Sodium glycerophosphate

All reactants, including p-nitrophenylphosphate (pNPP), bis p-nitrophenylphosphate (bis-pNPP), Thymidine 5′-monophosphate p-nitrophenylester sodium salt (TpNPP), calcium glycerophosphate (CaGP) and sodium glycerophosphate (NaGP) were purchased from Sigma-Aldrich. pNPP and glycerophosphate (GP) are phosphomonoesters while bis-pNPP and TpNPP are phosphodiesters.

The MSC-spheroid suspension was prepared using a 3D rotational culture system, as described previously [13 (link)–17 (link), 21 (link)]. In brief, the MSCs were detached from the tissue culture plate using 0.25 % trypsin/1 mM EDTA (Gibco Invitrogen, Grand Island, NY, USA) and resuspended at 1.0 × 10⁷ cells in 5-mL osteogenic medium for rotation culture. The osteogenic medium [22 (link)] comprised DMEM supplemented with 10 % FBS, 100 nM dexamethasone (Sigma, St. Louis, MO), 0.05 mM l-ascorbic acid-2-phosphate (Sigma), 10 mM sodium glycerophosphate (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. The cell suspension (MSCs in osteogenic medium) was placed in plates with an ultra-low attachment-coated polystyrene surface (Costar, 6-well cluster plates; Corning Costar Corp, Corning, NY). The dishes were placed on a shaker (Taitec, Saitama, Japan), rotated at a constant speed of 70 rpm, and incubated in a humidified 37 °C/5 % CO₂ incubator for 1 day. MSC-spheroids were formed within a day of the rotation culture. The spheroids were detached from the culture dishes by pipetting and examined by phase-contrast microscopy using an Olympus AX80 microscope (Olympus, Tokyo, Japan).

Streptozotocin, hCG, thyroid stimulating hormone (TSH) from bovine pituitary, thyrotropin-releasing hormone (TRH), forskolin, 5′-guanylylimidodiphosphate (GppNHp), creatine phosphate, creatine phosphokinase from the rabbit muscle, sodium glycerophosphate, sodium deoxycholate, phenylmethylsulfonyl fluoride, ortho-phenanthroline, pepstatin and other reagents were obtained from “Sigma-Aldrich” (St. Louis, MO, USA). Radiolabeled substrate for AC, [α-³²P]-ATP was obtained from “Izotope” (Moscow, Russia).

E13 Sprague-Dawley pregnant rats were purchased from Janvier Labs and allowed to acclimatize in the animal facility for 7 days. Water and standard food pellets were provided ad libitum. Thereafter, the rat dams were humanely euthanized via CO₂ on day 20 of gestation and the E20 embryos were obtained. After decapitation, the hind paws were collected and the 3 middle metatarsal bones were microdissected under an inverted microscope, as previously described [26 (link)]. Next, the metatarsals were transferred to 24-well plates and randomly distributed to the following groups: (i) control; (ii) venetoclax 3.75 μM; (iii) HNG 10, 20, or 40 μM; and (iv) the combination of venetoclax and HNG. The culture medium used was Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 50 μg/mL ascorbic acid (Sigma-Aldrich), 1 mM sodium glycerophosphate (Sigma-Aldrich), 0.2% bovine serum albumin (Sigma-Aldrich), and 20 μg/mL gentamicin, and the compounds of interest (venetoclax, HNG) were diluted in the medium from stock solutions. Medium changes together with image capture (Hamamatsu C4742-95 digital camera mounted on a Nikon SMZ-U microscope) and length measurements (Infinity Analyze software, Lumenera Corporation) were performed on days 0, 2, 5, 7, 9, and 12 (termination). Three independent experiments were performed, and each metatarsal bone was considered an experimental unit.

We isolated primary dental papilla mesenchymal cells (mDPCs) from embryonic day 18.5 (E18.5) first molar tooth germ. A dissection needle was employed to remove the dental epithelium after digestion using 0.75 mg/ml of dispase (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). The tooth germ was isolated from the mandible using forceps, dispersed, and digested with 0.25% trypsin-EDTA (Life Technologies, Carlsbad, CA, USA) at 37°C for 15 min. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Hyclone, Pittsburgh, PA, USA). To induce mineralization, mDPCs between passage 2 and 4 were cultured in an induction medium supplemented with 50 μg/ml of ascorbic acid (Sigma, St. Louis, MO, USA), 10 mmol/L sodium-glycerophosphate, and 10 nmol/L dexamethasone (Sigma). All the cells used in this study were maintained in 5% CO₂ at 37°C, and the medium was replenished every 2 days.

An osteogenic medium containing 10% FBS, 1% penicillin/streptomycin, 10 nM dexamethasone (Sigma, St. Louis, MO, USA), 10 mM sodium-glycerophosphate (Sigma, St. Louis, MO, USA), and 50 g/mL ascorbic acid (Sigma, St. Louis, MO, USA) was used to induce osteogenic differentiation. After treatment, osteogenic induction medium was used in the following experiment for six days (ALP staining, real-time PCR and western blot) and 14 days (Alizarin red staining).

Ethylene diamine tetraacetic acid (EDTA), ascorbic acid, sodium glycerophosphate, dexamethasone, cetylpyridinium bromide, alizarin red S and Triton-X100 were obtained from Sigma-Aldrich (USA). Foetal bovine serum (FBS), αMEM medium, non-essential amino acid (NEAA), β-mercaptoethanol, L-glutamax and penicillin/streptavidin were purchased from Gibco (USA). Cell culture plates and Matrigel were bought from Corning (USA). Methanol, absolute ethanol, chloroform, hydrochloric acid and isopropanol were obtained from BCIGC (China). Bovine serum albumin (BSA), phosphate buffer, N-hydroxysulfosuccinimide sodium salt (NHSS) and 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) were purchased from Aladdin (China). BCIP/NBT alkaline phosphatase colouring kit and ALP quantitative detection kit was acquired from CWBIO (China). SYBR Green I and TRIzol were bought from Takara (Japan). Paraformaldehyde was obtained from Solarbio (China). Quartz crystal microbalance chips were obtained from HRbio (China). Cell counting kit-8 (CCK8) was purchased from Dojindo (Japan). RevertAid™ First Stand cDNA Synthesis Kit was gained from Thermo (USA). Cell cycle assay reagent was obtained from KoradBio (China). E8 medium was acquired from Cellapy (China). DAPI stain was purchased from Roche (Switzerland). Carboxyl functionalized QCM chips were provided by Dongwei BiologicalTechnology Co., LTD (China).