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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.

Bone morphogenetic protein 9 (BMP9), as one of the most potent osteogenic factors, is a promising cytokine for bone tissue engineering. Wnt11 can regulate the development of the skeletal system and is related to high bone mass syndrome. However, the effect of Wnt11 on BMP9-induced osteogenic differentiation remains unknown. In this study, we investigated the relationship between Wnt11- and BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs). We recapitulated the osteogenic potential of BMP9 in C3H10T1/2 cells. The messenger RNA expression of Wnt11 is detectable in the available progenitor cells, and BMP9 can obviously increase the protein level of Wnt11 in these cells. Exogenous Wnt11 potentiates the effect of BMP9 on increasing alkaline phosphatase (ALP) activities, the expression of osteopontin (OPN), and Runt-related transcription factor 2 (Runx2), so does matrix mineralization in C3H10T1/2 cells. Although Wnt11 cannot increase the BMP9-induced ectopic bone formation, it can increase the bone density induced by BMP9 apparently. Wnt11 increases the level of p-Smad1/5/8, as well as p-p38. Meanwhile, Wnt11 promotes the effect of BMP9 on increasing the levels of p-Smad1/5/8 and p-p38. Inhibition of p38 decreases the BMP9-induced ALP activities, the expression of OPN, and the mineralization in C3H10T1/2 cells. However, all of these effects of the p38 inhibitor on BMP9-induced osteogenic markers can be almost reversed by the overexpression of Wnt11. Our findings suggested that Wnt11 can enhance the osteogenic potential of BMP9 in MSCs, and this effect may be partly mediated through enhancing BMPs/Smads and the p38 MAPK signal, which was induced by BMP9.

Correction for ‘Transgenic PDGF-BB/sericin hydrogel supports for cell proliferation and osteogenic differentiation’ by Feng Wang et al., Biomater. Sci., 2020, 8, 657–672, DOI: 10.1039/C9BM01478K.

Sericin has been exploited as a biomaterial due to its biocompatibility, biodegradability, and low-immunogenicity as an isolated polymer and support for cell adhesion. In the present study, human platelet-derived growth factor (PDGF-BB)-functionalized sericin hydrogels were generated using transgenic silkworms, where the as-spun silk incorporated engineered PDGF-BB (termed PDGFM) in the sericin layers of the cocoons. Sericin and PDGFM were simultaneously extracted from the silk fibroin cocoon fibers, and the soluble extract was then formed into a hydrogel via thermal exposure. The PDGFM sericin hydrogels exhibited increased β-sheet content and a compressive modulus of 74.91 ± 2.9 kPa comparable to chemically crosslinked sericin hydrogels (1.68–55.53 kPa) and a porous microstructure, which contributed to cell adhesion and growth. A 13.1% of total extracted PDGFM from the initial silk fibers was incorporated and immobilized in the sericin hydrogels during material processing, and 1.33% of PDGFM was released over 30 days from the hydrogels in vitro. The remaining PDGFM achieved long-term storage/stability in the sericin hydrogels for more than 42 days at 37 °C. In addition, the PDGFM sericin hydrogels were not immunogenic, were biocompatible and bioactive in promoting the support of cell proliferation. When combined with BMP-9, the PDGFM sericin hydrogels provided synergy to support the osteoblastic differentiation of mesenchymal stem cells (hMSCs) in vitro and in vivo. This study demonstrates that genetically functionalized PDGFM sericin hydrogels can provide useful biomaterials to support cell and tissue outcomes, here with a focus on osteogenesis.

Colon cancer is one of the most common malignancies. Although there has been great development in treatment regimens over the last few decades, its prognosis remains poor. There is still a clinical need to find new drugs for colon cancer. Evodiamine (Evo) is a quinolone alkaloid extracted from the traditional herbal medicine plant Evodia rutaecarpa. In the present study, CCK‑8, flow cytometry, reverse transcription quantitative polymerase chain reaction, western blot analysis and a xenograft tumor model were used to evaluate the anti‑cancer activity of Evo in human colon cancer cells and determine the possible mechanism underlying this process. It was revealed that Evo exhibited prominent anti‑proliferation and apoptosis‑inducing effects in HCT116 cells. Bone morphogenetic protein 9 (BMP9) was notably upregulated by Evo in HCT116 cells. Exogenous BMP9 potentiated the anti‑cancer activity of Evo, and BMP9 silencing reduced this effect. In addition, HIF‑1α was also upregulated by Evo. The anticancer activity of Evo was enhanced by HIF‑1α, but was reduced by HIF‑1α silencing. BMP9 potentiated the effect of Evo on the upregulation of HIF‑1α, and enhanced the antitumor effect of Evo in colon cancer, which was clearly reduced by HIF‑1α silencing. In HCT116 cells, Evo increased the phosphorylation of p53, which was enhanced by BMP9 but reduced by BMP9 silencing. Furthermore, the effect of Evo on p53 was potentiated by HIF‑1α and reduced by HIF‑1α silencing. The present findings therefore strongly indicated that the anticancer activity of Evo may be partly mediated by BMP9 upregulation, which can activate p53 through upregulation of HIF‑1α, at least in human colon cancer.

COX-2 specific inhibitor, which has been widely used, can delay bone fracture healing and reduce osteogenic potential of bone marrow stromal cells. However, it remains unknown how to prevent these side-effects of COX-2 inhibitor. In this study, we introduced BMP9-induced osteogenic differentiation as model to evaluate whether all-trans retinoic acid (ATRA) could ameliorate these adverse effects of COX-2 specific inhibitor on bone metabolism with in vitro and in vivo experiments, and uncover the possible mechanism underlying this process. Results showed that ATRA enhanced the potential of BMP9 to induce the osteogenic markers, such as alkaline phosphates (ALP) and mineralization; but retinoic acid receptor a (RARa) inhibitor showed the reversal effects. COX-2 specific inhibitor (NS398) reduced the osteogenic markers induced by BMP9, and ATRA almost eliminated the inhibitory effect of NS398. BMP9 up-regulated the protein level of β-catenin and promoted it translocate to nucleus, and both were reduced by NS398. On the contrary, ATRA notablely attenuated the inhibitory effect of NS398 on BMP9-increased β-catenin. Exogenous RXRa obviously ameliorated the inhibitory effect of silencing COX-2 on ectopic bone formation induced by BMP9. NS398 reduced the level of phosphorylated CREB, which was almost reversed by ATRA. Besides, RXRa interacted with phosphorylated CREB directly and both were recruited at β-catenin promoter region. Thus, we demonstrated that ATRA may reverse the side-effects of COX-2 inhibitor on bone metabolism through increasing the activation of Wnt/β-catenin pathway partly.

Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic factors, which may be a potential candidate for bone tissue engineering. However, the osteogenic capacity of BMP9 still need to be further enhanced. In this study, we determined the effect of Wnt10b on BMP9-induced osteogenic differentiation in mesenchymal stem cell (MSCs) and the possible mechanism underlying this process. We introduced the polymerase chain reaction (PCR), Western blot analysis, histochemical stain, ectopic bone formation, and microcomputed tomography analysis to evaluate the effect of Wnt10b on BMP9-induced osteogenic differentiation. Meanwhile, PCR, Western blot analysis, chromatin immunoprecipitation, and immunoprecipitation were used to analyze the possible relationship between BMP9 and Wnt10b. We found that BMP9 upregulates Wnt10b in C3H10T1/2 cells. Wnt10b increases the osteogenic markers and bone formation induced by BMP9 in C3H10T1/2 cells, and silencing Wnt10b decreases these effects of BMP9. Meanwhile, Wnt10b enhances the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) induced by BMP9, which can be reduced by silencing Wnt10b. On the contrary, Wnt10b inhibits adipogenic markers induced by BMP9, which can be decreased by silencing Wnt10b. Further analysis indicated that BMP9 upregulates cyclooxygenase-2 (COX-2) and phosphorylation of cAMP-responsive element binding (p-CREB) simultaneously. COX-2 potentiates the effect of BMP9 on increasing p-CREB and Wnt10b, while silencing COX-2 decreases these effects. p-CREB interacts with p-Smad1/5/8 to bind the promoter of Wnt10b in C3H10T1/2 cells. Our findings suggested that Wnt10b can promote BMP9-induced osteogenic differentiation in MSCs, which may be mediated through enhancing BMP/Smad signal and reducing adipogenic differentiation; BMP9 may upregulate Wnt10b via the COX-2/p-CREB-dependent manner.