JOURNALOF MATERIALS SCIENCE: MATERIALS IN MEDICINE 12 (2001) 939±944 In vivo/ex vivo cellular interactions with titanium and copper F. SUSKA 1 ,M.KA È LLTORP 1 , M. ESPOSITO 1 , C. GRETZER 1 , P. TENGVALL 2 P.THOMSEN 1 * 1 Institute of Anatomy and Cell Biology, Biomaterials Research Group, Go Èteborg University, Box 420, SE-405 30 Go Èteborg, Sweden 2 Laboratory of Applied Physics, Linko Èping University, SE-581 83 Linko Èping, Sweden E-mail: PeterThomsen@dof.se Machined, commercially pure titanium (Ti) disks were coated with approximately 400nm copper (Cu) by physical vapor deposition or left uncoated. The kinetics of in¯ammatory cell recruitment, distribution and viability was evaluated around Ti, Cu, and in sham sites after 1, 3, 12, 18, 24, and 48h in a rat subcutaneous (s.c.) model. Further analysis of the cells on implant surfaces was performed by ex vivo incubation of the disks. Ti and Cu stimulated an increased recruitment of in¯ammatory cells in comparison with sham sites. A markedly higher amount of cells, predominantly polymorpho-nuclear granulocytes (PMN), was detected around Cu after 18h and onwards. More cells were found at the implant surfaces than in the surrounding exudates after 18 h. The total amount of lactate dehydrogenase (LDH), an indicator of plasma membrane injury, was higher in Cu exudates after 18h in comparison with Ti and sham. In contrast, no differences in the proportion of dead cells (trypan blue dye uptake) were detected in the exudates. Further, LDH levels were higher around Ti than Cu during the initial 18h of ex vivo incubation. The results of this study indicate that the early in¯ammatory process associated with a cytotoxic material in soft tissuesislargelyattributedtotheinductionofamarkedlystrongandprolongedchemotactic response. In contrast, this process is characterized by a higher amount of in¯ammatory cells aroundabiocompatiblematerialthaninshamsites,butwithatransientcourseandtotalLDH similar to sham sites. # 2001 Kluwer Academic Publishers 1. Introduction The migration and accumulation of leukocytes consti- tutes a hallmark of the in¯ammatory response at an implant site. The surgical trauma and the presence of the implant stimulate leukocytes to migrate to the implant- tissue interface. The chemotactic response around titanium (Ti) is transient and lower in magnitude than for several other implant materials, which is of importance when designating a material the properties of biocompatibility (reviewed in Holgers et al. [1]). Copper (Cu) is a trace metal and an essential component of several enzymes. Cu insuf®ciency is associated with often severe pathologic alterations including impairment of blood, liver, and immune systems [2,3]. Cu ions stimulate endothelial cell proliferation [4] and migration [5] and modulate angiogenesis in vivo [6±8]. An extensive angiogenesis was a prominent feature around intra muscular (i.m.) implanted Cu disks [9]. On the other hand, corrosion of Cu implants with release of Cu ions may cause acute and chronic in¯ammation and in some cases even necrosis and sterile abscesses in tissues, including the brain [10±12], bone [13], abdomen and thorax and their organs [14±16], and muscle [17]. Cytotoxicity after Cu exposition has been studied mainly in vitro [18±23]. Cellular actions of Cu include destabilization of membranes via superoxide and hydroxyl radicals, af®nity to RNA and DNA and inhibition of the transcription process [24, 25], inhibition of calmodulin, a regulator of, e.g. intracellular Ca 2 [26], and induction of apoptosis [27]. Excess Cu within cells after in vivo implantation has been found in, e.g. hepatocytes [15]. In Wilson's disease and Menke's syndrome the accumulation of Cu is associated with cytotoxic effects [28,29]. The cytotoxicity of Cu has been used in cancer chemotherapy [30]. On the basis of the available literature on Ti and Cu these materials are associated with widely different biological outcomes. However, the early cellular responses at the implant- tissue interface in vivo have not been analyzed. The rat subcutaneous (s.c.) implantation model using the dorsum permits an analysis of cellular reactions as a function of time. Cells and molecules in the different compartments (material surface, exudate, and ®brous capsule) may be * Author to whom correspondence should be addressed. 0957±4530 # 2001 Kluwer Academic Publishers 939