Fertilized Leghorn eggs were obtained from the Avian Farm of the South China Agriculture University (Guangzhou, China) and were incubated in a humidified incubator (Hamburger and Hamilton) (Misske et al., 2007). PB (98% purity, Merck) were dissolved in 0.9% sterile saline and then stored in 4°C. The chick embryos were exposed to different concentrations of PB (0.04, 0.4, or 4 mM) or 0.9% sterile saline (control) for 15.5 days. Briefly, approximately 200 μL of 0.9% sterile saline or 0.04, 0.4, or 4 mM PB was carefully injected into a small hole made in the air chamber of the egg every other day from day 1.5 until day 17. The surviving embryos were harvested for skeleton staining (n = 6 for each group).

To visualize the skeleton, the chick embryos were stained with alcian blue and alizarin red as previously described (Schmitz et al., 2010a). Day-17 chick embryos were freed from adherent tissue, fixed in 95% ethanol for 3 days, stained for cartilage with alcian blue and counterstained for bone with alizarin red (Solarbio, Beijing, China). Long-bone tissues were carefully photographed using a stereomicroscope (Olympus MVX10, Japan). The length of the alizarin red-stained portion of each radius, ulna, tibia and phalanx was quantified using Image Pro-Plus 5.0 (Media Cybernetics).

The fertilized eggs were incubated for 14 days; then, the growth plates of phalanges were isolated and randomly used for control (0.9% sterile saline) or PB treatment (0.4 or 1.6 mM). The growth plates were cultured in F-12 (Myclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) containing PB or 0.9% sterile saline (control) at 37°C and 5% CO₂ (Galaxy S, RS Biotech, UK). After incubation for 72 h, the cultured growth plates were examined using semi-quantitative RT-PCR analysis (n = 3 for each group).

Fertilized eggs were incubated for 2.5 days and then placed into a sterilized glass dish with the YSM facing upward (n = 6 for each group). Two silicone rings were placed on top of the leading edge of the blood vessels marked with ink to indicate the starting position of the YSM within the ring. 50 μL of 0.9% sterile saline (control) was introduced into the ring located on the left side of the YSM, marked in black. Fifty microliter of 0.4 or 1.6 mM PB was introduced into the ring marked in red on the right side of the same embryo. The extent of the expansion of the blood vessel plexus inside the silicone rings was determined and photographed after incubation for 12–36 h. The density of blood vessels in the YSM was analyzed using Image Pro-Plus 5.0 software. The blood vessel density is expressed as the percentage of the blood vessel area in the whole stereomicroscopic field (He et al., 2014). The extended distance of blood vessels was also quantified. Some YSMs were also embedded in paraffin wax, serially sectioned at 5 μm (Leica RM2126RT, Germany) and stained with hematoxylin & eosin (H&E). The rest of the YSMs were harvested for RNA isolation.

Chick embryos were incubated until day 7.5 (n = 3 for each group), when the CAM is well developed. The embryos were treated with PB (0.4 or 1.6 mM) or 0.9% sterile saline (control) for 48 h, and all surviving embryos were harvested for analysis. The CAM and accompanying blood vessels in the control and PB-treated embryos were photographed using a Canon Powershot SX130 IS digital camera (12.1 M Pixels). The blood vessel density was quantified as described above for assessing angiogenesis in the YSM. The CAMs were also harvested for different biochemical assays as described below.

Seventeen-day-old embryos treated with PB were harvested and fixed in 4% paraformaldehyde (PFA). The phalanges of the embryos were decalcified using a 10% EDTA solution in 1 mM PBS (pH 7.4) for 7 days at 4°C and were then embedded in paraffin. The samples were serially sectioned at 5 μm thicknesses using a microtome (Leica RM2126RT, Germany). Longitudinal sections of these bones were produced and further stained with H&E using a standard protocol for histological observations (Schmitz et al., 2010b) (n = 4 for each group). The extent of apoptosis in the bone tissues was detected by TUNEL analysis using an in situ Cell Death Detection Kit (Roche, Switzerland) (n = 4 for each group). The staining was performed according to the manufacturer's protocol and was adapted for bone section labeling. Immunofluorescence staining was performed on some sections of the phalanges using a monoclonal primary antibody against p-Histone H3 (pH3, 1:400, Santa Cruz Biotechnology) or a rabbit polyclonal PCNA (1:100, Santa Cruz Biotechnology) at 4°C overnight, followed by Alexa Fluor 555-labeled anti-rabbit IgG secondary antibody (1:1000, Invitrogen, CA, USA) (n = 5.6 for each group). The sections were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 5 μg/mL; Life Tech, USA) to reveal the nuclei and were finally photographed using an Olympus IX51 microscope. Histomorphometry was performed on TUNEL-, pH3- and PCNA-immunofluorescent sections of phalange growth plates using Image Pro-Plus 5.0 software. Cell proliferation was evaluated as a percentage of pH3⁺ cells or PCNA⁺ cells relative to the corresponding cells of the control groups. Cell apoptosis was evaluated as a percentage of TUNEL⁺ cells relative to the corresponding cells of the control groups.

Human umbilical vein endothelial cells (HUVECs, a kind gift from Zhi Huang's lab) and MC3T3-E1 cells (a mouse pre-osteoblastic cell line that was a gift from Chao Wan's lab) were cultured in a humidified incubator at 5% CO₂ and 37°C in 6-well plates (1 × 10⁶ cells/ml) containing DMEM/F12 (Myclone, USA) supplemented with 10% FBS; cells were exposed to PB (0.4 or 1.6 mM) or control (0.9% sterile saline). The cells were photographed using an inverted fluorescence microscope (Nikon, Ti-u, Japan) linked to NIS-Elements F3.2 software. After incubation for 24 h, these cultures were incubated with p-Histone H3 primary antibody (pH3, 1:400, Santa Cruz Biotechnology) at 4°C overnight (n = 6 for each group). Then, Alexa Fluor 555-labeled anti-rabbit IgG secondary antibody was used for visualizing the primary antibody. For F-actin detection, cultured cells were stained using phalloidin-Alexa Fluor 555 (1:500, Invitrogen) at room temperature for 2 h. All the cells were counterstained with DAPI at room temperature for 1 h. Cell proliferation was evaluated as a percentage of pH3⁺ HUVECs or pH3⁺ MC3T3-E1 cells relative to the corresponding cells of the control groups.

The viability of HUVECs and MC3T3-E1 cells was assessed using a modified CCK8 assay (Dojindo Molecular Technologies, Japan). All of the cells were cultured in 96-well plates (2.5 × 10⁴ cells/ml) as described above and were exposed to PB (0.1, 0.2, 0.4, 0.8, or 1.6 mM) or the control (0.9% sterile saline). After 24 h, 10 μL of CCK8 (5 g/L) was added into the 96-well plates, followed by incubation for 4 h at 37°C. The absorbance values were measured at 450 nm using a Bio-Rad Model 450 Microplate Reader (Bio-Rad, CA, USA). Cell viability was indirectly established using the ratio of the absorbance value of PB-treated cells relative to the control (n = 6 for each group).

The mesenchymal cells were dissected from 4.5-day (Hamburger and Hamilton Stage 23, HH23) chick embryos and were insolated as previously described (Ahrens et al., 1977; San Antonio and Tuan, 1986; Delise and Tuan, 2002). Briefly, limb buds were dissected from HH23 chick embryos and treated with trypsin (0.25%; Life Tech, USA), the ectoderm was removed and limb buds were gently dissociated into single cells. The cells were cultured in 6-well plates (2.5 × 10⁴ cells/ml) containing DMEM supplemented with 10% FBS and were exposed to either PB (0.4 or 1.6 mM) or the control (0.9% sterile saline). Following treatment with different concentrations of PB for 2 weeks, the cultures were fixed in 95% ethanol for 20 min and then stained with 1% toluidine blue at room temperature overnight to demonstrate the chondrogenic differentiation.

Micromass cultures were produced from limb bud mesenchymal cells as previously described (Delise and Tuan, 2002). Briefly, limb buds were dissected from HH23 chick embryos and were treated with trypsin; the ectoderm was removed and limb buds were gently dissociated into single cells. The cells were suspended in DMEM (Life Tech, USA) with 10% FBS at a density of 2.0 × 10⁷ cells/mL and were spotted as 10 μL droplets per well on a 6-well plate. After 3 h of pre-incubation, all the wells were flooded with 500 μL of culture medium. The cells were incubated at 37°C and 5% CO₂ in an incubator (Galaxy S, RS Biotech, UK). PB (0.4 or 1.6 mM) dissolved in DMEM with 10% FBS was introduced on the second day after plating. The control cultures received 0.9% sterile saline only. The culture medium was changed every 2 days.After incubation for 3 days, the cultured cells were fixed in 95% ethanol and then stained with 1% alcian blue dye (pH 1.0) overnight at room temperature. The alcian blue-stained cartilage nodules that formed in the absence or presence of PB were photographed using an inverted microscope (Nikon Eclipse Ti-U, Japan). The average size (area) of the chondrogenic nodules was digitized as total stain intensity/nodule number (n = 4 for each group).

MC3T3-E1 cells were cultured as described above for the micromass cell cultures. After treatment with PB (0.4 or 1.6 mM) for 7 days, the cultures were fixed in 95% ethanol for 20 min and then were stained with 2% alizarin red dye (pH 4.2) at room temperature overnight to detect the calcium deposits (n = 3 for each group).

Each well of a 12-well plate was coated with 200 μL of a mixture of Matrigel (BD Biosciences, USA); then, the plate was incubated at 37°C for 30 min to promote gelling. HUVECs were resuspended in DMEM/F12 medium with 10% FBS in the absence or presence of PB (0.4 or 1.6 mM), and the final volume each well was 1 ml. Photographs were taken after incubation for 4–8 h using an inverted microscope (Nikon Eclipse Ti-U, Japan) at the middle of each well. The average number of tubules was calculated using the examinations of six separate microscopic fields. Tube formation in the presence of PB was compared to tube formation in media with 0.9% sterile saline as the control or the control vector (n = 3 for each group).

HUVECs were seeded in 6-well plates with DMEM/F12 medium. At confluence, a wound was induced by scratching the monolayer with a 1-mL pipette tip. The cells were then washed 3 times with sterile PBS. HUVECs were incubated in serum-free DMEM/F12 medium with PB (0.4 or 1.6 mM) or 0.9% sterile saline (control) at 5% CO₂. Images were acquired at 0, 12, 24, and 36 h post-scratching. The images were taken using an inverted microscope (Nikon Eclipse Ti-U, Japan) (n = 6 for each group).

Total RNA was extracted from the cells and tissues using a Trizol kit (Invitrogen, USA). First-strand cDNA was synthesized to a final volume of 25 μL using a SuperScript RIII first-strand kit (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed using chick-specific primers. The primers sequences are provided in Supplementary Figure 1. The PCR reactions were performed using a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA) as previously described (Ahir and Pratten, 2014). The resolved PCR products were visualized using a transilluminator (SYNGENE, UK), and photographs were captured using a computer-assisted gel documentation system (SYNGENE). The intensity of the fluorescently stained bands was measured and normalized using Image Pro-Plus.

Data analyses and construction of statistical charts were performed using Graphpad Prism 5 (Graphpad Software, CA, USA). The results were presented as mean ± SD. All comparisons among groups were made using ANOVA or Student's t-test.

To investigate the effect of PB on skeletal development, we performed alcian blue/alizarin red staining to examine skeletal development in detail (Figures 1A–C, Supplementary Figures 2A,B) and observed that exposing chick embryos to 0.4 mM PB caused a marked defect in the ossification of several cartilage-based structures. In the axial skeleton, the defects in endochondral ossification were evident in the vertebral column (Supplementary Figures 2A',B'). Simultaneously, in the appendicular skeleton at the level of the limbs, 0.4 mM PB treatment impaired endochondral ossification centers in the phalanges (Figures 1D,F,H), the radius and ulna (Supplementary Figures 2A”,B”), and the tibia (Supplementary Figures 2A”',B”'). The ossification of phalanges, however, was indistinguishable between the control embryos and the embryos treated with 0.04 mM PB, suggesting that lower concentrations of PB do not affect ossification (Figures 1E,H). We did not show the embryos treated with 4 mM PB because of their high mortality rates (Figure 1G). The length of the phalanges was measured in control and PB (0.4 mM) treated group (Figure 1I, Supplementary Table 1). For the ulna, the rate of alizarin red⁺ staining was measured between control and PB treatments and statistical analyzed (Supplementary Figure 2C, Supplementary Table 1). For the radius, the rate of alizarin red⁺ staining was measured between control and PB treatments and statistical analyzed (Supplementary Figure 2D, Supplementary Table 1). For the tibia, the rate of alizarin red⁺ staining was measured between control and PB treatments and statistical analyzed (Supplementary Figure 2E, Supplementary Table 1). The length of ulna was measured in control and PB (0.4 mM) treated group (Supplementary Figure 2F, Supplementary Table 1). The length of radius was measured in control and PB (0.4 mM) treated group (Supplementary Figure 2G, Supplementary Table 1). The length of tibia was measured in control and PB (0.4 mM) treated group (Supplementary Figure 2H, Supplementary Table 1). RT-PCR data showed that PB treatment down-regulated osteogenesis-related genes, including Runx-2, ALP-L and Col1α1 (Figure 1J, Supplementary Table 1).

Next, we used MC3T3-E1 cells and HUVECs to test the effect of PB on cell viability. MC3T3-E1 cell viability was inhibited by PB in a dose-dependent manner in comparison to that of the control (Figure 1K, Supplementary Table 2), as was HUVEC viability in comparison to that of the control group (Figure 1L, Supplementary Table 2). These results imply that PB treatment during embryogenesis shortened embryonic long bones and inhibited mineralization in vivo.

To investigate whether PB could affect mesenchyme differentiation, the limb buds of HH23 chick embryos were dissected into single cells and were then cultured in a monolayer in vitro for 2 weeks. The cells were stained with toluidine blue to verify that they were chondrocytes that had differentiated from mesenchyme (arrowheads in Figures 2A–C). In the presence of PB, the number of chondrocytes was reduced in comparison with that of the control group, suggesting that PB inhibits chondrogenesis. To further confirm this result, the high-density micromass culture system of limb bud mesenchymal cells was used. In this model, chondrogenesis is initiated when the mesenchymal cells start to condense and aggregate to form large nodules. These nodules appear morphologically similar to cartilage. After 3 days of culture, the nodules began to produce an extracellular matrix (Mello and Tuan, 1999). The size of alcian blue-positive cartilaginous nodules in PB was smaller than those of the control group (Figures 2D–F). We further measured the alcian blue-positive area in the absence or presence of PB and found that it was consistent with the results from the alcian blue staining (Figure 2G, Supplementary Table 3). RT-PCR showed that PB exposure down-regulated chondrogenesis-related genes, including SOX-9 (n = 3 for each group) and Col2α1 (Figure 2H, Supplementary Table 3). These observations indicate that PB treatment triggered a delay in chondrogenesis.

To explore the mechanism of delayed endochondral ossification in PB-treated chick embryos, we first performed H&E staining in the growth plate. The H&E stained phalanx sections showed that the HZ in the PB groups was longer than in the control group. Moreover, the PZ in the PB groups was shorter than in the control group (Figures 3A,B). For the growth plate, the rates in different zones are shown in Figure 3C and Supplementary Table 4.

To exclude the possibility that the expansion of HZ resulted from increased chondrocyte proliferation, we examined the proliferation rate of chondrocytes in the PZ or RZ using pH3 or PCNA immunofluorescence staining. The results revealed fewer pH3⁺ or PCNA⁺ cells in the RZ and PZ of the growth plates exposed to PB (Figures 3D–F,D'–E', Supplementary Figures 3A–F,A'–D', Supplementary Table 4). These results suggest that expanded HZ was not associated with increased cell proliferation, but it may be related to the decreased long-bone length in the PB-treated 17-day-old chick embryos.

To determine whether the expanded HZ was associated with the reduction of cell death, we examined apoptosis in RZ and PZ using the TUNEL assay. There was no apparent difference in the RZ or PZ between the PB and control groups (Supplementary Figure 3G–N,G'–L', Supplementary Table 4). These results suggest that an expanded HZ was not associated with decreased cell death. However, the percentage of apoptotic cells in the HZ in PB-treated group was less than that of the control group (Figures 3G–J, Supplementary Table 4), suggesting that PB might delay the ossification of long bones.

These data indicate that the expansion of the HZ in the PB growth plate is not caused by increased proliferation or decreased apoptosis of chondrocytes in RZ or PZ. Therefore, the shortened length of long bone may be due to the decreased proliferation, and the hypertrophic zone phenotype may be caused by the ossification defect.

To explore whether the expanded hypertrophic zone resulted from a defect in ossification, we performed H&E staining on the vertical sections of phalanges and measured the area of the mineralized zone of the phalanges (Figures 4A–C, Supplementary Table 4). We observed that the mineralized zone of the phalanges was narrowed by the PB treatment, suggesting that PB natively affects the ossification of long bones.

To further determine the inhibitory effect of PB on ossification, we used MC3T3-E1 cells to investigate the possible PB effects on the function of osteoblasts. We already showed that PB inhibited MC3T3-E1 cell viability in a dose-dependent manner. This inhibitory effect of PB was further confirmed by the pH3 immunofluorescent staining of PB-treated MC3T3-E1 cells (Figures 4D–G, Supplementary Table 5). Actin polymerization was determined using phalloidin staining. We observed that PB remarkably weakened the actin polymerization and caused the cells to lose their polarities (Figures 4H–J). The high-density micromass culture system stained with alizarin red dye also showed that PB significantly decelerated the mineralization of MC3T3-E1 cultures compared with that of the control culture (Figures 4K–N, Supplementary Table 5). Furthermore, we examined the expression of osteoblast markers using semi-quantitative RT-PCR analysis, including Col1α1, ALP-L, and OPN (Figure 4O, Supplementary Table 5). Taken together, these data reveal that the PB treatment caused a delay in the ossification of long bones and inhibited the cytoskeletal organization of MC3T3-E1 cells.

To discover whether the delayed ossification phenotype results from a defect of vascular invasion, we performed H&E staining on the marrow cavity of phalanges at day 17 and found that the number of blood vessels (arrowheads in Figures 5A'–B”) in the PB-treated group was less than that in the control group. This observation showed that PB delayed vascular invasion in long bones. Then, we isolated the growth plates from the 14-day-incubated embryos and cultured those in the absence or presence of PB for 72 h. The expression of Col10α1 and VEGFA was determined using semi-quantitative RT-PCR. The former is a specific marker of hypertrophic chondrocytes, and the latter is an angiogenesis-related gene (Figure 5C, Supplementary Table 5).

To further explore the role of PB in angiogenesis, we used HUVECs to conduct tube formation assays and scratch-wound assays. First, the tube formation assay showed that PB treatment restricted tube formation compared to that of the control group (Figures 5D–J, Supplementary Table 5). Meanwhile, the scratch-wound assay showed that PB treatment for 12 h (Figures 6A'–C'), 24 h (Figures 6A”–C”) or for 36 h (Figures 6A”'–C”') inhibited the cell migration distance toward the midline along with incubation time in comparison with that of the control group (Fig. 6D). Both the area (Figure 6E, Supplementary Table 5) and number of migrated cells toward the midline (Figure 6F, Supplementary Table 5) were reduced by 36 h of PB treatment. This inhibitory effect of PB was further confirmed by F-actin immunofluorescence staining of PB-treated HUVECs, and cytoskeletal organization was markedly weakened by PB treatment (Figs. 6G-I). Together, these findings suggest that the PB treatment indeed impaired vascular invasion and inhibited the migration ability of HUVECs.

To investigate the effect of PB on angiogenesis in vivo, we used the YSM angiogenesis model. PB or 0.9% sterile saline (control) was administered to the silicon rings. These rings are useful in retaining PB and sterile saline in one place on the YSM. The starting point of the YSM blood vessels was marked with red/black inks on the silicon rings and was kept constant in all replicates. We found that PB treatment significantly decreased the expansion velocity of the blood vessel plexus compared with that of the control embryos (Figures 7A–D,A'–D',A”–D”). This was indicated by the leading edges of the control blood vessel plexus reaching the rings after incubation for 24 h and reaching beyond the rings after incubation for 36 h. The blood vessel density was significantly decreased in PB-treated vessels after incubation for 36 h (Figure 7F, Supplementary Table 6). The extended distance of blood vessels was inhibited when exposed to PB for 36 h (Figure 7G, Supplementary Table 6). The area of transverse sections occupied by blood vessels was significantly decreased in the PB treated group compared to that of the control group (Figures 7E–E”,H, Supplementary Table 6). Furthermore, RT-PCR data showed that PB treatment down-regulated angiogenesis-related genes, including HIF-1α, MMP9, VEGFA, VEGF-R1, and VEGF-R2 (Figure 7I, Supplementary Table 6).

To further verify the observation above in a chick YSM model, we also used CAM, another angiogenesis model. Again we observed that the blood vessel density in chick CAM was suppressed after treatment with PB for 48 h (Supplementary Figures 4A–D,A'–C', Supplementary Table 6). Furthermore, RT-PCR data showed that PB treatment down-regulated the angiogenesis-related genes HIF-1α, VEGFA and VEGF-R1 (Supplementary Figure 4E, Supplementary Table 6). These data suggest that PB treatment indeed suppressed angiogenesis in the chick YSM and CAM models.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.