| Background and PurposesSevere periodontitis will eventually lead to periodontal supporting tissues loss. In recent years, tissue engineering technology has become a hot point for the therapy of periodontal diseases. There are three mainly factors in tissue engineering:Seed cells, biological scaffold materials and cytokines. However, due to the limited sources, low survival rate and immune rejection of seed cells, it cannot achieve the desired effect. The development of in situ tissue engineering will avoid the disadvantages of traditional tissue engineering by the application of chemokines that could recruit and home stem cells. Local administration of chemokines can facilitate the chemotaxis and migration of stem cells to the site of injury. In addition, systemic application of PTH can mobilize stem cells from bone marrow into circulation, which induce more stem cells migrate to the site of defect. The combination of PTH and SDF-1 can achieve this strategy.SDF-1 is an important chemokines which has been researched widely in other areas of medicine. CXCR4 (C-X-C chemokine receptor type4) is the receptor of SDF-1 that is expressed in the surface of a variety of stem/progenitor cells, and can be coupled with SDF-1 to mediate cell migration. Local administration of SDF-1 can facilitate the migration of stem cells to the defect area. Our previous study demonstrated that SDF-1 promoted the differentiation and proliferation of periodontal ligament stem cells. Local administration of SDF-1 can recruit stem cells to the periodontal defects, reduce inflammatory responses during the early phase of wound repair, and improve the quantity and quality of newly formed bone. PTH is a kind of anabolic drug for the treatment of osteoporosis. Intermittent PTH treatment can increase osteoblast numbers by converting lining cells to mature osteoblasts in vivo, promote the expression of osteogenesis related gene such as type I collagen and bone calcium protein to induce bone regeneration. PTH also has the ability to promote bone marrow-derived progenitor cells migrate to peripheral blood. Furthermore, studies show that PTH can raise the level of endogenous SDF-1, and then promote osteoblast derived from bone marrow homing to the site of defect by the chemotaxis of SDF-1.Based on the prophase research of our research group and the theory for the SDF-1 and PTH, we speculate that the combination of PTH and SDF-1 can promote the mobilization and homing of stem cells at the extreme. Systemic application of PTH can mobilize bone marrow-derived stem cells into peripheral blood and raise the level of endogenous SDF-1. Meanwhile, local administration of SDF-1 can facilitate the migration of stem cells to the site of injury. We established periodontal defects in rat models to explore the effects of PTH and SDF-1 combination on the number of stem cells, CXCR4 expression in periodontal wound, and periodontal regeneration in vivo.Materials and Methods1、The effect of PTH and SDF-1 combination on the mobilization of MSCs and CXCR4+cells in periodontal defectBilateral mandibular periodontal defect models were established in male Wistar rats, left-side defects were implanted with collagen membranes loaded with 5jig SDF-1, while the right-side defects were filled with collagen membranes loaded with PBS as the control group. Rats were numbered and divided randomly into two groups: Intraperitoneal injected with human PTH (1-34) at 40ug/kg or sodium chloride on alternate days. Mandibles were harvested at 3 days,1,2,4 weeks after surgery and made into paraffin section. Double immunofluorescence staining of CD90+/CD34-was performed to calculate the number of MSCs, and immunofluorescence staining of CXCR4 was performed to observe CXCR4+cells in the site of periodontal defect.2、The effect of PTH and SDF-1 on the regeneration of periodontal tissues in the defectBilateral mandibular periodontal defect models were established. The alveolar was obtained at 3 days,1,2,4,8 weeks after operation and samples were embedded in paraffin. H&E staining was performed to calculate the percentage of new bone area, determine the orientation of fibers, and observe the newly formed cementum, so that we can verify the effect of PTH and SDF-1 combination on the regeneration of alveolar bone, ligament and cementum in the process of periodontal defect healing. Meanwhile, Respectively, alkaline phosphatase (ALP), runt related transcription factor 2 (Runx2), collagen (Col I) immunohistochemical staining and TRAP staining were performed to compare the difference of osteogenesis and osteoclastogenesis between four groups in the periodontal defect healing process.Results1、The effect of PTH and SDF-1 combination on the mobilization of MSCs and CXCR4+cells in periodontal defectAfter surgery, all the animals survived without obvious inflammation and immune rejection. The species were taken to stain double immunofluorescence of CD90+/CD34-, the results showed that the number of MSCs in the PTH+SDF-1 group was more than other groups at each point in time (P<0.05). The immunofluorescence staining of CXCR4 showed that PTH and SDF-1 combination could facilitate the number of CXCR4+cells at the early stage of wound healing, and the differences were statistically significant compared with other groups (P<0.05).2、The effect of PTH and SDF-1 on the regeneration of periodontal tissues in the defectH&E staining of the periodontal defect showed that the combination of PTH and SDF-1 promote early osteogenesis, and the density of newly formed alveolar bone was higher than other groups, the results had statistically significant (P<0.05). Osteogenesis related protein immunohistochemical staining indicated that the combination of PTH and SDF-1 can promote the expression of ALP and Runx2 at the early stage of healing process, and the differences were statistically significant compared with other groups (P<0.05). At the late stage of the healing process, the level of Col I in PTH+SDF-1 group showed no difference with SDF-1 group (P>0.05), which was significantly higher than PTH and control group (P<0.05). TRAP staining showed that PTH and SDF-1 combination can induce early bone osteoclastogenesis. The regeneration of periodontal fibers and cementum appeared at the late stage of tissue healing process (4 and 8 weeks). Newly formed cementum appeared at 4 week in the PTH+SDF-1 group, and was obviously thicker than other groups at 8 weeks. Compared to the native mature ligament, we found that the orientation of PTH+SDF-1 group was similar to mature ligament at 8 weeks.Conclusion1、The combination of PTH and SDF-1 can recruit host-derived MSCs, promote the mobilization of CXCR4+cells, indicating that PTH can raise the level of SDF-1, and thus enhance its function.2、PTH combined with SDF-1 significantly promoted periodontal tissue regeneration. They can increase bone regeneration, induce early bone osteoclastogenesis, regulate the balance of bone anabolism, facilitate the expression of osteogenesis related protein, and promote the quality and quantity of regenerated bone. Moreover, the combination of PTH and SDF-1 can improve the quality of the newly formed periodontal ligament, and increase cementum formation. |
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