The Study On The Effect Of Prior Radiation On Fracture Healing And Its Mechanism

BackgroudRadiation therapy(also called radiotherapy)is a local curative treatment for cancer that uses ionizing radiation to control and kill tumor cells.As one of the three traditional cancer treatments,it plays an important role in cancer treatment.However,as well as kills the tumor cells,radiotherapy also damages the neighboring tissues,and radiation damage to the skeleton within the radiation field is a well-recognized late effect,causing problems such as osteoporosis,osteanabrosis,osteoradionecrosis,and fractures.Literature reports and our previous study show that radiotherapy can reduce the bone marrow MSC number,and promote MSC differentiate to adipocyte.Osteoblasts are radiosensitive and undergo cell apoptosis due to DNA damage after radiation.This leads to bone loss,causing osteoporosis,even fractures.Post-radiation fractures usually have longer healing time,and higher rate of nonunion.The fracture healing time in patients with soft tissue tumors exceeds a year and is compounded by a 45%nonunion rate,placing a substantial physical,medical,emotional and financial burden upon affected individuals and our society as a whole.So it is important to understand the mechanism of fracture healing after radiation and identifying an effective treatment to fracture non-union after radiation.Most animal studies analyzing radiation damage to bone either irradiate the entire animal or a large portion of the body.However,because of its extremely low dosage and extensive systemic effects,this type of radiation model might not truly mimic focal radiation commonly used to treat solid tumors in the clinic.The recently available Small Animal Radiation Research Platform(SARRP)developed in Johns Hopkins University provides a new opportunity to study the mechanisms of focal radiotherapy-induced bone damage in rodents.This irradiator is capable of delivering conformal and image-guided radiation with sub-millimeter accuracy,reducing the radiation to other tissues,it is widely used in research nowadays.Fracture healing is a physiological recovery process that leads to bone restoration of original quality and function.It combines many different biomechanical and biological factors.A recent study using lineage tracing method demonstrated that periosteal mesenchymal progenitors are the main contributors to callus cells during fracture healing.They can differentiate to osteoblast or chondrocyte,forming cartilage and woven bone.Periosteum can be divided into two layers:An outer fibrous layer and an inner cambium layer.Mesenchymal progenitors mainly locates in the cambium layer,and their function is affected by several factors,such as blood supply.Blood vessels supply oxygen and nutrients for the callus,and also facilitate recruitment of osteoblast and osteoclast into the callus.Researchers also find that oxygen concentration plays an important role in the differentiation of mesenchymal progenitors.Any steps going wrong during fracture healing will cause problems.It's reported that 5%-10%of fracture healing is delayed,or even nonunion.In this study,we investigated the effect of prior radiation on bone fracture healing using SARRP,trying to understand the mechanism of fracture healing after radiation.AimsTo establish a post-radiation fracture healing animal model using SARRP,mimicking the clinical radiotherapy.Through analyzing the fracture healing process,identify the outcome of post-radiation fracture healing(Part One),further understand the mechanism of fracture healing after radiation(Part Two).MethodsPart One1.Animal model:2-month old male C57BL/6 mice were first irradiated(8 Gy)at the midshaft of the right tibia using SARRP at day 1 and day 3.Two weeks later,transverse fractures were made within the radiated area and on the contralateral legs.Both tibiae were harvested at different time points for microCT,histology and mechanical assays.2.microCT scan:Tibiae harvested at different time points were scanned at the fracture sites by VivaCT 40 at a 10.5 ?m isotropic voxel size.2D and 3D microstructural analysis were used.To quantify vessel volume in the callus,mice were perfused with microfilm.Fractured tibiae were harvested and scanned before and after decalcification.3.Mechanical testing:Tibiae harvested at 6 weeks after fracture were placed on a 3-point bending fixture and loaded with mechanical force.The force to failure curve was recorded for analyzing peak load,stiffness,and energy to failure.4.Histology and immunohistochemistry:Tibiae harvested at different time points were prepared for paraffin or frozen embedding.The sections were stained with Safranin-O/Fast green,HE,picrosirius red,TRAP and IHC.5.Human nonunion scar tissue samples:Nonunion scar tissues were prepared from patients with long bone non-union fractures.Samples were imbedded in paraffin for Safranin-O/Fast green staining,HE staining,picrosirius red staining and IHC.6.Statistics,Data are expressed as means± standard error(SEM)and analyzed by paired Student's t-test for comparison of irradiated and non-irradiated contralateral bones or comparison of the proximal and distal sides of fracture,using Prism 5 software.Values of p<0.05 were considered significant.Part Two1.Animal model:2-month old male Col2/Tomato?aSMA/Tomato and Gli1/Tomato mice were first irradiated(8 Gy)at the midshaft of the right tibia using SARRP at day 1 and day 3.Two weeks later,transverse fractures were made within the radiated area and on the contralateral legs.Both tibiae were harvested at different time points for histology assays.2.Histology and immunohistochemistry:Tibiae harvested at different time points were prepared for paraffin or frozen embedding.The sections were stained with Safranin-O/Fast green,HE,picrosirius red and IHC.For EdU staining,mice received EdU injection at 3h before sacrifice and the staining was carried out according to the manufacturer's instructions.3.Periosteal mesenchymal progenitor culture:Periosteal mesenchymal progenitor culture was harvested from fresh mouse tibiae,and cultured at different environment(non-radiated and normoxia;non-radiated and hypoxia;radiated and mormoxia;radiated and hypoxia).Perform cell counting assay and differentiation assays by staining or real time RT-PCR.4.Statistics.Data are expressed as means ± standard error(SEM)and analyzed by paired Student's t-test for comparison of irradiated and non-irradiated contralateral bones or comparison of the proximal and distal sides of fracture,and by unpaired Student's t-test for comparison of cell culture samples followed by Bonferroni adjustment for multiple comparisons using Prism 5 software.Values of p<0.05 were considered significant.ResultsPart one:1.Prior focal radiation causes nonunion fracture in mouse long bones.In non-radiated tibiae,microCT scan showed the fracture healing has achieved by 6 weeks post fracture.However,the fracture line is still obvious in radiated tibiae.Fracture healing score and 3-point bending test further confirmed bone regeneration is greatly impeded in irradiated bones.We also found that the proximal side of radiated fracture still formed bone-containing callus despite of less callus volume compared to that in the corresponding site of non-irradiated bone fracture,however,we did not detect any callus and bone formation at the distal side.At week 6 post fracture,the proximal callus in irradiated bone grew past the fracture gap toward the distal site but never adhere to the cortical bone there.2.Instead of callus,fibrous tissue is formed at the distal side of fracture after radiation.Next we performed histology to investigate the underlying cellular mechanism.The images showed that the proximal side of irradiated bone had similar cartilage and bone structures in the callus as non-radiated tibiae,the distal side consisted of only fibroblastic-like cells surrounding the cortical bones from week 1 to 6 after fracture.Picrosirius red staining showed that these cells express abundant type I collagen,suggesting that they are fibrous tissue.3.Fibrous tissue lacks differentiation ability and vessel infiltration.We stained fracture sections with osteogenic(Osteocalcin and Osterix)and chondrogenic(type II collagen and Sox9)markers,the fibrous tissue were all negative.Endomucin staining and microfil perfusion identified the fibrous tissue lack vessel infiltration.4.Fibrous tissue mimics human nonunion samples.We collected waste scar tissue surrounding the fracture ends of nonunion patients for staining.Cells in those tissues showed typical fibroblastic morphology with no bone,cartilage,and vessels detected.Moreover,Picosirus red staining revealed abundant type I collagen matrix.Further staining also showed no osteogenic marker(Osteocalcin)expression.Therefore,the fibrous tissue in our mouse nonunion model closely mimics the pathology of human nonunion fracture.Part two:5.Radiation damages periosteal mesenchymal progenitors?In Col2/Tomato model,Tomato can mark the cambium layer of periosteum that continuously covers the cortical bone surface.At 2 weeks after radiation,Tomato+ cells within the radiation field was remarkably reduced while those in the neighboring area remained the same.Endomucin staining also showed the vessels in the radiation area is damaged.6.Radiation blunts the periosteum responses towards fracture.Three days after fracture,the periosteum layer in non-irradiated bones was greatly expanded.Radiated bones displayed significantly reduced periosteum expansion at both sides,EdU staining also showed prior radiated periosteum significantly less EdU+cell percentage,especially the distal side.In vitro experiments showed both radiation and hypoxia environment inhibit osteogenic differentiation ability,however,hypoxia can dramatically promote chondrogenic differentiation.7.Fibrous tissue in fracture nonunion originates from extraskeletal sources.We performed lineage tracing to investigate the source cells for fibrous tissue in nonunion fracture.We observed that Col2-Cre and aSMA-CreER label most cells in the inner layer but is entirely absent in the outer layer.On the contrary,Gli1-CreER labeled a small portion of cells in the inner layer and a majority of cells in the outer layer.The fibrous tissue at the distal end was Tomato-in Col2/Tomato and aSMA/Tomato mice,demonstrating that the pathological fibrotic cells do not originate from the cambium layer.Surprisingly,this tissue was Tomato+ in Glil/Tomato mice.However,we didn't observe the fibrous layer get expanded after fracture.We scraped the entire periosteum,including fibrous layer and cambium layer,then made fracture,there were same fibrous tissues formed at the place where the periosteum was gone,demonstrating that fibrous tissue originates from the Gli1 expressing cells of extraskeletal sources.Conclusions1.Prior focal radiation causes nonunion fracture in mouse long bones.Instead of callus,fibrous tissue is formed at the distal side of fracture.2.Fibrous tissue lacks differentiation ability and vessel infiltration,mimicking human nonunion samples.3.Radiation damages periosteal mesenchymal progenitors and blunts their injury responses?4.Fibrous tissue in fracture nonunion originates from extraskeletal Glil+cells.5.We established post-radiation fracture healing animal model and atrophic fracture nonunion animal model,which is highly reliable,nonsurgical,and clinically relevant,can be used for the study of relevant diseases.