Low Dose X-irradiation Promotes Mineralization Of Fracture Callus In A Rat Model

PartⅠEstablishment of a standard closed fracture model on rat femurIntroduction: Two distinct animal models, open osteotomy and closed fracture are used to study fracture healing. The results from studies using osteotomy models are often extrapolated to explain fracture repair, even though the healing process is affected by many variables including the extent of soft and hard tissue damage, vascular supply, and hematoma formation. A smooth cut surface with controlled angularity can be consistently reproduced with osteotomy, but it is accompanied by laceration of the peri-osseous soft tissues, including the periosteum, and by thermal damage to the cortical bone. While the closed fractures are easy to be established on small animals as rats with a special apparatus. Objective: To develop a technique for the production of a standard closed experimental fracture on rat femurs with a custom-built fracture apparatus. Methods: A reformed simplified fracture apparatus was designed and tested on 78 male Sprague-Dawley rats. First, the femur was treated with an intramedullary Kirschner's wire retrogradely through a medial parapatellar approach. The femoral diaphysis was then fractured by means of a blunt guillotine driven by a dropped weight. Results were assessed by plain radiographs and histographs. Results: A highly reproducible transverse fracture with a success rate of 85.9% was produced. Minimal comminution and angulation of the intramedullary wires were found radiographically. And after 12 weeks follow up, a typical secondary fracture healing process was observed histologically. Conclusions: The reformed apparatus is simple to build and easy to use. Through its application, a highly reproducible closed fracture model with minimal soft tissue damage is established.PartⅡLow Dose X-irradiation Promotes Mineralization of Fracture Callus in A Rat ModelIntroduction: X-irradiation at high dose impairs fracture healing. On the contrary, it has been reported that X-irradiation at low dose (LDI) promoted tissue revascularization, which critically precedes callus mineralization during fracture healing process. However, it still remains unclear whether LDI exerted beneficial effect on fracture callus mineralization, which provides local mechanical stability for early rehabilitation of fracture patients. Objective: To investigate the effect of LDI on fracture callus formation and mineralization evaluated using radiography, micro-CT, peripheral quantitative computed tomography (pQCT) and biomechanical test in a standardized rat femoral fracture model. Methods: 120 male Sprague-Dawley rats were subjected to standard closed fracture on right femur according to the way of the first part. Sixty rats of which were irradiated with low dose X-ray (1Gy as LDI group) for total body irradiation right before fracture induction. The remaining 60 rats without irradiation is regarded as SHAM-Group . The animals were euthanized at different time points: 2, 3, 4, 8 and 12 weeks post fracture. Fracture callus was assessed by using radiography and MicroCT for callus bridging, peripheral quantitative computed tomography (pQCT) for quantifying bone mineral content (BMC) and cross sectional area (CSA), confocal laser scanning microscopy for measuring area fraction of fluorescence labeling (AFFL), and four-point bending test for examining mechanical properties. Results: In both groups, the pQCT parameters of fracture callus continuously increased post operation and peaked at 3 weeks, then decreased at 4 weeks and kept stable at 8 and 12 weeks. With different pattern, the mechanical parameters continuously increased throughout experimental period.The CSA and AFFL were found 22% and 33% smaller in the LDI group compared to the SHAM group at 2 weeks (P<0.05 for both), whereas the BMC and AFFL were 15% and 34% higher in the LDI group at 3 weeks (P<0.05 for both). The changing patterns were consistent with the findings in 3-D MicroCT reconstructions. The mechanical parameters (Max-Load, Stiffness and Energy) were also 18%, 30% and 24% higher in the LDI group than in the SHAM group at 3 weeks (P<0.05 for all). At 4, 8 and 12 weeks, there was no difference found for all assessments between the two groups. The reduced CSA in the LDI-Group at 2 weeks could be explained by an acute inhibition effect of LDI on repairing progenitors, whereas the significantly enhanced mineralization in the LDI-Group at 3 weeks could be explained by delayed stimulation effect of LDI on mobilization of repairing progenitors and expression of growth factors required for angiogenesis hence osteogenesis reported by others. Conclusion: The results indicated LDI promoted mineralization at the stage of hard callus formation in a rat fracture model.PartⅢThe Possible Mechanisms of Promoted Callus Mineralization Induced by LDIIntroduction and Objective: At the study of second part, the results indicated LDI promoted mineralization at the stage of hard callus formation in a rat fracture model. Nevertheless, the possible mechanisms kept unclear. Fracture repair is highly related with blood flow and the number of osteoblastic progenitors, the present study will focus on assessing callus angiogenesis at a macroscopic level by vascular perfusion and MicroCT evaluation, and at the same time, examine the routine blood cells to know about the osteoblastic progenitors with an indirect way。Methods: Low dose irradiated fracture models on rats were established according to the method of the second part. Peripheral blood was assessed for blood cell accountings at the following intervals: immediately after model establishment (0.1 day), 7, 14, 21, 28, 56 and 84 days. After that, blood vessel casting with a radiopaque silicone rubber compound containing lead chromate and MicroCT scanning were performed on randomly selected rats(n=4). 3-D vascular images were reconstructed and then calculated for vessel volume, average diameter and vessel volume fraction. Results: The WBC and platelet numbers decreased 67% and 41% respectively right after LDI and fracture (LDI group). While those of the control groups(fracture without LDI) decreased much less than those of LDI group (P<0.05). The blood cells recovered slowly in both groups. On day 14 and 21, WBC increased more in LDI group than in control group. The platelet recovered much faster in both groups and on day 14 post operation the plated numbers have reached normal. At 1 week after model establishment, MicroCT images visibly showed reduced early neovascularization in LDI group, whereas at 2 weeks, the quantitative analysis revealed increased vessel volume and volume fraction in LDI group. No significant difference was found at 3 weeks though. Conclusions: LDI can induce cell mobilization and neovascularization on a rat fracture model, which, were the possible explanations for the accelerated callus mineralization.