Curr Osteoporos Rep (2010) 8:40–48 DOI 10.1007/s11914-010-0008-1

The Role of Microbial Biofilms in Osteonecrosis of the Jaw Associated with Bisphosphonate Therapy Satish K. S. Kumar & Amita Gorur & Christoph Schaudinn & Charles F. Shuler & J. William Costerton & Parish P. Sedghizadeh

Published online: 19 February 2010 # Springer Science+Business Media, LLC 2010

Abstract Microbial biofilms have been observed and described in bone specimens of patients with bisphosphonate (BP)-associated osteonecrosis of the jaw (BONJ) and investigators are more recently suggesting that this condition essentially represents an osteomyelitis of the jaw clinically, with greater susceptibility in some patients on BP therapy. This article explains the role of microbial biofilms in BONJ and also discusses associated factors in the disease pathogenesis, which include BP effects on bone remodeling, anti-angiogenesis, matrix necrosis, microcracks, soft tissue toxicity, and inflammation and wound healing. Recent findings suggest a key role for microbial

S. K. S. Kumar Clinical Dentistry, Orofacial Pain and Oral Medicine Center, Division of Diagnostic Sciences, School of Dentistry, University of Southern California, Los Angeles, CA, USA A. Gorur : C. Schaudinn Center for Biofilms, School of Dentistry, University of Southern California, Los Angeles, CA, USA C. F. Shuler School of Dentistry, University of British Columbia, Vancouver, Canada J. W. Costerton Microbiology Research, Department of Orthopedic Surgery, Allegheny General Hospital and Center for Genomics, Allegheny-Singer Research Institute, Pittsburgh, PA, USA P. P. Sedghizadeh (*) USC School of Dentistry, Center for Biofilms, 925 West 34th Street #4110A, Los Angeles, CA 90089-0641, USA e-mail: [email protected]

biofilms in the pathogenesis of BONJ; this has important therapeutic implications because biofilm organisms represent a clinical target for prevention and treatment efforts aimed at reducing the significant morbidity and costs associated with this condition. Keywords Bisphosphonate . BONJ . Osteonecrosis . Bone . Microcrack . Infection

Introduction Bisphosphonate (BP) drugs, in particular nitrogen-containing or amino-BP, have been associated with the complication of osteonecrosis of the jaws [1, 2]. Much of the knowledge related to this clinical problem has been derived from retrospective case studies and some institutional studies, and for the most part represent lower levels of evidence. Following unsubstantiated claims concerning disease pathogenesis in the initial years after it was reported in 2003, there has been a more recent emergence of well-designed, basic science to patient-related studies to explain the pathogenesis of BP-associated osteonecrosis of the jaw (BONJ) [3]. Infection is now believed to play an important role in this clinical problem [4, 5, 6•]. Microbial biofilms have been recently observed and described in BONJ specimens and investigators are suggesting that BONJ essentially represents an osteomyelitis of the jaw, with greater susceptibility in some patients on BP therapy [5, 6•]. This article focuses on the role of microbial biofilms in BONJ and also discusses associated factors in the disease pathogenesis. We also discuss the recent reports of jaw osteonecrosis occurring in patients on denosumab antiresorptive therapy for metastatic cancer, and discuss the implications of these findings for future nomenclature and investigation.

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Bisphosphonates BP compounds interfere with osteoclast-induced resorption and thus affect bone remodeling processes [7]. The drugs selectively absorb to mineral surfaces of bone and are subsequently released from the bone surface, where they inhibit osteoclast activities and reduce bone resorption and turnover through various biochemical mechanisms, ultimately leading to increased bone mass. Anti-angiogenic properties have been described in addition to anti-osteoclastic activities, and it has been recently proposed that these drugs may be prolonging the life span of osteoclasts and causing protracted apoptosis [8••]. Drug localization to the surface of bone likely plays a role in disease pathogenesis. BP therapy has been effectively used in managing osteoporosis, metastatic bone disease, multiple myeloma, Paget’s disease of bone, hypercalcemia of malignancy, and congenital bone abnormalities such as osteogenesis imperfecta [7, 9, 10]. BP drugs have been associated with several adverse short-term and long-term effects depending on administration route. For oral BP users, complications have been reported that include osteomalacia, musculoskeletal pain, gastrointestinal disturbances, and ocular inflammation; for intravenous BP users, complications include localized inflammation at the injection site, flu-like symptoms, hypocalcemia, hypophosphatemia, azotemia, and nephrotoxicity [7]. BONJ is a complication reported with oral and intravenous BP use, although the frequency is much higher in intravenous cases [3].

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tion of BP [14••]. However, because BONJ has not been defined consistently and pharmacovigilance remains uncoordinated with respect to these drugs, there is inherent limitation in identifying cases [15]. Emerging studies are showing that the risk of BONJ in osteoporosis patients and cancer patients may actually be higher than previously reported [16–18]. Dental extractions, denture trauma, and periodontitis have been implicated as major risk factors for BONJ [7, 19, 20••], and preventative dental treatment for at-risk patients on BP therapy has been shown to reduce the incidence of BONJ [20••, 21••]. This suggests that dental preventative treatment may have some efficacy even prior to initiating BP therapy, but welldesigned prospective studies are needed to assess this notion.

Pathogenesis The pathogenesis of osteonecrosis has not been clearly understood and is likely multifactorial [11]. Several novel and overlapping hypotheses have been published and summarized [3]; more recently, basic science and clinical research articles have been published to more accurately elucidate the pathogenesis of BONJ [8, 20••, 22••, 23•, 24•]. A synopsis of the prevailing suppositions for the pathogenesis of BONJ is presented in the subheadings that follow, with an emphasis on the role of infection in the disease process, which has important therapeutic implications.

BP-Associated Osteonecrosis of the Jaw

Bone Remodeling and Anti-angiogenesis

Osteonecrosis can generally affect any bone including the jaws due to various factors (eg, radiation, chemotherapy, steroid therapy, and BP therapy) [1, 2, 11]. BONJ is defined by the clinical appearance of nonhealing necrotic bone in the oral cavity for 6 to 8 weeks’ duration [3]. Clinical features may also include pain, erythema and ulceration of overlying mucosa, purulence, and malodor. Diagnosis in the early stage of the disease can be difficult and management can be elusive, protracted, and costly. The actual estimates of incidence and prevalence of BONJ have been largely based on retrospective data and include biases and discrepancies due to inconsistent definitions and inappropriate study populations [12]. In a recently published systematic review of the literature published before February 2008, the incidence of BONJ in cancer patients was estimated to be between 1% to 12% at 36 months of exposure, whereas the incidence was low in osteoporosis patients with an incidence of less than 1/100,000 person-years of exposure [13•]. The variation has been attributed to type, dose, and route of administra-

A recent analysis of BONJ bone using light microscopic and confocal scanning laser microscopic examination demonstrated prominent and dense woven bone formation with fewer and distant blood vessels along with reduced osteoclastic activity [25•]. This study also supports the notion that the anti-angiogenic effects by BP may not be necessary for the onset of BONJ as the new dense bone formation appears to cause ischemic necrosis due to the osteogenic and nonosteogenic expansion of the bone without actual increase in blood supply. The authors propose that this new dense bone with less blood supply would eventually lead to necrosis and secondary infection [25•]. To add to the knowledge of the role played by BPinduced anti-angiogenesis in BONJ, a retrospective analysis of 116 patients taking BP with and without antiangiogenesis agents were analyzed; there was a marked difference in the incidence of BONJ with 16% of patients affected with BONJ who were taking BP and antiangiogenic medications and only 1.1% affected who were taking BP alone [26•].

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Matrix Necrosis In one of the animal models of BONJ using beagle dogs, Burr and Allen [22••] showed that bone matrix necrosis occurs in bones with higher remodeling rates such as jaw bone that has been treated with clinical doses of alendronate and zoledronic acid; none of the control animals in their study acquired osteonecrosis. Although it is normal to see matrix necrosis in the skeleton, considering the fact that BP causes remodeling suppression, it is possible that the matrix necrosis will be cumulative over time and its removal may be impaired, contributing to the initiation of the clinical problem with BONJ in some cases [22••]. Microcracks A scanning electron microscopic study showed microcracks in 54% of patients with BONJ compared with 29% in patients without BONJ but taking BP, and 17% in osteoporosis patients not taking BP, whereas no microcracks were observed in patients with osteoradionecrosis of the jaw or osteomyelitis of the jaw without BP therapy [27•]. Soft Tissue Toxicity and Impaired Wound Healing An in vitro study showed that there is dose-dependent, gene-regulated apoptosis of gingival fibroblasts and keratinocyte cell lines by zoledronic acid, resulting in a direct soft tissue toxicity effect of BP, which may play an important role in BONJ induction prior to bone exposure and involvement [24•]. In a previously reported in vitro study using murine oral keratinocytes, pamidronate was shown to inhibit cell proliferation and wound healing at clinically relevant doses and these effects were not due to apoptotic activity [23•]. Inflammation Inflammation is believed to play an important role in BONJ with the initiation of osteonecrosis subclinically in an infection-free environment [14••]. However, inflammatory reactions could also be the result of microbial infection triggering host immune responses [28]. Significant research is needed to explore the role of inflammation and inflammatory mediators in BONJ and their correlation with infection. Infection Infection is believed to play a critical role in the pathogenesis of BONJ, and recent studies have shown bacterial colonization of affected bone in BONJ [4, 5, 6•, 29••]. BONJ is generally defined by the clinical appearance

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of necrotic bone (sequestrum) in the oral cavity; thus, infection naturally ensues on the bone exposed to the microbial flora of the oral cavity. Some authors have proposed that BONJ can be subclinical [27•], and BPinduced mucosal toxicity [24•] or dentoalveolar trauma exposes the subclinical BONJ (with no mucosal breakdown and bone exposure in the oral cavity) into a clinical BONJ (with mucosal breakdown and necrotic bone exposure in the oral cavity). However, almost 100% of these BONJ specimens are colonized with multispecies microbial biofilms [5, 6•, 30] whose source could have been from the spread of odontogenic or periodontal infection extending to the bone through fistulas in the absence of mucosal breakdown. Approximately 85% of affected patients have had periodontitis during the diagnosis of BONJ [30]. Therefore, microbial biofilms associated with teeth and periodontium that readily have access to underlying bone compromised by BP may play a crucial role in the etiopathogenesis of the majority of BONJ lesions [28, 31•]. Clinically, we observe a large majority of BONJ patients with obvious exposure and infection that is very similar to a presentation of osteomyelitis of the jaw without BP therapy, or in necrotic jaws secondary to high-dose therapeutic radiation (osteoradionecrosis) [32], trauma, or genetic bone pathology such as that seen in osteopetrosis [33]. Other bone conditions such as cemento-osseous dysplasia and Paget’s disease of bone may also develop into osteomyelitis or osteonecrosis after dental extractions, which has basically the same clinical course as seen in BONJ [34]. It is possible in some cases that subclinical osteonecrosis in the jaw helps in the progression of odontogenic and periodontal disease, which can lead to the unmasking of underlying osteonecrosis and cause jaw bone exposure and sequestration [7, 34]. Subclinical osteonecrosis may remain stable until complicated by infection or mucosal and dentoalveolar traumatic events, as has been seen in osteoradionecrosis patients in a long-term follow-up study [7, 32]. The odontogenic or periodontal infection in underlying osteonecrosis associated with BP can lead to chronic osteomyelitis in an asymptomatic, subclinical necrotic bone, eliciting further host inflammatory responses [6•, 29••, 31•, 35] and eventually causing mucosal breakdown, sequestration, and the clinical appearance of BONJ, which may be further complicated by impaired wound healing due to BP effects or patient comorbidities [23•]. This phenomenon has been observed in orthopedic patients with osteomyelitis and infected implants [36]. An MRI-based case series showed the origin of osteomyelitis around bone infarcts in long bones [37]. Another fact that may lead to this possibility is that BONJ is common in cancer patients who are on chemotherapy regimens including immunosuppressants and corti-

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costeroids, which give them greater susceptibility to dental diseases. Additionally, common medical comorbidities such as diabetes can be seen in BONJ patients, and studies have shown direct correlation with chronic periodontitis and impaired wound healing leading to bacteria-induced bone loss [38]. Until there are long-term prospective clinical studies on patients beginning to take BP and concurrent dental and medical history, the natural course of BONJ and compounding risk factors will remain unknown [39]. However, history, clinical, radiographic, and histopathologic findings, and similarities with bacterial osteomyelitis in patients with compromised bone remodeling secondary to several causes, argue for a fundamental role for microbial biofilms in the pathogenesis of BONJ. The orthopedic and bone research community has adopted biofilm theory to explain the pathogenesis of long bone osteomyelitis [36], and the dental community is following suit for jaw osteomyelitis [29••] to guide clinical management. The explanation of bacterial infection unmasking subclinical necrotic bone may not account for cases of bone exposure that has been reported to occur spontaneously without associated dental disease [7]. In this minority of cases of BONJ occurring spontaneously and also in BONJ seen in anatomical sites with thin mucosal covering such as mandibular and palatal tori [30], trauma or direct soft tissue toxicity [24•] by underlying BP may cause the mucosal breakdown and eventual exposure of subclinical necrotic bone to oral microorganisms. In other words, we suggest that both “inside-out” (matrix necrosis) and “outside-in” (microbial biofilm colonization of exposed jaw bone) processes may occur in tandem to produce the clinical appearance of BONJ [40]. Although the presence of bacterial biofilms may not directly cause necrosis initiation in some cases, it seems to have an essential contributing role in the clinical problem of BONJ. However, because the vast majority of BONJ cases involve exposed bone and comorbid conditions in patients that affect wound healing or immune function, BPcontaining bone is likely less resistant than normal bone to bacterial colonization and infection. If there is a subclinical osteonecrosis, imaging of the jaw bones in high-risk patients may be useful. A recent study showed that CT scans offer 96% detectability of BONJ lesions compared with MRI (92%) and panoramic radiographs (54%). However, these images were done in overt BONJ lesions and the extent of the lesions was not accurately determined [41]. Biomarkers and highly sensitive and specific imaging modalities for BONJ are needed to aid in earlier detection.

Microbial Biofilms in BONJ The clinical problem with BONJ is a chronic microbial biofilm infection of bone, or jaw osteomyelitis, in the

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context of BP therapy. A microbial biofilm is a community of microorganisms attached to a surface and surrounded by a matrix of extracellular polymeric substance [42••]. Biofilm organisms, compared to their free-floating or planktonic counterparts, demonstrate an altered genotype and phenotype with respect to growth and antimicrobial resistance, and survive effectively by chemical communication using signaling molecules referred to as quorum sensing compounds [42••] in addition to electrical communication via nanowires [43]. Microbial biofilms are believed to play a role in the etiopathogenesis of 65% to 80% of the chronic human infections treated by physicians [6•]. Complex mixed-species biofilms have been described and have been studied in detail in many diseases, including dental caries, chronic periodontitis [28], and apical periodontitis [31•]. Interspecies communication with quorum sensing mechanisms and community and polymicrobial synergistic behavior have all been described in these common dental infections [31•, 44]. Not surprisingly, most well-documented cases of bone necrosis in patients taking BP occur in patients with a pre-existing dental infection that is odontogenic or periodontal in origin and eventually spreads to the bone, culminating into BONJ [3]. Traditional methods of screening and culturing for infectious disease usually miss biofilm bacteria because these techniques are predicated on planktonic bacterial growth. Oral bacteria have evolved across millions of years in mixed biofilm communities, and many of these organisms are referred to as uncultivable or unculturable organisms. Furthermore, cultures or swabs from the oral cavity have high rates of contamination, and exposure to oxygen can kill many of the anaerobic bacteria that reside there. We have noted the presence of complex multispecies biofilms in BONJ and osteomyelitis of the jaw not related to BP use [6•, 29••]. A diagnostic criteria for a biofilm infection was proposed in 2003 by Parsek and Singh [45] and was modified by Hall-Stoodley and Stoodley (Table 1) [42••]. The authors correctly point out that a biofilm infection may not fulfill Koch’s postulates to be called an infection merely because of an absence of biofilm-specific markers and also animal models to study biofilms [42••]. Biofilm identification requires direct visualization methods with advanced microscopy or DNA- and RNA-based techniques as opposed to traditional culturing and antibiotic sensitivity testing studies. In the microbial biofilms noted in BONJ using advanced microscopic techniques for direct visualization [6•, 29••], the criteria listed for a biofilm infection were fulfilled. As an example, Fig. 1 shows a BONJ patient with conventional and advanced microscopic analysis of affected bone that demonstrates viable multispecies microbial biofilms in association with host inflammatory cells.

44 Table 1 Diagnostic criteria for biofilm infectionsa 1. Pathogenic microbes are associated with a surface 2. Direct examination of infected tissue demonstrates aggregated cells in cell clusters encased in a matrix, which is of microbial and host origin 3. Infection is confined to a particular site in the host 4. Recalcitrance to antibiotic treatment despite demonstrated susceptibility of planktonic bacteria. In the absence of culture, recalcitrance to antibiotic therapy may be inferred from the presence of live bacterial cells in the biofilm from in situ hybridization techniques with viability staining or reversetranscription polymerase chain reaction 5. Culture-negative result in spite of clinically documented high suspicion of infection (because localized bacteria in biofilms may be missed in a conventional blood sample or aspirate depending on the location and sample) 6. Ineffective host clearance evidenced by the location of bacterial cell clusters (macrocolonies) in discrete areas of the host tissue associated with host inflammatory cells a Parsek-Singh Criteria (Adapted from Hall-Stoodley and Stoodley [42••]; and from Parsek and Singh [45]; with permission.)

With the reduction of biofilm theory to the clinical problem of BONJ, it becomes comprehensible why cases of bone necrosis secondary to BP therapy occur almost exclusively in the jaws where oral bacteria have access to bone (eg, through saliva or odontogenic infection), especially after exposure of bone following a dental procedure such as an extraction (the most common dental procedure associated with BONJ). In the oral cavity compared with other parts of the body, bone can be easily colonized by the abundant flora of bacteria and yeast that have the potential to cause biofilm-mediated disease. Although routinely exposed to oral microbes, the jaws are usually resistant to microbial colonization. Typically, such colonization requires a notoriously pathogenic organism such as actinomyces, which predominates in cases of BONJ in addition to jaw osteomyelitis without BP therapy [29••]. Several morphotypes of actinomyces have been described from different anatomical sites in the oral cavity. Subspecies variation of genotypic, phenotypic, and physiologic characteristics among actinomyces have made classification challenging, and identification based only on phenotypic characteristics using conventional methods has become obsolete [46]. Additionally, in the oral cavity, if a pathologic condition such as an odontogenic infection, periodontal disease, or trauma occurs that exposes the jaw bone to oral organisms, local defenses and repair mechanisms can wall off and eradicate the necrotic bone via sequestration [29••]. Sequestration is the most common clinical feature in established cases of BONJ. Therefore, microbial biofilms play an important role in the pathogenesis of BONJ in the context of BP therapy. These findings may explain the identification of numerous microbial morphotypes in BONJ

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compared with jaw osteomyelitis without BP therapy, because the bone in BONJ is susceptible to a more diverse colonization of oral organisms that usually do not supplant bone (eg, superficial fungal organisms such as Candida species and some of the more benign bacterial morphotypes seen in BONJ cases) [29••]. One of the pivotal studies that supports the notion that BP-containing bone is more susceptible to bacterial colonization was performed by Ganguli et al. [47], who showed that bacterial adhesion to pamidronate-coated hydroxyapatite (HA) was 60-fold greater than uncoated HA and 90-fold greater than clodronate-coated HA. Because clodronate is a chlorine-based BP compound and not a nitrogen-based compound like pamidronate or most other BP drugs on the market, these findings suggest that the nitrogen moiety of the BP compound may play an important role in the pathogenesis of BONJ. Documented reports of BONJ occur in patients taking nitrogen-BP compared with non-nitrogen BP; therefore, the structure of these compounds is highly likely to play an important role in the disease process of BONJ [39], yet to date virtually nothing is known about their influence on BONJ pathogenesis. Elucidation of BP and biofilm structureactivity relationships on the bony surface where they colocalize will create a basis for novel drug design and therapies for prophylaxis or treatment of BONJ, minimizing the potential of this side effect. Future investigations will need to address the lack of systematic studies and a model system examining the influence of BP structure on biofilm colonization and inflammation. Several pathogenic bacteria found in the oral cavity and associated with BONJ cases can cause bone destruction through various direct and indirect mechanisms. Some of the proposed mechanisms include direct damage to bone matrix by bacterial substances, interaction and stimulation of bacterial factors directly with bone cells or indirectly through inflammatory mediators leading to bone resorption and inhibition of bone formation, and finally via direct invasion of osteoblasts by bacteria resulting in impairment of bone remodeling processes [48]. Bacterial-generated chemical mediators for bone resorption include proteins such as porins, or collagen-degrading enzymes such as collagenases, by which they secure necessary amino acids for growth or create anaerobic niches in the bone for further spread and growth [49].

Emerging Osteoporosis and Anti-cancer Therapies and Jaw Osteonecrosis In addition to using BPs to treat postmenopausal osteoporosis, there are emerging therapies that include antiresorptive and anabolic compounds that will allow clinicians to

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Fig. 1 a, Clinical digital image of a 65-year-old Hispanic female patient with a 3-year history of oral bisphosphonate (BP) therapy (alendronate, 70 mg/wk) for osteoporosis showing BP-associated osteonecrosis of the jaw of the maxilla with oral mucosal breakdown and exposed necrotic bony sequestrum. Clinically, the lesion was painful and recalcitrant to oral antibiotic therapy. b, Overview of a region of the affected bone from the same patient following sequestrectomy that demonstrates lack of osteocytes in lacunae (indicating necrotic bone) and extensive bacterial colonization of the surfaces (Hematoxylin and eosin stain, 10 × original magnification). c, Scanning electron micrograph of the same sample demonstrating significant resorption along the surface of the bone with biofilm bacteria occupying many of the resorption pits, in addition to colonizing the internal substance of the bone as a thickly layered biofilm that can be appreciated toward the bottom right of the image

(1000 × original magnification). d, Higher-power scanning electron micrograph of the bone showing various oral bacterial morphotypes in this matrix-enclosed mixed-species biofilm macrocolony that is associated with host inflammatory cells such as lymphocytes and erythrocytes, often referred to as a septic clot clinically (5000 × original magnification). e, Fluorescence in situ hybridization (FISH) of the necrotic bone with associated biofilms labeled with an oligonucleotide probe and fluorophore; the probe is complementary to a region of 16S rRNA, which is highly conserved and unique to bacteria. A significant number of intact ribosomes representing the biological activity of the tested cells are a prerequisite for the FISH method, so that predominantly vital bacteria are stained (EUB338 red probe, Technovit 8100 embedding, 20 × original magnification, optical slice 1.6 µm)

individualize and tailor osteoporosis therapies for their patients. Current antiresorptive therapies include oral daily, weekly, and monthly BP, which are now complemented by quarterly and yearly intravenous BP, nasal calcitonin, an approved estrogen agonist/antagonist and others in late clinical development, and a single anabolic agent, teriparatide or parathyroid hormone [50]. A novel drug called denosumab is in phase 3 clinical trials and uses a unique

pathway as its mechanism of antiresorptive action in bone. Denosumab is a human monoclonal antibody targeting the receptor activator of nuclear factor-κB ligand, a cytokine that is essential for the formation, function, and survival of osteoclasts [51•]. In the pivotal phase 3 denosumab FREEDOM trial (Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months), no cases of jaw osteonecrosis were reported [51•]. However, more recently,

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in a head-to-head phase 3 bone metastasis trial involving 2046 patients with advanced breast cancer and comparing denosumab versus BP (zoledronic acid), both treatment groups showed similar numbers of jaw osteonecrosis: 20 patients receiving denosumab (2.0%) compared with 14 patients (1.4%) receiving zoledronic acid [52••]. In the cancer trial, denosumab was given subcutaneously at a higher dose of 120 mg every 4 weeks compared with the osteoporosis FREEDOM trial, which involved subcutaneous injection of 60 mg of denosumab every 6 months for 36 months [51•, 52••]. These denosumab-related findings are significant because they indicate that antiresorptive therapy besides BP can be associated with jaw osteonecrosis, and that the dose and duration of antiresorptive therapy in addition to patient comorbidities such as cancer may play an important role in the disease process, which is already known with BONJ and osteoradionecrosis. These cases of denosumab-related jaw osteonecrosis have not yet been published or studied, ascertainment criteria has not been provided for adjudicated cases, patient comorbidities have not been made available, oral hygiene status or dental procedures have not been described, clinical or radiographic details are not available, nor has the bone been examined to determine whether infection and microbial biofilms are involved in this condition like they are in BONJ, osteoradionecrosis, or osteomyelitis. Therefore, any conclusions regarding pathogenesis or mechanisms of osteonecrosis induction in denosumab-related cases are premature at this point, but this should represent future avenues of investigation and pharmacovigilance and prevention efforts will be necessary. However, we suspect that similar to BONJ, osteoradionecrosis, or osteomyelitis of the jaw, microbial biofilms may play an important role in the pathogenesis of these denosumab-related cases given the drug-induced bone suppression or permutated effects on bone remodeling in the jaws, the compromised nature of affected patients, and the proximity of oral biofilm organisms. Perhaps a more appropriate classification as more evidence becomes available regarding jaw osteonecrosis associated with different antiresorptive therapies such as denosumab and BP may be antiresorptive-associated osteonecrosis of the jaw (ARONJ).

Prevention and Management Biofilm organisms have adopted advanced survival strategies and evade conventional treatment. Treatment of biofilms remains very challenging and several resistance mechanisms have been proposed (eg, the failure of antimicrobial agents to effectively penetrate beyond the outer biofilm matrix, which typically helps protect organisms in the deeper layers). Antimicrobial agents may be

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impaired with altered environment, such as altered pH, pCO2, or pO2, or may be destroyed by biofilm matrix. Slow growth shown by “persister” bacterial cells and expression of biofilm-specific resistance genes may make antimicrobial agents such as antibiotics ineffective, requiring up to 1000 times the minimum inhibitory concentration normally required to kill the same organisms in a planktonic state [53]. Some of the more recent strategies to overcome persistent biofilm infections include the use of anti-biofilm agents that causes destruction of biofilm matrix enzymes or persister cells, biofilm communication disruption methods, and oxygen-based antimicrobial methods (eg, the use of peroxides and gas-discharge plasma) [28, 53]. The osteomyelitis reported in osteopetrosis and Paget’s disease, both conditions that also have disturbances in bone remodeling with osteoclastic suppression, are similar to those noted in BONJ. The clinical, radiographic, and microscopic features of jaw osteomyelitis, jaw osteoradionecrosis, and BONJ all have the presence of infection; consequently, prevention and clinical management must be predicated on an understanding of biofilm-mediated infectious disease. Accordingly, current guidelines for treating BONJ are antibiofilm in nature and favor more conservative management with routine antimicrobial rinses, minor debridement, and sequestrectomy of the exposed necrotic bone instead of complete resection, with frequent follow-up evaluations and meticulous long-term wound management [20••]. Patients who are affected with BONJ tend to have a history of poor oral hygiene at some point, comorbid conditions such as cancer, or take medications that may impair wound healing or cause immunosuppression, and these factors may act as effect modifiers for the disease. Therefore, dental management in addition to medical management of patients should offer the greatest success for preventing and treating BONJ or ARONJ, and all associated studies in the future should be well documented to allow for accurate statistical analyses of effect modifiers or confounding variables. Finally, pharmacovigilance with respect to jaw osteonecrosis remains uncoordinated and this needs to be addressed.

Conclusions Treating and preventing the clinical problem of BONJ must be predicated on an understanding of the biofilm-mediated nature of the condition. Mixed-species pathogenic oral biofilms that essentially produce an osteomyelitis of the affected jaw bone in BONJ represent an important target for therapeutics, prevention efforts, and further investigations into the pathogenesis of this condition. Acknowledgments Owing to journal requirements for references, certain key contributions in the field of BONJ and microbial biofilms

Curr Osteoporos Rep (2010) 8:40–48 may not have been cited in this manuscript and the authors would like to acknowledge all such scientific contributions. Disclosure No potential conflicts of interest relevant to this article were reported.

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