Introduction
Osteoradionecrosis (ORN) is the most dangerous adverse clinical complication in patients with head and neck cancer following treatment with radiotherapy (RT), with an incidence of 5-15% (1-4). Most patients with head and neck cancer receive radiation therapy (5). Marx (6) reported that ORN occurs following bone tissue hypoxia, hypocellularity and hypovascularity in the early stage; however, the underlying mechanisms remain unclear. In the last decades, studies demonstrated that radiation-induced fibrosis promotes the development of ORN (7,8). Fibrosis is a common feature of end-stage ORN (9) and is rarely reversible. However, Xu et al (10) reported that reducing local blood flow and subsequent hypovascularity could lead to an imbalance in bone remodeling, suggesting that microvascular damage has a major impact on ORN early stage. However, whether IR directly causes bone cell death, triggers other factors that suppress bone cell function or results in a combination of both effects, remain unknown.
RT can induce the generation of high levels of reactive oxygen species (ROS), leading to microvascular structure necrosis, local ischemia and subsequent tissue loss (11). ROS overproduction also inhibits the survival of tissue-borne multipotent stromal cells in numerous tissues (12). In addition, Mazur et al (13) reported that irradiation (IR) can induce bone marrow cell apoptosis. Although the underlying mechanisms of radiation injury caused by ROS have been extensively studied, a valid medical therapy designed to prevent the deleterious side effects of radiation in patients is not currently available (14).
α2-macroglobulin (α2M) is an acute-phase protein that exerts radioprotective effects (15,16). Pretreatment of rats by total-body irradiation with α2M can significantly reduce radiation-induced DNA damage and completely restore liver function and body weight (17,18). In addition, a previous study reported that the rat acute-phase α2M protein serves a central role in amifostine-mediated radioprotection, increasing the protective effect by 45-fold (19). As demonstrated in a study from our laboratory, α2M maintains the osteogenic potential of human bone marrow mesenchymal stem cells (hBMMSCs) following IR (20). In particular, hBMMSCs are among the main cells damaged during bone tissue radiation (21,22). Radiation can alter hBMMSC proliferation, induce genomic DNA damage and micronucleus formation and inhibit the osteogenic differentiation of hBMMSCs (23).
The radioprotective effect of α2M in the late stage of ORN, with a notable effect on the fibrosis in bone marrow, has been demonstrated in our previous study (24). However, this treatment could not reverse the development of ORN disease process. The present study aimed therefore to examine whether α2M could exert a radioprotective effect on early-stage ORN in vivo and in vitro, and to explore its underlying mechanisms.
Materials and methods
Establishment of the ORN animal model
A total of 72 Sprague-Dawley male rats (12-week old, ~350 g) that were purchased from the Laboratory Animal Center of Sun Yat-sen University, were randomly assigned into four groups as follows: control group, α2M group, RT group and α2M + RT group (n=18 animals per group). Following anesthesia with pentobarbital (1%; 50 mg/kg, intraperitoneal injection), a subperiosteal injection of 0.5 ml of 2 mg/ml α2M (cat. no. 441251-10MG; EMD Millipore) was prophylactically administered to mice from the α2M and α2M + RT groups 30 min prior to IR, whereas the control and RT groups were injected with the same volume of physiological saline. Following preoperative anesthesia induction, a single dose of 30 Gy (voltage, 160 kV; current, 25 mA, 2.75 Gy/min, 10.91 min; Rad Source Technologies, Inc.) was delivered to the left mandible of mice from the RT and α2M + RT groups. Improved 3D-printed mandible fixtures and a lead sheet were used to precisely deliver radiation to the mandible and protect the surrounding tissues by shielding them from IR. The dosage was based on an extensive review of the literature (25,26) and previous work from our laboratory (20). All rats were fed a liquid diet during the first week after IR. The flow diagram for the experiment is shown in Fig. SI. Six rats in each group were sacrificed with pentobarbital (1%, 120 mg/kg, intraperitoneal injection) on day 7, 14 and 28 following IR. Death verification included cardiac arrest, respiratory arrest and disappearance of various reflexes. Unilateral mandible, tongue and buccal mucosal tissue were excised and examined immediately. In addition, humane endpoints included the following: 20-25% body weight loss, complete loss of appetite for 24 h or loss of appetite (<50% of normal) for 3 days; weakness (inability to eat or drink); dying (mental depression accompanied by hypothermia without anesthesia or sedation); rapid and abdominal breathing with pronounced effort; and severe infection. Six rats in RT group and three rats in α2M + RT group reached the humane and experimental endpoints exactly on day 7 after irradiation. After euthanasia by injection of pentobarbital (1%, 120 mg/kg), the observation time was 5-10 min to make sure animal died under fearless and painless condition. If no observation of cardiac and respiratory arrest over 10 min, an overdose of the drug would be given.
Histological staining and quantitative bone histomorphometry
Mandible bone, tongue and buccal mucosa tissues were collected from rats and immediately immersed in 4% formaldehyde at room temperature for 24 h. Tongue and buccal mucosa tissues were embedded into paraffin wax through dehydration and wax soaking. In addition, all bone samples were subjected to decalcification. Briefly, mandibular bone tissues were transferred from 4% paraformaldehyde into the decalcifying solution (cat. no. G1105; Servicebio) and sealed at 25-30°C, and the solution was replaced every three days until the bone softened. All tissues were then sliced into 0.5 mm sections. Sections were then subjected to hematoxylin and eosin (H&E) and Masson's trichrome staining. The samples were observed under a scanning electron microscope (NanoZoomer S360; Japan SLC, Inc.). The region of interest (ROI) was drawn from the first molar to the last molar, and both buccal and lingual cortices were visualized down to the nadir of the incisor root. With ×200 magnification, nine high-power-field (HPF) images were randomly selected from different sections of each specimen, and empty lacunae were counted by three independent reviewers, as previously described (27). The number of fat vacuoles was manually counted in the different images and reported as the mean number per HPF image in the marrow cavity (28).
Tartrate-resistant acid phosphatase (TRAP) staining and terminal uridine deoxynucleotidyl nick end labeling (TUNEL) staining
Osteoclasts in the four groups (control group, α2M group, RT group and α2M + RT group) were stained with a TRAP kit (cat. no. G1050; Servicebio) according to the manufacturers' instructions. TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts under a microscope (Leica DMi1; Leica Microsystems GmbH). TRAP-positive osteoclasts were quantified with Image J software (version 1.8.0; National Institutes of Health), in terms of osteoclast number related to total bone surface (N. Oc/BS) (29).
Apoptotic cells in the four groups (control group, α2M group, RT group and α2M + RT group) were quantified with a TUNEL apoptosis detection kit (cat. no. 11684817910; Roche Diagnostics) according to the manufacturers' instructions. Five images were randomly selected from each specimen, and the numbers of TUNEL-positive cells were counted in HPFs (magnification, ×400). The apoptosis index (AI) was determined as follows: AI = (number of TUNEL-positive cells/total number of counted cells) x 100%. Image-Pro Plus software (version 6.0, Media Cybernetics, Inc.) was used for automatic counting as previously described (30).
Immunohistochemistry of bone and soft tissues
The 0.5-mm thick embedded sections of four groups (control group, α2M group, RT group and α2M + RT group) were dewaxed by microwave-heating at 60°C for 2 h and hydrated using graded ethanol (100, 95, 90 and 80%) at room temperature for 5 min each time. Antigen retrieval was completed at 98°C for 20 min followed by quenching for 20 min. Sections were blocked with 1% bovine serum albumin (cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.) dissolved in double distilled H2O for 30 min at room temperature. The bone and tongue sections were incubated with primary antibodies against 8-OHdG (1:100; cat. no. ab26842; Abcam) whereas the buccal mucosa and tongue tissues were incubated with primary antibody against SOD2 (1:100; cat. no. 24127-1-AP; ProteinTech Group, Inc.) in a wet box at 4°C overnight. After washing twice in PBS for 5 min, sections were incubated with goat anti-rabbit IgG (1:100; cat. no. GAR007; Multi Sciences Biotech, Co., Ltd.) or goat anti-mouse IgG (1:100; cat. no. GAM007; Multi Sciences Biotech, Co., Ltd.) at room temperature for 30 min. Following incubation with DAB reagent (cat. no. DA1010; Beijing Solarbio Science & Technology Co., Ltd.) for 10 min at room temperature and counterstain in haematoxylin, sections were mounted with neutral balsam (cat. no. G8590; Beijing Solarbio Science & Technology Co., Ltd.). Images were captured using a light microscopy (Leica Microsystems GmbH). The relative optical density (ROD) was calculated using Image-Pro Plus software as the ratio of the integrated optical density (IOD) to the measured area, as previously described (31).
Cell proliferation and apoptotic sensitivity of irradiated hBMMSCs
The primary hBMMSCs obtained from ScienCell Research Laboratories, Inc. (cat. no. 7500) were cultured in Mesenchymal Stem Cell Medium (cat. no. 7501; ScienCell Research Laboratories, Inc.) supplemented with 1% mesen-chymal stem cell growth supplement (ScienCell Research Laboratories, Inc.), 1% penicillin/streptomycin solution (ScienCell Research Laboratories, Inc.) and 5% fetal bovine serum (ScienCell Research Laboratories, Inc.) and placed in a humidified 5% CO2 incubator at 37°C. Cells were seeded in 96-well plates at a density of 5×103 cells/well (6 duplicate wells per group) and were divided into four groups: Control group, no treatment; α2M group, 0.5 mg/ml α2M pretreatment for 24 h with no irradiation; RT group, no α2M pretreatment + irradiation with 8 Gy; and α2M + RT group, 0.5 mg/ml α2M pretreatment for 24 h + one dose of 8 Gy dose irradiation (20). After cell treatment for 24, 48 and 72 h, 10 µl of the Cell Counting Kit-8 solution (CCK-8; cat. no. C0038; Beyotime Institute of Biotechnology) was added to each well. After incubation for 2 h at 37°C (5% CO2), absorbance value was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).
Cell apoptosis was assessed with an Annexin V-FITC/PI apoptosis detection kit (cat. no. KGA108-1; Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturers' instructions. Briefly, hBMMSCs were seeded at the density of 2×105 cells/well in 6-well plates and incubated for 24 h, followed by 0.5 mg/ml α2M treatment 30 min before irradiation. Treated cells were cultured for 24 h and harvested. Cells were washed with cold PBS and resuspended in 100 µl of Annexin V-FITC binding buffer. Next, 5 µl of Annexin V-FITC and 10 µl of PI Staining Solution were added to the samples and incubated for 15 min in the dark at room temperature. Subsequently, 400 µl of 1X binding buffer was added, and cells were analyzed using flow cytometry (Beckman Cytomic FC500; Beckman Coulter). Data were analyzed using FlowJoTM software (v 10.6.2; BD Biosciences)..
ROS detection in irradiated hBMMSCs
The hBMMSCs were cultured in 6-well plates at a density of 2×105 cells/well in cell culture medium for 24 h before ROS detection. Cultured cells were divided into four groups: control group; α2M group; RT group; and α2M + RT group). ROS detection was performed with a ROS assay kit (cat. no. S0033; Beyotime Institute of Biotechnology) according to the manufacturers' instructions. Culture medium was removed and cells were incubated with 10 µmol/l DCFH-DA solution diluted in serum-free medium in the dark at 37°C for 20 min. Cells were washed three times with serum-free medium to remove excess dye. ROS intensity was observed through inverted fluorescence microscope (Olympus IX73; Olympus Soft Imaging Solutions GmbH).
ROS level was also detected by flow cytometry. At 24 h after IR, hBMMSCs were harvested and washed three times with PBS. DCFH-DA (cat. no. S0033; Beyotime Institute of Biotechnology) was added to each group at a final concentration of 10 µmol/l and incubated for 20 min at 37°C. Finally, the samples were washed and assessed using flow cytometer (BD FACSCanto II; BD Biosciences) and data were analyzed using FlowJo™ software (version 10.6.2; BD Biosciences).
Western blotting analysis of Akt, HO-1 and SOD2 expression
The expression of Akt, heme oxygenase-1 (HO-1), and SOD2 proteins was detected by western blotting. Briefly, cells were lysed using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) for 15 min on ice and centrifuged at 12,000 rpm for 15 min at 4°C. Protein concentration was measured using a BCA assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Proteins (50 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes.
Membranes were blocked with 5% skimmed milk at room temperature for 1 h and were incubated with primary antibodies presented in Table SII at 4°C overnight. Membranes were washed twice with TBST (cat. no. T1085; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min and were incubated with secondary antibodies presented in Table SII at room temperature for 2 h. Bands were detected with Immobilon western electrochemiluminescence HRO substrate (cat. no. P36599, Millipore) using a chemiluminescence equipment (Bio-Rad Laboratories, Inc.). The data were analyzed via densitometry using ImageJ software (version 1.8.0; National Institutes of Health).
Statistical analysis
Data were analyzed using one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. If the collected data were not normally distributed, a Kruskal-Wallis ANOVA with ranks was used. The results were analyzed using SPSS 20.0 software (IBM Corp.), and all data with a normal distribution are presented as the means ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
Results
α2M alleviates alopecia and mucositis at an early stage
Within 7 days and during the recovery phase, all IR rats exhibited signs and symptoms of IR side effects, including alopecia (Fig. 1A and B) and inflammation of the buccal mucosa and tongue (Fig. 1C and D). In addition, as presented in Fig. 1E, α2M-treated rats presented a tendency to gain weight earlier and recovered faster compared with rats in the RT group on days 7 and 14 after IR (P<0.05). As presented in Fig. 1F, the food intake of all IR rats began to decrease suddenly after IR and reached the lowest level on day 7 after IR, before returning to normal until the end of the study period.
α2M protect against IR-induced pathophysiological changes induced in bone and soft tissues
Compared with the control group, the cell loss and vascular obstruction in bone marrow was observed in the pathological sections from the RT group. However, compared with the RT group, the α2M + RT group presented a decreased cell loss and vascular obstruction following IR (Fig. 2A). The number of empty bone lacunae in the RT group was significantly increased compared with the control group; however, the α2M + RT group showed lower bone cell death within 28 days compared with the RT group (Fig. 2B). Results from Masson's trichrome staining demonstrated no fibrosis in the bone marrow cavity in the IR groups until day 28 (Fig. 2C). In particular, a 1.5-fold increase in fat vacuoles was observed in the marrow cavity of the RT group compared with the control group (Fig. 2D). Mucosal inflammation and ulcers were observed in the IR rats at the early stage but were significantly decreased in the α2M + RT group compared with the RT group in the buccal mucosa (Fig. 2E) and lingual mucosa (Fig. 2F).
α2M inhibits osteoclasts activity and apoptosis of cells in the bone tissue
As presented in Fig. 3A and B, a significant increase in the number of TRAP-positive multinucleated cells was observed in the RT group compared with the control group on days 7 and 14 following IR (P<0.05). In addition, the results from the TUNEL assay demonstrated a significant increase in the percentage of TUNEL-positive cells in the RT group compared with the control group on days 7 and 14 after IR (Fig. 3C and D; P<0.05).
SOD2 and 8-OHdG levels in bone and soft tissues
Pretreatment with α2M significantly inhibited the IR-induced increase in 8-OHdG level (P<0.05; Fig. 4A-D). Furthermore, following pretreatment with α2M, SOD2 expression was significantly increased in both the mucosa and tongue, as presented in Fig. 4E-H (P<0.05).
α2M reduced the apoptosis rate, improves the antioxidant capacity and increases Akt phosphorylation in IR-treated hBMMSCs
Four groups of cells were incubated in 6-well plates for 24 h after IR (Fig. 5A). IR-treated hBMMSCs had a lower proliferation rates than cells in the control group at 48 and 72 h (P<0.001). However, α2M pretreatment resulted in a higher proliferation rate compared with the RT group (P<0.01; Fig. 5B). In addition, significantly increased apoptosis rate was observed in the RT group compared with the control group (P<0.01), and pretreatment with α2M prior to IR decreased the apoptosis rate (P<0.05; Fig. 5C and D). Significant decrease in ROS levels were detected following IR in the α2M-treated group compared the RT group (P<0.05; Fig. 5E-H). Furthermore, the protective effects of α2M on the Akt signaling pathway were observed in the IR-treated hBMMSCs using Western blotting. Increased expression of SOD2 and HO-1 was observed in groups pretreated with α2M (Fig. 5I and J).
Discussion
Numerous studies have reported some changes in the rat mandible 3 months following IR (32,33). Other studies demonstrated changes in the soft tissues within two weeks after IR (34-36). However, the sequential changes observed in bone and soft tissues at the early stage after IR remain unclear. Furthermore, ORN is a disease that causes both bone and soft tissue injuries. To the best of our knowledge, the present study was the first to use a rat ORN model to investigate the early stage of ORN in bone and soft tissues.
As previously reported, IR with 30 Gy represents an ideal dose to induce a significant change in bone injury in rats (25,26). However, based on a previous study that applied graded doses of 2, 4, 8 and 12 Gy, the 8 Gy IR dose should be used to induce hBMMSC injury in vitro (20).
In the present study, cells in the marrow cavity were largely lost, vascular injury was significantly expanded and more fat vacuoles were present in the RT group compared with the control group. Conversely, the α2M + RT group presented resistance to IR-induced damage. As previously described by Xu et al (10), irreversible damage to the blood vessels caused by radiation serves a crucial role in the development of ORN. Bléry et al (25) reported an increase in the number of fat vacuoles in mandibular bone marrow following IR. In the present study, serious soft tissue injury was observed in the RT group, which was inhibited by α2M treatment, specifically by reducing ulceration and erosion. These findings were consistent with those from previous studies (18,19), and suggested that the potent effect of α2M may be due to its ability to decrease the IR-induced toxicity in the bone and the soft tissue.
Osteocytes serve an essential role in bone reconstruction and metabolism (37). The number of osteocytes undergoing cell death is commonly calculated by counting empty lacunae during histological observations (38). IR induces bone loss and increases osteoclast numbers and activity (39,40). In the present study, the number of empty lacunae increased after IR, and IR increased the activity of osteoclasts within the first week, which was consistent with previous in vivo studies describing significant increase in the number and activity of osteoclasts within the first three days after irradiation (41,42). However, after the first week, the number of osteoclasts decreased in the RT group, which was consistent with a previous in vivo study reporting that the cell number was decreased after 10 days (43). Because bone remodeling involves a balance between osteoclast resorption and osteoblasts activation, the findings from the present study suggested that the IR-induced disruption of this balance may contribute to the development of ORN. However, α2M treatment had a significant inhibitory effect on the number of TRAP-positive osteoclasts.
ROS levels are indicators of the degree of oxidative stress (44). The biomarker 8-OHdG was first reported by Kasai et al (45) in 1984 and is now commonly used as a sensitive indicator of oxidative stress-induced DNA damage (46). Furthermore, SOD2, which is a well-known enzyme located in the mitochondria, serves anti-apoptotic role by directly decreasing cellular ROS levels (47). α2M was previously reported to inhibit ROS production by increasing SOD2 activity and to reduce oxidative stress-induced DNA damage, confirming its protective effect on radiation-induced injury in liver tissue (48). The present study aimed therefore to determine whether the radioprotective effect of α2M could be associated with an antioxidant activity. The results reported increased ROS and 8-OHdG levels following IR, which was reversed after treatment with α2M. Furthermore, results from western blotting demonstrated an increased expression of SOD2 and HO-1 following α2M treatment, suggesting that α2M may possess some antioxidative effects. In addition, α2M stimulated Akt activation, which is known to be activated by oxidative stress (49,50).
The present study presented some promising results, which suggested the potential use of α2M prophylactically in clinical trials. The radioprotective effect of α2M has been demonstrated by pathological alteration of rat jaw bones in the early stage of irradiation, and not only physical signs of hard and soft tissues alteration were attenuated, but the pathological changes in irradiation jaw bone were also alleviated.
This study presented some limitations. Firstly, it was not clarified how α2M could regulate SOD2 expression, and further investigation is required. Regarding the radiation-induced impairment of oral epithelial cell proliferation and cell production, further investigation about the underlying mechanism of α2M on irradiated soft tissue is required. Secondly, no in vitro experiments on the effect of α2M on the apoptosis rate, antioxidant capacity and Akt pathway in oral epithelial cells was performed. Further in vivo experiments will explore the underlying mechanism of α2M in order to determine whether SOD2 expression was affected by gene knockout or Akt over-expression. In addition, the duration and time points of α2M management in patients must be further evaluated in order to determine its effectiveness.
The association between the early stage development of ORN and oxidative damage remains unclear. Our previous study discussed the radioprotective effect of α2M on an animal model of jaw osteonecrosis established by fraction dose irradiation (24). In the present study, a modified rat model of osteoradionecrosis with shorter experimental period, which in the early stage of osteoradionecrosis of the jaw (ORNJ), was successfully established by a single 30 Gy dose of radiation. The present study demonstrated that ORN was associated with oxidative damage, and that α2M may upregulate the activity of the antioxidant enzymes SOD2 and HO-1, leading to decreased levels of ROS and 8-OHdG. Furthermore, the expression of p-Akt in hBMMSCs was elevated following α2M administration, which indicated that Akt phosphorylation may serve a role in α2M effect against radiation-induced mandibular bone damage. The present study not only focused on the physical signs and pathological changes of irradiated bone tissues, but also on radiation-induced responses in soft tissues, at the early stage of ORN rat model. Based on these results, this study demonstrated that α2M may protect rats from IR-induced injury by improving the endogenous antioxidant system and inhibiting the oxidative damage.
In summary, the present study was the first to demonstrate that α2M may exert a radioprotective effect in early-stage ORN. The results from this study suggested that α2M may represent a novel radioprotective and therapeutic agent for ORN in patients with cancer. In addition, oxidative damage observed during early-stage ORN provides a new target for preclinical studies.