Stereochemistry Determines Immune Cellular Responses to Polylactide Implants

Abstract

Repeating L- and D-chiral configurations determine polylactide (PLA) stereochemistry which affects its thermal and physicochemical properties, including degradation profiles. Clinically, degradation of implanted PLA biomaterials promotes prolonged inflammation and excessive fibrosis, but the role of PLA stereochemistry is unclear. Additionally, although PLA of varied stereochemistries cause differential immune responses in-vivo, this observation has yet to be effectively modeled in-vitro. A bioenergetic model was applied to study immune cellular responses to PLA containing > 99% L-lactide (PLLA), > 99% D-lactide (PDLA) and a 50/50 melt-blend of PLLA and PDLA (stereocomplex PLA). Stereocomplex PLA breakdown products increased IL-1b, TNF-a and IL-6 protein levels but not MCP-1. Expression of these proinflammatory cytokines is mechanistically driven by increases in glycolysis in primary macrophages. In contrast, PLLA and PDLA degradation products selectively increase MCP-1 protein expression. Whereas both oxidative phosphorylation and glycolysis are increased with PDLA, only oxidative phosphorylation is increased with PLLA. For each biomaterial, glycolytic inhibition reduces proinflammatory cytokines and markedly increases anti-inflammatory (IL-10) protein levels; differential metabolic changes in fibroblasts were observed. These findings provide mechanistic explanations for the diverse immune responses to PLA of different stereochemistries, and underscore the pivotal role of immunometabolism on the biocompatibility of biomaterials applied in medicine.

Stereochemistry Determines Immune Cellular Responses to Polylactide Implants

“…19 According to methods outlined by the International Standard Organization (ISO 10993-5:2009�Biological evaluation of medical devices), extracts were made as previously described. 6 Briefly, we suspended 4 g of biomaterial pellets, having similar surface areas, in 25 mL of complete medium (see Section 2.5). After 12 days (d) at 250 rpm at 37 °C, the medium containing PLA breakdown products (called extracts) was decanted and used to treat cells for experiments.…”

“…20,21 In addition, NIH 3T3 cells with a Sleeping Beauty transposon plasmid (pLuBIG) having a bidirectional promoter driving an improved firefly luciferase gene (fLuc) and a fusion gene encoding a Blasticidinresistance marker (BsdR) linked to eGFP (BGL) 22 was used to assess ATP levels in live cells by making ATP the rate-limiting factor as previously described. 6 This allowed morphological and bioenergetic changes in live cells to be concurrently monitored. 23,24 In each well of a 96-well plate, 5,000 fibroblasts or 50,000 macrophages were cultured in complete medium containing DMEM, 10% heat-inactivated fetal bovine serum, and 100 U/mL penicillin−streptomycin (all from Thermo Fisher Scientific).…”

“…Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and lactate-linked proton efflux rate (PER) were measured using the Seahorse XFp and XFe-96 Extracellular Flux Analyzer (Agilent Technologies) as previously described. 6 After each time point, the medium in each well was washed off using Seahorse DMEM medium (pH 7.4); this removed any PLA breakdown products in contact with seeded cells. Afterward, the plates were incubated for an hour in a non-CO 2 incubator, according to the manufacturer's instruction.…”

“…5 Recently, an alternative mechanism for immune cell activation to bulk PLA and its hydrolytic degradation products was proposed using a bioenergetic in vitro model simulating in vivo events. 6 In the study, altered bioenergetic homeostasis and functional metabolic profiles were discovered to underlie adverse immune responses to amorphous and crystalline PLA degradation. Higher crystallinity delayed the onset of adverse immune responses, likely from faster degradation in amorphous compared to semicrystalline PLA.…”

“…Higher crystallinity delayed the onset of adverse immune responses, likely from faster degradation in amorphous compared to semicrystalline PLA. 6 However, spanning over decades, adverse responses to PLA have been controversial. While there have been some studies showing only mild proinflammatory responses to PLA degradation products, 7−9 other studies have demonstrated that PLA degradation is accompanied by severe adverse immune responses, 10−14 which could necessitate intervention in patients.…”