Comprehensive Physicochemical Characterization and in Vitro Human Cell Culture Studies of an Innovative Biocompatible and Biodegradable Silk-Derived Protein Hydrolysate, SDP-4.

SDP-4 is a soluble silk fibroin-derived protein hydrolysate extracted from the silkworm cocoon and is a novel first-in-class biopolymer that is biodegradable, biocompatible, and shown to have regenerative properties. SDP-4 is currently used as a commercial wetting agent in topical eye drops, but it has also been shown to have anti-inflammatory properties that could be utilized in other biomedical applications. The purpose of this study was to comprehensively characterize the physicochemical properties that are necessary to design formulations and examine cell viability in response to varying doses of SDP-4 on different human cell types, with a particular attention toward respiratory applications.

Silk-Derived Protein-4 Versus Vehicle Control in Treating Patients With Moderate to Severe Dry Eye Disease: A Randomized Clinical Trial.

Purpose: In this study the safety and efficacy of silk-derived protein 4 (SDP-4), also known as amlisimod, eye drops against a vehicle control formulation in patients with moderate to severe dry eye disease (DED) was assessed. SDP-4 is a novel, naturally derived, anti-inflammatory wetting agent that enhances coating on the ocular surface.

Design: Exploratory Phase 2, 12- and 8-week, serial cohort, multicenter, double-masked, randomized, vehicle-controlled study.

Effect of silk fibroin protein hydrolysis on biochemistry, gelation kinetics, and NF-kB bioactivity in vitro.

Fibroin is a structural protein derived from silk cocoons, which may be used in a variety of biomedical applications due to its high biocompatibility and controllable material properties. Conversely, fibroin solution is inherently unstable in solution, which limits its potential utility. Fibroin hydrolysates possess enhanced aqueous solubility and stability, with known anti-inflammatory bioactivity.

The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation.

We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal.

Micro- and Nanoscale Topographies on Silk Regulate Gene Expression of Human Corneal Epithelial Cells.

Purpose: Corneal basement membrane has topographical features that provide biophysical cues to direct cell adherence, migration, and proliferation. In this study, we hypothesize that varying topographic pitch created on silk films can alter epithelial cell morphology, adhesion, and the genetic expression involved in cytoskeletal dynamics-related pathways.

Methods: Silicon wafers with parallel ridge widths of 2000, 1000, and 800 nm were produced and used to pattern silk films via soft lithography.

Treatment with solubilized Silk-Derived Protein (SDP) enhances rabbit corneal epithelial wound healing.

There is a significant clinical need to improve current therapeutic approaches to treat ocular surface injuries and disease, which affect hundreds of millions of people annually worldwide. The work presented here demonstrates that the presence of Silk-Derived Protein (SDP) on the healing rabbit corneal surface, administered in an eye drop formulation, corresponds with an enhanced epithelial wound healing profile. Rabbit corneas were denuded of their epithelial surface, and then treated for 72-hours with either PBS or PBS containing 5 or 20 mg/mL SDP in solution four times per day.

Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation.

Purpose: The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury.

Silk film topography directs collective epithelial cell migration.

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation.

Human corneal limbal epithelial cell response to varying silk film geometric topography in vitro.

Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment and focal adhesion (FA) localization.

Silk fibroin as a biomaterial substrate for corneal epithelial cell sheet generation.

Purpose: To evaluate a silk fibroin (SF) biomaterial as a substrate for corneal epithelial cell proliferation, differentiation, and stratification in vitro compared with denuded human amniotic membrane (AM).

Methods: Primary human and rabbit corneal epithelial cells and immortalized human corneal limbal epithelial cells were cultured on the SF and denuded AM, respectively. The biological cell behavior, including the morphology, proliferation, differentiation, and stratification, on the two substrates was compared and analyzed.

Silk film culture system for in vitro analysis and biomaterial design.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment (1,2). These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications (3-8). In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications (9-13).

Effect of hydration on silk film material properties.

Effects of hydration on silk fibroin film properties were investigated for water-annealed and MeOH-treated samples. Hydration increased thickness by 60% for MeOH-immersed films, while water-annealed samples remained constant. MeOH-immersed films showed an 80% mass loss due to water, while water-annealed lost only 40%.

Silk film biomaterials for cornea tissue engineering.

Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture.

Effect of cyclic mechanical strain on glycosaminoglycan and proteoglycan synthesis by heart valve cells.

Heart valves are presumed to remodel their extracellular matrix upon application of mechanical strains. In this study, we investigated the effect of cyclic tensile strain on valvular interstitial cells' synthesis of glycosaminoglycans (GAGs) and proteoglycans (PGs), which are altered during myxomatous degeneration. Interstitial cells were isolated from mitral valve leaflets and chordate, and seeded separately within three-dimensional collagen gels.

Reversible secretion of glycosaminoglycans and proteoglycans by cyclically stretched valvular cells in 3D culture.

Mitral valve leaflets and chordae have been shown to contain different amounts and proportions of glycosaminoglycans (GAGs) and proteoglycans (PGs) corresponding to in vivo normal or diseased cyclic strain patterns. To understand the effect of cyclic strains on GAG/PG synthesis by valvular interstitial cells (VICs) isolated from valve leaflet and chordae separately, porcine VICs were seeded within collagen gels and alternately stretched or relaxed for 24 h periods for one week in a custom-designed tissue engineering bioreactor. We found cyclic-stretch-induced upregulation of total GAGs and of individual GAG classes secreted into the culture medium.

Bioactive silk protein biomaterial systems for optical devices.

Silk-based biomaterial systems have been previously explored for a variety of medical and nonmedical materials needs. The unique biophysical features of silks provide options to generate highly tailored structures and morphologies with this unique family of fibrous proteins. To exploit these features, we have optimized the all aqueous processing of silk fibroin into novel surface nanopatterned protein materials.