α2-Macroglobulin-Rich Serum As a Master Inhibitor of Inflammatory Factors Attenuates Cartilage Degeneration in a Mini Pig Model of Osteoarthritis Induced By “Idealized” Anterior Cruciate Ligament Reconstruction

doi:10.21203/rs.3.rs-1043859/v1

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Research article

α2-Macroglobulin-Rich Serum As a Master Inhibitor of Inflammatory Factors Attenuates Cartilage Degeneration in a Mini Pig Model of Osteoarthritis Induced By “Idealized” Anterior Cruciate Ligament Reconstruction

https://doi.org/10.21203/rs.3.rs-1043859/v1

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posted 10 Nov, 2021

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Background: α2-Macroglobulin (α2M) is important for chondral protection in post-traumatic osteoarthritis (PTOA). However, its injection into xenogeneic joint cavities has safety hazards, limiting clinical applications. Exploring serum α2M-enriching strategies and the therapeutic effect and mechanism of α2M-rich serum (α2MRS) autologous joint injection to treat PTOA has significant value.

Methods: A unique filtration process was used to concentrate α2M from serum. Human osteoarthritic chondrocytes induced with interleukin (IL)-1β were used to evaluate catabolic enzymes, cell proliferation, apoptosis, and gene expression 24h after α2MRS treatment. Eighteen mature female mini pigs were randomized to three groups, sham (n = 6), “idealized” anterior cruciate ligament autograft reconstruction (IACL-R) (n = 6), and IACL-R+α2MRS (n = 6). Expression of inflammatory factors in the synovial fluid (SF) was measured using Luminex assays. Gait features were recorded using the Tekscan Walkway system. The extent of PTOA progression was evaluated using imaging, real-time PCR , and histology 3 months post-surgery.

Results: The α2M concentration in α2MRS was higher than that in human and mini pig serum, respectively. In vitro, α2MRS significantly promoted human chondrocyte proliferation (p < 0.001) and reduced apoptosis (p < 0.001) and chondrocyte catabolic cytokine gene transcription (p < 0.001) and secretion (p < 0.001). In vivo, SF concentrations of all tested inflammatory factors were significantly lower in the IACL-R+α2MRS group than in the IACL-R group (p < 0.001). All gait parameters in the IACL-R+α2MRS group returned to normal significantly early compared to those in the IACL-R group (p < 0.05). Imaging , histology, and biochemistry data showed that cartilage degeneration in the IACL-R+α2MRS group was significantly diminished relative to that in the IACL-R group (p < 0.001).

Conclusion: Injecting α2MRS into the joint cavity after IACL-R can significantly delay articular cartilage degeneration.

Immunology

Rheumatology

Osteoarthritis

α2M-rich serum

anterior cruciate ligament

articular cartilage

inflammation

mini pig

Anterior cruciate ligament (ACL) rupture, one of the most common joint injuries in young people, is conventionally treated using surgical ACL reconstruction (ACL-R). However, even with the best surgical techniques available, these patients remain at a high risk for post-traumatic osteoarthritis (PTOA)[1–3]. Recently, researchers developed an “idealized” ACL autograft reconstruction (IACL-R) model[4, 5]. Notably, the authors found that cartilage degeneration still occurred despite this reconstruction and concluded that there was a significant correlation between the expression of inflammatory factors and cartilage injury. Moreover, other studies have indicated that catabolic proteases and cytokines reach their peak levels within 48 h after joint injury, initiating cell death and cartilage matrix degeneration[6–8]. Thus, early intervention to reduce the expression of these catabolic proteases and cytokines is critical to prevent or delay cartilage degeneration.

α2-macroglobulin (α2M), a tetrameric macromolecular glycoprotein, is mainly synthesized and secreted into the body fluids by liver epithelial parenchymal cells[9]. To date, α2M has been shown to play an important role in the diagnosis of diseases, prediction of liver fibrosis staging[10], non-invasive diagnosis of type II diabetes[11], and the treatment of various diseases including alleviating pain in subacromial bursitis, lateral epicondylitis, Achilles tendonitis, spinal intervertebral discogenic[12, 13] and jaw osteoradionecrosis[14]. Moreover, some studies have demonstrated that supplemental intra-articular α2M provides chondral protection in PTOA[15–17]. However, α2M is expensive. More importantly, the long-term injection of α2M, a blood protein component, into the xenogeneic joint cavity has safety hazards, such as immune rejection, which limits its clinical application. In the current study, we aimed to develop and use α2M-rich serum (α2MRS) to treat cartilage degeneration following IACL-R. We hypothesized that α2MRS could significantly reduce the expression of inflammatory factors in synovial fluid (SF), promote early recovery of the gait, and effectively attenuate cartilage degeneration.

Study Design

All procedures in this study were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (NO. SYDL2019001) and conformed to the ARRIVE guidelines. The mini pigs were purchased from the Beijing Shichuang Century Mini pig Breeding Base (Certificate number: SCXX(jing)2013-0008). Eighteen mature female mini pigs (age, 18 ± 1.55 months; weight, 43.3 ± 3.67 kg) were randomized into three groups based on animal ear numbers: sham (n = 6), IACL-R (n = 6), and IACL-R+α2MRS (n = 6). Unilateral surgery was performed on the right hind limbs of all mini pigs. All animals were housed at the China Institute for Radiation Protection (Certificate number: SYXK(Jin)2016-0002). All animals were euthanized with a pentobarbital overdose 3 months after surgery. Specific information on animal care can be found in the supplementary text (Text 1). All α2MRS used in in vitro experiments were derived from human serum and in vivo from autologous serum of mini pigs. All subjective scores were independently evaluated by two experienced examiners, who were blinded to the animal number and experimental condition.

α2M Concentrate from Human Serum

Whole blood (13 ml) was collected in a coagulation tube and centrifuged at 2,000 ´ g for 20 min to obtain 6 ml of serum, which was then added to the upper filter of the ultrafiltration tube (Cytonics Corporation, West Palm Beach, Florida, USA). The upper concentrate was obtained under different conditions of centrifugal force (3000, 4000, and 5000 ´ g) and time (20, 30, and 40 min). Finally, the best concentration conditions were determined based on the concentration of α2M in the upper concentrate.

Human Chondrocyte Isolation and Primary Culture

Human chondrocytes were isolated as previously described[18] and plated in 6-well culture plates at a density of 1×10⁶ cells/plate. At 90% confluence, the cells were cultured overnight under serum-free conditions and then treated with 10 ng/ml recombinant human interleukin (IL)-1β for 2 h before treatment with α2MRS. It was ensured that the concentration of α2M was 0.25 mg/ml in the culture medium. The culture medium and chondrocytes were collected and analyzed.

Elisa Assays

α2M concentrations in the upper concentrate under different centrifugal conditions were determined using ELISA (EK1118, Boster Bio, China). The human chondrocyte culture medium was collected 24h after α2MRS treatment and analyzed for the presence of matrix metalloproteinase 13 (MMP-13), tumor necrosis factor-α (TNF-α), and IL-6 using ELISA.

Human Chondrocyte Proliferation and Apoptosis Assays

Human chondrocyte proliferation was detected at 0, 24, 36, and 48 h using the Cell Counting Kit-8 (CCK-8) cell viability assay kit ( Boster Biological Technology, China). Human chondrocytes were collected 24h after α2MRS treatment and apoptosis was detected using a Terminal transferase dUTP end Labeling (TUNEL) assay kit (Key GEN Bio TECH, China) .The percentage of positive cells was determined. The detailed procedure was in accordance with the manufacturer’s protocol.

RNA Isolation and Real-time PCR Assays

mRNA levels of col-2, aggrecan, MMP-3, and MMP-13 in human chondrocyte samples and those plus col-10a1 and Runx-2 in minipig cartilage weight-bearing sites of the medial tibial plateau (MTP) were measured by real-time PCR. Primer pairs are listed in Supplementary Table 1. Levels of gene expression were normalized to 18S rRNA expression. The data were analyzed using the comparison Ct (2^−ΔΔCt) method and expressed as the fold-change relative to the respective control. The detailed PCR procedure previously described.[19]

Mini Pig α2MRS Reserve

With the help of a veterinarian, 120 ml of whole blood was collected from the anterior vena cava of each mini pig into coagulation tubes. According to the best concentration conditions (centrifugal force: 5000 ´ g; time: 30 min), 12–15 ml of α2MRS was obtained per mini pig (marked according to the mini pig ear number), and these samples were frozen at −80 °C.

Idealized ACL Autograft Reconstruction Surgery

All surgeries were performed under anesthesia via an intramuscular injection of 25 mg/ml tiletamine and 25 mg/ml zolazepam (Zoletil 50, 1 ml/15 kg; Virbac Group, Carros, France). Mini pigs in the IACL-R and IACL-R+α2MRS groups were subjected to surgery based on the methods described by researchers[4,5] (Fig 1). The minipigs in the sham group underwent arthrotomy, temporary patellar dislocation, and coring of one-third of the length of the lateral femoral condyle.

IACL-R and IACL-R+α2MRS groups. A: The stifle joint was open and the patella was dislocated to expose the ACL (arrow). B: The ACL reconstruction guide was positioned at a 45° angle (arrow) to the longitudinal axis of the femur. C: Before the hollow drill was about to penetrate the femoral tunnel, a Kirschner wire (diameter 1 mm) was drilled along outer edge of the tunnel to prevent the cartilage from splitting. D: The tunnel was gently penetrated by the same diameter thin-walled ring osteotomy to completely severv tendon-bone segment E: The tendon-bone segment was completely freed (arrow). F: The tendon-bone segment was fixed in situ with two crossed Kirschner wires.

SF Collection

SF from the right hind limbs of all animals was collected preoperatively (day 0) and postoperatively on days 3, 6, 14, 29, and 90. The detailed procedure previously described.[20]

Intra-articular Injections

Under general anesthesia, intra-articular injections were administered 2, 6, 14, and 29 days post-surgery using a sterile syringe. Under aseptic conditions, 2.5 ml autologous α2MRS was injected into the right hind limbs of mini pigs in the IACL-R+α2MRS group on the indicated days. Animals in the sham and IACL-R groups were administered an equivalent volume of saline.

Luminex Assay

The Millipore Porcine Cytokine Magnetic Bead Panel (EMD Millipore, No. PCYTMAG-23K) was used to measure the levels of IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-18, TNF-α, and granulocyte-macrophage colony-stimulating factor( GM-CSF). Luminex assays were performed as previously described[5].

Gait Assessment

Six gait indicators related to biomechanics—maximum force, contact area, peak force, impulse, stance time, and swing time—were determined using the Tekscan Walkway system[21, 22] (Tekscan Inc., USA). To rule out individual differences in learning skills and walking states, each animal was subjected to over 10 successful training sessions per day for 10 consecutive days before the surgery, and all indicators were expressed as a ratio of the average values for the left hind limb divided by the average values for the right hind limb [23]. Gait data were collected preoperatively (day 0) and postoperatively on days 7, 15, 30, 45, 60, 75, and 90. All the results obtained are the average of five successful repeated walkway trials performed at each time point for each animal.

Imaging Assessment

Three months after the surgery, the mini pigs were euthanized with a pentobarbital overdose, and their right hind limbs were severed from the hip joint. Each right hind limb semi flexed was immediately subjected to X-ray examination, computed tomography (CT), three-dimensional CT reconstruction (3D CT), and magnetic resonance imaging (MRI). The specific imaging parameters are listed in the supplementary text (Text 2).

We determined the Kellgren-Lawrence grade of the right hind limb of each animal by examining the X-ray image[24, 25]. The CT values and thickness of the subchondral bone plate were determined[26]. To avoid interference by metal artifacts, we obtained CT scans of only the middle sagittal plane from the medial femoral condyle (MFC) and MTP of the right hind limb. We also determined the whole-organ MRI score (WORMS) of the MFC and MTP of the right hind limb[27, 28].

Macroscopic Cartilage and Osteophyte Assessment

Macroscopic damage to the articular cartilage surfaces and osteophyte formation on the MFC, MTP, lateral femoral condyle, lateral tibial plateau, and trochlea were scored according to the Osteoarthritis Research Society International (OARSI) recommendations for sheep and goats[29]

Histological Assessment

Cartilage samples were obtained by drilling (φ8 mm; MOC Medizinische Gerate Gmbh, Fedderingen, Germany) the weight bearing site of the MFC. The cartilage tissue sections (6 µm) were stained with safranin O and fast green as previously described and scored according to the recommendations of OARS[29]. Furthermore, we collected synovium samples from inside the joint capsule. Synovium sections (4 µm) were stained with hematoxylin and eosin (H&E) as previously described and scored according to the OARSI recommendations[29]. Vertical meniscus slices from the middle region of the medial meniscus were processed and stained using H&E and scored according to the protocol detailed by Pauli et al[30].

Immunohistochemical Assessment

Cartilage tissue sections from the MFC were used to detect the distribution of collagen-2 (Col-2，ab34712, Abcam), MMP-3 (bs-0413R, Bioss), MMP-9 (bs-4593R, Bioss), MMP-13 (K009743P, Solarbio), Col-10 (bs-0554R, Bioss), and Runx-2 (ab76956, Abcam). Quantitative immunohistochemical analysis was performed using an imaging analyzer. The detailed immunohistochemical procedure previously described[31]

Statistical Analysis

SPSS statistical software (version 13.0) was used to analyze the collected data. Differences in gaits and inflammatory factor levels between the preoperative (day 0) and postoperative time points in the same group were analyzed using multiple comparisons of repeated measurement data. Differences in human chondrocytes, minipig gait, inflammatory factor levels, CT values, thicknesses of the subchondral bone plate T2 values, and quantitative immunohistochemical analysis at the same time point among the different groups were estimated using one-way analysis of variance. Differences in macroscopic cartilage and osteophyte scores; microscopic cartilage, synovium, and meniscus scores; and WORMS were estimated using nonparametric tests (Wilcoxon rank-sum test). Differences in Kellgren-Lawrence grades were estimated using Fisher probabilities. Statistical significance was determined at P < 0.05.

Concentration Analysis of α2M in Different Concentrates From Human and Mini pig

As the centrifugal force increased and centrifugation time was prolonged, Human α2M concentrations in the upper concentrate correspondingly increased (Table 1). Considering different factors, such as protein biological activity and concentrate volume, the concentration effect of α2M under the conditions of centrifugation at 5000 × g for 30 min was ideal. For human α2MRS, the concentration of α2M was 11.13 mg/ml, which was 4.88-fold higher than that in normal human serum. In mini pigs, the concentration of α2M was 12.32 mg/ml, which was 6.48-fold higher than that in normal pig serum (Fig. 2A).

Table 1 Human α2M concentrations in the upper concentrate under different centrifugal conditions（mg/ml）（Mean ± SD，n=6）

3000g 4000g 5000g

20min 4.82±0.75 6.98±1.15 8.23±1.12

30min 6.46±0.89 9.22±1.08 11.13±0.90

40min 7.39±0.98 9.92±1.17 11.93±1.53

Human Chondrocyte Culture Medium Analysis

ELISA results demonstrated that exogenous α2MRS significantly inhibited the induction of MMP-13 (P = 0.001), TNF-α (P = 0.005), and IL-6 (P < 0.001) activity in IL-1β-induced human primary osteoarthritis chondrocytes (Fig. 2B-D).

Human Chondrocyte Proliferation and Apoptosis Analysis

α2MRS promoted the proliferation and reduced the apoptosis of human chondrocytes in vitro. The results of the CCK-8 assay showed that the viability of chondrocytes was higher in the IL-1β + α2MRS group than in the IL-1β group (P < 0.001), and the viability gradually increased with treatment time (Fig. 2E). The results of the TUNEL assay showed that apoptosis was significantly reduced in the IL-1β + α2MRS group (18.33% ± 5.71%) relative to that in the IL-1β group (32.33% ± 7.23%) (P < 0.001;Fig. 2F,G).

Real-time PCR Analysis

Real-time PCR data indicated that supplementation with α2MRS reduced cartilage matrix catabolism and enhanced anabolic metabolism in vitro (Fig.3A-D) and in vivo (Fig.3E-J). mRNA levels of MMP-3 (P < 0.001), MMP-13 (P < 0.001), Col-10a1 (P= 0.001), and Runx-2 (P < 0.001)were lower in IL-1β+α2MRS and IACL-R+α2MRS groups than in IL-1β and IACL-R groups. In contrast, mRNA levels of Col-2 (P < 0.001) and aggrecan (P < 0.001)showed the opposite pattern. Both were increased in IL-1β+α2MRS and IACL-R+α2MRS groups as compared to levels inIL-1β and IACL-R groupsrespectively.

Inflammatory Factor Analysis

Postoperatively, changes in the levels of inflammatory factors in different groups, except IL-18 in the IACL-R group, showed similar trends; the levels markedly increased in the early stage and then decreased significantly. IL-2 concentration in the IACL-R group subsequently showed an increasing trend from 30 to 90 day. The concentrations of inflammatory factors, including IL-1β, IL-6, IL-18, TNF-α, and GM-CSF, in the IACL-R and IACL-R + α2MRS groups after surgery wer significantly higher than those before surgery (P < 0.001). The concentration of all tested inflammatory factors other than IL-1α after surgery was significantly lower in the IACL-R + α2MRS group than in the IACL-R group, and significant difference in peak concentration was observed in all factors (P < 0.001). Moreover, the peak concentrations of all detected inflammatory factors, other than IL-18 in the IACL-R group, appeared within 3–14 days after surgery (Fig. 4).

Gait Assessment

Across all groups, the ratios of all gait parameters of the left hind limb to those of the right hind limb initially showed an increasing trend, followed by a decreasing trend, except in the IACL-R group, which showed another increasing trend toward the end. In the IACL-R group, no gait parameters, from day 45 until day 75, differed significantly from their values on day 0 (P > 0.05). In the IACL-R + α2MRS group, no gait parameters, from day 30 until euthanasia, differed significantly from their values on day 0 (P > 0.05) (Fig. 5).

The ratios of left hind limb to right hind limb of all gait parameters were similar and close to 1 for symmetry and did not differ significantly in the all groups before surgery (P > 0.05). Meanwhile, the postoperative ratios of left hind limb to right hind limb of the gait parameters were significantly greater than 1 in all groups, and the values in the IACL-R group were significantly greater than those in the other two groups other than swing time, especially on days 7 and 15 (P < 0.001). The ratios of left hind limb to right hind limb of the gait parameters was close to 1 on days 45 and 60, indicating that there were no significant differences among the groups (P > 0.05). On day 75, this ratio increased only in the IACL-R group whereas it remained constant in the sham and IACL-R + α2MRS groups. On day 90, all gait parameters other than swing time (P = 0.345) significantly differed between the IACL-R group and the other two groups (P < 0.001; Fig. 5).

Imaging Assessment

The X-ray examinations showed that joint degeneration in the IACL-R group was relatively noticeable. The joints had a blurred border and mild osteophyte formation compared with those in the IACL-R + α2MRS group (Fig. 6A). The Kellgren-Lawrence grades did not significantly differ among the three groups (P > 0.05; Table 2). Three-dimensional CT reconstruction showed that all joint surfaces were relatively smooth and flat in the IACL-R + α2MRS group compared to IACL-R group. 0steophyte was seen on both sides of the patellofemoral joint in the IACL-R group (Fig. 6B). Both MFC (P <0.001) and MTP (P <0.001) showed significant differences in CT values in the subchondral bone plate between the sham and IACL-R groups. In addition, significant differences in CT values were found between the IACL-R + α2MRS and IACL-R groups in the MFC (P = 0.028; Fig. 6D). Significant differences in the thickness of the subchondral bone plate were found only between the sham and IACL-R groups in the MTP (P = 0.020; Fig. 6E). MRI OSag-fs PD showed that cartilage continuity was better without obvious local defects in the IACL-R + α2MRS group than in the IACL-R group (Fig. 6C, left). WORMS of the MFC (P = 0.006), MTP (P = 0.014) and sum (P = 0.004) were significantly lower in the IACL-R + α2MRS group than in the IACL-R group (Fig. 6G-I, Supplementary table 2). Osag T2MAP showed that regular orange-red layer were more obvious in the IACL-R + α2MRS group than in the IACL-R group (Fig. 6C, right). The T2 values of the MFC (P <0.001) and MTP (P <0.001) were significantly lower in the IACL-R + α2MRS group than in the IACL-R group (Fig. 6F).

Table 2 Kellgren-Lawrence grades on X-ray examination at 3 months (n = 6)

Group

Grade

sham

IACL-R

>0.05

IACL-R + α2M

Macroscopic Cartilage and Osteophyte Assessment

Compared to that in the IACL-R group, cartilage degeneration was relatively low and no obvious cartilage defects or large erosions were found in the IACL-R + α2MRS group (Fig. 7A). OARSI scores of macroscopic cartilage were significantly lower in the IACL-R + α2MRS group than in the IACL-R group (P = 0.031; Fig. 7B, Supplementary Fig. 1A-1E, Supplementary Table 2). Mild irregular protrusions were found only on sides of the trochlea in the IACL-R group (Fig. 7A). No differences were found in the OARSI sum scores of the osteophyte among the three groups (P = 0.438; Figure 7C, Supplementary Figure 1F-1J, Supplementary Table 2).

Histological Assessment

In the IACLR + α2-MRS group compared with those in the IACLR-group, less decreases in safranin O staining and surface fibrillation were observed in the IACL-R + α2MRS group (Fig. 8A). The microscopic OARSI cartilage scores were lower in the IACL-R + α2MRS group than in the IACL-R group (P = 0.015; Fig. 8B, Supplementary Fig. 2A-2E, Supplementary Table 2 ). Based on H&E staining of the synovium, we found mild intimal thickening, low inflammatory cell infiltration, and slight sub-intimal fibrosis and vascularity in the IACL-R + α2MRS group, relative to those in the IACL-R group (Fig. 8C). Thus, the microscopic OARSI synovium scores, both total scores (P = 0.002) and single indicator scores, showed that synovial damage was lesser in the IACL-R + α2MRS group than in the IACL-R group (Fig. 8D, Supplementary Figure 2F-2I, Supplementary Table 2). H&E staining of the meniscus also revealed mild surface fibrillation, normal cell distribution, and a normal collagen fiber organization (Fig. 8E), and the meniscus score was lower in the IACL-R + α2MRS group than in the IACL-R group (P <0.001; Fig. 8F, Supplementary Figure 2J-2L, Supplementary Table 2) .

Immunohistochemical Assessment

Both articular cartilage (Fig. 9A-B) and synovium (Fig. 9C-D) immunostaining showed that MMP-3 (P <0.001), MMP-9 (P <0.001), MMP-13 (P <0.001), Col -10 (P <0.001), and Runx-2 (P =0.001) staining significantly increased in the IACL-R group compared with the IACL-R + α2MRS group. In contrast, Col-2 (P <0.001) expression in articular cartilage was higher in the IACL-R + α2MRS group than in the IACL-R group.

α2M performs complex bodily functions including the regulation of cytokine and hormone levels. It can bind several cytokines, including basic fibroblast growth factor, platelet-derived growth factor, nerve growth factor, IL-1β, and IL-6, and regulate the levels of hepcidin and leptin[9]. The specific mechanism of α2M has been previously reported by Sottrup-Jensen [32]. Notably, recent studies showed that α2M can attenuate PTOA cartilage degeneration [15–17]. However, α2M is not present in SF at sufficient levels due to its large molecular weight, which prevents its migration from the blood into the SF to counteract the increased concentrations of catabolic factors that appear after joint injury[33]. Thus, introducing supplemental α2M in the joint cavity might be a strategy to attenuate cartilage degeneration. However, considering the expense and potential safety concerns of biosynthetic α2M, α2MRS is a promising alternative for PTOA treatment. The results of our study demonstrate, for the first time, that α2MRS, as a master inhibitor of inflammatory factors, can attenuate cartilage degeneration in vitro and in vivo.

First, our in vitro data clearly demonstrated that human α2MRS promotes the proliferation of human chondrocyte, reduces the apoptosis of these ,and decreases chondrocyte catabolic cytokine gene transcription and secretion, suggesting that α2MRS is a promising therapy. The ultimate goal of studying α2MRS is clinical application. Thus, its biological safety and effectiveness need to be accurately evaluated and verified in vivo.

Previous studies have demonstrated that that peak levels of cartilage catabolic enzymes could be detected on day 2 after joint injury [34]. Therefore, the mini pigs in the IACL-R + α2MRS group received the first α2MRS joint cavity injection 2 days after surgery, which inhibited the levels of various inflammatory factors. Luminex results revealed that not only were the peak concentrations of inflammatory factors in SF significantly reduced but the speed of decline was also faster in the IACL-R + α2MRS group than in the IACL-R group, which demonstrated the early effect of α2MRS. In addition, gait analysis showed that the gait ratios of left hind limb to right hind limb of the IACL-R + α2MRS group was significantly greater than 1 at 7 days post-surgery, but it was significantly lower than that in the IACL-R group, which further proves the effectiveness of α2MRS. Other research results³⁷ suggest that the activity of some cartilage catabolic enzymes might have two peaks. The first phase appears after the initial trauma to the joint. The second peak is associated with progressive cartilage degeneration at weeks 4 and 6 after surgery. Therefore, we repeated α2MRS supplementation, which constantly exerts an inflammation-inhibitory effect, and the inflammatory storm or waterfall effect was interrupted in time[35]. Therefore, in the later stage (30-90 day) of the experiment, the concentration of inflammatory factors in the SF in the IACL-R + α2MRS group was maintained at a low level, and no obvious rebound was observed. Gait analysis is a relatively sensitive test for abnormal biomechanics and pain in the knee joint [36–38]. Compared with preoperative parameters, the gait of the IACL-R group also returned to previous levels, showing no significant difference in the middle stage (30-60 day). We concluded that the IACL-R model can restore the normal gait parameters of the knee joint and indirectly speculate that the ACL might have relatively stable biomechanical and consistent functions in different groups.

The mini pigs in the IACL-R + α2MRS group received α2MRS joint cavity injection four times starting from 2 days after surgery, which resulted in long-lasting inflammation suppression, significantly slowing the degeneration of articular cartilage. Therefore, the final imaging assessment, macroscopic cartilage assessment, and microscopic histological analysis confirmed that the articular cartilage in the IACL-R + α2MRS group was only slightly degenerated. Moreover, our biochemistry data demonstrated that supplemental α2MRS not only inhibited catabolic factors, including MMP-3, MMP-13, Col-10, and Runx-2, but also enhanced Col-2 and aggrecan gene expression and protein synthesis. The increase in collagen and aggrecan suggests that α2MRS might have cartilage-repair functions. This finding is consistent with previous reports [17]. Moreover, the results of H&E staining of the synovium and meniscus proved that α2MRS injection into the joint cavity could significantly reduce inflammatory cell infiltration and vascularity, protecting the articular cartilage, synovium, and meniscus. In this study, α2MRS might act by binding cytokines in addition to directly neutralizing enzyme activities, but the exact mechanism is not clear. The relative contributions of these mechanisms will be addressed in future studies.

Our study has a few limitations. First, the state of tension in the ACL or biomechanical changes in the knee joint post-surgery are crucial. To date, there is no technology or instrument that can accurately detect the smaller motions between the femur and tibia that are controlled by the ACL. The gait analysis used in this study can only roughly or indirectly assess the biomechanical stability of the knee joint. Second, although the concentration of α2M in α2MRS was 6.48-fold higher than that in normal mini pig serum, α2MRS is still in fact a mixture containing extremely complex components. α2MRS was injected into the joint cavity, and proteins other than α2M might also play a role, but the exact mechanism is still unknown. Third, considering the side effects of the multiple anesthesia method used, we do not know the exact trend of inflammatory factor levels during the period from days 29 to 90 after surgery.

In summary, α2MRS is a promising bioinhibitor of catabolic proteases, and early and multiple injections in the joint cavity after IACL-R can significantly reduce the concentration of inflammatory factors in the joint SF of mini pigs and the degeneration of articular cartilage, exerting a chondroprotective effect.

α2M: α2-Macroglobulin; α2MRS: α2M-rich serum; PTOA: post-traumatic osteoarthritis; ACL: Anterior cruciate ligament; ACL-R: ACL reconstruction; IACL-R: “idealized” anterior cruciate ligament autograft reconstruction;SF :synovial fluid;ELISA: Enzyme-Linked ImmunoSorbent Assay;IL: interleukin ;MMP-13:matrix metalloproteinase 13; TNF-α:tumor necrosis factor-α ;CCK-8:Cell Counting Kit-8 ;TUNEL :Terminal transferase dUTP end Labeling; GM-CSF ：granulocyte-macrophage colony-stimulating factor；GAG, glycosaminoglycan ; CT:computed tomography; 3D CT: three-dimensional CT reconstruction; MRI :magnetic resonance imaging ; WORMS :whole-organ MRI score ; MFC:medial femoral condyle; LFC:lateral femoral condyle; MTP: medial tibial plateau; LTP:lateral tibial plateau;OARSI :Osteoarthritis Research Society International (OARSI);H&E :hematoxylin and eosin ; LH: left hind foot; RH: right hind foot

Ethics approval and consent to participate:

All procedures in this study were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University ( NO. SYDL2019001 ).

Consent for publication:

Not applicable

Availability of data and materials:

The datasets used and or analysed during the current study are available from the corresponding author on reasonable request

Competing interests:

The authors declare that they have no competing interests

Funding:

The project was funded by the International Science and Technology Cooperation Program of China [Grant No. 2015DFA33050]

Authors’ contributions:

RPZ,LAHparticipated in the study design, wrote the manuscript, performed most of the experiments, and analyzed data.XCW,LAH conceived the study, revised the manuscript.CMZ,HRW,CX,WPD,ZQD carried out the part of imaging and histological assessment and helped to perform the statistical analysi.WPD,HQL, CJL.YZ,performed the IACL-R of minipigs and gait Assessment .All authors read and approved the final manuscript.

Acknowledgments

We would like to thank Yi Xu and Feipeng Song for providing assistance with the imaging examination, and Yipeng Xue, a veterinarian at the Sciences of ShanXi Agricultural Academy Institute of Animal Husbandry & Veterinary, for teaching us aboutanimal management. Besides，We would like to thank Pengfei Han (Heping Hospital Affiliated to Changzhi Medical College), Xiaodong Gu (Bethune Hospital, Shanxi Medical University) and Pengcui Li( The Second Hospital of Shanxi Medical University) for providing assistance with the minipig model of IACL-R..

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α2-Macroglobulin-Rich Serum As a Master Inhibitor of Inflammatory Factors Attenuates Cartilage Degeneration in a Mini Pig Model of Osteoarthritis Induced By “Idealized” Anterior Cruciate Ligament Reconstruction

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Abstract

Figures

Background

Materials And Methods

Results

Concentration Analysis of α2M in Different Concentrates From Human and Mini pig

Human Chondrocyte Culture Medium Analysis

Human Chondrocyte Proliferation and Apoptosis Analysis

Real-time PCR Analysis

Inflammatory Factor Analysis

Gait Assessment

Imaging Assessment

Macroscopic Cartilage and Osteophyte Assessment

Histological Assessment

Immunohistochemical Assessment

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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