| [Background]Pathological calcification refers to the calcium deposition in tissues other than bones and teeth.The pathological calcification in blood vessels,muscles,cartilage and other tissues not only seriously affects the body function,but also endangers life.Trauma,abnormal force,infection and other causes can lead to pathological calcification of various tissues in the oral craniofacial region,such as myositis ossificans of masticatory muscle after trauma,cartilage calcification in temporomandibular joint osteoarthritis,heterotopic ossification of periarticular tissue after joint replacement,etc.This kind of pathological calcification will cause patients with maxillofacial pain,joint stiffness,limited movement and other functional disorders,and difficult to reverse once it occurs.It usually can only rely on surgical resection,but it is easy to relapse after resection,so it is extremely important to explore its prevention and treatment methods.Therefore,it is an important scientific problem in stomatology to study the pathogenesis and intervention strategies of pathological calcification systematically.Calcified vesicles were first discovered in the growth plates of cartilage.In recent years,more and more studies have been conducted on the physiological and pathological calcification of calcified vesicles,which participate in the calcification process through enrichment of calcium and phosphorus as the site of calcification nucleation.However,the understanding of calcified vesicles is still very limited,especially its role in pathological calcification,intracellular origin and formation mechanism still need to be further studied.As a common joint degenerative disease,OA seriously affects patients’quality of life,and TMJ is one of the joints most easily affected.The majority of TMJOA patients have cartilage calcification in diseased joints,which is an important factor in the disease progression,but the mechanism of cartilage calcification is unclear,so there is a lack of effective intervention methods.HO is the formation of ectopic bone in the soft tissue,and its pathogenesis is still unclear.Previous studies of our research group have confirmed that pathological calcification plays an important role in the occurrence and progression of both OA and HO,and interestingly,calcified vesicles have been found to participate in the progression of the above two diseases.Therefore,through studying the origin,occurrence and pathogenic mechanism of calcified vesicles in TMJOA and HO,etc.It is expected to further improve the knowledge and understanding of calcified vesicles and provide a new strategy for the clinical diagnosis and treatment of pathological calcification diseases.[Methods]In the first part of the experiment,we mainly took TMJOA cartilage calcification caused by abnormal biological force as the model.First,Alizarin red,Von Kossa and Hematoxylin-eosin staining and Micro-CT were performed for the macroscopic characterization of cartilage calcification,and the spatiotemporal relationship between cartilage pathological calcification and cartilage degeneration in TMJOA was explored.Then the pathological calcification was characterized by scanning electron microscopy,transmission electron microscopy,elemental energy spectrum analysis,and other technologies.The microscopic morphology of pathological calcification in TMJOA cartilage was explored,and the calcified vesicles were also identified and characterized in vivo and in vitro.In the second part,we further investigated the mechanism of TMJOA chondrocyte-derived calcified vesicles and their role in TMJOA cartilage calcification.We first determined the relationship between the autophagic flow block and the secretion of calcified vesicles by immunofluorescence staining and semi-quantitative analysis of condylar cartilage of TMJOA rats and their controls.Subsequently,a three-dimensional co-culture system of TMJOA chondrocytes with type II collagen was constructed in vitro,and the role of autophagy in OA pathological calcification was further explored by alizarin red staining and immunofluorescence staining.The relationship between chondrocyte-derived autophagosomes and calcified vesicles was identified by transmission electron microscopy,scanning electron microscopy and immune electron microscopy.At the same time,TMJOA chondrocyte-derived vesicles were extracted and characterized,and their autophagosome origin and pro-calcification function were confirmed.Secondly,m RFP-GFP-LC3adenovirus transfection and immunofluorescence staining were used to explore the upstream mechanism of the blocked autophagic flow in TMJOA chondrocytes,and immunofluorescence staining of microtubules and related pathway proteins were used to characterize the role of histone deacetylase 6（HDAC6）-mediated microtubule homeostasis.Finally,HDAC6 was used as a molecular intervention target to verify in vivo experiments,which confirmed that intervention of microtubule instability could effectively inhibit the release of autophagic calcified vesicles and prevent TMJOA disease progression.In the third part,we used traumatic heterotopic ossification（HO）as a model to investigate the effect of injury on HO heterotopic bone formation by Micro-CT and quantitative analysis.Subsequently,the extracellular matrix of the ligament of HO rats at different stages was detected by transmission electron microscopy,scanning electron microscopy and freezing electron microscopy,and the calcification of the ligament tissue was systematically characterized from the microscopic morphology.Secondly,single-cell sequencing combined with flow cytometry and immunofluorescence staining were used to explore the cell biological changes after ligament injury in rats,and the results of single-cell sequencing were verified by HE and immunofluorescence staining.At the same time,the apoptotic calcified vesicles found in vivo were characterized by scanning and transmission electron microscopy.Finally,the vesicles were isolated from the ligaments of the control and HO groups by enzymatic hydrolysis and gradient centrifugation.Flow cytometry,transmission electron microscopy and immunofluorescence staining were used to systematically identify and characterize the apoptotic calcified vesicles.In the fourth part,to further study the occurrence of fibroblast-derived apoptotic calcified vesicles and their role in pathological calcification,an in vitro fibroblast-derived osteogenesis model was established to simulate the in vivo environment of ligament injury to further observe and characterize the calcification process.Firstly,the apoptotic calcified vesicles were characterized in the in vitro model by TEM,immunofluorescence staining and flow cytometry.Then the apoptotic calcified vesicles were extracted and injected into the ligament to verify their in situ calcification function.Subsequently,mass spectrometry analysis,fluorescence microscopy and co-incubation of apoptotic calcified vesicles with two-dimensional collagen mineralization model were used to explore the specific mechanism of calcium and phosphorus enrichment in apoptotic calcified vesicles and induced calcification.Secondly,the atomic force microscopy combined with HE staining was used to characterize the changes in the physical and chemical properties of the extracellular matrix after early calcification of HO.Single-cell sequencing,immunofluorescence staining,in vitro model of soft and hard matrix,and quantitative polymerase chain reaction were used to further explore the effect of the hardness of the ligament matrix on the polarization of macrophages and the molecular mechanism of immuno-osteogenic effect.Finally,the effect of inhibiting the release of apoptotic calcified vesicles or eliminating macrophages on heterotopic bone formation in HO rats was evaluated by local injection of apoptosis inhibitor z-VAD-fmk or macrophage scavenger clodronate liposomes in vivo.[Results]1.Pathological calcification can occur in the early stage of TMJOA,accompanied by the production of calcified vesiclesIn TMJOA rats,a large number of calcification deposits were found in the junction area of cartilage and subchondral bone after 2 weeks of modeling,and the calcification deposits increased significantly after 8 weeks（P<0.001）.Microscopic characteristics showed that the collagen structure of cartilage matrix was covered by a large number of calcification deposits after 2 weeks of TMJOA modeling.At the same time,a large number of spherical particles containing calcium and phosphorus appeared on the surface and gap of collagen fibers,which gradually fused and aggregated to form larger calcified plaques scattered in the extracellular matrix.TEM results confirmed that chondrocytes released a large number of vesicles,and elemental energy spectrum detection confirmed that they contained calcium,phosphorus,oxygen and other elements,so we defined them as calcified vesicles.Vesicles in TMJOA were extracted by enzymatic hydrolysis and differential centrifugation to further confirm that calcified vesicles contained calcification precursors inside.Therefore,the above results suggest that calcified vesicles may be involved in the formation of pathological calcification of TMJOA cartilage.2.The microtubule instability of TMJOA chondrocytes leads to the blockage of autophagic flow,and the release of autophagosome-derived calcified vesicles leads to the formation of pathological cartilage calcificationTEM results showed that amorphous calcium and phosphorus precursors were contained in the autophagosome of TMJOA chondrocytes,and the autophagosome-lysosome fusion in the cells was significantly reduced（P<0.001）,and the gene expression of autophagosome-lysosome fusion-related factors（SNAP29,STX17,VAMP8）was significantly down-regulated（P<0.001）.Collagen type II chondrocytes culture system were constructed in vitro.Wortmannin,a reagent used to block autophagosome formation,significantly inhibited cartilage calcification（P<0.001）.Immunoelectron microscopy showed that autophagy-derived microtubule-associated protein 1A/1B light chain 3B（LC3）molecules aggregated along the periphery of the calcified vesicles,and these vesicles contained a large number of calcification precursors inside.LC3 positive calcified vesicles aggregated in the extracellular matrix（ECM）and formed calcification nodules.In vitro experiments confirmed that rapamycin,an autophagy agonist,could significantly increase the proportion of LC3-positive calcified vesicles secreted by OA chondrocytes（P<0.001）,while the ability of OA chondrocytes to induce calcification was significantly reduced after removing LC3-positive vesicles.The instability ofα-tubulin in TMJOA group chondrocytes was serious,and the acetylation level ofα-tubulin was significantly decreased（P<0.001）,and the activity of histone deacetylase 6（HDAC6）was significantly increased（P<0.001）.Therefore,deacetylation ofα-tubulin leads to microtubule instability,which leads to the failure of autophagosome to bind to lysosomes,and finally the autophagosome is released into the ECM as autophagic calcified vesicles.Intra-articular injection of tubacin（HDAC6inhibitor）in TMJOA rats showed that the proportion of LC3 positive calcified vesicles in TMJOA was significantly higher than that in the control group（P<0.001）,while the proportion of tubacin group was significantly decreased（P<0.001）,and cartilage calcification and degeneration were significantly alleviated.3.In the early stage of ligament injury,there was a large amount of calcification accompanied by the release of apoptotic calcified vesicles from PROCR positive fibroblastsEctopic bone nodules appeared 3 weeks after ligament injury in the rat model of HO,and the volume of bone nodules gradually increased with the extension of modeling time,and trabecular structure appeared 9 weeks later.Microscopic characterization results showed that the calcification deposits in the HO group appeared at 1 week,and the calcification deposits increased significantly and showed a diffuse distribution at 3 weeks.Six cell types were identified by the HO disease map based on single-cell transcriptome sequencing results.The number of fibroblasts was significantly reduced and the number of macrophages and endothelial cells was significantly increased after 1 and 3 weeks of ligament injury.Apoptosis score analysis defined the Cluster 1 fibroblast subset as the apoptotic preferential cell subset.The Procr gene was also highly expressed in a subset of Cluster 1 fibroblasts,and immunofluorescence staining and flow cytometry analysis further confirmed the presence of a PROCR-positive fibroblast subset.The results of HE and immunofluorescence staining confirmed that PROCR+fibroblasts underwent apoptosis after ligament injury,and TEM results showed that fibroblasts released a large number of apoptotic vesicles in the ECM.Energy spectrum analysis confirmed that they contained calcified precursors and therefore defined them as apoptotic calcified vesicles.Apoptotic calcified vesicles aggregated into larger calcified nodules in the ECM,and the number of apoptotic calcified vesicles peaked at 1 week after injury.We further confirmed the presence of apoptotic calcified vesicles using enzymatic hydrolysis and differential centrifugation to extract vesicles from rat HO ligaments.4.The apoptotic calcified vesicles caused matrix calcification and increased stiffness in the early stage of HO,which induced M2 polarization of macrophages and formed a pro-osteogenesis microenvironmentAn in vitro culture model of fibroblasts was constructed to simulate the in vivo model of HO.The results showed that fibroblasts underwent apoptosis and secreted apoptotic calcified vesicles,which aggregated to form calcified nodules in vitro.And the results of atomic force（AFM）showed that the hardness of calcified ECM increased significantly（P<0.05）.The calcification foci could be formed 1 week after the implantation of the apoptotic calcified vesicles into the ligament of rats,and the formation of ectopic bone was more obvious 3 weeks after the implantation.An in vitro mineralization model of apoptotic calcified vesicles and type I collagen fibers was established,and the results showed that apoptotic calcified vesicles could adsorb to collagen fibers and mediate collagen calcification.Apoptotic calcified vesicles can enrich calcium through Annexin channels,and the high internal concentration of calcium and phosphorus can promote the formation of internal crystal nuclei.Blockade of Annexin channel by LCKLSL can effectively inhibit Ca2+uptake by apoptotic calcified vesicles and their ability to induce calcification.AFM results showed that ligament calcification in the early stage of HO led to a significant increase in ECM stiffness（P<0.05）,accompanied by an enrichment of macrophages near the calcification.The results of single-cell sequencing and in vitro experiments showed that macrophages were a heterogeneous cluster in HO,and the increase in matrix stiffness caused by calcification deposition promoted the polarization of macrophages to M2 type,which initiated the osteogenic cascade of HO.Therefore,both apoptotic calcified vesicles and macrophages in the early stage of HO are potential targets for intervention.Animal experiments confirmed that both z-VAD-fmk（an apoptosis inhibitor）treatment and clodronate liposome injection to deplete macrophages could significantly reduce ectopic bone formation in HO rats（P<0.001）.[Conclusion]In this study,the cartilage calcification of TMJOA caused by abnormal biological force and heterotopic ossification of HO caused by trauma were used as models to study the source,pathogenic mechanism,and role of calcified vesicles in the formation of pathological calcification.The main conclusions can be summarized as follows:（1）Pathological cartilage calcification occurs in the early stage of TMJOA,and calcified vesicles are closely related to the progression of cartilage calcification.Secretory autophagosomes are the main pathway for the generation of calcified vesicles from OA chondrocytes,which is due to the blockage of autophagic flow in chondrocytes by HDAC6-mediated microtubule instability.（2）Inhibition of HDAC6 activity can block the release of autophagic calcified vesicles from TMJOA chondrocytes,thereby effectively reversing the calcification and degeneration of TMOA cartilage.（3）In the HO model,significant matrix calcification occurred in the early stage of ligament injury,and apoptotic calcified vesicles released by apoptotic PROCR+fibroblasts could be seen in the calcified area.Apoptotic calcified vesicles enriched calcium through Annexin,leading to ligament calcification and increased matrix stiffness,which initiated the polarization of peripheral M2 macrophages.It leads to the formation of ectopic bone.（4）Inhibition of the release of apoptotic calcified vesicles or removal of macrophages could effectively inhibit the progression of HO.The innovation of this research can be summarized as follows:（1）We took the lead in putting forward two new concepts of autophagic calcified vesicles and apoptotic calcified vesicles in the world,and preliminarily clarified their pro-calcification mechanism,which further improved the understanding of calcified vesicles（Theoretical innovation）.（2）We revealed that autophagic calcified vesicles secreted by chondrocytes mediate the pathological calcification of cartilage in the case of minor joint injury such as abnormal biological forces（Figure 1）,while apoptotic calcified vesicles secreted by PROCR+fibroblasts mediate the calcification of ligament in the case of major injury such as trauma（Figure 2）（Mechanism innovation）.（3）Inhibition of HDAC6 activity can block the release of autophagic calcified vesicles from TMJOA chondrocytes,thereby effectively reversing the calcification and degeneration of TMOA cartilage.Inhibition of the release of apoptotic calcified vesicles or removal of macrophages can effectively inhibit the progression of HO（Blocking strategy innovation）.Therefore,our findings improve the theoretical study of pathological calcification diseases,and provide a new perspective and potential therapeutic targets for the study of pathological calcification related diseases such as OA and HO. |
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