Cytoprotective effects of melatonin on zoledronic acid-treated human mesenchymal stem cells in vitro

Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 855e862

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Cytoprotective effects of melatonin on zoledronic acid-treated human mesenchymal stem cells in vitro Francisco Javier Rodríguez-Lozano a, b, *, 1, David García-Bernal a, 1,

a, Maria de los Angeles Ros-Roca a, Maria del Carmen Alguero b b ~ ate-Sa
nchez , Fabio Camacho-Alonso , Jose María Moraleda a Ricardo Elías On a

Hematopoietic Transplant and Cellular Therapy Unit, Hematology Department, Virgen de la Arrixaca Clinical University Hospital, IMIB, University of Murcia, Spain School of Dentistry, Faculty of Medicine, University of Murcia, Spain

b

a r t i c l e i n f o

a b s t r a c t

Article history: Paper received 16 December 2014 Accepted 10 April 2015 Available online 18 April 2015

Objective: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a common clinical complication in patients receiving bisphosphonate therapy. Furthermore, melatonin has been proposed as a therapeutic drug for the oral cavity due to its antioxidant properties. This study aimed to evaluate the cytoprotective effects of melatonin on zoledronic acid (ZA)-treated human mesenchymal stem cells from periodontal ligament (PDLSCs) and bone marrow (BMMSCs). Methods: PDLSCs and BMMSCs were exposed to ZA, melatonin or ZA þ melatonin for 72 h. Cell proliferation was measured by a colorimetric assay, whereas their mesenchymal phenotype was analyzed by flow cytometry. Results: Proliferation assays showed that BMMSCs presented higher ZA resistance than PDLSCs, as well as a difference in response to the simultaneous treatment of ZA þ melatonin. Using PDLSCs, high doses of melatonin significantly increased their proliferation, whereas lower concentrations were enough to enhance ZA-treated BMMSC proliferation. Moreover, PDLSCs displayed a CD90/CD105 downregulation and CD73 upregulation in response to ZA, which was more pronounced in response to melatonin. Furthermore, ZA or ZA þ low doses of melatonin induced a decrease of expression of CD90/CD105/CD73 on BMMSCs, while a higher concentration recovered CD73 levels. Conclusion: These results suggest that melatonin has a cytoprotective effect on ZA-treated PDLSCs and BMMSCs. Thus, it could be used for BRONJ prevention. © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Keywords: Bone marrow stem cells Melatonin Periodontal ligament stem cells Zoledronic acid

1. Introduction Bisphosphonates (BPs) are widely used compounds for the management of oncology and osteoporosis patients. They inhibit bone loss in osteoporosis, and the growth of bone tumors and metastasis, by limiting bone remodeling and also inhibiting the function of several cell types such as osteoclasts (Pavlakis et al., 2005; Wong et al., 2012). Although BPs are very effective in

* Corresponding author. Hematopoietic Transplant and Cellular Therapy Unit, Hematology Department, Virgen de la Arrixaca Clinical University Hospital, IMIB, University of Murcia, Ctra. Madrid-Cartagena. El Palmar. 30120, Murcia, Spain. Tel.: þ34 968 369532; fax: þ34 968 369088. E-mail address: [email protected] (F.J. Rodríguez-Lozano). 1 These authors equally contributed to this work.

reducing bone loss, pain and other skeletal clinical manifestations, they could also induce several adverse effects such as inflammation, esophageal ulcers and bisphosphonate-related osteonecrosis of the jaw (BRONJ) (Yoshiga et al., 2014; Bagan et al., 2014; Ruggiero, 2007, 2009). Zoledronic acid (ZA) is a nitrogen-containing intravenous BP and a highly potent inhibitor of bone resorption. Osteoclasts incorporate BPs by phagocytosis and pinocytosis, being rapidly adsorbed to bone hydroxyapatite (Rogers, 2003). Although BRONJ is a complication that appears not only after use of BPs, it has been previously reported that patients who received ZA treatment exhibited a significant 30-fold increase in the risk of developing BRONJ (Wessel et al., 2008). In the great majority of cases, BRONJ occurs in patients who have undergone dental extractions with active periodontal or periapical disease. Importantly, this clinical

http://dx.doi.org/10.1016/j.jcms.2015.04.012 1010-5182/© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

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complication could be also favored by the absence of suitable postoperative treatment (Marx et al., 2005, 2007). Some researchers have previously studied the role of periodontitis and the use of ZA in BRONJ development using different animal models (Aghaloo et al., 2011; Aguirre et al., 2012). In humans, some casereports suggested that periodontitis could predispose patients to develop BRONJ (Kos, 2014; Thumbigere-Math et al., 2014). On the other hand, melatonin is an endogenous antioxidant hormone synthesized from serotonin in the pineal gland and other
mez-Moreno et al., 2010; Reiter et al., 2014). Many of its tissues (Go effects differ to its primary neurohormonal functions and are due to its anti-inflammatory properties, its ability to be an effective freeradical scavenger and for the stimulation of several antioxidant enzymes (Allegra et al., 2003; Czesnikiewicz-Guzik et al., 2007). In response to the inflammation of periodontal disease, increased levels of plasma melatonin may be induced, leading to an increase in salivary melatonin, where this indoleamine may exert a protective role during the periodontal inflammatory process due to its
mez-Moreno et al., anti-inflammatory and anti-oxidant effects (Go
mez-Florit et al., 2013). 2007; Go Furthermore, several adult tissues such as bone marrow stroma and periodontal ligament contain a variety of mesenchymal stem cells (MSCs) that possess immunomodulatory properties and the ability to differentiate into adipogenic, osteogenic and chondrogenic cells (Rodríguez-Lozano et al., 2011). Their functional properties have been proven by several experimental and clinical studies using autologous bone marrow stem cells (BMMSCs) for wound healing, cell architecture repair, and recovery of local blood flow in injured and ischemic tissues (Sun et al., 2009). In addition, the osteogenic plasticity of BMMSCs is a very interesting and useful property in the field of reconstructive bone surgery, including oral
lvez-García et al., 2012, 2013). and maxillofacial surgery (Gonza Due to the role of periodontal ligament stem cells (PDLSCs) in periodontal regeneration and the possible application of BMMSCs as cell therapy in BRONJ (Gonz
alvez-García et al., 2013), PDLSCs and BMMSCs were chosen as cellular sources in this work. Many in vitro and in vivo studies have suggested beneficial effects of melatonin on bone metabolism including both anabolic and anti-resorptive effects. Also, melatonin promotes osteoblast proliferation and differentiation (Nakade et al., 1999; Roth et al., 1999); decreases osteoclast-related bone resorption (Koyama et al., 2002; Suzuki and Hattori, 2002); and accelerates osseointegration of ~ oz et al., 2012). It has been previously dental implants (Mun described that the effects of melatonin on bone cells may be mediated, at least in part, by a reduction of the cellular oxidative stress and the maintaining of the mitochondrial function (She et al., 2014). On these grounds, the aim of this in vitro study was to evaluate the possible cytoprotective effects of melatonin on zoledronic acidtreated human mesenchymal stem cells from different sources by analyzing cell proliferation and mesenchymal stem cell phenotype. 2. Material and methods 2.1. Isolation and culture of PDLSCs and BMMSCs Human periodontal ligaments (hPDL) were obtained from impacted third molars from 10 healthy donors. The hPDLs were scraped from the middle third region of the root surface, dissected, washed with Ca2þ/Mg2þ-free Hank's balance salt solution (HBSS) (Gibco Invitrogen, Paisley, Scotland), and incubated in the presence of collagenase A (3 mg/mL) (SigmaeAldrich, St. Louis, MO, USA) for 1 h at 37  C. After, cells were seeded into 25-cm2 plastic tissue culture flasks (BD Biosciences, San Diego, CA, USA) in Dulbecco's Modified Eagle Medium (DMEM) (Gibco Invitrogen) supplemented

with penicillin/streptomycin (PAA Laboratories, Pasching, Austria), (PAA Laboratories) and 10% fetal bovine serum (FBS) (Gibco Invitrogen) (complete medium) and incubated at 37  C in a humid atmosphere containing 7.5% CO2 for 3 days. The BMMSCs were isolated by percutaneous direct aspiration of bone marrow from the iliac crest of 10 healthy volunteers. The aspirated bone marrow was transferred to a sterile tube containing sodium heparin (20 U/ml), followed by a Ficoll density gradient-based separation and cell washing using the closed automated SEPAX System (Biosafe, Eysines, Switzerland) to obtain the mononuclear cell fraction. Then, cells were seeded into 75-cm2 tissue culture flasks (BD Biosciences) at a density of 1.6  105 cells/cm2 in complete medium and allowed to attach undisturbed for 7 days in the same culture conditions as the PDLSCs. For both types of culture, after visualizing adherent cells, erythrocytes and non-adherent cells were removed by washing with Ca2þ/Mg2þ-free phosphatebuffered saline (PBS) (Gibco Invitrogen). When cultures reached 80% of confluence, cells were washed with PBS and detached by incubating with 0.25% trypsin-EDTA solution (Gibco Invitrogen) for 3e5 min at 37  C. For subsequent subcultures, cells were seeded at a density of 5  103 cells/cm2, replacing the medium every 3 days. This study was approved by Ethical Committee of Murcia University (reference no. 300305440012). For obtaining all of the human samples, donors gave written, informed consent according to the guidelines of the Ethics Committee of our Institution. L-glutamine

2.2. Reagents and cell treatment Zoledronic acid (Zometa) was purchased from Teva Pharmaceutical Industries (Petah Tikva, Israel) and melatonin was obtained from Sigma Aldrich (St Louis, MO, USA). For the different experiments, BMMSCs and PDLSCs were resuspended in complete medium, added to wells at a density of 3.0  104 cells/cm2 and allowed to adhere for 24 h at 37  C. After this, cells were exposed to increasing concentrations of ZA and/or melatonin for an additional 72 h at 37  C. 2.3. Proliferation assays The effect of increasing concentrations of ZA and melatonin on the proliferation rate of BMMSC and PDLSCs was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay (MTT Cell Growth Kit, Chemicon, Rosemont, Illinois, USA). This assay is based on the ability of the mitochondrial dehydrogenase enzymes to convert the yellow water-soluble tetrazolium salt into colored compounds of formazan, whose absorbance is proportional to the number of living cells. Proliferation of BMMSCs and PDLSCs was analyzed after 72 h of culture. Briefly, wells were washed twice with PBS and 50 mL of MTT solution (1 mg/mL) were added to the culture and incubated for 4 h. After, each well was washed with 300 mL of PBS 1X, and 100 mL of dimethyl sulfoxide (DMSO) was added to extract and solubilize the formazan. Optical density at 570 nm (OD570) was measured by using an automatic microplate reader (ELx800; Bio-Tek Instruments, Winooski, VT, USA), using OD690 as the reference wavelength. Each experimental condition was analyzed in quintuplicate and subjected to statistical analysis. 2.4. Analysis of expression of mesenchymal stem cell markers by flow cytometry Changes in the expression of mesenchymal stem cell markers after treatment with zoledronic acid and/or melatonin were analyzed by flow cytometry. To carry out this objective, PDLSCs and BMMSCs were seeded and exposed to ZA and/or melatonin as

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described above. The cells were then detached using a 0.25% trypsin-EDTA solution and incubated with fluorescence-conjugated monoclonal antibodies for the mesenchymal stem cell markers CD73, CD90, and CD105 and hematopoietic markers CD14, CD20, CD34 and CD45 (Miltenyi Biotec, Bergisch Gladbach, Germany). The positive expression of CD73, CD90 and CD105 is recommended by the International Society of Cellular Therapy (ISCT) as essential to confirm the mesenchymal phenotype of the cells (Dominici et al., 2006; Horwitz et al., 2005). The cells were analyzed using a Beckman Coulter Navios flow cytometer (Fullerton, CA, USA) and data analyzed with Kaluza analysis software. Non-specific fluorescence was measured using specific isotype monoclonal antibodies. 2.5. Direct contrast-phase microscopic observation PDLSCs and BMMSCs were plated in 24-well plates as described above and cultured in the presence of different concentrations of ZA and/or melatonin for 72 h. After, microscopic images of the cells were acquired using an inverted contrast-phase microscope (Nikon, Tokyo, Japan). 2.6. Statistical analysis Statistical analysis was carried out using SPSS statistical software version 15.0 (SPSS, Inc., Chicago, IL, USA) and group comparisons were conducted using a Student's t-test. For all studies, a p < 0.05 was deemed significant. All values were expressed as mean ± SD. 3. Results 3.1. Direct contrast-phase microscopic observation In order to determine the effects of the in vitro treatment of ZA and/or melatonin on PDLSCs or BMMSCs cultures, cells were treated with different BP and melatonin concentrations for 72 h. Microscopic observation visually demonstrated that in the presence of ZA, cells detached from wells prior to undergoing apoptosis. In particular, high concentrations of ZA (10 mM) significantly decreased the number of attached cells, for both PDLSCs and BMMSCs (Fig. 1A and B). In the BMMSC cultures, the addition of 300 mM melatonin was enough to reduce the number of detached ZA-treated cells compared with cells only treated with ZA (Fig. 1B); whereas the addition of melatonin to PDLSC cultures had a lower cytoprotective effect from 5 mM ZA (Fig. 1A). 3.2. Effects of ZA and melatonin on MSC proliferation Cell proliferation and viability was examined by MTT assays following treatment with various concentrations of ZA, melatonin and ZA plus melatonin for 72 h. Doseeresponse experiments showed that BMMSCs presented higher resistance to ZA than PDLSCs. The proliferation rate of PDLSCs was significantly lower at 3 mM and 5 mM ZA compared with untreated cells (p < 0.01 and p < 0.001), whereas BMMSCs displayed a significantly decreased proliferation from 10 mM ZA (p < 0.001) (Fig. 2A and B). Using the higher concentration of ZA (30 mM), the proliferation rate of PDLSCs and BMMSCs showed a reduction of 72.8% and 44.8%, respectively. Importantly, after the simultaneous treatment of ZA and melatonin, PDLSCs and BMMSCs also displayed a different behavior. When PDLSCs were used, low doses of 100 mM melatonin were not enough to ameliorate the cytotoxic effect of ZA in cell proliferation, even at the minimum ZA concentration employed (1 mM). But melatonin concentrations 300 mM significantly increased the PDLSC proliferation up to 5 mM ZA when compared with levels

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obtained using same concentrations of ZA alone (p < 0.05, p < 0.01, p < 0.001) (Fig. 2A, left and 2C, top table). However, and due to their higher ZA resistance, melatonin-treated BMMSCs exhibited a significant increase of their proliferation rate from 10 mM ZA (p < 0.01, p < 0.001). Importantly, low concentrations of 100 mM of melatonin were enough to obtain the maximum cytoprotective effect on BMMSC proliferation (Fig. 2A, right, and 2C, bottom table). 3.3. Effect of ZA and melatonin on MSC phenotype of PDLSCs and BMMSCs To evaluate the possible regulatory effect of melatonin on ZAtreated MSC phenotype, the surface marker expression on PDLSCs and BMMSCs treated with different concentrations of both compounds was analyzed by flow cytometry. More than 95% of viable PDLSCs and BMMSCs were positive for the mesenchymal markers CD73, CD90 and CD105 (Fig. 3) and negative for the hematopoietic markers CD14, CD20, CD34 and CD45 (data not shown). After 72 h of treatment with increasing concentrations of ZA alone, viable PDLSCs showed a slight decrease in levels of expression (mean fluorescence intensity values) of CD90 and CD105, and a slight increase of expression of CD73, mainly from 5 mM ZA (Fig. 3A, upper histograms). Importantly, combined treatment with melatonin did not alter the level of expression of CD90 or CD105 on ZA-treated PDLSCs, but significantly increased CD73 expression, mainly at higher concentrations (500 mM) (Fig. 3A, middle and lower histograms). On the other hand, ZA-treated viable BMMSCs displayed a decrease of levels of expression of CD90, CD105 and CD73, mainly from 5 mM ZA, which was more pronounced when combined with low concentrations of melatonin (300 mM) (Fig. 3B, upper and middle histograms). Importantly, higher concentrations of melatonin (500 mM) induced a recovery of levels of expression of CD73 on BMMSCs, even at the higher ZA concentrations tested (Fig. 3B, lower histograms). 4. Discussion Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a multifaceted and common complication in patients subjected to long-term treatment with bisphosphonates (BPs). It was first reported in the early 2000s, when oral and maxillofacial surgeons began to report on cases of non-healing, exposed and necrotic bone in the maxillofacial region of these patients (Marx, 2003; Ruggiero et al., 2004; Dunstan et al., 2007). The clinical appearance of BRONJ is characterized by a breach in the oral mucosa that finally leads to a necrotic exposed area in the maxillary and/or mandibular bone (Ruggiero et al., 2014; Ruggiero, 2007, 2009, Rupel et al., 2014). The incidence and pathophysiology of BRONJ are still not well described (Sharma et al., 2013). However, many potential contributory factors have been identified, including the use of BPs. Most BRONJ lesions in adult patients appear after extraction of teeth that cannot be restored due to severe caries or periapical/periodontal disease. Thus, tooth extraction is the most frequent dental predisposing factor of BRONJ development (Thumbigere-Math et al., 2014; Ruggiero et al., 2014). It has been reported that oral or intravenous administration of BPs such as zoledronic acid, pamidronate or alendronate may have some adverse effects on cells other than osteoclasts, such as gingival/periodontal ligament fibroblasts and/or PDLSCs, causing a decrease in cell activity and viability (Cozin et al., 2011; Agis et al., 2009; Reid et al., 2007). The in vitro results presented here show that PDLSCs and other mesenchymal stem cells such as BMMSCs differ in their sensitivity to ZA cytotoxicity, which caused a decrease in cell proliferation/viability and modulation of mesenchymal stem cell markers. While PDLSCs showed a significant decrease of their

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Fig. 1. Direct contrast-phase microscopic observation of PDLSCs and BMMSCs cultured after ZA and melatonin treatment. PDLSCs (A) or BMMSCs (B) were cultured with different concentrations of ZA alone or combined with melatonin for 72 h at 37  C. Effects of both compounds were evaluated by direct-contrast phase microscopic observation. Representative images of each treatment are shown. Scale bar: 100 mm.

proliferation rate and viability from 3 mM ZA, BMMSCs were only sensitive from 10 mM ZA. However, BPs help and balance out the osteoblast and osteoclast homeostasis to give a positive outcome; furthermore, no other cells such as mesenchymal stem cells or osteoblast precursors should be affected. In fact, despite its toxic effects, it has been found that ZA is able to induce the osteoblastic differentiation of mesenchymal stem cells, a mechanism that potentially could help the recovery of bone homeostasis (Pan et al., 2004; Farshdousti Hagh et al., 2012; Ebert et al., 2009). Thus, the secondary effects of BP administration could be reduced by the concurrent use of cytoprotective drugs. Melatonin has been described as a cytoprotective and prosurvival agent that acts through both membrane receptordependent and independent mechanisms (Luchetti et al., 2014). Many of its effects differ to its primary neurohormonal functions due to its properties as an anti-inflammatory, free radical scavenger and stimulator of antioxidant enzymes (Rodriguez et al., 2004; Reiter et al., 2014, 2000). In this in vitro study, the cytoprotective effect of melatonin was confirmed in cultures of ZA-treated PDLSCs and BMMSCs. Melatonin displayed different levels of cytoprotective effects in ZA-treated MSCs. Low or medium doses of 100e300 mM of melatonin were enough to decrease the number of apoptotic BMMSCs in culture or increase their proliferation up to 30 mM ZA,

while in PDLSCs doses 300 mM of melatonin only protected up to 5 mM ZA. Importantly, it has been established that melatonin promotes osteoblast proliferation and differentiation, decreases osteoclastrelated bone resorption and stimulates osteointegration of dental implants (Nakade et al., 1999; Roth et al., 1999; Koyama et al., 2002; Cutando et al., 2008). On the other hand, expression of mesenchymal stem cell markers could be regulated by several stimuli. It has been previously reported that 50 -ecto-nucleotidase or CD73 could act as a regulator of osteogenic differentiation of MSCs. Using the CD73 inhibitor adenosine-50 -(a,b-methylene) diphosphate the mineral matrix deposition and expression of osteogenic markers such as alkaline phosphatase and osteocalcin were reduced, while CD73 overexpression induced an increase of osteocalcin, alkaline phosphatase and bone sialoprotein-2 expression (Ode et al., 2013). Here we show that ZA increased CD73 expression on PDLSCs that could hypothetically reflect a stimulation of osteogenic differentiation, as previously reported (Ebert et al., 2009). Importantly, melatonin further increased CD73 expression on ZA-treated PDLSCs and BMMSCs, being therefore a compound that could potentiate the osteoblast differentiation process. Moreover, ZA also induced a slight decrease of CD90 and CD105 levels on PDLSCs and BMMSCs. Confirming our results, it has also been found that expression levels

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Fig. 2. Effect of ZA and melatonin on PDLSCs proliferation. (A) PDLSCs and BMMSCs were treated with different concentrations of ZA and/or melatonin for 72 h at 37  C. Cell proliferation/viability of PDLSCs and BMMSCs were determined using the 3-(4,5-dimethyl-thiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. (B) Data represent the statistical analysis of the proliferation rate of PDLSCs and BMMSCs after treatment with different concentrations of ZA. Proliferation was significantly decreased compared with levels showed by untreated cells. (C) Data represent the statistical analysis of the proliferation rate of PDLSCs and BMMSCs after combined treatment of ZA and melatonin. Proliferation was significantly increased compared with levels showed by cells treated with same concentration of ZA alone. Level of statistical significance was obtained using Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

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Fig. 3. Mesenchymal immunophenotype analysis of PDLSCs and BMMSCs after ZA and melatonin treatment by flow cytometry. PDLSCs (A) and BMMSCs (B) were cultured in presence of the indicated concentrations of ZA and/or melatonin for 72 h at 37  C. After, PDLSCs and BMMSCs were labeled with fluorescence-conjugated specific antibodies for the mesenchymal markers CD73, CD90 and CD105 and hematopoietic markers CD14, CD20, CD34 and CD45 (not shown). Insert numbers represent the mean fluorescence intensity values from viable cells. Figure shows representative flow cytometry histograms obtained after three independent experiments.

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of both markers progressively declined in MSCs differentiating towards osteoblast-like cells (Chen et al., 1999; Wiesmann et al., 2006; Jin et al., 2009). On the other hand, stem cells of the orofacial region, including mandibular and maxillary jaws, differ from those of other bones. The jaws are derived from neural crest cells and undergo intramembranous, instead of endochondral, ossification (Chai and Maxson, 2006; Xu et al., 2013). Differences between mandibular and long bone osteoblastic and osteoclastic proliferation, differentiation, and function have been also reported (Aghaloo et al., 2010, 2011). This apparent close association of active dental disease with human BRONJ prompted us to explore the effects of ZA on PDLSCs and compare them with those obtained using BMMSCs. Our results confirmed that PDLSCs were more sensitive to ZA than BMMSCs. These results might explain the relationship between periodontal disease and the occurrence of BRONJ. However, in the presence of melatonin this cytotoxic effect was lower. It is interesting to note that a melatonin-induced increase in reactive oxygen species (ROS) has been described in cancer cells and appears to be associated with a reduction in tumor growth and proliferation (Bejarano et al., 2011; Sainz et al., 2003). In addition, melatonin was shown to have a pro-oxidant effect that is mediated by an increase in NF-kB activity and an upregulation or downregulation of several anti-oxidative or pro-oxidant enzymes, respectively (Cristofanon et al., 2009; Pozo et al., 1994; Gilad et al., 1998). 5. Conclusion The possible clinical use of cytoprotective agents such as melatonin in combination with BP therapy could represent a promising therapeutic alternative to avoid clinical complications such as BRONJ. Thus, preventive medication may be given for the whole time of the BP administration schedule and thereafter, covering the long half-life of the drugs. Prevention may also include giving the medication at events of imminent BRONJ risk (e.g., tooth extraction). Grants This work was supported by FIS EC07/90762 Grant and the Spanish Net of Cell Therapy (TerCel) provided by Carlos III Institute of Health (ISCiii) (PI13/02699 and EC11-009) together with the Junction Program for Biomedical Research in Advanced Therapies and Regenerative Medicine from ISCiii. Acknowledgments We thank the teeth donors for their generosity. This work was supported by FIS EC07/90762 Grant and the Spanish Net of Cell Therapy (TerCel) provided by Carlos III Institute of Health (ISCiii) (PI13/02699 and EC11-009) together with the Junction Program for Biomedical Research in Advanced Therapies and Regenerative Medicine from ISCiii. References Aghaloo TL, Chaichanasakul T, Bezouglaia O, Kang B, Franco R, Dry SM, et al: Osteogenic potential of mandibular vs. long-bone marrow stromal cells. J Dent Res 89: 1293e1298, 2010 Aghaloo TL, Kang B, Sung EC, Shoff M, Ronconi M, Gotcher JE, et al: Periodontal disease and bisphosphonates induce osteonecrosis of the jaws in the rat. J Bone Miner Res 26: 1871e1882, 2011 Agis H, Blei J, Watzek G, Gruber R: Is zoledronate toxic to human periodontal fibroblasts? J Dent Res 89: 40e45, 2009 Aguirre JI, Akhter MP, Kimmel DB, Pingel JE, Williams A, Jorgensen M, et al: Oncologic doses of zoledronic acid induce osteonecrosis of the jaw-like lesions

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