<meta charset="utf8"></head><body>使用外植体模型对骨形成进行多模态分析

<ol>
	<li>Feroz, S., Cathro, P., Ivanovski, S., Muhammad, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+bone+grafts+and+substitutes:+A+review+of+recent+advancements+and+applications.">Biomimetic bone grafts and substitutes: A review of recent advancements and applications.</a> Biomed Eng Adv. 6, 100107 (2023).</li><li>Tsuji, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP2+activity,+although+dispensable+for+bone+formation,+is+required+for+the+initiation+of+fracture+healing.">BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing.</a> Nat Genet. 38 (12), 1424-1429 (2006).</li><li>Hadjidakis, D. J., Androulakis, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boneremodeling.">Boneremodeling.</a> Ann N Y Acad Sci. 1092, 385-396 (2006).</li><li>Boivin, G., Meunier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+degree+of+mineralization+of+bone+tissue+measured+by+computerized+quantitative+contact+microradiography.">The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography.</a> Calcified Tiss Int. 70 (6), 503-511 (2002).</li><li>Boivin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+remodeling+on+the+mineralization+of+bone+tissue.">Influence of remodeling on the mineralization of bone tissue.</a> Osteoporo Int. 20 (6), 1023-1026 (2009).</li><li>Zanghellini, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+analysis+and+comparison+of+stoichiometric+and+structural+characteristics+of+parosteal+and+conventional+osteosarcoma+with+massive+sclerosis+in+human+bone.">Multimodal analysis and comparison of stoichiometric and structural characteristics of parosteal and conventional osteosarcoma with massive sclerosis in human bone.</a> J Str Biol. 216 (3), 108106 (2024).</li><li>Trento, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=formation+around+two+titanium+implant+surfaces+placed+in+bone+defects+with+and+without+a+bone+substitute+material:+A+histological,+histomorphometric,+and+micro-computed+tomography+evaluation.">formation around two titanium implant surfaces placed in bone defects with and without a bone substitute material: A histological, histomorphometric, and micro-computed tomography evaluation.</a> Clin Implant Dent Relat Res. 22 (2), 177-185 (2020).</li><li>Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiscale+analytical+approach+to+evaluate+osseointegration.">A multiscale analytical approach to evaluate osseointegration.</a> J Mater Sci Mater Med. 29 (5), 60 (2018).</li><li>Bud&#225;n, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+radiomics+evaluation+of+bone+formation+utilizing+multimodal+(SPECT/X-ray+CT)+in+vivo+imaging.">Novel radiomics evaluation of bone formation utilizing multimodal (SPECT/X-ray CT) in vivo imaging.</a> PLoS ONE. 13 (9), e0204423 (2018).</li><li>Hulsart-Billstr&#246;m, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surprisingly+poor+correlation+between+in+vitro+and+in+vivo+testing+of+biomaterials+for+bone+regeneration:+results+of+a+multicentre+analysis.">A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: results of a multicentre analysis.</a> Eur Cells Mater. 31, 312-322 (2016).</li><li>Cramer, E. E. A., Ito, K., Hofmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+bone+models+and+their+potential+in+preclinical+evaluation.">Ex vivo bone models and their potential in preclinical evaluation.</a> Curr Osteoporo Rep. 19 (1), 75-87 (2021).</li><li>Chan, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trabecular+bone+explant+model+of+osteocyte-osteoblast+co-culture+for+bone+mechanobiology.">A trabecular bone explant model of osteocyte-osteoblast co-culture for bone mechanobiology.</a> Cell Mol Bioeng. 2 (3), 405-415 (2009).</li><li>Doke, S. K., Dhawale, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+animal+testing:+A+review.">Alternatives to animal testing: A review.</a> Saudi Pharma J. 23 (3), 223-229 (2015).</li><li>Moussi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+formulation+of+injectable+and+degradable+calcium+phosphate/silanized+hyaluronic+acid+composite+foam:+Investigation+in+a+rabbit+model+of+long+bone+defect.">New formulation of injectable and degradable calcium phosphate/silanized hyaluronic acid composite foam: Investigation in a rabbit model of long bone defect.</a> SSRN. , (2024).</li><li>Par&#233;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+and+axially+vascularized+calcium+phosphate-based+implants+for+segmental+mandibular+defects:+A+promising+proof+of+concept.">Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept.</a> Acta Biomater. 154, 626-640 (2022).</li><li>Roschger, P., Fratzl, P., Eschberger, J., Klaushofer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+quantitative+backscattered+electron+imaging+for+the+measurement+of+mineral+density+distribution+in+human+bone+biopsies.">Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies.</a> Bone. 23 (4), 319-326 (1998).</li><li>Roschger, P., Plenk, H., Klaushofer, K., Eschberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+scanning+electron+microscopy+approach+to+the+quantification+of+bone+mineral+distribution:+backscattered+electron+image+grey-levels+correlated+to+calcium+K+alpha-line+intensities.">A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities.</a> Scanning Micro. 9 (1), 75-86 (1995).</li><li>Porter, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+and+inexpensive+paraffin-embedding+method+for+dynamic+bone+formation+analyses.">Quick and inexpensive paraffin-embedding method for dynamic bone formation analyses.</a> Sci Rep. 7, 42505 (2017).</li><li>Goldschlager, T., Abdelkader, A., Kerr, J., Boundy, I., Jenkin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undecalcified+bone+preparation+for+histology,+histomorphometry+and+fluorochrome+analysis.">Undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis.</a> J Vis Exp. (35), e1707 (2010).</li><li>Kazanci, M., Roschger, P., Paschalis, E. P., Klaushofer, K., Fratzl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+osteonal+tissues+by+Raman+spectral+mapping:+Orientation-composition.">Bone osteonal tissues by Raman spectral mapping: Orientation-composition.</a> J Str Biol. 156 (3), 489-496 (2006).</li><li>Unal, M., Ahmed, R., Mahadevan-Jansen, A., Nyman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compositional+assessment+of+bone+by+Raman+spectroscopy.">Compositional assessment of bone by Raman spectroscopy.</a> Analyst. 146 (24), 7464-7490 (2021).</li><li>Oliver, W. C., Pharr, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+technique+for+determining+hardness+and+elastic+modulus+using+load+and+displacement+sensing+indentation+experiments.">An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments.</a> J Mater Res. 7 (6), 1564-1583 (1992).</li><li>Proffen, B. L., McElfresh, M., Fleming, B. C., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+anatomical+study+of+the+human+knee+and+six+animal+species.">A comparative anatomical study of the human knee and six animal species.</a> Knee. 19 (4), 493-499 (2012).</li><li>Allen, M. J., Houlton, J. E. F., Adams, S. B., Rushton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surgical+anatomy+of+the+stifle+joint+in+Sheep.">The surgical anatomy of the stifle joint in Sheep.</a> Vet Surg. 27 (6), 596-605 (1998).</li><li>Kleuskens, M. W. A., van Donkelaar, C. C., Kock, L. M., Janssen, R. P. A., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+human+osteochondral+culture+model.">An ex vivo human osteochondral culture model.</a> J Ortho Res. 39 (4), 871-879 (2021).</li></ol>

<table><tbody><tr><td>0.20 filters</td><td>VWR&amp;nbsp;</td><td>28145-501</td><td></td></tr><tr><td>18 G needle (1,2x40 mm)</td><td>Sterican</td><td>4665120</td><td></td></tr><tr><td>3 mL syringe</td><td>HENKE-JECT</td><td>8300005762</td><td></td></tr><tr><td>37% hydrochloric acid&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>1.00317.1000&amp;nbsp;</td><td></td></tr><tr><td>Acetic acid (glacial)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A6283&amp;nbsp;</td><td></td></tr><tr><td>Acetone&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>20063-365&amp;nbsp;</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>VWR&amp;nbsp;</td><td>361186</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>VWR&amp;nbsp;</td><td>318612</td><td></td></tr><tr><td>Apatitic tricalcium phosphate&amp;nbsp;</td><td>Centre for Biomedical and Healthcare Engineering (Mines Saint Etienne, France)</td><td>TV26U&amp;nbsp;</td><td></td></tr><tr><td>Azophloxine</td><td>Sigma&amp;nbsp;</td><td>210633</td><td></td></tr><tr><td>Benzoyl peroxide</td><td>Sigma&amp;nbsp;</td><td>8.01641.0250</td><td></td></tr><tr><td>BMP2</td><td>Medtronic&amp;nbsp;</td><td>InductOs 1.5 mg/mL</td><td></td></tr><tr><td>Brillant crocein&amp;nbsp;</td><td>Aldrich&amp;nbsp;</td><td>2107507</td><td></td></tr><tr><td>CTVox&amp;nbsp;</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>DataViewer&amp;nbsp;</td><td>Skyscan</td><td>-</td><td></td></tr><tr><td>Diamond blade</td><td>Struers</td><td>MOD13</td><td></td></tr><tr><td>Diamond saw</td><td>Struers</td><td>Accutom-50</td><td></td></tr><tr><td>DiaPro Mol B3 diamond solution</td><td>Struers</td><td>40600379</td><td></td></tr><tr><td>DiaPro Nap B1 diamond solution</td><td>Struers</td><td>40600373</td><td></td></tr><tr><td>Dibasic sodium phosphate (Na2HPO4)</td><td>Sigma&amp;nbsp;</td><td>102404598</td><td></td></tr><tr><td>Dibutyl Phtalate</td><td>Chimie-Plus Laboratoires</td><td>28656</td><td></td></tr><tr><td>DragonFly software&amp;nbsp;</td><td>ORS</td><td>2022.1.0.1231.&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) high glucose, GlutaMAX(TM), pyruvate</td><td>ThermoFisher Scientific</td><td>31966-021</td><td></td></tr><tr><td>Eosine Y- Surgipath&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>1002830105</td><td></td></tr><tr><td>Erythrosin B</td><td>Sigma&amp;nbsp;</td><td>102141057</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR&amp;nbsp;</td><td>20820362</td><td></td></tr><tr><td>Eukitt&amp;nbsp;</td><td>Dutscher</td><td>6.00.01.0003.06.01.01</td><td></td></tr><tr><td>Falcon 50 mL</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>Ferric chloride hexahydrate (FeCl3, 6H&lt;sub&gt;2&lt;/sub&gt;O)&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>1.03943.0250</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Eurobio</td><td>CVFSVF00</td><td></td></tr><tr><td>Fuchsine acid</td><td>Merck&amp;nbsp;</td><td>1.05231.0025&amp;nbsp;</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Biosera</td><td>MS01NG100J</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma&amp;nbsp;</td><td>86.118.9&amp;nbsp;</td><td></td></tr><tr><td>Isostatic press</td><td>Nova Suisse</td><td>Pmax 1500 bars</td><td></td></tr><tr><td>Laser diffraction granulometry</td><td>&amp;nbsp;Malvern</td><td>Mastersizer 3000</td><td></td></tr><tr><td>Light green</td><td>Prolabo&amp;nbsp;</td><td>28947135</td><td></td></tr><tr><td>Lithium carbonate</td><td>Sigma&amp;nbsp;</td><td>A13149&amp;nbsp;</td><td></td></tr><tr><td>MD-Mol polishing cloth</td><td>Struers</td><td>40500077</td><td></td></tr><tr><td>Methylcyclohexane</td><td>VWR&amp;nbsp;</td><td>8.06147.1000&amp;nbsp;</td><td></td></tr><tr><td>Methylcyclohexane&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>8.06147.1000&amp;nbsp;</td><td></td></tr><tr><td>Methylcyclohexane&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>8.06147.1000&amp;nbsp;</td><td></td></tr><tr><td>Methylmethacrylate</td><td>Sigma&amp;nbsp;</td><td>8.00590.2500</td><td></td></tr><tr><td>Micro-CT, micro-scanner&amp;nbsp;</td><td>Bruker</td><td>Skyscan 1272</td><td></td></tr><tr><td>Monobasic sodium phosphate (NAH2PO4)</td><td>Sigma&amp;nbsp;</td><td>71496</td><td></td></tr><tr><td>Mortar</td><td>Fritsch&amp;nbsp;</td><td>Pulverisette 6</td><td></td></tr><tr><td>N,N, Dimethylanilin</td><td>Sigma&amp;nbsp;</td><td>803060</td><td></td></tr><tr><td>Nanoindentation station</td><td>Anton Paar</td><td>NHT2</td><td></td></tr><tr><td>ND-Nap polishing cloth</td><td>Struers</td><td>40500080</td><td></td></tr><tr><td>OATS Osteochondral Autograft Transfer System Set, 4,75 mm</td><td>Arthrex</td><td>AR-1981-04S</td><td></td></tr><tr><td>OATS Osteochondral Autograft Transfer System Set, 8 mm</td><td>Arthrex</td><td>AR-1981-08S</td><td></td></tr><tr><td>Orange G</td><td>Ral</td><td>M15</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td></td></tr><tr><td>Peel-a-way disposable embbedding moulds</td><td>Polysciences, Inc</td><td>18646C-1</td><td></td></tr><tr><td>Penicillin/Streptomycin (P/S)</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Phosphomolybdic acid&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>221856-100 g</td><td></td></tr><tr><td>Phosphotungstic acid</td><td>Aldrich&amp;nbsp;</td><td>12863-5&amp;nbsp;</td><td></td></tr><tr><td>Polishing machine</td><td>Sturers</td><td>Dap V</td><td></td></tr><tr><td>Poupinel</td><td>MEMMERT</td><td>TV26U&amp;nbsp;</td><td></td></tr><tr><td>Raman microspectrometer</td><td>Renishaw</td><td>InVia Qontor</td><td></td></tr><tr><td>Safran du G&amp;acirc;tinais&amp;nbsp;</td><td>Labonord&amp;nbsp;</td><td>11507737</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Carl Zeiss</td><td>Evo LS 10</td><td></td></tr><tr><td>SEM</td><td>Zeiss</td><td>Carl Zeiss Evo LS10</td><td></td></tr><tr><td>SiC foils/Grinding papers</td><td>Struers</td><td>40400008 (#320), 40400011 (#1000), 40400122 (#2000), 40400182 (#4000)</td><td></td></tr><tr><td>Silver paint</td><td>Electron microscopy sciences</td><td>12686-15</td><td></td></tr><tr><td>Standard stub with Faraday cup, carbon, aluminium and silicon standards</td><td>Micro-Analysis Consultants Ltd</td><td>8602</td><td></td></tr><tr><td>T25 flask</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>Xylene</td><td>VWR&amp;nbsp;</td><td>28975.325</td><td></td></tr><tr><td>Xylidine Ponceau</td><td>Aldrich&amp;nbsp;</td><td>19.976-1&amp;nbsp;</td><td></td></tr></tbody></table>

comprehensive characterization of tissue mineralization in an ex vivo model

<meta charset="utf8"></head><body>作者聚焦:在骨再生研究中表征组织矿化的高级技术

在离体模型中全面表征组织矿化

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

comprehensive-characterization-tissue-mineralization-an-ex-vivo

Research

JoVE Journal

Bioengineering

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

615 次观看.  Nantes Université, Univ Angers, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229. 我们的研究重点是了解和改善骨骼再生。在这里，我们旨在使用各种互补的技术方法广泛表征组织矿化，以促进骨再生疗法的有前途的候选者的早期识别。为了评估新矿化组织的质量和结构，我们使用互补方法，包括与深度学习分析相关的显微 CT 高分辨率 3D 成像。纳米压痕、组织学检查、背向散射扫描电子显微镜映射和拉曼光谱。修复骨缺损?...

视频: 作者聚焦:在骨再生研究中表征组织矿化的高级技术

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

医学

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

化学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

生物化学

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

生物学

HE 染色和 micro-CT 评估小鼠骨小梁微结构

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

作者聚焦:一种经济有效的小鼠骨质疏松症模型中骨微结构定量评估的方法 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

骨质疏松症小鼠模型中的骨小梁微观结构评估

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

在离体模型中全面表征组织矿化

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高分辨率三维成像 EX - VIVO生物样品显微CT

骨转移模型

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

一个简单的临界尺寸股骨缺损模型小鼠

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

高通量，矿化组织的多图像Cryohistology

骨质疏松症小鼠模型中的骨小梁微观结构评估

骨质疏松症小鼠模型中的骨小梁微观结构评估

骨质疏松症小鼠模型中的骨小梁微观结构评估