Evaluation of the in vitro cytotoxicity and modulation of the inflammatory response by the bioresorbable polymers poly(D,L-lactide-co-glycolide) and poly(L-lactide-co-glycolide)

Abstract

Bioresorbable polymers composed of poly(D,L-lactide-co-glycolide) (PDLLGA) and poly(L-lactide-co-glycolide) (PLLGA) have become increasingly popular for the preparation of bone substitute constructs. However, there are reports of a delayed inflammatory reaction occurring months or years after implantation. Due to the long polymer degradation times, in vitro tests carried out at physiological temperature, 37°C, tend to assess only the short-term biocompatibility of these materials. The aim of this work is to develop an in vitro protocol that can be used to assess the long-term cytotoxicity of bioresorbable polymers in a time efficient manner. This study used a previously developed and validated accelerated degradation protocol to obtain samples of PDLLGA and PLLGA at increasing levels of degradation. Samples were then applied to standard ISO 10993-5 direct contact cytotoxicity testing and it was found that PDLLGA samples showed increasing levels of cytotoxicity at the later stages of degradation, with PLLGA samples demonstrating significantly less cytotoxic behaviour. Following concern that accumulation of acidic degradation products in a closed multi-well culture environment could overestimate cytotoxicity, we developed and validated a new dynamic flow culture methodology, for testing the cytotoxicity of these degradable materials, by adapting a commercial "organ on a chip" flow culture system, Quasi Vivo®. In addition to cytotoxicity testing, we have carried out profiling of inflammatory cytokines released by cells in response to degraded PDLLGA and PLLGA, and have suggested mechanism by which lactide-based bioresorbable materials could modulate the inflammatory response through the G-protein coupled receptor (GPCR), hydroxycarboxylic acid receptor 1 (HCA₁). STATEMENT OF SIGNIFICANCE: Bioresorbable materials naturally disintegrate over time when implanted into the body. They are often used to make screws and clips for repair of broken bones. Unfortunately, some patients can react badly to the material, resulting in painful inflammation. Biomaterials scientists are interested in developing materials that are more compatible with the body. However, it is very difficult to predict the long-term compatibility of bioresorbable materials in the lab. In our study, we have developed a method that will allow us to study the effects of the materials as they continue to break down. This will help us understand why the materials may cause inflammation, and will support research into the development of new and improved materials for bone repair.

Keywords: Biocompatibility; Cytotoxicity; HCA1; Hydroxycarboxylic acid receptor 1; Lactate; Poly(lactide-co-glycolide); Quasi Vivo.