| Objective: Bisphosphonate-related osteonecrosis of the jaw(BRONJ)is a serious complication after the use of bisphosphonates.The main symptoms are non-union of soft tissue,bone exposure,swelling and pus in the maxillofacial region.At present,there is no effective treatment for BRONJ.Rapid and sufficient angiogenesis can create conditions for soft and hard tissue repair and regeneration,and is also for the key to treat BRONJ.Stem cells from apical papilla(SCAP)are derived from dental apical papilla in young permanent tooth which are rich in blood vessels.The exosomes,as a bioactive substance secreted by the cells,has similar biological functions with the SCAP and plays an important role in promoting angiogenesis.This study is aimed to investigate the therapeutic effects of exosomes derived from SCAP(SCAP-Exo)on BRONJ rats,and detect its effects on the proliferation,migration and angiogenesis ability of human umbilical vein endothelial cells(HUVECs)treated by zoledronic acid(Zol)through in vitro studies,which provides experimental foundations and theoretical basis for clinical transformation application with exosomes derived from dental stem cells.Methods:1.SCAP was extracted by enzyme digestion.The ability of multidirectional differentiation was detected by osteogenesis and adipogenesis induction,and the surface markers of stem cells were detected by flow cytometry.2.SCAP-Exo was extracted by ultrahigh-speed centrifugation.Morphological characteristics were observed by transmission electron microscope,particle size was analyzed by nanoparticle tracking analysis(NTA),and the expression of exosomespecific protein CD9,Alix,and endoplasmic reticulum-specific protein,calnexin,was detected by Western blot.3.Build the BRONJ rat model by intraperitoneal injection of Zol and extraction of the left maxillary first and second molars.Observe the wound healing of tooth extraction wound with photographs,and necrotic bone with hematoxylin-eosin(H&E)staining.4.The BRONJ rats were injected into the extraction socket with a concentration of 1μg/μL SCAP-Exo 150 μL(SCAP-Exo group)or an equivalent volume phosphate buffer solution(BRONJ group)immediately in the extraction socket,and injection of equal volume phosphate buffer solution is normal control group(Control group)in the extraction socket after tooth extraction of healthy rats.SCAP-Exo was labeled with PKH26 fluorescence,and the local retention of SCAP-Exo in tooth extraction wound was observed by tracing experiment.At 2 w and 4 w after operation,photographs were taken to observe the wound healing of the tooth extraction wound in rats.H&E staining was used to observe the blood vessels in the gingiva of the tooth extraction wound.Immunohistochemical staining was performed to observe the expression of CD31 in the gingiva of the tooth extraction wound.Immunofluorescence staining was used to observe the expression of VEGFA in the gingiva of the tooth extraction wound.Microcomputed tomography(Micro-CT)was used to evaluate the condition of alveolar bone,and calculate the bone volume/total volume(BV/TV)in the tooth extraction wound.H&E staining was used to observe the necrotic bone in tooth extraction wound,and calculate the number of empty bone lacunae and necrotic bone/total bone(NB/TB)in the alveolar bone of the tooth extraction wound.Masson staining was applied to observe the collagen fibers.5.The effect of Zol of different concentrations on the proliferation ability of HUVECs was detected by MTT to determine the pretreatment concentration of Zol.With 5 μM Zol-treated HUVECs for 48 h as Zol group,HUVECs in the SCAP-Exo group were pretreated with 5μM Zol for 24 h,and added SCAP-Exo(20 μg/m L)for another 24 h.The Control group was cultured normally.MTT and Ki-67 immunofluorescence staining were used to detect the proliferation ability of HUVECs.Scratch assay and Transwell cell migration assay were applied to identify cell migration ability.The total tube length,the number of meshes,the number of nodes and the number of junctions of the tubes formed by HUVECs were detected by tube formation assay.The expression level of angiogenesis related protein CD31 in HUVECs was detected by Western blot.The matrigel plug assay is aimed to detect the angiogenesis ability of HUVECs,and H&E staining was used to detect the numbers of blood vessels.The experimental data were statistically analyzed using SPSS version 22.0(IBM Corp,Armonk,NY,USA).The sample mean between the two groups that followed a normal distribution were compared by t test,and the cell proliferation of HUVECs at different Zol concentrations was compared by analysis of one-way analysis of variance.Non-parameter test was used to compare the number of blood vessels in the matrigel plugs.P < 0.05 indicates a statistically significant difference.Results:1.SCAP adhered to the dishes in a short spindle shape.After osteogenesis induction,alizarin red S staining showed that mineralized nodules were formed in SCAP.After adipogenesis induction,oil red O staining showed that lipid droplets were formed in SCAP.SCAP expressed the surface markers of mesenchymal stem cells,CD73,CD90,and CD105,but not the surface markers of hematopoietic stem cells,CD31,CD34,and CD45.2.Under the transmission electron microscope,SCAP-Exo was a round vesicle with a double-layer membrane structure.NTA showed that the peak of particle size was 130.1nm.Western blot showed that SCAP-Exo expressed CD9 and Alix but not calnexin.3.The BRONJ rat model was successfully constructed by intraperitoneal injection of Zol and tooth extraction.At 2 w after operation,no obvious healing in the tooth extraction wound was observed.H&E staining showed that necrotic bone existed in the alveolar bone.4.Immunofluorescence tracing showed that SCAP-Exo remained in the gingiva of BRONJ rats after tooth extraction at 1 w and 2 w after tooth extraction.From the photographs,the wound healing rate in the BRONJ group was significantly lower than that in the control group(2 w: P < 0.001;4 w: P < 0.001),and the wound healing rate in the SCAP-Exo group was significantly better than that in the BRONJ group(2 w: P< 0.05;4 w: P < 0.001);H&E staining and CD31 immunohistochemistry staining showed that compared with the control group,the BRONJ group showed a decrease in gingival angiogenesis(P < 0.01)and a decrease in the luminal structure with CD31 expression in vascular endothelial cells(P < 0.05);With SCAP-Exo treatment,the number of blood vessels(P < 0.05)and the number of the luminal structures of CD31-positive vascular endothelial cells(P < 0.05)in the gingiva of BRONJ rats increased significantly;VEGFA was expressed in the lamina propria of the gingiva of rats in the control group and SCAP-Exo group,only a small amount of VEGFA positive expression was seen in the proximal epithelium of rats in the BRONJ group.Micro-CT showed that,2 w after operation,the BV/TV in the tooth extraction wound of the SCAP-Exo group and the BRONJ group had no significant difference(P > 0.05),4 w after operation,the BV/TV in the tooth extraction wound of the SCAP-Exo group increased compared with the BRONJ group(P < 0.01);H&E staining showed that,the number of empty bone lacuna in the alveolar bone of tooth extraction wound in BRONJ group was significantly increased compared with the control group(2 w: P < 0.001;4w: P < 0.001).The number of empty bone lacuna in the alveolar bone of the SCAPExo group was significantly decreased(2 w: P < 0.001;4 w: P < 0.001),and NB/TB was significantly decreased(P < 0.001)compared with the BRONJ group;Masson staining showed that compared with Control group,the collagen fibers in the tooth extraction in the BRONJ group decreased(P < 0.001),and the collagen fibers in the tooth extraction wound in the SCAP-Exo group increased significantly compared with the BRONJ group(P < 0.001);5.MTT and Ki-67 immunofluorescence staining showed that compared with the control group,the proliferation rate and the rate of Ki-67 positive cells of HUVECs in the Zol group were significantly reduced(P < 0.05).There was no statistical difference in proliferation rate and Ki-67 positive cell ratio between the SCAP-Exo and Zol groups(P > 0.05).The scratch assay and transwell cell migration assay showed that the horizontal(P < 0.01)and vertical(P < 0.001)migration ability of HUVECs in Zol group decreased significantly compared with the control group,and the horizontal and vertical migration ability of SCAP-Exo group increased significantly compared with Zol group(P < 0.01);The tube formation assay showed that the total tube length(P <0.001),the number of meshes(P < 0.01),the number of nodes(P < 0.05)and the number of junctions(P < 0.01)of HUVECs in Zol group were significantly decreased compared with those in control group.The total tube length(P < 0.05),number of meshes(P < 0.01),number of nodes(P < 0.05)and number of junctions(P < 0.01)of HUVECs were significantly increased in the SCAP-Exo group than those in Zol group;Western blot showed that the expression level of CD31 in Zol group was lower than that in the control group(P < 0.05),while the expression level of CD31 in the SCAPExo group was significantly higher than that in Zol group(P < 0.01).The matrigel plug assay showed that the number of vessels in matrigel in Zol group was decreased compared with the control group(P < 0.01),and the number of vessels in matrigel plug in the SCAP-Exo group increased significantly compared with Zol group(P < 0.01).Conclusion: Local application of SCAP-Exo could treat BRONJ effectively by promoting gingival angiogenesis,and accelerating the gingival healing of the tooth extraction wound,thus increase the bone formation,and reduce bone necrosis in the tooth extraction wound in BRONJ rats.In vitro,SCAP-Exo could effectively restore the migration,tube formation,and angiogenesis ability of HUVECs treated by Zol,but had no effect on proliferation ability. |
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