Rapid, Fluorescence-based Assessment of in vitro Mineralization Using the New OsteoImage Mineralization Assay

Application Examples

Bone is a rigid, yet dynamic organ that is continuously molded, shaped, and repaired. Old bone is degraded by osteoclasts and replaced with new bone by osteoblasts, a process called remodeling or bone turnover.

This is the predominant metabolic process regulating bone structure and function during adult life. Aside from providing mechanical support, bone has other vital functions such as supporting hematopoiesis and regulating blood calcium levels. Osteoclasts and osteoblasts work together in healthy skeletal systems by releasing stored calcium into systemic circulation or by fixing circulating calcium into newly deposited extra-cellular organic matrix. The process by which osteoblasts add mineral to matrix is called mineralization.

Lonza offers several types of primary human cells useful for investigating in vitro osteogenesis. Clonetics NHOst- Normal Human Osteoblasts and Poietics hMSC-Human Mesenchymal Stem Cells can both be induced to produce bone-like material in culture. Mineralized material formed by cells in vitro is similar in composition to real bone, consisting of inorganic hydroxyapatite (HA), a crystalline substance of calcium, phosphates and hydroxyl ions and organic components including type I collagen and other proteins. Researchers studying stem cells or osteoblasts and their role in normal and pathologic bone remodeling frequently rely on assays to detect and quantitate in vitro mineralization.

Until now, only a few methods for detecting mineralization have been available to researchers, such as von Kossa staining, Alizarin Red staining and calcium quantitation through tedious extraction processes. The widely used histochemical von Kossa staining method is frequently presumed to label calcium phosphates when, in reality, it stains the anionic portion of phosphates, carbonates, and other salts and is therefore not calcium phosphate or HA specific1. The Alizarin Red method uses an anthraquinone dye that reacts with calcium cations to form a chelate, which can then be visualized by microscopy or measured using a plate reader after acid extraction2. Other available calcium quantitation kits also require this tedious step of acid extraction to measure calcium deposits in mineralizing stem cell and osteoblast cultures.

The OsteoImage Mineralization Assay is the newest addition to Lonza’s line of products for bone research. The assay is based on the specific binding of the fluorescent OsteoImage Staining Reagent to the hydroxyapatite portion of mineralized bone nodules in osteoblast or osteogenic stem cell cultures. The OsteoImage Assay allows researchers to assess in vitro mineralization both visually using fluorescent microscopy and quantitatively on a plate reader in the same easy three step protocol.

When using the OsteoImage Stain to visually assess mineralization, similar trends in nodule formation can be detected as with the von Kossa staining method.

Figure 1: Saos-2 cells were seeded into a 96-well plate at 3,000 cells/well and cultured in the presence of 0, 2.5, or 5 mM β-glycerophosphate (β-GP). After 12 days of culture, cells were fixed and either stained using the von Kossa method (A, B, C) or stained with OsteoImage (D, E, F). Both von Kossa and OsteoImage staining show no mineralization of cells treated with 0 mM β-GP (undifferentiated controls; A, D) and show increases in mineralization when treated with increasing β-GP: 2.5 mM (B, E) and 5.0 mM (C, F).

The osteoblast-like cell line, Saos-2 (ATCC HTB 85), shows increasing mineral deposition with increasing concentrations of β-glycerophosphate when viewed by microscopy as illustrated in Figure 1. Unlike the von Kossa stain, OsteoImage Assay allows quantitation of mineral deposits in cultures of differentiating osteoblasts. As shown in Figure 2A, the OsteoImage Assay is sufficiently sensitive to detect time-dependant increases in mineralization in cultures of differentiating NHOst. Differentiated NHOst produce increasing amounts of mineral over three weeks of culture as compared to undifferentiated control cells. This increase can also be detected when the same cultures stained with OsteoImage Staining Reagent are inspected visually (Figures 2B, C &D).

Figure 2A: Clonetics NHOst were seeded into a 96-well plate at 3,200 cells/well and cultured in the presence of 70 µM ascorbic acid, 10 mM β-glycerophosphate and 200 nM hydrocortisone. Medium was renewed twice a week and on days 7, 14, and 21 cells were fixed and stained using the OsteoImage Stain. Amount of mineralization was quantitated by reading plates on a fluorescent plate reader using 492ex/520em (A) above and visualized using fluorescent microscopy (B, C, D).

Figure 2B, C & D.

Whether using the stain to visually or quantitatively assess in vitro mineralization, the same simple OsteoImage Protocol is employed. Bone-forming cells can remain unfixed or be fixed with various cross-linking or denaturing fixation methods. The cells are then stained with the OsteoImage Staining Reagent and washed. Unlike current methods to quantitate calcium deposition, no tedious extraction steps are required. A plate reader with the appropriate fluorescence filters is the only requirement to generate quantitative data using the OsteoImage Assay. The OsteoImage Assay generates comparable results with significantly less effort when compared to assays requiring calcium extraction prior to quantitation. This is demonstrated in Figure 3, where the mineral deposited by human MSCs induced to undergo osteogenesis is analyzed using the OsteoImage Assay and an extraction-based calcium assay.

Figure 3: Poietics hMSCs were seeded into a 96-well plate at 3,200 cells/well and induced to undergo osteogenesis using Poietics hMSC Human Mesenchymal Stem Cell Osteogenic SingleQuots. Media was renewed twice a week and on days 7, 14, 21, and 28. Cells were either fixed and stained using OsteoImage Staining Reagent or calcium was extracted from the cells using 0.5N HCl and quantitated using the StanbioTotal Calcium LiquiColor.

In summary, Lonza’s new OsteoImage Mineralization Assay is a simple, fluorescence-based in vitro assay that can be used to assess bone formation both visually and quantitatively. OsteoImage Assay provides enough sensitivity to discern varying levels of mineralization by NHOst, hMSC, and cell lines such as Saos-2 (ATCC HTB 85).

The protocol has been optimized and is significantly faster and more convenient than other methods for detecting mineralization. OsteoImage Assay is also compatible with other biomarker staining techniques and can be incorporated into current protocols. This new tool for researchers is simple, versatile, and adaptable. The OsteoImage Assay will to enhance research techniques and save time.

References

1. BoneWald, L. F. et al. von Kossa Staining Alone is not Sufficient to Confirm that Mineralization in vitro Represents Bone Formation. Calcif. Tissue Int. 72, 537-547 (2003).

2. Wang, Y. H. et al. Examination of Mineralized Nodule Formation in Living Osteoblastic Cultures Using Fluorescent D yes. Biotechnol. Prog. 22, 1697-1701 (2006).