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A facile in vitro model to study rapid mineralization in bone tissues.

Deegan AJ, Aydin HM, Hu B, Konduru S, Kuiper JH, Yang Y - Biomed Eng Online (2014)

Bottom Line: Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield.The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size.Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours.

View Article: PubMed Central - PubMed

Affiliation: Institute for Science and Technology in Medicine, School of Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK. y.yang@keele.ac.uk.

ABSTRACT

Background: Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required.

Methods: We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT).

Results: Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture.

Conclusions: This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.

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The expression levels of four different genes associated with osteogenesis; collagen type I (COL1), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OCN) as the function of the culture conditions and time duration.
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Fig2: The expression levels of four different genes associated with osteogenesis; collagen type I (COL1), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OCN) as the function of the culture conditions and time duration.

Mentions: The four genes; COL1, ALP, OPN and OCN were expressed under all three cell culture conditions, but the expression pattern varied by culture duration and culture substrate. Except for COL1, non-coated aggregates had higher gene expression levels than the other two groups (Figure 2).Figure 2


A facile in vitro model to study rapid mineralization in bone tissues.

Deegan AJ, Aydin HM, Hu B, Konduru S, Kuiper JH, Yang Y - Biomed Eng Online (2014)

The expression levels of four different genes associated with osteogenesis; collagen type I (COL1), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OCN) as the function of the culture conditions and time duration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4228101&req=5

Fig2: The expression levels of four different genes associated with osteogenesis; collagen type I (COL1), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OCN) as the function of the culture conditions and time duration.
Mentions: The four genes; COL1, ALP, OPN and OCN were expressed under all three cell culture conditions, but the expression pattern varied by culture duration and culture substrate. Except for COL1, non-coated aggregates had higher gene expression levels than the other two groups (Figure 2).Figure 2

Bottom Line: Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield.The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size.Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours.

View Article: PubMed Central - PubMed

Affiliation: Institute for Science and Technology in Medicine, School of Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK. y.yang@keele.ac.uk.

ABSTRACT

Background: Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required.

Methods: We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT).

Results: Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture.

Conclusions: This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.

Show MeSH
Related in: MedlinePlus