Raman spectroscopic monitoring of the osteogenic differentiation of human mesenchymal stem cells

Abstract

The differentiation of stem cells into multi-lineages is essential to aid the development of tissue engineered materials that replicate the functionality of their tissue of origin. For this study, Raman spectroscopy was used to monitor the formation of a bone-like apatite mineral during the differentiation of human mesenchymal stem cells (hMSCs) towards an osteogenic lineage. Raman spectroscopy observed dramatic changes in the region dominated by the stretching of phosphate groups (950-970 cm(-1)) during the period of 7-28 days. Changes were also seen at 1030 cm(-1) and 1070 cm(-1), which are associated with the P-O symmetric stretch of PO(4)(3-) and the C-O vibration in the plane stretch of CO(3)(2-). Multivariate factor analysis revealed the presence of various mineral species throughout the 28 day culture period. Bone mineral formation was observed first at day 14 and was identified as a crystalline, non-substituted apatite. During the later stages of culture, different mineral species were observed, namely an amorphous apatite and a carbonate, substituted apatite, all of which are known to be Raman markers for a bone-like material. Band area ratios revealed that both the carbonate-to-phosphate and mineral-to-matrix ratios increased with age. When taken together, these findings suggest that the osteogenic differentiation of hMSCs at early stages resembles endochondral ossification. Due to the various mineral species observed, namely a disordered amorphous apatite, a B-type carbonate-substituted apatite and a crystalline non-substituted hydroxyapatite, it is suggested that the bone-like mineral observed here can be compared to native bone. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analyses for monitoring the various mineral species, degree of mineralisation and the crystallinity of hMSCs as they differentiate into osteoblasts.

Assessment of an osteoblast-like cell line as a model for human primary osteoblasts using Raman spectroscopy

“…Comparatively, the mineral to matrix ratio increased 2 fold with culture time, and also reached values that are similar to those reported in previous work [34]. This confirms that there is mineral present in the protein matrix as expected.…”

“…Further to this, the values reported here are similar to those calculated for hMSCs [34] and they further affirm that carbonate substitution occurs most rapidly between day 14 and 21.…”

“…The carbonate to phosphate ratios may not be in accordance with findings reported by various research groups [26,28,31,34]. However, the ratio is similar to the carbonate to phosphate ratio of native bone that was found to have a value of 3.84 ± 0.03.…”

“…From the univariate analysis, it is demonstrated that the mineral to matrix and carbonate to phosphate ratios for both cell types show significant differences. In the case of the primary osteoblasts, the mineral to matrix ratio increased with culture time, a trait that has been associated with the differentiation of stem cells into osteoblasts [27,34] and mineralising calvaria osteoblasts [28,31]. The carbonate to phosphate ratios show a ratio at day 7 that increases significantly through culture time.…”

“…A major aspect when characterising bone material is resolving changes in the phosphate band in terms of both frequency and peak position. Bone mineral crystallinity can be calculated from the full width half maximum (FWHM) of the PO 4 3 symmetric band, whereby the lower the FWHM the higher the crystallinity of the material [34] .…”