| Background/aims:Osteoarthritis(OA)is the most common chronic condition of the joints affecting approximately 27 million adults in the United States alone.Among the aging people over 60 years old,about 10%male or 18%female suffer from OA.According to a report,the expense on OA treatment or other things related is about 1%to 2.5%of the GDP in developed countries.Though a lot of hypothesis regarding the initiation and development of OA has been established,the biological mechanism of OA is still largely unknown.Epidermal growth factor receptor(EGFR)signaling is important for tissue homeostasis.Multiple ligands bind to and activate the EGFR,including epidermal growth factor(EGF),transforming growth factor-?(TGF?),amphiregulin,heparin-binding EGF(HBEGF),epiregulin,and betacellulin.Once activated,it stimulates an intracellular signal transd?CTion cascade to markedly influence cell behavior.Our current knowledge of EGFR function in articular cartilage mostly came from previous studies of its ligands and its intracellular inhibitor,mitogen-inducible gene 6(Mig-6).Large-scale gene profiling experiments have identified that the expression of TGF?(7)and HB-EGF(8)was increased in articular chondrocytes in both rodent surgical OA models and OA patients.Our pervious study demonstrated that EGFR cartilage specific knock out mice have spontaneous OA duo to articular cartilage defect.What's more,our study on growth plate indicated the importance of EGFR signal pathway in regulating the development of growth plate.We believe EGFR pathway plays an important role in cartilage.As a result,the first study is to demonstrate the function of EGFR in articular cartilage under normal situations.As the most common type of arthritis in the aging population,osteoarthritis(OA)is primarily characterized by progressive degeneration of knee articular cartilage.Mature articular cartilage consists of four zones:superficial,transitional,middle,and calcified zones.Among them,superficial layer is the first line of defense against OA initiation because of its surface location,unique composition and functions.It has 2-4 layers of flat cells expressing unique molecules,such as proteoglycan 4(Prg4,lubricin)and tenascin C,and contains a fine network of collagen fibrils that are oriented horizontally and parallel to the articular surface(2).Its multifaceted roles include,but not limited to,producing lubricant proteins,harboring chondroprogenitors,resisting shear stresses(2).In OA,degenerative changes initiate with cellular disorganization,gradual stiffening,and irregular surface of this layer(3-5).Despite the critical role of superficial chondrocytes in maintaining articular cartilage and in blocking OA,its regulation by growth factors and hormones are still largely unknown.In our previous study,we found EGFR activity is negatively correlated with the OA severity in OA patients,which indicates that EGFR plays an important role in OA,probably via protecting the superficial lay of articular cartilage.As a result,in the second part of my study,I used CKO mice and DMM model to study the function of EGFR in OA.To date,most researches seeking treatments for this disease focus on preventing cartilage damage and repairing degenerated cartilage at the cartilage level.However,clinical observations and animal studies over the past several decades have accumulated sufficient evidence to support that OA is a whole joint disease involving not only articular cartilage but also its neighboring tissues,such as subchondral bone,ligaments,menisci,periarticular muscles,peripheral nerves,and synovium.Indeed,the first radiographic criteria developed in 1950s for the diagnosis of OA include subchondral sclerosis as the hallmark of this disease.In the second part of my study,I observed the same thing that subchondral bone sclerosis occurred at late OA stage.Some researcher believe that subchondral sclerosis could accelerate the progression of overlying articular cartilage degradation.Some demonstrate that subchondral bone changes,such as bone marrow lesion found in MRI,occurs before the cartilage degradation and also could be used to predict OA.Though these points of views are not well accepted,yet they indicate that our study should not only focus on the cartilage,but also on the subchondral bone,especially on the mechanism of subchondral bone sclerosis and the relationship between subchondral bone and the cartilage.We expanded our study and focused on the subchondral bone changes and mechanism in our CKO OA model.In addition,in order to demonstrate that our conclusion is not restricted to our CKO mice.We tested our hypothesis on the WT mice only.As expected,we found the same changes that observed in our CKO mice model.Anatomically,subchondral bone consists of a layer of corticalized subchondral bone plate(SBP)and underneath subchondral trabecular bone(STB).It is important to distinguish these two subchondral bone components in OA research not only due to their intrinsic architectural and mechanical difference but also due to their different pathological changes at the late OA stage.The thickening of SBP is considered as the indisputable sign of OA.During OA progression,the bone remodeling rate in STB is drastically increased,leading to a deterioration of trabecular bone architecture.Several mechanisms have been suggested to explain this phenomenon,such as cellular signaling for micro-damage repair,stimulation of vascular invasion,and bone-cartilage crosstalks via channels in SBP.However,the molecular and cellular mechanisms for such changes are still largely unknown.The mechanism study of SBP and STB phenotypes based on the CKO mice is of vital importance.This is the final part of my study.While some studies have characterized subchondral bone changes in mouse OA models using micro-CT approaches,most of them scanned a thin proximal tibial area with a relatively low resolution(10-20 ?m)for 3D analysis.Moreover,measuring SBP thickness is usually performed on longitudinally sectioned micro-CT images in a 2D manner,which barely capture the overall changes of entire SBP.These results are highly variable depending on the sectioning angle and the temporal location of these sections within the tibial plateau.Thus,there is an urgent need of designing an unbiased 3D approach to accurately quantify bone parameters of SBP and STB in mice.The method is described in details in the third part.In addition,according to the conclusion of this study,we synthesized a fusion protein,named Fn3-Fc-TGF?,which can specifically bind to the chondrocytes to increase the EGFR activity.We got some promising data from our pilot experiment.However,it's still a long way to go.We hope we could make it in the future and benefit the OA patients.Method:Part 1.To study EGFR signaling in the articular cartilage,we first analyzed the gene expression of EGFR family members and its cognate ligands in mouse femoral head cartilage.In in vitro study,we compared the morphology of chondrocytes,their proliferation and differentiation ability and apoptosis after being treated with EGFR activator TGFa and inhibitor gefitinib.Using bovine cartilage explants to compare the amount of Prg4 and HA after TGFa and gefitinib treatment.Friction experiment further support our conclusion.Part 2:In the past,we established a chondrocyte-specific Egfr knockout mouse model(Col2-Cre EGFRwa5/flox)to demonstrate a pivotal role of EGFR signaling in growth plate development and secondary ossification center formation(SOC)(14,15).In this study,we used this model to investigate the function of EGFR in normal articular cartilage.A surgery model by destabilization of the medial meniscus(DMM)was applied to elucidate the action of EGFR in OA initiation in order to study the function of EGFR in maintaining chondrocyte number,its mechanical properties,and lubrication function.Part 3:we made an unbiased 3D approach to accurately quantify bone parameters of SBP in mice as the commonly used method didn't fit our purpose well.The method details are listed in this part.Part 4:We recently established a unique mouse model that develops the most severe OA symptoms,including a complete loss of cartilage and a drastic thickening of SBP,after surgical destabilization of the medial meniscus(DMM)(7).In this model,EGFR activity,was specifically suppressed in articular chondrocytes(Col2-Cre EgfrWat5/flox).Using the protocol listed in the part 3,we are able to measure SBP thickness at various locations within femoral condyle(anterior vs posterior,medial vs lateral)in a 3D manner.What's more,we explored the relationship of mechanical loading and SBP thickness under normal and pathological conditions.In addition,we discovered a novel mechanical and cellular mechanism to explain how cartilage depletion causes SBP sclerosis specifically underneath the cartilage damage site.At last,our conclusion was augmented by a computational simulation of finite elemental analysis.Result:EGFR signaling in healthy and diseased articular cartilage.To study EGFR signaling in the articular cartilage,we first analyzed the gene expression of EGFR family members and its cognate ligands in mouse femoral head cartilage.qRT-PCR revealed high levels of Egfr,its co-receptor Erbb2,and its ligands Tgfa,Hbegf,amphiregulin,and epiregulin mRNA,and relatively lower levels of other ligands Egf and betacellulin.While all chondrocytes within articular cartilage were positive for EGFR staining,EGFR activity,indicated by p-EGFR,was located mostly in surface chondrocytes and infrequently in calcified chondrocytes.DMM surgery induces OA-like phenotypes in mouse knees.At one month after DMM when articular cartilage had yet to show any morphological changes,EGFR staining remained throughout the cartilage but p-EGFR staining had already been diminished at the surface.In line with these data,healthy human articular cartilage exhibited strong EGFR staining throughout the cartilage(Fig.1Bb)and p-EGFR staining only at the superficial layer.At early OA stage when superficial zone(SZ)was still intact but proteoglycan staining was partially lost(Fig.1Bd),p-EGFR staining was remarkably reduced.These expression and activity profiles strongly implicate a potential role of EGFR signaling in the SZ of healthy and diseased articular cartilage.Deficiency in chondrogenic EGFR signaling leads to abnormal articular cartilage development.We next used a chondrogenic Egfr CKO(Col2-Cre EgfrWat5/flox)mouse model to investigate the action of EGFR in the articular cartilage.We previously demonstrated that EGFR activity in chondrocytes isolated from CKO mice and their sibling controls follows this sequence:WT>Wa5>CKO.While CKO mice expressed EGFR ubiquitously in the articular cartilage due to the presence of Wa5 allele,the amounts of p-EGFR and p-ERK,both EGFR signaling targets,were indeed drastically reduced compared to WT.Consistent to cell culture results,the staining patterns of p-EGFR and p-ERK in Wa5 cartilage were more similar to WT.At 2 months of age,Egfr CKO mice had slightly but significantly reduced total thickness of articular cartilage compared to those in WT and Wa5 mice.At 6 months of age,this reduction became much more evident in CKO mice,and Wa5 mice started to show similar changes.This drastic reduction of total thickness in CKO mice was accompanied by decreases in both uncalcified and calcified zones and an increase in the percentage of morphologically hypertrophied cells.In addition,CKO cartilage had less Safranin O staining and exhibited histological defects,such as minor fibrillation and clefting at the cartilage surface,resulting in an early OA-like phenotype with a Mankin score of 6.8±0.6.EGFR is required for maintaining superficial chondrocytes.Since cartilage surface possesses a high EGFR activity,we next took a close look at the SZ in CKO mice.Interestingly,while the structure of articular cartilage appeared normal in 2-week-old animals,the superficial layer in 2-month-old Wa5 and CKO mice had 56.6%and 88.5%,respectively,less cells than that in WT mice.Decreases in chondrocyte cellularity were also noticeable in rest of uncalcified cartilage but not as prominent as in the SZ.Those superficial cells in CKO mice had less Ki67 staining but more TUNEL staining and their deeper layer showed positive type X collagen,suggesting that EGFR signaling is important for regulating the proliferative ability and cell survival of superficial chondrocytes and hypertrophy of chondrocytes.Consistently,activating EGFR by TGF? stimulated proliferation of cultured superficial chondrocytes,and increased resistance to apoptosis upon TNF exposure under serum starvation.Upon chondrogenic differentiation,TGFa-treated cells maintained cell shape flat and the level of proteoglycan accumulation low while vehicle-treated cells became polygonal in shape and accumulated alcian blue-stained proteoglycan.Co-treatment of gefitinib,an EGFR inhibitor,abolished this effect of TGFa.TGFa also attenuated the expression of two cartilage matrix genes,Col2 and aggrecan.These data suggest a critical role of EGFR signaling in maintaining superficial chondrocytes as characterized by sustaining cell survival and inhibiting an increase in cartilage matrix prod?CTion.EGFR signaling promotes the lubrication function of articular cartilage.One key function of superficial chondrocytes is producing boundary lubricants,Prg4 and hyaluronic acid(HA).In WT joints,most cartilage and meniscus surfaces were covered by Prg4 and HA.Strikingly,Prg4 and HA signals were nearly absent at those surfaces in CKO joints.Note that their amounts in synovial membrane were not affected.These results are consistent with p-EGFR staining pattern in our CKO model in which EGFR activity was also reduced at meniscus surface but not in synovial membrane.The loss of lubricants in CKO articular cartilage could be a direct regulatory effect of EGFR signaling on Prg4 expression or an indirect effect due to decreased superficial chondrocyte number.Indeed,primary culture experiments showed that TGFa stimulates Prg4 expression in chondrocytes in a time-dependent fashion and that this up-regulation can be abolished by gefinitib.Bovine articular cartilage explant represents a superior model to study the actions of growth factors under controlled and physiologically relevant condition in a 3D environment.Using the uppermost 1 mm of bovine cartilage surface,we confirmed that TGFa increases Prg4 expression at both mRNA and protein levels in an EGFR-dependent manner,proving that EGFR autonomously regulates Pgr4 in the articular cartilage.Most importantly,tribological testing revealed that 3 days of TGFa treatment significantly reduces the equilibrium friction coefficient of articular surface by 28%,demonstrating that EGFR signaling is an important regulator of cartilage lubrication function.Deficiency of EGFR signaling weakens the articular cartilage.The principle function of articular cartilage is to provide load transmission and energy dissipative shock absorption during joint motion.Therefore,mechanical properties,mainly determined by cartilage extracellular matrix,are important parameters to characterize cartilage function.Under the polarized light microscope,we observed that WT mice had mostly horizontally aligned collagen fibrils at the cartilage surface and perpendicularly aligned fibrils with high collagen organization(high retardation values,in the deep layers of cartilage.In contrast,the collagen fibrillar structure changed remarkably in the middle and calcified zones of CKO mice,resulting in random orientation of collagen fibers and reduced collagen organization.Interestingly,these changes are very similar to the disruption of collagen fibril network in OA.WT articular cartilage developed dis-oriented collagen fibrils and reduced collagen organization in the middle zone of degenerated cartilage at 2 months after DMM.We recently developed an atomic force microscopy(AFM)-based nanoindentation approach to accurately measure mouse cartilage surface mechanical properties.In line with the above polarized light microscopy data,we detected remarkable decreases in the effective nanoindentation modulus,Eind,in both Wa5(58.7%)and CKO(74.7%)cartilage compared to WT.These data also match our current observation that DMM surgery rapidly reduced surface Eind of mouse cartilage.Taken together,our results clearly demonstrate that EGFR signaling regulates mechanical properties of articular cartilage.Loss of EGFR in chondrocytes remarkably accelerates cartilage damage after DMM surgery.Since the articular cartilage of Egfr CKO mice show structural,functional,and mechanical defects,they should be prone to develop OA after joint instability.One month after DMM,while WT joints remained intact,CKO joints showed prominent cartilage damages including surface fibrillation,clefts,and erosion,as well as remarkable reductions in uncalcified and calcified cartilage thicknesses.At 2 months post-surgery,CKO joints developed a more severe phenotype with a large part of articular cartilage eroded at the medial site,resulting in a full Mankin score.At 3 months post-surgery,CKO joints lost almost the entire articular cartilage with no measurements in both uncalcified and calcified cartilage thicknesses.Consistently with our previous report,Wa5 DMM joints had modestly accelerated OA development compared to WT but their symptoms were much milder compared to CKO.Consistent with these cartilage phenotypes,Egfr CKO mice developed several late OA phenotypes.First,while the lateral site of WT joints remained healthy,OA symptoms in CKO mice had propagated from medial to lateral site.Second,we observed a substantial increase in the thickness of the subchondral bone plate under the damaged cartilage area at the medial site in CKO mice,suggesting a subchondral sclerosis phenotype.Third,Eind of CKO DMM site increased remarkably to 4.3-fold of WT DMM joints,even more than WT sham joints,suggesting that the indented surface on CKO DMM joints is most likely calcified cartilage or even subchondral bone.Note that at the same time,DMM resulted in a 67.2%reduction in Eind of articular cartilage surface in WT miceAs OA further develops,DMM site of WT mice also exhibited same or elevated compared to sham site.Fourth,pain is the major symptom in OA.To detect mechanical allodynia,we performed von Frey assay and found that CKO DMM legs were extremely sensitive to the stimulus with a paw withdrawal threshold(PWT)only 29.7%of that observed in WT DMM legs,implicating that they experience much more pain than others after DMM.Lastly,while we did not observe any osteophyte in sham and DMM joints of 16 WT mice and 9 Wa5 mice,6 out of 18 sham and 8 out of 18 DMM joints of Egfr CKO had osteophytes originating from either tibial or femoral growth plate.Taken together,we demonstrated that Egfr CKO mice reached the late stage of OA at 2 months post-surgery through a pace much faster than WT mice.Additionally,we found that there are no changes in the trabecular bone structure in the metaphyseal area among WT,Wa5,and CKO mice regardless of surgery.Serum levels of bone formation marker(osteocalcin)and resorption marker(CTX)also remained the same among these mice(Supplementary Fig.8C).These data clearly demonstrate that our CKO model has no effect on overall bone structure.Subchondral bone quantification should be performed at the distal femur with SBP measurement at the posterior site.Examining 3D-reconstrpCTed microCT images of a mouse knee joint with a series of frontal sections revealed that the distal femoral subchondral region is much larger and taller than the proximal tibial subchondral region.On average,the height of tibial STB was 180 ?m across the entire plateau and the area suitable for analysis was between 2/6 and 5/6 of all frontal images as in images outside of this ranges SBP was fused with the growth plate at the middle of either medial or lateral sites.Assuming bones are scanned at the highest microCT resolution(6 ?m),only approximate 30 transverse-sectioned slides are available for trabecular bone structural analysis.Frontal or sagittal sectioning could provide more slides for analysis but the limited height of trabecular bone makes the contouring difficult to segregate SBP and STB in most images.SBP sclerosis at late OA stage make it even harder or even impossible to accurately contour SBP in proximal tibia.On the contrary,with an average of 600 ?m height,femoral subchondral region is a better site for accurately contouring STB for bone structural analysis despite sectioning directions.Unlike human knees almost straight when standing up,mouse knees are always bent at an angle between approximate 30 and 150 degree,resulting that only the posterior part,but not the anterior part,of distal femur is in contact with tibial plateau,and therefore is the area transducing mechanic loading during knee movement.Interestingly,sagittal sectioned images showing both anterior and posterior SBP revealed thicker SBP at posterior region compared to anterior region.To quantify SBP thickness in a 3D manner at any given surface area,we designed a novel protocol to transfer the microCT images into color-coded 3D images,in which the color at any position is linearly corresponded with its SBP thickness Using this method,we can manually define the region of interest within the SBP and calculated the average thickness within this region.Quantification confirmed that 4 month-old WT male mice have 2.0-fold thicker SBP in the posterior region of femoral condyle than that in the anterior region regardless of medial or lateral site.These results provide the first line of evidence that SBP thickness is positively correlated with loading strength.Due to the close proximity between SBP and growth plate,it is difficult to precisely contouring the SBP in tibiae using this approach.Taken together,our analysis of STB and SBP in the following studies focused on distal femur but not proximal tibia.Egfr CKO mice develop SBP sclerosis at the medial site of DMM joints.We previously reported that EGFR is critical for maintaining superficial chondrocyte number,promoting boundary lubricant secretion and cartilage surface lubrication.Strikingly,Egfr CKO(Col2-Cre EgfWa5/f)mice developed the most severe OA phenotype at 2-3 months after DMM with a complete depletion of articular cartilage layer at the medial site.MicroCT analysis of the entire femoral STB detected a modest,significant decrease in BV/TV,accompanied by reduced trabecular thickness,in CKO mice but not in WT mice after DMM surgery.The 2D frontal microCT images shown the SBP at the medial site of distal femurs of CKO mice,where the DMM surgery was performed and the most severe cartilage depletion was observed,is substantially thickened compared to the lateral site of DMM femurs and both medial and lateral sites of Sham femurs.By applying our new SBP measurement protocol,we precisely calculated the SBP thickness at different locations of femur epiphyseal region(posterior medial,posterior lateral,anterior medial,and anterial lateral)in WT and CKO mice with or without DMM.We observe a 31%increase in SBP thickness only at the posterior medial site in CKO mice but not all other sites,implicating that this localized SBP response is likely due to the depletion of overlaying articular cartilage.Most importantly,this thickened SBP in CKO mice showed much higher retardation than SBP in WT mice under polarized microscope,suggesting the collagen fiber composition or size might be altered in late OA stage.We previously reported that Wa5 mice are morphologically and functionally closer to WT mice than CKO mice during growth plate development,secondary ossification center formation,articular cartilage maintenance and OA progression,owning to the fact that the EGFR activity is decreased only modestly in Wa5 chondrocytes but drastically in CKO cells.Here,we found that is similar to those in WT mice,STB structural parameters,SBP thicknesses at all four sites,and the retardation of SBP in Wa5 mice are not altered by DMM surgery.Since Wa5 mice behaved similarly to WT mice throughout this study,we omitted Wa5 data in most figures.SBP thickening in the DMM knees of Egfr CKO mice is caused by increased bone formation at the bone marrow site of SBP and reduced sclerostin amount in SBP.To elucidate the mechanism for SBP thickening,we first investigated where the new bone is formed.Osteocalcin staining revealed that,compared to those in WT,the number of osteoblasts lining the SBP at the bone marrow site increased a 6.6-fold at the medial site of CKO femurs but not changed at the lateral site.Interestingly,this enhanced bone formation was accompanied by increased length of blood vessels aligning the SBP surface,which is likely to provide more nutrients for the high metabolic needs of active bone formation.Writs are potent signals for osteoblastic bone formation.Sclerostin is a Wnt pathway antagonist that is almost exclusively secreted by osteocytes.Like those in diaphyseal cortical bone,osteocytes in SBP express high amount of Sclerostin within the cell body and throughout dendritic processes,indicating that sclersotin might play an important role in maintaining SBP.In WT mice,DMM surgery did not altered Sclerostin amount at either medial or lateral site of femurs.Strikingly,compare to that in Sham site,Sclerostin amount was remarkably attenuated in SBP at the medial site,but not at the lateral site of CKO DMM joint.Quantification of the staining intensity of Sclerostin in these images confirmed our visual observation.These results indicate that a loss of Sclerostin in SBP underneath the damaged cartilage at late OA stage might be the cause of new bone formation at the bone marrow site,resulting SBP sclerosis.SBP thickening in aged Egfr CKO mice shares the same mechanism with Egfr CKO DMM model.Egfr CKO mice developed spontaneous OA with no SBP phenotype as early as 6 months of age.Interestingly,at 12 months of age,while OA symptoms in articular cartilage continued,their STB undergoes significant bone loss accompanied with deterioration in structural parameters.Note that different from surgery-induced OA,age-related OA exhibits cartilage damage at both medial and lateral sites.This resulted a similar level of SBP sclerosis across the entire knee of Egfr CKO mice at the posterior portion of both medial and lateral sites as shown by histology and microCT measurement.WT and Wa5 knees remained healthy at this age.Osteocalcin staining further confirmed that osteoblast number is elevated at 10-15 folds at medial and lateral sites,respectively,in these mice.Dynamic histomorphometry revealed that the newly formed bone surfaces are only observed at the bone marrow site but not at the cartilage site of SBP in all mice and that BFR was elevated 3.5-folds at medial and lateral sites,respectively,of CKO femurs.Furthermore,Sclerostin amount in SBP was significantly decreased at both medial and lateral sites of CKO mice compared to WT.We also tested our hypothesis in other WT late OA models,such as DMMH and 1 year after DMM model.SBP sclerosis in other late OA models.To confirm that our conclusion is not restricted to Egfr CKO mice,we tested two late OA models in WT mice.At 10 months after DMM surgery,WT mice exhibited severe OA symptoms including a complete depletion of articular cartilage and significant bone loss in the STB.In line with Egfr CKO DMM model,these mice displayed SBP thickening only at medial posterior site,but not at other 3 sites in the femoral condyle.Sclerostin staining confirmed a reduction in Sclerostin levels in osteocytes within the thickened SBP after DMM.Compared to DMM that only produces moderate OA,DMMH(DMM and hemisectomy of the meniscus)causes more severe OA.At 16 weeks post-surgery,WT joints had already developed late OA symptoms with partial cartilage depletion at the medial site.Consequently,those WT knees exhibited SBP thickening only at the medial site but not at the lateral site.Again,we observed a significant decrease in Sclerostin only at the medial site of DMMH knees but not at lateral site.Taken together,our data from these WT OA models further support that SBP sclerosis is the result of reduced osteocytic sclerostin in the SBP.Cartilage depletion in Egfr CKO mouse joints results in increased mechanical loads on SBP.Mechanical loading is a major regulator of Sclerostin in the cortical bone.However,currently it is impossible to measure mechanical stresses in live animal joints.Therefore,we adopted a simulation method using an asymmetric finite element model to estimate mechanical stress and strain distributions in the cartilage and subchondral bone plate after DMM surgery.Using the effective modulus values obtained from our ATF-nanoindentation experiments,we found that the largest strain in CKO sham joints was about 3.0-fold higher than that in WT sham joints,while the highest focal stress at joint contact sites in CKO and WT was the same.After DMM surgery,when the cartilage was still intact,both stress and strain values at the contact site increased regardless of the genotype due to the loss of meniscus support.At this point,the CKO cartilage surface experienced about 2.9-fold greater strain than WT joints suggesting that CKO cartilage is likely to be damaged more easily.DMM also increased the stress level on SBP.But with the intact cartilage,this increase was the same among different genotypes.However,when the cartilage was completely depleted,as in CKO joints at late OA stage,the highest focal stress on SBP drastically increased 14.6-fold higher than the knee joints with cartilage,indicating that large mechanical loads act on the SBP.Thus,using finite element analysis,we demonstrated that loss of cartilage at the medial site in CKO mice significantly elevates the mechanical stresses on SBP.This result provides a strong support for our experimental data showing reduced sclerostin and enhanced bone formation in the medial site of CKO SBP and explains why SBP sclerosis only occurs at the medial site of CKO joints.Conclusion:In short,our study is focused on 4 parts,namely 1.The function of EGFR signal pathway in normal articular cartilage.2.The function of EGFR signal pathway in OA initiation and progression.3.an unbiased 3D approach to accurately quantify bone parameters of SBP in mice.4.The mechanism and function of subchondral bone plate sclerosis at late OA.EGFR signaling is critical for maintaining the superficial layer of articular cartilage and preventing osteoarthritis initiation.Subchondral bone plate sclerosis during late osteoarthritis is caused by loading-induced reduction in Sclerostin... |
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