ORIGINAL ARTICLE Alendronate impairs epithelial adhesion, differentiation and proliferation in human oral mucosa E Donetti 1 , A Gualerzi 1 , A Sardella 2 , G Lodi 2 , A Carrassi 2 , C Sforza 1 1 Dipartimento di Scienze Biomediche per la Salute, Universit a degli Studi di Milano, Milan; 2 Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universit a degli Studi di Milano, Milan, Italy OBJECTIVE: This study aimed at evaluating from a mor- phological point of view the effects of alendronate (ALN), a widely used nitrogen-containing bisphosphonate for the chronic treatment of osteoporosis, on the oral epithelium of healthy keratinized human oral mucosa. Bisphospho- nate-related osteonecrosis of the jaw is a well-known severe consequence, but the effects during chronic ther- apy on the oral soft tissues are still matter of debate. MATERIALS AND METHODS: Six women over 60 year-old undergoing treatment of osteoporosis with 70 mg per week of oral ALN (lasting at least 2 years) were recruited and compared with a gender and age- matched group (n = 6). Proliferation, apoptosis, inter- cellular adhesion and terminal differentiation (TD) were investigated by immunofluorescence. In parallel, ultra- structural analysis was carried out. RESULTS: By immunofluorescence, a statistically signifi- cant decrease in keratinocyte proliferation was detected in the oral epithelium of the ALN group without any sign of apoptosis, but accompanied by a reduction in desmog- lein 1 and keratin 10 expressions. In the uppermost layers of the oral epithelium of the ALN group, thin desmo- somes were visible by transmission electron microscopy. CONCLUSION: Our results show that epithelial adhe- sion, TD and proliferation are affected by ALN thera- peutic doses in clinically healthy human oral mucosa. Oral Diseases (2013) doi:10.1111/odi.12154 Keywords: keratin; proliferation; desmosome; adherens junction; tight junction; transmission electron microscopy Introduction Osteoporosis is defined as low bone mineral density asso- ciated with skeletal fractures secondary to minimal or no trauma, most often involving spine, hip, and forearm. The decrease in bone mineral density is the consequence of an imbalance in bone remodelling process, consisting in higher bone resorption than bone formation. Osteoporosis affects predominantly postmenopausal women, but also older men, representing a considerable medical and socio- economic burden for modern societies (Sandhu and Hamp- son, 2011). So far, the therapeutic options for the treat- ment of osteoporosis have comprised mostly anti- resorptive drugs, in particular bisphosphonates (BPs) and more recently denosumab, but also calcitonin and, for women, oestrogens or selective oestrogen receptor modu- lators (Baron and Hesse, 2012). Bisphosphonates are synthetic analogues of endogenous pyrophosphates, showing a well-known affinity for hydroxyapatite in bones. They offer a safe and effective treatment to reduce fracture risk and can be administered either orally or i.v. Their pharmacological activity is due to the suppression of both the recruitment and the activity of osteoclasts, with consequent decrease in bone resorp- tion and hypercalcaemia (Mortensen et al, 2007). Since they were introduced in clinical practice, several questions have been raising about their possible side effects and, in particular, BP-related osteonecrosis of the jaw (BRONJ) (Watts and Diab, 2010; Diab and Watts, 2012), first described in 2003 (Marx, 2003). BRONJ is defined as the presence of exposed, necrotic bone in the oral cavity for more than 8 weeks in patients with no history of radiation therapy to the head and neck but with history of BP use (Ruggiero et al, 2009). Cumulative doses of BPs, dental extractions and/or minor oral surgery including photody- namic therapy, poorly fitting dental appliances or denture trauma are key risk factors in the development of BRONJ (Migliorati et al, 2011; Sardella et al, 2011). Originally, possible causes of BRONJ were considered the BP over-suppression of bone turnover, their anti- angiogenic activity and the acidic microenvironment neighbouring active osteoclasts, which can determine drug release from the bone. Raised BP concentration at the mu- coperiosteal interface would be responsible of the mucosal dehiscence and ulceration, which progresse in detectable bone exposure (Reid et al, 2007). Correspondence: Prof. Elena Donetti, Dipartimento di Scienze Biomedi- che per la Salute, Universit a degli Studi di Milano, via Mangiagalli 31, Milan 20133, Italy. Tel: +39 02 5031 5400, Fax: +39 02 5031 5387, E-mail: elena.donetti@unimi.it Received 1 March 2013; revised 24 May 2013; accepted 9 June 2013 Oral Diseases (2013) doi:10.1111/odi.12154 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com