Human osteoclast (OC) formation and activity was studied in cultures of peripheral blood mononuclear cells (PBMNC) from six healthy donors after stimulation with fetal calf serum (FCS), under the influence of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and the macrophage-colony stimulating factor (M-CSF). The results showed that selected FCS could stimulate OC formation without any medium supplementation with osteoclastogenic factors. The OC formation, investigated by quantification of multinucleated tartrate-resistant acid phosphatase-positive cells (TRAP+ cells), and the sensitivity of OC progenitors to RANKL and M-CSF, varied widely between individual donors. The OC resorption activity, measured in the "pit-assay" on dentine, was strictly dependent on the presence of RANKL and M-CSF in the medium and was also donor dependent. The considerable donor variability should be considered in culture studies investigating, e.g. the interactions of OC with biomaterials or the influence of cytokines, growth factors and drugs on osteoclastogenesis.

Zellkulturstudien wiesen nach, dass nichtsteroidale Antirheumatika osteogene Aktivitäten von Osteoblastenkulturen beeinflussen. Längerfristige Effekte auf die sich differenzierenden Osteoblasten wurden dabei allerdings nicht berücksichtigt. Deshalb wurde der Einfluss von Voltaren® mit dem nichtsteroidalen Wirkstoff Diclofenac auf die Proliferation und Genexpression von SaOS-2-Zellen 2, 9 und 16 Tage nach Inkubation untersucht.

Object. To study intervertebral disc cell mechanobiology, the authors developed experimental systems that allow the application of cyclic strain and intermittent hydrostatic pressure (IHP) on isolated disc cells under equal three-dimensional (3D) culture conditions. The purpose of the study was to characterize disc cell proliferation, viability, morphology, and gene expression in 3D collagen matrices. Methods. The effects of cyclic strain (1, 2, 4, and 8% strain; 1 Hz) and IHP (0.25 MPa, 0.1 Hz) on gene expression (real-time polymerase chain reaction) of anabolic and catabolic matrix proteins were investigated and compared with those derived from mechanically unstimulated controls. Intervertebral disc cells proliferated in the collagen gels (mean viability 91.6%) and expressed messenger RNA for collagen I, collagen II, aggrecan, matrix metalloproteinase (MMP)—2, and MMP-3. Morphologically, both spindle-shaped cells with longer processes and rounded cells were detected in the collagen scaffolds. Cyclic strain increased collagen II and aggrecan expression and decreased MMP-3 expression of anulus fibrosus cells. No significant difference between the four strain magnitudes was found. Intermittent hydrostatic pressure tended to increase collagen I and aggrecan expression of nucleus cells and significantly decreased MMP-2 and -3 expression of nucleus cells, whereas aggrecan expression of anulus cells tended to decrease. Conclusions. Based on these results, the collagen matrix appeared to be a suitable substrate to apply both cyclic strain and IHP to intervertebral disc cells under 3D culture conditions. Individual variations may be influenced by the extent of degeneration of the disc specimens from which the cells were isolated. This experimental setup may be suitable for studying the influence of degeneration on the disc cell response to mechanical stimuli.

The aim of the present study was to investigate the effect of cyclic uniaxial mechanical strain on a human osteoblastic precursor cell line (hFOB 1.19) in three-dimensional type I collagen matrices. Cell seeded collagen constructs were mechanically stretched by a daily application of cyclic uniaxial strain using a special motor-driven apparatus and compared to unstretched controls. Expression of genes involved in cell proliferation and osteoblastic differentiation as well as matrix production were investigated by analyzing the mRNA of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I (Col I) up to a cultivation period of 3 weeks using real-time PCR. Cyclic stretching of cell seeded Col I matrices at a magnitude occurring in healing bone increased cell proliferation and slightly elevated the expression of nearly all investigated genes over unstrained controls at various time points. It was concluded that mechanical load promotes the proliferation and differentiation of osteoblastic precursor cells in a Col I matrix and that the application of mechanical stimuli may have a beneficial effect on in vitro tissue formation.

Despite the long-standing use of metals as orthopedic implants there still are unsolved problems with these materials and open questions about their behavior in a biological environment. Cell-culture studies provide a useful tool for investigations. In addition to the determination of biochemical or molecular biological parameters, the morphology of adhering cells reflects their interaction with the substrata. This article describes an investigation of the morphology of human osteoblasts on stainless steel, cobalt chromium alloy, commercially pure titanium, Ti-6Al-4V, and Ti-6Al-7Nb with surface designs similar to those used as clinical implants. A cell culture plastic surface was used as a control material. The materials were examined by scanning electron microscopy at different points of time. The cells spread, proliferated, and formed nodules on all test substrates in a time-dependent manner, without signs of a disturbing influence from any of the materials. On the smooth surfaces the cells showed a flattened fibroblast-like morphology and only slight differences could be detected. Therefore, the cellular morphology seems not to be markedly affected by the different chemical material compositions. In contrast, the titanium alloy with a rough, sandblasted surface induced a three-dimensional growth. This three-dimensional cellular network could be the basis for the known earlier differentiation of osteoblasts on rough surfaces in vitro and a better osseointegration in vivo.

Tricalcium phosphate ceramics (TCPs) are increasingly used as bone substitutes. They demonstrate good biocompatibility and degrade relatively slowly. New glass ceramics based on calcium alkali orthophosphates (Ca(2)KNa(PO(4))(2)) were developed that degrade faster than TCP but could have reduced biocompatibility due to their high solubility. Therefore, they were modified by a neutralizing surface treatment. The aim of this study was to evaluate the biocompatibility of some of these ceramics, GB1a, GB9, and GB14, which differ in the amount of added Na, K, Mg, or Si ions, with standard and modified surfaces. The in vitro cytotoxicity of the ceramics GB1a, GB9, and GB14 was determined by the agar diffusion and filter test and the microculture tetrazolium (MTT) assay. In order to investigate the influence of surface modification, these three ceramics were compared to their surface-treated counterparts, GB1aN, GB9N, and GB14N. GB1a, the ceramic with the highest in vitro solubility, showed the strongest toxic influence in all cell culture tests. GB9 and GB14 produced better results. In contrast, the counterparts with modified surfaces exhibited no (GB9N, GB14N) or weak (GB1aN) signs of cytotoxicity. It is concluded that the toxicity of the ceramics GB1a, GB9, and GB14 depends on their solubility. A positive influence of the surface treatment on in vitro biocompatibility was demonstrated. Therefore, the surface-treated glass ceramics could be promising materials for bone replacement.

Commercially pure titanium (cpTi), titanium alloys, and steel are often used for dental and orthopedic implants. In these applications titanium is considered the "gold standard." However, tissue reactions around titanium implants and the changing trend to leave orthopedic devices in the body have led to a new examination of the preferred material. This in vitro study tested the behavior of osteoblasts on cpTi, Ti-6Al-7Nb, and stainless steel with surface designs similar to clinical implants. After surface characterization by scanning electron microscopy and profilometry, cell proliferation and the differentiation parameters of alkaline phosphatase (ALP) activity and osteocalcin were measured. For all materials tested, the growth curves showed a similar kinetic. On Ti-6Al-7Nb, ALP activity was significantly lower when compared with steel, and cpTi and did not change over the time. ALP activity increased moderately on steel and cpTi. Osteocalcin levels were higher on both titanium materials than on steel. Based on undisturbed cell growth and the relatively high alkaline phosphatase and osteocalcin levels, we suggest that cpTi provides the best biocompatibility with regard to proliferation, in addition to more reliable early and late differentiation markers of human osteoblasts in vitro.

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