Enhanced Osseointegration Of Implant In Osteoporosis Rat Through The Application Of Gingiva-derived Mesenchymal Stem Cell

Objective:The study assessed that whether systemic application of GMSCs can enhance osseointegration of a titanium implant in osteoporosis rats to put forward a new idea for promoting the osseo integration.Methods:1、GMSCs were isolated from GFP+transgenic C57/BL mice by enzymoly method and the cell surface markers were detected by flow cytometry analysis. Then GMSCs were mineralized induced and epithelial-like cell induced in vitro.2、Establish the osteoporosis model by ovariectomy:wistar female normal rats of three months were as control group, ovariectomic wistar rats were as experimental group. Three months later, The rats were sacrificed to evaluate whether the model were successfully built by analysis of ALP in serum and hematoxylin and eosin (HE) staining.3、The 63 wistar rats of three months were randomly divided into three groups:A group (normal+PBS) (n=21):PBS was administrated via tail vein 30min before the implant. B group (ovariectomic+PBS) (n=21):three months after ovariectomy, PBS was administrated via tail vein 30min before the implant. C group (ovariectomic+GMSCs) (n=21):three months after ovariectomy, GMSCs were administrated via tail vein 30min before the implant.4、Titanium implants were transplanted into the distal end of femurs after anesthesia. Six rats of every group were sacrificed 1、4 and 8 weeks after the implant. The mRNA expression of osteogenesis gene and inflammatory factors were measured by quantitative real-time RT-PCR. The undecalcified sections were stained with methylene blue-acid magenta to analyze the osseointegration. Three other rats were sacrificed to detect the GMSCs homing by immunohistochemical analysis and DAPI staining 4 weeks after the implant.Results:1、GMSCs isolated from GFP+ transgenic C57/BL mice appeared fibroblast-like spindle shape and the expression of GFP was stable. Flow cytometry analysis revealed that the GMSCs expressed CD29 (99.9%)、CD44 (92.6%) but not CD31 (4.0%)、 CD34 (5.1%)、 CD45 (0.8%)、 CDllb (1.5%).After osteogenesis induction, the mineralized nodules were visible and could be stained by Alizarin Red solution. After epithelial-like cell induction, the morphology of cells were polygonal like paving-stone.2、The osteoporosis model was established. The ALP in serum of experimental group was higher than the control group apparently (p<0.05). The hematoxylin and eosin (HE) staining revealed that compared to the control group, the bone trabecular became thinner and discontinuous.The number of bone trabecular reduced and the space became bigger in femurs of experimental group.3、The mRNA expression of osteogenesis gene and inflammatory factors1 week after implant:group A and C compared with group B, the expression of BSP was significantly higher(P<0.05) and the expression of IL-1 was significantly lower(P<0.05). The expression of OPN had statistical significance between group A and B only(P<0.05).4 weeks after implant:group A and C compared with group B, the expression of BSP and OPN was significantly higher(P<0.05), the expression of IL-1 was significantly lower(P<0.05).8 weeks after implant:group A and C compared with group B, the expression of BSP was a little higher(P>0.05), the expression of OPN was significantly higher and IL-1 was significantly lower(P<0.05).4、The rate of osseointegration in group A and group C were higher than group B and the difference was statistically significant(P<0.05) except the group B and C in 1 week.5、 GFP+ cells could be detected in the tissue around the implant by immuno-histochemical analysis and DAPI staining.Conclusion:1、GMSCs with stable expression of GFP can be isolated from transgenic C57/BL mice by enzymoly method. The process is easy and the purity is high as the cell passage increase.The GMSCs has the potential of osteogenesis differentiation and epithelial-like cell differentiation.2、The model of osteoporosis can be established by removing the bilateral ovaries. The method is simple and the outcome is stable.3、Systemically transplanted GMSCs can home to the bone defect, differentiate into osteoblasts and promote local bone regeneration to promote the osseointegration.