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Modified enzymatic collagen digestion-mediated isolation of osteocytes

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BIOCELL 2022年第4期46卷 1097-1104页

作者： WENJUAN XU GUANGMING DAI YIFEI LYU YINING ZHANG xiaolin tu Institute of Life and Science Research Chongqing Medical UniversityChongqing400016China Department of Bioengineering University of WashingtonSeattle98195USA

This study established a method for isolating large numbers of high-purity osteocytes from high-density *** fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times,and the digested cells and bone chips(BC)were cultured,digested,and passaged when cells were fully *** types of cells obtained were identified by morphology,viable cell counts,alkaline phosphatase staining,and biochemical activity analyses,and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain *** results showed that among the cells obtained from the third digestion(fractions 7–9)of wild mice tibias and femurs and the remaining BCs,85%–90%of the cells were ***,their morphology was approximately one-tenth to one-fifth the size of osteoblasts,star-shaped or polygonal,with a dendritic structure,negative for alkaline phosphatase staining,and showed a high expression of dmp1 and *** percent of the cells in fractions 1–3 were osteoblasts,and were fusiform or polygonal *** activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction,while the expression of osteocyte-specific dmp1 and sclerostin was not *** the second portion(fractions 4–6),a large number were osteoblasts,mixed with a small number of osteocytes,and had high alkaline phosphatase activity and osteocyte mRNA levels,a specific level of the osteocyte marker dmp1,and no sclerostin was *** in daβcatot mice were also successfully isolated by this method,and we found that Wnt signaling increased the proliferation of these *** proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone,which provides an avenue for the study of osteocyte function in vitro.

关键词： Osteocytes Cell culture Collagen protease Sclerostin Alkaline phosphatase

3D printing of osteocytic Dll4 integrated with PCL for cell fate determination towards osteoblasts in vitro

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Bio-Design and Manufacturing 2022年第3期5卷 497-511页

作者： Pengtao Wang Xiaofang Wang Bo Wang Xian Li Zhengsong Xie Jie Chen Tasuku Honjo xiaolin tu Laboratory of Skeletal Development and Regeneration Institute of Life SciencesChongqing Medical UniversityChongqing 400016China Medical Data Science Academy Chongqing Medical UniversityChongqing 400016China Department of Immunology and Genomic Medicine Graduate School of MedicineKyoto UniversityYoshida Konoe-choSakyo-kuKyoto 606-8501Japan

Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research *** this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast *** established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a *** PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day *** osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D *** Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 ***,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre *** data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards ***,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within *** study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards *** will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.

关键词： Integrated 3D printing PCL scaffold Cell-laden hydrogel Osteocytic Dll4 Bone marrow stromal cell Osteoblast differentiation Cell viability in hard material RBPjκ Notch signaling

Wnt3a-induced ST2 decellularized matrix ornamented PCL scaffold for bone tissue engineering

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BIOCELL 2022年第9期46卷 2089-2099页

作者： XIAOFANG WANG xiaolin tu YUFEI MA JIE CHEN YANG SONG GUANGLIANG LIU Laboratory of Skeletal Development and Regeneration The Institute of Life ScienceChongqing Medical UniversityChongqing400016China Department of Anatomy and Cell Biology Indiana University School of MedicineIndianapolisIN46202USA Bioengineering Collage Chongqing UniversityChongqing400044China

The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue ***3a activates the classicWnt/β-catenin signaling pathway which effects bone growth and development by the accumulation ofβ-catenin in the *** this study,we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix(Wnt3a-ST2-dCM-PCL)and ST2 decellularized matrix(ST2-dCM-PCL)by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized>90%DNA while preserved most *** to ST2-dCM-PCL,Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation,osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase(Alp),Runx2,type I collagen(Col 1)and osteocalcin(Ocn)mRNA *** 14 days of osteogenic induction,Wnt3a-ST2-dCM-PCL promoted ST2 *** results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials’osteoinductivity and osteoconductivity.

关键词： Wnt3a Decellularized matrix Bone marrow stromal cells Osteogenic differentiation Bone tissue engineering

A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis

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Bioactive Materials 2023年第3期21卷 110-128页

作者： Xiaofang Wang Yufei Ma Jie Chen Yujiao Liu Guangliang Liu Pengtao Wang Bo Wang Makoto M.Taketo Teresita Bellido xiaolin tu Laboratory of Skeletal Development and Regeneration Institute of Life SciencesChongqing Medical UniversityChongqing400016China Department of Pharmacology Graduate School of MedicineKyoto UniversityKyoto606-8501Japan Department of Physiology and Cell Biology University of Arkansas for Medical SciencesLittle RockAR72223USA Department of Anatomy and Cell Biology Indiana University School of MedicineIndianapolisIN46202USA

Cell source is the key to decellularized matrix(DM)*** study compared 3 cell types,osteocytes with/without dominant active Wnt/β-catenin signaling(daCO and WTO)and bone marrow stromal cells(BMSCs)for their DMs in bone *** removes all organelles and>95%DNA,and retained>74%collagen and>71%GAG,maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the *** produced higher cell survival rate(90%)and higher proliferative *** vitro,daCO-DM induces more and longer stress fibers in BMSCs,conducive to cell adhesion,spreading,and osteogenic differentiation.8-wk after implantation of the critical-sized parietal bone defect model,daCO-DM formed tight structures,composed of a large number of densely-arranged type-I collagen under polarized light microscope,which is similar to and integrated with host ***/TV(>54%)was 1.5,2.9,and 3.5 times of WTO-DM,BMSC-DM,and none-DM groups,and ***/***(3.2×10^(2)/mm^(2))was 1.7,2.9,and 3.3 *** 4-wk,daCO-DM induced osteoclastogenesis,2.3 times higher than WTO-DM;but BMSC-DM or none-DM didn'***-DM increased the expression of RANKL and MCSF,Vegfa and Angpt1,and Ngf in BMSCs,which contributes to osteoclastogenesis,angiogenesis,and neurogenesis,***-DM promoted H-type vessel formation and nerve markersβ3-tubulin and NeuN ***:daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone *** features are expected to achieve the effect of autologous bone transplantation,suitable for transformation application.

关键词： Decellularized matrix Osteocyte Wnt signaling 3D printing Regenerative repair Metabolic and neurovascular organoid bone

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代替陶瓷型芯生产空心铸件的精铸工艺

特种铸造及有色合金 2009年第12期29卷 1130-1131页

作者：屠晓林沈阳黎明航空发动机集团有限责任公司精密铸造厂

在精密铸造生产过程中,空腔结构铸件的内腔一般采用陶瓷型芯形成,但制造陶瓷型芯需专用工装,而专用工装制造成本高、周期长,而且陶瓷型芯制造工艺复杂、周期长。这种传统的空腔铸件成形工艺,往往不能满足新品研制周期的需要。通过对某... 详细信息

在精密铸造生产过程中,空腔结构铸件的内腔一般采用陶瓷型芯形成,但制造陶瓷型芯需专用工装,而专用工装制造成本高、周期长,而且陶瓷型芯制造工艺复杂、周期长。这种传统的空腔铸件成形工艺,往往不能满足新品研制周期的需要。通过对某燃机中介机匣的研制,提出了不用陶瓷型芯生产空心铸件的工艺方法,并在实际生产中得到了验证,取得了良好的效果。

关键词：陶瓷型芯空心铸件精密铸造

全反式维甲酸与VEGF联合应用诱导小鼠胚胎成纤维细胞定向成骨分化

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中国修复重建外科杂志 2020年第2期34卷 246-255页

作者：冯玮涂小林重庆医科大学生命科学研究院

目的研究全反式维甲酸(all-trans retinoic acid,ATRA)与VEGF联合应用对于小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)定向成骨分化的影响。方法取NIH孕鼠(孕12~15 d)子宫内的胎鼠,去掉头、心、肝等器官后,采用胰蛋白酶消化... 详细信息

目的研究全反式维甲酸(all-trans retinoic acid,ATRA)与VEGF联合应用对于小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)定向成骨分化的影响。方法取NIH孕鼠(孕12~15 d)子宫内的胎鼠,去掉头、心、肝等器官后,采用胰蛋白酶消化贴壁法分离培养MEFs。利用HEK-293细胞采用反复冻融法扩增重组腺病毒红色荧光蛋白(recombinant adenovirus-red fluorescent protein,Ad-RFP)及Ad-VEGF。采用ALP染色和ALP定量检测法检测单独ATRA或VEGF以及ATRA和VEGF联合应用培养MEFs第3、5天ALP活性变化。将第3~4代MEFs分为A、B、C、D 4组,分别加入DMSO+Ad-RFP、ATRA、Ad-VEGF、ATRA+Ad-VEGF。实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测第3、7天成骨相关标志物ALP、Ⅰ型胶原、骨桥蛋白(osteopontin,OPN)、骨钙素(osteocalcin,OCN)及成血管相关标志物VEGF、血管生成素1(angiopoietin 1,ANGPT1)、内皮黏蛋白(endomucin,EMCN)mRNA表达;免疫组织化学染色检测各组第3、5、7天OPN、VEGF蛋白表达;茜素红染色检测各组成骨诱导14、21 d钙盐沉积水平。取15只4~6周龄无胸腺雌性裸鼠,随机分为3组,每组5只,分别于背侧与腹侧皮下注射经ATRA、Ad-VEGF、ATRA+Ad-VEGF处理后的MEFs。植入后5周行X线片观察、大体观察及组织学染色(Masson、HE及番红O-固绿染色),观察各组裸鼠体内异位成骨情况。结果成功分离培养MEFs;扩增后的Ad-RFP及Ad-VEGF成功转染MEFs,转染效率约为50%和20%。ALP活性检测示,单独使用ATRA或VEGF均能增强MEFs中ALP活性,且ATRA作用较VEGF强;ATRA和VEGF联合使用较单独使用显著增强了MEFs中ALP活性(P<0.05)。qRT-PCR检测示,ATRA联合Ad-VEGF使用上调了早期成骨分化相关标志物ALP、OPN、Ⅰ型胶原mRNA相对表达量(P<0.05);7 d时成血管相关标志物VEGF、EMCN及ANGPT1 mRNA相对表达量有所上升(P<0.05)。免疫组织化学染色示,ATRA联合Ad-VEGF不仅增强了OPN蛋白表达,VEGF蛋白表达在第7天也有所增强。茜素红染色示,单独应用ATRA或Ad-VEGF诱导钙盐沉积作用较弱,二者联合应用明显增强了MEFs成骨晚期钙盐沉积的效应。裸鼠体内植入实验结果显示,X线片观察发现与其余两组相比,ATRA+Ad-VEGF组存在明显骨块,且骨块体积较大,组织学染色可见大量胶原及成熟骨小梁、骨基质形成以及胶原骨组织。结论ATRA与VEGF联合应用能诱导MEFs定向成骨分化。

关键词：全反式维甲酸 VEGF 小鼠胚胎成纤维细胞成骨分化成血管

An online monitor ionization chamber used in particle therapy

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Nuclear Science and Techniques 2009年第3期20卷 177-180页

作者： TANG Bin HU Zhengguo MAO Ruishi XU Zhiguo WANG Jiansong YUE Ke tu xiaolin WU Dapeng CHEN Jinda ZHANG Jie WANG Meng SUN Zhiyu ZHANG Xueheng LI Qiang XU Hushan XIOA Guoqing Institute of Modern Physics Chinese Academy of Sciences Lanzhou 730000 China Graduate University of Chinese Academy of Sciences Beijing 100049 China

The clinical trials of tumor therapy using heavy ions beam 12C are now in progress at Institute of Modern Physics in Lanzhou. In order to achieve the precise radiotherapy with the high energy 12C beam in active pencil beam scanning mode, we have developed an ionization chamber(IC) as an online monitor for beam intensity and also a dosimeter after calibration. Through the choosing of working gas and voltage, optimizing of the electrics and the read-out system, calibrating the linearity, the detector system provide us one of the simple and highly reliable way to monitoring the beam during the active pencil beam scanning treatments. The measurement results of this detector system show that it could work well under the condition of high energy 12C beam in active pencil beam scanning mode.

关键词：肿瘤治疗临床离子化粒子

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控制超薄细长无余量定向叶片变形的工艺措施

特种铸造及有色合金 2012年第2期32卷 149-151页

作者：王永明屠晓林中航工业沈阳黎明航空发动机(集团)有限责任公司

在发动机叶片定向无余量精铸件的研制过程中,通过对叶片变形的分析,制定预防变形的措施,设计成型浇注系统、组合夹具等工装,加强控制手段,解决了超薄细长定向无余量叶片变形问题,提高了叶片的尺寸精度,研制出符合设计要求的无余量叶片。

关键词：精密铸造无余量超薄细长叶片

体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响

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第三军医大学学报 2020年第9期42卷 891-898页

作者：谢映春涂小林重庆医科大学生命科学研究院骨发育与再生平台重庆400016

目的探究体外激活骨细胞Notch信号对骨髓基质细胞(bone marrow stromal cells,BMSCs)成骨分化的影响。方法设置Cre重组腺病毒(Ad-Cre)及对照腺病毒GFP(Ad-GFP),分别转染RosaNotch小鼠骨细胞后与野生C57BL/6小鼠BMSCs共培养为Ad-Cre组及Ad-GFP组,设置仅BMSCs为Blank组。碱性磷酸酶染色及生化定量测定碱性磷酸酶(ALP);qPCR检测转染后骨细胞Notch信号靶基因及共培养产物成骨相关标志物、Notch信号配体及成血管相关标志物,茜素红染色检测成骨诱导21 d钙盐沉积水平。结果Ad-Cre成功激活RosaNotch小鼠骨细胞Notch信号,ALP染色及生化定量结果显示Ad-Cre组ALP表达相较Ad-GFP组及Blank组显著降低(P<0.05),Ad-GFP组与Blank组间无统计学差异;qPCR结果显示Ad-Cre组共培养产物成骨标志物ALP、核心结合因子a1(Runx2)、特异性骨转化因子(Ostrix)及Notch配体Dll4 mRNA转录水平相较Ad-GFP组及Blank组显著降低(P<0.05),Jag1 mRNA转录水平显著降低(P<0.05);血管内皮生长因子(VEGF)、低氧诱导因子(hypoxia-inducible factor 1α,HIF1α)、血小板内皮细胞黏附分子(platelet endothelial cell adhesion molecule-1,CD31/PECAM-1)及内皮粘蛋白(endomucin,EMCN)mRNA转录水平相较Ad-GFP组及Blank组显著增高(P<0.05);茜素红染色结果显示Ad-Cre组在成骨诱导培养基作用21 d后钙盐沉积水平相较Ad-GFP组及Blank组显著降低。结论体外激活骨细胞Notch信号抑制骨髓基质细胞成骨分化。

关键词：骨细胞 Notch信号骨髓基质细胞成骨分化