Characterization of a Novel Collagen Scaffold for Corneal Tissue Engineering.

Globally millions are blind due to corneal disease, yet tissue for transplantation is a limited resource. This study characterizes the physical and biological properties of a novel collagen-based scaffold. Transparency, optical coherence tomography (OCT), and scanning electron microscopy (SEM) were used to analyze the structure of the scaffold, synthesized using rat tail collagen I. Water content was determined. The tensile strength was assessed using a micro-mechanical analyzer. In vitro biocompatibility was assessed by culturing the scaffold with epithelial or keratocyte spheres. The mean scaffold transmittance was 0.72 at 358 nm, 0.88 at 570 nm, and 0.92 at 900 nm. OCT imaging confirmed that the scaffold maintained a corneal shape, with a central thickness of 502 µm and a reflectivity profile comparable to that of a normal human cornea. SEM of the scaffold revealed multiple lamellae on cross section. The mean water content was 88.7% ± 0.7%. Ultimate tensile strength for the noncross-linked scaffold was 1.23 ± 0.27 MPa compared with 2.21 ± 0.70 MPa for the cross-linked scaffold (human corneal anterior stroma 1.53 ± 0.86 MPa) at a strain rate of 0.5%/s. Epithelial cells migrated over the scaffold to confluence. Keratocytes populated the scaffold and maintained a lamellar arrangement. The properties of this novel scaffold suggest that it has potential to be developed into a corneal tissue substitute for human transplantation.

Functional imaging: new views on lens structure and function.

1. We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2. Using this approach in the rat lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3. Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4. Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5. Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins.

Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.

PURPOSE: To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS: Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.