Isolation of mouse adipose-derived mesenchymal stem cells
To isolate murine AD-MSCs, adipose tissues were isolated from 8 ~ 10 weeks old male C57BL/6J mice. Adipose tissues were digested at 37°C for 30 min with 0.075% type I collagenase after washed with sterilized PBS. Enzyme activity was neutralized with α-MEM, containing 10% FBS and centrifuged at 200 g for 10 min to obtain a pellet. The pellet was incubated overnight at 37°C/5% CO2 in control medium (α-MEM, 10% FBS, 100 units/ml of penicillin, 100 µg/ml of streptomycin). Following incubation, the tissue culture plates were washed to remove residual nonadherent cells and maintained at 37°C /5% CO2 in control medium. When the monolayer of adherent cells has reached confluence (P0), cells were trypsinized (0.25% trypsin; Sigma), resuspended in α-MEM containing 10% FBS, and sub-cultured at a concentration of 2,000 cells/cm2. Cells of passage 3 ~ 5 were used in the following study. To collect peripheral blood, C57BL/6J mice were anesthetized with isoflurane. Peripheral blood was collected into a microcentrifuge tube containing heparin. To lyse red blood cells, harvested blood was treated with red blood cell lysis buffer (Bio Legend, San Diego, CA, USA). Collected cells were resuspended in complete culture medium containing α-minimum essential medium (α‐MEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 17% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin‐streptomycin-neomycin. Then, cells were seeded in 6‐well plates at a density of 0.5 × 106 cells/cm2. Culture medium was half changed every 3 d. Once cells reached a confluence of over 50%, cells were passaged regularly and cultured in complete culture medium supplemented with 10% FBS. Cells of passage 3 ~ 5 were used in the following assays.
Immunophenotypic analysis
Cells of passage 3 were enzymatically digested with 0.25% trypsin-EDTA (Thermo Fisher Scientific) for 30 s at room temperature, respectively. Cells were resuspended in resuspension buffer containing 1% BSA (Millipore Sigma) at 106 cells/ml. We transferred a volume of 300 µl cells into a 5‐ml flow cytometry tube. Cell aliquots were incubated with PE‐conjugated CD29 (SC018; R&D Systems, Minneapolis, MN, USA), APC‐conjugated Sca‐1 (stem cell antigen 1; SC018; R&D Systems, USA), peridinin chlorophyll protein (PerCP)‐conjugated CD45 (SC018; R&D Systems), or each corresponding isotype control antibody (SC018; R&D Systems), respectively, for 30 min at room temperature in the dark after washing with PBS. Cells were resuspended in 1000 µl resuspension buffer after washing with PBS. The cell suspension was analyzed by flow cytometric analysis using CellQuest software (BD Biosciences). BD FACS Canto II was used for data acquisition by adjusting voltage and compensation using appropriate excitation and detection channels. Data analysis was performed using Flow Jo (BD Biosciences) software.
Safranin O-fast green staining and Alcian Blue staining
Samples were fixed in 4% PFA and embedded in paraffin. Safranin O staining [0.1% (wt/vol) Safranin O in 0.1% (vol/vol) in acetic acid] or Alcian blue (Sigma-Aldrich) were applied. The staining results were viewed under a phase contrast microscope (Leica Microsystems, Wetzlar, Germany).
Destabilization of the medial meniscus surgery-induced Osteoarthritis Model
The surgical procedures were approved by Animal Experimental Ethical Committee of the Chinese University of Hong Kong and performed following the well-established protocol [24]. Twenty-eight C57BL/6J mice (8–10 weeks old male) were anesthetized with peritoneal injection of ketamine (50 mg/kg), and destabilization of the medial meniscus (DMM) surgery was performed with a microsurgical scalpel on the right knee by sectioning of the medial meniscotibial ligament (MMTL) that anchored the medial meniscus (MM) to the tibial plateau. The lateral meniscotibial ligament was identified and protected during the surgery. Contralateral knee joints received sham surgery in which the MMTL was exposed without sectioning. Mice were allowed completely free movement following DMM surgery. Eight weeks later, the mice were terminated and the knee joints were harvested for histological analysis and other examinations.
Intra-articular injections of MSCs
After DMM surgeries, mice were randomly allocated into three groups (n = 7 per group), which were treated with saline (10 µl, vehicle control), mouse PB-MSCs (PB-MSCs, 5 × 105 cells/mouse), mouse AD-MSCs (AD-MSCs, 5 × 105 cells/mouse) three times per week after surgery through 32-gauge micro-needles (Hamilton, Sigma-Aldrich). The mice underwent sham surgeries without injection were served as healthy controls (n = 7). To verify the accurate intra-articular injection, Alcian blue dye was injected in a pilot study. To avoid any leakage, the injections were performed by gently inserting the needle into the joint at a depth around 3 mm, waiting for 5 seconds and then slowly withdrawing the needle. The procedures were performed under isoflurane anesthesia.
3D microstructure of subchondral bone
Subchondral bone microstructure under medial side of tibial plateaus was determined by high-resolution micro-CT 40 system (Scanco Medical, Wangen-Brüttisellen, Switzerland). Besides the total subchondral bone, trabecular bone compartment was also segregated by selecting the region of interest at the cancellous bone and excluding the subchondral plate and the calcified growth plate. A total of one hundred 3D sagittal images of the tibiae medial subchondral bone (n = 7) were evaluated at global threshold (158 mg hydroxyapatite/cm3) and a Gaussian filter was used (sigma: 0.8, support: 2) to suppress image noise. Parameters including bone mineral density (BMD) and bone volume/ total tissue volume (BV/TV) were determined with a built-in program (Image Processing Language v4.29d, Scanco Medical) according to well-established protocols [25].
Histological assays for joint tissue sectioning
Joint tissue samples were fixed in 4% (wt/vol) paraformaldehyde (PFA), then subjected to 0.5 M EDTA at 4°C with constant shaking for decalcification. Decalcified tissue samples were paraffin embedded, sectioned at 5 µm, dewaxed, and rehydrated by standard procedures. The slides were stained with Hematoxylin Eosin (H & E) and Safranin O-fast green staining. The Osteoarthritis Research Society International (OARSI) OA scoring system was adopted to analyze the pathological changes in the joint as previously described [26]. Briefly, the depth of the cartilage damage as well as the extent of the damaged surface was scored in a blinded manner at two different locations in the rat knee joint, i.e., the lateral and medial tibial articular cartilage. The OA score was defined as the product of the multiplication of these two scores. The mean OA score was calculated using the scores for the two individual locations examined by three researchers.
Immunohistochemistry assays
To detect the expression of matrix metalloproteinase13 (MMP13) in articular cartilage, immunohistochemistry was performed. Tissue samples were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Paraffin sections were deparaffinized, blocked in 0.3% hydrogen peroxide in methanol for 20 min and rehydrated through graded alcohol. Antigen retrieval was performed by incubating in a 10 mM warm citrate buffer (pH 6.0) for 20 min at 60℃. The sections were incubated with primary antibodies (in blocking solution): anti-MMP13 (1:300; ab219620, Abcam, USA) overnight at 4℃ after blocked with blocking solution (5% animal serum in PBS /1% bovine serum albumin). 5 random microscopic fields were captured from each sample and images were captured under a phase-contrast microscope (Leica Microsystems, Wetzlar, Germany).
Enzyme-linked immunosorbent assays (ELISAs)
Peripheral sera were harvested through centrifugation of peripheral blood mixed with heparin. The secretion of serum inflammatory cytokines of Interleukin-6 (IL-6) in peripheral sera was measured using Mouse IL-6 Quantikine ELISA kits (SM6000B; R&D Systems, Minneapolis, MN, USA) following the kit instructions.
Statistical analysis
All statistical analysis was performed by the statistical software SPSS 15.0 (SPSS, Chicago, Illinois, USA). Data were presented as means ± SEM among groups. OA score in animal study was compared using Kruskal-Wallis test with Dunn’s post-test. Data from animal study except for OA score were analyzed by one-way analysis of variance (one-way ANOVA) followed by post hoc multiple comparison tests (Tukey’s test when equal variance was assumed, or Games-Howell test when equal variance was not assumed). A p-value < 0.05 was considered significant.