INTRODUCTION

The most common degenerative condition observed in temporomandibular joint disorder(TMD)is osteoarthritis(OA),which causes severe pain and discomfort on one or both sides of the face.1-2One percent of Hong Kong Chinese individuals have frequent TMD-related jaw pain,3and 14.56%of mainland Chinese patients with TMD exhibit radiographic signs of OA.4Temporomandibular joint OA(TMJ-OA)is a common condition that limits the quality of life of patients.5-6Its prevalence increases with occlusal disorder,which is characterised by marked changes in the condylar cartilage and altered composition and material properties of the cartilage matrix.7Despite previous investigations of the pathomechanism of TMJ-OA,the exact pathogenesis of TMJ-OA remains elusive.More importantly,there is no effective treatment approach for TMJ-OA.

在现代化教学不断深入发展的今天，发挥学生主体作用、实现以人为本已然成为高效教育教学的核心要点，符合新课程标准。教师应树立正确的教师观、教育观，为学生提供专业科学的启发引导，促使初中语文课堂发挥出新的活力，给予学生不同的学习体验，促使学生在高效教学中发挥主观能动性，实现不断进步。

Condylar cartilage,as secondary cartilage,differs from other cartilaginous tissue.It can be easily distinguished as a fibrous layer,a proliferative cell layer,a chondrocytic cell layer and a hypertrophic cell layer according to the cellular characteristics.8-9 Microscopically,mandibular condylar cartilage is dissimilar to articular cartilage,especially regarding its constituents.In general,articular cartilage is composed of hyaline cartilage,whereas mandibular condylar cartilage consists largely of fibrocartilage,with thick multilayers composed of several collagen fibre zones.10 Subchondral bone provides mechanical support for the overlying articular cartilage and forms the osteochondral unit together with articular cartilage.11-12Cartilage chondrocytes respond to alterations in mechanical loading,which cause changes in subchondral bone.13-14Chondrocyte catabolism can lead to articular cartilage damage in OA.15During the progression of TMJ-OA,a high concentration of type X collagen(Col X)is expressed in articular cartilage with chondrocyte hypertrophy.16-17The thickening of the hypertrophic layer was impressively represented in an OA rodent model.18Condylar cartilage degeneration with calcification and osteophyte formation has also been observed in OA.19 Numerous studies have shown that subchondral bone stiffness causes significant mechanical load and breakdown of the overlying cartilage.20-23However,subchondral bone in OA is subjected to a decrease rather than an increase in bone stiffness.24-25In addition,uncoupled boneremodelling byosteoclastsand osteoblasts in subchondral bone contributes to cartilage degeneration,which gradually results in TMJ-OA lesions.13,26

In recent years,transforming growth factor-β (TGF-β)has drawn increasing attention in the pathogenesis of OA.27 Increasing TGF-β1 signalling activity in knee joint OA causes severe cartilage degeneration,and a high level of TGF-β1 in chondrocytes was detected in models of OA.28The protein levels of TGF-β1 and phosphorylated Smad2/3(p-Smad2/3)were enhanced in the degenerative cartilage in the TMJ of a genetic form of OA.29Therefore,the interruption of TGF-β1 signalling in articular cartilage leads to articular and condylar cartilage degeneration.30Furthermore,a high concentration of TGF-β1 in subchondral bone was found in an anterior cruciate ligament transection(ACLT)OA mouse model.31The overexpression TGF-β1 signalling in subchondral bone leads to abnormal remodelling and is harmful to cartilage integrity.32-33Thus,TGF-β1 may play a critical role in the oetiology of TMJ-OA.34However,whether inhibition of TGF-β1 signalling in condylar cartilage and subchondral bone can rescue TMJ-OA progression remains to be elucidated.

In the present study,we used three different rodent models(TMD rats,Camurati-Engelmann disease(CED)mice and ageing mice)to represent obvious phenotypes of TMJ-OA and investigated the role of TGF-β1 signalling during TMJ-OA progression.Further analysis was performed to elucidate whether inhibition of TGF-β1 activity attenuates TMJ-OA.

(1)白垩纪晚世周田组(K2z)。勘查区广泛出露周田组，岩性主要由紫红色含钙页岩、粉砂质页岩夹砂岩、砂砾岩组成韵律性基本层序。岩层上部为紫红色砂岩，风化强烈，强风化厚度5～10 m，地层倾向260°～290°，倾角20°～55°。底部为紫红色巨厚层状砂砾岩，砂砾岩砾石成分为石英、石英砂岩、石英砾岩等，砾径1～20 cm。未见顶底，厚大于200.55 m。

RESULTS

Cartilage degeneration in the condyle of TMD and ageing models The TMD rat model was established by occlusal changes of the first molar(Fig.1a).Haematoxylin and eosin(HE)staining indicated that the mandibular condylar cartilage was divided into a fibrocartilage layer and a calcified cartilage layer depending on several collagen fibre zones.Compared with rats in the control group,rats in the TMD group showed a significantly decreased fibrocartilage layer and a thinner calcified cartilage layer(Figs.1b,c).Safranin O staining showed that the distribution of proteoglycans in controls was even and rich,whereas the TMD group exhibited cartilage degradation accompanied by an extensive loss of proteoglycans and a decreased total number of chondrocytes(Fig.1b).The Mankin and OARSI scores were increased,further confirming the degeneration of articular cartilage in TMD rats compared with controls(Figs.1d,e).Abnormal,upregulated expression Col X and p-Smad2/3 and an increased number of Osterix-positive cells in the cartilage layer in TMD rats were revealed by immunohistochemistry(Figs.1f,g).

Age-related changes were also detected in TMJ components.Compared with 12-wk-old mice,the fibrocartilage layer was thinner in 45-wk-old mice,whereas at 60 wks,mice only possessed a calcified cartilage layer with surface fibrillation(cracks)(Figs.2a,b).Mankin and OARSI scores were higher in 45-and 60-wk-old mice than in 12-wk-old mice(Figs.2c,d).Moreover,increased Col X and p-Smad2/3 expression and greater numbers of Osterix-positive cells were detected in the cartilage of 45-and 60-wk-old mice(Figs.2e,f).

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Occlusion disturbance is one of the most important factors in TMJ-OA.39Rat models provide adequate amounts of tissue and are easy to handle.Therefore,we established TMD rat models by inducing abnormal mechanical loading on the TMJ.In the initial phase of dental occlusion,there was predominant loss of subchondral bone in the TMJ condyles.16Cartilage degeneration in TMJ-OA is often accompanied by a high OARSI score and abnormally enhanced Col X and matrix metalloproteinase 13 expression.14,33-34,40In our study,Safranin O and HE staining revealed increased thickness of the calcified cartilage layer and cartilage matrix degeneration in the TMD models.Furthermore,bone volume decreased in the subchondral bone of TMD rats.Cartilagedamageandthelow subchondralbonevolume confirmed that we successfully established an early-onset TMJOA rat model.Col X is a specific marker of the hypertrophic layer.41 Osterix is essential for later-stage endochondral ossification and is involved in OA development.42The expression of Osterix and Col X,which were highly expressed in the hypertrophic layer of cartilage in the TMD groups,was detected in the cartilage.Therefore,high concentrations of Osterix and Col X contribute to the cartilage calcification degeneration in TMD.However,the Osterix level in subchondral bone differed from that in the cartilage of TMD rat models.The number of Osterix-positive osteoprogenitors and osteoblasts in subchondral bone was significantly decreased in TMD rats.These results indicate decreased bone formation ability and uncoupled bone formation and bone resorption in the subchondral bone of TMD rats.p-Smad2/3 was increased in subchondral bone and cartilage disrupted joint microarchitecture,supporting the pathogenesis of OA in TMD.

Active TGF-β1 in bone induces TMJ-OA

To examine whether high concentrations of active TGF-β1 in subchondral bone initiate TMJ-OA,we used a CED(40-wk-old)activation mutation mouse model in which TGF-β1 is activated after secretion by osteoblastic cells in subchondral bone marrow.HE staining revealed thinner cartilage in CED mice than in control mice.Only calcified cartilage was present in the cartilage of CED mice(Figs.4a,b).Safranin O staining(Fig.4a)and higher Mankin and OARSI scores(Figs.4c,d)revealed significant degeneration of the articular cartilage in CED mice compared with their WT littermates.Moreover,the percentage of p-Smad2/3-and Osterixpositive cells was increased in the cartilage of CED mice(Fig.4e).µCT images showed an uneven distribution of bone mass in the subchondral bone of the TMJ in CED mice,indicating disrupted bone formation.The volume of subchondral bone(%,BV/TV)was lower,but the Tb.Sp was higher in CED mice than in WT mice(Fig.4f).In CED mice,the percentage of p-Smad2/3-and TRAP-positive cells in subchondral bone was increased,but the percentage of Osterix-positive cells was lower than in WT mice(Fig.4g).

Inhibition of TGF-β signalling attenuates cartilage damage and enhances bone mass in subchondral bone

在历史教学过程中培养学生的核心素养，需要重视对学生历史思维的构建，教师在进行历史教学的过程中，首先需要改变传统教学模式中的填鸭式教学，传统的填鸭式教学会导致学生机械式的学习方式与接受能力，对历史事件缺乏自身的见解与思维。因此，教师需要改进教育教学方式，在创新教学模式的基础上，帮助学生进行探究学习和自主学习，在此基础上，教师对学生的学习进行适时正确的引导，带领学生以客观的思维进行历史事件和历史人物的分析。

To examine the effect of inhibition of TGF-β activity during TMJOA progression,we intraperitoneally injected a TGF-β receptor 1(TβRI)inhibitor or vehicle into TMD rats for 30 days.Upon injection of the TβRI inhibitor(TGF-I),the number of p-Smad2/3-positive cells was dramatically decreased in the cartilage and subchondral bone marrow(Figs.5b,d),indicating effective inhibition of TGF-β1 signalling.HE staining revealed a thicker fibrocartilage layer in rats injected with the TGF-I than in the vehicle-injected group(Figs.5a,b).Of note,cartilage degeneration was attenuated in TMD rats injected with the TGF-I,as determined by deeper Safranin O staining and higher Mankin and OARSI scores(Figs.5c,d).Although there was no difference in the number of Col X-positive cells between the TGF-I group and vehicle treatment group,the percentages of p-Smad2/3-and Osterix-positive cells in the cartilage layer were significantly decreased in the TGF-I group(Fig.5e).Moreover,injection of the TGF-I caused a significant increase in bone volume and a decrease in Tb.Sp relative to the effects of vehicle treatment(Fig.5f).In addition,the percentage of TRAP-positive cells was significantly reduced,while that of Osterix-positive cells was increased in subchondral bone marrow after TGF-I injection(Fig.5g).Thus,inhibition of TGF-β signalling prevented cartilage degeneration and bone resorption in early-stage TMJ-OA.

DISCUSSION

Severe malocclusion,ageing,psychological stress and skeletal jaw asymmetry cause TMJ-OA.35The limited understanding of its pathogenesis has led to ineffective therapies for restoring the structures of the TMJ with progressive OA.A critical approach to investigating the pathogenesis of TMJ-OA is the use of animal models.However,surgical,mechanical,chemical and gene TMJOA models are still unable to mimic its complex clinical conditions.36-38In our study,we used TMD and ageing models to mimic the clinical pathology and progression of TMJ-OA.

The TMJ subchondral bone of TMD rats was severely altered relative to that in sham-operated controls,as determined by micro-computed tomography(µCT)analysis(Fig.3a).In the TMD group,bonevolume(%,BV/TV)wassignificantlyreduced compared with that in controls,and the trabecular space(Tb.Sp)was significantly increased(Fig.3a).The number of tartrateresistant acid phosphatase(TRAP)-positive cells in subchondral bone was significantly increased and the number of Osterixpositive osteoprogenitors was significantly reduced in TMD rats compared with control animals.Similarly,in the ageing mouse model,µCT results revealed that the subchondral bone of 60-wkold mice was significantly affected,with a significant decrease in bone volume and an increase in Tb.Sp compared with 12-and 45-wk-old animals(Fig.3b).Moreover,60-wk-old mice showed a significantly higher percentage of TRAP-positive cells and a lower percentage of Osterix-positive cells in the subchondral bone region than 12-and 45-wk-old mice(Fig.3d).Notably,a significantly higher number of p-Smad2/3-positive cells was observed in the bone marrow in the TMD group and in ageing mice(60-wk-old mice)than in mice in the respective control groups(Figs.3c,d).

Subchondral bone loss in the TMJ of TMD and ageing models

Fig.1 Abnormal occlusion leads to cartilage degeneration in the temporomandibular joint.a Representative images of the first molar occlusion relationship in control and TMD rats.b HE(top)and Safranin O and fast green(bottom)staining analyses of glycosaminoglycan(red)in sagittal sections of the temporomandibular joint and mandibular condylar cartilage layers(FC fibrocartilage layer,CC:calcified cartilage layer).c FC/CC,d Mankin and e OARSI scores of control and TMD rats.f Immunohistochemical analyses of Col X,p-Smad2/3 and Osterix(brown)in the condylar cartilage.g Col X-,p-Smad2/3-and Osterix-positive cells(brown)were counted in the cartilage layer.Scale bars=20µm.n=6 per group.*P＜0.05,one-way ANOVA followed by Tukey′s test.All data are expressed as the mean±s.d.

Fig.2 Condylar cartilage degeneration in ageing mice at different time points(12 wks,45 wks and 60 wks).a HE(top)and Safranin O and fast green(bottom)staining analyses of glycosaminoglycan(red),black arrow indicates a crack.Mandibular condylar cartilage cell layers(FC fibrocartilage layer,CC calcified cartilage layer).b FC/CC,c Mankin and d OARSI scores of ageing mice.e Immunohistochemical analyses of Col X,p-Smad2/3 and Osterix(brown)in the mandibular condylar cartilage of ageing mice.f Col X-,p-Smad2/3-and Osterix-positive cells were counted in the cartilage layer.Scale bars=20µm.n=6 per group.*P＜0.05,**P＜0.01 one-way ANOVA followed by Tukey′s test.All data are expressed as the mean±s.d.

Ethics statement

Fig.3 Subchondral bone loss in the temporomandibular joint condyle.a RepresentativeµCT images and quantitative analysis showed significant bone loss at 8 wks after surgery in the temporomandibular joint condyle of rats.b RepresentativeµCT images and quantitative analysis showed increased bone loss in the temporomandibular joint condyle of ageing mice.Scale bar=300 μm.BV/TV(%),bone volume fraction;Tb.Sp(mm),trabecular separation.c,d TRAP staining and immunohistochemical(Osterix and p-Smad2/3)staining of the temporomandibular joint subchondral bone from control and TMD rats(c)and ageing mice(d).Quantitative analysis of TRAP and IHC staining is shown on the right.Scale bar=20 μm.n=6 per group.*P ＜ 0.05,one-way ANOVA followed by Tukey′s test.All data are expressed as the mean±s.d.

Fig.4 Transgenic activating mutation of TGF-β1 results in a TMJ-OA phenotype.a HE(left)and Safranin O and fast green(right)staining analyses of glycosaminoglycan(red)in the condylar cartilage of 10-month-old CED mice and WT littermates.Mandibular condylar cartilage cell layers(FC fibrocartilage layer,CC:calcified cartilage layer).b FC/CC,c Mankin and d OARSI scores of CED mice.e Immunohistochemical staining of p-Smad2/3 and Osterix(brown)in the mandibular condylar cartilage of ageing mice(left).p-Smad2/3-and Osterix-positive cells(brown)were counted in the cartilage layer(right).f RepresentativeµCT images of the condylar subchondral bone.Quantitative analysis of the structural parameters of subchondral bone is shown on the bottom.g TRAP and immunohistochemical(Osterix and p-Smad2/3)staining of the TMJ subchondral bone of CED mice and WT littermates.The percentage of positive cells is shown.Scale bar=20μm.n=6 per group.*P＜0.05,**P＜0.01 one-way ANOVA followed by Tukey′s test.All data are reported as the mean±s.d.

Fig.5 TβRI inhibitor stabilises the subchondral bone microarchitecture in TMJ-OA rats.a HE(top)and Safranin O and fast green(bottom)staining analyses of glycosaminoglycan(red)in the TMJ mandibular condyle from TMD rats treated with 1 mg/kg body weight of the TβRI inhibitor daily for 30 days.Mandibular condylar cartilage cell layers(FC fibrocartilage layer,CC calcified cartilage layer).b FC/CC,c Mankin and d OARSI scores of rats.c RepresentativeµCT images of the condylar subchondral bone and quantitative analysis of the structural parameters of subchondral bone.Scale bar=300 μm.e Immunohistochemical analyses of Col X,p-Smad2/3 and Osterix(brown)in the mandibular condylar cartilage of TMD rats(left).p-Smad2/3-and Osterix-positive cells(brown)were counted in the cartilage layer(right).f RepresentativeµCT images of the condylar subchondral bone.Quantitative analysis of the structural parameters of subchondral bone is shown on the right.g TRAP and immunohistochemical(Osterix and p-Smad2/3)staining of the TMJ subchondral bone.Quantitative analysis is shown on the right.Scale bar=20μm.n=6 per group.*P＜0.05,one-way ANOVA followed by Tukey′s test.All data are reported as the mean±s.d.

The TGF-I was used to inhibit TGF-β1 signalling to rescue cartilage damage induced by OA.56Moreover,the TGF-I ameliorated the abnormal role of high levels of active TGF-β1 in bone formation and directly attenuated Smad2/3 phosphorylation.57-58 In our study,injection of the TGF-I significantly decreased the number of p-Smad2/3-positive cells in the condylar cartilage and subchondral bone in the TMD model.The significant increase in Osterix-positive cells in the subchondral bone of the TGF-I group may trigger the bone formation process and lead to increased bone mass in subchondral bone.These results suggest that the TGF-I not only induced an increase in bone volume but also rescued cartilage degeneration.

OA osteoblasts produced a high level of TGF-β1 signalling,which plays a negative role in cartilage homeostasis and chondrocyte differentiation.27,51The ratio of the TGF-β1 receptor ALK1 to ALK5 was increased in the chondrocytes from aged and OA cartilage.52TGF-β1 plays a pivotal role in bone remodelling,and the TGF-β1 concentration is enhanced in subchondral bone in the ACLT mouse model of OA and in human OA patients.53 Interestingly,in both the TMD and ageing TMJ-OA models that exhibited the classic TMJ-OA phenotype,we observed condylar cartilage and subchondral bone with high levels of TGF-β1 signalling activity.These results are consistent with those from human OA studies.54-55High levels of active TGF-β1 signalling occur in the bone marrow microenvironment,leading to abnormal bone remodelling.31TGF-β1 overexpression in subchondral bone caused TMJ mandibular condyle degradation.33In this study,CED mice,an osteoblast-specific mutant TGF-β1 transgenic mouse model,were used to display abnormal active TGF-β1 participating in TMJ-OA progression.Similar to the TMD and ageing animal models,Osterix-positive osteoprogenitor cells and increasing numbers of TRAP-positive cells resulted in uncoupled bone formation in the condylar subchondral bone of CED mice.Cartilage degradation with increased Osterix,p-Smad2/3 and Col X expression were observed in 40-wk-old CED mice relative to age-matched WT mice.These results indicate that TGF-β1 overexpression in subchondral bone induces TMJ-OA.The expression and activity of TGF-β1 signalling significantly increase in the cartilage at the early degenerative stage of OA.30This observation indicates that abnormal activation of TGF-β1 signalling induces cartilage degeneration in TMJ-OA.

TMJ-OA is a common degenerative joint disease that limits the quality of life of patients.TMJ-OA oetiology is multifactorial and mainly includes malocclusion,ageing,joint injury and inflammation.We used ageing mice with spontaneous TMJ-OA to examine cartilage and subchondral bone lesions.The TMD models imitated the most common clinical oetiology of TMJ-OA.Furthermore,our results showed that an aberrant level of TGF-β1 signalling in the condylar cartilage induced cartilage degeneration in disordered occlusion animal models,age-related TMJ-OA mice and TGF-βoverexpressing mice.Moreover,significantly high TGF-β1 signalling in subchondral bone contributed to uncoupled bone remodelling in TMJ-OA.Thus,TGF-β1 can be considered a pathogenic factor in the onset of TMJ-OA.Our results suggest that the TGF-β1 concentration may be used as a diagnostic index of human TMJ-OA in clinical applications.Furthermore,it is possible to reverse aberrant TGF-β1 signalling to rescue cartilage degeneration and enhance subchondral bone volume in TMD models.Therefore,targeting the TGF-β1 signalling pathway might be an effective means of treating human TMJ-OA.Biological reagents that inhibit TGF-β1 signalling may be used as drugs to treat TMJ-OA in the clinic.

MATERIALS AND METHODS

As a degenerative disease,ageing is a major risk factor for human OA.Ageing-related changes in the musculoskeletal system may contribute to OA development and progression.43-45We established a spontaneous TMJ-OA model using 45-and 60-wkold C57BL/6J mice.Articular chondrocytes exhibited an agerelated decline in proliferation and synthetic capacity.46As age increased,the calcified cartilage layer became thicker.There was a higher number of Osterix-and Col X-positive chondrocytes in 45-and 60-wk-old mice than in 12-wk-old mice.Ageing not only reduced the cartilage thickness but also induced the condylar cartilage to form bone matrix like-tissue.47This result suggests that ageing may be an important factor in cartilage degeneration.In addition,the condylar subchondral bone of ageing mice displayed decreased bone volume,with more TRAP-positive cells and fewer Osterix-positive osteoprogenitor cells in subchondral bone marrow.With ageing,inevitable bone loss occurs,which is frequently the cause of osteoporosis,and inevitable bone and joint degeneration are observed,which often result in osteoarthrosis.48TGF-β levels become excessively high in the blood stream of older people and ageing mice.49-50In the ageing rodent models,p-Smad2/3-positive cells were significantly enhanced in the cartilage and subchondral bone.Therefore,ageing with abnormal TGF-β1 signalling activity aggravated condylar cartilage degeneration and subchondral bone resorption.

The current study was approved by the Ethical Committees of the West China School of Stomatology,Sichuan University,and the State Key Laboratory of Oral Diseases.All experimental methods and procedures were carried out in accordance with the approved guidelines.

首先，区块链的去中心化特性与IoT的分布式结构能够较好地融合。区块链系统是一种完全的去中心化结构，不依赖于任何形式的集中式管控，这恰恰与当前IoT系统的分布式架构具有较高的契合度。基于区块链的IoT技术不仅能够依靠共识机制实现对IoT设备的分布式管控，同时还可以使用智能合约技术实现对相关感知信息的自动反馈。

如何让调动好每个人的积极性，是企业家资源管控能力的表现。新生代企业家只有意识到每位人才对工作不同的定义，才能真正做到聆听员工的心声。要学会与各类人打交道，让不同类型的人都能拥有适合的岗位与任务，人尽其才。企业家不仅为大众提供工作岗位，更能让他们拥有一份令他们自己感到舒适与愉悦的工作，发挥他的社会价值。“舒适与愉悦”，即要找满足人们的真正需求，这种需求是一种人文关怀。由于80、90后的员工，内心世界丰富，对于他们的物质上的需求，新生代企业家是好把握的，但对于了解精神上的需求，则需要更贴近他们的生活习惯，满足已经养成的固定爱好，从人们的生活习惯中，找出每个人的真正需求。

Six-week-old male Sprague-Dawley(SD)rats(weighing 160-180g)were purchased from Chengdu Dossy Biological Technology Co.,Ltd.and randomly divided into sham-operated control(Con,operation was performed in a manner similar to the experimental group except without occlusal disorder)and TMD groups(n=18 rats/group).In the experimental group(TMD),disordered occlusion was created by abnormal dental occlusion force based on a previous report.16,59Brie fl y,an orthodontic ligation silk(0.25-mm diameter)was inserted between the first and second molars,and a ligation silk knot was created on the first molar of the maxillary to induce abnormal mechanical loading on the rat TMJ.TMJ samples from TMD rats were collected 8 wks later.For pharmacological treatment,4 wks after surgery,animals received intraperitoneal injections of a TGF-I(1mg/kg,SB-505124,Selleck Chemicals)or an equivalent volume of vehicle(DMSO,Dimethyl sulfoxide)daily for 30 days.31 Rats were euthanised 60 days after surgery.Eight-week-old male C57BL/6J mice were obtained from Chengdu Dossy Biological Technology Co.,Ltd.and randomly divided into three groups(n=9 mice/group).The mice were maintained in a temperature-controlled room(22°C)under artificial illumination(lights on from 800 to 1 800 hours)and provided access to food and water ad libitum.During the period of feeding,three mice from each experimental group died.Mice were euthanized at 12 weeks(n=6),45 weeks(n=6)and 60 weeks(n=6),and TMJ samples of ageing mice were collected.

CED mice(40 weeks old)were obtained from the Animal Facility of the Johns Hopkins University School of Medicine.The CED-derived TGF-β1 mutation,which contains the full-length TGF-β1 with a point mutation(H222D),is specifically expressed by osteoblastic cells driven by a 2.3-kb type I collagen promoter.60

µCT analysis

逆变器转化效率通常会受以下两种因素影响：①把直流电流快速转换成交流正弦波，在一定程度上会给功率半导体整体发热产生很大损失。②逆变器MPPT中的控制算法会严重影响整个转换效率[3]。光伏电池阵列中的输出电流与电压会随日照的温度变化发生巨大变化，且随用电设备负载率的变化发生较大波动。逆变器的MPPT算法在一定程度上可以对电流与电压进行有效控制，使光伏系统能够快速达到最大输出功率，并且能够在短时间内跟踪到最大电力点，转化效率也将得到进一步提高。当负载率在63%左右时，在此条件下的逆变器电气效转换效率呈现的是一种最高状态。

随着工业网络技术的不断发展和演进，未来工业内连接将被具有以太网物理接口的网络主导，并实现控制数据与信息数据同口传输；工业自动化生产开始在所有网络层次上横向与垂直集成，使得IT与OT节点（装备、物料等生产要素）直接互联；智能工厂+智能生产+智能物流，将智能设备、人和数据连接起来以智能的方式利用这些交换的数据组成更高层次的平台。

Samples were imaged on a µCT scanner(μCT50;SCANO,Switzerland).The samples were scanned at a voltage of 50kVp,a current of 200µA and a resolution of 5.0µm per pixel.Sagittal images of the TMJ condyle subchondral bone were used to perform threedimensional histomorphometric analysis.Two cubic regions of interest(each 0.5×0.5×0.5mm3)were selected from the middle of the centre and posterior third of the condylar subchondral bone.16Within the selected regions,bone volume fraction(%,BV/TV)and trabecular separation(Tb.Sp)were determined and compared between the experimental and control groups.

Animal models

Histological staining

At the time of euthanasia,the TMJ samples of all animals were dissected and fixed in 4%paraformaldehyde overnight.After decalcification in 10%EDTA(pH 7.2-7.4),samples were processed,embedded in paraffin and cut into 5-μm sections using a microtome(Leica,RM2235,Germany).Standard HE staining was used to examine tissue histology.Safranin O and fast green staining was performed to determine proteoglycan changes,and the histological data were further analysed by assessing the Mankin and Osteoarthritis Research Society International(OARSI)scores.The scoring methods were described previously.61-62 Semiquantitative Makin and OARSI scores were significantly correlated and positively associated with exercise duration.However,in the rodent model of early OA,the OARSI system was less sensitive than the Mankin sore in reflecting the progression of cartilage change.63We used two scoring systems to semiquantify the severity of TMJ-OA in the ageing and TMD models.TRAP staining was performed using a standard protocol(Sigma-Aldrich).

Immunohistochemistry

Immunohistochemical analyses of sections of each construct were performed using an Anti-Rabbit/Mouse HRP-DAB Cell&Tissue Staining Kit(R&D Systems,USA).Sections were subjected to epitope recovery in citrate buffer at 99°C for 30 min.Once room temperaturewasreached,slideswerewashed in triethanolamine-buffered saline,and nonspecific immunoglobulin binding was blocked with 5%(V/V)bovine serum albumin for 30 min at room temperature.Sections were incubated overnight at 4°C with the following primary antibodies:rabbit anti-Osterix(Santa Cruz Biotechnology,1:100,USA);rabbit anti-Col X(Abcam,1:100,ab58623)and a TGF-β pathway-specific antibody against p-Smad2/3(sc-11769,Santa Cruz Biotechnology,1:100,USA).All sections were incubated with a biotinylated secondary antibody,stained using an R&D HRP-DAB Staining Kit and counterstained with haematoxylin.After mounting,the slides were photographed with an Olympus BX53 microscope(Olympus,Japan).The numbers of Col X-positive cells in the cartilage layer,TRAP-positive osteoclasts,Osterix-positive cells and p-Smad2/3-positive cells in the posterior and middle condylar subchondralboneweredetermined.Thepercentagesof positive cells in all chondrocytes are shown.All sections were placed onto one slide and processed together under the same conditions.

Statistical analysis

All experiments were performed independently in triplicate.Comparisons between groups were evaluated with unpaired two-tailed Student's t-test between two groups or with one-way analysis of variance(ANOVA)followed by Tukey's test for multiple comparisons using SPSS 16.0 software(IBM,Armonk,NY,USA).*P＜0.05 was considered to indicate a significant difference between groups.

ACKNOWLEDGEMENTS

This study was supported by 2016JQ0054 and NSFC grants 81470711 to L.Z.,National Key Research and Development Program of China 2016YFC1102700 to X.Z.

ADDITIONAL INFORMATION

Competing interests:The authors declare no competing interests.

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