JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 7 (19961 77 83 Inhibition and stimulation of enzymatic activities of human fibroblasts by corrosion products and metal salts G.S. CARVALHO INEB-Instituto de Engenharia Biomedica, Praqa Coronel Pacheco, 1, 4000 Porto, Portugal Universidade do Minho, Portugal M. CASTANHEIRA, I. DIOGO, A.M. ABREU, J.P. SOUSA INEB-Instituto de Engenharia Biomodica, Praga Coronel Pacheco, 1, 4000 Porto, Portugal Departamento de Engenharia Qu[mica, FEUP, Porto, Portugal J.A. LOON, C.A. VAN BLITTERSWlJK Laboratory for Otobiology and Biocompatibifity, Biomaterials Research Group, University Hospital, 2333 Leiden, The Netherlands 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) M'I-I- 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 M'I-I" 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. 1. Introduction There is some concern over the long-term use of me- tallic biomaterials in orthopaedic surgery, based on the release of implant debris and metallic ions in tissues adjacent to implanted materials (reviewed in [1-4]). Several studies have demonstrated high metal concentrations in body fluids, including serum and urine, of patients with knee or hip prosthesis [3, 5 8]. Other reports have analysed the metal ions release from metallic alloys, their transport in the body and excretion m animal models [9-13]. Stainless steel (SS) corrosion products obtained by electrochemical means have been a matter of intense research at our laboratory. In fact, injection in mice of SS-corrosion products have shown to cause not only ultrastructural changes in male reproductive organs [14] and in liver parenchyma [15], but also accumula- tion of multinucleated giant cells and depletion of lymphocytes in spleen [16]. Further m vitro studies have demonstrated that such corrosion products in- 0957 4530 ,~', 1996 Chapman & Hall hibit the functional activity of human immune cells [17]. In the current study, in vitro cytotoxic evaluation of SS-corrosion products was performed on human fibroblasts cultured for 21 days. The cell culture para- meters analysed were: metal ion accumulation (atomic absorption spectrometry), cell morphology (neutral red assay), cell proliferation (DNA assessment), and enzyme activity (both MTT reduction and acid phos- phatase activity). The effects of SS-corroslon products were compared with the effects of iron, chromium and nickel ions used either separately (Fe, Cr, or Ni solu- tions) or combined (Fe + Cr + Ni solution). 2. Materials and methods 2.1. AISI 316L stainless steel corrosion products and metal salts Type AISI 316L stainless steel was anodically dis- solved in HBSS (Hank's Balanced Salt Solution, Sigma) through a chronoamperometric process as 77