富血小板血浆与透明质酸钠修复兔膝骨关节炎

doi:10.3969/j.issn.2095-4344.2015.38.012

摘要/Abstract

摘要：

背景：研究表明，透明质酸钠可抑制膝骨关节炎对软骨的破坏，加速软骨细胞的再生，达到稳定和修复关节软骨的目的。

目的：观察富血小板血浆与透明质酸钠治疗兔膝骨关节炎的疗效。

方法：将40只新西兰大白兔随机均分为5组，联合组、玻璃酸钠组、富血小板血浆组与模型组制作右侧膝骨关节炎模型，造模后分别向膝关节腔内注射透明质酸钠联合自体富血小板血浆、玻璃酸钠、自体富血小板血浆与生理盐水进行治疗，1次/周，连续5周；对照组不做任何处理，为正常对照。治疗完成1周后，ELISA法检测血液中白细胞介素1、白细胞介素6、肿瘤坏死因子α水平，光镜下观察关节软骨的变化。

结果与结论：与对照组比较，其余4组白细胞介素1、白细胞介素6、肿瘤坏死因子α水平均升高(P < 0.01)；联合组、透明质酸钠组、富血小板血浆组白细胞介素1、白细胞介素6、肿瘤坏死因子α水平均低于模型组(P < 0.01或P < 0.05)，且以联合组下降最显著。模型组关节软骨破坏明显，联合组、透明质酸钠组、富血小板血浆组关节软骨破坏程度轻于模型组，且以联合组破坏程度最轻。表明透明质酸钠配合自体富血小板血浆关节内注射可降低膝骨关节炎的炎症反应，保护关节软骨，效果优于单一药物注射。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 软骨生物材料, 膝骨关节炎, 富血小板血浆, 透明质酸钠, 炎症因子, 关节软骨

Abstract:

BACKGROUND: Studies have shown that sodium hyaluronate inhibits cartilage damage in osteoarthritis and accelerates regeneration of cartilage cells, to stabilize and repair the articular cartilage.

OBJECTIVE: To investigate the therapeutic effect of sodium-rich plasma combined with platelet-rich plasma (PRP) on rabbit knee osteoarthritis.

METHODS: Forty New Zealand white rabbits were randomly divided into five groups, control group, combined group, sodium hyaluronate group, PRP group and model group, and then an osteoarthritis model of the right knee was made in each rabbit. After modeling, sodium hyaluronate+PRP, sodium hyaluronate, autologous PRP and normal saline were given via the knee joint cavity in the latter four groups, respectively, once a week for 5 weeks. The control group received no treatment, as normal controls. At 1 week after treatment, ELISA assay was used to detect serum interleukin-1, interleukin-6, tumor necrosis factor-α levels, and changes of the articular cartilage were observed under a light microscope.

RESULTS AND CONCLUSION: Compared with the control group, the levels of interleukin-1, interleukin-6 and tumor necrosis factor-α were all increased in the other four groups (P < 0.01). Compared with the model group, the levels of interleukin-1, interleukin-6 and tumor necrosis factor-α were lowered significantly in the combined, sodium hyaluronate and PRP groups (P < 0.01 or P < 0.05), and the most significant decline was in the combined group. Articular cartilage damage was severest in the model group and mildest in the combined group. Experimental findings indicate that intra-articular injection of sodium hyaluronate+PRP can reduce inflammation and protect the articular cartilage in knee osteoarthritis, which is better than a single drug injection.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Osteoarthritis, Knee, Platelet-Rich Plasma, Cartilage, Articular

中图分类号:

R318

吉恒东，霍小燕，张厚庆，王余山，石瑄，霍雷. 富血小板血浆与透明质酸钠修复兔膝骨关节炎[J]. 中国组织工程研究, 2015, 19(38): 6133-6139.

Ji Heng-dong, Huo Xiao-yan, Zhang Hou-qing, Wang Yu-shan, Shi Xuan, Huo Lei. Platelet-rich plasma with sodium hyaluronate in repair of rabbit knee osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(38): 6133-6139.

图/表（结果） 2

Quantitative analysis of experimental animals
Forty rabbits were all included in result analysis.
ELISA detection of inflammatory factor levels in serum
Compared with the control group, the levels of interleukin-1, interleukin-6 and tumor necrosis factor-α were all increased in the other four groups (P < 0.01). Compared with the model group, the levels of interleukin-1, interleukin-6 and tumor necrosis factor-α were lowered significantly in the combined, sodium hyaluronate and PRP groups (P < 0.01 or P < 0.05), and the most significant decline was in the combined group (Table 1).

Light microscope observation of the articular cartilage
In the combined group, the cartilage tissues were stained uniformly, chondrocytes were full and arranged in neat that gathered together, some cells appeared to have karyopyknosis (Figures 1A, B). In the control group, the cartilage matrix was pink and stained uniformly, and cells were full and arranged in neat (Figures 1C, D). In the PRP group, the cartilage matrix was evenly colored, sparse cell arrangement was found, cell clusters were visible and some cells were concentrated (Figures 1E, F). In the sodium hyaluronate group, the cartilage tissues became thinning, some chondrocytes exhibited hypertrophy, cell arrangement was more disordered, there were cell clusters, even damaged cells, and some cells were concentrated (Figures 1G, H). In the model group, the cartilage tissues became significantly thinner, presenting with extensive surface fragmentation and exfoliation, fissures reached deep into the subchondral bone, the matrix was colored slightly, there were cell clusters and local cell damage, cell arrangement was the most disordered and sparse, a plurality of chondrocytes gathered together, and karyopyknosis was found (Figures 1I, J).

参考文献

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[3] Neogi T. Clinical significance of bone changes in osteoarthritis. Ther Adv MusculoskeletDis. 2012;4(4):259-567.

[4] Kapidzi?-Basi? N, Kikanovi? S, Hoti Hadziefendi? A, et al. Changes in social relations as a consequence of rheumatoid arthritis and osteoarthritis. Reumatizam. 2013;60(1):42-46.

[5] Wang SD, Wang YZ, Zeng LY, et al. Research progress of the induction and detection methods of chondrocyte sapoptosis in vitro. Zhongguo Guzhi Shusong Zazhi. 2014;(1):100-104．

[6] Ou Y, Tan C, An H, et al. Selective COX-2 inhibitor ameliorates osteoarthritis by repressing apoptosis of chondrocyte. Med Sci Monit. 2012;18(6):BR247-252.

[7] Zamli Z, Adams MA, Tarlton JF, et al. Increased chondrocyte apoptosis is associated with progression of osteoarthritis in spontaneous Guinea pig models of the disease. Int J Mol Sci. 2013;14(9):17729-17743.

[8] Intekhab-Alam NY, White OB, Getting SJ, et al. Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis? Cell Death Dis. 2013;4:e717.

[9] Zhao Z, Ji H, Jing R, et al. Extracorporeal shock-wave therapy reduces progression of knee osteoarthritis in rabbits by reducing nitric oxide level and chondrocyte apoptosis. Arch Orthop Trauma Surg. 2012;132(11):1547-1553.

[10] Turajane T, Tanavaree A, Labpiboonpong V, et al. Outcomes of intra-articular injection of sodium hyaluronate for the treatment of osteoarthritis of the knee. J Med Assoc Thai. 2007;90(9):1845-1852.

[11] Kato Y, Mukudai Y, Okimura A, et al. Effects of hyaluronic acid on the release of cartilage matrix proteoglycan and fibronectin from the cell matrix layer of chondrocyte cultures: interactions between hyaluronic acid and chondroitin sulfate glycosaminoglycan. J Rheumatol Suppl. 1995;43:158-159.

[12] Homandberg GA, Hui F, Wen C, et al. Hyaluronic acid suppresses fibronectin fragment mediated cartilage chondrolysis: I. In vitro. Osteoarthritis Cartilage. 1997;5(5):309-319.

[13] Conaghan PG. A turbulent decade for NSAIDs: update on current concepts of classification, epidemiology, comparative efficacy, and toxicity. Rheumatol Int. 2012;32(6):1491-1502.

[14] Hochberg MC, Altman RD, April KT, et al. American College of Rheumatology 2012 recommendations for the use of nonpharmacologic and pharmacologic therapies in osteoarthritis of the hand, hip, and knee. Arthritis Care Res (Hoboken). 2012;64(4):465-474.

[15] Ragle RL, Sawitzke AD. Nutraceuticals in the management of osteoarthritis : a critical review. Drugs Aging. 2012;29(9):717-731.

[16] Kahan A, Uebelhart D, De Vathaire F, et al. Long-term effects of chondroitins 4 and 6 sulfate on knee osteoarthritis: the study on osteoarthritis progression prevention, a two-year, randomized, double-blind, placebo-controlled trial. Arthritis Rheum. 2009;60(2):524-533.

[17] Lee KS, Wilson JJ, Rabago DP, et al. Musculoskeletal applications of platelet-rich plasma: fad or future? AJR Am J Roentgenol. 2011;196(3):628-636.

[18] Su JM, Jin Y, Qu Q, et al. The expression of microRNA130a during chondrogenic differentiation of rat bone mesenchymal stem cells. Jichu Yixue yu Linchuang. 2010;30(5):520-523.

[19] Moser C. Response to: cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection. Arthritis Res Ther. 2010;12(6):410.

[20] Pan HL, Yao Y, Wang GW, et al. Variety and significance of IL-1 levels in blood and syn ovial fluid of osteoarthritis model animals. Haerbin Yike Daxue Xuebao. 2001;35(3):192-194．

[21] Jiao YT, Wang DZ, Hu J. Effect of IL-1 and dexamethasone on proliferation, metabolization and precollagen gene expression of human condylar cartilage cells.Zhonghua Chuangshang Zazhi. 2000;16(2):94-95.

[22] Westacott CI, Sharif M. Cytokines in osteoarthritis: mediators or markers of joint destruction? Semin Arthritis Rheum.1996;25(4):254-272.

[23] Pelletier JP, DiBattista JA, Roughley P, et al. Cytokines and inflammation in cartilage degradation. Rheum Dis Clin North Am. 1993;19(3):545-568.

[24] Kotake S, Sato K, Kim KJ, et al. Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. J Bone Miner Res. 1996;11(1):88-95.

[25] Reboul P, Pelletier JP, Tardif G, et al. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. J Clin Invest. 1996;97(9):2011-2019.

[26] Zheng SM, Dong TH, Liu JE. Determination of tumor necrosis factor and interleukin-6 in kneen osteoarthritis patients. Zhonghua Jiaoxing Waike Zazhi. 1998;5(6):544.

[27] Neidal J, Sihulze M, Sova L, et al. Practical significance of cytokine determination in joint fluid in patients with arthroses or rheumatoid arthritis. Z Orthop Ihre Grenzgeb. 1996;134(4):381-385.

[28] Turajane T, Tanavaree A, Labpiboonpong V, et al. Outcomes of intra-articular injection of sodium hyaluronate for the treatment of osteoarthritis of the knee. J Med Assoc Thai. 2007;90(9):1845-1852.

引言

Osteoarthritis, also known as degenerative joint disease, often occurs in the elderly. Progressive articular cartilage degeneration and degradation are the typical pathological changes of osteoarthritis, the cartilage degradation is accompanied by compensatory bone hyperplasia, andosteophytes appear in the joint margins^[1]. Pathological basis of osteoarthritis is anabolic and catabolic imbalance of the cartilage so that articular cartilage degradation is faster than articular cartilage reconstruction. Large weight-bearing joints are more prone to develop osteoarthritis, such as knee and hip, and clinical manifestations are slow development of arthralgia, tenderness, stiffness, arthroncus, limited mobility and joint deformities, among which arthralgia and arthroncus may be directly associated with synovitis in early or advanced osteoarthritis^[2]. In the past, osteoarthritis was considered as a non-inflammatory hyperplastic joint disease, but with further research on the pathogenesis of osteoarthritis, osteoarthritis now has been defined as the disease under the influence of multiple factors, including genetic factor, environment, metabolism, increasing age, obesity, fatigue, trauma, congenital joint abnormalities, joint deformities, and so on.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Recent studies have found that interleukin-1, interleukin-6 and tumor necrosis factor-α are important neurotransmitters during the development of osteoarthritis, which are the initial factors to regulating inflammation. Interleukin-1 can suppress II, IX collagen synthesis and promote the synthesis of I, III collagen, thereby making chondrocyte degeneration and resulting in inhibition of chondrocyte proliferation and proteoglycan synthesis; interleukin-1 also can promote

chondrocytes to secrete metalloproteinases, increase protease activity in the cartilage matrix, and meanwhile can inhibit the synthesis of cartilage proteoglycan to degrade the articular cartilage. Tumor necrosis factor-α can activate interleukin-6 gene, cooperate with interleukin-1 to induce interleukin-6 production, which plays a key role in the development of osteoarthritis. Platelet-rich plasma (PRP) is rich in a variety of growth factors, based on which, stem cells can be induced to repair corresponding tissues. Sodium hyaluronate is a classic drug for treatment of osteoarthritis. Therefore, this study aimed to observe the effect of sodium hyaluronate with autologous RPR via intra-articular injection on inflammatory cytokines and cartilage in knee osteoarthritis, and to explore its mechanism.

材料方法

Design

A randomized controlled animal experiment.

Time and setting

The experiment was completed at BenQ Medical Centre, Nanjing Medical University from March 2014 to March 2015.

Materials

Animals: Forty New Zealand white rabbits, aged 5-8 months, male and female, clean level, weighing (2.25±0.50) kg, were provided by the Experimental Animal Center of Soochow University School of Medicine (license No. 2014-0029). All rabbits were subjected to adaptive feeding in animal rooms for 1 week, and then randomly divided into five groups, namely PRP group, sodium hyaluronate group, combined group (PRP+sodium hyaluronate), control group and model group. Rabbits were individually allotted to cages, fodder and drinking water were exchanged combined with daily cleaning of urination and defecation, and temperature, lighting, noise, ventilation and other conditions were controlled within a predetermined range.

Main reagents and instruments: Optical microscope, optical microscope imaging system (Olympus), clean bench, sodium hyaluronate injection (Bausch & Lomb, China).

Methods

Preparation of knee osteoarthritis models

According to modified Hulth method, 3% pentobarbital sodium (30 mg/kg) was injected into the ear vein of rabbits via the indwelling needle in the PRP, sodium hyaluronate, combined and model groups. Under anesthesia, the rabbits were placed in the supine position and fixed on the operating table to preserve the skin of the right knee followed by disinfection and draping strictly abided by aseptic technique. In the center of the medial tibial condyle, a longitudinal incision, about 2.5 cm, was made followed by isolation of the medial collateral ligament attached to the joint capsule, and then the medial collateral ligament was cut parallel to the inferior margin of the branch artery of the knee arterial network. After that, the severed ligament was proximally opened to expose the medial meniscus, and the anterior horn of the medial meniscus was cut off and pulled outward. The anterior cruciate ligament, whereafter, was isolated between the knee condyles using forceps, and the ligament was cut off with a scalpel blade. Subsequently, the knee joint was pulled up and down to shear the posterior cruciate ligament using an ophthalmic scissor. Finally, the knee joint was evaginated to cut the rest of the medial meniscus using the ophthalmic scissor; and the lateral meniscus was unprocessed.

Experimental models were checked through a drawer test. Positive results indicate the anterior and posterior cruciate ligaments are cut off completely. In particular, during the model preparation, it should be careful not to damage the articular cartilage surface, hemostasis should be done strictly. Firstly, 0.1% povidone iodine was used to rinse the joint cavity followed by washing with normal saline, and then the incision was sutured step by step and covered with sterilized dressing. After surgery, intramuscular injection of penicillin sodium 20×10⁴ U was given every day, the dressing was changed every other day, continuously for 7 days to prevent infection. At 2 weeks after surgery, the suture was removed, and rabbits were subjected to passive motion twice daily, each time for 30 minutes, and the journey was about 150 meters. After 4 weeks of passive motion, the rabbit model of knee osteoarthritis was made successfully. The control group received no treatment, as normal controls.

Preparation of platelet-rich plasma

Blood sample, 5 mL, was respectively extracted from the ear vein of rabbits in the combined and PRP groups and injected into a tube. Citrate glucose liquid, 0.4 mL, was added. The tube was centrifuged at 2 000 r/min for 15 minutes, and the whole blood sample in the test tube was divided into three layers. A plastic pipette was used carefully to draw the middle layer, 0.5 mL, to prepare PRP by addition of 0.02 mL calcium chloride.

Drug administration

At 1 week after modeling, 1% sodium hyaluronate (0.2 mL), autologous PRP (0.5 mL) and 1% sodium hyaluronate (0.2 mL)+autologous PRP (0.5 mL) were respectively intraarticularly injected into the knee joint cavity in the sodium hyaluronate, PRP and combined groups. Normal saline was injected in the model group. The injection was repeated weekly, totally for five times.

Main outcome measures

At 1 week after injection, 10 mL venous blood sample was extracted from the ear vein of each group, sealed, stored at 4 ℃ for 8 hours, and centrifuged at 6 000 r/min for 15 minutes. The upper layer was collected into an EP tube and stored at -20 ℃ ultra-low temperature freezer. Interleukin-1, interleukin-6, and tumor necrosis factor-α levels were detected using ELISA. The rabbits were sacrificed by injecting 20-40 mL air into the ear vein. Then, the knee joint was opened immediately under sterile conditions to cut cartilage specimens of medial femoral condyle and tibial plateau, with a thickness of about 3 mm. The cartilage specimens were stored in 10% formaldehyde solution and chondrocyte morphology was detected using hematoxylin-eosin staining.

Statistical analysis

Data were statistically analyzed using SPSS 3.0, and a value of P < 0.05 was considered significant.

讨论

Relationship between osteoarthritis and articular cartilage

The pathogenesis of osteoarthritis is complex and has not been yet fully understood. Studies have shown that injury, heredity, age, biomechanics and obesity can all lead to osteoarthritis. The pathogenesis involves imbalance of extracellular matrix metabolism, autoimmune responses of cartilage components, chondrocyte apoptosis^[3-4]. Progressive articular cartilage degeneration and degradation are the pathological changes typical of osteoarthritis. Cartilage degradation is accompanied by compensatory bone hyperplasia and osteophytes can appear in the joint margins. Currently, there is no common understanding on the specific pathogenesis of osteoarthritis; however, it is an indisputable fact that multifactor-induced chondrocyte apoptosis leads to further pathological changes of cartilage and subchondral bone^[5]. Ou et al. ^[6]observed apoptosis of chondrocytes from rats with knee osteoarthritis using TUNEL method, and they confirmed that the apoptotic rate was dramatically higher in rats with knee osteoarthritis than normal rats. Zamli and colleagues^[7]found that with the development of osteoarthritis, apoptotic chondrocyte increased. Intekhab-Alam et al ^[8] found that nitric oxide inhibitor could prevent the articular cartilage against knee osteoarthritis effectively, and stop chondrocyte apoptosis to some extent. A study from Zhao et al ^[9] showed that drugs for inhibition of chondrocyte apoptosis might be effective in treating osteoarthritis. Therefore, the key to cure and prevent osteoarthritis is to reduce and delay chondrocyte apoptosis and to speed up the regeneration of chondrocytes. In vitro cell culture and animal experiments have shown that sodium hyaluronate protect the cartilage from the damages of free radicals, enzymes and neurotransmitters, accelerate the regeneration of chondrocytes, to achieve the purpose of stabilizing and repairing the articular cartilage^[10]. Sodium hyaluronate inhibits the release of plasminogen and proteoglycan from the cartilage matrix. Plasminogen and proteoglycan can increase the viscosity of the cartilage surface by sugar-protein and sugar-sugar interactions, and the presence of the viscosity plays a fatal role to protect the articular cartilage^[11]. Sodium hyaluronate can significantly slow down in vitro N-terminal fragment of L-plasmin-mediated degradation of articular cartilage, and meanwhile, it has the potential ability to repair the cartilage^[12]. But it is found that this ability has a direct relationship with its role to block the penetration of N-terminal fragment of L-plasmin, and by contrast, the direct effect of sodium hyaluronate on the articular cartilage is even more limited.

Currently, the first-line drugs for osteoarthritis also include acetaminophen and non-steroidal anti-inflammatory drugs, but these drugs often brings a series of adverse reactions to

digestive, cardiovascular and urinary systems, seriously affecting the therapeutic effect on osteoarthritis^[13-14]. The second-line drugs for osteoarthritis mainly include chondroitin, collagen and glucosamine. Glucosamine and chondroitin are two kinds of drugs used more and reported most, which can effectively protect the articular cartilage and relieve symptoms of patients, but the existing evidence does not support glucosamine and chondroitin osteoarthritis as conventional drugs for treatment of osteoarthritis^[15-16]. There is no evidence to show these drugs can effectively reverse chondrocyte apoptosis and cartilage degradation. PRP is extracted platelet concentrates after blood centrifugation, containing a high concentration of platelets^[17], and activated platelets can release a variety of growth factors, including ascular endothelial growth factor, transforming growth factor-β, platelet-derived growth factor and insulin-like growth factor, which can effectively promote cartilage repair. Bone marrow mesenchymal stem cells are the most important source of seed cells in bone tissue engineering, and under appropriate inducing conditions, these cells can differentiate into a variety of tissues belonging to the mesoderm, including cartilage and bone. It has been reported that transforming growth factor-β1 can significantly promote the chondrogenic differentiation of bone marrow mesenchymal stem cells^[18]. Therefore, PRP with a variety of growth factors has become the key mechanism to the treatment of osteoarthritis.

Osteoarthritis and inflammatory factors

We have recognized aseptic inflammation plays an important role in the development of osteoarthritis, but there is no effective method for early intervention. The main pathological changes of osteoarthritis are articular cartilage degradation as well as synovial swelling and hyperplasia. With the continuous development of the molecular pathology, a series of inflammatory cytokines that mediate the progress of osteoarthritis have been gradually revealed, creating conditions for an in-depth study on the relationship between inflammatory cytokines and osteoarthritis. A variety of inflammatory cytokines are involved in the pathogenesis of osteoarthritis, including interleukin-1, interleukin-6, tumor necrosis factor-α, cyclooxygenase and matrix metalloproteinases^[19]. As a result of synovial inflammation, these inflammatory cytokines rapidly increase in the synovial fluid to further deteriorate the living environment of chondrocytes, creating a vicious cycle. So how to effectively reduce the release of inflammatory factors in the course of osteoarthritis is the key to block this vicious cycle. Interleukin-1 exists only in individual cells on the surface of normal cartilage. In 1983, the existence of interleukin-1 in the synovial fluid was first proposed, and soon afterwards, a higher concentration of interleukin-1 could be detected in the synovial fluid of osteoarthritis patients. In osteoarthritis patients, the expression of interleukin-1 was strongly positive in the upper-layer and intermediate chondrocytes as well as in the cartilage matrix. These findings indicate that interleukin-1 and osteoarthritis are closely correlated. Interleukin-1 exerts a role in target cells by binding to its two receptors, interleukin-1RI and interleukin-1RII. In osteoarthritis patients, the concentrations of these two receptors in chondrocytes are twice what they are in normal chondrocytes. Therefore, the synovial cells and cartilage have a high sensitivity to interleukin-1. In some animal experiments, the levels of interleukin-1 in the synovial fluid and serum of animal models of osteoarthritis were significantly higher than those in normal controls, indicating that the elevated level of interleukin-1 is highly correlated to the severity of osteoarthritis^[20]. Interleukin-1 exerts a series of effects on cartilage extracellular matrix degradation, structural and functional changes of chondrocytes, synovitis lesions, chondrocyte apoptosis and bone metabolism^[21]. Interleukin-6, also known as B cell differentiation factor, cannot be detected in normal synovial tissue but exists in the synovial tissue of patients with osteoarthritis^[22]. Whether interleukin-6 is involved in osteoarthritis is still unclear. In vitro experiments have shown that interleukin-6 can stimulate synovial cells and normal chondrocytes to produce collagenase and prostaglandin, and also increase responses of fibroblasts in the synovium of osteoarthritis patients to interleukin-1-stimulated matrix metalloproteinase^[23]. It is also found interleukin-6 combined with its soluble receptors can effectively promote the generation of osteoclasts. Osteoclasts exist both in normal subjects and patients with osteoarthritis, and there is no significant difference^[24]. Anti-soluble interleukin-6 receptor antibody can block the ability of the above process, which also confirms that interleukin-6 is associated with the generation of osteoclasts. Interleukin-6 can be extracted from chondrocytes of normal controls and osteoarthritis patients, suggesting that interleukin-6 is involved in the proliferation and cloning of chondrocytes. Chondrocytes from osteoarthritis patients can cause the synthesis of interleukin-6, and interleukin-6 promotes the proliferation of chondrocytes. Consequently, it is speculated that a mechanism of autocrine chondrocyte stimulation may exist in osteoarthritis, which may be associated with abnormal regeneration of chondrocytes in osteoarthritis patients. Tumor necrosis factor-α mainly comes from the mononuclear phagocyte system, with a similar role to interleukin-1. It can inhibit the production of chondrogen and synthesis of proteoglycan, which is closely related with cartilage damage and synovitis in osteoarthritis patients^[25]. Tumor necrosis factor-α and interleukin-1 work together to promote the absorption of cartilage, which mediates cartilage damage in osteoarthritis patients^[26]. In animal experiments, immunohistochemical staining showed that tumor necrosis factor-α and its receptor displayed significantly positive reactions in osteoarthritic cartilage matrix and cells, and moreover, the intensity and scope of the reactions was closely related to the severity of osteoarthritis^[27]. Hyaluronic acid is a linear polysaccharide widely distributed in the extracellular matrix of connective tissue in the body. Studies have shown that in patients with osteoarthritis, the concentration and molecular weight of hyaluronic acid are both significantly decreased in the synovial fluid. Peyron and co-workers^[25] first used hyaluronic acid in the treatment of osteoarthritis, and achieved good results. Hereafter, hyaluronic acid becomes a routine drug for clinical treatment of osteoarthritis. Various studies have demonstrated that hyaluronic acid can slow down cartilage degeneration and promote articular cartilage repair, and moreover, low-molecular weight hyaluronic acid has better effects than high-molecular weight hyaluronic acid^[26]. High-molecular weight hyaluronic acid is good in the mitigation of bradykinin-induced pain, and it can also protect chondrocytes from free radical damage^[27-28]. In addition, hyaluronic acid can reduce the chemotaxis of immune cells and inhibit the inflammatory response.

In this experiment, the model group always had higher levels of interleukin-1, interleukin-6 and tumor necrosis factor-α, indicating the above-mentioned three kinds of inflammatory cytokines are highly correlated to the severity of osteoarthritis. After the intra-articular injection of hyaluronic acid, the levels of interleukin-1, interleukin-6 and tumor necrosis factor-α were reduced. It is probably because that high-concentration hyaluronic acid combined with the receptors of above-mentioned three kinds of inflammatory cytokines inhibits the migration and chemokines of cytokines, thereby playing a protective effect on the articular cartilage. Due to the limitations of production methods, it is inevitable that the PRP contains some white blood cells concentrated and can induce varying degrees of inflammation rebound in the early stage of intra-articular injection. This phenomenon is also reflected in a variety of clinical studies. Intra-articular injection of PRP can reduce levels of inflammatory factors in serum, but the mechanism is unclear. In the present experiment, firstly, blood collection was done to beat the inflammation peak caused by PRP injection; secondly, a variety of growth factors in PRP induced cartilage regeneration to block the cycle of “cartilage disintegration-chondrocyte apoptosis- inflammatory cytokine release”. In the combined group, there was a more obvious effect on the reduction of inflammatory cytokine release, which probably resulted from the additive effects of two different mechanism by which the release of inflammatory cytokines was decreased. But the following aspects are to be further studied: whether there is an interaction of PRP and hyaluronic acid co-existing in the knee cavity, how to determine the sequence of injection and interval time between them, and how to choose the concentration of PRP and molecular weight of hyaluronic acid.
Findings from this experiment show that intra-articular injection of sodium hyaluronate with autologous PRP can reduce the levels of interleukin-1, interleukin-6 and tumor necrosis factor-α in rabbit knee osteoarthritis, reduce inflammation, protect the articular cartilage, which has better effects than single drug injection. 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程