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R. A. Charalel, K. Engberg, J. Noolandi, J. Cochran, C. Frank, C. N. Ta; Diffusion of Protein Through the Human Cornea. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1748.
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To determine the rate of diffusion of myoglobin and bovine serum albumin (BSA) through the human cornea. These small proteins have hydrodynamic diameters of approximately 44Å and 72Å for myoglobin and BSA, respectively. The diffusional properties of the human cornea are important to understand as part of the design for an artificial cornea that supports surface epithelialization. The epithelium and keratocytes of the cornea receive their nutrients from the aqueous chamber via diffusion through the corneal stroma. Therefore, to develop an artificial cornea that mimics the human cornea, it is desirable to understand whether or not protein diffusion is necessary.
Diffusion coefficients were measured using a diffusion chamber (Fig. 1) with a 5mg/ml solution of the protein of interest in chamber 1 and balanced salt solution (BSS) in chamber 2 separated only by an ex vivo human cornea. Protein concentrations in chamber 2 were then measured over at least one week using the Invitrogen Qubit system. Diffusion coefficients were calculated using basic diffusion equations derived from Fick’s law and conservation of mass in a closed system (Lee CJ, et al, 2006). These equations account for variable parameters such as thickness of the cornea, volume of solution in each chamber and surface area through which 1-D diffusion occurs.
Our experiments demonstrate that at 37 degrees Celsius, myoglobin diffuses through the cornea with a diffusion coefficient of 5.58E-8 cm2/s (n=8, SD = 2.44E-8 cm2/s, 95%CI: 3.89E-8 to 7.27E-8 cm2/s) whereas BSA does not diffuse through the cornea at any appreciable rate (n=5). Compared to the diffusion coefficient of glucose (~2E-6 cm2/s), the diffusion coefficient of myoglobin is about 100 fold less for the human cornea.
Our study suggests that neutral molecules smaller than 44Å are able to passively diffuse through the human cornea while molecules larger than 72Å do not. Further experiments are warranted to fully understand the limits of human corneal diffusion and its clinical relevance.
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