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Expression of RANKL in Osteolytic Membranes: Association with Fibroblastic Cell Markers
Samuel C. Ramage1; Nicole H. Urban, PhD1; William A. Jiranek, MD1; Aparna Maiti, PhD1; Matthew J. Beckman, PhD1
1 Departments of Biochemistry (S.C.R. and M.J.B.) and Orthopaedics (N.H.U., W.A.J., A.M., and M.J.B.), Orthopaedic Research Laboratory, Virginia Commonwealth University School of Medicine, 1112 East Clay Street, Richmond, VA 23298-0614. E-mail address for M.J. Beckman: mjbeckma@hsc.vcu.edu
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Disclosure: The authors did not receive any outside funding or grants in support of their research for or preparation of this work. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. Flow Cytometry and Imaging Facility paid or directed in any one year, or agreed to pay or direct, benefits of less than $10,000 to a research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which one or more of the authors, or a member of his or her immediate family, is affiliated or associated.
Investigation performed at the Departments of Biochemistry and Orthopaedics, Virginia Commonwealth University School of Medicine, Richmond, Virginia

The Journal of Bone and Joint Surgery, Incorporated
J Bone Joint Surg Am, 2007 Apr 01;89(4):841-848. doi: 10.2106/JBJS.F.00655
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Background: Aseptic loosening is often mentioned as the primary reason for costly revision of total joint arthroplasties. Receptor activator of nuclear factor-?B ligand (RANKL) appears to be a major factor in the bone resorption observed in periprosthetic osteolysis. RANKL plays an essential role in the recruitment, differentiation, and survival of the osteoclasts implicated in periprosthetic osteolysis. This study was performed in an effort to identify the cell type in the periprosthetic membrane responsible for expression of RANKL.

Methods: Tissues harvested from osteolytic lesions in nine patients undergoing total joint revision were serially sectioned for immunohistochemical analysis. Intercellular adhesion molecule-1 (ICAM-1) and prolyl 4-hydroxylase (5B5) antibodies were used to detect fibroblasts, and anti-CD-163 (Ber-MAC3) was used to detect macrophages. In addition, antibodies to osteoprotegerin (OPG), RANKL, and receptor activator of nuclear factor-?B (RANK) were utilized. The binding pattern of these antibodies was then viewed with confocal microscopy with the use of only secondary antibodies as method controls.

Results: Histological analysis was confined to areas of the membrane where cells were detected with use of Hoechst 34580 nuclear stain. In the membrane specimens from all nine patients, diffuse RANKL staining was localized to areas lacking cells and more intense staining was seen in areas containing nucleated cells. There was strong colocalization between RANKL and OPG, and there was weak but specific colocalization between RANKL and both 5B5 and ICAM-1. In contrast, there was complete separation of antibody staining of Ber-MAC3 and RANKL, indicating only generalized overlap of the myeloid markers with the RANKL.

Conclusions: RANKL expression was localized to cells that stained positively for fibroblast markers. The data also indicated that there is an intact RANKL/RANK/OPG system in the periprosthetic membrane that could regulate focalized bone resorption in osteolysis.

Clinical Relevance: Identifying the cell types responsible for RANKL production is critical to the development of a strategy to prevent periprosthetic osteolysis.

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