A new laboratory model using bull and boar spermatozoa and fluorescent beads to assess a membrane’s occlusive potential

The objective of the present study is to assess the potential of bull and boar spermatozoa and fluorescent beads to be used as a surrogate cell model to determine the cell occlusive potential in vitro using membranes of standardized porosities.

A two-chamber model system consisting of upper and lower chambers, which could be separated by membranes, was constructed. Isopore polycarbonate membranes with different standardized pore diameters were used to assess the mobile cellular penetration behavior of spermatozoa or the more passive non-cellular permeability of fluorescent particles (beads) of different diameter and color. In a first experiment, spermatozoa were placed in the lower chamber, whereas semen extender only was placed in the upper chamber. After 10 min of incubation at 37 °C, the sperm number was assessed in the latter. In a second experiment, a bead solution was drawn through resorbable collagen membranes from the upper into the lower chamber by vacuum using a syringe and bead number and size was analyzed by flow cytometry. All experiments were carried out in triplicates. A non-porous polyester membrane was used as negative control to assess the overall tightness of the setup.

Boar and bull spermatozoa had average cell body lengths and widths of 9 × 5 μm and were unable to pass through pores ≤2 μm, whereas they were detectable at pore sizes ≥3 μm. Their number increased with increasing pore diameters, i.e., from minimal concentrations of 0.1 × 10⁶/ml for boar and 0.5 × 10⁶/ml for bull spermatozoa at 3 μm to maximal concentrations of 2.1 × 10⁶/ml for boar and 13.1 × 10⁶/ml for bull spermatozoa at 8 μm. The fluorescent beads followed the expected pattern of permeability reliably correlating bead and pore diameter.

Within the limitations of this laboratory study and the xenogeneic cell surrogate material, the model allows to easily assess cell and particle penetration through porous structures like membranes. We hope to further assess, improve, and validate this model, which we aim to use for the screening of dental membranes after being exposed to different degradation methods.

Convenient and rapid test procedures to evaluate membranes for regenerative procedures are still warranted.