Hydrogel-hydroxyapatite-monomeric Collagen Type-Ⅰ Scaffold With Low-frequency Electromagnetic Field Treatment Enhances Osteochondral Repair In Rabbits

Objective:Cartilage damage is a common medical issue caused by trauma,aging,and disorders of cartilage itself and subchondral bone(including osteoarthritis,osteonecrosis,and osteochondritis dissecans).The chronic pain and dysfunction of affected joints significantly diminish the patient’s life quality.However,owing to the avascularity of cartilage and low metabolic activity of chondrocytes,such cartilage defects have limited capacity to heal spontaneously.Although there are several options for the treatment of cartilage defects,the complete repair of hyaline cartilage remains a major challenge in clinical practice owing to the inferior quality of regenerative tissue.Safe and non-invasive magnetic therapy combined with tissue engineering to repair cartilage may be a promising breakthrough.Methods:In this study,a composite scaffold made of Hydroxyapatite-Collagen type-Ⅰ(HAC)and PLGA-PEG-PLGA thermogel was produced to match the cartilage and subchondral layers in osteochondral defects,respectively.We synthesized the PLGA-PEG-PLGA thermogel by ring-opening polymerization approach.Then,we determined the temperature for the sol-to-gel transition of the thermogel.The influence of the thermogel on the viability of BMSCs was examined by live/dead staining at 1,4 and 7days.Next,we examined the in vivo effects of the PLGA-PEG-PLGA scaffolds loaded with BMSCs and stimulated with electromagnetic fields on the repair of osteochondral defects,using a rabbit model.We performed histological staining and used a quantitative scoring system in the subchondral bone evaluation to evaluate the effects of the scaffold on cartilage repair.Chondrogenesis and osteogenesis-related proteins,including Col2,Aggrecan,OCN and Col1,were examined using immunohistochemical staining to represent tissue regeneration.RT-PCR was also performed to evaluate the m RNA level of these markers in the regenerated tissue.Meanwhile,subchondral bone of the defect regions was also assessed by micro-CT and the bone volume per total volume was measured.Finally,we used western blot,RT-PCR,flow cytometry analysis and Immunofluorescence staining to explore the mechanism by which electromagnetic field treatment enhances osteochondral repair.Proteins involved in proliferation process,such as Cyclin D1,CDK4 and PCNA,were examined to evaluate the proliferation of BMSC,and the PI3K/AKT/m TOR pathway was examined to explain the effects of electromagnetic fields on BMSC proliferation.For the chondrogenic effects,chondrogenesis-related proteins(including Col2,Aggrecan and SOX9)were detected by immunofluorescence and Wnt1/LRP6/β?catenin pathway was evaluated as the potential mechanism.Results:The PLGA-PEG-PLGA copolymer was aqueous solution at 4 ℃ and converted into gel state without fluidity when heated up to 37 ℃.live/dead staining at revealed that the hydrogel did not affect activity and proliferation of BMSCs,and the cells were fairly evenly distributed in hydrogel.The boundary between the native and the regenerated tissue was almost eliminated by the application of electromagnetic fields,and the repaired tissue showed a similar appearance to the native hyaline cartilage.In the mechanical strength test,the electromagnetic fields group showed a lowest strain under the same stress,which indicated the new-formed tissue in the electromagnetic fields group possessed a satisfactory mechanical property with highest strength.The BV/TV was highest in the electromagnetic fields group in the repaired region.Histological evaluation revealed that the scores for the subchondral bone evaluation,including bone filling,bone morphology,and bone bonding were all highest in the electromagnetic fields group.The immunohistochemical staining for Col2,Aggrecan,Col X and OCN was the most positive within the chondral defect in the electromagnetic fields group.In accordance with the Immunohistochemical staining,the RT-PCR results showed that electromagnetic fields treatment significantly increase the RNA expression of Col2,Aggrecan,Col X,OCN and Col1.Electromagnetic fields significantly enhanced the expression of proliferation-related genes,including Cyclin D1,CDK4 and PCNA.Accordingly,the phosphorylation level of PI3 K,AKT and m TOR were increased remarkably in the presence of electromagnetic fields exposure.The PI3 K inhibitor(Dactolisib)or m TOR inhibitor(AZD8055)attenuated the increased expression of Cyclin D1,CDK4 and PCNA in the electromagnetic fields group.For the chondrogenic effects,the expression of Col2,Aggrecan and SOX9 proteins were all significantly strengthened by electromagnetic fields,according to the analysis of fluorescence intensity.PCR and western blot revealed increased expression of COL2,ACAN and SOX9 in the electromagnetic fields group compared to other groups.In addition,the gene expression of WNT1,LRP6 and β-catenin in electromagnetic fields groups were significantly higher than control groups.The inhibition of Wnt1 by SM04960 could partially downregulate the increased gene expression of Col2,Aggrecan and SOX9,which were induced by electromagnetic fields.Similar results were observed when the β-catenin inhibitor(LF3)was used.Conclusion:A composite scaffold was constructed using a HAC and PLGA-PEG-PLGA thermogel,which matched with cartilage and subchondral layers in osteochondral defects,respectively.The BMSCs encapsulated in the thermogel were stimulated by electromagnetic fields.Results from in vitro experiments suggested that electromagnetic fields could promote proliferation and chondrogenic differentiation of BMSCs by activating the PI3K/AKT/m TOR and Wnt1/LRP6/β-catenin pathways,respectively.In vivo evaluation further confirmed that electromagnetic fields could enhance repair of osteochondral defects,especially cartilage repair,combined with tissue engineering in rabbits.Therefore,this study significantly advances the potential application of electromagnetic fields or even other biophysical stimuli towards tissue regeneration.