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

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.

Adhesion of fibroblasts on micro- and nanostructured surfaces prepared by chemical vapor deposition and pulsed laser treatment.

The development of micro- and nanostructured surfaces which improve the cell-substrate interaction is of great interest in today's implant applications. In this regard, Al/Al2O3 bi-phasic nanowires were synthesized by chemical vapor deposition of the molecular precursor (tBuOAlH2)2. Heat treatment of such bi-phasic nanowires with short laser pulses leads to micro- and nanostructured Al2O3 surfaces. Such surfaces were characterized by scanning electron microscopy (SEM), electron dispersive spectroscopy and x-ray photoelectron spectroscopy. Following the detailed material characterization, the prepared surfaces were tested for their cell compatibility using normal human dermal fibroblasts. While the cells cultivated on Al/Al2O3 bi-phasic nanowires showed an unusual morphology, cells cultivated on nanowires treated with one and two laser pulses exhibited morphologies similar to those observed on the control substrate. The highest cell density was observed on surfaces treated with one laser pulse. The interaction of the cells with the nano- and microstructures was investigated by SEM analysis in detail. Laser treatment of Al/Al2O3 bi-phasic nanowires is a fast and easy method to fabricate nano- and microstructured Al2O3-surfaces for studying cell-surface interactions. It is our goal to develop a biocompatible Al2O3-surface which could be used as a coating material for medical implants exhibiting a cell selective response because of its specific physical landscape and especially because it promotes the adhesion of osteoblasts while minimizing the adhesion of fibroblasts.