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. 2021 Apr:145:115866.
doi: 10.1016/j.bone.2021.115866. Epub 2021 Jan 27.

Preventing or controlling periodontitis reduces the occurrence of osteonecrosis of the jaw (ONJ) in rice rats (Oryzomys palustris)

E J Castillo  1 , J G Messer  2 , A M Abraham  2 , J M Jiron  3 , A V Alekseyenko  4 , R Israel  5 , S Thomas  6 , G M Gonzalez-Perez  7 , S Croft  8 , A Gohel  9 , I Bhattacharyya  10 , J F Yarrow  11 , C M Novince  12 , D B Kimmel  2 , J I Aguirre  13
Affiliations

Preventing or controlling periodontitis reduces the occurrence of osteonecrosis of the jaw (ONJ) in rice rats (Oryzomys palustris)

E J Castillo et al. Bone. 2021 Apr.

Abstract

Introduction: Osteonecrosis of the jaw (ONJ) is an adverse event that requires association of both systemic risk factors, such as powerful anti-resorptives (pARs; e.g. zoledronic acid [ZOL]), and local oral risk factors (e.g. tooth extraction, periodontitis). Whereas optimal oral health prior to initiate pARs is recognized as critically important for minimizing ONJ risk, the efficacy of preventive/maintenance measures in patients who are taking pARs is understudied. Rice rats fed a standard diet (STD), rich in insoluble fiber, develop localized periodontitis. STD-rats with localized periodontitis treated with ZOL for 18-24 wk develop ONJ. Hence, we hypothesized that controlling/preventing localized periodontitis in the ZOL-treated rats, reduces ONJ occurrence.

Methods: We used two approaches to attempt reducing periodontitis prevalence: 1) periodontal cleaning (PC); and 2) replacing the STD-diet with a nutritionally-equivalent diet high in soluble fiber (SF). 75 four-week-old male rats were weight-randomized into five groups (n = 15) in a 24-week experiment. Three groups ate the STD-diet and two the high SF-diet. STD-diet groups received intravenous (IV) vehicle (VEH) q4wks (STD + VEH), 80 μg/kg ZOL q4wks IV (STD + ZOL), or ZOL plus PC q2wks (STD + ZOL + PC). The SF-diet groups received VEH (SF + VEH) or ZOL (SF + ZOL). Jaws were processed for histopathology and evaluated for ONJ prevalence and tissue-level periodontitis.

Results: 1) 40% of STD + VEH rats developed maxillary localized periodontitis with no ONJ; 2) 50% of STD + ZOL rats developed ONJ; 3) 7% of STD + ZOL + PC rats developed ONJ (p < 0.01 vs. STD + ZOL); and 4) one SF + ZOL rat developed localized periodontitis, and no SF + VEH or SF + ZOL rats developed ONJ (p < 0.001 vs. STD + ZOL).

Conclusions: 1) Periodontal cleaning in ZOL-treated rats decreases localized periodontitis severity and reduces ONJ prevalence; and 2) feeding a SF-diet to ZOL-treated rats reduces both incidence of localized periodontitis and ONJ. Our data indicates strong oral microbial community shifts according to oral health condition and trends in the shifts associated with diet.

Keywords: Controlling; ONJ; Oral microbiome; Periodontitis; Prevention; Rice rats.

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Conflict of interest statement

Declaration of competing interest

The authors have no conflicts of interest. AVA is a scientific advisory board member for Second Genome, Inc., which has not contributed to this research.

Figures

Fig. 1.
Fig. 1.. Prevalence and characteristics of gross oral lesions and by MicroCT.
A. Prevalence of maxillary lesions (GQG ≥ 0.5) rose quickly during Weeks 3–7 in groups receiving the STD diet and remained relatively stable thereafter. In contrast, rats receiving the high SF diet had an extremely low prevalence of such lesions. STD diet rats had a significantly greater prevalence during wks 7 to 23 compared to rats fed the SF diet (*p < 0.001) B. Severity of maxillary lesions in STD + ZOL rats was significantly greater at Weeks 17, 19, and 23 compared to both STD + VEH (*p < 0.005) and STD + ZOL + PC (†p < 0.005) rats; Mean ± SD. C. STD diet groups had a significantly greater prevalence of ex vivo maxillary lesions compared to the SF group (‡p < 0.001). There were no differences in prevalence among the three groups of STD diet fed rats or between the two groups of SF diet fed rats. D. Whereas ex vivo maxillary lesions with GQG ≥ 3 occurred only in the STD + ZOL group, every maxillary lesion in the STD + VEH and STD + ZOL + PC groups had GQG ≤ 2. Severity of maxillary lesions was significantly greater in STD + ZOL compared to STD + ZOL + PC and STD + VEH rats (†p = 0.0034 and *p = 0.009, respectively); Bars indicate Mean ± SD. E. Maxillary lesions were significantly larger in STD + ZOL rats compared to STD + ZOL + PC and STD + VEH rats, (†p = 0.031 and *p = 0.038, respectively). F. Representative high-resolution photographs and microCT images from each group are shown. Small discrete lesions (GQG = 1) restricted to the maxillary M2M3 interdental space are seen in STD + VEH and STD + ZOL + PC rats, while more severe lesions (up to GQG = 4) are seen only in the STD + ZOL group (black arrow). Note a large open wound involving gingival recession/ulceration around all three molars extending into the palatal mucosa towards the midline and buccal migration of M3. 3D MicroCT scans show that STD + VEH rats with PD have greater alveolar bone loss (demarcated in yellow) at the M2M3 interdental space compared to the M1M2 interdental space. STD + ZOL rats with ONJ have more severe alveolar bone loss, with a mottled appearance, at the M2M3 interdental space (white arrowheads), and extending onto the hard palate. STD + ZOL + PC rats show alveolar bone loss at the M2M3 interdental space and minimal bone loss at M1M2, similar to what is observed in STD + VEH rats with PD. Maxillae from rats in the STD + VEH, SF + VEH and SF + ZOL groups with no PD show minimal bone loss at both the M1M2 and M2M3 interdental spaces. Axial and sagittal slices show marked osteolysis (white arrows) at the M1M2 and M2M3 interdental regions of STD + ZOL rats with ONJ. Osteosclerosis (red Ω), erosion of the cortical borders (red arrow) and a scooped out or crater like defect (green arrow heads) with sequestered bone fragments are also seen is STD + ZOL rats with ONJ. Widening of the periodontal ligament (blue arrows) at both interdental regions, sequestered bone (yellow arrowhead) at the M2M3 interdental region, and increased number of bone marrow spaces are seen in STD + ZOL rats with ONJ. In contrast, STD + ZOL + PC and STD + VEH rats with PD show localized alveolar bone loss (orange arrows) constricted to the M2M3 region with no sequestrum present. STD + VEH, SF + VEH and SF + ZOL rats with no PD have normal PDL spaces with absence of osteolysis and sequestrum.
Fig. 2.
Fig. 2.. Histopathologic assessment of necrotic exposed bone and other features.
A. STD + ZOL rats had greater prevalence of ONJ compared to STD + VEH (*p = 0.02), STD + ZOL + PC (†p = 0.012) and both SF groups (‡p < 0.001). 50% of STD + ZOL rats developed ONJ compared to 7% and 0% of STD + ZOL + PC and STD + VEH rats, respectively. Both SF + VEH and SF + ZOL groups had 0% prevalence of ONJ. B–D. Photomicrographs showing features of ONJ found in the STD + ZOL group. b–d depict areas outlined in black in B–D, at a four-fold higher magnification. Maxillary oral lesions are shown at the M2M3 interdental space, in proximity to the palatal (B–C) and buccal (D) surfaces of STD + ZOL rats with ONJ. Ulcerated gingival epithelium (GE) accompanied by adherent bacterial colonies (*) and exposed, necrotic bone (black arrows) is present at the M2M3 interdental space. b–d. Exposed necrotic bone (Ω) with 10+ confluent empty osteocyte lacunae or osteocytes with pyknotic nuclei is seen next to vital bone (α) which has lacunae occupied by osteocytes with basophilic nuclei (demarcated in yellow). Severe inflammatory cell infiltration and fibrosis was often observed. E. The percentage of empty osteocyte lacunae was significantly greater in STD + ZOL rats with GQG≥1 lesions compared to the STD + ZOL subgroup with GQG = 0 lesions (Ωp < 0.001). STD + ZOL rats with GQG≥1 also had greater percentage of empty osteocyte lacunae compared to STD + VEH (*p = 0.001), STD + ZOL + PC (†p = 0.002), and both SF groups (‡p < 0.05). There are no significant differences in percentage of empty osteocyte lacunae among STD + VEH, STD + ZOL + PC, SF + VEH, and SF + ZOL. F. The number of empty osteocyte lacunae/bone area (B.Ar, mm2) was higher in STD + ZOL rats with GQG≥1 compared to STD + ZOL rats with GQG = 0 (Ωp = 0.005). STD + ZOL rats with GQG≥1 also had a greater number of empty osteocyte lacunae/bone area compared to STD + VEH (*p = 0.001) and STD + ZOL + PC rats with GQG≥1 (†p = 0.001) as well as SF + VEH and SF + ZOL rats with GQG = 0 (‡p < 0.05). G. Maxillary PD Score at the M2M3 interdental space. PD Scores ranged from gingivitis (PD Score 1) to mild periodontitis (PD Score 2) in the STD diet group subsets with GQG = 0. STD + VEH rats with GQG≥1 had mild to moderate periodontitis (PD Score 2–3) with a median score of 3. STD + ZOL rats had greater prevalence of periodontitis lesions compared to STD + VEH (*p = 0.002) and STD + ZOL + PC (†p = 0.005) rats with GQG = 0. STD + ZOL rats also had greater prevalence of periodontitis than SF + ZOL rats (‡p < 0.05). STD + VEH rats with GQG≥1 had greater prevalence of PD lesions compared to STD + VEH rats with GQG = 0 (*p > 0.05). Two-way ANOVA showed an effect in diet in which SF rats had less severe periodontitis lesions compared to STD rats (*p < 0.05). H. Maxillary PD Score at the M1M2 interdental space. There was a significantly higher prevalence and severity of periodontitis lesions in STD + ZOL rats with GQG≥1 compared to STD + ZOL rats with GQG = 0 (Ωp < 0.005). STD + ZOL also had a higher prevalence and severity of periodontitis lesions compared to STD + VEH (*p = 0.006), STD + ZOL + PC (†P = 0.006), SF + VEH (Ŧp = 0.017), and SF + ZOL (‡p = 0.016). Red or black transverse lines in each group of the graphs depict median values.
Fig. 3.
Fig. 3.. Maxillary alveolar bone loss and in situ analysis of TRAPc5b+ osteoclasts.
A. Alveolar bone loss was greater at the M2M3 interdental space in rats with GQG≥1 compared to rats with GQG = 0 of STD + VEH, STD + ZOL, and STD + ZOL + PC groups in each respective group (Ωp < 0.001). Alveolar bone loss was also significantly greater in STD rats with GQG≥1 compared to SF rats with GQG = 0 (‡p < 0.001). B. Alveolar bone loss was greater at the M1M2 interdental space in STD + ZOL rats with GQG≥1 compared to STD + VEH and SF + VEH groups (*p = 0.044; ‡p = 0.003, respectively). There were more TRAPc5b+ cells/B.Pm in STD + ZOL rats with GQG≥1 compared to STD + ZOL rats with GQG = 0 (Ωp < 0.001). STD + ZOL rats with GQG≥1 also had more TRAPc5b+ cells/B.Pm than STD + VEH (*p < 0.001), STD + ZOL + PC (†p < 0.001), SF + VEH, and SF + ZOL groups (‡p < 0.001). TRAPc5b+ cells/B.Pm was also greater in STD + VEH rats with GQG≥1 compared to STD + VEH rats with GQG = 0 (*p < 0.05) (C). Red or black transverse lines in each group of the graphs depict median values. Representative photomicrographs of TRAPc5b+ cells (red arrow heads) at alveolar bone surfaces of maxillae from STD + VEH (D), STD + ZOL (E), STD + ZOL + PC (F), SF + VEH (G), and SF + ZOL (H).
Fig. 4.
Fig. 4.. Oral microbiome composition and differences in bacterial families by diet and oral health condition after 24 wks of treatments.
Bar plots summarizing (A) the gingiva microbiome composition by diet; high sucrose and casein (HSC), high soluble fiber (SF) and standard diet (STD) and (B) the gingiva microbiome composition at family levels by oral health condition; maxillary localized periodontitis (PD), No-PD (healthy) and ONJ. Differences in bacterial families at the gingiva between: (C) rats fed the SF diet compared to rats fed the STD diet; (D) rats fed the HSC diet compared to rats fed the SF diet; and (E) rats fed the HSC diet compared to rats fed the STD diet. Differences in bacterial families at the gingiva between quadrants with: (F) PD and No-PD lesions; (G) PD and ONJ; and (H) No-PD and ONJ. AUC: area under the curve. Asterisk (*) depict significant differences (Kruskal-Wallis, p < 0.05).
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