Novel insights on genetics and epigenetics as clinical targets for paediatric astrocytoma.

Paediatric and adult astrocytomas are notably different, where clinical treatments used for adults are not as effective on children with the same form of cancer and these treatments lead to adverse long-term health concerns. Integrative omics-based studies have shown the pathology and fundamental molecular characteristics differ significantly and cannot be extrapolated from the more widely studied adult disease. Recent clinical advances in our understanding of paediatric astrocytomas, with the aid of next-generation sequencing and epigenome-wide profiling, have led to the identification of key canonical mutations that vary based on the tumour location and age of onset. These driver mutations, in particular the identification of the recurrent histone H3 mutations in high-grade tumours, have confirmed the important role epigenetic dysregulations play in cancer progression. This review summarises the current updates of the classification, epidemiology, pathogenesis and clinical management of paediatric astrocytoma based on their grades and the ongoing clinical trials. It also provides novel insights on genetic and epigenetic alterations as diagnostic biomarkers, highlighting the potential of targeting these pathways as therapeutics for this devastating childhood cancer.

Neuronal Replenishment via Hydrogel-Rationed Delivery of Reprogramming Factors.

The central nervous system's limited capacity for regeneration often leads to permanent neuronal loss following injury. Reprogramming resident reactive astrocytes into induced neurons at the site of injury is a promising strategy for neural repair, but challenges persist in stabilizing and accurately targeting viral vectors for transgene expression. In this study, we employed a bioinspired self-assembling peptide (SAP) hydrogel for the precise and controlled release of a hybrid adeno-associated virus (AAV) vector, AAVDJ, carrying the NeuroD1 neural reprogramming transgene. This method effectively mitigates the issues of high viral dosage at the target site, off-target delivery, and immunogenic reactions, enhancing the vector's targeting and reprogramming efficiency. In vitro, this vector successfully induced neuron formation, as confirmed by morphological, histochemical, and electrophysiological analyses. In vivo, SAP-mediated delivery of AAVDJ-NeuroD1 facilitated the trans-differentiation of reactive host astrocytes into induced neurons, concurrently reducing glial scarring. Our findings introduce a safe and effective method for treating central nervous system injuries, marking a significant advancement in regenerative neuroscience.

Calming the Nerves via the Immune Instructive Physiochemical Properties of Self-Assembling Peptide Hydrogels.

Current therapies for the devastating damage caused by traumatic brain injuries (TBI) are limited. This is in part due to poor drug efficacy to modulate neuroinflammation, angiogenesis and/or promoting neuroprotection and is the combined result of challenges in getting drugs across the blood brain barrier, in a targeted approach. The negative impact of the injured extracellular matrix (ECM) has been identified as a factor in restricting post-injury plasticity of residual neurons and is shown to reduce the functional integration of grafted cells. Therefore, new strategies are needed to manipulate the extracellular environment at the subacute phase to enhance brain regeneration. In this review, potential strategies are to be discussed for the treatment of TBI by using self-assembling peptide (SAP) hydrogels, fabricated via the rational design of supramolecular peptide scaffolds, as an artificial ECM which under the appropriate conditions yields a supramolecular hydrogel. Sequence selection of the peptides allows the tuning of these hydrogels' physical and biochemical properties such as charge, hydrophobicity, cell adhesiveness, stiffness, factor presentation, degradation profile and responsiveness to (external) stimuli. This review aims to facilitate the development of more intelligent biomaterials in the future to satisfy the parameters, requirements, and opportunities for the effective treatment of TBI.

Biofabrication of functional bone tissue: defining tissue-engineered scaffolds from nature.

Damage to bone leads to pain and loss of movement in the musculoskeletal system. Although bone can regenerate, sometimes it is damaged beyond its innate capacity. Research interest is increasingly turning to tissue engineering (TE) processes to provide a clinical solution for bone defects. Despite the increasing biomimicry of tissue-engineered scaffolds, significant gaps remain in creating the complex bone substitutes, which include the biochemical and physical conditions required to recapitulate bone cells' natural growth, differentiation and maturation. Combining advanced biomaterials with new additive manufacturing technologies allows the development of 3D tissue, capable of forming cell aggregates and organoids based on natural and stimulated cues. Here, we provide an overview of the structure and mechanical properties of natural bone, the role of bone cells, the remodelling process, cytokines and signalling pathways, causes of bone defects and typical treatments and new TE strategies. We highlight processes of selecting biomaterials, cells and growth factors. Finally, we discuss innovative tissue-engineered models that have physiological and anatomical relevance for cancer treatments, injectable stimuli gels, and other therapeutic drug delivery systems. We also review current challenges and prospects of bone TE. Overall, this review serves as guide to understand and develop better tissue-engineered bone designs.

Biomimetic triumvirate nanogel complexes via peptide-polysaccharide-polyphenol self-assembly.

Self-assembled peptide and polysaccharide nanogels are excellent candidates for bioactive delivery vectors. However, there are still significant challenges in the application of nanogels as delivery tools for bioactive elements. This study aims to deliver, and control the release of a hydrophobic bioactive flavonoid hesperidin. Using the self-assembling peptide (SAP) Fmoc-FRGDF, extracellular matrix mimicking nanofibrils were fabricated, which were decorated and bolstered with immunomodulatory polysaccharide strands of fucoidan and infused with hesperidin. The mechanical properties, secondary structure, and microscopic morphologies of the composite hydrogels were characterized using rheometer, FTIR, XRD, and TEM, etc. The encapsulation efficiency (EE) and release behavior of hesperidin were determined. Coassembly of the SAP with fucoidan improved the mechanical properties (from 9.54 Pa of Fmoc-FRGDF hydrogel to 7735 Pa of coassembly hydrogel at 6 mg/mL fucoidan concentration), formed thicker nanofibril bundles at 4 and 6 mg/mL fucoidan concentration, improved the EE of hesperidin from 72.86 % of Fmoc-FRGDF hydrogel to over 90 % of coassembly hydrogels, and showed effectively controlled release of hesperidin in vitro. Intriguingly, the first order kinetic model predicted an enhanced hydrogel retention and release of hesperidin. This study revealed a new approach for bioengineered nanogels that could be used to stabilize and release hydrophobic payloads.

Self-Assembled Peptide Habitats to Model Tumor Metastasis.

Metastatic tumours are complex ecosystems; a community of multiple cell types, including cancerous cells, fibroblasts, and immune cells that exist within a supportive and specific microenvironment. The interplay of these cells, together with tissue specific chemical, structural and temporal signals within a three-dimensional (3D) habitat, direct tumour cell behavior, a subtlety that can be easily lost in 2D tissue culture. Here, we investigate a significantly improved tool, consisting of a novel matrix of functionally programmed peptide sequences, self-assembled into a scaffold to enable the growth and the migration of multicellular lung tumour spheroids, as proof-of-concept. This 3D functional model aims to mimic the biological, chemical, and contextual cues of an in vivo tumor more closely than a typically used, unstructured hydrogel, allowing spatial and temporal activity modelling. This approach shows promise as a cancer model, enhancing current understandings of how tumours progress and spread over time within their microenvironment.

Extracellular Matrix Biomimetic Hydrogels, Encapsulated with Stromal Cell-Derived Factor 1, Improve the Composition of Foetal Tissue Grafts in a Rodent Model of Parkinson's Disease.

Clinical studies have provided evidence for dopamine (DA) cell replacement therapy in Parkinson's Disease. However, grafts derived from foetal tissue or pluripotent stem cells (PSCs) remain heterogeneous, with a high proportion of non-dopaminergic cells, and display subthreshold reinnervation of target tissues, thereby highlighting the need to identify new strategies to improve graft outcomes. In recent work, Stromal Cell-Derived Factor-1 (SDF1), secreted from meninges, has been shown to exert many roles during ventral midbrain DA development and DA-directed differentiation of PSCs. Related, co-implantation of meningeal cells has been shown to improve neural graft outcomes, however, no direct evidence for the role of SDF1 in neural grafting has been shown. Due to the rapid degradation of SDF1 protein, here, we utilised a hydrogel to entrap the protein and sustain its delivery at the transplant site to assess the impact on DA progenitor differentiation, survival and plasticity. Hydrogels were fabricated from self-assembling peptides (SAP), presenting an epitope for laminin, the brain's main extracellular matrix protein, thereby providing cell adhesive support for the grafts and additional laminin-integrin signalling to influence cell fate. We show that SDF1 functionalised SAP hydrogels resulted in larger grafts, containing more DA neurons, increased A9 DA specification (the subpopulation of DA neurons responsible for motor function) and enhanced innervation. These findings demonstrate the capacity for functionalised, tissue-specific hydrogels to improve the composition of grafts targeted for neural repair.

Matured Myofibers in Bioprinted Constructs with In Vivo Vascularization and Innervation.

For decades, the study of tissue-engineered skeletal muscle has been driven by a clinical need to treat neuromuscular diseases and volumetric muscle loss. The in vitro fabrication of muscle offers the opportunity to test drug-and cell-based therapies, to study disease processes, and to perhaps, one day, serve as a muscle graft for reconstructive surgery. This study developed a biofabrication technique to engineer muscle for research and clinical applications. A bioprinting protocol was established to deliver primary mouse myoblasts in a gelatin methacryloyl (GelMA) bioink, which was implanted in an in vivo chamber in a nude rat model. For the first time, this work demonstrated the phenomenon of myoblast migration through the bioprinted GelMA scaffold with cells spontaneously forming fibers on the surface of the material. This enabled advanced maturation and facilitated the connection between incoming vessels and nerve axons in vivo without the hindrance of a scaffold material. Immunohistochemistry revealed the hallmarks of tissue maturity with sarcomeric striations and peripherally placed nuclei in the organized bundles of muscle fibers. Such engineered muscle autografts could, with further structural development, eventually be used for surgical reconstructive purposes while the methodology presented here specifically has wide applications for in vitro and in vivo neuromuscular function and disease modelling.

Enhancing Peptide Biomaterials for Biofabrication.

Biofabrication using well-matched cell/materials systems provides unprecedented opportunities for dealing with human health issues where disease or injury overtake the body's native regenerative abilities. Such opportunities can be enhanced through the development of biomaterials with cues that appropriately influence embedded cells into forming functional tissues and organs. In this context, biomaterials' reliance on rigid biofabrication techniques needs to support the incorporation of a hierarchical mimicry of local and bulk biological cues that mimic the key functional components of native extracellular matrix. Advances in synthetic self-assembling peptide biomaterials promise to produce reproducible mimics of tissue-specific structures and may go some way in overcoming batch inconsistency issues of naturally sourced materials. Recent work in this area has demonstrated biofabrication with self-assembling peptide biomaterials with unique biofabrication technologies to support structural fidelity upon 3D patterning. The use of synthetic self-assembling peptide biomaterials is a growing field that has demonstrated applicability in dermal, intestinal, muscle, cancer and stem cell tissue engineering.

Tuneable Hybrid Hydrogels via Complementary Self-Assembly of a Bioactive Peptide with a Robust Polysaccharide.

Synthetic materials designed for improved biomimicry of the extracellular matrix must contain fibrous, bioactive, and mechanical cues. Self-assembly of low molecular weight gelator (LMWG) peptides Fmoc-DIKVAV (Fmoc-aspartic acid-isoleucine-lysine-valine-alanine-valine) and Fmoc-FRGDF (Fmoc-phenylalanine-arginine-glycine-aspartic acid-phenylalanine) creates fibrous and bioactive hydrogels. Polysaccharides such as agarose are biocompatible, degradable, and non-toxic. Agarose and these Fmoc-peptides have both demonstrated efficacy in vitro and in vivo. These materials have complementary properties; agarose has known mechanics in the physiological range but is inert and would benefit from bioactive and topographical cues found in the fibrous, protein-rich extracellular matrix. Fmoc-DIKVAV and Fmoc-FRGDF are synthetic self-assembling peptides that present bioactive cues "IKVAV" and "RGD" designed from the ECM proteins laminin and fibronectin. The work presented here demonstrates that the addition of agarose to Fmoc-DIKVAV and Fmoc-FRGDF results in physical characteristics that are dependent on agarose concentration. The networks are peptide-dominated at low agarose concentrations, and agarose-dominated at high agarose concentrations, resulting in distinct changes in structural morphology. Interestingly, at mid-range agarose concentration, a hybrid network is formed with structural similarities to both peptide and agarose systems, demonstrating reinforced mechanical properties. Bioactive-LMWG polysaccharide hydrogels demonstrate controllable microenvironmental properties, providing the ability for tissue-specific biomaterial design for tissue engineering and 3D cell culture.

Peptide Hydrogel Scaffold for Mesenchymal Precursor Cells Implanted to Injured Adult Rat Spinal Cord.

A unique, biomimetic self-assembling peptide (SAP) hydrogel, Fmoc-DIKVAV, has been shown to be a suitable cell and drug delivery system in the injured brain. In this study, we assessed its utility in adult Fischer 344 (F344) rats as a stabilizing scaffold and vehicle for grafted cells after mild thoracic (thoracic level 10 [T10]) contusion spinal cord injury (SCI). Treatments were as follows: Fmoc-DIKVAV alone, Fmoc-DIKVAV containing viable or nonviable rat mesenchymal precursor cells (rMPCs), and rMPCs alone. The majority of post-SCI treatments were administered at 11-15 days (mean 13.5 days) and the results then compared to SCI-only control (no treatment) rats. Postinjury behavior was quantified using open field locomotion (BBB) and LadderWalk analysis. After perfusion at 8 weeks, longitudinal spinal cord sections were immunostained with a panel of antibodies. Qualitatively, in the SAP-only treatment group, implanted gels contained regenerate axons as well as astrocytic, immune cell, and extracellular matrix (ECM) component profiles. Grafts of Fmoc-DIKVAV plus viable or nonviable rMPCs also contained numerous macrophages/microglia and ECM components, but astrocytes were generally confined to implant margins, and axons were rare. Quantitative analysis showed that, while average cyst size was reduced in all experimental groups, the decrease compared to SCI-only controls was only significant in the SAP and rMPC treatment groups. There was gradual improvement in functionality after SCI, but a consistent trend was only seen between the rMPC treatment group and SCI-only controls. In summary, after contusion SCI, implantation of Fmoc-DIKVAV hydrogel provided a favorable microenvironment for cellular infiltration and axonal regrowth, a supportive role that unexpectedly appeared to be compromised by prior inclusion of rMPCs into the gel matrix. Impact statement The self-assembling peptide hydrogel, Fmoc-DIKVAV, is a biomimetic scaffold that is an effective cell and drug delivery system in the injured brain. We examined whether this hydrogel, alone or combined with mesenchymal precursor cells, was also able to stabilise spinal cord tissue after thoracic contusion injury and improve morphological and behavioral outcomes. While improved functionality was not consistently seen, there was reduced cyst size and increased tissue sparing in some groups. There was regenerative axonal growth into hydrogels, but only in initially cell-free implants. This type of polymer is a suitable candidate for further testing in spinal cord injury models.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

A Programmed Anti-Inflammatory Nanoscaffold (PAIN) as a 3D Tool to Understand the Brain Injury Response.

Immunology is the next frontier of nano/biomaterial science research, with the immune system determining the degree of tissue repair. However, the complexity of the inflammatory response represents a significant challenge that is essential to understand for the development of future therapies. Cell-instructive 3D culture environments are critical to improve our understanding of the link between the behavior and morphology of inflammatory cells and to remodel their response to injury. This study has taken two recent high-profile innovations-functional peptide-based hydrogels, and the inclusion of anti-inflammatory agents via coassembly-to make a programmed anti-inflammatory nanoscaffold (PAIN) with unusual and valuable properties that allows tissue-independent switching of the inflammatory cascade. Here, extraordinary durability of the anti-inflammatory agent allows, for the first time, the development of a 3D culture system that maintains the growth and cytoskeletal reorganization of brain tissue, while also facilitating the trophic behavior of brain cells for 22 d in vitro. Notably, this behavior was confirmed within an active scar site due to the unprecedented resilience to the presence of inflammatory cells and enzymes in the brain. Efficacy of the culture system is demonstrated via novel insights about inflammatory cell behavior, which would be impossible to obtain via in vivo experimentation.

ENT audit and research in the era of trainee collaboratives.

Large surgical audits and research projects are complex and costly to deliver, but increasingly surgical trainees are delivering these projects within formal collaboratives and research networks. Surgical trainee collaboratives are now recognised as a valuable part of the research infrastructure, with many perceived benefits for both the trainees and the wider surgical speciality. In this article, we describe the activity of ENT trainee research collaboratives within the UK, and summarise how INTEGRATE, the UK National ENT Trainee Research Network, successfully delivered a national audit of epistaxis management. The prospective audit collected high-quality data from 1826 individuals, representing 94% of all cases that met the inclusion criteria at the 113 participating sites over the 30-day audit period. It is hoped that the audit has provided a template for subsequent high-quality and cost-effective national studies, and we discuss the future possibilities for ENT trainee research collaboratives.

Large and Small Assembly: Combining Functional Macromolecules with Small Peptides to Control the Morphology of Skeletal Muscle Progenitor Cells.

The material properties of natural tissues, such as skeletal muscle, are highly sophisticated and are synthetically challenging to mimic. Using natural biomacromolecules to functionalize self-assembled peptide (SAP) hydrogels has the potential to increase the utility of these materials by more closely reproducing the natural cellular environment. Here, to demonstrate that a conserved co-assembly pathway can retain distinct function, the biocompatible peptide derivative Fmoc-FRGDF was co-assembled with either a sulfated polysaccharide, fucoidan, or the provisional matrix proteoglycan, versican. Our results demonstrate that thermodynamically driven co-assembly with biologically active macromolecules is facile, stable, and does not affect the final assembled nanostructure. Biologically, the incorporation of these functionally distinct molecules had no effect on C2C12 myoblast proliferation and viability but strongly altered their morphology. The surface area of myoblasts cultured on the fucoidan scaffold was reduced at 24 and 72 h post seeding, with a reduction in the formation of multinucleated syncytia. Myoblasts cultured on versican scaffolds were smaller compared to cells grown on the empty vector scaffolds at 24 h but not 72 h post seeding, with multinucleated syncytia formation being unaffected. This work allows programmed and distinct morphological effects of cell behavior, paving the way for further mechanistic studies.

Temporally controlled growth factor delivery from a self-assembling peptide hydrogel and electrospun nanofibre composite scaffold.

Tissue-specific self-assembling peptide (SAP) hydrogels designed based on biologically relevant peptide sequences have great potential in regenerative medicine. These materials spontaneously form 3D networks of physically assembled nanofibres utilising non-covalent interactions. The nanofibrous structure of SAPs is often compared to that of electrospun scaffolds. These electrospun nanofibers are produced as sheets that can be engineered from a variety of polymers that can be chemically modified to incorporate many molecules including drugs and growth factors. However, their macroscale morphology limits them to wrapping and bandaging applications. Here, for the first time, we combine the benefits of these systems to describe a two-component composite scaffold from these biomaterials, with the design goal of providing a hydrogel scaffold that presents 3D structures, and also has temporal control over drug delivery. Short fibres, cut from electrospun scaffolds, were mixed with our tissue-specific SAP hydrogel to provide a range of nanofibre sizes found in the extracellular matrix (10-300 nm in diameter). The composite material maintained the shear-thinning and void-filling properties of SAP hydrogels that have previously been shown to be effective for minimally invasive material injection, cell delivery and subsequent in vivo integration. Both scaffold components were separately loaded with growth factors, important signaling molecules in tissue regeneration whose rapid degradation limits their clinical efficacy. The two biomaterials provided sequential growth factor delivery profiles: the SAP hydrogel provided a burst release, with the release rate decreasing over 12 hours, while the electrospun nanofibres provided a more constant, sustained delivery. Importantly, this second release commenced 6 days later. The design rules established here to provide temporally distinct release profiles can enable researchers to target specific stages in regeneration, such as the acute immune response versus sustained protection and survival of cells following injury. In summary, this novel composite material combines the physical advantages of SAP hydrogels and electrospun nanofibres, while additionally providing a superior vehicle for the stabilisation and controlled delivery of growth factors necessary for optimal tissue repair.

Peptide-Based Scaffolds Support Human Cortical Progenitor Graft Integration to Reduce Atrophy and Promote Functional Repair in a Model of Stroke.

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.

Large microplastic particles in sediments of tributaries of the River Thames, UK - Abundance, sources and methods for effective quantification.

Sewage effluent input and population were chosen as predictors of microplastic presence in sediments at four sites in the River Thames basin (UK). Large microplastic particles (1mm-4mm) were extracted using a stepwise approach to include visual extraction, flotation and identification using Raman spectroscopy. Microplastics were found at all four sites. One site had significantly higher numbers of microplastics than other sites, average 66 particles 100g-1, 91% of which were fragments. This site was downstream of a storm drain outfall receiving urban runoff; many of the fragments at this site were determined to be derived of thermoplastic road-surface marking paints. At the remaining three sites, fibres were the dominant particle type. The most common polymers identified included polypropylene, polyester and polyarylsulphone. This study describes two major new findings: presence of microplastic particles in a UK freshwater system and identification of road marking paints as a source of microplastics. CAPSULE: This study is the first to quantify microplastics of any size in river sediments in the UK and links their presence to terrestrial sources including sewage and road marking paints.

Temporally controlled release of multiple growth factors from a self-assembling peptide hydrogel.

Protein growth factors have demonstrated great potential for tissue repair, but their inherent instability and large size prevents meaningful presentation to biologically protected nervous tissue. Here, we create a nanofibrous network from a self-assembling peptide (SAP) hydrogel to carry and stabilize the growth factors. We significantly reduced growth factor degradation to increase their lifespan by over 40 times. To control the temporal release profile we covalently attached polysaccharide chitosan molecules to the growth factor to increase its interactions with the hydrogel nanofibers and achieved a 4 h delay, demonstrating the potential of this method to provide temporally controlled growth factor delivery. We also describe release rate based analysis to examine the growth factor delivery in more detail than standard cumulative release profiles allow and show that the chitosan attachment method provided a more consistent release profile with a 60% reduction in fluctuations. To prove the potential of this system as a complex growth factor delivery platform we demonstrate for the first time temporally distinct release of multiple growth factors from a single tissue specific SAP hydrogel: a significant goal in regenerative medicine.

Characterisation of minimalist co-assembled fluorenylmethyloxycarbonyl self-assembling peptide systems for presentation of multiple bioactive peptides.

UNLABELLED: 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.