Study On Xanthan Gum Injection And Its Pharmacodynamics And Action Mechanism On Experimental Osteoarthritis

Osteoarthritis (OA), also known as degenerative joint disease, is one of the most common joint disorders. It is characterized by the degradation and loss of articular cartilage. OA is one of the main cause of joint pain and functional disorders of elderly people. Multiple factors are known to affect the progression of OA, including increasing age, trauma, inflammation, occupation, obesity, metabolism and genetic factor. Current treatments for OA involve surgical treatment, pharmacotherapy and adjuvant therapy. Pharmacotherapy is recommended as the first line therapy on early and mid-term OA. Intro-articular (IA) injection of sodium hyaluronate (SH) is indicated as an effective treatment for OA due to its lubricating and cushioning properties. Furthermore, IA injection of SH can reduce joint pain. However, SH will be quickly degraded in vivo by hydrolytic or enzymatic reactions because of its instability. Therefore, a compound which is similar to SH in the structure and function, but with a longer effect in the joint, is needed. Xanthan gum (XG) is a natural microbial extracellular heteropolysaccharide. Typically, XG is made from fermentation of Xanthomonas campestris in well-agitated fermenter. The relative molecular mass (Mr) of XG distribution ranges from2×106to20×106. XG is similar to SH in rheology and viscosity. XG solution is very stable in wide ranges of pH, ionic concentration and temperature. Furthermore, XG will not be easily degraded in vivo. IA injection of XG is probably an effective therapeutic method for OA due to it's high viscosity. XG injection is also likely to have a long-lasting protective effect on articular cartilage to avoid numerous injections. In the present study, we determined the industrialized fermentation process of XG, obtained the XG with good quality and high Mr, evaluated the safety of IA injection of XG into knee joint of rabbit and the preliminary cytotoxicity of XG on rabbit chondrocytes, evaluated in vivo and in vitro pharmacodynamics and action mechanism of XG on experimental OA.1Preparation and quality control of the bulk drug of xanthan gumObjective:To determine the industrialized fermentation process of XG, obtain good quality and high Mr of XG and establish a quality standard which can control the samples. Methods:To optimize the carbon sources concentration and inoculum concentration in10L fermentation tank through investigating fermentation broth viscosity and XG yield. After determining the optimal fermentation condition, the fermentation processe in50L and10t fermentation tank were investigated. We firstly determined the main purification steps, and then investigated the key factors of each step through the orthogonal test, determined the optimal preparation process of XG eventually. We referred to the quality standard of the bulk drug of XG in ChP, USP, EP and SH to determine the method of quality control. Results:The optimal medium components were defined as:starch5%, ammonium sulfate0.1%, bean cake power0.3%, citric acid0.1%, MgSO40.01%, CaCO30.02%, inoculum concentration5%, temperature28.5℃, pH7.0. The preparation process consisted of the preparation and redissolution of raw XG samples, diatomite adsorption, cake filtration, enzymolysis, active carbon adsorption, cake filtration, fine filtration with microporous membrane filter, precipitation using isopropanol and stoving. The main items of quality standard involved characters, identification, tests (clear degree and color, pH, viscosity, pyruvic acid, absorbance at257nm and280nm, Mr and Mr distribution, protein content, nitrogen content, limit of isopropyl alcohol, loss on drying, ash content, heavy metals, arsenic, lead, endotoxin level, microbial enumeration tests) and assay. The results of quality tests indicated that all the test items were in accordance with the regulations. Conclusion:We determined the industrialized fermentation process of XG. The yield and quality of XG were stable in a large-scale production. XG with good quality and high Mr was obtained. The preparation process was simple, practical, and suitable for large-scale industrial production. Furthermore, we established a quality standard which can control XG. 2Preparation and quality control of xanthan gum injectionObjective:To prepare XG injection and establish the methods of quality control. Methods:The composition of formula and manufacturing technique were designed and the sterilization condition was investigated. XG injection was determined by3-phenylphenol colorimetric method. XG injection was compared with the national standard and the internal control standard of SH injection. Results:The formula composed of XG1%, sodium chloride0.5%, sodium dihydrogen phosphate0.44%and sodium hydrogen phosphate0.7%. XG injection was sterilized at121℃for15minutes. The main items of quality standard involved characters, identification, tests (pH, absorbance at257nm and280nm, osmolarity, Mr and Mr distribution, endotoxin level, and other items belong to the injection quality standard) and assay. The results of quality tests indicated that all the test items were in accordance with the regulations. Conclusion:The preparing technique was stable, simple and feasible. The quality standard could cotrol the quality of XG injection.3Safety evaluation of xanthan gum injectionObjective:To evaluate the safety of intra-articular injection of xanthan gum (XG) into knee joint of rabbit and the preliminary cytotoxicity of XG on rabbit chondrocytes. Methods:Rabbits were intra-articularly injected with0.1mL/kg of0.5%,1%,2%XG or0.9%NaCl in knee joints once a week for5weeks. The width of knee joint and the hematological and biochemical parameters were examined before and after treatment. The histopathological changes in liver, kidney and knee joint were observed. Rabbit chondrocytes were obtained through mechanical separation and enzymatic digestion, and cultured in DMEM/F12medium containing10%fetal bovine serum (FBS). The morphology of chondrocytes was observed by inverted phase microscopy, and chondrocytes were identified by histological examination of toluidine blue, safranin-O and type Ⅱ collagen immunofluorescence staining. After treatment with XG (10-2000μg/mL), the proliferation of cells was determined using MTT assay and the levels of MMP-1,-3,-13and TIMP-1proteins in media were determined using commercially available ELISA kits according to the manufacturer's instructions. Results:Compared to NS group, the width of knee joint and the hematological and biochemical parameters showed no significant change in XG groups with different doses, and no obvious histopathological changes were detected in liver, kidney and knee joint. The cells morphology and growth rhythm were in accordance with the chondrocytes' character. When stained with toluidine blue, safranin-O and anti-type Ⅱ collagen antibody, the results were positive. The color of the chondrocytes stained by toluidine blue was blue and by safranin-O was red. Type Ⅱ collagen immunofluorescence positive signals which presented with red fluorescence were localized in cytoplasm and cell membrane. Chondrocytes could proliferate actively in the presence of XG. XG did not significantly affect chondrocytes viability and the production of MMP-1,-3,-13and TIMP-1in chondrocytes in doses ranging from10to2000μg/mL. XG displayed no cytotoxicity to rabbit chondrocytes. Conclusion:When0.1mL/kg of0.5%-2%XG were intra-articularly injected into rabbit knee joint once a week for5weeks, there is no significant systemic and topical toxicity. XG (10-2000μg/mL) displayed no cytotoxicity to rabbit chondrocytes.4Pharmacodynamics and action mechanism evaluation of xanthan gum injection on experimental osteoarthritisObjective:To establish experimental OA models and evaluate in vivo and in vitro pharmacodynamics and action mechanism of XG on experimental OA. Methods: New Zealand white rabbits were randomly divided into four groups and0.1mL/kg of the mixed solutions of2%,5%or10%(w/v) papain with0.03mol/L L-cysteine were intra-articularly injected into the right knee on days1,3and5respectively. The rabbits of control group were injected intra-articularly with0.1mL/kg of NaCl0.9%(w/v). The rabbits were sacrificed at2,4,6weeks respectively after the initiation of papain injections and these OA models were evaluated through recording the width of knee joint, performing the morphological observation and histological evaluation of articular cartilage and synovium. Rats were randomly divided into four groups and60μL of5%,3.3%or1.7%(w/v) monosodium iodoacetate (MIA) were intra-articularly injected into the left knee respectively. The rats of control group were intra-articularly injected with60μL of NaCl0.9%(w/v). The pain related behaviors including weight bearing and hyperalgesia to punctate mechanical stimuli were tested on days3,7,14,21,28,35,42,49. Then the rats were sacrificed and these OA models were evaluated through performing the morphological observation and histological evaluation of articular cartilage.0.1mL/kg2%papain and0.03mol/L L-cysteine was intra-articularly injectioned into the right knees of all the rabbits three times on days1,3and5, respectively. Then these animals were randomly divided into three groups.0.1mL/kg of1%XG injection was given intra-articularly into the right knees in weeks2and4, while0.1mL/kg of0.9%NaCl was injected in weeks1,3and5.0.1mL/kg of0.9%NaCl and0.1mL/kg of1%SH were injected intra-articularly into the right knees once a week for5weeks respectively. All animals were injected intra-articularly with0.1mL/kg of0.9%NaCl into the left knees on days1,3and5during the induction (papain) phase and once a week in the treatment phase. The body weight and knee joint width were detected throughout the experimental period. After treatment, the contents of IL-1β, TNF-α and PG E2in synovial fluid were measured by enzyme-linked immunosorbent assay, the content of NO was measured by nitrate reductase assay. The gross morphological observation and histological evaluation of femoral condyle, tibial plateau and synovium were performed. The glycosaminoglycan (GAG) were measured using1,9-dimethylmethylene blue (DMB) colorimetric assay. The chondrocytes apoptosis was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. The MMPs and TIMP-1protein production were measured using immunohistochemistry and Western-blot. The MMPs and TIMP-1mRNA levels were measured using RT-PCR assay. IA injections of5%MIA60μL into the left knees of all rats were performed. Then these animals were randomly divided into four groups.60μL of1%XG injection was given intra-articularly into the left knees on days14and35, while60μL of0.9%NaCl was injected on days21,28and42(IA injection of XG once every two weeks for5weeks).60μL of1%SH injection was given intra-articularly into the left knees on days14and35, while60μL of0.9% NaCl was injected on days21,28and42(IA injection of SH once every two weeks for5weeks).60μL of1%SH injection (IA injection of SH once a week for5weeks) and0.9%NaCl (NS group) were given intra-articularly into the left knees on days14,21,28,35and42. The pain related behaviors including weight bearing and hyperalgesia to punctate mechanical stimuli were tested on days3,7,14,21,28,35,42,49. Then the rats were sacrificed and these OA models were evaluated through performing the morphological observation and histological evaluation of articular cartilage. The analgesic effect of XG was also evaluated through body torsion experiment in mice and the capillary permeability experiment was carried out to observe the inhibitory effect of XG on capillary permeability. The in vitro OA model was established by adding IL-1β(10ng/mL) into the cell culture medium. After treatment with XG (10-2000μg/mL), the proliferation of cells was determined using the MTT assay and the levels of MMP-1,-3,-13, and TIMP-1proteins in media were determined using commercially available ELISA kits according to the manufacturer's instructions. Results:The results of in vivo OA models evaluation indicated that the degenerative changes were demonstrated in rabbits and rats knee joints in all experimental groups, such as thinner articular cartilage, fibrillation and destroyed cartilage matrix, and inflammation, proliferation. All these changes were much worse with increased concentration and prolonged observation time. In all MIA-treated groups, there was a significant decrement in the paw withdrawal threshold (PWT) and weight bearing of the ipsilateral limb. The hyperalgesia in rats induced by MIA3mg was stable and lasted for the whole period of the experiment, however, the hyperalgesia induced by MIA1mg could gradually alleviate. The experimental results of XG on rabbit OA showed that, compared to NS group, IA injection of XG once every two weeks for5weeks significantly decreased the severity of swelling of the knee joint, reduced the damage of cartilage surfaces and loss of safranin-O staining intensity, inhibited cells hyperplasia and infiltration of mononuclear cells in the synovium, decreased the contents of IL-1β\TNF-α, PG E2and NO in synovial fluid, inhibited chondrocytes apoptosis and MMP-1,-3protein or mRNA levels in cartilage, enhanced GAG and TIMP-1production in cartilage. Furthermore, no significant differences between the XG-treated group (IA injection of XG once every two weeks for5weeks) and the SH-treated group (IA injection of SH once a week for5weeks) were observed. The experimental results of XG on rat OA showed that, compared to NS group, IA injection of XG once every two weeks for5weeks significantly reduced the damage of cartilage surfaces and loss of safranin-O staining intensity, significantly increased the weight distribution of the ipsilateral limb and the PWT in response to the von Frey monofilaments stimulus. Furthermore, these effects were much better than those in SH-treated group (IA injection of SH once every two weeks for5weeks), but no significant differences between the XG-treated group (IA injection of XG once every two weeks for5weeks) and the SH-treated group (IA injection of SH once a week for5weeks) were observed. The results of XG on body torsion experiment in mice and the capillary permeability experiment showed that intraperitoneal injection of XG could reduce the times of torsion and inhibit capillary permeability. XG could restore chondrocytes proliferation, suppressed protein expression of MMP-1,-3, and-13, increased TIMP-1production in a dose-dependent manner in doses ranging from100to1000μg/mL in IL-1β-induced rabbit chondrocytes. Conclution:Different severities of OA were established through giving injections into the knee including IA injections of2%,5%,10%papain or5%,3.3%,1.7%MIA. These models were characterized by short period and good reproducibility. XG showed significant analgesic effect and inhibited capillary permeability. XG also exhibited protective effect on rabbit chondrocytes in the presence of IL-1β.IA injection of XG could inhibit the continuous lesions on the cartilage in experimental OA, reduce inflammation, relieve pain and delay the progression of OA. Furthermore, compared with SH, fewer delivery times of XG could get the same treatment effect under current treatment regimen.