Effect Of Leptin Gene Modified Tissue Engineered Composites On Regenerationof Osteoporotic Periodontal Tissue

IntroductionOsteoporosis(OP) is kind of metabolic bone disease manifested as reduced bone mass and bone micro-structural damage, which increase bone fragility and risk of fracture. Periodontitis is an infection disease that causes the supporting tissue surrounding the tooth loss. OP not only can promote the absorption of alveolar bone in periodontitis, but also can affect the regeneration of periodontal tissue. Unfortunately, traditional periodontal treatment could hardly recover the total loss of the supporting tissue. Leptin(LEP), a member of cell factor family, is encoded by the obese gene and contributes mainly in the balance of food intake and energy. Recently, LEP was found can regulate bone metabolism by two pathways. Through the central nervous system, it will inhibit bone formation while it can significantly promote bone formation by peripheral application.The aim of the study was to explore whether the complex combined by human leptin(h LEP) gene modified osteoporotic rat bone marrow stromal cells(O-r BMSCs) and β-tricalcium phosphate(β-TCP) could enhance the ability of bone formation in nude mice and the repair of periodontal tissue in periodontal fenestration defects in osteoporotic rats. Methods and resultsFirstly, we established osteoporotic rat model and compared the ability of proliferation and osteogenic differentiation between O-r BMSCs and sham rat BMSCs(S-r BMSCs).The osteoporotic rat exhibited significant weight gain and bone density decrease, as well as histological sparse femur. Both kind of r BMSCs were successfully isolated and cultured with the manifestation that the proliferation and osteogenic differentiation of O-r BMSCs was weak.Secondly, we construct adenovirus that carrying the h LEP gene(Ad-h LEP-EGFP) and evaluate the gene transfection efficiency in O-r BMSCs followed by h LEP expression detection. Ad-h LEP-EGFP was successfully constructed and can efficiently transfect O-r BMSCs with the 72 h transfection efficiency of 84.5% under the optimal multiplicity of infection(100) by flow cytometry. The transfected O-r BMSCs showed strong green fluorescence under fluorescence microscope and can be detected effective h LEP gene and protein expression by RT-PCR, Western Blot, ELISA and immunocytochemistry, which proved the successful transfection of h LEP into O-r BMSCsThirdly, we compared the differences in the proliferation and osteogenic differentiation between the transfected and blank O-r BMSCs.After h LEP gene transfection, no obvious morphological changes were observed under inverted phase contrast microscope and fluorescence microscope. MTT assay suggested non-affected proliferation by transfection. However, increased alkaline phosphatase(ALP) activity, enhanced ability of mineralized nodules formation and upregulation of runt-related transcription factor 2(Runx2), ALP and collagen type ?(Col-?) gene expression were detected by ALP activity kit, alizarin red staining and real-time RT-PCR, respectively.Fourthly, we construct h LEP modified tissue engineered composite by cultured h LEP transfected O-r BMSCs inβ-TCP and evaluate the bone formation ability of the composite in vivo.Well adhering condition and growth of O-r BMSCs on β-TCP were observed under fluorescence microscope and scanning electron microscope, which proves the biocompatibility of β-TCP in vitro. After 4- and 8-week implantation into subcutaneous tissue of nude mice, the bone formation of all tissue engineered composites were evaluated by histological measurement with the result that the formed bone-like structure were counted most in Ad-h LEP-EGFP group(P <0.05).Lastly,we prepared periodontal fenestration defects in osteoporotic rats and evaluated whether the h LEP modified tissue engineered composite could enhance periodontal tissue regeneration in the periodontal defects.All tissue engineered composites or β-TCP alone were implanted into the periodontal fenestration defects and Periodontal regeneration was evaluated histomorphometrically after 10 and 28 days. We found that the area of newly formed alveolar bone(NB) in the Ad-h LEP-EGFP was the largest in both detective time-points while those in theβ-TCP group was the smallest(P<0.05). And no difference was showed between the Ad-EGFP and OVX group(P>0.05). As for the new periodontium(NP), no significant differences were presented between all the 4 groups(P>0.05). Conclusions 1. There is a diminished capacity of proliferation and osteogenic differentiation of BMSCs from osteoporosis individual. 2. Adenoviral vectors can mediate efficient expression of the h LEP gene in O-r BMSCs. 3. h LEP gene modification can improve osteogenic differentiation of O-r BMSCs. 4. h LEP genetically modified O-r BMSCs can grow well on the β-TCP and promote ectopic bone formation in vivo, suggesting that h LEP genetically modified O-r BMSCs might be expected as seed cells for tissue engineering. 5. h LEP gene modified tissue engineered composite can promote regeneration of periodontal defects in osteoporotic rats, which could be expected to be helpful for periodontal tissue engineering research and treatment.