Influence of Cross-Linker Concentration on the Functionality of Carbodiimide Cross-Linked Gelatin Membranes for Retinal Sheet Carriers

Carbodiimide cross-linking can easily regulate the functionality of gelatin carriers used for retinal sheet delivery. This paper investigates the effect of cross-linker concentrations (0–0.4 mmol EDC/mg gelatin membrane (GM)) on the properties of the chemically-modified GMs. ATR–FT-IR and ninhydrin analyses results consistently indicated that the EDC cross-linking reaction approaches saturation at concentrations around 0.02 mmol EDC/mg GM. The thermal stability and resistance to water dissolution and collagenase digestion were significantly enhanced with increasing cross-linker concentration from 0.001 to 0.02 mmol EDC/mg GM. In addition, the chemical cross-linking did not affect the ability to form a tissue-encapsulating structure at 37°C. Irrespective of their cross-linking degree, the GMs had an appropriate degradation rate sufficient to allow tissue integration. It was noted that, although high cross-linker concentrations can be used to improve the delivery efficiency of gelatin samples, the treatment with 0.1–0.4 mmol EDC/mg GM may lead to poor biocompatibility. Results of Live/Dead and pro-inflammatory cytokine expression analyses showed that the exposure of ARPE-19 cultures to the test materials cross-linked with a concentration ≥0.1 mmol EDC/mg GM induces significant cytotoxicity and high levels of interleukin-1β and interleukin-6. However, the presence of EDC cross-linked gelatin membranes in the culture medium had no effect on the glutamate uptake capacity. It is concluded that among the cross-linked gelatin samples studied, 0.02 mmol EDC/mg GM is the best cross-linker concentration for preparation of retinal sheet delivery carriers.     

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.     

Cross-linking and characterisation of gelatin matrices for biomedical applications

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOHgelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOHgelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40 degrees C.     

Cross-linking and characterisation of gelatin matrices for biomedical applications

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/ EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOH_gelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOH_gelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40°C.     

Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2)

Tissue integration between a tissue-engineered corneal equivalent and the host eye is of critical importance in ensuring long-term implant success. A novel dendrimer cross-linked collagen scaffold has previously shown good corneal epithelial cell compatibility in vitro particularly when the highly functional dendrimer cross-linkers were functionalized to introduce additional biological groups. Herein we investigated heparinization of these materials and their potential to facilitate the delivery of basic fibroblast growth factor (FGF-2) in an active form, ultimately for use as a corneal tissue scaffold. Collagen gels cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry, and varying amounts of polypropyleneimine octaamine generation 2 (G2) dendimer and heparin were synthesized. Swelling studies and differential scanning calorimetry characterization indicated higher gel stability with the introduction of dendrimer cross-linking, which was not compromised by heparin integration. Dendrimer cross-linked gels with or without heparin gave multiple denaturation peaks, as did the heparinized EDC gels. This is thought to be the result of the heterogeneous cross-linking possible between collagen, the dendrimer and heparin. Release of FGF-2 from collagen gels showed typical first-order kinetics, with an initial burst followed by a prolonged gradual release. Heparinized dendrimer cross-linked gels released approx. 40% of the growth factor over a 2-week period, with significance maintenance of growth factor activity. Incorporation of heparin resulted in somewhat prolonged release from these systems.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3- dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 °C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2)

Tissue integration between a tissue-engineered corneal equivalent and the host eye is of critical importance in ensuring long-term implant success. A novel dendrimer cross-linked collagen scaffold has previously shown good corneal epithelial cell compatibility in vitro particularly when the highly functional dendrimer cross-linkers were functionalized to introduce additional biological groups. Herein we investigated heparinization of these materials and their potential to facilitate the delivery of basic fibroblast growth factor (FGF-2) in an active form, ultimately for use as a corneal tissue scaffold. Collagen gels cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry, and varying amounts of polypropyleneimine octaamine generation 2 (G2) dendimer and heparin were synthesized. Swelling studies and differential scanning calorimetry characterization indicated higher gel stability with the introduction of dendrimer cross-linking, which was not compromised by heparin integration. Dendrimer cross-linked gels with or without heparin gave multiple denaturation peaks, as did the heparinized EDC gels. This is thought to be the result of the heterogeneous cross-linking possible between collagen, the dendrimer and heparin. Release of FGF-2 from collagen gels showed typical first-order kinetics, with an initial burst followed by a prolonged gradual release. Heparinized dendrimer cross-linked gels released approx. 40% of the growth factor over a 2-week period, with significance maintenance of growth factor activity. Incorporation of heparin resulted in somewhat prolonged release from these systems.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge.

This study was conducted to develop a new sponge type of biomaterial to be used for either wound dressing or scaffold for tissue engineering. We were able to prepare an insoluble matrix composed of gelatin and sodium hyaluronate (HA) by dipping the soluble sponge into 90% (w/v) acetone/water mixture containing a small amount of cross-linking agent, 1-ethyl-3-3-dimethylaminoproplycarbodiimide hydrochloride, EDC. To characterize the sponge, Fourier-transformed infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and Instron analysis were performed. The obtained results indicate that the chemically cross-linked sponge shows a cross-linking degree of 10-35%, a mean pore size of 40-160 microm, porosity of 35-67%, and a tensile strength of 10-30 gf/cm(2). Especially, the porosity measured by image analysis showed a tendency to increase with HA content, resulting in an increased water uptake. The resistance to collagenase degradation in vitro increased for up to 2 days. Silver sulfadiazine (AgSD)-impregnated gelatin-HA sponge was also prepared and compared with conventional vaseline gauze by applying it onto a dorsal skin defect of wistar rat for 5, 12, and 21 days. Histological results showed an enhancement of wound healing in AgSD-impregnated gelatin-HA sponge.     

In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods

This study is an investigation to evaluate the in vivo degradation of gelatin hydrogels in terms of their number of cross-links. Various hydrogels were prepared from acidic gelatin, extracted from bovine bone, porcine skin or fish scale, and basic gelatin, extracted from porcine skin, through four types of cross-linking methods, i.e., glutaraldehyde (GA) or dehydrothermal treatment and ultraviolet (UV) or electron beam irradiation. The water content of hydrogels and their number of cross-links, calculated from the tensile modulus of hydrogels, were evaluated as the measure of hydrogel cross-linking extent. Following subcutaneous implantation of 125I-labeled gelatin hydrogels into mice, the radioactivity remaining was measured at different time intervals to assess the in vivo degradability of hydrogels. Irrespective of the gelatin type and cross-linking method, a good correlation was found between the in vivo degradability of hydrogels and their number of cross-links, which is different from the correlation to their water content. This finding indicates that the degradability of hydrogels is governed by their number of cross-links.     

Cross-linking electrospun type II collagen tissue engineering scaffolds with carbodiimide in ethanol

In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked).     

Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase

In this study, we developed a method to fabricate large, perfusable, macroporous, cell-laden hydrogels. This method is suitable for efficient cell seeding, and can maintain sufficient oxygen delivery and mass transfer. We first loaded three types of testing cells (including NIH 3T3, ADSC and Huh7) into gelatin hydrogel filaments, then cross-linked the cell-laden gelatin hydrogel filaments using microbial transglutaminase (mTGase). In situ cross-linking by mTGase was found to be non-cytotoxic and prevented the scattering of the cells after delivery. The gelatin hydrogel constructs kept the carried cells viable; also, the porosity and permeability were adequate for a perfusion system. Cell proliferation was better under perfusion culture than under static culture. When human umbilical vein endothelial cells were seeded into the constructs, we demonstrated that they stably formed an even coverage on the surface of the hydrogel filaments, serving as a preliminary microvasculature network. We concluded that this method provides a viable solution for cell seeding, oxygen delivery, and mass transfer in large three-dimensional (3-D) tissue engineering. Furthermore, it has the potential for being a workhorse in studies involving 3-D cell cultures and tissue engineering.     

Studies of novel hyaluronic acid-collagen sponge materials composed of two different species of type I collagen

The authors have developed novel hyaluronic acid (HA)-collagen sponge materials (HACSMs) composed of various ratios of bird feet (BF) and pig skin (PS) collagen that are fabricated employing a combination of freezing, lyophilizing, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) crosslinking methods. Morphology, swelling ratio, resistance to collagenase, thermal stability, tensile strength, and free amine index are determined to evaluate the physical-chemical properties of various HACSMs. Different BF: PS ratios directly vary with the physical-chemical properties of HACSMs and control their biodegradability for multiple uses. Resistance to collagenase, thermal stability, and tensile strength of HACSMs increases as the ratio of BF collagen increases. On the contrary, the higher swelling ratio, free amine index, and pore size occur in materials composed of higher ratios of PS collagen. A linear relationship between the decreased ratio of PS collagen and the increase in tensile strength and biostability are observed. The materials of B4P1HA (BF: PS: HA=4: 1: 0.2) exhibit the highest value of tensile strength, but no significant difference exists between B4P1HA and B5P0HA (BF: PS: HA=5: 0: 0.2). These phenomena should be closely related to the BF collagen which contains a higher amount of carboxyl groups of glutamic or aspartic acid residues and forms more amine bonds under EDC cross-linking when compared to PS collagen. However, these results suggest that the B4P1HA and B5P0HA materials should be produced according to highest bio-stability and mechanical strength and, furthermore they may be suitable for artificial skin or drug delivery applications.     

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.     

Study on Chemical Cross-linking Modification of Hyaluronan and the Biocompatibility of its Derivatives

Objective: Prepare cross-linked HA gels with higher mechanical stability,lower degradation velocity and desirable biocompatibility,so as to extend the usage of HA.Method: 1.Test molecular weight of HA (MrHA) by viscosimetry;2.Prepare cross-linked HA gels by DVS,GTA,DEC;3.Discuss the cross-linking and degradation procedure;4,evaluate the biocompatibility of the best HA gels.Results: The mechanical stability and durability to degradation of HA-DVS gels are superior to those of other gels,and when HA :DVS = 40:1 (g/g),at 35℃ and in 0.2M NaOH solution,the HA-DVS gel shows the best mechanical stability,and its cytotoxicity reaches class I,hemolysis ratio is lower than 5 %.Conclusion:Our HADVS gel can be used to prepare biologic scaffolds.     

In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods

This study is an investigation to evaluate the in vivo degradation of gelatin hydrogels in terms of their number of cross-links. Various hydrogels were prepared from acidic gelatin, extracted from bovine bone, porcine skin or fish scale, and basic gelatin, extracted from porcine skin, through four types of cross-linking methods, i.e., glutaraldehyde (GA) or dehydrothermal treatment and ultraviolet (UV) or electron beam irradiation. The water content of hydrogels and their number of cross-links, calculated from the tensile modulus of hydrogels, were evaluated as the measure of hydrogel cross-linking extent. Following subcutaneous implantation of ¹²⁵I-labeled gelatin hydrogels into mice, the radioactivity remaining was measured at different time intervals to assess the in vivo degradability of hydrogels. Irrespective of the gelatin type and cross-linking method, a good correlation was found between the in vivo degradability of hydrogels and their number of cross-links, which is different from the correlation to their water content. This finding indicates that the degradability of hydrogels is governed by their number of cross-links.     

Study on Biocompatibility of Cross-linked Hyaluronic Acid Derivatives

Objective：The cross-linked production, which was prepared by HA and cross-linking agent STMP, EDC, GP through cross-linking reaction, might be used in drug delivery system（DDS）. To ensure the security of clinical application, the excellent properties such as none cell toxicity, nonirritant, none general toxicity, none immunological rejection are necessary. Methods ： In accordance with the request of GB/T 16886.1 on security evaluation of medical biomaterials, cell toxicity test, hemolysis test, intracutaneous stimulation test, acute toxicity test, and hypersensitive test were required. Results： Cell toxicity of HA-STMP, HA-EDC, HA-GP were all less than 1. All hypersensitive tests were eligible. But HA-EDC, HA-GP produced different degrees of slight thrill, slight toxicity, hemolysis rate,which were larger than the standard value. Conclusion：HA-STMP possesses favourable biocompatibility, which is a kind of ideal biomaterials and drug carriers.     

Formation and characterisation of a modifiable soft macro-porous hyaluronic acid cryogel platform

A facile method for the synthesis of cell supportive, highly macro-porous hyaluronic acid (HA) hydrogels via cryogelation is presented. Unmodified HA was chemically cross-linked via EDC/NHS zero-length cross-linking at sub-zero temperatures to yield cryogels with high porosity and high pore interconnectivity. The physical properties of the HA cryogels including porosity, average pore size, elasticity and swelling properties were characterised as a function of cryogelation conditions and composition of the precursor solution. The HA cryogels swell extensively in water, with the average porosities observed being ~90% under all conditions explored. The morphology of the cryogels can be controlled, allowing scaffolds with an average pore size ranging from 18 ± 2 to 87 ± 5 μm to be formed. By varying the cross-linking degree and HA concentration, a wide range of bulk elastic properties can be achieved, ranging from ~1 kPa to above 10 kPa. Preliminary cell culture experiments, with NIH 3T3 and HEK 293 cell lines, performed on biochemically modified and unmodified gels show the cryogels support cell proliferation and cell interactions, illustrating the biomedical potential of the platform.     

Novel Scaffolds Based on Poly(2-hydroxyethyl methacrylate) Superporous Hydrogels for Bone Tissue Engineering

In this study, poly(2-hydroxyethyl methacrylate) (pHEMA)-based superporous hydrogels were synthesized by radical polymerization of 2-hydroxyethyl methacrylate (HEMA) in the presence of a gas blowing agent, sodium bicarbonate. These hydrogels are: pHEMA, pHEMA–gelatin, glycerol phosphate (GP) cross-linked pHEMA–gelatin, glutaraldehyde (GA) cross-linked pHEMA–gelatin superporous hydrogels (SPHs) and pHEMA–hydroxyapatite (HA) superporous hydrogel composites (SPHCs). The hydrogels have a structure of interconnected pores with pore sizes of approx. 500 μm. Although the extent of swelling decreased when gelatin and HA were incorporated to the pHEMA structure, the time to reach the equilibrium swelling (approx. 20 s) was not affected so much. In the presence of gelatin and cross-linkers, mechanical properties significantly improved when compared with pHEMA SPH. Among all the synthesized hydrogels, pHEMA–HA SPHC showed great improvement in mechanical strength and its elastic modulus value was 0.027±0.002 N/mm². Osteogenic activities of pHEMA-based scaffolds were examined by preosteoblastic MC3T3-E1 cell-culture studies. The mitochondrial activity test (MTT) showed that gelatin-containing scaffolds stimulated cell proliferation compared with other scaffolds, while alkaline phosphatase levels (ALP) and mineralization were found highest for the GP cross-linked pHEMA–gelatin SPH. However, pHEMA SPH and pHEMA–HA SPHC did not support cell proliferation and also differentiation. In conclusion, pHEMA–gelatin SPH and GP cross-linked pHEMA–gelatin SPH can be considered as potential scaffolds for bone tissue-engineering applications.     

Swelling behavior and mechanical properties of chemically cross-linked gelatin gels for biomedical use.

Cross-linked gelatin gels are used as biomaterials in living tissues, either as bioadhesives or as devices for sustained drug release. As these applications involve surgical insertion of gels, the effect of cross-linking on mechanical properties is relevant. The effect of cross-linking on the gel water uptake is also relevant for the kinetics of drug release. Equally important is the influence of initial amounts of gelatin in the gelling solutions. We investigated the influence of cross-linking (with glutaraldehyde) and of gelatin content upon the equilibrium water content and tensile properties of resulting gels. A range of gels was prepared with gelatin contents of 10 to 40% wt, and cross-linked with 2.5 to 50% wt glutaraldehyde. The increase in gelatin content and degree of cross-linking reduced the water uptake of gels from about 60-65% wt to about 50% wt. The tensile characteristics were differentially affected. While the increase of cross-linking induced a decrease of toughness and elasticity and an increase of stiffness, it did not affect the ultimate strength of gels. On the contrary, the increase of gelatin content induced a definite increase of the ultimate strength but did not significantly affect the other properties.