title: Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK.
abstract: BACKGROUND
  Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa beta ligand /receptor activator of NF-kappa beta (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK.
  
  
  METHODS
  In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics.
  
  
  RESULTS
  In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients > or = 70 years (Fishers exact p < 0.01). Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was assayed in this study, but transmembrane RANKL may also be important in this system. Macrophages subjected to pressure (both alone and in co-culture) stained more strongly for RANK on immunohistochemstry than non-pressurized controls and 1,25-dihydroxyvitamin D3 (1,25 D3) further increased this. Immunocytochemical staining also demonstrated that more cells in pressurized co-cultures exhibited osteoclast markers (tartrate-resistant acid phosphatase, vitronectin receptor and multinuclearity) than did unpressurized controls.
  
  
  CONCLUSIONS
  These data show that in co-cultures of osteoblasts and macrophages the ratio of OPG : sRANKL was decreased by pressure in younger patients but increased in older patients. As falls in this ratio promote bone resorption, this finding may be important in explaining the relatively high incidence of osteolysis around orthopaedic implants in young patients. The finding that secretion of OPG and sRANKL by osteoblasts in monoculture was sensitive to hydrostatic pressure, and that hydrostatic pressure stimulated the differentiation of macrophages into cells exhibiting osteoclast markers indicates that both osteoblasts and preosteoclasts are sensitive to cyclic pressure. However, the effects of pressure on cocultures were not simply additive and coculture appears useful to examine the interaction of these cell types. These findings have implications for future therapies for aseptic loosening and for the development of tests to predict the development of this condition.
authors.affiliation: Laboratory & Regenerative Medicine, Stopford Building, The Medical School, University of Manchester, Oxford Road, Manchester M13 9PT, UK. carol.evans@man.ac.uk
authors.forename: C E
authors.initials: CE
authors.lastname: Evans
authors.forename: S
authors.initials: S
authors.lastname: Mylchreest
authors.forename: J G
authors.initials: JG
authors.lastname: Andrew
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000368:Aged; D000375:Aging; D015415:Biomarkers / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D006874:Hydrostatic Pressure; D007527:Isoenzymes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000378:metabolism; D012995:Solubility; D000071681:Tartrate-Resistant Acid Phosphatase; D014019:Tissue Donors
chemical_list: D015415:Biomarkers; D002352:Carrier Proteins; D006023:Glycoproteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase