Acetic acid and pepsin result in high yield, high purity and low macrophage response collagen for biomedical applications

Collagen based devices are frequently associated with foreign body response. Although several pre(e.g. species, state of animal, tissue) and post-(e.g. cross-linking, scaffold architecture) extraction method factors have a profound effect on foreign body response, little is known about which and how during the extraction process factors mediate foreign body response. In this study, we assessed the influence of acetic acid and hydrochloric acid and the utilisation or not of pepsin or salt precipitation during collagen extraction on the yield, purity, free amines, denaturation temperature, resistance to collagenase degradation and macrophage response. Acetic acid/pepsin extracted collagen exhibited the highest yield, purity and free amine content and the lowest denaturation temperature. No differences in resistance to collagenase digestion were detected between the groups. Although all treatments exhibited similar macrophage morphology comprised of round cells (M1 phenotype), elongated cells (M2 phenotype) and cell aggregates (foreign body response), significantly more elongated cells were observed on HC films. Although no differences in metabolic activity were observed between the groups, the DNA concentration was significantly lower for the hydrochloric acid treatments. Further, cytokine analysis revealed that hydrochloric acid treatments induced significantly higher IL-1 beta and TNF-alpha release with respect to acetic acid treatments. Salt precipitation did not influence the parameters assessed. Collectively, these data suggest that during the collagen extraction process variables should also be monitored as, evidently, they affect the physicochemical and biological properties of collagen preparations