Scientific Papers

The therapeutic effects of gingival mesenchymal stem cells and their exosomes in a chimeric model of rheumatoid arthritis | Arthritis Research & Therapy


GMSC isolation

Human gingival tissue samples were obtained as remnants of clinically healthy discarded gingiva following routine periodontal gingivectomy procedures at the College of Dentistry, The Ohio State University, under an approved Institutional Review Board (IRB) protocol. Gingiva-derived mesenchymal stem cells were isolated and cultured as previously described [29]. Briefly, GMSC were liberated from the tissue by sequential digestion with 2 mg/mL dispase II overnight and then 4 mg/mL collagenase IV for 2 h and then cultured in alpha-minimum essential medium (α-MEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% fetal bovine serum (FBS) (Clontech Lab, Inc., Mountain View, CA, http://www.clontech.com), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells from the fourth to sixth passages were used in experiments.

RASF

Human synovial tissue or synovial fluid samples were collected from RA patients during synovectomy, arthroplasty, or arthrocentesis under an approved IRB protocol. Tissue samples were digested with 4 mg/mL collagenase IV for 1 h and then mechanically dissociated with the GentleMACS (Miltenyi Biotec, Gaithersburg, MD). The cell suspension was diluted in DMEM and placed in culture for 2 days after which non-adherent cells were washed out and RASF were expanded. Experiments were performed with RASF between passages 2 and 6.

Exosome isolation and characterization

Exosomes were isolated from GMSC culture conditioned medium by ultracentrifugation as previously described [30]. Briefly, GMSC were cultured in exosome free medium for 2–3 days, the culture medium was then centrifuged at 300 g for 10 min, and the supernatant was subsequently centrifuged at 2000 g for 10 min; then, the supernatant was centrifuged at 10,000 g for 30 min. The supernatant was then centrifuged at 100,000 g for 70 min, and the pellet was washed with PBS and then centrifuged again at 100,000 g for 70 min. The exosomes utilized in these experiments were analyzed with a Nanosight NS300 and found to have an average size of 104.1 ± 5.5 nm and an average concentration of 6.17 × 107 ± 5.81 × 106 particles/mL.

Chimeric synovitis model

Animals were group housed in Allentown NexGen IVC rack caging system (Allentown, NJ) on corncob bedding with a compressed cotton nestlet. Cages were autoclaved before use, irradiated feed (Envigo Teklad 7912) was provided ad libitum and reverse osmosis, and chlorinated water was provided via automatic water valves. Cages were changed every 2 weeks in a biosafety cabinet, and light was provided on a 12:12 cycle. Inhaled isoflurane 1–5% in 100% oxygen was used for anesthesia. Peri-operative analgesia included an NSAID, either 0.1 ml meloxicam (0.5 mg/ml; 5 mg/ml Metacam (Boehringer Ingelheim) diluted in 0.9% sterile saline) SQ or ibuprofen (100 mg/5 ml (Children’s Motrin) diluted 1:100 in drinking water) for at least 3 days post-operative, 1 drop of 0.25% bupivacaine (0.5% diluted 1:1 with sterile saline) along the skin incision before closure, and 0.05 ml Buprenorphine SR-LAB (0.5 mg/ml, ZooPharm) SQ once at the time of surgery. Hair was removed from the dorsal cervical and scapular region using Nair (Church & Dwight Co., NJ) and the surgical site prepared using three alternating rounds of chlorhexidine and alcohol. A surgical plane of anesthesia was determined via unresponsiveness to a toe pinch, and surgery was performed using aseptic technique.

Surgery of female severe combined immunodeficiency disease (SCID) mice was performed as previously described [18, 20, 21] with the following modifications. Human joint samples were obtained from osteoarthritis patients undergoing joint replacement surgery at The Ohio State University, Department of Orthopedics, under an approved IRB protocol. The healthy articular cartilage/bone was cut away from the joint and then sliced into sections. Implants were assembled by inserting a 1–2-mm3 piece of cartilage/bone into a gelatin sponge (Pfizer, New York, NY) measuring approximately 3–5 mm3 which was wrapped in Bard surgical mesh (BD, Franklin Lakes, NJ) and sutured together. 5 × 105 RASF in 50 μL sterile PBS or 50 μL of sterile PBS was then allowed to absorb into the sponge of the implants which were then stored in a humidified container at room temperature until implantation (< 4 h). Each SCID mouse received two implants via a single transverse interscapular incision of approximately 5 mm followed by blunt dissection; the implants containing RASF were subcutaneously implanted in the right flank, while implants containing only PBS were implanted in the left flank. The incision was closed with 7 mm or 9 mm wound clips, and the entire procedure lasted less than 30 min. Mice were monitored daily for at least 5 days post-operative, and any animals that became moribund were removed and not included in the analysis. Dehisced surgical sites were reclosed once, or animals were removed from study. After 5–7 days, mice were treated with either 5 × 105 GMSC or 3 × 106 GMSC derived exosome particles via retro-orbital injection. Animals were euthanized via an overdose of carbon dioxide in accordance with the 2020 AVMA Euthanasia Guidelines. Implants were harvested at 60 days post-surgery, and the cartilage was isolated from the other components of the implant. The cartilage was then fixed with 10% neutral buffered formalin and stored in 70% ethanol until being further processed for histology. 40X H&E-stained digital slide images of cartilage sections were visualized and depth of penetration was measured by using Aperio ImageScope v12.1.0.5029.

Histology and IHC

All samples were either processed at The Ohio State University, College of Veterinary Medicine, Histology/Immunohistochemistry Core Lab, or at Histowiz (Brooklyn, NY) according to established protocols, and the resulting slides were scanned at 40X magnification.

Flow cytometry tracking of GMSC

GMSC were labeled with ViaFluor 405 (Biotium, Fremont, CA) and then injected intraperitoneally into female SCID mice that had received one implant containing cartilage and RASF 3 days earlier. Labeled GMSC were maintained in culture for the duration of the experiment. The implants were harvested at 4 days, and the cartilage was isolated and digested in collagenase to release adherent cells. The cell suspension was analyzed on a BD FACSCelesta flow cytometer (BD, Franklin Lakes, NJ), and data were analyzed by using FlowJo v10.8.1 (Ashland, OR).

The cultured ViaFluor 405-labeled GMSC were acquired on the cytometer on days 1, 2, 3, and 4 to track the fluorescence intensity of the dye over the course of the experiment to identify a positive cell gating strategy for the cells harvested from the implants on day 4.

In vitro co-incubation of RASF with GMSC

RASF were labeled with ViaFluor 405 according to the manufacturers’ protocol, and then 5000 RASF were plated per well in a 24-well plate. GMSC were labeled with CFSE, and then either 5000 or 500 GMSC were added to the appropriate wells of the 24-well plate. The cells were allowed to co-incubate for 3 days and were trypsinized, resuspended in AnnexinV binding buffer, stained with AnnexinV-APC (Biolegend, San Diego, CA), and then analyzed on a BD FACSCelesta flow cytometer. For the cell crosstalk experiment, the RASF were separately labeled with ViaFluor 405, and the GMSC were labeled with ViaFluor 488. The cells were washed extensively and incubated in complete media in suspension for 1 h to ensure no residual dye reactivity was present. The cells were then added to a 24-well plate at the indicated ratios and cultured for 2 days. For the transwell incubation, the RASF were added to the bottom well, and the GMSC were added to the top transwell chamber according to the manufacturer’s directions. The cells were cultured for 2 days and were then collected and analyzed on a BD FACSCelesta flow cytometer.

Statistics

Data were analyzed with GraphPad Prism 9.4.0 (San Diego, CA) by using two-way ANOVA followed by Tukey’s multiple comparison testing. P-values < 0.05 were considered significant.



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