Effects of ADAM12 abnormal expression on chondrocyte injury and IGFBP-related genes in patients with Kashin-Beck disease / 中华地方病学杂志

Objective:To investigate the role of a disintegrin and metalloprotease 12 (ADAM12) gene in chondrocyte injury in patients with Kashin-Beck disease (KBD) and its impact on genes related to insulin-like growth factor binding protein (IGFBP).Methods:Articular cartilage samples were obtained from 5 patients with KBD and 5 control subjects admitted to Honghui Hospital Affiliated to Xi'an Jiaotong University. Chondrocytes were extracted and cultured in vitro. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect the expression levels of ADAM12 mRNA and protein in chondrocytes of patients with KBD and control subjects, respectively. Subsequently, ADAM12 gene overexpression was performed using lentivirus in chondrocytes of patients with KBD. MTT assay was used to detect changes in cell viability after ADAM12 gene overexpression, and qRT-PCR was used to detect the mRNA expression levels of chondrocyte differentiation related genes SRY-box transcription factor 9 (SOX9) and type Ⅱ collagen (COLⅡ), apoptosis-related gene B-cell lymphoma/leukaemia-2-associated X protein (BAX), and anabolic related genes IGFBP3 and IGFBP5. Results:The expression levels of ADAM12 mRNA and protein in chondrocytes of patients with KBD (0.57 ± 0.05, 0.81 ± 0.07) were significantly lower than those of control subjects (1.00 ± 0.00, 1.00 ± 0.00), and the differences were statistically significant ( t = - 24.50, - 3.61, P < 0.05). The results of MTT assay showed that the cell viability of chondrocytes in ADAM12 overexpression group (1.09 ± 0.05) was higher than that in empty vector control group (1.00 ± 0.08), and the difference was statistically significant ( t = 4.12, P = 0.031). The results of qRT-PCR showed that compared with empty vector control group, the mRNA expression levels of IGFBP3 (2.35 ± 0.79 vs 0.96 ± 0.25), IGFBP5 (2.13 ± 0.30 vs 0.98 ± 0.34), SOX9 (2.92 ± 0.51 vs 0.94 ± 0.36) and COLⅡ (6.45 ± 2.81 vs 0.87 ± 0.19) in ADAM12 overexpression group were significantly increased, and the differences were statistically significant ( t = 3.19, 5.16, 6.27, 4.10, P < 0.05); while the expression level of BAX mRNA (0.31 ± 0.06 vs 1.02 ± 0.22) was significantly decreased, and the difference was statistically significant ( t = - 11.16, P < 0.001). Conclusion:The ADAM12 gene may have a role in inhibiting apoptosis and promoting differentiation in chondrocyte injury in patients with KBD, and its overexpression can increase expression of IGFBP3 and IGFBP5.

Effect of needle-knife on chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis based on CircSERPINE2-miR-1271-5P-ERG axis / 中国针灸

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.

Effect of pulsed electromagnetic fields on mesenchymal stem cell-derived exosomes in inhibiting chondrocyte apoptosis / 生物医学工程学杂志

The study aims to explore the effect of mesenchymal stem cells-derived exosomes (MSCs-Exo) on staurosporine (STS)-induced chondrocyte apoptosis before and after exposure to pulsed electromagnetic field (PEMF) at different frequencies. The AMSCs were extracted from the epididymal fat of healthy rats before and after exposure to the PEMF at 1 mT amplitude and a frequency of 15, 45, and 75 Hz, respectively, in an incubator. MSCs-Exo was extracted and identified. Exosomes were labeled with DiO fluorescent dye, and then co-cultured with STS-induced chondrocytes for 24 h. Cellular uptake of MSC-Exo, apoptosis, and the protein and mRNA expression of aggrecan, caspase-3 and collagenⅡA in chondrocytes were observed. The study demonstrated that the exposure of 75 Hz PEMF was superior to 15 and 45 Hz PEMF in enhancing the effect of exosomes in alleviating chondrocyte apoptosis and promoting cell matrix synthesis. This study lays a foundation for the regulatory mechanism of PEMF stimulation on MSCs-Exo in inhibiting chondrocyte apoptosis, and opens up a new direction for the prevention and treatment of osteoarthritis.

GPRC5B protects osteoarthritis by regulation of autophagy signaling

Osteoarthritis (OA) is one of the most common chronic diseases in the world. However, current treatment modalities mainly relieve pain and inhibit cartilage degradation, but do not promote cartilage regeneration. In this study, we show that G protein-coupled receptor class C group 5 member B (GPRC5B), an orphan G-protein-couple receptor, not only inhibits cartilage degradation, but also increases cartilage regeneration and thereby is protective against OA. We observed that Gprc5b deficient chondrocytes had an upregulation of cartilage catabolic gene expression, along with downregulation of anabolic genes in vitro. Furthermore, mice deficient in Gprc5b displayed a more severe OA phenotype in the destabilization of the medial meniscus (DMM) induced OA mouse model, with upregulation of cartilage catabolic factors and downregulation of anabolic factors, consistent with our in vitro findings. Overexpression of Gprc5b by lentiviral vectors alleviated the cartilage degeneration in DMM-induced OA mouse model by inhibiting cartilage degradation and promoting regeneration. We also assessed the molecular mechanisms downstream of Gprc5b that may mediate these observed effects and identify the role of protein kinase B (AKT)-mammalian target of rapamycin (mTOR)-autophagy signaling pathway. Thus, we demonstrate an integral role of GPRC5B in OA pathogenesis, and activation of GPRC5B has the potential in preventing the progression of OA.

Research progress on the role of chondrocyte mitochondrial homeostasis imbalance in the pathogenesis of osteoarthritis / 中国修复重建外科杂志

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.

Deeper Understanding the Pathogenesis of Osteoarthritis from Matrix Viscoelasticity and Calcium Signaling / 医用生物力学

Generally, extracellular matrix (ECM) has the characteristics of viscoelasticity. In osteoarthritis (OA), catabolic processes alter the viscoelastic properties of functional pericellular matrix (PCM) of chondrocytes. Chondrocytes sense and respond to their mechanical microenvironment via an array of mechanosensitive receptors and channels that activate a complex network of downstream signaling pathways to regulate several cell processes central to OA pathology. Advances in understanding the specific mechanosignalling mechanisms in articular cartilage will promote the development of cell microenvironment construction in cartilage tissue engineering and the targeted precision therapeutics for OA. In this review, the work on the mechanism of matrix viscoelasticity regulating chondrocytes mechanotransduction by Agarwal et al. was briefly commented, and the recent advances related with their work was also discussed.

The Role and Mechanisms of Mechanical Stimulation in Degeneration of Vertebral Cartilage Endplates / 医用生物力学

The important function of the endplate is to transmit stress and supply nutrition. Endplate degeneration might induce or promote degeneration of the intervertebral disc, causing a series of spine diseases that seriously impair people’s health and life quality. Endplate chondrocytes can respond to mechanical stimulation, which is an important factor affecting endplate degeneration. Inappropriate mechanical stimulation will accelerate endplate degeneration. This review summarized the effects of mechanical stimulation on vertebral endplate chondrocyte apoptosis, synthesis inhibition, calcification, and extracellular matrix degradation. The endplate degeneration induced by mechanical stimulation is regulated by a complex network of signal pathways composed of various signal transduction factors. The signal pathways involved in this review included NF-κB, Wnt, Hedgehog, MAPK, RhoA/Rock-1, AKT/mTOR, TGF-β signaling pathway and miRNA related signals. The interconnection of these pathways was highlighted and summarized. Multiple signaling pathways work together to regulate endplate chondrocyte metabolism, which ultimately leads to the endplate degeneration. This review might shed light on early diagnosis and precise treatment of cartilage endplate degeneration.

Mangiferin attenuates IL / 中南大学学报(医学版)

OBJECTIVES@#Chondrocyte apoptosis is an important process in the pathogenesis of osteoarthritis. Mangiferin exerts multiple pharmacological effects such as anti-inflammatory and anti-apoptosis. However, the role of mangiferin in chondrocyte apoptosis is not clear. In this study, we aimed to explore the role of mangiferin in IL-1β-induced chondrocyte apoptosis.@*METHODS@#ATDC5 cells were randomly divided into a control group, a IL-1β group, a MFN-L group, a MFN-M group, a MFN-H group and a MFN+LY294002 group. Cells in the control group were treated with IL-1β (10 ng/mL) for 24 h; cells in the MFN-L group, the MFN-M group and the MFN-H group were pretreated with 5, 10 and 20 μmol/L mangiferin for 1 h respectively, and then they were treated with IL-1β (10 ng/mL) for 24 h; cells in the MFN+LY294002 group were treated with LY294002 (25 μmol/L) for 1 h, then mangiferin (20 μmol/L) and IL-1β (10 ng/mL) for 1 h and 24 h, respectively. Cell viability was detected by CCK-8 assay and cell apoptosis was measured by flow cytometry. Colorimetric assay was conducted to measure the caspase-3 activity. The protein levels of Bcl-2, Bax, and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway related proteins were detected by Western blotting.@*RESULTS@#Compared to the control group, cell viability was significantly decreased; cell apoptosis, caspase-3 activity and Bax protein expression were significantly increased; the protein levels of Bcl-2, p-PI3K, and p-Akt were significantly decreased in the IL-1β group (all @*CONCLUSIONS@#Mangiferin could attenuate IL-1β-induced apoptosis of the mice chondrocytes, which is mediated by the activation of PI3K/Akt signaling pathway.

Chemerin, a pro-inflammatory adipokine, regulates chondrocyte proliferation and metabolism by increasing production of nitric oxide / 中国组织工程研究

BACKGROUND: The key pathological characteristics of osteoarthritis are manifested in the degeneration of the cartilage caused by inflammation, and chondrocytes are the only cells in cartilage tissues. Studies have shown that Chemerin can stimulate the migration of leukocytes to the inflammation site and increase the inflammation signal of chondrocytes, suggesting that Chemerin can play a role in arthritis. Our previous research indicated that the serum Chemerin level in patients with osteoarthritis was significantly increased, and the Chemerin level in the synovial fluid was related to the severity of osteoarthritis based on the Kellgren-Lawrence classification. Chemerin may be used as an inflammatory factor in osteoarthritis. OBJECTIVE: To investigate the effect of Chemerin on the proliferation and metabolism of chondrocytes. METHODS: The chondrocytes from neonatal mice were isolated by collagenase type II digestion, and then cultured. Cell growth curves were established and the range of concentrations of Chemerin that exhibited toxicity to normal chondrocytes was screened using an MTT assay. Subsequently, 10 μg/L interleukin-1β was used to stimulate the chondrocytes in order to establish an in vitro model of osteoarthritis induction. After the chondrocytes had been cultured in the presence of the drug for 2 days, cell morphology, proliferation and metabolism were evaluated by hematoxylin-eosin staining and diacetate fluorescein/ propidium iodide staining. In addition, the expression of inducible nitric oxide polymerase was analyzed by measuring the secretion of nitric oxide. Furthermore, qRT-PCR was used to quantify mRNA expression of proteoglycan, type II collagen α1, matrix metalloprotease-13 and nitric oxide synthase 2. RESULTS AND CONCLUSION: The chondrocytes cultured in vitro exhibited healthy activity and morphology. Furthermore, chemerin (50 μg/L) and interleukin-1β (10 μg/L) were able to reduce the synthesis of extracellular matrix, enhance the secretion of nitric oxide and increase chondrocyte apoptosis. More importantly, the qRT-PCR results indicated that Chemerin and interleukin-1β caused similar effects, by which the expression of cartilage-specific genes was downregulated and catabolism-related genes upregulated. As a pro-inflammatory factor, Chemerin can increase the generation of nitric oxide in chondrocytes, regulate cell metabolism, stimulate cell apoptosis and act synergistically with interleukin-1β.

Role and application of bone morphogenetic proteins in articular cartilage regeneration / 中国组织工程研究

BACKGROUND: Articular cartilage degeneration is the main cause of osteoarthritis. Bone morphogenetic proteins play an important role in cartilage regeneration and repair. OBJECTIVE: To review the research progress of bone morphogenetic protein in the process of articular cartilage regeneration. METHODS: A computer-based online search of PubMed and Elsevier databases was performed using the keywords “bone morphogenetic proteins, BMPs, arthritis, osteoarthritis, OA, cartilage, chondrocyte” in English. A total of 272 papers were retrieved, 96 of which were included in final analysis. Another 27 papers related to concepts were also included. Therefore, 123 papers are finally included. RESULTS AND CONCLUSION: Bone morphogenetic proteins participate in many biological processes including cell proliferation, differentiation, migration, and apoptosis, and play an important role in the formation of bone and cartilage. Bone morphogenetic proteins participate in a variety of signaling pathway cascades by binding to different receptors, which can protect articular cartilage from cartilage destruction caused by inflammation and trauma. Bone morphogenetic proteins alone or in combination with other cytokines can repair cartilage defects improve degenerative lesions, and promote the differentiation and regeneration of articular chondrocytes. However, there are still some practical problems that need to be solved for the widespread use of bone morphogenetic proteins in cartilage regeneration, such as the safety of drug transporters, the lack of effective biological scaffold materials, the optimal dosage and time point of use of biological agents, and their toxic and side effects. Future research will focus on how to solve the above problems. The widespread application of bone morphogenetic proteins will open a new era for targeted treatment of cartilage damage and cartilage degenerative diseases represented by osteoarthritis.

Ultrastructural changes of the white zone cells of the meniscus in a rat model of early osteoarthritis / 中国组织工程研究

BACKGROUND: The research focuses of knee osteoarthritis are on mainly articular cartilage, subchondral bone and synovium. There are few studies on the ultrastructure of the meniscus in animal models. OBJECTIVE: On the basis of observing the ultrastructure and types of chondrocytes in the white zone of the meniscus, to observe the ultrastructural changes of the white zone cells of the meniscus in an animal model of early osteoarthritis, in order to explore the relationship between the microstructural changes and the physiological functional changes in the meniscus. METHODS: Sprague-Dawley rats were randomly divided into a control group and a model group. Rats in the model group were made into the model of early knee osteoarthritis by intraarticular injection of papain. After 5 weeks, the meniscuses of three rat models in each group were taken, located and observed for the ultrastructure of the white zone cells through a transmission electron microscope. An ethics approval was obtained from the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine. RESULTS AND CONCLUSION: In the control group, most of cells were fusiform in the superficial layer of meniscus and were triangular-like in the deeper layer. They were rich in rough endoplasmic reticulum and mitochondria, with presence of Golgi and other organelles. Extracellular collagen fibrils were mainly type II collagen fibrils. The cells in the white zone of meniscus were chondrocytes. In the model group, the meniscal surface became rough, the cells were swollen, cytoplasmic organelles were reduced and swollen, glycogen was accumulated, and most of nuclei were abnormal with heterochromatin agglutination. Extracellular collagen fibrils became disordered and sparse. These findings indicate that the mild degeneration of chondrocytes and matrix in the meniscus can reduce the ability of cells to synthesize and secrete matrix components, which may lead to the physiological hypofunction.

Development of fibrocartilage layers in Achilles tendon enthesis in rabbits / Journal of Rural Medicine

Objective: The details regarding the development of fibrocartilage layers in Achilles tendon (AT) enthesis are unknown. Therefore, we evaluated the development of fibrocartilage layers in AT enthesis using a rabbit model.Materials and Methods: Forty-eight male Japanese white rabbits were used in this study. Six of them were euthanized at different stages (day 1, and 1, 2, 4, 6, 8, 12, and 24 weeks of age). The proliferation, apoptosis, Sox9-positivity rates, and chondrocyte number were evaluated. Additionally, safranin O-stained glycosaminoglycan (GAG) areas, width of AT enthesis, and calcaneus length were assessed. All parameters were compared to those at 24 weeks of age.Results: The level of chondrocyte apoptosis was high from 1 to 8 weeks of age, and high expression level of Sox9 was maintained from day 1 to 6 weeks of age, which decreased gradually. Safranin O-stained GAG areas increased up to 12 weeks, calcaneus length increased up to 6 weeks, and the width of AT enthesis increased up to 1 week of age.Conclusion: The changes in chondrocyte and extracellular matrix were completed by 8 and 12 weeks of age, respectively. The development of fibrocartilage layers in AT enthesis was completed by 12 weeks of age. Our results contribute to the administration of appropriate treatments based on age and aid in the development of novel methods for regenerating AT enthesis.

Effect of 17β-estradiol on the proliferation of condylar chondrocytes / 华西口腔医学杂志

OBJECTIVES@#To study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process.@*METHODS@#Condylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR.@*RESULTS@#E2 could significantly promote the proliferation of chondrocytes cultured @*CONCLUSIONS@#At a concentration of 10

Research progress of different mechanical stimulation regulating chondrocytes metabolism / 生物医学工程学杂志

As a kind of mechanical effector cells, chondrocytes can produce a variety of physical and chemical signals under the stimulation of multiaxial load

Effect of quercetin on chondrocyte phenotype and extracellular matrix expression / 中国天然药物

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.

Bone marrow mesenchymal stem cells combined with acellular dermal matrix repair beagle dog articular cartilage defects / 中国组织工程研究

BACKGROUND: Dermis-derived extracellular matrix, as a cartilage repair scaffold, provides a space for the growth of cartilage tissue, and promotes cell adhesion and proliferation. Bone marrow mesenchymal stem cells have the potential to differentiate into chondrocytes. Both of them used alone have disadvantages. OBJECTIVE: To evaluate the feasibility of bone marrow mesenchymal stem cells combined with calf acellular dermal matrix to repair beagle dog articular cartilage defects. METHODS: Beagle dog bone marrow blood was extracted from Beagle dogs. Bone marrow mesenchymal stem cells were obtained by density gradient centrifugation and passaged. Acellular dermal matrix was prepared from the dorsal dermis of neonatal calves by ultrasonic oscillation, freeze-drying and pepsin. 0. 2 mL of cell suspension was added to the surface of acellular dermal matrix until covered, then which was placed in a 5% CO2 incubator at 37 °C for 48 hours. Twelve adult beagle dogs were used to establish knee joint cartilage defect models, and then randomized into three groups: In the acellular dermal matrix combined with bone marrow mesenchymal stem cells group (combination group), cartilage defects were repaired with acellular dermal matrix combined with bone marrow mesenchymal stem cells. In the single acellular dermal matrix group, cartilage defects were repaired with acellular dermal matrix. The model control group received no treatment. At 12 weeks after surgery, the right knee joints were observed by stereomicroscope, hematoxylin-eosin staining and type II collagen immunohistochemical staining. RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that bone marrow mesenchymal stem cells adhered to and grew well in the acellular dermal matrix. (2) Hematoxylin-eosin staining revealed that the repaired surface in the combination group was slightly lower than that of the surrounding normal tissues, and the repaired tissues integrated well with the surrounding cartilages. The defects in the single acellular dermal matrix group were filled with fibrous tissues. Few surrounding tissues of defect were repaired in the model control group. (3) Type II collagen immunohistochemical staining showed that in the combination group, articular cartilage defects were filled with chondrocyte-like tissues. In the single acellular dermal matrix group, the defect was filled with fibrous tissues. No tissue was found in the model control group. (4) These results indicate that the new calf acellular dermal matrix has good biocompatibility and can promote the proliferation of bone marrow mesenchymal stem cells. Autologous bone marrow mesenchymal stem cells combined with acellular dermal matrix can effectively repair beagle dog knee joint cartilage defects.

Phenotypic maintenance of chondrocytes in vitro under tensile stress enhanced by the extract of clematis chinensis / 中国组织工程研究

BACKGROUND: Mechanical load is crucial for the degeneration of chondrocytes and the development of osteoarthritis. Clematis chinensis can improve the inflammatory microenvironment of osteoarthritis, but its effect on mechanical load-induced degeneration of chondrocytes has not been elucidated. OBJECTIVE: To study the effect and mechanism of the extracts of Clematis chinensis on the degenerative changes of chondrocytes induced by intermittent cyclic mechanical tension (ICMT) in vitro. METHODS: Chondrocytes of the rabbit knee joint were isolated by type II collagenase digestion method and identified by Alcian blue staining. There were five groups in the experiment: Blank group, ICMT group, high-, medium- and low-dose Clematis chinensis groups. There was no intervention in the blank group, and the other groups were subjected to ICMT (10% tensile strength, 0.5 Hz, 8 hours per day, for a total of 2 days) for inducing chondrocyte degeneration. Three Clematis chinensis groups were concurrently given 0.5, 1, 2 g/L extracts of Clematis chinensis, respectively. The intervention time was 48 hours. Cell counting kit-8 assay was used for detection of chondrocyte proliferation. FITC-phalloidin staining was used for observation of cytoskeleton morphology. Real-time quantitative PCR and western blot assay were used for determination of collagen type II, matrix metalloproteinase 13, and transforming growth factor β at protein and gene levels, respectively. RESULTS AND CONCLUSION: (1) Compared with the blank group, the cytoskeleton of chondrocytes stimulated by ICMT was long- stretched, the proliferation activity of chondrocytes decreased, and the expressions of collagen type II and transforming growth factor β were down-regulated, while the expression of matrix metalloproteinase 13 was up-regulated. (2) Compared with the ICMT group, the extract of Clematis chinensis could promote the proliferation of chondrocytes, up-regulate the expressions of transforming growth factor β and collagen II, and down-regulate the expression of matrix metalloproteinase 13 in a concentration-dependent manner. To conclude, the extract of Clematis chinensis can inhibit the catabolism of chondrocyte induced by ICMT through regulating the expression of transforming growth factor β, promote the synthesis of extracellular matrix of chondrocytes, and maintain the phenotypic stability of chondrocytes.

Protocatechuic acid alleviates degeneration of chondrocytes / 中国组织工程研究

BACKGROUND: Preliminary studies have shown that numerous antioxidants exhibit antiarthritic effects due to their inhibition on inflammatory factors. With the antioxidant and anti-inflammatory abilities whether protocatechuic acid is effective in the treatment of osteoarthritis has never been reported. OBJECTIVE: To investigate the biological effect of protocatechuic acid on interleukin-1β induced chondrocyte including phenotype and cellular metabolism in vitro, thereby providing a potential agent in osteoarthritis treatment, METHODS: The chondrocytes of neonatal rat femur were collected, intervened by 10 mg/L interleukin-1β to establish the degenerative model, and treated by 10, 30 and 50 mg/L protocatechuic acid. The study was approved by the Laboratory Animal Ethical Committee of Guangxi Medical University in October 2017, with the approval No. 201710008. RESULTS AND CONCLUSION: Protocatechuic acid effectively promoted chondrocyte growth in the range of 10-50 mg/L, while the dose of 30 mg/L was the strongest. Protocatechuic acid also enhanced the synthesis of the extracellular matrix and the mRNA expression of aggrecan, collagen II and Sox9, and downregulating the expression of the matrix metalloproteinase 13 (a marker of inflammatory factor). To conclude, protocatechuic acid exerts a positive effect on the proliferation and phenotypic maintenance of articular chondrocytes, providing reference for its use in the treatment of osteoarthritis and repair of degenerative articular cartilage in vivo.

Mechanism of DAIa2GIP inhibiting mitochondrial apoptosis in chondrocytes / 中国组织工程研究

BACKGROUND: The previous research of our group has shown that DAIa2GIP, a new analogue of glucose dependent insulin stimulating polypeptide, has a protective effect on chondrocytes, but its mechanism is not clear. OBJECTIVE: To observe the effect of DAIa2GIP on interleukin-1β (IL-1β) induced apoptosis of chondrocytes, and to explore its molecular biological mechanism. METHODS: The costal chondrocytes of Sprague-Dawley rats were extracted and identified by SABC immunohistochemistry. The third generation cells were divided into six groups: (1) normal control group; (2) IL-1β induction group; (3) DAIa2GIP+IL-1β induction group; (4) DAIa2GIP+IL-1β+pro3GIP (GIPR antagonists) induction group; (5) DAIa2GIP induction group; (6) pro3GIP induction group. After 48 hours of drug treatment, mitochondrial membrane potential was tested, cell apoptosis was detected using Annexin-V FITC Kit (phosphatidylserine eversion analysis), calcium overload of cells was determined under laser confocal microscope, and cytochrome C content were measured by ELISA. The study protocol was approved by the Animal Ethics Committee of Shanxi Medical University. RESULTS AND CONCLUSION: SABC immunohistochemical staining showed that collagen II could be developed as chondrocytes; the maintenance degree of mitochondrial membrane potential was significantly higher in the IL-1β+DAIa2GIP incubation group than the IL-1β induction group, but the potential in both groups was lower than that in the normal control group. The apoptotic rate of chondrocytes in the IL-1β+DAIa2GIP incubation group was significantly lower than that in the IL-1β induction group, but the apoptotic rate in both groups was higher than that in the normal control group. The degree of calcium overload was compared with fluorescence intensity, and the result showed that the IL-1β+DAIa2GIP incubation group had a significantly higher intensity than the IL-1β induction group, and the fluorescence intensity in both groups was higher than that in the normal control group. The release of chondrocyte mitochondrial cytochrome C in the IL-1β+DAIa2GIP incubation group was significantly lower than that in the IL-1β induction group (P < 0.01), and the release amount in both groups was significantly higher than that in the normal control group (P < 0.01). The above four indicators showed no significant difference between the IL-1β induction group and the DAIa2GIP+IL-1β+pro3GIP group, but the values in both groups were lower than those in the control group. There was no significant difference among the normal control group, DAIa2GIP induction group, and pro3GIP induction group. To conclude, DALa2GIP can effectively antagonize IL-1β induced mitochondrial dysfunction of rat chondrocytes, thus antagonizing chondrocyte apoptosis. In this process, DALa2GIP can effectively reduce the degree of calcium overload and the release of cytochrome C.

Effect of bone morphogenetic protein-7 combined with porous tantalum on chondrogenic differentiation and function of bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND: The establishment of coculture system combined with physical factors and scaffold materials and the Induction of cytokines have become the focus of chondrogenlc differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To observe the effect of bone morphogenetic protein 7 combined with porous tantalum on chondrogenlc differentiation of bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats (provided by Beijing Huafukang Biology) were Isolated and cultured. Group Intervention: (1) in the experimental group, porous tantalum tablet was added, while In the control group, porous tantalum tablet was not added. At 5 days after culture, cell growth on the surface of porous tantalum tablet was observed by phalloidin staining. At 1, 3,5, and 7 days after culture, CCK-8 method was used to detect cell proliferation. (2) Group A was added with chondrocyte Inducer; group B with chondrocyte Inducer and bone morphogenetic protein 7; group C with domestic porous tantalum material and chondrocyte Inducer; group D with domestic porous tantalum material and chondrocyte Inducer and bone morphogenetic protein 7. At 7,14 and 21 days after culture, the levels of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13 secreted by cells In each group was detected by ELISA. Western blot assay was used to detect the expression of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13. This study was approved by the Animal Experimental Ethics Committee of North China University of Science and Technology. RESULTS AND CONCLUSION: (1) The phalloidin staining results showed that bone marrow mesenchymal stem cells grew well on and around the porous tantalum surface. (2) At 3 and 5 days after culture, the proliferation of bone marrow mesenchymal stem cells was slower In the experimental group than in the control group (P 0.05). (3) At 7,14 and 21 days, the expression of type II collagen and SRY high mobility group protein Increased gradually among groups A, B, C and D (P 0.05). At 21 days, there was no significant difference among groups A, B, C and D (P > 0.05). (4) Western blot assay showed that at 7,14 and 21 days after culture, the expression level of type II collagen and SRY high mobility group protein Increased gradually In groups A, B, C and D (P 0.05). (5) The results showed that bone morphogenetic proteln-7 combined with domestic porous tantalum could Induce cartilage differentiation of bone marrow mesenchymal stem cells, facilitate the expression of type II collagen and SRY high mobility group protein, and Inhibit the expression of matrix metalloproteinase-13.