Stabilizing effect of carbodiimide and dehydrothermal treatment crosslinking on the properties of collagen/hydroxyapatite scaffolds

This paper reports the effect of the combined technique of dehydrothermal treatment (DHT) and a mixture of 1‐ethyl‐3(3‐dimethylaminopropyl) carbodiimide (EDC) and N‐hydroxysuccinimide (NHS) crosslinking on the physicochemical properties of collagen/hydroxyapatite materials. Collagen and collagen/hydroxyapatite porous scaffolds containing different amounts of collagen and hydroxyapatite were prepared with use of the freeze‐drying technique. All samples were capable of absorbing a large quantity of phosphate buffered saline. Samples crosslinked by DHT+EDC/NHS presented higher resistance to collagenase degradation (with slightly reduced degradation in DHT+EDC/NHS crosslinked scaffolds prepared from 2% collagen solution), whereas DHT scaffolds exhibited faster degradation. Mechanical testing results suggested that scaffolds crosslinked by DHT+EDC/NHS treatment have an improved compressive modulus compared with EDC/NHS crosslinking. The qualitative analysis of colour intensity resulting from the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) led to the conclusion that all samples, regardless of the crosslinking method, were well tolerated by cells. However, DHT and EDC/NHS crosslinked scaffolds seem to support better cell viability, in contrast to DHT+EDC/NHS crosslinked scaffolds that support cell differentiation instead. DHT+EDC/NHS crosslinked scaffolds markedly increase the specific alkaline phosphatase activity of cells, which may be of benefit in bone tissue engineering. © 2017 Society of Chemical Industry     

Surface treatment with amino acids of porous collagen based scaffolds to improve cell adhesion and proliferation

The purpose of this study was to improve the biocompatibility of glutaraldehyde (GA) cross‐linked chitosan coated collagen scaffold for cartilage tissue regeneration. In order to prevent the potential toxicity of GA, we treated the designed scaffold with either glutamic acid or glycine. Amino acid treated scaffolds were characterized by scanning electron microscopy (SEM) techniques. Afterward, chondrocyte interaction with the composite scaffold was investigated assessing cell adhesion and proliferation using Hoechst staining and MTT cell proliferation assay, respectively. The SEM analyses of the scaffolds’ surface and cross‐section confirmed the adhesion of amino acids on the surface of the scaffolds. We also observed that scaffolds’ porosity was reduced due to the coverage of the pores by chitosan and amino acids, leading to low porosity. The use of amino acid improved the chondrocyte adhesion and proliferation inside the scaffolds’ pores when cells were cultured onto the chitosan‐coated collagen scaffolds. Overall, our in vitro results suggest the use of amino acid to improve the biocompatibility of natural polymer composite scaffold being crosslinked with glutaraldehyde. Such scaffold has improved mechanical properties; biocompatibility thus may be useful for tissue regeneration such as cartilage.

     

Preparation and characterization of collagen‐based composite conduit for peripheral nerve regeneration

Collagen‐based composite nerve conduit scaffold was prepared by freeze‐drying steam‐extrusion method and modified chemically with glutaraldehyde (GTA) by adding chitosan into collagen. Fourier transform infrared spectroscopy showed that the collagen and chitosan are certainly crosslinked through GTA. It was observed under scanning electron microscope that the modified nerve conduit material is a porous three‐dimensional crosslinked structure and the quantity ratio of the collagen to chitosan has influence on the morphology. The cell proliferation experiment results showed that the collagen‐based composite scaffold prompts the adhesion and proliferation of cells, but as the chitosan increasing, the cell proliferation decreased slightly. The swelling property, the collagenase degradation, and the mechanical property of the scaffold are tested at the quantity ratios of collagen to chitosan 4 : 3, 3 : 1, and 4 : 1 and crosslinking time 0.5 and 1.0 h. The experiments show that the stability of the scaffold is enhanced with decreasing the quantity ratio of collagen to chitosan and increasing crosslinking time. Through the experimental investigations, the modifying technique parameters are discussed and the scaffold exhibits better physical and chemical properties at the quantity ratio of collagen to chitosan 3 : 1 and crosslinking time 0.5 h. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Synergistic effects of crosslinking and chitosan molecular weight on the microstructure,molecular mobility,thermal and sorption properties of porous chitosan/gelatin/hyaluronic acid scaffolds

In this study, synergistic effects of crosslinking and chitosan molecular weight on the microstructure, molecular mobility, thermal, and sorption properties of porous chitosan/gelatin/hyaluronic acid hybrid foams are reported. Fourier transform infrared spectroscopy has been utilized to confirm the covalent attachment of hyaluronic acid to gelatin and chitosan, and covalent chemical crosslinking between gelatin and chitosan. Detailed image analysis of scanning electron microscopy images of the porous scaffold hydrids reveal that the pore size of the materials formulated using either low‐ or high‐molecular‐weight chitosan increases significantly upon crosslinking using ethyl(dimethylaminopropyl) carbodiimide/N‐Hydroxysuccinimide. These microstructural changes are even more pronounced for the crosslinked hybrid scaffolds formulated using low‐molecular‐weight chitosan, highlighting a synergistic effect between crosslinking and the use of low‐molecular‐weight chitosan. Results obtained using differential scanning calorimetry demonstrate a significant reduction in molecular mobility reduction in molecular mobility for crosslinked scaffolds formed using high‐molecular‐weight chitosan compared to non‐crosslinked hybrids and crosslinked hybrids formulated using low‐molecular‐weight chitosan. Correspondingly, dynamic vapor sorption evidenced significantly lower water vapor sorption for crosslinked scaffolds formulated using high‐molecular‐weight chitosan. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44772.     

Polysaccharide‐based artificial extracellular matrix: Preparation and characterization of three‐dimensional,macroporous chitosan,and heparin composite scaffold

Scaffold‐guided tissue engineering based on synthetic and natural occurring polymers has gained many interests in recent year. In this study, the development of a chitosan‐heparin artificial extracellular matrix (AECM) is reported. Three‐dimensional, macroporous composite AECMs composed of heparin (Hep) and chitosan (Chito) were prepared by an interpolyelectrolyte complex/lyophilization method. The Chito‐Hep composite AECMs were, respectively, crosslinked with glutaraldehyde, as well as cocrosslinked with N,N‐(3‐dimethylaminopropyl)‐N′‐ethyl carbodiimide (EDC/NHS) and N‐hydroxysuccinimide (NHS). The crosslinking reactions were examined by FT‐IR analysis. In physiological buffer solution (PBS), the EDC/NHS‐crosslinked Chito‐Hep composite AECM showed a relative lower water retention ratio than its glutaraldehyde‐crosslinked counterparts. The EDC/NHS‐crosslinked Chito‐Hep composite AECMs showed excellent biocompatibility, according to the results of the in vitro cytotoxic test. This result suggested that the EDC/NHS‐crosslinked Chito‐Hep composite AECMs might be a potential biomaterial for scaffold‐guided tissue engineering applications. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Development and Characterization of Novel Biomimetic Composite Scaffolds Based on Bioglass‐Collagen‐Hyaluronic Acid‐Phosphatidylserine for Tissue Engineering Applications

Summary: Biomimetic scaffolds are appealing products for the repair of bone defects using tissue engineering strategies. The present study prepared novel biomimetic composite scaffolds with similar composite to natural bone using bioactive glass, collagen, hyaluronic acid, and phosphatidylserine. The microstructure, swelling ratio, biodegradability, and biomineralization characteristic of the composite scaffolds with and without hyaluronic acid and phosphatidylserine were compared and analyzed by SEM/EDAX, XRD, and FTIR techniques and in vitro test, and the properties can be influenced by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC)/N‐hydroxysuccinimide (NHS) crosslinking. The optimized properties of the crosslinked composite scaffolds observed in this study show the possibility of their use of bioactive and bioresorbable scaffolds in bone tissue engineering.

SEM micrographs of BG‐COL‐HYA‐PS composite scaffolds after immersion in SBF for 1 d.     

Air‐dried 3D‐collagen–chitosan biocomposite scaffold for tissue engineering application

Collagen–chitosan scaffolds of different compositions were developed using emulsion air‐drying method. The scaffolds prepared adding 10–30 wt% of chitosan to collagen improved the mechanical properties of the composite scaffold, and 7:3 ratio (collagen :chitosan) was found to be a better composite having a tensile strength of 13.57 MPa with 9% elongation at break. The water‐uptake characteristics were performed at different pH and found to be ameliorated for the composite scaffolds compared to pure collagen and chitosan scaffold, respectively. The pores ranging from 100 to 300 μm were well interconnected, and their distribution was fairly homogeneous in the scaffold as observed through scanning electron microscopy. Furthermore, the scaffold decreased the bacterial counts and supported fibroblasts attachment and proliferation, thus demonstrating this composite to be a good substrate for biomedical application.POLYM. COMPOS., 33:2029–2035, 2012. © 2012 Society of Plastics Engineers     

Development of fibrin conjugated chitosan/nano β‐TCP composite scaffolds with improved cell supportive property for bone tissue regeneration

In the present study, an attempt has been made to improve cell supportive property of chitosan/nano beta tri‐calcium phosphate (β‐TCP) composite scaffolds by modification of scaffold surface with fibrin using ethyl‐3‐(3‐dimethylaminopropyl) carbodimide (EDC) as crosslinking agent. The developed fibrin conjugated chitosan/nano β‐TCP composite scaffolds possess desired pore size and porosity in the range of 45–151 µm and 81.4 ± 4.1%, respectively. No significant change in compressive strength of scaffolds was observed before and after fibrin conjugation. The calculated compressive strength of fibrin conjugated and non‐conjugated chitosan/nano β‐TCP scaffolds are 2.71 ± 0.14 MPa and 2.67 ± 0.11 MPa, respectively. Results of cell culture study have further shown an enhanced cell attachment, cell number, proliferation, differentiation, and mineralization on fibrin conjugated chitosan/nano β‐TCP scaffold. The uniform cell distribution over the scaffold surface and cell infiltration into the scaffold pores were assessed by confocal laser scanning microscopy. Furthermore, higher expression of osteogenic specific genes such as bone sialo protein, osteonectin, alkaline phosphatase, and osteocalcin (OC) on fibrin conjugated scaffolds was observed when compared to scaffolds without fibrin. Altogether, results indicate the potentiality of developed fibrin conjugated composite scaffolds for bone tissue engineering applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41534.     

Chitosan/gelatin porous bone scaffolds made by crosslinking treatment and freeze‐drying technology: Effects of crosslinking durations on the porous structure,compressive strength,and in vitro cytotoxicity

In this study, freezing was used to separate a solute (polymer) and solvent (deionized water). The polymer in the ice crystals was then crosslinked with solvents, and this diminished the linear pores to form a porous structure. Gelatin and chitosan were blended and frozen, after which crosslinking agents were added, and the whole was frozen again and then freeze‐dried to form chitosan/gelatin porous bone scaffolds. Stereomicroscopy, scanning electron microscopy, compressive strength testing, porosity testing, in vitro biocompatibility, and cytotoxicity were used to evaluate the properties of the bone scaffolds. The test results show that both crosslinking agents, glutaraldehyde (GA) and 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide, were able to form a porous structure. In addition, the compressive strength increased as a result of the increased crosslinking time. However, the porosity and cell viability were not correlated with the crosslinking times. The optimal porous and interconnected pore structure occurred when the bone scaffolds were crosslinked with GA for 20 min. It was proven that crosslinking the frozen polymers successfully resulted in a division of the linear pores, and this resulted in interconnected multiple pores and a compressively strong structure. The 48‐h cytotoxicity did not affect the cell viability. This study successfully produced chitosan/gelatin porous materials for biomaterials application. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41851.     

Polysaccharide‐based artificial extracellular matrix: Preparation and characterization of three‐dimensional,macroporous chitosan and chondroitin sulfate composite scaffolds

Scaffold‐guided tissue engineering based on synthetic and natural occurring polymers has gained much interest in recent years. In this article, the development of a polysaccharide‐based artificial extracellular matrix (AECM) is reported. Three‐dimensional, macroporous composite AECMs composed of chondroitin sulfate (ChS) and chitosan (Chito) were prepared by an interpolyelectrolyte complex/lyophilization method. The ChS–Chito composite AECMs were crosslinked with glutaraldehyde and calcium ions (Ca²⁺) and cocrosslinked with N,N‐(3‐dimethylaminopropyl)‐N′‐ethyl carbodiimide (EDC) and N‐hydroxysuccinimide (NHS). The crosslinking reactions were examined with Fourier transform infrared analysis. Glutaraldehyde and Ca²⁺ crosslinked with Chito and ChS, respectively, to produce different types of ChS–Chito semi‐interpenetrated networks. In contrast, EDC/NHS crosslinked with both Chito and ChS to produce ChS–Chito connected networks. In physiological buffer solutions, the Ca²⁺‐crosslinked ChS–Chito composite AECMs showed a lower swelling ratio than their EDC/NHS‐ and glutaraldehyde‐crosslinked counterparts. The ChS–Chito composite AECMs showed excellent antibacterial capability and biocompatibility according to the results of the in vitro antibacterial test and cytotoxic assay. This result suggested that the ChS–Chito composite AECMs might be a potential biomaterial for scaffold‐guided tissue‐engineering applications. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

Bioactive monetite‐containing whisker‐like fibers reinforced chitosan scaffolds

The aim of this work was to develop bioactive chitosan scaffolds reinforced with monetite‐containing whisker‐like fibers. The fibers synthesized by homogeneous precipitation were characterized as monetite/hydroxyapatite short fibers (MAFs), using XRD, FTIR and SEM. The pure chitosan and MAFs/chitosan composite scaffolds were produced by freeze‐drying, and characterized with respect to porosity, pore size, swelling behavior, compressive strength and modulus, and in vitro bioactivity. The incorporation of MAFs in chitosan matrices led to increase the pore size, according to the evaluation by FE‐SEM, and decrease the porosity of composite scaffolds. The swelling ratio decreased as MAFs content of scaffolds increased. The compressive strength and modulus of scaffolds were improved by an increase in MAFs content. The noncross‐linked scaffolds with a chitosan: MAFs weight ratio of 1:1 (CW3) showed a porosity of 75.5%, and the strength and modulus of 259 kPa and 2.8 MPa in dry state, respectively. The crosslinking by glutaraldehyde resulted in improved mechanical properties. The strength and modulus of cross‐linked CW3 scaffolds in wet state reached to 345 kPa and 1.8 MPa, respectively. The in vitro bioactivity of the reinforced scaffolds, evaluated by FE‐SEM/EDS, XRD, and ATR‐FTIR, was confirmed by the formation of a carbonated apatite layer on their surfaces when they soaked in simulated body fluid (SBF). The results of this initial study indicate that the monetite‐containing whisker‐like fibers may be an appropriate reinforcement of chitosan scaffolds.     

Reinforcement of pH‐responsive γ‐poly(glutamic acid)/chitosan hydrogel for orally administrable colon‐targeted drug delivery

Orally administrable hydrogel was prepared by crosslinking chitosan (CS) with γ‐poly(glutamic acid) (γ‐PGA) for an excellent pH‐responsive colon‐targeted drug delivery system. The stable crosslinked amide bond appeared in the shifted region of FTIR spectroscopy, and the tensile strength and elastic modulus were also reduced by crosslinking of CS and γ‐PGA. The surfaces of crosslinked hydrogel have a homogeneous pore array with pore size corresponding to the varied blending ratio. The swelling ratio was dramatically changed by increasing the pH from 3 to 6, and the responsiveness of swelling ratio to the reversible pH changes between 3 and 10 was reliable for 72 h. The drug diffusion rate was mainly dependent on the pH, and a water‐soluble tetrazolium (WST‐1) assay indicated that cytocompatibility of the hydrogel was in an acceptable range. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci. 2013     

Bacterial cellulose/collagen composite: Characterization and first evaluation of cytocompatibility

The novel bacterial cellulose (BC)/collagen composites were prepared by immersing wet BC pellicle excreted by Acetobacter xylinum in collagen solution followed by freeze‐drying process. The product looks like a foam structure. The morphology of BC/collagen composite was examined by scanning electron microscope (SEM) and compared with pristine BC. SEM images showed that collagen molecules was not only coated on the BC fibrils surface but also could penetrate inside BC and hydrogen bond interactions were formed between BC and collagen. The prepared BC/collagen composite was also characterized by Fourier transform infrared (FTIR) spectroscopy, X‐ray diffraction (XRD), thermogravimetric analysis (TGA), and mechanical test. With the incorporation of collagen in the BC, no changes happened in the crystal structure but the thermal stability was improved. Tensile test results indicate that the Young's Modulus and tensile strength have a big increase while the elongation at break has a slight decrease. The cytocompatibility of composite was preliminarily evaluated by cell adhesion studies. The tests were carried out using 3T3 fibroblast cells. The cells incubated with BC/collagen scaffolds for 48 h were capable of forming cell adhesion and proliferation. It showed much better cytocompatibility than pure BC. So, the prepared BC/collagen scaffolds are bioactive and may be suitable for cell adhesion/attachment suggesting that these scaffolds can be used for wound dressing or tissue‐engineering scaffolds. Therefore, these results suggest that these novel BC/collagen scaffolds may have the potential to be sued for some biomedical applications. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Pervaporation separation of water/isopropanol mixtures through crosslinked carboxymethyl chitosan/polysulfone hollow‐fiber composite membranes

Carboxymethyl chitosan (CMCS)/polysulfone (PS) hollow‐fiber composite membranes were prepared through glutaraldehyde (GA) as the crosslinking agent and PS hollow‐fiber ultrafiltration membrane as the support. The permeation and separation characteristics for dehydration of isopropanol were investigated by the pervaporation method. Pure chitosan, carboxymethyl chitosan, and crosslinked carboxymethyl chitosan membranes were characterized by Fourier transform infrared (FT‐IR) spectroscopy and X‐ray diffraction (XRD) to study the crosslinking reaction mechanism and degree of crystallinity, respectively. The effects of feed composition, crosslinking agent, membrane thickness, and feed temperature on membrane performance were investigated. The results show that the crosslinked CMCS/PS hollow‐fiber composite membranes possess high selectivity and promising permeability. The permeation flux and separation factor for isopropanol/water is 38.6 g/m²h and 3238.5, using 87.5 wt % isopropanol concentration at 45°C, respectively. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1959–1965, 2007     

Synergistic strengthening of composite films by crosslinking graphene oxide reinforcement and poly(vinyl alcohol) with dicarboxylic acids

High‐strength plastic materials with excellent biodegradability, non‐toxicity and economically wide availability are in high demand. Herein, we demonstrate graphene oxide (GO) composite of poly(vinyl alcohol) (PVA) as a potential bioplastic material by chemical crosslinking. For a potential bioplastic material, PVA has to be addressed for its high water absorbing capacity along with improvement in tensile strength and thermal stability. These issues were addressed by enhancing the interfacial binding between PVA and GO, covalent bonds between the two being introduced by crosslinking with dicarboxylic acids, namely succinic acid (SuA) and adipic acid (AdA). Crosslinking of neat PVA with dicarboxylic acids also resulted in enhanced swelling resistance and thermal stability. The greatest improvement in tensile strength and swelling resistance was observed for a GO crosslinked with diacids due to the synergistic effect of reinforcement and crosslinking. Improvements of 225 and 234% in the tensile strength of PVA (31.19 MPa) were observed for 5% GO–PVA samples crosslinked with 6.25 mmol AdA and 7.5 mmol SuA, respectively. For the same samples, water uptake was 44 and 29%, respectively, compared to the non‐crosslinked PVA (359%). © 2017 Society of Chemical Industry     

A study on the fabrication of porous chitosan/gelatin network scaffold for tissue engineering

A novel porous chitosan/gelatin scaffold for tissue engineering was prepared via polyelectrolyte complex formation, freeze drying and post‐crosslinking with glutaraldehyde. The porosity and mean pore diameters could be controlled within 30∼100 µm by varying the original water content and the freezing conditions. Dipping the scaffolds in poly(lactic acid) provided good mechanical properties making it a promising candidate towards tissue engineering. © 2000 Society of Chemical Industry     

Tensile,Swelling, and Oxidative Degradation Properties of Crosslinked Polyvinyl Alcohol/Chitosan/Halloysite Nanotube Composites

Glutaraldehyde (GA) crosslinked polyvinyl alcohol (PVA)/chitosan (CS)/halloysite nanotube (HNT) composite films were prepared using a wet casting method. The tensile, morphology, thermal degradation, swelling, moisture, and oxidative degradation properties of crosslinked composite films were carried out. The presences of crosslinking in the composite films were confirmed by FTIR result. The tensile strength of the crosslinked composite films increased up to 0.5 wt% of HNTs loading. Increasing HNTs reduced the thermal degradation, swelling, and moisture properties of crosslinked composite films reduced with the increase of HNTs content. Results also indicated that the crosslinked composite films were degraded using Fenton reagent.     

Preparation and characteristic of a sodium alginate/carboxymethylated bacterial cellulose composite with a crosslinking semi‐interpenetrating network

A novel ionic crosslinking sodium alginate (SA)/carboxymethylated bacterial cellulose (CM‐BC) composite with a semi‐interpenetrating polymer network (semi‐IPN) structure was developed in this study. The composite was prepared through the blending of an SA gel with CM‐BC then crosslinking by Ca²⁺ followed by a freeze‐drying process. Scanning electron microscopy showed the composite matrix organized in a three‐dimensional network of CM‐BC interpenetrated against SA molecular chains with a quantity of calcium alginate microspheres upon the surface. The swelling ratios of the composite were enhanced by 183, 198, and 212% with the supplementation of CM‐BC weight fractions of 25, 50, and 75%, respectively; the swelling ratios changed with changing pH. The tensile modulus, tensile strength, and elongation at break of SA were enhanced by 165, 152, and 188%, respectively, with the addition of 50 wt % CM‐BC. This study demonstrated that the semi‐IPN structure dramatically changed the swelling and mechanical properties of the composite, and the semi‐IPN will be a promising candidate for biomedical applications such as wound dressings and skin tissue engineering. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39848.     

Star‐shaped polycaprolactone/chitosan composite hydrogels: fabrication and characterization

Star‐shaped polycaprolactone (stPCL)/chitosan composite hydrogel was fabricated by simply melt/solution blending between chitosan/dicarboxylic acid solution and melted stPCL, using 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride and N‐hydroxysuccinimide as conjugating agents to obtain a composite hydrogel. Here, stPCL and modified stPCL were investigated. The stPCL was modified to have a carboxyl‐terminated chain (stPCL‐COOH). The composite hydrogels were transparent. The network structure of the composite hydrogels was investigated. stPCL‐OH had no chemical bond to the chitosan network but stPCL‐COOH could co‐crosslink with the chitosan network. The porous structure and porosity of the composite hydrogels were similar to those of chitosan hydrogel. However, the hydrophobicity of stPCL resulted in a lower swelling ratio compared to chitosan hydrogel. The rheological analysis of the composite hydrogel exhibited a stable crosslinked network. Compression testing of the composite hydrogel obtained from stPCL‐COOH at a mole ratio of stPCL‐COOH and chitosan of 1:1 had optimum compressive mechanical properties comparable to chitosan hydrogel due to a synergistic effect of the flexibility in stPCL and the co‐crosslinking of stPCL‐COOH with the chitosan network. © 2020 Society of Chemical Industry     

Release of indomethacin from a novel chitosan microsphere prepared by a naturally occurring crosslinker: Examination of crosslinking and polycation–anionic drug interaction

Novel chitosan microspheres with lower cytotoxicity were fabricated in this study and their drug release characteristics were investigated. Genipin, a naturally occurring crosslinking reagent that has been used in herbal medicine and in the production of food dye, was used to prepare crosslinked chitosan microspheres by a water‐in‐oil dispersion method. The crosslinking mechanism examined by FTIR and ¹³C–NMR suggests that the crosslinking of chitosan by genipin leads to the formation of secondary amide and heterocyclic amino linkage. The polycation–anionic drug interaction between chitosan and indomethacin was pH dependent and could affect the dissolution property of indomethacin. By examination of the release profiles of the crosslinked chitosan microsphere, it was found that the release of indomethacin from the microsphere was sustainable and influenced by factors such as crosslinking of microsphere and chitosan–indomethacin interaction, thus establishing crosslinked chitosan microsphere as a very promising polymeric carrier for drug release. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 1700–1711, 2001