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).

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.

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.

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.

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.

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.

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.

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.