In Vitro Inflammatory Response of Bioresorbable Polymers at Clinically-Relevant Extents of Degradation

Introduction Research in orthopaedics is now moving away from permanent metallic implants and looking towards the use of bioresorbable polymers (e.g. PLA, PGA and related co-polymers) that, when implanted into the injured site, bioresorb as the tissue heals. However, reports of a delayed inflammatory response occurring in the late stages of polymer degradation has limited the wide scale use of these polymers1,2. Only a few studies assess the long-term biocompatibility of these polymers and, with an increasing market for bioresorbable materials, it is anticipated that this will be a future issue. This work aims to develop an experimental methodology that can be used to assess the delayed inflammatory response of poly(D,L-lactide-co-glycolide) (PDLLGA) and poly(L-lactide-co-glycolide) (PLLGA) using in vitro tests. This utilised an elevated temperature accelerated degradation test with the objective of determining the optimum temperature to achieve simulation of in vivo degradation behaviour. Degradation temperatures of 47oC, 57oC and 70oC were studied and compared to physiological temperature (37oC). This methodology was used to induce predetermined levels of degradation in PDLLGA and PLLGA, in order to mimic a range of clinically relevant in vivo implantation times of up to 6 months. This work can be applied to the optimisation of polymer degradation profiles to minimise late-stage inflammatory response and identification of potentially beneficial additives in this regard. Experimental Methods PDLLGA 85:15 (PURASORB PDLG 8531, Corbion, Gorinchem) and PLLGA 85:15 (PURASORB PLG 8531, Corbion, Gorinchem) samples were processed by compression moulding into 1mm thick sheets, followed by annealing (only for PLLGA) for 4h at 100oC, laser cut into 8mm diameter disc-shaped samples and electron beam sterilised (Steris, Westport, Ireland). Samples were degraded in PBS buffer, under sterile conditions, at 37oC, 47oC, 57oC and 70oC, with solution pH being monitored regularly. On retrieval, at predetermined time intervals, analysis included change in mass, molecular weight (gel-permeation chromatography, Agilent Technologies), thermal properties (differential scanning calorimetry) and molecular structure (nuclear magnetic resonance spectroscopy). Indicators of late stage inflammation will be assessed using an MTT cytotoxicity assay and multiple ELISA analysis for inflammatory factors, with mouse L929 fibroblasts and RAW264.7 macrophages. Results and Discussion The degradation of PDLLGA at 37oC, 47oC, 57oC and 70oC was modelled using the Arrhenius equation; the changes in molecular weight were used to calculate the hydrolysis rate at each temperature and then compared to physiological temperature. When samples were degraded at 47oC, 57oC and 70oC the degradation rate was increased by x3, x8 and x30 respectively. The results suggest that the degradation mechanism of PDLLGA at increased temperatures is similar to that of physiological temperature and can therefore be used as a method to accelerate its degradation. The degradation of PLLGA will also be modelled using a similar approach. Fig. 1 shows the changes in appearance of PDLLGA and PLLGA during degradation at 47oC. PDLLGA underwent significant changes to its appearance throughout degradation; after 12 days the samples had a soft, paste-like appearance. In contrast, PLLGA samples appeared volumetrically unchanged, however after 57days at 47oC the samples were fragile and disintegrated on handling. Fig. 2 compares the swelling ratio and mass loss of PDLLGA and PLLGA. Swelling in PDLLGA occurred immediately upon immersion in PBS buffer and increased rapidly throughout degradation. In comparison, swelling in PLLGA did not occur immediately; after 57days at 47oC polymer swelling was 16% and increased gradually to 49% after 78days. Unlike PDLLGA where swelling occurred without significant mass loss, mass loss in PLLGA almost replicated the amount of polymer swelling. It is expected that these polymers will cause different responses in vitro. The next stage of this study is to evaluate and compare the in vitro inflammatory response of both polymers at different stages of degradation. An MTT cytotoxicity has been used as an initial evaluation in the cellular response of the two polymers. Conclusion It is paramount that long-term performance of new bioresorbable devices can be demonstrated via internationally standardised methods. An accelerated degradation methodology has been validated in this study as a highly effective tool for pre-degrading PDLLGA and PLLGA bioresorbable polymers prior to evaluation of their long-term in vitro inflammatory response. It is proposed that 47oC is adopted by the research community when assessing the long-term response of these polymers. Further work will include an in vitro evaluation of PDLLGA and PLLGA with fibroblast and macrophage cell lines. References 1. Ginjupalli, K. et al. Poly(α-hydroxy acid) based polymers: A review on material and degradation aspects. Polym. Degrad. Stab.144, 520–535 (2017). 2. Böstman, O. & Pihlajamäki, H. Clinical biocompatibility of biodegradable orthopaedic implants for internal fixation: a review. Biomaterials21, 2615–2621 (2000). Acknowledgement Sterilisation by Steris is gratefully acknowledged