Microvesicles (MVs) are membrane vesicles secreted by cells and are present in the saliva of healthy individuals. It has various functions and has been reported to be a biomarker for malignant tumors. The changes in saliva levels of MVs associated with disease(s) is unclear. This study aimed to determine the proportion of salivary apoptotic MVs and their association with oral ulcer(s) in patients with non-healing oral ulcer(s) and reported oral cancer. Saliva (5 mL) was collected from patients with non-healing oral ulcer(s) and reported oral cancer (at the time of saliva collection, the participant have an oral ulcer(s) in the oral cavity and have an oral cancer lesion; n = 73) and healthy volunteers with oral ulcer(s) (n = 62). A standard differential centrifugation protocol was used for the purification of MVs. Dynamic light scattering and transmission electron microscopy were used to characterize MVs. Flow cytometry was used to quantify salivary apoptotic MVs. Immunocytochemistry was performed according to a standard protocol. None of patients with oral cancer has smoking and drinking habit. The majority of saliva samples derived from patients with non-healing oral ulcer(s) and reported oral cancer were more positive for the fluorescent dye carboxyfluorescein succinimidyl ester than those of healthy volunteers with oral ulcer(s). Salivary fluid obtained from patients had membrane-limited vesicles that were round and/or slightly elongated in shape, with diameters of 100–1,000 nm. The number of salivary apoptotic MVs was higher in patients with non-healing oral ulcer(s) than in those derived from healthy volunteers with oral ulcer(s) (p < 0.001). There was an association between salivary apoptotic MVs in patients with non-healing oral ulcer(s) and the degree or severity of oral ulcers (p < 0.001). Levels of salivary apoptotic MVs are elevated in patients with non-healing oral ulcer(s) and confirmed oral cancer. Elevated levels of salivary apoptotic MVs are associated with clinicopathological data of patients with oral cancer. Evidence level: IV. Technical efficacy: stage 3.

Background Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. Methods Microarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DANCR was screened out. Its effects on proliferation and osteogenic differentiation was explored by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DANCR on cell growth curve. To quantify the effects of DANCR on osteogenic differentiation of PDLSCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed. Results Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of lncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers’ upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. Conclusions The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs’ osteogenic differentiation.

Hypoxia-inducible factor (HIF)-1α protein, which is upregulated by hypoxia, serves an important role in angiogenesis during osteogenesis. The aim of the present study was to investigate the effect of HIF-1α on alveolar ridge preservation in a dog tooth extraction model. Six beagle dogs were used in the present study. The second and fourth premolar teeth of the lower jaws on both sides were extracted. Two unilateral extraction sockets were randomly selected and filled with Bio-Oss and Bio-Oss + HIF-1α. The contralateral sockets remained unfilled and served as the negative control. Micro-computed tomography examination and histological staining were performed to examine the difference of new bone formation among the three groups. Western blotting and reverse transcription-quantitative polymerase chain reaction analysis were used to detect the expression levels of osteogenesis- and angiogenesis-associated genes in the bone tissues of the three groups. Twelve weeks post-surgery, trabecular bone formation in the Bio-Oss + HIF-1α group was significantly increased compared with the other groups. The expression levels of osteogenesis-associated genes (runt-related transcription factor 2, osteoblast-specific transcription factor osterix and osteocalcin) and angiogenesis-associated genes (HIF-1α and vascular endothelial growth factor) were all significantly increased in the Bio-Oss + HIF-1α group compared with the other two groups (P<0.05). The present results indicated that Bio-Oss with HIF-1α can promote osteogenesis and angiogenesis in vivo and may be used as an effective treatment for the preservation of the alveolar ridge.

The aim of the present study was to evaluate the clinical efficacy of platelet-rich fibrin (PRF) in preserving the alveolar ridge following human tooth extraction. A total of 28 patients were divided into two groups: The experimental and control groups (n=14 each). Following tooth extraction, the experimental group was implanted with PRF membrane, whereas the control group was not. The gingival healing effect was assessed at 7 days, 1 and 3 months later. Cone-beam computed tomography was performed immediately and at 3 months following tooth extraction. The changes in alveolar ridge height, width, and bone mineral density were compared between the two groups. The alveolar bone was removed using the ring drill during the implant surgery at 3 months following tooth extraction. Histomorphometric evaluation was performed to compare new bone formation between groups. The patients in the experimental group reportedly felt better compared with the patients in the control group. The healing of gingival tissue was better in the experimental group than in the control group. A significantly greater novel bone area was observed in the PRF group compared with the control group (P<0.01). However, no statistically significant differences were observed in the mean value of buccal alveolar ridge height, lingual/palatal alveolar ridge height and alveolar ridge width between the two groups. These results suggested that PRF was advantageous in human alveolar ridge preservation with ease of use and simple handling. Histological analysis of novel bone formation confirmed that PRF increased the quality of the novel bone and enhanced the rate of bone formation, despite the effect of PRF was not significant to reduce alveolar bone resorption in the extraction socket alone.

Objective:To study Bio-oss bone with HIF-1α protein on tooth extraction bone healing.Methods:Under general anesthesia,mesial root of the 2nd,4th premolars in both sides of mandible of 3 Beagle dogs were extracted.Randomly,a wound was selected as the blank control group,and the rest were immediately implanted bio -oss,bio-oss with H IF-1 α protein of 100ng/ml.After six and twelve weeks,the beagle dogs were sacrificed.The gross speciments were examined by Micro-CT images,and mineralized specimen preparations were prepared.Results:Micro-CT scan analysis showed that each group had some differences in the number of trabecular,bone mineral density and BV/TV.After twelve weeks,bio-oss with HIF-1α group was significantly higher than the bio-oss group、the control group (P＜0.05).The difference was statistical significance.Tissue slides also revealed an effective osteogensis in those wounds implanted with bio-oss with HIF-1α.Conclusion:Bio-oss with H IF-1α protein 1 can promote the regeneration of alveolar bone.

PURPOSE To establish the model of alveolar ridge preservation after tooth extraction for dental implant replacement, and to observe the effect of tissue engineered bone on osseointegration. METHODS Isolated BMSCs were expanded and osteogenically induced in vitro. The tissue engineering complex was constructed with BMSCs/A-PCPC in vitro. Six extraction sockets, with three on each side, were created in the mandibles of four Beagle dogs by extracting the second, third and fourth premolars. BMSCs/A-PCPC were placed on one side of the extraction sockets, while autogenous bone, A-PCPC and nothing were placed on the other side as control. X-ray and CT scans were conducted 1day, 4 and 12 weeks after operation to detect the change of the alveolar ridge. The bone of sockets were harvested at 8-week post-implantation and subject to histological for evaluating. SPSS17.0 software package was used for data analysis. RESULTS Radiographs demonstrated higher radiodensity in group of complex than in simple materials group, autogenous bone group after 4 weeks. Hard tissue biopsy at 12-week showed that bone activity of BMSCs/A-PCPC complex was better than the other groups. Spiral CT analysis showed that alveolar ridge of each group experienced a certain degree of absorption. At 12-week, the alveolar ridge height reduction values in A-PCPC group was smaller than in A-PCPC group, autogenous bone group and blank group (P<0.05). CONCLUSION The BMSCs/A-PCPC complex is favorable for preservation of alveolar ridge.

目的 建立种植前牙槽嵴保存的动物模型,观察应用骨髓基质细胞/活性磷酸钙骨水泥复合物(bone marrow stromal cells/active porous calcium phosphate cement,BMSCs/A-PCPC)进行牙槽嵴保存后其对种植体骨结合的影响.方法 取Beagle犬4只,拔除双侧下颌第二、三、四前磨牙,随机选取一侧植入BMSCs/A-PCPC复合物作为实验组,另一侧作为对照组(空白对照组、自体骨组、单纯A-PCPC组).12周后,在原拔牙位点制备种植窝洞植入Atlas Fixture种植体(3.5×10mm).12周后,处死动物进行组织学观察.结果 BMSCs/A-PCPC复合物组骨结合率高于其它组,成骨活性也高于其它各组.结论 BMSCs/A-PCPC复合物可以有效地防止牙槽嵴的萎缩,促进种植体骨结合,可以作为种植前牙槽嵴保存的组织工程骨材料.

OBJECTIVE:We ascertained the effect of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) by a series of experiments: Proliferation and differentiation of bone marrow stromal cells (BMSCs) in vitro, ectopic and in situ bone formation and loaded porous calcium phosphate cement (CPC) on the repair of bone defects around dental implants.METHODS:BMSCs from Beagle dogs were cultured in vitro with basic culture medium containing BMP-2, bFGF, and BMP-2+bFGF. Proliferation and differentiation of BMSCs were quantified using methyl thiazolyl tetrazolium (MTT) and alkaline phosphatase (ALP) test. The CPC seeded with BMSCs and BMP-2, bFGF, combined BMP-2 with bFGF were implanted subcutaneously into nude rats in ectopic bone formation, and were implanted into critical-sized bone defects of Beagle dogs in the in situ bone formation. The bone formation was detected by histology examination and quantified using an image analysis system. Polychrome sequential fluorescent labels and fluorescence histological examinations of undecalcified sections were performed post-operatively.RESULTS:It was determined that BMP-2+bFGF promoted BMSCs statistically significant proliferation and differentiation compared to either BMP-2 or bFGF in vitro. The CPC with BMP-2+bFGF group yielded more bone than those with either BMP-2 or bFGF in ectopic bone formation test. The percentages of newly ectopic formed bone were higher in the BMP-2+bFGF group (48.79% +/- 11.31%) than those in other groups (BMP-2 group, 30.71% +/- 10.85%; bFGF group, 27.33% +/- 9.67%; and the control group, 10.65% +/- 6.05%). Undecalcified showed that new bone was actively formed in the BMP-2+bFGF group after 12 weeks in the in situ bone formation test. The bone mineralization apposition rate (MAR) was better in the BMP-2+bFGF group than in other groups (P<0.01).CONCLUSION:BMP-2 combined with bFGF are more effective than one alone in promoting the formation of new bone.

Objective: To examine the efficancy of active porous calcium phosphate cement together with bone marrow stromal cells(BMSCs) in alveolar ridge preservation.Methods: The 2nd,3rd,4th premolars in both sides of mandible of 4 beagle dogs were extracted and 24 extraction wounds were created.Randomly,one side of the mandible wounds were implanted by BMSCs with the growth factor/porous CPC composite.The opposite side of wounds were implanted by CPC,autologus bone,or keep void respectively.After four and ten weeks,the beagles were sacrificed.The gross specimens were examined,CT scan images,and mineralized specimen preparations were prepared.Results: CT analysis showed that alveolar ridge in each group experienced some degree of alveolar absorption.After ten weeks,the alveolar ridge height reduction values in active-CPC group was smaller than the CPC group,autogenous bone group,and blank group respectively(P0.05).The difference has statistical significance.Tissue slides also revealed an effective osteogensis in those wounds implanted with active-CPC.Conclusions: The BMSCs/active CPC complex is favorable for preservation of alveolar ridge.

PURPOSE:To study the osteogenic capability of the construct combined dog's bone stromal cells with active porous calcium phosphate cement (A-PCPC) in nude mice in vivo.METHODS:Isolated bone marrow stromal cells (BMSCs) were expanded and osteogenically induced in vitro. Their osteogenic phenotype was evaluated by cytochemistry. The tissue engineering complex was constructed with BMSCs/A-PCPC in vitro. After SEM scanning, the complex of BMSCs/A-PCPC was implanted into the subcutaneous tissue of the nude mice as experimental group, and A-PCPC as control group. The engineered bone was harvested 2,4,8weeks post-implantation and processed for HE staining, then evaluated by histology and histomorphometry.RESULTS:Cytochemistry showed alkaline phosphate activity, Von Kossa staining proved the formation of mineralization nodules. Scanning electron microscopy showed the cells adhered to the inner surface of the A-PCPC. HE staining showed a small group of woven bone formation 2 weeks later in the experimental group, while the formation of bone less in the control group. Woven bone turned into trabecular bone gradually at 4 weeks in the experimental group, while the control group showed a large number of bone-like tissue. Histomorphometry showed more mature bone in the experimental group than the control group at 8 weeks.CONCLUSIONS:The A-PCPC/BMSCs composites show good osteogenetic activity and could promote mineralization of the immature bone. It can be used as the bone tissue engineering scaffolds. Supported by Innovation Fund for Science and Technology Development of Pudong New District(Grant No. PKJ2009-Y19).

Purpose:To study the probability of Active pcpc that be used as a scaffold for bone tissue engineering.Material and Methods:After drawing out the bone marrow from the Beagle Dog,the bone marrow stroma cells were separated by adherent methods and induced with the conditional medium,the phenotype of the BMSCs was evaluated by immunocytochemistry.The induced passage 3 cells and active cpc were mixed to form a complex.After the SEM scanning,the complex was implanted into the subcutaneous parts of the beagle dog,histological observation were taken after 2 and 4 weeks.Reslts:ALP staining is positive,Von Kossa staining proofed the formation of mineralization nodules,immunocytochemical proofed the express of collagen I,Osteocalsin;Scanning electron micrograph showed the cells adhered to the inner surface of the Active pcpc;HE staining showed the new bone formation and vascularization 4 weeks later in group of complex.Dicussion:Activity CPC aperture is 200-500μm,small aperture is 1-5μm,porosity 70%.Active cpc is easy for osteoblast activity of BMSCs.Conclusion:Complex(Active cpc/BMSCs)showed new bone formation and vascularization in Beagle dog,the Active cpc can be be used as a scaffold for bone tissue engineering.

Objective To prepare seed cells for bone tissue engineering,establish the culturing condition and biological characteristics of canine bone marrow stromal cells(BMSCs),and proliferate them into osteoblasts in vitro.Methods Bone marrow was drew out from Beagle dogs and cultured with differentiation culture medium to harvest BMSCs.The morphology of the cells was studied with a phase contrast microscope.Von Kossa staining and alkaline phosphatase(ALP) activity test were employed to assess BMSCs osteoblastic differentiation and the generation of calcified extracellular matrix.The standard curves of cell-amount vs OD-value were drawn through spectrophotometer test and MTT assay.Results BMSCs could be observed after 18 days of primary culture.The viable characteristics of BMSCs were relatively stable,and these cells possessed bone formation potentials.Conclusion BMSCs can be cultured,differentiated and proliferated with active osteogenic function with differentiation culture medium in vitro,thus they can be candidate cells for bone tissue engineering.

背景:体外实验证明磷酸钙骨水泥/骨形态蛋白人工骨具有较强的骨诱导作用,但是磷酸钙骨水泥/骨形态蛋白人工骨能否作为骨髓基质细胞的生长支架报道较少。目的:验证磷酸钙骨水泥/骨形态蛋白人工骨做为骨组织工程支架材料的可行性。设计、时间及地点:观察实验,于2008-03/2009-03在上海市第九人民医院口腔组织工程实验室完成。材料:磷酸钙骨水泥/骨形态蛋白人工骨产品由瑞邦公司提供,大孔径200～500μm,小孔径1～5μm,孔隙率为70%。两岁的健康毕格犬1只。方法:抽取成年毕格犬的骨髓,贴壁法获得骨髓基质细胞,经成骨诱导培养液体外培养、扩增、诱导后观察细胞增殖情况。将培养的第2代细胞接种于多孔型活性磷酸钙骨水泥,进行超微结构观察,并将多孔型活性磷酸钙骨水泥/骨髓基质细胞复合物植入毕格犬背部皮下,2,4周后进行组织学检测。主要观察指标:倒置相差纤维镜下观察细胞的生长、增殖情况;碱性磷酸酶染色、Von Kossa染色、骨钙素免疫细胞化学染色鉴定骨细胞的形成标志;扫描电镜观察细胞材料复合情况;苏木精-伊红染色观察体内异位成骨情况。结果:碱性磷酸酶染色呈阳性;Von Kossa染色可见钙结节形成;骨钙素免疫细胞化学染色呈阳性;超微结构观察可见细胞生长附着于材料网孔内表面;组织学检测提示4周时复合物内有新骨形成。结论:多孔型活性磷酸钙骨水泥/骨髓基质细胞复合物显示良好的成骨活性,磷酸钙骨水泥/骨形态蛋白人工骨可以用于骨组织工程支架材料。