ARTICLE IN PRESS 1.75 1.50 0.50 0.00 Cell layer hyperplasia Cell hypertrophy Synovial linging cell reaction Fig 9. The evaluation of the synovial lining cell reaction was based on the addition of two individual parameters: cell hyperplasia and hypertrophia of the synovial lining cell. Results are presented for alumina( group B, n= 12). zirconia (2 group C, n=12)and for the control group ( E n=10). Values are expressed as mean+ SEM, p<0.05, cytocompatibility. Apart the control, the polished surface more device. According to several studies, bone cell functions may be preserved cell functions and proliferation than the roughness one, influenced by particles size, chemistry and shape 27,33 ven if the difference between these two materials was not statis- 35, 58,61,62] The clinical success of a new alumina-zirconia ceramic tically significant. This may confirm the real influence of the surface lies partly to the low production of wear particles. Because the topography on cell behavior described in many works (48, 56, 57 amount of particles we obtained after tribologic test was too low for If the cytocompatibility of the composite is the key element to be an in vitro evaluation, we decided to replace them by alumina and checked in order to use it in an implant, it is also known that wear zirconia constitutive powders. Germain et al. reported different debris induce bone loss and is an important cause of aseptic loos- toxicities for alumina wear debris and commercially or constituen ening of the hip prosthesis [27, 58-61. Thus, biological evaluation of particles if their size and volume are different [63]. Here, we have not these wear debris is needed to predict the long-term reliability of the verified these settings but we think that it is a reliable model especially in our condition of study. Indeed we tested in vitro twe powders of alumina and zirconia at two different concentrations with two cell types on many parameters like proliferation, viability 1.6 data not shown and ECm production. The concentrations of particles(100 and 1000 ug/mL) were chosen in accordance with 1.4 those used by gutwein in previous works [21, 38. Moreover, the results of tribological tests made us think they are both very 12 important compared to the amount that should be observed in clinical use. In parallel, the in vivo study came to reinforce the quality 1.0蕨 of this approach. If there were any significant toxicity, we would have underscored it. The present study has shown that alumina and zirconia constitutive powders at 100 ug/mL have no influence either 0.8 on the cell proliferation and viability(data not shown), or on the roduction of extra-cellular matrix. this was verified for both oste- oblasts and fibroblasts. similar results were observed with a concentration of 1000 ug/mL up to 10 days of culture. After this time, proliferation of osteoblasts decreased while that of fibroblasts 0.4 increased. These alterations were very light and associated only with an increased of the production of fibronectin for both cell types. This 0.2 is in agreement with literature reports of the very small detriment observed [21, 27, 38, 64,65. Furthermore, contrary to other works Macrophages Vascularisation about metal and polymer particles, we have not observed any gnificant decrease Fig- 10. Score of the evaluation of sub-synovial tissue reaction Macrophage and vas- 28, 29, 32, 66. Moreover, we have not observed any differences in acaeasetionswstre e anucted ed wings a degrees ed os maea n sEnt of ive eate cellular response in the presence of either alumina or zirconia valuated, n=12 for animals injected with alumina () or zirconia (50) and n=10 for powders. Their effect seems to depend on their concentration but the control group g and +++p<0.001 compared to the control for the same not on their chemical nature. The present study thus demonstrates the excellent in vitro cytocompatibility of the constitutive particles lease cite this article in press as: Roualdes O, et al, In vitro and in vivo evaluation of an., Biomaterials(2009), doi: 10.1016/ j biomaterials 2009. 11.107cytocompatibility. Apart the control, the polished surface more preserved cell functions and proliferation than the roughness one, even if the difference between these two materials was not statis￾tically significant. This may confirm the real influence of the surface topography on cell behavior described in many works [48,56,57]. If the cytocompatibility of the composite is the key element to be checked in order to use it in an implant, it is also known that wear debris induce bone loss and is an important cause of aseptic loos￾ening of the hip prosthesis [27,58–61]. Thus, biological evaluation of these wear debris is needed to predict the long-term reliability of the device. According to several studies, bone cell functions may be influenced by particles size, chemistry and shape [27,33– 35,58,61,62]. The clinical success of a new alumina–zirconia ceramic lies partly to the low production of wear particles. Because the amount of particles we obtained after tribologic test was too low for an in vitro evaluation, we decided to replace them by alumina and zirconia constitutive powders. Germain et al. reported different toxicities for alumina wear debris and commercially or constituent particles if their size and volume are different [63]. Here, we have not verified these settings but we think that it is a reliable model, especially in our condition of study. Indeed, we tested in vitro two powders of alumina and zirconia at two different concentrations with two cell types on many parameters like proliferation, viability (data not shown) and ECM production. The concentrations of particles (100 and 1000 mg/mL) were chosen in accordance with those used by Gutwein in previous works [21,38]. Moreover, the results of tribological tests made us think they are both very important compared to the amount that should be observed in clinical use. In parallel, the in vivo study came to reinforce the quality of this approach. If there were any significant toxicity, we would have underscored it. The present study has shown that alumina and zirconia constitutive powders at 100 mg/mL have no influence either on the cell proliferation and viability (data not shown), or on the production of extra-cellular matrix. This was verified for both oste￾oblasts and fibroblasts. Similar results were observed with a concentration of 1000 mg/mL up to 10 days of culture. After this time, proliferation of osteoblasts decreased while that of fibroblasts increased. These alterations were very light and associated only with an increased of the production of fibronectin for both cell types. This is in agreement with literature reports of the very small detrimental effect of nanosized particles despite the important endocytosis observed [21,27,38,64,65]. Furthermore, contrary to other works about metal and polymer particles, we have not observed any significant decrease on the production of type I collagen [28,29,32,66]. Moreover, we have not observed any differences in cellular response in the presence of either alumina or zirconia powders. Their effect seems to depend on their concentration but not on their chemical nature. The present study thus demonstrates the excellent in vitro cytocompatibility of the constitutive particles. Cell layer hyperplasia Score Cell hypertrophy Synovial linging cell reaction 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 + +++ ++ ++ +++ +++ Fig. 9. The evaluation of the synovial lining cell reaction was based on the addition of two individual parameters: cell hyperplasia and hypertrophia of the synovial lining cell. Results are presented for alumina ( , group B, n ¼ 12), zirconia ( , group C, n ¼ 12) and for the control group ( , n ¼ 10). Values are expressed as mean  SEM, þp < 0,05, þþp < 0,01, þþþp < 0.001 compared to control group for the same parameter. Macrophages Score Vascularisation 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 +++ +++ Fig. 10. Score of the evaluation of sub-synovial tissue reaction. Macrophage and vas￾cularisation were evaluated following 3 degrees: 0 ¼ normal, 1 ¼ slight or moderate increase, 2 ¼ strong increased. Values are expressed as mean  SEM of five fields evaluated, n ¼ 12 for animals injected with alumina ( ) or zirconia ( ) and n ¼ 10 for the control group ( ) and þþþp < 0.001 compared to the control for the same parameter. 10 O. Roualdes et al. / Biomaterials xxx (2009) 1–12 ARTICLE IN PRESS Please cite this article in press as: Roualdes O, et al., In vitro and in vivo evaluation of an..., Biomaterials (2009), doi:10.1016/ j.biomaterials.2009.11.107