Vitamin D delays intervertebral disc degeneration and improves bone quality in ovariectomized rats.

Known to be involved in bone-cartilage metabolism, Vitamin D (VD) may play a role in human's disc pathophysiology. Given that postmenopausal women are prone to suffer VD deficiency and intervertebral disc degeneration (IDD), this study is intended to investigate whether VD can delay IDD in ovariectomized rats by improving bone microstructure and antioxidant stress. Female Sprague-Dawley rats were randomly allocated into four groups: sham, oophorectomy (OVX)+VD deficiency (VDD), OVX, and OVX+VD supplementation (VDS). In vivo, after a 6-month intervention, imaging and pathology slice examinations showed that IDD induced by OVX was significantly alleviated in VDS and deteriorated by VDD. The expressions of aggrecan and Collagen II in intervertebral disc were reduced by OVX and VDD, and elevated by VDS. Compared with the OVX+VDD and OVX group vertebrae, OVX+VDS group vertebrae showed significantly improved endplate porosity and lumbar bone mineral density with increased percent bone volume and trabecular thickness. Furthermore, 1α,25(OH)2D3 restored the redox balance (total antioxidant capacity, ratio of oxidized glutathione/glutathione) in the disc. The cocultivation of 1α,25(OH)2D3 and nucleus pulposus cells (NPCs) was conducted to observe its potential ability to resist excessive oxidative stress damage induced by H2O2. In vitro experiments revealed that 1α,25(OH)2D3 reduced the senescence, apoptosis, and extracellular matrix degradation induced by H2O2 in NPCs. In conclusion, VDS exhibits protective effects in OVX-induced IDD, partly by regulating the redox balance and preserving the microstructure of endplate. This finding provides a new idea for the prevention and treatment of IDD.

[Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway].

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-ß-galactosidase(SA-ß-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1ß(IL-1ß), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1ß and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1ß and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process.

ETHNOPHARMACOLOGICAL RELEVANCE: The treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. METHODS: Using human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-ß1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated ß-Galactosidase staining. RESULTS: The results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 µg/mL, 0.3 µg/mL in vitro. In vivo, GLEXG at the dose of 0.3 µg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. CONCLUSION: we conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 µg, i.a.) rescued cartilage defects in rat OA knee model.

Nano-elemental selenium particle developed via supramolecular self-assembly of chondroitin sulfate A and Na2SeO3 to repair cartilage lesions.

Cartilage repair is a significant clinical issue due to its restricted ability to regenerate and self-heal after cartilage lesions or degenerative disease. Herein, a nano-elemental selenium particle (chondroitin sulfate A­selenium nanoparticle, CSA-SeNP) is developed by the supramolecular self-assembly of Na2SeO3 and negatively charged chondroitin sulfate A (CSA) via electrostatic interactions or hydrogen bonds followed by in-situ reducing of l-ascorbic acid for cartilage lesions repair. The constructed micelle exhibits a hydrodynamic particle size of 171.50 ± 2.40 nm and an exceptionally high selenium loading capacity (9.05 ± 0.03 %) and can promote chondrocyte proliferation, increase cartilage thickness, and improve the ultrastructure of chondrocytes and organelles. It mainly enhances the sulfation modification of chondroitin sulfate by up-regulating the expression of chondroitin sulfate 4-O sulfotransferase-1, -2, -3, which in turn promotes the expression of aggrecan to repair articular and epiphyseal-plate cartilage lesions. The micelles combine the bio-activity of CSA with selenium nanoparticles (SeNPs), which are less toxic than Na2SeO3, and low doses of CSA-SeNP are even superior to inorganic selenium in repairing cartilage lesions in rats. Thus, the developed CSA-SeNP is anticipated to be a promising selenium supplementation preparation in clinical application to address the difficulty of healing cartilage lesions with outstanding repair effects.

BSHXF-medicated serum combined with ADSCs regulates the TGF-ß1/Smad pathway to repair oxidatively damaged NPCs and its component analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lower back pain (LBP) is a common and frequent clinical condition, and intervertebral disc degeneration (IDD) is recognized as the leading cause of LBP, typically manifested by increased nucleus pulposus cell (NPC) senescence and death. In recent years, the treatment of IDD with stem cell injections has had great potential compared to surgical treatment. Combining the two may achieve better results, as BuShenHuoXueFang (BSHXF) is an herbal formula that improves the survival rate of transplanted stem cells and enhances their efficacy. AIM OF THE STUDY: We aimed to qualitatively and quantitatively analyze BSHXF-medicated serum and investigate the molecular mechanism of BSHXF-mediated serum in promoting the differentiation of adipose mesenchymal stem cells (ADSCs) into NPCs and delaying the senescence of NPCs by regulating the TGF-ß1/Smad pathway. MATERIALS AND METHODS: In this study, an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometer (UPLC-Q-TOF-MS) was used to establish a method for the analysis of rat serum samples to track the active components in vivo; the oxidative damage model of NPCs was induced by T-BHP, and a Transwell chamber was used to construct a coculture system of ADSCs and NPCs. Flow cytometry was used to determine the cell cycle; SA-ß-Gal staining was used to assess cell senescence; ELISA was used to detect IL-1ß, IL-6 inflammatory factors, CXCL-1, CXCL-3, CXCL-10 chemokines, and TGF-ß1 in the supernatants of ADSCs and NPCs. WB was used to detect COL2A1, COL1A1, and Aggrecan in ADSCs to assess the manifestation of NP differentiation in ADSCs, and the WB method was used to detect COL2A1, COL1A1, Aggrecan, p16, p21, p53, and p-p53 protein expression in NPCs to reflect the cellular senescence status and to detect TGF-ß1, Smad2, Smad3, p- Smad2, and p- Smad3 protein expression in NPCs to reflect the pathway condition. RESULTS: We finally identified 70 blood components and their metabolites, including 38 prototypes, from the BSHXF-medicated serum. Compared with that in the nonmedicated serum group, the TGF-ß1/Smad pathway was activated in the medicated serum group, ADSCs moved toward NPC characteristics, the number of NPCs in the S/G2M phase increased, the number of senescent NPCs decreased, IL-1ß and IL-6 inflammatory factors in the Transwell decreased, CXCL-1, CXCL-3, and CXCL-10 chemokines decreased, and the expression of p16, p21, p53 and p-p53 proteins in NPCs was inhibited. CONCLUSION: By regulating the TGF-ß1/Smad pathway, BSHXF-medicated serum promoted ADSCs to NPCs, effectively alleviated the cycle blockage of NPCs after oxidative damage, encouraged the growth and proliferation of NPCs, delayed the aging of NPCs, improved the deteriorating microenvironment around NPCs, and repaired oxidatively damaged NPCs. The combination of BSHXF or its compounds with ADSCs has great potential for the treatment of IDD in the future.

Effect of the addition of different concentrations of Solanum glaucophyllum desf. extract on chondrocyte cultures from the growth cartilage of newborn rats.

Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 µL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.

Chondroprotective Mechanism of Eucommia ulmoides Oliv.-Glycyrrhiza uralensis Fisch. Couplet Medicines in Knee Osteoarthritis via Experimental Study and Network Pharmacology Analysis.

Background: Knee osteoarthritis (KOA) is the primary prevalent disabling joint disorder among osteoarthritis (OA), and there is no particularly effective treatment at the clinic. Traditional Chinese medicine (TCM) herbs, such as Eucommia ulmoides Oliv. and Glycyrrhiza uralensis Fisch. (E.G.) couplet medicines, have been reported to exhibit beneficial health effects on KOA, exact mechanism of E.G. nevertheless is not fully elucidated. Purpose: We assess the therapeutic effects of E.G. on KOA and explore its underlying molecular mechanism. Methods: UPLC-Q-TOF/MS technique was used to analyze the active chemical constituents of E.G. The destabilization of the medial meniscus model (DMM) was employed to evaluate the chondroprotective action of E.G. in KOA mice using histomorphometry, µCT, behavioral testing and immunohistochemical staining. Additionally, network pharmacology and molecular docking were used to predict potential targets for anti-KOA activities of E.G., which was further verified through in vitro experiments. Results: In vivo studies have shown that E.G. could significantly ameliorate DMM-induced KOA phenotypes including subchondral bone sclerosis, cartilage degradation, gait abnormality and thermal pain reaction sensibility. E.G. treatment could also promote extracellular matrix synthesis to protect articular chondrocytes, which was indicated by Col2 and Aggrecan expressions, as well as reducing matrix degradation by inhibiting MMP13 expression. Interestingly, network pharmacologic analysis showed that PPARG might be a therapeutic center. Further study proved that E.G.-containing serum (EGS) could up-regulate PPARG mRNA level in IL-1ß-induced chondrocytes. Notably, significant effects of EGS on the increment of anabolic gene expressions (Col2, Aggrecan) and the decrement of catabolic gene expressions (MMP13, Adamts5) in KOA chondrocytes were abolished due to the silence of PPARG. Conclusion: E.G. played a chondroprotective role in anti-KOA by inhibiting extracellular matrix degradation, which might be related to PPARG.

Oroxin B alleviates osteoarthritis through anti-inflammation and inhibition of PI3K/AKT/mTOR signaling pathway and enhancement of autophagy.

Background: Osteoarthritis (OA) is a common aging-related degenerative joint disease with chronic inflammation as its possible pathogenesis. Oroxin B (OB), a flavonoid isolated from traditional Chinese herbal medicine, possesses anti-inflammation properties which may be involved in regulating the pathogenesis of OA, but its mechanism has not been elucidated. Our study was the first to explore the potential chondroprotective effect and elucidate the underlying mechanism of OB in OA. Methods: In vitro, primary mice chondrocytes were stimulated with IL-1ß along with or without the administration of OB or autophagy inhibitor 3-methyladenine (3-MA). Cell viability assay was measured with a cell counting kit-8 (CCK-8). The phenotypes of anabolic-related (Aggrecan and Collagen II), catabolic-related (MMP3, MMP13, and ADAMTS5), inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß), and markers of related signaling pathways in chondrocytes with different treatment were detected through western blot, RT-qPCR, and immunofluorescent staining. In vivo, the destabilized medial meniscus (DMM) operation was performed to establish the OA mice model. After knee intra-articular injection with OB for 8 weeks, the mice's knee joints were obtained for subsequent histological staining and analysis. Results: OB reversed the expression level of anabolic-related proteins (Aggrecan and Collagen II) and catabolic-related (MMP3, MMP13, and ADAMTS5) in IL-1ß-induced chondrocytes. Mechanistically, OB suppressed the inflammatory response stimulated by IL-1ß, as the inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß) markers were downregulated after the administration of OB in IL-1ß-induced chondrocytes. Besides, the activation of PI3K/AKT/mTOR signaling pathway induced by IL-1ß could be inhibited by OB. Additionally, the autophagy process impaired by IL-1ß could be rescued by OB. What's more, the introduction of 3-MA to specifically inhibit the autophagic process impairs the protective effect of OB on cartilage. In vivo, histological staining revealed that intra-articular injection of OB attenuated the cartilage degradation, as well as reversed the expression level of anabolic and catabolic-related proteins such as Aggrecan, Collagen II, and MMP13 induced in DMM-induced OA models. Conclusions: The study verified that OB exhibited the chondroprotective effect by anti-inflammatory, inhibiting the PI3K/AKT/mTOR signaling pathway, and enhancing the autophagy process, indicating that OB might be a promising agent for the treatment of OA.

Effects of Bushen Zhuangjin Decoction drug serum on SOX9 expression and Chondrocyte phenotype in vitro.

Introduction Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation.Hypothesis/Gap Statement Therefore, to explore the regulatory mechanism of Bushen Zhuangjin Decoction in chondrocytes will contribute to the repair of chondrocyte injury in OA, and may serve as a potential therapy for OA diseases.Aim To investigate the expression and distribution of SOX9 mediated by serum containing Bushen Zhuangjin Decoction (BZD) and its therapeutic effect on chondrocyte injury in rats.Methodology. The subcultured second-generation rat chondrocytes were randomly divided into four groups, and they were intervened with medium containing different serums, including: blank serum group, low-concentration BZD group, medium-concentration BZD group, and high-concentration BZD group. The viability, proliferation and apoptosis of chondrocytes were detected by MTT assay and flow cytometry. The gene and protein levels of SOX9, aggrecan and type II collagen genes were analysed by qRT-PCR and Western blot analysis. Immunofluorescence staining was used to analyse the expression and distribution of SOX9. Inflammatory factors in different culture mediums of chondrocytes were detected by ELISA.Results Compared with the control group, the activity of chondrocytes in the BZD drug-containing serum group was significantly enhanced, and the degree of apoptosis was significantly decreased. The gene and protein levels of SOX9, proteoglycan aggrecan and collagen II in chondrocytes increased significantly. The inflammatory factors in the culture medium also decreased significantly. And in the above experiments, the medium concentration group BZD drug-containing serum had the best effect.Conclusion Our research results show that BZD medicated serum can up-regulate the expression of SOX9, reduce the release of inflammatory factors, and promote changes in the phenotype of chondrocytes, which protects chondrocytes from damage.

[Therapeutic effects of alkaloids in Tibetan medicine Bangna (Aconiti Penduli et Aconiti Flavi Radix) on osteoarthritis rats and mechanisms].

This study aims to investigate the therapeutic effects of alkaloids in Tibetan medicine Bangna(Aconiti Penduli et Aconiti Flavi Radix) on osteoarthritis(OA) rats in vitro and in vivo and the underlying mechanisms. Chondrocytes were isolated from 2-3 week-old male SD rats and lipopolysaccharide(LPS) was used to induce OA in chondrocytes in vitro. Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of seven alkaloids(12-epi-napelline, songorine, benzoylaconine, aconitine, 3-acetylaconitine, mesaconitine, and benzoylmesaconine) to chondrocytes. Chondrocytes were classified into the control group, model group(induced by LPS 5 µg·mL~(-1) for 12 h), and administration groups(induced by LPS 5 µg·mL~(-1) for 12 h and incubated for 24 h). The protein expression of inflammatory factors cyclooxygenase-2(COX-2), inducible nitric oxide synthetase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1ß(IL-1ß) in each group were detected by Western blot, and the protein expression of matrix metalloprotease-13(MMP-13), aggrecan, collagen Ⅱ, fibroblast growth factor 2(FGF2) by immunofluorescence staining. For the in vivo experiment, sodium iodoacetate was used to induce OA in rats, and the expression of MMP-13, TNF-α, and FGF2 in cartilage tissues of rats in each group was detected by immunohistochemistry. The results showed that the viability of chondrocytes could reach more than 90% under the treatment of the seven alkaloids in a certain dose range. Aconitine, 12-epi-napelline, songorine, 3-acetylaconitine, and mesaconitine could decrease the protein expression of inflammatory factors COX-2, iNOS, TNF-α and IL-1ß compared with the model group. Moreover, 12-epi-napelline, aconitine, and mesaconitine could down-regulate the expression of MMP-13 and up-regulate the expression of aggrecan and collagen Ⅱ. In addition, compared with the model group and other Bangna alkaloids, 12-epi-napelline significantly up-regulated the expression of FGF2. Therefore, 12-epi-napelline was selected for the animal experiment in vivo. Immunohistochemistry results showed that 12-epi-napelline could significantly reduce the expression of MMP-13 and TNF-α in cartilage tissues, and up-regulate the expression of FGF2 compared with the model group. In conclusion, among the seven Bangna alkaloids, 12-epi-napelline can promote the repair of OA in rats by down-regulating the expression of MMP-13 and TNF-α and up-regulating the expression of FGF2.

Effect of Resveratrol on Serum Levels of Type II Collagen and Aggrecan in Patients with Knee Osteoarthritis: A Pilot Clinical Study.

Treatment of knee osteoarthritis (OA) remains a challenging concern. Preclinical studies provided accumulating evidence on resveratrol efficacy in ameliorating degenerative articular damage. The present study was conducted to evaluate the effects of resveratrol as monotherapy on the serum level of type II collagen (Coll 2-1) and aggrecan in patients with knee osteoarthritis. The study was an open-labeled noncontrolled clinical trial. Resveratrol 500 mg/day in a single oral dose was given to the patients with knee osteoarthritis for 90 days. The serum levels of Coll-2-1, aggrecan, and biomarkers of inflammation were measured pre- and posttreatment. Hematological profiles and both hepatic and renal function markers were investigated at the baseline and at the end of the treatment for evaluating the tolerability and safety of resveratrol. Visual Analog Scale (VAS) for pain and Knee injury and Osteoarthritis Outcome Score (KOOS) for disease activity were clinically assessed monthly. Administration of 500 mg resveratrol for three months led to a nonsignificant decrease in the serum level of Coll 2-1 while a significant increase in aggrecan serum level. Resveratrol significantly improves pain score measured by VAS and KOOS after 30 days. Improvements in patients' activity and functional status were also evident at day 30 and kept on for three months which was reflected by KOOS subscale scores and with a significant improvement in all KOOS areas. In conclusion, oral administration of resveratrol as a monotherapy provides a remarkable improvement in the clinical status of the patients but has no significant effect on serum levels of Coll 2-1.

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1ß, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3-/- mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Protective Effects of Hydrogen-Rich Water Against Cartilage Damage in a Rat Model of Osteoarthritis by Inhibiting Oxidative Stress, Matrix Catabolism, and Apoptosis.

BACKGROUND The aim of this study was to investigate the mechanisms underlying the potential effects of hydrogen-rich water (HW) on articular cartilage in a rat osteoarthritis (OA) model. MATERIAL AND METHODS A rat model of OA was established using the modified Hulth method, and rats were forced to exercise for 30 min every day 1 week after surgery for 7 weeks. Mankin's method was used to score the severity of OA. The animals were assigned into the OA group, OA+HW group, and sham operation group. After 8 weeks, the animals in the OA group had a Mankin score >8 points, and HW was administered into the knee joint. After 2 weeks of treatment, articular cartilage was obtained for pathological examination, consisting of hematoxylin and eosin, toluidine blue, and Hoechst staining, as well as quantitative real-time PCR and Western blot analyses. This combination of pharmacological and molecular biological analyses was performed to examine the mechanism underlying the protective effect of HW on articular cartilage. RESULTS The antioxidant effects of HW suppressed oxidative damage, which may have aided the inhibition of ECM-degrading enzymes (MMP3, MMP13, ADAMT4, and ADAMT5), the upregulation of Col II and aggrecan expression, and the downregulation of COX-2, iNOS, and NO expression. The results of HE staining indicated intra-articular treatment of HW attenuated cartilage degradation. However, Hoechst staining in the OA group indicated the nuclei of the fragmented chondrocytes were condensed compared to the sham operation group, and this effect was inhibited by HW. CONCLUSIONS HW showed a protective effect against the progression of OA in an animal model, which may have been mediated by its anti-oxidant and anti-apoptotic activities.

In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold.

BACKGROUND: Cartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP). METHODS: Human ADSCs and PRP were characterized. A silk fibroin scaffold with 500 µm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively. RESULTS: Cells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-ß1 (TGF-ß1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21. CONCLUSION: Our study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

Cladophora glomerata methanolic extract promotes chondrogenic gene expression and cartilage phenotype differentiation in equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome.

BACKGROUND: Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. OBJECTIVES: In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. METHODS: Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. RESULTS: The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. CONCLUSION: Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5∆cat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-∆cat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy.

OBJECTIVE: Intradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function. DESIGN: Human CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72 h in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared (FTIR) spectroscopy. RESULTS: Solute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity. CONCLUSIONS: CEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply.

Effects of malvidin, cyanidin and delphinidin on human adipose mesenchymal stem cell differentiation into adipocytes, chondrocytes and osteocytes.

BACKGROUND: Anthocyanidins are plant phytochemicals found at high concentrations in berries, vegetables and flowers. Anthocyanidins have been extensively investigated due to their antioxidative, antidiabetic and anti-inflammatory effects. Few studies show that anthocyanidins decrease obesity and improve bone density. However, the effects of anthocyanidins on tissue regeneration have not been sufficiently clarified. Human mesenchymal stem cells (MSCs) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Although MSCs are often used for screening of biologically active compounds, so far, the effect of anthocyanidins on MSC differentiation has not been addressed. PURPOSE: The aim of this study was to analyse the effect of anthocyanidins malvidin, cyanidin and delphinidin on adipose tissue-derived MSC differentiation into adipocytes, osteocytes and chondrocytes. STUDY DESIGN AND METHODS: Differentiation into adipocytes, osteocytes and chondrocytes was carried out in the defined cell culture conditions in the presence or absence of malvidin, cyanidin and delphinidin. The differentiation was confirmed by cytochemical staining and tissue-specific gene and protein expression. Antiobesity and anti-diabetes drug liraglutide was used as a reference drug in this study. RESULTS: Delphinidin inhibited MSC adipogenesis and downregulated FABP4 and adiponectin genes. Malvidin induced a significantly higher accumulation of calcium deposits in MSCs comparing to untreated MSCs, as well as upregulated the osteocyte-specific gene BMP-2 and Runx-2 expression and induced BMP-2 secretion. Cyanidin and delphinidin demonstrated a chondrogenesis stimulating effect by upregulation of Col2a1 and aggrecan. CONCLUSION: Altogether, our data show that anthocyanidins malvidin, cyanidin and delphinidin exert favourable effects on MSC osteogenesis and chondrogenesis whereas delphinidin inhibits adipogenesis. These results suggest that anthocyanidin effects on tissue regeneration could be further analysed in depth in vivo.

Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage.

OBJECTIVE: Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior. DESIGN: Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years. RESULTS: It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes. CONCLUSION: The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

[Phosphorylated Focal Adhesion Kinase, Phosphinositides 3 Kinase and Aggrecan Genes and Proteins in Cartilage Cells Are Probably Involved in Needle Knife Intervention Induced Improvement of Knee Osteoarthritis in Rabbits].

OBJECTIVE: We have demonstrated that needle knife (acupotomy) treatment can improve knee osteoarthritis (KOA) in rabbits. The present study was designed to examine its effect on expression of phosphorylated focal adhesion kinase (p-FAK), phosphinositides 3 kinase (p-PI 3 K) and Aggrecan genes and proteins in the knee-joint cartilage tissues of KOA rabbits, so as to explore its partial molecular mechanism underlying improvement of KOA. METHODS: Forty-nine New Zealand male rabbits were randomly divided into normal control, model, model +inhibitor, needle knife, needle knife+ inhibitor, electroacupuncture (EA), EA +inhibitor groups (n=7 in each). The KOA model was established by modified Videman method (left hindlimb extension immobilization and ankle dorsal flexion 60°). Acupotomy relaxing manipulation was applied to the lateral collateral ligament and patellar ligament of the left knee-joint, two times a week for 4 weeks, and EA (2 Hz/100 Hz, 3 mA) was applied to the left "Liangmen" (ST 21), "Xuehai" (SP 10), "Neixiyan " (EX-LE 4) and "Dubi" (ST 35) for 20 min, three times a week, for 4 weeks. About 2 h before every needle-knife or EA treatment or at the corresponding time-point, intra-articular cavity injection of PF-562271(a specific antagonist of FAK, 200 µmol/L, 0.5 mL)was performed in the three inhibitor groups. The expression levels of p-FAK, p-PI 3 K, Aggrecan genes and proteins in the cartilage tissues were measured with quantitative Real-time PCR and Western blot, separately. RESULTS: After modeling, the expression levels of p-FAK and p-PI 3 K genes and proteins were significantly up-regulated (P<0.05, P<0.01), while those of Aggrecan protein and mRNA considerably down-regulated in the model group in comparison with the normal group (P<0.01). Following 4 weeks' needle-knife or EA treatment, the expression levels of p-FAK and p-PI 3 K and Aggrecan proteins in both EA and needle-knife groups, and Aggrecan mRNA in the needle knife group were significantly up-regulated (P<0.05, P<0.01). After administration of p-FAK antagonist, modeling-induced upregulation of expression of p-FAK mRNA and protein and p-PI 3 K protein, as well as modeling-induced down-regulation of Aggrecan mRNA and protein were significantly suppressed in the model+inhibitor group (P<0.05, P<0.01), and needle knife-induced and EA-induced up-regulation of expression of p-FAK, p-PI 3 K and Aggrecan mRNAs and proteins was notably suppressed respectively in comparison with the needle knife and EA groups (P<0.05, P<0.01). The effect of needle knife was significantly superior to that of EA in up-regulating p-PI 3 K and Aggrecan mRNAs as well as p-FAK, p-PI 3 K and Aggrecan proteins (P<0.05, P<0.01). CONCLUSION: Needle knife intervention can up-regulate the expression levels of p-FAK, p-PI 3 K and Aggrecan proteins and mRNAs in the cartilage tissue of the knee-joint in KOA rabbits, suggesting an involvement of FAK-PI 3 K signaling in the needle knife-induced improvement of KOA.