Mathematical correlation between biomaterial and cellular parameters--critical reflection of statistics.

For mathematical modelling of the biomaterial-cell contact, it is necessary to find both parameters characterizing physical and chemical properties of the material surface and also such describing the reaction of the adhering cells. Only those material and cell parameters that correlate with each other are applicable to model this contact mathematically. Only few papers are dealing with this special problem. The aim of this paper is to present results of physical/chemical and biological investigations made on differently modified rough titanium implant surfaces in order to find out only the correlating parameters. Furthermore we discuss several ways to apply statistical methods to the correlation problem. Only few ones of all investigated parameters both on material and on cellular side were applicable for correlation. For example we found in our studies that fractal structure parameter topothesy has influence on the spreading behaviour of the osteoblastic cells. However the value of the correlation coefficient and its statistical significance heavily depend on the method of averaging the available data. Especially the biological data (spreading area) were afflicted with relatively high error up to 30%. Averaging of this data masks the true facts. That is why the correlation coefficient considerably decreases if the biological parameters are not averaged. On the other hand, the statistical reliability increases due to the higher number of investigated cases. Critical error discussion is necessary in statistical correlation between material and biological parameters. Often the results are heavily influenced by the statistical handling of data, especially if only few data are available. May be that new unconventional methods like bootstrap method can show a way out of this dilemma.

Spark plasma sintering synthesis of porous nanocrystalline titanium alloys for biomedical applications.

The reason for the extended use of titanium and its alloys as implant biomaterials stems from their lower elastic modulus, their superior biocompatibility and improved corrosion resistance compared to the more conventional stainless steel and cobalt-based alloys [Niinomi, M., Hattori, T., Niwa, S., 2004. Material characteristics and biocompatibility of low rigidity titanium alloys for biomedical applications. In: Jaszemski, M.J., Trantolo, D.J., Lewandrowski, K.U., Hasirci, V., Altobelli, D.E., Wise, D.L. (Eds.), Biomaterials in Orthopedics. Marcel Dekker Inc., New York, pp. 41-62]. Nanostructured titanium-based biomaterials with tailored porosity are important for cell-adhesion, viability, differentiation and growth. Newer technologies like foaming or low-density core processing were recently used for the surface modification of titanium alloy implant bodies to stimulate bone in-growth and improve osseointegration and cell-adhesion, which in turn play a key role in the acceptance of the implants. We here report preliminary results concerning the synthesis of mesoporous titanium alloy bodies by spark plasma sintering. Nanocrystalline cp Ti, Ti-6Al-4V, Ti-Al-V-Cr and Ti-Mn-V-Cr-Al alloy powders were prepared by high-energy wet-milling and sintered to either full-density (cp Ti, Ti-Al-V) or uniform porous (Ti-Al-V-Cr, Ti-Mn-V-Cr-Al) bulk specimens by field-assisted spark plasma sintering (FAST/SPS). Cellular interactions with the porous titanium alloy surfaces were tested with osteoblast-like human MG-63 cells. Cell morphology was investigated by scanning electron microscopy (SEM). The SEM analysis results were correlated with the alloy chemistry and the topographic features of the surface, namely porosity and roughness.

Calpain activation contributes to oxidative stress-induced pancreatic acinar cell injury.

Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca(2+)-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H(2)O(2))-generated oxidative stress in the presence or absence of the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H(2)O(2) provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms mu- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.

Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas expressed TF antigen on the syncytiotrophoblast layer, but with lower intensity compared to normal placentas. We found a significantly reduced co-expression of TF antigen and cytokeratin 7 in the decidua of abort placentas. These data suggested a reduction of extravillous trophoblast cells in the decidua of abort placentas. In addition, we found higher numbers of CD45-positive cells in the abort decidua compared to normal placentas.

Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material.

The interaction of cells with the extracellular matrix at the interface of an implant determines the biology of cells and tissues. We analysed components of cell adhesion and measured physico-chemical characteristics of structural modifications of titanium surfaces: polished, machined, glass particle-blasted, corundum-blasted, vacuum plasma-sprayed. Scanning electron microscopy and profilometry revealed a differentiated topography from smooth to rough surfaces, respectively. Osteoblastic MG-63 cells showed an increased spreading on surfaces with low roughness, although without a straight correlation with the surface topography. Integrin expression was increased on structured surfaces compared with polished material, and the organization of the actin cytoskeleton and fibronectin was impaired on extremely rough surfaces. Electrochemical methods, especially the electrochemical impedance spectroscopy (EIS) was used to evaluate physico-chemical characteristics, and the impedance curves revealed a dependence on the roughness of the material surfaces. Further analyses of the EIS results were performed using equivalent circuits which model the electrical flow through the interface. First indications for a correlation between parameters from the equivalent circuits with surface properties were obtained which promise a relevance for the biological response of the cells.