Studies On The Preparation Of Cross-linked Hyaluronic Acid, Its Properties And The Effects Treatment On Osteoarthritis

Osteoarthritis (OA) is one of the chronic degenerative joint diseases, which is especially harmful to the health of aged people. OA often occurs in knee joints. With the aging of social population, the incidence rate of OA is increasing. Nowadays, there is about 60 million to 70 million people suffering from this kind of disease in our country. According to the classification of American College of Rheumatology (ACR), OA is divided into primary OA and secondary OA. The primary OA is the focus in present investigation.Hyaluronic acid (HA) is a linear macromolecules dispersed glycosaminoglycan (GAG) consisting of repeating units of N-acetyl-β-D-glucosamine andβ-D-glucuronic acid, and is the important component of the matrix of synovial fluid and articular cartilage. In the seventies of the 20th century, Peyron first applicated intra-articular injection of HA to treatment OA and formed a viscoelastic replacement therapy. However, natural HA can be soluble easily in water, rapidly degraded in the body, and have a short lifetime, so repeated injections are needed. The carboxyl, hydroxyl and N-acetyl in the HA makes it available to form different types of cross-linked HA (CHA) with various cross-linking agents. The molecular weight and molecular size of CHA significantly increased. The CHA formed a macromolecular network structure, and it can swell in water but no longer be dissolved. The CHA not only maintaine a good biocompate and biodegrade, but also has better rheological properties, mechanical strength and longer retention in the body.This topic studied 1,4-butanediol diglycidyl ether (BDDE) with the HA to form a CHA gel, as well as its character of rheological, resistant abilities to free radicals and degradation by enzymes and other features. The effects and the mechanism of the preparation in rabbit knee osteoarthritis treatment were also studied. The main study contents and results are as follows.1 Preparation of CHA with BDDEStudy the preparation of different degree cross-linked HA with BDDE. The established technology have the advantage of more simple with higher purity products compared to that of other literature and patents.2 Investigation the properties of CHA gelThe rheological properties of CHA gel were studied. The shear viscosity of CHA and HA gel decreased with increasing frequency, the viscosity shows a pseudoplastic behavior. The water swelling polymer of CHA and HA belongs to the non-Newtonian fluid. The viscous modulus G" and elastic modulus G'of CHA were significantly higher than HA gel, and G'is much higher than G". The frequency dependence of G', G" is not obvious, and the angle 8 is relatively small. CHA has a high elasticity and strong gel characteristics.The abilities of resistant abilities to free radicals and degradation by enzymes of the CHA were also studied respectively. With the increase of cross-linked degree, the restistant abilities to free radical and hyaluronidase were also enhanced.3 Investigation of the effects of CHA on knee OA of rabbit modelsThe effects of the CHA in the treatment of OA were investigated in knee OA of rabbit models induced by papain. During the experiment, the CHA single injection was injected into the knee joint of the rabbits, HA and saline administered five times weekly as a control. After treatment for five weeks, the microscopic observation on articular cartilage and synovial membrane were made and the changes of articular cartilage and synovial membrane were studied. The results showed that CHA could significant reduce the symptoms of OA, the efficacy were significantly different with NS group.4 Study on the mechanism of CHA in the treatment of knee OA of rabbit modelsThe effects of CHA on the level of IL-1(3 and TNF-a in synovial fluid after treatment were also studied. The results showed that CHA injection could significantly inhibit the expression level of IL-1βand TNF-α. The efficacy of the CHA were better than that of HA injection.The content of chondroitin sulfate in the synovial fluid were also studied by high performance liquid chromatography (HPLC), CHA injection as well as HA injections can significantly reduce the content of chondroitin sulfate. It indicated that the CHA and HA may inhibite cartilage proteoglycan degradation and increase proteoglycan synthesis, play a role to maintain articular cartilage metabolism normally.The main achievements and conclusions of this paper as follows:(1) The technology of CHA with BDDE was studied, and the technology is suitable for industrial production. The process is simple and can obtain products with high purity.(2) The study also demonstrated the CHA has the strong resistant ability to hydroxyl radical and hyaluronidase.(3) The rheological study demonstrated that the CHA has higher viscous modulus and elastic modulus, and mainly dominated by elastic moduls.(4) The study also defined the appropriate buffer systems and pH condition to the stability of CHA. CHAⅠ, CHAⅡ, CHAⅢare stable in pH=5.0～9.0. And CHA II is stable in pH 8.0 borate buffer, pH 6.0, pH 7.0, pH 8.0 phosphate buffer, pH 7.0, pH 8.0 citrate buffer.(5) Animal model experiment showed that CHA could protect articular cartilage, relieve inflammation as well as other OA symptoms.(6) The mechanism of CHA from the viewpoint of molecular biology investigated. CHA injection can significant inhibit the expression level of IL-1βand TNF-α. After the analysis of the chondroitin sulfate in synovial fluid with HPLC, we found that CHA and HA can significant reduce the level of chondroitin sulfate in synovial fluid, which would play a role to delay the degradation of cartilage proteoglycan aggrecan and maintain the metabolism of articular cartilage normally.