Abstract
The currently available non-degradable implant materials may cause side effects like a stress shielding effect or an allergic reaction of patients to foreign implant materials. Finally, some of the non-degradable materials cannot remain in the body and must be removed via a second surgery. In order to counteract these undesired side effects, the degradable implant materials have already proven their effectiveness in the past. However, before new alloy materials can be tested and applied in vivo, a thorough in vitro investigation of these alloys is necessary. In order to create physiological osseous in vitro conditions as close as possible to in vivo (bone), a coculture of primary cells was established and applied in this study. The coculture consisted of peripheral blood mononuclear cells (PBMC) and human umbilical cord perivascular cells (HUCPV). The investigation of different experimental conditions has shown that at a ratio of 1:100 (HUCPV:PBMC), was optimal to obtain a differentiated coculture of osteoblasts and osteoclasts within 28 days. Based on this coculture, the aim of the study was to investigate the effects of different magnesium-based alloys. The reaction of the coculture to these alloys was investigated by different cellular, enzymatic and molecular assays. In addition, the function of osteoblasts was monitored by the formation of hydroxyapatite and the function of osteoclasts was demonstrated by a resorption assay. For a more comprehensive investigation, the alloys were examined in 3 different ways. Firstly (i), the reaction of the coculture to the corresponding ions (magnesium chloride, silver nitrate and gadolinium chloride) was investigated. Thus, it could be shown that the gadolinium ions increase the formation of hydroxyapatite. In addition, (ii) the different alloys were degraded in cell culture medium to prepare extract and the behaviour of the coculture was monitored in the presence of these extracts. The investigation of the Mg-8Ag alloy has shown that silver in high concentrations had a toxic effect on the coculture. Finally (iii), the coculture was applied directly to the materials (Mg-Pure, Mg-2Ag and Mg-10Gd) and incubated to determine, whether the cells could settle on these alloys despite degradation stress, grow, and whether direct contact with the material influenced the behaviour of the cells. The enhancing effect of gadolinium, both as a salt and as an extract, on mineralisation, is assumed to be mediated via the calcium sensing receptor found on both osteoblasts and osteoclasts. Calcium sensing receptor provides an affinity to positively charged ions and is an important component of bone homeostasis. By using a phospholipase C inhibitor, an important component of the calcium sensing receptor dependent pathway was inhibited and the influence of gadolinium on the formation of the calcium sensing receptor dependent pathways demonstrated by reducing hydroxyapatite formation.
IV In addition, Mg-Pure and magnesium-silver alloys have also been shown to interact with the calcium sensing receptor. This interaction led to an inhibition of the receptor signal. Nevertheless, a positive effect on bone formation of both magnesium and silver ions remained, indicating other implicated pathways. Indeed, magnesium positively influenced bone growth via the transient receptor potential cation channel 7 / phosphoinositide 3-kinase pathway. Furthermore, silver via the increased formation of reactive oxygen species supported alkaline phosphatase expression. Finally, a combination of the three materials magnesium, silver and gadolinium could be useful to combine their positive properties.
