Collagenase type 1 solution

Primary human osteoblasts were isolated according to the following protocol. Human bone specimens were obtained during hip or knee joint replacement surgeries. Cancellous bone fragments were removed with bone rongeurs from bone specimens. The isolated fragments were washed several times with PBS (Sigma-Aldrich, St. Louis, MO, USA) until a clear supernatant was achieved. The supernatant was discarded and 15 mL collagenase type I solution (1 mg/mL in medium 199, Gibco, ThermoFisher Scientific, Waltham, MA, USA) was added. Collagenase digestion was carried out under mechanical stirring in a water bath at 37 °C. After 45 min, the fragments were washed again several times with PBS (Sigma-Aldrich, St. Louis, MO, USA). The washed bone pieces were transferred into 6-well tissue culture plates with sterile forceps, followed by addition of DMEM/F-12 medium supplemented with 20% fetal calf serum (FCS) and 1% penicillin/streptomycin (PS). After the first passage, human osteoblasts were cultured in DMEM/Ham’s F12 (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum and antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich, St. Louis, MO, USA). The medium was changed twice a week. For osteoblast differentiation, the medium was supplemented with 10 nM dexamethasone, 3.5 mM b-glycerophosphate, and 50 μg/mL ascorbic acid.

ADMSCs were isolated as previously described [35 ]. Briefly, adipose tissues were harvested from the donor’s lower abdomen under general anesthesia. Then adipose tissue was minced into small pieces and digested using 200 unit/ml Collagenase type I solution (Gibco, Denmark) supplemented with 200 g/l Human Albumin (Baxter, USA) at 37 °C with agitation 300 rpm/min for 60 min. The mixture was centrifuged at 1500 rpm/min for 10 min to collect the stromal vascular fraction in the pellet. Finally, the cells were resuspended in PS (Pan-Biotech, Germany) supplemented with 1% Penicillin/Streptomycin (Gibco, USA). Cells were incubated at 37 °C and 5% CO₂ in a humidified atmosphere with medium exchange every three days. When reached 80–90% confluence, the cells were harvested using TrypLE™ Select Enzyme (Gibco, USA) and counted after staining with trypan blue (Life Technologies, USA).
Cells at passage 0 were cryopreserved in the Cryostor solution (Stemcell Technologies, Canada). All the cryovials were kept at -80 °C for no more than seven days and transferred to BioStore™ III Cryo (Brooks Life Sciences, USA) for long-term storage. After four months of cryopreservation, the cells were thawed rapidly at 37 °C and maintained in PS or SM (Miltenyi Biotec, Germany) to evaluate cell growth, phenotype, and functional properties.

Human dental pulp cells (hDPCs) were isolated from human healthy third molars (n = 10) that were extracted for surgical reasons; this process was previously approved by the Ethical Committee of the University of Murcia (ID: 2199/2018). The pulp tissues obtained were carefully examined and immersed in 3 mg/mL collagenase type I solution (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) at 37 °C in 5% CO₂ for 90 min, as previously reported [20 (link)]. Then, single-cell suspensions were obtained and cultured in DMEM (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fischer Scientific, Waltham, MA, USA), 1% L-glutamine (Lonza, Basel, Switzerland), and 100 μg/mL penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and then incubated at 37 °C in 5% CO₂. Cells at passages 2–4 were used for subsequent experiments.

All molars of the four quadrants were carefully extracted from the 4–5-week-old male SD rats under a dissection microscope (ZEISS Stemi 508, stereo microscope). The extracted teeth were immediately immersed in Dulbecco’s modified Eagle’s medium (DMEM (High glucose), Nacalai Tesque, Inc. Kyoto, Japan), supplemented with 10% fetal bovine serum (FBS, Biowest, Ireland) and 1% antibiotic solution (penicillin-streptomycin mixed solution, Nacalai Tesque, Inc. Kyoto, Japan). Following extraction, the teeth were washed thrice with phosphate buffered saline (PBS, Takara bio Inc. Shiga, Japan) and then dispersed in 3 mg/mL Collagenase type I solution (Gibco, Grand Island, NY 14072, USA) for 30 minutes at 37 °C and the tube was slightly tapped every 5 minutes. An equal amount of culture medium was added and the supernatant collected in another tube. The cells were washed and collected 3 times using the culture medium. Centrifugation was performed at 4 degrees and 1800 rpm for 3 minutes. Finally, the culture medium containing suspended PDL cells was cultured in a 35 mm dish. PDL cells between passage 3 and 5 were used for cell sheet fabrication.

Umbilical cords (UC) were collected from pregnant women after baby delivery at the surgery of Vinmec International Hospital. The UCs were washed three times with ethanol 70% and then 3–5 times with PBS to sterilize and remove blood. The UC was cut into small pieces and transferred into a 50 mL conical tube following by an incubation with 500 U collagenase Type I solution (Gibco, Massachusetts, USA) for 1 h at 37°C with shaking. After digestion, solution was diluted in chilled PBS and centrifuge at 1,400 × g/10 min at 4°C to collect cell pellet. The cell pellet was resuspended in a MSC culture media (StemMACS^TM MSC Expansion Media, Miltenyi Biotec, Bergisch Gladbach, Germany) and transferred into cell culture flasks (Nunc, Thermo Scientific, Massachusetts, United States) coated with solution CTS^TM CELLstart^TM substrate (diluted in PBS at ratio 1: 300) (Gibco, Massachusetts, USA) and incubated at 37°C and 5% CO₂ for cell expansion. With each 5 mg of UC, cell pellets were seed into 1 T25 cell culture flask. Culture media were replaced by every 2 days of culture. When the cells reached 80% confluency, the cells would be split by 0.05% trypsin for the next passage.