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W. M. Petroll, A. Kim, N. Lakshman; An in vitro Model for Investigating Intrastromal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4554.
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© ARVO (1962-2015); The Authors (2016-present)
We previously described a novel 3-D culture model using compressed collagen matrix that mimics corneal stromal mechanical properties, while supporting keratocyte differentiation and growth factor responsiveness. In this study, we investigate whether this model can be used as a platform for studying intrastromal wound healing in vitro.
Rabbit corneal keratocytes were isolated and cultured in serum-free media. To prepare compressed collagen matrices, a 6 ml solution containing cells and type I rat tail collagen (2 mg/ml) was poured into a 3x2x1 cm well, and allowed to polymerize for 1 hour at 37°C. Following polymerization, the matrix was compacted by using compression with a 130g stainless steel block for 5 minutes at room temperature. Following compression, 6mm diameter buttons were punched out of the compressed matrix and incubated in serum-free media for 24 hours to allow cell spreading. We then simulated an injury by either: (1) pushing on the surface near the center of the matrices for approximately 1 second using a 3mm diameter stainless steel probe, or (2) immersing the same probe in liquid nitrogen for 20 seconds, then placing the tip against the surface of the construct for 5 seconds using only gravitational force. After an additional 24 hours of culture in serum-free media, constructs were stained using a standard Live/Dead assay.
Corneal keratocytes maintained in serum-free media within compressed matrices had a broad, convoluted cell body and numerous thin dendritic processes. Following cell spreading, keratocytes within compressed matrices were susceptible to compressive loading. A region of cell death was observed in the center of the constructs, whereas the surrounding cells remained viable. Thermal injury induced a larger area of cell death within the matrix, and the transition between the live and dead cell regions was less abrupt than that observed following compressive injury. We also imaged samples 4 days after injury, and found a mixture of live and dead cells within the boundaries of the "artificial wound", suggesting activation and migration of surrounding viable cells into this area.
Refractive surgical procedures such as PRK and LASIK can induce localized keratocyte death within intact stromal tissue via apoptosis or necrosis. The response of the surrounding keratocytes can have a significant impact on corneal transparency and refractive outcome. Compressed collagen matrices support keratocyte differentiation and may provide a unique 3-D platform for investigating both the mechanisms of cell death and wound repopulation following these "intrastromal" wounds.
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