Reagents

BMP2 (R&D, Minnesota, USA, 355-BM-100), DKK1 (BioLegend, California, USA, 759602), 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, PRC, C0060), carboxyfluorescein succinimidyl ester (CFSE, eBioscience, California, USA, 65-0850-84) and gallocyanine (Selleck, Texas, USA, S5602) were used. The antibodies used in this study were monoclonal anti-YAP (Cell Signaling Technology, Boston, USA, 14074), anti-pYAP S127 (Cell Signaling Technology, Boston, USA, 13008), anti-actin alpha2, smooth muscle (anti-α-Sma, Servicebio, Wuhan, PRC, GB111364), anti-osteocalcin (anti-OCN, BioByt, Cambridge, UK, orb259644), tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit antibody (ABclonal, Wuhan, PRC, AS040), fluorescein 5-isothiocyanate (FITC)-conjugated anti-mouse antibody (ABclonal, Wuhan, PRC, AS001) and anti-β-actin (Sigma, St. Louis, USA, A1978). Tris-HCl, NaCl and other chemicals were purchased from Sigma.

Cell culture and differentiation

MSCs were obtained from bone marrow taken from C57BL/6 mice (BMSCs). Total bone marrow cells were cultured in stem cell cultures with 10% foetal bovine serum (FBS) from Cyagen (Guangzhou, PRC). After 48 h, the suspended cells were discarded, and the adherent cells were cultured for 2 weeks to obtain primary mouse BMSCs. The BMSCs used for differentiation assays were at less than eight passages. BMSCs from mice were cultured in differentiation media (α-MEM with 10% FBS, 50 µM ascorbic acid, and 100 mM β-glycerophosphate from Gibco) with 200 ng/mL BMP2 to induce osteoblast differentiation. After BMSCs were cultured in the differentiation media for 7 days, alkaline phosphatase (ALP) staining was conducted using the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime) to examine the early osteoblast differentiation of BMSCs. Furthermore, Alizarin red staining (Solarbio, Beijing, PRC) was conducted to examine the late osteogenic differentiation of BMSCs after BMSCs were cultured in differentiation media for 14–21 days. Mouse embryonic fibroblast (MEF) cells were extracted from 13.5-day embryos of C57BL/6 mice. MEFs, at less than five passages, were cultured in DMEM (Gibco, California, USA) with 10% FBS (Gibco, California, USA).

CFSE-labelled MSCs

BMSCs were resuspended in 1 mL of PBS with 2.5 µM CFSE. BMSCs were dyed in CFSE solution and shaken in a 37 °C water bath for 10 min. Then, the cells were washed with complete stem culture media twice before the following experiments.

Adenovirus infection of MSCs

The shRNA for YAP and constitutively active YAP (5SA) DNA were obtained from Cyagen (Guangzhou, PRC) and delivered via adenovirus. The shRNA sequence for YAP was 5′-ATGTGTCTCCAGGAGTAATAA-3′. The mutant form of YAP (5SA) had mutations at S61, S109, S127, S164 and S381. MSCs were infected with either the shRNA for YAP to knock down its expression or the constitutively active YAP (5SA) to overexpress it. Western blot analysis was performed 48–72 h after infection to confirm the expression levels of YAP. Equal numbers of MSCs with YAP knockdown/overexpression or controls were used for further differentiation assays.

Cell proliferation

MSCs (3 × 103) were cultured in a single well of a 96-well plate for 0 d, 1 d, 2 d and 3 d. Subsequently, cells were harvested and subjected to the CCK-8 assay. Absorbance at 450 nm was examined using a Molecular Devices SpectraMax i3X system. The experiments were conducted three times, and the average relative cell viability to Day 0 was calculated.

Quantitative analysis of cell proliferation under cell coculture conditions was performed as follows. After being labelled with the fluorescent dye CFSE, MSCs were cultured with the indicated treatment. Subsequently, cells were harvested and subjected to flow cytometry using BD Accuri C6. The percentage of MSCs with decreased fluorescence provided an indication of the proportion of proliferating MSCs.

Cell migration

A total of 5 × 103 MSCs labelled with CFSE and 5 × 103 MEFs in free DMEM were placed on the upper layer of a Corning (New York, USA) cell culture insert with an 8.0 μm polycarbonate membrane. DMEM with 10% FBS and 200 ng/mL BMP2 was placed below the cell permeable membrane. Following an incubation period of 24 h at 37 °C and 5% CO2, the cells that had migrated through the membrane were harvested and subjected to immunofluorescence. Images were taken using a Zeiss Axio Scope A1 FL for FISH microscopy.

Human samples

Human samples were obtained from patients with unhealed fractures over 6 months who were diagnosed with bone nonunion, which required surgical intervention in the clinic. During the operation, tissues in the gap of bone fracture were harvested and stored in formalin solution for further analysis. All experiments were approved by the Ethical Committee of First Affiliated Hospital, Fujian Medical University (MRCTA, ECFAH of FMU [2022]340), and all patients signed informed consent forms. Detailed characteristics of the donors are shown in Additional file 1: Table S1.

Mice

Female C57BL/6 mice, 6–8 weeks old, used in this study were bred and maintained in a specific pathogen-free animal facility at Fujian Medical University. All animal experiments were approved by the Animal Ethical Committee of Fujian Medical University (FJMU IACUC 2021-0506). To exclude the interference of in situ osteoblasts, we used only an ectopic bone formation model. C57BL/6 mice were anaesthetized with an intravenous injection of 1.5% pentobarbital sodium (40 mg/kg body weight, Sigma, USA). A 1 cm skin incision was made on the skin of each mouse’s back, and the myolemma was longitudinally split. BMP2-loaded collagen materials (7.5 µg BMP2/150 µL 3% rat tail collagen I solution) or control materials (vehicle/150 µL of 3% rat tail collagen I solution) were implanted into the intramuscular gap of the backs of the mice. The control group or the BMP2 group contained five animals each in one experiment. Animals in the same group were fed in the same cage. The operation was performed by the same researcher, and all the mice were maintained in the same environment to minimize potential confounders. The experiment was repeated three times, and a total of 30 mice were used. After the muscles and skin were sutured, 200,000 IU penicillin sodium was injected intramuscularly to prevent possible infection. Animals were sacrificed after 8 weeks, and no animals were excluded from the experiment. Approximately 0.5 cm3 tissues around the implanted sites were harvested for further examination.

Masson staining

The tissues were embedded in paraffin and cut into 2.5 μm tissue sections. Tissue sections were dewaxed with xylene and rehydrated with a gradient of 100%, 95% and 75% alcohol. A Masson staining kit (Solarbio, Beijing, PRC, G1346) was used to stain the sections. Then, a neutral resin was used to mount the sections. Photos were taken using a BX53 Olympus microscope.

Immunofluorescence

The protocol was the same as that of Masson staining before tissue sections were incubated with the primary antibodies. For cell samples, the samples were harvested and fixed with 4% paraformaldehyde (Sigma, Shanghai, PRC) for 20 min. Then, 10% BSA was used to block the cell samples for 20 min, and 0.25% Triton X-100 was used to permeate the samples for 5 min at room temperature. After that, the samples were incubated with primary antibodies overnight at 4 °C. On the second day, samples were further incubated with TRITC- or FITC-conjugated secondary antibodies for one hour at room temperature. DAPI (Solarbio, Beijing, PRC, C0060) was used to stain the nucleus. Images were taken using a Zeiss LSM 800 laser scanning confocal microscope.

Immunoblotting

Cells were lysed with TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, pH = 7.5) for the immunoblotting assay. The cell lysates were mixed with 4× loading buffer (40 mM Tris-HCl, 200 mM DTT, 4% SDS, 40% glycerol, 0.032% bromophenol blue, pH = 8.0) and run with 4% stacking gel and 10% separating gels. Proteins on the gels were transferred to nitrocellulose filter membranes for antibody incubation. The membrane exposure was performed using the Thermo Pierce ECL and FluorChem E (Protein Simple, California, USA) System.

Quantitative real-time PCR

Total cell RNA was isolated using TRIzol (Invitrogen, California, USA), and cDNA was synthesized using Revertra Ace (Promega, Madison, USA). Real-time PCR was performed using an ABI QuantStudio 5 system. The expression level of genes was measured using the comparative Ct method. Expression values were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) expression. The primer sequences are shown Additional file 1: Table S2.

RNA-seq

The RNA-seq data were obtained from the Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA). RNA library preparation was performed as described in the instructions provided in the QIAseq Stranded RNA Library Kits (QIAGEN, Dusseldorf, Germany). Total RNA was extracted from cells, and the mRNA was fragmented to an average insert size of 200–400 bp. The resulting RNA fragments were then converted into first-strand cDNA using reverse transcriptase (Thermo Fisher Scientific, Massachusetts, USA) and random primers. The first-strand cDNA was further converted into double-stranded DNA in the presence of dUTP, and these cDNA fragments were subjected to the addition of a single ‘A’ base and subsequent ligation of the adapter. The products were purified and enriched via PCR to generate the final library. The libraries were sequenced on the Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA). Raw sequences were mapped to the mouse genome mm10 by STAR (v2.5.4b_ENREF_32) [12]. The expression level FPKM values were obtained from Cuffnorm in the Cufflinks package (v2.2.1) [13]. The data could be assessed online at the Gene Expression Omnibus (GEO) (No. GSE205156).

ELISA

For ELISAs, 1 × 104 MSCs and MEFs were cultured in 1 mL of medium with 2% FBS. Supernatants from cell cultures were collected as samples and subjected to DKK1 ELISAs (Multi Sciences, Hangzhou, PRC, EK2111).

Statistical analysis

Statistical analysis was performed using Student’s t test, one-way ANOVA and two-way ANOVA. P values < 0.05 were considered statistically significant.