Microarray database reference datasets were downloaded from the ArrayExpress database (accession number E-GEOD-35959; available from https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-35959) by searching for osteoporosis, for which we obtained the gene expression profiles of 19 human MSC lines.
DLEPS was used for calculating the anti-osteoporosis score using the differentially expressed genes (DEG) described earlier. The drug with the lowest or highest score was likely to be effective for the selected disease, i.e., osteoporosis.
Culture, osteogenic induction of hBMMSCs
Primary hBMMSCs were acquired from ScienCell and cultivated in PM [α-minimum essential medium (α-MEM, Gibco) containing 10% (v/v) fetal bovine serum (FBS, ExCell Bio) and 1% (v/v) penicillin streptomycin (Gibco)]. The osteogenic differential induction medium (OM) contained α-MEM with 10% (v/v) FBS, 1% (v/v) penicillin streptomycin, 10 nM dexamethasone (Sigma), 200 μM vitamin C (Sigma), and 10 mM β-glycerophosphate (βGP, Sigma).
Concentrated Ataluren solution preparation
Ataluren (CSNpharm) was dissolved in DMSO (Sigma) and α-MEM to yield a concentrated solution of 0.1 mM. This was divided and used to detect the role of gradient concentrations of ataluren on hBMMSC differentiation and to select the best concentration for promoting osteoblastic differentiation.
The cytotoxicity of ataluren was determined by the CCK-8 assay. Briefly, cells were inoculated at 2,000 cells/well in clear, flat-bottomed, 96-well plates. Cells were then cultured in PM, or in PM at 10, 50 and 100 μM ataluren. cells from three wells of the same treatment were tested daily from day 1 to day 7 to determine the cytotoxicity of the relevant reagents by the CCK-8 assay. The cultures were then removed from the incubator and the absorbance at 570 nm was read.
Staining and quantification of alkaline phosphatase (ALP) and alizarin red S (ARS)
Early markers of osteoblast differentiation were examined with ALP staining and quantitative determination of ALP activity. Late markers of osteoblast differentiation were examined using ARS staining and semi-quantitative determination of mineralization. ALP staining was performed using an NBT/BCIP staining kit (CoWin Biotech) according to the manufacturer’s guidelines on day 7 after OM induction. ALP activity was tested with an ALP assay kit (Nanjing Jiancheng Bioengineering Institute). The ALP activity was calculated based on the absorbance in 520mm.
Fourteen days after OM induction, the cells were stained with 2% ARS buffer (Sigma). Mineral accumulation was quantified using 100 mM cetylpyridine solution (Sigma) in a multi-well sample plate. Mineral accumulation was quantitated based on the absorbance in 490mm.
Western blot analysis
The hBMMSCs were cultured in 60-mm dishes and incubated with 1× radioimmunoprecipitation assay lysis buffer (Huaxingbio) containing a mixture of PMSF (Huaxingbio) and protease inhibitor (Huaxingbio). After centrifugation on ice, the protein concentration in the supernatant was measured by the bicinchoninic acid approach. The same amount of protein was split on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto a PVDF membrane (Millipore). The membranes were incubated in rapid blocking solution to block nonspecific binding, then incubated with primary antibodies against BMP2, RUNX2, OSX, SMAD1, SMAD5, SMAD1/5 (Abcam), pSMAD1/5 (Cell Signaling Technology), or GAPDH (Abcam). Then, the membranes were incubated with the secondary antibody for 1 h. Finally, the immunoreacted protein bands were detected by enhanced chemiluminescence.
Quantitative real-time PCR (qRT–PCR)
Total cellular RNA was extracted from the hBMMSCs with TRIzol (Invitrogen) according to the manufacturer instruction manual. Complementary DNA (cDNA) was reverse-transcribed using a PrimeScript RT Reagent Kit (Takara). qRT-PCR was performed with SYBR Green Master Mix (YEASEN) on an ABI Prism 7500 RT-PCR System. The gene index was standardized to the GAPDH index, which was used as the housekeeping gene. The primer sequences used are presented in Additional file 1: TableS1. Each sample was taken in triplicate and fluorescence data was collected at the end of each cycle.
Small interfering RNA (siRNA) transfection
BMP2-targeting siRNAs and Scramble siRNA were acquired from Sangon Biotech. The siRNA sequences were as follows: sense, GAUCAUCUGAACUCCACUAAUTT and antisense, AUUAGUGGAGUUCAGAUGAUCTT. 24 hours prior to treatment, hBMMSCs were inoculated at 80,000 cells/well in 24-well plates. Cells were treated with siBMP2 or scramble in 300 μL of Opti-MEM (Gibco) containing 1 μL of lipo-2000 (Invitrogen) for 4 hours. Thereafter the medium was changed to PM. The cells were transfected with siBMP2 and harvested for RNA analyses and western blotting after 2 days. For osteogenic induction, the cells were transfected every 5 days in OM and harvested after 1 week.
Animals and experimental design and ethical statement
Female C57BL/6 mice were obtained from Charles River Laboratory Animal Technology Co., Ltd. They were housed in specific pathogen-free conditions at the Animal Center of Peking University School and Hospital of Stomatology. This study was authorized by the Peking University School of Medicine Institutional Committee for Animal Care and Use (LA2021040).
In vivo metabolism studies of ataluren suggested that the administration of ataluren in mice was by gavage at a concentration of 20mg/mL and a gavage dose of 15 mL/kg. Based on a mouse weight of 24 to 34 g, we can calculate the administered dose as 300 mg/kg(27).
Estrogen deficiency-induced bone loss model for primary validation：Twenty-five mice (56 days old) were placed in five groups(n = 5). After general anesthesia, bilateral OVX or sham surgery was performed with standardized techniques. Four weeks after surgery, 300 mg/kg/d ataluren (20 mg/mL, Macklin) mixed in 0.5% Carboxymethylcellulose (CMC, Sigma), 36.4 mg/kg/d natamycin (NATA, 15 mg/mL, Solarbio) mixed in 0.5% CMC and 0.26 mg/kg/d prucalopride (PRU, 0.0173 mg/mL, Mitachieve) mixed in 0.5% CMC was administered by gavage for 2 months (once every other day). The five groups were: (1) SHAM+CMC, (2) OVX+CMC, (3) OVX+ATA, (4) OVX+NATA, and (5) OVX+PRU.
Estrogen deficiency-induced bone loss model: Twenty mice (56 days old) were placed in four groups (n = 5). Four weeks after OVX or sham surgery, 300 mg/kg/d ataluren mixed in 0.5% CMC was administered by gavage for 2 months (once every other day). The four groups were: (1) SHAM+CMC, (2) SHAM+ATA, (3) OVX+CMC, and (4) OVX+ATA.
Age-related bone loss model: Ten mice (18 months old) were stochastically divided into two groups (n = 5): (1) Aged+CMC and (2) Aged+ATA. 300 mg/kg/d ataluren (20 mg/mL, Macklin) mixed in 0.5% CMC (Sigma) was administered by gavage for 2 months (once every other day).
All mice were euthanized, and the left femur and vital internal organs were collected and fixed in 10% formalin. The right femur was also collected and preserved from light. The serum was also collected from each mouse. The major organs were carefully collected and fixed in 10% formalin for H&E staining of tissue sections to observe the tissue toxicity of ataluren.
Serum enzyme-linked immunosorbent assay (ELISA)
Serum PINP and serum CTX-1 were used as markers of bone formation and bone resorption, respectively. The serum biomarkers were tested using an ELISA kit (Jiangsu Meimian Industrial Co., Ltd.).
Bone histomorphology analyses were performed according to a previously described protocol(28). For the measurement of dynamic bone parameters, the mice were injected with calcium xanthophyll green (10 mg/kg body weight) and alizarin complexes (10 mg/kg body weight) at 7 and 2 days before euthanasia, respectively. After euthanasia of the mice, the right femur was removed and stored away from light, dehydrated, and embedded for hard tissue sectioning. The double-labeling in the bone tissue was observed under fluorescence microscopy, and quantitative analysis was performed using Image J (National Institutes of Health). MAR (mineral apposition rates) represents the rate of new bone formation.
The left femurs were carefully collected and fixed in 10% formalin, dehydrated in 70% ethanol and embedded in methyl methacrylate. The femur was cut in half at the mid-axis and sectioned in the transverse plane of a 200 µm thick section for H&E staining and observed under the microscope.
Femoral specimens were scanned with the Inveon MM System (Siemens) micro-computed tomographer. The scanning conditions were 60 kV, 500 μA, and precision of 8.82 μm. Parametric analysis was performed using Inveon Research Workplace software (Siemens). The measurement area was 1 mm proximal to the epiphysis. The parameters analyzed were bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) by Inveon Research Workplace (Siemens).
All data are expressed as the mean ± standard deviation (SD) of at least 3 experiments per group. The statistics were analyzed with the Student t-test or one-way analysis of variance (ANOVA) via SPSS 23.0 (SPSS Inc.). P < 0.05 and <0.01 indicated statistical significance.