Calcein Binding to Assess Mineralization in Hydrogel Microspheres

Abstract

We describe a method to assess mineralization by osteoblasts within microspheres using calcein. Fluorescence imaging of calcein bound to the calcium in hydroxyapatite permits assessment of the mineralized portion of the extracellular matrix. Colorimetric imaging of Alizarin Red S complexed with calcium also gives measures of mineralization, and in tissue cultures calcein and Alizarin Red S have been shown to bind to the same regions of mineral deposits. We show that when the mineralization takes place within hydrogel microspheres, Alizarin Red S does not stain mineral deposits as consistently as calcein. As tissue engineers increasingly encapsulate osteoprogenitors within hydrogel scaffolds, calcein staining may prove a more reliable method to assess this mineralization.

Keywords: calcein; hydrogel; microsphere; mineralization; osteogenesis; polymer.

Figure 1

Phase contrast images hTERT-hMSCs at different time points following microencapsulation. Cells were imaged 24 h (A) and 3 weeks (B) after encapsulation using phase contrast microscopy. Cells appear bright and hydroxyapatite mineral deposits appear dark.

Figure 2

Calcium AM and ethidium homodimer-1 viability staining of microencapsulated hTERT-hMSCs over time. Green shows live and red shows dead. (A) Day 3, 90% ± 5% live cells. (B) Day 20, 78% ± 1% live cells. (C) Day 34, 50% ± 9% live cells. (D) Day 64, 4% ± 2% live cells. (E) Graph of viability over time.

Figure 3

Alkaline phosphatase activity in basal maintenance medium (A) or osteogenic differentiation medium (B). There was no observable difference between groups.

Figure 4

Representative Alizarin Red S stain of hydroxyapatite deposits at 2, 7, 32, and 50 days (A–D). (A–C) Complete maintenance basal media, (D) differentiation media. There were no observable differences between basal media and differentiation media, therefore (A–C) shows basal media.

Figure 5

Two weeks post microencapsulation in basal maintenance (A,C) or osteogenic differentiation (B,D) media. Microencapsulated hTERT-hMSCs show fluorescent labeling of calcium in hydroxyapatite (A,B) and when identical microspheres were stained with Alizarin Red S, there was no red stain. There was no observable difference between microspheres receiving basal or osteogenic media.