Spatiotemporal Distribution Of Microdamage During Fatigue Loading In Rat Ulnae Based On A Novel Mechanical Loading System

Stress fracture is a type of fatigue fracture induced by excessive and repetitive mechanical loading through an accumulative fatigue process on the musculoskeletal system.Fatigued muscles tend to lose the capacity of adequately absorbing and damping the external mechanical stress to the skeleton,which may lead to microdamage.Accelerated bone remodeling along with the accumulation of skeletal fatigue is able to induce the occurrence and accumulation of microdamage on the osseous surface,and finally leads to stress fracture.Stress fracture is a common condition in military recruits,athletes and dancers,seriously affecting their health,weakening the battle effectiveness of army,and shortening the career life of athletes and dancers.Thus,stress fracture is one of the most essential research topics of military and sports medicine.The current method for the treatment of stress fracture mainly included plaster immobilization and surgery once the stress fracture occurred,requiring long-time rest inbed for the patients;nevertheless,the prevention for stress fracture is much more essential than the treatment.In order to explore the effective preventive approaches of stress fracture,it is critical for establishing a scientific and reliable stress fracture animal model.In some previous Chinese literatures,scientists tried to use the electric cage or treadmill to induce stress fracture model.However,these two methods have some major limitations,including uncontrollable loads,long duration of model establishment,low positive rate,tendency of tolerance to loading and high death rate.Despite the high precision and consistency of loads along with the explicit damage location,three-and four-point bending cannot mimic the sports situation due to their not physiologically-related transverse loading direction.The method of axial cyclic mechanical loading via compressive or tensile loading is regarded as a new and effective approach to establish the stress fracture animal model.This method is capable of accurately controlling the loads and monitoring the strain with a real-time manner.However,in comparison with tensile loading,the occurrence and development of stress fracture induced by the axial compressive loading method are much closer to the situation of stress fracture in humans.Therefore,our current study aimed to develop a novel stress fracture animal model system based on axial cyclic compressive loading with controllable loads.This mechanical loading system was able to establish animal models of ulnar and tibial stress fracture with or without anesthesia,which could accurately detect the force and displacement at real time and provide a reliable experimental platform for exploring the effective countermeasures of stress fracture.Moreover,we also investigated the spatiotemporal distribution of microdamage during fatigue loading in rat ulnae based on this novel mechanical loading system.It was found that linear microcracks climbed up and then declined during the first week after fatigue loading and they mainly accumulated in tensile cortex.The whole project can be divided into the following two parts:Part Ⅰ: Development of a novel stress fracture animal model systemMethods: The novel stress fracture animal model system was comprised of threeparts,mechanical loading device,linear actuator control module and real-time data sampling module.Mechanical loading device consisted of linear actuator,linear guide,fixtures(including a fixed holder and a movable holder),load cell and displacement transducer.Ulnae and tibiae of rats were fixed using differently shaped fixtures,respectively.The axial mechanical loading was applied via a linear actuator based on the LabVIEW control program.During the loading process,a load cell and a laser displacement transducer were used to detect the force and displacement changes in the limbs,respectively.These two channels of signals were sampled at real-time manner by the PC-based LabVIEW data acquisition program.After fabrication of the mechanical loading device,compressive loading testing experiments were conducted on the rat tibia with or without anesthesia to examine the consistency and reliability of cyclic loads.To calibrate our loading system and evaluate the working precision,the developed mechanical loading system and a Bose mechanical testing machine were used to measure and compare the Young’s modulus of a standard ultra-high molecular weight polyethylene(UHMWPE)cylinder,respectively.Then,cyclic compressive loading was applied on the ulnae and tibiae of rats in vivo and ex vivo to verify the stability and reliability of the mechanical loading system.Results: The calibration experiment revealed the working accuracy of our developed mechanical loading system.The mechanical loading device was able to apply loads with the range of 0~90N and displacement of 0~1800μm.Compressive loading testing experiments demonstrated that the mechanical loading system was able to apply consistent and controllable loads on rat tibiae under the condition with anesthesia and without anesthesia.The positive results of SPECT/CT examination on rat tibia and Micro-CT imaging on rat ulna proved the success and reliability of the mechanical loading device on establishing stress fracture animal model.The ex vivo experiments showed that ulna was much easier to be compressed than tibia,and thus loads applied on ulna were easier to be set up and stabilized.Conclusion: The novel mechanical loading device is capable of generating controllable loads and accurately detecting the force and displacement at real-timemanner.Cyclic axial loading can be applied on animals with or without anesthesia.Ulnar and tibial stress fracture animal models can be successfully established with high precision,scientificity and reliability.Compression is approaching the real development situation of stress fracture,which may provide a reliable experimental basis for exploring the precautionary measures of stress fracture.Part Ⅱ: Spatiotemporal distribution of microdamage during fatigue loading in rat ulnae based on the novel mechanical loading systemMethods: Finite element analysis was performed based on the Micro-CT images of rat ulna to analyze the strain distribution on the surface of mid-diaphysis.Fatigue loading was applied on thirty male rats(3~4 months,554±99g body weight)after intraperitoneal anesthesia with pentobarbital(30mg/kg).The right ulna of the rat was stabilized between a fixed holder and a movable holder and then subjected to axial compressive loading in vivo.Loading was applied via a linear actuator and a 1N compressive pre-load was applied to fix the ulnae.Cyclic compressive loading was applied with a ramp-loading mode at the frequency of 0.67 Hz with the displacement change of 30% of the average fracture displacement(2.0±0.2mm).Right forelimbs of adult rats were loaded at a normalized peak force of 0.055N/g body weight until 6,000 cycles were reached.Left ulna was used as a control.Ten rats were killed on days 3,5 and 7 post the application of fatigue loading,respectively.Segments with the length of 2cm from mid-diaphysis were sectioned from both the loaded and contralateral ulnae.Specimens were bulk stained in basic fuchsin and embedded in poly-methylmethacrylate.Then,the embedded samples were sections using a Leica diamond saw microtome.Microcrack analyses were performed under optical microscopy.The other specimens were imaged using Micro-CT after Ba SO4 precipitation.Three-dimensional microdamage analyses and quantifications were performed.Results: Three-dimensional finite element analysis of rat ulnae based on Micro-CT imaging indicated that strains with the range of-5400~+2180με were detected on the surface of ulnar mid-diaphysis when displacement change was 30% of the average fracture displacement.Data from basic fuchsin bulk staining showed that microcrackdensity(Cr.Dn)in the fatigue-loaded group was significantly higher than that in the Control group(P=0.04,P=0.01 and P=0.03).The Cr.Dn level on days 5 was significantly higher in the mechanically loaded group than those in the Control group on days 3 and days 7(P<0.05).Cr.Dn in tensile cortex was significant greater than that in compression cortex(P=0.03,P=0.02 and P=0.01)and Cr.Dn in tensile cortex on days 5 was significantly higher than those on days 3 and days 7(P<0.05).Cr.Le in compression cortex on days 3 was significant greater than that in tensile cortex(P=0.04).Results from contrast-enhanced Micro-CT demonstrated that microdamage accumulated from periosteal to endosteal surface,from anterior and posterior cortex to medial cortex during the first week after fatigue loading.Data from contrast-enhanced Micro-CT were in line with those from basic fuchsin bulk staining.Conclusion: The positive rate of ulnar stress fracture based on the novel mechanical loading system is 100%.Linear microcracks climb up first and then decline during the first week after fatigue loading.Linear microcracks mainly accumulate in tensile cortex.These findings represent an important methodological innovation for investigating the potential mechanism and precautionary approaches of stress fracture.Moreover,the current study also provides important experimental evidence for enriching our understanding of the mechanism of the occurrence and development of bone microdamage.Thus,this study may be valuable for the future experimental studies and clinical application for resisting stress fracture.