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Time-lapse Raman imaging of osteoblast differentiation.

Hashimoto A, Yamaguchi Y, Chiu LD, Morimoto C, Fujita K, Takedachi M, Kawata S, Murakami S, Tamiya E - Sci Rep (2015)

Bottom Line: From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization.A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization.We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.

ABSTRACT
Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

No MeSH data available.


Related in: MedlinePlus

Time-lapse Raman images of β-carotene (1526 cm−1) and HA (956 cm−1) in two different areas of the osteoblasts.Merged images of 0 h β-carotene (yellow) and 24 h HA (red) Raman images are shown in the last column.
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f2: Time-lapse Raman images of β-carotene (1526 cm−1) and HA (956 cm−1) in two different areas of the osteoblasts.Merged images of 0 h β-carotene (yellow) and 24 h HA (red) Raman images are shown in the last column.

Mentions: The Raman images of β-carotene and HA at 0 h and 24 h, as well as the merged Raman images of 0 h β-carotene (yellow) and 24 h HA (red) in two different imaging areas are shown in Fig. 2. The β-carotene Raman signal in area I was stronger and more widely distributed than that in area II. This result indicates that more β-carotene was contained in area I than in area II because the spectral intensity is directly proportional to the concentration of the molecules. Similar to β-carotene, HA was more abundant and widely distributed in area I than in area II. This result suggests that the degree of osteoblastic mineralization is proportional to the accumulated β-carotene concentration. β-Carotene increases ALP activity and promotes the expression of osteopontin in a dose-dependent manner36. In addition, HA appeared to be localized close to where β-carotene was previously found in both areas. The data in Fig. 2 also support our hypothesis that β-carotene is a biomarker for the initial stages of mineralization.


Time-lapse Raman imaging of osteoblast differentiation.

Hashimoto A, Yamaguchi Y, Chiu LD, Morimoto C, Fujita K, Takedachi M, Kawata S, Murakami S, Tamiya E - Sci Rep (2015)

Time-lapse Raman images of β-carotene (1526 cm−1) and HA (956 cm−1) in two different areas of the osteoblasts.Merged images of 0 h β-carotene (yellow) and 24 h HA (red) Raman images are shown in the last column.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4515588&req=5

f2: Time-lapse Raman images of β-carotene (1526 cm−1) and HA (956 cm−1) in two different areas of the osteoblasts.Merged images of 0 h β-carotene (yellow) and 24 h HA (red) Raman images are shown in the last column.
Mentions: The Raman images of β-carotene and HA at 0 h and 24 h, as well as the merged Raman images of 0 h β-carotene (yellow) and 24 h HA (red) in two different imaging areas are shown in Fig. 2. The β-carotene Raman signal in area I was stronger and more widely distributed than that in area II. This result indicates that more β-carotene was contained in area I than in area II because the spectral intensity is directly proportional to the concentration of the molecules. Similar to β-carotene, HA was more abundant and widely distributed in area I than in area II. This result suggests that the degree of osteoblastic mineralization is proportional to the accumulated β-carotene concentration. β-Carotene increases ALP activity and promotes the expression of osteopontin in a dose-dependent manner36. In addition, HA appeared to be localized close to where β-carotene was previously found in both areas. The data in Fig. 2 also support our hypothesis that β-carotene is a biomarker for the initial stages of mineralization.

Bottom Line: From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization.A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization.We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.

ABSTRACT
Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

No MeSH data available.


Related in: MedlinePlus