Graphene oxide and electroactive reduced graphene oxide-based composite fibrous scaffolds for engineering excitable nerve tissue.

This study systematically investigates the role of graphene oxide (GO) and reduced GO (rGO)/silk-based composite micro/nano-fibrous scaffolds in regulating neuronal cell behavior in vitro, given the limited comparative studies on the effects of graphene family materials on nerve regeneration. Fibrous scaffolds can mimic the architecture of the native extracellular matrix and are potential candidates for tissue engineering peripheral nerves. Silk/GO micro/nano-fibrous scaffolds were electrospun with GO loadings 1 to 10 wt.%, and optionally post-reduced in situ to explore a family of electrically conductive non-woven silk/rGO scaffolds. Conductivities up to 4 × 10-5 S cm-1 were recorded in the dry state, which increased up to 3 × 10-4 S cm-1 after hydration. Neuronoma NG108-15 cells adhered and were viable on all substrates. Enhanced metabolic activity and proliferation were observed on the GO-containing scaffolds, and these cell responses were further promoted for electroactive silk/rGO. Neurite extensions up to 100 µm were achieved by day 5, with maximum outgrowth up to ~250 µm on some of the conductive substrates. These electroactive composite fibrous scaffolds exhibit potential to enhance the neuronal cell response and could be versatile supportive substrates for neural tissue engineering applications.

Modulation of Neuronal Cell Affinity on PEDOT-PSS Nonwoven Silk Scaffolds for Neural Tissue Engineering.

Peripheral nerve injury is a common consequence of trauma with low regenerative potential. Electroconductive scaffolds can provide appropriate cell growth microenvironments and synergistic cell guidance cues for nerve tissue engineering. In the present study, electrically conductive scaffolds were prepared by conjugating poly (3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT-PSS) or dimethyl sulfoxide (DMSO)-treated PEDOT-PSS on electrospun silk scaffolds. Conductance could be tuned by the coating concentration and was further boosted by DMSO treatment. Analogue NG108-15 neuronal cells were cultured on the scaffolds to evaluate neuronal cell growth, proliferation, and differentiation. Cellular viability was maintained on all scaffold groups while showing comparatively better metabolic activity and proliferation than neat silk. DMSO-treated PEDOT-PSS functionalized scaffolds partially outperformed their PEDOT-PSS counterparts. Differentiation assessments suggested that these PEDOT-PSS assembled silk scaffolds could support neurite sprouting, indicating that they show promise to be used as a future platform to restore electrochemical coupling at the site of injury and preserve normal nerve function.

Electroresponsive Silk-Based Biohybrid Composites for Electrochemically Controlled Growth Factor Delivery.

Stimuli-responsive materials are very attractive candidates for on-demand drug delivery applications. Precise control over therapeutic agents in a local area is particularly enticing to regulate the biological repair process and promote tissue regeneration. Macromolecular therapeutics are difficult to embed for delivery, and achieving controlled release over long-term periods, which is required for tissue repair and regeneration, is challenging. Biohybrid composites incorporating natural biopolymers and electroconductive/active moieties are emerging as functional materials to be used as coatings, implants or scaffolds in regenerative medicine. Here, we report the development of electroresponsive biohybrid composites based on Bombyx mori silkworm fibroin and reduced graphene oxide that are electrostatically loaded with a high-molecular-weight therapeutic (i.e., 26 kDa nerve growth factor-ß (NGF-ß)). NGF-ß-loaded composite films were shown to control the release of the drug over a 10-day period in a pulsatile fashion upon the on/off application of an electrical stimulus. The results shown here pave the way for personalized and biologically responsive scaffolds, coatings and implantable devices to be used in neural tissue engineering applications, and could be translated to other electrically sensitive tissues as well.

Human airway-like multilayered tissue on 3D-TIPS printed thermoresponsive elastomer/collagen hybrid scaffolds.

Developing a biologically representative complex tissue of the respiratory airway is challenging, however, beneficial for treatment of respiratory diseases, a common medical condition representing a leading cause of death in the world. This in vitro study reports a successful development of synthetic human tracheobronchial epithelium based on interpenetrated hierarchical networks composed of a reversely 3D printed porous structure of a thermoresponsive stiffness-softening elastomer nanohybrid impregnated with collagen nanofibrous hydrogel. Human bronchial epithelial cells (hBEpiCs) were able to attach and grow into an epithelial monolayer on the hybrid scaffolds co-cultured with either human bronchial fibroblasts (hBFs) or human bone-marrow derived mesenchymal stem cells (hBM-MSCs), with substantial enhancement of mucin expression, ciliation, well-constructed intercellular tight junctions and adherens junctions. The multi-layered co-culture 3D scaffolds consisting of a top monolayer of differentiated epithelium, with either hBFs or hBM-MSCs proliferating within the hyperelastic nanohybrid scaffold underneath, created a tissue analogue of the upper respiratory tract, validating these 3D printed guided scaffolds as a platform to support co-culture and cellular organization. In particular, hBM-MSCs in the co-culture system promoted an overall matured physiological tissue analogue of the respiratory system, a promising synthetic tissue for drug discovery, tracheal repair and reconstruction. STATEMENT OF SIGNIFICANCE: Respiratory diseases are a common medical condition and represent a leading cause of death in the world. However, the epithelium is one of the most challenging tissues to culture in vitro, and suitable tracheobronchial models, physiologically representative of the innate airway, remain largely elusive. This study presents, for the first time, a systematic approach for the development of functional multilayered epithelial synthetic tissue in vitro via co-culture on a 3D-printed thermoresponsive elastomer interpenetrated with a collagen hydrogel network. The viscoelastic nature of the scaffold with stiffness softening at body temperature provide a promising matrix for soft tissue engineering. The results presented here provide new insights about the epithelium at different surfaces and interfaces of co-culture, and pave the way to offer a customizable reproducible technology to generate physiologically relevant 3D biomimetic systems to advance our understanding of airway tissue regeneration.

Hierarchically Porous Silk/Activated-Carbon Composite Fibres for Adsorption and Repellence of Volatile Organic Compounds.

Fabrics comprised of porous fibres could provide effective passive protection against chemical and biological (CB) threats whilst maintaining high air permeability (breathability). Here, we fabricate hierarchically porous fibres consisting of regenerated silk fibroin (RSF) and activated-carbon (AC) prepared through two fibre spinning techniques in combination with ice-templating-namely cryogenic solution blow spinning (Cryo-SBS) and cryogenic wet-spinning (Cryo-WS). The Cryo-WS RSF fibres had exceptionally small macropores (as low as 0.1 µm) and high specific surface areas (SSAs) of up to 79 m2·g-1. The incorporation of AC could further increase the SSA to 210 m2·g-1 (25 wt.% loading) whilst also increasing adsorption capacity for volatile organic compounds (VOCs).

Thermoresponsive Stiffness Softening of Hierarchically Porous Nanohybrid Membranes Promotes Niches for Mesenchymal Stem Cell Differentiation.

Despite the attention given to the development of novel responsive implants for regenerative medicine applications, the lack of integration with the surrounding tissues and the mismatch with the dynamic mechanobiological nature of native soft tissues remain in the current products. Hierarchical porous membranes based on a poly (urea-urethane) (PUU) nanohybrid have been fabricated by thermally induced phase separation (TIPS) of the polymer solution at different temperatures. Thermoresponsive stiffness softening of the membranes through phase transition from the semicrystalline phase to rubber phase and reverse self-assembly of the quasi-random nanophase structure is characterized at body temperature near the melting point of the crystalline domains of soft segments. The effects of the porous structure and stiffness softening on proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBM-MSCs) are investigated. The results of immunohistochemistry, histological, ELISA, and qPCR demonstrate that hBM-MSCs maintain their lineage commitment during stiffness relaxation; chondrogenic differentiation is favored on the soft and porous scaffold, while osteogenic differentiation is more prominent on the initial stiff one. Stiffness relaxation stimulates more osteogenic activity than chondrogenesis, the latter being more influenced by the synergetic coupling effect of softness and porosity.

Development data associated with effects of stiffness softening of 3D-TIPS elastomer nanohybrid scaffolds on tissue ingrowth, vascularization and inflammation in vivo.

This DiB article contains data related to the research article entitled "Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS" (Wu et al., 2018). Thermoresponsive poly (urea-urethane) nanohybrid elastomer (PUU-POSS) scaffolds were implanted in rats for up to 3 months. The porous structure and tensile mechanical properties of the scaffolds are listed and compared before and after in vitro and in vivo tests. The details of the histological analysis of the explants with different initial stiffness and porous structures at various time points are presented. The images and data presented support the conclusion about the coupled effects of stiffness softening and the hierarchical porous structure modulating tissue ingrowth, vascularization and macrophage polarization in the article (Wu et al., 2018).

Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS.

Increasing evidence suggests the contribution of the dynamic mechanical properties of the extracellular matrix (ECM) to regulate tissue remodeling and regeneration. Following our recent study on a family of thermoresponsive 'stiffness memory' elastomeric nanohybrid scaffolds manufactured via an indirect 3D printing guided thermally-induced phase separation process (3D-TIPS), this work reports in vitro and in vivo cellular responses towards these scaffolds with different initial stiffness and hierarchically interconnected porous structure. The viability of mouse embryonic dermal fibroblasts in vitro and the tissue responses during the stiffness softening of the scaffolds subcutaneously implanted in rats for three months were evaluated by immunohistochemistry and histology. Scaffolds with a higher initial stiffness and a hierarchical porous structure outperformed softer ones, providing initial mechanical support to cells and surrounding tissues before promoting cell and tissue growth during stiffness softening. Vascularization was guided throughout the digitally printed interconnected networks. All scaffolds exhibited polarization of the macrophage response from a macrophage phenotype type I (M1) towards a macrophage phenotype type II (M2) and down-regulation of the T-cell proliferative response with increasing implantation time; however, scaffolds with a more pronounced thermo-responsive stiffness memory mechanism exerted higher inflammo-informed effects. These results pave the way for personalized and biologically responsive soft tissue implants and implantable device with better mechanical matches, angiogenesis and tissue integration. Statement of Significance This work reports cellular responses to a family of 3D-TIPS thermoresponsive nanohybrid elastomer scaffolds with different stiffness softening both in vitro and in vivo rat models. The results, for the first time, have revealed the effects of initial stiffness and dynamic stiffness softening of the scaffolds on tissue integration, vascularization and inflammo-responses, without coupling chemical crosslinking processes. The 3D printed, hierarchically interconnected porous structures guide the growth of myofibroblasts, collagen fibers and blood vessels in real 3D scales. In vivo study on those unique smart elastomer scaffolds will help pave the way for personalized and biologically responsive soft tissue implants and implantable devices with better mechanical matches, angiogenesis and tissue integration.

Porous, Aligned, and Biomimetic Fibers of Regenerated Silk Fibroin Produced by Solution Blow Spinning.

Solution blow spinning (SBS) has emerged as a rapid and scalable technique for the production of polymeric and ceramic materials into micro-/nanofibers. Here, SBS was employed to produce submicrometer fibers of regenerated silk fibroin (RSF) from Bombyx mori (silkworm) cocoons based on formic acid or aqueous systems. Spinning in the presence of vapor permitted the production of fibers from aqueous solutions, and high alignment could be obtained by modifying the SBS setup to give a concentrated channeled airflow. The combination of SBS and a thermally induced phase separation technique (TIPS) resulted in the production of macro-/microporous fibers with 3D interconnected pores. Furthermore, a coaxial SBS system enabled a pH gradient and kosmotropic salts to be applied at the point of fiber formation, mimicking some of the aspects of the natural spinning process, fostering fiber formation by self-assembly of the spinning dope. This scalable and fast production of various types of silk-based fibrous scaffolds could be suitable for a myriad of biomedical applications.

Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells.

The cellular microenvironment is dynamic, remodeling tissues lifelong. The biomechanical properties of the extracellular matrix (ECM) influence the function and differentiation of stem cells. While conventional artificial matrices or scaffolds for tissue engineering are primarily static models presenting well-defined stiffness, they lack the responsive changes required in dynamic physiological settings. Engineering scaffolds with varying elastic moduli is possible, but often lead to stiffening and chemical crosslinking of the molecular structure with limited control over the scaffold architecture. A family of indirectly 3D printed elastomeric nanohybrid scaffolds with thermoresponsive mechanical properties that soften by reverse self-assembling at body temperature have been developed recently. The initial stiffness and subsequent stiffness relaxation of the scaffolds regulated proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages over 4 weeks, as measured by immunohistochemistry, histology, ELISA and qPCR. hBM-MSCs showed enhanced chondrogenic differentiation on softer scaffolds and osteogenic differentiation on stiffer ones, with similar relative expression to that of human femoral head tissue. Overall, stiffness relaxation favored osteogenic activity over chondrogenesis in vitro.

Data of a stiffness softening mechanism effect on proliferation and differentiation of a human bone marrow derived mesenchymal stem cell line towards the chondrogenic and osteogenic lineages.

This article contains data related to the research article entitled "Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells" [1] (Wu et al., 2018). Cells respond to the local microenvironment in a context dependent fashion and a continuous challenge is to provide a living construct that can adapt to the viscoelasticity changes of surrounding tissues. Several materials are attractive candidates to be used in tissue engineering, but conventional manufactured scaffolds are primarily static models with well-defined and stable stiffness that lack the dynamic biological nature required to undergo changes in substrate elasticity decisive in several cellular processes key during tissue development and wound healing. A family of poly (urea-urethane) (PUU) elastomeric nanohybrid scaffolds (PUU-POSS) with thermoresponsive mechanical properties that soften by reverse self-assembling at body temperature had been developed through a 3D thermal induced phase transition process (3D-TIPS) at various thermal conditions: cryo-coagulation (CC), cryo-coagulation and heating (CC + H) and room temperature coagulation and heating (RTC + H). The stiffness relaxation and stiffness softening of these scaffolds suggest regulatory effects in proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages.

Bioactive Silk-Based Nerve Guidance Conduits for Augmenting Peripheral Nerve Repair.

Repair of peripheral nerve injuries depends upon complex biology stemming from the manifold and challenging injury-healing processes of the peripheral nervous system. While surgical treatment options are available, they tend to be characterized by poor clinical outcomes for the injured patients. This is particularly apparent in the clinical management of a nerve gap whereby nerve autograft remains the best clinical option despite numerous limitations; in addition, effective repair becomes progressively more difficult with larger gaps. Nerve conduit strategies based on tissue engineering approaches and the use of silk as scaffolding material have attracted much attention in recent years to overcome these limitations and meet the clinical demand of large gap nerve repair. This review examines the scientific advances made with silk-based conduits for peripheral nerve repair. The focus is on enhancing bioactivity of the conduits in terms of physical guidance cues, inner wall and lumen modification, and imbuing novel conductive functionalities.

Electroactive biomaterials: Vehicles for controlled delivery of therapeutic agents for drug delivery and tissue regeneration.

Electrical stimulation for delivery of biochemical agents such as genes, proteins and RNA molecules amongst others, holds great potential for controlled therapeutic delivery and in promoting tissue regeneration. Electroactive biomaterials have the capability of delivering these agents in a localized, controlled, responsive and efficient manner. These systems have also been combined for the delivery of both physical and biochemical cues and can be programmed to achieve enhanced effects on healing by establishing control over the microenvironment. This review focuses on current state-of-the-art research in electroactive-based materials towards the delivery of drugs and other therapeutic signalling agents for wound care treatment. Future directions and current challenges for developing effective electroactive approach based therapies for wound care are discussed.