Background: Aseptic loosening of total joint arthroplasty is characterised by osteolysis caused by osteoclasts and macrophages. Osteolysis occurs by acidification and dissolution of hydroxyapatite crystals then proteolysis of the bone collagen matrix. N-Telopeptide (NTx) and deoxypyridinolone (DPD) represent highly specific markers for bone resorption.

Aim: To investigate whether urinary NTx and DPD generated in-vivo can be used as bone markers in a small animal model of wear debris induced osteolysis.

Materials and Methods: 41 and 38 urinary samples were collected from mice at autopsy four weeks following either the implantation of clinically relevant ceramic particles or sham surgery into their femora and assayed for NTx and DPD respectively. Bone markers were corrected for urinary creatinine.

Results: The mean urinary NTx concentration for mice that underwent the implantation of clinically relevant ceramic particles was 95.0 nM BCE/mM creatinine compared to 85.3 nM BCE/mM creatinine for mice who had sham surgery (p = 0.8, 95%CI: −29.0 to 30.7). The mean urinary DPD concentration for mice that underwent the implantation of clinically relevant ceramic particles was 5.3 nM DPD/mM creatinine compared to 4.0 nM DPD/ mM creatinine for mice who had sham surgery (p = 0.07, 95%CI: −2.8 to 1.4).

Conclusion: The absolute values of NTx and DPD increased in mice that underwent the implantation of clinically relevant ceramic particles compared to sham surgery even though this was not statistically significant. Extending the post operative interval might allow both NTx and DPD to be utilised as bone markers of osteolysis in our small animal model of aseptic loosening.

Background: The commonest cause of long term failure of total joint arthroplasty is aseptic loosening. As a result, many patients will require complex revision surgery that is not only technically challenging but associated with poorer results. Revisions procedures are also associated with higher morbidity and costs.

Aim: To quantify osteolysis in a small animal model of aseptic loosening. This model can then be utilised for screening therapeutic agents to inhibit aseptic loosening.

Materials and Methods: 7 time mated female mice were injected with radioactive calcium 45 on day 14 of gestation. The 52 offsprings were divided into 2 equal groups and subjected to either the implantation of clinically relevant ceramic particles or sham surgery into their femora. The non-operated femora were used as control. Animals were killed 4 weeks following surgery. Femora were retrieved, dissolved and radioactivity measured as outcome (CPM/mg = Counts Per Minute per milligram). A Linear mixed effects model was utilised to examine the difference in outcome between the 2 groups.

Results: The mean scintillation count for sham surgery was 388 CPM/mg compared to 449 CPM/mg in the control femora. The mean scintillation count for ceramic particles was 351 CPM/mg compared to 420 CPM/mg in the control femora. The mean effect on outcome of surgery with ceramic particles relative to sham surgery was estimated at 16.7 CPM/mg (95CI%: 0.9 to 32.5 CPM/mg; p = 0.025).

Conclusion: We have successfully shown that this model can quantify osteolysis. However, the difference detected between sham surgery and ceramic particles was biologically small displaying the inert properties of ceramic. Extending the post surgery interval might show a larger difference between sham surgery and ceramic particles and permit quantitative analysis of therapeutic agents to be screened to inhibit aseptic loosening.

Introduction: Activated matrix metalloproteinases (MMPs) are responsible for the osteolytic process in aseptic loosening. Fluvastatin has been shown in previous studies to inhibit MMPs. The purpose of this study was to investigate the potential pharmacological effect of fluvastatin on aseptic loosening.

Materials and methods: A radiolabelled mouse calvaria and human interface membrane cells culture system was used to study the effect of fluvastatin on bone resorption induced by interface membrane cells. The experimental conditions were: 1) test culture consisted of culturing a dead radiolabelled bone disc with cells from human interface membrane in culture medium containing fluvastatin for 14 days 2) control culture consisted of similar culture system without fluvastatin and 3) baseline control culture consisted of bone disc only. The bone discs from each test and control groups comparison were from the same parietal bone to ensure equal amount of radioactive calcium in the bone discs at the start of the experiment. Supernatant were sampled on day 7, 10 & 14 for scintillation counting. The total Ca⁴⁵ remained in the bone discs at the completion of the culture were measured by scintillation counting. Eight sets of experiments were performed in this study. The results were expressed as the ratio of Ca⁴⁵ in the fluvastatin exposed culture over culture containing bone disc only. In the control, the results were expressed as ratio of bone disc exposed to cells over culture containing bone disc only.

Results: In the supernatant Ca⁴⁵, both the fluvastatin and control ratios increased with time confirming bone resorption in both culture. The fluvastatin culture consistently showed a lower ratio compared with control indicating an inhibitory effect. In the fluvastatin culture the mean ratios on day 7, 10 & 14 were 0.79, 1.53 and 2.55. The mean ratios in the control culture were 1.03, 1.81 and 3.20 (n = 8, p = 0.0001 ANOVA, General Linear Model). The mean ratio of total Ca⁴⁵ remaining in the bone disc in the fluvastatin culture was 0.87 and the control was 0.70 (p = 0.01, t test). This implies 55% less bone resorption in the presence of fluvastatin.

Conclusions: In this in vitro study we found fluvastatin has the ability to inhibit osteolysis by cells from interface membrane of aseptically loosened hips. We believe fluvastatin inhibit bone resorption by MMPs inhibition. This may have therapeutic implication in the treatment of patients with aseptic loosening of total joint replacements.

Background: Aseptic loosening of total joint arthroplasty is characterised by osteolysis, which is caused by osteoclasts and macrophages. Collagen cross link molecules N Telopeptides (NTx) are released during osteolysis and represents a highly specific marker for bone resorption. NTx could be a potential marker in the laboratory investigation of aseptic loosening with the advantage of being cheaper and easier to perform compared to present established markers. 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.

Methods: Cells from human interface membranes of aseptic loosened hip joints were cultured with dead radiolabelled (45Ca) mice calvaria discs and in the control culture no cells were added to the culture system. Calvaria discs used in each experiment comparison were from the same parietal bone. The supernatant culture medium were extracted on day 3,7,10 & 14 and assayed for NTx and by scintillation counting. On day 14 the remaining culture medium and cells were assayed by scintillation counting and the total remaining 45Ca in the bone were measured.

Results: All results were expressed as the ratio of bone exposed to cells (BC)/bone only (B). Supernatant samples for 45Ca and NTx showed a rise in BC/B ratio with time. These were 0.83, 0.88, 0.97 & 1.08 (p=0.0001) and 1.06, 1.21, 1.41 & 1.40 (p = 0.03) respectively. In the bottom sampling for 45Ca the mean ratio of BC/B was 1.8 (p=0.0001) and the mean BC/B ratio for the remaining 45Ca 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 = 0.88).

Discussion: The initial drop of calcium levels can be explained by calcium uptake by the cells. 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 radilabelled bone with NTx as a marker represents an important step towards simplifying and reducing the cost of an in in vitro model of particle induced osteolysis.

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