Experimental Study On Thermosensitive Hydrogel Containing KGN And TGF-β3 Combined With BMSCs To Promote Osteochondral Regeneration

Objective: Study on the induction of chondrogenic differentiation of BMSCs in vitro by KGN and TGF-β3.Preparation of the PLGA-PEG-PLGA thermosensitive hydrogel and study on the feasibility of the application of the thermosensitive hydrogel in osteochondral tissue engineering.To investigate the effect of PLGA-PEG-PLGA thermosensitive hydrogel coated with KGN and TGF-β3 combined with BMSCs on osteochondral defect repair in rabbits model.Method: 1.Cell pellet culture and two-dimensional monolayer culture was used to investigate the effect of KGN and TGF-β3 on chondrogenic induction of BMSCs.BMSCs were isolated from rabbit bone marrow and identified by flow cytometry and multidifferentiation.Then,BMSCs were seeded into confocal dishes and centrifuged into polypropylene tubes respectively for chondrogenic induction by different components chondrogenic induction medium for 21 days.After 21 days of chondrogenic induction,the cell pellets were sectioned into slices and stained with HE and alcian blue.Immunofluorescence staining of collagen Ⅱ and real-time polymerase chain reaction(RT-PCR)for cartilage related genes were performed to evaluate the effect of KGN and TGF-β3 on chondrogenic induction of BMSCs.2.The PLGA-PEG-PLGA triblock copolymers were synthesized.The critical gelling temperature of different concentration of PLGA-PEG-PLGA was tested by vial inverting test.The physical and chemical properties and biocompatibility of PLGA-PEG-PLGA thermogel were evaluated by swelling and degradation rate,cell proliferation and Live/Dead staining.KGN and TGF-β3 were encapsulated into the thermogel to study the release of KGN and TGF-β3 in vitro.3.Osteochondral defect models of New Zealand white rabbits were carried out for the reconstruction experiment in vivo.The rabbits were divided into four experimental groups: KGN+TGF-β3 group,the thermogel loading with BMSCs,KGN and TGF-β3 was implanted into the defect site;TGF-β3 groups,the thermogel loading with BMSCs and TGF-β3 was implanted into the defect site;blank thermogel group,the thermogel loading with BMSCs was implanted into the defect site;control group,without thermogel implantation.At the time points of 8 and 16 weeks,gross images,macroscopic score,histopathological analyses and histological score of the harvested femur condyles were taken to evaluated the cartilage regeneration.Results: 1.The BMSCs that we isolated were identified by flow cytometry and multidifferentiation.After 21 days of chondrogenic induction,histological staining,immunofluorescence staining and RT-PCR of cell pellets indicated that KGN and TGF-β3 can synergistically enhance chondrogenic differentiation.2.The critical gelling temperature of PLGA-PEG-PLGA thermogel increases with the increasing concentration.The critical gelling temperature of 20% PLGA-PEG-PLGA thermogel was 37℃.The swelling rate of 20% thermogel stabilized after 4 h.After immersed in PBS for 24 d,the thermogel degraded by 24%.The thermogel has no effect on cell growth and proliferation.In addition,the KGN or TGF-β3 thermogel showed a stable sustained release of KGN or TGF-β3.3.Compared with the other experimental groups,the cartilage regeneration in KGN+TGF-β3 group wan better,reflected in not only gross images but also histopathological staining.After 8 weeks and 16 weeks,the macroscopic and histological score of KGN+TGF-β3 group was higher than the other experimental groups(P<0.05).However,there was no significant difference in macroscopic and histological score between TGF-β3 group and blank thermogel(P>0.05).Conclusion: 1.In two-dimensional monolayer culture and pellet culture,KGN and TGF-β3 can synergistically promote chondrogenic differentiation of BMSCs.KGN can inhibit the high expression of cartilage related genes caused by TGF-β3.In addition,100 n M KGN has a better chondrogenic induction effect.2.20% PLGA-PEG-PLGA thermogel stably gelatinizes at 37℃ for 2min.The 20% thermogel has suitable swelling rate,degradation rate and good biocompatibility,which can load with BMSCs.The KNG and TGF-β3 thermogel can continuously release KGN and TGF-β3 at 37℃.3.KGN and TGF-β3 thermogel loading with BMSCs can promote the rabbit knee osteochondral defect repair.KGN and TGF-β3 are beneficial to the osteochondral defect repair.In vivo experiments,the combination of KGN and TGF-β3 had a synergically enhanced effect on cartilage repair,which provides a basis for the clinical application of KGN and TGF-β3.