Injectable Thermo-sensitive Chitosan/Collagen/β-glycerophosphate Composite Hydrogel For Bone Tissue Engineering:A Preliminary Study

In recent years, much attention was increasingly paid to fabricate an injectable sol-gel system for biomedical application. Therapeutic drugs, growth factors or cells which were pre-incorporated in liquid sol-gel solution could be directly infused into desired tissue, organ or body cavity in a minimally invasive manner and then formed into a gel scaffold in situ. Most studies of injectable materiel have focused on biopolymers so far.Chitosan, an amino-polysaccharide derived from chitin, is a kind of pH-dependent cationic polymer characterized by its biocompatible, biodegradable, and antimicrobial properties. In recent decades, chitosan was widely used in tissue engineering and regenerative medicine. Acid chitosan solution was commonly mixed with a basic beta-glycerophosphate (β-GP) solution to prepare a thermally responded pH-dependent sol-gel system. Chitosan share similar structure of glycosaminoglycans (GAGs) and its degradation time can be adjusted by changing its degree of deacetylation (DDA) to optimize its utility. Therefore, chitosan-based injectable hydrogels were also widely used for cartilage repair, nucleus pulposus regenerated, drug release and other biomedical utilities.In bone tissue engineering, chitosan has shown excellent biological properties to be used alone or combined with other bioceramics such as hydroxyapatite(HA) and tricalcium phosphate(TCP), chitosan also showed excellent osteoconductivity and could accelerate osteoblasts growth in the scaffold.So, we attempted to add another biomaterial, typeⅠcollagen, into the chitosan solution to fabricate a physical cross-linked chitosan/collagen/GP hydrogel. TypeⅠcollagen is a significant constituent of the natural bone extracellular matrix (ECM), which provides a structural framework for connective tissue and plays a key role in the temporal events cascade leading to formation of new bone. It was also proved that integrin-mediated adhesion to typeⅠcollagen could enhance osteodifferentiation of human BMSCs. TypeⅠcollagen was proposed to add into chitosan solution for better biological property beforeβ-GP initiated gelation.In this study, the noval thermo-sensitive injectable chitosan/collagen/β-GP hydrogel was fabricated and its thermo-sensitivity were tested. The BMSCs and HPLCs were seeded into the chitosan/collagen/β-GP composite hydrogel for cell viability test and marcomorphology observation. The BMSCs or HPLCs were also seeded into the chitosan/collagen/β-GP composite hydrogel for in vivo histological evaluation by the rude mice model. Its potential to be used for bone tissue engineering was evaluated.PartⅠ:Fabrication of a thermo-sensitive injectable chitosan/collagen/GP hydrogel scaffold, pH, gelation time and osmolality testAim:to fabricate a hydrogel scaffold which have injectability, easy to handle with and suitable to cell growth in it.Methods:the chitosan/collagen/GP hydrogel was fabricated by proper riato of mixed chitosan/collagen solution initialed by GP and temperature. PH meter and osmolality meter record the data. The gelation time was decided by invert the tube.Results:the pH of chitosan/collagen/GP hydrogel was between 7.09-7.21. The osmolality of the culture medium in contact with the hydrogel was range of 295-305 mmol/kg. Gelation time of chitosan/collagen/GP hydrogel was around 8 minutes.Conclusion:the good physical properties of chitosan/collagen/GP hydrogel made the composite scaffold suitable used as a cell carrier apply for tissue engineering.PartⅡ:Biocompatibility and osteogenic property of the BMSCs in chitosan/collagen/GP composite hydrogel scaffold:in vitro and in vivoAim:to test the biocompatibility of the scaffold to BMSCs. To evaluate the potential of this hydrogel used for bone tissue engineering.Methods:the BMSCs were seed into the chitosan/collagen/GP hydrogel, the cell growth in the scaffold was observed by live/dead cell toxic kit and SEM. The pre-osteodiffererntion BMSCs were seed into the hydrogel and be implanted to the subcutaneous of nude mice for 4 weeks. Chitosan/GP group was used for control.Results:More than 95% of cells were alive after 7 days culture by the live/dead test. From the SEM observation, it was founded the BMSCs were growth and expanded well in the chitosan/collagen/GP hydrogel. The bone mineral nodules were founded in the hydrogel after 28 days osteogenic culture. The history result showed the ectopic bone and vessel were formed.Conclusion:the chitosan/collagen/GP hydrogel have the huge potential to be used for bone tissue engineering.partⅢ:Biocompatibility and osteogenic property of the HPLCs in chitosan/collagen/GP composite hydrogel scaffold:in vitro and in vivoAim:to test the biocompatibility of the scaffold to HPLCs. To evaluate the potential of this hydrogel used for periodontal bone defect repair.Methods:the HPLCs were seed into the chitosan/collagen/GP hydrogel, the cell growth in the scaffold was observed by live/dead cell toxic kit and SEM. The BMP-2 and HPLCs were combined into the hydrogel and be implanted to the subcutaneous of nude mice for 2 weeks. Chitosan/GP group was used for control.Results:More than 95% of cells were alive after 7 days culture by the live/dead test. From the SEM observation, it was founded the HPLCs were growth and expanded well in the chitosan/collagen/GP hydrogel. The bone mineral nodules were founded in the hydrogel after 28 days osteogenic culture. The history result showed the cartilage and bone lacune were formed in the scaffold.Conclusion:the chitosan/collagen/GP hydrogel have the huge potential to be used for periodontal bone defect repair.