Human trabecular bone-derived osteoblasts support human osteoclast formation in vitro in a defined, serum-free medium.
While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1alpha,25-dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25-100 ng/ml) and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures, unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.
Atkins
Gerald J
GJ
Department of Orthopaedics and Trauma, University of Adelaide, and the Hanson Institute, Adelaide, South Australia, Australia. gerald.atkins@adelaide.edu.au
Kostakis
Panagiota
P
Welldon
Katie J
KJ
Vincent
Cristina
C
Findlay
David M
DM
Zannettino
Andrew C W
AC
D036541:ADP-ribosyl Cyclase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016723:Bone Remodeling / Q000502:physiology*; D001842:Bone and Bones / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002117:Calcitriol / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D058851:GPI-Linked Proteins; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D053244:Osteoprotegerin; D010281:Parathyroid Hormone / Q000494:pharmacology; D053245:RANK Ligand; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism
D015703:Antigens, CD; D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D058851:GPI-Linked Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D010281:Parathyroid Hormone; D053245:RANK Ligand; D012333:RNA, Messenger; 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; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D036541:ADP-ribosyl Cyclase; C087521:ADP-ribosyl cyclase 2; D002117:Calcitriol