Low-Concentration Gelatin Methacryloyl Hydrogel with Tunable 3D Extrusion Printability and Cytocompatibility: Exploring Quantitative Process Science and Biophysical Properties.

Three-dimensional (3D) bioprinting of hydrogels with a wide spectrum of compositions has been widely investigated. Despite such efforts, a comprehensive understanding of the correlation among the process science, buildability, and biophysical properties of the hydrogels for a targeted clinical application has not been developed in the scientific community. In particular, the quantitative analysis across the entire developmental path for 3D extrusion bioprinting of such scaffolds is not widely reported. In the present work, we addressed this gap by using widely investigated biomaterials, such as gelatin methacryloyl (GelMA), as a model system. Using extensive experiments and quantitative analysis, we analyzed how the individual components of methacrylated carboxymethyl cellulose (mCMC), needle-shaped nanohydroxyapatite (nHAp), and poly(ethylene glycol)diacrylate (PEGDA) with GelMA as baseline matrix of the multifunctional bioink can influence the biophysical properties, printability, and cellular functionality. The complex interplay among the biomaterial ink formulations, viscoelastic properties, and printability toward the large structure buildability (structurally stable cube scaffolds with 15 mm edge) has been explored. Intriguingly, the incorporation of PEGDA into the GelMA/mCMC matrix offered improved compressive modulus (∼40-fold), reduced swelling ratio (∼2-fold), and degradation rates (∼30-fold) compared to pristine GelMA. The correlation among microstructural pore architecture, biophysical properties, and cytocompatibility is also established for the biomaterial inks. These photopolymerizable bio(material)inks served as the platform for the growth and development of bone and cartilage matrix when human mesenchymal stem cells (hMSCs) are either seeded on two-dimensional (2D) substrates or encapsulated on 3D scaffolds. Taken together, this present study unequivocally establishes a significant step forward in the development of a broad spectrum of shape-fidelity compliant bioink for the 3D bioprinting of multifunctional scaffolds and emphasizes the need for invoking more quantitative analysis in establishing process-microstructure-property correlation.

Comprehensive functional outcome analysis and importance of bone remodelling on personalized cranioplasty treatment using Poly(methyl methacrylate) bone flaps.

Cranioplasty involves the surgical reconstruction of cranial defects arising as a result of various factors, including decompressive craniectomy, cranial malformations, and brain injury due to road traffic accidents. Most of the modern decompressive craniectomies (DC) warrant a future cranioplasty surgery within 6-36 months. The conventional process of capturing the defect impression and polymethyl methacrylate (PMMA) flap fabrication results in a misfit or misalignment at the site of implantation. Equally, the intra-operative graft preparation is arduous and can result in a longer surgical time, which may compromise the functional and aesthetic outcomes. As part of a multicentric pilot clinical study, we recently conducted a cohort study on ten human subjects during 2019-2022, following the human ethics committee approvals from the participating institutes. In the current study, an important aspect of measuring the extent of bone remodelling during the time gap between decompressive craniectomy and cranioplasty was successfully evaluated. The sterilised PMMA bone flaps were implanted at the defect area during the cranioplasty surgery using titanium mini plates and screws. The mean surgery time was 90 ± 20 min, comparable to the other clinical studies on cranioplasty. No signs of intra-operative and post-operative complications, such as cerebrospinal fluid leakage, hematoma, or local and systemic infection, were clinically recorded. Importantly, aesthetic outcomes were excellent for all the patients, except in a few clinical cases, wherein the PMMA bone flap was to be carefully customized due to the remodelling of the native skull bone. The extent of physiological remodelling was evaluated by superimposing the pre-operative and post-operative CT scan data after converting the defect morphology into a 3D model. This study further establishes the safety and efficacy of a technologically better approach to fabricate patient-specific acrylic bone flaps with improved surgical outcomes. More importantly, the study outcome further demonstrates the strategy to address bone remodelling during the patient-specific implant design.

3D bioprinting of multifunctional alginate dialdehyde (ADA)-gelatin (GEL) (ADA-GEL) hydrogels incorporating ferulic acid.

The present work explores the 3D extrusion printing of ferulic acid (FA)-containing alginate dialdehyde (ADA)-gelatin (GEL) scaffolds with a wide spectrum of biophysical and pharmacological properties. The tailored addition of FA (≤0.2 %) increases the crosslinking between FA and GEL in the presence of calcium chloride (CaCl2) and microbial transglutaminase, as confirmed using trinitrobenzenesulfonic acid (TNBS) assay. In agreement with an increase in crosslinking density, a higher viscosity of ADA-GEL with FA incorporation was achieved, leading to better printability. Importantly, FA release, enzymatic degradation and swelling were progressively reduced with an increase in FA loading to ADA-GEL, over 28 days. Similar positive impact on antibacterial properties with S. epidermidis strains as well as antioxidant properties were recorded. Intriguingly, FA incorporated ADA-GEL supported murine pre-osteoblast proliferation with reduced osteosarcoma cell proliferation over 7 days in culture, implicating potential anticancer property. Most importantly, FA-incorporated and cell-encapsulated ADA-GEL can be extrusion printed to shape fidelity-compliant multilayer scaffolds, which also support pre-osteoblast cells over 7 days in culture. Taken together, the present study has confirmed the significant potential of 3D bioprinting of ADA-GEL-FA ink to obtain structurally stable scaffolds with a broad spectrum of biophysical and therapeutically significant properties, for bone tissue engineering applications.

Osteogenesis, hemocompatibility, and foreign body response of polyvinylidene difluoride-based composite reinforced with carbonaceous filler and higher volume of piezoelectric ceramic phase.

Hybrid polymer-ceramic composites have been widely investigated for bone tissue engineering applications. The incorporation of a large amount of inorganic phase, like barium titanate (BaTiO3) with good dispersion, in a polymeric matrix using a conventional processing approach has always been challenging. Also, the comprehensive study encompassing the interactions of key components of living organisms (cell, blood, tissue) with such hybrid composites is not well explored in many published studies. Built on our earlier studies and recognizing the importance of poly(vinylidene fluoride) (PVDF) as a widely used polymer for a wide spectrum of biomedical applications, the present study reports the qualitative and quantitative analysis of the biocompatibility of PVDF composite (PVDF/30BT/3MWCNT) reinforced with large amounts of BaTiO3 (30 wt %) and tailored addition of multiwalled carbon nanotubes (MWCNT; 3 wt %). The melt mixing-extrusion-compression moulding-based processing approach resulted in an enhancement of ß-phase content, thermal stability, and wettability in the semi-crystalline PVDF composite. The enhanced hemocompatibility of PVDF/30BT/3MWCNT has been established conclusively by a series of in vitro blood-material interaction assays, including haemolysi, analysis of platelets attachment and activation, dynamic blood coagulation, and plasma recalcification time. The cytocompatibility study confirms an improved adhesion, proliferation, and migration of osteoprogenitor cells (preosteoblasts; MC3T3-E1) on PVDF/30BT/3MWCNT, in a manner better than neat PVDF, in vitro. When these cells were cultured in osteogenic differentiating media, the modulated osteogenesis, in terms of alkaline phosphatase activity, intracellular Ca2+ concentration, and calcium deposition on the PVDF/30BT/3MWCNT, was recorded. Following subcutaneous implantation of PVDF/30BT/3MWCNT in rat model, no apparent variation was recorded in the complete hemogram (blood hematology analysis) or serum biochemistry, post 30-, 60-, and 90-days surgery. Importantly, 90-days post-implantation, the fibrous capsule thickness was significantly reduced in the composites w.r.t PVDF alone, together with better blood vessel formation, indicating improved neovascularization around the composite. This study establishes the efficacy of inorganic fillers in enhancing the biocompatibility of PVDF, which could open up a wide range of biomedical applications.

Probing the Influence of Hybrid Thread Design on Biomechanical Response of Dental Implants: Finite Element Study and Experimental Validation.

This study aimed to perform quantitative biomechanical analysis for probing the effect of varying thread shapes in an implant for improved primary stability in prosthodontics surgery. Dental implants were designed with square (SQR), buttress (BUT), and triangular (TRI) thread shapes or their combinations. Cone-beam computed tomography images of mandible molar zones in human subjects belonging to three age groups were used for virtual implantation of the designed implants, to quantify patient-specific peri-implant bone microstrain, using finite element analyses. The in silico analyses were carried out considering frictional contact to simulate immediate loading with a static masticatory force of 200 N. To validate computational biomechanics results, compression tests were performed on three-dimensional printed implants having the investigated thread architectures. Bone/implant contact areas were also quantitatively assessed. It was observed that, bone/implant contact was maximum for SQR implants followed by BUT and TRI implants. For all the cases, peak microstrain was recorded in the cervical cortical bone. The combination of different thread shapes in the middle or in the apical part (or both) was demonstrated to improve peri-implant microstrain, particularly for BUT and TRI. Considering 1500-2000 microstrain generates in the peri-implant bone during regular physiological functioning, BUT-SQR, BUT-TRI-SQR, TRI-SQR-BUT, SQR, and SQR-BUT-TRI design concepts were suitable for younger; BUT-TRI-SQR, BUT-SQR-TRI, TRI-SQR-BUT, SQR-BUT, SQR-TRI for middle-aged, and BUT-TRI-SQR, BUT-SQR-TRI, TRI-BUT-SQR, SQR, and SQR-TRI for the older group of human patients.

Next-generation personalized cranioplasty treatment.

Decompressive craniectomy (DC) is a surgical procedure, that is followed by cranioplasty surgery. DC is usually performed to treat patients with traumatic brain injury, intracranial hemorrhage, cerebral infarction, brain edema, skull fractures, etc. In many published clinical case studies and systematic reviews, cranioplasty surgery is reported to restore cranial symmetry with good cosmetic outcomes and neurophysiologically relevant functional outcomes in hundreds of patients. In this review article, we present a number of key issues related to the manufacturing of patient-specific implants, clinical complications, cosmetic outcomes, and newer alternative therapies. While discussing alternative therapeutic treatments for cranioplasty, biomolecules and cellular-based approaches have been emphasized. The current clinical practices in the restoration of cranial defects involve 3D printing to produce patient-specific prefabricated cranial implants, that provide better cosmetic outcomes. Regardless of the advancements in image processing and 3D printing, the complete clinical procedure is time-consuming and requires significant costs. To reduce manual intervention and to address unmet clinical demands, it has been highlighted that automated implant fabrication by data-driven methods can accelerate the design and manufacturing of patient-specific cranial implants. The data-driven approaches, encompassing artificial intelligence (machine learning/deep learning) and E-platforms, such as publicly accessible clinical databases will lead to the development of the next generation of patient-specific cranial implants, which can provide predictable clinical outcomes. STATEMENT OF SIGNIFICANCE: Cranioplasty is performed to reconstruct cranial defects of patients who have undergone decompressive craniectomy. Cranioplasty surgery improves the aesthetic and functional outcomes of those patients. To meet the clinical demands of cranioplasty surgery, accelerated designing and manufacturing of 3D cranial implants are required. This review provides an overview of biomaterial implants and bone flap manufacturing methods for cranioplasty surgery. In addition, tissue engineering and regenerative medicine-based approaches to reduce clinical complications are also highlighted. The potential use of data-driven computer applications and data-driven artificial intelligence-based approaches are emphasized to accelerate the clinical protocols of cranioplasty treatment with less manual intervention and shorter intraoperative time.

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.

Dynamically crosslinked polydimethylsiloxane-based polyurethanes with contact-killing antimicrobial properties as implantable alloplasts for urological reconstruction.

A large population of patients is reported to suffer from urinary bladder-associated irreversible physiological disorders, rationalizing a continuous surge for structural and functional substitutes of urinary tissues, including ureters, bladder-wall, and urethra. The current gold standard for bladder reconstruction, an autologous gastrointestinal graft, is proven not to be an ideal substitute in the clinic. While addressing this unmet clinical need, a unique platform of antimicrobial polydimethyl siloxane-modified polyurethanes (TPU/PDMS) is designed and developed for its potential application as a urological implant. To the best of our knowledge, this study reports for the first time the successful integration of varying contents of PDMS within the molten polyurethane matrix using in situ crosslinking methodology. Thus, compatibilized binary blends possess clinically relevant viscoelastic properties to sustain high pressure, large distensions, and surgical manipulation. Furthermore, different chemical strategies are explored to covalently incorporate quaternized moieties, including 4-vinyl pyridine (4-VP), branched-polyethyleneimine (bPEI) as well as bPEI-grafted-(acrylic acid-co-vinylbenzyltriphenyl phosphonium chloride) (PAP), and counter urinary tract infections. The modified compositions, endowed with contact killing surfaces, reveal nearly three log reduction in bacterial growth in pathogenically infected artificial urine. Importantly, the antimicrobial TPU/PDMS blends support the uninhibited growth of mitochondrially viable murine fibroblasts, in a manner comparable to the medical-grade polyurethane. Collectively, the obtained results affirmed the newly developed polymers as promising biomaterials in reconstructive urology. STATEMENT OF SIGNIFICANCE: The clinical procedure for end-stage bladder disease remains replacement or augmentation of the bladder wall with a section of the patient's gastrointestinal tract. However, the absorptive and mucus-producing epithelium of intestinal segment is liable to short- and long-term complications. The dynamically crosslinked polydimethyl siloxane-based polyurethanes proposed herein, and the associated synthesis strategies to induce polycation grafted non-exhaustive contact-killing surfaces against uropathogents, have a significant clinical prospect in reconstructive urology. As an 'off-the-shelf' available alloplastic substitute, these blends offer the potential to circumvent the challenges associated with non-urinary autografts or scaffold based regenerative engineering and, thereby, shorten as well as simplify the surgical treatment. The targeted application has been conceived for a bladder patch to assist in various urinary diseases including, bladder carcinoma, refractory overactive bladder, interstitial cystitis, etc. However, given the ease of fabrication, moldability and the wide spectrum of mechanical properties that could be encompassed, these blends also present the possibility to be manifested into artificial ureteral or urethral conduits.

Evaluation of implant properties, safety profile and clinical efficacy of patient-specific acrylic prosthesis in cranioplasty using 3D binderjet printed cranium model: A pilot study.

There exists a significant demand to develop patient-specific prosthesis in reconstruction of cranial vaults after decompressive craniectomy. we report here, the outcomes of an unicentric pilot study on acrylic cranial prosthesis fabricated using a 3D printed cranium model with its clinically relevant mechanical properties. METHODS: The semi-crystalline polymethyl methacrylate (PMMA) implants, shaped to the cranial defects of 3D printed cranium model, were implanted in 10 patients (mean age, 40.8 ± 14.8 years). A binderjet 3D printer was used to create patient-specific mould and PMMA was casted to fabricate prosthesis which was analyzed for microstructure and properties. Patients were followed up for allergy, infection and cosmesis for a period of 6 months. RESULTS: As-cast PMMA flap exhibited hardness of 15.8 ± 0.24Hv, tensile strength of 30.7 ± 3.9 MPa and elastic modulus of 1.5 ± 0.1 GPa. 3D microstructure of the semi-crystalline acrylic implant revealed 2.5-15 µm spherical isolated pores. The mean area of the calvarial defect in craniectomy patients was 94.7 ± 17.4 cm2. We achieved a cranial index of symmetry (CIS -%) of 94.5 ± 3.9, while the average post-operative Glasgow outcome scale (GOS) score recorded was 4.2 ± 0.9. CONCLUSIONS: 3D printing based patient-specific design and fabrication of acrylic cranioplasty implant is safe and achieves acceptable cosmetic and clinical outcomes in patients with decompressive craniectomy. Our study ensured clinically acceptable structural and mechanical properties of implanted PMMA, suggesting that a low cost 3D printer based PMMA flap is an affordable option for cranioplasty in resource constrained settings.

Tissue-specific mesenchymal stem cell-dependent osteogenesis in highly porous chitosan-based bone analogs.

Among conventional fabrication techniques, freeze-drying process has widely been investigated for polymeric implants. However, the understanding of the stem cell progenitor-dependent cell functionality modulation and quantitative analysis of early osseointegration of highly porous scaffolds have not been explored. Here, we developed a novel, highly porous, multimaterial composite, chitosan/hydroxyapatite/polycaprolactone (CHT/HA/PCL). The in vitro studies have been performed using mesenchymal stem cells (MSCs) from three tissue sources: human bone marrow-derived MSCs (BM-MSCs), adipose-derived MSCs (AD-MSCs), and Wharton's jelly-derived MSCs (WJ-MSCs). Although cell attachment and metabolic activity [3-4,5-dimethylthiazol-2yl-(2,5 diphenyl-2H-tetrazoliumbromide) assay] were ore enhanced in WJ-MSC-laden CHT/HA/PCL composites, scanning electron microscopy, real-time gene expression (alkaline phosphatase [ALP], collagen type I [Col I], osteocalcin [OCN], and bone morphogenetic protein 4 [BMP-4]), and immunostaining (COL I, ß-CATENIN, OCN, and SCLEROSTIN [SOST]) demonstrated pronounced osteogenesis with terminal differentiation on BM-MSC-laden CHT/HA/PCL composites only. The enhanced cell functionality on CHT/HA/PCL composites was explained in terms of interplay among the surface properties and the optimal source of MSCs. In addition, osteogenesis in rat tibial model over 6 weeks confirmed a better ratio of bone volume to the total volume for BM-MSC-laden composites over scaffold-only and defect-only groups. The clinically conformant combination of 3D porous architecture with pore sizes varying in the range of 20 to 200 µm together with controlled in vitro degradation and early osseointegration establish the potential of CHT/HA/PCL composite as a potential cancellous bone analog.

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.

UHMWPE-MWCNT-nHA based hybrid trilayer nanobiocomposite: Processing approach, physical properties, stem/bone cell functionality, and blood compatibility.

The development of polymeric nanocomposites for biomedical applications remains a major challenge in terms of tailored addition of nanoparticles to realize the simultaneous enhancement of fracture resistance and cell/blood compatibility. To address this, the present work has been planned to determine whether small addition of surface functionalized multiwalled-carbon-nanotube, MWCNT (<1.5 wt%) and egg-shell derived nanosized hydroxyapatite, nHA (<10 wt%) to ultrahigh-molecular-weight-polyethylene (UHMWPE) can significantly improve the physical properties as well as biocompatibility. The difference in mouse osteoblast and human mesenchymal stem cell (hMSc) proliferation has been validated using both the monolithic composite and a trilayered composite with two different UHMWPE nanocomposites on either face with pure polymer at the middle. The combination of rheology and micro-CT with fractography reveals the homogeneous dispersion of nanofillers, leading to mechanical property enhancement. The quantitative analysis of cell viability and cell spreading by immunocytochemistry method, using vinculin and vimentin expression, establish significant cytocompatibility with hMSc and osteoblast cells onto the trilayer hybrid nanobiocomposite substrates. The hemocompatibility of the investigated composites under the controlled flow of rabbit blood in a microfluidic device reveals the signature of reduced thrombogenesis with reduction of platelet activation on UHMWPE nanocomposite w.r.t. unreinforced UHMWPE. An attempt has been made to discuss the blood compatibility results in the backdrop of the bovine serum albumin adsorption kinetics. Summarizing, the present study establishes that the twin requirement of mechanical property and cyto/hemo-compatibility can be potentially realized in developing trilayer composites in UHMWPE-nHA-MWCNT system.

Neurogenesis-on-Chip: Electric field modulated transdifferentiation of human mesenchymal stem cell and mouse muscle precursor cell coculture.

A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding on the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of murine myoblasts (C2C12) with hMScs [hMSc:C2C12=1:10] in a custom designed polymethylmethacrylate (PMMA) based microfluidic device with in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysical stimulation in the range of the endogenous physiological electric field strength (8 ±â€¯0.06  mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, the hMScs exhibited the neurosphere-like clusters with SOX2, nestin, ßIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signalling among the differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly supports neural-like behaviour for the electric field stimulated cells in coculture. The intriguing results are explained in terms of the paracrine signalling among the transdifferentiated cells in the electric field stimulated cellular microenvironment. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro.

Biophysical implications of Maxwell stress in electric field stimulated cellular microenvironment on biomaterial substrates.

A number of experimental studies have established the critical role of electric field stimulation on cell functionality modulation of various cell types on a wide range of biomaterial platforms in vitro. In particular, cell fate processes, morphological changes and electroporation are significantly modulated over a narrow range of electric field stimulation conditions. Although a few studies using electrical network theory, first principle simulations and electrohydrodynamic models are reported in the literature, the present study establishes a theoretical foundation to a new perspective that bioelectric stress can significantly influence cell morphological changes/electroporation and in a broader sense, the cell response to electric field on biomaterial substrates. A single cell is modelled as a spherical membrane separating the culture medium and the cytoplasm having different dielectric properties. The analytical solutions to the Laplace equation and Poisson equation for the system are adopted to quantitatively capture the potential distribution in the cellular microenvironment for different cases. These include a cell on a conducting substrate, on an insulating substrate and a cell with surface charge density, in electric field stimulated cellular microenvironment. The biophysical significance of the normal stress distribution has been discussed in terms of the variation in the cellular deformation, depending on the frequency of the electric field and substrate conductivity. A significant difference has been observed in the deformation behavior at higher frequencies (>109 Hz) compared to low frequency and DC electric fields. This theoretical study therefore unravels the significance of substrate conductivity in synergy with electric field parameters to modulate cell response. In addition, the tangential component of the Maxwell stress tensor (shear stress), a measure of the stretching force on the membrane, has been used to obtain estimates of the critical electric field required for membrane rupture. It has been predicted that a cell with surface charge density requires electric fields of the order of 10 kV/mm in order to undergo membrane rupture, which is in line with the experimental observations reported in the literature. Taken together, the presented analysis is expected to provide guidelines to develop next generation biomaterials and biomedical devices for regenerative medicine and cancer treatments.

Epithelial cell functionality on electroconductive Fe/Sr co-doped biphasic calcium phosphate.

In the perspective of dental restorative applications, co-doped bioceramics have not been explored much. From the clinical perspective, a successful dental implant is expected to interact with peri-prosthetic bones, gingival tissue, and surrounding connective tissues. The interaction of implant and implant coating materials with bone tissue is well studied. However, their interaction with surrounding epithelial components needs scientific validation. In this context, the present study aims at quantitative evaluation of the electrical properties of Fe/Sr co-doped biphasic calcium phosphate (BCP) samples and assessment of their cytocompatibility with epithelial (vero) cells. Sr/Fe co-doped BCPs were prepared by sol-gel synthesis technique, with different dopant concentration. Impact of co-doping on conductivity was assessed and interestingly an increase in conductivity with dopant amount was recorded in different co-doped BCPs. Cellular study showed the significant ( p = 0.01) increase in both cellular viability and functionality with increasing conductivity of samples. Higher epithelial cell adhesion indicates that (Sr/Fe) co-doped BCP would be favorable for faster epithelial sealing and also would reduce the chances of infection. Real-time PCR and immunofluorescence studies indicated that the expression of the epithelial marker (E-cadherin) significantly ( p = 0.01) increased in 10, 30 and 40 mol% co-doped samples in comparison to undoped BCP. In contrast to E-cadherin, fold change of ß-catenin remains unchanged amongst the co-doped ceramics, implying the absence of tumorigenic potential of (Sr/Fe) co-doped BCP. In addition, immune-fluorescence signatures for cellular polarity are established from enhanced expression PARD3 protein, which has major relevance for cellular morphogenesis and cell division. Summarizing, the present study establishes the efficacy of Sr/Fe co-doped BCPs as a dental implant coating material and its ability to modulate vero cell functionality.

Synergy of substrate conductivity and intermittent electrical stimulation towards osteogenic differentiation of human mesenchymal stem cells.

Human Mesenchymal Stem cells (hMSCs) have the unique potential to differentiate into multiple cell types. Depending on the cellular microenvironment (physical and biochemical cues), hMSCs can be directed to differentiate into osteogenic, chondrogenic, myogenic and adipogenic lineages. Among the strategies available to direct stem cell fate processes, electrical stimulation based approach has been extensively investigated in recent studies. In the present study, the conducting Hydroxyapatite-CaTiO3 (HA-CT) composites are used as electroconductive platforms to support the differentiation of hMSCs, in vitro. During culture without osteogenic supplements, intermittent electrical stimulation is provided every 24h over a period of 4weeks through parallel plate electrodes separated by a distance of 15mm and maintained at a static potential of 15V for 10min. In addition to cell morphological changes, the differentiation behavior of hMSCs after electrical stimulation is evaluated by mRNA expression analysis through polymerase chain reaction (PCR). Importantly, specific bone markers, in particular ALP, Col IA and Osteocalcin are expressed more significantly due to electrical stimulation, which also enhances the extent of extracellular matrix mineralization. Taken together, this study establishes the effectiveness of electroconductive HA-CT composites together with intermittent electrical stimulation to direct osteogenesis of hMSCs.

Engineered biomaterial and biophysical stimulation as combinatorial strategies to address prosthetic infection by pathogenic bacteria.

A plethora of antimicrobial strategies are being developed to address prosthetic infection. The currently available methods for implant infection treatment include the use of antibiotics and revision surgery. Among the bacterial strains, Staphylococcus species pose significant challenges particularly, with regard to hospital acquired infections. In order to combat such life threatening infectious diseases, researchers have developed implantable biomaterials incorporating nanoparticles, antimicrobial reinforcements, surface coatings, slippery/non-adhesive and contact killing surfaces. This review discusses a few of the biomaterial and biophysical antimicrobial strategies, which are in the developmental stage and actively being pursued by several research groups. The clinical efficacy of biophysical stimulation methods such as ultrasound, electric and magnetic field treatments against prosthetic infection depends critically on the stimulation protocol and parameters of the treatment modality. A common thread among the three biophysical stimulation methods is the mechanism of bactericidal action, which is centered on biophysical rupture of bacterial membranes, the generation of reactive oxygen species (ROS) and bacterial membrane depolarization evoked by the interference of essential ion-transport. Although the extent of antimicrobial effect, normally achieved through biophysical stimulation protocol is insufficient to warrant therapeutic application, a combination of antibiotic/ROS inducing agents and biophysical stimulation methods can elicit a clinically relevant reduction in viable bacterial numbers. In this review, we present a detailed account of both the biomaterial and biophysical approaches for achieving maximum bacterial inactivation. Summarizing, the biophysical stimulation methods in a combinatorial manner with material based strategies can be a more potent solution to control bacterial infections. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2174-2190, 2017.

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.

Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE Bionanocomposites.

The uniform dispersion of nanoparticles in a polymer matrix, together with an enhancement of interfacial adhesion is indispensable toward achieving better mechanical properties in the nanocomposites. In the context to biomedical applications, the type and amount of nanoparticles can potentially influence the biocompatibility. To address these issues, we prepared high-density polyethylene (HDPE) based composites reinforced with graphene oxide (GO) by melt mixing followed by compression molding. In an attempt to tailor the dispersion and to improve the interfacial adhesion, we immobilized polyethylene (PE) onto GO sheets by nucleophilic addition-elimination reaction. A good combination of yield strength (ca. 20 MPa), elastic modulus (ca. 600 MPa), and an outstanding elongation at failure (ca. 70%) were recorded with 3 wt % polyethylene grafted graphene oxide (PE-g-GO) reinforced HDPE composites. Considering the relevance of protein adsorption as a biophysical precursor to cell adhesion, the protein adsorption isotherms of bovine serum albumin (BSA) were determined to realize three times higher equilibrium constant (Keq) for PE-g-GO-reinforced HDPE composites as compared to GO-reinforced composites. To assess the cytocompatibility, we grew osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) on HDPE/GO and HDPE/PE-g-GO composites, in vitro. The statistically significant increase in metabolically active cell over different time periods in culture for up to 6 days in MC3T3 and 7 days for hMSCs was observed, irrespective of the substrate composition. Such observation indicated that HDPE with GO or PE-g-GO addition (up to 3 wt %) can be used as cell growth substrate. The extensive proliferation of cells with oriented growth pattern also supported the fact that tailored GO addition can support cellular functionality in vitro. Taken together, the experimental results suggest that the PE-g-GO in HDPE can effectively be utilized to enhance both mechanical and cytocompatibility properties and can further be explored for potential biomedical applications.

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.