title: Incorporation of RANKL promotes osteoclast formation and osteoclast activity on β-TCP ceramics.
abstract: β-Tricalcium phosphate (β-TCP) ceramics are approved for the repair of osseous defects. In large defects, however, the substitution of the material by authentic bone is inadequate to provide sufficient long-term mechanical stability. We aimed to develop composites of β-TCP ceramics and receptor activator of nuclear factor κ-B ligand (RANKL) to enhance the formation of osteoclasts and promote cell mediated calcium phosphate resorption. RANKL was adsorbed superficially onto β-TCP ceramics or incorporated into a crystalline layer of calcium phosphate by the use of a co-precipitation technique. Murine osteoclast precursors were seeded onto the ceramics. After 15 days, the formation of osteoclasts was quantified cytologically and colorimetrically with tartrate-resistant acidic phosphatase (TRAP) staining and TRAP activity measurements, respectively. Additionally, the expression of transcripts encoding the osteoclast gene products cathepsin K, calcitonin receptor, and of the sodium/hydrogen exchanger NHA2 were quantified by real-time PCR. The activity of newly formed osteoclasts was evaluated by means of a calcium phosphate resorption assay. Superficially adsorbed RANKL did not induce the formation of osteoclasts on β-TCP ceramics. When co-precipitated onto β-TCP ceramics RANKL supported the formation of mature osteoclasts. The development of osteoclast lineage cells was further confirmed by the increased expression of cathepsin K, calcitonin receptor, and NHA2. Incorporated RANKL stimulated the cells to resorb crystalline calcium phosphate. Our in vitro study shows that RANKL incorporated into β-TCP ceramics induces the formation of active, resorbing osteoclasts on the material surface. Once formed, osteoclasts mediate the release of RANKL thereby perpetuating their differentiation and activation. In vivo, the stimulation of osteoclast-mediated resorption may contribute to a coordinated sequence of material resorption and bone formation. Further in vivo studies are needed to confirm the current in vitro findings.
authors.affiliation: Group for Bone Biology and Orthopedic Research, Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland.
authors.forename: John
authors.initials: J
authors.lastname: Choy
authors.affiliation: Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, CH-3010 Bern, Switzerland.
authors.forename: Christoph E
authors.initials: CE
authors.lastname: Albers
authors.affiliation: Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, CH-3010 Bern, Switzerland.
authors.forename: Klaus A
authors.initials: KA
authors.lastname: Siebenrock
authors.affiliation: Group for Bone Biology and Orthopedic Research, Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland.
authors.forename: Silvia
authors.initials: S
authors.lastname: Dolder
authors.affiliation: Group for Bone Biology and Orthopedic Research, Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Hofstetter
authors.affiliation: Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, CH-3010 Bern, Switzerland. Electronic address: frank.klenke@insel.ch.
authors.forename: Frank M
authors.initials: FM
authors.lastname: Klenke
mesh_terms: D000818:Animals; D018786:Bone Substitutes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D002130:Calcium Phosphates / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D002454:Cell Differentiation; D002516:Ceramics / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000166:cytology*; D053245:RANK Ligand; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D018786:Bone Substitutes; D002130:Calcium Phosphates; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; C485817:beta-tricalcium phosphate
keywords: Beta-TCP ceramics; Bone substitute materials; Osteoclast; RANKL; Resorption