title: RANKL release from self-assembling nanofiber hydrogels for inducing osteoclastogenesis in vitro.
abstract: To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL.
  
  
  
  Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala)4-CONH2[(RADA)4] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers.
  
  
  
  NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL.
  
  
  
  This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures.
  
  
  
  Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth, facilitating the required changes in the surrounding alveolar bone through the process of bone remodelling. The RANKL system regulates alveolar bone remodelling and controls root resorption during OTM. The use of exogenous RANKL to accelerate OTM has not been attempted to date because large quantities of RANKL for systemic therapy may subsequently cause serious systemic loss of skeletal bone. The controlled and sustained local release of RANKL from a carrier matrix could maximize its therapeutic benefit whilst minimizing systemic side effects. In this study a NF-hydrogel was used for sustained and controlled release of RANKL and the release kinetics and biofunctionality of the released RANKL was characterized. Our results provide fundamental insight for further investigating the role of RANKL NF-hydrogel release systems for inducing osteoclastogenesis in vivo.
authors.affiliation: School of Dentistry, University of Alberta, Edmonton, Alberta T6G 1C9, Canada; Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada.
authors.forename: James Z
authors.initials: JZ
authors.lastname: Xing
authors.affiliation: National Research Council, National Institute for Nanotechnology, Edmonton, Alberta T6G 2M9, Canada; Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2G6, Canada.
authors.forename: Lei
authors.initials: L
authors.lastname: Lu
authors.affiliation: National Research Council, National Institute for Nanotechnology, Edmonton, Alberta T6G 2M9, Canada; Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2G6, Canada.
authors.forename: Larry D
authors.initials: LD
authors.lastname: Unsworth
authors.affiliation: School of Dentistry, University of Alberta, Edmonton, Alberta T6G 1C9, Canada.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Major
authors.affiliation: Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Doschak
authors.affiliation: School of Dentistry, University of Alberta, Edmonton, Alberta T6G 1C9, Canada. Electronic address: kaipatur@ualberta.ca.
authors.forename: Neelambar R
authors.initials: NR
authors.lastname: Kaipatur
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D065546:Drug Liberation; D006801:Humans; D020100:Hydrogels / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D018625:Microscopy, Atomic Force; D057139:Nanofibers / Q000737:chemistry* / Q000648:ultrastructure; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D017433:Protein Structure, Secondary; D053245:RANK Ligand / Q000737:chemistry / Q000494:pharmacology*; D000067996:RAW 264.7 Cells; D060888:Real-Time Polymerase Chain Reaction; D000071681:Tartrate-Resistant Acid Phosphatase / Q000378:metabolism
chemical_list: D020100:Hydrogels; D053245:RANK Ligand; D000071681:Tartrate-Resistant Acid Phosphatase
keywords: Controlled and sustained release; Drug delivery; Nanofiber hydrogel; Osteoclastogenesis; RANKL