Name: minced tissue in compressed collagen: a cell-containing biotransplant for single-staged reconstructive repair
Uploaded: 2016-02-24T13:00:00
Description: Watch this Scientific Journal Video about Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair at JoVE.com

The authors thank the Swedish Society for Medical Research, the Promobilia Foundation, the Crown Princess Lovisa Foundation, the Freemason Foundation for Children&#8217;s Welfare, the Swedish Society of Medicine, the Solstickan Foundation, Karolinska Institutet, and the Stockholm City Council for financial support.

Alla djurprotokoll var i förväg godkänts av Stockholms läns kommittén på djur och alla förfaranden överensstämde med reglerna för djuranvändning, samt relevanta federala lagar. 1. djurförsök <ol><li> Förbereda djur för kirurgi <ol><li> Förbered operationsbordet med alla material och instrument som behövs för verksamheten under sterila förhållanden. Utföra operationen uteslutande under sterila förhållanden för att minska risken för infektion och optimera förhållan...

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrarapid+engineering+of+biomimetic+materials+and+tissues:+fabrication+of+nano-+and+microstructures+by+plastic+compression.">Ultrarapid engineering of biomimetic materials and tissues: fabrication of nano- and microstructures by plastic compression.</a> Adv Funct Mater. 15, 1762-1770 (2005).</li><li>Fumagalli Romario, U., Puccetti, F., Elmore, U., Massaron, S., Rosati, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-gripping+mesh+versus+staple+fixation+in+laparoscopic+inguinal+hernia+repair:+a+prospective+comparison.">Self-gripping mesh versus staple fixation in laparoscopic inguinal hernia repair: a prospective comparison.</a> Surg Endosc. 27, 1798-1802 (2013).</li><li>Muangman, P., 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=Complex+Wound+Management+Utilizing+an+Artificial+Dermal+Matrix.">Complex Wound Management Utilizing an Artificial Dermal Matrix.</a> Annals of Plastic Surgery. 57, 199-202 (2006).</li><li>Ajalloueian, F., Zeiai, S., Fossum, M., Hilborn, J. 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Denna studie presenteras en enkel att använda tillvägagångssättet för att producera blåsväggen fläckar med autolog vävnad för transplantation vid operationsbordet. Plåstren är bildade genom kombinationen av en biologiskt nedbrytbar polymer stickning i mitten och kollagen med och utan malda vävnaden i de yttre ytorna i kombination med plast komprimering. Plast komprimering är en metod som tidigare beskrivits av andra författare och kan definieras som en snabb utdrivning av vätska från kollagengeler ...

De flesta vävnadstekniska studier transplantation till huden och urogenitalsystemet inkluderar autologa cell skördar från frisk vävnad och cellexpansion i specialutrustade cell odling anläggningar 1,2. Efter cellexpansion, cellerna vanligtvis lagras för senare användning när patienten är beredd att ta emot den kroppsegna. Kväve frysar tillåta långvarig lagring vid låga temperaturer av -150 ° C eller lägre. Processen för frysning måste vara försiktig och kontrollerad för att inte förlora cellerna. En risk av celldöd är kristallisation av intracellulärt vatten under upptiningsprocessen, vilket kan leda till brott på cellmembranen. Cell frysning utförs vanligtvis genom långsam och kontrollerad kylning (-1 ° C per min), med användning av en hög koncentration av celler, fetalt bovinserum, och dimetylsulfoxid. Efter upptining av cellerna behöver bearbetas på nytt genom att ta bort frysmedium och odling på cellodlings plast eller enbiomaterial innan transplantation tillbaka till patienten. Alla de ovan nämnda stegen är tidskrävande, arbetskrävande och kostsamma 3. Dessutom, alla in vitro-behandling av celler avsedda för patient transplantation är starkt reglerad och kräver välutbildad och ackrediterade personal och laboratorier 4. Allt som allt, att anskaffa en säker och tillförlitlig tillverkningsprocess, tekniken kan endast fastställas i ett mycket litet antal tekniskt avancerade centra och en bredare användning i vanliga kirurgiska sjukdomar är tveksamt. I syfte att övervinna begränsningarna hos cellodling i laboratoriemiljön, är konceptet med att transplantera malda vävnaden för cell expansionen in vivo infördes genom att använda kroppen själv som en bioreaktor. För dessa ändamål, skulle auto företrädesvis att transplanteras på en 3D-form enligt den form som behövs för den slutliga rekonstruktionen av organ intresset 5-7. Ursprungligen var tanken att transplantera malet epitel presenteras av Meek 1958 när han beskrev hur epitel växer från kanterna av ett sår. Han visade att en liten bit hud skulle öka sina marginaler och därmed dess potential för expansion cell med 100% genom att skära bit två gånger i vinkelräta riktningar (Figur 1) 8. Teorin har fått stöd genom användning av nät med partiell tjocklek hudtransplantation för hudtransplantation 9 och i huden sårläkningsmodeller 10. <img alt="Figur 1" src="/files/ftp_upload/53061/53061fig1.jpg" width="600" /> Figur 1:. Meek teori Enligt Meek teori, växer epitel från kanterna av ett sår. Genom att öka området exponeras av malningen teknik, epithelializes malet vävnad sår från många platser. Den aktuella studien är baserad på hypotesen att samma princip skulle kunna tillämpas på den subkutana vävnaden genom att placera malet epitel runt en form. Epitelcellerna skulle mobilisera från malet transplantationer (omorganisera), täcker de lindade områden (migrerar) och dividera (expandera) i syfte att bilda en kontinuerlig neoepithelium som täcker sårområdet och separerar det främmande föremålet (formen) från den inre kroppen ( Figur 2). <img alt="figur 2" src="/files/ftp_upload/53061/53061fig2.jpg" /> Figur 2:. Tecknad av en 3D-form med malet epitel för in vivo intrakorporeal expansions vävnad enligt teorin om Meek Genom att använda malda vävnaden placeras på en form och därefter transplanteras till den subkutana vävnaden, är hypotesen att epitelcellerna migrera från kanterna av den malda vävnaden, omorganisera, och expandera för att bilda en kontinuerlig neoepithelium som täcker sårområdet och separerar det främmande föremålet (formen) från den inre kroppen. Även tidigare in vivo-studier visar lovande resultat, kan ytterligare förbättringar uppnås genom att förstärka de auto så att regenere epitel kunde motstå mekanisk trauma bättre 7. För dessa ändamål, var viktiga förutsättningar för en framgångsrik biomaterial identifierats, såsom: enkel diffusion av näringsämnen och avfallsprodukter, möjlighet att mögel i en 3D sätt och enkelheten i kirurgisk behandling. Slutsatser gjordes att dessa behov kan tillgodoses genom att lägga till en sammansatt biomaterial till malet vävnaden. Den aktuella studien syftar till att utveckla en byggnadsställning som består av malet vävnad i plast-komprimerade kollagen innehållande en förstärkande kärna av en biologiskt nedbrytbar tyg. På detta sätt kan livskraftiga celler migrera från malet vävnadspartiklar och föröka sig med morfologiska egenskaper som kännetecknar den ursprungliga epitel (hud eller urothelium). Med hjälp av plast kompression, ställningen var att minskad i storlek från 1 cm till omkring 420 um som det malda partiklarna var innesluten i det övre skiktet kollagen. Stomfilten kan vara vilken som helst polymer men behöver modifieras med en hydrofil yta i syfte att koppla samman med de täckande kollagenskikt 11. Metoden gav en förbättrad ställnings integritet genom att införliva en stickad mesh bestående av poly (ε-kaprolakton) (PCL) inom två plast komprimerad kollagengeler använder den som en byggnadsställning för odling malet blåsan slemhinnan eller malet hud från svin. Konstruktet bibehölls i cellodlingsbetingelser i upp till 6 veckor in vitro, vilket demonstrerar framgångsrik bildning av en skiktad, flerskiktad urotelium eller skvamöst huden epitelet på toppen av en väl konsoliderad hybrid konstrukt. Konstruktionen var lätta att hantera och kan sys på plats för blås augmentation ändamål eller beläggning av skalet. Alla delar av vävnadsbyggnadsställning är FDA-godkända och teknikenskulle kunna användas för förfaranden enstegs genom vävnadsskörd, mals, plast kompression, och transplantera tillbaka till patienten som en enda-iscensatt ingripande. Det förfarande skulle kunna utföras för expansion vävnad och rekonstruktion under sterila betingelser i någon allmän kirurgi enhet.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Silicone catheter 10-French</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>DMEM 10X</td>
			<td>Gibco</td>
			<td>31885-023</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Falcon</td>
			<td>08-772-1</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>3&#39;3,&#39;5-Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>IRMM469</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Labmed Solutions</td>
			<td>200-001-8</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Histolab</td>
			<td></td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>ABC Elite kit: Biotin -Streptavidin detection kit</td>
			<td>Vector</td>
			<td>PK6102</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Histolab</td>
			<td>1399.01</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma-Aldrich</td>
			<td>A8626</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Atropine 25 &#956;g/kg&#160;</td>
			<td>Temgesic, RB Pharmaceuticals, Great Britain</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Azaperone 2&#8201;mg/kg&#160;</td>
			<td>Stresnil, Janssen-Cilag, Pharma, Austria</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Biosafety Level 2 hood&#160;</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Blocking solution:&#160; Normal serum from the same species as the secondary secondary antibody was generated in.</td>
			<td>Vector</td>
			<td>The blocking solution depends of the&#160; origin of&#160; first antibody</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Buprenorphine 45 &#956;g/kg</td>
			<td>Atropin, Mylan Inc, Canonsburg, PA</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Carprofen 3&#8201;mg/kg&#160;&#160;&#160;</td>
			<td>Rimadyl, Orion Pharma, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Chlorhexidine gluconate&#160;</td>
			<td>Hibiscrub 40&#8201;mg/mL, Regent Medical, England</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Cholera toxin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Coplin jar: staining jar for boiling</td>
			<td>Histolab</td>
			<td>6150</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Stainless mold&#160; (33x22x10 mm) custom made</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>3188-5023</td>
			<td>Plastic compression section 4. Keep on ice&#160; when using it in plastic compression</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Ethilon (non-absorbable monofilament for skin sutures)</td>
			<td>Ethicon</td>
			<td></td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10437-036</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Forceps (Adison with tooth)</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Gauze (Gazin Mullkompresse)&#160;</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Ham&#180;s F12</td>
			<td>Gibco</td>
			<td>31765-027</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Histolab</td>
			<td>1820</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>DALAB</td>
			<td></td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide Solution 30%</td>
			<td>Sigma-Aldrich</td>
			<td>H1009</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I3536</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Isoflurane, Baxter, Deerfield, IL</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Lidocaine 5 mg/ml</td>
			<td>Xylocaine, AstraZeneca, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Lucose 25&#8201;mg/mL&#160;</td>
			<td>Baxter, Deerfield, IL</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Marker pen pap pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821-1EA</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Medetomidine 25 &#956;g/kg&#160;</td>
			<td>Domitor, Orion Pharma, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Mincing device</td>
			<td>Applied Tissue Technologies LLC</td>
			<td></td>
			<td>&#160;Minced tissue preparation, section 2</td>
		</tr>
		<tr>
			<td>Monocryl (absorbable monofilament)</td>
			<td>Ethicon</td>
			<td></td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>NaOH 1N</td>
			<td>Merck Millipore</td>
			<td>106462</td>
			<td>Plastic compression section 4 and cell culture</td>
		</tr>
		<tr>
			<td>Nylon mesh, 110 uM thick pore size 0.04 sqmm</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Oculentum simplex APL: ointment for eye protection</td>
			<td>APL</td>
			<td>Vnr 336164</td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Phenobarbiturate 15&#8201;mg/kg&#160;</td>
			<td>Pentobarbital, APL, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>PLGA Knitted fabric</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Rat-tail collagen</td>
			<td>First LINK, Ltd, UK</td>
			<td>60-30-810</td>
			<td>Plastic compression section 4, keep on ice</td>
		</tr>
		<tr>
			<td>Scalpel blade - 15</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Shaving shears</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Stainless stell mesh, 400 uM thick pore size&#160;</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Steril gloves</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Sterile gowns</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Sterile drapes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilium</td>
			<td>Bode Chemie HAMBURG</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Suture Thread Ethilon</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>TE-solution (antigen unmasking solution) consist of 10 mM Tris and 1 mM EDTA, pH 9.0</td>
			<td></td>
			<td></td>
			<td>10 mM Tris/1 mM EDTA,&#160; adjust pH to&#160; 9.0</td>
		</tr>
		<tr>
			<td>Tiletamine hypochloride 2,5&#8201;mg/kg</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8158</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Trizma Base, H2NC&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Vector VIP kit: Enzyme&#160; peroxidase substrate&#160; kit</td>
			<td>Vector</td>
			<td>SK4600</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Vicryl (absorbable braded)</td>
			<td>Ethicon</td>
			<td></td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>Tris buffer pH 7.6 (washing buffer)</td>
			<td></td>
			<td></td>
			<td>TE solution: Make 10X&#160; (0,5M Tris, 1,5M NaCl) by mixing: 60,6 g Tris (Trizma Base, H2NC(CH2OH)3, M=121.14 g/mol), add 800 ml&#160; distilled water adjust the pH till 7.6, add 87,7 g NaCl and fill to 1000 ml with&#160; distilled water. Dilute to 1X with distilled water.</td>
		</tr>
		<tr>
			<td>X-tra solv (solvent)</td>
			<td>DALAB</td>
			<td>41-5213-810</td>
			<td>Immunocytochemistry; Section 6. Use under fume hood</td>
		</tr>
		<tr>
			<td>Zolazepam hypochloride</td>
			<td>Zoletil, Virbac, France</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Depilatory wax strips</td>
			<td>Veet</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Lundbeck</td>
			<td></td>
			<td>Termination, Section 3</td>
		</tr>
	</tbody>
</table>

minced tissue in compressed collagen: a cell-containing biotransplant for single-staged reconstructive repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Denna studie presenterar en metod som visar hur man kan producera ett biomaterial för transplantation med användning av plast komprimering av kollagen och malda vävnaden. Bladder slemhinna och hud kan skördas och sedan mekaniskt malet i små partiklar (Figur 3). Genom plastisk komprimering, är det malda partiklar inkorporeras inuti kompositbyggnadsställning består av en centralt placerad bionedbrytbar polymer som är mekaniskt stark inom ytterskikt av en kollagengel <...

Watch this Scientific Journal Video about Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair at JoVE.com

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Vävnadsteknik inkluderar ofta in vitro expansion i syfte att skapa autotransplantat för vävnadsregenerering. I denna studie en metod för vävnadsexpansion, regenerering, och återuppbyggnad in vivo utvecklades för att minimera behandlingen av celler och biologiska material utanför kroppen.

Malet Tissue i Compressed Kollagen: en cell som innehåller BioTransplant för enkel iscensatt rekonstruktiv Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

The overall goal of the following procedure is to transplant autologous tissue for its expansion during reconstructive surgery. This is accomplished by first harvesting a biopsy from the tissue of interest. Next, the biopsy is minced and a plastic compressed polycapro-lactone, or PCL, collagen autograft is constructed.The PCL and otologous minced tissues are then integrated to create the 3D scaffold. We have used a one to six expansion rate. Next, the scaffold is transplanted back into the subject.Ultimately, the regeneration of the autografted tissue can be assessed by immunohistochemical analysis of the proliferation and reorganization of the cells within the scaffolds and minced tissue particles. This method will be demonstrated in an animal model, on skin, and on urothelium. The implication of this technique extends toward the treatment of severely malformed tissues or after trauma in instances where there's severe lack of tissue for the surgical repair.The main advantage of this technique over existing methods, like in vitro culturing, is that this method can be performed as a one-stage surgical procedure. Visual demonstration of this method is critical, as the steps from the tissue harvest to the transplantation need to be performed under sterile conditions. To obtain a porcine bladder biopsy specimen, first, grasp the bladder with the forceps, and use a sterilized measuring tape to determine the dimensions of the tissue.Next, use a sterilized pen to mark an ellipsis shaped biopsy two by one centimeter, aiming for a one to six expansion rate. Then, excise the marked tissue with a scalpel, and place the bladder biopsy specimen in DMEM under sterile conditions. To harvest a skin biopsy specimen, use a dermatome to harvest a 0.3 millimeter partial thickness specimen.Then, place the tissue in DMEM and cover the wound area with a dressing. Next, wash the bladder biopsy twice in DMEM and transfer the tissue onto a sterilized dissecting plate, with the mucosa facing upwards. Using dissection pins, fix one side of the specimen to the plate and use fine scissors and forceps to separate the mucosal tissue from the detrusor muscle.Then, place a mincing device on top of the mucosa and pass it vertically and horizontally from one end of the tissue to the other to obtain 0.8 by 0.8 millimeter pieces of minced tissue. Note that the mincing device needs to be pressed on the tissue while running over it. To prepare a plastic compressed collagen autograft, first add one normal sodium hydroxide drop by drop into 10X DMEM supplemented with collagen type I until the medium turns from intense yellow to pink.After confirming a pH of 7.4 to 8, carefully mix two milliliters of DMEM into the solution. Then, plate approximately two milliliters of collagen into a well of a 20x30x10 millimeter steel rectangular mold and incubate the collagen for ten minutes. When the collagen has set, plate the appropriate volume of PCL biomaterial on top of the gelled collagen and pour the remaining six milliliters of collagen on top of the PCL.Place the mold back into the incubator for another 20 minutes. Next, stack a thick layer of gauze pads onto a sterile surface. Then, place a 400 micron thick stainless steel mesh on top of the gauze, followed by a 110 micron thick sheet of nylon mesh.Gently transfer the collagen gel onto the nylon mesh and carefully remove the rectangular steel mold. Transfer the minced tissue onto the collagen. Here, we aim at a one to six expansion rate.Then, place a new layer of nylon mesh on top of the collagen gel and tissue, then place a second steel mesh on top of the nylon mesh, followed by a loading plate weighing a minimum of 120 grams. After five minutes, remove the weight, steel meshes, and nylon from the autograft. For in vitro culture of the autograft, first cut the thinned construct into pieces small enough to fit into 12 well plates.Then, add one milliliter of keritinocyte medium to each well and incubate the tissues for three to six weeks. To transplant an autograft with minced bladder mucosa to a pig bladder, moisten the graft with DMEM. Then, graft the construct to the healthy tissue with fine running monofilament sutures.Confirm that the graft is water tight by filling the bladder with saline through the indwelling urinary catheter. To transplant an autograft with minced skin epidermis to a full thickness wound, moisten the graft with DMEM. Then, using interrupted sutures in the corners and middle of the autograft, attach the construct to the bottom of the skin full thickness wound.Finally, cover the wound with a plastic dressing to keep it moist. As observed in this image of a minced skin autotransplant, the composite biograft can sustain grasping, suturing, and surgical handling. Immunohistochemistry at different time points during the in vitro culture reveals that the cells proliferate, migrate, and reorganize into a continuous epithelium with a microarchitecture typical for the cell phenotype.For example, at two weeks, the cell layer on the surface of the skin autograft is approximately two layers thick. To the right, bladder epithelium is about eight layers thick after the same culture period. After five to six weeks, the continuous epithelium is approximately eight to ten cell layers thick in both minced skin epithelium and minced bladder mucosa autografts.Compared to tissue expansion in vitro, this method is quick, less labor intensive, and does not require sophisticated laboratory resources. In addition, all parts are FDA approved, allowing their use in standard surgical setting. While attempting this procedure, it's important to remember to prepare the gel, biotransplants, and autologous tissues under sterile conditions.Following this procedure and other methods like in vitro culture of the cells can be performed to answer additional questions about cell proliferation and differentiation. After its development, this technique may pave the way for researchers in the field of regenerative medicine. By these means, we could treat severe malformations in the skin or urinary system with autologous tissue engineered transplants.

Research

JoVE Journal

Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Electric Field-kontrollerad Riktad Migration av neurala stamceller i 2D-och 3D-miljöer

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Tillverkning och tillämpning av Rose Bengal-kitosan Films i Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

An Improved Method for the Preparation of Type I Collagen From Skin

En förbättrad metod för framställning av typ I kollagen från huden

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Imaging Denaturerat Kollagen Strands<em&gt; In vivo</em&gt; Och<em&gt; Ex vivo</em&gt; Via Foto-utlöst Hybridisering av Caged Collagen Mimetic peptider

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Analys av Cell Migration inom en tredimensionell kollagenmatris

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Generering av ett tredimensionellt Full Tjocklek hudekvivalent och automatiserade sårar

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Ingenjörs 3D Cellularized kollagengeler för kärlvävnad Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Engineered 3D Silk-kollagenbaserad modell av polariserat Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Skikt för skikt kollagendeponering i mikroflödessystem enheter för Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

tillämpningar av<em&gt; In Vivo</em&gt; Provning av Rat tibialis anterior för utvärdering vävnadstekniska skelettmuskulatur Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...