Name: visualizing angiogenesis by multiphoton microscopy in vivo in genetically modified 3d-plga/nhap scaffold for calvarial critical bone defect repair
Uploaded: 2017-09-07T12:30:00
Description: Watch this Scientific Journal Video about Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair at JoVE.com

Denne studien ble støttet av Shenzhen Peacock programmet, Kina (nr. 110811003586331), Shenzhen grunnleggende forskningsprogrammet (nr. JCYJ20150401150223631, nr. JCYJ20150401145529020 og nr. JCYJ20160331190714896), Guangdong offentlig forskning og kapasitetsbygging spesielle programmet (nr. 2015A020212030), National Natural Science Foundation i Kina (nr. 81501893), National Major Basic forskningsprogrammet Kina (2013CB945503), og Netthinnen innovasjon Program for utmerket unge forskere (Y5G010).

dyr omsorg var i samsvar med veiledningen og bruk av laboratoriet dyr av Guangdongprovinsen. Alle prosedyrer ble utført under veiledning og godkjenning av den etiske komiteen for dyr forskning, Shenzhen institutter for avansert teknologi, kinesiske Academy of Sciences.
 1. lentiviral (LV) produksjon <ol><li>klone pdgf-b cDNA i en lentiviral uttrykk vektor (pLenti6/5-eGFP eller LV-eGFP) på en tilpasset flere-kloning området nedstrøms til cytomegalovirus promoter bruker Spe jeg o...

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E., Mosheiff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+approaches+for+bone+repair:+concepts+and+evidence.">Tissue engineering approaches for bone repair: concepts and evidence.</a> Injury. 42 (6), 609-613 (2011).</li><li>Chiarello, 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=allograft+and+bone+substitutes+in+reconstructive+orthopedic+surgery.">allograft and bone substitutes in reconstructive orthopedic surgery.</a> Aging Clin Exp Res. 25, S101-S103 (2013).</li><li>Elangovan, S., 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=The+enhancement+of+bone+regeneration+by+gene+activated+matrix+encoding+for+platelet+derived+growth+factor.">The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor.</a> Biomaterials. 35 (2), 737-747 (2014).</li><li>Bouyer, M., 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=Surface+delivery+of+tunable+doses+of+BMP-2+from+an+adaptable+polymeric+scaffold+induces+volumetric+bone+regeneration.">Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration.</a> Biomaterials. 104, 168-181 (2016).</li><li>Rezwan, 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=Biodegradable+and+bioactive+porous+polymer/inorganic+composite+scaffolds+for+bone+tissue+engineering.">Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering.</a> Biomaterials. 27 (18), 3413-3431 (2006).</li><li>Burg, K. 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D., 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=Angiogenesis+in+bone+regeneration.">Angiogenesis in bone regeneration.</a> Injury. 42 (6), 556-561 (2011).</li><li>Schmidt, C., 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=Rapid+three-dimensional+quantification+of+VEGF-induced+scaffold+neovascularisation+by+microcomputed+tomography.">Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography.</a> Biomaterials. 30 (30), 5959-5968 (2009).</li><li>Perng, C. 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Ben er en svært vascularized vev med en unik evne til å helbrede og renovere av en individuell1kontinuerlig. Endometrial blodkar er viktig for osteogenesis og defekt reparasjon. Lav endometrial blodkar begrenser bredt klinisk anvendelse av vev-konstruert ben. Konstruere en svært vascularized vev-konstruert Ben ifølge teorien om biomimetics har blitt et verktøy for å reparere stort segment bein mangler. Ulike typer stillasene er vellykket brukt for å reparere forholdsvis lite osseous mangler...

Ben er en svært vascularized vev som fortsetter å remodel i løpet av en individuell1. Rask og effektiv benet fornyelse av store bein mangler fra traumer, nonunion, svulst resections eller craniofacial misdannelser er en kompleks fysiologisk prosess. Tradisjonelle behandlingsmetoder brukes for bein defekt reparasjon inkluderer autograft og allograft implantasjon, men bruken innebærer flere problemer og begrensninger som begrenset tilgjengelighet, betydelig donor området sykelighet, høy risiko for infeksjon, og vert uimottakelig avvisning2,3. Men tilbyr kunstig bein grafts et alternativ for å lindre disse begrensningene. De kan være laget av biologisk nedbrytbart materiale, er lett å være dikte med en egnet porestørrelse, og kan være genetisk modifisert4,5.
Foreløpig har ulike vev engineering stillaser vært ansatt i utviklingen av vev-konstruert Ben6,7. For å indusere bein reparasjon og regeneration mer effektivt, har konstruert biologisk materiale kombinert med vekstfaktorer dukket opp og oppnådd gode resultater8,9. Dessverre, kort halveringstid, lett å miste aktivitet og supraphysiological dosering av vekstfaktorer for terapeutiske effekten begrense deres klinisk anvendelse10. For å overvinne disse problemene, har levering av vekstfaktor gener i stedet for vekst faktorer vist som en effektiv tilnærming til å opprettholde bioactivity for behandling av osseous feil og sykdommer11,12. Viral vektorer er lovende levering verktøy for vev gjenfødelse på grunn av sin høye uttrykke effektivitet13.
Blant vekstfaktorer, ble blodplater-avledet vekst faktor (PDGF-BB) valgt i denne studien fordi det er ikke bare en mitogen og chemoattractant for mesenchymal og osteogenic celler, men også en stimulerende for angiogenese14,15 . Tidligere prekliniske og kliniske studier viste at PDGF-BB kan trygt og effektivt fremme bein reparasjon i periodontal osseous feil16,17. Nyere studier viser at PDGF-BB stimulerer angiogenese av motiverende endothelial celle migrasjon og spredning i vivo18,19. Videre kan PDGF-BB også gjengi stamceller (MSCs) i stand til å skille i endotelceller20, og denne flere høydepunkter MSCs potensielle rolle i neovascularization. Derfor er inducing de novo dannelsen av blodkar i stillaser med PDGF-BB et viktig skritt for reparasjon av vev vokst til stillaser i bein vev engineering.
Bein defekt healing er en dynamisk vev Morfogenetiske prosess som krever koordinert osteogenesis og angiogenese på reparere posisjoner21. Neoangiogenesis i implantert vev-konstruert stillasene er en viktig forutsetning for å forsyne celler med næringsstoffer og oksygen for vekst og overlevelse og fjerner metabolske avfall. Brukte imaging metoder, beregnet inkludert X-ray mikro-tomografi (microCT), magnetisk resonans imaging (MRI), skanning elektronmikroskop (SEM), optical coherence tomografi (OCT) og AC confocal laserskanning mikroskopi, brukes i stedet for histologiske undersøkelser for angiogenese informasjon22,23. Men møte metodene ulike hindringer i visualisere og måler neovasculature i 3D stillaser i bein vev engineering. Multiphoton mikroskopi (MPM) er en relativt ny bio-tenkelig teknikk som har klar fordel av å visualisere celler, ekstracellulær matrix, og rundt vaskulære nettverk samtidig i vivo. Det har en iboende tredimensjonale imaging evnen for dype vev penetrasjon og forårsaker lav photodamage. Derfor, i det siste tiåret, MPM har fått mye oppmerksomhet i biomedisinsk studier24, inkludert i nevrovitenskap, immunologi og stamcelleforskningen dynamics. Men er det knapt brukt i Ortopedisk forskning.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly(D,L-lactide-co-glycolide) (PLGA)</td>
			<td>Sigma</td>
			<td>P1941</td>
			<td>L/G ratio 75:25, MW 66000-107000</td>
		</tr>
		<tr>
			<td>Hydroxyapatite nanoparticles</td>
			<td>Sigma</td>
			<td>702153</td>
			<td>Average diameter &#60; 200nm</td>
		</tr>
		<tr>
			<td>Chloroquine diphosphate salt</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated 250-kD dextran</td>
			<td>Sigma</td>
			<td>FD250S</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-dioxane</td>
			<td>lingfeng,Shanghai</td>
			<td></td>
			<td>0.45 micron</td>
		</tr>
		<tr>
			<td>Stericup filters</td>
			<td>Merck Millipore Corporation</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB Cdna</td>
			<td>Sino Biological, Inc</td>
			<td>MZ50801-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PDGF-BB mouse polyclonal antibody</td>
			<td> BioVision, Inc</td>
			<td>5489-30T</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB recombinant protein</td>
			<td></td>
			<td>4489-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium-phosphate transfection solution</td>
			<td>Promega Corporation</td>
			<td>E1200</td>
			<td></td>
		</tr>
		<tr>
			<td>L-DMEM</td>
			<td>Hyclone</td>
			<td>SH30021.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell </td>
			<td>Corning</td>
			<td>3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Male BALB/c mice</td>
			<td>Guangdong Medical Laboratory Animal Center </td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pentobarbital </td>
			<td>Merck</td>
			<td>1063180500</td>
			<td></td>
		</tr>
		<tr>
			<td>multiphoton microscopy</td>
			<td></td>
			<td></td>
			<td>A homemade in Shenzhen Institutes of Advanced Technology to detect two-photon excited fluorescence (TPEF) and second harmonic generation signal (SHG).</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Keyuan, Shandong</td>
			<td>401750169</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Mix (Perfect Real Time)</td>
			<td>Takara</td>
			<td>RR420B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq (Tli RNaseH Plus)</td>
			<td>Takara</td>
			<td>RR036B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin</td>
			<td>Beyotime</td>
			<td>C0105</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica </td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge OPtima L-100XP</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-temperature printer </td>
			<td>Tsinghua university</td>
			<td></td>
			<td>A homemade in Tsinghua university</td>
		</tr>
		<tr>
			<td>LightCycler 480 instrument </td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>microCT</td>
			<td>Bruker</td>
			<td>1176</td>
			<td></td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing angiogenesis by multiphoton microscopy in vivo in genetically modified 3d-plga/nhap scaffold for calvarial critical bone defect repair

Gjenoppbygging av kritisk størrelse bein mangler forblir en alvorlig klinisk problem på grunn av dårlig angiogenese i vev-konstruert stillaser under reparasjon, som gir opphav til en mangel på tilstrekkelig blodtilførsel og fører til nekrose av nye vev. Rask endometrial blodkar er en viktig forutsetning for nye vev overlevelse og integrasjon med eksisterende vert vev. De novo generering av blodkar i stillasene er en av de viktigste trinnene i å gjøre bein gjenfødelse mer effektivt, slik at reparasjon vev til å vokse til et stillas. For å håndtere dette problemet, brukes den genetisk modifisering av en biomateriale stillaset å akselerere angiogenese og osteogenesis. Imidlertid er visualisere og spore i vivo blodkar formasjonen i sanntid og tredimensjonale (3D) stillaser eller nye benvev fortsatt en hindring for bein vev engineering. Multiphoton mikroskopi (MPM) er en roman bio-imaging modalitet som kan skaffe volumetriske data fra biologiske strukturer på en høy oppløsning og minimal-invasiv måte. Målet med denne studien var å visualisere angiogenese med multiphoton mikroskopi i vivo i en genetisk modifisert 3D-PLGA/nHAp stillaset calvarial kritisk bein defekt reparasjon. PLGA/nHAp stillaser var functionalized for vedvarende levering av et vekstfaktor pdgf-b gen bærer lentiviral vektorer (LV -pdgfb) for å lette angiogenese og å styrke bein gjenfødelse. I et stillas-implantert calvarial kritisk bein defekt musemodell, blodkar områder (BVAs) i PHp stillaser var betydelig høyere enn i PH stillaser. I tillegg økt uttrykk for pdgf-b og angiogenese-relaterte gener, vWF og VEGFR2, tilsvarende. MicroCT analyse indikerte at nye bein dannelsen i gruppen PHp dramatisk forbedret i forhold til andre grupper. Vi vet er dette første gang multiphoton mikroskopi ble brukt i bein vev utvikling for å etterforske angiogenese i en 3D bio-nedbrytbar stillaset i vivo og i sanntid.

Sylindrisk porøse PLGA/nHAp stillaser 0.6 mm inne høyde og 4 mm i diameter, ble laget med en 3D-skriver. Morphologies av stillasene ble analysert via skanning elektronmikroskop og microCT. Figur 1A viser fotografiet av implantert stillaset. MicroCT skanning viste at mer enn 85% av porene hadde størrelser fra 200 til 400 µm (figur 1B). SEM imaging vist at overflaten av stillaset hadde en grov microtopography, med micropores (diameter ca 5-10 µm) som s...

Watch this Scientific Journal Video about Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair at JoVE.com

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Her presenterer vi en protokoll for å visualisere blodkar formasjonen i vivo og i sanntid i 3D stillaser av multiphoton mikroskopi. Angiogenese i genmodifiserte stillaser ble studert i murine calvarial kritisk bein feil modell. Flere nye blodkar ble oppdaget i behandling gruppen enn i kontroller.

Visualisere angiogenese av Multiphoton mikroskopi I Vivo i genmodifiserte 3D-PLGA/nHAp stillaset Calvarial kritisk bein defekt reparasjon

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

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Bioengineering

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

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Visualisere Proteiner og makromolekylær komplekser ved Negative Stain EM: fra Grid Forberedelse til Image Acquisition

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En Full huddefekt modell for å evaluere vaskularisering av biomaterialer<em&gt; In Vivo</em

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Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

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Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Særpreget Handlekraft av mikrokanaler i Bone-lignende Maler kan forbedre Rekruttering av celler for Restaurering av Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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Stive innebygging av fast og farget, hele, Millimeter skala prøver for delen-gratis 3D Histology av mikro-beregnet tomografi

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

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In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...