GTA, DTBP and acyl azide treated matrices were equally resistant to collagenase degradation with approximately 6% solubilization after 5h while the DMS treated was least stable with 52.4% solubilization after the same time period. The spatial orientation of amino acid side chain residues on collagen plays an important role in determining the crosslinking density and consequent physical properties of the collagen matrix.     

Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.

In order to develop a scaffolding material for tissue regeneration, porous matrices containing collagen and hyaluronic acid were fabricated by freeze drying at -20 degrees C, -70 degrees C or -196 degrees C. The fabricated porous membranes were cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in a range of 1-100 mM concentrations for enhancing mechanical stability of the composite matrix. Scanning electron microscope (SEM) views of the matrices demonstrated that the matrices obtained before cross-linking process had interconnected pores with mean diameters of 40, 90 or 230 microm and porosity of 58-66% according to the freezing temperature, and also the porous structures after cross-linking process were retained. The swelling test and IR spectroscopic measurement of different cross-linked membranes were carried out as a measure of the extent of cross-linking. The swelling behavior of cross-linked membranes showed no significant differences as cross-linking degree increased. FT-IR spectra showed the increase of the intensity of the absorbencies at amide bonds (1655, 1546, 1458 cm(-1)) compared to that of CH bond (2930 cm(-1)). In enzymatic degradation test, EDC treated membranes showed significant enhancement of the resistance to collagenase activity in comparison with 0.625% glutaraldehyde treated membranes. In cytotoxicity test using L929 fibroblastic cells, the EDC-cross-linked membranes demonstrated no significant toxicity.