**3. Results of our experience**

#### **3.1. Regarding the risk factors**

All the patients of this study visited the Master of Oral Medicine, Surgery and Implantology of the Faculty of Dentistry of the University of Santiago de Compostela. The patients were derived from the Unit of Oncology of the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS) for a dental examination prior to treatment with intravenous bisphosphonates. The study was approved by the Clinical Research Ethics Committee of Galicia, in addition to the gaining informed consent from the patients.

Patients treated with zoledronic acid for a period of 7 years (2006–2013) and patients with a history of treatment of head and neck radiotherapy were excluded.

**Local risk factors:** Only those patients who underwent exodontics during bisphosphonate treatment had a statistically significant risk (P > 0.001), as well as a direct relationship according to the periodontal status of the patient, since no patient with periodontal health developed MRONJ (P = 0.001). As previously described in the literature, research showed that the mandible is more affected than the upper maxilla and is also statistically significant (P < 0.001)

**Systemic risk factors:** There were no significant differences in terms of gender (P = 0.063). Age did not show significant differences but there were more cases of osteonecrosis in elderly patients, probably because these patients are more compromised at the systemic level; in fact, 86.6% of them were polymedicated. The type of cancer, arterial hypertension and treatment with chemotherapy or corticosteroids did not show statistically significant differences, although the literature describes them as obvious risk factors. We could only conclude that patients without diabetes did not develop MRONJ (P = 0.048). In our study we did not factor in their race, since 100% of the patients were Caucasian and from a specific population. As described by Wessel et al. [29] in their study, 100% of our patients had bone metastases, which justified the use of intravenous bisphosphonates and, therefore, also implied an increased risk of complications.

**Drug-related factors:** Regarding potency, all patients were treated with the same bisphosphonate (zoledronic acid, 4 mg). The cumulative dose showed that patients with more than three years of treatment had a higher risk of developing MRONJ, which was statistically significant (P < 0.001) (**Table 5**).

#### **3.2. Bacterial role in osteonecrosis of the jaws**

causes rapid and prolonged decreases in bone exchange markers without any change in bone formation, which gives it antireabsorptives characteristics [124]. It has also shown better clinical results compared to bisphosphonates in the treatment of osteoporosis and cancer with a higher increase in bone density and suppression of bone remodeling markers, with a proven efficacy even in patients who had been previously resistant to bisphosphonates [117, 125, 126]. These drugs also produce osteonecrosis of the jaws with a prevalence of 0.7–19% [127, 128], which is very similar to the osteonecrosis from bisphosphonate treatments [117]. Since the first case of maxillary osteonecrosis due to this drug published in 2010 [129], several studies have been published but only one of them describes histopathologic characteristics [130]. The fragments of necrotic bone showed empty osteocytic lacuna and absence of osteocytes, osteoblasts and osteoclasts. The authors suggest that these characteristics are very similar to

These drugs hinder the development of new blood vessels and block the cascade of angio-

All the patients of this study visited the Master of Oral Medicine, Surgery and Implantology of the Faculty of Dentistry of the University of Santiago de Compostela. The patients were derived from the Unit of Oncology of the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS) for a dental examination prior to treatment with intravenous bisphosphonates. The study was approved by the Clinical Research Ethics Committee of Galicia, in

Patients treated with zoledronic acid for a period of 7 years (2006–2013) and patients with a

**Local risk factors:** Only those patients who underwent exodontics during bisphosphonate treatment had a statistically significant risk (P > 0.001), as well as a direct relationship according to the periodontal status of the patient, since no patient with periodontal health developed MRONJ (P = 0.001). As previously described in the literature, research showed that the mandible is more affected than the upper maxilla and is also statistically significant (P < 0.001)

**Systemic risk factors:** There were no significant differences in terms of gender (P = 0.063). Age did not show significant differences but there were more cases of osteonecrosis in elderly patients, probably because these patients are more compromised at the systemic level; in fact,

**Bevacizumab:** Monoclonal antibodies that stop the growth factor.

**Suntinib and Sorafenib:** Tyrosine kinase inhibitors.

addition to the gaining informed consent from the patients.

history of treatment of head and neck radiotherapy were excluded.

**3. Results of our experience**

**3.1. Regarding the risk factors**

bisphosphonate-related osteonecrosis [131]

*2.5.2. Antiangiogenics*

genesis [132].

68 Osteonecrosis

We selected 28 patients (16 men and 12 women) with a mean age of 71.96 years, all of whom were treated in the Oncology Department of the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS). They were referred to the Unit of Oral Surgery and Implantology of the Faculty of Medicine and Dentistry of Santiago de Compostela for a prior dental examination and for the follow-up of possible complications after treatment.

All patients treated with both oral and intravenous bisphosphonates and those undergoing head and neck radiotherapy within an 8-year period were included (2006–2014). The diagnostic criteria followed were those determined by the AAOMS [15] for patients treated with bisphosphonates, while in the case of those undergoing radiotherapy, we included those who had bone exposure for more than 8 weeks.

Samples of exudates from bone exposure were sent to the Department of Microbiology to be processed under Gram staining and seeded in liquid medium (thioglycolate broth) and in solids (agar-blood and agar-chocolate for the growth of aerobic bacteria, Sabouraud agar for yeast growth and Schaedler agar for anaerobic growth). The seeded plates were incubated for 48 h at 37°C in a CO2 atmosphere and 72 h in anaerobic chambers. Identification of microorganisms was done using the Vitek 2 system (Bio-Merieux, Marcy l'Etoile, France) and Microscam (Siemens, Erlangen, Germany) in the case of aerobes; and API-ANA (Biomeriex) in the case of anaerobes. Antimicrobial susceptibility testing was performed by the e-test method (AB biodisk) and the Clinical Laboratory Standards Institute (CLSI) interpretation criteria were followed.

Biopsies of bone sequestration were sent to the Pathological Anatomy Service where they were fixed in 10% buffered formaldehyde and embedded in paraffin following standard processing. For evaluation, they were stained with hematoxylin/eosin, PAS and methenamine silver to visualize the colonies of Actinomyces. Their presence was evaluated semi-quantitatively and divided into scarce, moderately abundant or very abundant. In addition, the presence of acute inflammation (when polymorphonuclear neutrophil cells were observed), which was also quantified in three degrees: mild, moderate and intense, and the presence of chronic inflammation (indicated by the presence of lymphocytes and plasma cells).


**Table 5.** Risk factors in the occurrence of ONJ.

The collected data were analyzed with the SPSS statistical system, version 20.0 for Windows. The discontinuous quantitative or discreet variables were analyzed through descriptive statistics, expressing the results in mean, deviation and standard. The frequency tables and percentages were used for qualitative variables. For the study of the association of variables we employed the chi-squared test, the T-Student test or the ANOVA factor test, depending on the application conditions. Values in which P ≤ 0.05 were considered statistically significant.

#### *3.2.1. Clinical results*

**Characteristics Total patients with ONJ (%) Total patients without ONJ (%) P Gender** 0.063

**Systemic Risk Factor** 0.214

Diabetes 168 (86.6) 26 (13.4) 0.048 Polymedicated. 94 (48.5) 100 (51.5) 0.139 HTA 42 (21.6) 152 (78.4) 0.704 Cortisone 65 (33.5) 129 (66.5) 0.234 QTP 11 (5.7) 54 (27.8) 0.462

Extraction Before 8 (4.1) 86 (44.3) 0.078 Extraction during 5 (2.6) 1 (0.5) <0.001 Extraction after 4 (2) 7 (3.6) 0.017 Prosthesis 5 (2.6) 33 (17) 0.936 periodontal state 19 (9.8) 70 (36) 0.001 **Toxic factors** 0.998

Unifocal mandible 13 (6.7) – <0.001

Men 14 (7.2) 125 (64.4) Women 11 (5.7) 44 (22.7)

Cancer 12 (6.2) 95 (49) Prostate 9 (4.6) 34 (17.5) Breast 4 (2) 11 (5.7) Myeloma 0 (0) 18 (9.3) Lung 0 (0) 2 (1) Bladder 0 (0) 5 (2.6) Colon 0 (0) 3 (1.5) Kidney 18 (9.3) 176 (90.7)

Tobacco 2 (1) 13 (6.7) Alcohol 3 (1.5) 19 (9.8)

Unifocal maxilla 5 (2.6) – Multifocal mandible 3 (1.5) – Maxilla and mandible 4 (2) –

**Table 5.** Risk factors in the occurrence of ONJ.

**Local Risk Factor**

70 Osteonecrosis

**Location**

Of the 28 patients, 16 were men (57.1%) and 12 women (48.8%) with a mean age of 71.96 years (SD 8.94). According to the risk factors analyzed, 8 patients (28.5%) were diabetic, 15 (53.6%) were undergoing chemotherapy, 4 were smokers (14.3%), 14 was hypertensive (50%) and 9 (32.1%) were taking corticosteroids. The reason for treatment with bisphosphonates was oral cancer (14.3%), breast cancer (25%), prostate cancer (39.2%), multiple myeloma (10.7%) and osteoporosis (10.7%).

The most affected region of the mouth was the mandible (67.8%) followed by the upper jaw (21.4%) or both (10.7%).

The degree of affectation was variable depending on the type of treatment. Patients on intravenous bisphosphonates had all stages of ONJ, whereas patients undergoing the oral treatment had only stage II ONJ and patients treated with radiotherapy showed both stage II (5%) and stage III (75%).

#### *3.2.2. Histological results*

A total of 24 of 28 patients underwent a histological study with a biopsy of a bone sequestration lesion. Of these, in 21 patients (87.5%) we proved the presence of Actinomyces within the 3 degrees of osteonecrosis of the jaws. The amount of Actinomyces present was quantified semi-quantitatively by the pathologist. The pathogen count was very abundant in degrees I and II, while in degree III the patients had lower amounts of Actinomyces (**Figure 4**).

The degree of acute and/or chronic inflammation was also evaluated. In 54.2% of the patients, the presence of Actinomyces was not accompanied by any sign of inflammation; while in the rest of the patients, it was observed that as age increased, the intensity of the inflammation also increased; therefore a lower mean age accounted for the absence of inflammation (65.31; SD: 7.91) contrasting with abundant inflammation (81.00; SD: 2.83). Such differences were statistically significant (F = 5.270, P = 0.005). There was acute inflammation in 37.5% of the patients being quantified as mild inflammation (two patients), moderate (five patients) and severe (two patients); the latter two were present in patients with grade II osteonecrosis, exclusively. There were only two chronic inflammation cases, one patient with grade II and another with grade III ONJ.

The relationship between the amount of Actinomyces present in the histological sections and the degree of inflammation observed in bone sequestration was evaluated. Despite not having statistically significant data, it was observed that the high amount of Actinomyces could trigger either null or an abundant inflammatory response (**Table 6**).

**Figure 4.** Histological image of bone sequestration. Histopathologic examination revealed different combinations of Actinomyces and the inflammatory response. Some cases showed abundant colonies of Actinomyces, but lacked any inflammatory infiltration (A); while in other examples this microorganism was identified along with a dense PMN infiltrate (B). PMNs were also seen in some cases, in the absence of Actinomyces (C). Last of all, in some patients, the only change consisted of fibrosis and the scarce inflammatory response was composed mainly of lymphocytes (D).

#### *3.2.3. Microbiological results*

Regarding the isolation of bacteria obtained through suppuration of the necrosed area, all the bacteria described by the microbiologist were recorded, which were later classified according to their aerobic or anaerobic metabolism. Aerobic bacteria were mostly found (85%) in patients with grade I and II of ONJ, being statistically significant (P = 0.002). However, anaerobic bacteria were present in 56% of the patients in the three stages of ONJ. Although all the cases of grade III presented anaerobic bacteria, this data were not statistically significant.


**Table 6.** Relationship of the amount of Actinomyces and the inflammatory response.

There was practically no significance in the families of bacterial species specific to the different degrees of osteonecrosis, except *Streptococcus* sp. which was very abundant in grade II of ONJ. Only three bacteria showed statistically significant differences in relation to the ONJ stages (**Table 7**, **Figure 5**).

Antibiograms were also performed for each of the species found, in order to guide the antibiotic pattern of these patients. We studied the six most common antibiotics, as well as the specific ones for the pathology described in the literature, we observed a variable bacterial behavior among the patients with osteonecrosis of the jaws.

Penicillin G did not show complete sensitivity in any patient against all the bacteria isolated in the cultures, in addition, there was much variability among patients regarding the response to this antibiotic (**Figure 6**).

The combination of amoxicillin and clavulanic acid showed good sensitivity in most patients (82.6%) although this was not statistically significant (**Figure 7**).

Clindamycin was effective in 40% of patients and resistant in 14.5%, while 28% of the patients showed variability in the response to this antibiotic. Azithromycin was effective in a few patients (38%) and the response was highly variable, without showing complete sensitivity to isolated bacteria (**Figure 8**).

Levofloxacin was effective in 42.8% of the patients, which showed good sensitivity in most cases (88.8%). Last of all, gentamicin, another antibiotic which is less frequent in our daily practice, showed good sensitivity although it was effective in few patients (38%).


**Table 7.** Relationship between the isolated type of bacteria and the degree of ONJ.

*3.2.3. Microbiological results*

72 Osteonecrosis

**Inflammation**

Regarding the isolation of bacteria obtained through suppuration of the necrosed area, all the bacteria described by the microbiologist were recorded, which were later classified according to their aerobic or anaerobic metabolism. Aerobic bacteria were mostly found (85%) in patients with grade I and II of ONJ, being statistically significant (P = 0.002). However, anaerobic bacteria were present in 56% of the patients in the three stages of ONJ. Although all the cases of grade III presented anaerobic bacteria, this data were not statistically significant.

**Actinomyces Null Mild Moderate Abundant Chronic** Null 0 0 1 0 0 Scarce 1 0 1 0 1 Moderate 8 1 1 0 1 Abundant 4 1 2 2 0

**Table 6.** Relationship of the amount of Actinomyces and the inflammatory response.

**Figure 4.** Histological image of bone sequestration. Histopathologic examination revealed different combinations of Actinomyces and the inflammatory response. Some cases showed abundant colonies of Actinomyces, but lacked any inflammatory infiltration (A); while in other examples this microorganism was identified along with a dense PMN infiltrate (B). PMNs were also seen in some cases, in the absence of Actinomyces (C). Last of all, in some patients, the only change consisted of fibrosis and the scarce inflammatory response was composed mainly of lymphocytes (D).

**Figure 5.** Relationship between the isolated type of bacteria and the degree of ONJ.

**Figure 6.** Antibiogram of Penicillin G.

Osteonecrosis of the Jaws. Prevalence, Risk Factors and Role of Microbiota and Inflammation... http://dx.doi.org/10.5772/intechopen.69315 75

**Figure 7.** Antibiogram of the association of amoxicillin with clavulanic acid.

**Figure 8.** Clindamycin antibiogram.

**Figure 5.** Relationship between the isolated type of bacteria and the degree of ONJ.

**Figure 6.** Antibiogram of Penicillin G.

74 Osteonecrosis
