| Osteoradionecrosis of jaws(ORNJ), which is considered to be the most devastating long-term complications of head and neck radiotherapy. ORNJ can lead to intolerable pain, ulceration, trismus, sequestration of devitalized bone, fistulae, and pathologic fracture, making oral feeding impossible and sometimes even death can occur. While the clinical symptoms are severe,no effective methods existed to prevent and cure ORNJ, only surgical methods, such as sequestrectomy and bone grafting, can be used. Once happened, it is irreversible.The obscure pathogenesis and ambiguous definition of ORNJ limited its effective prevention and treatment.The pathophysiology of ORNJ has been a controversial topic since it was first recognized, and various theories have evolved with sequential changes in treatment,such as radiation, trauma, infection theory and hypoxic-hypocellular-hypovascular theory.The radiation-induced fibroatrophic(RIF) theory has arisen in recent years to elucidate the pathogenesis of ORN, and some antifibrotic medicines(pentoxifylline, tocopherol) have been used to treat ORNJ in clinical trial, with promising results.Without experimental evidence, the specific fibrotic process has not been fully clarified.As the key effector cell in the fibroticprocess,it is widely agreed that the myofibroblast is the most responsible for accumulation of the interstitial matrix and the consequent structural deformations associated with fibrosis, and it is also a therapeutic target in fibrotic disease treatment. Given that radiation-induced fibrosis is essentially a type of fibrosis, myofibroblast may also play an important role in the pathogenesis of ORNJ. However, as far as we know, no literature has been published on the relationship between myofibroblastand ORNJ yet.Therefore, to further clarify the radiation-induced fibrotic mechanism and to provide new ideas and methods for the prevention and treatment of ORNJ, this study established a clinically translational animal model of ORNJ according to the new clinical diagnostic standards, and observed the expression, distribution, differentiation and cell source of myofibroblasts in the affected tissues. Four parts were included in this study:Part 1:Establishment and evaluation of ORNJ animal modelObjectives:According to the new clinical diagnostic standards, we want to establish an osteoradionecrosis of jaws(ORNJ) rat model which could be used to simulate ORNJ at different clinical stages. Methods:Thirty six male SD rats were divided into 6 groups randomly. For group a1, b1 and c1, the left mandible of each rat were irradiated at doses of 7.0 Gy for 5 fractions, which were equivalent to total doses of 70 Gy. Group a0, b0 and c0 were sham irradiated, serving as control groups. Six weeks after irradiation, rats in group a0 and a1 were sacrificed, meanwhile, left mandibular molars of rats in group b1, b0, c1 and c0 were extracted. 12 weeks and 18 weeks after irradiation, the rats in group b0, b1 and in group c0, c1 were sacrificed respectively. All the rats' mandibles were taken. All samples were examined by clinical manifestation, micro-CT and histology methods. Results:Alopecia and retardation of central incisor growth,at the irradiated site were seen in group a1. Exposed necrotic bone was found in all irradiated mandibles of group b1 and c1. Reduced condyle volume, dental sepsis and obviously loose, necrotic bone of the irradiated mandible were found in group c1. Fibrosis and inflammtions were found in medulla in all irradiated groups. In group b1, small pieces of sequestrum were seen around the tooth root. And large sequestrum, pathological fracture, overwhelming fibrosis and disrupted incisor root were seen in group c1. Significant increment of empty lacunae and decrement of osteocytes were observed in all irradiated groups, accompanying with the decreased BV/TV( a1>b1>c1).Conclusions:A combined rat model that can stimulate three different stages of ORNJ was successfully established. And this model could be served as a new platform for future studies on pathogenesis and treatment of ORNJ.Objective: To explore the expression and distribution character of MFB in ORNJ, and to study the expression level of the molecules related to MFB differentiation and fibrotic formation in ORNJ.Methods:Immunohistochemical staining of ?-SMA, the specific molecular marker of MFB, was used on irradiated tissue samples to detect the existence of MFB. And the positive expression was calculated by semi-quantitative analysis.The number changes of positive staining, tissue localization of MFB, the time dependent alteration of MFB expression were observed.The expression level of TGF-?1, CTGF, Col?and Col?in ORNJ tissues were also detected by Western-Blot. Results:MFB was in stromal spindle shape and positive for ?-SMA staining. Overwhelming accumulation of MFB was located at the fibrosis medullary,around the sequestrum, in the devital alveolar bone, and invading to the root and parodontium of the incisor.The extent of tissue damage,?-SMA positive cell number and staining intensity, and the expression level of CTGF, Col?and Col? were increased over time, but the expression level of TGF-?1 was decreased. Conclusion:As other fibrotic diseases, ORNJ showed large number of MFB accumulation, and the expression level of MFB related to time, which indicated that MFB may also play an important role in the process of ORNJ formation, and provided more evidence for the RIF theory of ORNJ. The differentiation of MFB may be induced by TGF-?1 in the early time, while CTGF may play a more important role in the differentiation of MFB at later period.Part 3:Isolation, culture,and observation of MFB from the ORNJ tissuesObjective:To successfully culture MFB cells from the ORNJ tissues, which can be served as an in vitro cytological platform for further study of the RIF theory.Methods:MFB cells were cultured with tissue pieces harvested from the irradiated region of mandibles, and the fibroblasts were used as control. The cultured cells were detected by immunofluorescence staining of ?-SMA, and the expression level of ?-SMA, CTGF, Col?and Col? in cultured cells were observed by Western-Blot.The effects of TGF-?1 on the expression level of ?-SMA, CTGF, Col?and Col? were also observed.Results:Cells were observed growing from the irradiated explants within 1 week.At confluence, MFB cell cultures had uniform stromal spindle cell morphology, and the cell morphology was retained after several passages.Compared to the fibroblasts, the MFB cell cultures showed high-level immunofluorescence staining for ?-SMA, and the express level of CTGF, Col?and Col? were increased. After treatment with TGF-?1, MFB cells showed increased expression of ?-SMA, Col?and Col?.Conclusion:MFB Part 2:The expression characteristic of MFB in ORNJ tissuescan be successfully cultured from the ORNJ tissues, and the cells can secrete CTGF, Col?and Col? in vitro. TGF-?1 promoted the proliferation of MFB, and the expression of ?-SMA, CTGF, Col?and Col?.This in vitro study can further supported that MFB may be a key factor to promote the fibrotic process of ORNJ.Part 4: preliminary study in the cell source of MFB in ORNJObjective:To investigate whether the bone marrow mesenchymal stem cells(BMSCs) can be served as the local original cell source of MFB.Methods:BMSCs were isolated from rats' mandibles,cultivated, and identified. TGF-?1 solution was used to induce the differentiation of BMSCs. The cell proliferation, and the expression of ?-SMA, Col?and Col? were also detected.Results:Being induced by TGF-?1, the expression of ?-SMA in BMSCs increased significantly, and the cells secreted more Col?and Col?. Conclusion:BMSCs cultured from the mandibles can be induced into MFB,suggesting that local BMSCs were one of the cell sources of MFB in ORNJ. |
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