Electrically driven intracellular and extracellular nanomanipulators evoke neurogenic/cardiomyogenic differentiation in human mesenchymal stem cells

Nanomechanical intervention through electroactuation is an effective strategy to guide stem cell differentiation for tissue engineering and regenerative medicine. In the present study, we elucidate that physical forces exerted by electroactuated gold nanoparticles (GNPs) have a strong influence in regulating the lineage commitment of human mesenchymal stem cells (hMSCs). A novel platform that combines intracellular and extracellular GNPs as nano-manipulators was designed to trigger neurogenic/cardiomyogenic differentiation in hMSCs, in electric field stimulated culture condition. In order to mimic the native microenvironment of nerve and cardiac tissues, hMSCs were treated with physiologically relevant direct current electric field (DC EF) or pulsed electric field (PEF) stimuli, respectively. When exposed to regular intermittent cycles of DC EF stimuli, majority of the GNP actuated hMSCs acquired longer filopodial extensions with multiple branch-points possessing neural-like architecture. Such morphological changes were consistent with higher mRNA expression level for neural-specific markers. On the other hand, PEF elicited cardiomyogenic differentiation, which is commensurate with the tubelike morphological alterations along with the upregulation of cardiac specific markers. The observed effect was significantly promoted even by intracellular actuation and was found to be substrate independent. Further, we have substantiated the participation of oxidative signaling, G0/G1 cell cycle arrest and intracellular calcium Ca2+] elevation as the key upstream regulators dictating GNP assisted hMSC differentiation. Thus, by adopting dual stimulation protocols, we could successfully divert the DC EF exposed cells to differentiate predominantly into neural-like cells and PEF treated cells into cardiomyogenic-like cells, via nanoactuation of GNPs. Such a novel multifaceted approach can be exploited to combat tissue loss following brain injury or heart failure. (C) 2015 Elsevier Ltd. All rights reserved

Unraveling the mechanistic effects of electric field stimulation towards directing stem cell fate and function: A tissue engineering perspective

Electric field (EF) stimulation can play a vital role in eliciting appropriate stem cell response. Such an approach is recently being established to guide stem cell differentiation through osteogenesis/neurogenesis/cardiomyogenesis. Despite significant recent efforts, the biophysical mechanisms by which stem cells sense, interpret and transform electrical cues into biochemical and biological signals still remain unclear. The present review critically analyses the variety of EF stimulation approaches that can be employed to evoke appropriate stem cell response and also makes an attempt to summarize the underlying concepts of this notion, placing special emphasis on stem cell based tissue engineering and regenerative medicine. This review also discusses the major signaling pathways and cellular responses that are elicited by electric stimulation, including the participation of reactive oxygen species and heat shock proteins, modulation of intracellular calcium ion concentration, ATP production and numerous other events involving the clustering or reassembling of cell surface receptors, cytoskeletal remodeling and so on. The specific advantages of using external electric stimulation in different modalities to regulate stem cell fate processes are highlighted with explicit examples, in vitro and in vivo. (C) 2017 Elsevier Ltd. All rights reserved

Absence of systemic toxicity in mouse model towards BaTiO3 nanoparticulate based eluate treatment

One of the existing issues in implant failure of orthopedic biomaterials is the toxicity induced by the fine particles released during long term use in vivo, leading to acute inflammatory response. In developing a new class of piezobiocomposite to mimic the integrated electrical and mechanical properties of bone, bone-mimicking physical properties as well as in vitro cytocompatibility properties have been achieved with spark plasma sintered hydroxyapatite (HA)-barium titanate (BaTiO3) composites. However, the presence of BaTiO3 remains a concern towards the potential toxicity effect. To address this issue, present work reports the first result to conclusively confirm the non-toxic effect of HA-BaTiO3 piezobiocomposite nanoparticulates, in vivo. Twenty BALB/c mice were intraarticularly injected at their right knee joints with different concentrations of HA-BaTiO3 composite of up to 25 mg/ml. The histopathological examination confirmed the absence of any trace of injected particles or any sign of inflammatory reaction in the vital organs, such as heart, spleen, kidney and liver at 7 days post-exposure period. Rather, the injected nanoparticulates were found to be agglomerated in the vicinity of the knee joint, surrounded by macrophages. Importantly, the absence of any systemic toxicity response in any of the vital organs in the treated mouse model, other than a mild local response at the site of delivery, was recorded. The serum biochemical analyses using proinflammatory cytokines (TNF-alpha and IL-1 beta) also complimented to the non-immunogenic response to injected particulates. Altogether, the absence of any inflammatory/ adverse reaction will open up myriad of opportunities for BaTiO3 based piezoelectric implantable devices in biomedical applications

Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro

In designing and developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of the substrate conductivity on cell fate processes is rather limited. In order to address this issue, spark plasma sintered HA-CaTiO3 (Hydroxyapatite-Calcium titanate) has been used as a model material system to showcase the effect of varying conductivity on cell functionality. Being electroactive in nature, mouse myoblast cells (C2C12) were selected as a model cell line in this study. It was inferred that myoblast adhesion/growth systematically increases with substrate conductivity due to CaTiO3 addition to HA. Importantly, parallel arrangement of myoblast cells on higher CaTiO3 containing substrates indicate that self-adjustable cell patterning can be achieved on conductive biomaterials. Furthermore, enhanced myoblast assembly and myotube formation were recorded after 5 days of serum starvation. Overall, the present study conclusively establishes the positive impact of the substrate conductivity towards cell proliferation and differentiation as well as confirms the efficacy of HA-CaTiO3 biocomposites as conductive platforms to facilitate the growth, orientation and fusion of myoblasts, even when cultured in the absence of external electric field

Magnetic field assisted stem cell differentiation - role of substrate magnetization in osteogenesis

Among the multiple modulatory physical cues explored to regulate cellular processes, the potential of magneto-responsive substrates in magnetic field stimulated stem cell differentiation is still unperceived. In this regard, the present work demonstrates how an external magnetic field can be applied to direct stem cell differentiation towards osteogenic commitment. A new culture methodology involving periodic delivery of 100 mT static magnetic field (SMF) in combination with HA-Fe3O4 magnetic substrates possessing a varying degree of substrate magnetization was designed for the study. The results demonstrate that an appropriate combination of weakly ferromagnetic substrates and SMF exposure enhanced cell viability, DNA synthesis and caused an early switchover to osteogenic lineage as supported by Runx2 immunocytochemistry and ALP expression. However, the mRNA expression profile of early osteogenic markers (Runx2, ALP, Col IA) was comparable despite varying substrate magnetic properties (diamagnetic to ferromagnetic). On the contrary, a remarkable upregulation of late bone development markers (OCN and OPN) was explicitly detected on weak and strongly ferromagnetic substrates. Furthermore, SMF induced matrix mineralization with elevated calcium deposition on similar substrates, even in the absence of osteogenic supplements. More specifically, the role of SMF in increasing intracellular calcium levels and in inducing cell cycle arrest at G0/G1 phase was elucidated as the major molecular event triggering osteogenic differentiation. Taken together, the above results demonstrate the competence of magnetic stimuli in combination with magneto-responsive biomaterials as a potential strategy for stem cell based bone tissue engineering

Optically transparent polymer devices for in situ assessment of cell electroporation

In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 mu l was quantified in situ as a function of applied voltage from 10 to 90 V in a series of 2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10 % glycerol)

Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells

Surface interactions at the biomaterial–cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for <i>in vitro</i> adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications

Pigmented Silk Nanofibrous Composite for Skeletal Muscle Tissue Engineering

Skeletal muscle tissue engineering (SMTE) employs designed biomaterial scaffolds for promoting myogenic differentiation of myoblasts to functional myotubes. Oxidative stress plays a significant role in the biocompatibility of biomaterials as well as in the fate of myoblasts during myogenesis and is also associated with pathological conditions such as myotonic dystrophy. The inherent electrical excitability of muscle cells inspired the use of electroactive scaffolds for SMTE. Conducting polymers attracted the attention of researchers for their use in muscle tissue engineering. However, poor biocompatibility, biodegradability and development of oxidative stress associated immunogenic response limits the extensive use of synthetic conducting polymers for SMTE. In order to address the limitations of synthetic polymers, intrinsically electroactive and antioxidant silk fibroin/melanin composite films and electrospun fiber mats were fabricated and evaluated as scaffolds for promoting myogenesis in vitro. Melanin incorporation modulated the thermal stability, electrical conductivity of scaffolds, fiber alignment in electrospun mats and imparted good antioxidant properties to the scaffolds. The composite electrospun scaffolds promoted myoblast assembly and differentiation into uniformly aligned high aspect ratio myotubes. The results highlight the significance of scaffold topography along with conductivity in promoting myogenesis and the potential application of silk nanofibrous composite as electoractive platform for SMTE

Competing Roles of Substrate Composition, Microstructure, and Sustained Strontium Release in Directing Osteogenic Differentiation of hMSCs

Strontium releasing bioactive ceramics constitute an important class of biomaterials for osteoporosis treatment. In the present study, we evaluated the synthesis, phase assemblage, and magnetic properties of strontium hexaferrite, SrFe₁₂O₁₉, (SrFe) nanoparticles. On the biocompatibility front, the size- and dose-dependent cytotoxicity of SrFe against human mesenchymal stem cells (hMSCs) were investigated. After establishing their non-toxic nature, we used the strontium hexaferrite nanoparticles (SrFeNPs) in varying amount (<i>x</i> = 0, 10, and 20 wt %) to consolidate bioactive composites with hydroxyapatite (HA) by multi-stage spark plasma sintering (SPS). Rietveld refinement of these spark plasma sintered composites revealed a near complete decomposition of SrFe₁₂O₁₉ to magnetite (Fe₃O₄) along with a marked increase in the unit cell volume of HA, commensurate with strontium-doped HA. The cytocompatibility of SrHA-Fe composites with hMSCs was assessed using qualitative and quantitative morphological analysis along with phenotypic and genotypic expression for stem cell differentiation. A marked decrease in the stemness of hMSCs, indicated by reduced vimentin expression and acquisition of osteogenic phenotype, evinced by alkaline phosphatase (ALP) and collagen deposition was recorded on SrHA-Fe composites in osteoinductive culture. A significant upregulation of osteogenic marker genes (Runx2, ALP and OPN) was detected in case of the SrHA-Fe composites, whereas OCN and Col IA expression were similarly high for baseline HA. However, matrix mineralization was elevated on SrHA-Fe composites in commensurate with the release of Sr²⁺ and Fe²⁺. Summarizing, the current work is the first report of strontium hexaferrite as a non-toxic nanobiomaterial. Also, SrHA-based iron oxide composites can potentially better facilitate bone formation, when compared to pristine HA