Milliscale Substrate Curvature Promotes Myoblast Self-Organization and Differentiation.

Biological tissues comprise complex structural environments known to influence cell behavior via multiple interdependent sensing and transduction mechanisms. Yet, and despite the predominantly nonplanar geometry of these environments, the impact of tissue-size (milliscale) curvature on cell behavior is largely overlooked or underestimated. This study explores how concave, hemicylinder-shaped surfaces 3-50 mm in diameter affect the migration, proliferation, orientation, and differentiation of C2C12 myoblasts.

Biomechanical Modulation Therapy-A Stem Cell Therapy Without Stem Cells for the Treatment of Severe Ocular Burns.

Ocular injuries caused by chemical and thermal burns are often unmanageable and frequently result in disfigurement, corneal haze/opacification, and vision loss. Currently, a considerable number of surgical and pharmacological approaches are available to treat such injuries at either an acute or a chronic stage. However, these existing interventions are mainly directed at (and limited to) suppressing corneal inflammation and neovascularization while promoting re-epithelialization.

Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation.

Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction.

Autogenous Biofabrication of Nativelike, Scaffold-Free Human Skin Equivalents Using a Smart, Enzyme-Degradable Tissue Templating Coating.

In this study, we used tissue templating technology to direct human dermal fibroblasts to biofabricate large-area tissues that closely emulate the natural dermis. This technology also allowed the new tissues to promote their own release from the template surface, thus facilitating their recovery as self-sustained, scaffold-free dermal equivalents solely comprising human cells and their own extracellular matrix. The structure and composition of these dermal self-lifting autogenous tissue equivalents (SLATEs) were evaluated in detail and were shown to closely correlate to normal tissue function.

Template Curvature Influences Cell Alignment to Create Improved Human Corneal Tissue Equivalents.

To accurately create corneal stromal equivalents with native-like structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with cornea-like shape and composition. Specifically, tissues self-released from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stroma-characteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.

Controlling the 3D architecture of Self-Lifting Auto-generated Tissue Equivalents (SLATEs) for optimized corneal graft composition and stability.

Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues.

Assessing corneal biomechanics with Brillouin spectro-microscopy.

A new Brillouin spectro-microscope was designed and built to investigate the mechanical properties of bovine and human corneas. This instrument integrates a single-stage virtually imaged phased array spectrometer with a novel adaptive-optics interferometric filter to achieve unprecedented rejection of the elastic background signal. As a result, highly-resolved, reproducible data from both thin and thick collagen-based materials were obtained.

Application of retinoic acid improves form and function of tissue engineered corneal construct.

Retinoic acid has recently been shown to control the phenotype and extracellular matrix composition of corneal stromal cells cultured in vitro as monolayers. This study set out to investigate the effects of retinoic acid on human corneal keratocytes within a 3D environment. Human corneal keratocytes were encapsulated in collagen gels, which were subsequently compressed under load, and cultured in serum-free media supplemented with 10 µM retinoic acid or DMSO vehicle for 30 days.

Bio-fabrication and physiological self-release of tissue equivalents using smart peptide amphiphile templates.

In this study we applied a smart biomaterial formed from a self-assembling, multi-functional synthetic peptide amphiphile (PA) to coat substrates with various surface chemistries. The combination of PA coating and alignment-inducing functionalised substrates provided a template to instruct human corneal stromal fibroblasts to adhere, become aligned and then bio-fabricate a highly-ordered, multi-layered, three-dimensional tissue by depositing an aligned, native-like extracellular matrix. The newly-formed corneal tissue equivalent was subsequently able to eliminate the adhesive properties of the template and govern its own complete release via the action of endogenous proteases.

Peptide Amphiphiles in Corneal Tissue Engineering.

The increasing interest in effort towards creating alternative therapies have led to exciting breakthroughs in the attempt to bio-fabricate and engineer live tissues. This has been particularly evident in the development of new approaches applied to reconstruct corneal tissue. The need for tissue-engineered corneas is largely a response to the shortage of donor tissue and the lack of suitable alternative biological scaffolds preventing the treatment of millions of blind people worldwide.

Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions.

The avascular cornea is a uniquely-isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose in the behaviour of human corneal keratocytes has been overlooked.

A self-assembling fluorescent dipeptide conjugate for cell labelling.

Derivatives of fluorophore FITC (fluorescein isothiocyanate) are widely used in bioassays to label proteins and cells. An N-terminal leucine dipeptide is attached to FITC, and we show that this simple conjugate molecule is cytocompatible and is uptaken by cells (human dermal and corneal fibroblasts) in contrast to FITC itself. Co-localisation shows that FITC-LL segregates in peri-nuclear and intracellular vesicle regions.

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs.

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac).

New self-assembling multifunctional templates for the biofabrication and controlled self-release of cultured tissue.

The need to source live human tissues for research and clinical applications has been a major driving force for the development of new biomaterials. Ideally, these should elicit the formation of scaffold-free tissues with native-like structure and composition. In this study, we describe a biologically interactive coating that combines the fabrication and subsequent self-release of live purposeful tissues using template-cell-environment feedback.

The bioactivity of composite Fmoc-RGDS-collagen gels.

The incorporation of small bioactive peptide motifs within robust hydrogels constitutes a facile procedure to chemically functionalise cell and tissue scaffolds. In this study, a novel approach to utilise Fmoc-linked peptide amphiphiles comprising the bio-functional cell-adhesion RGDS motif within biomimetic collagen gels was developed. The composite scaffolds thus created were shown to maintain the mechanical properties of the collagen gel while presenting additional bio-activity.

Differential nuclear expression of Yap in basal epithelial cells across the cornea and substrates of differing stiffness.

Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells, followed by migration and maturation centripetally across the ocular surface. The present study sets out to explore the role tissue stiffness (compliance) may have in directing both differentiation and centripetal migration of limbal epithelial stem cells during homeostasis. For that, we analysed the localization of the Yes-associated protein (Yap), a transcriptional co-activator previously shown to mediate cellular response and mechanical stimuli.

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions.

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems.

The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions.

Purpose: Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions.

Methods: Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle.

New RGD-peptide amphiphile mixtures containing a negatively charged diluent.

Here, we studied the self-assembly of two peptide amphiphiles, C16-Gly-Gly-Gly-Arg-Gly-Asp (PA 1: C16-GGG-RGD) and C16-Gly-Gly-Gly-Arg-Gly-Asp-Ser (PA 2: C16-GGG-RGDS). We showed that PA 1 and PA 2 self-assemble into nanotapes with an internal bilayer structure. C16 chains were highly interdigitated within the nanotape cores, while the peptide blocks formed water-exposed 13-sheets too.

Kinetic analysis of L1 homophilic interaction: role of the first four immunoglobulin domains and implications on binding mechanism.

L1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding.

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells.

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5-6 (L1/Ig5-6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO.