Name: computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts
Uploaded: 2015-12-22T13:00:00
Description: Watch this Scientific Journal Video about Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts at JoVE.com

The authors acknowledge funding from the NIDCR (Grant No. DE019902) and from the Israeli Science Foundation (Grant No. 382/13).

Protokollen følger retningslinjene i den institusjonelle dyr omsorg og bruk komité (IACUC) fra Det hebraiske universitetet i Jerusalem, Israel (Request No. MD-12-13524-4), en AAALAC godkjent anlegg, og ved Cedars-Sinai Medical Center IACUC (Request No. 3770). Dyrene ble behandlet i streng overholdelse NIH retningslinjer. 1. Utarbeidelse av Bone allotransplantater <ol><li> Avlive 7 til 8 uker gamle Balb / C-mus, eller en hvilken som helst stamme forskjellig fra mottakeren, ved hjelp av st...

<ol>
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Formålet med multimodale avbildningsfremgangsmåter som er beskrevet her, er å muliggjøre grundig undersøkelse av angiogenese-osteogenesis aksen i sammenheng med kraniebentransplantering. Neovascularization ble avbildes med en μCT protokollen, som tillot en nøyaktig høyoppløselig 3D-demonstrasjon av vaskulær treet fôring hele kranie defekt. μCT data lett kan analyseres ved hjelp av avanserte verktøy som IPL programvare. For eksempel kan tykkelsen analysen er vist i figur 3C ...

Kraniofaciale bentap på grunn av traumer, tumor reseksjon, decom kraniotomi, og medfødt defekt sjelden helbreder seg selv og presenterer et klart udekket klinisk behov. Autologe bein grafts og allogene bein grafts er mye brukt til å behandle disse forholdene 1. Det er allment akseptert at osteogenesis er tett koblet med angiogenese 2,3. Således bør fullstendig studium av et slått terapi for benregenerering inkludere en omfattende undersøkelse av det vaskulære treet danner hele defekte område. Det er flere tilgjengelige metoder for å karakter vaskularisering i forskningsmodeller. Vaskulære treet kan undersøkes ved histologisk analyse. Siden histologi er avhengig av seksjonering vev, er det stor sannsynlighet for at det resulterende bildet vil bli forvrengt. For å løse dette problemet, kan intramikroskopi utføres til bilde intakt blodkar 4; Imidlertid er denne metodenbegrenset til en plan-imaging. μCT scanning av prøver hentet fra et dyr dynket med kontrastmiddel tillater 3D avbildning av det vaskulære nettverket som mater regenerering nettstedet fem. Denne tilnærmingen muliggjør en meget detaljert demonstrasjon av et organ vaskulatur som helhet, samt en grundig analyse av blodkarfordelingen. Videre gjør μCT differensiering mellom ulike diametre av blodkar, som karakteriserer de ulike subtyper av blodkar. Vi antok at implantering av et calvarial autograft vil indusere neovaskularisering er større enn implantasjon av en allograft, og denne økte neovaskularisering fører i sin tur til økt bein formation.To følge denne hypotesen vi benyttet en rekke teknikker. Vi undersøkte mønstre av den nydannede vaskulær treet ved å utføre en μCT basert analyse. Vi målte blodperfusjon bruker en blod-pool fluorescerende probe. Neste, vi eslersed benvev mineralisering av FLI av hydroksyapatitt styrt sonde og μCT analyse. Til slutt overvåkes vi stamcelle rekruttering og differensiering, utføre BLI i transgene mus som luciferase er uttrykt i osteocalcin-positive celler.

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			<td height="21" style="height:21px;width:216px;">isoflurane (furane)</td>
			<td style="width:112px;">Baxter</td>
			<td align="right" style="width:119px;">1001936040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS kinetics</td>
			<td style="width:112px;">Xenogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beetle luciferin</td>
			<td style="width:112px;">Promega</td>
			<td style="width:119px;">E160A</td>
			<td></td>
		</tr>
	</tbody>
</table>

computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts

En viktig parameter å bestemme suksessen til et bein pode prosedyren er vaskularisering av området rundt pode. Vi antok at implantasjon av et bein autograft ville indusere større benregenerering av frodig blodkardannelse. For å undersøke effekten av graftet i neovaskulariseringen i det defekte område, har vi utviklet en mikro-computertomografi (μCT) tilnærming til å karakterisere nylig danne blodkar, som involverer systemisk perfusjon av dyret med et polymeriserende kontrastmiddel. Denne metoden gjør det mulig detaljert vaskulær analyse av et organ i sin helhet. I tillegg ble blodperfusjon vurdert ved hjelp av fluorescens imaging (FLI) av en blodbårne fluorescerende agent. Bendannelse ble kvantifisert ved FLI bruke en hydroksyapatitt-målrettet sonde og μCT analyse. Stamcelle rekruttering ble overvåket ved bioluminescens imaging (BLI) av transgene mus som uttrykker luciferase under kontroll av osteocalcin promoteren.Her beskriver og demonstrerer fremstillingen av allograft, calvarial defekt kirurgi, μCT skanning protokoller for neovaskularisering studier og bendannelse analyse (inklusive in vivo perfusjon av kontrastmiddel), og protokollen for dataanalyse. 3D høy oppløsning analyse av vaskulaturen viste signifikant større angiogenese hos dyr med implantert autografts, særlig med hensyn til arterioler formasjonen. Følgelig blodperfusjon var signifikant høyere i autograft gruppen ved den 7. dagen etter operasjonen. Vi observerte overlegen beinmineralisering og målt større beindannelse hos dyr som fikk autografts. Autograft implantasjon indusert bosatt stamcelle rekruttering til graft-host bein sutur, der cellene differensiert i bendannende celler mellom 7. og 10. postoperative dag. Dette funnet betyr at økt beindannelse kan tilskrivesaugmented vaskulær fôring som karakteriserer autograft implantasjon. Metodene avbildet kan tjene som et optimalt verktøy for å studere benregenerering i form av tett avgrenset beindannelse og neovascularization.

Neovascularization ble vurdert av μCT volumetrisk analyse og ved FLI bruker en fluorescerende blodbårne agent for å kvantifisere blodperfusjon. Syv dager etter operasjonen, viste μCT scanning et betydelig høyere volum av små og mellom diameter blodkar i mus som hadde fått autografts enn i mus som hadde fått allografter høstet fra C57BL / 6 (figur 3A). Interessant, i autograft gruppen den nydannede vaskulære tre syntes å nå hele defekte område, mens det i allograft gruppen blodkar viste seg ...

Watch this Scientific Journal Video about Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts at JoVE.com

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Implantasjon av autologe og allogene bein grafts utgjør akseptert tilnærminger til å behandle store kraniofaciale bentap. Likevel effekten av graft sammensetning på samspillet mellom neovascularization, celledifferensiering og beindannelse er uklart. Vi presenterer en multimodal bildebehandling protokoll forsøkte å belyse angiogenese-osteogenesis gjensidig avhengighet på pode nærhet.

Computertomografi og optisk avbildning av Osteogenesis-angiogenese Kobling til Vurdere Integrering av kraniet autografts og allografter

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

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