Tissue responses to bone-implant biomaterials

Abstract

The purpose of the studies was to understand the mechanisms which underlie the interaction between tissue and bone-implant biomaterials. Tissue and naturally occurring aragonite nacre interaction was systematically studied for the first time by combined histological examination, cellular and molecular biological approaches. The performance of titanium, its surface oxide layer and titanium ion in vitro were also within the scope of the present thesis.

A matched resorption of nacre and ingrowth of new bone was found by histological examination in vivo. Bone was formed in an osteoconductive manner and tightly bonded with nacre, as for with the bioactive hydroxyapatite. An intermediate phosphorous-rich layer was detected at the bone-nacre interface. The different reactions of bone to nacre and to titania-hydroxyapaptite indicated that the surface of implant material exerted effect on both osteogenic and soft-tissue cells as examined by in situ hybridisation. However, the nacre implant surface did not exert a significant stimulatory effect on osteogenesis, and the bone-nacre bonding was achieved by the favourable surface provided by nacre for bone cell proliferation and bone matrix to attach. Degradation of nacre was mediated by a combination of cellular and physico-chemical dissolution processes. Macrophages and multinucleated giant cells were shown to be responsible for the cellular degradation process. These cells exhibited different molecular profiles when compared in soft and osseous tissue, i.e., the cells expressed high level of osteopontin mRNA in soft tissue and less at the osseous site. The multinucleated giant cells did not represent typical osteoclasts, since their size, degradation efficiency and expression of tartrate resistance acid phosphate mRNA and tartrate resistance acid phosphate enzyme activity were different from that of osteoclasts.

We also examined the structural and compositional influence of cultured osteoblasts on titanium surface, together with the effect of hydrogen peroxide treatment. By a combination of electrochemical impedance spectroscopy and x-ray photoelectron spectroscopy, we found that phosphate, calcium ions and carboxyl groups were incorporated into microscopic pores in the titanium oxide film, with surface hydroxyl groups being involved, precipitating an hydroxylcarbonated apatite layer on the titanium oxide film during the mineralization. Hydrogen peroxide treatment resulted in a thickening of the porous oxide layer and large amounts of surface hydroxyl groups, which facilitated the incorporation of phosphate and calcium ions. When culturing osteoblasts on such a surface, the morphology of cells and mineralization of the nodules was changed, indicating a beneficial effect of hydrogen peroxide treatment on bone formation.

The potential risk of titanium ions was studied by using the rat calvaria cell culture. We found that the titanium ions was toxic to calvaria cells in vitro at concentration above 1Oppm. Titanium ion also had an inhibitory effect on mineralization in a concentration-dependent manner. Under the challenge of 5ppm titanium ion, the ability of osteoid nodules formation was retained, however, the properties of the nodule matrix were altered, resulting in incomplete mineralization. By the analysis of specific gene expressions, we revealed that titanium ion altered the proportional composition of cellular mRNA, which lacked osteonectin and osteopontin, and preserved osteocalcin sequences.

In conclusion, the combination of molecular biological and cell culture techniques has been shown to be a useful approach to understanding the complexity of tissue responses to bone implant materials, and by studying such responses, it also envisaged that our knowledge of bone biology will increase in general.