To explore the effects of miR-101-3p on IL-1β-induced chondrocyte injury and its underlying mechanisms.
 Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1β group, a negative control group (IL-1β+miR-NC group), and a miR-101-3p group (IL-1β+miR-101-3p group), which were treated with IL-1β after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1β (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1β+miR-101-3p group), an over-expression control group (IL-1β+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1β+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II.
 Results: Compared with the 0 ng/mL IL-1β, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1β (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1β group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1β group compared with the NC group (both P<0.05). Compared with the IL-1β+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1β+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1β+miR-101-3p group compared with the IL-1β+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1β-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes.
 Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1β-induced chondrocyte injury.
Cell Proliferation ; Chondrocytes ; Glycoproteins ; metabolism ; Interleukin-1beta ; metabolism ; MicroRNAs

OBJECTIVE: To assess the efficacy of GelMA hydrogel loaded with bone marrow stem cell-derived exosomes for repairing injured rat knee articular cartilage. METHODS: The supernatant of cultured bone marrow stem cells was subjected to ultracentrifugation separate and extract the exosomes, which were characterized by transmission electron microscopy, particle size analysis and Western blotting of the surface markers. The changes in rheology and electron microscopic features of GelMA hydrogel were examined after loading the exosomes. We assessed exosome release from the hydrogel was detected by BCA protein detection method, and labeled the exosomes with PKH26 red fluorescent dye to observe their phagocytosis by RAW264.7 cells. The effects of the exosomes alone, unloaded hydrogel, and exosome-loaded hydrogel on the polarization of RAW264.7 cells were detected by q-PCR and immunofluorescence assay. We further tested the effect of the exosome-loaded hydrogel on cartilage repair in a Transwell co-culture cell model of RAW264.7 cells and chondrocytes in a rat model of knee cartilage injury using q-PCR and immunofluorescence assay and HE and Masson staining. RESULTS: GelMA hydrogel loaded with exosomes significantly promoted M2-type polarization of RAW264.7 cells (P < 0.05). In the Transwell co-culture model, the exosome-loaded GelMA hydrogel significantly promoted the repair of injured chondrocytes by regulating RAW264.7 cell transformation from M1 to M2 (P < 0.05). HE and Masson staining showed that the exosome-loaded hydrogel obviously promoted cartilage repair in the rat models damage. CONCLUSION GelMA hydrogel loaded with bone marrow stem cell-derived exosomes can significantly promote the repair of cartilage damage in rats by improving the immune microenvironment.
Animals ; Bone Marrow Cells ; Cartilage ; Chondrocytes ; Exosomes ; Hydrogels/metabolism* ; Rats

OBJECTIVE: To summarize the role of chondrocyte mitochondrial homeostasis imbalance in the pathogenesis of osteoarthritis (OA) and analyze its application prospects. METHODS: The recent literature at home and abroad was reviewed to summarize the mechanism of mitochondrial homeostasis imbalance, the relationship between mitochondrial homeostasis imbalance and the pathogenesis of OA, and the application prospect in the treatment of OA. RESULTS: Recent studies have shown that mitochondrial homeostasis imbalance, which is caused by abnormal mitochondrial biogenesis, the imbalance of mitochondrial redox, the imbalance of mitochondrial dynamics, and damaged mitochondrial autophagy of chondrocytes, plays an important role in the pathogenesis of OA. Abnormal mitochondrial biogenesis can accelerate the catabolic reaction of OA chondrocytes and aggravate cartilage damage. The imbalance of mitochondrial redox can lead to the accumulation of reactive oxygen species (ROS), inhibit the synthesis of extracellular matrix, induce ferroptosis and eventually leads to cartilage degradation. The imbalance of mitochondrial dynamics can lead to mitochondrial DNA mutation, decreased adenosine triphosphate production, ROS accumulation, and accelerated apoptosis of chondrocytes. When mitochondrial autophagy is damaged, dysfunctional mitochondria cannot be cleared in time, leading to ROS accumulation, which leads to chondrocyte apoptosis. It has been found that substances such as puerarin, safflower yellow, and astaxanthin can inhibit the development of OA by regulating mitochondrial homeostasis, which proves the potential to be used in the treatment of OA. CONCLUSION The mitochondrial homeostasis imbalance in chondrocytes is one of the most important pathogeneses of OA, and further exploration of the mechanisms of mitochondrial homeostasis imbalance is of great significance for the prevention and treatment of OA.
Humans ; Reactive Oxygen Species/metabolism* ; Chondrocytes/metabolism* ; Osteoarthritis/metabolism* ; Homeostasis ; Mitochondria/metabolism* ; Cartilage, Articular/metabolism*

OBJECTIVE: To investigate whether Salvianolic acid A (SAA) can restore cartilage endplate cell degeneration of intervertebral discs and to identify the mechanism via regulation of micro-RNA. METHODS: Cartilage endplate cells were isolated from lumbar intervertebral disc surgical samples and were treated with serum containing a series of concentrations of SAA (2, 5, and 10 ?M) for 24, 48, and 72 h to identify a proper dose and treatment time of SAA. The effect SAA on interlenkin-1β (IL-1β)-induced extracellular matrix degradation of cartilage endplate cells were analyzed by Alcian blue staining and assessment of the expression levels of ADAMTS-5, MMP3 and Col2a1. Further, the potential target miRNAs were preliminarily screened by micro-RNA sequencing combining qRT-PCR and Western blot, and then, the miRNAs mimics and inhibitors were used to verify the regulatory effect of SAA on potential target miRNAs. RESULTS: The 10 μM SAA treatment for 48 h significantly enhanced the viability of cartilage endplate cells, and increased Col2a1 expression and glycosaminoglycan accumulation that were repressed by IL-1β, and reduced the effect of IL-1β on ADAMTS-5, and MMP3. Screening analysis based on micro-RNA sequencing and Venny analysis identified the downstream micro-RNAs, including miR-940 and miR-576-5p. Then, the miR-940-mimic or miR-576-5p-mimic were transfected into CEPCs. Compared with the SAA group, the expression of ADAMTS-5 and MMP3 increased significantly and the expression of COL2A1 obviously decreased after overexpression of miR-940 or miR-576-5p in CEPCs. CONCLUSION Salvianolic acid A attenuated the IL-1β-induced extracellular matrix degradation of cartilage endplate cells by targeting regulate the miR-940 and the miR-576-5p.
Humans ; Apoptosis ; Cartilage/metabolism* ; Chondrocytes/metabolism* ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 3/metabolism* ; MicroRNAs/metabolism*

The Wnt signaling exists in every kinds of species and regulates a variety of biological processes including cell fate, proliferation and function, immunity, stress, apoptosis and so on. During the researching, Wnt signaling also plays an important role in chondrocyte differentiation and maturation. So it has been the new spot in pathogenesis of osteoarthritis study.
Animals ; Chondrocytes ; metabolism ; pathology ; Humans ; Osteoarthritis ; metabolism ; pathology ; Signal Transduction ; Wnt Proteins ; metabolism

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology, proliferation, adhesion and differentiation, and even determining the cell fate. Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it. Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment, their regulatory network is not completely clear. In the current study, we generated silicon-based elastomer polydimethylsiloxane (PDMS) substrates with stiff (15:1, PDMS elastomer vs. curing agent) and soft (45:1) stiffnesses, which showed the Young's moduli of ~450 kPa and 46 kPa, respectively, and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein laminin β1, focal adhesion complex protein FAK to microfilament bundling. We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses. We then found the changes of secreted extracellular linkage protein, laminin β1, and focal adhesion complex protein, FAK, in chondrocytes in response to different substrate stiffnesses. These two proteins were shown to be directly interacted by Co-IP and colocalization. We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators. Taking together, this modulation axis of laminin β1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing, mechanotransduction, and cytoskeleton re-organization.
Cell Adhesion ; Chondrocytes ; Cytoskeleton/metabolism* ; Elastomers/metabolism* ; Laminin/metabolism* ; Mechanotransduction, Cellular

OBJECTIVETo investigate the protein profile after treatment of the cyclic uniaxial compressive stress on the rat condylar chondrocyte in vitro.

METHODSThe third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats, and a cellular compressive stress device (self-made four-point bending system) was used to apply stress on cells at 2000 microstrain and 4000 microstrain (0.5 Hz frequency) for 60 min. The early effects of cyclic uniaxial compressive stress on the protein profile of the rat mandibular condylar chondrocytes were examined by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

RESULTSThe results showed that the protein profile of the condylar chondrocyte did not change statistically in 2000 microstrain group. In 4000 microstrain group, the protein profile of the condylar chondrocyte was changed. Three new proteins appeared. Five proteins disappeared. Twenty-two proteins were down-regulated and 7 proteins were up-regulated (P < 0.05). The eight different protein spots were identified by MALDI-TOF-MS. It included cytoskeleton protein (gamma-actin and vimentin), glycometabolism protein (alpha enolase and stress-70 protein) and signal transduction protein (Raf kinase inhibited protein, RKLP).

CONCLUSIONSThere were significant alternations of the protein profile in the rat condylar chondrocyte after the 4000 microstrain cyclic uniaxial compressive stress loading for 60 min. These different proteins might take part in the early response to the cyclic uniaxial compressive stress.

Animals ; Cells, Cultured ; Chondrocytes ; metabolism ; Mandibular Condyle ; cytology ; Proteome ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical

Pericellular matrix (PCM) is a narrow tissue region surrounding chondrocytes, which "chondron" with its enclosed cells. A number of studies suggested that PCM is rich in proteoglycans, collagen and fibronectin, and plays an important role in regulating microenvironment of chondrocytes. Direct measures of PCM properties through micropipette aspiration technique showed that PCM was different from mechanical property of chondrocytes and nature extracellular matrix. However, the function of PCM is not clear, and need further study.
Animals ; Biomechanical Phenomena ; Chondrocytes ; chemistry ; cytology ; metabolism ; Extracellular Matrix ; chemistry ; metabolism ; Humans

BACKGROUNDThe mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint. The pericellular matrix, cellular membrane of the chondrocytes, and their cytoskeletal structures are key elements in the mechanical environment. The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties.

METHODSThree age groups of rabbit knees were evaluated: (1) young (2 months, n = 10); (2) adult (8 months, n = 10); and (3) old (31 months, n = 10). Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods. Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation. The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining.

RESULTSIn response to an applied constant 0.3 - 0.4 kPa of negative pressure, all chondrocytes exhibited standard linear viscoelastic solid properties. Model predictions of the creep data showed that the average equilibrium modulus (E(∞)), instantaneous modulus (E(0)), and apparent viscosity (m) of old chondrocytes was significantly lower than the young and adult chondrocytes (P < 0.001); however, no difference was found between young and adult chondrocytes (P > 0.05). The adult and old chondrons generally possessed a thicker pericellular matrix (PCM) with more enclosed cells. The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure (1.0 - 1.1 kPa) without the deformation seen in the old chondrons. The viscoelastic properties (E(∞), E(0), and m) of young and adult chondrons were significantly greater than that observed in young and adult cells, respectively (P < 0.001). The adult chondrons were stiffer than the young chondrons under micropipette aspiration (P < 0.001).

CONCLUSIONSOur findings provide a theoretical model to measure the viscoelastic properties of the chondrons as a whole unit by micropipette aspiration, and further suggest that the properties of the chondrocytes and PCM have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with aging.

Aging ; physiology ; Animals ; Cartilage, Articular ; metabolism ; Chondrocytes ; metabolism ; Elasticity ; Rabbits ; Viscosity

OBJECTIVE: To observe the effect of needle-knife on the chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis (KOA) based on the CircSERPINE2-miR-1271-5P-E26 specific transformation-related gene (ERG) axis, and to explore the mechanism of needle-knife for KOA. METHODS: Thirty-six New Zealand white rabbits were randomly divided into a normal group, a model group, a needle-knife group and a sham needle-knife group, 9 rabbits in each group. The rabbits in the model group, the needle-knife group and the sham needle-knife group were treated with modified Videman method to prepare KOA model. After successful modeling, the rabbits in the needle-knife group were treated with needle-knife at cord adhesion and nodules near quadriceps femoris tendon and internal and external collateral ligament on the affected knee joint; the rabbits in the sham needle-knife group were treated with sham needle-knife baside the needle insertion point of the needle-knife group (needle-knife was only inserted, without any operation). The treatment was given once a week, 3 times in total. The Lequesne MG behavioral score was used to evaluate the knee joint damage in each group before and after intervention. After intervention, HE staining and transmission electron microscopy were used to observe the cartilage tissue morphology and ultrastructure of chondrocytes in the knee joint in each group; TUNEL method was used to detect the level of chondrocyte apoptosis in the knee joint; real-time fluorescence quantitative PCR was used to detect the expression of CircSERPINE2, miR-1271-5P and ERG mRNA in knee cartilage tissue in each group. RESULTS: After intervention, compared with the normal group, the Lequesne MG behavioral score in the model group was increased (P<0.01). Compared with the model group and the sham needle-knife group, the Lequesne MG behavioral score in the needle-knife group was decreased (P<0.01). In the model group and the sham needle-knife group, the number of chondrocytes and organelles was decreased, the cell nucleus was shrunk, mitochondria was swelling or disappeared; in the needle-knife group, the number of chondrocytes and organelles was increased, the cell nucleus was not obviously shrunk and the mitochondria was not obviously swelling. Compared with the normal group, the level of chondrocyte apoptosis in the model group was increased (P<0.01); compared with the model group and the sham needle-knife group, the level of chondrocyte apoptosis in the needle-knife group was decreased (P<0.01, P<0.05). Compared with the normal group, the expression of CircSERPINE2 and ERG mRNA in the model group was decreased (P<0.01), and the expression of miR-1271-5P mRNA was increased (P<0.01); compared with the model group and the sham needle-knife group, the expression of CircSERPINE2 and ERG mRNA in the needle-knife group was increased (P<0.01), and the expression of miR-1271-5P mRNA was decreased (P<0.01). CONCLUSION Needle-knife could reduce the knee joint damage and chondrocyte apoptosis in KOA rabbits, which may be related to up-regulating the expression of CircSERPINE2 and ERG mRNA, and inhibiting the expression of miR-1271-5P mRNA.
Rabbits ; Animals ; Osteoarthritis, Knee/metabolism* ; Chondrocytes/metabolism* ; Knee Joint/surgery* ; Apoptosis ; MicroRNAs/genetics*