J. P. Sousa

In vitro biomineralization by osteoblast-like cells I. Retardation of tissue mineralization by metal salts

The cytocompatibility of stainless steel 316L (SS 316L) corrosion products was investigated with particular focus on the dose- and time-effect of electrochemically dissolved SS and the corresponding separate metal ions on osteogenic bone marrow derived cells. Type AISI 316L stainless steel (Fe 63.9%, Cr 18.0%, Ni 12.5%, Mo 2.8%, Si 1.2%, Mn 1.6% and C 0.025%, weight for weight) was anodically dissolved in Hanks Balanced Salt Solution (HBSS) and diluted to the following concentrations: 500 microg ml(-1) of Fe, 122 microg ml(-1) of Cr and 101 microg ml(-1) of Ni, as estimated by atomic absorption spectrometry. Similarly, salt solutions containing 50 microg ml(-1) of Fe (FeCl3 x 6H2O), 122 microg ml(-1) of Cr (CrCl3 x 6H2O) or 101 microg ml(-1) of Ni (NiNO3) were prepared. All solutions were diluted 1:10(3), 1:10(4) and 1:10(5) and their effects on cell proliferation and function of rabbit bone marrow cells were studied up to 28 days of culture. Bone marrow cells (second subculture) were cultured in alpha-Minimal Essential Medium (alpha-MEM) supplemented with 10% fetal bovine serum 10(-8) mol l(-1) dexamethasone, 2.52 x 10(-4) mol l(-1) ascorbic acid and 10(-2) mol l(-1) beta-glycerophosphate. The osteoblast response to the presence of metal ions was evaluated by biochemical assays (enzymatic reduction of MTT for evaluation of cell viability/proliferation, and estimation of alkaline phosphatase (ALP) activity) and histochemical assays (identification of ALP positive cells and calcium and phosphates deposits). Results suggest a decrease in the expression of the osteoblast phenotype in the presence of ion and alloy solutions. Stainless steel corrosion products elicited slight effects but the corresponding metal ions produced pronounced effects on the osteoblast phenotype, namely an alteration in the levels and temporal expression of ALP and lower and retarded tissue mineralization ability.

Effects of AISI 316L corrosion products in in vitro bone formation

Rat bone marrow cells were cultured in experimental conditions that favour the proliferation and differentiation of osteoblastic cells (i.e., 2.52 x 10(-4) mol l(-1) ascorbic acid, 10(-2) mol l(-1) beta-glycerophosphate and 10(-8) mol l(-1) dexamethasone) in the absence and in the presence of stainless-steel corrosion products, for a period of 18 days. An AISI 316L stainless-steel slurry (SS) was obtained by electrochemical means and the concentrations of the major metal ions, determined by atomic absorption spectrometry, were 8.78 x 10(-3) mol l(-1) of Fe, 4.31 x 10(-3) mol l(-1) of Cr and 2.56 x 10(-3) mol l(-1) of Ni. Bone marrow cells were exposed to 0.01, 0.1 and 1% of the SS and at the end of the incubation period, control and treated cultures were evaluated by histochemical assays for the identification of the presence of alkaline phosphatase and also calcium and phosphate deposition. Cultures were further observed by scanning electron microscopy. Levels of total and ionised calcium and phosphorus in the culture media collected from control and metal exposed cell cultures were also quantified. Histochemical staining showed that control cultures presented a strong reaction for the presence of alkaline phosphatase and exhibited formation of calcium and phosphates deposits. The presence of 0.01% SS caused no detectable biological effects in these cultures, 0.1% SS impaired osteoblastic behaviour and, 1% SS resulted in cell death. In the absence of bone cells, levels of total and ionised calcium and phosphorus in the control and metal added culture medium were similar throughout the incubation period. A significant decrease in the levels of ionised calcium and phosphorus were observed in the culture medium of control cultures and also in cultures exposed to 0.01% SS after two weeks of incubation, an event related with the formation of mineral calcium phosphate deposits in these cultures. In cultures grown in the presence of 0.1 and 1% SS corrosion products, levels of calcium and phosphorus were similar to those observed in the absence of cells. Results showed that stainless-steel corrosion products above certain concentrations may disturb the normal behaviour of osteoblast-like rat bone marrow cell cultures.

In vitro osteoblastic differentiation of human bone marrow cells in the presence of metal ions

For periods up to 21 days human bone marrow was cultured in control conditions that favor the proliferation and differentiation of osteoblastic cells. The effect of AISI 316L corrosion products and the corresponding major separate metal ions (Fe, Cr, and Ni) were studied in three different phases of the culture period in order to investigate the effects of metal ions in cell populations representative of osteoblastic cells in different stages of differentiation. Toxicity consequences of the presence of metal ions in bone marrow cultures were evaluated by biochemical parameters (enzymatic reduction of MTT, alkaline phosphatase activity, and total protein content), histochemical assays (identification of ALP-positive cells and Ca and phosphates deposits), and observation of the cultures by light and scanning electron microscopy. Culture media were analyzed for total and ionized Ca and P and also for metal ions (Fe, Cr, and Ni). The presence of AISI 316L corrosion products and Ni salt in bone marrow cultures during the first and second weeks of culture significantly disturbs the normal behavior of these cultures, interfering in the lag phase and exponential phase of cell growth and ALP expression. However, the presence of these species during the third week of culture, when expression of osteoblastic functions occurs (mineralization process), did not result in any detectable effect. Fe salt also disturbs the behavior of bone marrow cell cultures when present during the lag phase and proliferation phase, and a somewhat compromised response between the normal pattern (control cultures) and intense inhibition (AISI 316L corrosion products and Ni salt-added cultures) was observed. Fe did not affect the progression of the mineralization phase. Osteogenic cultures exposed to Cr salt (Cr3+) presented a pattern similar to the controls, indicating that this element does not interfere, in the concentration studied, in the osteoblastic differentiation of bone marrow cells. Quantification of metal ions in the culture media showed that Cr (originated from AISI 316L corrosion products but from not Cr3+ salt) and Ni (originated from AISI 316L corrosion products and Ni salt) appear to be retained by the bone marrow cultures.

Evaluation of nickel toxicity on liver, spleen, and kidney of mice after administration of high-dose metal ion

The toxic effects caused by nickel (Ni) per si were explored by performing in vivo studies on mice following subcutaneous administration of a metallic solution of nickel at 1, 2, 3, and 4 weeks to evaluate the side effects of this metal ion when released from stainless steel implants. Other groups were similarly injected with HBSS and used as controls. Accumulation of Ni ions on liver, spleen, and kidney was assessed by an electrochemical method, adsorptive stripping voltammetry (AdSV) using microelectrodes, and atomic absorption spectrometry (AAS). Alterations of those organs induced by Ni ions were studied, showing that several histological changes had been induced. Chemical analysis and histological features indicate that Ni is partially accumulated in the study organs.

Decreased consumption of Ca and P during in vitro biomineralization and biologically induced deposition of Ni and Cr in presence of stainless steel corrosion products

The purpose of this study was to investigate the effects of 316L stainless steel (SS) corrosion products on the in vitro biomineralization process, because tissue necrosis, bone loss, impaired bone mineralization, and loosening of orthopedic implants are associated with ions and debris resulting from biodegradation. Rat bone marrow cells were cultured in experimental conditions that favored the proliferation and differentiation of osteoblastic cells and were exposed to SS corrosion products obtained by electrochemical means for periods ranging from 1 to 21 days. Quantification of total and ionized Ca and P, as well as Fe, Cr, and Ni, ions in the culture media of control and metal added cultures during the incubation period was performed to study the influence of corrosion products on the Ca and P consumption that occurs during the mineralization process. Control cultures and metal effects on cultures were evaluated concerning DNA content, enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity. Histochemical detection of ALP, Ca, and phosphate deposition, and examination of the cultures by scanning and transmission electron microscopy (SEM and TEM) were also performed. The presence of SS corrosion products resulted in impairment of the normal behavior of rat bone marrow cultures. Levels of Cr and Ni in the medium of cultures exposed to 316L SS corrosion products decreased throughout the incubation period, suggesting a regular deposition of these species; these results were supported by TEM observation of the cultures. Cultures exposed to the corrosion products presented lower DNA content, MTT reduction, and ALP activity and failed to form mineralized areas. These cultures showed negative staining on histochemical reactions for the identification of calcium and phosphate deposition and SEM and TEM examination did not show mineral globular structures or mineralization foci, respectively, which is characteristic of cultures grown in control conditions. These results suggest that metal ions associated with 316L SS are toxic to osteogenic cells, affecting their proliferation and differentiation.

Chromium accumulation and ultrastructural changes in the mouse liver caused by stainless steel corrosion products

Stainless steel (SS) corrosion products were obtained by electrochemical dissolution of SS type AISI 316L. Mice were injected subcutaneously with 0.5 ml of SS solution (containing 283 μg Fe, 69.3 μg Cr and 57 μg Ni) each 72 h, for 10 days or 14 days. After the treatment time, livers were removed and were analysed for: (a) liver wet weight; (b) contents in Fe, Cr and Ni; (c) histological and ultrastructural alterations. Results showed that the percentage of liver weight per animal body weight was significantly higher (p < 0.05) in SS-injected animals than in the control animals. The atomic absorption spectrometry analysis of dry livers showed that chromium, but not iron or nickel, had a significant increase (p < 0.05) in SS-treated mice compared to the control animals. No histopathological differences between 10 and 14 days of SS-injection could be detected, however, massive hepatic degeneration was observed in both groups when compared to the control. These histological changes in SS-treated mice were confirmed at the ultrastructural level, as hepatocytes exhibited an augmentation of vacuoles in their cytoplasm. These actual liver morphological alterations suggest that the hepatocyte function may be hampered, which constitutes a matter of some concern since liver is a blood filtering organ.

Mouse inflammatory response to stainless steel corrosion products

Corrosion occurs regularly following long-term implantation of stainless steel. Little is known about the inflammatory and immunological potential of stainless steel corrosion products. AISI 316L stainless steel was anodically dissolved in a physiologically solution, HBSS, through a chronoamperometric process by imposing an external constant current of 0.5 mA. The solution, containing 245 μg of Fe, 112 μg of Cr, 75 μg of Ni and 13 μg of Mo, was injected in the peritoneal cavity of male C57BL/6 mice. Five animals were used per survival period of time: 4, 16, 24, and 48 h, 1, 2, 4, 8 and 16 weeks. Three control mice per survival period of time were injected with HBSS. For each assay, peritoneal samples were analysed not only for the total number of cells but also for the percentages of macrophages, lymphocytes and polymorphonuclear (PMN) cells, which were estimated by differential counting on Wright-stained cytocentrifuge preparations. Our follow-up study showed that stainless steel corrosion products induced an acute inflammatory response for a period of one week as demonstrated by the influx of PMN cells and macrophages. In contrast neither a chronic inflammation nor an immune response was observed indicating that the stainless steel solution caused a minor tissue response.

Chromium Determination in Osteoblast-Like Cell Culture Medium by Catalytic Cathodic Stripping Voltammetry with a Mercury Microelectrode

A catalytic cathodic stripping voltammetric procedure for the determination of total chromium in osteoblast-like cell culture medium using a mercury film microelectrode (MFM) was optimised. The method is based on the pre-concentration of the Cr(III)-diethylenetriaminepentaacetic acid (DTPA) complex by adsorption at the potential of-1.00 V (vs. Ag/AgC1) in the presence of 10 x 10(-3) mol/L DTPA, 0.70 mol/L sodium nitrate, 0.04 mol/L sodium acetate and 1.0 x 10(-3) mol/L potassium permanganate at pH 5.9-6.0. The limit of detection obtained for a 40 s collection time was 2.80 x 10(-10) mol/L of chromium. The results achieved by stripping voltammetry using the MFM were compared to those obtained by atomic absorption spectrometry (AAS) to ensure the reliability of the electrochemical method. This procedure proved to be an alternative to AAS and valuable in biocompatibility studies performed in vitro using osteoblast-like cells.

Inhibition and stimulation of enzymatic activities of human fibroblasts by corrosion products and metal salts

Fibroblasts from normal human skin were cultured for a period of 21 days in the absence or in the presence of metal ions. The effects of stainless steel (SS) corrosion products were compared to the effects of iron, chromium and nickel ions used either separately (Fe, Cr, or Ni solutions) or combined (Fe+Cr+Ni solution). At several periods of time (4, 7, 14 and 21 days) the cell cultures were analysed for the following parameters: (a) metal ion accumulation by atomic absorption spectrometry; (b) cell morphology and viability by the neutral red assay; (c) cell proliferation by DNA assessment, and enzyme activity by both (d) MTT reduction and (e) acid phosphatase activity. Results showed that SS-corrosion products and the corresponding metal ions combined at the same concentrations, Fe+Cr+Ni solution, had opposite effects on fibroblast cultures. In fact, SS-corrosion products caused no apparent effects on cell morphology nor on cell proliferation whereas Fe+Cr+Ni solution stimulated both neutral red uptake and cell proliferation. The enzymatic assays showed that SS-corrosion products caused inhibition of both MTT reduction and acid phosphatase activity in contrast to Fe+Cr+Ni solution which stimulated their activity. Furthermore, in all biological parameters studied, a strong association was observed between the effects of Fe+Cr+Ni solution and Cr alone, suggesting that Cr was the metal ion mostly involved in the stimulatory effects of the combined solution.

Individual study of chromium in the stainless steel implants degradation: An experimental study in mice

To study the accumulation and the histological effects in mice organs caused by hexavalent chromium, one of the corrosion products released from AISI 316L stainless steel implants, mice groups were subcutaneously injected with a metallic solution of chromium during a certain period of time. Similar injections were made with HBSS (Hanks Balanced Salt Solution) in other groups to be used as controls. The levels of chromium found in the liver, kidney and spleen of the control and the treated animals were obtained by atomic absorption spectrometry (AAS) and were compared to those obtained by AdSV (adsorptive stripping voltammetry) to test the accuracy of the results. During the experimental period, the liver and spleen showed a progressive and significant accumulation of chromium whereas in the kidney the significant accumulation found after the first week practically remained unchanged during the four weeks. Apparently, the histological analysis of these tissues did not evidence any relevant morphological alteration induced by the chromium accumulations during the four weeks of treatment.