Cell contact guidance via sensing anisotropy of network mechanical resistance.

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*| -1 - |X*| -1 increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.

Pediatric tri-tube valved conduits made from fibroblast-produced extracellular matrix evaluated over 52 weeks in growing lambs.

There is a need for replacement heart valves that can grow with children. We fabricated tubes of fibroblast-derived collagenous matrix that have been shown to regenerate and grow as a pulmonary artery replacement in lambs and implemented a design for a valved conduit consisting of three tubes sewn together. Seven lambs were implanted with tri-tube valved conduits in sequential cohorts and compared to bioprosthetic conduits. Valves implanted into the pulmonary artery of two lambs of the first cohort of four animals functioned with mild regurgitation and systolic pressure drops <10 mmHg up to 52 weeks after implantation, during which the valve diameter increased from 19 mm to a physiologically normal ~25 mm. In a second cohort, the valve design was modified to include an additional tube, creating a sleeve around the tri-tube valve to counteract faster root growth relative to the leaflets. Two valves exhibited trivial-to-mild regurgitation at 52 weeks with similar diameter increases to ~25 mm and systolic pressure drops of <5 mmHg, whereas the third valve showed similar findings until moderate regurgitation was observed at 52 weeks, correlating to hyperincrease in the valve diameter. In all explanted valves, the leaflets contained interstitial cells and an endothelium progressing from the base of the leaflets and remained thin and pliable with sparse, punctate microcalcifications. The tri-tube valves demonstrated reduced calcification and improved hemodynamic function compared to clinically used pediatric bioprosthetic valves tested in the same model. This tri-tube valved conduit has potential for long-term valve growth in children.

Prevascularized hydrogels with mature vascular networks promote the regeneration of critical-size calvarial bone defects in vivo.

Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8 mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors.

3D Printing of Microgel-Loaded Modular Microcages as Instructive Scaffolds for Tissue Engineering.

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based 3D printing strategy is used to fabricate a novel miniaturized modular microcage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional toy interlocking plastic blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-microcage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized microcage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance.

Biologically-engineered mechanical model of a calcified artery.

Vascular calcification is a commonly occurring pathological process and is recognized as an independent prognostic marker for cardiovascular morbidity and mortality. Recent progress in developing novel therapies to modify vascular calcification is critically hampered due to the lack of reliable in vitro experimental models that recapitulate the structural and mechanical attributes of calcified arteries. In this study, we show the ability to model the behavior of diffuse vascular calcification in vitro using biologically-engineered grafts approximating the composition, structure, and mechanical properties of arteries. Transmural calcification was achieved by exposing the acellular grafts of collagenous ECM to complete medium containing elevated Calcium (Ca) and Phosphate (P) concentrations. It was found that increasing the serum concentration from 2% to 10% increased the extent and degree of calcification based on histochemical, ultrastructural, chemical and thermal analyses. The presence of variably-sized spherical calcific deposits within the matrix further confirmed its morphological similarity to pathologic calcification. Mechanical testing demonstrated up to a 16-fold decrease in compliance due to the calcification, consistent with prior reports for calcified arteries. The model developed thus has potential to improve the design and development of interventional devices and therapies for the diagnosis and treatment of arterial calcification. STATEMENT OF SIGNIFICANCE: The presence of extensive vascular calcification makes angiographic/interventional procedures difficult due to reduced arterial compliance. Current attempts to develop safe and effective non-surgical adjunctive techniques to treat calcified arteries are largely limited by the lack of a physiologically relevant testing platform that mimics the structural and mechanical features of vascular calcification. Herein, we developed an off-the-shelf calcified artery model, with the goal to accelerate the pre-clinical development of novel therapies for the management of arterial calcification. To the extent of our knowledge, this is the first report of an in vitro tissue-engineered model of diffuse arterial calcification.

Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization.

Bone tissue, by definition, is an organic-inorganic nanocomposite, where metabolically active cells are embedded within a matrix that is heavily calcified on the nanoscale. Currently, there are no strategies that replicate these definitive characteristics of bone tissue. Here we describe a biomimetic approach where a supersaturated calcium and phosphate medium is used in combination with a non-collagenous protein analog to direct the deposition of nanoscale apatite, both in the intra- and extrafibrillar spaces of collagen embedded with osteoprogenitor, vascular, and neural cells. This process enables engineering of bone models replicating the key hallmarks of the bone cellular and extracellular microenvironment, including its protein-guided biomineralization, nanostructure, vasculature, innervation, inherent osteoinductive properties (without exogenous supplements), and cell-homing effects on bone-targeting diseases, such as prostate cancer. Ultimately, this approach enables fabrication of bone-like tissue models with high levels of biomimicry that may have broad implications for disease modeling, drug discovery, and regenerative engineering.

Carvacrol/ß-cyclodextrin inclusion complex inhibits cell proliferation and migration of prostate cancer cells.

Carvacrol, a phenolic monoterpene derived from thyme oil has gained wide interest recently because of its anticancer activities. To improve the solubility of carvacrol, the formation of inclusion complexes with ß-cyclodextrin was performed by ultrasound and freeze-drying methods and characterized using thermal analysis, FTIR, XRD, SEM, NMR and HPLC analysis. From these results, carvacrol was successfully complexed within ß-cyclodextrin cavity. Moreover, HPLC analysis demonstrated a higher entrapment efficiency for freeze-drying method (81.20 ±â€¯0.52%) in contrast to ultrasound method (34.02 ±â€¯0.67%). Hence, freeze-drying inclusion complex was evaluated for its antiproliferative effect and cytotoxicity against prostate cancer cell line (PC3) in vitro. Further, freeze-drying complex led to a dose-dependent inhibition in tumor cell growth in 2D and 3D cell culture systems. Altogether, the inclusion of carvacrol in ß-cyclodextrin led to the formation of stable complexes with potent antiproliferative effects against PC3 cells, in vitro. Such an improved cytotoxic effect can be attributed to the enhanced the aqueous solubility and bioavailability of carvacrol by effective complexation in ß-cyclodextrin.

The influence of osteopontin-guided collagen intrafibrillar mineralization on pericyte differentiation and vascularization of engineered bone scaffolds.

Biomimetically mineralized collagen scaffolds are promising for bone regeneration, but vascularization of these materials remains to be addressed. Here, we engineered mineralized scaffolds using an osteopontin-guided polymer-induced liquid-precursor mineralization method to recapitulate bone's mineralized nanostructure. SEM images of mineralized samples confirmed the presence of collagen with intrafibrillar mineral, also EDS spectra and FTIR showed high peaks of calcium and phosphate, with a similar mineral/matrix ratio to native bone. Mineralization increased collagen compressive modulus up to 15-fold. To evaluate vasculature formation and pericyte-like differentiation, HUVECs and hMSCs were seeded in a 4:1 ratio in the scaffolds for 7 days. Moreover, we used RT-PCR to investigate the gene expression of pericyte markers ACTA2, desmin, CD13, NG2, and PDGFRß. Confocal images showed that both nonmineralized and mineralized scaffolds enabled endothelial capillary network formation. However, vessels in the nonmineralized samples had longer vessel length, a larger number of junctions, and a higher presence of αSMA+ mural cells. RT-PCR analysis confirmed the downregulation of pericytic markers in mineralized samples. In conclusion, although both scaffolds enabled endothelial capillary network formation, mineralized scaffolds presented less pericyte-supported vessels. These observations suggest that specific scaffold characteristics may be required for efficient scaffold vascularization in future bone tissue engineering strategies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1522-1532, 2019.

A dentin-derived hydrogel bioink for 3D bioprinting of cell laden scaffolds for regenerative dentistry.

Recent studies in tissue engineering have adopted extracellular matrix (ECM) derived scaffolds as natural and cytocompatible microenvironments for tissue regeneration. The dentin matrix, specifically, has been shown to be associated with a host of soluble and insoluble signaling molecules that can promote odontogenesis. Here, we have developed a novel bioink, blending printable alginate (3% w/v) hydrogels with the soluble and insoluble fractions of the dentin matrix. We have optimized the printing parameters and the concentrations of the individual components of the bioink for print accuracy, cell viability and odontogenic potential. We find that, while viscosity, and hence printability of the bioinks, was greater in the formulations containing higher concentrations of alginate, a higher proportion of insoluble dentin matrix proteins significantly improved cell viability; where a 1:1 ratio of alginate and dentin (1:1 Alg-Dent) was most suitable. We further demonstrate high retention of the soluble dentin molecules within the 1:1 Alg-Dent hydrogel blends, evidencing renewed interactions between these molecules and the dentin matrix post crosslinking. Moreover, at concentrations of 100 µg ml-1, these soluble dentin molecules significantly enhanced odontogenic differentiation of stem cells from the apical papilla encapsulated in bioprinted hydrogels. In summary, the proposed novel bioinks have demonstrable cytocompatibility and natural odontogenic capacity, which can be a used to reproducibly fabricate scaffolds with complex three-dimensional microarchitectures for regenerative dentistry in the future.

Photopolymerization of cell-laden gelatin methacryloyl hydrogels using a dental curing light for regenerative dentistry.

Photopolymerized hydrogels, such as gelatin methacryloyl (GelMA), have desirable biological and mechanical characteristics for a range of tissue engineering applications. OBJECTIVE: This study aimed to optimize a new method to photopolymerize GelMA using a dental curing light (DL). METHODS: Lithium acylphosphinate photo-initiator (LAP, 0.05, 0.067, 0.1% w/v) was evaluated for its ability to polymerize GelMA hydrogel precursors (10% w/v) encapsulated with odontoblast-like cells (OD21). Different irradiances (1650, 2300 and 3700mW/cm2) and photo-curing times (5-20s) were tested, and compared against the parameters typically used in UV light photopolymerization (45mW/cm2, 0.1% w/v Irgacure 2959 as photoinitiator). Physical and mechanical properties of the photopolymerized GelMA hydrogels were determined. Cell viability was assessed using a live and dead assay kit. RESULTS: Comparing DL and UV polymerization methods, the DL method photopolymerized GelMA precursor faster and presented larger pore size than the UV polymerization method. The live and dead assay showed more than 80% of cells were viable when hydrogels were photopolymerized with the different DL irradiances. However, the cell viability decreased when the exposure time was increased to 20s using the 1650mW/cm2 intensity, and when the LAP concentration was increased from 0.05 to 0.1%. Both DL and UV photocrosslinked hydrogels supported a high percentage of cell viability and enabled fabrication of micropatterns using a photolithography microfabrication technique. SIGNIFICANCE: The proposed method to photopolymerize GelMA cell-laden hydrogels using a dental curing light is effective and represents an important step towards the establishment of chair-side procedures in regenerative dentistry.

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.

Biomaterials for Craniofacial Bone Regeneration.

Functional reconstruction of craniofacial defects is a major clinical challenge in craniofacial sciences. The advent of biomaterials is a potential alternative to standard autologous/allogenic grafting procedures to achieve clinically successful bone regeneration. This article discusses various classes of biomaterials currently used in craniofacial reconstruction. Also reviewed are clinical applications of biomaterials as delivery agents for sustained release of stem cells, genes, and growth factors. Recent promising advancements in 3D printing and bioprinting techniques that seem to be promising for future clinical treatments for craniofacial reconstruction are covered. Relevant topics in the bone regeneration literature exemplifying the potential of biomaterials to repair bone defects are highlighted.

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 (x = 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.

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 in vitro 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.

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 tube-like 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 [Ca(2+)]i 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.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro.

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)-10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

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 intra-articularly 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-α and IL-1ß) 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.

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 µl 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 45.6 ± 0.5 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).