4:15 PM - 4:30 PM
▼ [14p-3A-7] Raman and immunofluorescence imaging analysis of mineralization process in mouse osteoblasts
Keywords:mineralization process,osteoblast,Raman imaging
Mineralization process is the early process of bone formation. In the process, osteoblasts initiate production of hydroxyapatite (HA: Ca10(PO4)6(OH)2), which is major component of bone. Through several biological processes osteoblasts calcify surrounding extracellular matrix and then hard bone tissue is finally formed. With traditional biological assays it was revealed that various biomolecules are involved in the process of osteoblastic mineralization. Especially immunofluorescence imaging possesses great ability to visualize the distribution of bone matrix proteins such as type I collagen. Although traditional assays greatly contribute to understanding bone formation, they are unable to identify the accurate location of HA without harm to sample cells. Therefore we have employed Raman imaging to observe the accurate localization of HA in the osteoblasts. Raman imaging is a non-destructive and label-free technique that provides contrast images based on vibrational frequencies that are derived to target molecules. Previously, we accomplished time-lapse observation of the mineralization process in the same osteoblast culture with 4 hour time resolution by Raman imaging. In this report, we tried to reveal the relationship between HA localization and type I collagen distribution during the mineralization process. Raman image of HA and immunofluorescence image of type I collagen were obtained from the same mouse osteoblasts. Formation of uniform type I collagen network was observed in the tissue around mineralized nodules, while the collagen network was not uniform or not exist in the tissue which was not contain mineralized nodules. This result suggests that wide and uniform type I collagen network may be important for the initiation of the mineralization process.