Regenerative bioelectronics: A strategic roadmap for precision medicine.

In the past few decades, stem cell-based regenerative engineering has demonstrated its significant potential to repair damaged tissues and to restore their functionalities. Despite such advancement in regenerative engineering, the clinical translation remains a major challenge. In the stance of personalized treatment, the recent progress in bioelectronic medicine likewise evolved as another important research domain of larger significance for human healthcare. Over the last several years, our research group has adopted biomaterials-based regenerative engineering strategies using innovative bioelectronic stimulation protocols based on either electric or magnetic stimuli to direct cellular differentiation on engineered biomaterials with a range of elastic stiffness or functional properties (electroactivity/magnetoactivity). In this article, the role of bioelectronics in stem cell-based regenerative engineering has been critically analyzed to stimulate futuristic research in the treatment of degenerative diseases as well as to address some fundamental questions in stem cell biology. Built on the concepts from two independent biomedical research domains (regenerative engineering and bioelectronic medicine), we propose a converging research theme, 'Regenerative Bioelectronics'. Further, a series of recommendations have been put forward to address the current challenges in bridging the gap in stem cell therapy and bioelectronic medicine. Enacting the strategic blueprint of bioelectronic-based regenerative engineering can potentially deliver the unmet clinical needs for treating incurable degenerative diseases.

Electrical stimulation waveform-dependent osteogenesis on PVDF/BaTiO3 composite using a customized and programmable cell stimulator.

Directing cellular functionalities using biomaterial-based bioelectronic stimulation remains a significant constraint in translating research outcomes to address specific clinical needs. Electrical stimulation is now being clinically used as a therapeutic treatment option to promote bone tissue regeneration and to improve neuromuscular functionalities. However, the nature of the electrical waveforms during the stimulation and underlying biophysical rationale are still not scientifically well explored. Furthermore, bone-mimicking implant-based bioelectrical regulation of osteoinductivity has not been translated to clinics. The present study demonstrates the role of the electrical stimulation waveform to direct differentiation of stem cells on an electroactive polymeric substrate, using monophasic direct current (DC), square waveform, and biphasic waveform. In this regard, an in-house electrical stimulation device has been fabricated for the uninterrupted delivery of programmed electrical signals to stem cells in culture. To provide a functional platform for stem cells to differentiate, barium titanate (BaTiO3 , BT) reinforced poly(vinylidene difluoride) (PVDF) has been developed with mechanical properties similar to bone. The electrical stimulation of human mesenchymal stem cells (hMSCs) on PVDF/BT composite inhibited proliferation rate at day 7, indicating early commitment for differentiation. The phenotypical characteristics of DC stimulated hMSCs provided signatures of differentiation towards osteogenic lineage, which was subsequently confirmed using alkaline phosphatase assay, collagen deposition, matrix mineralization, and genetic expression. Our findings suggest that DC stimulation induced early osteogenesis in hMSCs with a higher level of intracellular reactive oxygen species (ROS), whereas the stimulation with square wave directed late osteogenesis with a lower ROS regeneration. In summary, the present study critically analyzes the role of electrical stimulation waveforms in regulating osteogenesis, without external biochemical differentiation inducers, on a bone-mimicking functional biomaterial substrate. Such a strategy can potentially be adopted to develop orthopedic implant-based bioelectronic medicine for bone regeneration.

Tunable Substrate Functionalities Direct Stem Cell Fate toward Electrophysiologically Distinguishable Neuron-like and Glial-like Cells.

Engineering cellular microenvironment on a functional platform using various biophysical cues to modulate stem cell fate has been the central theme in regenerative engineering. Among the various biophysical cues to direct stem cell differentiation, the critical role of physiologically relevant electric field (EF) stimulation was established in the recent past. The present study is the first to report the strategy to switch EF-mediated differentiation of human mesenchymal stem cells (hMSCs) between neuronal and glial pathways, using tailored functional properties of the biomaterial substrate. We have examined the combinatorial effect of substrate functionalities (conductivity, electroactivity, and topography) on the EF-mediated stem cell differentiation on polyvinylidene-difluoride (PVDF) nanocomposites in vitro, without any biochemical inducers. The functionalities of PVDF have been tailored using conducting nanofiller (multiwall-carbon nanotube, MWNT) and piezoceramic (BaTiO3, BT) by an optimized processing approach (melt mixing-compression molding-rolling). The DC conductivity of PVDF nanocomposites was tuned from ∼10-11 to ∼10-4 S/cm and the dielectric constant from ∼10 to ∼300. The phenotypical changes and genotypical expression of hMSCs revealed the signatures of early differentiation toward neuronal pathway on rolled-PVDF/MWNT and late differentiation toward glial lineage on rolled-PVDF/BT/MWNT. Moreover, we were able to distinguish the physiological properties of differentiated neuron-like and glial-like cells using membrane depolarization and mechanical stimulation. The excitability of the EF-stimulated hMSCs was also determined using whole-cell patch-clamp recordings. Mechanistically, the roles of intracellular reactive oxygen species (ROS), Ca2+ oscillations, and synaptic and gap junction proteins in directing the cellular fate have been established. Therefore, the present work critically unveils complex yet synergistic interaction of substrate functional properties to direct EF-mediated differentiation toward neuron-like and glial-like cells, with distinguishable electrophysiological responses.

Functionalized Fluoropolymer-Compatibilized Elastomeric Bilayer Composites for Osteochondral Repair: Unraveling the Role of Substrate Stiffness and Functionalities.

The osteochondral lesions and osteoarthritis-related complications continue to be clinically relevant challenges to be addressed by the biomaterials community. Hydrogel-based scaffolds have been widely investigated to enhance osteochondral regeneration, but the inferior mechanical properties together with poor functional stability are the major constraints in their clinical translation. The development of osteochondral implants with natural tissue-mimicking mechanical properties remains largely unexplored. In this perspective, the present study demonstrates a strategy to develop a bilayer osteochondral implant with an elastically stiff composite (poly(vinylidene difluoride)-reinforced BaTiO3, PVDF/BT) and elastically compliant composite (maleic anhydride-functionalized PVDF/thermoplastic polyurethane/BaTiO3, m-PVDF/TPU/BT). The compositional variation in polymer composites allowed the elastic modulus of the hybrid bilayer construct to vary from ∼2 GPa to ∼90 MPa, which enabled a better understanding of the substrate-stiffness-dependent cellular behavior and maturation of preosteoblasts and chondrocytes. The cellular functionalities on PVDF-based polymer matrices have been benchmarked against ultrahigh-molecular-weight polyethylene (UHMWPE), which is clinically used for a wide spectrum of orthopedic applications. The increased alkaline phosphatase (ALP) activity, collagen synthesis, and matrix mineralization confirmed the early differentiation of preosteoblasts on the PVDF/BT matrix with subchondral bone-like mechanical properties. On the contrary, the upregulated chondrogenic functionalities were recorded on m-PVDF/TPU/BT with an elevated level of collagen content, glycosaminoglycans, and proteoglycans. Emphasis has been laid on probing the regulation of the osteochondral behavior using tailored substrate stiffness and functionalities using compatibilized fluoropolymer-based elastomeric composites. Taken together, the results of this work conclusively establish the efficacy of the hybrid bilayer composite with natural tissue-mimicking mechanical properties for the functional repair of osteochondral defects.

Faster Biomineralization and Tailored Mechanical Properties of Marine-Resource-Derived Hydroxyapatite Scaffolds with Tunable Interconnected Porous Architecture.

Although hydroxyapatite (HA)-based porous scaffolds have been widely researched in the last three decades, the development of naturally derived biomimetic HA with a tunable elastic modulus and strength together with faster biomineralization properties has not yet been achieved. To address this specific issue, we report here a scalable biogenic synthesis approach to obtain submicron HA powders from cuttlefish bone. The marine-resource-derived HA together with different pore formers can be conventionally sintered to produce physiologically relevant scaffolds with porous architecture. Depending on pore formers, the scaffolds with a range of porosity of up to 51% with a larger range of pore sizes up to 50 µm were fabricated. An empirical relationship between the compression strength and the elastic modulus with fractional porosity was established. A combination of moderate compressive strength (12-15 MPa) with an elastic modulus up to 1.6 GPa was obtained from cuttlefish-bone-derived HA with wheat flour as the pore former. Most importantly, the specific HA scaffold supports the faster nucleation and growth of the biomineralized apatite layer with full coverage within 3 days of incubation in simulated body fluid. More importantly, the marine-species-derived HA supported better adhesion and proliferation of murine osteoblast cells than HA sintered using powders from nonbiogenic resources. The spectrum of physical and biomineralization properties makes cuttlefish-bone-derived porous HA a new generation of implantable biomaterial for potential application in cancellous bone regeneration.

A New Approach to Compute the Porosity and Surface Roughness of Porous Coated Capillary-Assisted Low Pressure Evaporators.

The fundamental characteristics of metal coatings that influence heat transfer are porosity and surface roughness. It is a challenge to analyze the porosity and surface roughness due to the inadequate amount of copper per coated area. In this study, a new approach to non-invasively determine the porosity of metal films utilizing a helium pycnometer and computed micro-tomography (CMT) is presented. Furthermore, a telescope-goniometer is used to measure the surface roughness. Experiments are conducted on four varieties of thin film samples coated with copper powder using wire flame and plasma thermal spray coating methods. The porosities of the thin films were determined to be between 39 and 43%. The thermal spray coating increased the hydrophobicity of the surface and the plasma coating created super-hydrophobic surfaces. The new approach establishes that the porosity of thin films can be non-invasively determined and may also be applied to a wide variety of coated surfaces.

Controlled Shear Flow Directs Osteogenesis on UHMWPE-Based Hybrid Nanobiocomposites in a Custom-Designed PMMA Microfluidic Device.

The combinatorial influence of a biophysical cue (substrate stiffness) and biomechanical cue (shear flow) on the osteogenesis modulation of human mesenchymal stem cells (hMSCs) is studied for bone regenerative applications. In this work, we report stem cell differentiation on an ultra high molecular weight polyethylene (UHMWPE)-based hybrid nanobiocomposite [reinforced with a multiwalled carbon nanotube (MWCNT) and/or nanohydroxyapatite (nHA)] under a physiologically relevant shear flow (1 Pa) in a custom-built microfluidic device. Using a genotypic assessment with qRT-PCR and phenotypic assessment through analysis of cytoskeletal remodelling and marker proteins, the role of shear on the progression of osteogenesis modulation has been quantitatively established with statistically significant differences between nHA-reinforced and MWCNT-reinforced UHMWPE. Early-stage (alkaline phosphatase activity at day 8), middle-stage (matrix collagenation at day 14), and late-stage (matrix calcification at day 20) events were analyzed using mRNA expression changes of a limited cell volume after microfluidic culture experiments. The conventional Petri dish culture (static) exhibited an increased osteogenesis for nanoparticle-reinforced UHMWPE, irrespective of the type of nanoparticle. The shear-mediated culture experiments resulted in noticeable differences in the degree of osteogenesis with MWCNT being more effective than nHA reinforcement. The shear-mediated osteogenesis has been attributed to the skewed cellular morphology with a higher cell adhesion (vinculin expression) on UHMWPE and nHA than that of UHMWPE and MWCNT. The signatures of the cytoskeletal changes are reflected in terms of left-to-right (L-R) chirality as well as alignment and pattern of actin fibers. Moreover, stemness (vimentin expression) was found to be decreased because of differentiation. The electrophysiological analysis using patch clamp experiments also revealed a higher inward calcium current and intracellular calcium activity for the cells grown on the UHMWPE and nHA nanobiocomposite under shear. Overall, the present study conclusively establishes the synergistic role of substrate stiffness and shear on osteogenesis of hMSCs, in vitro.