Strenuous Running Enhances Subchondral Bone Remodeling And Induces Articular Cartilage Degeneration Of Knee Joint

Osteoarthritis(OA),which frequently produces pain and disability of joints,is one of the most popular degenerative diseases middle-aged and elderly in the global.An epidemiological investigation showed that the morbidity of primary OA in people over 40 years is 46.3%and the data grows with age.From now on,conservative treatments and their curative effect are limited.Most patients eventually have to receive joint replacement.However,the complications of the surgeries are disastrous to patients.The treatments and the disability resulted from this disorder bring financial and psychological burden to patients.In this context,it is urgent to find precautionary measures and effective treatment for OA.It is well known that OA initiates from the alterations of subchondral bone,which are characterized by enhanced bone remodeling,subchondral bone sclerosis,thickening of subchondral plate,modification of trabecular architecture,bone attrition and so on.From a biomechanical point of view,the thickening of the subchondral bone leads to increased joint stiffness,so the loads imposed on the articular cartilage would be less efficiently transmitted to the bone,resulting in augmented cartilage strain and damage.Nevertheless,numerous studies suggest that the mineralization of the subchondral bone is relative insufficient in combined with the increased bone remodeling.Therefore,the elasticity of the subchondral bone decreases and the newly formed bone offers less resistance and has greater compliance than the thinner subchondral bone that underlies the normal joint.In this theory,the overlying cartilage is more prone to deformation and cracking.The mechanisms of the subchondral bone remodeling increasing induced from the altered mechanical environment and the subsequently developed cartilage degeneration are still uncertain.It is still debated that whether heavy physical activity,elite level competition,and long-distance or marathon running could induce irreversible cartilaginous damages.Although the epidemiological study demonstrates there is no difference of the morbidity of primary OA between long-distance runner and nonathletes group,numerous animal studies and clinical investigations have revealed that abnormal manifestations which are related to arthralgia could be detected from magnetic resonance imaging(MRI).Furthermore,not only the high-intensity exercises,but also the repetitive and passive movement can induce altered anabolism in subchondral bone and articular cartilage degeneration,which are crucial to OA initiation and progressing.Therefore,this study was conducted in order to confirm the strenuous exercises,strenuous running especially,could induce articular cartilage damage and look further into its potential mechanism.We divided this study into 3 parts:The first part is to identify whether strenuous running could promote subchondral bone remodeling and induce articular cartilage damage.The second part is to explore the mechanism of osteoprogenitor cells(OPCs)recruitment resulting in enhanced subchondral bone remodeling under the strenuous running.The third part is to explore the mechanism of the cytokines transport from subchondral bone to chondrocytes inducing phenotype changes in articular cartilage.Chapter 1 Strenuous running promotes subchondral bone remodeling and induces articular cartilage damage[Background]Whether elite level competition,such as marathon running,could induce knee joint damage is still debated.[Objective]To identify the response of subchondral bone and articular cartilage to strenuous running.[Methods and Results]C57BL/6 mice were divided into 3 groups according to the intensity of exercises forced to conduct:control group,moderate running group and strenuous running group.T2-weighted MRI with fat suppression was acquired after 4-week running to detect and calculated the signal intensity in condyle marrow of right knees.The results indicated the signal intensity in femoral and tibial condyle marrows grew with the intensity of running.The relative signal intensity of strenuous group was significantly higher than control group((P=0.017 and P=0.024).All animals were sacrificed in the fifth week.Micro-CT scans were performed for knee joints to analyze morphological characteristics and found trabecular thickness(Tb.Th)(P=0.031 and P=0.0034)and trabecular number(Tb.N)(P<0.001)of subchondral bone in strenuous group were both higher than the other groups.Trabecular space(Tb.Sp)in strenuous group was found increased compared with control and moderate groups(P<0.001).Safranin O/Fast green and HE staining to evaluate the articular matrix and subchondral bone alterations and found surface discontinuity and massive proteoglycan loss of articular cartilage in strenuous running group,while the other two groups showed intact cartilage.Moreover,the thickness and the area of calcified cartilage zone,number of hypertrophic chondrocyte increased in comparison with control and moderate running groups(P<0.05).In addition,thicker subchondral bone plate of tibia was also observed after 5-week strenuous running in comparison with control(P<0.001)and moderate running groups(P=0.009).[Conclusions]Strenuous running generates bone marrow lesions(BMLs)in subchondral bone of knee joints,promotes subchondral bone remodeling and alters the overlying cartilage phenotypes.Further researches should be conducted to identify its intrinsic pathomechanism.Chapter 2 Abnormal recruitment of osteoprogenitor cells alters subchondral bone anabolism after strenuous running[Background]We have proved that strenuous running can promote subchondral bone remodeling in chapter 1,while its intrinsic pathomechanism is unclear.Previous studies have demonstrated that OPCs recruitment in subchondral bone marrow is important to its anabolism.Therefore,we hypothesized that the enhanced osteogenic activity in subchondral bone after strenuous running may be caused from the corrupted recruitment of OPCs.[Objective]To explore the mechanisms of corrupted recruitment of OPCs inducing altered subchondral bone remodeling after strenuous running.[Methods and Results]Goldner staining was performed for the paraffin sections acquired from chapter 1 and found the osteogenic activity increased compared the other two groups while amounts of osteoid and unmineralized tissue were also observed in strenuous running group.On the other hand,tartrate resistant acid phosphatase(TRAP)staining indicated the number of osteoclasts was not statistically different between groups(P=0.40).We then performed flow cytometry(FCM)for cells in subchondral bone marrow after 5-week intervening to calculate the numbers of OPCs and bone marrow mesenchymal stem cells(BMSCs).The results showed the proportion of OPCs in strenuous group increased in comparison with control group(P=0.034).Moreover,cells isolated from subchondral bone marrow were cultured in vitro and the total area of colony forming units(CFUs)of OPCs in strenuous group was higher than control(P=0.028)and moderate groups(P=0.037),while CFUs of BMSCc in strenuous group was decreased with relative to control(P=0.048)and moderate groups(P=0.044).OPCs were isolated from subchondral bone marrow and cultured under cyclic tensile strain at different intensities:elongation of 0%with frequencies of 0 Hz(0%,0 Hz,control),10%,0.5 Hz,and 20%,1 Hz for 6 hours every day.Wound healing assay revealed that the migration of OPCs was inhibited in a mechanical intensity dependent manner.To identify whether the excessive retention of OPCs onto trabecular bone was caused from the dyspoiesis of primary cilium after strenuous running,FCM was then performed to detect the a-Tubulin,the specific and important component of primary cilium,expression level in OPCs.We found positive OPCs was decreased in strenuous group.Moreover,using real-time polymerase chain reaction(RT-PCR)and western blot,KIF3A,a driven factor of a-Tubulin,was also inhibited(P=0.0029)and its activating factors,PKA-C,and AC6 and cAMP,were all downregulated in strenuous running group(P<0.05).[Conclusions]Strenuous running initiatively inhibits AC6 expression in subchondral bone and cAMP/PKA/KIF3A signal path activity is resultantly downregulated.Following that,a-Tubulin expression decreases and the formation and function of primary cilium of OPC are corrupted.This may be the crucial process to the enhanced osteogenic activity in subchondral bone after strenuous running.Chapter 3 Strenuous running induces cartilage degeneration by reducing transport of PDGF-AA from subchondral bone to articular cartilage[Background]We have confirmed that strenuous running can alter the overlying cartilage phenotypes.It is well proved that the molecular transport across the osteochondral interface plays crucial roles in cartilage degeneration and OA development.Platelet derived growth factors(PDGFs)are crucial cytokines that promote cell proliferation and survival and regulate cell differentiation.However,the role of PDGF/Akt signaling in the cross-talk between subchondral bone and articular cartilage is still unknown.[Objective]To explore the mechanism that how anabolic changes in subchondral bone induced from strenuous running accelerate cartilage degeneration and the role of PDGF/Akt signaling in the cross-talk between subchondral bone and articular cartilage.[Method and Results]To identify the phenotype changes of articular cartilage after strenuous running,immunohistochemical analyses for COL 10 and MMP13 were performed and the results showed that both the COL10 positive and MMP13 positive chondrocytes in articular cartilage were increased in strenuous group(P<0.001 compared with control group,P=0.0052 and<0.001 compared with moderate group).Moreover,the number of apoptotic cells in strenuous group was larger than control group(P=0.002)and moderate group(P=0.007)according to the TUNEL assay.Subchondral bone tissues harvested from sacrificed mice were proceeded to mRNA extraction and RT-PCRs were performed thereafter.Expression of Pdgfa mRNA from subchondral bone decreased in strenuous group(P<0.01)compared with the other two groups,which was also confirmed by immunohistochemical staining.In order to prove that suppression of PDGF-AA synthesis in subchondral bone after strenuous running leads to terminal differentiation of the overlying cartilage,preosteoblast MC3T3 were cultured in vitro and exposed to cyclic tensile strain at different intensities.We found both the mRNA and protein levels of PDGF-AA decreased in an intensity dependent manner(P<0.05).On the other hand,the mechanic-treated(MT)medium was harvested and used to co-culture for primary chondrocytes.Both of the expressions of Col10al and MMP13 were upregulated in chondrocytes cultured with the MT medium(20%,1 Hz)(P<0.05).This effect was attenuated by cultured with addition of 10 ng/mL PDGF-AA(MT+PDGF)(P<0.05).Similar results were observed in Ethidium bromide/acridine orange staining.Percentage of apoptotic cells increased remarkably in chondrocytes cultured with the MT medium(20%,1 Hz)and that addition of PDGF-AA attenuated this proapoptotic effect(P<0.05).These results suggested that the transport of PDGF-AA from subchondral bone to articular cartilage was suppressed by strenuous running,which may initiate the cartilage degeneration.To explore for the further mechanism of initiating the cartilage degeneration in this model,we detected the activation of PDGF/Akt signaling.Accompanied by the suppressed expression of PDGF-AA in osteoblasts,the p-Akt positive chondrocytes located in calcified cartilage zone decreased significantly in strenuous running group(P=0.018).The phosphorylation level of Akt was also down-regulated in primary chondrocytes cultured with the MT medium(20%,1 Hz)and addition of PDGF-AA recovered its activation.[Conclusion]These revealed that,after strenuous running,PDGF/Akt signaling pathway in chondrocytes was down-regulated(inhibited)by reducing the transports of PDGF-AA from subchondral bone to calcified cartilage.