Elizabeth Orwin

Biomechanical and Optical Characteristics of a Corneal Stromal Equivalent

Cell matrix interactions are important in understanding the healing characteristics of the cornea after refractive surgery or transplantation. The purpose of this study was to characterize in more detail the evolution of biomechanical and optical properties of a stromal equivalent (stromal fibroblasts cultured in a collagen matrix). Human corneal stromal fibroblasts were cultured in a collagen matrix. Compaction and modulus were determined for the stromal equivalent as a function of time in culture and matrix composition. The corneal stromal fibroblasts were stained for alpha-smooth muscle actin expression as an indicator of myofibroblast phenotype. The nominal modulus of the collagen matrix was 364 +/- 41 Pa initial and decreased initially with time in culture and then slowly increased to 177 +/- 75 Pa after 21 days. The addition of chondroitin sulfate decreased the contraction of the matrix and enhanced its transparency. Cell phenotype studies showed dynamic changes in the expression of alpha-smooth muscle actin with time in culture. These results indicate that the contractile behavior of corneal stromal cells can be influenced by both matrix composition and time in culture. Changes in contractile phenotype after completion of the contraction process also indicate that significant cellular changes persist beyond the initial matrix-remodeling phase.

The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods

The field of corneal tissue engineering has made many strides in recent years. The challenges of engineering a biocompatible, mechanically stable, and optically transparent tissue are significant. To overcome these challenges, researchers have adopted two basic approaches: cell-based strategies for manipulating cells to create their own extracellular matrix, and scaffold-based strategies for providing strong and transparent matrices upon which to grow cells. Both strategies have met with some degree of success. In addition, recent advances have been made in innervating a tissue-engineered construct. Future work will need to focus on further improving mechanical stability of engineered constructs as well as improving the host response to implantation.

Office-based optical coherence tomographic imaging of human vocal cords.

Optical coherence tomography (OCT) is an evolving noninvasive imaging modality and has been used to image the larynx during surgical endoscopy. The design of an OCT sampling device capable of capturing images of the human larynx during a typical office based laryngoscopy examination is discussed. Both patients and physicians movements were addressed. In vivo OCT imaging of the human larynx is demonstrated. Though the long focal length limits the lateral resolution of the image, the basement membrane can still be readily distinguished. Office-based OCT has the potential to guide surgical biopsies, direct therapy, and monitor disease. This is a promising imaging modality to study the larynx.

Characterizing the effects of aligned collagen fibers and ascorbic acid derivatives on behavior of rabbit corneal fibroblasts

The cornea is responsible for functional optical activity of the mammalian eye, as it must remain transparent in order to focus light onto the retina. Corneal disease is the second leading cause worldwide of vision loss [1]. Human donor tissue transplantation in the cornea is associated with problems such as immunorejection and recurring graft failures [1]. Tissue engineering offers a promising alternative to using human donor tissues in treating corneal diseases. A viable tissue-engineered cornea must be mechanically resilient to protect the fragile intraocular components of the eye, and optically transparent to refract light onto the retina. In the native cornea, transparency is maintained by both the cells in the stromal layer and the high organization of the extracellular matrix (ECM). This study aims to combine the effects of aligned collagen fibers and ascorbic acid derivatives to control corneal fibroblast behavior to not only express the appropriate proteins, but also to deposit aligned, small diameter collagen fibers that resemble the highly organized structure of the natural ECM. Results from this study suggest that the combined effect of an aligned scaffolding material and ascorbic acid supplements can create a cell-matrix construct that both downregulates expression of the light scattering protein a-smooth muscle actin (α-sma) and supports an increased number of cell layers.

Immunogold labeling to enhance contrast in optical coherence microscopy of tissue engineered corneal constructs

Our lab has used an optical coherence microscope (OCM) to assess both the structure of tissue-engineered corneal constructs and their transparency. Currently, we are not able to resolve cells versus collagen matrix material in the images produced. We would like to distinguish cells in order to determine if they are viable while growing in culture and also if they are significantly contributing to the light scattering in the tissue. In order to do this, we are currently investigating the use of immunogold labeling. Gold nanoparticles are high scatterers and can create contrast in images. We have conjugated gold nanoparticles to antibodies specific to the /spl alpha//sub 5//spl beta//sub 1/ integrins expressed in some corneal cells. This choice of target should allow assessment of the phenotypic behavior of the cells in the tissue, as different integrins are expressed in different phenotypes. This study applies the immunogold technique to study cultured corneal cells using the OCM with the ultimate goal of monitoring cellular behavior in engineered tissue and corroborating results from standard histological methods.

Optical coherence microscopy for the evaluation of a tissue-engineered artificial cornea

A transparent artificial cornea derived from biological material is the ultimate goal of corneal research. Attempts at artificial corneal constructs produced from synthetic polymers have proved unsuccessful due to lack of biocompatibility and ability to integrate into the tissue. We have designed a corneal model derived from collagenous biological materials that has several advantages: it has low antigenicity and therefore small chance of eliciting an immune reaction, it can be broken down by the bodys own cells and gradually replaced over time by natural materials, and it may contain signaling information for native cells, thereby inducing normal phenotype and behavior. In addition, a transparent corneal model has the potential to be used for testing of novel ophthalmic drugs or gene therapy approaches, eliminating the need for animal testing. We have used an optical coherence microscope (OCM) to evaluate both the structure of our tissue constructs over time in culture and the optical properties of the tissue itself. This imaging technique promises to be an important diagnostic tool in our efforts to understand the influence of mechanical forces, cell phenotype, and soluble factors on the transparency of corneal tissue.

Physical attributes and assembly of PEG-linked immuno-labeled gold nanoparticles for OCM image contrast in tissue engineering and developmental biology

Excessive nonspecific binding often occurs when labeling cells with immuno-labeled gold nanoparticles (IgG-AuNPs). We have investigated the physical properties of IgG-AuNPs assembled with three different protocols in an attempt to understand and eliminate this non-specific binding. One of these protocols involves conjugating the secondary antibody AP124F via van der Waals (vdW) and/or electrostatic forces to the AuNPs, and the other two employ a PEG-linker, OPSS-PEG-NHS (OPN). In all three protocols we follow with PEG-SH to provide protection against aggregation in saline solution. OPN and PEG-SH chains of varying molecular weights were examined in different combinations to determine the optimally protective layer. The hydrodynamic radius and surface plasmon resonance (SPR) were monitored at each stage of assembly using a dynamic light scattering (DLS) instrument and spectrophotometer, respectively. SPR measurements indicate a different physical structure near the gold surface when the PEG-linker is bound to gold first and then bound to the antibody second (AP124F-[OPN-Au]) rather than vice versa ([AP124F-OPN]- Au). These observed structural differences may lead to differences in the amount of non-specific binding observed when immuno-labeling cells. SPR measurements also yielded a half-life of 27 minutes for the binding of the PEG-linker to the surface of the AuNPs and a half-life of 133 minutes for the hydrolysis of the NHS functional groups on the OPN molecule. These different reaction rates led us to add AP124F 40 minutes after the linker began binding to the AuNPs, so that the antibody can bind covalently to the correct end of the OPN linker.