Does a Change in Bone Mineral Density Occur in the Mandible of Göttingen Minipigs After Irradiation in Correlation With Radiation Dose and Implant Surgery?



Does a Change in Bone Mineral Density Occur in the Mandible of Göttingen Minipigs After Irradiation in Correlation With Radiation Dose and Implant Surgery?




Journal of Oral and Maxillofacial Surgery, 2014-11-01, Volume 72, Issue 11, Pages 2149-2156, Copyright © 2014 American Association of Oral and Maxillofacial Surgeons


Purpose

To date, studies have not agreed on the effects of irradiation on bone mineral density. The aim of our study was to investigate the changes in mandibular bone mineral density after irradiation at various doses with and without surgery.

Materials and Methods

We implemented a descriptive animal experiment. The sample included 16 female Göttingen Minipigs, randomly assigned to 4 groups and irradiated with equivalent doses of 0, 25, 50, and 70 Gy to the mandibular region. At 3 months after irradiation, the mandibular left premolars and molars were removed, and dental implants were placed. Computed tomography scans were taken before and 6 months after irradiation. The measured bone density was related to a bone phantom to calculate the bone mineral density quotient (BMDQ). The outcome variable was the BMDQ. Other study variables were the radiation dose and surgery. Descriptive and univariate analyses were computed, and significance was set at P ≤ .05.

Results

In the left hemimandible, compared with the control group, a significant decrease in BMDQ was observed: 0.01 at 0 Gy, −0.01 at 25 Gy, −0.06 at 50 Gy, and −0.11 at 70 Gy ( P = .023). The right hemimandible compared with the control group also showed a significant decrease in BMDQ: −0.02 at 0 Gy, −0.08 at 25 Gy, −0.09 at 50 Gy, and −0.11 at 70 Gy ( P = .007).

Conclusions

The present study used a large animal model to simulate the tissue reactions induced by various radiation doses in the mandible. We found a significant decrease in the BMDQ after irradiation, but no significant correlation could be found between the irradiation dose and a decrease in the BMDQ.

Radiotherapy (RT) is one of the treatment options for malignancies of the head and neck region. However, RT results in side effects on healthy tissue. The most serious complication in the head and neck region is the development of osteoradionecrosis (ORN). The definition most widely used for ORN has been “irradiated bone, which becomes devitalized and is exposed through the overlying skin or mucosa and does not heal within a period of three months, without tumor recurrence.” Patient factors that will increase the risk of the development of ORN include age, gender, health, nutritional status, and the use of tobacco and alcohol. The treatment of ORN is challenging, because it often involves major surgery and causes high morbidity.

RT-induced ORN of the mandible was described as early as 1930. However, when modern photon therapy with megavolt energy levels is applied in RT, the high-energy doses are absorbed differently by the bone compared with the kilovolt energy doses used previously. Before modern technology, the incidence of ORN in the mandible was reported as high as 22%. Current data have reported an incidence of mandibular ORN after RT of approximately 5%, most likely because of more precise and refined irradiation protocols.

Various hypotheses have been proposed about the pathophysiology of ORN. According to Marx and Marx and Johnson, ORN is caused by a combination of vascular and cellular changes in the mandibular bone. Histologic evaluation has shown acellularity and avascularity of the bone marrow and a nonviable periosteum. This condition develops in irradiated areas, leading to tissue hypoxia. Hypoxic conditions cause direct damage to the bone cells, the osteoclasts and osteoblasts.

The cell damage is initiated by 2 different pathways: the direct effect of radiation on the bone and tissue cells and the effect of free radicals damaging the healthy cells. Tissue repair due to iatrogenic or noniatrogenic trauma will no longer be possible. Tissue breakdown, however, can also be seen spontaneously, without any trauma.

According to Delanian et al and Delanian and Lefaix, 3 phases can be discerned in the tissue reaction to irradiation. This reaction has been termed “radiation-induced fibroatrophy” (RIF). The initial prefibrotic phase includes damage to the endothelial cells. The loss of the endothelial cells results in exposure of the connective tissue cells to normally foreign stimuli, which could trigger fibroblastic activation. After the initial phase, RIF induces tissue growth composed of active fibroblasts (myofibroblasts), senescent fibroblasts (fibrocytes), and extracellular matrix. The combined damage to the endothelial cells and the connective tissue cells, amplified by the action of cytokines, leads to a permanent state of RIF. Finally, remodeling of the extracellular matrix progressively increases the tissue density, leaving friable tissue with poor vascularization and cellularized fibroatrophy. These areas are prone to developing ORN.

How these pathophysiologic processes affect bone quality and density is not clear. To date, studies have not agreed on the changes in bone mineral density (BMD) after RT. It is still unclear whether BMD measurements will aid in assessing the severity of bone damage.

The purpose of the present study was twofold. First, we intended to introduce a large animal model to investigate the BMD changes in the mandibles of Göttingen Minipigs irradiated with various radiation doses. Second, we wished to investigate a possible correlation between BMD changes and the radiation dose, with and without surgical intervention. Our hypothesis was, according to the pathophysiology and findings of Verdonck et al, that we would expect a small increase in the BMD quotient (BMDQ) after irradiation. The specific aim of our study was to develop a tool to measure the quality of the irradiated bone. We tried to more closely emulate the clinical human patient in the animal model by using a split mouth model to be able to compare both sides of the mandible, performing surgery on the left side only. The surgical intervention consisted of extractions and implant placement to simulate clinical reality. The bone density was measured adjacent to the implants to prove the clinical concept of implant-based prostheses in the animal model. Quantitative computed tomography (CT) was used to evaluate the BMD changes.


Materials and Methods

The experiment was performed in accordance with the European Community Guidelines for the Protection of (Laboratory) Animals. The local animal ethical committee approved the study.


Study Design

The study design was an animal experiment. For the present study, 16 healthy adult 18-month-old female Göttingen Minipigs were used. There were no additional inclusion or exclusion criteria.


Study Variables

The predictor variable in the present study was the irradiation dose group. Four pigs were randomly assigned to each of the 4 research groups stratified by the radiation dose received (0, 25, 50, and 70 Gy, respectively). The primary outcome variable was the BMDQ. The other study variables were radiation dose and surgery (yes or no). The primary predictor variables were radiation dose and surgery. Other variables, such as age and gender, were the same for all the pigs. No other variables were related to the outcome.


Data Collection Methods

For RT planning purposes, a preparatory planning CT scan was taken with the pigs under general anesthesia with endotracheal intubation. A thermoplastic immobilization mask was used for optimal fixation of the minipig's head. A Siemens Sensation Open CT scanner was used for radiologic documentation (Siemens, Erlangen, Germany). Axial slices of 1 mm were taken. The CT scans were performed with a calibrated bone phantom placed in the mouth of the minipig to calculate the relative BMDQ and absolute bone density. At 26 weeks after irradiation, another native CT scan was taken. The procedure was the same as that before irradiation.

The minipigs underwent irradiation under general anesthesia with endotracheal intubation. They were positioned in an individualized thermoplastics mold for reproducible and steady positioning. Irradiation was delivered with a 6-megavolt linear accelerator with 2 opposing lateral beams (Siemens). The entire body of the mandible was irradiated in 2 fractions with an equivalent dose of 0, 25, 50, or 70 Gy (2 fractions of 0, 6.5, 9.7, and 11.8 Gy). To calculate the equivalent dose, an α/β ratio for late-responding tissues was used. We assumed that the α/β ratio was 3. The second fraction was given 48 hours after the first fraction.

At 13 weeks after irradiation, surgery was performed with the minipigs under general anesthesia with endotracheal intubation and mechanical ventilation. Established protocols for animal care were used to guarantee maximum safety.

To simulate a translational clinical situation, the surgical procedure on the mandible involved extraction of all premolars and molars on the left side. Next, 4 submerged BioComp dental implants (diameter 4.0 mm and length 12 mm) were placed in each pig (BioComp Industries BV, Vught, The Netherlands). The pigs received intramuscular antibiotics (amoxicillin) and intramuscular pain medication (buprenorphine), which were continued for 3 days postoperatively.

Using SimPlant 15 (Materialize, Leuven, Belgium), the data from the 2 CT scans were analyzed. To measure the BMD, we used a region of interest (ROI) of 5 to 10 mm 2 . The ROI tool for measuring bone density was placed on 5 different predetermined locations of the mandibular cortex on both sides of the mandible ( Fig 1 ). Cortical bone was deliberately chosen to eliminate measurement errors caused by dental or implant material. Because the cortical bone contains less collagenous tissue than bone marrow, its measurements of BMD have been considered to be more accurate.

Location of the circular region of interest. A, Preirradiation right mandible, B, left mandible, C, postirradiation right mandible, and D, implanted left mandible (SimPlant 15, Materialise, Leuven, Belgium). Std. Dev., standard deviation.
Figure 1
Location of the circular region of interest.
A, Preirradiation right mandible,
B, left mandible,
C, postirradiation right mandible, and
D, implanted left mandible (SimPlant 15, Materialise, Leuven, Belgium). Std. Dev., standard deviation.

Two of us (L.P., G.B.) performed the measurements independently from one another. The calibrated bone phantom had a predetermined density given in g/mL. The BMDQ was calculated by dividing the measurement by the average BMD of the phantom ( Fig 2 ). The BMDQ in the present study was used to quantitatively compare the changes in the BMD. The BMD values represented the degree of mineralization of bone. The BDM is known to be relatively low in immature newly formed bone compared with the mature bone matrix.

Photograph of the bone phantom used in this experiment.
Figure 2
Photograph of the bone phantom used in this experiment.


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Does a Change in Bone Mineral Density Occur in the Mandible of Göttingen Minipigs After Irradiation in Correlation With Radiation Dose and Implant Surgery? Lucas J. Poort MD, DDS , Gerhard K.P. Bittermann MD , Roland A. Böckmann MD, DDS , Frank J. Hoebers MD, PhD , Ruud Houben MSc , Alida A. Postma MD, PhD and Peter A.W.H. Kessler MD, DDS, PhD Journal of Oral and Maxillofacial Surgery, 2014-11-01, Volume 72, Issue 11, Pages 2149-2156, Copyright © 2014 American Association of Oral and Maxillofacial Surgeons Purpose To date, studies have not agreed on the effects of irradiation on bone mineral density. The aim of our study was to investigate the changes in mandibular bone mineral density after irradiation at various doses with and without surgery. Materials and Methods We implemented a descriptive animal experiment. The sample included 16 female Göttingen Minipigs, randomly assigned to 4 groups and irradiated with equivalent doses of 0, 25, 50, and 70 Gy to the mandibular region. At 3 months after irradiation, the mandibular left premolars and molars were removed, and dental implants were placed. Computed tomography scans were taken before and 6 months after irradiation. The measured bone density was related to a bone phantom to calculate the bone mineral density quotient (BMDQ). The outcome variable was the BMDQ. Other study variables were the radiation dose and surgery. Descriptive and univariate analyses were computed, and significance was set at P ≤ .05. Results In the left hemimandible, compared with the control group, a significant decrease in BMDQ was observed: 0.01 at 0 Gy, −0.01 at 25 Gy, −0.06 at 50 Gy, and −0.11 at 70 Gy ( P = .023). The right hemimandible compared with the control group also showed a significant decrease in BMDQ: −0.02 at 0 Gy, −0.08 at 25 Gy, −0.09 at 50 Gy, and −0.11 at 70 Gy ( P = .007). Conclusions The present study used a large animal model to simulate the tissue reactions induced by various radiation doses in the mandible. We found a significant decrease in the BMDQ after irradiation, but no significant correlation could be found between the irradiation dose and a decrease in the BMDQ. Radiotherapy (RT) is one of the treatment options for malignancies of the head and neck region. However, RT results in side effects on healthy tissue. The most serious complication in the head and neck region is the development of osteoradionecrosis (ORN). The definition most widely used for ORN has been “irradiated bone, which becomes devitalized and is exposed through the overlying skin or mucosa and does not heal within a period of three months, without tumor recurrence.” Patient factors that will increase the risk of the development of ORN include age, gender, health, nutritional status, and the use of tobacco and alcohol. The treatment of ORN is challenging, because it often involves major surgery and causes high morbidity. RT-induced ORN of the mandible was described as early as 1930. However, when modern photon therapy with megavolt energy levels is applied in RT, the high-energy doses are absorbed differently by the bone compared with the kilovolt energy doses used previously. Before modern technology, the incidence of ORN in the mandible was reported as high as 22%. Current data have reported an incidence of mandibular ORN after RT of approximately 5%, most likely because of more precise and refined irradiation protocols. Various hypotheses have been proposed about the pathophysiology of ORN. According to Marx and Marx and Johnson, ORN is caused by a combination of vascular and cellular changes in the mandibular bone. Histologic evaluation has shown acellularity and avascularity of the bone marrow and a nonviable periosteum. This condition develops in irradiated areas, leading to tissue hypoxia. Hypoxic conditions cause direct damage to the bone cells, the osteoclasts and osteoblasts. The cell damage is initiated by 2 different pathways: the direct effect of radiation on the bone and tissue cells and the effect of free radicals damaging the healthy cells. Tissue repair due to iatrogenic or noniatrogenic trauma will no longer be possible. Tissue breakdown, however, can also be seen spontaneously, without any trauma. According to Delanian et al and Delanian and Lefaix, 3 phases can be discerned in the tissue reaction to irradiation. This reaction has been termed “radiation-induced fibroatrophy” (RIF). The initial prefibrotic phase includes damage to the endothelial cells. The loss of the endothelial cells results in exposure of the connective tissue cells to normally foreign stimuli, which could trigger fibroblastic activation. After the initial phase, RIF induces tissue growth composed of active fibroblasts (myofibroblasts), senescent fibroblasts (fibrocytes), and extracellular matrix. The combined damage to the endothelial cells and the connective tissue cells, amplified by the action of cytokines, leads to a permanent state of RIF. Finally, remodeling of the extracellular matrix progressively increases the tissue density, leaving friable tissue with poor vascularization and cellularized fibroatrophy. These areas are prone to developing ORN. How these pathophysiologic processes affect bone quality and density is not clear. To date, studies have not agreed on the changes in bone mineral density (BMD) after RT. It is still unclear whether BMD measurements will aid in assessing the severity of bone damage. The purpose of the present study was twofold. First, we intended to introduce a large animal model to investigate the BMD changes in the mandibles of Göttingen Minipigs irradiated with various radiation doses. Second, we wished to investigate a possible correlation between BMD changes and the radiation dose, with and without surgical intervention. Our hypothesis was, according to the pathophysiology and findings of Verdonck et al, that we would expect a small increase in the BMD quotient (BMDQ) after irradiation. The specific aim of our study was to develop a tool to measure the quality of the irradiated bone. We tried to more closely emulate the clinical human patient in the animal model by using a split mouth model to be able to compare both sides of the mandible, performing surgery on the left side only. The surgical intervention consisted of extractions and implant placement to simulate clinical reality. The bone density was measured adjacent to the implants to prove the clinical concept of implant-based prostheses in the animal model. Quantitative computed tomography (CT) was used to evaluate the BMD changes. Materials and Methods The experiment was performed in accordance with the European Community Guidelines for the Protection of (Laboratory) Animals. The local animal ethical committee approved the study. Study Design The study design was an animal experiment. For the present study, 16 healthy adult 18-month-old female Göttingen Minipigs were used. There were no additional inclusion or exclusion criteria. Study Variables The predictor variable in the present study was the irradiation dose group. Four pigs were randomly assigned to each of the 4 research groups stratified by the radiation dose received (0, 25, 50, and 70 Gy, respectively). The primary outcome variable was the BMDQ. The other study variables were radiation dose and surgery (yes or no). The primary predictor variables were radiation dose and surgery. Other variables, such as age and gender, were the same for all the pigs. No other variables were related to the outcome. Data Collection Methods For RT planning purposes, a preparatory planning CT scan was taken with the pigs under general anesthesia with endotracheal intubation. A thermoplastic immobilization mask was used for optimal fixation of the minipig's head. A Siemens Sensation Open CT scanner was used for radiologic documentation (Siemens, Erlangen, Germany). Axial slices of 1 mm were taken. The CT scans were performed with a calibrated bone phantom placed in the mouth of the minipig to calculate the relative BMDQ and absolute bone density. At 26 weeks after irradiation, another native CT scan was taken. The procedure was the same as that before irradiation. The minipigs underwent irradiation under general anesthesia with endotracheal intubation. They were positioned in an individualized thermoplastics mold for reproducible and steady positioning. Irradiation was delivered with a 6-megavolt linear accelerator with 2 opposing lateral beams (Siemens). The entire body of the mandible was irradiated in 2 fractions with an equivalent dose of 0, 25, 50, or 70 Gy (2 fractions of 0, 6.5, 9.7, and 11.8 Gy). To calculate the equivalent dose, an α/β ratio for late-responding tissues was used. We assumed that the α/β ratio was 3. The second fraction was given 48 hours after the first fraction. At 13 weeks after irradiation, surgery was performed with the minipigs under general anesthesia with endotracheal intubation and mechanical ventilation. Established protocols for animal care were used to guarantee maximum safety. To simulate a translational clinical situation, the surgical procedure on the mandible involved extraction of all premolars and molars on the left side. Next, 4 submerged BioComp dental implants (diameter 4.0 mm and length 12 mm) were placed in each pig (BioComp Industries BV, Vught, The Netherlands). The pigs received intramuscular antibiotics (amoxicillin) and intramuscular pain medication (buprenorphine), which were continued for 3 days postoperatively. Using SimPlant 15 (Materialize, Leuven, Belgium), the data from the 2 CT scans were analyzed. To measure the BMD, we used a region of interest (ROI) of 5 to 10 mm 2 . The ROI tool for measuring bone density was placed on 5 different predetermined locations of the mandibular cortex on both sides of the mandible ( Fig 1 ). Cortical bone was deliberately chosen to eliminate measurement errors caused by dental or implant material. Because the cortical bone contains less collagenous tissue than bone marrow, its measurements of BMD have been considered to be more accurate. Figure 1 Location of the circular region of interest. A, Preirradiation right mandible, B, left mandible, C, postirradiation right mandible, and D, implanted left mandible (SimPlant 15, Materialise, Leuven, Belgium). Std. Dev., standard deviation. Two of us (L.P., G.B.) performed the measurements independently from one another. The calibrated bone phantom had a predetermined density given in g/mL. The BMDQ was calculated by dividing the measurement by the average BMD of the phantom ( Fig 2 ). The BMDQ in the present study was used to quantitatively compare the changes in the BMD. The BMD values represented the degree of mineralization of bone. The BDM is known to be relatively low in immature newly formed bone compared with the mature bone matrix. Figure 2 Photograph of the bone phantom used in this experiment. Statistical Analysis The data were incrementally entered during the study period into an electronic sheet (Excel, Microsoft Office 2010, Microsoft, Redmond, WA) and then processed using IBM SPSS Statistics, version 20 (IBM, Armonk, NY). First, to test the reproducibility of the measurements of the 2 independent investigators, we calculated the agreement between the measurements using the Bland-Altman procedure. The statistical significance of the changes in BMD in relation to the radiation dose was determined using the Kruskal-Wallis test. The statistical significance of the changes in bone density per location was determined using the Wilcoxon signed rank test. The Kruskal-Wallis test for independent samples was chosen to evaluate the relationship between the radiation dose and the BMDQ in different locations. We also compared the BMDQ of all the locations before and after RT using the Wilcoxon signed rank test. Because of the small sample and corresponding small power, we used only univariate analyses and nonparametric tests, because the data could not be assumed to be normally distributed, and significance was set at P ≤ .05. Results The BMDQ values of the nonirradiated and irradiated mandibles of the minipigs are listed in Table 1 . These values represent the difference in the BMD of the mandible compared with a calibrated bone phantom. The absolute values should not be extrapolated to those of other BMD data from human patients. Table 1 Bone Mineral Density Quotient Before and After Irradiation Irradiation Mandible Left (Surgical Intervention) Mandible Right (No Surgical Intervention) Before After δ (%) Before After δ (%) None Median 1.72 1.73 0.01 1.71 1.69 −0.02 Range 1.63-1.74 1.61-1.77 0.6 1.64-1.79 1.55-1.74 −1.2 25 Gy Median 1.64 1.63 −0.01 1.71 1.63 −0.08 Range 1.44-17 1.52-1.64 −0.6 1.63-1.78 1.52-1.74 −4.7 50 Gy Median 1.63 1.57 −0.06 1.73 1.64 −0.09 Range 1.62-1.78 1.48-1.63 −3.7 1.45-1.79 1.34-1.72 −5.2 70 Gy Median 1.67 1.56 −0.11 1.73 1.62 −0.11 Range 1.57-1.72 1.54-1.66 −6.6 1.63-1.79 1.40-1.64 −6.4 P = .134 and P = .599 for comparisons among the irradiation groups after irradiation for the left mandible and right mandible, respectively; P = .023 and P = .007 for comparisons between before and after irradiation for the left mandible and right mandible, respectively; differences were statistically significant at P < .05. Abbreviations: BMDQ, bone mineral density quotient (calculated using the average of a known bone phantom (measured Hounsfield unit value of the area divided by the known value of the phantom); δ, difference between the BMDQ value before and after irradiation. In our experiment, a significant decrease was found in the BMDQ after RT in the left (surgically altered) mandible, with a significant decrease in the right (nonsurgically altered) mandibular side. Between the left and right side of the mandible, no significant differences ( P = .023 for the left mandible, P = .007 for the right mandible, and P = .564 for the difference between the left and right mandibles) were found. In the nonirradiated mandible, no significant change was seen in the BMDQ between the first and second CT scan. Although no significant correlation was found between the radiation dose and the decrease in BMDQ on both sides (left side, P = .134; right side, P = .599), a progressive decrease in the BMDQ was visible on both sides. In Figure 3 , the Bland-Altman graph is shown and demonstrated the agreement of the measurements of the 2 independent investigators. Nearly all the measurements lay within the calculated standard deviation (SD ± 2 ranges; ie, the so-called limits of agreement). Thus, an adequate agreement was present between the measurements of the 2 investigators. Figure 3 Bland-Altman limits of agreement plot showing the agreement between the measurements of the 2 investigators. The standard deviation +2 and −2 was calculated, and the differences between both measurements were plotted. Two pigs developed clinical ORN: 1 in the 50-Gy and 1 in the 70-Gy group. Both pigs developed bone sequestration on the surgically altered mandibular side. Despite this clinical finding, the BMDQ of these individual pigs did not differ significantly from the remaining pigs in their groups. Discussion The purpose of the present study was to introduce a large animal model to investigate BMD changes in the mandibles of Göttingen Minipigs irradiated with various doses. Furthermore, our purpose was to investigate a possible correlation between the BMD changes and radiation dose, with and without surgical intervention. Our hypothesis was that we expected a small increase in the BMDQ after irradiation. The specific aim of the present study was to develop a tool to measure the quality of the irradiated bone. We succeeded in defining a large animal model to simulate the tissue reactions induced by various radiation doses in the mandible. At 6 months after irradiation, a significant decrease had occurred in the BMDQ compared with that in the nonirradiated animals, independent of surgical intervention. However, the BMDQ changes did not correlate with the individual radiation doses applied, making a translational correlation from the results of this experiment difficult. To the best of our knowledge, we report the first experimental animal study regarding the effects of various radiation doses on BMD. Our hypothesis stated that an increase in the BMD would occur because of the effects of irradiation on bone. However, in our experiment, an evident decrease was found, and we did not find a significant correlation between the radiation dose and the BMDQ. On both the surgically altered left side and the nonsurgically altered right side of the mandible, the bone had a significant decrease in the BMDQ. Because no surgical intervention had occurred in the right mandibular side, these results prove a general loss of bone density after irradiation of the mandible, regardless of any additional surgical intervention. In contrast to our findings, Verdonck et al found an insignificant increase in the BMDQ after irradiation. In their study of Göttingen Minipigs, all the molars and premolars were removed. The pigs were allowed to heal for 3 months before irradiation (Cobalt-60, 3 weekly fractions of 8 Gy). CT scans were performed 3 months after irradiation. We believe that in that study's duration, reparatory mechanisms could have caused the BMDQ to increase slightly. Their results can be interpreted as a mechanism seen, for instance, in fractures of the femoral head in which, in adjacent bone, the density will have increased 3 months after a fracture has been treated. After a certain period, the BMD will then return to normal. One must also accept that despite surgical intervention, animals never rest their mandibles. Reparation and wound healing always occur under physiologic load. This phenomenon could be another explanation for the findings of Verdonck et al. It is possible that the post-trauma remodeling was partially arrested by irradiation, causing the relatively mild increase in BMD. In accordance with our experimental results, Chen et al found no changes in BMD in a clinical setting. Several years after therapeutic pelvic irradiation, they compared the BMD in irradiated L5 vertebrae with the nonirradiated L2 vertebrae of the same individuals using dual-energy x-ray absorptiometry. Several explanations are possible for the mechanism of this seemingly paradoxical maintenance or even increase in BMD after irradiation. Some investigators believe that a greater attenuation occurs in the bone resorbing osteoclasts compared with the osteoblasts, causing a net increase of bone apposition, which is interpreted as hypermineralization. Other investigators have described an inflammatory response to irradiation, causing a sclerotic effect on bone density despite the pathophysiologic proof of hypocellularity induced by RT. In their experiment on irradiation and distraction osteogenesis, Fregene et al found a significant increase in low-mineralized, immature bone and a significant decrease in highly mineralized, mature bone in the irradiated bony callus. Overall, the densitometric values were unchanged. The irradiation induced a diminution in cell function, impairing optimal bone regeneration and resulting in growth of dens of unripe bone. Because of these mineralization pattern variations, it was difficult to detect impaired bone regeneration capacity using only BMD measurements using large bone volume test methods. The decrease in BMD found in our experiment compared with the insignificant increase found in the same animal species under similar experimental conditions used by Verdonck et al requires an explanation. We believe that the differences in BMD measurements could have resulted from measuring different locations in the mandible and, possibly, the presence of some low mineralized immature bone as a reaction to the irradiation. Additionally, the variable timing of tooth extractions and implant placement can help to explain this difference. Finally, the different irradiation pattern of applying cobalt-60 in 3 weekly fractions of 8 Gy might help to explain the difference. We believe that our irradiation schedule, based on a realistic high-dose photon irradiation protocol, resembled the clinical reality better than the scheme used by Verdonck et al. The clinical reaction to irradiation of the test animals was close to the acute and chronic tissue reactions observed in irradiated patients with head and neck cancer. The formation of radiation-induced bone sequestration in 2 pigs supports our hypothesis that the experimental setting presented can be used as a large animal model for the evaluation of RT-induced tissue changes. The definition of RT-related risks for the development of ORN is impossible from the data we have presented, because a direct correlation between the radiation doses applied and the changes in BMD could not be proved. The number of pigs per group was relatively small, but the decrease in the BMDQ in general was still significant. The radiation doses given were clinically relevant, because 2 pigs developed ORN at dose levels known to cause ORN. Evaluating the measurements, we found a wide dispersion of the points. We interpreted this finding as inevitable variation, because it was impossible for the 2 independent investigators to measure exactly in the same location on the CT scans. In addition, the size of the measured area varied from 5 to 10 mm. Despite these challenges, it is absolutely possible to perform reproducible measurements in this manner. The methods used in our experiment were reproducible and precise enough to detect significant changes in the BMDQ after irradiation. We did not observe scattering from the titanium implants. Because the gantry was oriented coronally instead of axially (such as it is for humans), the problem of scattering was greatly reduced ( Fig 1 ). The major drawback was that we could not prove significant BMDQ changes in relation to the radiation dose applied. A possible significant relation between the radiation dose and BMDQ might be achieved with larger groups. The changes in the BMD measured using the BMDQ mirrors the effect of the pathophysiologic reactions of the bone tissue to irradiation on the bone matrix mineral content. Because some studies have found an increase in BMD after irradiation and others have found a decrease in BMD, we are still not sure which part of these pathophysiologic reactions is responsible for BMD changes nor of the clinical consequences. In conclusion, we have succeeded in defining a large animal model to simulate the tissue reactions induced by various radiation doses in the mandible. At 6 months after irradiation, a significant decrease in the BMDQ compared with the nonirradiated pigs was found, independent of surgical intervention. However, the BMDQ changes did not correlate with the individual radiation doses applied, making a translational correlation from the results of this experiment difficult. Future research should aim to investigate the pathophysiologic reactions of bone tissue after irradiation and imaging or measuring these reactions of the bone tissue, for instance, with magnetic resonance imaging of the bone marrow. 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