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 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.
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.
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 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.
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.
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.
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 . 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.
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. 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
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. 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. 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.
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
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.