Research Of The Bi-layer Hydrogel Based On PVA For Osteochondral Tissue Engineering

With the continuous development of aging process,the incidence of osteochondral defects keeps rising because of degenerative diseases,inappropriate exercise patterns,osteoarthropathy and so on.The osteochondral defects already have caused a serious impact on patients’ physical and mental health.Osteoarthritis,which is formed by the natural wear and tear of cartilage due to aging,is one of the major reasons leading to cartilage damage.The natural articular cartilage presents limited self-repair and regeneration capability for its avascular and aneural inner structure.Meanwhile,articular cartilage and subchondral bone are interconnected in the anatomical structure and affect each other in the biological function,resulting in the fine and complex structure of osteochondral interface.On the whole,osteochondral tissue is a complex functional unit,but each layer owns different structural,mechanical and biological characteristics.Therefore,damage to either cartilage or subchondral bone would affect the entire osteochondral tissue.Currently,the treatments for osteochondral defects are not ideal but palliative.As a promising alternative,osteochondral tissue engineering might provide significant advantages than current traditional therapies.The main obstacle of osteochondral tissue engineering lies in the simultaneous fabrication of an integrated bionic scaffold with different functions,structures and good interface adhesion.Thus,this paper is focused on fabricating an integrated osteochondral repair hydrogel and analysing the related performances of the bi-layer hydrogel.The main research contents are as follows:1.Firstly,the different content ratio of PVA/Col-II composite hydrogels were fabricated which were further modified by certain content CS as articular cartilage hydrogel implantation.The hydrogels were fully cross-linked by physical crosslinking,without any chemical crosslinking agent.After the related physico-chemical performance analysis of the hydrogels,we found that the related properties of the composited hydrogels were the best,when the PVA and Col-II content reached to 1:1.The compression mechanical modulus of PVA/Col-II(1:1)reached to the highest(11 ± 1.7 k Pa),compared with pure PVA group(4.9 ± 0.6 k Pa).Furthermore,we extracted the murine chondrocytes and seeded them onto the hydrogels.The results showed that the appropriate amount addition of COL-Ⅱ would improve chondrocytes viability,increase the proliferation rate and reinforce the spreading morphology in the PVA composite hydrogel.But excessive COL-Ⅱ addition leaded to PVA composite hydrogel structure change,which resulted in the decrease of its mechanical properties.Meanwhile,cell viability,spreading morphology and proliferation ability were also influenced.2.Secondly,the bone hydrogels were fabricated.We prepared BCP micro-nano particles and analyzed their composition and morphology.The results showed that the BCP particles were made by 40% HA and 60% β-TCP,and there were no other impurities.Then,different content CNTs were adopted to reinforce PVA/BCP,and the related physico-chemical performances were observed.The results presented that the mechanical property of the composited hydrogel reached to the highest about 0.081 ± 0.006 MPa,when the CNTs content was 0.25%.Furthermore,we seeded MC3T3-E1 cells onto the composite hydrogels to analyse the biological performance.We found that the BCP addition improved the MC3T3-E1 cells spreading,viability and proliferation abilities.And BCP could also induce early bone differentiation through the ALP experiment.Furthermore,the appropriate addition of CNTs could enhance the biological properties of composite hydrogels.3.Owing to the complex inner structure of osteochondral tissue,we adopted PVA/Col-II(1:1)as cartilage hydrogel layer,and used PVA/BCP/0.25%CNTs hydrogel layer as the subchondral bone hydrogel implantation.The bi-layer hydrogel was successfully prepared by circulating freeze-thaw method and its physico-chemical properties were analyzed.In terms of microstructure,the overall structure of the bi-layer hydrogel was porous with large and small pore connecting with each other,and the overall pore size of the bi-layer hydrogel decreased from upper layer to lower layer.Then,non-restrictive compression and tensile tests were carried out,and the mechanical strength of bi-layer hydrogel was found to be between the upper and lower hydrogels(0.014 ± 0.002 MPa and 7.14 ± 3 MPa respectively).Meanwhile,the bi-layer hydrogel’s tensile fracture surface was observed,and it was found that the fractures always appeared at the upper hydrogels,which proved that the prepared bi-layer hydrogel owned good interface adhesion.Meanwhile,in vitro degradation experiments were conducted on the bi-layer hydrogel,and statistical analysis was conducted on the mechanical properties of the bi-layer hydrogel during degradation.Furthermore,the self-heal ability of the bi-layer hydrogel was tested.The results confirmed that the bi-layer hydrogel had certain self-repairing ability.4.In this part,the prepared bi-layer hydrogel was implanted into the rabbit knee joint osteochondral defect model for in vivo repair.The repair time was lasted up to 12 weeks and the joint samples were took out in 4 weeks and12 weeks for correlation analysis.Through Micro-CT,immunofluorescence,immunohistochemical,push-out and mechanics performance tests,it was showed the max load was 67.24 ± 36.06 N and the compression mechanical modulus was 0.39 ± 0.1 MPa in bi-layer hydrogel implanted group.Furthermore,we found that the bi-layer hydrogel owned favorable biocompatibility,which could promote the early bone-cartilage formation,more stable integration between neo-tissue within host and much higher stiffness at regenerative area.The results confirmed the bi-layer hydrogel as a potential application in the osteochondral tissue engineering yield.