Effect Of CEMP1 Gene Modified Bone Marrow Stromal Cell Sheet On Periodontal Regeneration In Osteoporotic Rats

ObjectiveWe use lentivirus that carrying the CEMP1 gene(LV-CEMP1-EGFP)transfect BMSCs,then we construct the cell sheets and implanted into the periodontal defect of rats with osteoporosis to investigate the effects of CEMP1 modified bone marrow stromal cell sheets on periodontal regeneration in osteoporotic rat.MethodsWe use whole bone marrow culture to isolated and cultured BMSCs in vitro and then the cell surface markers were detected by flow cytometry.The ability of multidirectional differentiation was observed after induction of osteogenesis and lipogenesis.The cell sheet was constructed by vitamin C continuous induction method,and its structure was observed by histological staining and scanning electron microscopy.The expression of extracellular matrix was detected by immunofluorescence assay.BMSCs were transfected with lentivirus LV-CEMP1-EGFP carrying CEMP1 gene and the cell sheets were constructed.The transfection efficiency was detected by flow cytometry,and the expression of CEMP1 gene and protein in the BMSCs membrane was detected by RT-PCR and Western Blot.The osteoporotic rat was established by bilateral oophorectomy,the weight of the rats and the morphological identification of femur and uterus were compared three months after the operation.Acute periodontal defect was constructed in the mandible of osteoporotic rat.Then the CEMP1 gene modified BMSCs cell sheets were implanted into the periodontal defect.Rats were sacrificed 28 days after surgery.Specimens were decalcified,embedded in paraffin,making pathological section,HE and Masson stain.The effect of CEMP1 gene modified BMSCs sheet on regeneration of periodontal tissue under PMO was evaluated by histological observation and measurement.ResultsBMSCs were isolated and cultured.After 21 days induction of osteogenesis and lipogenesis,alizarin red staining showed mineralized nodules and vacuole-like droplets were observed after oil red O staining.Surface marker were detected by flow cytometry,and the expression of CD29,CD90 and CD105 was high,while the expression of CD31,CD45 and CD34 was low.The BMSCs cell sheets were successfully constructed,and the cell sheet presented a translucent membrane shape.The live or dead fluorescence staining showed that the BMSCs cell sheets were mostly composed of active cells.HE and Masson stain showed that cell sheets were composed of multilayer cells arrange overlapping,rich in extracellular matrix,immunofluorescence staining results in high expression of FN,PN and Col-?.BMSCs cell sheets transfected with LV-CEMP1-EGFP were successfully constructed,and the transfection efficiency detected by flow cytometry(FCM)was 85.2%.Dense green fluorescence expression could be observed under fluorescence inverted microscope.RT-PCR and Western Blot results showed that CEMP1 gene and protein were highly expressed in the transfected BMSCs cell sheets.Osteoporotic model were successively established.Compared with SHAM group,OVX group had significantly increased body weight,decreased uterine volume,atrophy of glands,and sparse cancellous bone trabeculae with significantly reduced number.After implantation of CEMP1 gene-modified BMSCs cell sheets into PMO rats with periodontal defect,it was found that the LV-CEMP1-EGFP group had the largest new born cementum,alveolar bone and periodontal tissue area(P<0.05).There was no significant difference between LV-EGFP group and BMSCs cell sheet group(P>0.05).There was no significant difference in the width of new born periodontal ligament between each group(P>0.05).ConclusionThe BMSCs cell sheets were successfully constructed by vitamin C continuous induction,and the cell sheet is rich in extracellular matrix.CEMP1 gene modified BMSCs cell sheet can promote periodontal tissue regeneration in osteoporotic rat.