A Preliminary Investigation into the Effects of X-Ray ... - Springer Link

Calcif Tissue Int (2009) 84:379–387 DOI 10.1007/s00223-009-9217-y

LABORATORY INVESTIGATIONS

A Preliminary Investigation into the Effects of X-Ray Radiation on Superficial Cranial Vascularization Sophie Desmons Æ Michal Heger Æ Caroline Delfosse Æ Guillaume Falgayrac Æ Thierry Sarrazin Æ Claire Delattre Æ Sylvain Catros Æ Serge Mordon Æ Guillaume Penel

Received: 11 October 2008 / Accepted: 6 January 2009 / Published online: 4 February 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Radiation therapy (RT) is an established treatment modality for malignant neoplasms. RT induces tissue damage that may lead to osteoradionecrosis in more severe cases. Suitable animal models to study RT-induced changes in membranous craniofacial bone are currently not available. The aim of this study was therefore to quantify RT-induced changes in cranial microcirculation using a newly developed calvaria chamber model and to relate these changes to RT-induced histological damage. New Zealand white rabbits received a total radiation dose of 18.75 Gy through the calvaria chamber, and the number of vessels, the vessel length density (VLD), and angiogenic

S. Desmons (&) C. Delfosse G. Falgayrac G. Penel EA 4032, School of Dentistry, Lille University Hospital, IFR 114, IMPRT, Place Verdun, 59000 Lille, France e-mail: [email protected] S. Desmons S. Mordon U703 INSERM, Lille University Hospital, Pavillon Vancostenobel, 59000 Lille, France M. Heger Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands T. Sarrazin Department of Radiotherapy, Centre Oscar Lambret, Lille University Hospital, 3 Rue Fre´de´ric Combemalle, BP307, 59020 Lille, France C. Delattre Department of Pathology, Lille University Hospital, 59000 Lille, France S. Catros U577 INSERM, Bordeaux University Hospital, 146 Rue Le´o-Saignat, 33076 Bordeaux Cedex, France

sprouting were quantified on a weekly basis during a 12week period. At the end of 12 weeks, the RT-treated (n = 5) or control (n = 5) calvarias were biopsied for histopathological analysis. RT resulted in a steep reduction in the number of vessels and the VLD during the first 3 weeks, particularly in larger-diameter vessels, followed by a flat stabilization/remodeling phase in the subsequent 9 weeks that never restored to baseline values. Histomorphometric analysis revealed a high degree of osteocytic depletion, prominent hypocellularity in the lacunae and intraosseous vasculature, enlarged and nonconcentric Haversian systems, and a severely disorganized bone matrix in the RT-treated calvarias. Despite the prevalence of some angiogenic potential, the RT-induced effects in the early phase persisted in the intermediate to late phase, which may have contributed to the poor recovery of the RT-treated bone. Keywords Radiation therapy Rabbit calvaria chamber model Vasculature Vessel length density Histomorphometry

Radiation therapy (RT) is an established treatment modality for malignant neoplasms in the head and neck region. Despite its relative efficacy in treating these malignancies, radiation therapy is often associated with adverse side effects in soft and hard tissues in the exposed area. One of the late effects of RT is osteoradionecrosis [1], which is a condition of metabolically compromised osseous (and surrounding) tissue as a result of RT-induced cumulative progressive endarteritis and hypocellularity [2–4]. It has been demonstrated that the bone healing process requires an intact pericranial vascular plexus to support osteoprogenitor cell function [5]. At the vascular

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S. Desmons et al.: Effects of X-Ray Radiation on Superficial Cranial Vascularization

level, RT induces early inflammatory processes that likely lead to thrombosis, cell death, and fibrosis. The latter culminate in marked hypovascularity, a reduced flow through the Haversian and Volkmann’s canals, and hypoxia [6]. Consequently, RT-induced vascular lesions can definitively disturb bone remodeling [4]. In the case of craniofacial defect reconstruction by grafting techniques, substantial recovery of bone vascularization is crucial [7]. Indeed, an optimal recovery should contain an extensive vascular bed for the rapid re-establishment of perfusion and a solid collagenous matrix to resist in the early phase of hypoperfusion [8]. Currently, relatively little is known about the recovery potential of membranous craniofacial bone following RT. The superficial microvasculature may constitute an ideal indicator for the overall state of RT-affected bone and reflects the degree to which the tissue has been able to recuperate following injury [9]. To study the effects of RT on the pericranial microcirculation in its native state, an in vivo rabbit calvaria chamber model was developed. The model was then used to quantify RT-induced changes in the number of vessels, vessel length density (VLD), and angiogenic sprouting in superficial cranial sites over a period of 12 weeks. Additionally, histomorphometric analysis was performed to assess the possible relationship between the RT-induced vascular dynamics and pathophysiology.

Materials and Methods Animal Preparation Animal experiments were approved by the Veterinary Department of the French Ministry of Agriculture. The animals were treated in accordance with the guidelines established by the Department of Experimental Research of the University of Lille and the Guide for the Care and Use of Laboratory Animals (NIH publication 93–23), and were given standard care throughout the study. Experiments were performed on 1-year-old New Zealand white female rabbits (CEGAV, St. Mars-d’Egrenne, France) with a mean ± SD weight of 3.5 ± 0.3 kg (n = 10). Animals were anesthetized by subcutaneous injection of 500 lg/kg medetomidine (Domitor; Orion, Espoo, Finland) and intramuscular injection of 25 mg/kg ketamine (Imalgen; Rhoˆne Me´rieux, Lyon, France). A bone chamber was implanted on the calvaria as described in [10]. A 3-cm incision was made in the skin and the pericranium at the top of the calvaria. Both layers were carefully retracted. The exposed bone was flattened by grinding down to a depth of*50 lm with a 5-mm calibrated cylindrical burr fitted in a low-speed drill, inasmuch as a flat geometry was

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imperative for optimal securement of the chamber. The titanium chamber was fastened to the calvaria with three osteosynthetic microscrews (Modus; Medartis, Basel, Switzerland). A sterile glass coverslip secured inferiorly in the chamber served to seal off the wound. Three titanium screwposts (Anthogyr, Sallanches, France) were implanted in the calvaria behind the bone chamber. The screwposts were embedded in dental polymethyl methacrylate (Tab 2000; Kerr, Salerno, Italy) in order to secure two nuts required for the locking of the digital camera. The fixed position of the chamber and screwposts ensured that the same region of interest (ROI) was being analyzed photographically. Soft tissue was closed with Vicryl 3-0 sutures (Johnson&Johnson, New Brunswick, NJ, USA). Postoperatively, antibiotics (enrofloxacin, 0.2 mL/kg/day; Bayer, Leverkusen, Germany) and analgesics (carprofen, 0.06 mL/ kg/day; Vericore, Dundee, Scotland) were subcutaneously administered for 5 days. In preliminary experiments we found that implantation of the chamber was associated with a postsurgical variability in superficial vascular density that persisted for 8– 10 weeks and that the stabilization period differed among the animals. Consequently, experimentation was initiated after a relatively persistent vascular density had been maintained for 3 weeks. A 12-week follow-up period was chosen to examine the possible long-term persistence of RT-induced changes as pointed out in [11]. Since the turnover of bone remodeling is approximately 6 weeks [12], a 12-week follow-up provided ample time to study the overall remodeling of the observed bone site. Five animals comprised the control group (implantation of the chamber without RT), and five animals received RT as described below. X-Ray Radiation Before RT the animals were anesthetized as described above. X-ray radiation was administered using a lowenergy X-ray generator (Darpac 2000; Gulmay Medical, Surrey, UK). The beam was passed through a collimator and a neutral density filter to deliver a power of 40 keV 8.6 mA within a spot size 1 cm in diameter through the open bone chamber onto the exposed calvaria. Five animals received a total radiation dose of 18.75 Gy at 2.49 Gy/min in a single pass to obtain*15 Gy at a depth of*2 mm [13]. The dose distribution in the rabbit calvaria conformed to the dose distribution as described in [14], a study that focused on RT-induced impairment of osseous healing in rat femurs. Determination of Vascular Plexus Localization A confocal Raman microspectrometer (Labram; HORIBA Jobin Yvon, Lille, France) was used as described in [10] to


determine the anatomical location of the vascular plexuses that were imaged pre- and post-RT. The animals (n = 3 per group) were sacrificed by intravenous injection of 0.3 mL/kg T-61 (Tanax; Sanofi Aventis, Bridgewater, NJ, USA) and the bone chamber and underlying calvaria were excised and immobilized to a metallographic microscope setup (BX40; Olympus, Tokyo). A helium-neon laser (kex = 632.8 nm) was used for excitation at an incident power of 8 mW, whereby the laser light was separated from the Raman signal by a notch filter. The long working distance of the 920 microscope objective produced a spot size*5 lm in diameter with an axial resolution of*25 lm. The pinhole was set to 100 lm. Raman spectra were acquired with a spectrograph equipped with an air-cooled CCD detector in the range of 200–1200 Dcm-1 at a spectral resolution of 2 Dcm-1. The integration time was 600s (10 accumulations of 60s) for each spectrum. Acquisition data were processed using LabSpec software (HORIBA Jobin Yvon). Background fluorescence was subtracted from baseline computation and polynomial filtering was performed. In addition to Raman microspectrometry, the chambercalvaria interface was examined histologically. Calvarial bone segments with the affixed bone chamber were excised, dehydrated in ethanol (70%, 80%, 90%, 100%) and 100% isopropanol, and embedded in a methyl-metacrylate butyl-metacrylate (4:1 ratio) solution in accordance with Wolf’s methodology [15]. Sections (80 lm thick) were cut using a microsaw (model 106; Leica, Wetzlar, Germany) and stained with the light Light Green SF of Masson’s Trichrome. Software-Assisted Analysis of Vascular Parameters Following a 3-week stabilization period, images of the vascular network were acquired every week on the same day for up to 12 weeks. Before image acquisition, the animals were placed in a restrainer without prior administration of anesthesia. Digital pictures were taken of the pericranial vascular plexus with a modified digital camera (Coolpix 5700; Nikon, Tokyo) equipped with a 50-mm lens (Nikon) and 10 diodes for uniform lighting. Aphelion software (ADCIS, He´rouville Saint-Clair, France) was used for quantification of the number of vessels (classified into four groups according to their diameter), vessel length density (VLD), and number of nodes. The RGB images of the ROI were converted to binary images in which vascular pixels and perivascular pixels were discriminable based on grayscale intensity. The number of branches (vessels) and nodes were obtained with a segmentation algorithm preprogrammed in the software. The VLD was defined as the cumulative length of the branches in the ROI divided by the ROI pixel area. The

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extent of angiogenic sprouting was extrapolated by calculating the number of branches per node in the ROI. The data sets were normalized to the mean values at t = 0 (before RT or baseline) for each measured parameter and are expressed as a percentage of the baseline. Histomorphometric Analysis of X-Ray-Treated Calvaria At the end of the 12-week monitoring the animals were sacrificed following the administration of anesthesia as previously described. The portion of the calvaria that had been irradiated through the bone chamber was biopsied for histomorphometric analysis. For control, nonirradiated adjacent calvarial tissue was biopsied and processed in the same manner as the irradiated specimens. The biopsies were fixed in 10% buffered formalin, pH = 7.4, and decalcified in 10% (v/v) nitric acid for 48 h. After complete decalcification, the specimens were embedded in paraffin and cut into 5-lm-thick sections parallel to the axis of radiation. Sections were routinely stained with Mayer’s hematoxylin and eosin (H&E) and visualized by light microscopy (model DMLB; Leica) and linearly polarized light microscopy (model 9901; Carl Zeiss, Jena, Germany). Both microscopes were equipped with a Leica DC200 CCD camera controlled by Qwin image acquisition software (Leica). Statistical Analysis Statistical analysis (means, standard deviations, and independent homoscedastic Student’s t-tests) were performed with the Statistical Package for Social Sciences 12.0 (SSPS, Chicago, IL, USA). A p-value of B0.05, designated by an asterisk in the figures, was considered statistically significant. A p-value B0.01 is indicated by a doubleasterisk.

Results The implantation of the calvaria chamber was associated with minimal morbidity and the bone chamber was generally well tolerated throughout the entire experiment. No animals were excluded from the study as a result of experiment-related concerns. Anatomical Location of the Superficial Vascular Plexus The bone implantation procedure included a grinding step to flatten the bone, which caused the removal of soft tissue such as pericranium and ensured direct contact between bone and glass surfaces (Fig. 1a). Noninvasive in situ

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Fig. 1 a Cross-sectional view of the bone chamber attached to the calvaria, prepared in accordance with Wolf’s methodology [15]. Note the direct contact between the glass coverslip and the bone (green). b Raman spectra of an inferiorly positioned vessel (top spectrum) and a superiorly positioned vessel (bottom spectrum). The green lines indicate the respective vessels from which the spectra were derived. c Vascular anatomy of X-ray irradiated calvaria as observed through the bone chamber. The sudden ‘terminations’ (arrows) or the vertical orientation (inset; encircled) of blood vessels are indicative of a continuation of blood vessels in the vertical orientation and thus intact perfusion of the superficial calvaria

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Raman microspectrometry was employed to derive an anatomical location of the observed blood vessels. Raman spectra were acquired of glass (control), bone sites visibly lacking superficial blood vessels, and blood vessels. The Raman spectrum of glass exhibited no peaks in the range of 600–1200 Dcm-1 (not shown). Positioning the focal plane within a superficial blood vessel of a superiorly situated vascular plexus (Fig. 1b; lower spectrum) resulted in main Raman peaks characteristic of red blood cells, i.e., bands at 1170, 1122, and 755 cm-1 [16, 17]. Concomitantly, peaks attributable to apatite (959 and 1073 cm-1) were observed [10, 18–20]. The band at 1073 cm-1 suggests a type B substitution (CO32– substitution of PO43–) in the crystal lattice [10, 19], as is always the case in biological apatites such as bone [21, 22]. These bands were also present in Raman spectra acquired form bone sites lacking superficial vasculature (not shown). The concurrence of blood and bone bands strongly suggests that the probed blood vessel resided in the bone rather than in the soft tissue layer at the cranium-glass interface. Raman spectra were subsequently acquired on a neighboring blood vessel of the inferiorly situated vascular plexus while keeping the focal plane unchanged (by xytranslation of the microscope stage only). The deeper-situated plexus could be captured by the camera as a result of the extensive focal depth (*0.8 mm), but the blood vessels appear hazier as a result of increased light scattering in the overlaying bone. The upper spectrum in Fig. 1b clearly shows that the red blood cell-specific bands either attenuated (755 cm-1) or dissipated (1170 and 1122 cm-1) and that bone-specific Raman bands became more pronounced (e.g., 959 cm-1). Those data indicate an inferior localization of the blood vessel and thus its position in the bone. Additionally, distinct features of the vascular plexus anatomy revealed that the imaged blood vessels either constitute or contribute to the perfusion of the Haversian and Volkmann systems. This was evinced by sudden ‘terminations’ of the vascular tubes (Fig. 1c; arrows), which suggest a continuation of the blood vessel in the vertical orientation. This phenomenon is illustrated in the inset in Fig. 1c. Consequently, the blood vessels that were imaged and analyzed represent functional, physiologically relevant structures. Radiation-Induced Changes in Superficial Calvaria Vasculature Calvarial segments were subjected to RT through the bone chamber aperture. The consequential changes in the number of vessels, VLD, and number of branches per node were quantified on a weekly basis.


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X-ray-treated calvarias exhibited a more profound reorganization of the superficial vasculature than controls, as evidenced by the vast structural differences in the vascular plexus at t = 0 versus t = 84 days (Fig. 2). RT-induced changes in the number of vessels were characterized by a rapid depletion phase in the first 21 days, followed by a stabilization/remodeling phase during the remainder of the experimental period (Fig. 3a). The mean reduction in the number of vessels at the deflection point (Fig. 3a; demarcated area) was 36%, which recovered to 81% of baseline by 84 days. Figure 3b shows that the mean VLD in the control

group significantly exceeded that in the RT group, indicating that the vessels in the latter were not only fewer in number but also shorter. The depletion phase manifested itself in a mean decrease of 53% in the fraction of large vessels ([50 lm) and a 32% reduction in capillary-level vasculature (Fig. 3c). The stabilization/remodeling phase (days 28– 84) was characterized by diametrical expansion of residual vasculature, as evidenced by the persistent leveling-off of capillaries and recovery of the larger-diameter vessels (Fig. 3c). When interpreted in the context of the vessel quantity and VLD data, it can be concluded that the post-RT

Fig. 2 Vascular dynamics portrayed as a function of time in days (d; upper-right corner) for control animals and RT-treated animals following an 11-13-week stabilization period. A marked RT-induced depletion of vasculature and lumenal narrowing can be observed. The

latter phenomenon is highlighted in the top-right (pre-RT) and bottom-left (9d post-RT) panels, where the lumen demarcated by the arrows is traced in time to accentuate the RT-induced decrease in vascular diameter

Fig. 3 Software-assisted quantification of a the number of vessels in the field of view, normalized to t = 0 days; b the vascular length density (VLD), normalized to t = 0 days; c the number of vessels categorized according to diameter, normalized to t = 0 days; and d the number of branches per node, which serves as an indicator for angiogenesis. Values are plotted as a function of time for the control (dotted lines) and the RT (RT at t = 0 days; solid lines) group following an 11- to 13-week stabilization period

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calvarial vasculature embodies significantly less blood volume compared to untreated animals for at least 12 weeks. To assess the extent of angiogenic remodeling, the number of branches per node were computed (Fig. 3d). The results justify the trends exhibited in the RT group with respect to the number of vessels and the VLD. Although there was a greater degree of angiogenic sprouting in RTtreated vascular plexuses relative to control, the extent to which this occurred was minimal and did not substantially outweigh capillary dropout rates (Fig. 2), resulting in minimal vascular recovery following RT. The gross vasculometric parameters in the control group exhibited no significant alterations during the 12-week period (Fig. 3a–d). Histomorphometric Analysis of X-Ray-Treated Calvaria At the end of the 12-week follow-up, the superior layer of the calvaria (directly adjacent to the bone chamber) had formed de novo as evidenced by the presence of viable osteocytes, the differential chromatism, and the corollary appearance of a ‘demarcation’ border in H&E-stained

Fig. 4 Histological sections of calvaria subjected to a single dose of 18.75 Gy administered over 7.5 min. a H&E-stained section illustrating empty lacunae (arrowheads) and intraosseous vasculature (black arrow). The white arrow points to a region of membranous bone that was likely formed de novo as evidenced by the healthy, nonaffected features of the tissue. H—Haversian system, which was often empty and acellular. b RT-treated calvaria imaged by linear polarized light microscopy. Note the lack of birefringence (arrows) around most Haversian systems, suggesting damage/reorientation of

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sections (Fig. 4a; white arrow), as well as the strong birefringence in polarized microscopy images (Fig. 4b; upward-pointing arrowhead). Twelve weeks after RT, several marked RT-induced histopathological features could be observed in deepersituated tissue. The foremost finding was that little evidence for bone regeneration was found, given the high degree of osteocytic depletion. The non-Haversian bone matrix was highly disorganized and exhibited hypocellularity with respect to the lacunae (Fig. 4a; arrowheads) and intraosseous vasculature (Fig. 4a; black arrow). The majority of Haversian canals was empty and acellular in the RT-treated specimens (Fig. 4a [‘‘H’’], c, and d). Moreover, numerous Haversian systems were enlarged and nonconcentric (Fig. 4a and c; red demarcations) and contained empty and expanded lacunae. Most RT-treated Haversian systems lacked an endothelial lining, and some Haversian canals contained semi- or entirely occlusive eosinophilic fibrin-like deposits with a basophilic perimeter (Fig. 4c; arrows). The lack of polarization (birefringence) in several Haversian systems (Fig. 4b; arrows vs. control [inset]) is indicative of a reorientation of or damage to matrix collagen and, in conjunction with

collagen. Birefringence was present in some regions of non-Haversian bone (arrowheads) and in control Haversian systems (inset). c H&Estained section that clearly depicts the non-concentricity of most Haversian systems (red; encircled). Some Haversian canals contained semi- or entirely occlusive eosinophilic fibrin-like deposits with a basophilic perimeter (arrows). d H&E-stained section showing a depletion of acidic matrix components (arrowheads), which are likely noncollagenous proteins, that may have occurred as a result of RT


the heterogeneity in birefringence patterns (Fig. 4b; arrows vs. downward-pointing arrowheads), suggests that RT imposed a profound effect on matrix collagen. These phenotypic aberrations were common in all the irradiated calvarias.

Discussion Our study aimed to evaluate the effect of X-ray irradiation on superficial membranous bone vascularization. The calvaria bone chamber model enabled the in vivo long-term observation of RT-induced changes in vascular dynamics and bone remodeling in flat membranous bone. Although the leveling-off of bone through the grinding step induced some local pathological effects, the subsequent 11- to 13week recovery period allowed the cranial vasculature to stabilize. Visual assessment of the digital photographs confirmed that the vascular plexuses in control animals were highly dynamic, exhibiting continuous vascular remodeling. Histological and Raman data, respectively, attested the viability of the tissue and the intraosseous localization of the vasculature. On the basis of these results the bone underlying the optical chamber could be considered a well-vascularized osteogenic organ [23] and, hence, is suitable for studying RT-induced damage in vivo. Damage caused by ionizing radiation essentially entails three critical effects: the production of free radicals irreversibly disrupts DNA replication that leads to mitotic cell death [24, 25] and results in (per)oxidation of fatty acids [26] and membrane lipids [27]. Radiation also causes stasis and occlusion of small blood vessels within a few hours after RT [28, 29], which may in part be ascribable to the thrombogenic nature of free radicals [30, 31]. As was expected, a single dose of 18.75 Gy administered over 7.5 min resulted in a substantial depletion of osteocytes, a highly disorganized bone matrix, and aberrant bone remodeling 12 weeks after RT. These results are in general conformity with the critical effects outlined above as well as with comparable studies on other types of bone [11, 32, 33]. Furthermore, vasculometric analysis evinced that the RT-induced pathophysiology is revealed at the level of the cranial microcirculation. During the early phase (first 3 weeks), RT was associated with a substantial reduction in the number of vessels and the VLD. The vasculaturerelated pathophysiological conditions induced in the first 3 weeks prevailed during the subsequent 9 weeks, which may have further thwarted the recovery of the affected tissue. In this respect, Takahashi et al. [11] found a strong positive correlation (Pearson’s r = 0.839, p \ 0.001) between the capillary density and the number of viable osteocytes following X-ray irradiation of rabbit knee joints

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with a single dose of 25, 50, or 100 Gy, with a follow-up period of 4, 12, 24, and 52 weeks, underscoring the importance of intact vasculature in the healing process. It is therefore possible that the partial devitalization of intraosseous vasculature caused and/or accentuated the histomorphometric aberrations found in this study and by others [11, 30, 34–37] during the intermediate to late phase (weeks 3–12). Perfusion defects [25] and hypoxia [24] have a debilitating impact on the recovery potential of cells and tissue as well as their survival due to the reduced supply of compounds required to meet metabolic needs. Moreover, obstructed vascular lumens hinder the infiltration by osteoprogenitor cells and endothelial progenitor cells and thereby deter the replenishment of the affected bone with viable cells that contribute to the remodeling process. At a cumulative dosage of 18.75 Gy, the vasculature-related pathophysiological conditions induced in the early phase prevailed during the intermediate to chronic phase, which may have further thwarted the recovery of the affected tissue beyond the 12-week time frame. How the bone recovers in the long term is currently unknown, but the possibility exists that the recovery process is very long or forestalled altogether. Takahashi et al. [11] have shown that 1 year after a single dose of 25 Gy no recovery was observed in either the number of viable osteocytes or the capillary density. The microcirculatory insufficiency that was the result of our irradiation protocol, despite the minimally prevailing angiogenic potential of RT-treated bone, may therefore have contributed to or caused the underlying histopathological effects. Based on these results and extensive literature, future therapeutic modalities should focus on minimizing the chronic effects of RT, since these seem to exert the most profound impact on post-RT bone remodeling. The most straightforward approach is to reduce the cumulative dosage, inasmuch as the observed effects are known to be dose-dependent [34, 37–39]. Additional experiments using fractionated X-ray radiation modalities are required to elucidate the effects of different singular or fractioned doses on superficial bone vasculature. Others have focused on alleviating the antiangiogenic response by the posttherapeutic administration of angiogenic cytokines such as basic fibroblast growth factor [25] or by protection of cells of osteoblastic origin from the toxicity of ionizing radiation, e.g., by administration of interleukin-12 [40]. Alternatively, a novel approach is laser preconditioning of the region to be subjected to RT, which seems to impart significant protective effects [41] without administration of (bio)pharmaceuticals. It also constitutes a promising application to diminish the incidence of (mandibular) osteoradionecrosis.

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In conclusion, RT leads to a profound reduction in superficial bone vascularization in the early phase that is accompanied by substantial histopathological effects in the underlying bone. Despite the prevalence of some angiogenic potential, the RT-induced effects in the early phase persisted in the intermediate to late phase, which may have contributed to the poor recovery of the RT-treated bone.

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18. Acknowledgments We are grateful to Prof. Vincent Everts (Academic Center for Dentistry Amsterdam, Vrije Universiteit) for assistance with histological analysis and Dr. Jan van Marle (Department of Cell Biology and Histology, Academic Medical Center) for assistance with the microscopic techniques.

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