| Background: Traditional treatments of bone deficiency caused by trauma, inflammation and tumor need the application of autograft or allograft transplantation and tissue engineering. However, both autograft and allograft transplantation have inevitable drawbacks which limit the application. The use of bone tissue engineering technology in manufacturing new bone to repair bone defects is a hot topic in recent years, but traditional bone tissue engineering using the bioreactor often need the application of stem cells and growth factors. However, this methodology is often unable to regenerate bone tissue with the vascularization and hierarchical organization found in native bone tissues which often leads to bone repair insufficient or failure. Accordingly, investigators have focused on in vivo bone tissue engineering which use the body’s own bone regeneration ability to create new bone without the application of exogenous growth factors and stem cells. But it is still a big problem in finding appropriate anatomic sites and methods to construct in vivo tissue engineering bone sufficiently.Objective: Construct two novel different in vivo bioreactors: osteo-regenerator in New Zealand rabbits based on the principle of in vivo bone tissue engineering. Compare the two osteo-regenerators in new bone formation and scaffolds degeneration in order to find a better one which could be used in future bone defect repair.Methods: 1. Semi-cylinder porous titanium alloy based on three-dimensional print technology: Design the three dimensional model of the semi-cylinder porous titanium alloy by CAD and input the data such as pore size, interval and length into 3D printing machine. Then construct the semi-cylinder porous titanium alloy with Ti6Al4 V powder.2. Thirty-Six New Zealand white rabbits in female with an average weight of 3±0.5 kg were randomly divided into group A and group B. Each group had three time points which are at 4 weeks, 8 weeks and 12 weeks. Each time point was randomly assigned with 6 New Zealand white rabbits. A longitudinal incision about 4.0 cm in length was made through full-thickness of the femur skin, and the muscles were bluntly separated by forceps. Subsequently, the femur was exposed to our vision. In group A, we drilled two perforative holes in 1mm diameter and 5 mm interval in the shaft of femur. After that, one semi-circular porous titanium shield filled with β-TCP granules in 1mm-2.5mm was put down below the femur, making sure the two holes were just in the middle of the shield. Next, we put another titanium shield with β-TCP granules to fit well with the previous one. Then two steel wires were used to fasten the two semi-circular titanium shields which just around the shaft of the femur. However, in group B, there was no difference with group A except that no drilled holes on the shaft of the femur. Instead, we incised the periosteum of femur. The rabbits were sacrificed at 4, 8 and 12 weeks postoperatively, and the implants were harvested and used for fluorochrome labeling, Micro-CT evaluation and histological examination and quantitative histological analysis.Results:(1)A rabbits died after surgery, probably because of the anesthesia-related complications. We did another rabbit for supplement. Then all the other New Zealand rabbits survived the surgery and the incision healed at primary intention. We did not found any infections in the incised sites and other place. The X ray showed that all the osteo-regenerators were fastened around the femur of rabbits.(2).The general observation showed that there were connective tissue grew into the osteo-regenerator from the holes in the titanium alloy. There was no leak of scaffolds.(3). Image examination: Micro-CT analysis showed that the bone volume fraction in the osteo-regenerator increased significantly both in group A and group B during the study. When compared group A with B at 8 and 12 weeks, we found that the group A had significant high new bone formation than that in group B. We could found that the new bone was nearly occupied the whole osteo-regenerator in group A at 12 weeks after implantation. There is no significant difference in material degradation at 4 and 8 weeks between two groups. However, at 12 weeks after implantation, the amount of residual material in group A was less than that in group B(P<0.05).(4). Histological examination: The quantitative analysis of fluorochrome markers interval showed that the calcification deposition in group A was significantly higher than that in group B at 12 weeks after implantation. VG staining results show that moderate regenerative bone tissue was observed around the β-TCP granules near the shaft of femur after implantation for 4 weeks and abundant connective tissue was detected growing cross the holes in the titanium shields into the osteo-regenerator. As time went on, new bone gradually grew from the femur to the Semi-cylinder porous titanium alloy and the fibrous connective tissue decreased. After implantation for 12 weeks, the osteo-regenerator in group A was occupied with new bone which was more than group B(P<0.05).Conclusion: The newly designed two kinds of osteo-regenerators with β-TCP granules in 1mm-2.5mm could facilitate the new bone formation without the application of other exoteric stem cells and growth factors. The osteo-regenerator with two perforative holes in the femur had better ability in new bone generation than that with incision in perioeteum. This new technology could potentially serves as treatment in bone defect in the future. |
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