Layer-by-Layer Analysis of In Vitro Skin Models.

In vitro human skin models are evolving into versatile platforms for the study of skin biology and disorders. These models have many potential applications in the fields of drug testing and safety assessment, as well as cosmetic and new treatment development. The development of in vitro skin models that accurately mimic native human skin can reduce reliance on animal models and also allow for more precise, clinically relevant testing. Recent advances in biofabrication techniques and biomaterials have led to the creation of increasingly complex, multilayered skin models that incorporate important functional components of skin, such as the skin barrier, mechanical properties, pigmentation, vasculature, hair follicles, glands, and subcutaneous layer. This improved ability to recapitulate the functional aspects of native skin enhances the ability to model the behavior and response of native human skin, as the complex interplay of cell-to-cell and cell-to-material interactions are incorporated. In this review, we summarize the recent developments in in vitro skin models, with a focus on their applications, limitations, and future directions.

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.

Rational design of potent ultrashort antimicrobial peptides with programmable assembly into nanostructured hydrogels.

Microbial resistance to common antibiotics is threatening to cause the next pandemic crisis. In this context, antimicrobial peptides (AMPs) are receiving increased attention as an alternative approach to the traditional small molecule antibiotics. Here, we report the bi-functional rational design of Fmoc-peptides as both antimicrobial and hydrogelator substances. The tetrapeptide Fmoc-WWRR-NH2-termed Priscilicidin-was rationally designed for antimicrobial activity and molecular self-assembly into nanostructured hydrogels. Molecular dynamics simulations predicted Priscilicidin to assemble in water into small oligomers and nanofibrils, through a balance of aromatic stacking, amphiphilicity and electrostatic repulsion. Antimicrobial activity prediction databases supported a strong antimicrobial motif via sequence analogy. Experimentally, this ultrashort sequence showed a remarkable hydrogel forming capacity, combined to a potent antibacterial and antifungal activity, including against multidrug resistant strains. Using a set of biophysical and microbiology techniques, the peptide was shown to self-assemble into viscoelastic hydrogels, as a result of assembly into nanostructured hexagonal mesophases. To further test the molecular design approach, the Priscilicidin sequence was modified to include a proline turn-Fmoc-WPWRR-NH2, termed P-Priscilicidin-expected to disrupt the supramolecular assembly into nanofibrils, while predicted to retain antimicrobial activity. Experiments showed P-Priscilicidin self-assembly to be effectively hindered by the presence of a proline turn, resulting in liquid samples of low viscosity. However, assembly into small oligomers and nanofibril precursors were evidenced. Our results augur well for fast, adaptable, and cost-efficient antimicrobial peptide design with programmable physicochemical properties.

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.

Towards bioengineered skeletal muscle: recent developments in vitro and in vivo.

Skeletal muscle is a functional tissue that accounts for approximately 40% of the human body mass. It has remarkable regenerative potential, however, trauma and volumetric muscle loss, progressive disease and aging can lead to significant muscle loss that the body cannot recover from. Clinical approaches to address this range from free-flap transfer for traumatic events involving volumetric muscle loss, to myoblast transplantation and gene therapy to replace muscle loss due to sarcopenia and hereditary neuromuscular disorders, however, these interventions are often inadequate. The adoption of engineering paradigms, in particular materials engineering and materials/tissue interfacing in biology and medicine, has given rise to the rapidly growing, multidisciplinary field of bioengineering. These methods have facilitated the development of new biomaterials that sustain cell growth and differentiation based on bionic biomimicry in naturally occurring and synthetic hydrogels and polymers, as well as additive fabrication methods to generate scaffolds that go some way to replicate the structural features of skeletal muscle. Recent advances in biofabrication techniques have resulted in significant improvements to some of these techniques and have also offered promising alternatives for the engineering of living muscle constructs ex vivo to address the loss of significant areas of muscle. This review highlights current research in this area and discusses the next steps required towards making muscle biofabrication a clinical reality.

Replace and repair: Biomimetic bioprinting for effective muscle engineering.

The debilitating effects of muscle damage, either through ischemic injury or volumetric muscle loss (VML), can have significant impacts on patients, and yet there are few effective treatments. This challenge arises when function is degraded due to significant amounts of skeletal muscle loss, beyond the regenerative ability of endogenous repair mechanisms. Currently available surgical interventions for VML are quite invasive and cannot typically restore function adequately. In response to this, many new bioengineering studies implicate 3D bioprinting as a viable option. Bioprinting for VML repair includes three distinct phases: printing and seeding, growth and maturation, and implantation and application. Although this 3D bioprinting technology has existed for several decades, the advent of more advanced and novel printing techniques has brought us closer to clinical applications. Recent studies have overcome previous limitations in diffusion distance with novel microchannel construct architectures and improved myotubule alignment with highly biomimetic nanostructures. These structures may also enhance angiogenic and nervous ingrowth post-implantation, though further research to improve these parameters has been limited. Inclusion of neural cells has also shown to improve myoblast maturation and development of neuromuscular junctions, bringing us one step closer to functional, implantable skeletal muscle constructs. Given the current state of skeletal muscle 3D bioprinting, the most pressing future avenues of research include furthering our understanding of the physical and biochemical mechanisms of myotube development and expanding our control over macroscopic and microscopic construct structures. Further to this, current investigation needs to be expanded from immunocompromised rodent and murine myoblast models to more clinically applicable human cell lines as we move closer to viable therapeutic implementation.

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.