Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture

In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al. 1996, 2001). Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al. 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells. In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts. In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM). The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM. The cells also extended fine fibrillar processes into the ECM. With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM. The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine. The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells. They extended slender processes into the new ECM, which is characteristic of tubular matrix. Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced. Furthermore, the tubular matrix became mineralized under prolonged culture. These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro.     

熊果酸对酒精性骨质疏松大鼠骨形成、骨矿化的影响

目的：探讨熊果酸对酒精所致骨质疏松大鼠骨形成、骨矿化的影响。方法：雄性Wistar大鼠60只,按体重随机分为空白对照组、熊果酸对照组、模型组、熊果酸低、中、高剂量组,同时分别给予生理盐水、150 mg/kg 熊果酸、50%酒精,50 mg/kg 熊果酸,100mg/kg 熊果酸,150 mg/kg 熊果酸灌胃。熊果酸对照组生理盐水剂量同空白组,熊果酸低、中、高剂量组酒精剂量同模型组。灌胃共持续8 周。磷钼酸法检测血清磷(P)含量,比色法检测血清钙(Ca)含量,酶联免疫吸附（ELISA）法检测血清骨钙素(BGP)、骨形成蛋白-2(BMP-2)浓度;HE 染色法观察股骨结构的病理学变化。结果：与空白对照组相比较,模型组血清BGP、BMP-2 和Ca、P 均明显降低,且有统计学差异(P < 0.05),但熊果酸对照与空白对照组各项指标结果相近。熊果酸中、高剂量组大鼠血清BGP、Ca 和P 水平均较模型组有显著升高,差异具有统计学意义(P < 0.05),但仅熊果酸高剂量组血清BMP-2 显著升高(P < 0.05)。股骨组织HE染色结果显示,空白对照组骨小梁致密、规则且较粗,粗细均匀;模型组骨小梁稀松、不规则、粗细不均匀,甚至可见骨小梁断裂;熊果酸中、高剂量组骨小梁致密、规则、较厚、粗细均匀,未见骨小梁断裂。结论：熊果酸能够促进酒精性骨质疏松大鼠的骨形成,抑制骨矿物质的流失,在改善酒精致骨质疏松方面有一定的保护作用。     

The assessment of trabecular bone parameters and cortical bone strength: A comparison of micro-CT and dental cone-beam CT

This study compared the capabilities of micro-computed tomography (micro-CT) and dental cone-beam computed tomography (CBCT) in assessing trabecular bone parameters and cortical bone strength. Micro-CT and CBCT scans were applied to 28 femurs from 14 rats to obtain independent measurements of the volumetric cancellous bone mineral density (vCanBMD) in the femoral head, volumetric cortical bone mineral density (vCtBMD) in the femoral diaphysis, cross-sectional moment of inertia (CSMI), and bone strength index (BSI) (=CSMI×vCtBMD). Five structural parameters of the trabecular bone of the femoral head were calculated from micro-CT images. A three-point bending test was then conducted to measure the fracture load of each femur. Bivariate linear Pearson analysis was conducted to calculate the correlation coefficients (r values) of the micro-CT, dental CBCT, and three-point bending measurements. The statistical analyses showed a strong correlation between vCanBMD values obtained using micro-CT and dental CBCT (r=0.830). There were strong or moderate correlation between vCanBMD measured using dental CBCT and five parameters of trabecular structure measured using micro-CT. Additionally, the results were satisfactory regardless of whether micro-CT or dental CBCT was used to measure the femoral diaphysis vCtBMD (r=0.733 and 0.680, respectively), CSMI (r=0.756 and 0.726, respectively), or BSI (r=0.846 and 0.847, respectively) to predict fracture loads. This study has yielded a new method for using dental CBCT to evaluate bone parameters and bone strength; however, further studies are necessary to validate the use of dental CBCT on humans.     

流感疫苗单向免疫扩散试验中抗血清制备新方法的研究

流感疫苗血凝素含量检测方法为单向免疫扩散试验,其抗血清通常由WHO参比实验室提供,通过纯化病毒、蛋白酶切获得血凝素主要抗原片段,然后再免疫动物获得抗血清。该方法制备时间较长,是制约流感疫苗研发及检测的主要因素。以RT-PCR方法获取甲型H1N1流感病毒血凝素中主要抗原片段基因,构建重组质粒并在大肠杆菌中表达,表达产物经纯化、复性后,以蛋白电泳及免疫印迹方法进行了鉴定。以纯化蛋白免疫家兔,制备了相应的抗体,初步证明可用于常规的单向免疫扩散试验。以该法可以快速获得抗血清,尤其是在大流行流感疫苗的研发中,加快疫苗研发进程。     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

Osteoblastic γ-aminobutyric acid, type B receptors negatively regulate osteoblastogenesis toward disturbance of osteoclastogenesis mediated by receptor activator of nuclear factor κB ligand in mouse bone

The prevailing view is that signaling machineries for the neurotransmitter GABA are also expressed by cells outside the CNS. In cultured murine calvarial osteoblasts, mRNA was constitutively expressed for both subunits 1 and 2 of metabotropic GABA(B) receptor (GABA(B)R), along with inhibition by the GABA(B)R agonist baclofen of cAMP formation, alkaline phosphatase (ALP) activity, and Ca(2+) accumulation. Moreover, baclofen significantly inhibited the transactivation of receptor activator of nuclear factor-κB ligand (RANKL) gene in a manner sensitive to a GABA(B)R antagonist, in addition to decreasing mRNA expression of bone morphogenetic protein-2 (BMP2), osteocalcin, and osterix. In osteoblastic MC3T3-E1 cells stably transfected with GABA(B)R1 subunit, significant reductions were seen in ALP activity and Ca(2+) accumulation, as well as mRNA expression of osteocalcin, osteopontin, and osterix. In cultured calvarial osteoblasts from GABA(B)R1-null mice exhibiting low bone mineral density in tibia and femur, by contrast, both ALP activity and Ca(2+) accumulation were significantly increased together with promoted expression of both mRNA and proteins for BMP2 and osterix. No significant change was seen in the number of multinucleated cells stained for tartrate-resistant acid phosphatase during the culture of osteoclasts prepared from GABA(B)R1-null mice, whereas a significant increase was seen in the number of tartrate-resistant acid phosphatase-positive multinucleated cells in co-culture of osteoclasts with osteoblasts isolated from GABA(B)R1-null mice. These results suggest that GABA(B)R is predominantly expressed by osteoblasts to negatively regulate osteoblastogenesis through down-regulation of BMP2 expression toward disturbance of osteoclastogenesis after down-regulation of RANKL expression in mouse bone.     

抗癫痫药物左乙拉西坦对大鼠骨骼的物理及生化指标的影响

本研究的目的是以性腺完整的动物为模型调查左乙拉西坦(LEV)是否对于骨矿物质密度、骨结构和骨代谢生化指标产生影响,探究抗癫痫药物左乙拉西坦(LEV)是否对骨骼健康存在显著风险。本研究将实验大鼠分为对照组和试验组,每组10只。对照大鼠接受标准实验室饮食(SLD),而试验组中的大鼠喂食富含LEV的实验室饮食12周,并以双能X-线吸收仪测量全身、股骨和腰椎的骨密度。在骨组织中检查骨标记的浓度,股骨和胫骨均用于生物力学测试。研究结果表明:在LEV组中,脂肪组织的绝对值和相对值显著降低,全身骨密度增高,骨碱性磷酸酶(BALP)、Ⅰ型胶原C末端肽(CTX-1)和Ⅰ型前胶原氨基端前肽(PINP)浓度显著增加。本研究初步得出结论:在性腺完整的大鼠模型中长期施用LEV对骨质不具有负面影响。骨矿物质密度(BMD)的显著增加可能表明LEV对骨质可能有正面影响。     

The Free Fatty Acid Receptor G Protein-coupled Receptor 40 (GPR40) Protects from Bone Loss through Inhibition of Osteoclast Differentiation

The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40^−/− mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/β) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40^−/− primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40^−/− mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications.     

miR-30 family members negatively regulate osteoblast differentiation

miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3' untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.     

BamHI DNA甲基化酶的提纯及物理化学性质

 通过硫酸铵盐析,DEAE-纤维素柱层析,磷酸纤维素亲和层析及SephadexG-100凝胶过滤法,从噬淀粉芽孢杆菌HI(Bacillus amyloliguefaciens HI)提纯了DNA甲基化酶。用聚丙烯酰胺凝胶电泳检查,已达电泳均一,比活力提高了326倍。并用聚丙烯酰胺梯度凝胶电泳和Sephadex G-100凝胶过滤法测得其天然酶的分子量为273000,又用SDS聚丙烯酰胺凝胶电泳测得它的亚基分子量为34500,故该酶有8个分子量相同的亚基。用凝胶电聚焦法测得其pI_(22 c)=9.0。     

光学相干层析用于牙齿病变的检测

阐述了适用于牙齿结构成像的光学相干层析成像(OCT)系统。系统光源中心波长为1 310 nm,成像分辨率10μm,在牙内成像深度2 mm,成像速度1幅/秒,系统信噪比100 dB。利用此OCT仪清晰检测到牙齿样品的牙釉质和牙本质,观察到牙釉质与牙本质的分界面以及正常牙齿牙釉质与龋齿牙齿牙釉质OCT图像的区别。进一步设计研制了适用于口腔内探测的小型OCT探头。     

以甘草为原料的食品天然防腐剂的研制

采用不同的溶剂和提取条件,提取甘草中的抗菌有效成分,然后通过抑菌试验筛选出抗菌力最强的制剂,通过测定该制剂对常见食品污染菌的最小抑菌浓度(MIC),证实该制剂对多种常见的食品污染菌均有较强的抑制作用,保存试验证实本制剂能有效地延长食品的保质期。     

Identification of a diverse synthetic abietane diterpenoid library and insight into the structure-activity relationships for antibacterial activity

A diverse natural product-like (NPL) synthetic abietane diterpenoid library containing 86 compounds were obtained and the SARs were studied based on their antibacterial potential. Further in vitro cytotoxic and in silico drug-like properties evaluation showed that the potent antibacterial compound 84 had good drug-like properties and displayed low cytotoxicity toward noncancerous mammalian cells, indicating the study of AA and DHAA might be a good starting point for the search of novel antimicrobial molecules. Future work should be focused on the optimization of their potency and selectivity.     

Activation And Deactivation Of Quinones Catalyzed By Dt-Diaphorase. Evidence For Bioreductive Activation Of Diaziquone (Azq) In Human Tumor Cells And Detoxification Of Benzene Metabolites In Bone Marrow Stroma

Bioactivation of diaziquone (AZQ) in HT-29 human colon carcinoma cells and detoxification of benzene metabolites in bone marrow stromal cells were used as examples of the potential role of DT-diaphorase in both activation and deactivation processes. HT-29 cell cytosol contained high levels of DT-diaphorase activity and removed AZQ in the presence of either NADH or NADPH. Prior boiling of cytosol. omission of NADH or NADPH or inclusion of dicoumarol. an inhibitor of DT-diaphorase. inhibited removal of AZQ. AZQ-inouced cytotoxicity in HT-29 cells was also inhibited by dicoumarol. Chemical reduction of AZQ in a cell free system enhanced formation of a GSH conjugate of AZQ. Two of the major cell types in bone marrow stroma are macrophages and fibroblastoid stromal cells. A fibroblastoid cell line derived from stromal cells contained approximately fourfold higher levels of DT-diaphorase than macrophages. Inclusion of dicournarol in incubations containing ¹⁴C-hydroquinone and the respective stromal cell type, significantly increased covalent binding of radiolabel to macromolecules in stromal fibroblasts but not in macrophages