| Background and objectivesRadiotherapy in combination with surgery is the treatment commonly used for patients with head and neck cancer. Surgical treatment may result in anatomical deformities. Dental implants can be used to replace missing teeth and anchor facial prostheses, allowing for reconstruction of tumor defects. However, radiotherapy influences the survival of dental implants adversely as bone quality is compromised by radiation. Hyperbaric oxygen(HBO) treatment has been used to promote osseointegration in irradiated bone, but its efficacy is still controversial. Some researchers focuse on implant surface modification, but no effective solution has been formed. In our previous research, we used the cell-sheet method to construct a marrow mesenchymal stem cells(MSC)-implant complex. The results demonstrated that it had osteogenic ability. But it is difficult to promote angiogenesis in the irradiated bone, which is critical for osseointegration. Endothelial progenitor cells(EPCs) could facilitate new vessel formation by differentiating into endothelial cells, incorporating into neovessels or producing paracrine signals. EPCs have been used for complex bone defects to improve bone regeneration. In the present study, cell sheet-implant complexes were fabricated by co-cultured BMSCs and EPCs, aiming to find an effective way to promote bone regeneration around implants in the irradiated bone and enhance osseointegration. Methods1.BMSCs were isolated and cultured by the whole bone marrow adherent method. BMSCs were identified by cell surface markers, osteogenic and adipogenic differentitation abilities. EPCs were isolated and cultured by density gradient centrifugation and differential adhesion. EPCs were further identified by surface markers. The abilities of capillary tube formation, Dil-Ac-LDL uptake and FITC-UEA-1 bingding were also tested.2. EPCs and BMSCs were co-cultured at ratios of 1:0, 10:1,5:1, 1:1, 1:5, 1:10 and 0:1. The ratio of co-culture for further study were choosen according to the expression of osteogenesis related genes and VEGF. Cell sheets of co-cultured cells were induced using a continuous Vc inducing method.3. Cell sheets were wrapped around the implants to construct cell sheet-complexes. To assess the in vitro osteogenesis of cell sheet-complexes, they were incubated with osteogenic medium for 5 days. The collagen secretion, alkaline phosphatase(ALP) production, extracellular matrix(ECM) mineralization as well as expressions of osteogenesis-related genes and proteins were tested.4. To assess ectopic osteogenesis of cell sheet-complexes, the cell sheet-complexes were transplanted subcutaneously into the backs of nude mice. Bone formation were tested by micro-CT and examination of hard tissue slices 8 weeks after transplantation.5. To assess the osseointegration of cell sheet-complexes in irradiated bone, irradiated tibias of rats were used for the implantation. Bone formation around implants were tested by Micro-CT and examination of hard tissue slices. Bone fragments around implants were analyzed for gene expression. Results1. BMSCs positively expressed MSC markers such as CD29, CD44, CD90 and were negative for CD31, CD34. BMSCs showed multipotent differentiation ability by differentiating into osteoblasts and adipocytes when cultured in induction media. EPCs positively expressed EPC markers such as CD31, CD144 and VEGFR2. They exhibited a tube-like structure when seeded on Matrigel. They were also positive for Weible-Palade bodies, Dil-Ac-LDL uptake and FITC-UEA-1 bingding.2. The co-culture ratio of 10:1 showed the optimal gene expression of osteogenesis markers and VEGF, thus were choosen for further study. The co-cultured cells could survive in the Vc containing medium and form cell sheets.3. The co-cultured group showed enhanced osteogenic differentiation by denser extracellular matrix mineralization, up-regulated expression of osteogenesis related genes and proteins. Gene expression of VEGF was also higher in the co-cultured group.4. In the study of ectopic osteogenesis, the co-cultured group showed obvious new bone formation around implants as indicated by micro-CT and Van Gieson staining.5. After implanted in irradiated tibias of rats, the co-cultured group showed enhanced osseointegration. The new bone formation in the co-cultured group was better than the other groups as indicated by Micro-CT, sequential fluorescent labeling and Van Gieson's staining. Expression of osteogenesis related genes and VEGF was the highest in the co-cultured group. Conclusion1. Co-cultured EPCs and BMSCs at the ratios of 10:1 could enhance the expression of osteogenic related genes and VEGF. The co-cultrued cells could form cell sheets.2. Co-cultured cell sheets of EPCs and BMSCs showed enhanced osteogenic differentiation around titanium materials in vitro and enhanced ectopic bone formation in vivo. 3. Co-cultured cell sheets enhanced osseointegration in irradiated rat tibias. |
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