Electrospun-Reinforced Suturable Biodegradable Artificial Cornea.

Despite rigorous investigations, the hydrogels currently available to replace damaged tissues, such as the cornea, cannot fulfill mechanical and structural requirements and, more importantly, cannot be sutured into host tissues due to the lack of hierarchical structures to dissipate exerted stress. In this report, solution electrospinning of polycaprolactone (PCL), protein-based hydrogel perfusion, and layer-by-layer stacking are used to generate a hydrogel-microfiber composite with varying PCL fiber diameters and hydrogel concentrations. Integrating PCL microfibers into the hydrogel synergistically improves the mechanical properties and suturability of the construct up to 10-fold and 50-fold, respectively, compared to the hydrogel and microfiber scaffolds alone, approaching those of the corneal tissue. Human corneal cells cultured on composites are viable and can spread, proliferate, and retain phenotypic characteristics. Moreover, corneal stromal cells migrate into the scaffold, degrade it, and regenerate the extracellular matrix. The current hydrogel reinforcing system paves the way for producing suturable and, therefore, transplantable tissue constructs with desired mechanical properties.

Critical media attributes in E-beam sterilization of corneal tissue.

When ionizing irradiation interacts with a media, it can form reactive species that can react with the constituents of the system, leading to eradication of bioburden and sterilization of the tissue. Understanding the media's properties such as polarity is important to control and direct those reactive species to perform desired reactions. Using ethanol as a polarity modifier of water, we herein generated a series of media with varying relative polarities for electron beam (E-beam) irradiation of cornea at 25 kGy and studied how the irradiation media's polarity impacts properties of the cornea. After irradiation of corneal tissues, mechanical (tensile strength and modulus, elongation at break, and compression modulus), chemical, optical, structural, degradation, and biological properties of the corneal tissues were evaluated. Our study showed that irradiation in lower relative polarity media improved structural properties of the tissues yet reduced optical transmission; higher relative polarity reduced structural and optical properties of the cornea; and intermediate relative polarity (ethanol concentrations = 20-30% (v/v)) improved the structural properties, without compromising optical characteristics. Regardless of media polarity, irradiation did not negatively impact the biocompatibility of the corneal tissue. Our data shows that the absorbed ethanol can be flushed from the irradiated cornea to levels that are nontoxic to corneal and retinal cells. These findings suggest that the relative polarity of the irradiation media can be tuned to generate sterilized tissues, including corneal grafts, with engineered properties that are required for specific biomedical applications. STATEMENT OF SIGNIFICANCE: Extending the shelf-life of corneal tissue can improve general accessibility of cornea grafts for transplantation. Irradiation of donor corneas with E-beam is an emerging technology to sterilize the corneal tissues and enable their long-term storage at room temperature. Despite recent applications in clinical medicine, little is known about the effect of irradiation and preservation media's characteristics, such as polarity on the properties of irradiated corneas. Here, we have showed that the polarity of the media can be a valuable tool to change and control the properties of the irradiated tissue for transplantation.

Systematic optimization of visible light-induced crosslinking conditions of gelatin methacryloyl (GelMA).

Gelatin methacryloyl (GelMA) is one of the most widely used photo-crosslinkable biopolymers in tissue engineering. In in presence of an appropriate photoinitiator, the light activation triggers the crosslinking process, which provides shape fidelity and stability at physiological temperature. Although ultraviolet (UV) has been extensively explored for photo-crosslinking, its application has been linked to numerous biosafety concerns, originated from UV phototoxicity. Eosin Y, in combination with TEOA and VC, is a biosafe photoinitiation system that can be activated via visible light instead of UV and bypasses those biosafety concerns; however, the crosslinking system needs fine-tuning and optimization. In order to systematically optimize the crosslinking conditions, we herein independently varied the concentrations of Eosin Y [(EY)], triethanolamine (TEOA), vinyl caprolactam (VC), GelMA precursor, and crosslinking times and assessed the effect of those parameters on the properties the hydrogel. Our data showed that except EY, which exhibited an optimal concentration (~ 0.05 mM), increasing [TEOA], [VA], [GelMA], or crosslinking time improved mechanical (tensile strength/modulus and compressive modulus), adhesion (lap shear strength), swelling, biodegradation properties of the hydrogel. However, increasing the concentrations of crosslinking reagents ([TEOA], [VA], [GelMA]) reduced cell viability in 3-dimensional (3D) cell culture. This study enabled us to optimize the crosslinking conditions to improve the properties of the GelMA hydrogel and to generate a library of hydrogels with defined properties essential for different biomedical applications.

Tuning gelatin-based hydrogel towards bioadhesive ocular tissue engineering applications.

Gelatin based adhesives have been used in the last decades in different biomedical applications due to the excellent biocompatibility, easy processability, transparency, non-toxicity, and reasonable mechanical properties to mimic the extracellular matrix (ECM). Gelatin adhesives can be easily tuned to gain different viscoelastic and mechanical properties that facilitate its ocular application. We herein grafted glycidyl methacrylate on the gelatin backbone with a simple chemical modification of the precursor, utilizing epoxide ring-opening reactions and visible light-crosslinking. This chemical modification allows the obtaining of an elastic protein-based hydrogel (GELGYM) with excellent biomimetic properties, approaching those of the native tissue. GELGYM can be modulated to be stretched up to 4 times its initial length and withstand high tensile stresses up to 1.95 MPa with compressive strains as high as 80% compared to Gelatin-methacryloyl (GeIMA), the most studied derivative of gelatin used as a bioadhesive. GELGYM is also highly biocompatible and supports cellular adhesion, proliferation, and migration in both 2 and 3-dimensional cell-cultures. These characteristics along with its super adhesion to biological tissues such as cornea, aorta, heart, muscle, kidney, liver, and spleen suggest widespread applications of this hydrogel in many biomedical areas such as transplantation, tissue adhesive, wound dressing, bioprinting, and drug and cell delivery.

Toward electron-beam sterilization of a pre-assembled Boston keratoprosthesis.

PURPOSE: To evaluate the effects of electron-beam (E-beam) irradiation on the human cornea and the potential for E-beam sterilization of Boston keratoprosthesis (BK) devices when pre-assembled with a donor cornea prior to sterilization. METHODS: Human donor corneas and corneas pre-assembled in BK devices were immersed in recombinant human serum albumin (rHSA) media and E-beam irradiated at 25 kGy. Mechanical (tensile strength and modulus, and compression modulus), chemical, optical, structural, and degradation properties of the corneal tissue after irradiation and after 6 months of preservation were evaluated. RESULTS: The mechanical evaluation showed that E-beam irradiation enhanced the tensile and compression moduli of human donor corneas, with no impact on their tensile strength. By chemical and mechanical analysis, E-beam irradiation caused a minor degree of crosslinking between collagen fibrils. No ultrastructural changes due to E-beam irradiation were observed. E-beam irradiation slightly increased the stability of the cornea against collagenase-induced degradation and had no impact on glucose diffusion. The optical evaluation showed transparency of the cornea was maintained. E-beam irradiated corneal tissues and BK-cornea pre-assembled devices were stable for 6 months after room-temperature preservation. CONCLUSIONS: E-beam irradiation generated no detrimental effects on the corneal tissues or BK-cornea pre-assembled devices and improved native properties of the corneal tissue, enabling prolonged preservation at room temperature. The pre-assembly of BK in a donor cornea, followed by E-beam irradiation, offers the potential for an off-the-shelf, ready to implant keratoprosthesis device.

Electron Beam Sterilization of Poly(Methyl Methacrylate)-Physicochemical and Biological Aspects.

Electron beam (E-beam) irradiation is an attractive and efficient method for sterilizing clinically implantable medical devices made of natural and/or synthetic materials such as poly(methyl methacrylate) (PMMA). As ionizing irradiation can affect the physicochemical properties of PMMA, understanding the consequences of E-beam sterilization on the intrinsic properties of PMMA is vital for clinical implementation. A detailed assessment of the chemical, optical, mechanical, morphological, and biological properties of medical-grade PMMA after E-beam sterilization at 25 and 50 kiloGray (kGy) is reported. Fourier transform infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry studies indicate that E-beam irradiation has minimal effect on the chemical properties of the PMMA at these doses. While 25 kGy irradiation does not alter the mechanical and optical properties of the PMMA, 50 kGy reduces the flexural strength and transparency by 10% and 2%, respectively. Atomic force microscopy demonstrates that E-beam irradiation reduces the surface roughness of PMMA in a dose dependent manner. Live-Dead, AlamarBlue, immunocytochemistry, and complement activation studies show that E-beam irradiation up to 50 kGy has no adverse effect on the biocompatibility of the PMMA. These findings suggest that E-beam irradiation at 25 kGy may be a safe and efficient alternative for PMMA sterilization.

Photo-cross-linked Gelatin Glycidyl Methacrylate/N-Vinylpyrrolidone Copolymeric Hydrogel with Tunable Mechanical Properties for Ocular Tissue Engineering Applications.

Corneal transplantation is currently the primary treatment for corneal blindness. However, severe global scarcity of donor corneas is driving the scientific community to find novel solutions. One potential solution is to replace the damaged tissue with a biocompatible artificial cornea. Here, gelatin glycidyl methacrylate (GM) and N-vinylpyrrolidone (VP) were cocrosslinked to afford a hybrid bicomponent copolymeric hydrogel with excellent mechanical, structural, and biological properties. Our studies showed that the GM/VP ratio can be adjusted to generate a construct with high tensile modulus and strength of 1.6 and 1.0 MPa, respectively, compared to 14 and 7.5 MPa for human cornea. The construct can tolerate up to 22.4 kPa pressure before retention sutures can tear through it. Due to the presence of a synthetic component, it has a significantly higher stability against collagenase induced degradation, yet it is biocompatible and promotes cellular adhesion, proliferation, and migration under in vitro settings.

Graphene-Lined Porous Gelatin Glycidyl Methacrylate Hydrogels: Implications for Tissue Engineering.

Despite rigorous research, inferior mechanical properties and structural homogeneity are the main challenges constraining hydrogel's suturability to host tissue and limiting its clinical applications. To tackle those, we developed a reverse solvent interface trapping method, in which organized, graphene-coated microspherical cavities were introduced into a hydrogel to create heterogeneity and make it suturable. To generate those cavities, (i) graphite exfoliates to graphene sheets, which spread at the water/ heptane interfaces of the microemulsion, (ii) heptane fills the microspheres coated by graphene, and (iii) a cross-linkable hydrogel dissolved in water fills the voids. Cross-linking solidifies such microemulsion to a strong, suturable, permanent hybrid architecture, which has better mechanical properties, yet it is biocompatible and supports cell adhesion and proliferation. These properties along with the ease and biosafety of fabrication suggest the potential of this strategy to enhance tissue engineering outcomes by generating various suturable scaffolds for biomedical applications, such as donor cornea carriers for Boston keratoprosthesis (BK).

Sputter Deposition of Titanium on Poly(Methyl Methacrylate) Enhances Corneal Biocompatibility.

Purpose: To evaluate titanium (Ti) sputtering of the poly(methyl methacrylate) (PMMA) stem of the Boston Keratoprosthesis (BK) as a method to enhance interfacial adhesion between the PMMA and the recipient corneal tissue. Methods: PMMA specimens were plasma treated with Ar/O2 and coated with Ti using a DC magnetron sputtering instrument. The topography and hydrophilicity of the surfaces were characterized using atomic force microscopy and a water contact angle instrument, respectively. Scratch hardness and adhesion of the Ti film were measured using a mechanical tester. Biocompatibility assessments were performed using cultured human corneal fibroblasts and whole blood ex vivo. The optical quality of the Ti sputtered BK was evaluated using a custom-made optical bench. Results: By contact angle studies, the Ti coating improved PMMA hydrophilicity to match that of medical-grade Ti (Ti-6Al-4V-ELI). Ti sputtering of contact surfaces resulted in a plate-like morphology with increased surface roughness, without impacting the transparency of the BK optical component. Scratch testing indicated that the mechanical behavior of the Ti coating was similar to that of casted Ti, and the coating was stable in pull-off adhesion testing. Sputtered Ti film was highly biocompatible based on tests of cell viability, adhesion, proliferation, differentiation, collagen deposition, and keratocan expression, the properties of which exceeded those of uncoated PMMA and did not induce increased complement activation. Conclusions: Titanium coating of the BK stem generated a mechanically and biologically favorable interface, which may help to enhance corneal stromal adhesion and biocompatibility. Translational Relevance: Improving the biocompatibility of the BK PMMA stem may improve long-term outcomes of implantation.