| Since the highly biocompatible nature of Titanium (Ti) was found in the 50s last century, the use of dental implants has been developed so much and become an ideal oral rehabilitation and retention method of care in dentistry, with osseointegration the key of application.However, some uncontrolled systemic diseases like diabetes are associated with many complications and have some negative effect on bone healing, which may lead to implant falures, and thus always been contradiceated for implant rahabilitations. The reasons may be attributed to compromised bone response and delayed wound healing. Glycemic control has been reported the best treatment method containing oral administration of antidiabetic drugs and insulin applications. Numerous studies have shown that insulin therapy can stimulates the amount of neoformed bone directly and indirectly and relieves the neglect effect of diabetes on bone formation around the implants. However, the whole positive effect of insulin has been contradicted, in addition, insulin may not restore all the pathological effects caused by diabetes while the disease becomes medium or serious.As a result, the problem how to improve the implant success rate of diabetic patients has become a critical point. In this study, we tried to prepare an MSC-implant complex by cell sheet to act as a modified surface implant, hoping to improve osseointegration in a type 2 diabetic rat model. The MSC-implant complex represents a novel biomaterials-based tissue engineering strategy to aid in bone regeneration of type 2 diabetic patients. Major methods and results:1. Animal model of type 2 diabetes50 Male Wistar rats (200–250 g) were divided into two groups randomly. A high-fat diet and low-dose streptozotocin(STZ) intraperitoneal injection were administered to 40 rats to induce type 2 diabetes. The remaining 10 rats fed with normal diet served as controls. After consuming the diets for 4 weeks, rats in the diabetes mellitus (DM) group were intraperitoneally injected with 30 mg/kg of STZ, while those of the control group with 0.25 mL/kg vehicle citrate buffer (pH 4.4). One week later, the plasma glucose levels (PGL) of the rats in the DM group were measured. The rats with PGL < 16.7 mmol/L were injected with STZ again (30 mg/kg). The PGL and the body weight of the animals were measured every week. Four weeks following the STZ injections, rats with blood glucose levels of≥16.7 mmol/L were considered diabetic. Finally, to ensure the onset of the disease, an intraperitoneal glucose tolerance test (IPGTT) and an insulin tolerance test (ITT) were carried out on both groups. The diabetic rats were allowed to continue to feed on a high-fat diet until the end of the study. The results showed that, hyperglycemia in the rats was stable at between 16.7mmol/L and 20mmol/L from four weeks to eight weeks after STZ injections. Four rats were excluded from experiments, and the remaining 36 rats continued on to the follow-up tests. Futhermore,the IPGTT and ITT demonstrated that the rats in the DM group presented hyperglycemia and insulin resistance compared that of control group. All of these data indicate that a high-fat diet associated with 30 mg/kg STZ injection produced a diabetic model analogous to human type 2 diabetes mellitus.2. Fabrication of the MSC-implant complex by cell sheet.Based on inserted implants, the rats were divided into MSC-implant and titanium implant groups. Rat MSCs were isolated from Wistar rats weighing 60 g and cultured until we get purified P2 generation MSCs by regular passaging using standard tissue culture protocols. Confluent cells in flasks were cultured in osteogenic medium until MSCs sheets formed and could be detached intact from the substratum using a cell scraper. The sheets were then wrapped closely around the implant. The MSC-implant complexes were then cultured in static osteogenic media and maintained in a thermostatic oscillation incubator. From the SEM, on the titanium surface, there was a porous oxide layer composed of small craters covered the rough titanium material surface, and meanwhile, a SEM examination of the MSC sheet culture revealed a stratified extracellular–matrix complex composed of crosslinked collagen fiber networks and cells. Due to collagen fibers providing the majority of the mechanical support for cell attachment, the density of cells grown on the rough surfaces was increased. The sheet was composed of many small patches made up of flattened cells with extensive extracellular matrix deposits, cells that produced extracellular matrix, differentiating cells and cells that bound the material surface with their synapses.3. In vivo implantation and osseointegration evaluationAfter pentobarbital sodium solution intra-abdominally injection, one site 5-8 mm below the knee joints on the right side was incised and prepared for the implant cavity. Next, the two kinds of implants were inserted into position. A post-operative antibiotic treatment, was injected twice daily into the rats for four days. General body scanning using micro-computed tomography at a resolution of 110μm was conducted to identify suitable implantation sites and also for live animal scanning. After four weeks weeks of healing, nine rats in each group were anaesthetized and scanned by local Micro-CT at a scanning resolution of 14.97μm to evaluate the dynamic changes in peri-implant tissue. At a three-dimensional level, the Hounsfield Unit (HU) of this newly formed bone area was determined with an Inveon Research Workplace V2.2.0 (Siemens AG, Germany). Trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone surface/bone volume (BS/BV), and bone volume/total volume (BV/TV) were all determined and recorded.After CT scan, rats were scarificed. Right tibiae were removed for histological analysis. Using a high-speed precision microtome. All sections were ground and polished to a thickness of 35μm. And stained with Ponceau Tri-Chrome.Eight weeks after healing, the protocols of the other 18 rats in the two groups were the same as those of four weeks after healing.The results are found as following:1. From MicroCT images, 4 weeks after implantation, the implants were integrated with the host bone tissues in the two groups. However, the thickness of the newly formed bone surrounding the implants was obviously higher in the MSC-implant group than in the control group. The well-organized supporting bone in the MSC-implant group was formed around the implant, compared with the control group which showed fewer trabeculae in the peri-implant area after 8 weeks of healing. The new bone formation around the control implant was inconsistent and sparse, and the bone mass was much less than what was observed in the MSC-implant group.2. From MicroCT data, compared with the control group, the BIC rate of the MSC-implant complexes was 59±3.5% after 4 weeks of healing, which is higher than that of the titanium implant (P<0.05). After 8 weeks of healing, the BIC, BV/TV, and the Tb.Th of the MSC-implant complex group were higher and the Tb.Sp were significantly lower than those of the implant group (P<0.05). No statistically significant changes were found in Tb.N and BS/BV values between the two groups.3. From histologic images, in the titanium implant group, trabecular bone around the implants was loosely organized with less new bone formation around the implant than was observed around the MSC-implants at 4 weeks after implantation. At 8 weeks post-implantation, the bone tissue which remained loosely organized and was located far away from the implant surface in the titanium implant group, appeared to be more immature and less continuous than the MSC-implant. However, the lateral bone reconstitution appeared to be more rapid in the MSC-implant group compared to the titanium implant group, and there was a higher bone density and more new bone formation around the MSC-implants than in the titanium implant group. In rats with the MSC-implants, the bone was in close contact with the implant, and a thin lining of bone around the implant could be seen.The supporting bone around the MSC-implant group had uniform consistency and adhered closely to the implant surface after 8 weeks healing.4. Among 18 MSC-implant complexes specimens, histomorphometry revealed that the BIC rate of the MSC implant complexes group was 67.3±3.1%, higher than that of the titanium implant group (37.6±3.9%, P<0.05) after four weeks healing in vivo. At eight weeks after implantation, the BIC rate of the MSC implant complexes (78.4±5.25%) was still considerably higher than that of the titanium implant group (54.5±4.28%, P<0.05).Conclusion:This study demnonstrated that cell sheets composed of multilayered rat bone mesenchymal stem cells (BMCs) assembled with titanium implant (surface-modified titanium) coplexes could improve the peri-implant osseointegration in the tibiae of type 2 diabetic rats induced by high fat diet combining low dose STZ intraperitoneal injection. |
PDF Full Text Request