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.

모든 동물 프로토콜은 동물의 스톡홀름 카운티위원회의 사전 승인을하고, 모든 절차는 동물의 사용뿐만 아니라 관련 연방법의 규정을 따른다. 1. 동물 절차 <ol><li> 수술에 대한 동물 준비 <ol><li> 무균 조건 하에서 작동에 필요한 모든 재료와 도구로 수술 테이블을 준비합니다. 감염의 위험을 감소시키고 생존 수술 조건을 최적화하기 위해 멸균 조건하에 단독으로 수술을 수?...

<ol>
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이 연구는 수술 테이블에 이식자가 조직과 방광 벽에 ​​패치를 생성하기 위해 사용하기 쉬운 방법을 제공합니다. 패치와 플라스틱의 압축과 함께 외면 다진 조직없이 중간 및 콜라겐 분해성 중합체 편성의 조합에 의해 형성된다. 압축 성형 방법은, 이전에 다른 저자에 의해 기재된 콜라겐 겔 (12, 13)에서 유체의 급속 제적으로 정의 될 수있다. 방광 점막이나 피부 조직 다진이 지지체에 ?...

피부와 비뇨 기관에 이식에 대부분의 조직 공학 연구는 특별히 장착 된 세포 배양 시설 1, 2의 건강한 조직과 세포의 확장에서자가 세포 수확을 포함한다. 환자 이식술을 수신 할 준비가되었을 때 셀 전개 후, 세포는 일반적으로 나중에 사용하기 위해 저장된다. 질소 냉동고 -150 ° C 이하의 낮은 온도에서의 장기간 저장을 허용한다. 동결 프로세스는 신중하고 세포 손실하지 않도록 제어되어야한다. 세포 사멸의 한 위험 세포막의 파괴로 이어질 수 해동 과정 중 세포 내 물이 결정화된다. 동결 세포는 일반적으로 세포를 소 태아 혈청 및 디메틸 술폭 시드의 고 농도를 사용하여 천천히 제어 냉각 (-11 ° C 당 분)에 의해 수행된다. 해동 후, 세포를 동결 배지를 제거하고 세포 배양 용 플라스틱 또는 배양에 의해 다시 처리 될 필요다시 환자에게 이식하기 전에 생체 재료. 모든 상기 언급 된 단계는 시간 소모, 번잡하고 고비용 3. 또한, 환자의 이식을위한 세포의 모든 체외 처리는 매우 규제하고 잘 훈련 된 공인 인력과 실험실 4 필요합니다. 전부, 기술은 기술적으로 진보 센터 극소수에 설립 될 수 있고, 일반적인 수술 질환을 넓게 사용하기 어렵다, 안전하고 신뢰할 수있는 제조 공정을 조달. 실험실 환경에서 세포 배양의 한계를 극복하기 위해, 생체 내에서 세포 확장 다진 조직 이식의 개념은 바이오 리액터로서 본체 자체를 사용하여 도입된다. 이러한 목적을 위해,자가 이식 우선적 I의 장기의 최종 재구성에 필요한 형상에 따라 3D 금형에 이식 될5-7 nterest. 원래, 다진 상피 세포를 이식의 아이디어는 그가 상피 세포가 상처의 가장자리에서 성장하는 방법을 설명 할 때 1958 년 온순한 제시했다. 그 피부의 작은 조각 (도 1) (8)를 수직 방향으로 두 조각을 절단하여 100 %로함으로써, 셀 확장 가능성을 마진을 증가한다고 입증 하였다. 이론은 피부 이식 9 맞물린 부분 층 피부 이식의 사용에 의해 지원 피부 모델 10 상처 치유되었다. <img alt="그림 1" src="/files/ftp_upload/53061/53061fig1.jpg" width="600" /> 그림 1 :. 온순한 이론은 미크의 이론에 따르면, 상피 세포는 상처의 가장자리에서 성장. 닦지 기술에 의해 노출되는 면적을 증가시킴으로써, 다진 조직은 많은 점에서 상처를 epithelializes. 본 연구는 저자에 기초동일한 원리가 주형 주위 다진 상피 배치하여 피하 조직에 적용될 수 논문. (상기 내부 몸체로부터 이물 (금형) 상처 면적 분리 연속 neoepithelium을 형성하기 위해 상피 세포는 상처 영역 (마이그레이션) 커버 다진 이식 (재구성)로부터 동원 것이고 분할기 (확장) 그림 2). <img alt="그림 2" src="/files/ftp_upload/53061/53061fig2.jpg" /> 그림 2. 선량의 이론에 따른 생체 intracorporal 조직 확장 다진 상피 3D 금형 만화 피하 조직에 금형에 배치 한 후 이식 다진 조직을 이용하여 상기 가설은 상피 세포에서 마이그레이션 있다는 다진 조직의 가장자리는 재구성 및 상처 면적 본체 내부에서 이물질 (몰드)을 분리하는 연속 neoepithelium을 형성하도록 확장. 이전의 생체 내 연구는 유망한 결과를 보여하지만 재생 된 상피 더 나은 7 기계적 충격에 견딜 수 있도록, 더 개선은자가 이식을 강화함으로써 달성 될 수있다. 쉬운 영양소와 폐기물의 확산 가능성 금형 3D 방식 외과 취급 용이성 이러한 목적을 위해 성공적인 생체 중요한 전제 같은 확인되었다. 결론이 요구 다진 티슈 복합 생체 재료를 첨가함으로써 충족 될 수 있다고 하였다. 에 다진 조직으로 구성된 인공 지지체의 개발을 목표로 현재 연구 생분해 성 섬유의 보강 코어를 포함하는 콜라겐을 플라스틱 압축. 이러한 방법으로, 실행 가능한 세포는 다진 조직의 입자에서 마이그레이션 수 있고 원래의 상피 (피부 또는 요로 상피)의 형태 학적 기능 특성으로 확산. 플라스틱 압축을 사용하여 비계 감소했다약 420 μm의 다진 입자로에 1cm의 크기 d는 상위 계층 콜라겐에 싸여 있었다. 코어 섬유는 중합체 일 수 있지만, 피복 콜라겐 층 11 인터링크하기 위해 친수성 표면으로 변형 될 필요가 있었다. 이 방법은 다진 방광 점막 또는 돼지에서 다진 피부를 배양하기위한 발판으로 사용하여 두 개의 플라스틱 압축 된 콜라겐 젤 내에서 폴리 (ε 카프로 락톤을)로 구성된 니트 메쉬 (PCL)를 통합하여 향상된 발판 무결성을 제공했다. 구조는 잘 통합 하이브리드 구조물의 위에 층화 다층 요로 상피 또는 피부 편평 상피의 성공적인 형성을 입증하는 시험 관내에서 6 주 동안 세포 배양 조건에서 유지되었다. 구조는 취급이 용이이었고, 방광 확대 술의 목적이나 피부 결점 커버 장소에 봉합 될 수있다. 조직 골격의 모든 부분은 FDA 승인 및 기술되어있다티슈 닦지 수확 플라스틱 압축 및 단일 스테이지 개입 같은 환자에게 다시 이식하여 단일 단계 과정에 이용 될 수있다. 절차는 임의의 외과 부 멸균 조건 하에서 조직 팽창과 복원에 대해 수행 될 수있다.

<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.

이 연구는 콜라겐과 다진 조직의 플라스틱 압축을 사용하여 이식 생체 재료를 생산하는 방법을 보여줍니다 방법을 제시한다. 방광 점막 및 피부는 수확 후 기계적으로 작은 입자 (그림 3)에 다진 할 수 있습니다. 플라스틱 압축하여 다진 입자 콜라겐 겔 (도 4)의 외층 내에 기계적으로 강한 중앙 배치 생분해 성 고분자로 이루어지는 복합체 지지체 내?...

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

조직 공학은 종종 조직 재생을위한자가 이식을 만들기 위해 체외 확장에 포함되어 있습니다. 본 연구에서는 조직 확장 재생 및 생체 내에서 재구성하기위한 방법이 체외 세포 및 생체 물질의 처리를 최소화하기 위해 개발되었다.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

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

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

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

연구

생체공학

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

제조 및 레이저 조직 복구에 장미 벵골 - 키토산 영화의 응용

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

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

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

변성 콜라겐 물가 이미징<em&gt; 생체 내</em&gt;와<em&gt; 예 생체 내</em&gt; 리테이너 타입 콜라겐 미메시스 펩티드의 사진 트리거 하이브리드를 통해

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

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

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

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

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

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

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

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델

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

Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

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

의 응용 프로그램<em&gt; 생체</em&gt; 조직 평가를위한 쥐 앞정 전방의 기능 테스트는 골격근 수리 엔지니어링

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