Pathological cartilage calcification plays an important role in osteoarthritis progression but in which the origin of calcified extracellular vesicles (EVs) and their effects remain unknown. Here, we demonstrate that pathological cartilage calcification occurs in the early stage of the osteoarthritis in which the calcified EVs are closely involved. Autophagosomes carrying the minerals are released in EVs, and calcification is induced by those autophagy-regulated calcified EVs. Autophagy-derived microtubule-associated proteins 1A/1B light chain 3B (LC3)–positive EVs are the major population of calcified EVs that initiate pathological calcification. Release of LC3-positive calcified EVs is caused by blockage of the autophagy flux resulted from histone deacetylase 6 (HDAC6)–mediated microtubule destabilization. Inhibition of HDAC6 activity blocks the release of the LC3-positive calcified EVs by chondrocytes and effectively reverses the pathological calcification and degradation of cartilage. The present work discovers that calcified EVs derived from autophagosomes initiate pathological cartilage calcification in osteoarthritis, with potential therapeutic targeting implication.
Introduction: Sensory nerves and vessels are critical for skeletal development and regeneration, but crosstalk between neurovascular network and mineralization are not clear. The aim of this study was to explore neurovascular changes and identify bioactive regulators during in situ osteogenesis.Method:In situ osteogenesis model was performed in male rats following Achilles tenotomy. At 3, 6 and 9 weeks after surgery, mineralization, blood vessels, sensory innervation, and bioactive regulators expression were evaluated via micro-computed tomography, immunofluorescent staining, histology and reverse transcriptase-polymerase chain reaction analyses.Result: In the process of in situ osteogenesis, the mineral density increased with time, and the locations of minerals, nerves and blood vessels were highly correlated at each time point. The highest density of sensory nerve was observed in the experimental group at the 3rd week, and then gradually decreased with time, but still higher than that in the sham control group. Among many regulatory factors, semaphorin 3A (Sema3A) was highly expressed in experimental model and its expression was temporally sequential and spatially correlated sensory nerve.Conclusion: The present study showes that during in situ osteogenesis, innervation and angiogenesis are highly correlated, and Sema3A is associated with the position and expression of the sensory nerve.
Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic bodies (CABs), which are secreted by PROCR+ fibroblasts in the early stage of HO, lead to increased stiffness of the extracellular matrix. Specifically, single-cell transcriptome analyses of different stages of HO revealed a PROCR+ fibroblast population that released CABs in the early stage of HO. CAB aggregation produced calcified nodules with high concentrations of calcium and phosphate, similar to those in calcified tendons. Annexin channels mediate calcium influx into CABs, which absorb to collagen I via electrostatic interaction. Functional inhibition of CABs significantly decreased the early stage microcalcification and inhibited HO of Achilles tendons. Thus, we revealed a pathological mechanism of HO initiation and identified CABs from PROCR+ fibroblasts as the initiating factor of local microcalcification, creating an osteogenic microenvironment for HO.
Objective
To investigate the significant role of fibrocytes in pathogenesis of fibrous epulis.
Material
Human epulis specimens and human peripheral blood mononuclear cells (PBMCs) were used in this study.
Methods
Different subtypes of human fibrous epulides and normal gingival tissue specimens were collected for histological and immunofluorescence analyses. Electron microscopy and elemental analysis were used to characterize the extracellular microenvironment in different subtypes of fibrous epulides. Human PBMCs were subsequently isolated from peripheral blood to identify the factor that trigger fibrocyte differentiation in vitro.
Results
We demonstrated the presence of circulation-derived fibrocytes in fibrous epulides. These fibrocytes differentiate into myofibroblasts or osteoblasts under the local inflammatory environment in fibrous epulides. TGF-β1 promotes fibrocytes differentiation into myofibroblasts in a concentration-dependent manner. The TGF-β1 along with a high calcium and phosphorus extracellular environment stimulates the fibrocytes to differentiate into osteoblasts. The fibrocytes-derived myofibroblasts and osteoblasts are responsible for the fibrogenesis and osteogenesis of fibrous epulides, respectively. The persistent local inflammatory environment drived the differentiation of circulation-derived fibrocytes and drived the recurrence of fibrous epulides.
Conclusions
Fibrocytes play an important role in the fibrogenesis and osteogenesis in fibrous epulis, and might serve as a therapeutic target for the recurrence of fibrous epulides.
Heterotopic ossification (HO) severely affects people's lives; however, its pathological mechanism remains poorly understood. Although extracellular DNA (ecDNA) has been shown to play important roles in pathological calcification, its effects in HO development and progression remain unknown. The in vivo rat Achilles tendon injury model and in vitro collagen I calcification model were used to evaluate the effects of ecDNA in the ectopic calcifications and the main cell types involved in those pathological process. Histology, immunofluorescent staining, reverse transcriptase-polymerase chain reaction analysis and micro-computed tomography were used to identify the distribution of macrophage-derived ecDNA and elucidate their roles in HO. The results showed that the amount of ecDNA and ectopic calcification increased significantly and exhibited a strong correlation in the injured tendons of HO model compared with those of the controls, which was accompanied by a significantly increased number of M2 macrophages in the injured tendon. During in vitro co-culture experiments, M2 macrophages calcified the reconstituted type I collagen and ectopic bone collected from the injured tendons of HO rats, while those effects were inhibited by deoxyribonuclease. More importantly, deoxyribonuclease reversed the pathological calcification in the injured rat tendon HO model. The present study showed that ecDNA from M2 macrophages initiates pathological calcification in HO, and the elimination of ecDNA might be developed into a clinical strategy to prevent ectopic mineralization diseases. The use of deoxyribonuclease for the targeted degradation of ecDNA at affected tissue sites provides a potential solution to treat diseases associated with ectopic mineralization.
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