Collagenase type 1

Isolation of primary pituitary cells were performed as previously described 43 (link). In brief, human pituitary adenoma and rat normal pituitary tissues were minced and incubated with 0.5% collagenase type I and 0.05% DNase I (Solarbio, Beijing, China) at 37℃ for 45 min. The resulting mixture was centrifuged and the supernatant was collected. Then the tissues were incubated once more with 0.25% trypsin at 37℃ for 15 min, followed by centrifugation. Supernatant was collected, pooled with the former supernatant and flushed over a 70 μm filter. The filtrate was centrifuged and the pellets (pituitary cells) were collected. Cells were then cultured in DMEM medium (Life technologies, Waltham, MA) with 10% fetal bovine serum (FBS) (Gibco, New York) and 1% penicillin/streptomycin (Gibco, New York).

Specific pathogen-free (SPF) chicken eggs were obtained from Sais Poultry Co. Ltd., Jinan, China, and incubated at 37.8 °C under a relative humidity of 60%.
Chicken fetal myoblasts (CFMs) were isolated from chicken embryos (n = 20) at 13 days of age and minced in 0.2% collagenase type I (Solarbio, Beijing, China) for digestion. The resulting suspension was dispersed, filtered, centrifuged, and plated in cell culture plates. The cultures were enriched in myoblasts by placing the cells in an incubator twice for 40 minutes to remove fibroblasts. The enriched culture was grown in DMEM/F12 medium (Gibco, Shanghai, China) supplemented with 20% FBS (Gibco, Shanghai, China) at 37 °C 5% CO₂. The CFMs were induced to differentiate by replacing the growth medium containing 20% fetal bovine serum with differentiation medium containing 2% horse serum.
As non-muscle control cells, primary chicken embryonic fibroblasts (CEFs) were isolated from chicken embryos (n = 8) at 9 days of age. The heads, wings, legs, and abdominal organs were removed from the chicken embryos, and the remaining tissues were minced and digested with 0.25% trypsin (Gibco, Shanghai, China). The resulting suspension was filtered, centrifuged, and plated in cell culture plates. CEFs were grown in DMEM/F12 medium (Gibco, Shanghai, China) supplemented with 5% FBS (Gibco, Shanghai, China) at 37 °C 5% CO₂.

Cell culture-grade chemicals were used in this study. Low glucose Dulbecco’s Modified Eagle’s Medium (LG-DMEM) and FBS were acquired from Bio West, Nuaillé, France. Penicillin–streptomycin, Amphotericin-B, and trypsin-EDTA (0.5% and 5.3 mM w/v, respectively) were obtained from Caisson, Smithfield, UT, USA. Minimum essential medium-alpha (α-MEM) was purchased from Gibco, Carlsbad, CA, USA, and Ex-Cyte from Millipore, Billerica, MA, USA. Insulin (3.5 mg [100 IU]/mL) was obtained from Novo Nordisk, Søborg, Denmark. MTT dye, collagenase type I, and TRIZOL (TriQuick, Catalogue #R1100) reagent were obtained from Solarbio, Fengtai, China. Dimethyl sulfoxide (DMSO), formalin, Triton X–100, isobutylmethylxanthine (IBMX), Dulbecco phosphate buffer saline (DPBS^−/−; without Ca²⁺ and Mg²⁺), and Alizarin Red S stain (ARS) were procured from Sigma-Aldrich, Taufkirchen, Germany. The antifade mounting media was obtained from Vecta Shield, St. Neots, UK. The cDNA synthesis kit (Catalogue # cDSK01-100) was purchased from Vivantis Technologies, Selangor, Malaysia. SYBR green master mix and Oil red O (ORO) were obtained from Thermo Scientific, Chino, CA, USA. T-25, and T-75 cell culture flasks, serological pipettes, 6-well, 24-well, and 48-well cell culture plates, and cell strainers were received from Corning, NY, USA.

Human apical papilla tissues were obtained from immature third permanent molars extracted from patients aged 16–20 years for orthodontic reason at the School and Hospital of Stomatology, Shandong University. This project was approved by the Ethics Committee of the School and Hospital of Stomatology, Shandong University. The consent was obtained from the patients or their parents. The apical papilla was separated from the third molar and digested with 3 mg/mL Collagenase Type I (Solarbio, Beijing, China) and 4 mg/mL Dispase (Roche, Indianapolis, IN, United States) at 37°C for 1 h. Then cells were cultured in α-minimum essential medium (HyClone, Logan, UT, United States) supplemented with 20% fetal bovine serum (FBS) (Gibco, Grand Island, NY, United States), 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone) incubated at 37°C in a 5% CO₂ atmosphere. The cells from passage 2–5 were used for subsequent experiments.
Human embryonic kidney 293T (HEK 293T) cells were cultured in DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.

Gelatin (Type B from bovine bone, average molecular weight 80,000 Da) was obtained from Dongbao Bio-tech (Baotou, China). Methacrylic anhydride (MA), poly (ethylene glycol) diacrylate (PEGDA), 2-Hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959), and deuterium oxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase Type I, FITC-BSA were purchased from Solarbio (Beijing, China). MC3T3-E1 (Mouse osteoblast cell line, 6 passages), fetal bovine serum, Alpha Modification Eagle Medium (α-MEM), and PBS buffer (pH 7.4) were purchased from Union Hospital (Beijing, China). The live/dead assay kit was purchased from ABcam (Britain, UK). All other reagents and solvents were of reagent grade.

GelMA (degree of modification 76%) was self-made [29 (link)], poly (ethylene glycol) diacrylate (PEGDA), 2-hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959), and deuterium oxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). nHA (<100 nm particle size) was purchased from Aladdin (Shanghai, China). Collagenase type I were purchased from Solarbio (Beijing, China). MC3T3-E1 (mouse osteoblast cell line, six passages), fetal bovine serum, Alpha Modification Eagle Medium (α-MEM), and PBS buffer (pH 7.4) were purchased from Union Hospital (Beijing, China). The live/dead assay kit was purchased from ABcam (Britain, UK). All other reagents and solvents were of reagent grade.