α minimal essential medium α mem

Primary osteocytes (pOC) cells were harvested from mice²⁷ (Sino-British Sippr/BK Lab Animal Co., Ltd, Shanghai, China) long bones (femora, and tibia) using 300 U/mL collagenase (Sigma-Aldrich) dissolved in α-minimal essential medium (α-MEM) (Gibco, Shanghai, China) and were cultured in α-MEM medium supplemented with 10% fetal bovine serum (Gibco) at 37 °C in a CO₂ incubator (Shanghai Hengyue Medical Instruments Co., Ltd, Shanghai, China), respectively. Animal study protocols and procedures were approved by the Shanghai Ocean University institutional animal care and use committee (Permit Number: 13-0012). All methods were employed in accordance with the relevant guidelines and regulations of Scientific and Ethical Care and Use of Laboratory Animals of Shanghai Ocean University.
Mouse bone marrow-mesenchymal stem cells (ZQ0465) (MMSC-bm) were purchased from the Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd, Shanghai, China and were cultured in mesenchymal stem cell culture medium (brand: Sciencell, Cat. No. 7501) containing 10% fetal bovine serum (FBS), (brand: Sciencell, Cat. No. 0025), mesenchymal stem cell growth supplement (1% MSCGS, Cat. No. 7552) and 1% penicillin/streptomycin (10,000 units/ml of Penicillin and 10,000 μg/ml of Streptomycin in a saline solution) (Cat. No. 0503) at 37 °C in a CO₂ incubator.

Six-week-old male mice were purchased from Dae Han Bio Link (Chungbuk, Korea). All animal experiments were approved by the committees on the care and use of animals in research at Kyungpook National University and were conducted in accordance with the guidelines for the care and use of laboratory animals (KNU 2016-0147). PF-3845 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and α-minimal essential medium (α-MEM) were purchased from Gibco BRL (Grand Island, NY, USA). Recombinant M-CSF and RANKL were obtained from R & D Systems (Minneapolis, MN, USA).

Frozen vials of ASCs were thawed and cultured on 150 cm² culture dishes (Nunc, Rochester, NY) in 25 ml complete culture medium (CCM), which consisted of α-minimal essential medium (αMEM; GIBCO; Grand Island, NY), 20% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 100 units per ml penicillin/100 μg/ml streptomycin (P/S; GIBCO), and 2 mM L-glutamine (GIBCO). The cells were then incubated at 37°C with 5% humidified CO₂. After 24 hours, viable cells were harvested with 0.25% trypsin/1 mM EDTA and replated at 100–200 cells/cm² in CCM. The medium was changed every 2-3 days. For all experiments, subconfluent cells (≤70% confluent) between passages 2–6 were used. Characterization of stem cells was previously performed and published [26 (link)].

Primary cultures of OCs were obtained from the peripheral blood mononucleated cells (PBMCs) as previously described [31 (link)]. PBMCs were isolated by density gradient centrifugation (Histopaque 1077, Sigma Chemical, St Louis, MO) and diluted at 1×10⁶ cells/mL in α-Minimal Essential Medium (α-MEM) (Gibco, Uxbridge, UK) and supplemented with 10% fetal bovine serum (FBS) (Euroclone, Siziano, Italy), 100 IU/mL penicillin, 100 g/mL streptomycin (Gibco, Uxbridge, UK) and L-glutamine. Successively, PBMCs were plated in 24-well plates for 21 days in the presence of 25 ng/mL recombinant human macrophage colony-stimulating factor (rh-MCSF) (Peprotech, London, UK) and 50 ng/mL RANK-L (Peprotech, London, UK) to obtain differentiated OCs. Culture medium was replaced every three days. OCswere incubated at 37°C in a 5% CO₂ humidified atmosphere and in normoxic conditions.

We isolated periodontal ligament stem cells from periodontitis patients and healthy controls (P-PDLSCs and H-PDLSCs) as previously described [12 (link), 13 (link)]. Briefly, periodontal ligament tissues were isolated from the middle third of the root surface and digested in 3 mg/ml collagenase I (Sigma-Aldrich, St Louis, MO, USA) for 2 h at 37°C. Periodontal ligament stem cells were cultured in 6-well culture dishes (Costar; Corning, NY, USA) in α-minimal essential medium (α-MEM; Gibco, Gaithersburg, MD, USA) supplemented with 0.292 mg/ml L-glutamine (Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Sijiqing, Zhejiang, China), 100 mg/ml streptomycin (Invitrogen), and 100 unit/ml penicillin (Invitrogen) in 5% CO₂ at 37°C. Limiting dilution technique purified the stem cells into single cell colony cultures and then pooled and expanded. Cells from passages 2-5 were used in the following experiments. An identified ER stress inhibitor, 4-phenyl butyric acid (4-PBA, 5 mM; Sigma-Aldrich) was used to treat cells for 24 h, and lipopolysaccharide (LPS, 10 μg/ml) was acquired from Pepro-Tech (Pepro-Tech, NJ, USA).

Murine periosteum-derived cells (mPDC) were isolated from the long bones of 8 week-old male mice as previously described [21] (link). In short, the femurs and tibias isolated from 8 week old male C57BL/6J mice were dissected and digested with collagenase-dispase after protecting the epiphyses with low melting point agarose. After a filtration and washing step, the cells were plated at 1×10⁴ cells per square centimeter and replated when reaching 80–90% confluency. After isolation, cells were pooled per 2–3 mice and cultured in a humidified incubator at 37°C with 5% CO₂ in α-minimal essential medium (α-MEM) supplemented with 2 mM glutaMAX-I, 1% penicillin/streptomycin (100 units/ml and 100 µg/ml respectively) and 10% fetal bovine serum (all from Gibco, Life Technologies, Gent, Belgium). When reaching 80–90% confluency, cells were trypsinized and reseeded at 7500 cells/cm².

Primary MSCs were classified as advanced therapy medicinal products (ATMPs), qualified according to standards set by the International Society of Cellular Therapy [20 (link)] and manufactured in the GMP-licensed Cell Therapy Facility (Department of Clinical Pharmacy at the UMC Utrecht), as described earlier [21 (link)–24 (link)]. Briefly, MSCs were isolated from third-party non-HLA-matched healthy bone marrow donors as approved by the Dutch Central Committee on Research Involving Human Subjects (CCMO, Biobanking bone marrow for MSC expansion, NL41015.041.12) and all samples were obtained with written informed consent from the bone marrow donor or parent/legal guardian of the donor. MSCs were grown in α-minimal essential medium (α-MEM) (Gibco) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin (Gibco), 1% L-glutamine (Gibco), basic fibroblast growth factor (bFGF) (1ng/ml; Gibco), and L-ascorbic acid phosphate (200 µM; Sigma-Aldrich) at 37 °C and 5% CO₂. Experiments were performed with sub-confluent MSCs at passage 5–9. Description of MSC donors used in this study are listed in Additional file 1: Table S1.