Transgenic PDGF-BB sericin hydrogel potentiates bone regeneration of BMP9-stimulated mesenchymal stem cells through a crosstalk of the Smad-STAT pathways

Abstract

Silk as a natural biomaterial is considered as a promising bone substitute in tissue regeneration. Sericin and fibroin are the main components of silk and display unique features for their programmable mechanical properties, biocompatibility, biodegradability and morphological plasticity. It has been reported that sericin recombinant growth factors (GFs) can support cell proliferation and induce stem cell differentiation through cross-talk of signaling pathways during tissue regeneration. The transgenic technology allows the productions of bioactive heterologous GFs as fusion proteins with sericin, which are then fabricated into solid matrix or hydrogel format. Herein, using an injectable hydrogel derived from transgenic platelet-derived GF (PDGF)-BB silk sericin, we demonstrated that the PDGF-BB sericin hydrogel effectively augmented osteogenesis induced by bone morphogenetic protein (BMP9)-stimulated mesenchymal stem cells (MSCs) in vivo and in vitro, while inhibiting adipogenic differentiation. Further gene expression and protein-protein interactions studies demonstrated that BMP9 and PDGF-BB synergistically induced osteogenic differentiation through the cross-talk between Smad and Stat3 pathways in MSCs. Thus, our results provide a novel strategy to encapsulate osteogenic factors and osteoblastic progenitors in transgenic sericin-based hydrogel for robust bone tissue engineering.

Keywords: BMP9; biomaterials; growth factor; osteogenesis.

Graphical abstract

Figure 1.

Properties of the PDGF-BB sericin hydrogel. (A) Prepared injectable PDGF-BB sericin hydrogel. (B) Viscosity–shear rate of WT (black triangle) and PDGF-BB (red dot) sericin hydrogels. (C) Micromorphological structure of the freeze-dried PDGF-BB sericin hydrogel. (D) FTIR spectral analysis of WT (black curve) and PDGF-BB (red curve) sericin hydrogels scanned from 1000 cm⁻¹–1700 cm⁻¹. Three strong peaks are indicated by the dotted lines representing typical amide I (C=O stretching vibrations), amide II (N-H bending) and amide III (C–N stretching vibrations) regions. The blue dotted lines show the absorption peaks of β-sheet structures in the sericin hydrogels, while the orange dotted lines indicate those of random coils. (E) Cytotoxicity of NIH/3T3 cells incubated with WT or PDGF-BB sericin hydrogel. Live cells were stained by the calcein-AM dye and showed green fluorescence (ex/em ∼495 nm/∼515 nm), while dead cells were stained by the EthD-1 dye and showed red fluorescence (ex/em ∼495 nm/∼635 nm). The magnification is ×100.

Figure 2.

Effects of the PDGF-BB std and PDGF-BB sericin hydrogel on MSCs osteogenesis. (A–B) Relative ALP activities in Ad-BMP9-MSCs treated by different concentrations of either PDGF-BB std or PDGF-BB sericin hydrogel for 5 days. The ALP activities in Ad-GFP-MSCs stimulated with PDGF-BB std or PDGF-BB sericin hydrogel were used as controls. The responses of Ad-BMP9-MSCs treated either by 1× PBS or WT sericin hydrogel were used as controls. The results collected from Ad-BMP9-MSCs were normalized by those from Ad-GFP-MSCs. Tukey’s test has been performed to compare the tests versus control. a, P < 0.05; b, P < 0.001. (C) Quantification of the relative ALP activities in MSCs showed early osteogenesis on day 7 after treated by various stimuli. The treatments were as shown in groups 1–8. 3.44 × 1 0⁻³μg/mL PDGF-BB sericin hydrogel and 3.5 × 1 0⁻³μg/mL PDGF-BB std were used in testes. (D) Alizarin Red S staining on day 18 showed the effects of 3.5 × 1 0⁻³μg/mL PDGF-BB std and 3.44 × 1 0⁻³μg/mL PDGF-BB sericin hydrogel on Ad-BMP9-MSCs. (E) Quantification of Alizarin Red S staining. Data are shown as the means ± SD. Error bar = SD. Data were analyzed by an ANOVA followed by Tukey’s multiple comparisons test. The significant differences shown in (C) and (E) were as follows: a, groups 2–8 versus group 1 (control), P < 0.001; b, groups 3–8 versus group 2, P < 0.001; c, groups 4–8 versus groups 3, P < 0.001; d, groups 5, 7–8 versus group 4, P < 0.001; e, group 6 versus group 4, P < 0.05; f, groups 6–8 versus groups 5, P < 0.001; g, groups 7 and 8 versus group 6, P < 0.001; h, group 8 versus group 7, P < 0.001.

Figure 3.

The expressions of osteogenic markers in MSCs induced by BMP9 and PDGF-BB sericin hydrogel. (A, C and E) Relative expression of Runx 2, OPN and OCN mRNA detected by real-time PCR. (B, D and F) Western blots showing the Runx 2, OPN and OCN protein expression. Data are shown as the means ± SD. Error bar = SD. Tukey’s test was used for multiple comparisons. The significance values are as follows: a, groups 2–4 versus group 1 (control), P < 0.001; b, groups 3 and 4 versus group 2, P < 0.001; c, group 4 versus group 3, P < 0.001.

Figure 4.

Ectopic bone formation enhanced by PDGF-BB sericin hydrogel. (A) 3D reconstruction of bone masses. Scale bar = 1 mm. (B) Data analysis of the bone mass detected by micro-CT. BV/TV, bone/tissue volume; BS/BV, bone surface area/bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. (C) Histology of the bone matrix stained by H&E and Masson’s trichrome stains. MB, mineralized bone matrix; OB, osteoblast; OM, osteoid matrix. All data were shown as the means ± SD. Error bar = SD. Student’s t-test was used for pairwise comparison. The significance values are as follows: a, P < 0.05; b, P < 0.001.

Figure 5.

Effect of PDGF-BB on the adipogenic differentiation of Ad-BMP9-MSCs. (A) Adipocyte formation was assessed by Oil Red O staining. (B) The A_450nm evaluation of Oil Red O staining. (C) (E, G and H) Relative expression levels of PPARγ, C/EBPα, FABP4 and LPL mRNA detected by real-time PCR. (D and F) The protein expressions of PPARγ and C/EBPα detected by Western blots. Data are shown as the means ± SD. Error bars = SD. Tukey’s test was used for multiple comparisons. Significance values are shown as follows: a, groups 2–4 versus group 1 (control), P < 0.001; b, groups 3 and 4 versus group 2, P < 0.001; c, group 4 versus group 3, P < 0.001; d, groups 2–4 versus group 1, P < 0.05; e, group 3 and 4 versus group 2, P < 0.05.

Figure 6.

Signaling crosstalk between STAT and Smad pathways during MSCs differentiation. (A) Western blots showing the expression levels of p-Smad 1/5/9 and Stat3 following various treatments. (B) The interaction between p-Smad 1/5/9 and p-Stat3 detected by IP assay. (C) A ChIP assay revealed the recruitment of p-Smad 1/5/9 and p-Stat3 to the promoter regions of PPARγ. Data are shown as the means ± SD. Error bars = SD. Tukey’s test was used for multiple comparisons. Significance values are shown as follows: a, groups 2–4 versus group 1 (control), P < 0.001; b, group 3 and 4 versus group 2, P < 0.001; c, group 4 versus group 3, P < 0.001.