Analysis of collagen types I & II at bone fracture healing tissue using polarization SHG

Discriminating type I and type II collagen is important owing to its dominating presence in cartilage and connective tissues where an alteration of collagen matrix is observed in several diseases including osteogenesis imperfecta and osteoarthritis. For non-destructive investigation of the molecular level properties of collagen, a non-invasive Dual-liquid crystal based polarization-resolved second harmonic (SHG) microscopy is utilized to facilitate the quantitative characterization of collagen types I and II in fracture healing tissues. In this study, we extend an existing approach allowing the quick generation of any desired linear polarization states without any mechanical parts to quantify the characteristics of collagen types using pitch angle and anisotropy parameter. Furthermore, data reliability is ensured by using right and left-hand circular polarization imaging centered circular dichroism analysis. Our findings indicate that the effective pitch angle for the collagen at fracture healing tissue is 48.4° and 49.9° at two weeks and four weeks of repair respectively where type II collagen dominates in the former and type I in the latter. The mean SHG-CD response of the articular cartilage is 0.271 and 0.183 at the rich zone of collagen types II and I, respectively. These findings are correlated to the values obtained from the non-fractured control bone tissue. The measurements obtained reflect the different types of collagen in the molecular fibril assembly. Therefore, these methods demonstrate a powerful tool to provide new insights on understanding the role of collagen in ECM structure and on the development of cartilage repair.