BMSC isolation and culture

Human bone marrow samples were extracted by bone marrow puncture. Mouse BMSCs were harvested from femurs and tibias. Then, BMSCs were isolated and cultured as described in our previous studies [10]. For induction of osteogenesis, BMSCs were cultured in osteogenic medium composed of 10% FBS DMEM, 100 IU/mL penicillin, 100 IU/mL streptomycin, 0.1 μmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 μmol/L ascorbic acid. For induction of adipogenic differentiation, the medium included 10% FBS DMEM, 1 μM dexamethasone, 10 μg/mL insulin, 0.5 mM IBMX, 0.2 mM indomethacin, and 100 IU/mL penicillin–streptomycin. The osteogenic medium and adipogenic medium were replaced every three days.

RNA Interference

Gene silencing was achieved via small interfering RNAs (siRNAs) synthesized by GenePharma. For gene silencing in 1 × 105 BMSCs, 0.2 nmol of siRNA and 4 μL of Lipofectamine RNAiMAX (Invitrogen, 13778150) were individually incubated in 100 μL of Opti-MEM Reduced Serum Medium (Thermo Fisher, 31985070). The two were combined, incubated for 30 min at 37 °C, and then added to the cells. The cells were cultured in Opti-MEM Reduced Serum Medium for 6 h before switching to DMEM containing 10% FBS. Three days post-transfection, the knockdown efficacy was assessed, and the cells were utilized in subsequent experiments.

Lentivirus construction and transfection

ALG5, SLC6A9-WT, and SLC6A9-Mut overexpression lentiviruses were constructed and added to BMSCs in DMEM supplemented with 5 μg / mL polybrene for 24 h. After 48 h post-transfection, the transfection efficacy was evaluated, and subsequent experiments were conducted.

Alizarin Red S (ARS) staining and alkaline phosphatase (ALP) assays

Before staining, BMSCs were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. For ARS staining, BMSCs were incubated with 1% ARS solution (pH 4.2) (Solarbio, G8550) for 15 min at room temperature. After nonspecific staining with PBS was removed, images of the stained BMSCs were captured. ARS staining was extracted with 10% cetylpyridinium chloride monohydrate (Sigma Aldrich, 8400080100) for quantification at a spectrophotometric absorbance of 562 nm. For ALP staining, BMSCs were stained with a BCIP/NBT alkaline phosphatase kit (Beyotime, C3206). For determination of ALP activity, BMSCs were lysed in RIPA buffer (Sigma Aldrich, R0278), and ALP activity kits (Nanjing Jiancheng Biotech, A059-2) were used to assess ALP activity via quantification of the spectrophotometric absorbance at 405 nm.

Oil red O (ORO) assays

ORO was dissolved in isopropyl alcohol to a concentration of 12 mM, and the ORO working solution was diluted with deionized water to a concentration of 7.2 mM. After fixation with 4% PFA for 30 min at room temperature, the BMSCs were stained with ORO working solution for 30 min. After nonspecific staining with PBS was removed, images of the stained BMSCs were acquired under a microscope. For quantification of the staining, ORO extraction was performed by adding isopropanol, followed by measuring the absorbance at 520 nm.

BMSC differentiation assays in vivo

For osteogenic induction in vivo, BMSCs were transplanted onto hydroxyapatite (HA)/tricalcium phosphate (TCP) for 24 h after culture in osteogenic medium for 5 days. HA/TCP grafts containing BMSCs were implanted into the subcutaneous dorsal region of eight-week-old BALB/c nude mice. Eight weeks post-implantation, the mice were euthanized by cervical dislocation, and the grafts were harvested for histological analyses, including hematoxylin and eosin (HE) staining, Masson staining, and histochemistry. For adipogenic differentiation in vivo, BMSCs were mixed with Matrigel, and the mixture was injected subcutaneously into the backs of nude mice after the cells were cultured in adipogenic medium for 5 days. After 6 weeks, the implants were collected and analyzed by HE staining.

Immunohistochemical assay

Bone tissues and other samples were collected as described above. Following deparaffinization and rehydration, the sections were subjected to antigen retrieval by incubation in citrate buffer (10 mm) and microwaving at 750 W for 20 min. After the sections were cooled to room temperature, they were treated with 3% H2O2 for 25 min, followed by a 30-min incubation in 5% normal goat serum. Subsequently, the sections were separately incubated overnight at 4 °C with anti-collagen I (Abcam, ab34710), anti-ALG5 (Proteintech, 16046-1-AP), and anti-SLC6A9 (SAB, 47583) antibodies. The following day, secondary antibodies were applied, and color development was achieved using an SP Rabbit & Mouse HRP Kit (CWBIO, CW2069S). Microscopy images were then captured.

Immunofluorescence assay

The collected samples were subjected to fixation, decalcification, rehydration and antigen retrieval as described above. After treatment with a 0.5% Triton X-100 solution for 30 min, the sections were blocked with 5% normal goat serum and then incubated with primary antibodies overnight at 4 °C. The following day, after PBS washes, the sections were incubated with fluorescein-labeled secondary antibodies (Abcam, ab150114 and ab150077) for 1 h. DAPI antifade mounting medium (Beyotime, P0131) was used for mounting. Images were captured under a microscope.

Protein extraction and western blot assay

The cells were collected and treated with RIPA buffer containing protease and phosphatase inhibitors for 30 min on ice. After centrifugation at 14,000 rpm at 4 °C for 10 min, the lysates were collected and mixed with 5% sodium dodecyl sulfate (SDS) loading buffer. The protein lysates were subjected to SDS‒PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% nonfat dry milk in TBST (150 mM NaCl, 50 mM Tris–HCl, and 0.05% Tween 20) for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4 °C. The cells were subsequently incubated with secondary antibodies (Cell Signaling Technology, 7074 and 7076) for 1 h at room temperature. Protein levels were visualized using chemiluminescent reagents (Millipore, WBKLS0500) per the manufacturer's instructions.

Proteomic examination

Following siRNA treatment of BMSCs, protein lysates were collected and processed as outlined above. Subsequently, label-free quantitative proteomics analysis was performed using an OrbitrapTM AstralTM high-resolution mass spectrometer. Differential expression was assessed utilizing DESeq2, employing the criteria of |log2Fold Change|> 1 and Q value < 0.05 for analysis.

Measurement of glycine uptake

Serum was collected from OVX mice and control mice, and serum glycine levels were measured using a mouse glycine enzyme-linked immunosorbent assay (ELISA) kit (JSBOSSEN, BS-E8790M1). Cell culture supernatants were harvested and measured with a human glycine ELISA kit (JSBOSSEN, BS-E6687H1).

Determination of GSH and NADH/NAD+ levels

GSH levels in BMSCs were measured with GSH and GSSG Assay Kits (Beyotime, S0053). The NADH/NAD+ ratio was measured by an NADP + /NADPH Assay Kit with WST-8 (Beyotime, S0179).

OVX mice

OVX mice were generated by performing bilateral ovariectomy or sham surgery on 2-month-old female wild-type (WT) C57BL/6 mice. After a 2-month recovery period post-ovariectomy, the mice were sacrificed and subjected to various assays.

Bone-targeting ALG5 overexpression in vivo

Bone-targeting rAAV9-ALG5 was designed and constructed by OBiO Technology (Shanghai). Seven days after ovariectomy, the mice were administered rAAV9-ALG5 via tail vein injection. Three months later, the mice were euthanized, and their femora were analyzed for bone mass using microcomputed tomography (micro-CT), HE staining, and Masson staining.

Femoral bone defects in mice

For generation of bone defects in the femur, 8-week-old mice were euthanized and sterilized. The femur was exposed and subjected to surgery with a 1.0-mm electric bone drill. After a 2-week interval, femurs were collected for micro-CT analysis.

Micro‑CT analysis

The micro-CT assay was carried out with the Inveon MM system (Siemens) to evaluate bone structures. The images were captured across 360 rotational steps, with a pixel size of 8.82 μm, a voltage of 80 kV, a current of 500 μA, and an exposure time of 1500 ms. Key parameters, including bone volume/tissue volume (BV/TV) ratio, trabecular thickness (Tb. Th), trabecular number (Tb. N), cortical thickness (Ct. Th), and trabecular separation (Tb. Sp), were calculated using Inveon Research Workplace software from Siemens.