Preparation And Properties Of Collagen-based Directional Pore Hydrogel Cross-linked By DAG

The skin,muscles,nerves,bones which are anisotropic tissues have important functions such as protecting the body,movement,behavior control,and mechanical support.However,it will seriously endanger people’s life and health once these tissues are injured or diseased in a large area,due to their relatively poor self-healing ability.Hydrogels are three-dimensional soft materials composed of a large amount of water and polymer networks formed by physical or chemical crosslinking.They worth well in the field of tissue engineering scaffold materials.The aligned 3D structure of directional hydrogel could effectively guide the directional growth of muscle cells and nerve cells in vitro,which play an important role in solving the problems and challenges of directional tissue repairation.Collagen(COL)is the main structural protein of extracellular matrix.It has been widely studied and applied in the field of biomaterials attributed to its good biocompatibility,biodegradability and low immunogenicity.Guar gum(GG)is a natural plant polysaccharide with excellent water retention,thickening properties,biological stability and biocompatibility.The aim of this thesis is to prepare the directional collagen-based hydrogels using type I collagen and guar gum as raw materials.The natural biopolymer crosslinker dialdehyde guar gum(DAG)was prepared by the oxidation of guar gum.Directional structure collagen based hydrogels were fabricated by adjusting the Schiff’s base reaction between collagen and DAG using directional freezing-low temperature curing crosslinking method.And the structure,physical and chemical properties as well as biological properties of hydrogels were systematacially investigated.In this work,the following three main parts of reaserch have been carried out:1.DAG was prepared by the specific oxidation of ortho hydroxyl group in GG molecule using Na IO4.DAG with different aldehyde group contents were prepared by controlling the p H value,the amount of Na IO4 additive,the reaction temperature and time in the reaction system.The results showed that low p H value could promote the reaction by providing H+ needed for the catalytic reaction.The oxidation efficiency of GG could be improved by appropriately increasing the molar ratio of Na IO4 to GG and turning temperature up.However,too high temperature triggered hemiacetal reaction,and even caused a large number of GG molecular chain broken.The amount of aldehyde groups in the DAG could be increased by extending the oxidation time.Nevertheless,the aldehyde group formed slowly and the molecular chain of DAG was significantly degraded when the reaction time was longer than 4 h.Considering the aldehyde group content of DAG and the degradation degree of its molecular chain,the optimized reaction condition for the preparation of DAG is as follows: the p H=3,the molar ratio of Na IO4 to guar gum was 1.5:1,the reaction temperature was 40 oC and the oxidation time was 4 h.The aldehyde of DAG prepared by the above optimal parameter was 23.30%.In this part,the influence of preparation process on the molecular structure and aldehyde group content of DAG was studied in detail,which was significative to the research of oxidative modification mechanism of galactomannan and the development of natural biological macromolecular aldehyde crosslinking agents.2.Type I collagen was extracted from fresh bovine achilles tendon by acetic acid-pepsase method.The dialdehyde guar gum-collagen(DAG-COL)hydrogel with directional pore structure were prepared by using directional freezing-low temperature curing crosslinking technology.The Schiff’s base chemical crosslinking reaction was achieved between the aldehyde groups of DAG molecule and the amino groups of collagen molecule during preparation procedure.The results found that the extracted collagen retained a relatively complete triple-helical structure,indicating that the undenatured type I collagen was obtained in this work.The crosslinking degree of DAG-COL hydrogel increased with the increase dosage of DAG and more than about 77.88%.Significantly,it was found that the DAG-COL hydrogels prepared by directional freezing-low temperature curing crosslinking technology showed directionally oriented pore structure,and the their pore diameter was decreased gradually with the increasing dosage of DAG.In the process of directional freezing and low temperature solidification crosslinking,the unidirectional cold source caused the water in the solution to form ice crystals along the cooling direction,which promoted the collagen and the crosslinker in the liquid microphase to come close to each other due to the action of concentration and ice crystal extrusion.As a result,directional DAG-COL hydrogel with anisotropic structure formed by entangling and cross-linking between DAG and collagen.This work provided new ideas and methods for the preparation of hydrogels with directionally oriented pore structure and excellent physical and chemical properties.3.The DAG-COL hydrogels with directionally oriented pore structure was used as cell scaffolds,and their mechanical property,water retention capacity,swelling property,thermal stability,blood compatibility,cytotoxicity,in vitro biodegradability were systematically studied.Moreover,the effect of DAG-COL hydrogels cell scaffolds on inducing and regulating the proliferation and migration direction of L-929 fibroblast epidermal cells was also investigated.The results showed that the mechanical properties,water retention capacity and thermal stability of DAG-COL hydrogels were significantly improved,while their swelling degree was decreased by the chemical crosslinking of DAG.Therefore,DAG-COL hydrogel as a tissue engineering materia can effectively avoid the compression of surrounding tissues by absorbing tissue fluid,which further broadens the application field of collagen-based hydrogels.The hemolysis rate of the DAG-COL hydrogels cell scaffold was less than 5%,which met the application requirements of biomedical materials.The bovine serum albumin adsorption capacity of DAG-COL hydrogels was slightly weakened,which made great contribution for reducing the potential risk of thrombosis once contact with blood.It indicated that DAG-COL hydrogel cell scaffolds had good blood compatibility.The in vitro biodegradation rate of DAG-COL hydrogels cell scaffolds was significantly reduced on account of the formed three-dimensional network structure achieved by the crosslinking between DAG and collagen.The improved degradation stability of DAG-COL hydrogel cell scaffolds is beneficial to guide tissue repair in vivo.The survival rates of the L-929 cells were all over 80% after culturing with the extracting liquid of DAG-COL hydrogel cell scaffolds.It demonstrated that DAG-COL hydrogel cell scaffolds were non or low cytotoxic.It should be noted that the DAG-COL hydrogel cell scaffolds had a significant inducing effect on the migration direction of L-929 cells,and it promoted the growth of cells in a specific direction along the directional structure of the scaffolds.Overall,the DAG-COL hydrogel cell scaffolds with a directional structure showed potential application prospects to repair anisotropic tissues in tissue engineering.