CAN NTX BE USED AS AN IN VITRO MARKER OF WEAR PARTICULATE INDUCE OSTEOLYSIS

Abstract

Aseptic loosening of total joint arthroplasty is characterised by osteolysis that is caused by osteoclasts and macrophages. The mechanism of bone resorption is by acidification, dissolution of hydroxyapatite crystals then proteolysis of the bone collagen matrix. The collagen cross-link molecules are cleaved by osteoclasts exposing the N terminal of the cross-link protein - N Telo-peptides (NTx). This represents a highly specific marker for bone resorption. Previously described bone resorption models include radiolabelled animal bones which require the use of animals and radioactive materials or thin dentine slice resorption pits which are only semi-quantitative and technically difficult to produce. NTx could be a potential osteolysis marker in the laboratory investigation of aseptic loosening with the advantage of being cheaper and easier to perform compared to present established marker and also does not require animals or radioactive materials. The aim of this study was to show that NTx generated during osteolysis by cells extracted from human interface membranes of aseptically loosened hips correlates with the established radiolabelled ⁴⁵Ca bone resorption model.

Cells from human interface membranes of aseptic loosened hip joints were extracted from the tissue following enzyme digestion. These cells were cultured with dead radiolabelled (⁴⁵Ca) mice calvaria discs in the presence of 1,25 dihydroxyvitamin D₃, hydrocortisone, RANKL and M-CSF. In the control culture no cells were added to the culture system. Calvaria discs used for each experiment comparison were from the same parietal bone. The supernatant culture medium were extracted on day 3, 7, 10 and 14 and assayed for NTx and by scintillation counting. On day 14 the remaining culture medium and cells were assayed by scintillation counting. The remaining bone samples were decalcified and the total remaining ⁴⁵Ca in the bone was measured.

All results were expressed as the ratio of bone exposed to cells (BC)/bone only (B). Supernatant samples for ⁴⁵Ca showed a rise in BC/B ratio with time 0.83, 0.88, 0.97 and 1.08 (p= 0.0001). Supernatant samples for NTx also showed a rise with time 1.06, 1.21, 1.41 and 1.40 (p=0.03). In the bottom sampling for ⁴⁵Ca the mean ratio of BC/B was1.8 (p=0.0001) and the BC/B ratio for the remaining radioactivity in the bone at the end of the culture was 0.81(p=0.0007). There was a strong correlation between ⁴⁵Ca and NTx (r= 0.9).

The absolute values of ⁴⁵ Ca dropped initially due to the uptake of calcium by the cells and this explains our previously unsuccessful attempt to use non radioactive calcium as a marker of bone destruction. We believe this is the first time human interface membrane cells have been shown to release NTx during osteolysis in an in vitro model. Replacing ⁴⁵Ca radiolabelled bone with NTx as a marker represents an important step towards simplifying and reducing the cost of an in vitro model of particle induced osteolysis