Name: Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model
Uploaded: 2023-09-08T14:10:00
Description: Watch this Scientific Journal Video about An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model at JoVE.com

Dette arbeidet ble støttet av Sichuan Provincial Administration of Traditional Chinese Medicine (2021YJ0175) og Graduate Research Innovation Project ved School of Clinical Medicine (LCYJSKT2023-11), Chengdu University of Traditional Chinese Medicine.

Dyreprotokollen er godkjent av Animal Ethical Committee of Chengdu University of Traditional Chinese Medicine (Rekordnummer: 2020-34). Hunnmus C57BL/6J (12-ukers alder, n = 14) ble tilfeldig delt inn i to grupper, en narreoperert gruppe (Sham group, n = 7) og en modellgruppe (OVX group, n = 7). Dyrene ble kjøpt fra en kommersiell leverandør (se Materialfortegnelse). Alle mus ble holdt i individuelle merder ved 22-26 °C med 45% -55% fuktighet, fikk lov til å tilpasse seg sitt nye miljø i 1 uke, og ga...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

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Osteoporose kan føre til hyppige brudd, som er kostbare, kan forårsake smerte, funksjonshemming eller til og med død, og alvorlig påvirke livskvaliteten til pasienter20. Gjennom årene har ovariektomimodellen blitt anerkjent som en av standardmetodene for å studere osteoporose21. Den vanligste prekliniske dyremodellen for osteoporose er ovariektomisert (OVX) rotte. Til tross for dette har flertallet av forskningen på mekanismene for beinforstyrrelser, inkludert osteop...

Osteoporose er en utbredt metabolsk beinsykdom, særlig blant eldre, og er forbundet med økt risiko for skjørhetsbrudd. Etter hvert som osteoporose blir mer vanlig i Kina1, vil det være en økende etterspørsel etter å studere beinstrukturer hos små dyr 2,3. De tidligere metodene for måling av bentap er avhengige av resultatene av todimensjonal dual-energy røntgenabsorptiometri. Dette fanger imidlertid ikke opp endringene i den arkitektoniske mikrostrukturen til det trabekulære beinet, som er en nøkkelfaktor for skjelettstyrke4. Mikrostrukturen av bein påvirker dens styrke, stivhet og bruddmotstand. Ved å sammenligne beinmikroarkitektur i normale og patologiske tilstander, kan endringer i beinvevsmorfologi, struktur og funksjon forårsaket av osteoporose identifiseres. Denne informasjonen bidrar til forståelsen av utviklingen av osteoporose og dens tilknytning til andre sykdommer.
Mikrocomputertomografi (Micro-CT) avbildning har nylig blitt en populær teknikk for vurdering av beinmorfologi, der den kan gi nøyaktige og omfattende data om beinstruktur og tetthetsparametere som benvolumfraksjon, tykkelse og separasjon 5,6. Samtidig kan Micro-CT-resultatene påvirkes av analyseprogrammet7. Ulike metoder for bildeinnsamling, evaluering og rapportering brukes av ulike kommersielle Micro-CT-systemer. Denne inkonsekvensen gjør det vanskelig å sammenligne og tolke resultatene rapportert av ulike studier5. Dessuten kan det for tiden ikke erstatte beinhistomorfometri ved å gi forskere informasjon om parametere på cellenivå i skjelettsystemet8. I mellomtiden tillater histologiske teknikker direkte observasjon og måling av mikroskopisk morfologi av bein. Farging av hematoksylin og eosin (HE) er en vanlig fargeteknikk som brukes i histologi for å visualisere den generelle strukturen til celler og vev. Det brukes til å identifisere tilstedeværelsen av beinvev og dets mikroarkitektur.
Denne artikkelen bruker Micro-CT kombinert med vevskuttingsteknikk (Hematoxylin-Eosin [HE] farging) for å samle beinvevsbilder og utføre kvantitativ analyse av trabekulært bein for å evaluere endringene i beinmikrostruktur i en osteoporose musemodell.

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			<td>4% Paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
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			<td>Adobe Photoshop</td>
			<td>Adobe Inc.</td>
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			<td>Chengdu Kolon Chemical Co., Ltd</td>
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			<td>Analyze 12.0</td>
			<td>AnalyzeDirect, Inc</td>
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			<td>Anatomical Forceps</td>
			<td>Jinzhong surgical instrument Co., Ltd</td>
			<td>J3C030</td>
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			<td>Anhydrous Ethanol</td>
			<td>Chengdu Kolon Chemical Co., Ltd</td>
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			<td>Automatic Dyeing Machine</td>
			<td>Thermo scientific</td>
			<td>Varistain&#8482; Gemini ES</td>
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			<td>Bone Microarchitecture Analysis Add-on</td>
			<td>AnalyzeDirect, Inc</td>
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			<td>C57BL/6J mice</td>
			<td>SPF (Beijing) Biotechnology Co., Ltd.</td>
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			<td>Carrier Slides</td>
			<td>Nantong Mei Wei De Experimental Equipment Co., Ltd</td>
			<td>220518001</td>
			<td></td>
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			<td>Coverslips</td>
			<td>Nantong Mei Wei De Experimental Equipment Co.</td>
			<td>220518001</td>
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			<td>Decalcification Solution</td>
			<td>Wuhan Xavier Biotechnology Co., Ltd</td>
			<td>CR2203047</td>
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			<td>Delicate Scissors</td>
			<td>Jinzhong surgical instrument Co., Ltd</td>
			<td>ZJA010</td>
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			<td>Embedding box marking machine</td>
			<td>Thermo scientific</td>
			<td>&#160;PrintMate AS</td>
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			<td>Embedding Machine</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-P5</td>
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			<td>Fiji: ImageJ</td>
			<td>National Institutes of Health, USA</td>
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			<td></td>
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			<td>Film Sealer</td>
			<td>Thermo scientific</td>
			<td>Autostainer 360</td>
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			<td>Freezing Table</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-L5</td>
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			<td>H&#38;E Staining Kit</td>
			<td>Leagene</td>
			<td>DH0020</td>
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			<td>Hydrochloric Acid Solution</td>
			<td>Sichuan Xilong Science Co., Ltd</td>
			<td>210608</td>
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			<td>ImageJ2 Plugin</td>
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			<td>BoneJ 7.0.16</td>
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			<td>Medical Gauze</td>
			<td>Shandong Ang Yang Medical Technology Co.</td>
			<td></td>
			<td></td>
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			<td>Mersilk 3-0 Silk Braided Non-Absorbable Sutures</td>
			<td>Ethicon, Inc.</td>
			<td>SA84G</td>
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			<td>Needle Holder</td>
			<td>Jinzhong surgical instrument Co., Ltd</td>
			<td>J32010</td>
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			<td>Neutral Balsam</td>
			<td>Sinopharm Group Chemical Reagent Co., Ltd</td>
			<td>10004160</td>
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			<td>Oven</td>
			<td>Shanghai Yiheng Scientific Instruments Co., Ltd</td>
			<td>DHG-9240A</td>
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			<td>PANNORAMIC Digital Slide Scanners</td>
			<td>3DHISTECH Ltd.&#160;</td>
			<td>PANNORAMIC DESK/MIDI/250/1000</td>
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			<td>PBS buffer</td>
			<td>Biosharp</td>
			<td>G4202</td>
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			<td>Povidone-iodine solution 5%</td>
			<td>Chengdu Yongan Pharmaceutical Co., Ltd</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Quantum GX2 microCT Imaging System</td>
			<td>PerkinElmer, Inc.</td>
			<td></td>
			<td></td>
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			<td>Rotary Microtome</td>
			<td>Thermo scientific</td>
			<td>HM325</td>
			<td></td>
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		<tr>
			<td>Scalpel</td>
			<td>Quanzhou Excellence Medical Co., Ltd</td>
			<td>20170022</td>
			<td></td>
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			<td>Scan &#38; Browse Software</td>
			<td>3DHISTECH Ltd.&#160;</td>
			<td>CaseViewer2.4</td>
			<td></td>
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			<td>Single-Use Sterile Rubber Surgical Gloves</td>
			<td>Guangdong Huitong Latex Products Group Co., Ltd</td>
			<td>22B141EO</td>
			<td></td>
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			<td>Sodium Chloride Solution 0.9%</td>
			<td>Sichuan Kelun Pharmaceutical Co., Ltd</td>
			<td></td>
			<td></td>
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			<td>Sterile Hypodermic Syringes for Single Use</td>
			<td>Shandong Weigao Group Medical Polymer Products&#160; Co., Ltd</td>
			<td></td>
			<td></td>
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			<td>Sterile Medical Suture Needles</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.&#160;</td>
			<td>PW8068</td>
			<td></td>
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			<td>Tissue Processor</td>
			<td>Thermo scientific</td>
			<td>STP420 ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Spreading and Baking Machine</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JK-6</td>
			<td></td>
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		<tr>
			<td>Tribromoethanol</td>
			<td>Nanjing Aibei Biotechnology Co., Ltd</td>
			<td>M2920</td>
			<td></td>
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			<td>Wax Trimmer</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JXL-818</td>
			<td></td>
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			<td>Xylene</td>
			<td>Chengdu Kolon Chemical Co., Ltd</td>
			<td>2022051901</td>
			<td></td>
		</tr>
	</tbody>
</table>

trabecular bone microarchitecture evaluation in an osteoporosis mouse model

Benmikrostruktur refererer til arrangementet og kvaliteten på beinvev på mikroskopisk nivå. Å forstå skjelettets beinmikrostruktur er avgjørende for å få innsikt i patofysiologien ved osteoporose og forbedre behandlingen. Imidlertid kan håndtering av beinprøver være komplisert på grunn av deres harde og tette egenskaper. For det andre gjør spesialisert programvare bildebehandling og analyse vanskelig. I denne protokollen presenterer vi en kostnadseffektiv og brukervennlig løsning for analyse av trabekulær beinmikrostruktur. Detaljerte trinn og forholdsregler er gitt. Micro-CT er en ikke-destruktiv tredimensjonal (3D) avbildningsteknikk som gir høyoppløselige bilder av trabekulær beinstruktur. Det muliggjør objektiv og kvantitativ evaluering av beinkvalitet, og det er derfor det er allment ansett som gullstandardmetoden for vurdering av beinkvalitet. Imidlertid forblir histomorfometri uunnværlig, da det gir viktige parametere på cellulært nivå, som bygger bro over gapet mellom todimensjonale (2D) og 3D-vurderinger av beinprøver. Når det gjelder de histologiske teknikkene, valgte vi å avkalke beinvevet og deretter utføre tradisjonell parafininnebygging. Oppsummert kan kombinasjon av disse to metodene gi mer omfattende og nøyaktig informasjon om beinmikrostruktur.

Osteoporosis is a prevalent metabolic bone disease, especially among the elderly, and is associated with an increased risk of fragility fractures. As osteoporosis becomes more common in China1, there will be a growing demand for studying the bone structures of small animals2,3. The previous methods of measuring bone loss rely on the outcomes of two-dimensional dual-energy x-ray absorptiometry. However, this does not capture the changes in the architectural microstructure of the trabecular bone, which is a key factor for skeletal strength4. The microstructure of bone affects its strength, stiffness, and fracture resistance. By comparing bone microarchitecture in normal and pathological states, changes in bone tissue morphology, structure, and function caused by osteoporosis can be identified. This information contributes to the understanding of the development of osteoporosis and its association with other diseases. 
Micro-computed tomography (Micro-CT) imaging has recently become a popular technique for bone morphology assessment, where it can provide accurate and comprehensive data on bone structure and density parameters such as bone volume fraction, thickness, and separation5,6. At the same time, the Micro-CT results can be affected by the analysis software7. Different methods of image acquisition, evaluation, and reporting are used by various commercial Micro-CT systems. This inconsistency makes it hard to compare and interpret the results reported by different studies5. Also, it cannot currently replace bone histomorphometry in providing researchers with information on cellular-level parameters in the skeletal system8. Meanwhile, histologic techniques allow direct observation and measurement of the microscopic morphology of bone. Hematoxylin and eosin (HE) staining is a common staining technique used in histology to visualize the general structure of cells and tissues. It is used to identify the presence of bone tissue and its microarchitecture.
This article uses Micro-CT combined with tissue slicing technique (Hematoxylin-Eosin [HE] staining) to collect bone tissue images and perform quantitative analysis of trabecular bone to evaluate the changes of bone microstructure in an osteoporosis mouse model.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

Evaluering av trabekulær benmikroarkitektur i en osteoporosemusemodell

Our research focused on the effects of traditional Chinese medicine, or TCM, on osteoporosis. We are trying to explore the mechanism, efficacy, and safety of TCM intervention for osteoporosis. Micro-computed tomography imaging has recently become a popular technique for bone morphology assessment.It can provide accurate and comprehensive data on bone structure and density parameters, such as bone volume, fracture thickness, and separation. The skeletal system is the most unique tissue structure in terms of the physical properties of the body. It contains the inorganic component arginine calcium phosphate, and it is hard and stiff as a result.The process of hurting bone symptoms is tedious. We provided a complete and detailed process from modeling to osteoporosis in evaluation for researchers. We believe the application of the technology will undoubtedly bring great improvements to osteoporosis research in the context of ethnomedicine exploration.

Micro-CT-analyse Vi målte trabekulære mikroarkitektoniske parametere hos mus fra begge gruppene og rapporterte deres gjennomsnittsverdier og SD i tabell 1. Fordelingen av noen parametere (dvs. forholdet mellom benvolum og totalt vevsvolum, trabekulær tykkelse, trabekulær separasjon) innenfor hver gruppe er illustrert i figur 3.
Disse resultatene indikerer signifikante forskj...

Watch this Scientific Journal Video about An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model at JoVE.com

Denne protokollen presenterer en økonomisk og effektiv metode for kvantitativ evaluering av beinmikroarkitektur i en musemodell av osteoporose ved å kombinere hematoksylin-eosin (HE) farging og mikrocomputertomografi (Micro-CT) teknikker.

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

Vår forskning fokuserte på effekten av tradisjonell kinesisk medisin, eller TCM, på osteoporose. Vi prøver å utforske mekanismen, effekten og sikkerheten til TCM-intervensjon for osteoporose. Mikro-computertomografi avbildning har nylig blitt en populær teknikk for bein morfologi vurdering.Det kan gi nøyaktige og omfattende data om beinstruktur og tetthetsparametere, for eksempel beinvolum, bruddtykkelse og separasjon. Skjelettsystemet er den mest unike vevstrukturen når det gjelder kroppens fysiske egenskaper. Den inneholder den uorganiske komponenten arginin kalsiumfosfat, og det er hardt og stivt som et resultat.Prosessen med å skade bein symptomer er kjedelig. Vi ga en komplett og detaljert prosess fra modellering til osteoporose i evaluering for forskere. Vi tror anvendelsen av teknologien utvilsomt vil gi store forbedringer til osteoporoseforskning i sammenheng med etnomedisinsk utforskning.