Properties Of Glucosamine-loading Photocrosslinked Gelatin Hydrogels

Osteoarthritis (OA) is a kind of degenerative disease of articular cartilage caused by many factors such as the increasing age, joint strain and injury. Injectable hydrogel-based tissue engineering uses three-dimensional (3D) complex of cells and biomaterials to restore the form, structure and function of damaged tissue, which gives a new way for arthritis treatment. The 3D network of hydrogels can help stem cells differentiate into chondrocytes and be used as a drug carrier to support the transportation and release of drugs for OA in situ quantitatively.In our research, gelatin was selected as substrate of the hydrogels which is the hydrolysis product of collagen, the main composition of articular cartilage. Glucosamine molecules were covalently crosslinked into the photocrosslinked gelatin hydrogels after the chemical modification of glucosamine and gelatin. The physichemical properties of photocrosslinked gelatin hydrogels, the characteristics of glucosamine release and biocompatibility of the system were investigated. Detailed results of our research include:1. Double bonds were introduced to gelatin and glucosamine molecules through acrylation. N-acryloyl-glucosamine (AGA) and gelatin methacrylate (GelMA) were controllable crosslinked to hydrogels in the condition of ultra violet (UV) irradiation. The physichemical properties of GelMA-AGA hydrogels were investigated and analyzed and it was demonstrated that more radicals could be released in the system when more AGA were added. So the activity and extent of the crosslinking reaction was increased and hydrogel with much denser network was formed. These results illustrated reaction mechanism of the photocrosslinked system and structure unit of the product.2. A simple, accurate and validated reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of glucosamine and its derivatives. The HPLC separation was achieved on an amino column using a mobile phase of acetonitrile-phosphate buffer and UV detection of 194 nm. The method was validated for specificity, linearity, accuracy, extraction recovery, and solution stability. The method was successfully used for the analysis of released properties of glucosamine and AGA. The results showed that the release rate of covalently crosslinked AGA was much lower than the physically encapsulated glucosame. The amount of released AGA increased with the increasement of GelMA concentration in the system. Moreover, the relationship between GelMA concentration and the degree of polymerization was investigated under the condition of equal AGA concentration in the photocrosslinked system by means of determination of relative volume swelling ratio and degradation curves.3. Cytocompatibility of the glucosamine-loading photocrosslinked gelatin hydrogels was evaluated in this dissertation. The influence of constituents of the hydrogels and the released drugs on the cell viability was investigated and the adhesion and growth of bone mesenchymal stem cells (BMSCs) on the surface of hydrogels with different AGA concentration was compared. The growth and viability of BMSCs in the 3D network of photocrosslinked hydrogels was also studied. It was shown in the results that the modification of glucosamine molecules and the change of their release from photocrosslinked hydrogels could improve the cytocompatibility and reduce the apoptosis of BMSCs. It was observed that BMSCs exhibited better adhesion and more stretchable morphology on the surface of hydrogels with higher AGA concentration. The increase of AGA concentration could make the hydrogels harder, however, BMSCs still kept relative good viability in 3D hydrogel networks with the highest AGA concentration.The novelty of our research includes:1) Injectable hydrogels with the composition similar to articular cartilage were fabricated through photocrosslinking of gelatin and glucosamine.2) The theory was proposed that small AGA molecules could produce more active radicals under UV light which were able to increase the degree of polymerization of the photocrosslinked hydrogels.3) A HPLC/UV method without pre-column derivatization was developed to determine the concentration of glucosamine and its derivatives and the released properties of glucosamine and AGA from photocrosslinked gelatin hydrogels were investigated, which is the basis and precondition of OA treatment using in situ GelMA-AGA hydrogels.In conclusion, we have made a deep and basic research on the properties of glucosamine-loading photocrossed gelatin hydrogels. The results can provide early theoratical foundation for the application of this noval photocrosslinked hydrogel in the field of OA treatment.