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Histopathologic and Histomorphometric Analysis of Irradiation Injury in Bone and the Surrounding Soft Tissues of the Jaws


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Histopathologic and Histomorphometric Analysis of Irradiation Injury in Bone and

the Surrounding Soft Tissues of the Jaws

Marcos Martins Curi, DDS, MSc, PhD,*Camila Lopes Cardoso, DDS, MSc, PhD,y Heliton Gustavo de Lima, DDS, MSc,zLuiz Paulo Kowalski, MD, MSc, PhD,x

and Manoela Domingues Martins, DDS, MSc, PhDk

Purpose: Surgery of irradiated tissue has an increased complication rate because of the development of hypovascular, hypocellular, and hypoxic tissue. This study was undertaken to perform histopathologic and histomorphometric analyses of irradiation tissue injury in bone and the surrounding soft tissues.

Material and Methods: The histopathologic findings of 40 human mandibular bones and the surround- ing soft tissue specimens obtained from different patients who underwent surgical procedures for treat- ment of osteoradionecrosis of the jaws were reviewed.

Results: Histopathologic examination showed 7 processes in the following order of appearance: hyper- emia, endarteritis, thrombosis, cell loss, hypovascularity, increase of fat in the bone marrow cavity, and fibrosis. Histomorphometric analysis showed significant hypocellularity (P = .007), hypovascularity (P < .001), and fibrosis (P < .001) in irradiated specimens compared with control specimens.

Conclusion: These results showed that radiation injuries affect the bone and surrounding soft tissues.

However, the irradiation-induced injuries, such as cellular loss (hypocellularity) and fibrosis, were more expressive in bone tissue than in the surrounding soft tissues.

Ó 2016 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 74:190-199, 2016

Surgery and radiation have been the standard treat- ment for advanced cancers of the head and neck. Os- teoradionecrosis (ORN) of the jaws is one of the most severe late complications of radiation therapy.1-4It is defined as exposed irradiated bone tissue that fails to heal over a period of 3 months without signs of primary tumor, recurrence, or metastatic disease.5,6 Marx5found that ORN was characterized by progres- sive obliterative endarteritis and the development of hypovascular, hypocellular, and hypoxic tissues (‘‘3-H concept’’), in which there is an imbalance between cell death and collagen breakdown exceeding the

normal homeostasis of cell repair and collagen synthe- sis, which can undergo necrosis spontaneously or in response to trauma. Micro-organisms play only a contaminating role in ORN.7

A new theory was proposed by Delanian and Lefaix8 based on the radiation-induced fibro-atrophic process to explain the damage to normal tissues, including bone and soft tissues, constituting a local and unavoid- able sequela to high-dose radiotherapy. They described 3 distinct phases. An initial prefibrotic phase is characterized mainly by changes in endothe- lial cells. Cytokines, released in response to injury,

*Chair, Department of Stomatology, Hospital Santa Catarina, S~ao Paulo, Brazil; Professor, Department of Oral Surgery, University Sagrado Corac¸~ao, Bauru, Brazil.

yProfessor, Department of Oral Surgery, University Sagrado Corac¸~ao, Bauru, Brazil.

zPhD Student, Department of Oral Medicine, Area of Pathology, Bauru School of Dentistry, University of S~ao Paulo, Bauru, Brazil.

xChair, Department of Head and Neck Surgery and Otorhinolaryngology, Cancer Hospital A.C. Camargo, S~ao Paulo, Brazil.

kProfessor, Department of Oral Pathology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil.

Conflict of Interest Disclosures: None of the authors reported any disclosures.

Address correspondence and reprint requests to Dr de Lima:

Department of Oral Medicine, Bauru School of Dentistry, University of S~ao Paulo, Al Dr Otavio Pinheiro Brisolla 9-75, CEP 17.012-901, Bauru, SP, Brazil; e-mail:heliton.lima@usp.br

Received October 7 2014 Accepted July 10 2015

Ó 2016 American Association of Oral and Maxillofacial Surgeons 0278-2391/15/01008-3




attract leukocytes to the site of injury that trigger an acute inflammatory response characterized by increased vascular permeability with local edema for- mation, destruction of endothelial cells, and an associ- ation with vascular thrombosis. This can lead to necrosis of microvessels, local ischemia, and tissue loss. Currently, it is not fully understood how fibro- blasts are activated to become myofibroblasts. The sec- ond constitutive organized phase is characterized by the radiation-induced fibro-atrophic tissue composed of fibroblasts and the extracellular matrix. The reactive oxygen species–mediated release of cytokines results in a high density of active fibroblasts (myofibroblasts) characterized by high rates of proliferation and secre- tion of abnormal products in a disorganized extracel- lular matrix with a decreased capacity to degrade these components. The third phase, late fibroatrophy, can last for decades after radiotherapy and consists of poorly vascularized and cellular tissue with few fibro- blasts and a dense extracellular matrix. An attempted tissue remodeling occurs with the formation of fragile healed tissues with a high inherent risk of reactivated inflammation in the event of local injury.8

On histopathologic evaluation, hyperemia, inflam- mation (endarteritis), thrombosis, cellular damage, hy- povascularity, and fibrosis are observed after radiation exposure.9In general, hyperemia, endarteritis, cellular damage, and vascular thrombosis begin soon after radi- ation exposure and is maintained for an additional 6 months. Hypo-vascularization and fibrosis occur 6 to 12 months after radiotherapy and represent the end stage of radiation tissue injury.9

Although specific treatments for ORN have not been well defined, surgical approaches are necessary in most cases, from conservative intervention to invasive surgery.1,10 ORN requires treatment when there is pain, impaired function, or active infection. There are limited options for treatment of refractory and persistent cases of ORN other than complete bone surgical resection or multimodal therapy in which hyperbaric oxygen therapy (HBO) is combined with surgical resection of necrotic bone. However, the exact physiologic mechanisms of HBO are not completely understood. No consensus exists regarding its prophylactic use, its mechanism of action, and its effectiveness in the prevention and treatment of ORN.11-22 There is some evidence that HBO improves outcome to prevent the development of ORN after dental extraction in patients irradiated for head and neck tumors.23There also is some evi- dence that HBO might improve outcome in irradiated patients who need resection and reconstruction sur- gery.24 Two recent randomized controlled trials concluded that HBO was of little or no benefit for the treatment of ORN, especially in patients who un- derwent free tissue transfer.25,26 A recent Cochrane

meta-analysis suggested that for patients with late radi- ation tissue injury, HBO therapy is associated with an improved outcome.19 In summary, the efficacy of HBO in the management of irradiated patients has been contradictory and there is a lack of randomized controlled double-blinded trials and further studies are needed to evaluate the indication of this ther- apy.15-22 Based on the new theory of radiation- induced fibro-atrophic tissue in which the main event in the progression of ORN is the activation and dysre- gulation of fibroblastic activity that leads to atrophic tissue within a previously irradiated tissue, some clin- ical studies have suggested the use of pentoxifylline combined with tocopherol (vitamin E) to prevent and treat ORN.8,27

Despite the better understanding of the pathogen- esis of ORN and advances in its treatment modalities, some important clinical dilemmas remain. Marx and Johnson9 mentioned the uncertainty about the risk of ORN as a function of time since radiation and as a result of oral and maxillofacial surgical procedures.

The data from their tissue biopsy study showed that af- ter 6 months, the greater extent radiation tissue in- juries indicated a higher risk for ORN. They found evidence of progressive vascular damage causing en- darteritis, thrombosis, and fibrosis in animal and hu- man histologic studies.9 In addition, they believed that vascular damage is responsible for many of the late effects observed in ORN tissues.9However, this tissue biopsy study was based on qualitative histology.

Few studies thus far have been undertaken to eluci- date the importance of the relation between radiation tissue injury and the risk for ORN and to create clinical guidelines for irradiated patients. Studies in this field could improve the management of patients with head and neck cancer before and after irradiation.

The purpose of this study was to perform histopath- ologic and histomorphometric analyses of the effects of irradiation injury on mandibular bone and the sur- rounding soft tissues.

Material and Methods

This study was approved by the Cancer Hospital Center institutional review board and all participants signed an informed consent agreement. The study sub- jects consisted of 40 irradiated specimens obtained from different patients who were treated for ORN.

Fifteen nonirradiated mandibular bone and surround- ing soft tissue samples obtained from different patients who were treated for head and neck tumors were used as control specimens. Irradiated and control (nonirra- diated) specimens were similar for age, gender, and race, and there were no major differences between them. The diagnostic criteria for ORN was a slow- healing radiation-induced necrosis of bone with


associated soft tissue necrosis for at least 3 months with the absence of local primary tumor necrosis, recurrence, or metastatic disease.

Radiation therapy consisted of external beam radia- tion in all irradiated specimens. The specimens received megavoltage delivery (linear accelerator, 4 MeV). The average total dose of radiation for these specimens was 5,942 cGy (range, 5,040 to 7,040 cGy). The average dose rate per day for these specimens was 174 cGy (range, 109 to 200 cGy).

The average time of specimen evaluation after comple- tion of radiation treatment was 22.1 months (range, 2 to 108 months).

Individual cases were excluded from the study if they had a history of local or systemic disorders related to arterial changes, such as diabetes mellitus, rheu- matic fever, arterial vasculitis, or collagen diseases.

Furthermore, patients who had local diseases of the mandible, such as tumors, tumor-like lesions, and cysts, were excluded by histopathologic examination.

Patient records and clinical follow-up provided the following information: age, gender, race, initial tumor diagnosis and staging, treatment sequence, total radia- tion exposure, period of radiation therapy, and date of specimen evaluations.


All bone and soft tissues specimens had been fixed in formalin and embedded in paraffin. Bone specimens were decalcified in a solution of 10% buffered formalin with 10% ethylenediaminetetra-acetic acid. Five- micrometer-thick sections were obtained from the original paraffin blocks and then stained with hema- toxylin and eosin for light microscopic histopatholog- ic and histomorphometric observations. The histopathologic and histomorphometric examinations were performed in a blinded manner by 2 pathologists.

The amount of hyperemia, endarteritis (inflamma- tion), thrombosis, and fat in the bone marrow cavity were categorized as abundant, moderate, rare, or none for each specimen. Serial sections were used for immunohistochemical evaluation.


Histomorphometric measurements were performed with a computer-supported histometric system con- sisting of a microcomputer and software program (IMAGELAB, S~ao Paulo, Brazil) for processing the microscopic appearance. Measurements were per- formed using an objective of10 and a zoom of 2.5.

Histomorphometry of cellularity and fibrosis was measured and calculated for 3 areas with greater vascular density. The mean percentages for the mentioned parameters were calculated for each area

in the irradiated specimens and compared with the mean value for the control specimens. Areas of inflam- mation and necrosis were excluded.

IMMUNOHISTOCHEMISTRY PROTOCOL FOR CD34 Endothelial vascular immunostaining was per- formed using the streptavidin, biotin, and immunoper- oxidase (DAKO, Hamburg, Germany) method.

Deparaffinized and dehydrated 5-mm-thick sections were incubated with 3% H2O2 in distilled water for 15 minutes to block endogenous peroxide activity. An- tigen retrieval was performed with a pressure-cooking method in sodium citrate buffer 0.1 mol/L (pH 6.0).28 After pretreatment, sections were blocked with 10%

normal goat serum and Tris-buffered saline (TBS) for 15 minutes and then incubated with monoclonal anti- body QBEnd10 to CD34 (DAKO) at a 1:50 dilution for 1 hour at room temperature. The secondary bio- tinylated antibody and the streptavidin-and- peroxidase conjugate were applied according to the manufacturer’s instructions (Super Sensitive System, BioGenex, San Ramon, CA).28A chromogenic precipi- tate was obtained by immersing the sections for 10 mi- nutes in a ready-to-use AEC substrate solution (DAKO).

After counterstaining with Mayer hematoxylin, the sections were cover-slipped with glycerol gelatin. All incubations were performed at room temperature and all washings between incubations were per- formed in TBS.


Vessels were counted according to a standard in which 3 areas with dense vascularity were identified and selected in each tissue section under low-power magnification (100). In each of these ‘‘hot spots,’’ mi- crovessels (capillaries and small venules) were counted at 400 magnification. Any brown-staining endothelial cell or endothelial cell cluster that was clearly separate from adjacent microvessels and other connective tissue elements was considered a single countable microvessel.29Vessel lumens, although usu- ally present, were not necessary for a structure to be defined as a microvessel, and red blood cells were not used to define a vessel lumen.30Areas of inflamma- tion and necrosis were excluded. The ratio of vascular area per bone or soft tissue area was estimated by point vessel counting using an integrating 25-point eyepiece (integration plate, 10; Carl Zeiss GmbH, Jena, Germany) on a400 field (40 lens and 10 eyepiece, 0.616 mm2 per field area). Vessels coinci- dent with the grid intersection points were counted.

Three fields of higher vascular density previously iden- tified in a100 magnification image were analyzed in each section, making up a total of 100 points. The


percentage of vascular area was calculated as: ([num- ber of coincidences/total of counted points] 100).


All statistical analyses were performed with SPSS 10.0 for Windows (SPSS, Inc, Chicago, IL). Ninety- five percent confidence intervals were calculated for specimen groups. Statistical differences between mean percentages of cellularity, fibrosis, and vascular density were determined by the Student t test for un- paired observations. The Pearson correlation test was used to evaluate linear correlation between the mentioned parameters and time from radiotherapy in irradiated specimens with and without ORN. In all tests,a errors equal to or less than to 5% were consid- ered significant.


The histopathologic examination showed 7 pro- cesses: hyperemia, endarteritis, thrombosis, hypocel- lularity, loss of vascular content (hypovascularity), increase of fat in the bone marrow cavity, and fibrosis.

In general, hyperemia and endarteritis were early ef- fects of radiation and observed for up to 6 months after radiotherapy (Fig 1A, B). Thrombosis was seen only years after radiation and thrombi were densely fibrous (Fig 1C). Cell loss occurred rapidly after radiation and remained progressive through the years. Loss of vascular content, increase of fat in the bone marrow cavity, and fibrosis showed a linear relation with time after radiation and were considered the end stage of radiation tissue injury (Fig 1D-F).

There were significant differences between the mean cellularity values of irradiated specimens and control specimens (P = .007). The mean cellularity of irradiated specimen was 1.9% (range, 0.3 to 5.2%).

The mean cellularity of control specimens was 7.8%

(range, 1.7 to 30.5%;Fig 2). Cell loss was greater in bone than in soft tissues. When only bone tissue was evaluated, there was a significant decrease in mean cellularity in the irradiated specimens (1.6%) compared with the control specimens (8.1%;

P = .003). When evaluating only the soft tissues, there was a decrease in cell loss in irradiated specimens (2.4%) compared with control soft tissues specimens (7.6%), but this was not statistically significant (P = .168).

There were significant differences between the mean fibrosis values of irradiated specimens and con- trol specimens (P = .027). The mean fibrosis of irradi- ated specimens was 72.7% (range, 57.4 to 83.7%). The mean fibrosis of control specimens was 65.7% (range, 42.0 to 88.0%; Fig 3). Similarly, fibrosis was more evident in bone than in the soft tissues. When evalu- ating only the bone tissue, there was a significant in-

crease in fibrosis in the irradiated specimens (74.9%) compared with the control specimens (62.4%;

P = .007). No significant difference in soft tissues was found between irradiated and control specimens (P = .796).

For time-dependent irradiation tissue injury, irradi- ated specimens observed at 6 months after radio- therapy exhibited significant cell loss compared with irradiated specimens in the first 6 months after irradi- ation (P = .049). Similarly, examination of irradiated specimens at 6 months after irradiation showed a sig- nificant decrease in mean vascular density compared with irradiated specimens in the first 6 months after irradiation (P < .001). There was a trend toward statis- tical significance of an increase in fibrosis in irradiated specimens at 6 months after irradiation compared with irradiated specimens in the first 6 months after irradiation (P = .072).

Mean vascular density values of irradiated speci- mens and control specimens showed significant differ- ences (P < .001). The mean vascular density of irradiated specimen was 3.0% (range, 1.7 to 4.7%).

The mean vascular density of control specimens was 15.1% (range, 10.3 to 24.0%;Fig 4). Six months after radiation, all specimens showed considerable hypo- vascularity. However, no meaningful differences in mean vascular density values were found between those in irradiated bone and those in surrounding soft tissues. A sample of CD34 immunohistochemical staining of irradiated specimen is shown inFigure 5.


There is still uncertainty about the pathophysiology of irradiated tissue injury and the exact nature of this process is not fully understood. The basic pathophys- iology of irradiation tissue injury is normal tissue cell death and sublethal normal tissue cell damage leading to a wound healing defect.31

In the literature, there is a scarcity of clinical studies on the evaluation of radiation tissue injury in humans.

In the present study, hyperemia and endarteritis began early in the radiation sequence and persisted for up to 6 months after radiotherapy. Thrombosis appeared to be a late effect in the radiation sequence, and the thrombi became fibrous in later years compared with the early thrombi, which were basically blood clots. Hypovascularity and fibrosis began at approxi- mately 6 months after radiation and progressively worsened over time. Similar histologic findings have been reported in other bone tissue locations.9,32,33 In addition to these histopathologic processes, the present histologic examination identified an increase of fat in the bone marrow cavity in irradiated bone specimens. This phenomenon could be related to retardation of the normal process of bone turnover,


because the mandible would be considered skeletally mature in this period. Although there is a tendency to recover from this event, bone formation appears to take a longer time to do so than does bone resorption. This large fatty bone marrow cavity would be considered an end stage of radiation tissue injury. Similar findings have been reported in studies of radiation in skeletally mature animals.34

Marx and Johnson9proposed the concept of hypo- vascular, hypocellular, and hypoxic tissue formation

as the major problem related to irradiated wounds.

The present results of measurements of irradiation tis- sue effects confirm the findings of these previous studies. In the present study, the measurement of cell loss (hypocellularity) in irradiated specimens indi- cated a significant decrease compared with the control specimens (P = .007). It seems that cell loss begins to a small degree just after radiation therapy and then pro- gressively worsens throughout the years.9 This phe- nomenon was observed in the present study, which

FIGURE 1. Photomicrographs of irradiated specimens stained with hematoxylin and eosin showing radiation tissue injuries.A, Early histologic effects of radiation exhibiting hyperemia and endarteritis (magnification,200). B, Early histologic effects of radiation exhibiting hyperemia, endarteritis, and cellular thrombosis formation. Note inflammatory cells inside the vessel lumens (magnification,200). (Fig 1 continued on next page.)

Curi et al. Analysis of Irradiation Injury of the Jaws. J Oral Maxillofac Surg 2016.


showed an increase in cell loss in irradiated specimens at 6 months after radiation (1.8%) compared with irra- diated specimens in the first 6 months after radiation (2.4%), and this was statistically significant (P = .049). There has been a debate about whether the effect of irradiation is mainly on the bone or on the soft tissues. Regarding cell loss, bone specimens showed greater hypocellularity than soft tissue speci- mens. When considering the radiosensitivity of bone tissue, the particular physical condition with respect to the absorption of ionizing radiation must be consid-

ered. Because of its high calcium content, bone can absorb 30 to 40% more radiation than the surrounding soft tissues, and this factor could account for an in- crease in secondary radiation produced in bone under certain conditions.9 Thus, the actual absorption of a given dose of irradiation is considerably greater in bone than in the overlying mucous membrane.31 Therefore, bone cells would receive primary radiation and the maximum effect of the secondary rays.31 Another point related to this fact is the importance of recognizing the differences in the quality of

FIGURE 1 (cont’d). C, Early histologic effects of radiation exhibiting hyperemia, endarteritis, and cellular thrombosis formation. Note inflam- matory cells inside the vessel lumens (magnification,200). D, Late histologic effect of radiation. Note the large and fatty bone marrow cavity (magnification,200). (Fig 1 continued on next page.)

Curi et al. Analysis of Irradiation Injury of the Jaws. J Oral Maxillofac Surg 2016.


radiation (kilovolt vs megavolt irradiation), because of the difference in absorption of radiation energy be- tween bone and soft tissues. When using high- energy photons, the absorbed dose is approximately the same in bone and soft tissues.27

Hypocellularity is frequently present in irradiated tissue and is often associated with a disproportionate accumulation of collagen.9,35 Marx and Tursun35 compared the histopathologic features of suppurative osteomyelitis of the jaws, bisphosphonate-induced os- teonecrosis of the jaws, and ORN of the jaws, and all 3 conditions evidenced the common finding of necrotic bone with empty osteocytic lacunae and Haversian

and Volkmann canals, but each showed a distinctive histopathologic pattern indicating a different disease mechanism. ORN exhibited considerable marrow fibrosis, a shortage of cells, and the ghosts of old blood vessels. A recent theory for the pathogenesis of ORN has suggested that damage to bone is caused by radiation-induced fibrosis.8 Thus, when ORN occurs in the jaw bone, there is a decrease in the bone matrix and its replacement with fibrous tissue.8,36 The present results of the measurement of fibrosis in irradiated specimens indicated a significant increase compared with the control specimens (P = .027).

Similarly, fibrosis also has been noted to appear at

FIGURE 1 (cont’d). E, F, Late histologic effects of radiation. Note the characteristic hypovascular and hypocellular fibroses in the bone spec- imens (magnification,40 and 100, respectively).

Curi et al. Analysis of Irradiation Injury of the Jaws. J Oral Maxillofac Surg 2016.


approximately 6 months and then progressively worsen, being an end stage of radiation tissue injury.9In the present study, fibrosis showed a great tendency to statistical significance in irradiated speci- mens at 6 months after radiation therapy (67.3%) compared with irradiated specimens in the first 6 months after radiotherapy (60.1%; P = .072). Fibrosis also was more evident in bone than in the surrounding soft tissues. As described earlier, bone might receive more radiation because of secondary rays, and recov-

ery from this process appears to take a much longer time than for soft tissues.

The CD34 antigen is a sensitive marker of vascular endothelium and angiogenesis. The examination of CD34 has been reported in several physiologic and pathologic events.37-39 The present study found radiation-induced hypovascularity using antihuman CD34 monoclonal antibody. These results for the mea- surement of vascular density in irradiated specimens indicated a significantly decreased vascular density

FIGURE 2. Mean cellularity values of irradiated specimens and control specimens.

Curi et al. Analysis of Irradiation Injury of the Jaws. J Oral Maxillofac Surg 2016.

FIGURE 3. Mean fibrosis values of irradiated specimens and control specimens.

Curi et al. Analysis of Irradiation Injury of the Jaws. J Oral Maxillofac Surg 2016.


compared with the control specimens (P < .001). A previous study has shown the inverse index of vascular density in the irradiated tissue and a direct risk for ORN.9The same study showed a linear relation with time indicating loss of vascular component and, therefore, loss of oxygen tension perfusion with time. In the present study, there was no evidence of spontaneous revascularization with time.

The present findings support previous findings that irradiation affects bone and the surrounding soft tis- sues. Although there has been a debate about whether the effect of irradiation is mainly on the bone or on the soft tissues, the present findings showed that irradiation-induced injuries, such as cellular loss (hy-

pocellularity) and fibrosis, were more evident in bone than in the surrounding soft tissues. There also has been a discussion about whether the effect of irra- diation is mainly on the cells or on the fine vasculature in irradiated tissue. The present results showed that the 2 mechanisms are evident and play an important role in the pathologic process of irradiation tissue injury.


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In this way, economic yield and surplus were explained in religious terms and religious institu- tions were able to play an important role in the economy and every-

Giant cells are also seen in other diseases like giant cell granuloma of the jaws, traumatic bone cyst, aneurysmal bone cyst, and jaw tumor of

DWI findings of thyroglossal duct cysts were not similar to those of ranulas, as 2 of the 3 lesions had heterogeneous high signal intensity (see Figure 3 and Table

In this respect, the aim of the present study was to as- sess volumetric as well as morphological surface changes of the orbital cavity in patients treated with both tooth- borne

Animal or vegetable fats and oils and their fractiors, boiled, oxidised, dehydrated, sulphurised, blown, polymerised by heat in vacuum or in inert gas or otherwise chemically

Milk and cream, in powder, granule or other solid form, of a fat content, by weight, exceeding 1.5%, not containing added sugar or other sweetening matter.

Although Taiwan stipulates explicit regulations governing the requirements for organic production process, certification management, and the penalties for organic agricultural

Consistent with the negative price of systematic volatility risk found by the option pricing studies, we see lower average raw returns, CAPM alphas, and FF-3 alphas with higher