Zusammenfassung Polyvinyl-Pyrrolidon-Jod (Polyvinyl-Pyrrolidon-Jod-Komplex, PVP-Jod, CAS 25655-41-8) ist wegen seines breiten anti-mikrobiellen Wirkungsspektrums und des vergleichsweise geringen Allergierisikos ein weitverbreitetes Antiseptikum, das bereits klinisch zur Spülung offener Gelenke eingesetzt wird, um Entzündungen vorzubeugen. Ergebnisse aus tierexperimentellen Studien und Untersuchungen an Organkulturen liefern widersprüchliche Ergebnisse über die knorpelschädigende Wirkung von PVP-Jod. In-vitro-Untersuchungen über den Einfluss von PVP-Jod auf Knorpelzellkulturen liegen derzeit nicht vor. Ziel dieser Studie war es deshalb, die Wirkung von PVP-Jod auf das Zellwachstum und die Stoffwechselaktivität humaner Knorpelzellen in verschiedenen Konzentrationen und bei unterschiedlichen Expositionszeiten zu untersuchen. Zunächst wurden an fibroblastären Zellen (BALB3T3) mittels Vitalitätstest (MTT-Test) und Proliferationstest (BrdU-ELISA) geeignete Konzentrationen und Einwirkungszeiträume identifiziert, mit denen der Einfluss von PVP-Jod auf die Proteoglykan- und DNA-Synthese von primären humanen Knorpelzellen untersucht wurde. Konzentrationen bis 1 % PVP-Jod hatten keinen Einfluss auf die Proteoglykan- und DNA-Synthese von Knorpelzellen nach Inkubation über 30 min. Eine Einwirkungsdauer von 24 h hatte ab 0,2 % PVP-Jod einen deutlich hemmenden Effekt auf die DNA- und Proteoglykansynthese. Die DNA-Syntheserate war allerdings schon nach 10-minütiger Inkubation mit 0,2 % PVP-Jod beeinträchtigt und mit 1 % PVP-Jod vollständig gehemmt. BALB3T3 reagierten empfindlicher als Knorpelzellen. Die Vitalität und Proliferationsrate war nach gleicher Einwirkungsdauer schon bei 0,5 % PVP-Jod völlig gehemmt. Allerdings erholten sich die Zellen bereits 24 h nach 30-minütiger Inkubation mit 0,5 bzw. 1 % PVP-Jod wieder. Nach Einwirkung von 5 % PVP-Jod erholten sich die Zellen nicht mehr. Zusammenfassend sprechen die Ergebnisse für eine kurze Anwendung (30 min) von PVP-Jod in geringen Konzentrationen (< 1 %) und ergänzen damit Ergebnisse aus Studien über die antimikro-bielle Wirksamkeit geringer PVP-Jod-Konzentrationen und deren Wirksamkeit bei der Behandlung des durch Mikroorganismen geschädigten Gewebes.

Protein kinase C (PKC), protein kinase A (PKA), prostaglandin synthesis, and various mitogen-activated protein kinases (MAPKs) have been reported to be activated in bone cells by mechanical loading. We studied the involvement of these signal transduction pathways in the downregulation of HB-GAM expression in osteoblastic cells after cyclic stretching. Specific antagonists and agonists of these signal transduction pathways were added to cells before loading and to non-loaded control cells. Quantitative RT-PCR was used to evaluate gene expression. The data demonstrated that the extracellular signal-regulated kinase (ERK) 1/2 pathway, PKC, PKA, p38, and c-Jun N-terminal kinase MAPK participated in the mechanical downregulation of HB-GAM expression, whereas prostaglandin synthesis did not seem to be involved.

Povidone-iodine (polyvinyl-pyrrolidone-iodine complex, PVP-iodine, CAS 25655-41-8) is a commonly used antiseptic because of its broad spectrum of antimicrobial effect and its comparatively low allergic risk. It is also used for open joint lavage. Animal and organ culture studies provide controversial results about the risk of cartilage damage due to povidone-iodine. There is a paucity of in vitro study data concerning the effect of povidone-iodine on chondrocyte cultures are still missing. The aim of this study was therefore to investigate the effect of different concentrations and exposition times of povidone-iodine on cell growth and differentiation of human chondrocytes. Using of a vitality test (MTT) and a proliferation assay (BrdU) in the fibroblast-like cell line BALB3T3, suitable concentrations and incubation times were identified to investigate the influence of povidone-iodine on proteoglycan synthesis and DNA synthesis of primary human chondrocytes. Concentrations of up to 1% povidone-iodine had no significant effect on proteoglycan and DNA synthesis of chondrocytes after incubation for 30 min. An incubation time of 24 h did not inhibit DNA- and proteoglycan synthesis, until a concentration of 0.2% povidone-iodine was used. DNA synthesis rate was impaired after 10 min incubation with 0.2% and fully inhibited with 1% povidone iodine. BALB3T3 reacted more sensitively than chondrocytes. Vitality and proliferation rate were fully inhibited at a concentration of 0.5% after the same exposition time. However, cells recovered 24 h after 30 min incubation with 0.5% povidone-iodine. After incubation with 5% povidone iodine cells did not recover. From the results it can be concluded that low concentrations of povidone-iodine (< 1%) and short incubation times (< 30 min) have no damaging influence on chondrocytes. Previous studies have reported the antimicrobial effectiveness of low concentrations of povidone-iodine on the reduction of tissue damage by microorganisms. Data from previus studies and the current findings from this investigation support the clinical use of povidone-iodine at low concentrations and short incubation times for antiseptic treatment of cartilage tissues.

Several in vivo and in vitro studies with different loading regimens showed that mechanical stimuli have an influence on proliferation and differentiation of bone cells. Prerequisite for this influence is the transduction of mechanical signals into the cell, a phenomenon that is termed mechanotransduction, which is essential for the maintenance of skeletal homeostasis in adults. Mechanoreceptors, such as the integrins, cadherins, and stretch-activated Ca2+ channels, together with various signal transduction pathways, are involved in the mechanotransduction process that ultimately regulates gene expression in the nucleus. Mechanotransduction itself is considered to be regulated by hormones, the extracellular matrix of the osteoblastic cells and the mode of the mechanical stimulus.

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.

BACKGROUND:The aim of the present study was to investigate the effect of mechanical strain on human osteoblastic precursor cells in a three-dimensional scaffold.METHODS:Osteoblastic precursor cells were seeded in a collagen type I gel and mechanically stretched by daily application of cyclic uniaxial strain. The expression of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I was investigated by analysing the mRNA. Cell and matrix orientation were investigated by scanning electron microscopy.RESULTS:Cyclic stretching increased cell proliferation. The expression of osteogenic markers was slightly increased by mechanical strain. The cells and matrix were strictly oriented in the stress direction.CONCLUSION:The application of mechanical load might have a beneficial effect on the quality and quantity of generated bone tissue and might be a important factor in tissue engineering of bone.

Ziel der Studie war die Untersuchung der Differenzierung osteoblastärer Zellen in einer dreidimensionalen Matrix nach mechanischer Belastung.

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.

UNLABELLED:The influence of mechanical tissue strain caused by flexible fracture fixation on the systemic occurrence of systemic mitogens during callus healing was investigated. For this purpose the mitogenic capacity and growth factor concentration of sera from patients undergoing fracture treatment were determined. Sera from 9 patients whose fractures had been stabilized by external fixation were collected before and during fracture treatment. The sera were added to cell culture media of the osteoblastic cell line SaOS-2. After 5-6 days cell proliferation was measured. Transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-I (IGF-I) concentrations were analyzed in serum samples from different healing stages. STATISTICS:paired Wilcoxon-test. Sera from fracture patients decreased SaOS-2 proliferation in the first week after surgery (p<0.05) compared to sera obtained prior to surgery. In the fourth or fifth week proliferation increased significantly (p<0.03). The increased proliferation of the SaOS-2 cells was associated with elevated levels of TGF-beta and IGF-I (p<0.05). The higher mitogenic activity of sera suggests an increased level of circulating mitogens. In a previous study this increase had also been observed in patients during distraction osteogenesis treatment but not in patients with primary bone healing by a stable fixated plate. It is therefore assumed that their release from the fracture site is a consequence of mechanical stimulation by interfragmentary movement of fracture ends.

We tested the hypothesis whether the number of applied load cycles and the frequency of uniaxial strain have an effect on proliferation of human bone derived osteoblast-like cells. A new approach was developed in order to differentiate between the effects of frequency and the effects of cycle number and strain duration. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along there longitudinal axes. Strain was applied over 2 days varying the number of applied load cycles (4-3600) at a constant frequency (1Hz) or varying the frequency (0.1-30Hz) at a constant number of applied cycles (1800) or at a constant strain duration (5min). At a constant frequency, proliferative response increases (103%) with the number of applied cycles until a cycle number maximum (1800 cycles) was reached. 3600 cycles reduced cell number (43%) in contrast to the maximum. The variation of the frequency of applied strain tended to result in slight differences with regard to cell proliferation when cycle number was left constant. However, combined with an appropriate number of cycles there was an optimal frequency (1Hz) as stimulus for bone cell proliferation (84%). A higher frequency (30Hz) in combination with a high cycle number (9000) reduced cell number to control level (4%). This study demonstrates a frequency and cycle number dependent proliferative response of human osteoblast-like cells. It could be shown that effects of the frequency should not be considered separately from the effects of the cycle number.

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.

The cell activity of human bone derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human bone derived cells were stretched in rectangular silicone dishes with cyclic uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 mustrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human cancellous bone derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25% and 5-32% respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

The cell activity of human-bone-derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human-bone-derived cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 μstrain. Cyclic stretching of the cells resulted in an increased proliferation (10–48%) and carboxyterminal collagen type I propeptide release (7–49%) of human-cancellous bone-derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9–25 and 5–32%, respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

Zusammenfassung Es wurde die Zellaktivität humaner Knochenzellen nach Stimulation mit zyklischer, mechanischer Dehnung in physiologischer Größenordnung untersucht. Subkonfluente Monolayer aus humanen Knochenzellen wurden hierzu in rechteckigen Silikonschalen mit uniaxialer Bewegung gedehnt. Die Dehnung wurde an 2 aufeinanderfolgenden Tagen jeweils 30 min lang bei einer Frequenz von 1 Hz und einer Dehnungsamplitude von 1000 μstrain appliziert. Es kam zu einer Zunahme der Zellproliferation (10–48 %) und der Freisetzung an Typ-I-Kollagen-Propeptid (7–49 %). Die alkalische Phosphataseaktivität und die Osteocalcinfreisetzung waren signifikant reduziert (9–25 % bzw. 5–32 %). Diese Ergebnisse deuten darauf hin, dass zyklische Dehnungen bei physiologischer Amplitude zu einer Zunahme derjenigen Osteoblastenaktivitäten führen, die mit der Matrixbildung einhergehen. Die Zellaktivitäten, die für den differenzierten Osteoblasten charakteristisch sind und zur Matrixreifung führen, nehmen hingegen ab.