Otolaryngology -- Head and Neck Surgery

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Standardized Analysis of Mandibular Osteoradionecrosis in a Rat Model Matthew Tamplen, Kelley Trapp, Ichiro Nishimura, Bob Armin, Michael Steinberg, John Beumer, Elliot Abemayor and Vishad Nabili Otolaryngology -- Head and Neck Surgery 2011 145: 404 originally published online 3 March 2011 DOI: 10.1177/0194599811400576 The online version of this article can be found at: http://oto.sagepub.com/content/145/3/404

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Original Research—Facial Plastic and Reconstructive Surgery

Standardized Analysis of Mandibular Osteoradionecrosis in a Rat Model Matthew Tamplen1, Kelley Trapp1, Ichiro Nishimura, DDS, DMSc, DMD2, Bob Armin, MD1, Michael Steinberg, MD3, John Beumer, DDS, MS2, Elliot Abemayor, MD, PhD1, and Vishad Nabili, MD1

No sponsorships or competing interests have been disclosed for this article.

Abstract Objective. To develop a rat model of mandibular osteoradionecrosis (ORN) that uses novel micro-computed tomography bone volume analysis and detailed histology to provide a more effective, quantifiable, and standardized way to study ORN in vivo. Study Design. Animal model. Setting. Academic medical center. Subjects and Methods. Modifications to our previously published rat model of mandibular ORN were done to develop an ideal protocol consisting of 10 rats (6 experimental and 4 controls) with their left middle mandibular molar removed 7 days after either 20 Gy high dose rate brachytherapy or sham irradiation. Rats were sacrificed 21 days after extraction for landmark defined bone volume and histologic analysis. Results. A standardized method of quantification was achieved in all samples. The radiated group (XRT) had a mean bone volume/total volume (BV/TV) of 13.8% compared to 65.9% for controls (P \ .001). There were increases in osteoclasts and fibrosis, decreases in osteoblasts, and less bone in radiated samples with a mean (SD) of 5.91 (3.77) osteoclasts/high-powered field (HPF) and 4.00 (1.83) osteoblasts/HPF in XRT samples compared to 1.08 (1.08) osteoclasts/HPF and 22.49 (6.00) osteoblasts/HPF for controls (P \.001). Conclusion. Our updated model continues to be clinically analogous to human mandibular ORN and improves the radiologic and histologic analysis of bony defects, providing a method for quantification of bone loss. Further cellspecific staining, including immunohistochemistry, can be used with this model to study potential cellular mechanisms of mandibular ORN and test any future therapeutic options. Keywords osteoradionecrosis, animal model, mandible, irradiation Received September 30, 2010; revised January 21, 2011; accepted January 25, 2011.

Otolaryngology– Head and Neck Surgery 145(3) 404–410 Ó American Academy of Otolaryngology—Head and Neck Surgery Foundation 2011 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0194599811400576 http://otojournal.org

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steoradionecrosis (ORN) of the mandible is a serious complication of radiation therapy to the head and neck where irreversible bone loss ensues. Despite continued advances in radiation therapy for head and neck cancer, patients continue to suffer side effects as severe as mandibular ORN. The severity of mandibular ORN is highlighted by a recent series by Suh et al,1 which demonstrated the recurrence of ORN after resection and microvascular free flap reconstruction, thus proposing the possibility of mandibular ORN being an incurable disease. Clinically, the problem becomes debilitating for patients who have already undergone head and neck cancer therapy and will now need extensive mandibular resection and reconstruction even though they remain cancer free. The many previously proposed attempts at defining mandibular ORN have relied purely on clinical criteria without clearly investigating the mechanisms underlying this disease. Current clinical criteria include exposed bone for at least 2 to 6 months, a history of radiation therapy to the region of exposed bone, the presence of necrotic or devitalized bone, and no evidence of tumor recurrence.2-6 However, there remains no defined radiologic criterion for mandibular ORN, and reports of posttreatment histologic analysis are limited without a defined mechanism for the pathogenesis of the disease. Because the pathophysiology of mandibular ORN remains unknown, there is yet to be a clear definition of the disease in current literature, and treatment options remain limited. Many previous animal models have tried to demonstrate the clinical criteria of mandibular ORN.7-9 Recently, 1

Division of Head and Neck Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 2 UCLA School of Dentistry, Weintraub Center for Reconstructive Biotechnology, Los Angeles, California, USA 3 Department of Radiation Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA This article was presented at the 2010 AAO-HNSF Annual Meeting & OTO EXPO; September 26-29, 2010; Boston, Massachusetts. Corresponding Author: Matthew Tamplen, Division of Head and Neck Surgery, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave., CHS 62-132, Los Angeles, CA, USA Email: [email protected]


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Niehoff et al10 successfully demonstrated the use of a more clinically analogous high dose rate (HDR) brachytherapy in a rat model of radiogenic bone damage. Our group then used this method of HDR brachytherapy to mimic clinical ORN localized to the rat mandibular bone, incorporating postradiation tooth extractions, plus gross, histologic, and radiologic analysis for a more complete description of the radiogenic defects.2 Despite advancements in the rat model, a major shortcoming was the lack of a standardized protocol providing the anatomical orientation necessary to reproducibly measure and study a defined area of the radiated mandible. A detailed and standardized animal model clinically analogous to mandibular ORN is a valuable tool that can be used to investigate the underlying pathophysiology of the disease and evaluate any potential treatment modalities. However, previous animal models of ORN have had limited success because of both the lack of standardized methods that allow for comparison across multiple studies and the need for a more comprehensive and detailed approach that provides a complete description of the pathologic defects on both clinical and cellular levels. This is why it was our objective to develop a rat model of mandibular ORN that uses both a novel micro-computed tomography (micro-CT) bone volume analysis method and detailed histology to provide a more effective, quantifiable, and standardized method that can be reproducibly used to study the pathogenesis of and potential treatment for mandibular ORN in vivo.

Materials and Methods Experimental Design Approval for the research protocol was obtained from the University of California Los Angeles Chancellor’s Animal Research Committee. Ten male Sprague-Dawley rats 6 weeks of age were used in this study. On the basis of previous pilot models, we performed a power calculation demonstrating the ability to have 80% power to detect an effect size (ES) of 1.80 using a 2-sample t test with a 0.05 2-sided significance when comparing the percent bone volume remaining between radiated and nonradiated groups with 10 animals. Rats were obtained from Charles River Laboratories (Wilmington, Massachusetts). The rats were kept in pairs and given a standard pelleted rodent diet and water ad libitum in accordance with the requirements of the US Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Our previously published animal model protocol was used for irradiation, tooth extraction, and radiologic and histologic analysis except for the modifications described below.2

Irradiation Modification A single dose of 20 Gy as opposed to previously described 30-Gy radiation was applied with an HDR afterloading remote machine with predefined isodose lines.

Figure 1. Axial micro-CT section taken at the level of the crownroot junction of the posterior molar demonstrating placement of the standardized circle (diameter = .31 cm) in the center of the extraction socket.

Tooth Extraction Modification Under isoflurane anesthesia, all rats underwent atraumatic extraction of a single left middle mandibular molar 7 days following irradiation (or sham catheter placement) as opposed to previously described removal of all 3 mandibular molars on the day after irradiation.

Radiologic Analysis Using a desktop cone-beam micro-CT scanner (mCT 40, Scanco Medical, Bru¨ttisellen, Switzerland) and mCT Evaluation Software v6.0 (Scanco Medical), a standardized method for micro-CT volume analysis using a single-tooth extraction method was developed. A cylinder with a defined total volume was created to determine the volumes of bone in the extracted single-tooth sockets. A consistent landmark was noted at the crown-root junction of the posterior molar to start analysis. At this junction, the average length between the anterior and posterior molars and width between the lingual and buccal cortex were used to create a circle measuring .31 cm in diameter centered in the extraction socket. This circle was saved in the evaluation program and automatically applied to the next 45 sections in an inferior direction (each slice = 36 mm). The depth of 45 slices was calculated from the average depth from the crown-root junction to the base of the root socket (0.16 cm). With these length, width, and height measurements, the software was able to create a reproducible, standardized cylindrical volume measuring 11.157 mm3, serving as the total volume (TV) of analysis for all of the animals (Figure 1).


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Figure 2. Hematoxylin and eosin–stained section of left hemimandible at 43 demonstrating the standardized anatomically oriented area used for histologic analysis.

expressed as a ratio (BV/TV) (Table 1). A 2-tailed t test was used to compare each of these variables between radiated and nonradiated rat mandibles. The level of significance was set at 0.05. The average (SD) BV/TV in the irradiated group was 13.8% (7.0%) as compared with 65.9% (8.0%) for controls (P \ .001). Three-dimensional (3D) CT reconstructions of the rat mandibles were created using m-CT Evaluation Software (Scanco Medical), allowing for ultrastructural viewing of bone regrowth in the tooth extraction socket sites and qualitative comparisons between radiated and nonirradiated samples (Figure 4). The 3D reconstructions demonstrate gross reductions in cortical width, decreased bone formation in extraction sockets, and increased resorption both in the traumatic and atraumatic areas of irradiated mandibles compared to controls.

Decalcified Histologic Analysis Histologic Analysis The 10 specimens (6 irradiated and 4 controls) were decalcified in 10% EDTA (pH 7.4) for 7 days, fixed in formalin overnight, and paraffin embedded by the UCLA Translational Pathology Core Laboratory. Then, 4-mm sagittal sections of the left hemimandibles were stained with hematoxylin and eosin (H&E) and viewed for both qualitative and quantitative histologic analysis using cameraassisted light microscopy (Nikon Labophot-2; Nikon, Tokyo, Japan). The metabolizing surface of the lower cortical bone between the anterior and posterior molar roots was used as a standardized area for quantification, as shown in Figure 2. Quantification of osteoclasts and osteoblasts was done at 403. Three high-power fields within the landmark defined tooth socket were examined, and the mean value was calculated for each animal. Fibrosis was quantified as mild (filling \25% of tooth socket), moderate (filling 25%90% of tooth socket), or severe (filling more than 90% of tooth socket) (Table 1).

Results Gross Findings All 10 rats survived the study period. All the rats from the irradiated group demonstrated adverse effects of targetspecific HDR radiation as evidenced by unilateral left cheek skin alopecia in 100% of the group, whereas all of the animals in the control group had complete hair regrowth. Likewise, ipsilateral incisor growth was also retarded in the radiated animals as compared with nonirradiated animals. Evaluation of the intraoral inferior alveolar ridges using high-resolution digital photography demonstrated exposed bone at the site of dental extraction in 100% of the irradiated rats. Complete wound mucosalization was seen in all of the animals in the control group (Figure 3).

Micro-CT Evaluation Bone volume (BV) was measured in relation to the TV of the mandibular middle molar extraction sockets and

The radiated specimens showed a qualitative reduction of bone formation and an increased presence of fibrosis and inflammation, as well as a quantifiable increase of osteoclasts and decrease of osteoblasts within the extraction sockets when compared to controls. All of the radiated samples demonstrated severe fibrosis; only 1 control animal demonstrated moderate fibrosis, and the rest had mild fibrosis. Moreover, the control samples specifically had increased bone formation in the standardized region of evaluation with several areas lined completely with osteoblasts and only minimal inflammation (Figure 5). There was a mean (SD) of 4.00 (1.83) osteoblasts/high-powered field (HPF) in the radiated group (XRT) compared to 22.49 (6.00) osteoblasts/HPF for controls (P \ .001). There was a mean of 5.91 (3.77) osteoclasts/HPF in XRT samples compared to a mean of 1.08 (1.31) osteoclasts/HPF for controls (P = .046) (Table 1).

Discussion ORN is a debilitating, severe disease with high morbidity, recurrence risk, and potential incurability.1 Previous animal and human models have relied on purely clinical criteria providing little insight into the pathogenesis of the disease. Without a clear understanding of the pathophysiology on a cellular level, treatment options have remained limited, and our patients continue to suffer significant morbidity and even mortality from mandibular ORN. A reproducible and standardized model of mandibular ORN has not been previously described. This is why we have made it a priority to develop a clinically analogous and standardized animal model of mandibular ORN that can be used to study the cellular mechanisms of the disease, as well as any potential therapeutic strategies. Our previously published animal model laid the groundwork for studying mandibular ORN.2 Although it addressed many of the issues with previous models, it had several shortcomings. One of the shortcomings was the lack of reproducibility. The area for both micro-CT and histologic analysis was variable between animals and not


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Table 1. Micro-CT Data of Hemimandible Volume Analysis Rat

20 Gy BV, mm3 TV, mm3 BV/TV, % Fibrosis Osteoblasts/HPF Osteoclasts/HPF

1

2

3

4

5

6

7

8

9

10

XRT 1.2819 11.157 11.5 Severe 2.66 10.66

XRT 1.5998 11.157 14.3 Severe 1.33 4.00

XRT 0.6605 11.157 5.9 Severe 6.66 2.00

XRT 1.2495 11.157 11.2 Severe 4.67 7.00

XRT 3.2622 11.157 29.2 Severe 4.67 4.00

XRT 1.2071 11.157 10.8 Severe 4.00 1.00

Control 6.6615 11.157 59.7 Moderate 30.00 0

Control 7.1435 11.157 64.0 Mild 15.33 0

Control 7.3889 11.157 66.2 Mild 22.00 2.66

Control 7.9858 11.157 71.6 Mild 22.66 1.67

Abbreviations: BV, bone volume; HPF, high-powered field; TV, total volume; XRT, radiated group. TV: standardized volume of 11.157 mm3. XRT: mean (SD) BV/TV = 13.83 (7.03). No XRT: mean (SD) BV/TV = 65.94 (8.02), P \.001. Severe fibrosis, filling more than 90% of entire tooth socket; moderate fibrosis, filling 25% to 90% of tooth socket; mild fibrosis, \25% of the tooth socket. There was a mean (SD) of 4.00 (1.83) osteoblasts/HPF in the XRT group compared to 22.49 (6.00) osteoblasts/HPF for controls (P \.001). There was a mean (SD) of 5.91 (3.77) osteoclasts/HPF in XRT samples compared to a mean of 1.08 (1.31) osteoclasts/HPF for controls (P = .046).

Figure 3. Localized clinical manifestations of irradiation in the rat model, including alopecia of the left facial skin, retardation of central incisor growth, and exposed bone at the middle molar extraction site.

standardized.2 A standardized way to measure a defined space with the same total volume was needed to determine the amount of bone remaining in the extraction site that could be compared among animals and across studies. Some of the difficulty in creating a reproducible area for evaluation is because the rat mandible is a complex 3D structure with variable height, width, and thickness, as well as a large central incisor that spans the inferior mandibular body that can confound true bone volume measurements. In our previous model, the extensive variability in mandibles between each animal was confounded by lack of orientation and anatomical landmarks. The variabilities were evident in that the TV of analysis was different in the evaluation of every animal.2 To create a standardized space measurement, better orientation of the area of interest (ie, the dental extraction socket) was needed to improve orientation for micro-CT and histologic analysis. By only extracting the middle mandibular molar, it allowed the posterior and

anterior molars to serve as clear landmarks for micro-CT and histologic evaluation. Furthermore, the central molar is smaller than the anterior molar and, as such, can be extracted with much ease, eliminating the risk of a retained root that can also confound bone volume measurements as seen in our previous model.2 More important, axial and coronal orientation of the depth and location of the extraction socket in relation to the remaining anterior and posterior molar roots aids in micro-CT and histologic analysis, as described earlier. To successfully create a standardized model of mandibular ORN, the entire protocol must be standardized, including the radiation delivery and tooth extraction protocol. Many have tried to mirror the human radiation treatments in animal models going as far back as the 1960s, including the recent use of fractionated radiotherapy in a rat model by Fenner et al.11 The problem with these methods has been with reliably reproducing the area of radiation damage in


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Figure 4. Three-dimensional micro-CT reconstruction of irradiated mandible (XRT) compared to control (no XRT).

a safe way that minimizes the anesthesia risk and incidence of early mortality. The difficulty of safely using fractionated methods is highlighted by the 5 early mortalities in the pilot study by Fenner et al.11 In our previous model, we used a single 30-Gy radiation insult with tooth extractions the day after irradiation in an effort to maximize bony necrosis and minimize the need for anesthesia. However, the 30-Gy insult was shown to be excessive, leading to an early mortality.2 Jacobsson et al12 previously established that a maximal bone reduction is achieved at doses above 15 Gy, and Niehoff et al10 demonstrated the success of a single reproducible 20-Gy irradiation dose to the rat hemimandible to maximize the diminution in bone regeneration and minimize animal morbidity. On the basis of these previous studies, we chose a single irradiation dose of 20 Gy that allowed for maximal animal safety and reproducibility not feasible with current fractionated methods. This further clearly demonstrates a safe and effective way to deliver radiation to a defined area of the rat mandible that optimally creates the desired bone defect and limits animal morbidity. As for the timing of tooth extractions, we felt that our previous method of tooth extraction on the day after irradiation is unlikely to be clinically relevant, and the initial inflammation seen with extraction may lead to confounding factors in the analysis of radiation damage. It has been shown that tooth extractions after radiation therapy have the highest risk for the development of ORN and thus are more

useful for the development of an animal model.13-16 More recently, tooth extractions 1 week after radiation therapy were found to be optimal for a rat model of maxillary ORN to maximize the possibility of ORN occurrence.9 The lack of a clearly defined pathogenesis of mandibular ORN continues to limit the development of new therapeutic strategies and remains a large void in the current literature. Historically, many hypotheses to describe the pathogenesis of ORN have been attempted, but none are proven to date. From the 1920s to the 1980s, 3 factors were thought to contribute to ORN, including radiation, trauma, and infection. The 3-H theory proposed that radiation causes an endarteritis, resulting in hypoxia, hypocellularity, and hypovascularity, leading to chronic nonhealing wounds in the mandibular bone. This theory was the basis for using hyperbaric oxygen (HBO) to treat mandibular ORN, with a variety of mixed results in the literature.4,17-26 In 2004, a French randomized trial put the 3-H theory and the usage of HBO in question as a placebo-treated group (surgical debridement) had a better response than HBO.27 Most recently, cellular alterations causing ORN are under investigation. One new theory involves a fibroatrophic process leading to ORN. Again, this theory is based on clinical improvements from a phase II trial using antifibrosis agents in ORN.28 The mechanism behind this theory has yet to be proven, but the results from this treatment method appear promising and point to a cellular role of ORN involving fibroblasts. The recent theories on the pathogenesis of ORN have provided increasing evidence for a cellular defect underlying the disease process.28,29 These theories have yet to be substantiated by an in vivo model, which is why it is crucial that any model of mandibular ORN includes detailed cellular quantification of bony defects, something lacking in our previous model.2 Using the same anatomical landmarks we used for radiologic evaluation, we were able to create a standardized and easily reproducible area for histologic analysis. Focusing on the metabolizing surface of the lower cortical bone in the extraction socket, the area most damaged in the irradiated group on micro-CT, we were able to quantify the amount of bone-metabolizing cells present in the landmarkdefined area (Figure 5). There was a significant increase in osteoclasts and decreases in osteoblasts in the XRT group compared to controls. Previous models have suggested an increase in bone resorption but have not described a consistent way to measure the effect ORN has on cell number or function.9 Our model demonstrates a reproducible and standardized way to measure any potential imbalance in cell number at the surface of cortical bone and highlights the possibility of an imbalance underlying the pathogenesis of this disease. It is our belief that cellular changes happening immediately after the insults to mandibular bone lead to the effects of ORN, and in effort to capture these effects, we chose a 21-day follow-up period for analysis. Our previous model demonstrated significant changes in bone metabolism already present at 28 days after tooth extractions, indicating


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Figure 5. Hematoxylin and eosin–stained sections of metabolizing surface of cortical bone within standardized area of evaluation of tooth extraction socket sites from a radiated (XRT) and control (no XRT) group at 403 magnification. The radiated sample demonstrates increased osteoclasts and fewer osteoblasts with a reduction in bone formation when compared with the control sample.

early manifestations of ORN.2 According to previous published literature on rat metabolism, 3 weeks is analogous to 3 years in humans and represents an adequate time point for development of an analogous animal model of mandibular ORN.2,9 It is a future goal to use this standardized model to evaluate possible cellular manifestations of ORN at several time points from 1 week to up to a year postradiation, but to do that, we first need to clearly define a standardized protocol for the animal model. Our model is clinically analogous to human mandibular ORN based on clinical definitions used today and provides the anatomic orientation necessary for standardized bone volume and histologic analysis. We demonstrated the first standardized method for mandibular bone volume analysis with micro-CT, making our model better equipped to define the pathogenesis of this disease. Our standardized protocols provide a detailed description for the radiogenic defects on clinical, radiologic, and cellular levels. These protocols can be used to compare results across several animal models, allowing for quicker scientific discovery in a field that is long overdue. Our model demonstrates significant bone loss after radiation and dental extraction, successfully limits the amount of animal morbidity, increases experimental feasibility, and limits the animals needed for experimentation, as well as provides the histologic evidence for a cellular imbalance mechanism in the pathogenesis of ORN. Further cellspecific histologic staining and immunochemistry can be used with this model to study the potential cellular mechanisms of mandibular ORN and serve as a valuable tool for future studies to define ORN and delineate its pathogenesis. Author Contributions Matthew Tamplen, substantial contributions to conception and design, acquisition of data and analysis and interpretation of data, drafting the article and revising it critically for important intellectual content, and final approval of the version to be published;

Kelley Trapp, substantial contributions to conception and design, acquisition of data, revising article critically for important intellectual content, and final approval of the version to be published; Ichiro Nishimura, substantial contributions to conception and design, analysis and interpretation of data, revising article critically for important intellectual content, and final approval of the version to be published; Bob Armin, substantial contributions to conception and design, acquisition of data and analysis and interpretation of data, revising article critically for important intellectual content, and final approval of the version to be published; Michael Steinberg, substantial contributions to conception and design, analysis and interpretation of data, revising article critically for important intellectual content, and final approval of the version to be published; John Beumer, substantial contributions to conception and design, analysis and interpretation of data, revising article critically for important intellectual content, and final approval of the version to be published; Elliot Abemayor, substantial contributions to conception and design, acquisition of data and analysis and interpretation of data, drafting the article and revising it critically for important intellectual content, and final approval of the version to be published; Vishad Nabili, substantial contributions to conception and design, acquisition of data and analysis and interpretation of data, drafting the article and revising it critically for important intellectual content, and final approval of the version to be published.

Disclosures Competing interests: None. Sponsorships: None. Funding source: None.

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