Enhancement Of Bone Regeneration Through Cyclically Mechanical Stimulation Combined With Local Hypoxia In A Rat Femoral Distraction Osteogenesis Model

Objective: Distraction osteogenesis(DO),has become a widely applicated technique in orthopedic and reconstructive surgery for deformity correction and bone defect treatment,this technique provides advantages including avoidance of local or distant donor site morbidity,and tissue concurrent generation using the local endogenous substrate.Although satisfactory outcomes have been achieved in abundant cases,the most important limitation of DO is the absence or delayed callus formation in the distraction zone,which can prolong the duration with a bulky frame needed for the regenerate bone to finally consolidate,resulting in additionally unfavorable psychological impact and negative complications.Various efforts have been developed to expedite bone regeneration following DO.The purpose of this study was to establish a reproducible model of femoral distraction osteogenesis in rats to investigate the effectiveness and potential mechanism of different amplitude,rate,and rhythm of cyclic distraction-compression technique(accordion technique,AT)on bone regeneration,and to determine the optimal intervention timing of hypoxia administration during DO for the acceleration of bone regeneration.Furthermore,the synergistic osteogenesis-contributing effect of cyclically mechanical stimulation combined with local hypoxia during DO was verified.Methods: In section one,a custom monolateral distraction external fixator was installed on the right femur of a male SpragueDawley(SD)rat weighing approximately 420 g followed by a mid-diaphysis transverse osteotomy to establish a reproducible DO model(n=12).After a five-day latency,the distraction was started at a rate of 0.25mm/12 h for 10 days,producing a cumulative final gap distance of 5.0 mm followed by a consolidation duration of six weeks.The DO model was evaluated by digital radiographic imaging,HE(Hematoxylin and eosin)staining,and Safranin O staining.In section two,75 rats were divided into five groups(n=15)randomly according to the various interventions after distraction: control group,general AT group,larger amplitude AT group,slower rate AT group,and the same day distraction-compression AT group.The effectiveness and potential mechanism of different amplitude,rate,and rhythm of AT on bone regeneration were assessed by radiographic evaluation,biomechanical test,histological assessment,and molecular biology detection.In section three,the effect of hypoxia on osteogenic differentiation in rat bone marrow mesenchymal stem cells(r BMSCs)was verified by cell experiments in vitro firstly.And then,63 rats were divided into three groups(n=21)randomly according to the various interventions after distraction: control group,group of hypoxia in distraction phase,and group of hypoxia in consolidation phase.The effectiveness of different hypoxia administration timing on bone formation was assessed by radiographic evaluation,biomechanical test,histological assessment,and molecular biology detection.In section four,84 rats were divided into four groups(n=21)randomly according to the various interventions after distraction: control group,AT group,hypoxia group,and group of cyclically mechanical stimulation combined with local hypoxia.The synergistic osteogenesis-contributing effect of cyclically mechanical stimulation combined with local hypoxia during DO was also verified by radiographic evaluation,biomechanical test,histological assessment,and molecular biology detection.Results: In section one,rats recovered from surgical procedures completely and survived to the termination of the experiment.All rats achieved normal ambulation,and there was no significant difficulty in daily activities.The general inspection,radiographic imaging,and histological assessment of the specimens at each time point were consistent with the characteristics of regenerated tissues during DO.In section two,the expression of HIF-1α and VEGF was increased after the accordion technique.The quality of bone regeneration,mechanical properties of the regenerated bone,and the expression of osteogenic and angiogenic protein factors in all experimental groups were better than those in the control group.In addition,the larger amplitude AT group and slower rate AT group was better than the general AT group respectively,while the same day distractioncompression AT group was worse than the general AT group.In section three,the results of invitro experiments revealed that hypoxia contributed to the osteogenic differentiation and calcium deposition of r BMSCs.In vivo,the quality of bone regeneration,mechanical properties of the regenerated bone,and the expression of osteogenic and angiogenic protein factors in all experimental groups were better than those in the control group.Furthermore,the group of hypoxia in distraction phase was better than the group of hypoxia in consolidation phase.In section four,the quality of bone regeneration,mechanical properties of the regenerated bone,and the expression of osteogenic and angiogenic protein factors in all experimental groups were better than those in the control group.Moreover,the group of cyclically mechanical stimulation combined with local hypoxia was better than the AT group and the hypoxia group respectively.Conclusion: Firstly,this study successfully established a reproducible model of femoral distraction osteogenesis in rats,paving a way for many future works aiming to make the DO process more efficient.Secondly,the cyclic distractioncompression technique has benefits on the enhancement of bone regeneration during distraction osteogenesis,and the mechanism may be due to the tissue hypoxia activating the HIF pathway followed by the augmentation of osteogenic-angiogenic coupling.Better outcomes may be achieved by moderately increasing the amplitude and slowing down the rate of the AT,while the rhythm of “distraction and compression are completed on the same day” is not recommended.Thirdly,hypoxia contributes to the osteogenic differentiation and calcium deposition of r BMSCs.Local hypoxia in the distraction zone can accelerate the process of bone formation and consolidation during DO,especially in the distraction period.Fourthly,the synergistic osteogenesis-contributing effect of cyclically mechanical stimulation combined with local hypoxia on bone regeneration was better than the single intervention respectively.