| dc.description.abstract | In the present study, a novel method was designed to prepare chitosan-alginate, chondroitin sulfate and heparin complex based artificial extracellular matrix (scaffolds) for wound repairing. Tissue engineering is a newly developed specialty involves the construction of temporary scaffolds to serve as a three-dimensional (3-D) template for initial cell attachment and subsequent tissue formation. Ideally, a scaffold should be fabricated from biocompatible and bioresorbable materials conducive to cell attachment, proliferation, and differentiation. It should also be high porosity with an interconnected pore network for cell growth and transport of nutrients/metabolic waste. However, even if cells are distributed throughout a scaffold, there is a need for a vascular supply to nourish the cells in the interior of the scaffold. Thus, stimulation of blood vessel ingrowth into the scaffold would assure tissue survival and function. It was reported that basic fibroblast growth factors (bFGF) played an important role for angiogenesis. The vascularization could be promoted by bFGF to provide sufficient nutrient transport for the transplanted cells. One promising way to enhance in vivo efficacy of growth factors is the controlled release at the site of action over an extended time period by incorporating the growth factor into an appropriate bFGF-binding material.
Heparin and chondroitin sulfate, the sulfated glycosaminoglycans, can stabilize an active conformation of bFGF to protect them from proteolysis and enhance their interaction with specific cellular receptors. Alginate is a negatively charged polysaccharider. The carboxyl groups on alginate appear to bind with basic amino acid residues in the FGFs. Chitosan is a copolymer of glucosamine and N-acetylglucosamine obtained by N-deacetylation of chitin, which has structural characteristics similar to extracellular glucosaminoglycan. Chitosan-based biomaterials have been noted for its wound-healing acceleration, cartilage repairing and bone-forming ability in several studies. It is believed that the combination of chitosan-alginate, chondroitin sulfate and heparin is of benefit to binding bFGF for tissue repairing. Since alginate, chondroitin sulfate and heparin were very soluble in water, we want to couple the polysaccharides to chitosan respectively using glutaraldehyde, EDC and calcium ion for the preparation of stable polysaccharides complex scaffolds.
The process involves the construction of three-dimensional (3-D) porous films based on polysaccharides complex. To evaluate the interaction of growth factors (EGF, IP=4.6; bFGF, IP=9.6) with the polysaccharides complex scaffolds, the adsorption and release properties of EGF and bFGF-conjugated scaffolds are examined by ELISA studies. The bFGF or EGF releasing from the polysaccharides complex scaffolds retain its biological activity as examined by the in vitro proliferation of human fibroblast and in vitro histological examination of regenerative tissue.
The lyophilized product of the polysaccharides complex scaffolds show interconnected porous structures with pore size of 100-200μm. After one weeks of soaking in PBS solution, the weight loss of non-crosslinked polysaccharides complex scaffolds approach to 100%. Less than 40% of weight loss is observed from the chitosan-heparin (chitosan/heparin=2:1) and chitosan-alginate scaffold; however, almost 80% of weight loss can be found from the chitosan-heparin (chitosan/heparin=1:1) and chitosan-chondroitin sulfate scaffold. The protonation of amino groups and ionization of the carboxylic acid and sulfonate groups are responsible for the stability of prepared polysaccharides complex scaffolds. Weight loss of the polysaccharides complex scaffolds were reduced to less than 30% after deprotonation of amino groups. On the contrary, the polysaccharides complex scaffolds still retain more than 60% of weight loss after reduction of carboxylic ions.
After crosslinked by glutaraldehyde, all serious of chitosan/anion polysaccharide scaffolds were appeared anion polysaccharide losses seriously and brittle properties obvious with crosslinking time increased. The phenomenon due to amine group reaction with aldehyde to form semi-IPN and then anion polysaccharide were liberated. From literature report the crosslinked type of EDC was showed into full interpenetrating, but using in chitosan/ anion polysaccharide scaffolds will induce unstable during the initial stage due to its polyion chains destroyed by carboxyl group reaction to imime group of EDC. This phenomenon indicated both chitosan and anion polysaccharide with losses through EDC reaction, and weight loss increased obvious in three type chitosan/anion polysaccharide scaffolds by EDC reaction time increased through one week. But in this case, we can find that sulfate element content were raised after EDC crosslinked 24hours in chitosan/ chondroitin sulfate and chitosan/heparin scaffolds, the higher sulfate element content will benefit to absorption basic fibroblast growth factor. The third type crosslinked agent is calcium ion. Even though divalent calcium ion will stable aion polysaccharides (carboxyl and sulfuric group), but its competition reactions with amine on chitosan would resulting into chitosan and anion polysaccharide weight losses during the primary stage.
Due to the decrease of electronic groups, the efficiencies for the adsorption of growth factors (EGF and bFGF) to the cross-linked polysaccharides complex scaffolds were less than that of the non-crosslinked polysaccharides complex scaffolds. By cross-linked with glutaraldehyde, the bFGF- and EGF-adsorption efficiencies of the polysaccharides complex scaffolds decrease with the increase of reaction time for crosslinking. The bFGF-adsorption efficiencies of the polysaccharides complex scaffolds increase with the increase of reaction time for crosslinking; however, the EGF-adsorption efficiencies decrease with the increase of reaction time for crosslinking. In case of EDC crosslinked, the Basic-FGF absorption efficiencies increase through EDC reaction time increase; on the contrary, the EGF absorption amounts decrease with EDC reaction time increase. In other hand, divalent calcium ion crosslinked type polysaccharide scaffolds, such as chitosan/alginate and chitosan/chondroitin sulfate had same absorption model in Basic-FGF and EGF which efficiencies increase with the increase of reaction time for crosslinking. But both growth factors absorption efficiencies increase with the increase of reaction time for calcium ion crosslinking under chitosan/heparin scaffolds.
The release of growth factors (EGF and bFGF) determined by ELISA assay indicates that the release profiles are dependent on the electrostatic interaction between the polysaccharides complex scaffolds and growth factors. There are 1~12% of EGF release and 5~20% of bFGF release from the polysaccharides complex scaffolds. The biological activity of released EGF and bFGF are examined by the continued proliferation of human fibroblasts. The result indicates that the released bFGF retained its biological activity to enhance the proliferation of human fibroblasts, within one week of incubation.
By the animal experiment results show that the polysaccharide scaffold combination with mEGF and Basic-FGF will promote capillaries newborn and than modulate the fibroblast to accelerate the collagen hyperplasia in period of 30days. Compare with the control(inflammation reaction) in 60 days, the scaffold combine with growth factor can also accelerate the cell move into scaffold and then decompose the polysaccharide by enzyme and replace with newborn one, this phenomenon can be utilized to tissue repair or induce new-born organ regenerates of damaging. Final effects will benefit in the development of the tissue engineering research. | en_US |