In Vitro Evaluation of the Permeability of Different Resorbable Xenogeneic Membranes after Collagenolytic Degradation

Abstract

In this in vitro study, we compare the penetration of cells through different resorbable collagen membranes, which were collagenolytically degraded over different time periods. Three different resorbable collagen membranes were evaluated, including two non-cross-linked (NCL) membranes—namely, a porcine (NCL-P) and an equine (NCL-E) membrane—and an enzymatically cross-linked porcine (ECL-B) membrane. A special two-chamber model was fabricated, allowing for the placement of separating membranes, and a non-porous polyester membrane was used as a negative control (C), in order to verify the impermeability of the experimental chamber device. Round membrane samples with a diameter of 16 mm were fabricated. Eighteen membranes of each type were punched and placed on polyethylene nets as carriers. The membranes were then biodegraded—each on its carrier—in 12-well polystyrene plates: three samples of each membrane type were degraded for 1.5, 3, 6, or 12 h in 2 mL of a buffered collagenase solution, at 37 °C. For control purposes, three samples of each membrane type were not degraded, but only immersed in buffer solution for 1.5, 3, 6, or 12 h, at 37 °C. Another three samples of each type of membrane were degraded until complete dissolution, in order to determine the full hydroxyproline content for comparison. Liquid-preserved boar semen (containing at least 120 million sperm cells per milliliter) was used to test the cell occlusivity of the degraded membranes. At baseline and initial degradation, all tested membranes were tight, and no penetration was observed with up to 30 min of incubation time (results not shown). After 1.5 h, cells were partially capable of penetrating the NCL-E membrane only. One sample showed leakage, with a sperm volume of 1.7 million cells/mL over all samples. No penetration occurred in the test, NCL-P, and ECL-B groups. After a degradation time of 3 h, the NCL-P and ECL-B membranes remained occlusive to cells. All the membranes and measurements indicated leakage in the NCL-E group. After 6 h, four NCL-P measurements showed the first signs of cell penetration, as boar spermatozoa were detectable in the lower chamber (64 million cells/mL). The ECL-B membranes remained completely cell occlusive. After 12 h, four NCL-P measurements were cell penetration positive (14.6 million cells/mL), while the ECL-B group remained tight and showed no cell penetration. As the findings of our study are well in accordance with the results of several previous animal studies, it can be concluded that the surrogate model is capable of performing rapid and cheap screening of cell occlusivity for different collagen membranes in a very standardized manner. In particular, claims of long degradation resistance can be easily proven and compared. As the boar spermatozoa used in the present report had a size of 9 × 5 μm, smaller bacteria are probably also able to penetrate the leaking membranes; in this regard, our proposed study set-up may provide valuable information, although it must be acknowledged that sperm cells show active mobility and do not only translocate by growth.