Phenotypic characterization of mononuclear inflammatory cells following equine hydroxyapatite/collagen block grafting
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Objective. To measure inflammatory changes associated with the implantation of an equine hydroxyapatite and collagen-containing block graft (eHAC block) in a rodent model system. Materials and Methods. An eHAC block graft was implanted subcutaneously in Wistar rats. Control groups included saline (negative control), turpentine oil (a standard model of acute-phase inflammation), and a commercially available human mineralized particulate allograft (hMPA). Animals were sacrificed and tissue samples obtained after 3 days and 1, 2, 4 and 8 weeks. A panel of 6 monoclonal antibodies was used to identify circulating monocytes (ED1), resident tissue macrophages (ED2), lymphoid macrophages (ED3), Ia-antigen expression (OX6), T-lymphocytes (OX19), and B-lymphocytes (OX33). Immunocytochemical staining was performed and cells identified by each antibody were enumerated. Sera obtained 8 wks after grafting were used in immunoblotting assays to detect the presence of systemic anti-equine antibodies. Statistical analysis was performed via two-tailed analysis of variance (F-tests) using the Bonferroni correction for multiple analyses. Results. A transient increase in ED1-positive macrophages at 3 d and 1 wk was observed in all groups, but was significantly higher in the turpentine control (P<0.0001). The influx of ED1-positive cells following turpentine administration resolved by 2 wks. The hMPA was associated with significantly greater numbers of monocytes compared to saline controls after 1 wk (P<0.05). At all other times, there were no statistically significant differences in numbers of ED1-positive cells among groups. A significant increase in numbers of ED2-positive cells was observed in the turpentine group, compared to the other groups (3 d to 4 wks; P<0.0001). A statistically significant, but smaller, difference was observed at 8 wks. After 8 wks, significance decreased to P<0.05 when comparing turpentine to saline and hMPA, and to P<0.001 when comparing turpentine to eHAC block graft. A significant increase in numbers of ED3-positive cells was observed with turpentine, compared to other groups at all time periods (P<0.0001). At all times, there were no statistically significant differences in numbers of ED3-positive cells between saline, hMPA and eHAC block groups except after 1 wk; ED3-positive cell numbers were significantly higher in the hMPA group compared to saline and eHAC block graft (P<0.0001). Significantly more OX6-positive cells were observed in the turpentine group, compared to other groups (3 d to 1 wk; P<0.0001). CD5-positive T-lymphocytes were essentially absent except for rats given turpentine (1 wk after grafting). No B-lymphocyte response was found with clone OX33. None of the rats developed systemic anti-equine antibodies as determined by immunoblotting. Conclusion. These data indicate that a cellular immune response is not elicited following implantation with the eHAC block graft material, and that eHAC might present a possible alternative for periodontal regeneration and alveolar ridge reconstruction. This project was supported in part by a grant from E. Geistlich, Ltd., Switzerland.