Professional Education

  • Bachelor of Technology, Vellore Institute of Technology University, India, Biotechnology (2010)
  • Master of Technology, Indian Institute of Technology, Kharagpur (2012)
  • Doctor of Philosophy, University of Nebraska Lincoln (2019)

Stanford Advisors

All Publications

  • Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways. Stem cell research & therapy Sahu, N. n., Budhiraja, G. n., Subramanian, A. n. 2020; 11 (1): 6


    Continuous 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.Human 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.cLIUS 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.Collectively, 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.

    View details for DOI 10.1186/s13287-019-1532-2

    View details for PubMedID 31900222

  • Continuous Low-Intensity Ultrasound Promotes Native-to-Native Cartilage Integration TISSUE ENGINEERING PART A Sahu, N., Miller, A., Viljoen, H. J., Subramanian, A. 2019; 25 (21-22): 1538–49


    Failure of the host/graft interface to integrate impedes the success of cartilage repair protocols. Continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed as a treatment modality for promoting native-to-native cartilage integration in vitro. Cylindrical incisions (4 mm) simulating chondral discontinuity were made in bovine cartilage and osteochondral explants, and maintained under cLIUS stimulation (14 kPa [5 MHz, 2.5 Vpp], 20 min, four times/day) for 28 days. Incised cartilage and osteochondral explants were categorized into three study groups; Group I: cLIUS was applied immediately upon incision; Group II: cLIUS was applied after 14 days following incision; Group-III: after 14 days following incision, explants were treated with 0.1% hyaluronidase and 30 U/mL collagenase VII. As a separate study group, incised osteochondral explants were treated immediately with cLIUS at a nonresonant frequency of 2 MHz (14 kPa [2 MHz, 6 Vpp], 20 min, four times/day). Cellular migration was analyzed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage/hydrogel constructs. Explants under cLIUS (5 MHz) displayed higher percent apposition along with gap closures when compared with untreated controls and explants treated with cLIUS at 2 MHz. cLIUS (5 MHz)-treated explants were immunopositive for type II collagen. The strength of native-to-native cartilage integration was higher (p = 0.005) in cLIUS-treated cartilage explants at 0.19 ± 0.08 MPa as compared with 0.05 ± 0.03 MPa in untreated controls. Enhanced cartilage phenotype coupled with increased cellular migration were noted under cLIUS (5 MHz), alluding to the observed integration between cartilage interfaces. Collectively, cLIUS at cell resonant frequency promoted integrative cartilage repair, therefore, has the potential to improve cartilage repair outcomes. Impact Statement Lack of integration between the host and graft cartilage interfaces impedes the success of cartilage repair techniques. Continuous low-intensity ultrasound (cLIUS) is documented to induce chondrogenesis and chondrocyte phenotype. However, integrative cartilage repair under cLIUS has not been evaluated. Our results demonstrated integration between cartilage interfaces, increased percent apposition, increased strength of integration, and maintenance of cartilage phenotype under cLIUS (5 MHz). Integrative repair under cLIUS (5 MHz) stemmed from enhanced migration of cells and increased expression of cartilage-specific genes, namely SOX9 and COL2A1. Thus, cLIUS has the potential to improve the outcomes of grafting protocols for cartilage repair.

    View details for DOI 10.1089/ten.tea.2018.0355

    View details for Web of Science ID 000492782600001

    View details for PubMedID 31190618

  • Continuous low-intensity ultrasound attenuates IL-6 and TNF alpha-induced catabolic effects and repairs chondral fissures in bovine osteochondral explants BMC MUSCULOSKELETAL DISORDERS Sahu, N., Viljoen, H. J., Subramanian, A. 2019; 20: 193


    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.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.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.cLIUS repaired chondral fissures, and elicited pro-anabolic and anti-catabolic effects, thus demonstrating the potential of cLIUS in improving cartilage repair outcomes.

    View details for DOI 10.1186/s12891-019-2566-4

    View details for Web of Science ID 000466965000004

    View details for PubMedID 31054572

    View details for PubMedCentralID PMC6499975

  • A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells STEM CELL REPORTS Lin, H., Du, Q., Li, Q., Wang, O., Wang, Z., Sahu, N., Elowsky, C., Liu, K., Zhang, C., Chung, S., Duan, B., Lei, Y. 2018; 11 (2): 454–69


    Endothelial 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.

    View details for DOI 10.1016/j.stemcr.2018.07.001

    View details for Web of Science ID 000441583100013

    View details for PubMedID 30078557

    View details for PubMedCentralID PMC6092882

  • Low-Intensity Ultrasound Upregulates the Expression of Cyclin-D1 and Promotes Cellular Proliferation in Human Mesenchymal Stem Cells BIOTECHNOLOGY JOURNAL Budhiraja, G., Sahu, N., Subramanian, A. 2018; 13 (4): e1700382


    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-2 ) 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.

    View details for DOI 10.1002/biot.201700382

    View details for Web of Science ID 000430116900005

    View details for PubMedID 29283212

  • Chondrocyte primary cilium is mechanosensitive and responds to low-intensity-ultrasound by altering its length and orientation INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY Subramanian, A., Budhiraja, G., Sahu, N. 2017; 91: 60–64


    The primary cilium has been implicated in mechanotransduction, however, its mechanosensory role in transducing signals under low-intensity ultrasound (LIUS) which has the potential to repair fractures and cartilage, remains uninvestigated. This study examined the impact of continuous-wave US, at the cell resonance frequency of 5.0MHz and pressure amplitude of either 14 or 60kPa, on the incidence, length and orientation of primary cilium in bovine articular chondrocytes. Visualization of primary cilium with acetylated α-tubulin staining demonstrated that the primary cilium was elongated, bent under US and these changes were reversible. Basal expression of phospho-ERK1/2 was lower in deciliated chondrocytes, thus implicating the role for the primary cilium in transducing signals via the MAPK/ERK pathway. This study demonstrates that the chondrocyte primary cilium is mechanosensitive and responds to US by altering its length and orientation.

    View details for DOI 10.1016/j.biocel.2017.08.018

    View details for Web of Science ID 000413880900008

    View details for PubMedID 28870737

  • Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture BIOMED RESEARCH INTERNATIONAL Sahu, N., Pal, S., Sapru, S., Kundu, J., Talukdar, S., Singh, N., Yao, J., Kundu, S. C. 2016: 7461041


    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.

    View details for DOI 10.1155/2016/7461041

    View details for Web of Science ID 000380701800001

    View details for PubMedID 27517047

    View details for PubMedCentralID PMC4969515

  • Nonmulberry Silk Fibroin Scaffold Shows Superior Osteoconductivity Than Mulberry Silk Fibroin in Calvarial Bone Regeneration ADVANCED HEALTHCARE MATERIALS Sahu, N., Baligar, P., Midha, S., Kundu, B., Bhattacharjee, M., Mukherjee, S., Mukherjee, S., Maushart, F., Das, S., Loparic, M., Kundu, S. C., Ghosh, S., Mukhopadhyay, A. 2015; 4 (11): 1709–21


    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.

    View details for DOI 10.1002/adhm.201500283

    View details for Web of Science ID 000359382600014

    View details for PubMedID 26084249