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. In particular, this technique is capable of rigorously measuring the relative stiffness of different areas of human corneas, thus providing a true non-contact method to characterise the fundamental mechanical features of both live and fixed biological tissue samples.

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). In addition, when used in solution at 5-150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

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. Tissues recovered through this method were structurally stable, easily handled, and carrier-free. Furthermore, topographical and mechanical analysis by atomic force microscopy showed that tissue equivalents formed on the alignment-inducing PA template had highly-ordered, compact collagen deposition, with a two-fold higher elastic modulus compared to the less compact tissues produced on the non-alignment template, the PA-coated glass. We suggest that this technology represents a new paradigm in tissue engineering and regenerative medicine, whereby all processes for the bio-fabrication and subsequent self-release of natural, bio-prosthetic human tissues depend solely on simple template-tissue feedback interactions.

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. Using both models of ocular surface compliance and normal bovine corneas we evaluated the nuclear/cytoplasmic expression ratio of Yap. Expression levels within corneal epithelial cells were compared in situ between the limbus and central cornea, and in vitro between limbal epithelial stem cells expanded upon biomimetic collagen gels of increasing stiffness. Nuclear expression of Yap was shown to increase within the expanded cells upon substrates of increasing stiffness. Subsequently, Yap was used as a novel molecular probe to investigate the mechanical microenvironment within a normal ocular surface. The in situ localization of Yap was predominantly cytoplasmic within basal limbal epithelial cells and nuclear within basal central corneal epithelial cells. Furthermore, nuclear p63 expression was not co-localized with Yap in basal limbal epithelial cells. In conclusion, the current investigation provides new insights into the relationship between Yap and distinct cell populations across the ocular surface indicating that cells experience a different mechanical environment between the limbus and central cornea. A new hypothesis is put forward, in which centripetal differences in substrate stiffness drives the migration and differentiation of limbal epithelial stem cells, thus controlling corneal epithelium homeostasis.

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. Notably, these milliscale cues significantly affect cell responses compared with planar substrates, with myoblasts grown on surfaces 7.5-15 mm in diameter showing prevalent migration and alignment parallel to the curvature axis. Moreover, surfaces within this curvature range promote myoblast differentiation and the formation of denser, more compact tissues comprising highly oriented multinucleated myotubes. Based on the similarity of effects, it is further proposed that myoblast susceptibility to substrate curvature depends on mechanotransduction signaling. This model thus supports the notion that cellular responses to substrate curvature and compliance share the same molecular pathways and that control of cell behavior can be achieved via modulation of either individual parameter or in combination. This correlation is relevant for elucidating how muscle tissue forms and heals, as well as for designing better biomaterials and more appropriate cell-surface interfaces.

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. Reconstruction of the ocular surface represents a suitable but last-option recourse in cases where epithelial healing is severely impaired, such as due to limbal stem cell deficiency. In this concise review, we discuss how biomechanical modulation therapy (BMT) may represent a more effective approach to promoting the regeneration of ocular tissues affected by burn injuries via restoration of the limbal stem cell niche. Specifically, the scientific basis supporting this new therapeutic modality is described, along with our growing understanding of the role that tissue biomechanics plays in stem cell fate and function. The potential impact of BMT as a future treatment option for the management of injuries affecting tissue compliance is also further discussed.

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. Specifically, dermal SLATEs were shown to be composed of a dense collagen-based matrix interwoven with dermal-characteristic elastic fibers. In addition, the mechanical properties of these tissues (i.e., robustness, elastic modulus, and resistance to contraction and enzymatic degradation) were comparable to those of the natural human dermis. Furthermore, dermal SLATEs were capable of constituting tissues with a higher-order complexity by serving as a substrate to support the growth of keratinocytes into stratified epithelia with distinct layers of differentiation. This work thus illustrates the great potential of tissue templating technologies and how these can pave the way for the biofabrication of easily retrievable, scaffold-free human skin tissues with a structure, composition, and function suitable for both clinical and nonclinical applications.

YAP, ΔNp63, and ß-Catenin Signaling Pathways Are Involved in the Modulation of Corneal Epithelial Stem Cell Phenotype Induced by Substrate Stiffness.

Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche's distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and ß-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/ß-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.

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. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue's mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration.

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 = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo.

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. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.

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. Above a critical aggregation concentration, the conjugate is shown to self-assemble into beta-sheet nanostructures comprising molecular bilayers.

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. This review is focused on recent developments in corneal tissue engineering, specifically on the use of self-assembling peptide amphiphiles for this purpose. Recently, peptide amphiphiles have generated great interest as therapeutic molecules, both in vitro and in vivo. Here we introduce this rapidly developing field, and examine innovative applications of peptide amphiphiles to create natural bio-prosthetic corneal tissue in vitro. The advantages of peptide amphiphiles over other biomaterials, namely their wide range of functions and applications, versatility, and transferability are also discussed to better understand how these fascinating molecules can help solve current challenges in corneal regeneration.

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. Cell proliferation was quantified on selected days, while the expression of several important keratocytes markers was evaluated at day 30 using RT-PCR and immunoblotting. The weight and size of the collagen constructs were measured before and after hydration and contraction analyses. Retinoic acid enhanced keratocyte proliferation until day 30, whereas cells in control culture conditions showed reduced numbers after day 21. Both gene and protein expressions of keratocyte-characteristic proteoglycans (keratocan, lumican and decorin), corneal crystallins and collagen type I and V were significantly increased following retinoic acid supplementation. Retinoic acid also significantly reduced the expression of matrix metalloproteases 1, 3 and 9 while not increasing α-smooth muscle actin and fibronectin expression. Furthermore, these effects were also correlated with the ability of retinoic acid to significantly inhibit the contractility of keratocytes while allowing the build-up of corneal stromal extracellular matrix within the 3D constructs. Thus, retinoic acid supplementation represents a promising strategy to improve the phenotype of 3D-cultured keratocytes, and their usefulness as a model of corneal stroma for corneal biology and regenerative medicine applications.

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. As such, we sought to investigate the effects of low-glucose formulations on the phenotype of human corneal stromal cells. Cells cultured in low-glucose were able to survive for extended periods when compared to high-glucose, serum-free conditions. Furthermore, low-glucose enhanced their reversal to a keratocyte-characteristic phenotype. Specifically, cells within low-glucose medium assumed dendritic morphologies, with bean-shaped condensed nuclei, absence of alpha-smooth muscle actin or stress fibres, and a corresponding reduction in migratory and contractile activities when compared with high-glucose, serum-free conditions. Moreover, cells within low-glucose uniquely recovered the ability to express a robust keratocyte-characteristic marker, CD34, while still expressing elevated levels of other representative phenotypic markers such as keratocan, lumican, ALDH1A1, and ALDH3A1. These results indicate that low-glucose enhances keratocyte-characteristic phenotype above and beyond established media formulations and thus has important implications for corneal biology in health and disease.

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. This smart coating was formed from a self-assembling peptide amphiphile comprising a protease-cleavable sequence contiguous with a cell attachment and signaling motif. This multifunctional material was subsequently used not only to instruct human corneal or skin fibroblasts to adhere and deposit discreet multiple layers of native extracellular matrix but also to govern their own self-directed release from the template solely through the action of endogenous metalloproteases. Tissues recovered through this physiologically relevant process were carrier-free and structurally and phenotypically equivalent to their natural counterparts. This technology contributes to a new paradigm in regenerative medicine, whereby materials are able to actively direct and respond to cell behavior. The novel application of such materials as a coating capable of directing the formation and detachment of complex tissues solely under physiological conditions can have broad use for fundamental research and in future cell and tissue therapies.

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. In particular, these materials enhanced the adhesion and proliferation of viable human corneal stromal fibroblasts by 300% compared to non-functionalised gels. Furthermore, the incorporation of Fmoc-RGDS nanostructures within the collagen matrix significantly suppressed gel shrinkage resulting from the contractile action of encapsulated fibroblasts once activated by serum proteins. These mechanical and biological properties demonstrate that the incorporation of peptide amphiphiles provides a suitable and easy method to circumvent specific biomaterial limitations, such as cell-derived shrinkage, for improved performance in tissue engineering and regenerative medicine applications.

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. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 × 10-3 M, the bioactive PAs were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts (hCSFs), while improving the cell phenotype. These PAs also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene (PTFE), significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

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. PA 1 nanotapes were characterized by one spacing distribution, corresponding to a more regular internal structure than that of PA 2 nanotapes, which presented two different spacing distributions. We showed that it is possible to obtain homogeneous nanotapes in water by co-assembling PA 1 or PA 2 with the negatively charged diluent C,16-Glu-Thr-Thr-Glu-Ser (PA 3: C16-ETTES). The homogeneous tapes formed by PA 1-PA 3 or PA 2-PA 3 mixtures presented a structure similar to that observed for the corresponding pure PA 1 or PA 2 nanotapes. The mixed nanotapes, which were able to form a stabilized matrix containing homogeneously distributed cell adhesive RGD groups, represent promising materials for designing new cell adhesion substrates.

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. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1-4 interacted homophilically in trans, contrary to mutants L1/Ig1-3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 +/- 2 and 130 +/- 6 nm for L1/ECD-L1/ECD and L1/ECD-L1/Ig1-4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1-4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1-4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1-Ig4 in a horseshoe conformation.