Proliferation Of Human Chondrocytes Research Articles

Impact of gadolinium-based MRI contrast agent and local anesthetics co-administration on chondrogenic gadolinium uptake and cytotoxicity

The gadolinium-based contrast agent DOTA-Gd is clinically used in combination with local anesthetics for direct magnetic resonance arthrography. It remains unclear whether gadolinium uptake into cartilage is influenced by co-administration of bupivacaine or ropivacaine and whether DOTA-Gd alters their chondrotoxicity. Gadolinium quantification of chondrogenic spheroids revealed enhanced gadolinium uptake after simultaneous exposure to local anesthetics. Analyses of the spatial gadolinium distribution using synchrotron X-ray-fluorescence scanning indicates gadolinium exposed chondrocytes. In vitro exposure to DOTA-Gd does not alter viability and proliferation of human chondrocytes and the chondrotoxic potential of the anesthetics. Reduced viability induced by ropivacaine was found to be reversible, while exposure to bupivacaine leads to irreversible cell death. Our data suggest that ropivacaine is more tolerable than bupivacaine and that DOTA-Gd exposure does not alter the cytotoxicity of both anesthetics. Enhanced gadolinium uptake into cartilage due to co-administration of anesthetics should find attention.

Evaluation of the effect of mesenchymal stromal cells from different sources on human chondrocyte proliferation

Objective: to study the effect of a conditioned medium of mesenchymal stromal cells (MSCs) from different sources on human chondrocyte proliferation.Materials and methods. To confirm functional activity, chondrocytes were cultured in a cartilage cell-engineered construct (CEC), including 5 × 105 cells and 5 mg of tissue-specific matrix from decellularized cartilage. The conditioned medium was obtained after culturing MSCs derived from human adipose tissue (AT), MSCs derived from the pulp of primary teeth and MSCs isolated from umbilical cord-derived Wharton’s jelly in a complete cell growth medium (CCGM). To evaluate the effect of MSC-derived secretome on chondrocyte proliferation, the conditioned medium, diluted 1 : 1 with CCGM, was added to wells containing chondrocytes. The effect of MSCs on human chondrocyte proliferation was studied by indirectly coculturing cells in CCGM using Transwell inserts. 5 × 104 MSCs were applied to the bottom of the lower chamber, and 5 × 104 human chondrocytes and 5 mg of matrix were placed in the upper chamber. Chondrocyte proliferation was assessed at days 7 and 14 by DNA quantification. Interleukin-6 content was determined as a marker of secretory activity of MSCs in the conditioned medium. The morphology of the samples was studied using histological staining methods.Results. The ability of chondrocytes to produce cartilage-specific extracellular matrix was confirmed when forming cartilage CEC with tissue-specific matrix in a chondrogenic differentiation medium. When comparing the effect of the conditioned medium of MSCs obtained from different sources on the growth of human chondrocytes in vitro, increased proliferation was observed in all samples compared to controls. Indirect co-culture of MSCs with chondrocytes as part of CEC showed increased DNA amount in all samples at day 14, with the amount of DNA in the sample with MSC conditioned medium significantly higher than the control.Conclusion. Studies on the effect of MSC conditioned medium on chondrocyte proliferation in 2D culture indicate a possible regenerative potential of MSCs for cartilage tissue repair. Within the scope of this work, we did not identify significant differences in the effect of secretome derived from MSCs that were obtained from different sources on chondrocyte proliferation. However, additional in vivo studies are warranted in the future.

Cytotoxic effects of different mouthwash solutions on primary human articular chondrocytes and normal human articular cartilage – an in vitro study

ObjectivesTo compare the cytotoxicity of octenidine dihydrochloride and chlorhexidine gluconate at different concentrations on primary human articular chondrocytes and cartilage.Materials and methodsPrimary cultures of human normal adult articular chondrocytes were exposed to octenidine dihydrochloride (0.001562%, 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%), chlorhexidine gluconate (0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, and 0.2%), and control (Dulbecco’s modified Eagle medium or phosphate-buffered saline) for 30 s. Normal human articular cartilage explants were exposed to octenidine dihydrochloride (0.1% versus control) and chlorhexidine gluconate (0.1% versus control) for 30 s. The viability of human articular chondrocytes was measured by Trypan blue staining, Cell Proliferation Reagent WST-1, and Live/Dead staining. The proliferation of human chondrocytes was measured using the Cell Proliferation Reagent WST-1. The viability of human articular cartilage explants was measured by using Live/Dead staining.ResultsOctenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability and proliferation in a dose-dependent manner in primary human articular chondrocytes. Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability in human articular cartilage explant cultures.ConclusionThe degree of toxicity varied between octenidine dihydrochloride and chlorhexidine gluconate, with chlorhexidine gluconate being less toxic than octenidine dihydrochloride at the same concentration. Additionally, both octenidine dihydrochloride and chlorhexidine gluconate evaluation had cytotoxic effects on human articular cartilage. Therefore, dosing for the antimicrobial mouthwash ingredients administration would ideally be determined to remain below IC50.Clinical relevanceThese data support the in vitro safety of antimicrobial mouthwashes on primary adult human articular chondrocytes.

Adeno-Associated Virus-Delivered Fibroblast Growth Factor 18 Gene Therapy Promotes Cartilage Anabolism.

To determine the characterization of chondrogenic properties of adeno-associated virus type 2 (AAV2)-delivered hFGF18, via analysis of effects on primary human chondrocyte proliferation, gene expression, and in vivo cartilage thickness changes in the tibia and meniscus. Chondrogenic properties of AAV2-FGF18 were compared with recombinant human FGF18 (rhFGF18) in vitro relative to phosphate-buffered saline (PBS) and AAV2-GFP negative controls. Transcriptome analysis was performed using RNA-seq on primary human chondrocytes treated with rhFGF18 and AAV2-FGF18, relative to PBS. Durability of gene expression was assessed using AAV2-nLuc and in vivo imaging. Chondrogenesis was evaluated by measuring weight-normalized thickness in the tibial plateau and the white zone of the anterior horn of the medial meniscus in Sprague-Dawley rats. AAV2-FGF18 elicits chondrogenesis by promoting proliferation and upregulation of hyaline cartilage-associated genes, including COL2A1 and HAS2, while downregulating fibrocartilage-associated COL1A1. This activity translates to statistically significant, dose-dependent increases in cartilage thickness in vivo within the area of the tibial plateau, following a single intra-articular injection of the AAV2-FGF18 or a regimen of 6 twice-weekly injections of rhFGF18 protein relative to AAV2-GFP. In addition, we observed AAV2-FGF18-induced and rhFGF18-induced increases in cartilage thickness of the anterior horn of the medial meniscus. Finally, the single-injection AAV2-delivered hFGF18 offers a potential safety advantage over the multi-injection protein treatment as evidenced by reduced joint swelling over the study period. AAV2-delivered hFGF18 represents a promising strategy for the restoration of hyaline cartilage by promoting extracellular matrix production, chondrocyte proliferation, and increasing articular and meniscal cartilage thickness in vivo after a single intra-articular injection.

α2-macroglobulin-rich serum as a master inhibitor of inflammatory factors attenuates cartilage degeneration in a mini pig model of osteoarthritis induced by "idealized" anterior cruciate ligament reconstruction.

Post-traumatic osteoarthritis is a special type of osteoarthritis and a common disease, with few effective treatments available. α2-Macroglobulin (α2M) is important to chondral protection in post-traumatic osteoarthritis. However, its injection into xenogeneic joint cavities involves safety hazards, limiting clinical applications. Exploring serum α2M-enriching strategies and the therapeutic effect and mechanism of α2M-rich serum (α2MRS) autologous joint injection to treat post-traumatic osteoarthritis has significant value. In the present study, a unique filtration process was used to obtain α2MRS from human and mini pig serum. We evaluated the potential of α2MRS in protecting against post-surgery cartilage degeneration. We identify the potential of α2MRS in reducing the expression of inflammatory cytokines and factors that hasten cartilage degeneration in post-operative conditions leading to post-traumatic osteoarthritis. The potential of α2MRS was analyzed in interleukin-1β induced human chondrocytes and mini pig models. In the chondrocyte model, α2MRS significantly promoted human chondrocyte proliferation and reduced apoptosis and chondrocyte catabolic cytokine gene transcription and secretion. The anterior cruciate ligament autograft reconstruction model of mini pigs was randomized into groups, operated on, and injected with α2MRS or saline. The results showed that α2MRS injection significantly suppressed the levels of inflammatory factors, improved gait, and showed significantly lower cartilage degeneration than the groups that did not receive α2MRS injections. This study highlights the chondroprotective effects of α2MRS, elucidated its potential applications against cartilage degeneration, and could provide a basis for the clinical translation of α2MRS.

Depletion of circ_0128846 ameliorates interleukin-1β-induced human chondrocyte apoptosis and inflammation through the miR-940/PTPN12 pathway

BackgroundPrevious evidence has suggested that circular RNA (circRNA) is abnormally expressed in osteoarthritis (OA). However, the underlying mechanism of circRNA in OA progression remains unclear. The study aims to reveal the mechanism of circ_0128846 regulating OA. MethodsHuman chondrocytes (C28/I2 cells) were treated with interleukin-1β (IL-1β) to mimic an OA cell model. The expression levels of circ_0128846, miR-940 and protein tyrosine phosphatase 12 (PTPN12) were detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation, and apoptosis were analyzed by a cell counting kit-8 assay, 5-Ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α) and IL-6 was determined by an enzyme-linked immunosorbent assay (ELISA). The binding relationship between miR-940 and circ_0128846 or PTPN12 was identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. ResultsCirc_0128846 and PTPN12 expression were significantly upregulated, whereas miR-940 was downregulated in the cartilage tissues of OA patients and IL-1β-treated C28/I2 cells compared with controls. IL-1β treatment inhibited C28/I2 cell proliferation and induced cell apoptosis and the production of inflammatory factors, TNF-α and IL-6; however, these effects were partly reversed after circ_0128846 depletion. In terms of mechanism, circ_0128846 acted as a miR-940 sponge, and miR-940 combined with PTPN12. Also, circ_0128846 depletion partly ameliorated IL-1β-induced C28/I2 cell disorders through miR-940. PTPN12 overexpression also partly relieved miR-940-mediated effects in IL-1β-treated C28/I2 cells. Further, circ_0128846 induced PTPN12 expression by interacting with miR-940. ConclusionCirc_0128846 regulated human chondrocyte proliferation, apoptosis and inflammation through the miR-940/PTPN12 pathway in OA.

Synoviocyte-Derived Extracellular Matrix and bFGF Speed Human Chondrocyte Proliferation While Maintaining Differentiation Potential.

Improving the ability of human chondrocytes to proliferate, while maintaining their differentiation potential, has presented a great challenge in cartilage tissue engineering. In this study, human chondrocytes were cultured under four unique growth conditions at physiologic oxygen tension: tissue culture plastic (TCP) only, synoviocyte matrix (SCM)–coated flasks only, SCM-coated flasks with bFGF media supplement, and TCP with bFGF media supplement. The results indicated that, compared to standard TCP, all test conditions showed significantly increased cell expansion rates and an increase in both glycosaminoglycan (GAG) and collagen content during redifferentiation culture. Specifically, the combined SCM + bFGF growth condition showed an additive effect, with an increase of approximately 36% more cells per passage (5–7 days) when compared to the SCM alone. In conclusion, the results of this study demonstrate that bFGF and SCM can be used as supplements to enhance the growth of human chondrocytes both as individual enhancers and as a combined additive.

Freeze-Dried Curdlan/Whey Protein Isolate-Based Biomaterial as Promising Scaffold for Matrix-Associated Autologous Chondrocyte Transplantation-A Pilot In-Vitro Study.

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of its micro- and macrostructures as well as evaluation of its mechanical stability, and its ability to undergo enzymatic degradation in vitro. Subsequently, the cytocompatibility of the biomaterial towards human chondrocytes (isolated from an orthopaedic patient) was assessed. It was demonstrated that the novel freeze-dried curdlan/whey protein isolate-based biomaterial possessed a porous structure and a Young’s modulus close to those of the superficial and middle zones of cartilage. It also exhibited controllable degradability in collagenase II solution over nine weeks. Most importantly, this biomaterial supported the viability and proliferation of human chondrocytes, which maintained their characteristic phenotype. Moreover, quantitative reverse transcription PCR analysis and confocal microscope observations revealed that the biomaterial may protect chondrocytes from dedifferentiation towards fibroblast-like cells during 12-day culture. Thus, in conclusion, this pilot study demonstrated that novel freeze-dried curdlan/whey protein isolate-based biomaterial may be considered as a potential scaffold for matrix-associated autologous chondrocyte transplantation.

Influence of pore sizes in 3D-scaffolds on mechanical properties of scaffolds and survival, distribution, and proliferation of human chondrocytes

Articular cartilage has weak intrinsic self-healing capacity. Tissue engineering is an appropriate option for cartilage regeneration. This research was designed to evaluate the effects of pore size in scaffolds on mechanical properties and chondrocyte-scaffold interactions. PCL scaffolds were fabricated with large, medium, and small pore sizes. The constructs were analyzed by SEM, swelling tests, mechanical tests, MTT assay, and H&E staining after chondrocyte seeding. Mechanical features of the scaffolds were near to human articular cartilage. Our findings suggest that the PCL scaffold with medium pore sizes provides suitable mechanical strength and better chondrocyte-scaffold interactions simultaneously for application in cartilage.

Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR-874-3p/SMAD3 signalling pathway in idiopathic short stature.

Idiopathic short stature (ISS) is a main reason for low height among children. Its exact aetiology remains unclear. Recent findings have suggested that the aberrant expression of circRNAs in peripheral blood samples is associated with many diseases. However, to date, the role of aberrant circRNA expression in mediating ISS pathogenesis remains largely unknown. The up‐regulated circANAPC2 was identified by circRNA microarray analysis and RT‐qPCR. Overexpression of circANAPC2 inhibited the proliferation of human chondrocytes, and cell cycle was arrested in G1 phase. The expressions of collagen type X, RUNX2, OCN and OPN were significantly down‐regulated following circANAPC2 overexpression. Moreover, Von Kossa staining intensity and alkaline phosphatase activity were also decreased. Luciferase reporter assay results showed that circANAPC2 could be targeted by miR‐874‐3p. CircANAPC2 overexpression in human chondrocytes inhibits the expression of miR‐874‐3p. The co‐localization of circANAPC2 and miR‐874‐3p was confirmed in both human chondrocytes and murine femoral growth plates via in situ hybridization. The rescue experiment demonstrated that the high expression of miR‐874‐3p overexpression antagonized the suppression of endochondral ossification, hypertrophy and chondrocyte growth caused by circANAPC2 overexpression. A high‐throughput screening of mRNA expression and RT‐qPCR verified SMAD3 demonstrated the highest different expressions following overcircANAPC2. Luciferase reporter assay results indicated that miR‐874‐3p could be targeted by Smad3, thus down‐regulating the expression of Smad3. Subsequent rescue experiments of SMAD3 further confirmed that circANAPC2 suppresses endochondral ossification, hypertrophy and chondrocyte growth through miR‐874‐3p/Smad3 axis. The present study provides evidence that circANAPC2 can serve as a promising target for ISS treatment.

Hsa_circularRNA_0079201 suppresses chondrocyte proliferation and endochondral ossification by regulating the microRNA‑140‑3p/SMAD2 signaling pathway in idiopathic short stature.

Circular (circ)RNAs are an important group of non-coding RNAs involved in different pathological and physiological functions, such as longitudinal bone growth. However, the effects of an increase or decrease in circRNA expression on idiopathic short stature (ISS) remain largely unknown. The present study compared the circRNA expression patterns of patients with ISS and healthy individuals to identify differentially expressed circRNAs involved in the regulation of ISS pathogenesis and their target microRNAs (miR). Microarray analysis revealed that 145 circRNAs were differentially expressed in patients with ISS, including 83 up- and 62 downregulated circRNAs. Reverse transcription-quantitative PCR confirmed that hsa_circRNA_0079201 was increased in patients with ISS compared with that in the normal individuals, whilst hsa_circRNA_0079201 overexpression in human chondrocytes was shown to significantly suppress their proliferation, hypertrophy and endochondral ossification abilities. Luciferase reporter assays identified that circRNA_0079201 acted as an miR-140-3p sponge. In situ hybridization confirmed the co-localization of circRNA_0079201 and miR-140-3p in the human chondrocyte and neonatal femur growth plate of C57 mice, while rescue experiments demonstrated that miR-140-3p overexpression reversed the inhibition of human chondrocyte proliferation, hypertrophy and endochondral ossification, caused by circRNA_0079201 overexpression. Bioinformatics analysis and luciferase reporter assays revealed that SMAD2 was a potential target gene of miR-140-3p. Furthermore, overexpressing circRNA_0079201 in human chondrocytes suppressed miR-140-3p and increased SMAD2 protein expression level. Taken together, chondrocyte proliferation, hypertrophy and endochondral ossification in ISS was suppressed by a novel regulatory axis consisting of the hsa_circRNA_0079201/miR-140-3p/SMAD2 pathway. The present study provided evidence that hsa_circRNA_0079201 may be a potential target for ISS therapy.

MicroRNA-675-3p regulates IL-1β-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GNG5

Growing evidence has indicated that microRNAs (miRNAs) are modulators of osteoarthritis (OA) development and progression. In this study, we first evaluated the anti-apoptosis and chondroprotective effects of microRNA-675-3p (miR-675-3p) on interleukin-1β (IL-1β)-stimulated human chondrocytes. The overexpression of miR-675-3p inhibited apoptosis and cartilage matrix degradation and promoted cell proliferation in human chondrocytes. Target gene prediction and luciferase reporter assays suggested that G-protein subunit γ 5 (GNG5) may be the target gene of miR-675-3p. The overexpression of miR-675-3p inhibited IL-1β-stimulated chondrocyte apoptosis, and this effect was reversed by the overexpression of GNG5. Finally, we used bioinformatic tools and biological methods to show that the long noncoding RNA X-inactive specific transcript (lncRNA XIST) could bind to miR-675-3p, which affects the expression of GNG5 mRNA. Our findings may substantiate miR-675-3p as a new treatment for OA.

Melatonin Prevents Osteoarthritis-Induced Cartilage Degradation via Targeting MicroRNA-140.

Osteoarthritis (OA) is characterized by the progressive destruction of articular cartilage, which is involved in the imbalance between extracellular matrix (ECM) synthesis and degradation. MicroRNA-140-5p (miR-140) is specifically expressed in cartilage and plays an important role in OA-induced matrix degradation. The aim of this study was to investigate (1) whether intra-articular injection of melatonin could ameliorate surgically induced OA in mice and (2) whether melatonin could regulate matrix-degrading enzymes at the posttranscriptional level by targeting miR-140. In an in vitro OA environment induced by interleukin-1 beta (IL-1β), melatonin treatment improved cell proliferation of human chondrocytes, promoted the expression of cartilage ECM proteins (e.g., type II collagen and aggrecan), and inhibited the levels of IL-1β-induced proteinases, such as matrix metalloproteinase 9 (MMP9), MMP13, ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), and ADAMTS5. Both the microarray and polymerase chain reaction (PCR) experiments revealed that miR-140 was a melatonin-responsive microRNA and melatonin upregulated miR-140 expression, which was suppressed by IL-1β stimulation. In vivo experiments demonstrated that intra-articular injection of melatonin prevented disruptions of cartilage matrix homeostasis and successfully alleviated the progression of surgery-induced OA in mice. Transfection of miR-140 antagomir completely counteracted the antiarthritic effects of melatonin by promoting matrix destruction. Our findings demonstrate that melatonin protects the articular cartilage from OA-induced degradation by targeting miR-140, and intra-articular administration of melatonin may benefit patients suffering from OA.

Effect of Hyperbaric Oxygen on Proliferation and Gene Expression of Human Chondrocytes: An In Vitro Study.

The present study investigated the effects of hyperbaric oxygen (HBO) on human chondrocyte proliferation and gene expression patterns. Chondrocyte cultures were transferred to a HBO chamber and exposed to 100% oxygen for 7 consecutive days. Within groups, pressure was varied between 1 and 2 atm and duration of HBO administration was varied among 60, 90, and 120 minutes. Cell counts were performed using the WST-1 assay at 1, 3, 5, and 7 days after initiation of HBO treatment to obtain data to plot a growth curve. Gene expression of apoptosis markers PARP and caspase 3, as well as cartilage specific proteins collagen II and COMP, were detected by reverse transcription polymerase chain reaction. The experiments showed that in vitro administration of HBO inhibit chondrocyte growth. When applied compression was increased up to 2 atm, chondrocyte cell count was reduced by half at days 3 and 7 in association with an upregulation of the apoptosis markers PARP and caspase 3 as well as the cartilage specific proteins collagen II and COMP. No significant differences were monitored from varied duration of daily treatment. Chondrocyte growth was inhibited in vitro by treatment of HBO. This inhibitory effect was even increased by elevating the applied pressure, while molecular testing showed reduced chondrocyte growth. Higher levels of HBO inhibited cell growth even more, but up-regulation of apoptosis specific markers and cartilage specific proteins were seen during administration of high oxygen levels. Thus, it has to be evaluated that there is a critical level of hypo-/hyperoxia required to stimulate or at least maintain chondrocyte cell proliferation.

Development of arginine-glycine-aspartate-immobilized 3D printed poly(propylene fumarate) scaffolds for cartilage tissue engineering

Poly(propylene fumarate) (PPF) has known to be a good candidate material for cartilage tissue regeneration because of its excellent mechanical properties during its degradation processes. Here, we describe the potential application of PPF-based materials as 3D printing bioinks to create macroporous cell scaffolds using micro-stereolithography. To improve cell-matrix interaction of seeded human chondrocytes within the PPF-based 3D scaffolds, we immobilized arginine-glycine-aspartate (RGD) peptide onto the PPF scaffolds. We also evaluated various cellular behaviors of the seeded chondrocytes using MTS assay, microscopic and histological analyses. The results indicated that PPF-based biocompatible scaffolds with immobilized RGD peptide could effectively support initial adhesion and proliferation of human chondrocytes. Such a 3D bio-printable scaffold can offer an opportunity to promote cartilage tissue regeneration.

MALAT1/miR-127-5p Regulates Osteopontin (OPN)-Mediated Proliferation of Human Chondrocytes Through PI3K/Akt Pathway.

Osteoarthritis (OA) is characterized by progressive destruction of articular cartilage, resulting in significant disability. Chondrocytes present in various types of cartilage and are responsible for the growth and maintenance of the tissue. Over-proliferation of human chondrocytes may contributes to OA pathological process. Previously, we revealed that miR-127-5p could inhibit the proliferation of human chondrocytes through osteopontin (OPN). In the present study, we used online tools to figure out several candidates lncRNAs which were potentially correlated with miR-127-5p. Through assessing the expression levels of the candidates lncRNAs, metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was chosen as a further research subject. MALAT1 knockdown significantly repressed human OA chondrocyte proliferation, as well as the protein levels of OPN, p-PI3K, and p-Akt in OA chondrocytes. As verified by luciferase assays, MALAT1 directly bound to miR-127-5p to inhibit miR-127-5p expression. Then we achieved miR-127-5p inhibition through miR-127-5p inhibitor transfection; the miR-127-5p inhibition could promote chondrocyte proliferation, as well as the protein levels of OPN, p-PI3K, and p-Akt; in addition, the MALAT1 knockdown partially reversed the promotive effect of miR-127-5p inhibition on chondrocyte proliferation, OPN and PI3K/Akt signaling-related protein levels. Taken together, MALAT1 could directly bind to miR-127-5p to inhibit its expression, so as to rescue OPN expression and promote chondrocyte proliferation through PI3K/Akt pathway. Targeting MALAT1 so as to rescue miR-127-5p expression in OA might help to inhibit chondrocyte proliferation through miR-127-5p-mediated OPN regulation and downstream PI3K/Akt pathway. J. Cell. Biochem. 119: 431-439, 2018. © 2017 Wiley Periodicals, Inc.

Glucose adsorption to chitosan membranes increases proliferation of human chondrocyte via mammalian target of rapamycin complex 1 and sterol regulatory element-binding protein-1 signaling.

Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d-glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucose's effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS-Glc), and the study aimed to investigate whether CTS-Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS-Glc-regulated proliferation of human chondrocytes. We found that CTS-Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose- or chitosan-only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS-Glc-increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP-1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin-dependent kinase-4 and -6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS-Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS-Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients.

MicroRNA-34a affects chondrocyte apoptosis and proliferation by targeting the SIRT1/p53 signaling pathway during the pathogenesis of osteoarthritis.

Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors such as ageing, obesity and trauma. Previously, it was reported that the inhibition of microRNA-34a (miR-34a) may reduce rat chondrocyte apoptosis induced by IL-1β, whereas the molecular mechanism and the role of miR-34a in human chondrocyte as well as in OA progression remains to be determined. In the current study, using MTT, luciferase reporter assays and western blot analysis we identified that miR-34a was upregulated while silent information regulator 1 (SIRT1) was inhibited in chondrocytes from 12 OA patients compared with healthy chondrocytes from 10 trauma amputees. Overexpression of miR-34a promoted apoptosis and inhibited cell proliferation in human chondrocytes. Transfection with miR-34a mimic inhibited SIRT1 expression, which attenuated the deacetylation of p53, leading to the upregulation of Bax and downregulation of Bcl-2. Furthermore, results from the western blot analysis and luciferase reporter assay demonstrated that SIRT1 was directly regulated by miR-34a in human chondrocytes. A rat model of OA was induced through anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx). The results showed that the intra-articular injection of lentiviral vector encoding anti-miR-34a sequence effectively ameliorated the progression of OA. The results suggest that miR-34a has a crucial role in the pathogenesis of OA through direct regulation of the SIRT1/p53 signaling pathway and serves as a potential therapeutic target of OA.

MicroRNA-127-5p regulates osteopontin expression and osteopontin-mediated proliferation of human chondrocytes.

The aim of this study was to determine the specific microRNA (miRNA) that regulates expression of osteopontin (OPN) in osteoarthritis (OA). The potential regulatory miRNAs for OPN messenger RNA (mRNA) were predicted by miRNA prediction programs. Among eight potential regulatory miRNAs, miR-220b, miR-513a-3p and miR-548n increased, while miR-181a, miR-181b, miR-181c, miR-181d and miR-127-5p decreased in OA patients. miRNA-127-5p mimics suppressed OPN production as well as the activity of a reporter construct containing the 3′-UTR of human OPN mRNA. In addition, mutation of miR-127-5p binding site in the 3′-UTR of OPN mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with miR-127-5p inhibitor increased reporter activity and OPN production. Interestingly, miR-127-5p inhibited proliferation of chondrocytes through OPN. In conclusion, miRNA-127-5p is an important regulator of OPN in human chondrocytes and may contribute to the development of OA.

Topography-Guided Proliferation: Distinct Surface Microtopography Increases Proliferation of Chondrocytes In Vitro.

Chondrocyte-based cartilage repair techniques require control of articular chondrocyte expansion ex vivo. Articular chondrocytes have limited availability, and prolonged culturing to obtain a cell number sufficient for clinical use often results in phenotypic alterations and increased costs. In this study, we applied a screening library consisting of micrometer-sized topographical features, termed biosurface structure array (BSSA), to identify specific topographical microstructures affecting the proliferation of human chondrocytes in passage 1 (P1) or 2 (P2). The BSSA library comprised 10 patterns and 16 combinations of pillar size (X) and interpillar gap size (Y). Specific microstructures significantly increased the chondrocytes' proliferative responsiveness in term of patterns, X and Y for P2 compared with P1. The P1 and P2 chondrocytes responded independently to similar patterns after 4 days of culturing, whereas only chondrocytes at P2 responded to specific microstructures with Y = 1 μm and X = 2, 4 μm by a 2.3- and 4.4-fold increased proliferation, respectively. In conclusion, these findings indicate that specific surface topographies promote chondrocyte proliferation and may, indeed, be a tool to control the behavior of chondrocytes in vitro.