Alpha-2-Macroglobulin Attenuates Posttraumatic Osteoarthritis Cartilage Damage by Inhibiting Inflammatory Pathways in a Modified Intra-Articular Drilling Yucatan Minipig Model

Abstract

Background:

Posttraumatic osteoarthritis (PTOA) arises secondarily to joint trauma and is driven by catabolic inflammatory pathways. Alpha-2-macroglobulin (α₂M) is a naturally occurring proteinase inhibitor found in human serum and synovial fluid that binds proteases as well as pro-inflammatory cytokines, such as IL-1β, involved in PTOA pathogenesis.

Hypothesis/Purpose:

(1) To investigate the therapeutic potential of intra-articular α₂M injections during the acute stages of PTOA by inhibiting inflammatory pathways driven by the cytokines expressed by the synovium in a large pre-clinical Yucatan minipig model (2) To determine if three intra-articular α₂M injections has greater chondroprotective effects than one.

Study Design:

Controlled Laboratory Study

Methods:

48 Yucatan minipigs were randomized into four groups (n=12): (1) modified intra-articular drilling (mIAD)+saline, (2) mIAD and one intra-articular α₂M injection (mIAD+α₂M-1), (3) mIAD and three α₂M injections (mIAD+α₂M-3), and (4) sham control. Surgical hind limbs were harvested 15 weeks post-surgery. Cartilage degeneration, synovial changes, inflammatory gene expression, and matrix metalloproteinase (MMP) levels were evaluated. Gait asymmetry was measured before and after surgery using a pressure-sensing walkway system.

Results:

Macroscopic lesion areas and microscopic cartilage degeneration scores were lower in mIAD+α₂M-1 and mIAD+α₂M-3 compared with mIAD+saline (p<0.05) and similar to the sham (p>0.05). Synovial membrane scores of mIAD+α₂M-1 and mIAD+α₂M-3 were lower than mIAD+saline (p<0.05) and similar to the sham (p>0.05). IL-1β, NF-κB, and TNF-α mRNA expression in the synovium and MMP-1 levels in the synovial fluid were significantly lower in mIAD+α₂M-1 and mIAD+α₂M-3 compared with mIAD+saline (p<0.05). No significant differences were observed between mIAD+α₂M-1 and mIAD+α₂M-3 for all measured outcomes. In general, there were no significant gait changes within groups from preoperative to postoperative time points.

Conclusion:

Animals receiving early α₂M treatment exhibited less cartilage damage, milder synovitis, and lower inflammation than animals with no α₂M treatment. These results exemplify the early anti-inflammatory effects of α₂M and provide evidence that intra-articular α₂M injections may slow the progression of PTOA.

Clinical Relevance:

In patients presenting with an acute joint injury, early intervention with α₂M may have the potential to reduce cartilage degeneration from catabolic pathways and delay PTOA development.

INTRODUCTION

Posttraumatic osteoarthritis (PTOA) disproportionately affects the youth and young adults and arises secondarily to joint trauma^23,34. Despite advancements in surgical techniques to restore joint mechanics, patients are still at high risk of developing PTOA, possibly due to uncontrolled inflammation that initiates catabolic pathways^6,17. Within one week of injury, the joint hosts an acute inflammatory response characterized by an increase in cytokines, including IL-1β, IL-6, IL-8, and TNF-α, and enzymes such as matrix metalloproteinases (MMPs) in the synovial fluid^3,7,15.

The synovium, which maintains the composition of the synovial fluid and provides nutrition to the articular cartilage, is a particularly critical tissue that mediates this process^6,16,36. Acute synovial inflammation results in elevated secretion of catabolic factors into the synovial fluid, leading to the breakdown of articular cartilage^25,36. Due to various factors, such as the severity of initial injury, imbalance of catabolic and anabolic pathways, and repeated damage signaling, the acute inflammatory reaction may evolve into chronic inflammation and PTOA development even after surgical repair^1,4,17. An emerging area of focus is the potential of anti-inflammatory agents administered during the early stages of injury to control the acute inflammatory response of the synovium and attenuate future PTOA development.

Alpha-2-macroglobulin (α₂M) is a large 720 kDa tetrameric protein that inhibits active proteases such as MMPs and binds a wide array of inflammatory mediators involved in PTOA^{19,26,29–31}. While most protease inhibitors act at the protease active site, α₂M forms a cage-like tetramer that physically entraps the protease². It is speculated that α₂M inhibits cytokines through non-covalent trapping in the molecular cage and covalent thiol-group interactions³¹. Although α₂M is found in both serum and synovial fluid, the synovial fluid concentration is only 15% of that in serum, a level insufficient to counteract the high levels of inflammation in the injured joint^31,35. Given the overlapping profile of α₂M substrates and biological PTOA drivers, our group previously demonstrated the effectiveness of intra-articular α₂M injections in decreasing cartilage degeneration in rats that underwent ACL transection (ACLT)^35,38. It is necessary to verify small animal findings in a large preclinical animal model, such as Yucatan minipig, before moving on to clinical trials^14,21,24,33. In addition, previous small studies varied the concentration of α₂M in each injection but not the number of injections^35,38. In this study, we investigate the effectiveness of a singular α₂M injection during the acute post-injury period compared with three serial injections to better characterize the “golden window” of treatment to attenuate future PTOA development.

Our group previously developed the modified intra-articular drilling (mIAD) model, involving drilling osseous tunnels adjacent to the ACL attachments to induce PTOA through inflammatory pathways without causing mechanical changes in the Yucatan minipig²⁷. This model was created as an alternative to the “idealized” ACL reconstruction developed by Heard et al., which entails coring out and immediately reattaching the femoral attachment of the ACL⁸. Similar to the “idealized” ACL reconstruction, the mIAD procedure models the PTOA-driving effects of inflammation⁸. However, the mIAD procedure is less invasive and, unlike the “idealized” ACL reconstruction, may avoid inducing additional trauma to the lateral extra-articular structures or cutting some fibers of the normal ACL during surgery²⁷. By isolating the effects of inflammation, the mIAD model allows for the specific investigation of the anti-inflammatory effects of therapeutic agents²⁷.

The primary objective of this study was to determine if intra-articular α₂M injections following mIAD effectively attenuate PTOA development in the Yucatan minipig by inhibiting inflammatory mediators. The second objective was to determine if three α₂M treatments were more effective in reducing PTOA development than one. We hypothesized that groups treated with either one or three α₂M intra-articular injections would have milder cartilage damage, synovitis, and inflammation than the untreated mIAD group but worse outcomes compared with the sham arthrotomy group. In addition, we hypothesized that there would be no changes in joint loading, as measured by gait analysis. Lastly, we hypothesized that the group receiving three α₂M injections would have less cartilage damage, synovitis, and inflammation than the group treated with one α₂M injection.

MATERIALS AND METHODS

Study Design

The study was approved by our Institutional Animal Care and Use Committee and the Animal Care and Use Review Office from the Department of Defense (Protocol number: 19–04-0001). The study was designed to meet ARRIVE guidelines²². Forty-eight Yucatan minipigs (24 castrated males, 24 females, age 15–16 months old, weight 54.6±5.8kg) were randomly allocated to four groups, each with 6 males and 6 females (n=12): (1) mIAD with control saline injections (mIAD+saline), (2) mIAD and one α₂M injection (mIAD+α₂M-1), (3) mIAD and three α₂M injections (mIAD+α₂M-3), and (4) sham surgery with no α₂M injection (negative control). Data on mIAD+saline and sham animals were reported in a previous study establishing the mIAD model²⁷. However, all surgeries, injections, and experiments were performed concurrently with the α₂M-treated animals as part of this present study. All animals were housed individually in adjacent pens for 15 weeks after surgery. The minimum pen size was 2.1m².

Surgical Technique

Surgeries were performed by a fellowship-trained, board certified orthopedic sports medicine surgeon (BDO). A medial arthrotomy was performed under general anesthesia to access the joint space of the left hind knee. Two tunnels 2mm in diameter and 15mm deep were drilled into the tibial bone adjacent to the anterior and posterior edges of the ACL insertion using Kirschner wires (Cordless Driver 4; Stryker, Kalamazoo, MI, USA). The drilling was repeated at the anterior medial and posterior lateral edges of the femoral ACL insertion. They were drilled at 6 and 12 o’clock positions on each footprint, allowing for reproducibility similar to an ACL reconstruction. These holes did not violate the articular cartilage. The joint was irrigated with sterile saline and closed in layers with buried sutures (Vicryl, Ethicon, Raritan, NJ, USA): Arthrotomy: 0 Vicryl, interrupted; Bursa: 2–0 Vicryl, running; Subcutaneous tissue: 2–0 Vicryl, interrupted; and Subcuticular layer: 3–0 Vicryl, interrupted. Animals were treated with an anti-vomiting agent (Odansetron) immediately after the operation. A fentanyl patch was placed on the dorsum of the pig for 3 days to manage post-operative pain. The sham group underwent the same arthrotomy procedure but without intra-articular drilling. The animals were not forced to exercise but appeared active when checked daily. The animals were euthanized (Beuthanasia-D Special, Merck, Madison, WI, USA) 15 weeks after surgery, and surgical hind knees were harvested.

Intra-articular Injections

Full-length human-derived α₂M, with molecular weight 725 kDA, was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Under general anesthesia, all animals received intra-articular injections of either α₂M or a control saline solution at three time points: immediately after surgery and at two and four weeks. 3mL of saline with a concentration of 2mg α₂M /mL was used to create the α₂M intra-articular injection solution. 3mL of saline was used as the control injection solution. The mIAD+α₂M-1 group received one α₂M injection immediately after surgery and two saline injections at two and four weeks. The mIAD+α₂M-3 group received α₂M injections immediately after surgery and at two and four weeks. The mIAD+saline and sham groups received intra-articular saline injections at the three time points. All animals received a total of 3 intra-articular injections. Intra-articular injections were performed via the medial infrapatellar site under sterile conditions with a 20-gauge needle. After injection, animals were treated with an anti-vomiting agent (Ondansetron). One dose of buprenorphine was given for pain.

Macroscopic Cartilage Assessment

The medial and lateral articulating surfaces of the tibial plateau and femoral condyle of the hind limbs were stained with India Ink. The length and width of the stained lesions were measured using an electronic caliper. The lesion area was approximated as an ellipse¹³.

Cartilage Preparation

Osteochondral specimens were taken from the medial and lateral compartments of the tibial plateau and femoral condyle in the surgical knee (10mm x 10mm x 25mm). One specimen was taken from each compartment for a total of 4 per knee per animal. The specimens were fixed in 10% formalin for 48 hours and stored in 70% ethanol. The tissue was rinsed with running tap water for 30 minutes, placed in cassettes, and transferred to a buffered formic acid decalcification solution (20% formic acid, 10% sodium citrate in dH₂O) at room temperature. The solution was changed every 3–4 days, and the decalcification progress was monitored using X-ray. This process took between 7–10 days, depending on the tissue size. After decalcification, the tissue was rinsed under running tap water for 30 minutes and transferred to 70% ethanol at room temperature. An automated tissue processor was used to dehydrate and infiltrate the tissue with paraffin under pressure. After infiltration, the tissue was trimmed to fit into embedding molds, ensuring that the two halves were embedded bifurcation-side down in the mold. Embedded samples were sectioned with a rotary microtome at 6μm and placed on positively charged slides. Slides were stained with Safranin O-Fast Green.

Microscopic Cartilage Assessment

Slides stained with Safranin O-fast Green were scored by 6 blinded evaluators (CS, KC, LW, BF, MH, and MP) using the Osteoarthritis Research Society International (OARSI) grading system for large animals ^18,32. Four features were evaluated: structure (0–10), chondrocyte density (0–4), cell cloning (0–4), and Interterritorial Safranin O-fast Green (0–4)^18,32. The total scores for the medial femoral condyle (MFC), medial tibial plateau (MTP), lateral femoral condyle (LFC), and lateral tibial plateau (LTP) were summed and averaged between reviewers.

Synovial Tissue Preparation

Samples of the synovial membrane were taken from the suprapatellar fold to obtain consistent sub-intimal fibrosis for comparison amongst the animals. Frozen synovium sections (10μm) were stained with hematoxylin & eosin. Three blinded evaluators (CS, GP, and LW) evaluated the synovium features using a modified OARSI grading system: intimal hyperplasia (0–3), inflammatory cell infiltration (0–3), and vascularity (0–3)¹⁸. For each observation, three areas of the synovium were randomly selected and scored. The average total scores for the three areas were averaged between reviewers.

Quantitative Real-Time PCR for Inflammatory Gene Analysis

The mRNA expression levels of IL-1β, NF-κB, and TNF-α in the synovial membrane were assessed using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) following the manufacturer’s instructions (iQ SYBR Green Supermix, Bio-Rad, Hercules, CA, USA). For this analysis, synovial membrane specimens from each group were randomly paired (n=6 for each group) and homogenized in TRIzol reagent (cat# 15596026, Invitrogen, Waltham, MA, USA) using a homogenizer. The total RNA was then extracted using the TRIzol reagent and purified with the RNeasy Mini Kit (cat# 74004, Qiagen, Hilden, Germany). The 28S:18S ratio for all samples met the 1.8:1 to 2:1 ratio determined by NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) to ensure RNA integrity. Gene expression was measured through first-strand cDNA synthesis using a reverse transcription kit (cat# 1708890, Bio-Rad, Hercules, CA, USA) and qRT-PCR using the iQ^™ SYBR Green Supermix kit (cat# 170–8887, Bio-Rad, Hercules, CA, USA) in a real-time PCR system (CFX Connect, Bio-Rad, Hercules, CA, USA).

Priming was conducted at 25°C for five minutes followed by reverse transcription at 40°C for 20 minutes. Reverse transcriptase inactivation was done at 95°C for one minute. For qRT-PCR testing, 18S was used as an internal control. Porcine-specific primers were designed and synthesized by Integrated DNA technologies (IDT, Coralville, IA, USA) as specified in Supplementary Table 1. PCR was performed for 40 cycles after an initial denaturation step at 95°C for 3 minutes. Each cycle involved an additional denaturation step for 15 seconds at 95°C, annealing for 60 seconds at 55°C or 58°C, and extension for 40 seconds at 72°C. The reaction was terminated at 70°C after a 10-minute extension. Three independent PCR experiments were performed to obtain the relative level of expression for each gene.

Synovial Lavage Collection

Immediately following euthanasia, 10mL of isotonic saline was injected into the knees of each animal, and the knees were flexed and extended 10 times before aspiration³⁷. 2–3 mL of synovial lavage from each animal were collected. The synovial fluid was centrifuged at 2,000g for 15 minutes at room temperature to remove cells and debris. The isolated synovial fluid was frozen at −80°C until analysis.

Enzyme-Linked Immunosorbent Assay

The porcine MMP-1 ELISA Kit (catalog #MBS2701168, MyBioSource, CA, USA) was performed following the manufacturer-provided protocol to quantify MMP-1 levels in synovial fluid (n=7 for each group). Samples were diluted 1:100 in each well with the provided sample buffer and incubated at 37°C for 2.5 hours. Wells were washed four times with wash buffer (10X dilution with ddH₂O, 0.3ml per well). After washing, the provided conjugate solution was added (0.1ml per well) and incubated with gentle shaking at room temperature for 60 minutes. After a second wash, Streptavidin-HRP solution was added (0.1ml per well) and incubated at room temperature for 45 minutes. After a third wash, TMB substrate was added (0.1ml per well) and incubated for 30 minutes at room temperature in the dark with gentle shaking until the substrate turned blue. The stop solution was then added (0.05ml per well). The plate was tapped gently to mix. The solution in the well changed from blue to yellow, and the absorbance values of sample wells were measured immediately at 450 nm within 30 minutes of adding the stop solution. The background absorbance was subtracted from all data points prior to plotting, and the concentrations for samples and control were obtained from the standard curve. A standard curve of MMP-1 protein was obtained for each assay.

Gait Assessment

To evaluate joint instability, six biomechanical gait parameters were measured: maximum force, contact area, peak pressure, impulse, stance time, and swing time³⁹. A pressure-sensing walkway system (23″× 130″) (HRV6 Walkway, Tekscan, Boston, MA, USA) was used to measure load asymmetry during gait. Load asymmetry was measured before (week 0) and four times after surgery (weeks 4, 8, 12, and 15). Gait assessment was conducted prior to intra-articular injections at 0, 4, and 8 weeks. Before gait analysis, the walkway system was calibrated following the manufacturer’s guidelines³⁹. The loading curves were checked to ensure the system was operating properly during data collection. At each time point, five successful walkway trials were performed and recorded. The differential hind limb loading between the left (surgery) and right (contralateral) hind limb was calculated across gait cycles and expressed as a ratio for the six parameters.

Statistical Analysis

Data were imported into SAS version 9.4 (SAS Institute Inc., Cary, NC) for hypothesis testing. Generalized linear mixed models were used to compare macroscopic lesion area, microscopic scores, synovial membrane changes, qRT-PCR mRNA levels, ELISA protein levels, and gait ratios across experimental conditions. Generalized linear mixed models were used to allow for the analysis of data with repeated and longitudinal measurements as well as subjects with missing observations. A Gaussian distribution was used to analyze macroscopic damage area, qRT-PCR mRNA in the synovium, ELISA protein levels in the synovial fluid, and gait measurements. A binomial distribution was used for microscopic cartilage damage and microscopic synovial membrane scores. The Holm test was used for multiple comparisons, and an adjusted p-value of 0.05 was used as the threshold for statistical significance. Power for the microscopic OARSI damage scores was estimated to be 80% for detecting a 6-point difference using a one-tailed pairwise comparison. A sample size of 12 minipigs per group allowed us to maintain at least 80% power for all hypothesis tests.

RESULTS

Macroscopic Cartilage Assessment

In the mIAD+saline group, all animals showed macroscopic cartilage degeneration (Figure 1A). Six animals showed minor damage in the mIAD+α₂M-1 group. Eight animals showed minor degeneration in the mIAD+α₂M-3 group. Six animals showed no macroscopic damage in the sham group. Animals receiving one (p<0.001) and three (p=0.001) treatments of α₂M as well as sham animals (p=0.016) had significantly lower lesion areas than the untreated mIAD animals (Figure 1B). The total lesion area was statistically similar among the mIAD+α₂M-1, mIAD+α₂M-3, and sham groups.

Figure 1.

Macroscopic Cartilage Damage. (A) Images of median lesion areas stained with India Ink for each group: modified intra-articular drilling (mIAD)+saline, mIAD+α₂M-1, mIAD+α₂M-3, and sham. Red arrows indicate areas of India Ink-stained lesions. (B) Total lesion area for each group. Error bars represent the standard deviation. An * indicates significant differences.

In all animals, most of the damage was localized in the medial femoral condyle (mIAD+saline: 17.4±13.5mm²; mIAD+α₂M-1: 2.3±4.6mm²; mIAD+α₂M-3: 3.7±6.3mm²; sham: 8.2±15.0mm²) and medial tibial plateau (mIAD+saline: 19.7±16.9mm²; mIAD+α₂M-1: 1.5±4.5mm²; mIAD+α₂M-3: 0.9±1.2mm²; sham: 4.2±8.1mm²). Minimal lesions were present in the lateral femoral condyle (mIAD+saline: 4.5±7.0mm²; mIAD+α₂M-1: 0.1±0.2mm²; mIAD+α₂M-3: 1.3±4.5mm²; sham: .0±.0mm²) and lateral tibial plateau (mIAD+saline: 2.9±6.9mm²; mIAD+α₂M-1: 1.5±4.5mm²; mIAD+α₂M-3: 0.8±2.7mm²; sham: 2.9±10.mm²).

Microscopic Cartilage Assessment

In the mIAD+saline group, we observed degeneration affecting 10–25% of the cartilage surface area, severe surface irregularities, decrease in chondrocyte density, increased chondrocyte cell cloning, decreased interterritorial Safranin-O staining to the middle zone, and occasional erosions to the deep zone (Figure 2A). On the other hand, in the mIAD+α₂M-1 (Figure 2B), mIAD+α₂M-3 (Figure 2C), and sham groups (Figure 2D), we observed degeneration affecting less than 10% of the cartilage surface area, slight surface irregularities, minimal changes in chondrocyte density, minimal chondrocyte cell cloning, and no changes in interterritorial Safranin-O staining. The severity of microscopic cartilage damage score was significantly lower in the mIAD+α₂M-1 (9.3±8.1, p=0.008), mIAD+α₂M-3 (11.0±9.7, p=0.043) and sham groups (9.9±10.2, p=0.043) compared with the mIAD+saline (22.6±7.9) (Figure 2E). There were no significant differences in OARSI scores among the mIAD+α₂M-1, mIAD+α₂M-3, and sham groups.

Figure 2.

Microscopic cartilage damage. Histological images of the median medial femoral condyle and medial tibial plateau stained with Safranin O-fast green for (A) modified intra-articular drilling (mIAD)+saline, (B) mIAD+α₂M-1, (C) mIAD+α₂M-3, and (D) sham. Boxed areas indicate regions of cartilage degeneration. (E) The Osteoarthritis Research Society International (OARSI) scores measuring structure, chondrocyte density, cell cloning, and interterritorial staining are shown. Error bars represent the standard deviation. An * indicates significant differences.

Changes in the Synovial Membrane

We observed moderate diffuse hyperplasia, diffuse inflammatory cell infiltration, and the presence of vascular elements in the mIAD+saline synovium (Figure 3A). Though less severe, we observed mild hyperplasia and mild inflammatory cell infiltration but no changes in vascularity in the mIAD+α₂M-1 (Figure 3B) and mIAD+α₂M-3 (Figure 3C). The sham group had minimal changes in the synovial features (Figure 3D). The synovial membrane scores of the mIAD+α₂M-1 (2.5±0.8, p<0.0001), mIAD+α₂M-3 (1.9±1.1, p<0.0001), and sham (0.7±0.3, p<0.0001) animals were significantly lower than the mIAD+saline (5.9±1.8) (Figure 3E). While the synovial membrane scores of mIAD+α₂M-1 and mIAD+α₂M-3 were statistically similar, the scores of the sham were significantly lower than mIAD+α₂M-1 (p<0.0001) and mIAD+α₂M-3 (p<0.0001).

Figure 3.

Synovitis from the modified intra-articular drilling (mIAD) surgery. (A) Histological images of the median synovial membrane from the suprapatellar fold for mIAD+saline, (B) mIAD+α₂M-1, (C) mIAD+α₂M-3, and (D) sham. (E) Microscopic synovial membrane scores measuring hyperplasia, inflammatory cell infiltration, and vascularity. Error bars represent the standard deviation. An * indicates significant differences.

IL-1β, NF-κB, and TNF-α mRNA levels in the synovial membrane of mIAD+saline were approximately twice the levels in mIAD+α₂M-1 (IL-1β: p=0.0004; TNF-α: p= 0.0002; NF-κB: p=0.0004), twice the mIAD+α₂M-3 (IL-1β: p=0.0004; TNF-α: p=0.0006; NF-κB: p= 0.0030), and three times the sham (IL-1β: p=0.0003; TNF-α: p<0.0001; NF-κB: p=0.0004) (Figure 4). Levels of IL-1β, NF-κB, and TNF-α mRNA expression were not significantly different among mIAD+α₂M-1, mIAD+α₂M-3, and sham groups.

Figure 4.

Differential mRNA expression of inflammatory mediators in each treatment group measured by reverse transcription-quantitative polymerase chain reaction. (A) Fold change of IL-1β mRNA, (B) NF-κB mRNA, and (C) TNF-α mRNA mRNA levels in the synovial membrane. Error bars represent the standard deviation. An * indicates significant differences.

MMP-1 levels in the synovial fluid of the mIAD+α₂M-1 (0.08±0.02 ng/mL, p=0.0292), mIAD+α₂M-3 (0.08±0.03 ng/mL, p=0.0292), and sham (0.03±0.01 ng/mL, p<0.0001) groups were significantly lower compared with the mIAD+saline group (0.13±0.05 ng/mL) (Figure 5). However, MMP-1 levels were significantly higher in the mIAD+α₂M-1 (p=0.0292) and mIAD+α₂M-3 (p=0.0292) than the sham group. There were no significant differences between mIAD+α₂M-1 and mIAD+α₂M-3.

Figure 5.

Alpha-2-macroglobulin treated groups had lower levels of MMP-1 in the synovial fluid compared with the untreated modified intra-articular drilling group but higher levels than the sham surgery group. Error bars represent the standard deviation. An * indicates significant differences.

Gait Analysis

In the mIAD+saline group, there were no significant changes in gait ratios between preoperative (week 0) and postoperative (weeks 4, 8, 12, and 15) measurements for maximum force, impulse, stance time, and swing time (Figure 6). An increase in the peak pressure ratio (p=.0045) and a decrease in the contact area ratio (p=.0013) was detected between week 0 and week 8. In the mIAD+α₂M-1 group, a decrease in the impulse ratio was detected between week 0 and week 8 (p=.0342). Similarly, in the mIAD+α₂M-3 group, a decrease in the maximum force was detected between week 0 and week 4 (p=.0044). Furthermore, in the mIAD+α₂M-3 group, all 12 animals experienced a limp after the second and third injections that started after treatment but resolved within 3–5 days. There were no significant differences between preoperative and postoperative time points for all gait indicators in the sham group. Moreover, all gait ratios were centered around 1.

Figure 6.

Line charts of the six gait indicators expressed as a ratio of left to right hind limb (A) Maximum force, (B) contact area, (C) peak pressure, (D) impulse (E) stance time, and (F) swing time. Error bars show the mean±SD. An * indicates significant differences between gait ratios of preoperative and postoperative time points.

DISCUSSION

Although surgical treatments for an injured joint attempt to restore native biomechanics, evidence suggests that they do not eliminate PTOA risk (13–15). The upregulation of pro-inflammatory cytokines and proteases during the acute stage of injury continue to linger and promote long-term catabolic alterations^5,8,11,17. Therefore, treatments targeted against a single catabolic mediator are unlikely to effectively halt PTOA pathogenesis^10,12. To explore the therapeutic potential of a broad-spectrum agent, we investigated the effects of intra-articular α₂M injections in minipigs undergoing a previously developed intra-articular drilling model²⁷, which induces a significant amount of early-stage PTOA through inflammatory pathways without causing mechanical changes. The results of this study support the primary hypothesis that intra-articular α₂M injections in minipigs undergoing intra-articular drilling effectively reduce cartilage degeneration and synovial inflammation at 15 weeks. However, the results of this study did not support the secondary hypothesis that three injections confer greater chondroprotective benefits than one. Collectively, these findings exemplify the therapeutic potential of α₂M as well as the importance of early anti-inflammatory intervention.

The present gross and histological findings demonstrate that both one and three intra-articular α₂M injections reduce cartilage degeneration to a degree similar to the sham surgery group. Previous small animal studies using ACL transection have similarly documented stark decreases in cartilage degeneration following weekly intra-articular α₂M injections^35,38. However, unlike the present study, the degree of cartilage degeneration remained significantly higher than that of the sham surgery group^35,38. This difference may be attributed to the chronic instability of an ACL transected knee, which results in excessive anterior tibial translation and subsequent cartilage damage²⁰. As a result, the ACLT model may not capture the clinically restabilized joint, and the mechanical cartilage damage in these studies may overshadow the therapeutic benefits of α₂M. Furthermore, these small animal findings must be validated in a larger animal model before clinical trials. As such, this study utilized the adolescent Yucatan minipig, an established pre-clinical PTOA animal model with significant anatomic and functional resemblances to the human knee^14,21,24,33.

The attenuation of cartilage degeneration observed in the present and previous studies^35,38 is likely a product of the anti-inflammatory effects of α₂M on the synovial membrane¹⁶. When inflamed, the synovium secretes pro-inflammatory cytokines and proteases into the synovial fluid, which mediate further inflammation in intra-articular tissues and destruction of articular cartilage^25,28,36. At 15 weeks post-surgery, the synovium from untreated minipigs demonstrated significant synovitis and increased IL-1β, NF-κB, and TNF-α mRNA expression that dovetailed the magnitude of observed cartilage degeneration. Minipigs treated with α₂M had similar IL-1β, NF-κB, and TNF-α mRNA levels as the sham. These results are reflected histologically, with α₂M treated minipigs having reduced synovitis compared to untreated minipigs. Similarly, α₂M significantly reduced MMP-1 protein levels in the synovial fluid of α₂M treated animals. Given that α₂M inhibits a variety of cytokines and proteases, the reductions in IL-1β, NF-κB, and TNF-α mRNA and MMP-1 protein levels are likely reflective of a combination of upstream intracellular inhibition of inflammatory signaling and downstream binding of translated proteins^{19,26,30,31,35}. Ultimately, the alteration of the synovial environment to a less catabolic phenotype likely reduced the susceptibility of the articular cartilage to degeneration⁹. Although this study focused on the therapeutic effects derived from reducing synovial inflammation due to its key upstream role in mediating the heightened inflammatory response to injury, previous studies suggest additional chondroprotective mechanisms. Sun et al. found that in chondrocytes incubated with IL-1β, α₂M enters chondrocytes and intracellularly binds IL-1β, decreasing NF-κB, TNF-α, MMP-1, MMP-3, and MMP-13 mRNA expression while simultaneously increasing collagen 2 and aggrecan expression²⁶.

Our results did not support the secondary hypothesis that three α₂M injections would have greater chondroprotective effects than one. There were no significant differences in amount of cartilage damage, synovitis, inflammatory mRNA expression, or MMP-1 levels between mIAD+α₂M-1 and mIAD+α₂M-3 groups. Previous studies in humans have demonstrated that within one week of injury, levels of cytokines, including IL-1β, IL-6, IL-8, and TNF-α, and MMPs are increased in the joint^3,7,15. These acute changes develop into long-term alterations, noted by decreases in Il-1 receptor antagonist and increases in TNF-α³. The changes in joint chemistry have led researchers to propose the existence of a “golden window” of treatment during the acute stages, which may attenuate PTOA development. The similarity in mIAD+α₂M-1 and mIAD+α₂M-3 outcomes suggests that one singular injection of α₂M may be sufficient to control the acute inflammatory response and prevent chronic pathology.

To the best of our knowledge, this study is the first to investigate the effects of the number and timing of α₂M injections by treating minipigs with either three injections immediately after surgery and 2 and 4 weeks after surgery or just one injection immediately after surgery. Previous study designs involved treatment with an equal number of injections at identical time points but with different concentrations or variants of α₂M (21,22). For instance, in chondrocytes derived from humans with osteoarthritis, Wang et al. found that treatment with greater concentrations of exogenous α₂M had a greater inhibitory effect on IL-1 and MMP-13³⁵. Similarly, rats treated with 2 IU α₂M/kg demonstrated significantly lower cartilage degeneration than rats treated with 1 IU/kg³⁵. Future work should evaluate the chondroprotective and anti-inflammatory effects of varying concentrations in a singular intra-articular α₂M injection immediately after surgery.

This study has several limitations. First, the α₂M used was derived from human plasma. Although no meaningful changes in gait were detected in the mIAD+α₂M-3 group, we observed consistent limping in all 12 animals that started after the second and third α₂M injections but resolved within 3–5 days. This limping was not captured in the gait analysis because gait measurements were collected prior to intra-articular injections for all 48 animals. This potentially indicates that animals in the mIAD+α₂M-3 group developed an immune reaction to human-unique epitopes of the allogenic α₂M. This speculated immunologic reaction may have temporarily increased inflammation and reduced the therapeutic properties of multiple α₂M treatments as we observed in this study. Unfortunately, we did not collect blood samples and could not test for anti-human α₂M antibodies. Second, the development of PTOA was monitored only until 15 weeks, and at this time point, α₂M dramatically attenuated PTOA development. However, if the mild synovitis present at 15 weeks in α₂M treated pigs worsened and increased catabolic drivers in the joint, we could expect to observe gradual increases in cartilage degeneration at later follow-up times. To address these limitations, future studies should use autogenic α₂M to avoid potential immunologic confounders and increase the study period to measure long-term outcomes.