Hyperbaric oxygen stimulates vascularization and bone formation in rat calvarial defects

Int. J. Oral Maxillofac. Surg. 2013; 42: 907–914 http://dx.doi.org/10.1016/j.ijom.2013.01.003, available online at http://www.sciencedirect.com

Research Paper Bone Healing

Hyperbaric oxygen stimulates vascularization and bone formation in rat calvarial defects T. O. Pedersen, Z. Xing, A. Finne-Wistrand, S. Hellem, K. Mustafa: Hyperbaric oxygen stimulates vascularization and bone formation in rat calvarial defects. Int. J. Oral Maxillofac. Surg. 2013; 42: 907–914. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Hyperbaric oxygen (HBO) therapy is used to treat or prevent tissue necrosis in patients undergoing irradiation. Many such patients require reconstructive surgery, but little is known of the effects of HBO on bone vascularization and regeneration. In this study, copolymer poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) scaffolds were implanted into critical-sized calvarial defects in Wistar rats. The animals were randomly allotted to hyperbaric or normobaric oxygen groups. The treatment group received five sessions weekly for 90 min at increased atmospheric pressure, for up to 4 weeks. Samples were retrieved at weeks 2 and 8, i.e. after a total of 10 and 20 sessions, respectively. The samples were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and histology at week 2, and radiographically and histologically at week 8. At week 2, defects treated with HBO exhibited greater numbers of cells positive for the endothelial marker CD31, up-regulated gene expression of osteogenic markers, and down-regulated expression of pro-inflammatory cytokines. At week 8, radiographic examination revealed that calvarial defects subjected to HBO exhibited a higher percentage of radiopacities than normobaric controls, and histological examination disclosed enhanced bone healing. These results confirmed that HBO treatment was effective in stimulating vascularization and bone formation in rat calvarial defects.

Reconstruction of bone defects through autologous grafting is associated with donor site morbidity and is not feasible in all patients. As a basis for translational research in oral and maxillofacial bone reconstruction,1 a series of studies using stem cells in combination with biodegradable synthetic scaffolding materials has been undertaken. Two major challenges in this field of research are inadequate vascularization and the dependence of 0901-5027/070907 + 08 $36.00/0

tissue regeneration on adequate recruitment of endogenous cells.2 Hyperbaric oxygen (HBO) therapy, where the patient breathes 100% oxygen at increased atmospheric pressure, leads to both arterial and tissue oxygen tension.3 The production of reactive oxygen species activates various signalling molecules and has been attributed a key role in the therapeutic effects of HBO.4 Stimulated homing of vasculogenic stem cells has been

T. O. Pedersen1,2, Z. Xing1, A. Finne-Wistrand3, S. Hellem1,4, K. Mustafa1 1

Department of Clinical Dentistry, Center for Clinical Dental Research, University of Bergen, Norway; 2Department of Biomedicine, University of Bergen, Norway; 3 Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, Stockholm, Sweden; 4 Department of Clinical Dentistry, Section for Oral and Maxillofacial Surgery, University of Bergen, Norway

Key words: hyperbaric oxygen; vascularization; copolymer scaffold; osteogenesis; osteoprogenitor cells. Accepted for publication 9 January 2013 Available online 9 February 2013

found in skin wounds,5 and a review by Goldman6 concluded that there is good evidence for using HBO to promote wound healing. Nilsson et al.7 found an effect on bone regeneration in a rabbit bone harvest chamber, and more recently the healing of critical-sized calvarial defects in rabbits were shown to increase after HBO treatment.8 Sirin et al.9 investigated healing of rat tibia defects filled with b-tricalcium phosphate (b-TCP) and

# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

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calcium phosphate-coated bovine bone, where only the former showed improved bone healing compared to control defects. Following HBO, bone defects filled with biphasic calcium phosphate have also shown a more favourable bone healing response than defects filled with demineralized bone matrix.10 Many patients needing major bone reconstruction have impaired local vascularization due to tumour surgery and radiation therapy, and HBO has been proposed as a post-grafting treatment modality. Sawai et al.11 grafted corticocancellous bone from the iliac crest to the mandible in rabbits, followed by post-operative HBO. The results suggested that the union of autogenous bone grafts was accelerated in the treatment group. Furthermore, the reduction of bone mineral content in autografts following HBO has also been found, possibly attributable to increased angiogenesis within the graft.12 Recent studies have shown that copolymer poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLAco-DXO)) scaffolds are able to serve as a framework for cellular proliferation and differentiation,13–15 and the chemical versatility of the material enables degradation and mechanical properties to be tailored to meet the intended application.16 HBO might be a part of the treatment regime in the rehabilitation of patients recovering from malignant disease, especially if reconstructive surgery is required. However, limited knowledge exists about the biological effects of HBO in the presence of biodegradable scaffolding materials aimed at bone tissue engineering. In the present work, cell- and materialderived osteoinductive factors were not included in order to study the isolated effects of treatment. The aim of the present study was to test the hypothesis that HBO stimulates vascular ingrowth in calvarial bone defects filled with poly(LLAco-DXO) scaffolds, leading to mobilization of endogenous progenitor cells and improved bone healing. Materials and methods Scaffolds

Production of the copolymer poly(LLAco-DXO) scaffolds has been described previously.13 Briefly, polymerization was performed in bulk at 110 8C for 72 h and precipitated three times in cold hexane and methanol. Scaffolds were made through the salt-leaching method whereby dissolved poly(LLA-co-DXO) was casted in a mould with NaCl at a ratio of 10:1. After repeated soaking in water,

Table 1. Experimental groups. HBO

NBO Time-point (weeks) Number of exposures Exposure time (min) Ambient pressure (atm) Gas

2 0 0 1 Air

8 0 0 1 Air

2a 10a 90 2.5 O2

8b 20b 90 2.5 O2

NBO, non-treated; HBO, treated with hyperbaric oxygen therapy. a HBO was administered five times weekly for 2 weeks; the animals were then euthanized. b HBO was administered five times weekly for 4 weeks, followed by an interval of 4 weeks without treatment before the animals were euthanized at week 8.

the salt-free scaffolds were vacuum-dried and sterilized with electron beam radiation.

Animal procedures

Skeletally mature 16 week-old female Wistar rats were anaesthetized with isoflurane (IsobaVet1; Schering-Plough, Kenilworth, NJ, USA) combined with O2 using a custom-made mask. The scalp was shaved and disinfected. A midline incision was made through skin, temporalis muscle and periosteum and flaps were reflected laterally. A trephine burr was used under saline irrigation to create bilateral 6-mm bone defects in the parietal calvaria. The dura mater was exposed and inspected for bleeding. Supradural placement of scaffolds of the same size as the defects was carefully performed, leaving the material in contact with the bony margins of the defects. The wound was closed in two layers with resorbable sutures (vicryl rapid 4-0; Ethicon, Somerville, NJ, USA). A topical antibiotic was applied on the sutured skin wounds to prevent postoperative infections. Postoperatively, the animals were placed in separate cages for recovery, after which they were returned to their original cages as before surgery. One rat had a partially open wound on the first postoperative day, which was re-sutured. At weeks 2 and 8, animals were euthanized with an overdose of CO2. At week 2, scaffolds were dissected out and halved, one for reverse transcriptase polymerase chain reaction (RT-PCR) analysis and the other for histological analysis. Samples intended for paraffin embedding were fixed in 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany), while those intended for RT-PCR and O.C.T.TM compound (Sakura Finetek, Tokyo, Japan) embedding were kept in RNALater (Invitrogen, Carlsbad, CA, USA) and frozen within 3 h. Samples retrieved at week 8 were fixed in 4% PFA.

Hyperbaric oxygen chamber

An Oxycom 250 Arc pressure chamber was used (Hypcom Oy, Tampere, Finland), with a volume of 27 l, an inner diameter of 25 cm, and inner length of 55 cm. A standard prophylactic protocol for osteoradionecrosis was applied. The chamber was filled with 100% O2 for 15 min, and pressure was raised to 1.5 bar (2.5 atm) over a period of 10– 15 min. Pressure was maintained for 90 min, and no more than four rats were treated simultaneously. To maintain >97% O2 at all times, the chamber was flushed with pure oxygen for 5 min every 10 min. The rats were decompressed for 10–15 min. As shown in Table 1, HBO was administered to the experimental group from the first day post-operatively and every weekday for 2 or 4 weeks: the 4 weeks of treatment were followed by an interval of 4 weeks without treatment before the animals were euthanized. Thus the animals in the experimental group that were euthanized at week 2 received a total of 10 HBO treatments, and those euthanized at week 8 received 20 treatments over the first 4 weeks.

Real-time RT-PCR

An E.Z.N.A.1 Total RNA Kit (Omega Bio-Tek, Norcross, GA, USA) was used to extract RNA from the retrieved scaffolds. A NanoDrop Spectrophotometer (ThermoScientific NanoDrop Technologies, Wilmington, DE, USA) was used to quantify RNA and determine the purity of RNA. A high capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, CA, USA) was used for the reverse transcription reaction. Total RNA (1000 ng) was mixed with nuclease-free water, reverse transcriptase buffer, random primers, dNTP, and MultiScribe reverse transcriptase. The real-time RT-PCR was performed on a StepOneTM Real-Time PCR System (Applied Biosystems) under standard enzyme and cycling conditions. cDNA corresponding to 10 ng mRNA

Hyperbaric oxygen in bone regeneration was used in each reaction, prepared in duplicate for each target gene. Taqman1 gene expression assays were used: alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2), bone sialoprotein (BSP), osteocalcin (OC), interleukin 1 (IL-1), interleukin 6 (IL-6), and interleukin 10 (IL-10). A comparative cycle threshold (Ct) method was used for data analysis, and b-actin served as endogenous control. Histological evaluation

Among the samples retrieved for histology at week 2, half were fixed in 4% PFA and embedded in paraffin and the remainder were embedded in O.C.T. compound (Sakura Finetek) and maintained at 80 8C. The paraffin-embedded samples were sectioned to 8-mm thickness with a microtome (Leica Microsystems, Wetzlar, Germany), while frozen samples were cryosectioned with a Leica CM 3050S at 24 8C. Samples retrieved at week 8 were decalcified for 3 weeks with 10% ethylenediaminetetraacetic acid (EDTA) in 0.1 M Tris buffer and 7.5% polyvinylpyrrolidone (PVP) (Merck) before embedding in paraffin, sectioning, and staining with haematoxylin–eosin (HE) or Masson’s trichrome. The slides were passed through a graded alcohol and xylol series before mounting with Eukitt (O. Kindler, Freiburg, Germany). ALP staining was performed with a 2-h incubation time at room temperature with freshly made substrate solution (Sigma–Aldrich, St. Louis, MO, USA) containing 100 mM Tris–maleate buffer, 8 mg/ml naphthol AS-TR, and 2 mg/ml diazonium salt fast red violet LB. The slides were washed with distilled water and counterstained with 0.1% fast green. CD31 staining was performed with a polyclonal CD31 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 in phosphate-buffered saline (PBS) with 5% rabbit serum for 4 h at room temperature. The secondary antibody was a tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit antigoat antibody diluted 1:1000, incubated for 2 h at room temperature. The slides were mounted with Prolong1 Gold Antifade Reagent (Invitrogen) before imaging. Radiographic evaluation

A dental X-ray machine (Gendex, Hatfield, PA, USA) was used to obtain radiographs from the retrieved calvaria at week 8, before decalcification. Exposure conditions were 10 mA, 0.08 ms, and 60 kV. NIS-Elements BR 3.07 software (Nikon,

Tokyo, Japan) was used for image analysis. A common threshold was applied to all defects and the sum percentage of radiopacity was determined for each defect. Sectional images were obtained with a BrightField CT Scanner (GE Healthcare, Waukesha, WI, USA), and OsiriX DICOM Viewer version 5.0.2 was applied for image processing. Statistical analysis

The sample size for radiographic evaluation was eight. For histology and real-time RT-PCR, the results presented are from a minimum of six parallels of treated (HBO) and non-treated (NBO) samples. For statistical evaluation, the Shapiro–Wilk test was first applied to test for normality, in which H0 of normally distributed values could not be rejected. Next, a two-sample F-test with n  1 degrees of freedom was performed, which also failed to reject H0, in this case s 21 ¼ s 22 . Based on this, the independent sample t-test was selected. SPSS Statistics 19.0 (IBM, Armonk, NY, USA) was applied for statistical processing and analysis, and the significance level was set to P < 0.05. The results are presented as mean  standard deviation. A post hoc power analysis was performed specifically for the radiographic measurements using the non-central t-distribution. The effect size d for this parameter was calculated to be 1.957, and the non-centrality parameter d was 3.916, with the critical t being 2.145 with 14 degrees of freedom ((n1 + n2)  2). With a set to 0.05, the statistical power defined as the probability of 1  b error was calculated to be 0.953. The respective formulas for calculating effect size and the non-centrality parameter were:

x¯ 1  x¯ 2 and srffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n1 n2 d¼d  n1 þ n2 d¼

Results Vascularization and mobilization of osteoprogenitor cells

At week 2, positive staining for ALP was found adjacent to areas of osteoid formation in the HBO group (Fig. 1A), and gene expression levels were significantly upregulated (Fig. 1C). Cells positive for the mesenchymal stem cell marker CD90 were detected at the edge of the defects in the treated animals (Fig. 1B). ALP is a well-established biomarker for

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early osteogenic differentiation, and the combination of up-regulated levels of ALP and CD90-positive cells suggests a higher presence of osteoprogenitor cells in samples harvested from the treatment group at week 2. Vascularized connective tissue filled the space within scaffolds (Fig. 1D), with cells positive for the endothelial cell marker CD31 (Fig. 1E), which accounted for a greater area fraction in the treatment group (Fig. 1F). These findings suggest a higher vascular density in the connective tissue stroma within implanted scaffolds in the phase before mesenchymal condensation and osteogenesis. Osteogenic and inflammatory gene expression

At week 2, expression of BMP-2 (Fig. 2A), BSP (Fig. 2B), and OC (Fig. 2C) was up-regulated in the HBO group. Increased levels of BMP-2 suggest the presence of active cellular signalling to attract osteogenic cells, whereas the extracellular matrix components BSP and OC indicate greater deposition of bone matrix in the treatment group. Levels of IL-1 (Fig. 2D) and IL-6 (Fig. 2E) were down-regulated, whereas expression of IL-10 was up-regulated (Fig. 2F). These findings suggest a decreased inflammatory response in defects at week 2. Bone formation

The percentage of radiopacities for samples analyzed at week 8 is presented in Table 2. Only minor ingrowth of new bone could be detected in control defects (Fig. 3A and B) compared to defects subjected to HBO (Fig. 3E and F). In the treatment group, new bone formation observed as woven bone was detected from the edge of the defects (Fig. 3C), and individual islands could also be found (Fig. 3G). A higher percentage of radiopacities was detected in the treatment group at week 8 (Fig. 3D), which was confirmed by histology (Fig. 4). A sectional view of the bone formation as evaluated by computed tomography (CT) is shown in Fig. 5. At the end of the experimental period, improved vascular ingrowth, up-regulated osteogenic activity, and down-regulated inflammatory cytokines were associated with enhanced bone healing. Discussion

This study demonstrates that bone formation into a poly(LLA-co-DXO) scaffold could be accelerated by the administration

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Fig. 1. Vascularization and mobilization of osteoprogenitor cells at week 2. (A) Representative light micrograph (40) showing osteoid formation surrounded by alkaline phosphatase (ALP) positive staining (purple) in the HBO group. (B) Representative fluorescent micrograph of CD90-positive cells at the border of defects in the treatment group. (C) Relative gene expression (mean  standard deviation) of ALP; *P < 0.05. (D) Representative light micrograph from the treatment group showing highly vascularized loose connective tissue inside the scaffold material. (E) Representative fluorescent micrograph of CD31-positive cells in the treatment group. (F) Total area fraction of CD31-positive cells; *P < 0.05.

Fig. 2. Expression of osteogenic and inflammatory markers at week 2. Relative gene expression (mean  standard deviation) of the biomarkers: (A) bone morphogenetic protein 2 (BMP-2), (B) bone sialoprotein (BSP), (C) osteocalcin (OC), (D) interleukin 1 (IL-1), (E) interleukin 6 (IL-6), and (F) interleukin 10 (IL-10). Significant differences shown in the treatment group are relative to the normobaric controls; *P < 0.05, **P < 0.01.

Hyperbaric oxygen in bone regeneration Table 2. Percentage of radiopacities at week 8. Sample

NBO

HBO

1 2 3 4 5 6 7 8 Mean SD

13.9 1.6 14.1 10.3 13.9 3.4 6.9 18.2 10.3 5.8

38.4 7.1 39.3 28.3 30.6 27.9 63.8 33.5 33.6 15.8

NBO, non-treated; HBO, treated with hyperbaric oxygen therapy; SD, standard deviation.

of HBO from the first postoperative day and five times weekly for a total number of 20 treatment sessions. In the treatment group, samples retrieved at week 2 showed enhanced vascularization, upregulated expression of osteogenic markers, and down-regulated expression of pro-inflammatory cytokines. The present literature on the effects of HBO on inflammation is inconsistent, with little information available about the inflammatory response in bone defect models. HBO has been proposed as an alternative anti-inflammatory treatment for arthritis, with effects similar to acetylsalicylic acid.17 Animal studies have shown that HBO contributes to dissociation of acute inflammation with a subsequent reduction in oedema and improved

response to mechanical stimulation.18–21 IL-1 and IL-6 are both secreted by tissue macrophages as well as by mesenchymal cells from the periosteum, and have important roles in the acute local inflammatory response.22 The release of IL-6 is also up-regulated in multiple chronic inflammatory conditions.23 Weisz 24 et al. found decreased levels of IL-1 and IL-6 in monocyte supernatants isolated from patients suffering from Crohn’s disease after HBO. However, after treatment was discontinued, these pro-inflammatory cytokines returned to a level closer to the baseline values. In the present study, intermittent HBO was administered, starting the day after surgical trauma. At week 2, expression of IL-1 and IL-6 had decreased significantly, suggesting early cessation of the inflammatory response. The up-regulated expression of IL-10, which is associated with resolution of inflammation,25 would tend to support this interpretation of the results. Fluctuations in cells present in defects might be expected depending on the time elapsing between treatment and the collection of samples. We administered HBO on the same day as the animals were euthanized, and this might have influenced the expression of genes compared to a normal situation. However, rapid sample collection after HBO could be beneficial for investigating local effects of treatment at the molecular level, with morphological characteristics of the tissue intact. Acute

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inflammation following surgical trauma should normally have subsided by week 2, and cross-sectional analysis provides only limited data about the time-course of the inflammatory response. Earlier time-points might be of interest to further map the release-curves of pro- and antiinflammatory mediators. However, the evaluation at week 2 suggests that resolution of the initial inflammation is enhanced by HBO treatment. Persistent inflammation inhibits tissue maturation, remodelling, and regeneration, and a rapid cessation of the inflammatory response might therefore be beneficial for osteogenesis.26 Staining for the endothelial cell surface marker CD31 disclosed a higher area fraction of positive cells in the treatment group. This finding supports previous work where HBO therapy resulted in increased microvascular perfusion in dermal wound healing at 10 days.27 Increased neoangiogenesis in burn wound healing has also been reported, particularly in the first 7 days after injury.18 In contrast to early stimulatory effects, prolonged exposure to HBO has been shown to decrease vascularization.28 The beneficial effects might therefore be attributed to the intermittent nature of the therapy. We administered HBO once daily as opposed to twice in the aforementioned study, which could have contributed to the discrepancies in results. The release of proangiogenic cytokines such as vascular

Fig. 3. Bone regeneration in calvarial defects at week 8. (A) Representative radiograph of sample 4 from the control group (10.3% healing) at week 8 showing minor bone formation, compared to (E) a representative radiograph of sample 4 from the treatment group (28.3% healing). Thresholding of radiographs with the circle surrounding the edge of the original 6-mm defects is shown in (B) and (F), with the area of new bone formation in red. (C) Representative light micrograph (10) from the treatment group showing new bone ingrowth seen from the edge of the defects (black line) (scale bar = 200 mm) with interspersed islands of woven bone (G) (20, scale bar = 100 mm). (D) The total percentage (mean  standard deviation) of radiopacities was higher in the treatment group compared to the normobaric control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4. Histological appearance of calvarial defects at week 8. Representative light micrographs (4) of new bone formation at week 8 in (A) untreated and (B) treated defects. Masson’s trichrome staining. Scale bar = 1 mm.

endothelial growth factor (VEGF) is a well-known effect of tissue hypoxia following tissue damage. Hopf et al.29 found that angiogenesis is stimulated by hyperoxia and inhibited by hypoxia. The release of VEGF has also been shown to increase after HBO.30 However, the development of a functional vasculature depends on attenuated pro-angiogenic stimulation allowing vessel maturation and recruitment of perivascular cells,31 and increased vascular maturity after treatment has also been reported.32 Stimulated release of endothelial progenitor cells into the systemic circulation has been shown in both mice and humans.33,34 Thom et al.34 found an eight-fold increase in vascular progenitor cells, while the total number of circulating white blood cells (WBCs) did not increase

significantly. A larger percentage of vascular progenitors in circulating WBC might explain the coexistence of increased vascularization and anti-inflammatory effects. Although HBO is an established treatment and prophylaxis for osteoradionecrosis, only limited data are available on its effects on bone vascularization and healing. Enhancement of the osteogenic phenotype of osteoblasts has been shown in vitro,35 and the present work reports upregulated expression of both early and late osteogenic markers at week 2. ALP is associated with osteogenic differentiation at an early stage, whereas BSP and OC are both central components of bone matrix deposition and mineralization. This finding suggests the increased presence of osteogenic cells at several stages of

differentiation after 2 weeks of treatment. This is in accordance with previous reports of enhanced osteoblastic activity after HBO in irradiated bone subjected to distraction osteogenesis.36 Bone formation has been accelerated by HBO through delivered BMP-2 in an ectopic bone model37; the up-regulated expression of BMP-2 found in the present study might enhance the attraction of osteogenic cells. Further investigation is warranted into the mechanisms underlying enhanced osteogenic cell activity in bone defects subjected to HBO. HBO has been shown to increase bone healing in rat tibia defects using a b-TCP scaffold, an effect not observed with calcium phosphate-coated bovine bone, whereby the authors suggested that larger gaps within materials influenced the effect of HBO on bone healing.9 The poly(LLAco-DXO) scaffold used in this study is highly porous (90%) and has shown an adequate ability to support cell proliferation and differentiation.13,14 However, the primary function of polymer materials in bone regeneration is to facilitate cell growth and tissue development, and its osteoinductive properties are limited. Thus, the bone healing achieved in this study is encouraging, as it occurred in the absence of an osteoinductive material, added osteogenic cells, or growth factors. Positive effects of HBO on bone ingrowth in critical- and supra-critical-sized empty defects have been reported, as well as in a bone harvest chamber.7,8 Chen et al. studied healing of 3-mm  3-mm osteochondral

Fig. 5. Sectional view of bone ingrowth. A series of CT slides of sample 4 from the treatment group showing the distribution of mineralized bone. Slides obtained were of 625-mm thickness.

Hyperbaric oxygen in bone regeneration defects in rabbits and were able to achieve complete healing after 12 weeks.38 These results suggest that non-critical-sized defects can heal without the need for exogenous cells or growth factors, and healing of empty defects subjected to HBO has been found comparable to non-vascularized grafts.12 When combining autografts with HBO, decreased mineral content was observed after transplantation. This might be explained by increased neovascularization of defects, suggesting that the combination with HBO might enhance graft resorption. Although HBO treatment improved bone healing in the present study, full healing had not been achieved by week 8, suggesting a need for additional osteoinductive factors. On the basis of these results, investigation is warranted into further enhancement of bone healing by a combination of HBO and exogenous osteogenic cells or growth factors, as an alternative to nonvascularized bone grafts. In conclusion, in poly(LLA-co-DXO) scaffolds implanted into critical-sized rat calvarial defects, HBO led to improved vascularization and mobilized osteoprogenitor cells at week 2, with accelerated bone healing at week 8. These findings suggest a role for HBO, not only in preventing and treating radiation-induced tissue necrosis and fibrosis, but also for improving the overall outcome of bone reconstruction in the oral and maxillofacial region. Funding

This study was supported by the University of Bergen (fellowships to TOP and ZX), and the L. Meltzers Foundation (project grant, TOP) and the VascuBone Project (EU FP7; No. 242175). Competing interests

None declared. Ethical approval

Animal experiments were approved by the Norwegian Animal Research Authority and conducted according to the European Convention for the Protection of Vertebrates used for Scientific Purposes (local approval number 3674). Acknowledgements. The authors thank R. Greiner-Simonsen and S.H. Østvold from the Department of Clinical Dentistry for excellent technical assistance, L. Stuhr and I. Moen from the Department of

Biomedicine for consultations on the hyperbaric chamber, M.V. Jonsson from the Department of Clinical Dentistry for providing the radiological facilities, Y. Sun from the Royal Institute of Technology for producing the scaffolds, J.F. Owe and T. Nedrebø from the Hyperbaric Medical Unit for consultations on the study design, and J. Bevenius-Carrick for constructive comments on the manuscript.

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References 1. Arvidson K, Abdallah BM, Applegate LA, Baldini N, Cenni E, Gomez-Barrena E, et al. Bone regeneration and stem cells. J Cell Mol Med 2011;15:718–46. 2. Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev 2011;63:300–11. 3. Thom SR. Hyperbaric oxygen: its mechanisms and efficacy. Plast Reconstr Surg 2011;127(Suppl. 1):131S–41S. 4. Thom SR. Oxidative stress is fundamental to hyperbaric oxygen therapy. J Appl Physiol 2009;106:988–95. 5. Thom SR, Milovanova TN, Yang M, Bhopale VM, Sorokina EM, Uzun G, et al. Vasculogenic stem cell mobilization and wound recruitment in diabetic patients: increased cell number and intracellular regulatory protein content associated with hyperbaric oxygen therapy. Wound Repair Regen 2011;19:149–61. 6. Goldman RJ. Hyperbaric oxygen therapy for wound healing and limb salvage: a systematic review. Phys Med Rehabil 2009;1:471– 89. 7. Nilsson P, Albrektsson T, Granstrom G, Rockert HO. The effect of hyperbaric oxygen treatment on bone regeneration: an experimental study using the bone harvest chamber in the rabbit. Int J Oral Maxillofac Implants 1988;3:43–8. 8. Jan AM, Sandor GK, Iera D, Mhawi A, Peel S, Evans AW, et al. Hyperbaric oxygen results in an increase in rabbit calvarial critical sized defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:144–9. 9. Sirin Y, Olgac V, Dogru-Abbasoglu S, Tapul L, Aktas S, Soley S. The influence of hyperbaric oxygen treatment on the healing of experimental defects filled with different bone graft substitutes. Int J Med Sci 2011;8:114–25. 10. Jan A, Sandor GK, Brkovic BB, Peel S, Kim YD, Xiao WZ, et al. Effect of hyperbaric oxygen on demineralized bone matrix and biphasic calcium phosphate bone substitutes. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:59–66. 11. Sawai T, Niimi A, Takahashi H, Ueda M. Histologic study of the effect of hyperbaric oxygen therapy on autogenous free bone

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

913

grafts. J Oral Maxillofac Surg 1996;54:975–81. Jan A, Sandor GK, Brkovic BB, Peel S, Evans AW, Clokie CM. Effect of hyperbaric oxygen on grafted and nongrafted calvarial critical-sized defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:157–63. Danmark S, Finne-Wistrand A, Wendel M, Arvidson K, Albertsson AC, Mustafa K. Osteogenic differentiation by rat bone marrow stromal cells on customized biodegradable polymer scaffolds. J Bioact Compat Polym 2010;25:207–23. Schander K, Arvidson K, Mustafa K, Hellem E, Bolstad AI, Finne-Wistrand A, et al. Response of bone and periodontal ligament cells to biodegradable polymer scaffolds in vitro. J Bioact Compat Polym 2010;25:584– 602. Xing Z, Xue Y, Danmark S, Schander K, Østvold SH, Arvidson K, et al. Effect of endothelial cells on bone regeneration using poly(L-lactide-co-1,5-dioxepan-2-one) scaffolds. J Biomed Mater Res A 2011;96:349– 57. Andronova N, Albertsson AC. Resilient bioresorbable copolymers based on trimethylene carbonate, L-lactide, and 1,5-dioxepan-2one. Biomacromolecules 2006;7:1489–95. Wilson HD, Toepfer VE, Senapati AK, Wilson JR, Fuchs PN. Hyperbaric oxygen treatment is comparable to acetylsalicylic acid treatment in an animal model of arthritis. J Pain 2007;8:924–30. Bilic I, Petri NM, Bezic J, Alfirevic D, Modun D, Capkun I, et al. Effects of hyperbaric oxygen therapy on experimental burn wound healing in rats: a randomized controlled study. Undersea Hyperb Med 2005;32:1–9. Sumen G, Cimsit M, Eroglu L. Hyperbaric oxygen treatment reduces carrageenaninduced acute inflammation in rats. Eur J Pharmacol 2001;431:265–8. Wilson HD, Wilson JR, Fuchs PN. Hyperbaric oxygen treatment decreases inflammation and mechanical hypersensitivity in an animal model of inflammatory pain. Brain Res 2006;1098:126–8. Zhang Q, Chang Q, Cox RA, Gong X, Gould LJ. Hyperbaric oxygen attenuates apoptosis and decreases inflammation in an ischemic wound model. J Invest Dermatol 2008;128:2102–12. Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D, et al. Expression of osteoprotegerin, receptor activator of NFkappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res 2001;16:1004–14. Feghali CA, Wright TM. Cytokines in acute and chronic inflammation. Front Biosci 1997;2:d12–26. Weisz G, Lavy A, Adir Y, Melamed Y, Rubin D, Eidelman S, et al. Modification of in vivo

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25.

26.

27.

28.

29.

30.

Pedersen et al.

and in vitro TNF-alpha, IL-1, and IL-6 secretion by circulating monocytes during hyperbaric oxygen treatment in patients with perianal Crohn’s disease. J Clin Immunol 1997;17:154–9. Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765. Thomas MV, Puleo DA. Infection, inflammation, and bone regeneration: a paradoxical relationship. J Dent Res 2011;90:1052–61. Sheikh AY, Rollins MD, Hopf HW, Hunt TK. Hyperoxia improves microvascular perfusion in a murine wound model. Wound Repair Regen 2005;13:303–8. Roth V, Herron MS, Bueno Jr RA, Chambers CB, Neumeister MW. Stimulating angiogenesis by hyperbaric oxygen in an isolated tissue construct. Undersea Hyperb Med 2011;38:509–14. Hopf HW, Gibson JJ, Angeles AP, Constant JS, Feng JJ, Rollins MD, et al. Hyperoxia and angiogenesis. Wound Repair Regen 2005;13:558–64. Sheikh AY, Gibson JJ, Rollins MD, Hopf HW, Hussain Z, Hunt TK. Effect of hyper-

31. 32.

33.

34.

35.

36.

oxia on vascular endothelial growth factor levels in a wound model. Arch Surg 2000;135:1293–7. Jain RK. Molecular regulation of vessel maturation. Nat Med 2003;9:685–93. Sakata N, Chan NK, Ostrowski RP, Chrisler J, Hayes P, Kim S, et al. Hyperbaric oxygen therapy improves early posttransplant islet function. Pediatr Diabetes 2010;11:471–8. Goldstein LJ, Gallagher KA, Bauer SM, Bauer RJ, Baireddy V, Liu ZJ, et al. Endothelial progenitor cell release into circulation is triggered by hyperoxia-induced increases in bone marrow nitric oxide. Stem Cells 2006;24:2309–18. Thom SR, Bhopale VM, Velazquez OC, Goldstein LJ, Thom LH, Buerk DG. Stem cell mobilization by hyperbaric oxygen. Am J Physiol Heart Circ Physiol 2006;290:H1378–86. Wu D, Malda J, Crawford R, Xiao Y. Effects of hyperbaric oxygen on proliferation and differentiation of osteoblasts from human alveolar bone. Connect Tissue Res 2007;48:206–13. Muhonen A, Haaparanta M, Gronroos T, Bergman J, Knuuti J, Hinkka S, et al. Osteo-

blastic activity and neoangiogenesis in distracted bone of irradiated rabbit mandible with or without hyperbaric oxygen treatment. Int J Oral Maxillofac Surg 2004;33:173–8. 37. Okubo Y, Bessho K, Fujimura K, Kusumoto K, Ogawa Y, Iizuka T. Effect of hyperbaric oxygenation on bone induced by recombinant human bone morphogenetic protein-2. Br J Oral Maxillofac Surg 2001;39:91–5. 38. Chen AC, Lee MS, Lin SS, Pan LC, Ueng SW. Augmentation of osteochondral repair with hyperbaric oxygenation: a rabbit study. J Orthop Surg Res 2010;5:91.

Address: Torbjorn O. Pedersen Department of Clinical Dentistry Center for Clinical Dental Research University of Bergen A˚rstadveien 19 N-5009 Bergen Norway Tel: +47 55586540; Fax: +47 55586487 E-mail: [email protected]