lable at ScienceDirect

Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 214e219

Contents lists avai

Journal of Cranio-Maxillo-Facial Surgery

journal homepage: www.jcmfs.com

Hyperbaric oxygen therapy as a prevention modality for radiationdamage in the mandibles of mice

Linda Spiegelberg*, Joanna A.M. Braks, Lisanne C. Groeneveldt, Urville M. Djasim,Karel G.H. van der Wal, Eppo B. WolviusDepartment of Oral and Maxillofacial Surgery (Head: Eppo B. Wolvius, MD, DMD, PhD), Erasmus University Medical Center, 3000 CA,Rotterdam, The Netherlands

a r t i c l e i n f o

Article history:Paper received 22 April 2014Accepted 12 November 2014Available online 18 November 2014

Keywords:Hyperbaric oxygen therapyRadiation therapyBoneMandibleRadiation-Induced DamageMicrocomputed tomography

* Corresponding author. Department of Oral & MUniversity Medical Center, Room Ee 15.93, PO Box 20Netherlands. Tel.: þ31 (0) 10 7043788; fax: þ31 (0) 1

E-mail address: l.spiegelberg@erasmusmc.nl (L. Sp

http://dx.doi.org/10.1016/j.jcms.2014.11.0081010-5182/© 2014 European Association for Cranio-M

a b s t r a c t

Background: Radiation therapy (RT) as part head and neck cancer treatment often leads to irradiation ofsurrounding normal tissue, such as mandibular bone. A reduced reparative capacity of the bone can leadto osteoradionecrosis (ORN). Hyperbaric oxygen therapy (HBOT) is used to treat ORN, based on its po-tential to raise the oxygen tension in tissues. However, prevention of radiation-induced damage is ofgreat interest. Our purpose was to investigate whether HBOT could prevent radiation-induced damage tomurine mandibles.Methods: Twenty-eight mice were irradiated in the head and neck region with a single dose (15 Gy) andhalf of them were subsequently subjected to HBOT. Another 14 mice did not receive any treatment andserved as controls. Ten and 24 weeks after RT, mandibles were harvested and analysed histologically andby microcomputed tomography (micro-CT).Results: Micro-CT analysis showed a reduction in relative bone volume by RT, which was partly recov-ered by HBOT. Trabecular thickness and separation were also positively influenced by HBOT. Morpho-logically, HBOT suppressed the osteoclast number, indicating decreased resorption, and decreased theamount of lacunae devoid of osteocytes, indicating increased bone viability.Conclusions: HBOT was able to partly reduce radiation-induced effects on microarchitectural parameters,resorption, and bone viability in mouse mandibles. HBOT could therefore potentially play a role in theprevention of radiation-induced damage to human mandibular bone.

© 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rightsreserved.

1. Introduction

Radiation therapy (RT) is a standard component in the protocoltreatment of head and neck cancer. Inevitably, normal surroundingtissues, including maxillofacial bones such as the mandible, willalso be exposed to RT. Radiation damages small arteries, reducingthe circulation to the already relatively poor vascularizedmandible.This causes an impaired remodelling capacity of the bone and canlead to a reduction in bone mass and bone density, and thus tomore vulnerable bone (Pacheco and Stock, 2013). The impairedreparative capacity of the bone especially poses a risk when trauma,such as subsequent surgery, biopsy, or tooth extraction andconsecutive implant placement, is inflicted on the previously

axillofacial Surgery, Erasmus40, 3000 CA Rotterdam, The0 7044685.iegelberg).

axillo-Facial Surgery. Published by

irradiated bone. Due to the vascular damage, a hypoxic and hypo-vascular environment exists in which bone is more prone toinflammation, which can eventually lead to destruction of bone, socalled osteoradionecrosis (ORN) (Marx, 1983). ORN of the mandibleis a serious long-term side effect in patients who receive radiationas part of the treatment for head and neck cancer. It is a very painfulcondition that can present itself even years after RT and is difficultto treat. Treatment regimens depend on the grade of ORN; lowergrade can be treated effectively by long-term oral antibiotic ther-apy, whereas for more severe cases, removal of the affected bonemight be necessary. Hyperbaric oxygen therapy (HBOT) is used asan adjuvant therapy to treat ORN, or is used in a preventive mannerwhen minor or major surgery is performed in irradiated bone. Therationale is that HBOT, in which patients breathe 100% oxygenunder elevated pressure, raises the oxygen tension and can therebypositively affect the healing process. Although clinical studiesreport positive effects, a general consensus about the effectiveness

Elsevier Ltd. All rights reserved.

L. Spiegelberg et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 214e219 215

of the therapy remains to be achieved (Spiegelberg et al., 2010;Lubek et al., 2013). Experimental studies on the effects of HBOTon the treatment or prevention of ORN are especially scarce,although these kinds of studies would provide a better under-standing of the effects and working mechanism of HBOT. Further-more, the potential of HBOT to protect bone from radiation damagebefore complications have arisen has not been investigatedthoroughly.

In this study, we investigated the effects of HBOT, when givendirectly after RT, on irradiated mandibular bone of mice, by meansof microecomputed tomography (micro-CT) and histology. Wewere particularly interested in the effect on bonemicroarchitectureand viability in nontraumatised bone, to assess whether HBOT isable to prevent radiation-induced bone damage.

2. Methods

2.1. Animals

Female C3H mice (Harlan Netherlands BV, Horst, theNetherlands), 7e9 weeks old at the start of experimentation, werekept under standard housing conditions with free access to foodpellets and acidified water. The mice were divided into threegroups: control, radiation therapy (RT), and radiation therapy fol-lowed by hyperbaric oxygen therapy (RT þ HBOT). At 10 and 24weeks after RT, 7mice of each groupwere sacrificed, andmandibleswere harvested for ex vivo micro-CT scanning, after which theywere used for histology. Animals were weighed frequently andgiven soft crushed food pellets to allow sufficient food intake afterRT. The experimental protocol was approved by the Animal CareCommittee of the Erasmus MC, Rotterdam, the Netherlands, underthe National Experiments on Animals Act and adhered to the ruleslaid down in this national law that serves the implementation of“Guidelines on the Protection of Experimental Animals” by theCouncil of Europe (1986), Directive 86/609/EC.

2.2. Radiation- and hyperbaric oxygen therapy

Radiation therapy and hyperbaric oxygen therapy were given aspreviously described (Spiegelberg et al., 2014). In short, RT con-sisted of a single 15-Gy dose administered to the head and neckregion of anesthetized mice.

Mice in the RTþHBOTgroup received the first HBOT session theday after RT. In an HBOT session, mice breathed 100% oxygen at2.4 atm absolute during 1 h in a hyperbaric chamber suitable forsmall laboratory animals (Djasim et al., 2012). Twenty consecutivesessions were carried out daily, except at Saturdays and Sundays.

2.3. Micro-CT scanning

Immediately after mice were sacrificed by CO2 asphyxiation, at10 and 24 weeks after RT, mandibles were harvested and fixated in10% buffered formalin. micro-CT was used to analyse bone pa-rameters. micro-CT scans of the tissue blocks were made with aSkyScan 1076 in vivo microCT scanner (SkyScan, Aartselaar,Belgium) and the manufacturer's scanning software. Examinationconsisted of a scout view, selection of region of interest, off-linereconstruction, and evaluation. Serial transverse scan imageswere made at a resolution of 18 mm. Nrecon 1.3 (SkyScan, Aartse-laar, Belgium) and CT Analyser 1.3.2.2 (SkyScan, Aartselaar,Belgium) software were used to reconstruct the data for analysis. A3-dimensional volume of interest was created by applying inter-polation between 2-dimensional free-hand selections of themandibular bones. Within the volume of interest, the relative bonevolume (BV/TV) was determined to quantify new bone formation,

as well as trabecular number (TbN), trabecular thickness (TbTh),and trabecular separation (TbSp).

2.4. Histology

After micro-CT scanning, mandibles were decalcified in 10%ethylenediaminetetraacetic acid (EDTA) for 12 days, after whichthey were dehydrated and embedded in paraffin blocks. Slides(5 mm) were sagitally cut and standard haematoxylin eosin (HE)staining was performed. Per slide, the amount of empty lacunaeand osteocytes were counted in 2e3 fields (�20 magnification).Empty lacunae were expressed as a percentage of the total count ofosteocytes. Adipocyte density in bone marrow (number and area ofadipocytes per square millimeter (mm2) of bone marrow) wasquantified using ImageJ version 1.45b (National Institutes of Health,Bethesda, MD, USA). Tartrate-resistant acid phosphatase (TRAP)staining was used to stain osteoclasts. Slides were first incubatedfor 20 min in 0.2 M acetate buffer with 50 mM L(þ) tartaric acid(ICN Biomedicals Inc, Aurora, IL, USA). Then, 0.5 mg/ml naphtholAS-MX phosphate (SigmaeAldrich Corp., St. Louis, MO, USA) and1.1 mg/ml fast red TR salt (SigmaeAldrich Corp., St. Louis, MO, USA)were added, after which slides were incubated for 60 min at 37 �C,rinsed in distilled water, counterstained with haematoxylin, andembedded with vectamount. The number of positively stained os-teoclasts per millimeter of bone marrow perimeter was counted.

2.5. Statistical analysis

All data are expressed as mean values with standard error of themean (SEM), andwere analysed using SPSS PASW21.0 forWindows(SPSS Inc., Chicago, IL, USA). The ShapiroeWilk test was used to testfor normality, followed by the ManneWhitney U test for thecomparison of nonenormally distributed data, whereas the Stu-dent t test was used for normally distributed data. Values ofp < 0.05 indicated significant differences.

3. Results

3.1. Micro-CT

Fig. 1 shows bone parameters quantified by micro-CT scanningin the mandibles of mice, 10 and 24 weeks after radiation therapy.Fig. 1A shows the volume of interest, between the red lines, thatwas selected. At 10 weeks post-RT, no differences between groupswere found in bone volume (Fig. 1B), trabecular thickness (Fig. 1C),trabecular separation (Fig. 1D), and trabecular number (Fig. 1E).However, porosity (Fig. 1F) was significantly increased in the RTgroup (36.6% ± 1.1%) compared to controls (31.3% ± 2.0%; p < 0.05).HBOT given after RT reduced the percentage of porosity towardscontrol levels (30.7% ± 2.0%; p < 0.05). Twenty-four weeks after RT,no differences in the percentage of porosity were present anymore.However, the effect of RT became evident in the other parametersmeasured. Relative bone volume (BV/TV) significantly decreased inthe RT-group (57.2% ± 0.6%) compared to control (63.0% ± 1.1%;p < 0.01). When HBOT was given after RT, relative bone volumeincreased towards control levels (62.2 ± 3.0; p < 0.05 for RT vs.RTþHBOT). The same patternwas seen for the trabecular thickness(control 0.171 ± 0.005 mm; RT 0.145 ± 0.003 mm; RT þ HBOT0.157 ± 0.007 mm; p < 0.001 for control vs. RT; p < 0.05 for RT vs.RT þ HBOT). Trabecular separation was inversely affected, with ahigher separation of trabeculae in RT (0.108 ± 0.002) compared tocontrol (0.100 ± 0.002 mm; p < 0.05) and oppositely a reduction intrabecular separation due to HBOT (0.096 ± 0.008 mm; p < 0.05).Trabecular number was increased by RT (3.96 ± 0.06 vs. 3.70 ± 0.04

Fig. 1. micro-CT analysis. micro-CT analysis was performed in mandibles of mice; the volume of interest is indicated between red lines (A). Relative bone volume (B), trabecularthickness (C), trabecular separation (D), trabecular number (E), and percentage porosity (F) were calculated, at 10 and 24 weeks after radiation therapy. RT ¼ radiation therapy,HBOT ¼ hyperbaric oxygen therapy. Lines indicate significant differences between groups.

L. Spiegelberg et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 214e219216

in controls; p < 0.01), whereas the administration of HBOT after RTdid not influence trabecular number (3.95 ± 0.04) compared to RT.

3.2. Histology

The amount of empty lacunae, devoid of osteocytes, reflectsbone viability and has been reported to be increased after RT(Fenner et al., 2010; Damek-Poprawa et al., 2013). In our study, thepercentage of empty lacunae increased at 24 weeks after RT(3.69% ± 0.41% vs. 1.26% ± 0.43% in controls; p < 0.01), whereas noeffect was seen at 10 weeks post-RT (Fig. 2A). HBOT had a positiveeffect, as a lower percentage of empty lacunae was found at 24weeks post-RT (2.34% ± 0.31%; p < 0.05 for RT vs. RT þ HBOT).

Adipocyte density (Fig. 2B) of bonemarrowwas increased by RT,at both 10 weeks (62.1 ± 14.6 vs. 8.2 ± 4.4 in controls; p < 0.05) and24 weeks post-RT (49.88 ± 13.5 vs. 13.8 ± 7.2 in controls; p < 0.05).HBOT did not have an effect on adipocyte density.

The number of bone-resorbing osteoclasts (Fig. 2C) wasincreased by RTat both time-points, although not significantly at 24weeks post-RT (1.68 ± 0.34 vs. 0.07 ± 0.04 in controls at 10 weeks,p < 0.05; 1.48 ± 0.55 vs. 0.66 ± 0.17 in controls at 24 weeks). HBOTdecreased osteoclast number at 24 weeks post-RT compared to RTtreatment alone (0.38 ± 0.17 in RT þ HBOT vs. 1.48 ± 0.55 in RT;p < 0.05).

4. Discussion

Radiation therapy, used as a standard component in the protocoltreatment of head and neck cancer, inevitably exposes surroundingnormal tissues to radiation. Maxillofacial bones that are in the fieldof irradiation become more vulnerable to fractures and have adecreased regenerative capacity. Reconstructive surgery on irradi-ated bones therefore often gives suboptimal results (Ferrari et al.,2013; Lye et al., 2013). Treatments that can improve the conditionof tissues with radiation-induced damage are desired. HBOT is usedin the treatment of osteoradionecrosis, as well as necrosis of thebone due to bisphosphonates (Voss et al., 2012). It is also used in apreventive manner when dental alveolar surgery and implantologyneed to be performed in previously irradiated mandibular bone.However, its effectiveness remains debatable, with an increasingamount of studies reporting a lack of effect and advocating the needfor randomized controlled trials (Jacobson et al., 2010; Esposito andWorthington, 2013; Heyboer et al., 2013; Lubek et al., 2013). Also,the mechanism of action is not fully understood (Spiegelberg et al.,2010; Phi-Nga and Thom, 2011).

In the present study, we investigated whether HBOT was able toprotect the mandibular bone from long-term radiation effects. Forthis purpose, microarchitecture and morphology were assessed at10 and 24 weeks after RT and compared with those in controls.

Fig. 2. Morphological analysis. Quantification of morphological parameters, with pictures from different groups at 24 weeks after radiation therapy (RT). The percentage of emptylacunae is shown in A. White arrows depict empty lacunae, which are more abundant in the RT group. Scale bars represent 50 mm. Adipocyte density is shown in B, with anincreased density in both RT-treated groups. Scale bars represent 100 mm. C represents the number of osteoclast per millimeter of bone marrow perimeter. Osteoclasts are stainedred by TRAP staining and are more abundant in the RT-group. Scale bars represent 100 mm. HBOT ¼ hyperbaric oxygen therapy; TRAP ¼ tartrate-resistant acid phosphatase. Linesindicate significant differences between groups.

L. Spiegelberg et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 214e219 217

micro-CT analysis is a powerful and nondestructive tool toquantify bone properties on a microstructural level. Its advantageover histologic analysis is that it enables the measurement ofvarious parameters in larger volumes of bone, whereas histologicalassessment of bone microarchitecture relies on the extrapolation ofmeasurements performed in a few slides (Lee et al., 2013). Relativebone volume (BV/TV), trabecular number (TbN), trabecular thick-ness (TbTh), and trabecular separation (TbSp) are the most com-mon parameters used to describe bone microarchitecture. BV/TV,TbN, and TbTh have been shown to decrease in response to radia-tion in animal models, whereas TbSp increases after RT (Greenet al., 2012; Chandra et al., 2013; Damek-Poprawa et al., 2013; Leeet al., 2013). These changes in bone microarchitecture result inreduced strength of the bone, which thus becomesmore vulnerableto fractures. Our study is in accordance with previous studies andshowed a decrease of BV/TV and TbTh 24 weeks after radiationtherapy, whereas TbSp was increased. Surprisingly, TbN wasincreased due to radiation. Apart from the percentage of porosity,no effect was seen at 10 weeks after RT, whereas other studiesshowed changes in microstructure at the same or earlier time-points (Green et al., 2012; Chandra et al., 2013; Damek-Poprawaet al., 2013; Lee et al., 2013). This could be due to the use ofdifferent animal models with varying RT dose and administrationin these studies, making it difficult to compare time-points. Moststudies are performed in hind limbs, while the effects of RT onmandibular bone are less well studied. Damek-Poprawa and

colleagues (Damek-Poprawa et al., 2013) have established that theonset of osteoradionecrosis is more rapid in the mandibles than inthe tibias of rats. A 50-Gy single dose, administered locally to themandible, was used in this study, whereas we used a 15-Gy singledose that comprised the head and neck area. These differencescould account for the fact that in our study, effects on bonemicroarchitecture were only visible 24 weeks after RT, as comparedto effects seen on 10 weeks after RT in the study of Damek-Poprawaet al.

In addition to the observed microarchitectural changes,morphological changes due to RT were also observed. Adipocytedensity in the bone marrow increased at both time points studied.The increase of adipocytes in bone marrow as a consequence ofradiation has been reported (Casamassima et al., 1989). Mesen-chymal stem cells (MSCs) in the bone marrow can differentiate intoosteoblasts and adipocytes (Yang et al., 2010). Radiation probablytargets the MSCs and disrupts the balance between MSCs osteo-blast- and adipocyte differentiation. Adipocytes secrete anti-osteogenic factors and therefore decrease bone formation andbone mass (Harada and Rodan, 2003), probably due to an inductionof apoptosis of bone-forming osteoblasts and increased prolifera-tion and differentiation of bone-resorbing osteoclasts (Liu et al.,2011). Osteoclast number was also increased after RT in ourstudy, indicating increased bone resorption.

The number of empty lacunae, which represents loss of osteo-cytes, increased under the influence of RT at 24 weeks post-RT.

L. Spiegelberg et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 214e219218

Osteocytes derive from osteoblasts that have become ‘trapped’ inthe matrix that they have produced themselves. They maintaincontact with each other and with the osteoblasts and osteoclaststhat reside at the bone surface by cytoplasmic extensions calleddendrites, that extend into channels in the matrix (Rochefort et al.,2010). In this way, osteocytes send signals for bone resorption andformation to osteoclasts and osteoblasts, respectively, and there-fore play an essential role in bone maintenance and remodelling(Bonewald, 2007). Dying or dead osteocytes have been suggested tostimulate resorption (Aguirre et al., 2006; Jilka et al., 2013). Variousstudies on the effects of radiation on bone use the increasedamount of empty lacunae as a measure for radiation damage(Fenner et al., 2010; Tchanque-Fossuo et al., 2011, 2012; Damek-Poprawa et al., 2013; Donneys et al., 2014), and our resultsconfirm the radiation-induced loss of osteocytes.

Effects of hyperbaric oxygen on (irradiated) bone have not beenstudied extensively. Experimental animal studies are mainly con-ducted in either distracted, previously irradiated, bone or includethe placement of implants. Moderately positive effects of HBOT onosteoblastic activity have been reported in irradiated distractedbone (reviewed in Spiegelberg et al., 2010). Implantology is usuallyperformed in the hind legs of rats, mice, or rabbits, which differfrom the predominantly cortical bones of the head and neck region,as they consist mainly of cancellous bone. Small improvements inbone formation, implant-bone contact, and force needed to un-screw implants due to HBOT were reported (Spiegelberg et al.,2010). Williamson et al. subjected rats to RT followed by HBOTand investigated bone 36 weeks after RT (Williamson, 2007). Theycounted the number of lacunae filled with osteocytes and reporteda positive effect (i.e., higher number of filled lacunae) in the grouptreated with RT and HBOT compared with the group that wasirradiated. Our findings, at 24 weeks after RT, correspond withthese results, as HBOT was also able to positively influence osteo-cyte count and thus the viability of mandibular bone. In addition,we found positive effects of HBOT on microstructural parametersthat were negatively influenced by RT, and a suppression ofosteoclasts.

The effects of HBOT on osteogenic cells, i.e., bone-forming os-teoblasts and bone-resorbing osteoclasts, have been studiedin vitro. Positive effects of HBOT on proliferation and/or differen-tiation of osteoblasts have been described (Tuncay et al., 1994; Wuet al., 2007), although Wong et al. found no effect of HBOT onnormal or irradiated osteoblasts (Wong et al., 2008). Osteoclastformation has been shown to be suppressed due to HBOT (Al Hadiet al., 2013). In our in vivo study, osteoclast number was decreasedby HBOT, at 24 weeks after RT, indicating a reduction of boneresorption. Together with the decreased number of empty lacunae,this may have led to the increased relative bone volume seen bymicro-CT analysis.

The clinical relevance of our results remains to be elucidated. Itis to be defined if the single radiation dose of 15 Gy (biologicallyequivalent to a cumulative dose of 32 Gy) that was used in ourexperimental model leads to the same degree of bone damage asthe fractionated radiation dosing schemes that are clinically used.Furthermore, it is clinically important to investigate whether theobserved HBOT induced changes cause stronger bone and animproved regenerative capacity when trauma is inflicted. Furtherexperimental investigation followed by randomized clinical trials istherefore desired.

5. Conclusion

Radiation irreversibly damages bone and other specialized tis-sues. Therefore, the prevention of radiation-induced damage tobone would be preferable to the treatment of complications. Our

results showed that HBOT was able to positively influence micro-architectural parameters of irradiated mandibular bone in mice, tosuppress osteoclast number, and to increase bone viability. Thiscould indicate that HBOT has the potential to be used as a means ofpreventing radiation-induced bone damage. Further research willbe necessary to evaluate whether the observedmicrostructural andmorphological changes of irradiated bone can indeed cause stron-ger bone that is less vulnerable to microfractures or other traumathat can lead to ORN.

Source of supportThis research was financially supported by Fonds NutsOhra

(grant number 0801-77).

Conflict of interestNone declared.

Acknowledgements

The authors would like to thank Bastiaan Tuk for technicaladvice and ErwinWaarsing for helpwith themicro-CT scanner. Thisresearch was financially supported by Fonds NutsOhra (grantnumber 0801-77).

References

Aguirre JI, Plotkin LI, Stewart SA, Weinstein RS, Parfitt AM, Manolagas SC, et al:Osteocyte apoptosis is induced by weightlessness in mice and precedes oste-oclast recruitment and bone loss. J Bone Miner Res 21: 605e615, 2006

Al Hadi H, Smerdon GR, Fox SW: Hyperbaric oxygen therapy suppresses osteoclastformation and bone resorption. J Orthop Res 31: 1839e1844, 2013

Bonewald LF: Osteocytes as dynamic multifunctional cells. Ann N Y Acad Sci 1116:281e290, 2007

Casamassima F, Ruggiero C, Caramella D, Tinacci E, Villari N, Ruggiero M: He-matopoietic bone marrow recovery after radiation therapy: MRI evaluation.Blood 73: 1677e1681, 1989

Chandra A, Lan S, Zhu J, Lin T, Zhang X, Siclari VA, et al: PTH prevents the adverseeffects of focal radiation on bone architecture in young rats. Bone 55: 449e457,2013

Damek-Poprawa M, Both S, Wright AC, Maity A, Akintoye SO: Onset of mandibleand tibia osteoradionecrosis: a comparative pilot study in the rat. Oral Surg OralMed Oral Pathol Oral Radiol 115: 201e211, 2013

Djasim UM, Spiegelberg L, Wolvius EB, van der Wal KG: A hyperbaric oxygenchamber for animal experimental purposes. Int J Oral Maxillofac Surg 41:271e274, 2012

Donneys A, Tchanque-Fossuo CN, Blough JT, Nelson NS, Deshpande SS, Buchman SR:Amifostine preserves osteocyte number and osteoid formation in fracturehealing following radiotherapy. J Oral Maxillofac Surg 72: 559e566, 2014

Esposito M, Worthington HV: Interventions for replacing missing teeth: hyperbaricoxygen therapy for irradiated patients who require dental implants. CochraneDatabase Syst Rev 9: CD003603, 2013

Fenner M, Park J, Schulz N, Amann K, Grabenbauer GG, Fahrig A, et al: Validation ofhistologic changes induced by external irradiation in mandibular bone. Anexperimental animal model. J Craniomaxillofac Surg 38: 47e53, 2010

Ferrari S, Copelli C, Bianchi B, Ferri A, Poli T, Ferri T, et al: Rehabilitation withendosseous implants in fibula free-flap mandibular reconstruction: a case se-ries of up to 10 years. J Craniomaxillofac Surg 41: 172e178, 2013

Green DE, Adler BJ, Chan ME, Rubin CT: Devastation of adult stem cell pools byirradiation precedes collapse of trabecular bone quality and quantity. J BoneMiner Res 27: 749e759, 2012

Harada S, Rodan GA: Control of osteoblast function and regulation of bone mass.Nature 423: 349e355, 2003

Heyboer 3rd M, Wojcik SM, Grant WD, Faruqi MS, Morgan M, Hahn SS: Professionalattitudes in regard to hyperbaric oxygen therapy for dental extractions inirradiated patients: a comparison of two specialties. Undersea Hyperb Med 40:275e282, 2013

Jacobson AS, Buchbinder D, Hu K, Urken ML: Paradigm shifts in the management ofosteoradionecrosis of the mandible. Oral Oncol 46: 795e801, 2010

Jilka RL, Noble B, Weinstein RS: Osteocyte apoptosis. Bone 54: 264e271, 2013Lee JH, Lee HJ, Yang M, Moon C, Kim JC, Jo SK, et al: Establishment of a murine

model for radiation-induced bone loss using micro-computed tomography inadult C3H/HeN mice. Lab Anim Res 29: 55e62, 2013

Liu HY, Wu AT, Tsai CY, Chou KR, Zeng R, Wang MF, et al: The balance betweenadipogenesis and osteogenesis in bone regeneration by platelet-rich plasma forage-related osteoporosis. Biomaterials 32: 6773e6780, 2011

L. Spiegelberg et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 214e219 219

Lubek JE, Hancock MK, Strome SE: What is the value of hyperbaric oxygen therapyin management of osteoradionecrosis of the head and neck? Laryngoscope 123:555e556, 2013

Lye KW, Chin FK, Tideman H, Merkx MA, Jansen JA: Effect of postoperative radiationtherapy on mandibular reconstruction using a modular endoprosthesis e a pilotstudy. J Craniomaxillofac Surg 41: 487e495, 2013

Marx RE: Osteoradionecrosis: a new concept of its pathophysiology. J Oral Max-illofac Surg 41: 283e288, 1983

Pacheco R, Stock H: Effects of radiation on bone. Curr Osteoporos Rep 11: 299e304,2013

Phi-Nga JL, Thom SR: Mechanistic perspective for experimental and acceptedindications of hyperbaric oxygen therapy. J Exp Integr Med 1: 207e214,2011

Rochefort GY, Pallu S, Benhamou CL: Osteocyte: the unrecognized side of bonetissue. Osteoporos Int 21: 1457e1469, 2010

Spiegelberg L, Braks J, Djasim U, Farrell E, van der Wal K, Wolvius E: Effects ofhyperbaric oxygen therapy on the viability of irradiated soft head and necktissues in mice. Oral Dis 20: e111e119, 2014

Spiegelberg L, Djasim UM, van Neck HW, Wolvius EB, van der Wal KG: Hyperbaricoxygen therapy in the management of radiation-induced injury in the head andneck region: a review of the literature. J Oral Maxillofac Surg 68: 1732e1739,2010

Tchanque-Fossuo CN, Donneys A, Razdolsky ER, Monson LA, Farberg AS,Deshpande SS, et al: Quantitative histologic evidence of amifostine-induced

cytoprotection in an irradiated murine model of mandibular distractionosteogenesis. Plast Reconstr Surg 130: 1199e1207, 2012

Tchanque-Fossuo CN, Monson LA, Farberg AS, Donneys A, Zehtabzadeh AJ,Razdolsky ER, et al: Dose-response effect of human equivalent radiation in themurine mandible: part I. A histomorphometric assessment. Plast Reconstr Surg128: 114e121, 2011

Tuncay OC, Ho D, Barker MK: Oxygen tension regulates osteoblast function. Am JOrthod Dentofacial Orthop 105: 457e463, 1994

Voss PJ, Joshi Oshero J, Kovalova-Muller A, Veigel Merino EA, Sauerbier S, Al-Jamali J, et al: Surgical treatment of bisphosphonate-associated osteonecrosis ofthe jaw: technical report and follow up of 21 patients. J Craniomaxillofac Surg40: 719e725, 2012

Williamson RA: An experimental study of the use of hyperbaric oxygen to reducethe side effects of radiation treatment for malignant disease. Int J Oral Max-illofac Surg 36: 533e540, 2007

Wong AK, Schonmeyr BH, Soares MA, Li S, Mehrara BJ: Hyperbaric oxygen inhibitsgrowth but not differentiation of normal and irradiated osteoblasts. J CraniofacSurg 19: 757e765, 2008

Wu D, Malda J, Crawford R, Xiao Y: Effects of hyperbaric oxygen on proliferation anddifferentiation of osteoblasts from human alveolar bone. Connect Tissue Res 48:206e213, 2007

Yang Y, Tao C, Zhao D, Li F, Zhao W, Wu H: EMF acts on rat bone marrow mesen-chymal stem cells to promote differentiation to osteoblasts and to inhibit dif-ferentiation to adipocytes. Bioelectromagnetics 31: 277e285, 2010