Exosome secreted by human gingival fibroblasts in radiation therapy inhibits osteogenic differentiation of bone mesenchymal stem cells by transferring miR-23a.

Radiation-induced fibrosis is recently established as a main reason for osteoradionecrosis of the jaw (ORNJ), anti-eradiation fibrosis drugs achieve satisfactory therapeutic effects. However, the molecular mechanism remain to be fully elucidated. In this study, we found the inhibitory effect of irradiation activated gingival fibroblasts on osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). Moreover, irradiation-activated-fibroblasts significantly increased miR­23a expression in hBMSCs. Decreased miR­23a enhanced osteogenic differentiation of BMSCs, and elevated miR­23a inhibited this process via directly targeting CXCL12. Finally, exosome released from irradiation-activated-fibroblasts inhibited osteogenic differentiation of BMSCs, and these exosome mediated delivery of miR-23a and further regulated miR-23a/CXCL12 axis in hBMSCs. Therefore, our findings suggest that by transferring miR-23a, exosome secreted by human gingival fibroblasts in radiation therapy serves a vital role in osteogenic differentiation of hBMSCs, which may provide novel clinical treatments for ORNJ.

Osteoclastic expression of higher-level regulators NFATc1 and BCL6 in medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy: a comparison with osteoradionecrosis and osteomyelitis.

BACKGROUND: With an increasing indication spectrum of antiresorptive drugs, the medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy [MRONJ (BP)] is continuously gaining clinical relevance. Impaired osteoclast function, accompanied by altered cell morphology and expression of osteoclastic effector proteins, contributes to the pathogenesis of MRONJ (BP). However, the underlying regulatory mechanisms at a transcriptional level are unaddressed so far. These mechanisms are crucial to the development of disease-characteristic osteoclastic anomalies, that contribute to the pathogenesis of MRONJ (BP). NFATc1 is considered a master upstream osteoclastic activator, whereas BCL6 acts as osteoclastic suppressor. The present study aimed to elucidate the NFATc1 and BCL6 mediated osteoclastic regulation and activity in MRONJ (BP) compared to osteoradionecrosis (ORN) and osteomyelitis (OM) and normal jaw bone. METHODS: Formalin-fixed jaw bone specimens from 70 patients [MRONJ (BP) n = 30; OM: n = 15, ORN: n = 15, control: n = 10] were analyzed retrospectively for osteoclast expression of NFATc1 and BCL6. The specimens were processed for H&E staining and immunohistochemistry. The histological sections were digitalized and analyzed by virtual microscopy. RESULTS: Osteoclastic expression of NFATc1 and BCL6 was significantly higher in MRONJ (BP) specimens compared to OM and control specimens. NFATc1 and BCL6 labeling indices revealed no significant differences between MRONJ (BP) and ORN. The ratio of nuclear BCL6+ osteoclasts to cytoplasmic BCL6+ osteoclasts revealed significantly higher values for MRONJ (BP) specimens compared to OM and controls. CONCLUSION: This study displays that osteoclasts in MRONJ (BP) tissues feature increased expression of the higher-level regulators, paradoxically of both NFATc1 and BCL6. These observations can help to explain the genesis of morphologically altered and resorptive inactive osteoclasts in MRONJ (BP) tissues by outlining the transcriptional regulation of the pathomechanically relevant osteoclastic effector proteins. Furthermore, they strengthen the etiological delineation of MRONJ (BP) from OM and extend the osteoclast profiles of MRONJ (BP), OM and ORN and thus could lead to a better histopathological differentiation that can improve treatment decision and motivate new therapeutic concepts.

Protective role of α2-macroglobulin against jaw osteoradionecrosis in a preclinical rat model.

OBJECTIVE: We have previously demonstrated the effect of alpha-2-macroglobulin (α2M) in the remediation of radiation-induced cellular damage. Here, we investigated the protective effects of α2M in a preclinical rat model of jaw osteoradionecrosis (ORN). METHODS: Eighteen rats were divided randomly into three groups: the control group, the radiation therapy (RT) alone group, and the radiated mandibles pretreated with α2M (α2M + RT) group. One month after radiation, all left molar teeth were extracted. After another 3 months, the animals were sacrificed and body weight, histopathology, microcomputed tomography and immunofluorescence were evaluated in all groups. RESULTS: The RT group showed serious alopecia, bone exposure, inflammation, necrosis, fibrosis, and the absence of new bone formation within the socket. The α2M + RT group exhibited less alopecia than the RT group and slight inflammation and fibrosis in the bone marrow cavity. The cortical bone was similar to normal bone tissue. Interestingly, compared with RT group, serum superoxide dismutase levels in the α2M + RT group increased at the 1th day (P = 0.037), 14th day (P = 0.012), while reactive oxygen species levels clearly decreased at the 1th day (P< 0.001), 14th day (P = 0.007), and 28th day (P = 0.013). CONCLUSIONS: A clinically translational model of jaw ORN was successfully established and the application of α2M prior to radiation protected the bone from being injured by the radiation, possibly related to oxidative stress.

Protective effects of α­2­macroglobulin on human bone marrow mesenchymal stem cells in radiation injury.

Osteoradionecrosis of the jaws (ORNJ) is a complication of oral and maxillofacial malignancy that arises following radiotherapy; progressive jaw necrosis severely decreases the quality of life of patients. Human bone marrow mesenchymal stem cells (hBMMSCs) are a cell type with self­renewal and pluripotent differentiation potential in the bone marrow stroma. These cells are associated with bone tissue regeneration and are one of the primary cell types affected by bone tissue radiation injury. α­2­macroglobulin (α2M) is a glycoprotein­rich macromolecule that interacts with cytokines, growth factors and hormones to serve a variety of biological roles. In addition, α2M possesses radio­protective effects. The aim of the present study was to investigate whether α2M has protective effects against radiation injury of hBMMSCs. Cell counting kit­8 and colony formation assays were used to monitor cell proliferation. Western blot analysis and reverse transcription­quantitative polymerase chain reaction were used to detect Beclin1, microtubule­associated protein 1A/1B, sex determining region Y, Nanog, runt­related transcription factor 2, osteoglycin and manganese superoxide dismutase expression. The formation of calcium nodules was evaluated by Alizarin red staining after osteogenic induction. Flow cytometric analysis of Annexin­V and propidium iodide double staining was used to detect changes in apoptosis rate. Alkaline phosphatase and superoxide dismutase activity were determined using colorimetric assays. Reactive oxygen species levels were detected using 2',7'­dichlorodihydrofluorescein diacetate. The results of the present study revealed that α2M increased the rate of proliferation, reduced autophagy, alleviated pluripotent differentiation injury, increased the osteogenic differentiation ability and decreased the rate of apoptosis in hBMMSCs following irradiation via an antioxidative pathway. In conclusion, α2M exhibited protective effects against radiation injury in hBMMSCs and may be considered a potential therapeutic agent for the prevention and treatment of ORNJ.

Osteo-radio-necrosis (ORN) and bisphosphonate-related osteonecrosis of the jaws (BRONJ): the histopathological differences under the clinical similarities.

OBJECTIVES: Both osteoradionecrosis (ORN) and bisphosphonate associated osteonecrosis of the jaws (BRONJ) present clinically as regions of exposed necrotic bone. The study aimed to demonstrate the histopathological differences behind the observed clinical similarities. STUDY DESIGN: Ten ORN specimens and ten BRONJ specimens were used, as well as ten samples of normal mandibular bone as control. Two bone-specific stainings were used, i.e. Sirius Red for the study of the relative presence of collagen types I and III and toluidine blue for the study the osteon density. RESULTS: The Red Green Blue (RGB)-analysis of the specimens stained with Sirius Red identified significant differences between the chromatic patterns observed in bone preparations of patients suffering from ORN when compared to both BRONJ and control samples. Moreover, the osteon density of the BRONJ samples was significantly lower when compared to ORN and normal bone samples. CONCLUSIONS: The demonstrated differences in the bone architecture and in the bone collagen content between the two pathological conditions most likely reflect underlying pathophysiological differences.

Osteoradionecrosis in head-and-neck cancer has a distinct genotype-dependent cause.

PURPOSE: We performed a case-control study to establish whether the development of osteoradionecrosis (ORN) was related to a variant allele substituting T for C at -509 of the transforming growth factor-ß1 gene (TGF-ß1). PATIENTS AND METHODS: A total of 140 patients, 39 with and 101 without ORN, who underwent radiotherapy for head-and-neck cancer with a minimum of 2 years follow-up, were studied. None of the patients had clinical evidence of recurrence at this time. DNA extracted from blood was genotyped for the -509 C-T variant allele of the TGF-ß1 gene. RESULTS: There were no significant differences in patient, cancer treatment, or tumor characteristics between the two groups. Of the 39 patients who developed ORN, 9 were homozygous for the common CC allele, 19 were heterozygous, and 11 were homozygous for the rare TT genotype. Of the 101 patients without ORN, the distribution was 56 (CC), 33 (CT), and 12 (TT). The difference in distribution was significant, giving an increased risk of ORN of 5.7 (95% CI, 1.7-19.2) for homozygote TT patients (p = 0.001) and 3.6 (95% CI, 1.3-10.0) for heterozygotes (p = 0.004) when compared with patients with the CC genotype. Postradiotherapy dentoalveolar surgery preceding the development of ORN was associated with the CC genotype (p = 0.02). CONCLUSIONS: Our findings support the postulate that the development of ORN is related to the presence of the T variant allele at -509 within the TGF-ß1 gene.

Persistent F-18 deoxyglucose and Tc-99m methylene diphosphonate uptake in a lower extremity radiation port.

Four years after radiation therapy to the lower extremity for a bone metastasis, persistent F-18 FDG and Tc99m MDP uptake were noted in the radiation port. Presumably ongoing inflammatory cellular processes accounts for this finding.

In vivo tetracycline labeling of bone: an intraoperative aid in the surgical therapy of osteoradionecrosis of the mandible.

In this technical approach, we demonstrate that preoperative tetracycline administration helps distinguish between viable and necrotic bone in osteoradionecrosis (ORN) during surgery by exciting tetracycline fluorescence by black light. The difficult aspect of the operative approach lies in the decision of the extent of the debridement in order to prevent an iatrogenic fracture or progression of ORN. With this technique, vital bone, in contrast to necrotic bone, showed detectable fluorescence. Therefore, tetracycline fluorescence labeling may be used in attempts to facilitate the intraoperative decision making in the surgical therapy of osteoradionecrosis.

Measurement of oxygen partial pressure in the mandibular bone using a polarographic fine needle probe.

In this study, the oxygen partial pressure in the cancellous bone substance of the mandible was measured for the first time with a polarographic fine needle probe. This has so far only been established in soft tissue. The aim was to prove and to test the feasibility in principle of this method of measurement in order to ascertain the normal values for the O2 partial pressure in healthy bone. These values were afterwards compared with the results of measurements in areas of different pathological bone conditions in order to assess the clinical suitability of the method for "mapping" during mandibular resection. Measurements of oxygen partial pressure were made in a total of 42 patients (16 women, 26 men). Of these, 12 patients with clinically normal bones served as a control group. Seventeen patients had osteoradionecrosis following radiation treatment, and 13 patients presented with chronic osteomyelitis of the mandible. All measurements were carried out with a polarographic fine needle probe applied to the cancellous bone substance. The statistical analysis included a comparison of the mean values of the oxygen partial pressures measured. No statistical correlation between oxygen partial pressure and pH and hemoglobin values could be detected. The average oxygen partial pressure in the healthy mandibular bone was 71.4 mmHg. In non-healthy bone, the value fell to an average of 30.6 mmHg (osteoradionecrosis 32.3 mmHg, chronic osteomyelitis of the mandibular bone 28.4 mmHg). Statistically, the differences in the group values ascertained were highly significant (P < 0.005). The data found show that this method can be successfully used to detect poorly perfused bone. The values are reproducible and reflect the clinical situation. In the long term, the method appears to be a suitable diagnostic tool for assessing the oxygen supply in bone in studying various clinical problems related to bone surgery.

Expression of bone morphogenic protein 2/4, transforming growth factor-beta1, and bone matrix protein expression in healing area between vascular tibia grafts and irradiated bone-experimental model of osteonecrosis.

PURPOSE: For the surgical treatment of osteoradionecrosis after multimodal therapy of head-and-neck cancers, free vascular bone grafts are used to reconstruct osseous structures in the previously irradiated graft bed. Reduced, or even absent osseous healing in the transition area between the vascular graft and the irradiated graft bed represents a clinical problem. Inflammatory changes and fibrosis lead to delayed healing, triggered by bone morphogentic protein 2/4 (BMP2/4) and transforming growth factor (TGF)-beta(1). Given the well-known fibrosis-inducing activity of TGF-beta(1), an osteoinductive effect has been reported for BMP2/4. However, the influence of irradiation (RT) on this cytokine expression remains elusive. Therefore, the aim of the present in vivo study was to analyze the expression of BMP2/4, TGF-beta(1), collagen I, and osteocalcin in the transition area between the bone graft and the graft bed after RT. METHODS AND MATERIALS: Twenty Wistar rats (male, weight 300-500 g) were used in this study. A free vascular tibia graft was removed in all rats and maintained pedicled in the groin region. Ten rats underwent RT with 5 x 10 Gy to the right tibia, the remainder served as controls. After 4 weeks, the previously removed tibia grafts were regrafted into the irradiated (Group 1) and nonirradiated (Group 2) graft beds. The interval between RT and grafting was 4 weeks. After a 4-week osseous healing period, the bone grafts were removed, and the transition area between the nonirradiated graft and the irradiated osseous graft bed was examined histomorphometrically (National Institutes of Health imaging program) and immunohistochemically (avidin-biotin-peroxidase complex) for the expression of BMP2/4, TGF-beta(1), collagen I, and osteocalcin. RESULTS: Absent or incomplete osseous healing of the graft was found in 9 of 10 rats after RT with 50 Gy and in 1 of 10 of the rats with nonirradiated osseous grafts. Histomorphometrically, the proportion of osseous healing in the transition area was 17% in Group 1 and 48% in Group 2 (p = 0.001). Compared with the nonirradiated rats, reduced enchondral and perichondral ossification was found in the healing area after RT, with a reduction of BMP2/4 and osteocalcin expression. TGF-beta(1) and collagen I expression in the transition area to the irradiated osseous graft bed was significantly increased compared with that in the nonirradiated osseous graft bed. CONCLUSION: After RT, osseous healing of vascular bone grafts is significantly reduced and may be a result of radiation-induced inhibition of BMP2/4 and osteocalcin expression. In addition, induction of TGF-beta(1) and collagen I expression occurs. Because the effects of the TGF-beta superfamily are manifold and partially unknown, additional research directions could be in the exogenous application of BMP2/4 and inhibition of TGF-beta(1) by antibody treatment to search for appropriate therapeutic approaches for improving osseous healing in the irradiated graft bed.

Osteoradionecrosis (ORN) of the mandible: a laser Raman spectroscopic study.

Laser Raman spectroscopy has been used in this study to characterize mandibular bone samples from patients who had undergone radiation therapy for oral cancer. The paper discusses spectral changes resulting in osteoradionecrosis (ORN) of the mandibular bone, a serious complication that may occur after radiation therapy. Histopathological studies normally reveal the radiation damage on vascular canals and loss in bone cells, but will not reveal any structural or biochemical changes. All radiation-induced side effects are attributed to this hypovascularity and hypocellularity caused by early- and/or late-delayed effects. Our Raman studies on normal and ORN bone and on bone exposed to radiation, but not in the ORN state, show that irradiation produces immediate structural changes in the inorganic bone matrix with a slight loss in cells. ORN bone, in addition to the structural changes that had already occurred on radiation exposure, shows almost complete loss of cellular components. Since bone tissue is continuously being remodeled (dissolved and rebuilt) under normal conditions, our results suggest that the immediate structural changes in the calcium hydroxy apatite mineral part is not repaired in ORN, due to loss of the highly transient osteoblasts and osteoclasts resulting from destruction of stem cells. The spectral studies also show changes in the organic matrix, which is mostly type I collagen.

Therapeutic ultrasound for osteoradionecrosis: an in vitro comparison between 1 MHz and 45 kHz machines.

Mandibular osteoradionecrosis is a serious chronic complication which may follow radiotherapy. Therapeutic ultrasound is a highly effective, inexpensive and readily available means of promoting revascularisation and healing. 'Long wave' ultrasound increases penetration depth and, therefore, seems to be more appropriate than traditional high frequency ultrasound. The aim of this study was to compare a new treatment using 45 kHz with the current standard 1 MHz machine. A traditional 1 MHz machine, pulsed 1:4, at intensities of 0.1, 0.4, 0.7 and 1.0 W/cm2(SAPA) was compared with a long wave machine, 45 kHz, at intensities of 5, 15, 30 and 50 mW/cm2(SA). The ultrasound was applied to human gingival fibroblasts and mandibular osteoblasts in vitro. Cell proliferation (DNA synthesis) and collagen and non-collagenous protein synthesis assays were performed, using radiolabelled thymidine and proline, respectively. Controls were sham-insonated and all readings were given as a percentage of controls. Fibroblast proliferation increased by 47% at 0.7 W/cm2 (1 MHz) and by 43% at 50 mW/cm2 (45 kHz), and osteoblast proliferation increased by 52% at 1.0 W/cm2 (1 MHz), and by 35% at 30 mW/cm2 (45 kHz). Fibroblast collagen production increased by 48% at 0.1 W/cm2 (1 MHz), and by 44% at 15 mW/cm2 (45 kHz) and osteoblast collagen production increased by 55% at 0.1 W/cm2 (1 MHz) and by 112% at 30 mW/cm2 (45 kHz). Long wave ultrasound was, therefore, capable of inducing a comparable or even higher enhancement of bone formation compared with traditional ultrasound, which, with its greater penetration, may accelerate the healing effect of ultrasound on osteoradionecrosis. The suggested intensity for 45 kHz ultrasound is 30 mW/cm2.

The investigation of osteoradionecrosis of the mandible by 99mTc-methylene diphosphonate radionuclide bone scans.

The need for a reliable non-invasive investigation in osteoradionecrosis (ORN) of the mandible is discussed. The results of 99mTc-methylene diphosphonate bone scans using single photon emission tomography, and measuring dynamic uptake in 13 patients are presented. These suggest that sites of osteoradionecrosis may be defined by a paradoxically increased uptake of 99mTc-methylene diphosphonate.

The investigation of osteoradionecrosis of the mandible by near infrared spectroscopy.

The results of near infrared spectroscopy in eight patients with osteoradionecrosis of the mandible are presented. These suggest that sites of osteoradionecrosis may be marked by decreased amounts of deoxygenated haemoglobin. The advantages and potential of near infrared spectroscopy are discussed but the technique requires further refinement.

Studies in the radiobiology of osteoradionecrosis and their clinical significance.

The radiobiology of osteoradionecrosis is a complex of cellular death and cellular functional impairments from radiation energy transfers. Four studies of irradiated patients and a data base from 536 patients with osteoradionecrosis revealed separate pathophysiologic conditions for osteoradionecrosis induced by early trauma, osteoradionecrosis induced by late trauma, and spontaneous osteoradionecrosis. A large body of data suggested useful clinical guidelines for the management of irradiated patients. The guidelines, in part, include a recommendation for deferring radiation treatment for 21 days after tissue wounding, if possible; a relative contraindication to wounding tissue during a radiation course; a recommendation for the use of hyperbaric oxygen before wounding; and a strong recommendation to provide comprehensive dental care to the irradiated patient.