Intra-articular Injection of Hyaluronic Acid and Alpha-2-Macroglobulin to Ameliorate Knee Posttraumatic Osteoarthritis: A Rat Model

Intra-articular Injection of Hyaluronic Acid and Alpha-2-Macroglobulin to Ameliorate Knee Posttraumatic Osteoarthritis: A Rat Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intra-articular Injection of Hyaluronic Acid and Alpha-2-Macroglobulin to Ameliorate Knee Posttraumatic Osteoarthritis: A Rat Model Zhenyu Wang, Chaoji Huangfu, Chongwei Chen, Mengbo Zhu, Yanjing Guo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1779669/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Posttraumatic osteoarthritis (PTOA) correlates with a dramatic increase in multiple inflammatory factors after joint injury. There is a broad spectrum proteinase inhibitor known as alpha-2-macroglobulin (A2M) that plays an important role in protecting against inflammatory injury. Endogenous A2M is abundant in serum but insufficient in synovial fluid due to its large molecular weight and has a limited effect on articular cartilage inflammation. We hypothesize that intra-articular injection of A2M has a better clinical effect than the commonly used hyaluronic acid (HA) injection therapy. Furthermore, A2M could be a safer method than alternative treatments because it is a native substance in the human body. Method The in vivo effects of A2M and HA on cartilage degeneration were evaluated in rats with surgery-induced anterior cruciate ligament transection (ACLT) OA. One hundred rats were randomly divided into four groups (N = 25/group): (a) sham surgery + saline (Sham + S), (b) ACLT + A2M, (c) ACLT + HA, or (d) ACLT + saline (ACLT + S). Intra-articular injections of A2M were given immediately and 3 days after surgery, and then 20 µl was injected each weekly in each joint. The animals were sacrificed 12 weeks after surgery. Indian ink staining, safranin O staining and immunohistochemical staining were performed to assess cartilage damage. The extent of OA progression was graded by the Osteoarthritis Research Society International Osteoarthritis Cartilage Histopathology Assessment System (OOCHAS) (OA score = Grade × Stage; range, 0 to 24). The concentrations of matrix metalloproteinase-13 (MMP-13) and sGAG in synovial fluid lavage were measured by ELISA and spectrophotometric quantitative determination. OA-related gene expression was quantified by qPCR. Results Indian ink staining showed that the articular cartilage surface in rats treated with A2M was relatively intact compared with that in the animals treated with ACLT plus saline or HA injection, but there was no significant difference between the ACLT + HA group and the ACLT + S group. Histological staining indicated that early intra-articular injection of A2M attenuated OA pathogenesis in the rat ACLT model compared with that in animals treated with saline and HA. However, intra-articular injection of HA did not significantly protect cartilage against posttraumatic OA compared with saline treatment. The ELISA results showed that A2M reduced the concentration of MMP-13 in synovial fluid compared with that in the HA treatment group and other groups. RT–qPCR indicated that intra-articular A2M inhibited catabolism and enhanced anabolic metabolism, while there was no significant difference in the expression of OA-related genes between the ACLT + HA group and the ACLT + S group. Discussion In rat model, intra-articular injection of A2M had obvious protective effects against cartilage degeneration compared with HA treatment. The inflammatory factor MMP-13 provides strong evidence for this inhibitory effect. Moreover, we found no significant alleviation of articular cartilage pathogenesis in the HA-treated group, which suggests that the efficacy of HA is questionable and possibly transient, although it is still extensively used in clinical practice. Osteoarthritis Alpha-2-macroglobulin Hyaluronic Acid MMP-13 Cartilage degeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Osteoarthritis (OA) is characterized by progressive destruction of the articular cartilage that lines the knee joints, subchondral bone surfaces, and synovium. [1] Initially, joint pain is the main symptom of osteoarthritis. As the disease continues to advance, patients undergo reductions in function and might even lose the ability to perform activities. [2, 3] A wide range of studies have confirmed that joint injury is a critical risk factor for the development of OA. [4] Intra-articular inflammation induced by joint injury frequently leads to chronic progressive cartilage degeneration, namely, posttraumatic OA. In the acute phase (0–1 days postinjury), relatively high levels of inflammatory mediators, such as IL-1β, IL-6, IL-8 and TNFα, can be found in synovial fluid and joint tissues. [5] These inflammatory mediators remain high for a long time after the trauma. The overexpression of SF IL-1, the cardinal inducer of intra-articular inflammation, lasts for three months after injury, and the overexpression of IL-6 and IL-8 lasts for six months. [6] Although the levels of protective cytokines also increase after joint injury, this protective effect occurs only in the early stage of injury. The SF IL-10 level drops to normal two weeks post-trauma, and the SF level of the IL-1 receptor antagonist (IL-1Ra) is below normal at three weeks. [7] Additionally, matrix metalloproteinases (MMPs) produced by chondrocytes and synovial cells result in the loss of normal cartilage tissue, exacerbating progressive cartilage degeneration. Intensive studies have demonstrated that MMP levels dramatically increase in injured joints, and this finding has been validated in various animal models. [8–12] In addition to the degeneration of cartilage, monocyte chemoattractant protein 1 (MCP-1) contributes to joint pain in PTOA. [13, 14] Therefore, the development of PTOA can be considered the result of persistent inflammation after trauma. Inflammatory mediators leading to the destruction of cartilage and a lack of inflammatory control ultimately result in PTOA. [15] Thus, it is thought that by removing destructive inflammatory mediators and enzymes, we can reduce the chronic inflammation and cartilage degeneration caused by intra-articular injury. A2M is a broad-spectrum proteinase inhibitor found in both serum and synovial fluid that can be used to attenuate cartilage degeneration. Proteinases that induce chronic inflammation can be captured by A2M, and the A2M-proteinase complex will be rapidly purged from the serum. This protective effect of A2M benefits from its molecular structure, which is capable of blocking almost all kinds of proteinases. [16] Unfortunately, the concentration of A2M in synovial fluid is much lower than that in the serum of normal individuals, as well as OA patients. Thus, endogenous A2M in the joint is not sufficient to diminish joint inflammation. [17, 18] One widely used clinical therapy is visco-supplementation, and the most common method the injection of HA into the joint to supplement the natural joint lubricant. [19] Hyaluronic acid (HA) received FDA approval in 1997 for the treatment of osteoarthritis (OA) in the United States. In 2009, the American Academy of Orthopedic Surgeons (AAOS) conducted a meta-analysis regarding HA treatment and found that the evidence of efficacy was inconclusive. [20, 21] As of early 2014, the AAOS did not find adequate evidence to support listing HA as indicated for the treatment of knee OA. [22] However, by this same thinking, it is possible that injecting A2M instead of HA as an intra-articular supplementation would be helpful in slowing cartilage degeneration in the knee during OA development, and A2M would make intra-articular injection therapy more effective and safe. The objective of this study was to compare the therapeutic effects of A2M and HA on OA in rats with knee arthritis. Materials And Methods This study was approved by the Institutional Review Board and the Institutional Animal Care and Use Committee of Shanxi Medical University. Animals One hundred 10-week-old male Wistar rats (180–230 g/each) were obtained from the Animal Center of Shanxi Medical University (Shanxi, China). The study was performed according to a protocol approved by the Shanxi Medical University Animal Research Committee (ARC). Seventy-five animals received ACL transection (ACLT) surgery on the right knee joint and were divided into 3 groups (n = 25 per group): (1) ACLT plus intra-articular saline injection (ACLT + Saline), (2) ACLT plus intra-articular hyaluronic acid injection (ACLT + HA), and (3) ACLT plus intra-articular A2M injection (ACLT + A2M). The remaining twenty-five animals received sham surgery on the right knee joint and intra-articular saline injection (Sham + Saline). A2M (Cat. 10602442001, Roche Life Science, USA) was dissolved in saline and administered to rats at a dose of 2 IU/kg (20 µL). [17] Intra-articular injections were performed immediately following and 3 days after ACLT and then weekly for 6 weeks. All animals were administered 20 µL of liquid in their right knee joint and were euthanized using a standard CO 2 chamber 12 weeks after surgery. In each group, 10 animals were used for India ink staining for gross evaluation of cartilage lesions; the tibiae of 15 animals were studied histologically, and the femurs were used for real-time polymerase chain reaction (PCR) analysis. Surgical procedures ACLT was surgically performed on the right hind knee joints of the animals as previously described. [23] Once they were anesthetized, a 1.5 cm midline incision was made over the anterior knee. The skin was mobilized to expose the patellar tendon. An incision through the joint capsule was made immediately lateral to the tendon. The ACL was cut with a scalpel. Manual laxity testing verified functional loss. The joint capsule, fascia, and skin were closed in layers. The right hind knee joints of rats in the sham group were sham-operated using the same approach but without ACLT. Postoperative analgesia using buprenorphine hydrochloride (0.05 mg/kg SQ) was maintained for at least 3 days. Animals were allowed to bear weight on the limbs as tolerated. Histology After the rats were killed with carbon dioxide, gross morphologic lesions on the femur condyle and tibia plateau (n = 10 per group) were visualized with India ink staining. [24] Tibiae in the rest of the knee joints (n = 15 per group) were fixed in 10% formalin for 72 hours, followed by decalcification in 10% EDTA solution, and the femurs were used for real-time qPCR analysis. Then, the tibiae were hemisected in the midsagittal plane, and each half was embedded in a single block of Paraplast X-tra (Sigma–Aldrich, St Louis, MO, USA). Serial 6-µm-thick sections were cut at intervals of 0 µm, 100 µm, and 200 µm and collected on positively charged glass slides (Superfrost Plus; CITOGLAS). Safranin-O/fast green staining was performed, and the severity of cartilage damage was assessed using the OARSI Assessment System (OA score = grade × stage; range, 0 to 24). [25] Three independent blinded observers scored each section, and the scores for all of the sections within each joint were averaged. Immunohistochemical staining Immunohistochemistry was carried out using a Histostain-SP kit (Cat. 959943B Invitrogen) to examine the distribution of type II collagen and MMP-13 in cartilage. The sections were digested with 5 mg/mL hyaluronidase in phosphate-buffered saline (PBS) (Sigma–Aldrich) for 20 minutes. Nonspecific protein binding was blocked by incubation with a serum blocking solution. Thereafter, the sections were incubated with specific antibodies against rat type II collagen (Santa Cruz Biotechnology) and MMP-13 (Santa Cruz Biotechnology) at 4°C overnight. The negative control sections were incubated with isotype-matched control serum (2 µg/mL) (R&D Systems, Minneapolis, MN, USA) in PBS. Subsequently, the sections were treated sequentially with biotinylated secondary antibodies and SP conjugate and then developed with DAB chromogen. Photomicrographs were taken with a Nikon E800 microscope (Nikon, Melville, NY, USA). Rat articular cartilage real-time qPCR The cartilage samples from the rat femur condyles were ground with a mortar and pestle in liquid nitrogen. Total RNA was isolated from rat cartilage tissues using an RNeasy isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Cartilage samples from 5 rats were dissected using a scalpel and pooled together; thus, there were 3 pooled samples per group. Total RNA was reverse transcribed into first-strand cDNA using the iScript™ cDNA synthesis kit (Bio–Rad, Hercules, CA, USA). RT–PCR was performed on a 96-well ABI Prism 7500 system (Applied Biosystems, Foster City, CA) using SuperReal PreMix reagent (Qiagen, Valencia, CA) according to the manufacturer’s protocols. The total volume (20 µL) of each PCR contained 10 µL SuperReal PreMix, 7 µL of ddH2O, 2 µL of cDNA, and 1 µL of the forward and reverse primers (10 µM ) (Table 1 ). The amplification conditions were as follows: 2 min of preincubation at 50°C; 10 min at 95°C for enzyme activation; and 40 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 30 s and extension at 72°C for 30 s. Gene expression was normalized to 18S mRNA levels. The comparative threshold cycle (Ct) method (2-ΔΔCt method) was used to calculate fold changes. [26] Table 1 Primers for real-time qPCR used in this study Gene Primer Sequence 5' to 3' Type II Collagen Forward AAG-GGA-CAC-CGA-GGT-TTC-ACT-GG Reverse GGG-CCT-GTT-TCT-CCT-GAG-CGT Aggrecan Forward CAG-TGC-GAT-GCA-GGC-TGG-CT Reverse CCT-CCG-GCA-CTC-GTT-GGC-TG Runx2 Forward CCG-CAC-GAC-AAC-CGC-ACC-AT Reverse CGC-TCC-GGC-CCA-CAA-ATC-TC MMP-3 Forward TTG-TCC-TTC-GAT-GCA-GTC-AG Reverse AGA-CGG-CCA-AAA-TGA-AGA-GA MMP-13 Forward GGA-CCT-TCT-GGT-CTT-CTG-GC Reverse GGA-TGC-TTA-GGG-TTG-GGG-TC Type X Collagen Forward CCA-GGT-GTC-CCA-GGA-TTC-CC Reverse CAA-GCG-GCA-TCC-CAG-AAA-GC 18S RNA Forward CGG-CTA-CCA-CAT-CCA-AGG-AA Reverse GCT-GGA-ATT-ACC-GCG-GCT Synovial Fluid Collection and Analysis Synovial fluid lavages were collected from the knees as soon as euthanasia was performed. [23] The knee was shaved and prepped with betadine. Fifty microliters of isotonic saline was injected into the right knees of each animal through the inferior patellar tendon. The joint capsule was visibly distended. The knee was then manually cycled through flexion and extension 10 times to distribute the fluid within the joint before collection via joint aspiration. The procedure was repeated twice. This technique typically yielded 80–100 µl of fluid from each animal. Then, the synovial fluid was centrifuged at 2,000 × g for 10 minutes to remove cells and debris and frozen at -80°C until analysis. Once thawed, MMP-13 and sulfated-glycosaminoglycans (sGAG) in the synovial fluid lavages were measured. MMP-13 was measured using ELISA according to the instructions of the manufacturer (Uscn Life Science). The colorimetric density in the developed plates was measured using a microplate reader (Model BF10000; Packard) at a wavelength of 450 nm. ELISA analysis of each sample was performed in duplicate. To evaluate matrix PG release, we used the metachromatic dye 1,9-dimethylmethylene blue (DMMB) (Sigma-Aldrich, Gillingham, UK) assay to quantify the amount of sulfated glycosaminoglycan (sGAG) in SF samples according to the manufacturer’s instructions. The concentrations of sGAG were determined by a spectrophotometric reader at 540 nm (Thermo Scientific Microplate Reader, UK). Statistical analysis Statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to compare the concentrations of MMP-13 and levels of mRNA in the different groups. All data are expressed as the mean ± SD, and p values less than 0.05 were considered statistically significant. Results Gross morphological cartilage lesions and fibrillation in the rat tibia plateau were visualized by India ink staining. We found that intra-articular HA did not attenuate posttraumatic OA compared to A2M treatment, as determined macroscopically. There was a significant decrease in the OA scores of A2M-treated rats compared with rats that underwent ACLT and HA/saline treatment. After treatment with A2M, decreased India ink staining and a smoother surface were observed compared to those in the HA-treated groups (Fig. 1A). India ink staining and the Meacham grading system indicated that the A2M-treated group had fewer cartilage lesions and fibrillation than the HA-treated or saline-treated group (p = 0.001) but more than the sham control group (p = 0.008) (Fig. 1B). There was no significant difference between the HA-treated group and the saline-treated group (p = 0.378). Then, we evaluated the effect of various treatments by histological staining. Intra-articular A2M was superior to HA in attenuating posttraumatic OA pathogenesis in a rat model of ACLT. First, we found a significant decrease in the OA score in A2M-treated rats compared with rats that underwent ACLT and saline/HA treatment. After treatment with A2M, stronger safranin O staining, a more intact surface and more cellularity but less chondrocyte cloning, and less fibrillation were observed than in the saline/HA-treated groups than in the other groups, but there was weaker staining than that in control rats that underwent sham operation (Fig. 2A). Cartilage from rats that were administered HA had almost similar staining and surfaces compared with cartilage from untreated rats (Saline group) (Fig. 2A). The OARSI histologic grading system scores in the A2M-treated group suggested mild degeneration (mean ± SD, 9.82 ± 4.0), while cartilage damage in the saline-treated group and HA-treated group was significantly worsened (18.2 ± 3.2 in the ACLT + saline group and 16.1 ± 3.8 in the ACLT + HA group; P < 0.01). It should be noted that there were no significant differences between the ACLT + saline group and the ACLT + HA group (P = 0.062). Cartilage from rats that underwent sham operation had the least amount of damage (0.43 ± 0.4; P < 0.01) (Fig. 2B). Next, immunohistochemistry was performed to determine the expression of type II collagen and MMP-13. Type II collagen levels were higher in cartilage in the ACLT + A2M and Sham + Saline groups than in the ACLT + HA group and ACLT + saline group (Fig. 3A). In contrast, MMP-13 was elevated in cartilage in the HA-treated group compared to the A2M-treated group, suggesting that the group with higher MMP-13 levels suffered critical cartilage injury (Fig. 3B). Additionally, cartilage damage was associated with changes in the levels of MMP-13 in joint lavage fluid. The MMP-13 level (mean ± SD )in rats that underwent ACLT and A2M treatment was 0.63 ± 0.05 µg/ml, which was higher than that in the sham operation group (0.266 ± 0.03 µg/ml; P = 1.85E-05) but much lower than that in rats that were administered HA (1.168 ± 0.18 µg/ml; P = 4.65E-05) or saline (1.34 ± 0.19 µg/ml, P = 1.27E-06). However, there was no significant difference between the ACLT + HA group and the ACLT + saline group (Fig. 4). In addition, we examined sulfated glycosaminoglycan (sGAG) levels in the synovial fluid in the different groups (Fig. 5). sGAG is an integral component of the cartilage matrix. However, the presence of sGAG in synovial fluid indicates the destruction of the articular cartilage. The sGAG level in the A2M-treated group was 331.68 ± 75.47 µg/ml, which was higher than that in the sham group (213.74 ± 44.14 µg/ml; P = 0.008). However, it is noteworthy that the level of sGAG in synovial fluid in the A2M group was significantly lower than that in the untreated group (567.70 ± 75.23 µg/ml; P = 0.001) (Fig. 5). This result demonstrated the protective effect of intra-articular A2M injection. We found that ACLT rats in the groups that were treated saline or HA exhibited higher levels of synovial fluid sGAG (576.56 ± 44.14 µg/ml) than those treated with A2M (P = 9.33E-05), which was also much higher than that of rats in the control group (P = 2.77E-07) (Fig. 5). Moreover, there was no significant difference between the ACLT + saline group and the ACLT + HA group (P = 0.825) (Fig. 5). Additionally, we performed qPCR to investigate the expression of related genes. The qPCR results indicated that the mRNA expression of type II collagen and aggrecan increased in the group that was treated with intra-articular A2M, while the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen were decreased (Fig. 6). In the HA treated group, the levels of aggrecan and type II collagen mRNA were obviously low; in contrast, the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen were increased (Fig. 6). The type II collagen mRNA level in rats that underwent ACLT and A2M treatment was significantly higher than that in rats that underwent ACLT plus HA or saline injection, and there was no significant difference between the latter two groups. In the groups that underwent sham operation and were treated with A2M, aggrecan mRNA levels were significantly higher than those in the groups that were injected with saline and HA. In contrast, the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen in rats that underwent ACLT and saline/HA treatment were much higher than those in the other two groups (Fig. 6). Discussion Post-traumatic osteoarthritis is a longterm disease initiated by joint injury and consequent intra-articular inflammation, and its progression is irreversible. [27, 28] Thus, delaying cartilaginous degeneration is the main purpose of nonsurgical treatment for osteoarthritis. Chronic inflammation is a critical risk factor for cartilage destruction during the process of osteoarthritis; therefore, it is believed that reducing intra-articular inflammation slows the degeneration of cartilage. [28] Accordingly, we thought that intra-articular injection of A2M, an autogeneic broad spectrum proteinase inhibitor, could ameliorate cartilage destruction induced by inflammation. We used four groups to compare the protective effects of A2M and HA, and the two experimental groups that received ACLT were treated separately with A2M and HA injection. The India ink staining and safranin-O/fast green staining results were consistent. All groups that underwent ACLT showed articular cartilage defects and subchondral bone outcroppings, which are typical pathological changes in osteoarthritis. [29] However, there was little normal cartilage remaining in the HA-treated group, indicating that cartilage damage in the HA group and untreated group was not significantly different; thus, the protective effect of HA is weak. This outcome echoed the results of clinical trials and meta-analyses, suggesting the uncertainty of the curative effect of HA. [30–32] In the A2M-treated group, cartilage damage occurred similarly but was obviously less severe than that in the HA group and untreated group. The A2M-treated group had a relatively intact surface and more chondrocyte cloning than the untreated group and HA-treated group. Although degenerative cartilage damage still existed after A2M injection treatment, the protective effect of A2M was still very evident. Notably, the OARSI scores in the A2M-treated group also showed a significant decrease, suggesting mild degeneration. The major pathological change in PTOA is chronic inflammation secondary to joint injury. Due to the increased production of proinflammatory cytokines and matrix metalloproteinases, the inflammatory damage of the articular tissue is dysregulated. Thus, a relatively strong way to analyze and compare the protective effects of HA and A2M is to study whether treatment can inhibit the levels of destructive cytokines and enzymes. Accordingly, we examined additional biological indicators of cartilage degeneration. Immunohistochemistry was performed to examine the distribution of type II collagen and MMP-13 in cartilage. Type II collagen is produced by chondrocytes, and the level of type II collagen in degenerated cartilage with reduced chondrocyte cloning will be reduced. [33] MMP-13 is an enzyme that catalyzes the destruction of cartilage. MMP-13 induces the degradation of proteoglycan and type II collagen; thus, it is used as an important indicator of cartilage damage. [34–36] Higher type II collagen levels and lower MMP-13 levels in the A2M injection group than in the other groups; thus, the protective effect of A2M injection was confirmed. In addition, we examined sGAG concentrations in synovial fluid in each group. Compared to those in the untreated group, synovial fluid sGAG levels in the A2M group were reduced. Quantitatively assessing sGAG levels in synovial fluid can be used to reflect the destruction of cartilage extracellular matrix. [37] It is likely that the protective effect of A2M led to reduced sGAG levels. The qPCR results clearly showed that A2M injection reduced the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen. However, the mRNA levels of these factors in rats that underwent ACLT and saline/HA treatment were much higher than those in the other two groups. These data suggested that A2M could protect cartilage in vivo by decreasing the gene expression of catabolic factors and hypertrophic markers, as well as by increasing anabolic gene expression. It is also apparent that HA failed to achieve the desired therapeutic effect. Immunohistochemical staining showed that cartilage degeneration in rat knees after HA injection was still severe, while the MMP-13 level was much higher than that in the sham operation group and A2M group. Quantitative analysis of MMP-13 levels in synovial fluid showed no significant disparity between the HA injection group and the untreated group. Similarly, there was no difference between sGAG levels in the HA group and the untreated group. The qPCR results also indicated that HA treatment failed to prevent cartilage destruction. In many studies, HA cannot exert a protective effect on injured joint tissue. A commonly accepted view is that intra-articular injection of HA can work via visco-supplementation. [19] However, as early as the 1980s, a study suggested that injecting HA did not increase the specific viscosity of synovial fluid in experimentally damaged joints. [38] One of the important reasons is that high molecular weight HA (HMW-HA) will be hydrolyzed to low molecular weight HA (LMW-HA) during the wound healing process, and LMW-HA cannot increase the specific viscosity of synovial fluid. [39] Furthermore, many studies suggest that LMW-HA is a proinflammatory factor that can induce metalloproteinase production. [40–42] In addition, there are proteoglycans called aggrecan on articular cartilage that bind to HA, providing elasticity to cartilage. [43] When PTOA occurs, aggrecan on joint cartilage is dissociated, leading to the loss of HA, cartilage erosion, and ultimately cartilage degeneration. [44] There is no evidence that supplementation with HA without aggrecan contributes to cartilage repair. Additionally, a previous study suggested that HA had no influence on the proteoglycan concentrations in PTOA. [45] Our study also concluded that there was no significant difference in the mRNA level of aggrecan in the HA-treated group and the untreated group. Although intra-articular injection of HA supplies HMW-HA, the change is temporary. Through HA injection therapy, synovial fluid is difficult to restore to normal standards. Any change in the molecular weight and concentration of HA in synovial fluid will exacerbate articular cartilage damage. For these reasons, HA treatment is no longer recommended by the American Academy of Orthopedic Surgeons (AAOS). [22] We decided to use A2M as an alternative therapy to replace HA therapy. A2M is not only able to protect articular cartilage against chronic inflammation but also has high biosafety and few side effects. There are many studies focused on resolving inflammation caused by cartilage destruction in osteoarthritis. In this context, common treatment methods are intra-articular calcium channel blocking anesthetics, corticosteroid injection (IACI), mesenchymal stem/stromal cells (MSCs), and platelet-rich plasma. [46, 47] However, some of these treatments cannot slow cartilage degeneration; some are not suitable for prolonged use in the clinic. A2M, which is a novel therapeutic drug for knee OA, has good prospects in solving these shortcomings. Compared with other drugs, A2M is an autologous proteinase inhibitor that has no autoimmune rejection. Additionally, A2M treatment contains only one active ingredient but inhibits various inflammatory factors and degenerative proteinases due to the broad-spectrum anti-inflammatory effect of A2M. Therefore, A2M injection eliminates the abovementioned limitations. As a result, safety is guaranteed, and risk is controllable. New technologies allowed us to concentrate A2M molecules from the autologous plasma of patients and then inject A2M-rich preparations into joints to compensate for the low level of A2M in synovial fluid. [48] Furthermore, the variant of A2M known as CYT-108 can be synthesized according to the molecular properties of A2M. Synthetic A2M is likely more economical and convenient than the parent compound, and the inhibitory effect of CYT-108 A2M will far exceeded that of wild-type A2M. [49] Conclusion This study examined the uncertainty of HA treatment in animal models. The protective effect of A2M on articular cartilage in osteoarthritis was tested. The results demonstrated that A2M could effectively postpone retrograde degeneration in joint cartilage. As expected, HA treatment did not have an obvious protective effect. These observations indicate that A2M is a promising anti-inflammatory treatment for osteoarthritis and can be used as a replacement for HA. Abbreviations PTOA Posttraumatic osteoarthritis A2M alpha-2-macroglobulin HA hyaluronic acid ACLT anterior cruciate ligament transection OOCHAS Osteoarthritis Cartilage Histopathology Assessment System MMP-13 matrix metalloproteinase-13 OA Osteoarthritis MCP-1 monocyte chemoattractant protein 1 AAOS the American Academy of Orthopedic Surgeons ARC Animal Research Committee sGAG sulfated-glycosaminoglycans OARSI Osteoarthritis Research Society International IACI intra-articular calcium channel blocking anesthetics, corticosteroid injection MSCs mesenchymal stem/stromal cells Declarations Ethics approval and consent to participate We confrmed that all experimental protocols were approved by the Animal Ethics Committee of Shanxi Medical University. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. Consent for publication Not applicable. Availability of data and materials The data that support our findings are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This project was supported by the National Natural Science Foundation of China (Grant No. 81572207, 81201435), Health Commission of Shanxi Province (Grant Nos. 2020XM54, 2020075), and Shanxi Scholarship Council of China (grant numbers 2017-063). contributions SW, YM and CH designed this study; CC, MZ and YG finished rat studies; XC and YL finished Histology and Immunohistochemical staining; ZW and MZ finished Synovial Fluid Collection and Analysis; XC and ZW finished real-time qPCR studies; ZW, CC and YG performed the statistical analysis; All authors contributed to the article and approved the submitted version. Corresponding author Correspondence to Shaowei Wang [email protected] and Yuyuan Ma [email protected] . Acknowledgements Not applicable. 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Attia E, Brown H, Henshaw R, George S, Hannafin JA: Patterns of gene expression in a rabbit partial anterior cruciate ligament transection model: the potential role of mechanical forces. Am J Sports Med 2010, 38(2):348-356. Aini H, Ochi H, Iwata M, Okawa A, Koga D, Okazaki M, Sano A, Asou Y: Procyanidin B3 prevents articular cartilage degeneration and heterotopic cartilage formation in a mouse surgical osteoarthritis model. PLoS One 2012, 7(5):e37728. Haslauer CM, Elsaid KA, Fleming BC, Proffen BL, Johnson VM, Murray MM: Loss of extracellular matrix from articular cartilage is mediated by the synovium and ligament after anterior cruciate ligament injury. Osteoarthritis Cartilage 2013, 21(12):1950-1957. Nagai T, Sato M, Kobayashi M, Yokoyama M, Tani Y, Mochida J: Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis. Arthritis Res Ther 2014, 16(5):427. Cuellar JM, Scuderi GJ, Cuellar VG, Golish SR, Yeomans DC: Diagnostic utility of cytokine biomarkers in the evaluation of acute knee pain. J Bone Joint Surg Am 2009, 91(10):2313-2320. Cuellar VG, Cuellar JM, Golish SR, Yeomans DC, Scuderi GJ: Cytokine profiling in acute anterior cruciate ligament injury. Arthroscopy 2010, 26(10):1296-1301. Lieberthal J, Sambamurthy N, Scanzello CR: Inflammation in joint injury and post-traumatic osteoarthritis. Osteoarthritis Cartilage 2015, 23(11):1825-1834. Sottrup-Jensen L: Alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 1989, 264(20):11539-11542. Wang S, Wei X, Zhou J, Zhang J, Li K, Chen Q, Terek R, Fleming BC, Goldring MB, Ehrlich MG et al: Identification of α2-Macroglobulin as a Master Inhibitor of Cartilage-Degrading Factors That Attenuates the Progression of Posttraumatic Osteoarthritis. Arthritis & Rheumatology 2014, 66(7):1843-1853. Demirag B, Sarisozen B, Durak K, Bilgen OF, Ozturk C: The effect of alpha-2 macroglobulin on the healing of ruptured anterior cruciate ligament in rabbits. Connect Tissue Res 2004, 45(1):23-27. Altman RD, Manjoo A, Fierlinger A, Niazi F, Nicholls M: The mechanism of action for hyaluronic acid treatment in the osteoarthritic knee: a systematic review. BMC Musculoskelet Disord 2015, 16:321. Lo GH, LaValley M, McAlindon T, Felson DT: Intra-articular hyaluronic acid in treatment of knee osteoarthritis: a meta-analysis. JAMA 2003, 290(23):3115-3121. Arrich J, Piribauer F, Mad P, Schmid D, Klaushofer K, Mullner M: Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee: systematic review and meta-analysis. CMAJ 2005, 172(8):1039-1043. Cook R, Davidson P, White A, Centre ND: Partial knee replacement could be first choice for some patients with osteoarthritis. BMJ 2019, 367:l5994. Wei L, Fleming BC, Sun X, Teeple E, Wu W, Jay GD, Elsaid KA, Luo J, Machan JT, Chen Q: Comparison of differential biomarkers of osteoarthritis with and without posttraumatic injury in the Hartley guinea pig model. J Orthop Res 2010, 28(7):900-906. Meachim G: Light microscopy of Indian ink preparations of fibrillated cartilage. Ann Rheum Dis 1972, 31(6):457-464. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, Salter D, van den Berg WB: Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 2006, 14(1):13-29. Wang S, Wei X, Zhou J, Zhang J, Li K, Chen Q, Terek R, Fleming BC, Goldring MB, Ehrlich MG et al: Identification of alpha2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis. Arthritis Rheumatol 2014, 66(7):1843-1853. Yamada EF, Bobinski F, Martins DF, Palandi J, Folmer V, da Silva MD: Photobiomodulation therapy in knee osteoarthritis reduces oxidative stress and inflammatory cytokines in rats. J Biophotonics 2020, 13(1):e201900204. Irie K, Uchiyama E, Iwaso H: Intraarticular inflammatory cytokines in acute anterior cruciate ligament injured knee. Knee 2003, 10(1):93-96. Takeda H, Nakagawa T, Nakamura K, Engebretsen L: Prevention and management of knee osteoarthritis and knee cartilage injury in sports. Br J Sports Med 2011, 45(4):304-309. Bannuru RR, McAlindon TE, Sullivan MC, Wong JB, Kent DM, Schmid CH: Effectiveness and Implications of Alternative Placebo Treatments: A Systematic Review and Network Meta-analysis of Osteoarthritis Trials. Ann Intern Med 2015, 163(5):365-372. Bannuru RR, Schmid CH, Kent DM, Vaysbrot EE, Wong JB, McAlindon TE: Comparative effectiveness of pharmacologic interventions for knee osteoarthritis: a systematic review and network meta-analysis. Ann Intern Med 2015, 162(1):46-54. Niemela TM, Tulamo RM, Hielm-Bjorkman AK: A randomised, double-blinded, placebo-controlled clinical study on intra-articular hyaluronan treatment in equine lameness originating from the metacarpophalangeal joint. BMC Vet Res 2016, 12:60. Poole AR, Kobayashi M, Yasuda T, Laverty S, Mwale F, Kojima T, Sakai T, Wahl C, El-Maadawy S, Webb G et al: Type II collagen degradation and its regulation in articular cartilage in osteoarthritis. Ann Rheum Dis 2002, 61 Suppl 2:ii78-81. Gu R, Shi Y, Huang W, Lao C, Zou Z, Pan S, Huang Z: Theobromine mitigates IL-1beta-induced oxidative stress, inflammatory response, and degradation of type II collagen in human chondrocytes. Int Immunopharmacol 2020, 82:106226. Alamgeer, Hasan UH, Uttra AM, Qasim S, Ikram J, Saleem M, Niazi ZR: Phytochemicals targeting matrix metalloproteinases regulating tissue degradation in inflammation and rheumatoid arthritis. Phytomedicine 2020, 66:153134. Burrage PS, Mix KS, Brinckerhoff CE: Matrix metalloproteinases: role in arthritis. Front Biosci 2006, 11:529-543. Jin Y, Chen X, Gao ZY, Liu K, Hou Y, Zheng J: The role of miR-320a and IL-1beta in human chondrocyte degradation. Bone Joint Res 2017, 6(4):196-203. Hilbert BJ, Rowley G, Antonas KN, McGill CA, Reynoldson JA, Hawkins CD: Changes in the synovia after the intra-articular injection of sodium hyaluronate into normal horse joints and after arthrotomy and experimental cartilage damage. Aust Vet J 1985, 62(6):182-184. Aviad AD, Houpt JB: The molecular weight of therapeutic hyaluronan (sodium hyaluronate): how significant is it? J Rheumatol 1994, 21(2):297-301. Chen WY, Abatangelo G: Functions of hyaluronan in wound repair. Wound Repair Regen 1999, 7(2):79-89. Stern R, Asari AA, Sugahara KN: Hyaluronan fragments: an information-rich system. Eur J Cell Biol 2006, 85(8):699-715. Jiang D, Liang J, Noble PW: Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol 2007, 23:435-461. Tortorella MD, Malfait AM, Deccico C, Arner E: The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation. Osteoarthritis Cartilage 2001, 9(6):539-552. Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, Rosenfeld SA, Copeland RA, Decicco CP, Wynn R et al: Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science 1999, 284(5420):1664-1666. Brandt KD, Smith GN, Jr., Simon LS: Intraarticular injection of hyaluronan as treatment for knee osteoarthritis: what is the evidence? Arthritis Rheum 2000, 43(6):1192-1203. Xie X, Zhang C, Tuan RS: Biology of platelet-rich plasma and its clinical application in cartilage repair. Arthritis Res Ther 2014, 16(1):204. Richards MM, Maxwell JS, Weng L, Angelos MG, Golzarian J: Intra-articular treatment of knee osteoarthritis: from anti-inflammatories to products of regenerative medicine. Phys Sportsmed 2016, 44(2):101-108. Cuellar JM, Cuellar VG, Scuderi GJ: alpha2-Macroglobulin: Autologous Protease Inhibition Technology. Phys Med Rehabil Clin N Am 2016, 27(4):909-918. Zhang Y, Wei X, Browning S, Scuderi G, Hanna LS, Wei L: Targeted designed variants of alpha-2-macroglobulin (A2M) attenuate cartilage degeneration in a rat model of osteoarthritis induced by anterior cruciate ligament transection. Arthritis Res Ther 2017, 19(1):175. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1779669","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":118325323,"identity":"c2465f3c-6567-42e8-8249-80b3484b0f36","order_by":0,"name":"Zhenyu Wang","email":"","orcid":"","institution":"Shanxi Key Lab of Bone and Soft Tissue Injury Repair，Department of Orthopedics, The Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Wang","suffix":""},{"id":118325326,"identity":"d30045df-c1d0-4d28-b070-cb4bf0e06f10","order_by":1,"name":"Chaoji Huangfu","email":"","orcid":"","institution":"NMPA Key Laboratory for Quality Control of Blood Products, Institute of Health Service and Transfusion Medicine, Academy of Military Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chaoji","middleName":"","lastName":"Huangfu","suffix":""},{"id":118325328,"identity":"793913c6-98a7-4433-a187-f91a50961fad","order_by":2,"name":"Chongwei Chen","email":"","orcid":"","institution":"Shanxi Key Lab of Bone and Soft Tissue Injury Repair，Department of Orthopedics, The Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chongwei","middleName":"","lastName":"Chen","suffix":""},{"id":118325331,"identity":"52276659-8a22-4cca-a5d7-056f78c1b646","order_by":3,"name":"Mengbo Zhu","email":"","orcid":"","institution":"Shanxi Key Lab of Bone and Soft Tissue Injury Repair，Department of Orthopedics, The Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengbo","middleName":"","lastName":"Zhu","suffix":""},{"id":118325332,"identity":"9db097b1-b70a-4da4-8107-232f0ea44849","order_by":4,"name":"Yanjing Guo","email":"","orcid":"","institution":"Shanxi Key Lab of Bone and Soft Tissue Injury Repair，Department of Orthopedics, The Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanjing","middleName":"","lastName":"Guo","suffix":""},{"id":118325334,"identity":"f383a3c9-e31f-4bf3-b35f-67e07d1e106d","order_by":5,"name":"Xinping Chen","email":"","orcid":"","institution":"Shanxi Key Lab of Bone and Soft Tissue Injury Repair，Department of Orthopedics, The Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinping","middleName":"","lastName":"Chen","suffix":""},{"id":118325335,"identity":"c8e31f66-e907-4f4a-bb0c-6549e3ab9857","order_by":6,"name":"Yue Li","email":"","orcid":"","institution":"Department of Biochemistry, Basic Medical College, Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Li","suffix":""},{"id":118325336,"identity":"51fc0cf8-6be7-4c53-b231-f86fbff8fae1","order_by":7,"name":"Yuyuan Ma","email":"","orcid":"","institution":"NMPA Key Laboratory for Quality Control of Blood Products, Institute of Health Service and Transfusion Medicine, Academy of Military Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuyuan","middleName":"","lastName":"Ma","suffix":""},{"id":118325337,"identity":"c5d28ce9-824a-4a07-9b76-398a51d7a8d9","order_by":8,"name":"Shaowei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYLACxgYw2fggwUBCjo29+QDRWpoNPhTYGPPxHEsgVgsDm+SMD2mJ8yRyFPCqNjh+9vDLnzvs5AyONzdI8xgcTm9jyGFg+FGxDbeWM3lp1rxnko0NzhxsMAZqyW1jOHuAsefMbZxazA7kmBkztjEnbruR2JAM1sLYl8DM2IZHy/k3ZoY/2+oTt91/2HAY5DA2Zh4D/Fpu5Bg/4G07DLSFsbFxhkFaAhsbAS32N96YMfO2HTe2P5PYzPDBwMawjYct4SA+v0j25xh//NlWLSfZfvz5j4Q/EvLy8x8ffPCjArcWIGCTwBA6gE89EDB/IKBgFIyCUTAKRjoAANszXzLh2uDXAAAAAElFTkSuQmCC","orcid":"","institution":"Shanxi Key Lab of Bone and Soft Tissue Injury Repair，Department of Orthopedics, The Second Hospital of Shanxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shaowei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-06-21 08:29:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1779669/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1779669/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23763689,"identity":"eb2f3b8d-4ddc-4003-97af-c0da0153f52e","added_by":"auto","created_at":"2022-07-12 15:08:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":690514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntra-articular HA could not attenuate posttraumatic OA macroscopically\u003c/strong\u003e. (A) Decreased India ink staining and a smoother surface were detected in the articular cartilage of A2M-treated animals as compared to the ACLT and saline/HA treatment groups. (B) The Indian ink Meachim Grade score indicated that cartilage damage was much severe in rats that underwent ACLT and saline/HA treatment, while cartilage in rats that underwent sham operation had the least damage. Cartilage damage was also reduced in rats that received A2M as compared to rats that received the HA treatment. Values are the mean±SD.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/fea8ac79f76e6734a7b5b2f9.png"},{"id":23763692,"identity":"25770b07-6400-41a3-9781-3c17ebdfe7ff","added_by":"auto","created_at":"2022-07-12 15:08:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":919219,"visible":true,"origin":"","legend":"\u003cp\u003ePosttraumatic OA rats modeled by ACLT suggested that the A2M has more advantage than HA in attenuating the injury. (A) A smoother surface with stronger safranin O staining was detected in the articular cartilage of A2M-treated animals as compared to HA-treated animals and untreated controls (the bottom panels are higher-magnification views of the boxed areas in the top panels). (B) The Osteoarthritis Research Society International Osteoarthritis Cartilage Histopathology Assessment System (OOCHAS) score indicated that cartilage damage was mild in rats that underwent A2M treatment, while cartilage in rats that underwent sham operation had the least damage. Cartilage damage was aggravated in rats that received the ACLT operation and HA/Saline treatment. And there was no statistical significance between the ACLT+saline group and the ACLT+HA group. Values are the mean±SD.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/3a1d403909ef4ea8d9e0b28e.png"},{"id":23763691,"identity":"1f648d90-aaf1-495f-98ad-f409202e6a13","added_by":"auto","created_at":"2022-07-12 15:08:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1201699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMMP\u003c/strong\u003e-\u003cstrong\u003e13 expression was reduced in A2M treated rats, but type II collagen was preserved\u003c/strong\u003e. (A) Type II collagen expression in articular cartilage was higher in the A2M-treated and the sham-operated rats than in rats that underwent ACLT and HA treatment. In contrast, MMP-13 staining was elevated in rats that underwent ACLT and HA treatment, but was lower in the A2M-treated and sham-operated rats, which is consistent with reduced OA damage in these rats. In A and B, the bottom panels are higher-magnification views of the boxed areas in the top panels (20x objective lens).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/c3cb6763da6418cf06bd08a2.png"},{"id":23763690,"identity":"4231838b-fee0-47ab-a7ab-95c6b008f6d3","added_by":"auto","created_at":"2022-07-12 15:08:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101817,"visible":true,"origin":"","legend":"\u003cp\u003eIn HA-treated rats, the concentration of MMP-13 in SF was much higher than that in rats that underwent ACLT and A2M treatment and was similar to that in ACLT and saline treated rats. Values are the mean±SD.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/476851708735be7d21d38cda.png"},{"id":23764213,"identity":"9216d4b3-9532-488a-821a-81201b46250e","added_by":"auto","created_at":"2022-07-12 15:13:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":274678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACLT rats treated by HA had shown more articular cartilage destruction than those treated by A2M.\u003c/strong\u003e The sGAG concentrations were tested separately in four groups. In HA-treated rats, the concentration of sGAG in SF was much higher than that in rats that underwent ACLT and A2M treatment. No statistical difference was found between the ACLT+saline group and the ACLT+ HA group. Values are the mean±SD.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/fd3e8908029ba52f6f35dbd1.png"},{"id":23763693,"identity":"7a9f6983-7126-4e84-a78b-3827a8712efd","added_by":"auto","created_at":"2022-07-12 15:08:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":192656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental intra-articular A2M has the advantage over HA in inhibiting catabolism and enhancing anabolic metabolism in a rat model of ACLT.\u003c/strong\u003e Levels of mRNA for type II collagen and aggrecan were increased in rats that were administered A2M as compared to rats that underwent ACLT and saline/HA treatment, suggesting that A2M has a positive impact on anabolic metabolism. In contrast, Runx2, MMP-3, MMP-13, and type X collagen showed the opposite pattern. These genes were expressed at a lower level in rats that were administered A2M as compared to rats that underwent ACLT and saline/HA treatment. Values are the mean±SEM. #: P\u0026lt;0.01 versus the ACLT and saline treatment group; \u0026amp;: P\u0026lt;0.01 versus the ACLT and HA treatment group.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/0fd8f2ac6aecd91e8f09a0dd.png"},{"id":28319472,"identity":"5b39cf25-9225-4a65-a0c5-92d281f42eeb","added_by":"auto","created_at":"2022-10-27 10:44:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4551232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1779669/v1/c6782f3a-dd57-40fa-90a7-2acb0f2fb3b2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intra-articular Injection of Hyaluronic Acid and Alpha-2-Macroglobulin to Ameliorate Knee Posttraumatic Osteoarthritis: A Rat Model","fulltext":[{"header":"Background","content":"\u003cp\u003eOsteoarthritis (OA) is characterized by progressive destruction of the articular cartilage that lines the knee joints, subchondral bone surfaces, and synovium. [1] Initially, joint pain is the main symptom of osteoarthritis. As the disease continues to advance, patients undergo reductions in function and might even lose the ability to perform activities. [2, 3] A wide range of studies have confirmed that joint injury is a critical risk factor for the development of OA. [4] Intra-articular inflammation induced by joint injury frequently leads to chronic progressive cartilage degeneration, namely, posttraumatic OA. In the acute phase (0\u0026ndash;1 days postinjury), relatively high levels of inflammatory mediators, such as IL-1β, IL-6, IL-8 and TNFα, can be found in synovial fluid and joint tissues. [5] These inflammatory mediators remain high for a long time after the trauma. The overexpression of SF IL-1, the cardinal inducer of intra-articular inflammation, lasts for three months after injury, and the overexpression of IL-6 and IL-8 lasts for six months. [6] Although the levels of protective cytokines also increase after joint injury, this protective effect occurs only in the early stage of injury. The SF IL-10 level drops to normal two weeks post-trauma, and the SF level of the IL-1 receptor antagonist (IL-1Ra) is below normal at three weeks. [7]\u003c/p\u003e \u003cp\u003eAdditionally, matrix metalloproteinases (MMPs) produced by chondrocytes and synovial cells result in the loss of normal cartilage tissue, exacerbating progressive cartilage degeneration. Intensive studies have demonstrated that MMP levels dramatically increase in injured joints, and this finding has been validated in various animal models. [8\u0026ndash;12] In addition to the degeneration of cartilage, monocyte chemoattractant protein 1 (MCP-1) contributes to joint pain in PTOA. [13, 14] Therefore, the development of PTOA can be considered the result of persistent inflammation after trauma. Inflammatory mediators leading to the destruction of cartilage and a lack of inflammatory control ultimately result in PTOA. [15]\u003c/p\u003e \u003cp\u003eThus, it is thought that by removing destructive inflammatory mediators and enzymes, we can reduce the chronic inflammation and cartilage degeneration caused by intra-articular injury. A2M is a broad-spectrum proteinase inhibitor found in both serum and synovial fluid that can be used to attenuate cartilage degeneration. Proteinases that induce chronic inflammation can be captured by A2M, and the A2M-proteinase complex will be rapidly purged from the serum. This protective effect of A2M benefits from its molecular structure, which is capable of blocking almost all kinds of proteinases. [16] Unfortunately, the concentration of A2M in synovial fluid is much lower than that in the serum of normal individuals, as well as OA patients. Thus, endogenous A2M in the joint is not sufficient to diminish joint inflammation. [17, 18]\u003c/p\u003e \u003cp\u003eOne widely used clinical therapy is visco-supplementation, and the most common method the injection of HA into the joint to supplement the natural joint lubricant. [19] Hyaluronic acid (HA) received FDA approval in 1997 for the treatment of osteoarthritis (OA) in the United States. In 2009, the American Academy of Orthopedic Surgeons (AAOS) conducted a meta-analysis regarding HA treatment and found that the evidence of efficacy was inconclusive. [20, 21] As of early 2014, the AAOS did not find adequate evidence to support listing HA as indicated for the treatment of knee OA. [22] However, by this same thinking, it is possible that injecting A2M instead of HA as an intra-articular supplementation would be helpful in slowing cartilage degeneration in the knee during OA development, and A2M would make intra-articular injection therapy more effective and safe. The objective of this study was to compare the therapeutic effects of A2M and HA on OA in rats with knee arthritis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e This study was approved by the Institutional Review Board and the Institutional Animal Care and Use Committee of Shanxi Medical University.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eOne hundred 10-week-old male Wistar rats (180\u0026ndash;230 g/each) were obtained from the Animal Center of Shanxi Medical University (Shanxi, China). The study was performed according to a protocol approved by the Shanxi Medical University Animal Research Committee (ARC). Seventy-five animals received ACL transection (ACLT) surgery on the right knee joint and were divided into 3 groups (n\u0026thinsp;=\u0026thinsp;25 per group): (1) ACLT plus intra-articular saline injection (ACLT\u0026thinsp;+\u0026thinsp;Saline), (2) ACLT plus intra-articular hyaluronic acid injection (ACLT\u0026thinsp;+\u0026thinsp;HA), and (3) ACLT plus intra-articular A2M injection (ACLT\u0026thinsp;+\u0026thinsp;A2M). The remaining twenty-five animals received sham surgery on the right knee joint and intra-articular saline injection (Sham\u0026thinsp;+\u0026thinsp;Saline). A2M (Cat. 10602442001, Roche Life Science, USA) was dissolved in saline and administered to rats at a dose of 2 IU/kg (20 \u0026micro;L). [17] Intra-articular injections were performed immediately following and 3 days after ACLT and then weekly for 6 weeks. All animals were administered 20 \u0026micro;L of liquid in their right knee joint and were euthanized using a standard CO\u003csub\u003e2\u003c/sub\u003e chamber 12 weeks after surgery. In each group, 10 animals were used for India ink staining for gross evaluation of cartilage lesions; the tibiae of 15 animals were studied histologically, and the femurs were used for real-time polymerase chain reaction (PCR) analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSurgical procedures\u003c/h2\u003e \u003cp\u003eACLT was surgically performed on the right hind knee joints of the animals as previously described. [23] Once they were anesthetized, a 1.5 cm midline incision was made over the anterior knee. The skin was mobilized to expose the patellar tendon. An incision through the joint capsule was made immediately lateral to the tendon. The ACL was cut with a scalpel. Manual laxity testing verified functional loss. The joint capsule, fascia, and skin were closed in layers. The right hind knee joints of rats in the sham group were sham-operated using the same approach but without ACLT. Postoperative analgesia using buprenorphine hydrochloride (0.05 mg/kg SQ) was maintained for at least 3 days. Animals were allowed to bear weight on the limbs as tolerated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eAfter the rats were killed with carbon dioxide, gross morphologic lesions on the femur condyle and tibia plateau (n\u0026thinsp;=\u0026thinsp;10 per group) were visualized with India ink staining. [24] Tibiae in the rest of the knee joints (n\u0026thinsp;=\u0026thinsp;15 per group) were fixed in 10% formalin for 72 hours, followed by decalcification in 10% EDTA solution, and the femurs were used for real-time qPCR analysis. Then, the tibiae were hemisected in the midsagittal plane, and each half was embedded in a single block of Paraplast X-tra (Sigma\u0026ndash;Aldrich, St Louis, MO, USA). Serial 6-\u0026micro;m-thick sections were cut at intervals of 0 \u0026micro;m, 100 \u0026micro;m, and 200 \u0026micro;m and collected on positively charged glass slides (Superfrost Plus; CITOGLAS). Safranin-O/fast green staining was performed, and the severity of cartilage damage was assessed using the OARSI Assessment System (OA score\u0026thinsp;=\u0026thinsp;grade \u0026times; stage; range, 0 to 24). [25] Three independent blinded observers scored each section, and the scores for all of the sections within each joint were averaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was carried out using a Histostain-SP kit (Cat. 959943B Invitrogen) to examine the distribution of type II collagen and MMP-13 in cartilage. The sections were digested with 5 mg/mL hyaluronidase in phosphate-buffered saline (PBS) (Sigma\u0026ndash;Aldrich) for 20 minutes. Nonspecific protein binding was blocked by incubation with a serum blocking solution. Thereafter, the sections were incubated with specific antibodies against rat type II collagen (Santa Cruz Biotechnology) and MMP-13 (Santa Cruz Biotechnology) at 4\u0026deg;C overnight. The negative control sections were incubated with isotype-matched control serum (2 \u0026micro;g/mL) (R\u0026amp;D Systems, Minneapolis, MN, USA) in PBS. Subsequently, the sections were treated sequentially with biotinylated secondary antibodies and SP conjugate and then developed with DAB chromogen. Photomicrographs were taken with a Nikon E800 microscope (Nikon, Melville, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRat articular cartilage real-time qPCR\u003c/h2\u003e \u003cp\u003eThe cartilage samples from the rat femur condyles were ground with a mortar and pestle in liquid nitrogen. Total RNA was isolated from rat cartilage tissues using an RNeasy isolation kit (Qiagen, Valencia, CA) according to the manufacturer\u0026rsquo;s instructions. Cartilage samples from 5 rats were dissected using a scalpel and pooled together; thus, there were 3 pooled samples per group. Total RNA was reverse transcribed into first-strand cDNA using the iScript\u0026trade; cDNA synthesis kit (Bio\u0026ndash;Rad, Hercules, CA, USA). RT\u0026ndash;PCR was performed on a 96-well ABI Prism 7500 system (Applied Biosystems, Foster City, CA) using SuperReal PreMix reagent (Qiagen, Valencia, CA) according to the manufacturer\u0026rsquo;s protocols. The total volume (20 \u0026micro;L) of each PCR contained 10 \u0026micro;L SuperReal PreMix, 7 \u0026micro;L of ddH2O, 2 \u0026micro;L of cDNA, and 1 \u0026micro;L of the forward and reverse primers (10 \u0026micro;M ) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The amplification conditions were as follows: 2 min of preincubation at 50\u0026deg;C; 10 min at 95\u0026deg;C for enzyme activation; and 40 cycles of denaturation at 95\u0026deg;C for 10 s, annealing at 55\u0026deg;C for 30 s and extension at 72\u0026deg;C for 30 s. Gene expression was normalized to 18S mRNA levels. The comparative threshold cycle (Ct) method (2-ΔΔCt method) was used to calculate fold changes. [26]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers for real-time qPCR used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence 5' to 3'\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType II Collagen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAG-GGA-CAC-CGA-GGT-TTC-ACT-GG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGG-CCT-GTT-TCT-CCT-GAG-CGT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggrecan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAG-TGC-GAT-GCA-GGC-TGG-CT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCT-CCG-GCA-CTC-GTT-GGC-TG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRunx2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCG-CAC-GAC-AAC-CGC-ACC-AT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGC-TCC-GGC-CCA-CAA-ATC-TC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTG-TCC-TTC-GAT-GCA-GTC-AG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGA-CGG-CCA-AAA-TGA-AGA-GA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP-13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGA-CCT-TCT-GGT-CTT-CTG-GC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGA-TGC-TTA-GGG-TTG-GGG-TC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType X Collagen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCA-GGT-GTC-CCA-GGA-TTC-CC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAA-GCG-GCA-TCC-CAG-AAA-GC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18S RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGG-CTA-CCA-CAT-CCA-AGG-AA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCT-GGA-ATT-ACC-GCG-GCT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSynovial Fluid Collection and Analysis\u003c/h2\u003e \u003cp\u003eSynovial fluid lavages were collected from the knees as soon as euthanasia was performed. [23] The knee was shaved and prepped with betadine. Fifty microliters of isotonic saline was injected into the right knees of each animal through the inferior patellar tendon. The joint capsule was visibly distended. The knee was then manually cycled through flexion and extension 10 times to distribute the fluid within the joint before collection via joint aspiration. The procedure was repeated twice. This technique typically yielded 80\u0026ndash;100 \u0026micro;l of fluid from each animal. Then, the synovial fluid was centrifuged at 2,000 \u0026times; g for 10 minutes to remove cells and debris and frozen at -80\u0026deg;C until analysis. Once thawed, MMP-13 and sulfated-glycosaminoglycans (sGAG) in the synovial fluid lavages were measured. MMP-13 was measured using ELISA according to the instructions of the manufacturer (Uscn Life Science). The colorimetric density in the developed plates was measured using a microplate reader (Model BF10000; Packard) at a wavelength of 450 nm. ELISA analysis of each sample was performed in duplicate. To evaluate matrix PG release, we used the metachromatic dye 1,9-dimethylmethylene blue (DMMB) (Sigma-Aldrich, Gillingham, UK) assay to quantify the amount of sulfated glycosaminoglycan (sGAG) in SF samples according to the manufacturer\u0026rsquo;s instructions. The concentrations of sGAG were determined by a spectrophotometric reader at 540 nm (Thermo Scientific Microplate Reader, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to compare the concentrations of MMP-13 and levels of mRNA in the different groups. All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and \u003cem\u003ep\u003c/em\u003e values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eGross morphological cartilage lesions and fibrillation in the rat tibia plateau were visualized by India ink staining. We found that intra-articular HA did not attenuate posttraumatic OA compared to A2M treatment, as determined macroscopically. There was a significant decrease in the OA scores of A2M-treated rats compared with rats that underwent ACLT and HA/saline treatment. After treatment with A2M, decreased India ink staining and a smoother surface were observed compared to those in the HA-treated groups (Fig. 1A). India ink staining and the Meacham grading system indicated that the A2M-treated group had fewer cartilage lesions and fibrillation than the HA-treated or saline-treated group (p\u0026thinsp;=\u0026thinsp;0.001) but more than the sham control group (p\u0026thinsp;=\u0026thinsp;0.008) (Fig. 1B). There was no significant difference between the HA-treated group and the saline-treated group (p\u0026thinsp;=\u0026thinsp;0.378).\u003c/p\u003e\n\u003cp\u003eThen, we evaluated the effect of various treatments by histological staining. Intra-articular A2M was superior to HA in attenuating posttraumatic OA pathogenesis in a rat model of ACLT. First, we found a significant decrease in the OA score in A2M-treated rats compared with rats that underwent ACLT and saline/HA treatment. After treatment with A2M, stronger safranin O staining, a more intact surface and more cellularity but less chondrocyte cloning, and less fibrillation were observed than in the saline/HA-treated groups than in the other groups, but there was weaker staining than that in control rats that underwent sham operation (Fig. 2A). Cartilage from rats that were administered HA had almost similar staining and surfaces compared with cartilage from untreated rats (Saline group) (Fig. 2A). The OARSI histologic grading system scores in the A2M-treated group suggested mild degeneration (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, 9.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0), while cartilage damage in the saline-treated group and HA-treated group was significantly worsened (18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 in the ACLT\u0026thinsp;+\u0026thinsp;saline group and 16.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 in the ACLT\u0026thinsp;+\u0026thinsp;HA group; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). It should be noted that there were no significant differences between the ACLT\u0026thinsp;+\u0026thinsp;saline group and the ACLT\u0026thinsp;+\u0026thinsp;HA group (P\u0026thinsp;=\u0026thinsp;0.062). Cartilage from rats that underwent sham operation had the least amount of damage (0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eNext, immunohistochemistry was performed to determine the expression of type II collagen and MMP-13. Type II collagen levels were higher in cartilage in the ACLT\u0026thinsp;+\u0026thinsp;A2M and Sham\u0026thinsp;+\u0026thinsp;Saline groups than in the ACLT\u0026thinsp;+\u0026thinsp;HA group and ACLT\u0026thinsp;+\u0026thinsp;saline group (Fig. 3A). In contrast, MMP-13 was elevated in cartilage in the HA-treated group compared to the A2M-treated group, suggesting that the group with higher MMP-13 levels suffered critical cartilage injury (Fig. 3B).\u003c/p\u003e\n\u003cp\u003eAdditionally, cartilage damage was associated with changes in the levels of MMP-13 in joint lavage fluid. The MMP-13 level (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD )in rats that underwent ACLT and A2M treatment was 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;g/ml, which was higher than that in the sham operation group (0.266\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;g/ml; P\u0026thinsp;=\u0026thinsp;1.85E-05) but much lower than that in rats that were administered HA (1.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;g/ml; P\u0026thinsp;=\u0026thinsp;4.65E-05) or saline (1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u0026micro;g/ml, P\u0026thinsp;=\u0026thinsp;1.27E-06). However, there was no significant difference between the ACLT\u0026thinsp;+\u0026thinsp;HA group and the ACLT\u0026thinsp;+\u0026thinsp;saline group (Fig. 4).\u003c/p\u003e\n\u003cp\u003eIn addition, we examined sulfated glycosaminoglycan (sGAG) levels in the synovial fluid in the different groups (Fig. 5). sGAG is an integral component of the cartilage matrix. However, the presence of sGAG in synovial fluid indicates the destruction of the articular cartilage. The sGAG level in the A2M-treated group was 331.68\u0026thinsp;\u0026plusmn;\u0026thinsp;75.47 \u0026micro;g/ml, which was higher than that in the sham group (213.74\u0026thinsp;\u0026plusmn;\u0026thinsp;44.14 \u0026micro;g/ml; P\u0026thinsp;=\u0026thinsp;0.008). However, it is noteworthy that the level of sGAG in synovial fluid in the A2M group was significantly lower than that in the untreated group (567.70\u0026thinsp;\u0026plusmn;\u0026thinsp;75.23 \u0026micro;g/ml; P\u0026thinsp;=\u0026thinsp;0.001) (Fig. 5). This result demonstrated the protective effect of intra-articular A2M injection. We found that ACLT rats in the groups that were treated saline or HA exhibited higher levels of synovial fluid sGAG (576.56\u0026thinsp;\u0026plusmn;\u0026thinsp;44.14 \u0026micro;g/ml) than those treated with A2M (P\u0026thinsp;=\u0026thinsp;9.33E-05), which was also much higher than that of rats in the control group (P\u0026thinsp;=\u0026thinsp;2.77E-07) (Fig. 5). Moreover, there was no significant difference between the ACLT\u0026thinsp;+\u0026thinsp;saline group and the ACLT\u0026thinsp;+\u0026thinsp;HA group (P\u0026thinsp;=\u0026thinsp;0.825) (Fig. 5).\u003c/p\u003e\n\u003cp\u003eAdditionally, we performed qPCR to investigate the expression of related genes. The qPCR results indicated that the mRNA expression of type II collagen and aggrecan increased in the group that was treated with intra-articular A2M, while the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen were decreased (Fig. 6). In the HA treated group, the levels of aggrecan and type II collagen mRNA were obviously low; in contrast, the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen were increased (Fig. 6). The type II collagen mRNA level in rats that underwent ACLT and A2M treatment was significantly higher than that in rats that underwent ACLT plus HA or saline injection, and there was no significant difference between the latter two groups. In the groups that underwent sham operation and were treated with A2M, aggrecan mRNA levels were significantly higher than those in the groups that were injected with saline and HA. In contrast, the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen in rats that underwent ACLT and saline/HA treatment were much higher than those in the other two groups (Fig. 6).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePost-traumatic osteoarthritis is a longterm disease initiated by joint injury and consequent intra-articular inflammation, and its progression is irreversible. [27, 28] Thus, delaying cartilaginous degeneration is the main purpose of nonsurgical treatment for osteoarthritis. Chronic inflammation is a critical risk factor for cartilage destruction during the process of osteoarthritis; therefore, it is believed that reducing intra-articular inflammation slows the degeneration of cartilage. [28] Accordingly, we thought that intra-articular injection of A2M, an autogeneic broad spectrum proteinase inhibitor, could ameliorate cartilage destruction induced by inflammation.\u003c/p\u003e \u003cp\u003eWe used four groups to compare the protective effects of A2M and HA, and the two experimental groups that received ACLT were treated separately with A2M and HA injection. The India ink staining and safranin-O/fast green staining results were consistent. All groups that underwent ACLT showed articular cartilage defects and subchondral bone outcroppings, which are typical pathological changes in osteoarthritis. [29] However, there was little normal cartilage remaining in the HA-treated group, indicating that cartilage damage in the HA group and untreated group was not significantly different; thus, the protective effect of HA is weak. This outcome echoed the results of clinical trials and meta-analyses, suggesting the uncertainty of the curative effect of HA. [30\u0026ndash;32]\u003c/p\u003e \u003cp\u003eIn the A2M-treated group, cartilage damage occurred similarly but was obviously less severe than that in the HA group and untreated group. The A2M-treated group had a relatively intact surface and more chondrocyte cloning than the untreated group and HA-treated group. Although degenerative cartilage damage still existed after A2M injection treatment, the protective effect of A2M was still very evident. Notably, the OARSI scores in the A2M-treated group also showed a significant decrease, suggesting mild degeneration.\u003c/p\u003e \u003cp\u003eThe major pathological change in PTOA is chronic inflammation secondary to joint injury. Due to the increased production of proinflammatory cytokines and matrix metalloproteinases, the inflammatory damage of the articular tissue is dysregulated. Thus, a relatively strong way to analyze and compare the protective effects of HA and A2M is to study whether treatment can inhibit the levels of destructive cytokines and enzymes.\u003c/p\u003e \u003cp\u003eAccordingly, we examined additional biological indicators of cartilage degeneration. Immunohistochemistry was performed to examine the distribution of type II collagen and MMP-13 in cartilage. Type II collagen is produced by chondrocytes, and the level of type II collagen in degenerated cartilage with reduced chondrocyte cloning will be reduced. [33] MMP-13 is an enzyme that catalyzes the destruction of cartilage. MMP-13 induces the degradation of proteoglycan and type II collagen; thus, it is used as an important indicator of cartilage damage. [34\u0026ndash;36] Higher type II collagen levels and lower MMP-13 levels in the A2M injection group than in the other groups; thus, the protective effect of A2M injection was confirmed. In addition, we examined sGAG concentrations in synovial fluid in each group. Compared to those in the untreated group, synovial fluid sGAG levels in the A2M group were reduced. Quantitatively assessing sGAG levels in synovial fluid can be used to reflect the destruction of cartilage extracellular matrix. [37] It is likely that the protective effect of A2M led to reduced sGAG levels.\u003c/p\u003e \u003cp\u003eThe qPCR results clearly showed that A2M injection reduced the mRNA levels of Runx2, MMP-3, MMP-13, and type X collagen. However, the mRNA levels of these factors in rats that underwent ACLT and saline/HA treatment were much higher than those in the other two groups. These data suggested that A2M could protect cartilage in vivo by decreasing the gene expression of catabolic factors and hypertrophic markers, as well as by increasing anabolic gene expression.\u003c/p\u003e \u003cp\u003eIt is also apparent that HA failed to achieve the desired therapeutic effect. Immunohistochemical staining showed that cartilage degeneration in rat knees after HA injection was still severe, while the MMP-13 level was much higher than that in the sham operation group and A2M group. Quantitative analysis of MMP-13 levels in synovial fluid showed no significant disparity between the HA injection group and the untreated group. Similarly, there was no difference between sGAG levels in the HA group and the untreated group. The qPCR results also indicated that HA treatment failed to prevent cartilage destruction.\u003c/p\u003e \u003cp\u003eIn many studies, HA cannot exert a protective effect on injured joint tissue. A commonly accepted view is that intra-articular injection of HA can work via visco-supplementation. [19] However, as early as the 1980s, a study suggested that injecting HA did not increase the specific viscosity of synovial fluid in experimentally damaged joints. [38] One of the important reasons is that high molecular weight HA (HMW-HA) will be hydrolyzed to low molecular weight HA (LMW-HA) during the wound healing process, and LMW-HA cannot increase the specific viscosity of synovial fluid. [39] Furthermore, many studies suggest that LMW-HA is a proinflammatory factor that can induce metalloproteinase production. [40\u0026ndash;42]\u003c/p\u003e \u003cp\u003eIn addition, there are proteoglycans called aggrecan on articular cartilage that bind to HA, providing elasticity to cartilage. [43] When PTOA occurs, aggrecan on joint cartilage is dissociated, leading to the loss of HA, cartilage erosion, and ultimately cartilage degeneration. [44] There is no evidence that supplementation with HA without aggrecan contributes to cartilage repair. Additionally, a previous study suggested that HA had no influence on the proteoglycan concentrations in PTOA. [45] Our study also concluded that there was no significant difference in the mRNA level of aggrecan in the HA-treated group and the untreated group.\u003c/p\u003e \u003cp\u003eAlthough intra-articular injection of HA supplies HMW-HA, the change is temporary. Through HA injection therapy, synovial fluid is difficult to restore to normal standards. Any change in the molecular weight and concentration of HA in synovial fluid will exacerbate articular cartilage damage. For these reasons, HA treatment is no longer recommended by the American Academy of Orthopedic Surgeons (AAOS). [22]\u003c/p\u003e \u003cp\u003eWe decided to use A2M as an alternative therapy to replace HA therapy. A2M is not only able to protect articular cartilage against chronic inflammation but also has high biosafety and few side effects.\u003c/p\u003e \u003cp\u003eThere are many studies focused on resolving inflammation caused by cartilage destruction in osteoarthritis. In this context, common treatment methods are intra-articular calcium channel blocking anesthetics, corticosteroid injection (IACI), mesenchymal stem/stromal cells (MSCs), and platelet-rich plasma. [46, 47] However, some of these treatments cannot slow cartilage degeneration; some are not suitable for prolonged use in the clinic. A2M, which is a novel therapeutic drug for knee OA, has good prospects in solving these shortcomings. Compared with other drugs, A2M is an autologous proteinase inhibitor that has no autoimmune rejection. Additionally, A2M treatment contains only one active ingredient but inhibits various inflammatory factors and degenerative proteinases due to the broad-spectrum anti-inflammatory effect of A2M. Therefore, A2M injection eliminates the abovementioned limitations. As a result, safety is guaranteed, and risk is controllable.\u003c/p\u003e \u003cp\u003eNew technologies allowed us to concentrate A2M molecules from the autologous plasma of patients and then inject A2M-rich preparations into joints to compensate for the low level of A2M in synovial fluid. [48] Furthermore, the variant of A2M known as CYT-108 can be synthesized according to the molecular properties of A2M. Synthetic A2M is likely more economical and convenient than the parent compound, and the inhibitory effect of CYT-108 A2M will far exceeded that of wild-type A2M. [49]\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study examined the uncertainty of HA treatment in animal models. The protective effect of A2M on articular cartilage in osteoarthritis was tested. The results demonstrated that A2M could effectively postpone retrograde degeneration in joint cartilage. As expected, HA treatment did not have an obvious protective effect. These observations indicate that A2M is a promising anti-inflammatory treatment for osteoarthritis and can be used as a replacement for HA.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePTOA\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePosttraumatic osteoarthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eA2M\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealpha-2-macroglobulin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eHA\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehyaluronic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eACLT\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanterior cruciate ligament transection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eOOCHAS\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteoarthritis Cartilage Histopathology Assessment System\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMMP-13\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ematrix metalloproteinase-13\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eOA\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteoarthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMCP-1\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonocyte chemoattractant protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAAOS\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethe American Academy of Orthopedic Surgeons\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eARC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnimal Research Committee\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003esGAG\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esulfated-glycosaminoglycans\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eOARSI\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteoarthritis Research Society International\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIACI\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintra-articular calcium channel blocking anesthetics, corticosteroid injection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMSCs\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emesenchymal stem/stromal cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confrmed that all experimental protocols were approved by the Animal Ethics Committee of Shanxi Medical University. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support our findings are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the National Natural Science Foundation of China (Grant No. 81572207, 81201435), Health Commission of Shanxi Province (Grant Nos. 2020XM54, 2020075), and Shanxi Scholarship Council of China (grant numbers 2017-063).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003econtributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSW, YM and CH designed this study; CC, MZ and YG finished rat studies; XC and YL finished Histology and Immunohistochemical staining; ZW and MZ finished Synovial Fluid Collection and Analysis; XC and ZW finished real-time qPCR studies; ZW, CC and YG performed the statistical analysis; All authors contributed to the article and approved the submitted version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Shaowei Wang [email protected] and Yuyuan Ma [email protected].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMinguzzi M, Cetrullo S, D\u0026apos;Adamo S, Silvestri Y, Flamigni F, Borzi RM: Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis. 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Osteoarthritis Cartilage 2001, 9(6):539-552.\u003c/li\u003e\n\u003cli\u003eTortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, Rosenfeld SA, Copeland RA, Decicco CP, Wynn R et al: Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science 1999, 284(5420):1664-1666.\u003c/li\u003e\n\u003cli\u003eBrandt KD, Smith GN, Jr., Simon LS: Intraarticular injection of hyaluronan as treatment for knee osteoarthritis: what is the evidence? Arthritis Rheum 2000, 43(6):1192-1203.\u003c/li\u003e\n\u003cli\u003eXie X, Zhang C, Tuan RS: Biology of platelet-rich plasma and its clinical application in cartilage repair. Arthritis Res Ther 2014, 16(1):204.\u003c/li\u003e\n\u003cli\u003eRichards MM, Maxwell JS, Weng L, Angelos MG, Golzarian J: Intra-articular treatment of knee osteoarthritis: from anti-inflammatories to products of regenerative medicine. Phys Sportsmed 2016, 44(2):101-108.\u003c/li\u003e\n\u003cli\u003eCuellar JM, Cuellar VG, Scuderi GJ: alpha2-Macroglobulin: Autologous Protease Inhibition Technology. Phys Med Rehabil Clin N Am 2016, 27(4):909-918.\u003c/li\u003e\n\u003cli\u003eZhang Y, Wei X, Browning S, Scuderi G, Hanna LS, Wei L: Targeted designed variants of alpha-2-macroglobulin (A2M) attenuate cartilage degeneration in a rat model of osteoarthritis induced by anterior cruciate ligament transection. Arthritis Res Ther 2017, 19(1):175.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, Alpha-2-macroglobulin, Hyaluronic Acid, MMP-13, Cartilage degeneration","lastPublishedDoi":"10.21203/rs.3.rs-1779669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1779669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePosttraumatic osteoarthritis (PTOA) correlates with a dramatic increase in multiple inflammatory factors after joint injury. There is a broad spectrum proteinase inhibitor known as alpha-2-macroglobulin (A2M) that plays an important role in protecting against inflammatory injury. Endogenous A2M is abundant in serum but insufficient in synovial fluid due to its large molecular weight and has a limited effect on articular cartilage inflammation. We hypothesize that intra-articular injection of A2M has a better clinical effect than the commonly used hyaluronic acid (HA) injection therapy. Furthermore, A2M could be a safer method than alternative treatments because it is a native substance in the human body.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eThe in vivo effects of A2M and HA on cartilage degeneration were evaluated in rats with surgery-induced anterior cruciate ligament transection (ACLT) OA. One hundred rats were randomly divided into four groups (N\u0026thinsp;=\u0026thinsp;25/group): (a) sham surgery\u0026thinsp;+\u0026thinsp;saline (Sham\u0026thinsp;+\u0026thinsp;S), (b) ACLT\u0026thinsp;+\u0026thinsp;A2M, (c) ACLT\u0026thinsp;+\u0026thinsp;HA, or (d) ACLT\u0026thinsp;+\u0026thinsp;saline (ACLT\u0026thinsp;+\u0026thinsp;S). Intra-articular injections of A2M were given immediately and 3 days after surgery, and then 20 \u0026micro;l was injected each weekly in each joint. The animals were sacrificed 12 weeks after surgery. Indian ink staining, safranin O staining and immunohistochemical staining were performed to assess cartilage damage. The extent of OA progression was graded by the Osteoarthritis Research Society International Osteoarthritis Cartilage Histopathology Assessment System (OOCHAS) (OA score\u0026thinsp;=\u0026thinsp;Grade \u0026times; Stage; range, 0 to 24). The concentrations of matrix metalloproteinase-13 (MMP-13) and sGAG in synovial fluid lavage were measured by ELISA and spectrophotometric quantitative determination. OA-related gene expression was quantified by qPCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIndian ink staining showed that the articular cartilage surface in rats treated with A2M was relatively intact compared with that in the animals treated with ACLT plus saline or HA injection, but there was no significant difference between the ACLT\u0026thinsp;+\u0026thinsp;HA group and the ACLT\u0026thinsp;+\u0026thinsp;S group. Histological staining indicated that early intra-articular injection of A2M attenuated OA pathogenesis in the rat ACLT model compared with that in animals treated with saline and HA. However, intra-articular injection of HA did not significantly protect cartilage against posttraumatic OA compared with saline treatment. The ELISA results showed that A2M reduced the concentration of MMP-13 in synovial fluid compared with that in the HA treatment group and other groups. RT\u0026ndash;qPCR indicated that intra-articular A2M inhibited catabolism and enhanced anabolic metabolism, while there was no significant difference in the expression of OA-related genes between the ACLT\u0026thinsp;+\u0026thinsp;HA group and the ACLT\u0026thinsp;+\u0026thinsp;S group.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eIn rat model, intra-articular injection of A2M had obvious protective effects against cartilage degeneration compared with HA treatment. The inflammatory factor MMP-13 provides strong evidence for this inhibitory effect. Moreover, we found no significant alleviation of articular cartilage pathogenesis in the HA-treated group, which suggests that the efficacy of HA is questionable and possibly transient, although it is still extensively used in clinical practice.\u003c/p\u003e","manuscriptTitle":"Intra-articular Injection of Hyaluronic Acid and Alpha-2-Macroglobulin to Ameliorate Knee Posttraumatic Osteoarthritis: A Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-12 15:08:23","doi":"10.21203/rs.3.rs-1779669/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c2e17d8-28f9-48d1-8621-f3ec07b72e83","owner":[],"postedDate":"July 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-10-27T10:44:20+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-12 15:08:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1779669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1779669","identity":"rs-1779669","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}