Integrin β1, PDGFRβ and type II collagen are essential for meniscus regeneration by synovial mesenchymal stem cells in rats

Animals

All animal care and experiments were conducted in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and the institutional guidelines of the Animal Committee which includes the first author as a member. Male/female Lewis rats (6-10 weeks old) were purchased from Jackson Laboratory Japan, Inc. (Yokohama, Japan) (total of 130 animals). All rats were allowed free access to food, water, and activity. Rats were maintained under a 12 h dark-light cycle at controlled temperature (20-26°C) and humidity (30-70%).

Rat Synovial MSC Isolation

Male rats were euthanized by bleeding from the inferior vena cava under isoflurane anesthesia. The synovium was harvested from the infrapatellar fat pad of both knees of 10 rats, pooled and minced. The minced synovium was then digested with 0.3% collagenase V solution (Merck KGaA, Darmstadt, Germany) in a 37°C water bath for 2 h. Cells were cultured in α-minimum essential medium (αMEM; Thermo Fisher Scientific, Waltham, MA, USA) with 20% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA) for 8 days under 5% CO2 and 37°C. Cells were then harvested, prepared as the original stocks, and cryopreserved in a freezer (CLN-1700CWE, Nihon Freezer, Tokyo, Japan) at -150°C. For use, synovial MSCs from the original stocks were grown for 7 days, harvested and combined. The cells were used for further analyzes without any sorting. The cells showed surface antigens and multidifferentiation abilities similar to those of MSCs, as previously reported36.

Neutralizing MSCs treated with antibodies

Neutralizing antibodies were anti-integrin β1 antibody (purified anti-mouse/rat CD29 antibody; BioLegend Inc., San Diego, CA, USA), anti-PDGF Receptor β antibody (R&D Systems Inc., Minneapolis, MN, USA ), and anti-CD44 antibodies (Absolute Antibody Ltd., Cleveland, UK). Controls were Purified Armenian Hamster Isotype Ctrl IgG (BioLegend Inc.), Goat Normal IgG Control (R&D Systems Inc.), and Isotype Ctrl IgG (Thermo Fisher Scientific, MA, USA), respectively. . Synovial MSCs at 5 × 106 cells were suspended in 900 μL PBS with 2% FBS to neutralize integrin β1 or 450 μL PBS with 2% FBS to neutralize PDGFRβ. A sample of 50 μg of neutralizing antibodies for integrin β1 and CD44 and 100 μg for PDGFRβ were added to the suspensions, and the cells were left to react for 1 h on ice, washed and corrected. The final volume was then adjusted with vehicle to give a concentration of 5 × 106 cells/50 μL.

Cell adhesion assay

Untreated MSCs, IgG-treated MSCs, and 1 × 10 β1 integrin-neutralizing antibody treated MSCs5 were suspended in 10 µL of PBS with 10 mM of MgCl2 and placed for 10 min on 8-well culture slides coated with type I collagen (Corning Inc., Corning, NY, USA). After washing the slides with PBS containing 10 mM MgCl2the number of cells in a high power field (HPF) was counted20.

Cell proliferation test

Untreated MSCs, IgG-treated MSCs, and PDGFRβ neutralizing antibody-treated MSCs were plated at 1 × 103 cells/100 μL in complete medium containing 20% ​​FBS in 96-well plates (Corning). The next day, the medium was replaced with 100 μL of αMEM supplemented with 0.5% FBS and 4 ng/mL of PDGF-BB (R&D Systems Inc.). Untreated MSCs were also cultured in αMEM supplemented with 0.5% FBS but without PDGF-BB. These MSCs in the four experimental conditions were cultured at 37°C for 6 days37. Cell proliferation rate was determined by ATP assay using Cell Titer Glo (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol.

KO of Col2a1, Vcam1and Tnfr1 genes in synovial MSCs

Synovial MSCs from the original stock were cultured in 24-well plates (Corning Inc.) at a density of 8 × 104 cells/well in 500 μL of complete medium for 1 day. For Cas9 protein transfection, 50 μL of mix containing TrueCut Cas9 Protein v2 (Invitrogen, cat# A36498), Lipofectamine CRISPRMAX Cas9 Transfection Reagent (Thermo Fisher Scientific), Lipofectamine Cas9 Plus Reagent (Thermo Fisher Scientific), Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and 240 ng Custom Modified TruGuide gRNA (Thermo Fisher Scientific) for the Col2a1, Vcam1and Tnfr1 genes (Table S1) were added and the plates were incubated for 2 days. Target gene editing was confirmed by extracting DNA from 1 × 105 cells, amplifying the target gene by PCR and detecting the mutant gene using the GenArt Genomic Cleavage Detection Kit (Thermo Fisher Scientific). For cell cloning, cells with mutant genes were collected, plated in 96-well plates at a concentration of 0.5 cells/well, and cultured to provide more than 108 cells for transplantation. To confirm gene inactivation, DNA was extracted from 1 × 105 cloned cells and its sequences were confirmed by Sanger sequencing (Fig. S10, Table S2–S4).

In vitro chondrogenesis test

In a 15 mL polypropylene tube, 2.5 × 105 synovial MSCs were suspended in 1 mL of chondrogenic medium composed of high glucose DMEM (Thermo Fisher Scientific), including 10 ng/mL TGF-β3 (R&D Systems Inc.), 3.92 μg/mL dexamethasone (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), 50 μg/mL L-ascorbic acid 2-phosphate (Cayman Chemical Company, Ann Arbor, MI, USA), 40 μg/mL L-proline (MP Biomedicals, Irvine, CA, USA), 1 μg/mL sodium pyruvate (Thermo Fisher Scientific), ITS-X Supplement 1% (×100) (FUJIFILM Wako Pure Chemical Corp.) and 0.5 μg/mL BMP- 2 (R&D Systems Inc.). The cells were pelleted by centrifugation at 450 g for 10 min. The pellets were cultured for 3 weeks, the medium being changed every 3 to 4 days12,19,29. For histological analysis, pellets were embedded in paraffin, cut into sections 5 μm thick, stained with safranin-O and toluidine blue, and immunostained for type II collagen with using purified anti-hCL(II) IgG antibody (KYOWA PHARMA CHEMICAL CO., LTD., Toyama, Japan)15.38.

MSC meniscectomy and synovial graft

Female rats were anesthetized with isoflurane. The right and left knee joints were operated. A medial parapatellar incision and lateral patellar tendon dislocation were performed to expose the medial meniscus. The anterior insertion ligament of the medial meniscus was cut to dislocate the medial meniscus anteriorly, and the medial meniscus was resected at the level of the medial collateral ligament. The capsule was closed using nylon sutures and 5 × 10 MSC6 cells/50 µL or vehicle were injected into the knee joint using a 28G needle. (All MSCs and vehicle were stored on ice until administration). The knee joint was moved three times and the skin sutured15. Surgical treatment and administration were performed together by condition rather than randomly, in the order of cell preparation. The time required was approximately 40 min per animal for both knees. Rats were allowed to walk freely in their cages after surgery. Eight rats per condition were used for the Integrin β1 and PDGFRβ experiments, 6 or 7 for the CD44 experiments, and 7 for each of the Vcam1 IS, Tnfr1 KO and Col2a1 knockout experiences.

Evaluation of the regenerated meniscus

Three weeks after MSC injection, the knee joint was removed and the medial meniscus was photographed. The regenerated area of ​​the meniscus was measured with the J 1.53e image (National Institutes of Health, Bethesda, MD, USA). The meniscus was immersed in 10% neutral buffered formalin solution and decalcified with 0.5% EDTA (pH 7.5) for 3 days at 4°C, followed by replacement of the gradient with 20% sucrose for 24 h at 4°C. The center of the regenerated area of ​​the meniscus was sectioned radially and observed histologically with safranin-O staining.

statistical analyzes

Comparisons between the two groups were made using an unpaired t-test, and comparisons between the three or more groups were made using a one-way ANOVA with Bonferroni’s multiple comparison test . Statistical tests were performed using GraphPad Prism ver. 5.04 (GraphPad Software Inc., San Diego, CA, USA). A P a value < 0.05 was considered statistically significant. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p< 0.0001.

Ethics approval

All experimental protocols and studies have been approved by the animal care and use committee (reference number: A-1–200 136, A-1–210 027, A-1–210 031, A-1– 210033, A-1–210069, A-1–210072) of FUJIFILM Corporation. All animal care and experiments were performed in accordance with the institutional guidelines of the Animal Committee of FUJIFILM Corporation.

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