Recent years have witnessed the advancement of silk biomaterials in bone tissue engineering, although clinical application of the same is still in its infancy. In this study, the potential of pure nonmulberry Antheraea mylitta (Am) fibroin scaffold, without preloading with bone precursor cells, to repair calvarial bone defect in a rat model is explored and compared with its mulberry counterpart Bombyx mori (Bm) silk fibroin. After 3 months of implantation, Am scaffold culminates in a completely ossified regeneration with a progressive increase in mineralization at the implanted site. On the other hand, the Bm scaffold fails to repair the damaged bone, presumably due to its low osteoconductivity and early degradation. The deposition of bone matrix on scaffolds is evaluated by scanning electron and atomic force microscopy. These results are corroborated by in vitro studies of enzymatic degradation, colony formation, and secondary conformational features of the scaffold materials. The greater biocompatibility and mineralization in pure nonmulberry fibroin scaffolds warrants the use of these scaffolds as an "ideal bone graft" biomaterial for effective repair of critical size defects.

BackgroundContinuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS.MethodsHuman bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting.ResultscLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2.ConclusionsCollectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1532-2) contains supplementary material, which is available to authorized users.

SummaryEndothelial cells (ECs) are of great value for cell therapy, tissue engineering, and drug discovery. Obtaining high-quantity and -quality ECs remains very challenging. Here, we report a method for the scalable manufacturing of ECs from human pluripotent stem cells (hPSCs). hPSCs are expanded and differentiated into ECs in a 3D thermoreversible PNIPAAm-PEG hydrogel. The hydrogel protects cells from hydrodynamic stresses in the culture vessel and prevents cells from excessive agglomeration, leading to high-culture efficiency including high-viability (>90%), high-purity (>80%), and high-volumetric yield (2.0 × 107 cells/mL). These ECs (i.e., 3D-ECs) had similar properties as ECs made using 2D culture systems (i.e., 2D-ECs). Genome-wide gene expression analysis showed that 3D-ECs had higher expression of genes related to vasculature development, extracellular matrix, and glycolysis, while 2D-ECs had higher expression of genes related to cell proliferation.

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

Background Molecular mechanisms underlying inflammation-associated breast tumor growth are poorly studied. S100A7, a pro-inflammatory molecule has been shown to enhance breast cancer growth and metastasis. However, the S100A7-mediated molecular mechanisms in enhancing tumor growth and metastasis are unclear. Methods Human breast cancer tissue and plasma samples were used to analyze the expression of S100A7, cPLA2, and PGE2. S100A7-overexpressing or downregulated human metastatic breast cancer cells were used to evaluate the S100A7-mediated downstream signaling mechanisms. Bi-transgenic mS100a7a15 overexpression, TNBC C3 (1)/Tag transgenic, and humanized patient-derived xenograft mouse models and cPLA2 inhibitor (AACOCF3) were used to investigate the role of S100A7/cPLA2/PGE2 signaling in tumor growth and metastasis. Additionally, CODEX, a highly advanced multiplexed imaging was employed to delineate the effects of S100A7/cPLA2 inhibition on the recruitment of various immune cells. Results In this study, we found that S100A7 and cPLA2 are highly expressed and correlate with decreased overall survival in breast cancer patients. Further mechanistic studies revealed that S100A7/RAGE signaling promotes the expression of cPLA2 to mediate its oncogenic effects. Pharmacological inhibition of cPLA2 suppressed S100A7-mediated tumor growth and metastasis in multiple pre-clinical models including transgenic and humanized patient-derived xenograft (PDX) mouse models. The attenuation of cPLA2 signaling reduced S100A7-mediated recruitment of immune-suppressive myeloid cells in the tumor microenvironment (TME). Interestingly, we discovered that the S100A7/cPLA2 axis enhances the immunosuppressive microenvironment by increasing prostaglandin E2 (PGE2). Furthermore, CO-Detection by indEXing (CODEX) imaging-based analyses revealed that cPLA2 inhibition increased the infiltration of activated and proliferating CD4+ and CD8+ T cells in the TME. In addition, CD163+ tumor associated-macrophages were positively associated with S100A7 and cPLA2 expression in malignant breast cancer patients. Conclusions Our study provides new mechanistic insights on the cross-talk between S100A7/cPLA2 in enhancing breast tumor growth and metastasis by generating an immunosuppressive TME that inhibits the infiltration of cytotoxic T cells. Furthermore, our studies indicate that S100A7/cPLA2 could be used as novel prognostic marker and cPLA2 inhibitors as promising drugs against S100A7-overexpressing aggressive breast cancer.

Human mesenchymal stem cells (hMSCs) hold great potential for cellular based therapeutics and tissue engineering applications and their expansion is an interesting prospect due to their low availability from in vivo sources. Therefore, this study investigated the effect of continuous-wave low-intensity ultrasound (LIUS) at 5.0-MHz and 14.0-kPa (<20 mW cm ) on the proliferative capacity, colony-formation efficiency, genetic stability, and differentiation potential of hMSCs. Additionally, potential signaling pathways involved in LIUS-mediated proliferation of hMSCs are studied. Compared to non-stimulated controls, LIUS-treated hMSCs shows a 1.9-fold greater colony-forming efficiency and 2.5-fold higher rate of cell proliferation, respectively. Differential staining and qRT-PCR analysis for selective chondrogenic, osteogenic, and adipogenic markers further confirmed that the LIUS treatment did not impact the multipotency of hMSCs. LIUS-treated hMSCs expressed normal male karyotype. The synthesis of cyclin-D1, a master regulator of cellular proliferation, is upregulated under LIUS and its enhanced mRNA expression under LIUS is noted to be mediated by the activation of both MAPK/ERK and PI3K/AKT pathways. In conclusion, LIUS promotes proliferation and self-renewal capacity of hMSCs.

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Background Cartilage repair outcomes are compromised in a pro-inflammatory environment; therefore, the mitigation of pro-inflammatory responses is beneficial. Treatment with continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed for the repair of chondral fissures under pro-inflammatory conditions. Methods Bovine osteochondral explants, concentrically incised to create chondral fissures, were maintained under cLIUS (14 kPa (5 MHz, 2.5 Vpp), 20 min, 4 times/day) for a period of 28 days in the presence or absence of cytokines, interleukin-6 (IL-6) or tumor necrosis factor (TNF)α. Outcome assessments included histological and immunohistochemical staining of the explants; and the expression of catabolic and anabolic genes by qRT-PCR in bovine chondrocytes. Cell migration was assessed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage-hydrogel constructs. Results Both in the presence and absence of cytokines, higher percent apposition along with closure of fissures were noted in cLIUS-stimulated explants as compared to non-cLIUS-stimulated explants on day 14. On day 28, the percent apposition was not significantly different between unstimulated and cLIUS-stimulated explants exposed to cytokines. As compared to non-cLIUS-stimulated controls, on day 28, cLIUS preserved the distribution of proteoglycans and collagen II in explants despite exposure to cytokines. cLIUS enhanced the cell migration irrespective of cytokine treatment. IL-6 or TNFα-induced increases in MMP13 and ADAMTS4 gene expression was rescued by cLIUS stimulation in chondrocytes. Under cLIUS, TNFα-induced increase in NF-κB expression was suppressed, and the expression of collagen II and TIMP1 genes were upregulated. Conclusion cLIUS repaired chondral fissures, and elicited pro-anabolic and anti-catabolic effects, thus demonstrating the potential of cLIUS in improving cartilage repair outcomes. Electronic supplementary material The online version of this article (10.1186/s12891-019-2566-4) contains supplementary material, which is available to authorized users.