Physical, mechanical and degradation properties, and schwann cell affinity of cross-linked chitosan films

Three kinds of cross-linked chitosan films were prepared with hexamethylene diisocyanate (HDI), epichlorohydrin (ECH) and glutaraldehyde (GA) as cross-linking agents, respectively. The physical and mechanical properties, biodegradability and Schwann cell affinity of the cross-linked films were investigated. A significant decrease in the degradation rate in lysozyme solution and a large change in the mechanical properties were observed compared with non-cross-linked chitosan films. The protein adsorption on chitosan films was determined by means of enzyme-linked immunosorbent assay (ELISA). In comparison with the non-cross-linked films, the chitosan films cross-linked with HDI showed a significant increase (up to 40-50%) in both fibronectin and laminin adsorption, while the protein adsorption on the other two kinds of cross-linked films was similar to that on non-crosslinked films. In addition, cell culture revealed that the HDI cross-linked chitosan films enhanced the spread and proliferation of Schwann cells while the other cross-linked films delayed the cell proliferation. These results suggest that HDI cross-linking of chitosan films provides a combination of physical properties, biodegradability and Schwann cell affinity suitable for peripheral nerve regeneration.     

Effects of the degree of deacetylation on the physicochemical properties and Schwann cell affinity of chitosan films

Chitosan is a potential material for the preparation of nerve repair conduits. In order to find a better chitosan for the application in peripheral nerve regeneration, the effects of the degree of deacetylation (DD) on the physicochemical properties and Schwann cell affinity of chitosan films have been evaluated. Six kinds of chitosan samples with similar molecular weight, but various DD in a range from 70.1 to 95.6% were prepared from one stock chitosan material and fabricated into films. X-ray diffraction analysis showed that there were more crystalline regions in the higher DD chitosan films. Swelling and mechanical property measurements revealed that the swelling index of chitosan films decreased and their elastic modulus and tensile strength increased with the increase in DD. The adsorption amount of fibronectin and laminin on chitosan films was measured by means of enzyme-linked immunosorbent assay (ELISA). Culture of adult rat Schwann cells on the films showed that the chitosan films with higher DD provided better substrata for Schwann cell spreading and proliferation. In conclusion, DD of chitosan plays an important role in their physicochemical properties and affinity with Schwann cells. The results suggest that chitosan with a DD higher than 90% is considered as a promising material for application in peripheral nerve regeneration.     

戊二醛交联对壳聚糖/羟基磷灰石-庆大霉素缓释材料性能的影响

背景：交联是骨组织工程材料改性的一种常用方法,但目前仍缺乏交联剂对载药人工骨材料性能影响的相关研究与报道。 目的：研究戊二醛交联对壳聚糖/羟基磷灰石-庆大霉素载药人工骨材料力学性能、降解性能及体外药物缓释行为的影响。 方法：分别制备壳聚糖质量分数为10%,20%,30%的壳聚糖/羟基磷灰石-庆大霉素载药人工骨材料与戊二醛交联壳聚糖/羟基磷灰石-庆大霉素载药人工骨材料,检测各组材料的机械强度、吸水率、降解率及体外药物释放行为。 结果与结论：壳聚糖含量为10%,20%,30%壳聚糖/羟基磷灰石-庆大霉素的抗压强度分别为(10.16±1.17),(28.40±0.64),(23.28±1.30) MPa,经戊二醛交联后材料的抗压强度分别增大至(36.30±1.20),(51.60±2.08),(36.90±3.22) MPa。壳聚糖含量为10%,20%,30%壳聚糖/羟基磷灰石-庆大霉素交联后的吸水率与降解率均低于交联前。在体外缓释的第1天,30%壳聚糖/羟基磷灰石-庆大霉素的药物释放量为42.2%,材料经戊二醛交联处理后药物释放量降至33.6%,在随后的9 d,交联壳聚糖/羟基磷灰石-庆大霉素的总释放量均低于壳聚糖/羟基磷灰石-庆大霉素。表明戊二醛交联赋予了材料更好的生物稳定性,减缓了材料降解速率,显著改善了药物突释现象。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Cross-Linking of Gelatin and Chitosan Complex Nanofibers for Tissue-Engineering Scaffolds

The aim of this study is to investigate cross-linked gelatin–chitosan nanofibers produced by means of electrospinning. Gelatin and chitosan nanofibers were electrospun and then cross-linked by glutaraldehyde (GTA) vapor at room temperature. Scanning electron microscopy (SEM) images showed that the cross-linked mats could keep their nanofibrous structure after being soaked in deionized water at 37° C. The cross-linking mechanism was discussed based on FT-IR results. The two main mechanisms of cross-linking for chitosan and gelatin–chitosan complex are Schiff base reaction and acetalization reaction. For gelatin, the mechanism of cross-linking was Schiff base reaction. The mechanical properties of nanofibrous mats were improved after cross-linking. The biocompatibility of electrospun nanofibrous mats after cross-linking was investigated by the viability of porcine iliac endothelial cells (PIECs). The morphologies of PIECs on the cross-linked nanofibrous mats were observed by SEM. In addition, proliferation of PIECs was tested with the method of methylthiazol tetrazolium (MTT) assay. The results indicate that gelatin–chitosan nanofibrous mats could be a promising candidate for tissue-engineering scaffolds.     

Branched chitosans II: Effects of branching on degradation,protein adsorption and cell growth properties

The demand for biodegradable implant materials has fueled interest in chitosan as a biomaterial. In previous work, branched chitosans were synthesized and structurally characterized. In this study the biological properties of branched chitosans were explored. Branched chitosans were synthesized by grafting low molecular weight chitosan chains (1.6, 16 and 80 kDa) to high molecular weight (600 kDa) linear chitosans via reductive amination. Films of the branched materials were evaluated with regard to: lysozyme-mediated degradation; protein adsorption; cell adhesion and proliferation. Branched chitosan with a 1.6 kDa branch length exhibited higher degradation rates than either linear or higher branch length materials. Branched chitosans also exhibited reduced adsorption of bovine serum albumin that was more pronounced with thicker films. Branched chitosans supported proliferation of rat endothelial cells, but growth rates were significantly lower than on linear chitosan. The results of this study demonstrate that control of many aspects of chitosan’s physical and biological properties can be achieved by changes in molecular architecture.     

Nano and micro mechanical properties of uncross-linked and cross-linked chitosan films

The aim of this study is to determine the nano and micro mechanical properties for uncross-linked and cross-linked chitosan films. Specifically, we looked at nanoindentation hardness, microhardness, and elastic modulus. It is important to study the nano and microscale mechanical properties of chitosan since chitosan has been widely used for biomedical applications. Using the solvent-cast method, the chitosan films were prepared at room temperature on the cleaned glass plates. The chitosan solution was prepared by dissolving chitosan in acetic acid 1% (v/v). Tripolyphosphate (TPP) was used to create the cross-links between amine groups in chitosan and phosphate groups in TPP. In this study, atomic force microscopy was used to measure the nanoindentation hardness and surface topography of the uncross-linked and cross-linked chitosan films. Elastic modulus was then calculated from the nanoindentation results. The effective elastic modulus was determined by microhardness with some modifications to previous theories. The microhardness of the chitosan films were measured using Vicker's hardness meter under three different loads. Our results show that the microhardness and elastic modulus for cross-linked chitosan films are higher than the uncross-linked films. However, the cross-linked chitosan films show increased brittleness when compared to uncross-linked films. By increasing the load magnitude, the microhardness increases for both uncross-linked and cross-linked chitosan films.     

Properties and biocompatibility of chitosan films modified by blending with PEG

Chitosan (beta-1,4-D-glucosamine), a polysaccharide with excellent biological properties, has been widely used in biomedical fields, but many barriers still exist to its broader usage due to its chemical and physical limitations. Further work is needed to improve these properties, but changes of the chemical and physical properties will influence its biocompatibility, so the biological attribute of modified chitosan must be evaluated. In this study, the biocompatibility of chitosan modified by several methods was carefully evaluated at the cellular and protein levels using different physical and biological methods. The results provide a theoretical basis for screening biomaterials. We studied the properties of five kinds of materials made by blending chitosan with different types of polyethylene glycol (PEG). The properties included physical and chemical properties, such as mechanical strength, static contact angle, spectroscopy, thermodynamic attributes and so on. The mechanical properties were slightly improved with the proper amount of PEG, but the improvement was not obvious and was destroyed by the wrong proportion of PEG. Cultures of the cells and amounts and structures of the adsorbed proteins on different materials showed that the PEG effectively improved the biocompatibility of the materials. The PEG enhanced the protein adsorption, cell adhesion, growth and proliferation, but the effects were impaired by excessive PEG. The experiments also demonstrated that the optimum PEG concentration helped to maintain the natural structure of the protein adsorbed on the materials and that maintaining the natural structure benefited cell growth. Analysis of the results based on the intramolecular and intermolecular interaction forces leads to a basic theory for the modification of biomaterials.     

Characterization of a microbial transglutaminase cross-linked type II collagen scaffold

This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of epsilon-(gamma-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 +/- 0.03 kPa) compared to controls (0.21 +/- 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase crosslinked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications.     

Cross-linking of amniotic membranes

Human amniotic membrane was cross-linked with chemical and radiation methods to investigate the effect of cross-linking on its physicochemical and biodegradation properties. Radiation cross-linking was performed with gamma-ray and electron beam while chemical cross-linking was with glutaraldehyde (GA). Both gamma-ray and electron beam irradiation decreased the tensile strength and elongation at break of the amniotic membrane with an increase in the irradiation dose, whereas GA cross-linking had no effect on the tensile properties. This is probably due to the scission of collagen chains through irradiation. No significant change was observed on the water content of cross-linked amniotic membranes for any of the crosslinking methods and in marked contrast with cross-linking of a gelatin membrane. A permeation study revealed that protein permeation through the amniotic membrane was not influenced by the GA concentration at cross-linking. These findings are ascribed to the structure characteristic of the amniotic membrane. The membrane is composed of a fibrous mesh structure from an assemblage of collagen fibers. It is possible that cross-linking takes place in the interior of the fiber assembly without impairing the mesh structure, resulting in no change of the water content and protein permeability. In vitro degradation of cross-linked amniotic membranes revealed that radiation cross-linking appeared to be much less effective than GA cross-linking in retarding the degradation, probably because of low cross-linking densities. GA-cross-linked amniotic membranes were degraded more slowly as the GA concentration at cross-linking increased. When the GA-cross-linked amniotic membrane was subcutaneously implanted in the rat, the tissue response was mild, similar to that of the non-cross-linked native membrane.     

Tailoring the Properties of Cholecyst-Derived Extracellular Matrix Using Carbodiimide Cross-Linking

Modulation of properties of extracellular matrix (ECM) based scaffolds is key for their application in the clinical setting. In the present study, cross-linking was used as a tool for tailoring the properties of cholecyst-derived extracellular matrix (CEM). CEM was cross-linked with varying cross-linking concentrations of N,N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Shrink temperature measurements and ATR–FT-IR spectra were used to determine the degree of cross-linking. The effect of cross-linking on degradation was tested using the collagenase assay. Uniaxial tensile properties and the ability to support fibroblasts were also evaluated as a function of cross-linking. Shrink temperature increased from 59°C for non-cross-linked CEM to 78°C for the highest EDC cross-linking concentration, while IR peak area ratios for the free –NH₂ group at 3290 cm^?1 to that of the amide I band at 1635 cm^?1 decreased with increasing EDC cross-linking concentration. Collagenase assay demonstrated that degradation rates for CEM can be tailored. EDC concentrations 0 to 0.0033 mmol/mg CEM were the cross-linking concentration range in which CEM showed varied susceptibility to collagenase degradation. Furthermore, cross-linking concentrations up to 0.1 mmol EDC/mg CEM did not have statistically significant effect on the uniaxial tensile strength, as well as morphology, viability and proliferation of fibroblasts on CEM. In conclusion, the degradation rates of CEM can be tailored using EDC-cross-linking, while maintaining the mechanical properties and the ability of CEM to support cells.     

Cross-linking of amniotic membranes

Abstraet-Human amniotic membrane was cross-linked with chemical and radiation methods to investigate the effect of cross-linking on its physicochemical and biodegradation properties. Radiation cross-linking was performed with γ-ray and electron beam while chemical cross-linking was with glutaraldehyde (GA). Both γ-ray and electron beam irradiation decreased the tensile strength and elongation at break of the amniotic membrane with an increase in the irradiation dose, whereas GA cross-linking had no effect on the tensile properties. This is probably due to the scission of collagen chains through irradiation. No significant change was observed on the water content of cross-linked amniotic membranes for any of the crosslinking methods and in marked contrast with cross-linking of a gelatin membrane. A permeation study revealed that protein permeation through the amniotic membrane was not influenced by the GA concentration at cross-linking. These findings are ascribed to the structure characteristic of the amniotic membrane. The membrane is composed of a fibrous mesh structure from an assemblage of collagen fibers. It is possible that cross-linking takes place in the interior of the fiber assembly without impairing the mesh structure, resulting in no change of the water content and protein permeability. In vitro degradation of cross-linked amniotic membranes revealed that radiation cross-linking appeared to be much less effective than GA cross-linking in retarding the degradation, probably because of low cross-linking densities. GA-cross-linked amniotic membranes were degraded more slowly as the GA concentration at cross-linking increased. When the GA-cross-linked amniotic membrane was subcutaneously implanted in the rat, the tissue response was mild, similar to that of the non-cross-linked native membrane.     

Surface modification and characterization of chitosan or PLGA membrane with laminin by chemical and oxygen plasma treatment for neural regeneration

Attachment to and proliferation on the substrate are deemed important considerations when Schwann cells (SCs) are to be seeded in synthetic nerve grafts. Good attachment is a prerequisite for the SCs to survive. Fast proliferation will yield large numbers of SCs in a short time, which appears to be promising for stimulating peripheral nerve regeneration. However, surface properties are the dominating factor in influencing the interactions between cells and synthetic nerve grafts. The aim of this study was to investigate the surface effects of laminin modified PLGA and chitosan membranes after chemical method and plasma treatment. Laminin, the extracellular matrix protein, is a permissive protein for SCs adhesion used in neural regeneration. The surface properties of laminin modified membranes were assayed by BCA, FTIR and XPS analysis. Results showed that laminin was covalently bonded onto the surface of both PLGA and chitosan membranes either by chemical method or by oxygen plasma treatment. The cell affinity of the laminin modified membranes was verified by Schwann cells culturing. Our results also indicate that oxygen plasma is indeed a better method to incorporate laminin onto the surface of membrane. Laminin-modified chitosan membrane significantly increases SCs attachment and affinity for directing peripheral nerve regeneration.     

Preparation and characterization of keratin-chitosan composite film.

Keratin-chitosan composite film was prepared by casting the mixed solution of both biopolymers in 75% acetic acid. Although keratin film without any additive is very fragile, 10-30 wt% of chitosan addition gave strong and flexible film (ultimate strength: 27-34 MPa, ultimate elongation: 4-9%). Glycerol (20 wt%) also afforded flexibility to keratin film (ultimate strength: 1 MPa, ultimate elongation: 28%). Further addition of chitosan to glycerol-containing keratin film increased the ultimate strength to 9-14 MPa but gave little effect on ultimate elongation. These data suggest that mechanical properties of keratin film are adjustable by appropriately adding chitosan and glycerol. Waterproof characteristics such as swelling behavior and mechanical properties after swelling were much ameliorated for the composite film compared with keratin and chitosan films, respectively. Furthermore, keratin-chitosan composite film as well as chitosan film decreased bacteria number when the bacteria suspension was treated with a film owing to the irreversible adsorption of bacteria onto the film. The composite film as well as keratin and chitosan films supported fibroblast attachment and proliferation, demonstrating to be a good substrate for mammalian cell culture.     

Novel soy protein wound dressings with controlled antibiotic release: mechanical and physical properties

Naturally derived materials are becoming widely used in the biomedical field. Soy protein has advantages over various types of natural proteins employed for biomedical applications due to its low price, non-animal origin and relatively long storage time and stability. In the current study soy protein isolate (SPI) was investigated as a matrix for wound dressing applications. The antibiotic drug gentamicin was incorporated into the matrix for local controlled release and, thus, protection against bacterial infection. Homogeneous yellowish films were cast from aqueous solutions. After cross-linking they combined high tensile strength and Young’s modulus with the desired ductility. The plasticizer type, cross-linking agent and method of cross-linking were found to strongly affect the tensile properties of the SPI films. Selected SPI films were tested for relevant physical properties and the gentamicin release profile. The cross-linking method affected the degree of water uptake and the weight loss profile. The water vapor transmission rate of the films was in the desired range for wound dressings (∼2300 g m⁻² day⁻¹) and was not affected by the cross-linking method. The gentamicin release profile exhibited a moderate burst effect followed by a decreasing release rate which was maintained for at least 4 weeks. Diffusion was the dominant release mechanism of gentamicin from cross-linked SPI films. Appropriate selection of the process parameters yielded SPI wound dressings with the desired mechanical and physical properties and drug release behavior to protect against bacterial infection. These unique structures are thus potentially useful as burn and ulcer dressings.     

Superporous hydrogels containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks

Superporous hydrogels containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks (SPH-IPNs) were prepared by cross-linking O-carboxymethyl chitosan (O-CMC) with glutaraldehyde (GA) after superporous hydrogel (SPH) was synthesized. The structures of the SPH-IPNs were characterized with FT-IR, 13C-NMR and DSC. SEM, CLSM and light images revealed that the SPH-IPNs possessed both the IPN network and large numbers of pores and the cross-linked O-CMC molecules were located on the peripheries of these pores. The swelling behavior of SPH-IPNs was dependent on the O-CMC content, GA amount and cross-linking time. Due to the cross-linked O-CMC network, in vitro muco-adhesive force and mechanical properties, including compression and tensile modulus, of the SPH-IPN were greatly improved when compared with the CSPH. An enhanced loading capacity for insulin could be obtained by the SPH-IPNs as compared to non-porous hydrogel and CSPH, and more than 90% of the insulin was released within 1 h. With the improved mechanical properties, in vitro muco-adhesive force and loading capacities, the SPH-IPN may be used as a potential muco-adhesive system for peroral delivery of peptide and protein drugs.     

壳聚糖/聚己内酯共混膜的制备及性能

背景：壳聚糖/聚己内酯共混材料在生物材料领域具有广泛的应用前景,但与蛋白、细胞反应机制尚不明确。 目的：观察壳聚糖/聚己内酯共混膜表面蛋白黏附和细胞活性。 方法：将不同配比的壳聚糖/聚己内酯混合溶液旋转涂膜法成膜。分别通过原子力显微镜、滴形分析仪、石英晶体天平和MTT比色法测量膜的表面形貌、亲疏水性、蛋白吸附和细胞增殖活性。 结果与结论：膜的表面形貌、亲疏水性、蛋白吸附和细胞增殖活性在很大程度上取决于壳聚糖和聚己内酯的质量配比。细胞在壳聚糖膜上具有较好的伸展形态,在聚己内酯膜上具有较高的增殖活性。     

In vitro evaluation of a chitosan membrane cross-linked with genipin

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties, antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

Influence of mechanical properties of alginate-based substrates on the performance of Schwann cells in culture

In tissue engineering, artificial tissue scaffolds containing living cells have been studied for tissue repair and regeneration. Notably, the performance of these encapsulated-in-scaffolds cells in terms of cell viability, proliferation, and expression of function during and after the scaffold fabrication process, has not been well documented because of the influence of mechanical, chemical, and physical properties of the scaffold substrate materials. This paper presents our study on the influence of mechanical properties of alginate-based substrates on the performance of Schwann cells, which are the major glial cells of peripheral nervous system. Given the fact that alginate polysaccharide hydrogel has poor cell adhesion properties, in this study, we examined several types of cell-adhesion supplements and found that alginate covalently modified with RGD peptide provided improved cell proliferation and adhesion. We prepared alginate-based substrates for cell culture using varying alginate concentrations for altering their mechanical properties, which were confirmed by compression testing. Then, we examined the viability, proliferation, morphology, and expression of the extracellular matrix protein laminin of Schwann cells that were seeded on the surface of alginate-based substrates (or 2D culture) or encapsulated within alginate-based substrates (3D cultures), and correlated the examined cell performance to the alginate concentration (or mechanical properties) of hydrogel substrates. Our findings suggest that covalent attachment of RGD peptide can improve the success of Schwann cell encapsulation within alginate-based scaffolds, and provide guidance for regulating the mechanical properties of alginate-based scaffolds containing Schwann cells for applications in peripheral nervous system regeneration and repair.     

In vitro evaluation of a chitosan membrane cross-linked with genipin.

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties. antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

Controlled compaction with ruthenium-catalyzed photochemical cross-linking of fibrin-based engineered connective tissue

Tissue engineering utilizing fibrin gel as a scaffold has the advantage of creating a completely biological replacement. Cells seeded in a fibrin gel can induce fibril alignment by traction forces when subjected to appropriate mechanical constraints. While gel compaction is key to successful tissue fabrication, excessive compaction can result due to low gel stiffness. This study investigated using ruthenium-catalyzed photo-cross-linking as a method to increase gel stiffness in order to minimize over-compaction. Cross-links between the abundant tyrosine molecules that comprise fibrin were created upon exposure to blue light. Cross-linking was effective in increasing the stiffness of the fibrin gel by 93% with no adverse effects on cell viability. Long-term culture of cross-linked tubular constructs revealed no detrimental effects on cell proliferation or collagen deposition due to cross-linking. After 4 weeks of cyclic distension, the cross-linked samples were more than twice as long as non-cross-linked controls, with similar cell and collagen contents. However, the cross-linked samples required a longer incubation period to achieve a UTS and modulus comparable to controls. This study shows that photo-cross-linking is an attractive option to stiffen the initial fibrin gel and thereby reduce cell-induced compaction, which can allow for longer incubation periods and thus more tissue growth without compaction below a useful size.