Jaw osteoradionecrosis (ORN) is a common and serious complication of radiation therapy for head and neck cancers. Bone marrow mesenchymal stromal cells (BMMSCs) are multipotent postnatal stem cells and have been widely used in clinical therapies. In the present study, we generated the mandibular ORN model in swine using a combination of single-dose 25-Gy irradiation and tooth extraction. A typical ORN phenotype, including loss of bone regeneration capacity and collagen collapse with the obliteration of vessels, gradually appeared after irradiation. After autologous BMMSC transplantation, new bone and vessels were regenerated, and the advanced mandibular ORN was treated successfully. In summary, we developed a swine model of jaw ORN, and our results indicate that autologous BMMSC transplantation may be a promising therapeutic approach for ORN.
Introduction
Osteoradionecrosis (ORN) of the mandible is a common and serious complication of radiation therapy of head and neck cancers (26,30), with an incidence ranging from 4% to 30% (3,4). Treatment of ORN, especially advanced ORN, is a challenging clinical issue, and there is no well-established large animal model for basic and clinical studies (30).
Recent studies have demonstrated that bone marrow mesenchymal stromal cells (BMMSCs), which are multipotent postnatal stem cells with the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, and neural cells (7,9,22,31,32), have therapeutic potential in irradiated tissues (2,15,37). The most significant differentiating pathway of BMMSCs is development into osteogenic lineages and vascular tissues, which has been demonstrated in vitro and in vivo (9,12). When transplanted subcutaneously into immunocompromised mice, BMMSCs generate organ-like structures containing newly formed bone and associated hematopoietic marrow components (10,24). BMMSCs also have the potential to regulate immune and inflammatory responses (23,25,35). BMMSCs have been used to treat a variety of medical conditions in humans, including bone fracture, severe aplastic anemia, systemic lupus erythematosus, and acute graft-versus-host disease (1,16,29).
BMMSCs have therapeutic potential in irradiated tissues (2,15,37); therefore, we used swine (miniature pigs) as a model to generate advanced ORN to determine if the condition could be ameliorated by treatment with autologous BMMSCs. Because of the high similarity between swine and humans in terms of histology and functions of the orofacial tissues (18,33,34), this experimental design may yield important preclinical information about the application of stem cell-based therapy for treating human mandibular ORN.
Materials and Methods
Animal and Irradiation Process
Fifteen inbred miniature pigs, 7–8 months old and 25–30 kg, were obtained from the Institute of Animal Science of the Chinese Agriculture University, Beijing. The animals were housed under conventional conditions with free access to water and food. This study was reviewed and approved by the Animal Care and Use Committee of Capital Medical University. The miniature pigs were anesthetized with a combination of 6 mg/kg ketamine chloride and 0.6 mg/kg xylazine before all experimental procedures. Computed tomography (CT, GE Medical Systems HiSpeed NX/i, General Electric Company, Waukeska, WI, US) was used to assist in designing of the irradiation plan (Fig. 1A). The center of the mandibular body was guaranteed to take up the full radiation dose. A single dose of 25 Gy equal to conventional fractionation of 65 Gy in 32 fractions of 2 Gy/day irradiation was delivered via the Varian Clinac 600C Clinac Linear Accelerators (Varian Oncology Systems, Palo Alto, CA, US; Department of Radiation Oncology, Beijing Friendship Hospital, Capital Medical University, Beijing, China) with 6 mV photon energy at 3.2 Gy/min using three-dimensional conformal radiotherapy (3D-CRT) (ADAC Pinnacle3 7.0 g; CA, US).
Figure 1. Generation of osteoradionecrosis (ORN) in swine. (A) Computed tomography (CT) was performed to aid in the design of the irradiation plan. Scale bar: 1 cm. Left: The box area was the target of irradiation. A single dose of 25 Gy equal to conventional fractionation of 65 Gy in 32 fractions of 2 Gy/irradiation was delivered via an electronic linear accelerator. (B) Conventional radiograph (scale bar: 1 cm) showing that tooth extraction in the irradiated area 2 months postirradiation resulted in destruction and defects in mandibular bone at 3 months postirradiation that had progressed at 5 months postirradiation (left, white arrow). No signs of mandibular ORN were observed in the group without tooth extraction (control group). (C) Two months after irradiation, ulceration on the surface of the jaw region was detected in both groups (arrow). In the tooth extraction group, exposure of necrotic bone was observed 3 months postirradiation (arrow). Scale bar: 1 cm. (D) Coronal CT images (scale bar: 1 cm) revealed rarefaction of cortical bone (dotted box) 2 months postirradiation and destruction and defects in cortical bone (dotted box) in the tooth extraction group 3 months postirradiation, in contrast to the contralateral nonirradiated sides of mandible.
Fifteen miniature pigs were used in this study, and all of the animals were irradiated. Three animals without tooth extraction served as the control group. The other 12 animals had their first molar in the irradiated field extracted 2 months after irradiation. Three pigs of the 12 ORN animals were used only to obtain dynamic samples for pathological and ultrastructural observations at scheduled points including before irradiation and 2, 3, and 5 months postirradiation. The other nine animals with ORN were randomly divided into a treatment group (n = 5) and control group (n = 4). The treatment group underwent BMMSC transplantation using hydroxyapatite/tricalcium phosphate (HA/TCP) particles (Sichuan University, Chengdu, China) as the carrier vehicle. The four control group animals were implanted with HA/TCP particles only. During the study, oral conditions such as periodontal tissue destruction before and after tooth extraction were observed in the irradiated animals.
Radiographic Observation
For radiographic observation, the miniature pigs were anesthetized before examination. Mandible bones were examined by radiography and CT analysis at scheduled time points.
Bone Formation Measurement
Bone formation was determined by calculation of the bone formation area over tissue area per field in hematoxylin and eosin (H&E)-stained sections at 200x magnification. Samples for bone formation determination were taken from the irradiated field of the mandible; three random samples (size, 0.5 cm3) were collected from each mandible. After H&E staining, the sections were photographed and the bone formation area was measured by Image-Pro Plus version 6.0 software. We counted three mandibular bone sections for each animal, with an average of 15 fields per section.
Electron Microscope Examination
Samples were cut into approximately 1-mm3 cubes and immediately immersed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 4 h, followed by decalcification with 5% EDTA with 0.2 M sucrose (pH 7.4) at 4°C for 2 weeks. We then washed the samples with sodium dimethyl arsenate buffer, postfixed in 1% osmium tetroxide, and dehydrated with gradient alcohol. The alcohol was replaced with isoamyl acetate, and the samples were dried and coated with gold. Samples were examined under an S-520 scanning electron microscope and transmission electron microscope (Hitachi Company, Japan).
Immunohistochemical Staining
For immunohistochemistry, sections were dewaxed and preincubated with 0.3% hydrogen peroxide in methanol for 30 min and blocked with 5% goat serum in PBS at room temperature for 1 h. Then they were incubated in a 1:100 dilution of mouse anti-Factor VIII antibody (Thermo Fisher Scientific, US). Next, the primary antibody was detected with an Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen, US). Sections were finally counterstained with 4,6 diamidino-2-phenylindole (DAPI) and viewed using fluorescent microscopy. Pictures were taken at 200x magnification.
Isolation, Culture, and Identification of BMMSCs
BMMSCs were obtained from bone marrow aspirated from the ilium of irradiated miniature pigs (n = 9) 5 months after irradiation (3 months after tooth extraction). After centrifugation for 10 min, cells were resuspended in culture medium and plated in 25-ml flasks. The culture medium contained Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin (Peking Union Medical College, Beijing, China). Cells were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO2. The medium was changed on day 5 and twice a week afterward. For identification of mesenchymal stromal cells, BMMSCs were subcultured in 24-chamber slides. Cells were fixed in 4% paraformaldehyde for 15 min, blocked with nonspecific antibodies, and incubated with either anti-Stro-1 (R&D, Minneapolis, MN, US) at dilutions of 1:200 to 1:500 or anti-vimentin (Chemicon, Temecula, CA, USA) at a dilution of 1:500 for 1 h according to the manufacturer's protocol. Samples were subsequently incubated with goat secondary antibodies for 45 min and observed under fluorescence microscopy. Nonimmune serum served as the negative control. Subsequently, sections were counterstained with DAPI. For flow cytometric analysis of BMMSCs, detached cells were permeabilized with PBS containing 0.1% (wt/v) saponin at room temperature for 20 min. After being blocked with normal serum, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated Stro-1 antibodies (R&D Systems) or FITC-conjugated CD146 antibodies (R&D Systems) for 30 min at room temperature. After three washes with PBS containing 0.1% saponin, fluorescence was analyzed by a FACS Calibur flow cytometer with CellQuest software (BD Bioscience, Palo Alto, CA, US). Positive cells were identified by comparison with the corresponding isotype controls (FITC-conjugated IgG); a false-positive rate of < 2% was accepted.
Autologous BMMSC Transplantation
BMMSCs were cultured for 1 month in vitro before transplantation. At 6 months after irradiation, the ORN areas were surgically operated on prior to BMMSC transplant. In this operation, bone sequestrum and necrotic soft tissues were removed, 3% hydrogen peroxide and saline were used to irrigate, and then orocutaneous fistulae was ablated and trimmed and the intraoral wound was closed. After irrigating of the wound with gentamicin, we filled the coloboma with BMMSC-HA/TCP mixture (or HA/TCP alone in the control group). Approximately 107 of ex vivo fifth expanded miniature pig BMMSCs were mixed with 4 g of HA/TCP particles (Sichuan University, Chengdu, China) and transplanted into the surgically treated ORN area of miniature pigs in the treatment group (n = 5). In the control group, 4 g of HA/TCP particles alone were delivered (n = 4). After the operation, the animals were moved to a climate- and light-controlled environment and allowed free access to food and water. Clinical follow-up observation was performed after transplantation by gross observation and CT. Six months after transplantation, all the miniature pigs were sacrificed, and transplanted bone samples were harvested for further examinations.
Statistical Analysis
All data are with a normal distribution and presented as the mean ± standard deviation (SD) of three independent experiments. The data were statistically analyzed with one-way analysis of variance (ANOVA) using SPSS 13.0; each value was compared with the control values. A value of p < 0.05 was regarded as statistically significant and was adjusted by the Bonferroni method to allow for the multiple comparisons.
Results
Generation of the ORN Model in Swine
There was no sign of ORN 2–5 months post-25 Gy irradiation in the swine without tooth extraction. However, following a combination of irradiation and tooth extraction (at 2 months postirradiation), mandibular bone destruction and defect was observable at 3 months postirradiation (Fig. 1B, C). We did not find any periodontal tissue destruction in these animals over the course of the experiments. CT images confirmed cortical bone defects in animals with ORN induced by irradiation (Fig. 1D). At the histological and cellular levels, we found abnormal changes in the bone matrix, ingrowth of fibrotic tissue in the bone marrow compartment, and loss of osteocytes in the mandible ORN area 3 months postirradiation (Fig. 2A). The vessels in the mandibular sections of both the ORN and control groups were obstructed and had begun to undergo vitreous degeneration (Fig. 2B). The bone formation area over tissue area decreased significantly 3 months after irradiation in the ORN group (30.18% ± 6.2% of ORN group vs. 90.98% ± 4.26% of baseline) (p < 0.05, n = 3) and it was lower than in the control group (30.18% ± 6.2% vs. 64.59% ± 9.43% of control group) (p < 0.05, n = 3) (Fig. 2C), indicating that these animals possessed the phenotype typical of mandibular ORN (6). Following irradiation, osteocytes exhibited dissolved nuclei, condensed chromosomes, and cell death (Fig. 2D), and the collagen fibers also lost their architectural integrity characteristic to bone (Fig. 2E).
Figure 2. Pathological observation of ORN in swine. (A) Histogram (H&E staining; scale bar: 50 μm) showed loss of bone and ingrowth of fibrotic or demineralization tissue in the bone marrow compartment in ORN group animals 3 months after irradiation. The coalescence of widened osteoblasts causes polycylic cavities in tissue sections from the control group 3 months after irradiation. (B) At 3 months postirradiation, vessels in mandibular sections of both ORN and control groups were obstructed and began to show signs of vitreous degeneration. (C) The bone formation area over tissue area decreased significantly 3 months after irradiation in the ORN group (30.18% ± 6.2% of ORN group vs. 90.98% ± 4.26% of baseline) (p<0.05, n = 3) and was lower than control group (30.18% ± 6.2% vs. 64.59% ± 9.43% of control group) (p < 0.05, n = 3). (D) Transmission electron microscope analysis showed normal osteocytes with organized nuclei and cytoplasm at baseline and loss of nuclear membrane and mixed nuclei and cytoplasm 3 months postirradiation. (E) Scanning electron microscope analysis showed normal collagen at baseline, and collapsed collagen at 3 months postirradiation.
BMMSC-Mediated Tissue Regeneration Cured ORN
Autologous BMMSCs were isolated and identified, as our previous reports (8,16,35), and then transplanted to the ORN lesion in the swine. Six months after transplantation, all of the BMMSC-treated swine exhibited complete recovery of skin and soft tissue defects. In contrast, transplantation of HA/TCP particle alone as a control failed to demonstrate any therapeutic effect (Fig. 3A). Irradiation-induced cortical bone disruption was also ameliorated in the BMMSC group, but not in the HA/TCP-only group (Fig. 3B, C). Moreover, histological examination revealed regeneration of normal bone/marrow structure following BMMSC transplantation, but there was fibrotic tissue following HA/TCP-only transplantation (Fig. 4A). In addition, new blood vessels (which express Factor VIII-related antigen) (19) were regenerated in the BMMSC group, while very few regenerated vessels were observed in the control group (Fig. 4B). The percentage of bone volume over tissue volume in the BMMSC transplantation group was almost the same as in the normal controls and was significantly higher than in the HA/TCP group (Fig. 4C). Microvessel density was significantly recovered in the BMMSC group (Fig. 4D). These findings indicate that autologous BMMSC transplantation was associated with the healing of ORN lesions in swine.
Figure 3. BMMSC transplantation cures ORN in swine. (A) After surgical removal of bone and soft tissue necrosis debris, 107 autologous bone marrow mesenchymal stromal cells (BMMSCs) were transplanted into the ORN site using hydroxyapatite/tricalcium phosphate (HA/TCP) as a carrier vehicle. Scale bar: 1 cm. Three months posttransplantation, orocutaneous fistulae (arrow) had disappeared, and soft tissues had completely recovered. Transplantation of HA/TCP alone did not cure ORN, as demonstrated by the existence of orocutaneous fistulae and damaged skin tissue (arrow). (B) Coronal CT imaging (scale bar: 1 cm) revealed organized mandibular bone reconstructed in the ORN region in the BMMSC-HA/TCP 6 months after treatment. In the HA/TCP-only trans plantation group, there were still defects in mandibular bone. (C) Mandibular reconstruction occurred in the BMMSC-HA/TCP group. Scale bar: 1 cm. The magnified buccal surface of a BMMSC-HA/TCP transplant mandible (shown in yellow box) was smoother than comparable surfaces in the HA/TCP-only group. The mandible is split horizontally alone the dotted line. Right: Horizontal surface of the bone. Blue arrows point to the buccal surface, and red arrows point to the lingual surface. In the HA/TCP-only group, we observed fibrous tissues occupying the ORN region along with unabsorbed HA/TCP particles and limited bone regeneration (purple circle). In contrast, most of the ORN region was reconstructed in BMMSC-HA/TCP group. Moreover, the mandibular marrow cavity (black dotted area) of the HA/TCP group animals revealed less mineralization than BMMSC-HA/TCP group animals.
Figure 4. BMMSC-mediated tissue regeneration in ORN. (A) H&E staining showed the regeneration in the ORN region treated with BMMSC-HA/TCP or HA/TCP alone. Scale bar: 50 μm. Bottom: Area in black box is magnified. Regenerated tissue with bone formation (yellow arrow heads, top and middle) and a number of organizing hematopoietic marrow elements (*) were observed in the ORN region of animals in the BMMSC-HA/TCP group. In the HA/TCP-only group, we observed fibrotic tissue and a small amount of bone-like tissue (+) around HA/TCP particles (#) and a lack of bone formation in HA/TCP transplants. Scale bar: 50 μm. Osteoclasts were observed in both groups (black arrow, bottom). (B) Newly regenerated blood vessels (yellow arrows, fluorescein isothiocyanate [FITC] labeled factor VIII) were found in the new bone tissues after BMMSC-HA/TCP transplantation. Limited regeneration of microvessels was seen in HA/TCP-only transplants. Scale bar: 50 μm. (C) Bone formation area over tissue area analysis indicated that mean amounts of regenerated bone tissues in the BMMSC-HA/TCP group (85.12% ± 3.31%) were almost the same as normal mandible bone (86.3% ± 2.54%) and significantly higher than the HA/TCP-only group (8.92% ± 1.07%) (p < 0.05, n = 3). (D) Microvascular density (MVD) assay based on immunodetection of factor VIII showed that MVD in the BMMSC-HA/TCP group was significantly higher than in the HA/TCP group (76.23 ± 8.9 of BMMSC-HA/TCP group vs. 27.54 ± 5.7 of HA/TCP group) (p < 0.05, n = 3) and slightly higher than in normal mandible bone group (55.6 ± 6.5).
Discussion
In the present study, we generated a mandibular ORN model in swine using a combination of single-dose 25-Gy irradiation and tooth extraction. After autologous BMMSC transplantation, new bone and vessels were regenerated, and the advanced mandibular ORN was treated successfully. The development of a large-animal model of ORN will provide researchers with the opportunity to elucidate the mechanisms underlying ORN and to explore potential therapeutic approaches, particularly those that are difficult to implement in small animal models such as rodent due to the small size of the orofacial region. The miniature pig is increasingly used as a large animal model for a variety of biomedical studies (11,14,17,28,36). In the present study, we used an advanced clinical radiotherapy technology, the electronic linear accelerator controlled by 3D-CRT, accurately delivered irradiation to the target area in swine to generate mandibular ORN. Although this is not an irradiation regimen that is used clinically, the calculated biologically effective dose of a single-dose of 25-Gy irradiation roughly equates to the delivery of a total dose of 65 Gy in conventional fractionation. Because of the high similarity between swine and humans in terms of the histology and functions of the orofacial tissues (9,11,17,18,28,33,34), we considered this approach to be optimal for generating a reliable ORN model in this large animal.
ORN can be either spontaneous or the result of an insult. It can occur through direct trauma (e.g., tooth extraction or cancer surgery or biopsy) or by exposure of irradiated bone to the hostile environment of the oral cavity secondary to overlying soft tissue necrosis. In daily activities, particularly during chewing, jaw bones are cyclically strained, leading to microdamage (8). This damage, which takes the form of numerous micro-cracks, can cause fracture because of the decreased capacity for repair of irradiated bone. As a result, spontaneous ORN, which is 10% of all ORN (5), may occur not only because of a bone remodeling deficiency but also as the result of an accumulation of microdamage. This could act as a type of “chronic trauma” that leads to ORN symptoms long after irradiation, as spontaneous ORN does not develop for at least 2 years in humans. In the present study, we found that irradiation alone did not generate observable ORN in the mandible 5 months postirradiation. Only the combination of irradiation and tooth extraction reliably generate ORN in swine. Trauma (tooth extraction in this study) could serve as a portal of entry for oral bacteria into the underlying bone, which may develop mandibular ORN. Although the mechanism by which tooth extraction contributes to ORN is unknown, this phenomenon is similar to what has been observed in human ORN (6).
If ORN is diagnosed early, local debridement and antibiotic treatment may be successful. In advanced ORN, the preferred treatment is hyperbaric oxygen coupled with necrotic bone resection, although there is some controversy regarding the efficacy of hyperbaric oxygen (21). The use of free tissue transfer has also been proposed as an effective treatment for advanced ORN (4,27). Because it is difficult to control advanced ORN in the long term, new, highly effective therapeutic approaches are required. In this study, we used mandibular ORN in swine as a model to investigate the potential of BMMSC-mediated tissue regeneration for the treatment of bone disease. HA/TCP not only osteoconducts but can also bond new bone to host bone, making it an ideal tissue-engineering scaffold. When there is adequate blood circulation, HA/TCP can even lead to regeneration of local bone defects in the presence or absence of osteogenic factors. However, after high doses of radiation, local osteoblasts may be lost and the blood supply may decrease so that the bone receives less oxygen than is required. Under these conditions, bone defects are unlikely to be resolved by HA/TCP alone. Moreover, the cumulative progressive endarteritis caused by radiotherapy results in insufficient blood supply (tissue oxygen delivery), which may affect normal wound healing. In the present study, animals were operated on by the same experienced surgeons under the same conditions, and antibiotics were used for both animal groups postoperation. Because it is difficult to cure advanced ORN by resection of orocutaneous fistulae and debridement alone without the transfer of an osteogenic, appropriately vascularized flap to the affected site, skin defects persisted in the HA/TCP-only group after transplantation. However, in the BMMSC plus HA/TCP swine, both soft and bone tissue healed. These results indicate that BMMSCs plus HA/TCP may potentially be an effective therapy for advanced ORN.
Our study demonstrates that substantial reconstruction had occurred 6 months after the BMMSC-HA/TCP complexes were implanted. A recent clinical study reported a case of regenerative reconstruction in the terminal stage of ORN following treatment with BMMSCs and progenitor cells (20). BMMSC transplantation may treat ORN via two mechanisms: by active tissue regeneration, in which the BMMSCs assist in the recovery of surgically removed necrotic bone tissue, and by organization of recipient-origin bone marrow, in which the BMMSCs assist in tissue revitalization. Furthermore, marked microvessel regeneration was evident in BMMSC-mediated tissue regeneration in ORN, suggesting that microvessel regeneration is critical in the treatment of ORN. A previous study performed by us demonstrated that green fluorescent protein (GFP)-labeled mesenchymal stromal cells (MSCs) could repair mandibular defects in swine; new bone tissues were derived from both GFP-labeled MSCs and host osteoblasts (38). Further studies are required for tracing transplanted MSCs in the long term and for the role of cytokines secreted by MSCs in ORN treatment. In a clinical study, about 37.5% of ORN patients appeared to have undergone hyalinized vessel obliteration (13). The mineralized tissues of the irradiated mandible were also damaged in this study; thus, microvessel regeneration may be very important for healing, because MSCs in the adjacent nonirradiated field could be carried to the site by blood vessels.
In summary, a single 25-Gy dose of irradiation combined with tooth extraction successfully generated ORN in swine model. Miniature pigs offer an excellent translational model for the use of autologous BMMSCs to treat ORN, and they may be of use in the development of a similar treatment approach in humans.
Acknowledgments
The authors thank Drs. Y.L. Sun and X.Y. Liu of Capital Medical University for their assistance. This work was supported by grants from the National Key Basic Research Program of China (No. 2007CB947304 and No. 2010CB944801 to S.W.); Funding Project for Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality (PHR20090510 to S.W.); the Funding Project to Science Facility in Institutions of Higher Learning Under the jurisdiction of Beijing Municipality (PXM 2009-014226-074691 to S.W., PXM 2011-014226-07-000066 to Z.F.); the National Natural Science Foundation of China (grant 30428009 to S.S., S.W.); Beijing Major Scientific Program (D0906007000091 to S.W.); and California Institute for Regenerative Medicine (RN 1-00572-1 to S.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflicts of interest.
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These authors provided equal contribution to this work.
Molecular Laboratory for Gene Therapy and Tooth Regeneration, Capital Medical University School of Stomatology, Tian Tan Xi Li No. 4, Beijing 100050, China. Tel: +86-10-67062012; Fax: +86-10-83911708; E-mail: [email protected]
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