Assessment of <i>in vitro</i> markers of apoptosis after citalopram treatment.

<p>A. Caspase 3/7 functional assay results from MC3T3 cells treated with serial dilutions of citalopram (10⁻⁴ mol through 10⁻¹⁰ mol). No significantly significant differences were found by day, dose, or interaction terms. B. Gene expression markers of apoptosis from MC3T3 cells treated with media only or media supplemented with 10⁻⁴ mol citalopram. There were no statistically significant gene expression changes.</p

Quantitative qRT-PCR TaqMan Probe/Primer Set Data.

<p>Quantitative qRT-PCR TaqMan Probe/Primer Set Data.</p

Histological markers of apoptosis in control and citalopram exposed mouse cranial sutures.

<p>A & B. Coronal sutures from control (A) and citalopram exposed (B) 15 day old C57BL6 mice stained for Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) activity (green). Periosteum, dura and osteogenic front (OF) are as marked, and the sutural area has been outlined. Inset lower magnification of TUNEL staining with a 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). Note the highly cellularized (blue stained) area of the suture abutting the less cellularized (darker) area of the osteogenic front. C. Quantification of percent TUNEL positive staining as compared to DAPI stain in coronal sutures. Significant decreases in TUNEL activity were noted due to citalopram exposure at the periosteal and dural areas of the sutures. Midsutral space and osteogenic fronts did not demonstrate significantly different staining between unexposed and citalopram exposed sutures. D & E. Sagittal sutures from unexposed (control) (D) and citalopram exposed (E) 15 day old C57BL6 mice stained for TUNEL activity (green). Inset lower magnification of TUNEL staining with a DAPI nuclear counterstain (blue) F. Quantification of percent TUNEL positive staining as compared to DAPI stain. A significant increase in TUNEL staining was noted in the periosteal area while a significant decrease in staining was noted at the osteogenic front of citalopram exposed sagittal sutures.</p

Histological markers of proliferation in control and citalopram exposed mouse cranial sutures.

<p>A & B. Coronal sutures from control (A) and citalopram exposed (B) 15 day old C57BL6 mice stained for Proliferating Cell Nuclear Antigen (PCNA) (black arrows = example of positive cell, white arrows = example of negative cells). Periosteum, dura and osteogenic front (OF) are as marked, and the sutural area has been outlined. C. Quantification of percent PCNA stain. PCNA activity shows slight decreases for all but dural associated areas, but no statistical differences. D & E. Sagittal sutures from control (D) and exposed (E) 15 day old C57BL6 mice stained for PCNA. F. Quantification of percent PCNA stain. Overall, PCNA activity increased in all areas assessed in citalopram exposed sagittal sutures.</p

Assessment of <i>in vitro</i> markers of proliferation after citalopram treatment.

<p>A. MTS functional assay results from MC3T3 cells treated with serial dilutions of citalopram (10⁻⁴ mol through 10⁻¹⁰ mol). Significant time and dose effects are noted. B. Gene expression markers of proliferation from MC3T3 cells treated with media only or media supplemented with 10⁻⁴ mol citalopram. All three markers indicate a biological change due to treatment by 7 days. <i>Jun</i> was found to have significantly greater expression due to treatment at both 3 and 7 days.</p

Systemic effects of ZA treatment.

<p>A: No significant difference in kidney histology or BUN serum level between control and ZA-treated animals. 9B: No significant difference in liver histology or ALT activity, used to evaluate liver function between the two groups. 9C: No significant difference in body weights between the two groups.</p

A Model for Osteonecrosis of the Jaw with Zoledronate Treatment following Repeated Major Trauma

<div><p>This study aims to develop a reproducible rat model for post-traumatic bisphosphonate-related osteonecrosis of the jaw (BRONJ). In our previous studies using dental extraction as an inducing factor, only 30% - 60% of zoledronate-treated animals fulfilled the definition of clinical BRONJ. We modified the zoledronate regimen and introduced repeated surgical extraction to illicit quantifiable BRONJ in all animals. Eighty retired-breeder female Sprague-Dawley rats were divided between the treatment (IV zoledronate; 80 μg/kg/week for 13 weeks) and control (saline) groups. On week 13, the left mandibular first molar was surgically extracted, followed by the second molar a week later. Animals were euthanized at 1-week, 2-weeks, and 8-weeks following extraction. The occurrence and severity of BRONJ were scored in each animal based on gross and MicroCT analysis. Parameters of bone formation and osteoclast functions at the extraction site were compared between groups. All zoledronate-treated animals developed a severe case of BRONJ that fulfilled the clinical definition of the condition in humans. Osteoclast attachment continued to be defective eight weeks after stopping the treatment. There were no signs of kidney or liver toxicity. Our data confirmed that repeated surgical extraction (major trauma) by itself consistently precipitated massive bone necrosis in ZA-treated animals, eliminating the need to induce pre-existing infection or comorbidity. These results will be the basis for further studies examining the <i>in-vivo</i> pathogenesis and prevention of BRONJ.</p></div

Intraoral images of extraction sites in control vs. ZA-treated rats, showing persistent exposure of necrotic bone in the ZA-treated animals 8 weeks after extraction.

<p>Untreated animals showed complete healing.</p

Systemic effects of ZA treatment.

<p>A: No significant difference in kidney histology or BUN serum level between control and ZA-treated animals. 9B: No significant difference in liver histology or ALT activity, used to evaluate liver function between the two groups. 9C: No significant difference in body weights between the two groups.</p

Analysis of alveolar bone at the extraction site, excluding any sequestered bone.

<p>The numbers of empty lacunae over total number of osteocyte lacunae were used to calculate the percentage of dead osteocytes within the bone. There was a significant increase in the percentage of empty lacunae (arrows) on the extraction sites on ZA-treated animals. There was also a significant decline in the numbers of empty lacunae in the control group from week 1 to week 8. The un-operated sites in the ZA-treated animals did not show a significant difference in bone viability, compared to the un-operated sites in control animals, signifying that surgical extraction was necessary to illicit bone necrosis.</p