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Summary Stem cell transplants offer significant hope for brain repair following ischemic damage. Pre-clinical work suggests that therapeutic mechanisms may be multi-faceted, incorporating bone-fide circuit reconstruction by transplanted neurons, but also protection/regeneration of host circuitry. Here, we engineered hydrogel scaffolds to form “bio-bridges” within the necrotic lesion cavity, providing physical and trophic support to transplanted human embryonic stem cell-derived cortical progenitors, as well as residual host neurons. Scaffolds were fabricated by the self-assembly of peptides for a laminin-derived epitope (IKVAV), thereby mimicking the brain’s major extracellular protein. Following focal ischemia in rats, scaffold-supported cell transplants induced progressive motor improvements over 9 months, compared to cell- or scaffold-only implants. These grafts were larger, exhibited greater neuronal differentiation, and showed enhanced electrophysiological properties reflective of mature, integrated neurons. Varying graft timing post-injury enabled us to attribute repair to both neuroprotection and circuit replacement. These findings highlight strategies to improve the efficiency of stem cell grafts for brain repair. [ABSTRACT FROM AUTHOR]

The local inflammatory environment of the cell promotes the growth of epithelial cancers. Therefore, controlling inflammation locally using a material in a sustained, non-steroidal fashion can effectively kill malignant cells without significant damage to surrounding healthy cells. A promising class of materials for such applications is the nanostructured scaffolds formed by epitope presenting minimalist self-assembled peptides; these are bioactive on a cellular length scale, while presenting as an easily handled hydrogel. Here, we show that the assembly process can distribute an anti-inflammatory polysaccharide, fucoidan, localized to the nanofibers within the scaffold to create a biomaterial for cancer therapy. We show that it supports healthy cells, while inducing apoptosis in cancerous epithelial cells, as demonstrated by the significant down-regulation of gene and protein expression pathways associated with epithelial cancer progression. Our findings highlight an innovative material approach with potential applications in local epithelial cancer immunotherapy and drug delivery. [ABSTRACT FROM AUTHOR]

The nanofibrillar structures that underpin self-assembling peptide (SAP) hydrogels offer great potential for the development of finely tuned cellular microenvironments suitable for tissue engineering. However, biofunctionalisation without disruption of the assembly remains a key issue. SAPS present the peptide sequence within their structure, and studies to date have typically focused on including a single biological motif, resulting in chemically and biologically homogenous scaffolds. This limits the utility of these systems, as they cannot effectively mimic the complexity of the multicomponent extracellular matrix (ECM). In this work, we demonstrate the first successful co-assembly of two biologically active SAPs to form a coassembled scaffold of distinct two-component nanofibrils, and demonstrate that this approach is more bioactive than either of the individual systems alone. Here, we use two bioinspired SAPs from two key ECM proteins: Fmoc-FRGDF containing the RGD sequence from fibronectin and Fmoc-DIKVAV containing the IKVAV sequence from laminin. Our results demonstrate that these SAPs are able to co-assemble to form stable hybrid nanofibres containing dual epitopes. Comparison of the co-assembled SAP system to the individual SAP hydrogels and to a mixed system (composed of the two hydrogels mixed together post-assembly) demonstrates its superior stable, transparent, shear-thinning hydrogels at biological pH, ideal characteristics for tissue engineering applications. Importantly, we show that only the coassembled hydrogel is able to induce in vitro multinucleate myotube formation with C2C12 cells. This work illustrates the importance of tissue engineering scaffold functionalisation and the need to develop increasingly advanced multicomponent systems for effective ECM mimicry. Statement of Significance Successful control of stem cell fate in tissue engineering applications requires the use of sophisticated scaffolds that deliver biological signals to guide growth and differentiation. The complexity of such processes necessitates the presentation of multiple signals in order to effectively mimic the native extracellular matrix (ECM). Here, we establish the use of two biofunctional, minimalist self-assembling peptides (SAPs) to construct the first co-assembled SAP scaffold. Our work characterises this construct, demonstrating that the physical, chemical, and biological properties of the peptides are maintained during the co-assembly process. Importantly, the coassembled system demonstrates superior biological performance relative to the individual SAPs, highlighting the importance of complex ECM mimicry. This work has important implications for future tissue engineering studies. [ABSTRACT FROM AUTHOR]

Abstract: Assessment of axonal infiltration and guidance within neural tissue engineering scaffolds, along with the characterisation of the inflammatory response, is critical in determining these scaffolds'' potential for facilitating neural repair. In this study, the extent of microglial and astrocytic response was measured following implantation of electrospun poly(ɛ-caprolactone) (PCL) scaffolds into the caudate putamen of the adult rat brain. The inflammation peaked at around 4 days (microglia) and 7 days (astrocytes) and subsided to homeostatic levels by 60 days. There was no evidence of microglial encapsulation and indeed neurites had infiltrated the implants, evidence of scaffold-neural integration. Whilst the inflammatory response was uninfluenced by the degree of PCL fibre alignment, the extent of neurite entry was. Large porosity, as was the case with the randomly orientated polymer fibres, enabled neurite infiltration and growth within the scaffold. However, neuronal processes could not penetrate scaffolds when fibres were partially aligned and instead, preferentially grew perpendicular to the direction of PCL fibre alignment at the implant-tissue interface i.e. perpendicular, not parallel, contact guidance was provided. This investigation shows that electrospun PCL fibres are compatible with brain tissue and provide preliminary insights regarding the influence of microglia and astrocytes in neural integration within such scaffolds. [Copyright &y& Elsevier]

Abstract: Cells respond to their environment in complex and sometimes poorly understood ways. Protein, peptide and synthetic peptidomimetic ligands may all be used to stimulate cells via receptor signaling, using interactions that are often highly specific. Polymer substrates that present these ligands provide a promising way to control cell development, both for applications in biotechnology and for fundamental studies of cell biology. Here we review a large range of techniques that have been employed to create and characterize ligand-functionalized substrates, with a particular focus on techniques that allow specific and consistent stimulation. [Copyright &y& Elsevier]

Abstract: In this work we show that sonication alone can be used to scission bulk electrospun membranes into short fibres. The mechanism of such scission events is bubble cavitation stimulated by the ultrasonic probe, followed by bubble implosion. The tendency of polymer nanofibres to undergo failure by such a scission process appears to primarily depend on the ductility of the polymer, with brittle, electrospun polymer membranes such as poly(styrene) and poly(methyl methacrylate) readily producing short fibres of approximately 10 μm length. More ductile polymers such as poly(l-lactide) or poly(acrylonitrile) require additional processing after electrospinning and before sonication, to make them conducive to such sonication-based scission. Both the initial diameter of the fibres and the degree of nanofibre alignment of the electrospun membrane influence the final length of the resultant short fibres. It was found that the chemical and physical properties of the short nanofibres were unaltered by the sonication process. We are thus able to demonstrate that sonication is a promising method to produce significant quantities of short fibres of nanometre diameter and microns in length. [Copyright &y& Elsevier]

Abstract: With the brain''s limited capacity for repair, new and innovative approaches are required to promote regeneration. While neural transplantation for a number of neural disease/injuries have been demonstrated, major limitations in the field include poor cell survival and integration. This, in part, is due to the non-conducive environment of the adult brain, failing to provide adequate chemical and physical support for new neurons. Here we examine the capacity of fibrous poly ε-caprolactone (PCL) scaffolds, biofunctionalised with immobilised glial cell-derived neurotrophic factor (GDNF), to influence primary cortical neural stem cells/progenitors in vitro and enhance integration of these cells following transplantation into the brain parenchyma. Immobilisation of GDNF was confirmed prior to in vitro culturing and at 28 days after implantation into the brain, demonstrating long-term delivery of the protein. In vitro, we demonstrate that PCL with immobilised GDNF (iGDNF) significantly enhances cell viability and neural stem cell/progenitor proliferation compared to conventional 2-dimensional cultureware. Upon implantation, PCL scaffolds including iGDNF enhanced the survival, proliferation, migration, and neurite growth of transplanted cortical cells, whilst suppressing inflammatory reactive astroglia. [Copyright &y& Elsevier]

Biomedical implants have been widely used in various orthopedic treatments, including total hip arthroplasty, joint arthrodesis, fracture fixation, non-union, dental repair, etc. The modern research and development of orthopedic implants have gradually shifted from traditional mechanical support to a bioactive graft in order to endow them with better osteoinduction and osteoconduction. Inspired by structural and mechanical properties of natural bone, this review provides a panorama of current biological surface modifications for facilitating the interaction between medical implants and bone tissue and gives a future outlook for fabricating the next-generation multifunctional and smart implants by systematically biomimicking the physiological processes involved in formation and functioning of bones. [ABSTRACT FROM AUTHOR]

With the brain's limited capacity for repair there is a need for new and innovative therapies to promote regeneration. Stem/progenitor cell transplantation has received increasing attention, and whilst clinical trials demonstrating functional integration exist, inherent variability between patients has hindered development of this therapy. Variable outcomes have largely been attributed to poor survival and insufficient reinnervation of target tissues due in part to the suboptimal host environment. Here we examined whether improving the physical properties of the host milieu, by way of bioengineered scaffolds, may enhance engraftment. We developed a composite scaffold, incorporating electrospun poly( l -lactic acid) short nanofibers embedded within a thermo-responsive xyloglucan hydrogel, which could be easily injected into the injured brain. Furthermore, to improve the trophic properties of the host brain, glial derived neurotrophic factor (GDNF), a protein known to promote cell survival and axonal growth, was blended into and/or covalently attached onto the composite scaffolds to provide controlled delivery. In vitro we confirmed the ability of the scaffolds to support ventral midbrain (VM) dopamine progenitors, and provide sustained delivery of GDNF – capable of eliciting effects on cell survival and dopaminergic axon growth. In Parkinsonian mice, we show that these composite scaffolds, whilst having no deleterious impact on the host immune response, enhanced the survival of VM grafts and reinnervation of the striatum, an effect that was augmented through the scaffold delivery of GDNF. Taken together, these functionalized composite scaffolds provide a means to significantly improve the milieu of the injured brain, enabling enhanced survival and integration of grafted neurons. [ABSTRACT FROM AUTHOR]

Macrophages play a key role in tissue regeneration following peripheral nerve injury by preparing the surrounding parenchyma for regeneration, however, they can be damaging if the response is excessive. Interleukin 10 (IL-10) is a cytokine that promotes macrophages toward an anti-inflammatory/wound healing state (M2 phenotype). The bioactive half-life of IL-10 is dependent on the cellular microenvironment and ranges from minutes to hours in vivo. Our objective was to extend the in vivo bioavailability and bioactivity of IL-10 by attaching the protein onto nanofibrous scaffolds and demonstrating increased expression levels of M2 macrophages when placed around healthy intact peripheral nerves. IL-10 was adsorbed and covalently bound to electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffolds. In vivo bioavailability and bioactivity of IL-10 was confirmed by wrapping IL-10 conjugated nanofibres around the sciatic nerves of Wistar rats and quantifying M2 macrophages immunohistochemically double labelled with ED1 and either arginase 1 or CD206. IL-10 remained immobilised to PCL scaffolds for more than 120 days when stored in phosphate buffered saline at room temperature and for up to 14 days when implanted around the sciatic nerve. IL-10 conjugated nanofibres successfully induced macrophage polarisation towards the M2 activated state within the scaffold material as well as the adjacent tissue surrounding the nerve. PCL biofunctionalised nanofibres are useful for manipulating the cellular microenvironment. Materials such as these could potentially lead to new therapeutic strategies for nervous tissue injuries as well as provide novel investigative tools for biological research. [ABSTRACT FROM AUTHOR]

Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neuritemorphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules. [ABSTRACT FROM AUTHOR]

Highlights: [•] Substrate–plasma polymer film interfaces were exposed allowing the underside of the films to be examined and compared with the top side. [•] Both sides of the plasma polymer films were examined using X-ray photoelectron spectroscopy (XPS) and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. [•] High input power during polymerization produced plasma polymer films with chemical gradients, which contain more ether units at the initial formation stage compared with the top layer. [•] Film chemistry was dependent on the type of substrate used for the plasma polymerization deposition. [Copyright &y& Elsevier]

Non-oxidized cellulose nanofibers (CNFs) were produced using ball-milling of cellulose. The optimized nanofibers with high aspect ratio and specific surface area induced robust clotting in thromboelastomtry by increasing fibrin formation and trapping of platelets. [Display omitted] • Non-oxidized cellulose nanofibers made by ball-milling induce effective clotting. • The promotion of hemostasis is dependent on the size and morphology of nanofibers. • Nanofibers with a higher specific surface area enhance plasma coagulation. • Nanofibers with a higher aspect ratio entrap and activate platelets. Hemorrhage remains a significant cause of morbidity and mortality following trauma and during complex surgeries. A variety of nanomaterials, including oxidized cellulose nanofibers (OCNFs), have been studied to overcome the disadvantages of current commercial topical hemostats. However, the relationship between nano-structural characteristics and hemostatic efficacy of non-oxidized cellulose nanofibers (CNFs) has not been elucidated. Herein, we present the first report of the correlation between structure and hemostatic performance of CNFs. In vitro thromboelastometry studies on CNFs, synthesized by ball-milling, showed that there is an optimum balance point between the aspect ratio (AR) and specific surface area (SSA) of nanofibers in terms of their maximum contribution to platelet function and plasma coagulation. The optimized CNFs with high SSA (17 m2/g) and a high AR (166) shortened normal whole blood clotting time by 68 %, outperforming cellulose-based hemostats. Additionally, CNFs reduced clotting time in platelet-deficient blood (by 80 %) and heparinized blood (by 54 %). [ABSTRACT FROM AUTHOR]