In situ Raman imaging of osteoblastic mineralization

Abstract

Hydroxyapatite (HA) is synthesized at early stages of bone formation by osteoblasts. Nondestructive observation of early stages of osteoblastic mineralization provides crucial information for biological mechanism of bone formation. Raman microscopy serves as an ideal tool to observe the osteoblastic mineralization process because it shows the chemical information of the sample at a minimally invasive level. In addition, HA is a marker for osteoblastic mineralization, and HA Raman signal is strong enough to identify mineralized spots in osteoblasts. In this research, we visualized the distribution of HA in cultured mouse osteoblasts by Raman imaging and observed the location of the mineralized spots in the culture. We monitored HA Raman signal from osteoblast culture for 3 days after administrating the osteogenic differentiation medium and observed Raman signal associated with HA. We identified mineralized spots of KUSA‐A1 by Raman imaging constructed from the distribution of HA Raman signal. We successfully visualized the distribution of the mineralized spots in the culture of KUSA‐A1. We compared our Raman images with Alizarin red S staining assay, which was a conventional method to evaluate the mineralization process. Raman imaging of the KUSA‐A1 culture visualized the mineralized spots more accurately than Alizarin red S staining assay. Raman imaging of HA serves as a powerful tool to identify the mineralized spots in an in vitro culture of osteogenic lineage cells. Copyright © 2014 John Wiley & Sons, Ltd.

Wide-field Raman imaging for bone detection in tissue

“…3). By using the unique Raman stokes signature of the Phosphate (PO 4 3-), specifically the tetrahedral symmetric stretching of the P-O bonds [1][2][3][4], a successful detection of the inorganic constituent within the bone structure is possible. Phosphate is a major building block of hydroxyapatite, the calcium mineral found in bone and heterotopic ossification, and is not found in significant quantities in other tissues.…”

“…Most Raman techniques are timeconsuming, require expensive and bulky systems, and are destructive in nature due to large illumination fluence [1][2][3]. There exist many disorders that cannot be detected or imaged via traditional methods and could use the high specificity that Raman signals offer.…”

“…There exist many disorders that cannot be detected or imaged via traditional methods and could use the high specificity that Raman signals offer. The detection of bone tissue that forms outside the skeleton hypothetically is an ideal candidate disorder, as the Phosphorus-Oxygen bond provides a strong unique Raman signature [1][2][3][4]. This mineralization growth occurs in traumatic wounds of the limbs where the injury affects soft tissue and is prevalent in combat wounds [5][6][7][8].…”

Synergistic influence of topomimetic and chondroitin sulfate-based treatments on osteogenic potential of Ti-6Al-4V

“…Therefore the results of Von Kossa staining were not enough reliable to be reported here. Generally, we consider that Raman microspectroscopy is a more accurate technique than the common nonspecific Von Kossa or Alizarin Red calcium staining methods, which may overestimate the extent of mineralization and even, retrieve false positives …”

Changes in chemical composition of bone matrix in ovariectomized (OVX) rats detected by Raman spectroscopy and multivariate analysis

“…On the other hand, vibrational spectroscopic techniques, Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy have been applied to analyze bone quality on both the mineral and the proteins directly or non-destructively 1,2,6 . Remarkably, Raman spectroscopy enables to perform high spatial resolution measurement in vitro, ex vivo and in vivo [6][7][8][9] . Here we demonstrated Raman spectroscopic studies of osteoporosis model rats.…”