The Mechanism Study Of Osteoarthritic Cartilage Calcification Mediating TMJOA Disease Progression Through Lysosomal Destabilization

Osteoarthritis(OA)is the most common degenerative joint disease and one of the leading causes of disability globally.The degeneration of joint cartilage is the most significant pathological changes in OA,and the entire disease process of OA is closely related to inflammatory responses.OA not only occurs in large joints such as the knees or hips.The temporomandibular joint(TMJ)in the oral and maxillofacial system is also one of the most common sites of OA.TMJOA is a severe manifestation of temporomandibular disorder(TMD),which is the fourth largest category of oral and maxillofacial diseases.The occurrence of TMD/TMJOA is often accompanied by abnormalities in functions such as chewing and speech in the stomatognathic system,thereby affecting patients’ quality of life and even endangering their psychological health.However,the clinical management of OA is still challenging.Contemporary mainstream therapies are limited to palliative treatments such as pain or inflammation reduction,with no effective methods to retard or reverse the progression of OA.The lack of understanding of the source and mechanisms of inflammation in OA progression may be an important reason for this clinical dilemma.Pathological calcification of cartilage has been recognized as a hallmark of OA and has been considered to initiate OA in recent years.The degree of cartilage calcification is significantly positively correlated with the severity of OA.However,no strategies are available to prevent crystal deposition and permit mineral dissolution in osteoarthritic cartilage so far.Pathological calcification often leads to inflammatory reactions and tissue damage,and the cartilage calcification is also closely related to inflammatory responses in OA.However,the specific mechanism by which pathological calcification of cartilage initiates inflammatory reactions remains controversial.Interleukin-1β(IL-1β)is one of the most important pro-inflammatory factors mediating cartilage degeneration in OA,which can lead to the degradation of extracellular matrix of cartilage.However,clinical trials targeting IL-1β for the treatment of OA have not yielded the desired results.This contradictory result reminds us that the cascade of inflammatory responses triggered by upstream signals of IL-1β in OA may be more complex than we know.NLRP3(NOD-like receptor protein-3)inflammasome is a classic upstream regulator of IL-1β,and it has been shown to be closely related to the occurrence of OA-related inflammatory responses.Moreover,in diseases such as silicosis and atherosclerosis,NLRP3 inflammasome can be activated by different types of endogenous and exogenous crystals deposited in the body,thereby triggering inflammatory reactions;In some cases,it may even induce pyroptosis to amplify inflammatory reactions,ultimately exacerbating disease progression.However,whether the cartilage calcification can activate NLRP3 inflammasome and induce chondrocyte pyroptosis in the progression of OA has not been reported.Furthermore,lysosomes are hydrolases-loaded intracellular organelles involved in the digestion of external agents.Different types of extracellular crystals deposited in tissues can trigger lysosomal destabilization characterized by "lysosomal membrane permeabilization(LMP)," thereby releasing various hydrolytic enzymes into the cytoplasm.As one of the important hydrolytic enzymes in lysosomes,Cathepsin B(CTSB)can effectively activate NLRP3 inflammasome.However,it is still unclear whether the above pathological processes are involved in the progression of OA.In summary,this study proposes the following hypothesis: pathological calcification of condylar cartilage in TMJOA activates NLRP3 inflammasome by causing lysosomal destabilization and induces chondrocyte pyroptosis,thereby exacerbating the degeneration of condylar cartilage and the progression of TMJOA.【Objectives】Using the unilateral anterior crossbite(UAC)induced mouse model of TMJOA established by our research group and the primary condylar chondrocytes,this study aimed to explore:1.Reveal the specific components of pathological calcification in condylar cartilage of TMJOA and whether the calcification of condylar cartilage is a key factor in exacerbating the progression of TMJOA.2.Elucidate the specific mechanisms by which pathological calcification of condylar cartilage exacerbates the progression of TMJOA,and clarify whether lysosomal destabilization,NLRP3 inflammasome,and pyroptosis play critical roles in it.3.Screen for effective targets to counteract the pathogenic effects of pathological calcification of condylar cartilage,thereby providing new insights into the treatment of TMJOA.【Methods and Result】Section One: HAp crystals,the main component of pathological calcification in condylar cartilage,is an important factor in promoting the progression of TMJOA.After establishing a mouse TMJOA animal model,we first performed Safranin O staining,von Kossa staining,modified OARSI histological scoring,scanning electron microscopy(SEM)combined with energy dispersive X-ray spectroscopy(EDX),atomic force microscopy(AFM),and micro-Fourier transform infrared spectroscopy(Micro-FTIR)detection on mouse temporomandibular joint(TMJ)tissue specimens.We determined that the main component of pathological calcification in OA condylar cartilage is HAp crystals,and the deposition of HAp crystals in condylar cartilage gradually increases with the progression of TMJOA.Subsequently,through immunohistochemical staining and ELISA detection of IL-1β,as well as the correlation analysis,we confirmed that the degree of pathological calcification in condylar cartilage is strongly positively correlated with the expression of IL-1β in cartilage and the severity of TMJOA.Finally,based on the above results,we attempted to inhibit the occurrence of pathological calcification of condylar cartilage in OA mice by intraperitoneal injection of high-dose alendronate sodium.Using the same experimental methods,we ultimately found that high-dose alendronate sodium successfully inhibited the deposition of HAp crystals in mouse OA condylar cartilage.The reduction of HAp crystal deposition significantly alleviated the expression of IL-1β in condylar cartilage and the progression of TMJOA.However,high-dose alendronate sodium also led to severe skeletal side effects in mice.In summary,this section clarifies that HAp crystals are the main component of pathological calcification in OA condylar cartilage,and the deposition of HAp crystals in condylar cartilage is an important factor exacerbating the expression of IL-1β and the progression of TMJOA.Section Two: HAp crystals deposited in OA condylar cartilage can induce chondrocyte pyroptosis by activating the NLRP3 inflammasome.After establishing the mouse TMJOA animal model,we first performed immunofluorescence staining(NLRP3,Caspase1 p20),TUNEL staining,immunohistochemical staining(N-GSDMD),and transmission electron microscopy(TEM)on mouse TMJ tissue specimens.The results showed that with the increasing deposition of HAp crystals in condylar cartilage,the activation of NLRP3 inflammasomes and the occurrence of chondrocyte pyropyosis were also significantly enhanced.Subsequently,on the basis that high-dose alendronate sodium effectively inhibited the deposition of HAp crystals in the OA condylar cartilage,using the same detection methods,we found that the reduction of HAp crystals in condylar cartilage effectively inhibited the activation of NLRP3 inflammasomes and the occurrence of chondrocyte pyroptosis,thereby confirming that the HAp crystal deposited in condylar cartilage is a key factor in activating the NLRP3 inflammasome and inducing chondrocyte pyroptosis.Finally,to further clarify the causal relationship between them,we used co-cultured commercialized HAp crystals with primary condylar chondrocytes fo in vitro combined with NLRP3 inflammasome inhibition experiments(using NLRP3-specific inhibitor MCC950 and Caspase1 inhibitor VX-765 to inhibit the NLRP3 inflammasome in chondrocytes),through transmission electron microscopy(TEM),SEM,Raman spectroscopy,Western blot,Hoechst/PI staining,LDH cytotoxicity detection,ELISA and other detection methods.We ultimately confirmed that HAp crystals activated the NLRP3 inflammasome to induce chondrocyte pyroptosis,thereby exacerbating the progression of TMJOA.Section Three: The lysosomal destabilization and the subsequent cytosolic release of CTSB caused by HAp crystals are key factors triggering NLRP3inflammasome-mediated chondrocyte pyroptosis.Using the mouse TMJOA animal model and the co-culture system of HAp crystals and primary chondrocytes,we began to explore how extracellular HAp crystals activate intracellular NLRP3 inflammasomes.First,using TEM combined with EDX and selected area electron diffraction,calcein labeling of HAp crystals,TRITC Phalloidin staining,Lysotracker staining,immunofluorescence staining(Galectin-3,LAMP2),we investigated the interaction between HAp crystals and chondrocytes in vivo and in vitro,and found that HAp crystals could be engulfed by chondrocytes and subsequently ingested by lysosomes,leading to lysosomal destabilization characterized by lysosomal membrane permeabilization.To further clarify the causal relationship between HAp crystals and lysosomal destabilization,we inhibited the internalization of HAp crystals by chondrocytes and the deposition of HAp crystals in OA condylar cartilage using cytochalasin D and high-dose alendronate sodium,respectively.Through the same methods,we confirmed that HAp crystals in OA condylar cartilage were the key cause of lysosomal destabilization in condylar chondrocytes.In addition,through immunofluorescence staining(CTSB),Western blot,and ELISA,we determined that the occurrence of lysosomal destabilization would release the hydrolytic enzyme CTSB,which is an agonist NLRP3 inflammasome,into the cytoplasm.Finally,by reducing the expression of CTSB protein in chondrocytes through small interfering RNA and inhibiting the internalization of HAp crystals in chondrocytes by cytochalasin D,combined with immunofluorescence staining(CTSB),Western blot,Hoechst/PI staining,LDH cytotoxicity detection and other detection methods,we found that the reduction of cytoplasmic CTSB effectively inhibited HAp crystal-induced NLRP3inflammasome-mediated chondrocyte pyroptosis.In summary,this section of the experiment firstly confirmed in TMJOA that the pathological calcification of OA condylar cartilage is a key factor in inducing lysosomal destabilization and subsequently releasing CTSB protein into the cytoplasm,which is the key to trigger the NLRP3inflammasome-mediated chondrocyte pyroptosis.Section Four: Alleviating lysosomal destabilization effectively inhibits the pathogenic effects of HAp crystals and alleviates the progression of TMJOA.Given the above findings,we attempted to treat mouse TMJOA model by alleviating lysosomal destabilization of OA condylar cartilage.First,we investigated the therapeutic effect of the small molecule inhibitor CA074 me on the co-culture system of HAp crystals and primary condylar chondrocytes.Through immunofluorescence staining(CTSB,Galectin-3,LAMP2),Western blot,Hoechst/PI staining,and ELISA,we found that CA074 me effectively inhibited CTSB expression while alleviating the lysosomal destabilization caused by HAp crystals in chondrocytes,thereby also inhibiting NLRP3inflammasome-mediated chondrocyte pyroptosis.Therefore,we performed intra-articular injection of CA074 me in the mouse TMJOA model and simultaneously compared its efficacy with intra-articular injection of MCC950.The results showed that under the existence of HAp crystal deposition in OA condylar cartilage,CA074 me effectively inhibited the expression of CTSB and alleviated the occurrence of lysosomal destabilization in OA cartilage,but MCC950 did not produce the above effects.However,both CA074 me and MCC950 significantly inhibited the activation of the NLRP3 inflammasome and the occurrence of chondrocyte pyroptosis in condylar cartilage,ultimately alleviating the progression of TMJOA.In addition,the efficacy of CA074 me was better than that of MCC950.【Conclusion】1.Successfully identified the main component of pathological calcification in TMJOA condylar cartilage was hydroxyapatite crystals(HAp),whose deposition in cartilage increases with the progression of TMJOA.2.It was clarified that the deposition of pathological calcification in condylar cartilage promotes the progression of TMJOA.3.It was clarified that the deposition of pathological calcification in condylar cartilage activates the NLRP3 inflammasome to induce chondrocyte pyroptosis,thereby exacerbating the progression of TMJOA.4.For the first time in TMJOA,the pathological link between pathological calcification in condylar cartilage and chondrocyte lysosomal destabilization was discovered,and the key role of lysosomal destabilization in inducing NLRP3 inflammasome-mediated chondrocyte pyroptosis was confirmed.5.It was verified that alleviating lysosomal destabilization by the small molecule inhibitor CA074 me effectively inhibited NLRP3 inflammasome-mediated chondrocyte pyroptosis and ultimately alleviated the progression of TMJOA in animal model.6.Compared with MCC950,an NLRP3 inhibitor that has entered clinical trials for the treatment of arthritis,CA074 me has shown better efficacy in the treatment of TMJOA in animal model.Through this study,we have further elucidated the pathogenic mechanism of condylar cartilage calcification in the progression of TMJOA,For the first time,we clarified the central role of lysosomal destabilization in the pathogenic effects of cartilage calcification and confirmed the therapeutic effect of alleviating lysosomal destabilization on TMJOA.Therefore,we innovatively proposed that the prevention of lysosomal destabilization maybe a conceptual and promising target for retarding TMJOA and even other joint OA progression.