Basic Research—Biology
Osseous Characteristics of Mice Lacking Cannabinoid Receptor 2 after Pulp Exposure Elizabeth P. Nikolaeva, DDS,* Timothy C. Cox, PhD,†‡§ and Natasha M. Flake, DDS, PhD, MSD* Abstract Introduction: Endogenous cannabinoid compounds are involved in many physiological processes, including bone metabolism. Cannabinoid receptor 2 (CB2) plays a role in modulating bone density, but published research results are conflicting. Furthermore, the specific role of CB2 in inflammation-induced bone resorption and craniofacial bone density has not been reported. The objective of this study was to assess the role of CB2 in dental pulp exposure–induced periapical bone loss and mandibular bone density. Methods: Adult female wild-type (WT) and CB2 homozygous knockout (KO) mice were used. Pulp exposures were created unilaterally in the mandibular first molars, and the pulp was left exposed to the oral cavity to induce periapical lesion formation. Mandibles were harvested 26 days after pulp exposure. Mandibular bone mineral density and periapical lesion volume were assessed using micro–computed tomographic imaging. Results: Periapical lesion volume measured on the mesial root of the pulp-exposed first molar was significantly less in CB2 KO than WT mice (P < .05). No significant difference was detected between KO and WT mice in the size of the PDL space measured on the mesial root of the contralateral intact first molar. CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05). Conclusions: CB2 plays a role in mandibular bone metabolism. Increased bone density in CB2 KO mice may contribute to the smaller periapical lesion size observed after pulp exposure in KO compared with WT mice. Additional experiments are needed to further elucidate the function of CB2 and clinical implications of cannabinoids on bone and periapical pathosis. (J Endod 2015;41:853–857)
Key Words Bone density, cannabinoid, cannabinoid receptor 2, micro–computed tomographic imaging, periapical lesion, pulp exposure
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ndogenous cannabinoid compounds (endocannabinoids) are involved in many physiological processes, including pain, appetite, immune responses, and bone metabolism. Cannabinoid signaling involves endocannabinoids, cannabinoid receptors, and enzymes that synthesize and break down endocannabinoids. Three main cannabinoid receptors have been identified: cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2), and the G protein–coupled receptor 55 (GPR55). CB2 agonists are of therapeutic interest because they are analgesic but lack psychoactive effects (1). CB2 is also of therapeutic interest because it plays a role in regulating bone density (2–8). CB2 is expressed in many monocyte-derived cells, including circulating macrophages, microglia, and dendritic cells, as well as osteoblasts, osteoclasts, and osteocytes (1, 2). Although CB2 has been shown to play a role in modulating bone density, published results from both in vivo and in vitro research are conflicting (3, 4, 6). Furthermore, few studies have investigated the bones of the craniofacial region. The specific role of CB2 in inflammation-induced bone resorption and craniofacial bone density has not been reported. Bone metabolism plays a critical role in endodontics in the development and healing of apical periodontitis. Thus, identifying the molecular events that regulate bone resorption and apposition will provide insight into the mechanisms regulating the development and healing of apical periodontitis. There is a great body of literature on the effects of cigarette smoking on periodontal disease, periodontal treatment, and dental implants (9–12). Furthermore, the effect of cigarette smoking on apical periodontitis and endodontic outcomes has been reported in the endodontic literature (13–16). In contrast, there remains no published research on the effects of cannabinoids and cannabis on endodontic treatment, apical periodontitis, and/or endodontic outcomes. Marijuana is the most commonly used illegal drug in the United States (17). With the increase in medicinal marijuana use and the legalization of recreational marijuana in the states of Colorado and Washington, it is likely that patients will be more forthcoming in reporting their use of marijuana. Thus, investigations into the effects of the cannabinoid system, and both endogenous and exogenous compounds that affect this system, on apical periodontitis and bone metabolism are warranted. The objective of this study was to assess the role of CB2 in mandibular bone metabolism using a CB2 knockout (KO) mouse model and an established model of inflammation-induced periapical bone resorption. The role of CB2 in dental pulp exposure–induced periapical bone loss and mandibular bone density was assessed. Two null hypotheses were tested: 1. There is no difference in the size of pulp exposure–induced periapical lesions between wild-type (WT) and CB2 KO mice. 2. There is no difference in mandibular bone mineral density between WT and CB2 KO mice.
From the Departments of *Endodontics and †Pediatrics, University of Washington, Seattle, Washington; ‡Center for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington; and §Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia. Address requests for reprints to Dr Natasha M. Flake, Department of Endodontics, University of Washington, Box 357448, Seattle, WA 98195-7448. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.01.030
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Basic Research—Biology Methods Animals All protocols were approved by the University of Washington Institutional Animal Care and Use Committee. Adult female mice were used for all experiments. WT (C57BL/6J, n = 10) and CB2 homozygous KO mice (B6.129P2-Cnr2tm1Dgen/J, n = 10) were obtained from The Jackson Laboratory (Bar Harbor, ME; stock numbers: 000664 and 005786, respectively). Experiments were started when mice were 8 to 10 weeks old. Mice were housed in a room with a 12-hour dark/12-hour light cycle. Pulp Exposure Surgery Mice were anesthetized by intraperitoneal injection of ketamine/ xylazine/acepromazine (KXA; 117 mg/kg ketamine, 7.2 mg/kg xylazine, 3 mg/kg acepromazine). Pulp chambers of the mandibular first molars were exposed unilaterally under magnification using a ¼ round bur. A #08 endodontic file was used to verify the pulp exposure and allow contamination of the pulp with oral microorganisms. The pulp was left exposed to the oral cavity to induce periapical lesion formation. Using this model, periapical lesions are detectable by 2 weeks after the pulp exposure surgery (18, 19). Sample Collection Twenty-six days after pulp exposure surgery, mice were anesthetized with KXA and euthanized by transcardial perfusion with 4% paraformaldehyde. The mandibles were dissected and post-fixed in 4% paraformaldehyde overnight at 4 C and then stored in phosphatebuffered saline at 4 C.
Micro–computed Tomographic Imaging Micro–computed tomographic scans were performed at the Small Animal Tomographic Analysis (SANTA) facility located at the Seattle Children’s Research Institute, Seattle, WA, using a SkyScan 1076 instrument (SkyScan, Antwerp, Belgium). Scans were performed at an isotropic resolution of 17.63 mm using the following settings: 65 kV, 150 mA, 1.0-mm aluminum filter, 460-millisecond exposure, rotation step of 0.7o, 180o scan, and 3 frame averaging. Raw data were reconstructed using NRecon V1.6.0 software (SkyScan) and the data resliced in the coronal plane to simplify subsequent delineation of regions of interest. The 3-dimensional rendered images of each data set were generated using Drishti V2 Volume Exploration software (http://sf.anu.edu. au/Vizlab/drishti). Analysis of Periapical Lesion Size Using CTan software (SkyScan), a polygonal region of interest was chosen to outline the space between the apical portion of the mesial root and the alveolar bone (20). The volume of the periapical lesion or periodontal ligament (PDL) was determined from the area of the space within 20 consecutive sections with the first slice starting at the bottom of the tooth socket (Fig. 1A and B). An example of the volume measured in each case is visually represented by rendering the volume of interest with the entire mandibular volume but using different render settings (ie, transfer functions). Volumes were calculated for the periapical lesion on the pulp-exposed side and the PDL on the nonexposed side for all 20 specimens, with the nonexposed sides used as controls. All measurements were taken blind to the genotype of the specimen.
Figure 1. Images of a mandibular first molar with pulp exposure obtained by micro–computed tomographic imaging. (A) Three-dimensional rendered image. The green area represents a periapical volume of interest consistent with a periapical lesion. (B) Slices in the axial plane. The red areas represent examples of the region of interest used to calculate periapical lesion volume. Every fifth slice from the first slice starting at the bottom of the tooth socket is shown (slices #1, 6, 11, 16, and 20). (C) Three-dimensional rendered image. The red area represents a sphere in the mandibular ramus where bone density was measured. (D) A slice in the coronal plane. The red area represents an example of a region of interest used to calculate bone density.
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Basic Research—Biology Results To assess the role of CB2 in periapical lesion size, micro– computed tomographic imaging was used to measure lesion size 26 days after pulp exposure surgery in WT and CB2 KO mice. Reconstructed images of mandibles showed that all teeth treated with pulp exposure had a verified exposure of the pulp chamber without further damage to the tooth or alveolus (Fig. 2A–C). Periapical lesion size measured on the mesial root of the pulp-exposed first molar was significantly greater in WT mice than CB2 KO mice (P < .05) (Fig. 3A). As a control, the size of the PDL space on the mesial root of the contralateral intact first molar was measured. No significant difference was observed between WT and CB2 KO mice in the intact PDL space (Fig. 3B). To determine whether mandibular bone density is affected by CB2 receptor expression, bone mineral density of the mandibular ramus was assessed, and CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05) (Fig. 4).
Discussion Figure 2. Representative images of pulp-exposed teeth from the (A) mesialdistal, (B) buccal-lingual, and (C) occlusal views.
Analysis of Bone Mineral Density Bone mineral density was measured from a region of interest sphere created within the mandibular ramus using CTan software following calibration with commercially supplied calcium hydroxyapatite phantoms of known density (0.25 [minimum] and 0.75 [maximum] g/cm3 [Skyscan]) (Fig. 1C and D). An area distant from the site of the pulp-exposed tooth was chosen to ensure that there was no local effect of inflammation on bone density. The area measured was located posteriorly to the mandibular incisor and at the widest portion of the ramus when visualized in the coronal plane. All measurements were taken blind to the genotype of the specimen. Statistical Analyses Differences in periapical lesion or PDL size between WT and CB2 KO mice were assessed using the t test (n = 10 per group). To calculate bone density, the mean of the measurements from the right and left side of the mandible was calculated for each mouse. Differences in bone density between WT and CB2 KO mice were assessed using the MannWhitney rank sum Test because the 2 data sets did not have equal variances (n = 10 per group). Statistical significance was assessed at P < .05.
Our results show that CB2 receptor expression influences the size of periapical lesions after pulp exposure. We also show that CB2 plays a role in mandibular bone mineral density. Several studies have suggested a role for CB2 in bone metabolism. Administration of a CB2 antagonist to ovariectomized WT mice has been shown to completely prevent bone loss that normally occurs after ovariectomy in vivo (4). In this same study, Idris et al (4) showed that CB2 antagonists in vitro inhibited osteoclast formation and stimulated osteoclast apoptosis. In contrast, Ofek et al (6) reported that CB2 KO mice had decreased trabecular bone density and increased cortical expansion of the femoral metaphysis. The differences between WT and CB2 KO mice were greater with increased age, an effect similar to changes observed in human osteoporosis. Ofek et al also reported that bone in CB2 KO mice was characterized by an increased number of osteoclasts and both mineral apposition and bone formation rates, suggesting that the low bone mass was attributable to a high bone turnover rate. Additionally, they showed that a CB2 agonist decreased ovariectomy-induced bone loss and increased osteoblast number and activity (6). In contrast to the findings of Ofek et al, Idris et al (3) reported greater ovariectomy-induced bone loss of the tibial metaphysis in WT compared with CB2 KO mice and a CB2 antagonist/inverse agonist protected against ovariectomy-induced bone loss in WT mice. In their in vitro studies, a CB2-selective antagonist/inverse agonist inhibited osteoclast formation and activity, and CB2-selective agonists stimulated osteoclast formation (3). Recent reports suggest that some of the differences in results observed between studies may be caused by differences in the genetic background of the
Figure 3. (A) Periapical lesion size was significantly greater in WT mice than in CB2 KO mice (P < .05). (B) No significant difference was detected between WT and CB2 KO mice in the size of the PDL space in intact first molars. Data are means standard error of the mean. JOE — Volume 41, Number 6, June 2015
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Figure 4. CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05). Data are represented as box plots (median, 10th, 25th, 75th, and 90th percentiles).
mice studied (7, 8). The results of these studies in mice are likely relevant to humans because a single nucleotide polymorphism in the CB2 gene has been associated with osteoporosis in postmenopausal osteoporotic women (5). The results of the present study suggest that CB2 is important in both modulating bone density and the response of bone to inflammation-induced bone resorption. Mice lacking CB2 receptors had greater mandibular bone density and smaller periapical lesion sizes after pulp exposure compared with WT mice. These results are consistent with the report that ovariectomy-induced bone loss occurs to a greater extent in WT than CB2 KO mice (3). This is the first report of the effect of CB2 on inflammation-induced bone loss. The increased mandibular bone density in CB2 KO mice may contribute to the smaller periapical lesion size observed after pulp exposure in CB2 KO compared with WT mice. The effect may be mediated by the altered formation, activity, or apoptosis of osteoclasts and/or osteoblasts. Furthermore, this is the first study to investigate bone from the craniofacial region in CB2 KO mice, and it is possible that there are site-specific effects of CB2 on bone (7). Several differences have been documented in bone metabolism and physiology between the mandible and other bones (21). For example, it has been hypothesized that the high remodeling rate of bone in the jaw contributes to bisphosphonate-associated osteonecrosis at this site (22). Thus, the variations in results between studies could be caused by the genetic background of the mice used, the age of the mice, the stimulus to induce bone resorption, the specific bone studied, or a combination of these factors. Further experiments are required to elucidate the mechanism by which a lack of CB2 contributes to decreased periapical lesion size. In addition to the effects of CB2 on bone, effects of CB2 on the immune response may also play a role in modulating periapical lesion size after pulp exposure. CB2 is expressed by immune cells, including B cells, NK cells, monocytes, polymorphonuclear cells, T8-cells, and T4-cells (23). Furthermore, CB2 has been shown to play a role in immunity and inflammatory diseases in both animal models and humans (24). The role of immune cells and the inflammatory response in periapical pathosis has been widely documented; thus, the effect of CB2 on the development of apical periodontitis may be mediated by these cells. 856
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Multiple investigations of the role of CB2, cannabinoids, and marijuana have been reported in the periodontal and implant dentistry literature. These publications have investigated both bone and periodontal soft tissues. CB2 has been shown to be expressed in primary cultures of human periodontal ligament cells, and treatment of these cells with a CB2 agonist modulated osteogenic gene expression, suggesting that CB2 may play a role in alveolar bone metabolism (25). Chronic treatment with a synthetic cannabinoid decreased alveolar bone loss in a rat model of induced periodontitis (26). Cannabidiol, a component of cannabis, has also been shown to decrease alveolar bone loss in another rat model of experimental periodontitis (27). Qian et al (28) hypothesized that CB2 may be useful to facilitate osseointegration of dental implants. However, chronic inhalation of marijuana smoke decreased cancellous bone fill around titanium implants in a rat model (29). Gingival crevicular fluid has been shown to contain the endogenous cannabinoid anandamide, and both CB1 and CB2 were upregulated in human gingival fibroblasts in cases of gingivitis and periodontitis (30). Kozono et al (31) reported up-regulation of CB1 and CB2 in granulation tissue in a rat model of periodontal wound healing and also saw an increase in anandamide in gingival crevicular fluid in human patients after periodontal surgery. Furthermore, chronic marijuana use has been associated with gingival enlargement in case reports in humans (32–35). Thus, although the roles of CB2 and marijuana use are not completely elucidated in periodontics, the field has begun to address some of the mechanisms by which these compounds may impact patients. Investigations of the effects of CB2 and marijuana use on endodontic patients are also warranted. In summary, mice lacking CB2 had increased mandibular bone density and decreased periapical lesion size after pulp exposure. These results suggest that CB2 plays a role in mandibular bone metabolism. Additional research is needed to further elucidate the function of CB2 and clinical implications of cannabinoids on bone metabolism in response to endodontic pathosis.
Acknowledgments The authors thank Dr Margie Byers for use of essential resources. Supported by the American Association of Women Dentists and the American Association of Endodontists Foundation. The authors deny any conflicts of interest related to this study.
References 1. Atwood BK, Straiker A, Mackie K. CB(2): therapeutic target-in-waiting. Prog Neuropsychopharmacol Biol Psychiatry 2012;38:16–20. 2. Idris AI, Ralston SH. Role of cannabinoids in the regulation of bone remodeling. Front Endocrinol (Lausanne) 2012;3:136. 3. Idris AI, Sophocleous A, Landao-Bassonga E, et al. Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology 2008;149:5619–26. 4. Idris AI, van ’t Hof RJ, Greig IR, et al. Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat Med 2005;11:774–9. 5. Karsak M, Cohen-Solal M, Freudenberg J, et al. Cannabinoid receptor type 2 gene is associated with human osteoporosis. Hum Mol Genet 2005;14:3389–96. 6. Ofek O, Karsak M, Leclerc N, et al. Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc Natl Acad Sci U S A 2006;103:696–701. 7. Sophocleous A, Idris AI, Ralston SH. Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover. Calcif Tissue Int 2014;94:259–68. 8. Sophocleous A, Sims AH, Idris AI, Ralston SH. Modulation of strain-specific differences in gene expression by cannabinoid type 2 receptor deficiency. Calcif Tissue Int 2014;94:423–32. 9. Sherwin GB, Nguyen D, Friedman Y, Wolff MS. The relationship between smoking and periodontal disease. Review of literature and case report. N Y State Dent J 2013;79:52–7.
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Basic Research—Biology 10. Mikkilineni H, Reddy DM, Jayanth N. Effects of smoking on implant failure—a review. J N J Dent Assoc 2013;84:14–5. 11. Chambrone L, Preshaw PM, Rosa EF, et al. Effects of smoking cessation on the outcomes of non-surgical periodontal therapy: a systematic review and individual patient data meta-analysis. J Clin Periodontol 2013;40:607–15. 12. Snider TN, Cottrell D, Batal H. Summary of current consensus on the effect of smoking on implant therapy. J Mass Dent Soc 2011;59:20–2. 13. Lopez-Lopez J, Jane-Salas E, Martin-Gonzalez J, et al. Tobacco smoking and radiographic periapical status: a retrospective case-control study. J Endod 2012;38: 584–8. 14. Segura-Egea JJ, Castellanos-Cosano L, Velasco-Ortega E, et al. Relationship between smoking and endodontic variables in hypertensive patients. J Endod 2011;37: 764–7. 15. Duncan HF, Pitt Ford TR. The potential association between smoking and endodontic disease. Int Endod J 2006;39:843–54. 16. Doyle SL, Hodges JS, Pesun IJ, et al. Factors affecting outcomes for single-tooth implants and endodontic restorations. J Endod 2007;33:399–402. 17. Banta-Green CJ. Marijuana Use: Impact in Washington State. Seattle: Alcohol & Drug Abuse Institute, University of Washington; 2013. 18. Alshwaimi E, Purcell P, Kawai T, et al. Regulatory T cells in mouse periapical lesions. J Endod 2009;35:1229–33. 19. Fouad A, Barry J, Russo J, et al. Periapical lesion progression with controlled microbial inoculation in a type I diabetic mouse model. J Endod 2002;28:8–16. 20. von Stechow D, Balto K, Stashenko P, Muller R. Three-dimensional quantitation of periradicular bone destruction by micro-computed tomography. J Endod 2003;29: 252–6. 21. Matsuura T, Tokutomi K, Sasaki M, et al. Distinct characteristics of mandibular bone collagen relative to long bone collagen: relevance to clinical dentistry. Biomed Res Int 2014;2014:769414. 22. Allen MR, Burr DB. The pathogenesis of bisphosphonate-related osteonecrosis of the jaw: so many hypotheses, so few data. J Oral Maxillofac Surg 2009;67(5 Suppl):61–70.
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23. Galiegue S, Mary S, Marchand J, et al. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 1995;232:54–61. 24. Malfitano AM, Basu S, Maresz K, et al. What we know and do not know about the cannabinoid receptor 2 (CB2). Semin Immunol 2014;26:369–79. 25. Qian H, Zhao Y, Peng Y, et al. Activation of cannabinoid receptor CB2 regulates osteogenic and osteoclastogenic gene expression in human periodontal ligament cells. J Periodontal Res 2010;45:504–11. 26. Ossola CA, Surkin PN, Pugnaloni A, et al. Long-term treatment with methanandamide attenuates LPS-induced periodontitis in rats. Inflamm Res 2012;61:941–8. 27. Napimoga MH, Benatti BB, Lima FO, et al. Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats. Int Immunopharmacol 2009;9:216–22. 28. Qian H, Jin Z, Li S, et al. Activation of CB2 cannabinoid receptors: a novel therapeutic strategy to accelerate osseointegration of dental implants. Med Hypotheses 2009;72: 311–3. 29. Nogueira-Filho Gda R, Cadide T, Rosa BT, et al. Cannabis sativa smoke inhalation decreases bone filling around titanium implants: a histomorphometric study in rats. Implant Dent 2008;17:461–70. 30. Nakajima Y, Furuichi Y, Biswas KK, et al. Endocannabinoid, anandamide in gingival tissue regulates the periodontal inflammation through NF-kappaB pathway inhibition. FEBS Lett 2006;580:613–9. 31. Kozono S, Matsuyama T, Biwasa KK, et al. Involvement of the endocannabinoid system in periodontal healing. Biochem Biophys Res Commun 2010;394:928–33. 32. Layman FD. Marijuana: harmful or not? Tex Dent J 1978;96:6–8. 33. Baddour HM, Audemorte TB, Layman FD. The occurrence of diffuse gingival hyperplasia in a patient using marijuana. J Tenn Dent Assoc 1984;64:39–43. 34. Rawal SY, Dabbous M, Tipton DA. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor beta. J Periodontal Res 2012;47:320–9. 35. Rawal SY, Tatakis DN, Tipton DA. Periodontal and oral manifestations of marijuana use. J Tenn Dent Assoc 2012;92:26–31. quiz 31–22.
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Osseous Characteristics of Mice Lacking Cannabinoid Receptor 2 after Pulp Exposure Elizabeth P. Nikolaeva, DDS,* Timothy C. Cox, PhD,†‡§ and Natasha M. Flake, DDS, PhD, MSD* Abstract Introduction: Endogenous cannabinoid compounds are involved in many physiological processes, including bone metabolism. Cannabinoid receptor 2 (CB2) plays a role in modulating bone density, but published research results are conflicting. Furthermore, the specific role of CB2 in inflammation-induced bone resorption and craniofacial bone density has not been reported. The objective of this study was to assess the role of CB2 in dental pulp exposure–induced periapical bone loss and mandibular bone density. Methods: Adult female wild-type (WT) and CB2 homozygous knockout (KO) mice were used. Pulp exposures were created unilaterally in the mandibular first molars, and the pulp was left exposed to the oral cavity to induce periapical lesion formation. Mandibles were harvested 26 days after pulp exposure. Mandibular bone mineral density and periapical lesion volume were assessed using micro–computed tomographic imaging. Results: Periapical lesion volume measured on the mesial root of the pulp-exposed first molar was significantly less in CB2 KO than WT mice (P < .05). No significant difference was detected between KO and WT mice in the size of the PDL space measured on the mesial root of the contralateral intact first molar. CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05). Conclusions: CB2 plays a role in mandibular bone metabolism. Increased bone density in CB2 KO mice may contribute to the smaller periapical lesion size observed after pulp exposure in KO compared with WT mice. Additional experiments are needed to further elucidate the function of CB2 and clinical implications of cannabinoids on bone and periapical pathosis. (J Endod 2015;41:853–857)
Key Words Bone density, cannabinoid, cannabinoid receptor 2, micro–computed tomographic imaging, periapical lesion, pulp exposure
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ndogenous cannabinoid compounds (endocannabinoids) are involved in many physiological processes, including pain, appetite, immune responses, and bone metabolism. Cannabinoid signaling involves endocannabinoids, cannabinoid receptors, and enzymes that synthesize and break down endocannabinoids. Three main cannabinoid receptors have been identified: cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2), and the G protein–coupled receptor 55 (GPR55). CB2 agonists are of therapeutic interest because they are analgesic but lack psychoactive effects (1). CB2 is also of therapeutic interest because it plays a role in regulating bone density (2–8). CB2 is expressed in many monocyte-derived cells, including circulating macrophages, microglia, and dendritic cells, as well as osteoblasts, osteoclasts, and osteocytes (1, 2). Although CB2 has been shown to play a role in modulating bone density, published results from both in vivo and in vitro research are conflicting (3, 4, 6). Furthermore, few studies have investigated the bones of the craniofacial region. The specific role of CB2 in inflammation-induced bone resorption and craniofacial bone density has not been reported. Bone metabolism plays a critical role in endodontics in the development and healing of apical periodontitis. Thus, identifying the molecular events that regulate bone resorption and apposition will provide insight into the mechanisms regulating the development and healing of apical periodontitis. There is a great body of literature on the effects of cigarette smoking on periodontal disease, periodontal treatment, and dental implants (9–12). Furthermore, the effect of cigarette smoking on apical periodontitis and endodontic outcomes has been reported in the endodontic literature (13–16). In contrast, there remains no published research on the effects of cannabinoids and cannabis on endodontic treatment, apical periodontitis, and/or endodontic outcomes. Marijuana is the most commonly used illegal drug in the United States (17). With the increase in medicinal marijuana use and the legalization of recreational marijuana in the states of Colorado and Washington, it is likely that patients will be more forthcoming in reporting their use of marijuana. Thus, investigations into the effects of the cannabinoid system, and both endogenous and exogenous compounds that affect this system, on apical periodontitis and bone metabolism are warranted. The objective of this study was to assess the role of CB2 in mandibular bone metabolism using a CB2 knockout (KO) mouse model and an established model of inflammation-induced periapical bone resorption. The role of CB2 in dental pulp exposure–induced periapical bone loss and mandibular bone density was assessed. Two null hypotheses were tested: 1. There is no difference in the size of pulp exposure–induced periapical lesions between wild-type (WT) and CB2 KO mice. 2. There is no difference in mandibular bone mineral density between WT and CB2 KO mice.
From the Departments of *Endodontics and †Pediatrics, University of Washington, Seattle, Washington; ‡Center for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington; and §Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia. Address requests for reprints to Dr Natasha M. Flake, Department of Endodontics, University of Washington, Box 357448, Seattle, WA 98195-7448. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.01.030
JOE — Volume 41, Number 6, June 2015
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Basic Research—Biology Methods Animals All protocols were approved by the University of Washington Institutional Animal Care and Use Committee. Adult female mice were used for all experiments. WT (C57BL/6J, n = 10) and CB2 homozygous KO mice (B6.129P2-Cnr2tm1Dgen/J, n = 10) were obtained from The Jackson Laboratory (Bar Harbor, ME; stock numbers: 000664 and 005786, respectively). Experiments were started when mice were 8 to 10 weeks old. Mice were housed in a room with a 12-hour dark/12-hour light cycle. Pulp Exposure Surgery Mice were anesthetized by intraperitoneal injection of ketamine/ xylazine/acepromazine (KXA; 117 mg/kg ketamine, 7.2 mg/kg xylazine, 3 mg/kg acepromazine). Pulp chambers of the mandibular first molars were exposed unilaterally under magnification using a ¼ round bur. A #08 endodontic file was used to verify the pulp exposure and allow contamination of the pulp with oral microorganisms. The pulp was left exposed to the oral cavity to induce periapical lesion formation. Using this model, periapical lesions are detectable by 2 weeks after the pulp exposure surgery (18, 19). Sample Collection Twenty-six days after pulp exposure surgery, mice were anesthetized with KXA and euthanized by transcardial perfusion with 4% paraformaldehyde. The mandibles were dissected and post-fixed in 4% paraformaldehyde overnight at 4 C and then stored in phosphatebuffered saline at 4 C.
Micro–computed Tomographic Imaging Micro–computed tomographic scans were performed at the Small Animal Tomographic Analysis (SANTA) facility located at the Seattle Children’s Research Institute, Seattle, WA, using a SkyScan 1076 instrument (SkyScan, Antwerp, Belgium). Scans were performed at an isotropic resolution of 17.63 mm using the following settings: 65 kV, 150 mA, 1.0-mm aluminum filter, 460-millisecond exposure, rotation step of 0.7o, 180o scan, and 3 frame averaging. Raw data were reconstructed using NRecon V1.6.0 software (SkyScan) and the data resliced in the coronal plane to simplify subsequent delineation of regions of interest. The 3-dimensional rendered images of each data set were generated using Drishti V2 Volume Exploration software (http://sf.anu.edu. au/Vizlab/drishti). Analysis of Periapical Lesion Size Using CTan software (SkyScan), a polygonal region of interest was chosen to outline the space between the apical portion of the mesial root and the alveolar bone (20). The volume of the periapical lesion or periodontal ligament (PDL) was determined from the area of the space within 20 consecutive sections with the first slice starting at the bottom of the tooth socket (Fig. 1A and B). An example of the volume measured in each case is visually represented by rendering the volume of interest with the entire mandibular volume but using different render settings (ie, transfer functions). Volumes were calculated for the periapical lesion on the pulp-exposed side and the PDL on the nonexposed side for all 20 specimens, with the nonexposed sides used as controls. All measurements were taken blind to the genotype of the specimen.
Figure 1. Images of a mandibular first molar with pulp exposure obtained by micro–computed tomographic imaging. (A) Three-dimensional rendered image. The green area represents a periapical volume of interest consistent with a periapical lesion. (B) Slices in the axial plane. The red areas represent examples of the region of interest used to calculate periapical lesion volume. Every fifth slice from the first slice starting at the bottom of the tooth socket is shown (slices #1, 6, 11, 16, and 20). (C) Three-dimensional rendered image. The red area represents a sphere in the mandibular ramus where bone density was measured. (D) A slice in the coronal plane. The red area represents an example of a region of interest used to calculate bone density.
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Basic Research—Biology Results To assess the role of CB2 in periapical lesion size, micro– computed tomographic imaging was used to measure lesion size 26 days after pulp exposure surgery in WT and CB2 KO mice. Reconstructed images of mandibles showed that all teeth treated with pulp exposure had a verified exposure of the pulp chamber without further damage to the tooth or alveolus (Fig. 2A–C). Periapical lesion size measured on the mesial root of the pulp-exposed first molar was significantly greater in WT mice than CB2 KO mice (P < .05) (Fig. 3A). As a control, the size of the PDL space on the mesial root of the contralateral intact first molar was measured. No significant difference was observed between WT and CB2 KO mice in the intact PDL space (Fig. 3B). To determine whether mandibular bone density is affected by CB2 receptor expression, bone mineral density of the mandibular ramus was assessed, and CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05) (Fig. 4).
Discussion Figure 2. Representative images of pulp-exposed teeth from the (A) mesialdistal, (B) buccal-lingual, and (C) occlusal views.
Analysis of Bone Mineral Density Bone mineral density was measured from a region of interest sphere created within the mandibular ramus using CTan software following calibration with commercially supplied calcium hydroxyapatite phantoms of known density (0.25 [minimum] and 0.75 [maximum] g/cm3 [Skyscan]) (Fig. 1C and D). An area distant from the site of the pulp-exposed tooth was chosen to ensure that there was no local effect of inflammation on bone density. The area measured was located posteriorly to the mandibular incisor and at the widest portion of the ramus when visualized in the coronal plane. All measurements were taken blind to the genotype of the specimen. Statistical Analyses Differences in periapical lesion or PDL size between WT and CB2 KO mice were assessed using the t test (n = 10 per group). To calculate bone density, the mean of the measurements from the right and left side of the mandible was calculated for each mouse. Differences in bone density between WT and CB2 KO mice were assessed using the MannWhitney rank sum Test because the 2 data sets did not have equal variances (n = 10 per group). Statistical significance was assessed at P < .05.
Our results show that CB2 receptor expression influences the size of periapical lesions after pulp exposure. We also show that CB2 plays a role in mandibular bone mineral density. Several studies have suggested a role for CB2 in bone metabolism. Administration of a CB2 antagonist to ovariectomized WT mice has been shown to completely prevent bone loss that normally occurs after ovariectomy in vivo (4). In this same study, Idris et al (4) showed that CB2 antagonists in vitro inhibited osteoclast formation and stimulated osteoclast apoptosis. In contrast, Ofek et al (6) reported that CB2 KO mice had decreased trabecular bone density and increased cortical expansion of the femoral metaphysis. The differences between WT and CB2 KO mice were greater with increased age, an effect similar to changes observed in human osteoporosis. Ofek et al also reported that bone in CB2 KO mice was characterized by an increased number of osteoclasts and both mineral apposition and bone formation rates, suggesting that the low bone mass was attributable to a high bone turnover rate. Additionally, they showed that a CB2 agonist decreased ovariectomy-induced bone loss and increased osteoblast number and activity (6). In contrast to the findings of Ofek et al, Idris et al (3) reported greater ovariectomy-induced bone loss of the tibial metaphysis in WT compared with CB2 KO mice and a CB2 antagonist/inverse agonist protected against ovariectomy-induced bone loss in WT mice. In their in vitro studies, a CB2-selective antagonist/inverse agonist inhibited osteoclast formation and activity, and CB2-selective agonists stimulated osteoclast formation (3). Recent reports suggest that some of the differences in results observed between studies may be caused by differences in the genetic background of the
Figure 3. (A) Periapical lesion size was significantly greater in WT mice than in CB2 KO mice (P < .05). (B) No significant difference was detected between WT and CB2 KO mice in the size of the PDL space in intact first molars. Data are means standard error of the mean. JOE — Volume 41, Number 6, June 2015
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Figure 4. CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05). Data are represented as box plots (median, 10th, 25th, 75th, and 90th percentiles).
mice studied (7, 8). The results of these studies in mice are likely relevant to humans because a single nucleotide polymorphism in the CB2 gene has been associated with osteoporosis in postmenopausal osteoporotic women (5). The results of the present study suggest that CB2 is important in both modulating bone density and the response of bone to inflammation-induced bone resorption. Mice lacking CB2 receptors had greater mandibular bone density and smaller periapical lesion sizes after pulp exposure compared with WT mice. These results are consistent with the report that ovariectomy-induced bone loss occurs to a greater extent in WT than CB2 KO mice (3). This is the first report of the effect of CB2 on inflammation-induced bone loss. The increased mandibular bone density in CB2 KO mice may contribute to the smaller periapical lesion size observed after pulp exposure in CB2 KO compared with WT mice. The effect may be mediated by the altered formation, activity, or apoptosis of osteoclasts and/or osteoblasts. Furthermore, this is the first study to investigate bone from the craniofacial region in CB2 KO mice, and it is possible that there are site-specific effects of CB2 on bone (7). Several differences have been documented in bone metabolism and physiology between the mandible and other bones (21). For example, it has been hypothesized that the high remodeling rate of bone in the jaw contributes to bisphosphonate-associated osteonecrosis at this site (22). Thus, the variations in results between studies could be caused by the genetic background of the mice used, the age of the mice, the stimulus to induce bone resorption, the specific bone studied, or a combination of these factors. Further experiments are required to elucidate the mechanism by which a lack of CB2 contributes to decreased periapical lesion size. In addition to the effects of CB2 on bone, effects of CB2 on the immune response may also play a role in modulating periapical lesion size after pulp exposure. CB2 is expressed by immune cells, including B cells, NK cells, monocytes, polymorphonuclear cells, T8-cells, and T4-cells (23). Furthermore, CB2 has been shown to play a role in immunity and inflammatory diseases in both animal models and humans (24). The role of immune cells and the inflammatory response in periapical pathosis has been widely documented; thus, the effect of CB2 on the development of apical periodontitis may be mediated by these cells. 856
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Multiple investigations of the role of CB2, cannabinoids, and marijuana have been reported in the periodontal and implant dentistry literature. These publications have investigated both bone and periodontal soft tissues. CB2 has been shown to be expressed in primary cultures of human periodontal ligament cells, and treatment of these cells with a CB2 agonist modulated osteogenic gene expression, suggesting that CB2 may play a role in alveolar bone metabolism (25). Chronic treatment with a synthetic cannabinoid decreased alveolar bone loss in a rat model of induced periodontitis (26). Cannabidiol, a component of cannabis, has also been shown to decrease alveolar bone loss in another rat model of experimental periodontitis (27). Qian et al (28) hypothesized that CB2 may be useful to facilitate osseointegration of dental implants. However, chronic inhalation of marijuana smoke decreased cancellous bone fill around titanium implants in a rat model (29). Gingival crevicular fluid has been shown to contain the endogenous cannabinoid anandamide, and both CB1 and CB2 were upregulated in human gingival fibroblasts in cases of gingivitis and periodontitis (30). Kozono et al (31) reported up-regulation of CB1 and CB2 in granulation tissue in a rat model of periodontal wound healing and also saw an increase in anandamide in gingival crevicular fluid in human patients after periodontal surgery. Furthermore, chronic marijuana use has been associated with gingival enlargement in case reports in humans (32–35). Thus, although the roles of CB2 and marijuana use are not completely elucidated in periodontics, the field has begun to address some of the mechanisms by which these compounds may impact patients. Investigations of the effects of CB2 and marijuana use on endodontic patients are also warranted. In summary, mice lacking CB2 had increased mandibular bone density and decreased periapical lesion size after pulp exposure. These results suggest that CB2 plays a role in mandibular bone metabolism. Additional research is needed to further elucidate the function of CB2 and clinical implications of cannabinoids on bone metabolism in response to endodontic pathosis.
Acknowledgments The authors thank Dr Margie Byers for use of essential resources. Supported by the American Association of Women Dentists and the American Association of Endodontists Foundation. The authors deny any conflicts of interest related to this study.
References 1. Atwood BK, Straiker A, Mackie K. CB(2): therapeutic target-in-waiting. Prog Neuropsychopharmacol Biol Psychiatry 2012;38:16–20. 2. Idris AI, Ralston SH. Role of cannabinoids in the regulation of bone remodeling. Front Endocrinol (Lausanne) 2012;3:136. 3. Idris AI, Sophocleous A, Landao-Bassonga E, et al. Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology 2008;149:5619–26. 4. Idris AI, van ’t Hof RJ, Greig IR, et al. Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat Med 2005;11:774–9. 5. Karsak M, Cohen-Solal M, Freudenberg J, et al. Cannabinoid receptor type 2 gene is associated with human osteoporosis. Hum Mol Genet 2005;14:3389–96. 6. Ofek O, Karsak M, Leclerc N, et al. Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc Natl Acad Sci U S A 2006;103:696–701. 7. Sophocleous A, Idris AI, Ralston SH. Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover. Calcif Tissue Int 2014;94:259–68. 8. Sophocleous A, Sims AH, Idris AI, Ralston SH. Modulation of strain-specific differences in gene expression by cannabinoid type 2 receptor deficiency. Calcif Tissue Int 2014;94:423–32. 9. Sherwin GB, Nguyen D, Friedman Y, Wolff MS. The relationship between smoking and periodontal disease. Review of literature and case report. N Y State Dent J 2013;79:52–7.
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Basic Research—Biology 10. Mikkilineni H, Reddy DM, Jayanth N. Effects of smoking on implant failure—a review. J N J Dent Assoc 2013;84:14–5. 11. Chambrone L, Preshaw PM, Rosa EF, et al. Effects of smoking cessation on the outcomes of non-surgical periodontal therapy: a systematic review and individual patient data meta-analysis. J Clin Periodontol 2013;40:607–15. 12. Snider TN, Cottrell D, Batal H. Summary of current consensus on the effect of smoking on implant therapy. J Mass Dent Soc 2011;59:20–2. 13. Lopez-Lopez J, Jane-Salas E, Martin-Gonzalez J, et al. Tobacco smoking and radiographic periapical status: a retrospective case-control study. J Endod 2012;38: 584–8. 14. Segura-Egea JJ, Castellanos-Cosano L, Velasco-Ortega E, et al. Relationship between smoking and endodontic variables in hypertensive patients. J Endod 2011;37: 764–7. 15. Duncan HF, Pitt Ford TR. The potential association between smoking and endodontic disease. Int Endod J 2006;39:843–54. 16. Doyle SL, Hodges JS, Pesun IJ, et al. Factors affecting outcomes for single-tooth implants and endodontic restorations. J Endod 2007;33:399–402. 17. Banta-Green CJ. Marijuana Use: Impact in Washington State. Seattle: Alcohol & Drug Abuse Institute, University of Washington; 2013. 18. Alshwaimi E, Purcell P, Kawai T, et al. Regulatory T cells in mouse periapical lesions. J Endod 2009;35:1229–33. 19. Fouad A, Barry J, Russo J, et al. Periapical lesion progression with controlled microbial inoculation in a type I diabetic mouse model. J Endod 2002;28:8–16. 20. von Stechow D, Balto K, Stashenko P, Muller R. Three-dimensional quantitation of periradicular bone destruction by micro-computed tomography. J Endod 2003;29: 252–6. 21. Matsuura T, Tokutomi K, Sasaki M, et al. Distinct characteristics of mandibular bone collagen relative to long bone collagen: relevance to clinical dentistry. Biomed Res Int 2014;2014:769414. 22. Allen MR, Burr DB. The pathogenesis of bisphosphonate-related osteonecrosis of the jaw: so many hypotheses, so few data. J Oral Maxillofac Surg 2009;67(5 Suppl):61–70.
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23. Galiegue S, Mary S, Marchand J, et al. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 1995;232:54–61. 24. Malfitano AM, Basu S, Maresz K, et al. What we know and do not know about the cannabinoid receptor 2 (CB2). Semin Immunol 2014;26:369–79. 25. Qian H, Zhao Y, Peng Y, et al. Activation of cannabinoid receptor CB2 regulates osteogenic and osteoclastogenic gene expression in human periodontal ligament cells. J Periodontal Res 2010;45:504–11. 26. Ossola CA, Surkin PN, Pugnaloni A, et al. Long-term treatment with methanandamide attenuates LPS-induced periodontitis in rats. Inflamm Res 2012;61:941–8. 27. Napimoga MH, Benatti BB, Lima FO, et al. Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats. Int Immunopharmacol 2009;9:216–22. 28. Qian H, Jin Z, Li S, et al. Activation of CB2 cannabinoid receptors: a novel therapeutic strategy to accelerate osseointegration of dental implants. Med Hypotheses 2009;72: 311–3. 29. Nogueira-Filho Gda R, Cadide T, Rosa BT, et al. Cannabis sativa smoke inhalation decreases bone filling around titanium implants: a histomorphometric study in rats. Implant Dent 2008;17:461–70. 30. Nakajima Y, Furuichi Y, Biswas KK, et al. Endocannabinoid, anandamide in gingival tissue regulates the periodontal inflammation through NF-kappaB pathway inhibition. FEBS Lett 2006;580:613–9. 31. Kozono S, Matsuyama T, Biwasa KK, et al. Involvement of the endocannabinoid system in periodontal healing. Biochem Biophys Res Commun 2010;394:928–33. 32. Layman FD. Marijuana: harmful or not? Tex Dent J 1978;96:6–8. 33. Baddour HM, Audemorte TB, Layman FD. The occurrence of diffuse gingival hyperplasia in a patient using marijuana. J Tenn Dent Assoc 1984;64:39–43. 34. Rawal SY, Dabbous M, Tipton DA. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor beta. J Periodontal Res 2012;47:320–9. 35. Rawal SY, Tatakis DN, Tipton DA. Periodontal and oral manifestations of marijuana use. J Tenn Dent Assoc 2012;92:26–31. quiz 31–22.
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