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Permanent orthopedic implants are becoming increasingly important due to the demographic development. Their optimal osseointegration is key in obtaining good secondary stability. For anchorage dependent cells, topographic features of a surface play an essential role for cell adhesion, proliferation, differentiation and biomineralization. We studied the topographical effect of nanostructured alumina surfaces prepared by chemical vapor deposition on osteogenic differentiation and growth of human osteoblasts. Chemical vapor deposition of the single source precursor (tBuOAIH2)2 led to synthesis of one dimensional alumina nanostructures of high purity with a controlled stoichiometry. We fabricated different topographic features by altering the distribution density of deposited one dimensional nanostructures. Although the topography differed, all surfaces exhibited identical surface chemistry, which is the key requirement for systematically studying the effect of the topography on cells. Forty-eight hours after seeding, cell density and cell area were not affected by the nanotopography, whereas metabolic activity was reduced and formation of actin-fibres and focal adhesions was impaired compared to the uncoated control. Induction of osteogenic differentiation was demonstrated via up-regulation of alkaline phosphatase, bone sialoprotein, osteopontin and Runx2 at the mRNA level, demonstrating the potential of nanostructured surfaces to improve the osseointegration of permanent implants.

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.