The Influence Of TiO₂ Nanotubes On The Biological Behaviors Of Periodontal Ligament Stem Cells And Jawbone Mesenchymal Stem Cells

Dental implant surgery is one of the important methods to repair the missing teeth or dentition defect. The direct contact between implant and periodontal tissue lacking the periodontal ligament like structure as a buffer may increase the moment of the implant and lead to the failure of implant operation due to the poor or long time combination between the implant and bone. Lots of literatures reported that stem cell-based tissue engineering regeneration technology can restore damaged periodontal tissues effectively. Periodontal ligament stem cells and jaw bone mesenchymal stem cells as the seed cells in periodontal regeneration, which possess the capacity of multilineage differentiation and self-renew, play an important role in periodontal tissue regeneration.The surface morphology of pure titanium nanotubes that can simulate the natural bone tissue structure may show an excelent biological activity from the perspective ofbionics. The morphology of TiO2 nanotubes has been commonly used to modulate the function of a variety of cells, such as osteoblast cells, chondrocyte cells, fibroblast cells, endothelial cells, muscle cells, skin cells and mesenchymal stem cells. However, no investigation has been reported on the the influence of Ti O2 nanotubes on the biological behaviors of human origin periodontal ligament stem cells and jaw bone mesenchymal stem cells. In the present study, a systemic research is conducted on the effects caused by Ti O2 nanotube morphology on the biological behavior of those two kinds human stem cells. This study may provide comprehensively theoretical instruction on the optimization of implant surface structures as well as the combination promotion between the implant and periodontal tissues.Part I Fabrication of Ti O2 nanotubular topography on titanium surface[Objective] To fabricate different diameter sizes of TiO2 nanotubular topographies on titanium surface for the following biological experiments.[Methods] Pure Ti plates were polished with different roughness Si C sandpapers, then were anodized in an electrolyte containing 0.5wt% hydrofluoric acid and 1 M phosphoric acid for 1 h with a DC power supply and a platinum cathode at 5, 10 and 20 V to fabricate the NTs of different tube size. After ultrasonic cleaning, the morphology of the samples was inspected by field-emission scanning electron microscopy.[Results] The titanium surface treated by etching and anodic oxidation formed neatly arranged nanotube structures. The diameter sizes of nanotubes are positively correlated with the anodic oxidation voltage, i.e. 5, 10 and 20 V voltage formed 25, 50 and 90 nm diameters, respectively.[Conclusions] TiO2 nanotube morphology of different diameters on titanium surface can be formed by the etching and anodization methods.Part II The influence of Ti O2 nanotubes on periodontal tissue formation of periodontal ligament stem cells(PDLSCs)[Objective] To evaluate the influence of Ti O2 nanotubes on PDLSCs biological changes, including morphology, cell adhesion, proliferation and the capability to form periodontal tissues.[Methods] The PDLSCs were obtained using the limiting-dilution technique, and then cultured on different diameters Ti O2 nanotubes. The morphology of PDLSCs was observed by FE-SEM. Using hoechst staining to observe the cells adhesion, CCK-8 kit to detect the cells proliferation, ALP kit to detect the cells activity, sirius red staining to observe the collagen secretion, alizarin red staining to observe the sample surface extracellular matrix mineralization, flow cytometry to detect the cells cycle, and real time quantitative PCR to detect the cells genetic level.[Results] Compared with Ti control group, PDLSCs grown in spindle shape on the surface of the different diameters nanotubes with abundant long lamellipodia. The nanotube morphology facilitated the early adhesion of PDLSCs. The cell cycle results showed that NT10 and NT20 slightly promoted PDLSCs mitosis. Meanwhile, the CCK-8 proliferation results showed that the nanotube morphology inhibit the proliferation of PDLSCs. The opposite result may be due to antagonistic reaction during the cell proliferation and differentiation or the different experimental methods of processing these cells. Nanotube morphology promotes the collagen secretion and the expression of Col-I, Col-III gene of PDLSCs. In the condition of non-osteogenic induction, nanotube morphology can not promote PDLSCs osteogenic differentiation. The NT5 group promoted the up regulation of periostin and S100A4 expression.[Conclusions] Different diameters Ti O2 nanotube morphology can induce the PDLSCs to form different cell cytoskeletons. Nanotube morphology promoted the early adhesion, collagen secretion and related genes expression of PDLSCs.Part III The influence of Ti O2 nanotubes on the biological behaviors of jawbone mesenchymal stem cells(BMSCs)[Objective] To evaluate the influence of TiO2 nanotubes on BMSCs biological changes, including morphology, cell adhesion, proliferation and the capability of osteogenic differentiation.[Methods] The BMSCs were separated and purified, and then cultured on different diameters Ti O2 nanotubes. The morphology of BMSCs was observed by FE-SEM. Using hoechst staining to observe the cells adhesion, CCK-8 kit to detect the cells proliferation, ALP kit to detect the cells activity, sirius red staining to observe the collagen secretion, alizarin red staining to observe the sample surface extracellular matrix mineralization, and real time quantitative PCR to detect the cells genetic level.[Results] Compared with Ti control group, BMSCs stretched rapidly on the surface of the different diameters nanotubes with abundant long lamellipodia. The nanotube morphology facilitated the early adhesion of BMSCs. The adhesion cell numbers of NT5 and NT10 groups showed significant differences compared with the control group. Cell proliferation results showed that NT5 and NT10 groups promoted the cell proliferation, which indicated that the small diameter nanotubes possess a good proliferation ability and with increasing diameter of the nanotubes its cell proliferation ability getting weakened. In the both conditions of non-osteogenic induction and osteogenic induction, the NT20 morphology promotes the ALP activity, collagen secretion and the expression of Col-I, Col-III gene of BMSCs. In the non-osteogenic induction condition, nanotube morphology did not significantly promote the osteoblast differentiation of BMSCs. Gene expression results showed that NT20 could up regulation of ALP and RUNX2.[Conclusions] The nanotube morphology promoted the the early adhesion of BMSCs. Small diameter nanotubes promoted cell proliferation, while large diameter nanotubes promoted the cell ALP activity, collagen secretion and the expression of osteogenic genes.Part IV Studies on periodontal tissue regeneration of composite cell sheet around the Ti O2 nanotubes in vivo[Objective] To establish the PDLSCs and BMSCs composite cell sheet and investigate its periodontal tissue regeneration ability around the Ti O2 nanotubes in vivo.[Methods] The PDLSCs and BMCSs were obtained using the limiting-dilution technique. The flow cytometry was used to detect surface molecule of two kinds of stem cells. The clone capacity was observed by the cloning formation experiment. Using CCK-8 kit to detect the stem cells proliferation. Using alizarin red staining and oil red O staining to observe the potential multi-directional differentiation abilities of osteogenic and adipogenic differentiation. Vitamin C was used to induce these two types of stem cells to formed cell sheets. The composite cell sheets coated with Ti and HA were cultured in vitro and then were detected for the expression of periodontal related genes after one month. The Ti rods with different diateter Ti O2 nanotubes were coated with the the two kinds of cell sheets were placed into the HA hole and then were planted into the back of nude mice. After 8 weeks, the implantations were got out and cutted into hard tissue sections which were treated with VG staining to evaluate their ectopic periodontal regeneration ability.[Results] The PDLSCs and BMSCs were obtained using the limiting-dilution technique. These two kinds of stem cells were identified with the abilities of both clone forming and proliferation. These two kinds of stem cells were proved to be mesenchymal stem cells by detecting the surface molecules by flow cytometry. These two kinds of stem cells showedgood potential of osteogenic and adipogenic differentiation by alizarin red staining and oil red O staining. Gene expression results showed the up regulation of Col-I、Col-III and S100A4. Though not all of the nanotube morphology promoted upregulation of the expression, the results still indicated that the morphology could promote the periodontal differentiation of the composite cell sheets. In vivo experimental results of nude mice showed that the nanotube morphology could promote periodontal regeneration of the composite cell sheets. Cementum like structure was found in the cell sheets/NT10/HA group. In contrast, continuous structure can not be found in the groups of non-coated by HA. The resluts also indicated that the composite cell sheets showed a better regeneration ability than the single source cell sheets.[Conclusions] PDLSCs and BMSCs obtained using the limiting-dilution technique showed a good ability of multilineage differentiation and self-renewal potential. The nanotube morphology can promote periodontal regeneration of composite cell sheets in vivo.Innovations and significance:1. The present research is the first study of the influence of Ti O2 nanotubes on the biological behaviors of human origin periodontal ligament stem cells and jawbone mesenchymal stem cells. The results showed the effects of nanotube morphology with different diameters on the biological behavior of these stem cells.2. The periodontal regeneration model of different sources cell sheets around the nanotube morphology was established. The results indicated that the regeneration ability of multiple sources cell sheets was stronger than single source cell sheets.3. In vivo experiments indicated that the nanotube morphology could promote the periodontal tissue regeneration, among which NT10 nanotube is best one that close to the natural bone tissue morphology. The results provides a theory instruction for the periodontal regeneration, and also shows a certain application prospect.