Studies of cellular responses to implanted biomaterials have become the focus of many recent investigations as biopolymers are used increasingly to improve function and/or provide replacement structures within the human system. To illustrate, new generations of polyisobutylene (PIB)-based thermoplastic elastomers (TPEs) have been developed with enhanced drug-delivery capabilities for localized control of in-stent restenosis. In order to assess the efficacy of these drug-delivery polymers the effects of both the drug-loaded and non-drug loaded polymers upon proliferation of vascular wall cells had to be assessed. There literally exists a need for a battery of tests to assess cell/material interactions that includes both in vitro and in vivo tests.
My investigation specifically focused on the development of a procedure to evaluate the growth and attachment of cells in vitro to three novel (PIB)-based TPEs that have the potential for unique in vivo applications to improve human health. Two of the test materials (TPE-1 and 06DNX040) were block co-polymers with arborescent cores that differed only in polystyrene content. The third test material (SIBS073T) was linear.
The PIB-based TPEs were characterized by 1H NMR and Size Exclusion Chromatography (SEC) and the softness of each film was analyzed using Durometer type A (ASTM D2240). For the in vitro tests of cell growth and attachment, multiplicates of 0.01 mm thick films of each polymer were inserted into modified BEEM® capsules which were sterilized with ethylene oxide. Before performing the attachment and proliferation tests, a standard in vitro growth curve was determined using aortic advential fibroblasts (AoAF) seeded onto polystyrene tissue culture dishes to understand the in vitro growth pattern of these cells under usual in vitro conditions. Thereafter, 3.2 x 104 AoAF cells were inoculated onto each film of each test polymer and the films incubated in vitro for 11 days to measure growth curve dynamics. Replicate samples from each polymer were counted and studied (using a Scanning Electron Microscope (SEM), hemocytometer, and inverted light microscope) on Days 1, 3, 5, 7, 9 and 11 to establish growth curves. Growth curves of cells on test polymers were compared to growth curves of AoAF seeded onto polystyrene, silicone and polyvinyl chloride (PVC). Data from the curves were compared statistically using the Student t test and one way Analysis of Variance (ANOVA).
The growth curves derived from counts of cells seeded on TPE-1, SIBS073T, silicone and polystyrene did not differ (p > 0.05) from each other. Cell growth on 06DNX040 mimicked that on the other test polymers through Day 5. By Day 7, however, cells on this polymer were mis-shapen and detached. Cells did not attach or grow on PVC. Growth of AoAf on TPE-1 and SIBS073 was similar to that observed on silicone and polystyrene, suggesting that these materials are non toxic with vascular wall cells. The poor outcome for 06DNX040 is likely attributable to the accumulation of leachates.
We infer from these in vitro data that TPE-1 and SIBS073T will not affect adversely vascular cell wall proliferation in vivo. Additional manipulation and evaluation of 06DNX040 will be required in order to minimize the observed negative effects of this polymer upon cell proliferation.