Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability

Abstract

Purpose: To evaluate porcine sclera as a model of human sclera for in vitro studies of transscleral drug delivery of both low and high molecular weight compounds.

Methods: Human and porcine scleras were characterized for thickness and water content. The tissue surface was examined by scanning electron microscopy (SEM), and the histology was studied with hematoxylin-eosin staining. Comparative permeation experiments were performed using three model molecules, acetaminophen as the model compound for small molecules; a linear dextran with a molecular weight of 120 kDa as the model compound for high molecular weight drugs; and insulin, which was chosen as the model protein. Permeation parameters such as flux, lag time, and permeability coefficient were determined and compared.

Results: Human and porcine scleras have a similar histology and collagen bundle organization. The water content is approx 70% for both tissues while a statistically significant difference was found for the thickness, porcine sclera being approximately twofold thicker than human sclera. Differences in thickness produced differences in the permeability coefficient. In fact, human sclera was found to be two to threefold more permeable toward the three molecules studied than porcine sclera.

Conclusions: The results obtained in the present paper prove that porcine sclera can be considered a good model for human sclera for in vitro permeation experiments of both low and high molecular weight compounds. In fact, if the different tissue thickness is taken into account, comparable permeability was demonstrated. This suggests a possible use of this model in the evaluation of the transscleral permeation of new biotech compounds, which currently represent the most innovative and efficient therapeutic options for the treatment of ocular diseases.

Figure 1

Histological microscopic sections of human and porcine sclera stained with hematoxylin-eosin. Empty lacunae between fibers are an artifact due to tissue preparation.  Original magnification was 10X in panel A and 4X in panel B. In both human and porcine scleras, scattered small fibrocyte nuclei are dispersed between the bundles of interwoven collagen fibers. In porcine sclera, thicker and more disorganized collagen bundles are visible. From the 4X magnification (B) it is possible to appreciate differences in thickness between human and porcine sclera.

Figure 2

SEM image of the outer region of human and porcine sclera. Original magnification was 5,000X in panel A and 20,000X in panel B. Scale bar=10 µm in both cases. From the pictures at lower magnification (A) it is possible to observe branching and anastomosis of the collagen bundles to form dense connective tissue. The bundles were of varying thickness and width and often intertwined with each other. Porcine bundles looked thicker at lower magnification, but still showed a similar arrangement. Moreover, higher magnification (B) did not show any difference between human and porcine sclera in the diameter of the single collagen fibers

Figure 3

Permeation profiles of acetaminophen, FD-150, and insulin for porcine and human sclera. Profiles for acetaminophen (A), FD-150 (B), and insulin (C) through porcine (○)  and human (●) sclera are shown. Open squares (□) in panel A indicate the permeation profile of acetaminophen through fresh porcine sclera. Transscleral fluxes obtained for all three molecules tested were higher for human sclera. In particular, the fluxes through human sclera were 2 times greater than through pig sclera in the case of acetaminophen and insulin, and 3 times greater in the case of FD-150. Permeability of acetaminophen through fresh porcine sclera confirmed that the freezing procedure did not have any effect on tissue permeability. Mean value ±SEM