Designing Novel Biomaterials for Cornea Replacement

Artificial corneas, also known as keratoprostheses (KPros), are an alternative cornea replacement therapy when donor cornea is either inappropriate or inaccessible to a patient. KPro recipients often have preexisting ocular diseases, ocular tissue injury, or a history of corneal graft failure. While a number of KPro models are available on the market, each fails to completely address one or more of the following vital parameters: host integration, mass transport, tissue epithelialization, or innervation. This study presents biomaterials that can potentially improve stable host integration and long term vision restoration. KPros made with poly (2-hydroxyethyl methacrylate) (PHEMA) - poly (methyl methacrylate) (PMMA) (PHEMA-PMMA) and copolymers made with of PHEMA and PEG (polyethylene glycol) are structurally, mechanically, and biologically appropriate for corneal replacement. The center region of the KPro is optically transparent to permit vision; the outer rim of the device is porous to permit tissue ingrowth. Following the addition of cell adhesion components (collagen type I) human corneal fibroblasts proliferated on the porous skirt structures. Mechanical tensile data indicated that the KPro structures are strong enough to resist rupture under the mechanical forces of the eye. PHEMA-PMMA based KPros are mechanically stable enough to be sutured in as full thickness corneal replacement devices. PHEMA and PEG copolymers, mechanically weaker than PHEMA-PMMA, are better suited for suture-less cornea replacement strategies. SEM and Micro CT data show that the pores in the skirts are large enough to permit cell ingrowth. In vivo, healthy tissues penetrate the voids of the porous copolymers. Since diseased ocular tissue is more reluctant to colonize synthetic biomaterials compared to natural-synthetic hybrids, the each skirt’s amenability to cell adhesion component addition is vital to the survival of the KPro in the target patient population. A cell adhesive KPro skirt with a high density of cell permeable pores and physiologically relevant mass transport could potentially facilitate the stable integration of a KPro into ocular tissue

Tensile testing showed that the porogens can significantly alter the mechanical properties.

<p>The PHEMA-PEGDA made with water porogen was significantly stiffer than samples made with benzyl alcohol porogen. Low damping factors (tan (δ)) show that the PHEMA-PEGDA samples are viscoelastic with dominant elastic properties (B; n = 3). Tension was applied until rupture to determine the ductility and overall strength of the scaffolds (C). “X” marks the failure or rupture point of each sample. Horizontal bars identify samples with a statistically significant difference (p<0.05).</p

SEM images of PHEMA-PEGDA scaffolds in low (A) and high (B) magnification.

<p>By increasing the content of the total deionized water content by 7%, average pore area was substantially increased. Data from alamarBlue shows that HCFs proliferated significantly in this structure from day 4 to 20 (C, p<0.05, n = 3). Fluorescent images show that, at day 8, live HCFs were clearly visible in this scaffold with essentially minimal cell death (D).</p

Collagen type I coating the PHEMA-PEGDA scaffolds was immunostained using collagen antibody.

<p>Scaffolds were removed from the tissue culture wells and rinsed with PBS. (A) Collagen gelled within the micron sized surface pores of PHEMA-PEGDA using the water porogen. Collagen fibers were evident in the sucrose and benzyl alcohol PHEMA-PEGDA scaffolds (B and C, respectively). At week 2, HCFs were detected in the three different PHEMA-PEDGA scaffold types, and average DNA contents were quantitatively measured (D; n = 6). “*” indicates p<0.05, and error bars represent standard error. Cell viability staining at day 7 shows both live (green) and a significant number of dead (red) HCFs in the benzyl alcohol PHEMA-PEGDA scaffold (E).</p

Porogens used to generate pores in the PHEMA-PEGDA include water (W), sucrose solution (S), and benzyl alcohol (BOH).

<p>This table shows the % v/v quantity of components used to create the PHEMA-PEGDA structures. The deionized water content (% v/v) listed shows the total aqueous content of the final pre-polymerized mix of monomers and other agents. As porogen types and volumes were adjusted, the quantities of PHEMA and PEGDA monomers remained constant.</p

SEM images show dehydrated PHEMA-PEGDA.

<p>Water (A), sucrose (B), and benzyl alcohol (C) were used as porogens. Low magnification SEM images are displayed in the top row. Corresponding SEM images with higher magnification are shown in the bottom row. Image analysis was used to measure pore areas. Histograms display pore areas for the scaffolds made with water (D, n = 558), sucrose (E, n = 12,533), or benzyl alcohol (F, n = 11,485) porogens.</p

NMR spectroscopy results indicate that PEG chains appear to have leached out of the PHEMA-PEGDA scaffold following 3 days soaking in PBS.

<p>The signal for the middle O-CH2 group of PEGDA (A) and the middle O-CH2 group of PEG (B) were both found at approximately 70 ppm. Signals that represent the repeating side groups in PHEMA were not observed in PBS, however, indicating neither HEMA nor PHEMA leached out.</p