Increased Chondrocyte Proliferation Research Articles

Osteophyte Cartilage as a Potential Source for Minced Cartilage Implantation: A Novel Approach for Articular Cartilage Repair in Osteoarthritis.

Osteoarthritis (OA) is a common joint disorder characterized by cartilage degeneration, often leading to pain and functional impairment. Minced cartilage implantation (MCI) has emerged as a promising one-step alternative for large cartilage defects. However, the source of chondrocytes for MCI remains a challenge, particularly in advanced OA, as normal cartilage is scarce. We performed in vitro studies to evaluate the feasibility of MCI using osteophyte cartilage, which is present in patients with advanced OA. Osteophyte and articular cartilage samples were obtained from 22 patients who underwent total knee arthroplasty. Chondrocyte migration and proliferation were assessed using cartilage fragment/atelocollagen composites to compare the characteristics and regenerative potential of osteophytes and articular cartilage. Histological analysis revealed differences in cartilage composition between osteophytes and articular cartilage, with higher expression of type X collagen and increased chondrocyte proliferation in the osteophyte cartilage. Gene expression analysis identified distinct gene expression profiles between osteophytes and articular cartilage; the expression levels of COL2A1, ACAN, and SOX9 were not significantly different. Chondrocytes derived from osteophyte cartilage exhibit enhanced proliferation, and glycosaminoglycan production is increased in both osteophytes and articular cartilage. Osteophyte cartilage may serve as a viable alternative source of MCI for treating large cartilage defects in OA.

Synergistic roles of CBX4 chromo and SIM domains in regulating senescence of primary human osteoarthritic chondrocytes

BackgroundCellular senescence is a critical factor contributing to osteoarthritis (OA). Overexpression of chromobox homolog 4 (CBX4) in a mouse system was demonstrated to alleviate post-traumatic osteoarthritis (PTOA) by reducing cellular senescence. Additionally, replicative cellular senescence of WI-38 fibroblasts can be attenuated by CBX4. However, the mechanisms underlying this senomorphic function of CBX4 are not fully understood. In this study, we aimed to investigate the role of CBX4 in cellular senescence in human primary osteoarthritic chondrocytes and to identify the functional domains of CBX4 necessary for its function in modulating senescence.MethodsChondrocytes, isolated from 6 individuals undergoing total knee replacement for OA, were transduced with wild-type CBX4, mutant CBX4, and control lentiviral constructs. Senescence-related phenotypic outcomes included the following: multiple flow cytometry-measured markers (p16INK4A, senescence-associated β-galactosidase [SA-β-gal] activity and dipeptidyl peptidase-4 [DPP4], and proliferation marker EdU), multiplex ELISA-measured markers in chondrocyte culture media (senescence-associated secretory phenotypes [SASPs], including IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-3, and MMP-9), and PCR array-evaluated senescence-related genes.ResultsCompared with control, CBX4 overexpression in OA chondrocytes decreased DPP4 expression and SASP secretion and increased chondrocyte proliferation confirming CBX4 senomorphic effects on primary human chondrocytes. Point mutations of the chromodomain domain (CDM, involved in chromatin modification) alone were sufficient to partially block the senomorphic activity of CBX4 (p16INK4A and DPP4 increased, and EdU decreased) but had minimal effect on SASP secretion. Although having no effect on p16INK4A, DPP4, and EdU, deletion of two small-ubiquitin-like-modifier-interaction motifs (CBX4 ΔSIMs) led to increased SASP secretion (IL-1β, TNF-α, IL-8). The combination CBX4 CDMΔSIMs altered all these measures adversely and to a greater degree than the single domain mutants. Deletion of the C-terminal (CBX4 ΔC-box) involved with transcriptional silencing of polycomb group proteins increased IL-1β slightly but significantly but altered none of the other senescence outcome measures.ConclusionsCBX4 has a senomorphic effect on human osteoarthritic chondrocytes. CDM is critical for CBX4-mediated regulation of senescence. The SIMs are supportive but not indispensable for CBX4 senomorphic function while the C-box is dispensable.

PG545 Prevents Osteoarthritis Development by Regulating PI3K/AKT/mTOR Signaling and Activating Chondrocyte Autophagy

Introduction: Osteoarthritis (OA) is a degenerative disease common in the elderly and is characterized by joint pain, swelling, and restricted movement. In recent years, heparanase has been reported to play an important role in the development of osteoarthritic cartilage. PG545 is a heparan sulfate mimetic with heparanase inhibitory activity. In this study, the therapeutic effects and possible mechanisms of PG545 were investigated in a chondrocyte injury model induced by interleukin-1β (IL -1β). Methods: Following treatment with PG545 or the autophagy inhibitor 3-methyladenine (3-MA), chondrocyte viability was detected using Cell Counting Kit-8 and fluorescein diacetate/propidium iodide double staining. The apoptosis rate of chondrocytes was determined by flow cytometry. Expression of light chain 3 and P62 was monitored by immunofluorescence labeling. Western blot, lentivirus infection with red fluorescent protein and green fluorescent protein, and quantitative real-time polymerase chain reaction were used to determine the expression levels of chondrocyte markers, apoptosis-related factors, autophagy proteins, and key proteins of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. The expression and activity of stress-specific enzymes such as malondialdehyde, superoxide dismutase, and catalase (CAT) were investigated. Chondrocytes with ATG5 knockdown were used to investigate the relationship between the therapeutic effect of PG545 and autophagy. The therapeutic effect of PG545 was verified in vivo. Results: PG545 had a significant protective effect on chondrocytes by reducing oxidative stress, apoptosis, and degradation of chondrocytes and increasing chondrocyte proliferation. PG545 was effective in inducing autophagy in IL-1β-treated cells, while 3-MA attenuated the effect. The PI3K/Akt/mTOR pathway may be involved in the promotion of autophagy and OA treatment by PG545. Conclusion: PG545 was able to restore impaired autophagy and autophagic flux via the PI3K/Akt/mTOR pathway, thereby delaying the progression of OA, suggesting that PG545 may be a novel therapeutic approach for OA.

OR18-5 A Prospective Clinical Trial of Vosoritide in Selected Genetic Causes of Short Stature

Abstract Objectives Vosoritide is a C-type natriuretic peptide analog which binds its receptor on chondrocytes, leading to increased chondrocyte proliferation and differentiation via its inhibition of the ERK1/2-MAPK pathway. It was recently approved for increasing linear growth in children with achondroplasia. Our study aims to assess the safety and efficacy of vosoritide in children with genetic mutations in 6 categories: hypochondroplasia, Rasopathies (including Noonan syndrome), aggrecan deficiency, carriers of heterozygous NPR2 mutations, CNP deficiency, and SHOX deficiency. Methods This is a prospective, Phase II study with a goal N of 35 subjects. All subjects must have a documented genetic mutation in one of the 6 categories, be prepubertal between the ages of 3 and 11 for boys and 3 and 10 for girls, and have a height < -2.25 SD. Subjects are followed for a 6-month observation period to establish a baseline annualized growth velocity (AGV) and then receive daily subcutaneous vosoritide (15 mcg/kg/day) for 12 months. The primary outcomes are rate of AEs and change in AGV from baseline. Pharmacokinetic studies and pharmacodynamic studies, using cGMP production as a pharmacodynamic marker, are completed at Day 1 and Months 6 and 12. Results To date, 27 subjects have enrolled in the trial (20 hypochondroplasia, 3 Noonan, 2 NPR2, 2 Aggrecan). Median baseline height is -3.1 SD (IQR -3.6, -2.6). 14 subjects have initiated on vosoritide, and 10 completed 6 months of therapy. Median increase in AGV was 3.9 cm/yr (IQR 2.2, 4.6). The two subjects with NPR2 mutations had increased AGVs of 4.4 and 9.2 cm/yr. The two subjects with Noonan syndrome had an increase in AGV of 7.5 and 3.3 cm/yr. One subject with Noonan syndrome, a 4-year-old male, had previously been treated with growth hormone (GH) for 2 years. His AGV was 6.7 cm/yr while on GH, 4.1 cm/yr during the 6-month observation period, and 11.6 cm/year during the first 6 months of vosoritide treatment. There were no serious adverse events related to vosoritide treatment. One subject had a brief episode of syncope with her first injection which self-resolved, and she continued on therapy with no further incidents. PK parameters were similar to previously published parameters for vosoritide in children with achondroplasia. In preliminary data, vosoritide Cmax and AUC correlated strongly with peak increase in cGMP (r=0.70, p=0.01;r=0.84, p<0.01, respectively) but not with increase in AGV (r=0.35, p=0.32). Conclusions This is the first clinical trial of vosoritide for children with genetic short stature who do not have achondroplasia. Vosoritide treatment may work as a precision therapy to improve growth in multiple genetic conditions which interact with the ERK1/2-MAPK pathway. Presentation: Monday, June 13, 2022 12:00 p.m. - 12:15 p.m.

Trp53 controls chondrogenesis and endochondral ossification by negative regulation of TAZ activity and stability via β-TrCP-mediated ubiquitination

Transformation-related protein 53 (Trp53) is a critical regulator of cell fate determination by controlling cell proliferation and differentiation. Ablation of Trp53 signaling in osteoblast lineages significantly promotes osteogenesis, bone formation, and bone remodeling. However, how Trp53 regulates chondrogenesis and endochondral bone formation is undefined. In this study, we found that Trp53 expression gradually decreased in tibia growth plates during embryonic development in vivo and during chondrogenesis in vitro. By deleting Trp53 in chondrocyte lineage using Col2-Cre transgenic line, we found that loss of Trp53 in chondrocytes significantly increased growth plate growth and bone formation by increasing chondrocyte proliferation, matrix production and maturation, and bone dynamic formation rate. Mechanistically, our data revealed loss of Trp53 significantly promoted TAZ transcriptional activity through inhibition of TAZ phosphorylation and nuclear translocation, whereas its activity was pronouncedly inhibited after forced expression of Trp53. Furthermore, Co-IP data demonstrated that Trp53 associated with TAZ. Moreover, Trp53 decreased the stability of TAZ protein and promoted its degradation through β-TrCP-mediated ubiquitination. Ablation of TAZ in Col2-Cre;Trp53f/f mice rescued the phenotypes of enhanced chondrogenesis and bone formation caused by Trp53 deletion. Collectively, this study revealed that Trp53 modulates chondrogenesis and endochondral ossification through negative regulation of TAZ activity and stability, suggesting that targeting Trp53 signaling may be a potential strategy for fracture healing, heterotopic ossification, arthritis, and other bone diseases.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

Objective: CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. Methods: The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. Results: It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. Conclusion: CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

Intra-articular injection of loaded sPL sustained-release microspheres inhibits osteoarthritis and promotes cartilaginous repairs

BackgroundOsteoarthritis is a chronic inflammatory disease of the joints associated with significant morbidity and lower quality of life. Current treatment strategies focus on reducing cartilage degeneration but fail to restore their proliferative ability. Super-activated platelet lysate (sPL) is an enhanced form of platelet-rich plasma that can be easily inactivated. The purpose of this study is to evaluate whether sPL-loaded PLGA/chitosan/gelatin microspheres can prevent and treat osteoarthritis.MethodsFeatures of biological microspheres were detected by SEM and ELISA. Osteoarthritis chondrocytes were co-cultured with hydrogel loaded with sPL. The effect of biological microspheres on chondrocyte proliferation was evaluated using a CCK-8 cell proliferation test. Cell morphology and cell necrosis were measured with a microscope. The gene expression levels of cartilage-related markers type 2 collagen, aggrecan (ACAN), and SRY type high mobility group box-9 (SOX9) were determined by real-time quantitative polymerase chain reaction (Rt-PCR). A rat osteoarthritis model was established. Micro-CT was used to characterize cartilaginous changes after the injection of biological microspheres. Histopathological HE staining, Safranin-O Fast Green staining and staining scores, type II collagen staining, and proteoglycan staining were used to evaluate the degree of cartilaginous repair.ResultsBiological microspheres were able to continuously release biological factors. Exposure to loading sPL microspheres significantly increased chondrocyte proliferation, reduced cell necrosis, and increased the expression of cartilage markers type 2 collagen, ACAN, and SOX9 in osteoarthritic chondrocytes. In vivo experiments found that biological microspheres also smoothen cartilage surfaces, promote the expression of proteoglycan and type 2 collagen while also increasing cartilaginous integrity as evaluated using Safranin-O Fast Green staining.ConclusionsPLGA/chitosan/gelatin hydrogel loaded with sPL is a promising tool for effective and non-invasive articular cartilage repair in osteoarthritis.Graphic abstractBiological microspheres loaded with sPL release various biological factors to promote chondrocyte proliferation and upregulate chondrocyte functionalization genes (SOX9, CoX II, ACAN), leading to an overall enhanced cartilaginous matrix.

Effect of fluoride on bone and growth plate cartilage

The use of fluoride (F) for therapeutic purposes is controversial and its toxicity is a health problem. The aim of this study was to evaluate the effect of F on endochondral ossification in growing rats. Twenty-four rats of 21 days were divided into 4 groups which received 0, 20, 40 or 80 μmol F/100 g body weight/day for 30 days, through an orogastric tube. Histological evaluation of growth plate cartilage (GPC) and primary and secondary bone were analyzed on sections of the metaphysis of tibias. Total thickness of the GPC (GPC.Th), thickness of resting zone (RZ.Th), proliferative zone (PZ.Th) and hypertrophic zone (HZ.Th); bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and apoptosis by the TUNEL were measured. A hyperplasia of the proliferative zone and a significant increase in PZ.Th with 40 and 80 μmol F without changes in GPC.Th were found. In the secondary trabecular bone, presence of immature trabeculae, peritrabecular inflammatory foci and sinusoidal dilatation were observed. A significant decrease in BV/TV was also found due to a decrease in Tb.Th and a progressive increase was observed in the number of apoptotic nuclei as the dose of F increased. In conclusion, results suggest that prolonged administration (30 days) of F negatively affect the endochondral ossification with increased chondrocyte proliferation and delayed maturity of new bone, causing inflammatory damage, edema, and increased apoptotic bone cells.

Hypertonic Dextrose Stimulates Chondrogenic Cells to Deposit Collagen and Proliferate.

Hypertonic dextrose (HD) injections (prolotherapy) for osteoarthritis are reported to reduce pain. Cartilage regeneration is hypothesized as a mechanism. This in vitro study identifies an HD concentration that stimulates chondrogenic cells to increase metabolic activity and assesses whether this concentration affects collagen deposition and proliferation. ATDC5 chondrogenic cells were cultured in normoglycemic DMEM/F12 medium, treated with concentrations of HD (4-400 mM), and assessed with PrestoBlue. Advanced light microscopy was used to conduct live imaging of collagen deposition through second harmonic generation microscopy (SHG) and proliferation via 2-photon excitation microscopy. Proliferation was additionally assessed with hemocytometer counts. A linear regression model found that, relative to the 4 mM baseline control, cells treated with 200 mM had a higher mean absorbance (P = 0.023) and cells treated with 250 mM were trending toward a higher mean absorbance (P = 0.076). Polynomial regression interpolated 240 mM as producing the highest average absorbance. Hemocytometer counts validated 250 mM as stimulating proliferation compared with the 4 mM control (P < 0.01). A concentration of 250 mM HD led to an increase in collagen deposition compared with that observed in control (P < 0.05). This HD concentration also led to increases in proliferation of ATDC5 cells relative to that of control (P < 0.001). A 250 mM HD solution appears to be associated with increased metabolic activity of chondrocytes, increased collagen deposition, and increased chondrocyte proliferation. These results support clinical prolotherapy research suggesting that intra-articular HD joint injections reduce knee pain. Further study of HD and cellular processes is warranted.

Chondrocytic and pharmacokinetic properties of Phlpp inhibitors

ObjectiveThe pleckstrin homology domain leucine-rich repeat protein phosphatases (Phlpp1/2) were recently identified as potential therapeutic targets for cartilage regeneration in osteoarthritic joints. Phlpp inhibitors NSC 117079 and NSC 45586 increase chondrocyte proliferation and matrix production, but the pharmacodynamics and pharmacokinetics of these compounds are not known. DesignChondrocytic effects of Phlpp inhibitors, NSC 117079 and NSC 45586, were measured by western blotting of Phlpp substrates, glycosaminoglycan (GAG) assays, and transcriptomic assays. Liquid chromatography/mass spectroscopy assays were established to measure NSC 117079 and NSC 45586 in vitro and in vivo. The effects of NSC 117079 and NSC 45586 on articular cartilage structure in vivo after intra-articular injection were determined by histology. ResultsThe Phlpp inhibitors NSC 117079 and NSC 45586 were highly stable in vitro and stimulated GAG, Sox9, proteoglycan 4 and collagen 2 production in maturing but not more differentiated chondrocytes in vitro. Both molecules reduced Phlpp1/2 levels and suppressed matrix degradation to functionally extend their inhibitory effect on these phosphatases. In vivo, NSC 117079 was eliminated from the bloodstream within 4 ​h after intravenous injection, while NSC 45586 was eliminated in 8 ​h and had a higher volume distribution. Both molecules increased articular cartilage area on lateral and medial tibial plateaus and femoral condyles by 15% in C57Bl/6 mice between four and five weeks of age. ConclusionThese data advance our understanding of how Phlpp inhibitors promote and preserve cartilage formation and provide a basis for understanding their safety and activity in vivo.

Low-intensity pulsed ultrasound promotes osteoarthritic cartilage regeneration by BMSC-derived exosomes via modulating the NF-κB signaling pathway

Osteoarthritis is the most common disabling joint disease throughout the world, and the effect of therapy on its course is still unsatisfactory in clinical practice. Recent studies have shown that mesenchymal stem cell (MSC)-derived exosomes can promote cartilage repair and regeneration in osteoarthritis, indicating that these exosomes could be a novel and promising strategy for treating osteoarthritis. This study investigated whether low-intensity pulsed ultrasound (LIPUS) enhances the effects of bone marrow MSC (BMSC)-derived exosomes on cartilage regeneration in osteoarthritis and examined the underlying mechanism. Our results revealed that BMSC-derived exosomes display the typical morphological features of exosomes. LIPUS-mediated BMSC-derived exosomes promoted cartilage regeneration, increased chondrocyte proliferation and extracellular matrix synthesis, suppressed inflammation, and inhibited the interleukin (IL)-1β-induced activation of the nuclear factor kappa B (NF-κB) pathway. In brief, LIPUS enhances the promoting effects of BMSC-derived exosomes on osteoarthritic cartilage regeneration, mainly by strengthening the inhibition of inflammation and further enhancing chondrocyte proliferation and cartilage matrix synthesis. The underlying mechanism could be related to the inhibition of the IL-1β-induced activation of the NF-κB pathway.

Activation of autophagy contributes to the protective effects of lycopene against oxidative stress-induced apoptosis in rat chondrocytes.

Oxidative stress is commonly associated with osteoarthritis (OA). Lycopene (LYC), a natural carotenoid compound, is an effective antioxidant with potential cartilage-protecting actions. However, how it affects hydrogen peroxide (H2 O2 )-induced damage to the cartilage is unclear. In this study, an in vitro oxidative stress model was developed via treating primary chondrocytes with H2 O2 . Western blot, immunohistochemistry, and quantitative RT-PCR (qRT-PCR) were used to assess the levels of related factors. Reactive oxygen species (ROS) and apoptosis levels were analyzed by the use of appropriate probes and flow cytometry. The expression and activity of stress-specific enzymes (malondialdehyde, superoxide dismutase, and catalase) were also assessed. The role of autophagy was explored by using the inhibitor, 3-methyladenine (3-MA), as well as monodansylcadaverine staining, western blotting, and red fluorescent protein-green fluorescent protein-light chain 3 lentivirus infection. The result showed LYC exerted significant chondrocyte-protective effects, including reduced inflammation and chondrocyte degradation, increased chondrocyte proliferation, apoptosis inhibition, and reduced ROS production. LYC could effectively induce autophagy in the H2 O2 treatment group, and this effect could be attenuated by 3-MA. In terms of mechanism, LYC played a role in inhibiting MAPK and PI3K/Akt/NF-κB axis, which down-regulates levels of mTOR and had a potential therapeutic significance for cartilage degeneration.

Osteopontin\u2010induced lncRNA HOTAIR expression is involved in osteoarthritis by regulating cell proliferation

BackgroundOsteopontin plays critical roles in osteoarthritis (OA) by regulating the functions of osteoclasts. It is known that osteopontin can induce the expression of lncRNA HOX transcript antisense RNA (HOTAIR), indicating the involvement of HOTAIR in OA. This study was carried out to investigate the role of HOTAIR in OA.MethodsSynovial fluid was extracted from both OA patients (n = 58) and healthy controls (n = 58). Expression of osteopontin and HOTAIR in synovial fluid was determined by RT-qPCR. Osteopontin was used to treat chondrocytes at dosages of 0, 1, 5 and 10 µg/ml, followed by measurement of HOTAIR expression by RT-qPCR. The role of osteopontin and HOTAIR overexpression, as well as HOTAIR knockdown in regulating the proliferation of chondrocytes was analyzed by cck-8 assay.ResultsHOTAIR was upregulated in OA. A positive correlation between HOTAIR and osteopontin was observed. In the primary chondrocytes, osteopontin treatment increased HOTAIR expression, while HOTAIR overexpression and knockdown failed to significantly affect osteopontin expression. In addition, osteopontin and HOTAIR overexpression increased chondrocyte proliferation, while HOTAIRE knockdown decreased chondrocyte proliferation. In addition, HOTAIR knockdown reduced the effects of osteopontin treatment on cell proliferation.ConclusionsOsteopontin-induced HOTAIR expression is involved in osteoarthritis by regulating cell proliferation.

BMP2 Is Required for Postnatal Maintenance of Osteochondral Tissues of the Temporomandibular Joint.

Bone morphogenetic protein 2 (BMP2) plays important roles in cartilage growth and development. Paradoxically, elevated levels of BMP2 leads to hypertrophic differentiation and osteoarthritis of cartilage. We examined the in vivo loss of BMP2 in cells expressing aggrecan of the mandibular condyle and knee. Three-week-old BMP2 flox/flox-CreER-positive mice and their Cre-negative littermates were treated with tamoxifen and raised until 3 or 6 months. We also investigated the direct effects of BMP2 on chondrocytes in vitro. Cells from the mandibular condyle of mice were treated with recombinant human BMP2 (rhBMP2) or rhNoggin (inhibitor of BMP2 signaling). Conditional deletion of BMP2 caused breakage of the cartilage integrity in the mandibular condyle of mice from both age groups, accompanied by a decrease in cartilage thickness, matrix synthesis, mineralization, chondrocyte proliferation, and increased expression of degeneration markers, while the effects at articular cartilage were not significant. In vitro results revealed that rhBMP2 increased chondrocyte proliferation, mineralization, and differentiation, while noggin induced opposite effects. In conclusion, BMP2 is essential for postnatal maintenance of the osteochondral tissues of the mandibular condyle.

HEMA and alginate-based chondrogenic semi-interpenetrated hydrogels: synthesis and biological characterization

Cartilage tissue engineering (CTE) has the general objective of restoring and improving damaged cartilage. A very interesting strategy of CTE is to combine different polymers to obtain a viscoelastic material. In this study, we have evaluated the applicability of poly(2-hydroxyethyl methacrylate) networks semi-interpenetrated with sodium alginate for CTE. Alginate-containing hydrogels show an increase in scaffold porosity and swelling capacity, when compared with nonporous poly(2-hydroxyethyl methacrylate) scaffolds. Primary chondrocytes from young rats were cultured on the hydrogels, and an increase in chondrocyte proliferation and chondrocytic markers was observed in alginate-containing hydrogels. Chondrocytic phenotype was preserved on hydrogels containing the lowest amount of crosslinker and initiator (SEMI 3 and SEMI 4). In addition, nitric oxide production by RAW264.7 macrophages grown on hydrogels was tested, and none of the hydrogels showed high levels of this inflammatory marker after 2 days. These results indicate that our alginate-containing hydrogels could be useful for CTE.

Prolotherapy upregulates Fibroblast Growth Factor‐2 (FGF‐2) and Insulin‐like Growth Factor‐1 (IGF‐1) mRNA which may increase chondrocyte proliferation and mediates osteoarthritic inflammation

Osteoarthritis is a highly prevalent disease that is progressively generated and has no known cure. Current treatments, such as opioids, have highly adverse effects. A more effective treatment can be the form of proliferative therapy (prolotherapy), a complementary medical intervention with promising clinical evidence. The purpose of the present study is to investigate the molecular basis of prolotherapy as a treatment option for osteoarthritis. Specifically, the study investigates whether prolotherapy upregulates proliferation‐enhancing cytokines (FGF‐2 and IGF‐1) in chondroprogenitor cells. In vitro stimulation of chondroprogenitor cells was carried out with prolotherapy treatment followed by qRT‐PCR quantification to determine the mRNA expression of the genes FGF‐2, IGF‐1, CCND‐1, TGF‐β1, AKT, STAT1, and BMP2. Results indicated a likely upregulation of cytokines that enhanced cell proliferation in chondroprogenitor cells with statistically higher levels of expression for FGF‐2 (p&lt;0.001, p&lt;0.05, p&lt;0.01), IGF‐1 (p&lt;0.001), and CCND‐1 (p&lt;0.05) relative to controls. Results in conjunction with prolotherapy and molecular‐focused inflammation theory suggest that prolotherapy has the potential to transform chondroprogenitor cells that are dysregulated by osteoarthritic inflammation. This would contribute to cartilage degradation back into chondroprogenitor cells that proliferate and balance catabolic/anabolic roles to repair and maintain cartilage.P2G (1.5%) upregulates FGF‐2 mRNA expression in chondroprogenitor cells. A Exploratory Trial 1 suggests P2G effects FGF‐2 gene expression (Welch’s one‐way ANOVA, p&lt;0.01). B Confirmatory Trial 2 indicates that relative to time‐specific controls, chondrocytes treated with P2G exhibit increased levels of FGF‐2 at hours 24, 30, and 38 (Welch’s t‐tests yield p&lt;0.001, p&lt;0.05, and p&lt;0.01). qRT‐PCR quantifies mRNA expression. NS p&gt;0.05, * p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001Figure 1P2G (1.5%) upregulates CCND‐1 (Cyclin D) mRNA expression at hour 30 in chondroprogenitor cells in the confirmatory Trial 2 (directional Welch’s t‐test yields p&lt;0.05). CCND‐1 mRNA upregulation at hour 30 suggests subsequent entry chondroprogenitor cell proliferation. qRT‐PCR quantifies mRNA expression. NS p&gt;0.05, * p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001Figure 2

Deletion of p21 expression accelerates cartilage tissue repair via chondrocyte proliferation

Articular cartilage tissue has a poor healing potential, and when subjected to traumatic damage this tissue undergoes cartilage degeneration and osteoarthritis. The association between the regulation of cell cycle checkpoints and tissue regeneration has been previously investigated, and p21 was initially identified as a potent inhibitor of cell cycle progression. However, the effects of p21 defects on damaged tissue remain controversial. Therefore, the aim of the present study was to evaluate the effects of p21 deficiency on cartilage repair. A mouse model of articular cartilage repair was generated by inducing a patellar groove scratch in 8‑week‑old p21‑knockout (KO) mice and C57Bl/6 wild‑type (WT) mice. Mice were sacrificed at 4and8weeks post‑surgery. The present study also investigated the effect of p21 deficiency on cartilage differentiation in ATDC5 cells invitro. Safranin O staining results indicated that cartilage repair initially occurred in p21 KO mice. In addition, immunohistochemical analysis demonstrated that p21 KO upregulated proliferating cell nuclear antigen and increased cell proliferation. However, typeII collagen and Sox9 expression levels remained unchanged in p21 KO and WT mice. Moreover, it was identified that p21 downregulation did not affect Sox9 and typeII collagen expression levels invitro. Furthermore, p21 deficiency promoted healing of articular cartilage damage, which was associated with cell proliferation invivo, and increased chondrocyte proliferation but not differentiation invitro. Therefore, the present results suggested that p21 does not affect Sox9 or typeII collagen expression levels during cartilage differentiation in the repair of cartilage defects.

Implications of Anti-Inflammatory Nature of Exosomes in Knee Arthritis.

Exosomes are extracellular vesicles (EVs) released from cells that are a part of many biological and pathological processes, especially in intercellular communication. These vesicles are involved cell signaling, influence tissue and immune response, and serve as biomarkers for diseases. Most interesting are the exosomes that are released from mesenchymal stem cells (MSCs) for inflammation in joint diseases. Preliminary studies have demonstrated the advantages of using EVs rather than MSCs for cell free therapy. Research on exosomes have shown promising results as biomarkers for tracking the pathogenesis and prognosis of inflammatory arthritis. Therapeutically, animal studies have demonstrated immunosuppression, reversing inflammation, increasing chondrocyte proliferation, and drug delivery properties. The field of exosomes continues to develop and more basic science and clinical studies with safety and efficacy studies are needed.

Promotion of G1/S Transition and Inhibition of Inflammatory Cytokine Production by Hydroxypyridinone-Coumarin in Osteoarthritis Rats.

BackgroundOsteoarthritis is a joint disorder characterized by articular cartilage degradation leading to joint stiffness and pain. The present study investigated the effect of hydroxypyridinone-coumarin on proliferation of chondrocytes.Material/MethodsChondrocyte proliferation was assessed by MTT assay, and distribution of cells in various phases of the cell cycle was determined using flow cytometry. RT-PCR and Western blot assays were used for assessment of mRNA and protein levels, respectively. Osteoarthritis was induced in the rats by injecting monosodium iodoacetate (5 mg/kg) by the intra-articular route. The rats in the treatment groups were intraperitoneally injected with 5, 10, or 15 mg/kg doses of hydroxypyridinone-coumarin alternately for 1 month.ResultsThe proliferation of chondrocytes was increased significantly (P<0.05) by treatment with hydroxypyridinone-coumarin in a concentration-based manner. The increase in chondrocyte proliferation by hydroxypyridinone-coumarin was maximum at 50 μM. Treatment with hydroxypyridinone-coumarin markedly increased chondrocyte population in S and G2/M phases, with subsequent reduction in G0/G1 phase. The cyclin D1, CDK4, and CDK6 levels in the chondrocytes were increased by treatment with hydroxypyridinone-coumarin. The production of IL-6, TNF-α, and IL-1β in the osteoarthritis rats was markedly suppressed by hydroxypyridinone-coumarin. Treatment of the OA rats with hydroxypyridinone-coumarin markedly reduced the expression of IκB-α and NF-κB p65.ConclusionsThe present study revealed that the proliferative potential of chondrocytes is increased by hydroxypyridinone-coumarin through acceleration of G1/S transition. Moreover, hydroxypyridinone-coumarin treatment reduced inflammatory cytokine production in the osteoarthritis rats. Therefore, hydroxypyridinone-coumarin should be evaluated further for possible use in the treatment of osteoarthritis.

Suv39h1 promotes facet joint chondrocyte proliferation by targeting miR-15a/Bcl2 in idiopathic scoliosis patients

BackgroundIdiopathic scoliosis (IS) is a complex disease with an unclear etiology, and the worldwide prevalence is approximately 2–3%. As an important link between environmental factors and phenotypic differences, epigenetic changes, such as lncRNA, miRNA, and DNA methylation, have recently been reported to be associated with the development of IS. However, the correlation between histone methylation, another classical epigenetic mechanism, and IS has not been determined. In this study, we investigated the morphological changes, alterations in the levels of histone methylation, and cell proliferation-related pathway in inferior facet joint cartilage in 11 IS patients and 10 comparable controls.ResultsCompared with the control group, narrowed facet joint cartilage but increased proliferative chondrocytes and upregulated collagen type II (COL2A1) and B-cell lymphoma-2 (Bcl2) were observed in IS patients. Additionally, tri-methylation levels of H3K9 (H3K9me3) rather than other lysine sites were significantly increased in IS patients, coinciding with the upregulation of its specific enzyme, suppressor of variegation 3-9, drosophila homolog of 1 (SUV39H1). In addition, Bcl2-targeted miR-15a was downregulated in IS patients, and the level of H3K9me3 in the promoter region of the miR-15a host gene was remarkably increased in IS patients compared with the control group. Moreover, overexpressing SUV39H1 in ATDC5 cells with increased H3K9me3 levels led to similar changes, with increased expression of COL2A1 and Bcl2, decreased expression of miR-15a, and increased cell proliferation.ConclusionsThus, our study suggests that increased chondrocyte proliferation occurs in the facet joint cartilage of IS patients compared with the control group and may be promoted by the elevated levels of H3K9me3 and SUV39H1, which regulate the miR-15a/Bcl2 pathway. This dysregulation of chondrocyte proliferation could result in abnormal spinal growth and may additionally participate in the development and progression of IS.