Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture

Abstract

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as "mini tissues" to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D(3) induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D(3) has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D(3) and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

Vitamin D Enhanced the Osteogenic Differentiation of Cell Spheroids Composed of Bone Marrow Stem Cells

“…Several studies described vitamin D's utility to enhance osteogenesis in primary murine osteoblasts and MC3T3-E1 cell lines [3][4][5]. Replenishing the cell culture medium containing vitamin D induces osteocalcin expression in osteoblasts [6]. Addition of both vitamin D and osteogenic factors resulted in an osteoblast phenotype which expresses alkaline phosphatase activity, secretes osteocalcin, and deposits calcium [6].…”

“…Replenishing the cell culture medium containing vitamin D induces osteocalcin expression in osteoblasts [6]. Addition of both vitamin D and osteogenic factors resulted in an osteoblast phenotype which expresses alkaline phosphatase activity, secretes osteocalcin, and deposits calcium [6]. Mechanical testing showed that vitamin D induced a stiffer osteosphere compared with control [7].…”

Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids

“…As already described earlier (Metzger et al, 2011(Metzger et al, , 2013, the LOT is both, a powerful and easy technique to generate exactly one spheroid per cavity under highly standardized conditions. The size of the spheroids could easily be determined by adjusting the cellular concentrations in the seeding suspension while keeping the seeding volume of 100 µl per cavity constant (Figure 2).…”

Journey into Bone Models: A Review

“…They are relatively easy to produce, handle and offer potential for medium- to high-throughput applications. Therefore, spheroids are an increasingly attractive approach in organoid generation, most often to mimic tissues like liver [ 69 , 70 ], brain [ 71 ], tumour models [ 72 , 73 ] but also with regard to in vitro bone models [ 74 , 75 , 76 , 77 ]. Generally, the differentiation potential of MSCs is evidently enhanced in 3D spheroid cultures [ 68 , 78 , 79 ].…”