The Role And Mechanism Of Hoxc10 In The Fate Of Chondrogenic Bone Formation Of BMSCs After Transdermal Free Transplantation

Objective:Large maxillofacial bone defects are a common clinical bone repair challenge,and despite the invention of many excellent biological materials,autologous bone grafting is still considered the gold standard for the treatment of bone defects.However,we found that limb bone grafting of the mandible requires a longer healing period to complete the fusion and reconstruction of the free bone block and the implanted bed of the mandible,and is prone to bone discontinuity.Hox9 to Hox13 are highly expressed in mesodermal-derived limb bones and regulate the development and remodeling of limb bones,thus they are called germline-specific genes.The status of Hox genes characterizes the embryonic origin of bone and determines the fate of bone regeneration.On this basis,we hope to reveal whether the originally highly expressed Hox gene changes after limb bone is grafted to mandible bone and whether the expression status of this gene affects the osteogenesis of free limb bone grafted to mandible bone,thus causing differences in bone healing.Methods:1.To prepare the animal model of trans-embryonic free autogenous bone grafting:the femur and mandible of SD rats were transplanted to each other and orthotopic,respectively.The formation of cartilage and bone after Bone grafting was evaluated by H&E staining,Masson staining,and safranine fixation green staining.The Transcriptome of the tissue at the suture of the bone graft was removed and sequenced to explore the possible mechanism of the difference in bone healing after transplantation.2.In vitro study of chondrogenic differentiation of BMSCs:（1）Acquisition and identification of limb bone BMSCs（L-BMSCs）and mandible BMSCs（M-BMSCs）:SD rat limb bone and mandible were collected under aseptic conditions for primary cell culture,and cells were passaged when the cell wall was greater than 90%,and the P₃-P₅ generation cells were selected for the experiment.（2）Transwell co-culture system was established:the upper and lower layers of the Transwell chambers were composed of femoral BMSCs;the upper and lower layers were composed of mandible BMSCs;the upper layer was composed of femoral BMSCs and the lower layer was composed of mandible BMSCs.The expression of cartilage-related genes,such as SRY-box transcription factor 9（Sox9）and collagen type II alpha1 chain（Col2a1）,was detected by real-time quantitative polymerase chain reaction（RT-PCR）.The RT-PCR was performed to validate the transcriptome sequencing results and detect the expression of Hoxc10 gene.（3）The overexpression adenovirus of Hoxc10 was constructed,and the adenoviral vector was transferred into M-BMSCs.The overexpression efficiency was demonstrated by fluorescence staining,and the upregulation efficiency of Hoxc10 was detected by RT-PCR to study the function of Hoxc10 in the process of chondrogenic differentiation of BMSCs and its effect on differentiation potential.Results:1.In vivo H&E staining,Masson staining,and Safranine O-Fast Green Stain demonstrated high bone maturity after mandible bone graft to mandible bone defect area healing,a large amount of cartilage formation after femoral bone graft to femur,and a small amount of red mature bone tissue and a certain amount of red cartilage matrix could be seen after femoral bone graft to mandible bone.RNA sequencing showed that Hoxc10 expression was most significantly different after femur transplantation into the jaw environment compared with jaw orthotopic transplantation;2.Successfully isolated L-BMSCs and M-BMSCs,and found no significant differences in the expression of chondrogenic marker genes Sox9 and Col2a1 and embryo specific gene Hoxc10 between L-BMSCs cultured in the microenvironment of M-BMSCs and those in the microenvironment of L-BMSCs.3.Adenovirus successfully overexpressed Hoxc10,RT-PCR experiments demonstrated elevated expression of cartilage-related genes Sox9 and Col2a1 after high expression of Hoxc10,and Alcian Blue experiments demonstrated that Hoxc10 showed more blue-stained proteoglycans after Hoxc10 high expression.Conclusion:Extremity bone shows more cartilage formation after trans-germline grafting to mandible bone compared to mandible bone in situ.The germ layer-specific gene Hoxc10 affects the osteogenic pattern of the limb bone implant block and new bone after bone grafting,and Hoxc10-positive high expression limb bone grafted into Hoxc10-negative low expression mandible environment retains the chondrogenic osteogenic pattern and affects bone graft healing.