Study On Regeneration Of Alveolar Bone Defect By Adipose Tissue-derived Stem Cells Combined With Allogeneic Acellular Cartilage Matrix Particles

Objective:To investigate the biocompatibility of rabbit acellular cartilage matrix particles（ACM）and to clarify its promoting effect on adipose tissue-derived stem cells（ADSCs）endochondral osteogenesis in SD rats.Methods:The ear cartilage of 3-month-old New Zealand white rabbits was collected and prepared by grinding and soaking method.The primary ADSCs were extracted for three-dimensional differentiation and flow cytometry to detect cell dryness,and CCK-8 cell proliferation assay to detect cell proliferation activity.The cells were divided into ADSC control group and ADSC+ACM experimental group.The experimental group was cultured for 3 days for Calcein-AM/PI fluorescence staining experiment,and the microscopic morphology of the control group and the experimental group was observed under scanning electron microscopy.Real-time quantitative reverse transcription PCR（q-PCR）was used to detect m RNA of type 2 collagen（COL-2）,type X collagen（COL-X）,SOX-9,vascular endothelial growth factor（VEGF）,alkaline phosphatase（ALP）and Runt-related Transcription Factor 2（RUNX-2）expression level in ADSCs of each group at 7 and 14 days of chondrogenic induction.In vivo experiment,rats were divided into four groups: ADSC+ACM group,ACM group,ADSC+PLGA group and blank control group.The alveolar bone defect model of rats was constructed,and materials and cells were implanted into the defect group.Three months later,the maxilla of rats in each group was detected by Micro CT and HE staining,and the heart,liver,spleen,lung and kidney of each group were stained by HE staining to observe the tissue toxicity of scaffold materials.Results:Rabbit ACM was successfully prepared.The ADSCs could differentiate in three ways and had specific surface markers of stem cells.Compared with the control group,rabbit ACM had no cytotoxicity and promoted cell proliferation（P<0.05）,and adipose stem cells could adhere to ACM.q-PCR results showed that the m RNA expression levels of VEGF,ALP and RUNX-2 in ADSCs in ADSC+ACM group were higher than those in ADSC group at 7 days（P<0.001）.At day 14,compared with ADSC group,the m RNA expression of COL-2,VEGF,ALP and RUNX-2in ADSC+ACM group was increased（P<0.05）,and the m RNA expression of ALP and RUNX-2 was increased（P<0.001）.COL-X expression was decreased（P<0.05）.Compared with 7 days,the expression level of ALP m RNA,RUNX-2 m RNA and COL-2 m RNA of ADSCs in ADSC group at 14 days decreased（P<0.001）,and increased（P<0.001）.The COL-X m RNA expression level was increased（P<0.05）;compared with 7 days,the expression level of COL-2 m RNA and COL-X m RNA of ADSCs in ADSC+ACM group was increased at 14 days（P<0.001）,and the expression level of COL-2 m RNA was increased at 14days（P<0.05）.The m RNA expression levels of VEGF,ALP and RUNX-2decreased（P<0.001）.Western blot showed that VEGF,ALP and RUNX-2were highly expressed at 7 days.Micro CT results showed that the experimental group had the most new bone formation and the highest bone density.The bone density of ACM group was slightly lower than that of the experimental group.The amount of new bone formation in positive control group was lower than that in ACM group,and the bone mineral density was slightly lower than that in negative control group.In blank control group,no new bone was formed and bone resorption to perforation.The results of bone HE staining showed that in the experimental group,the bone was almost repaired to the original bone surface,and there were more vascular tissues in the new bone.The number of blood vessels in the ACM group was slightly less.Almost no neovascularization was observed in the ADSC+PLGA group.HE staining of heart,liver,spleen,lung and kidney showed no significant difference among the four groups.Conclusion:Rabbit acellular cartilage matrix particles have three-dimensional pore structure and good biocompatibility.Rabbit acellular cartilage matrix particles have chondrogenic induction ability,which is beneficial to the occurrence of endochondral osteogenesis.In vivo experiments showed that rabbit ACM scaffold combined with SD rat ADSCs can effectively reconstruct bone defects,which is a valuable new method for bone regeneration.