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

BioMedical Engineering OnLine

Anthony J Deegan¹  Halil M Aydin³  Bin Hu¹  Sandeep Konduru²  Jan Herman Kuiper¹  Ying Yang¹ 

[1] Institute for Science and Technology in Medicine, School of Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK

[2] Orthopedics Department, University Hospital of Staffordshire, Stoke-on-Trent ST4 6QG, UK

[3] Environmental Engineering Department & Bioengineering Division and Center for Bioengineering, Hacettepe University, Ankara 06800, Turkey

关键词: Aggregate size;    Cell aggregates;    Matrix mineralization;    Rapid bone formation;    Osteoblasts;   

Others  :  1084383
DOI  :  10.1186/1475-925X-13-136

 received in 2014-07-07, accepted in 2014-09-09,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Deegan et al.; licensee BioMed Central Ltd.

【 图 表 】

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