Huarong Shao

Culture-expanded allogenic adipose tissue-derived stem cells attenuate cartilage degeneration in an experimental rat osteoarthritis model

Mesenchymal stem cell (MSC)-based cell therapy is a promising avenue for osteoarthritis (OA) treatment. In the present study, we evaluated the efficacy of intra-articular injections of culture-expanded allogenic adipose tissue-derived stem cells (ADSCs) for the treatment of anterior cruciate ligament transection (ACLT) induced rat OA model. The paracrine effects of major histocompatibility complex (MHC)-unmatched ADSCs on chondrocytes were investigated in vitro. Rats were divided into an OA group that underwent ACLT surgery and a sham-operated group that did not undergo ACLT surgery. Four weeks after surgery mild OA was induced in the OA group. Subsequently, the OA rats were randomly divided into ADSC and control groups. A single dose of 1 × 106 ADSCs suspended in 60 μL phosphate-buffered saline (PBS) was intra-articularly injected into the rats of the ADSC group. The control group received only 60 μL PBS. OA progression was evaluated macroscopically and histologically at 8 and 12 weeks after surgery. ADSC treatment did not cause any adverse local or systemic reactions. The degeneration of articular cartilage was significantly weaker in the ADSC group compared to that in the control group at both 8 and 12 weeks. Chondrocytes were co-cultured with MHC-unmatched ADSCs in trans-wells to assess the paracrine effects of ADSCs on chondrocytes. Co-culture with ADSCs counteracted the IL-1β-induced mRNA upregulation of the extracellular matrix-degrading enzymes MMP-3 and MMP-13 and the pro-inflammatory cytokines TNF-α and IL-6 in chondrocytes. Importantly, ADSCs increased the expression of the anti-inflammatory cytokine IL-10 in chondrocytes. The results of this study indicated that the intra-articular injection of culture-expanded allogenic ADSCs attenuated cartilage degeneration in an experimental rat OA model without inducing any adverse reactions. MHC-unmatched ADSCs protected chondrocytes from inflammatory factor-induced damage. The paracrine effects of ADSCs on OA chondrocytes are at least part of the mechanism by which ADSCs exert their therapeutic activity.

The protective effect of xanthan gum on interleukin-1β induced rabbit chondrocytes

We have previously shown that intra-articular injection of xanthan gum (XG) could protect the joint cartilage and reduce osteoarthritis progression. In this study, we investigated the preliminary cytotoxicity of XG on chondrocytes, evaluated the effects of XG on the proliferation and the protein expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinase-1 (TIMP-1) in interleukin-1β (IL-1β)-induced rabbit chondrocytes. Primary rabbit chondrocytes were cultured. After treatment with various concentrations of XG with or without 10 ng/mL IL-1β, the proliferation of chondrocytes was evaluated using the MTT assay and the expression levels of MMPs and TIMP-1 were evaluated using ELISA. The results showed that XG alone displayed no adverse effects on cell viability and reversed significantly IL-1β-reduced cell proliferation in a dose-dependent manner. Furthermore, XG showed a dose-dependent inhibition in the IL-1β-induced release of MMPs while increasing TIMP-1 expression. These results strongly suggest that XG affords protection on IL-1β induced rabbit chondrocytes.

Xanthan gum protects rabbit articular chondrocytes against sodium nitroprusside-induced apoptosis in vitro.

We have previously reported that intra-articular injection of xanthan gum (XG) could significantly ameliorate the degree of joint cartilage degradation and pain in experimental osteoarthritis (OA) model in vivo. In this present study, we evaluated the protective effect of XG against Sodium nitroprusside (SNP)-induced rabbit articular chondrocytes apoptosis in vitro. Rabbit articular chondrocytes were incubated with various concentrations of XG for 24h prior to 0.5mmol/L SNP co-treatment for 24h. The proliferation of chondrocytes was analyzed using MTT assay. The chondrocytes early apoptosis rates were evaluated using Annexin V-FITC/PI flow cytometry. The morphology of apoptosis chondrocytes were observed by scanning electron microscopy (SEM). The loss/disruption of mitochondrial membrane potential was detected using rhodamin 123 by confocal microscope. The concentration of prostaglandin E2 (PGE2) in cell culture supernatants was evaluated using ELISA assay. The results showed that XG could significantly reverse SNP-reduced cell proliferation and inhibited cell early apoptosis rate in a dose-dependent manner. XG alleviated loss/disruption of mitochondrial membrane potential and decreased the PGE2 level of chondrocytes cell culture supernatants in SNP-induced chondrocytes. These results of the present research strongly suggest that XG can protect rabbit articular chondrocytes against SNP-induced apoptosis in vitro.

Intra-articular injection of xanthan gum reduces pain and cartilage damage in a rat osteoarthritis model.

The objective of this study was to evaluate the alleviative effect of intra-articular (IA) injection of xanthan gum (XG) on pain and cartilage degradation in a model of monosodium iodoacetate (MIA)-induced knee osteoarthritis (OA). The rheological study and hyaluronidase (HAse) degradation analysis of XG injection were presented. The effect of pain relief was determined by measurements of paw withdrawal threshold and weight bearing by hind limbs. The protective effect on the cartilage was evaluated by gross morphological observation and histological evaluation of knee joints. The effect was investigated in two protocols: a therapeutic treatment protocol, and a prophylactic treatment protocol. Our results showed that HAse had no effect on the rheological properties of XG injection. Local XG administration in both protocols could reduce OA pain and alleviate the joint cartilage degradation induced by MIA. IA injection of XG might be an effective method for OA treatment in human.

Low molecular weight xanthan gum for treating osteoarthritis

Osteoarthritis (OA) is one of the most common chronic diseases and characterized by degradation of articular cartilage. We have previously reported xanthan gum (XG) injection preparation with high molecular weights (Mw) in ranging from 3×106Da to 5×106Da (HM-XG) could enhance the viscosity of synovial fluid, protect joint cartilage in rabbit, and the therapeutical effect has no significance difference with an existing clinical medication (sodium hyaluronate, SH) at the same injection frequency (once weekly for 5 weeks). Herein, we prepared a XG injection preparation with a low Mw (LM-XG) in ranging from 1×106Da to 1.5×106Da, and evaluated the therapeutical effect for OA therapy at once every 2 weeks for 5 weeks with an SH at once weekly for 5 weeks as reference. The model of OA was induced using anterior cruciate ligament transection (ACLT) in a rabbit in vivo and also using sodium nitroprusside (SNP) in cell culture in vitro. The results showed that LW-XG could also protect cartilage from damage, decrease the concentration of nitric oxide (NO) in synovial fluid and reverse the amplification of the knee joint width similar to HM-XG as our previously reported. At the cellular level, LW-XG promotes proliferation while decreases apoptosis of chondrocytes. Mechanistically at the molecular level, these effects are elicited via down-regulation of the protein levels of caspase-3 and bax and up-regulation of the protein levels of bcl-2 in cartilage in both in vivo and in vitro. These results showed that LW-XG maybe become an excellent candidate long-acting drug for treating OA.

Immunomodulatory effects of xanthan gum in LPS-stimulated RAW 264.7 macrophages

In this study, we evaluated the immunomodulatory effects of xanthan gum (XG) in RAW264.7 macrophages and the underlying molecular mechanisms. We used scanning electron microscopy (SEM) to analyze the morphology of XG-treated RAW264.7 cells with and without lipopolysaccharide (LPS) stimulation and investigated the subsequent effects on nitric oxide (NO), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) levels in LPS-activated mouse RAW264.7 macrophages. We also analyzed the binding affinity of XG to Toll-like receptor 4 (TLR4) with surface plasmon resonance (SPR) analysis and observed that XG decreased NO, IL-6 and TNF-α secretion into the culture medium and iNOS and COX-2 protein levels induced by LPS. This study reveals a two-way immunomodulatory effect of XG on inflammatory mediators in RAW264.7 macrophages that may involve the TLR4 signal pathway, providing a pharmacological basis for the use of XG in the control of inflammatory disorders.

Development, validation and influence factor analysis of a near-infrared method for the molecular weight determination of xanthan gum

A practical molecular weight determination model of xanthan gum (XG), based on near-infrared (NIR) spectroscopy, was built in this study. Two sample measurement modules, integrating sphere module and fiber-optic probe module, were compared, and the best partial least square (PLS) regression model was based on fiber-optic probe module. The values of coefficient of determination in calibration (R(2)c), coefficient of determination in prediction (R(2)p), residual predictive deviation (RPD) and root mean square error of prediction (RMSEP) were 0.967, 0.975, 6.028 and 0.250×10(6)Da, respectively. The molecular weight range, linearity, accuracy and precision of the established method were also validated. Furthermore, influence factors on this method were discussed in order to establish an appropriate measurement protocol. Results showed that the proposed NIR method may be suitable for practical applications in manufacturing plants and probably be accepted as a good alternative approach for fast determination of molecular weight of XG in production process.

Xanthan gum inhibits cartilage degradation by down-regulating matrix metalloproteinase-1 and -3 expressions in experimental osteoarthritis:

We previously reported that intra-articular injection of xanthan gum protected the joint cartilage and reduced osteoarthritis progression. In this study, the effects of xanthan gum on chondrocytes apoptosis were evaluated using the labeling assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, to determine the protein expression of matrix metalloproteinase-1, 3, and tissue inhibitors of metalloproteinase-1 using immunohistochemistry and Western blot assay in cartilage of papain-induced rabbit osteoarthritis model. Compared to the negative control group, intra-articular injection of xanthan gum, once every 2 weeks for 5 weeks significantly inhibited chondrocytes apoptosis and matrix metalloproteinase-1 and -3 protein expression levels and also enhanced the tissue inhibitors of metalloproteinase-1 production in cartilage. No significant differences between the xanthan gum-treated group and the sodium hyaluronate-treated group (intra-articular injection of sodium hyaluronate only once a week for 5 weeks) were observed.

Lower range of molecular weight of xanthan gum inhibits cartilage matrix destruction via intrinsic bax-mitochondria cytochrome c-caspase pathway

We have previously reported an application of lower range of molecular weight of xanthan gum (LRWXG) for inhibiting cartilage matrix destruction and preventing mitochondrial damage in rabbit osteoarthritis (OA) model. However, whether LRWXG exerts its anti-OA activity through intrinsic bax-mitochondria cytochrome c-caspase signaling pathway in OA still requires further study. To address this problem, the OA model was induced by anterior cruciate ligament transection (ACLT) in rabbit and then treated with LRWXG. The results showed that LRWXG could inhibit the loss of collagen in cartilage matrix, protect trabecular bone in subchondral, decrease the apoptosis of chondrocytes, down-regulate the expressions of active caspase-9, active caspase-3 and bax, and up-regulate the expression of bcl-2. In addition, LRWXG could up-regulate the expression of cyt-c in mitochondria, while down-regulate the expression of cyt-c in cytoplasm. These findings show that LRWXG inhibits cartilage degradation via an intrinsic bax-mitochondria cytochrome c-caspase pathway in OA.