$386 Journal of Biomechanics 2006, Vol. 39 (Suppl 1)

used to test hypotheses about the failure mechanisms of the tissues supporting the nerves at the ONH as we explore the mechanistic causes for loss of vision in glaucoma.

20.1.2. Biomechanics and Cell and Tissue Engineering of the Anterior Seg- ment

7217 Mo, 11:00-11:15 (P9) Traditional and ultrasonic mechanical analysis of porcine iris

J.E. Whitcomb 1, D. Liu 2, E. Ebbini 3, V.H. Barocas 2. 1Dept efMechanical Engineering, University of Minnesota, Minneapolis, MN, USA, 2Dept of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA, 3 Dept of Electrical Engineering, University of Minnesota, Minneapolis, MN, USA

The iris is known to deform considerably due to the combined effects of anterior-posterior pressure difference, near-contact with the lens, and mus- cular activity. Our goal of understanding how these various effects contribute to the iris contour requires a good mechanical description of the iris. Me- chanical characterization is extremely difficult, however, and the best available data to date were from cadaveric bovine tissue two days post mortem. In the current study, we have used porcine tissue tested shortly post mortem, and two different techniques were used. First, direct pulling of the iris was employed, providing access to large strains but presenting difficulty because of the inherently non-uniform strain field. A second strategy was also used, in which a focused ultrasound wave was used to apply a small localized force within the tissue, and a second ultrasonic transducer was used to monitor the resulting local displacement field. The force~:fisplacement relationship was then combined with a linear elastic model (displacements o f -10urn) , and results compared to the traditional measurements. An additional attractive feature of the ultrasonic measurement is that the tests could be performed on an intact globe, which allowed less damage to the tissue during preparation and made possible administration of drugs to study how they affect the mechanical properties of the iris. A major concern with the ultrasound method is that the analysis is based on the assumption of an infinite medium, but the porcine iris is only a few hundred microns thick; finite element simulations were performed to generate an approximate correction factor for thin samples.

5433 Mo, 11:15-11:30 (P9) Modeling the human cornea - a stromal tissue constitutive model based on measured collagen architecture P.M. Pinsky 1 , D. van der Heide 1 , D. Chernyak 2, K.M. Meek 2, C. Boote 2. 1Stanford University, Stanford, CA, USA, 2AMO Inc., Santa Clara, CA, USA, 3 Cardiff University, Cardiff, UK

Disturbances of the stromal microstructure, occurring in refractive surgical procedures such as LASIK, may create unexpected and undesired changes to the vision quality of the eye. With the advent of wavefront-guided LASIK, enabling highly precise measurement and treatment of the defects of the eye's entire visual system, it has become crucial that surgical planning include knowl- edge of the cornea's mechanical response to the procedure. The mechanical properties of the cornea derive from the specialized architectural arrangement of its collagen fibrils. The preferred orientation of collagen fibrils throughout the cornea, limbus, and adjacent sclera has recently been mapped using synchrotron X-ray scattering and shows the collagen to have a striking degree of anisotropy in its arrangement. A mathematical description of the data has been derived providing a probability distribution for collagen fibril orientation. A constitutive model for a corneal lamella, containing parallel arrays of collagen fibrils, is based on a mixture theory which accounts for the strain energy of the oriented collagen, the water-like extra-fibrillar matrix and the cross-linking energy of the proteoglycans. The stromal tissue model is found by weighting the lamella strain energy by the position-dependent probability distribution for the collagen fibril orientation. The stromal elasticity tensor then has an anisotropy that reflects the geometric anisotropy of the collagen orientation. The elastic constants were calibrated using clinical data for radial keratotomy. The cornea is a prestressed by the action of the lOP. A finite element modeling approach has been developed to determine the prestress and allow true mod- eling of the surgical process whereby stress is redistributed by surgical cutting of stressed tissue. Results are expressed in Zernike coefficients, obtained via an SVD approach, and provide measures of optical power and aberrations in the optic zone. The model has been employed to simulate: (i) tunnel incisions in the sclera made for cataract extraction, (ii) microkeratome cutting of LASIK flaps, and (iii) myopic LASIK procedures.

Oral Presentations

6983 Mo, 11:30-11:45 (P9) Spatial organization of engineered corneal stroma: Is there a need for contact guidance or direct mechanical stimulus? J.D. Zieske 1 , X.Q. Guo 1 , S.A. Melotti 2, A.E. Hutcheon 1 , J.W. Ruberti 1,2. 1Schepen's Eye Research Institute, Boston, MA, USA, 2Mechanical and Industrial Engineering, Northeastern University, Boston, MA, USA

Purpose: The corneal stroma comprises a nematic stack of alternating collagen lamellae which contain arrays of aligned, evenly-spaced, uniform collagen fibrils. This architecture, which is organized locally and globally, is responsible for the mechanical strength (global organization) and transparency (local organization) of the cornea. A significant challenge in corneal tissue engineering is controlling both the local and global organization of secreted corneal stromal matrix. In this investigation, we characterize the intrinsic ability of primary human corneal fibroblasts (stimulated with 2-O-a-D-Glucopyranosyl- L-Ascorbic acid) to assemble a locally and globally organized ECM. Methods: Primary human corneal fibroblasts were cultured onto polyester transwells and stimulated to produce matrix with a stable form of ascorbic acid. The cell and matrix organization in the living tissue were characterized by confocal and differential interference contrast (DIC) imaging at four, eight and twelve weeks. Results: Confocal and DIC microscopy detected consistent 3-dimensional vertical organization within the stromal constructs. There were 4 identifiable strata, beginning at the transwell membrane (1) a thin, dense, locally aligned layer of spindle shaped cells (2) a thick layer populated with few cells and comprising aligned "lamellar" sheets of matrix (3) several layers of cells with random orientation (4) a layer of loosely attached cells. Construct total thickness averaged 36 (sd 6.6) microns at four weeks and 50 (sd 17.7) microns thick at eight weeks (not statistically significant). The layer of aligned lamellar matrix was 25 microns both at four weeks (sd 6.4) and at eight weeks (sd 12.4). At twelve weeks, lateral scans using DIC imaging demonstrated that the local uniform alignment of the cells adjacent to the transwell membrane did not hold globally. Conclusions: DIC and confocal imaging revealed that the primary human corneal fibroblasts produce constructs with consistent vertical stratification and local organization. However, lateral scans on the twelve week specimens indicate that the local orientation of cells adjacent to the transwell membrane (which potentially control matrix organization) varies over longer distances. These results imply that an externally applied organizing signal such as contact guidance or mechanical load might be necessary to control cell orientation globally.

5342 Mo, 11:45-12:00 (P9) Nucleation, growth and alignment of collagen fibrils produced by shear-influenced self-assembly for corneal tissue engineering templates N. Saeidi, J. Ruberti. Northeastern University, Mechanical and Industrial Engineering, Boston, USA

Purpose: The corneal stroma comprises a nematic stack of highly monodis- perse, uniformly-spaced, oriented, collagen fibrils arranged in thin lamellae which alternate in direction. To duplicate this structure de neve for the purpose of corneal tissue engineering requires exquisite control over the self-assembly of collagen. Our efforts to shear-align assembling collagen via spin coating have been hampered by the lack of data on collagen self-assembly and alignment kinetics in flow fields. This investigation demonstrates direct optical observation of collagen self-assembly on glass under the influence of a shearing flow. Methods: Extracted type I bovine collagen monomers (3.0 mg/mL) were in- jected into a temperature-controlled microfluidics chamber mounted on the stage of an inverted microscope. Following an initial nucleation period, flow was restarted to produce varying shear rates. Nucleation, growth and alignment of collagen fibrils on the surface of the glass bottom of the chamber were followed dynamically with Differential Interference Contrast (DIC) imaging. The results of the flow chamber experiment were used to guide a second experimental series designed to produce aligned collagen via spin-coating. Results: Dynamic DIC imaging demonstrated that observable nucleation (in the absence of flow) begins at approximately 150 seconds. At shear rates of (264/sec) growth rates for fibrils were 8.79 microns/min (±3.2) and fibril alignment was observed. This information was used to design a spin coating experiment where collagen monomers were injected onto a glass disk, allowed to prenucleate for 4 minutes, followed by spinning (@200 rpm) to produce shearing flow (264/sec). DIC imaging of collagen arrays on the spin-disk demonstrated large areas of uniform, aligned fibrils. Conclusions: Using dynamic DIC imaging of "shear-influenced" collagen assembly to guide spin coating experiments has the potential to accelerate and refine our production of highly-organized natural templates and scaffoldings for corneal tissue engineering.