title: Zoledronic acid impairs re-epithelialization through down-regulation of integrin αvβ6 and transforming growth factor beta signalling in a three-dimensional in vitro wound healing model.
abstract: This study examined the negative effects of zoledronic acid on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model. A living oral mucosa equivalent was constructed by seeding a mixture of primary human oral keratinocytes and fibroblasts, at a cell density of 1.5 × 10(5)cm(2) each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2mM Ca(2+) EpiLife for 5 days, and then in an air-liquid interface for 14 days. The equivalent was wounded by excising a linear 2-mm-wide epithelial layer on day 8 and subsequently incubated with 10 μM zoledronic acid for an additional 11 days. Histological and immunohistochemical observations revealed zoledronic acid to significantly suppress the epithelial thickness and Ki-67-labelling index. Zoledronic acid also abolished integrin αvβ6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-β1) production into the supernatant, but down-regulated TGF-β receptor types I and II expression and Smad3 phosphorylation, as was also confirmed by immunofluorescence microscopy. This study therefore showed zoledronic acid to abrogate integrin αvβ6 expression, cause the down-regulation of TGF-β/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan; Division of Oral and Maxillofacial Surgery, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Saito
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan. Electronic address: izumik@dent.niigata-u.ac.jp.
authors.forename: K
authors.initials: K
authors.lastname: Izumi
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Shiomi
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan; Division of Oral and Maxillofacial Surgery, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Uenoyama
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan; Division of Oral and Maxillofacial Surgery, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Ohnuki
authors.affiliation: Department of Oral and Maxillofacial Surgery, University of Michigan, Ann Arbor, MI, USA.
authors.forename: H
authors.initials: H
authors.lastname: Kato
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Terada
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nozawa-Inoue
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kawano
authors.affiliation: Division of Oral and Maxillofacial Surgery, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Takagi
authors.affiliation: Division of Oral Anatomy, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Maeda
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D050071:Bone Density Conservation Agents / Q000009:adverse effects*; D002465:Cell Movement; D004164:Diphosphonates / Q000009:adverse effects*; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000009:adverse effects*; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D016023:Integrins / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D008297:Male; D009061:Mouth Mucosa / Q000187:drug effects* / Q000378:metabolism*; D062326:Re-Epithelialization / Q000187:drug effects*; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014945:Wound Healing / Q000187:drug effects*; D055815:Young Adult; D000077211:Zoledronic Acid
chemical_list: D000951:Antigens, Neoplasm; D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D007093:Imidazoles; D016023:Integrins; D016212:Transforming Growth Factor beta; C073878:integrin alphavbeta6; D000077211:Zoledronic Acid
keywords: 3D in vitro model; TGF-β signalling; bisphosphonate; oral mucosa; re-epithelialization