Bone morphogenetic protein 9 enhances osteogenic and angiogenic responses of human amniotic mesenchymal stem cells cocultured with umbilical vein endothelial cells through the PI3K/AKT/m-TOR signaling pathway

Characterization of hAMSCs and HUVECs

Primary cultured hAMSCs adhered to the culture flask within 24 h and its morphology observed after 24 h. hAMSCs at P1, P2, and P3 exhibited a spindle-shaped extrinsic feature. During primary culture, hAMSCs reached 80-90% confluence at 5 d (Figure 1A). Proliferation ability of hAMSCs and HUVECs were determined by the CKK-8 assay. The hAMSCs and HUVECs went through a logarithmic phase for 2-3 d after 1 d of incubation period, where the multiplication rate was recorded. The doubling times of the HUVECs and hAMSCs were 33.5 h and 28 h, respectively (Figure 1B). Flow cytometry (FCM) assay showed that hAMSCs, positively expressed specific surface markers of MSCs including CD73, CD90, CD105, and CD44, while negatively possessed markers CD19, CD45, CD34, and HLA-DR at P3 (Figure 1C). The immunofluorescence staining assay showed that HUVECs greatly expressed angiogenic relative markers CD31, VEGF, Endomucin (EMCN), and vWF (Figure 1D). Moreover, multiple-directional differentiation of hAMSCs showed that isolated cells be able to differentiated into osteoblastic, chondrogenic and adipogenic cells after being cultured in specifically appointed differentiation culture medium at 21 d (Figure 1E). In the isolation procedures of hAMSCs, the human amniotic epithelial cells (hAECs) are interfused with hAMSCs. In hAECs, CK-19 exists as a specific marker and vimentin exists as a specific marker in hAMSCs. The immunofluorescence staining assay revealed that hAMSCs positively expressed vimentin which negatively expressed CK-19 at P3 (Figure 1F).

Figure 1. Characterization of isolated hAMSCs and HUVECs. hAMSCs at P0 to P3 showing a fibroblast-like morphology and spindle shaped configuration (×40, bar: 200 μm) (A); CCK-8 assay showed the doubling time for hAMSCs and HUVECs was 28 h and 33.5 h, respectively (*P < 0.05) (B); Flow cytometry results showed phenotypical identities of hAMSCs at P3 (C); Phenotypic properties of HUVECs and immunofluorescence staining showed HUVECs at P3 highly expressed the surface markers of endothelial cells (×100, bar: 50 μm) (D); Multi-lineage differentiation potential of hAMSCs in vitro. Alizarin Red S staining of hAMSCs for 21 d. Alcian Blue staining of hAMSCs for 21 d. Oil Red O staining of hAMSCs for 21 d (×100, bar: 50 μm) (E); hAMSCs at P3 scarcely expressed CK-19 and highly expressed vimentin. CK-19 and vimentin were stained green by FTIC, and cell nuclei were stained blue by 4’, 6-diamidino-2-phenylindole (DAPI) (×100, bar: 50μm) (F). FITC: Fluorescein isothiocyanate; PE: Phycoerythrin.

BMP9 enhances osteoblastic differentiation of cocultures of hAMSCs and HUVECs

We observed the effect of cocultures of hAMSCs and HUVECs on BMP9-induced osteoblastic differentiation. Then we constructed an adenovirus Ad-BMP9 to effectively overexpress BMP9 and showed that Ad-BMP9 is capable of transfecting hAMSCs and HUVECs with high efficiency (Figure 2A). The RT-qPCR results revealed that compared with the control group, BMP9 expression in hAMSCs and HUVECs increased more than 17-fold and 12-fold, respectively, after transfection with Ad-BMP9 for 48 h (Figure 2B).

Figure 2. BMP9 enhances osteoblastic differentiation of hAMSCs cocultured with HUVECs in vitro. The adenovirus Ad-BMP9 (green) was shown to successfully transfect hAMSCs and HUVECs for 24 h (×100, bar: 50μm) (A); Ad-BMP9 upregulated the gene expression level of BMP9 over 17 times and 12 times of hAMSCs and HUVECs at 48 h, respectively (B); Overexpressed BMP9 enhanced ALP activity in hAMSCs cocultured with HUVECs at 3, 5 and 7 d (C); ALP biochemical quantification results showed BMP9 increased the expression of ALP in cocultured hAMSCs and HUVECs at 3, 5 and 7 d, respectively (D); The bone mineralization in each group were detected by Alizarin Red S staining assay under the treatment as shown at 14 and 21 d (E); The quantification of mineralization in each group were analyzed under the treatment as shown at 14 and 21 d (F) (*P < 0.05).

Thereafter, ALP staining was used to investigate changes in ALP activity, which is an early osteoblastic indicator. Alizarin Red S staining was to assess the alterations in bone mineralization and matrix deposition, which are late osteogenic indicators. When hAMSCs were cocultured with HUVECs, we found that ALP activity significantly increased at 3, 5, and 7 d in Group C, when compared with Group A and B; Group B exhibited the lowest ALP activity at the same time points (P<0.05). The ALP staining demonstrated that BMP9 notably increased ALP activity in Group F compared with Group D and E at 3, 5, and 7 d (Figure 2C). Quantitatively, overexpression of BMP9 in hAMSCs cocultured with HUVECs could lead to a considerable increase in ALP activity for Group D and E at 3, 5, and 7 d (Figure 2D). Alizarin Red S staining results exhibited a significant increase in bone matrix mineralization in hAMSCs cocultured with HUVECs (Group C) than Group A and B at 14 and 21 d, respectively (Figure 2E). Moreover, we found that BMP9 enhanced bone matrix mineralization and calcium deposition in Group F at 14 and 21 d, respectively, compared to other groups (Figure 2F). Collectively, the results suggest that BMP9 could greatly enhance osteoblastic differentiation of cocultured hAMSCs and HUVECs in vitro, suggesting that determining the incongruous expression level of BMP9 may be crucial for osteoblastic differentiation of MSCs and endothelial cells.

BMP9 upregulates the expression of osteogenic and angiogenic-related factors of cocultured hAMSCs and HUVECs

Furthermore, we performed RT-qPCR and western blotting analyses to investigate mRNA expression levels and protein secretion of osteogenic and angiogenic relative factors after transfection with Ad-BMP9. Runt-related transcription factor 2 (Runx2) is a primary transcription factor that acts as a critical protein regulating bone specific genes and co-regulatory proteins for osteogenesis. Runx2, OPN, OCN, collagen type I (COL-I) and binding sialoprotein (BSP) are vital indicators for osteoblastic differentiation of hAMSCs. Hence, we investigated the effects of BMP9 on gene and protein expression of Runx2, COL-I, BSP, OPN and OCN. The RT-qPCR results indicated that the mRNA expression of Runx2, OPN, COL-I and BSP was significantly up-regulated in Group F compared to other groups at 5 d (P<0.05). These factors were also significantly upregulated in Group C than Group A and B at the same time points. The mRNA expression of these factors greatly increased in Group D compared to Group A; however, BMP9 did not exert significant effects on HUVECs (P<0.05) (Figure 3A).

Figure 3. BMP9 upregulates the expression of osteogenic and angiogenic related factors of cocultured hAMSCs and HUVECs in vitro. RT-qPCR assay was performed to detect the mRNA expression level of BMP9 induced osteogenic relative factors COL-I, Runx2, OPN, and BSP in each group at 5 d (A); The mRNA expression level of BMP9 induced angiogenic relative factors including ANT1, VEGF, CD31, and vWF in each group at 5 d (B); Western blot was used to determine the protein expression of OPN (C), OCN (D), and Runx2 (E) of hAMSCs cocultured HUVECs at 5 d in vitro; Quantification analysis of Western blot showed that BMP9 significantly increased the protein expression of osteogenic relative factors OPN, OCN, and Runx2 in hAMSCs cocultured with HUVECs at days 5 (F) (*P < 0.05).

Our previous study suggested that BMP9 could mediate angiogenic differentiation of MSCs [29]. However, it is not clear whether BMP9 had any effects on the expression level of angiogenic factors on cocultured hAMSCs and HUVECs. Hence, we investigated the effects of BMP9 on the expression of angiogenic factors including angiopoietin 1 (ANT1), VEGF, CD31, and vWF in hAMSCs cocultured with HUVECs. Our results demonstrated that BMP9 was able to significantly upregulate the mRNA expression level of ANT1, VEGF, CD31, and vWF in Group F than in other groups at 5 d. BMP9 notably increased the gene expression level of angiogenic factors in HUVECs (Group E) than Group B. Though BMP9 upregulated the expression of these factors in Group D compared to Group A at 5 d, the upregulated level was lower than Group E and F (P<0.05) (Figure 3B).

Similarly, the western blotting and quantification results revealed that BMP9 boosted protein expression of osteoblastic relative factors OPN, OCN and Runx2 in cocultured hAMSCs and HUVECs at 5 d. The expression of OPN, OCN and Runx2 were upregulated by BMP9 significantly in Group F than with other groups (P<0.05) (Figure 3C–3F). Collectively, these results demonstrated that BMP9 exerts a positive-going regulatory effect on the expression of osteoblastic and angiogenic relative factors on cocultured hAMSCs and HUVECs.

BMP9 reinforced the ectopic bone formation of cocultured hAMSCs and HUVECs

With the above conclusion that BMP9 enhanced osteogenic differentiation of cocultured hAMSCs and HUVECs in vitro, we further confirmed whether BMP9 played such a role in vivo by ectopic bone formation in nude mice. The general observation showed ectopic osteogenesis in nude mice (Figure 4A). Size differences were detected by three-dimensional reconstruction analysis of micro-CT. The heat image analysis of mineral density revealed that BMP9 enhanced the mineral density of bone masses formed by cocultured hAMSCs and HUVECs (Group F) than other groups, yet, the average mineral density was lower in Group C than Group D (Figure 4B). The retrieved samples were further performed to histologic analysis and quantification, including values of bone volume/total volume (BV/TV%), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), and bone mineral density (BMD). These values were evidently increased in Group F as compared with Group C and D (P<0.05). There were no significant differences in trabecular Tb. Sp (Figure 4C), histologic analysis including H&E staining and Masson staining demonstrated that the number and quality of trabecular bone formation (red in H&E staining; blue in Masson Trichrome staining) in Group F increased (Figure 4D). Collectively, these results illustrated that BMP9 exerts a positive regulatory effect on osteoblastic differentiation and BMP9 augments ectopic bone formation of cocultured hAMSCs and HUVECs in vivo.

Figure 4. BMP9 increases subcutaneous ectopic bone formation of hAMSCs cocultured with HUVECs. Representative images of subcutaneous ectopic bone formation of nude mice (A); Subcutaneous ectopic osteogenic bone formation were detected by micro-CT scanning to investigate the 3D iso-surface and the heat map of average mineralization density at 4 weeks. In the heat map, white represents the highest average mineral density and black represents the lowest (B); Quantified analysis results of micro-CT scanning show the effect of BMP9 on cocultured hAMSCs and HUVECs induced ectopic bone formation, and the relative values of bone volume/total volume (BV/TV), trabecular bone number (Tb. N), trabecular bone separation (Tb. Sp), trabecular bone thickness (Tb.Th), and bone mineral density (BMD) were demonstrated (C); The retrieved bone masses were subjected to Trichrome-Masson staining and H&E staining to determine the formation of trabecular bone and bone matrix (×400, bar: 50μm) (D) (*P < 0.05).

BMP9 augments osteogenic and angiogenic relative factors in subcutaneous ectopic bone formation

The immunofluorescence results and quantification of ectopic bone formation indicated that the expression level of osteogenic relative factor OPN was significantly increased in Group F compared with control group (P<0.05). The expression level of OPN was higher in Group D than that in Group C. Furthermore, we investigated the effect of BMP9 on angiogenic relative factor VEGF. Similarly, BMP9 could potentiate the expression of VEGF in Group F as compared with Group C and D. The protein expression level of VEGF was lower in Group C than that in Group D (Figure 5A, 5B). In summary, these results suggest that BMP9 could increase protein expression of osteogenic and angiogenic relative factors in subcutaneous ectopic bone formation.

Figure 5. Immunofluorescence staining and quantification analysis of osteogenic and angiogenic differentiation of cocultured hAMSCs and HUVECs in vivo. Immunofluorescence and quantified analysis of ectopic bone masses to investigate the synthesis of osteogenic relative factors OPN (green) and VEGF (green) (×400, bar: 20 μm) (A); The protein expression of OPN and VEGF were analyzed by immunofluorescence and quantified analysis (B) (*P < 0.05).

Effects of BMP9 on vessel invasion in cocultured hAMSCs and HUVECs

The hAMSCs and HUVECs were treated according to the experimental design and seeded on PLGA scaffolds for 48 h. The cells on scaffolds were implanted on the subcutaneous tissues of nude mice (Figure 6A). The topological properties of PLGA itself with cells for 24 h were detected by scanning electron microscope (SEM) (Figure 6C). After implantation for 5 weeks, mice were euthanized. The implants were retrieved and detected by immunofluorescence staining. The general graft was observed (Figure 6B) and immunofluorescence staining and its quantification data demonstrate the expression of CD31 was significantly higher in Group C than Group A and B, and Group F expressed the highest level among groups (P<0.05) (Figure 6D, E). Collectively, BMP9 not only strengthened angiogenesis of cocultured hAMSCs and HUVECs in vitro, it also potentiated in vivo subcutaneous vessel invasion ability in the cocultivation of both cells.

Figure 6. Effect of BMP9 induced in vitro angiogenic differentiation and subcutaneous vascular invasion of PLGA-cells composite in vivo. Illustrative diagram demonstrating cells treated according to the experimental design and seeded on electron-spun PLGA scaffold, which were implanted into the dorsal subcutaneous space of mice (A); Representative image of the macromorphological observation of PLGA implants located on subcutaneous tissue at 5 weeks (B); The topological structure of PLGA and cell-PLGA-hybrids were observed by scanning electron microscopy (SEM) (magnification ×1000) (C); Immunofluorescence staining and its quantification analysis were performed to detect the CD31 expression (green) in PLGA scaffold subcutaneously (×400, bar: 20 μm) (D, E) (*P < 0.05).

The PI3K/AKT/mTOR axis mediates induced osteoblastic and angiogenic differentiation induced by BMP9 in cocultivation of hAMSCs and HUVECs

In order to investigate the mechanism underlying osteogenic and angiogenic differentiation induced by BMP9 in cocultured hAMSCs and HUVECs, we determined the protein expression variation of VEGF in each group induced by BMP9. Protein expression of VEGF was greatly increased in Group F than in other groups at 5 d, assessed by the ELISA assay (P<0.05). The protein level in cocultured hAMSCs and HUVECs (Group C) was higher than Group A and B (Figure 7A). The western blotting and quantification results illustrated that BMP9 notably increased the expression of Smad1/5/8 (p-Smad1/5/8) of Group F among groups (P<0.05). Compared with Group C, D and F, the p-Smad1/5/8 expression did not alert Group A, B and C without BMP9 induction. Additionally, BMP9 also increased the expression of HUVECs (Group E) than Group B. Hence, it remains clear that BMP9 on the expression of p-Smad1/5/8 in cocultured hAMSCs and HUVECs (Figure 7B, 7F).

Figure 7. Molecular mechanism and signaling pathways underlying BMP9 enhanced osteogenesis and angiogenesis in cocultured hAMSCs with HUVECs. ELISA was adopted to detect the effect of BMP9 that regulates protein expression of VEGF in cocultured hAMSCs and HUVECs (A); Western blot and quantification analysis were used to determine the expression of p-Smad1/5/8, Smad1/5/8, p-PI3K, PI3K, p-AKT, AKT, and mTOR in each group as shown at 5 d after transfection. The results suggested that BMP9 enhanced osteogenesis and angiogenesis of cocultured hAMSCs and HUVECs by activating the BMP/Smad and PI3K/AKT/m-TOR signaling pathways through upregulating the level of phosphorylation of Smad1/5/8 and of PI3K, AKT, and protein expression of m-TOR (B–F) (*P < 0.05).

PI3K/Akt/mTOR signaling was involved in multiple pathways and played a vital role in proliferation, migration and angiogenesis of tumor cells [30]. The mechanism by which BMP9 induces cocultured hAMSCs and HUVECs is ambiguous, so we further investigated the expression of the PI3K/Akt/mTOR.

The western blotting and quantification results demonstrated that phosphorylation of PI3K, AKT and of mTOR was increased after the induction of BMP9. The phosphorylation level of PI3K (p-PI3K), AKT (p-AKT) and mTOR in Group C was notably higher than Group A and B, which indicated that cocultured hAMSCs and HUVECs could enhance the intensity and phosphorylation level than hAMSCs and HUVECs alone. The expression level of p-PI3K, p-AKT and mTOR remarkably increased in Group F than other groups, which suggested that BMP9 could drastically up-regulate the intensity and phosphorylation level of cocultured hAMSCs and HUVECs (P<0.05). Moreover, BMP9 increased the phosphorylation of Smad1/5/8 and either p-PI3K, p-AKT and mTOR in HUVECs (Figure 7C–7F) (Supplementary Figure 1). In summary, these results may point to the effects of osteogenic and angiogenic differentiation induced by BMP9 on cocultivation with hAMSCs and HUVECs, and the mechanism may be transduced by strengthening BMP/Smad1/5/8 and BMP/PI3K/Akt/mTOR signaling pathways.

Study groups

All experiments were performed in triplicate. The hAMSCs were used as Group A and HUVECs as Group B; Group C was composed of cocultured hAMSCs and HUVECs, and Group D, hAMSCs infected with Ad-BMP9. Group E was composed of HUVECs infected with Ad-BMP9. Group F was composed of cocultures of hAMSCs and HUVECs, in which, both were infected with Ad-BMP9.

Animals and ethics statements

This experiment was approved by the Research Ethics Committee of the First Affiliated Hospital of Zunyi Medical University. Human placentas were obtained from the Obstetrics Department of the First Affiliated Hospital of Zunyi Medical University and informed consent was provided by all patients before surgical retrieval and application. hAMSCs were isolated from five full-term puerperants, and their basic information is demonstrated in Table 1. Informed consent was provided by patients before delivery. Surgical procedures were performed on thirty-three adult male athymic nude mice (four to six weeks old, weighing 15-20 g).

Isolation and culture of hAMSCs

The amniotic membrane was bluntly peeled from the placenta and washed three times with phosphate-buffered saline (PBS) with 1% penicillin and streptomycin. The amniotic membrane was minced with sterile scissors into 1-2 mm² sized pieces, and digested twice (30 min each) with 0.05% trypsin/0.02%. Then, the amniotic pieces were incubated with 0.75 g/L collagenase, digested at 37° C in a water bath for 60 min. hAMSCs were collected and passed through a 300-mesh filter, suspended in culture dishes containing Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1%penicillin and streptomycin, 1%L-glutamine, and nonessential amino acids. The medium was replaced every 48h, and morphology were recorded. Cells were passaged upon attaining 80% confluence, and seeded at a 1:2 ratio for subculture. hAMSCs at P3 were used for experiments.

Phenotypic identification of hAMSCs

Specific surface markers

In accordance with the minimal criteria for defining MSCs proposed by the International Society for Cellular Therapy (ISCT) [65], the phenotypic markers were evaluated using flow cytometry. Cells were adjusted to 2 × 10⁶ cells/mL were incubated with FITC-conjugated anti-CD90,-CD44, anti-CD105, and -CD73. Cells were combined with negative control antibodies, including PE-conjugated anti-CD34, anti-CD19, anti-CD45, anti-CD11b, and anti-HLA-DR in the dark for 30 min, washed by the addition of 2mL. The labeled cells were washed with PBS three times and resuspended in 250μL of flow buffer, and expression of surface markers was analyzed by BD Software (BD Accuri^TM C6 Plus Corporation, USA).

Multilineage differentiation of hAMSCs

HAMSCs at P3 were adjusted to 10⁵ cells/mL. When cells reached 50% confluence then were cultivated in osteogenic, chondrogenic and adipogenic differentiation medium (Gibico™, USA). The osteogenic differentiation was detected by Alzarin Red S staining (Solarbio, Beijing, China). The chondrogenic differentiation was assessed by Alcian Blue staining (Solarbio, Beijing, China). Oil Red O (Solarbio, Beijing, China) staining was performed to determine the adipogenic differentiation.

Isolation and identification of HUVECs

The umbilical cord was removed and rinsed three times in 4° C PBS with 1% penicillin and streptomycin. The hematoma was removed from the umbilical cord with PBS, then pre-heated PBS was injected to flush the blood in the umbilical vein. A hemostat was used to clamp the end of the umbilical cord and 1 g/L pre-heated type 2 collagenase was injected using a syringe into the other end of the umbilical vein to fill the lumen. The umbilical cord was placed into a petri dish containing the pre-warmed PBS, incubated at 37° C for 30 min. The digestive mixture was collected in a centrifuge tube containing 200 mL/L FBS complete medium. The cells were replaced with endothelial conditioned medium containing 10% FBS, 2 mM/L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin and 1% endothelial cell growth supplement (ECGS) (ScienCell, CA, USA) after 24 h adherence, and cultured every 2 d. Immunofluorescence staining was used to identify specific markers of HUVECs, including CD31, VEGF, EMCN, and vWF.

Cell counting kit- (CCK-8) assays

Cells were trypsinized and prepared into a cell suspension at 105 cells/mL. One hundred microliters of cell suspension was added to each well. Marginal wells were filled with sterile PBS and incubated at 37° C with 5% CO₂. Thereafter, cells were cultured for 7 days. Ten microliters of CCK-8 solution was added to each well, the plate mixed by gentle tapping for 2 h. The absorbance was detected at 450 nm using a microplate reader, and the cell proliferation activity was calculated.

Immunofluorescence staining

Cells were seeded on cover slips at 10⁴ cells/mL, and treated as the experimental design. Cells were washed with PBS for 10 min each, then fixed with 4% paraformaldehyde at 37° C for 15 min at the indicated time point. The cover slips were gently washed with PBS then washed with 0.4% Triton X-100 for 30 min. Thereafter cells were blocked by using goat serum for 30 min, then incubated with the first antibodies overnight. Incubation with the corresponding FITC antibodies for 60 min. Slips were washed with PBS for 10 min and stained with DAPI for 5-10 min. Results were observed by fluorescence microscope and images were assessed by Image Pro Plus software.

Construction and infection of recombinant adenovirus

By using Ad-Easy technology as described previously, the recombinant adenoviruses were constructed [66, 67]. Briefly, the coding areas were amplified with the real time polymerase chain reaction and cloned into adenoviral vectors. The shuttle vectors were recombined with BJ5183/AdEasy-1 cells. Then, the correct recombinant vectors were linearized and transduced into HEK293 cells to package recombinant adenoviruses. The resulting adenoviruses were designated as Ad-BMP9 [68] and the Ad-BMP9 expresses GFP (green fluorescent protein). The multiplicity of infection (MOI) of transduction of hAMSCs and HUVECs was adjusted to 40% and the cell ratios of two type of cells in co-culture system was 1:1.

ALP activity assays

Cells were adjusted into 30-40% confluence and treated as the experimental design. ALP activities were detected quantitatively by a modified Great Escape SEAP Chemiluminescence assay (Beyotime, Beijing, China) as described according to the manufacturer’s protocol [69, 70]. For the chemiluminescence assay, the results were repeated in three independent experiments.

Alizarin S staining and mineralization assay

Cells were treated with recombinant adenovirus as the experimental design. Cells were cultivated with osteogenesis conditioned medium containing 50 mg/L vitamin C, 0.1 μmol/L dexamethasone, and 10 mmol/L β-glycerol phosphate disodium for 14 and 21 d. Mineralization was determined by Alizarin Red S staining as described previously [71, 72].

RNA isolation and RT-qPCR reaction

Total RNA was collected to synthesize cDNA by using reverse transcription reaction kit (RR047a, Takara, Japan) following the manufacturer’s instructions. Thereafter, the cDNA were diluted 5-fold and as templates for detection by PCR. The reactions were run on a Bio-Rad CFX-96 system. All samples were normalized with the level of glyceraldehyde phosphate dehydrogenase (GAPDH). Custom-specific primers (Sangon Biotech, Shanghai, China) are as shown in Table 2. The relative expression level of mRNAs in groups was calculated using the 2^ΔΔCT method and was normalized to the gene expression of the control.

Western blotting analysis

Total protein was collected after cleared lysates were denatured by boiling for 10 min. Protein were separated by 8% or 10% (wt/vol) SDS-PAGE as manufacturers’ described [73]. Proteins were separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was blocked with 5% milk for 2 h and incubated overnight with first antibodies. Then the membranes were probed with secondary antibody. Immune-reactive signals were detected and captured using Bio-Rad. The β-ACTIN (60004-1; Proteintech) antibody was used as the internal control. Concentrations of proteins were quantified by using Image-Pro Plus software.

ELISA

Cells were treated according to the experimental design. The cell culture medium were centrifuged to remove debris. Supernatants were diluted 5-fold. Protein level of VEGF (DVE00, R&D, MN, USA) were detected by ELISA. Standard and sample were added per well and covered with the adhesive strip and the plate was incubated for 30 min. Standard concentration holes were added as follows: 0, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 ng/mL, in accordance with the manufacturer’s instructions.

Cell implantation, ectopic ossification and micro computed tomographic (Micro-CT) assay

Cells were treated with specific adenoviruses and cocultured according to the experimental design. Then the ectopic bone masses were collected for injection (5 × 10⁶ cells) from the flanks of nude mice (4-6-week old males, adult male athymic nude mice). After injection for 4 weeks, the nude mice were euthanized the samples were collected for micro-CT (Bruker Company, Belgian) and histologic staining.

Retrieved samples were decalcified for 14 days and then processed for paraffin embedding. The parameters including trabecular bone volume (BV/TV, %), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th) and bone mineral density (BMD) were detected.

Histochemical staining

The tissues were decalcified with EDTA, washed with PBS and fixed in 4% paraformaldehyde overnight at 37° C, and embedded in paraffin. Serial sections of embedded bone masses were stained with (H&E), and Masson Trichrome staining Solarbio Company, Beijing, China carried out as previously described [69, 74].

Implantation to evaluate angiogenesis in vivo

Cells were seeded at 5 × 10⁵ cells/mL on PLGA scaffolds (diameter 3.5 mm, thickness 200 μm) (Lepton Company, China) for 24 h. The morphology of PLGA scaffold seeded with cells was detected by scanning electron microscopy. Eighteen mice (6 week old males; BALB/cAnN, Beijing, China), weighing 20 g, were anesthetized with 1% Pentobarbital Sodium, and the cells-PLGA compounds were implanted into the subcutaneous skin. The composite were analyzed after 5 weeks. The composite was retrieved and fixed in 4% paraformaldehyde, immunohistochemical staining was performed for CD31 (ab28364, Abcam, Cambridge, MA, USA), then by incubation with secondary FITC antibodies. The results were analyzed using an Olympus auxiliary system.

Statistical analysis

All experiments were performed in three times. The data are reported as the mean ± SD and statistical analyses using the software package SPSS 14.0. Student q tests were used to commit significant differences. Statistical significance was set P < 0.05.