Background: Both cellular and matrix components of healthy bone are permanently renewed in a balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases. Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix proteins.Objectives: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to proton secretion expressed on osteoblasts.Methods: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer.Results: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is associated partially with proton pump ATPases.Conclusion: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone degradation.

The light-addressable potentiometric sensor (LAPS) is a silicon-based detector which allows for measuring the electric potential distribution along its surface. The point of measurement is determined by illumination with a pulsed light source, and therefore the surface potential distribution can be measured spatially resolved by raster scanning a light-pointer along the surface. In this study electrically excitable neuronal cells (N1-E115 cell line) were grown on top of the surface of the LAPS. Individual cells were electrically stimulated using a patch clamp pipette. The light-pointer was focused on the stimulated cell and the electric potential of the silicon surface below the stimulated cell was recorded with the LAPS. Averaged extracellularly recorded signals were found to correspond with the intracellularly recorded patch clamp signals. Although it was possible to record averaged ionic currents of individual cells with the LAPS technique, the low signal-to-noise ratio so far hampers practical applications.

Metabolic activity of cultured cells can be monitored by measuring changes in the pH of the surrounding medium caused by metabolic products such as protons, carbon dioxide or lactic acid. Although many systems designed for this purpose have been reported, almost all of them are based on bulk measurements, where the average metabolic activity of all cells in contact with the device is recorded. Here, we report on a novel biosensor, based on a modified light-addressable potentiometric sensor (LAPS) device, which enables the metabolic activity of cultured cells to be measured with spatial resolution. This is demonstrated here by detecting the differential sensitivity to a cholinergic receptor agonist of two different co-cultured cellular populations. By making simultaneous measurements of the metabolic activity of different cell types seeded on different segments of one sensor, this device not only provides a rapid means of assessing cellular specificity of pharmaceutical compounds but also has the potential of being used to non-invasively monitor humoral as well as synaptic communication between different cell populations in co-culture. The temporal and spatial resolution of the device were investigated and are discussed.

The chemical instability of the gallium arsenide surface poses a serious limitation to its use under ambient conditions, such as in air or aqueous solution. It is shown that both bare GaAs and GaAs coated with self-assembled monolayers of organic alkane-thiols are continuously etched in aqueous environments, which limits their use as biosensor devices in physiological environments. A corrosion protection material having long-term stability ("chemical passivation") and biocompatibility ("biological passivation") with the GaAs surface was found to be interfacial layers of polymerized organic mercaptosilanes a few tens of nanometers thick. The mercaptosilanes not only provided a nearly perfect corrosion protection of GaAs in water, but they also have the potential to introduce chemical groups that allow easy, further surface functionalization. Characterization of the inter-facial polymer layer and its protective role was done by atomic force microscopy (AFM), ellipsometry, contact angle measurements, and atomic absorption spectroscopy (AAS). The interfacial polymer layers fully suppressed the release of arsenic into the electrolyte buffer and also provided an adhesion-promoting interface, which allowed the cultivation of electrically excitable cells, normal rat kidney (NRK) fibroblasts, on GaAs. The electrical performance of GaAs and GaAs/InGaAs heterostructures in water was monitored via cyclic voltammetry and the IU characteristics of field-effect channels. GaAs was significantly stabilized by the insulating polymeric surface coatings, even under moderate electrochemical loads. These findings are promising for, e.g., the implementation of GaAs technology in future cell-semiconductor hybrids.

Background Metabolic activation of synovial fibroblasts (SF,1), prosthesis loosening fibroblasts (PLF,2) and osteoblasts (OB,3) results in an enhanced acid secretion as determined by cytosensor microphysiometer. In addition, PLF were shown to degrade bone in absence of infiltrating macrophages or osteoclasts. Objectives We were interested to study if the proton secretion was associated with the activity of specific proton pump, especially with the activity of V-type H ± ATPases. Methods Fibroblasts or osteoblasts were expanded in complete medium containing 10% FCS and antibiotics. For cytosensor analysis cells were seeded at 30 000 cells/ml in microcups, mounted into the cytosensor. For metabolic activation the medium was enriched with cytokines such as TNF-alpha, IL-1 (Roche) or different agents such as ionomycin or PMA (Calbiochem) at different concentrations. To block V-ATPase activity, cells were incubated with Bafilomycin A1 or Amiloride (Calbiochem). Results Metabolic activation as determined by enhanced acid secretion was observed upon stimulation of the fibroblasts with TNF-alpha, IL-1, PDGF, ionomycin or PMA but not with IL-6 or bFGF. Osteoblasts were activated by TNF-alpha, ionomycin and PMA; bFGF, IL-1, IL-6 or PDGF are not tested yet. Acid secretion occurred as early as 20–30 min. after initial stimulation. Under identical conditions, immortalised synovial fibroblast clones secreted less acid when compared to normal fibroblasts. As metabolic activation stimulates glycolysis, the acidification may be simply a consequence of carbohydrate catabolism generating acetate, lactate or carbonate. Therefore fibroblasts were incubated in medium containing Bafilomycin A1, a specific V-type H ± ATPase blocker. Bafilomycin reduced the proton secretion in a time course experiment within 20 min. irreversibly. Interestingly, Amiloride inhibited the proton secretion reversibly. Conclusion Metabolic activation of fibroblasts or osteoblasts by pro-inflammatory cytokines results in enhanced acid secretion. As pro-inflammatory cytokines enhance the expression of matrix degrading proteases as well, we conclude that the acidification of the pericellular milieu by mesenchymal cells accelerates matrix degradation. In addition, we provide for the first time experimental evidence that the acid secreted upon metabolic activation is not only a product of anaerobe (lactate, acetate) or aerobe (carbonate) glycolysis, but may be associated with a specific V-ATPase located in vacuolar membranes in the cytoplasm or on the surface of the cells. Preliminary data corroborate this conclusion, since a V-ATPase was located on the cell surface by immunohistochemistry and a V-ATPase encoding mRNA was detected by RT/PCR in the respective cells. References Parak, et al. Metabolic activation stimulates acid production in synovial fibroblasts. J Rheumatol. 2000;27:2312 Sainsbury, et al. Cathepsin K expression by activated fibroblasts at the bone interface of the pseudosynovium in aseptic prosthesis loosening. Proc. ORS Conv. Anaheim # 27, 1999 Mast A. Charakterisierung von Knochenzellen mit biophysikalischen Methoden auf zellulärem Niveau, Master Thesis, Fakult. of Physics, University of Tübingen, 1999