Alpha-2-Macroglobulin Attenuates Posttraumatic Osteoarthritis Cartilage Damage by Inhibiting Inflammatory Pathways With Modified Intra-articular Drilling in a 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 proinflammatory cytokines involved in the pathogenesis of PTOA.

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 preclinical Yucatan minipig model and (2) to determine if 3 intra-articular α₂M injections have greater chondroprotective effects compared with 1 intra-articular injection.

Study design: Controlled laboratory study.

Methods: A total of 48 Yucatan minipigs were randomized into 4 groups (n = 12 each): (1) modified intra-articular drilling (mIAD) and saline (mIAD + saline), (2) mIAD and 1 intra-articular α₂M injection (mIAD +α₂M-1), (3) mIAD and 3 α₂M injections (mIAD +α₂M-3), and (4) sham control. Surgical hindlimbs were harvested at 15 weeks after surgery. Cartilage degeneration, synovial changes, inflammatory gene expression, and matrix metalloproteinase 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 the mIAD +α₂M-1 and mIAD +α₂M-3 groups compared with the mIAD + saline group (P < .05) and similar to those in the sham group (P > .05). Synovial membrane scores of the mIAD +α₂M-1 and mIAD +α₂M-3 groups were lower than that of the mIAD + saline group (P < .05) and higher than that of the sham group (P < .05). Interleukin-1 beta, nuclear factor kappa B, and tumor necrosis factor alpha mRNA expression in the synovium and matrix metalloproteinase-1 levels in synovial fluid were significantly lower in the mIAD +α₂M-1 and mIAD +α₂M-3 groups compared with the mIAD + saline group (P < .05). No significant differences were observed between the mIAD +α₂M-1 and mIAD +α₂M-3 groups for all measured outcomes. There were early changes in gait (P < .05) between preoperative and postoperative time points for the mIAD + saline, mIAD +α₂M-1, and mIAD +α₂M-3 groups that normalized by 15 weeks.

Conclusion: Animals receiving early α₂M treatment exhibited less cartilage damage, milder synovitis, and lower inflammation compared with 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, an early intervention with α₂M may have the potential to reduce cartilage degeneration from catabolic pathways and delay the development of PTOA.

Keywords: alpha-2-macroglobulin; inflammation; posttraumatic osteoarthritis; synovium.

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.

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.

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.

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.

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.

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.