EJ Castillo1, JG Messer1, JM Jiron1, AM Abraham1, SM Thomas1, JF Yarrow2, DB Kimmel1, JI Aguirre1

1University of Florida, Department of Physiological Sciences Gainesville, FL, USA2VA Medical Center, Research Service, VA Medical Center, Gainesville, FL

Preventing Periodontitis or Controlling its Progression Reduces the Development of Medication-Related Osteonecrosis of the

Jaw (MRONJ) in Rice Rats (Oryzomys Palustris)

This research was supported by NIH grant RO1DE023783-01A National Institute of Dental and Craniofacial Research (NIDCR)

MRONJ

• >8 wk without healing• Systemic medications

• Powerful anti-resorptives [(pARS) eg. Bisphosphonates (Zoledronic acid, ZOL) or anti-RANKL inhibitors]

• Anti – angiogenics (eg. anti- vascular endothelial growth factors.

• No history of radiation therapy or apparent metastases to head and neck

• Local oral risk factors• Recent tooth extractions• Periodontitis (PD), periapical infection• Mucosal trauma

Necrotic bone

H&E

Bacterial colonies

• Exposed necrotic bone in the oral cavity

• Elimination/Reduction of oral and dental risk factors

Preventative Measures can reduce MRONJ

Tooth Extraction

Unfitting Removable Denture

Peri-implantitis

Periodontitis

STD rodent chow

Rice rat (O. palustris) M1

M2

M3

hard palate

soft palate

Food-impaction induced localized PD (FILP) lesions

(Messer et al 2017)

+Zoledronicacid (ZOL)+

MRONJ-like lesionmaxillae

M2M3

alveolar bone

alveolar bone

necrotic bone

Oral cavity

Preclinical Model of PD and MRONJ

(Messer et al 2018)

60-80% rice rats develop FILP lesions at 16-34 wks of age

Around 94% of FILP lesions occur in the maxilla

A dietary modification or mechanical dental cleaning in rice rats will prevent or control PD, and hence will reduce the prevalence of MRONJ

Determine the efficacy of preventing or controlling PD in the development of MRONJ by:

1) Oral mechanical cleaning of Lesions

2) Diet modification

Hypothesis

STD diet

VEH

ZOL 80μg/Kg

ZOL 80μg/Kg +Dental cleaning (DC)

Necropsy

In vivo oral exams under ISO anesthesia (q2wks)

High Soluble Fiber (HSF) (7.5% inulin and 10% fructo-oligosaccharides)

SF diet

OUTCOMES: • In vivo analysis of the jaws• High Resolution Photographs of Jaws

(necropsy = 24 wks). Gross Quadrant Grade (GQG)

• MicroCT• Histopathology

• decalcified, serially sectioned, and H&E stained

• Immunohistochemical TRAP staining

Materials and Methods

VEH

IV q4wks

ZOL 80μg/Kg

n=15/ groupPD lesions

n=15/ groupNo PD lesions

Rice rat (O.Palustris)

In Vivo Analysis of Maxillary Quadrants

0102030405060708090

100

0 5 10 15 20 25

Prev

alen

ce (%

)

Treatment Duration (wks)

Prevalence of Maxillary Gross Oral Lesions

STD+VEH STD+ZOL STD+ZOL+DC

SF+VEH SF+ZOL

• SF+VEH and SF+ ZOL rats had significantly lower prevalence of oral lesions than STD rats

• STD+ZOL rats that received dental cleanings had significantly reduced severity of PD lesions.

0

1

2

3

0 5 10 15 20 25Gro

ss Q

uadr

ant

Gra

de (G

QG

)

Treatment Duration (wks)

Maxillary Lesion severity

STD+VEH STD+ZOL STD+ZOL+DC

✢* ✢*✢*

SFSF+VEH

M1

SF+ZOL

M1

Ex-Vivo Gross Analysis of Oral Lesions

M1

3D reconstruction of MicroCT slices

M1

STD+VEH STD+ZOL

M1

STD+ZOL+DC

M1

STD

M1M1 M1

STD

STD+VEH STD+ZOL STD+ZOL+DC

M1 M1

SFSF+VEH SF+ZOL

High Resolution Photographs

0

1

2

3

4

Gross Lesion Severity based on Maxillary Quadrants

Gro

ss Q

uadr

ant G

rade

STD+VEH STD+ZOL STD+ZOL+DC

** P <0.05

0

2

4

6

8

10

12

14

16

18

20

STD+VEH STD+ZOL STD+ZOL+DC PD

Area

%

Lesion area/Total area(Ex Vivo)

P =0.004**

0

200

400

600

800

1000

1200Maxillary ABL M2M3

CEJ

-ABC

dis

tanc

e(u

m)

no PD PD no PD ONJ no PD PD no PD no PD

STD+VEH STD+ZOLSTD+ ZOL+DC SF+VEH SF+ZOL

Alveolar Bone Loss

ZOL, ONJ

M3M2M1

ABC

ABC

CEJ

500 µm * *

500 µm

STD+VEH no PD

M1M2 M3

CEJ

ABC

ABC

STD+VEH PD

M1M2 M3CEJ

ABC

ABC

500 µm

STD+ZOL+DC PD

M2M1 M3

CEJ

ABC

ABC

500µm

SF+VEH no PDM1

M3

M2

CEJ

ABCABC

500 µm

SF+ZOL no PDM1

M2M3CEJ

ABCABC

Immunohistochemical staining of TRAP+ Cells

0

2

4

6

8 TRAP+ cells/B.Pm

#/m

m2

No PD PD No PD No PD No PD No PDONJ PD

STD+VEH STD+ZOL STD+ZOL+DC SF+VEH SF+ZOL

STD +VEH STD+ZOL STD+ZOL+DC

STD

SFSF+ZOL no PDSF+VEH no PD

Immunohistochemical TRAP stained sections. Red arrow indicates TRAP+ osteoclast

Quantification of osteoclast number/mm2

0

1

2

3

4

5

Maxillary Histological PD Scores at 24 weeks (M2M3)

STD VEH STD ZOL STD ZOL+DC SF VEH SF ZOL

PD s

core

Histopathologic Assessment

ONJ

0

20

40

60

80

100 Empty Osteocyte Lacunae

Perc

ent

STD+VEH PD STD+ZOL MRONJ STD+ZOL+DC PD

*Epi

bone

*

200 µm

*M2

M3

50 µm

*

*

bone• Exposed necrotic bone• Lack of overlying gingival epithelium• ≥10 confluent empty osteocyte lacunae

0

20

40

60

80

STD+VEH STD+ZOL STD+ZOL+DC SF+VEH SF+ZOL

Prev

alen

ce (%

)

Treatment

* **

*

MRONJ Prevalence

Conclusions

• Dental cleaning reduces gross severity and extension but does not resolve PD lesions in rice rats.

• Preventing progression of PD reduces occurrence of MRONJ.

• SF diet prevented the development of PD and MRONJ regardless of treatment

• These findings provide direct preclinical evidence to support current guidelines2 concerning maintenance of good oral hygiene in pAR patients.