Additive Manufacturing of Bovine Serum Albumin-Based Hydrogels and Bioplastics.

Biosourced and biodegradable polymers for additive manufacturing could enable the rapid fabrication of parts for a broad spectrum of applications ranging from healthcare to aerospace. However, a limited number of these materials are suitable for vat photopolymerization processes. Herein, we report a two-step additive manufacturing process to fabricate robust protein-based constructs using a commercially available laser-based stereolithography printer. Methacrylated bovine serum albumin (MA-BSA) was synthesized and formulated into aqueous resins that were used to print complex three-dimensional (3D) objects with a resolution comparable to a commercially available resin. The MA-BSA resins were characterized by rheometry to determine the viscosity and the cure rate, as both parameters can ultimately be used to predict the printability of the resin. In the first step of patterning these materials, the MA-BSA resin was 3D printed, and in the second step, the printed construct was thermally cured to denature the globular protein and increase the intermolecular noncovalent interactions. Thus, the final 3D printed part was comprised of both chemical and physical cross-links. Compression studies of hydrated and dehydrated constructs demonstrated a broad range of compressive strengths and Young's moduli that could be further modulated by adjusting the type and amount of co-monomer. The printed hydrogel constructs demonstrated good cell viability (>95%) after a 21 day culture period. These MA-BSA resins are expected to be compatible with other vat photopolymerization techniques including digital light projection and continuous liquid interface production.

Dynamically Tunable Cell Culture Platforms for Tissue Engineering and Mechanobiology.

Human tissues are sophisticated ensembles of many distinct cell types embedded in the complex, but well-defined, structures of the extracellular matrix (ECM). Dynamic biochemical, physicochemical, and mechano-structural changes in the ECM define and regulate tissue-specific cell behaviors. To recapitulate this complex environment in vitro, dynamic polymer-based biomaterials have emerged as powerful tools to probe and direct active changes in cell function. The rapid evolution of polymerization chemistries, structural modulation, and processing technologies, as well as the incorporation of stimuli-responsiveness, now permit synthetic microenvironments to capture much of the dynamic complexity of native tissue. These platforms are comprised not only of natural polymers chemically and molecularly similar to ECM, but those fully synthetic in origin. Here, we review recent in vitro efforts to mimic the dynamic microenvironment comprising native tissue ECM from the viewpoint of material design. We also discuss how these dynamic polymer-based biomaterials are being used in fundamental cell mechanobiology studies, as well as towards efforts in tissue engineering and regenerative medicine.

Prolonged nerve blockade delays the onset of neuropathic pain.

Aberrant neuronal activity in injured peripheral nerves is believed to be an important factor in the development of neuropathic pain. Pharmacological blockade of that activity has been shown to mitigate the onset of associated molecular events in the nervous system. However, results in preventing onset of pain behaviors by providing prolonged nerve blockade have been mixed. Furthermore, the experimental techniques used to date to provide that blockade were limited in clinical potential in that they would require surgical implantation. To address these issues, we have used liposomes (SDLs) containing saxitoxin (STX), a site 1 sodium channel blocker, and the glucocorticoid agonist dexamethasone to provide nerve blocks lasting ~1 wk from a single injection. This formulation is easily injected percutaneously. Animals undergoing spared nerve injury (SNI) developed mechanical allodynia in 1 wk; nerve blockade with a single dose of SDLs (duration of block 6.9 ± 1.2 d) delayed the onset of allodynia by 2 d. Treatment with three sequential SDL injections resulting in a nerve block duration of 18.1 ± 3.4 d delayed the onset of allodynia by 1 mo. This very prolonged blockade decreased activation of astrocytes in the lumbar dorsal horn of the spinal cord due to SNI. Changes in expression of injury-related genes due to SNI in the dorsal root ganglia were not affected by SDLs. These findings suggest that formulations of this kind, which could be easy to apply clinically, can mitigate the development of neuropathic pain.

Synthetic ligand-coated magnetic nanoparticles for microfluidic bacterial separation from blood.

Bacterial sepsis is a serious clinical condition that can lead to multiple organ dysfunction and death despite timely treatment with antibiotics and fluid resuscitation. We have developed an approach to clearing bacteria and endotoxin from the bloodstream, using magnetic nanoparticles (MNPs) modified with bis-Zn-DPA, a synthetic ligand that binds to both Gram-positive and Gram-negative bacteria. Magnetic microfluidic devices were used to remove MNPs bound to Escherichia coli , a Gram-negative bacterium commonly implicated in bacterial sepsis, from bovine whole blood at flows as high as 60 mL/h, resulting in almost 100% clearance. Such devices could be adapted to clear bacteria from septicemic patients.

Macroporous nanowire nanoelectronic scaffolds for synthetic tissues.

The development of three-dimensional (3D) synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot electrically probe the physicochemical and biological microenvironments throughout their 3D and macroporous interior, although this capability could have a marked impact in both electronics and biomaterials. Here, we address this challenge using macroporous, flexible and free-standing nanowire nanoelectronic scaffolds (nanoES), and their hybrids with synthetic or natural biomaterials. 3D macroporous nanoES mimic the structure of natural tissue scaffolds, and they were formed by self-organization of coplanar reticular networks with built-in strain and by manipulation of 2D mesh matrices. NanoES exhibited robust electronic properties and have been used alone or combined with other biomaterials as biocompatible extracellular scaffolds for 3D culture of neurons, cardiomyocytes and smooth muscle cells. Furthermore, we show the integrated sensory capability of the nanoES by real-time monitoring of the local electrical activity within 3D nanoES/cardiomyocyte constructs, the response of 3D-nanoES-based neural and cardiac tissue models to drugs, and distinct pH changes inside and outside tubular vascular smooth muscle constructs.

Nanocomposite gold-silk nanofibers.

Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNF(seed)). Following gold reduction, there was a 2-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFs(Au)). The Young's modulus of the SNFs(Au) was almost 70% higher than that of SNFs. SNFs(Au) were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNF(Au) had a more than 2-fold larger cell area compared to the cells cultured on bare SNFs; SNF(Au) also increased cell size. This approach may be used to alter the cell-material interface in tissue engineering and other applications.

Nanoparticles targeting the infarcted heart.

We report a nanoparticulate system capable of targeting the heart after myocardial infarction (MI). Targeting is based on overexpression of angiotensin II type 1 (AT1) receptor in the infarcted heart. Liposomes 142 nm in diameter were conjugated with a ligand specific to AT1. The nanoparticles were able to specifically target cardiac cells in vitro, and in the infarcted heart after intravenous injection in vivo. This system may be useful for delivering therapeutic agents specifically to the infarcted heart.

Fabrication of nanomolded Nafion thin films with tunable mechanical and electrical properties using thermal evaporation-induced capillary force lithography.

In this paper, we report a simple and facile method to fabricate nanomolded Nafion thin films with tunable mechanical, and electrical properties. To achieve this, we combine a novel thermal evaporation-induced capillary force lithography method with swelling process to obtain enhanced pattern fidelity in nanomolded Nafion films. We demonstrate that structural fidelity and mechanical properties of patterned Nafion thin films can be modulated by changing fabrication parameters such as swelling time, Nafion polymer concentration, and curing temperature. Interestingly, we also find that impedance properties of nanomolded Nafion thin films are associated with the Nafion polymer concentration and curing temperature. In particular, 20% Nafion thin films exhibit greater impedance stability and lower impedance values than 5% Nafion thin films at lower frequencies. Moreover, curing temperature-specific impedance changes are observed. These results suggest that capillary lithography can be used to fabricate Nafion nanostructures with high pattern fidelity capable of modifying mechanical and electrical properties of Nafion thin films.

3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix.

3D bioprinting is a powerful technique for engineering tissues used to study cell behavior and tissue properties in vitro. With the right formulation and printing parameters, bioinks can provide native biological and mechanical cues while allowing for versatile 3D structures that recapitulate tissue-level organization. Bio-based materials that support cellular adhesion, differentiation, and proliferation - including gelatin, collagen, hyaluronic acid, and alginate - have been successfully used as bioinks. In particular, decellularized extracellular matrix (dECM) has become a promising material with the unique ability to maintain both biochemical and topographical micro-environments of native tissues. However, dECM has shown technical limitations for 3D printing (3DP) applications posed by its intrinsically low mechanical stability. Herein, we report hydrogel bioinks composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). The Laponite facilitated extrusion-based 3DP, while PEG-DA enabled photo-polymerization after printing. Improving upon previously reported bioinks derived from dECM, our bioinks combine extrudability, shape fidelity, rapid cross-linking, and cytocompatibility in a single formulation (> 97% viability of encapsulated human cardiac fibroblasts and > 94% viability of human induced pluripotent stem cell derived cardiomyocytes after 7 days). The compressive modulus of the cured hydrogel bioinks was tunable from 13.4-89 kPa by changing the concentration of PEG-DA in the bioink formulation. Importantly, this span of mechanical stiffness encompasses ranges of tissue stiffness from healthy (compressive modulus ~5-15 kPa) to fibrotic (compressive modulus ~30-100 kPa) cardiac tissue states. The printed constructs demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue.

Tunable electroconductive decellularized extracellular matrix hydrogels for engineering human cardiac microphysiological systems.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) offer tremendous potential when used to engineer human tissues for drug screening and disease modeling; however, phenotypic immaturity reduces assay reliability when translating in vitro results to clinical studies. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cell and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces and had increased expression of genes that regulate contractile function. Improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. dECM-rGO hydrogels could also be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced electrophysiological function of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Hybrid gold/DNA nanowire circuit with sub-10 nm nanostructure arrays.

We report on a simple and efficient method for the selective positioning of Au/DNA hybrid nanocircuits using a sequential combination of electron-beam lithography (EBL), plasma ashing, and a molecular patterning process. The nanostructures produced by the EBL and ashing process could be uniformly formed over a 12.6 in2 substrate with sub-10 nm patterning with good pattern fidelity. In addition, DNA molecules were immobilized on the selectively nanopatterned regions by alternating surface coating procedures of 3-(aminopropyl)triethoxysilane (APS) and diamond like carbon (DLC), followed by deposition of DNA molecules into a well-defined single DNA nanowire. These single DNA nanowires were used not only for fabricating Au/DNA hybrid nanowires by the conjugation of Au nanoparticles with DNA, but also for the formation of Au/DNA hybrid nanocircuits. These nanocircuits prepared from Au/DNA hybrid nanowires demonstrate conductivities of up to 4.3 × 105 S/m in stable electrical performance. This selective and precise positioning method capable of controlling the size of nanostructures may find application in making sub-10 nm DNA wires and metal/DNA hybrid nanocircuits.

Conductive Silk-Polypyrrole Composite Scaffolds with Bioinspired Nanotopographic Cues for Cardiac Tissue Engineering.

We report on the development of bioinspired cardiac scaffolds made from electroconductive acid-modified silk fibroin-poly(pyrrole) (AMSF+PPy) substrates patterned with nanoscale ridges and grooves reminiscent of native myocardial extracellular matrix (ECM) topography to enhance the structural and functional properties of cultured human pluripotent stem cells (hPSC)-derived cardiomyocytes. Nanopattern fidelity was maintained throughout the fabrication and functionalization processes, and no loss in conductive behavior occurred due to the presence of the nanotopographical features. AMSF+PPy substrates were biocompatible and stable, maintaining high cell viability over a 21-day culture period while displaying no signs of PPy delamination. The presence of anisotropic topographical cues led to increased cellular organization and sarcomere development, and electroconductive cues promoted a significant improvement in the expression and polarization of connexin 43 (Cx43), a critical regulator of cell-cell electrical coupling. The combination of biomimetic topography and electroconductivity also increased the expression of genes that encode key proteins involved in regulating the contractile and electrophysiological function of mature human cardiac tissue.

Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization.

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.

Electroconductive Nanopatterned Substrates for Enhanced Myogenic Differentiation and Maturation.

Electrically conductive materials provide a suitable platform for the in vitro study of excitable cells, such as skeletal muscle cells, due to their inherent conductivity and electroactivity. Here it is demonstrated that bioinspired electroconductive nanopatterned substrates enhance myogenic differentiation and maturation. The topographical cues from the highly aligned collagen bundles that form the extracellular matrix of skeletal muscle tissue are mimicked using nanopatterns created with capillary force lithography. Electron beam deposition is then utilized to conformally coat nanopatterned substrates with a thin layer of either gold or titanium to create electroconductive substrates with well-defined, large-area nanotopographical features. C2C12 cells, a myoblast cell line, are cultured for 7 d on substrates and the effects of topography and electrical conductivity on cellular morphology and myogenic differentiation are assessed. It is found that biomimetic nanotopography enhances the formation of aligned myotubes and the addition of an electroconductive coating promotes myogenic differentiation and maturation, as indicated by the upregulation of myogenic regulatory factors Myf5, MyoD, and myogenin (MyoG). These results suggest the suitability of electroconductive nanopatterned substrates as a biomimetic platform for the in vitro engineering of skeletal muscle tissue.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells.

Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700-1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies.

Combined effects of substrate topography and stiffness on endothelial cytokine and chemokine secretion.

Endothelial physiology is regulated not only by humoral factors, but also by mechanical factors such as fluid shear stress and the underlying cellular matrix microenvironment. The purpose of the present study was to examine the effects of matrix topographical cues on the endothelial secretion of cytokines/chemokines in vitro. Human endothelial cells were cultured on nanopatterned polymeric substrates with different ratios of ridge to groove widths (1:1, 1:2, and 1:5) and with different stiffnesses (6.7 MPa and 2.5 GPa) in the presence and absence of 1.0 ng/mL TNF-α. The levels of cytokines/chemokines secreted into the conditioned media were analyzed with a multiplexed bead-based sandwich immunoassay. Of the nanopatterns tested, the 1:1 and 1:2 type patterns were found to induce the greatest degree of endothelial cell elongation and directional alignment. The 1:2 type nanopatterns lowered the secretion of inflammatory cytokines such as IL-1ß, IL-3, and MCP-1, compared to unpatterned substrates. Additionally, of the two polymers tested, it was found that the stiffer substrate resulted in significant decreases in the secretion of IL-3 and MCP-1. These results suggest that substrates with specific extracellular nanotopographical cues or stiffnesses may provide anti-atherogenic effects like those seen with laminar shear stresses by suppressing the endothelial secretion of cytokines and chemokines involved in vascular inflammation and remodeling.

Thermoresponsive nanofabricated substratum for the engineering of three-dimensional tissues with layer-by-layer architectural control.

Current tissue engineering methods lack the ability to properly recreate scaffold-free, cell-dense tissues with physiological structures. Recent studies have shown that the use of nanoscale cues allows for precise control over large-area 2D tissue structures without restricting cell growth or cell density. In this study, we developed a simple and versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and the gel casting method for the fabrication of scaffold-free 3D tissues. Our TNFS allows for the structural control of aligned cell monolayers which can be spontaneously detached via a change in culture temperature. Utilizing our gel casting method, viable, aligned cell sheets can be transferred without loss of anisotropy or stacked with control over individual layer orientations. Transferred cell sheets and individual cell layers within multilayered tissues robustly retain structural anisotropy, allowing for the fabrication of scaffold-free, 3D tissues with hierarchical control of overall tissue structure.

Nanopatterned muscle cell patches for enhanced myogenesis and dystrophin expression in a mouse model of muscular dystrophy.

Skeletal muscle is a highly organized tissue in which the extracellular matrix (ECM) is composed of highly-aligned cables of collagen with nanoscale feature sizes, and provides structural and functional support to muscle fibers. As such, the transplantation of disorganized tissues or the direct injection of cells into muscles for regenerative therapy often results in suboptimal functional improvement due to a failure to integrate with native tissue properly. Here, we present a simple method in which biodegradable, biomimetic substrates with precisely controlled nanotopography were fabricated using solvent-assisted capillary force lithography (CFL) and were able to induce the proper development and differentiation of primary mononucleated cells to form mature muscle patches. Cells cultured on these nanopatterned substrates were highly-aligned and elongated, and formed more mature myotubes as evidenced by up-regulated expression of the myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). When transplanted into mdx mice models for Duchenne muscular dystrophy (DMD), the proposed muscle patches led to the formation of a significantly greater number of dystrophin-positive muscle fibers, indicating that dystrophin replacement and myogenesis is achievable in vivo with this approach. These results demonstrate the feasibility of utilizing biomimetic substrates not only as platforms for studying the influences of the ECM on skeletal muscle function and maturation, but also to create transplantable muscle cell patches for the treatment of chronic and acute muscle diseases or injuries.

Synergistic effects of matrix nanotopography and stiffness on vascular smooth muscle cell function.

Vascular smooth muscle cells (vSMCs) retain the ability to undergo modulation in their phenotypic continuum, ranging from a mature contractile state to a proliferative, secretory state. vSMC differentiation is modulated by a complex array of microenvironmental cues, which include the biochemical milieu of the cells and the architecture and stiffness of the extracellular matrix. In this study, we demonstrate that by using UV-assisted capillary force lithography (CFL) to engineer a polyurethane substratum of defined nanotopography and stiffness, we can facilitate the differentiation of cultured vSMCs, reduce their inflammatory signature, and potentially promote the optimal functioning of the vSMC contractile and cytoskeletal machinery. Specifically, we found that the combination of medial tissue-like stiffness (11 MPa) and anisotropic nanotopography (ridge width_groove width_ridge height of 800_800_600 nm) resulted in significant upregulation of calponin, desmin, and smoothelin, in addition to the downregulation of intercellular adhesion molecule-1, tissue factor, interleukin-6, and monocyte chemoattractant protein-1. Further, our results allude to the mechanistic role of the RhoA/ROCK pathway and caveolin-1 in altered cellular mechanotransduction pathways via differential matrix nanotopography and stiffness. Notably, the nanopatterning of the stiffer substrata (1.1 GPa) resulted in the significant upregulation of RhoA, ROCK1, and ROCK2. This indicates that nanopatterning an 800_800_600 nm pattern on a stiff substratum may trigger the mechanical plasticity of vSMCs resulting in a hypercontractile vSMC phenotype, as observed in diabetes or hypertension. Given that matrix stiffness is an independent risk factor for cardiovascular disease and that CFL can create different matrix nanotopographic patterns with high pattern fidelity, we are poised to create a combinatorial library of arterial test beds, whether they are healthy, diseased, injured, or aged. Such high-throughput testing environments will pave the way for the evolution of the next generation of vascular scaffolds that can effectively crosstalk with the scaffold microenvironment and result in improved clinical outcomes.

Microfluidics-assisted in vitro drug screening and carrier production.

Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development.