Neeraj Raghuraman Rajagopalan

Bio: Neeraj Raghuraman Rajagopalan is an academic researcher from University of Massachusetts Amherst. The author has contributed to research in topics: Medicine & Electroporation. The author has co-authored 1 publications.

Morphology and cellular-traction of fibroblasts on 2D silk-fibroin hydrogel substrates

Abstract: Development of clinically amenable bio-implants with silk-fibroin (SF) necessitates characterization of cellular-traction generated between cells and the substrate. However, studies on the biomecha...

Macrophage activity at the site of tumor ablation can promote murine urothelial cancer via transforming growth factor-β1

Abstract: Cell death and injury at the site of tumor ablation attracts macrophages. We sought to understand the status and activity of these cells while focusing on transforming growth factor-β1 (TGF-β1), a potent immunosuppressive and tumorigenic cytokine. Patients with urothelial cancer who underwent ablation using electrocautery or laser demonstrated increased infiltration and numbers of CD8+ T cells, along with FoxP3+ regulatory T cells, CD68+ macrophages and elevated levels of TGF-β1 in recurrent tumors. Similar findings were reproduced in a mouse model of urothelial cancer (MB49) by partial tumor ablation with irreversible electroporation (IRE). Stimulation of bone marrow derived macrophages with MB49 cell debris produced using IRE elicited strong M2 polarization, with exuberant secretion of TGF-β1. The motility, phenotypic markers and cytokine secretion by macrophages could be muted by treatment with Pirfenidone (PFD), a clinically approved drug targeting TGF-β1 signaling. MB49 cancer cells exposed to TGF-β1 exhibited increased migration, invasiveness and upregulation of epithelial-mesenchymal transition markers α-Smooth Muscle Actin and Vimentin. Such changes in MB49 cells were reduced by treatment with PFD even during stimulation with TGF-β1. IRE alone yielded better local tumor control when compared with control or PFD alone, while also reducing the overall number of lung metastases. Adjuvant PFD treatment did not provide additional benefit under in vivo conditions.

Acute ATP loss during irreversible electroporation mediates caspase independent cell death.

Abstract: Irreversible electroporation (IRE) has been reported to variably cause apoptosis, necrosis, oncosis or pyroptosis. Intracellular ATP is a key substrate for apoptosis which is rapidly depleted during IRE, we sought to understand whether intracellular ATP levels is a determinant of the mode of cell death following IRE. A mouse bladder cancer cell line (MB49) was treated with electric fields while increasing the number of pulses at a fixed electric field strength, and pulse width. Cell proliferation and viability and ATP levels were measured at different timepoints post-treatment. Cell death was quantified with Annexin-V/Propidium Iodide staining. Caspase activity was measure with a fluorometric kit and western blotting. A pan-caspase (Z-VAD-FMK) inhibitor was used to assess the impact of signal inhibition. We found cell death following IRE was insensitive to caspase inhibition and was correlated with ATP loss. These findings were confirmed by cell death assays and measurement of changes in caspase expression on immunoblotting. This effect could not be rescued by ATP supplementation. Rapid and acute ATP loss during IRE interferes with caspase signaling, promoting necrosis. Cell necrosis from IRE is expected to be immunostimulatory and may be effective in cancer cells that carry mutated or defective apoptosis genes.

Bioinspired silk fibroin materials: From silk building blocks extraction and reconstruction to advanced biomedical applications

Abstract: Silk fibroin has become a promising biomaterial owing to its remarkable mechanical property, biocompatibility, biodegradability, and sufficient supply. However, it is difficult to directly construct materials with other formats except for yarn, fabric and nonwoven based on natural silk. A promising bioinspired strategy is firstly extracting desired building blocks of silk, then reconstructing them into functional regenerated silk fibroin (RSF) materials with controllable formats and structures. This strategy could give it excellent processability and modifiability, thus well meet the diversified needs in biomedical applications. Recently, to engineer RSF materials with properties similar to or beyond the hierarchical structured natural silk, novel extraction and reconstruction strategies have been developed. In this review, we seek to describe varied building blocks of silk at different levels used in biomedical field and their effective extraction and reconstruction strategies. This review also present recent discoveries and research progresses on how these functional RSF biomaterials used in advanced biomedical applications, especially in the fields of cell-material interactions, soft tissue regeneration, and flexible bioelectronic devices. Finally, potential study and application for future opportunities, and current challenges for these bioinspired strategies and corresponding usage were also comprehensively discussed. In this way, it aims to provide valuable references for the design and modification of novel silk biomaterials, and further promote the high-quality-utilization of silk or other biopolymers.

Trends in mechanobiology guided tissue engineering and tools to study cell-substrate interactions: a brief review

Abstract: Sensing the mechanical properties of the substrates or the matrix by the cells and the tissues, the subsequent downstream responses at the cellular, nuclear and epigenetic levels and the outcomes are beginning to get unraveled more recently. There have been various instances where researchers have established the underlying connection between the cellular mechanosignalling pathways and cellular physiology, cellular differentiation, and also tissue pathology. It has been now accepted that mechanosignalling, alone or in combination with classical pathways, could play a significant role in fate determination, development, and organization of cells and tissues. Furthermore, as mechanobiology is gaining traction, so do the various techniques to ponder and gain insights into the still unraveled pathways. This review would briefly discuss some of the interesting works wherein it has been shown that specific alteration of the mechanical properties of the substrates would lead to fate determination of stem cells into various differentiated cells such as osteoblasts, adipocytes, tenocytes, cardiomyocytes, and neurons, and how these properties are being utilized for the development of organoids. This review would also cover various techniques that have been developed and employed to explore the effects of mechanosignalling, including imaging of mechanosensing proteins, atomic force microscopy (AFM), quartz crystal microbalance with dissipation measurements (QCMD), traction force microscopy (TFM), microdevice arrays, Spatio-temporal image analysis, optical tweezer force measurements, mechanoscanning ion conductance microscopy (mSICM), acoustofluidic interferometric device (AID) and so forth. This review would provide insights to the researchers who work on exploiting various mechanical properties of substrates to control the cellular and tissue functions for tissue engineering and regenerative applications, and also will shed light on the advancements of various techniques that could be utilized to unravel the unknown in the field of cellular mechanobiology.

The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model

Abstract: Background/Aim: Fibrosis is an essential process for wound healing, but excessive fibrosis, such as keloids and hypertrophic scars, can cause cosmetic and functional problems. These lesions are caused by abnormal deposition and shrinkage of collagen fibers. The light chain of FIX, a plasma protein essential for hemostasis, has the amino acid sequence CXDXXXXYXCXC in the EGF domain. Peptides containing this sequence inhibited stromal growth in a mouse transplant tumor model. In this study, the effect of the FIX light chain on wound healing was studied. Materials and Methods: A full-layer wound was made on the back of each mouse, and cDNA encoding the light chain of mouse FIX (F9-LC) in an expression vector was injected locally once each week using a non-viral vector. Histochemical analysis of the wound was then performed to assess the effects on wound healing. Moreover, the effect of F9-LC on fibroblasts was studied in vitro. Results: Macroscopic observation showed that wounds with forced expression of F9-LC appeared flatter and had fewer wrinkles than control wounds. Tissue collagen staining and immunostaining revealed that administration of F9-LC suppressed collagen 1 and 3 deposition and decreased α–smooth muscle actin expression. Electron microscopy revealed sparse and disorganized collagen fibers in the F9-LC–treated mice. In experiments using fibroblasts, addition of a recombinant protein of the FIX light chain disrupted the typical spindle shape and alignment of fibroblasts. Conclusion: F9-LC is a new candidate for use in treatments to regulate excessive fibrosis and contraction in wound healing.