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.

動物ストックホルム郡委員会とすべての手続きにより事前承認された全ての動物プロトコルは、動物用の規制だけでなく、関連する連邦政府の法令に準拠しました。 1.動物の手順<ol><li>手術のために動物を準備<ol><li>無菌条件下での操作に必要なすべての材料と器具で手術台を準備します。感染の危険性を減少させ、生存手術における条件を最適化するために、無菌条?...

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この研究は、手術台での移植のための自家組織と膀胱壁のパッチを作成するための使いやすいアプローチを提示します。パッチは、プラスチック製の圧縮と組み合わせて、外面で細分化した組織とない中・コラーゲン中の生分解性ポリマー編みの組み合わせによって形成されています。プラスチックの圧縮は、以前に他の著者によって記載された方法で、コラーゲンゲル12,13からの流...

皮膚と泌尿生殖器管への移植のほとんどの組織工学の研究は、特別装備し、細胞培養施設1,2における健康な組織や細胞増殖の自己細胞の収穫が含まれます。 患者は、自家移植片を受け取るように準備される場合、細胞増殖の後、細胞は、通常、後の使用のために格納されます。窒素冷凍庫は-150℃以下の低温での長期保存を可能にします。凍結のプロセスは、慎重かつ細胞を失わないために制御しなければなりません。細胞死の一つのリスクは、細胞膜の破裂につながることが融解プロセス中に細胞内の水の結晶です。細胞の凍結は、通常、細胞、ウシ胎児血清、およびジメチルスルホキシドの高い濃度を用いて、ゆっくりと制御冷却（-1°Cあたり分）によって行われます。解凍後、細胞を凍結培地を除去し、細胞培養プラスチックまたはで培養することによって再び処理される必要があります戻って患者に移植する前に生体材料。 すべての上記の手順は、時間がかかり、面倒であり、高価な3。また、患者の移植のために意図された細胞のすべてのin vitroでの処理は非常に規制されているとよく訓練されたと認定された担当者や研究室4が必要です 。すべてのすべてで、安全で信頼性の高い製造プロセスを調達するために、技術は技術的に高度なセンターの非常に少ない数で確立することができ、一般的な外科的疾患における普​​及は疑問です。 実験室環境での細胞培養の限界を克服するために、in vivoでの細胞増殖のために細分化した組織を移植するという概念は、バイオリアクターとしての本体自体を使用することによって導入されます。これらの目的のために、自家移植は、優先的に私の臓器の最終的な再構築のために必要とされる形状に応じて3Dモールド上に移植されることになります5-7 nterest。 もともと、みじん切り上皮を移植するという考えは、彼が上皮は、創傷の縁から成長方法を説明したときに1958年にミークによって発表されました。彼は皮膚の小片は、垂直方向（ 図1）8に二回ピースを切断することにより、100％によって、その余白とそれによって細胞の拡大のためにその可能性を増大させることを実証しました。理論は、皮膚移植9用のメッシュ構造部分層皮膚移植片の使用によってサポートされており、肌にモデル10を創傷治癒されています。 <img alt="図1" src="/files/ftp_upload/53061/53061fig1.jpg" width="600" /> 図1：ミーク理論はミークの理論によれば、上皮は、創傷の縁から成長。ミンチ技術により露出面積を大きくすることで、細分化した組織は、多くのスポットから傷をepithelializes。 本研究は、ハイポに基づいています同じ原理は、金型の周囲にミンチ上皮を配置することによって、皮下組織に適用することができる論文。 （内側本体から異物（金型）を創傷領域をカバーし、分離連続neoepitheliumを形成するために、上皮細胞は、創傷領域は（移行）をカバー、ミンチ移植（再編成）から動員だろうと除算は（拡大します） 図2）。 <img alt="図2" src="/files/ftp_upload/53061/53061fig2.jpg" /> 図2：ミークの理論によるin vivoでの体内組織拡大のためのみじん切り上皮と3Dモールドの漫画の皮下組織に金型に入れ、次いで移植細分化した組織を使用することにより、仮説は、上皮細胞から移行することです細分化した組織の縁は、再編成、および創傷領域をカバーし、内側本体から異物（金型）を分離連続neoepitheliumを形成するように拡張します。 インビボ研究において以前は有望な結果を示しているが、再生上皮が良い7機械的外傷に耐えることができるように、更なる改良が自家移植片を強化することによって達成することができます。簡単に栄養素や老廃物の拡散、可能性金型に3Dの方法で、手術取り扱いの容易さ：これらの目的のために、成功した生体材料のための重要な前提条件は、次のような、同定されました。結論は、これらの必要性を細分化した組織への複合生体材料を添加することによって達成することができたと判断しました。 生分解性生地の補強芯を含むプラスチック - 圧縮コラーゲンに細分化した組織で構成される足場を開発することを目的とした今回の研究。これらの手段によって、生存細胞を細分化した組織粒子から移行する可能性があり、元の上皮（皮膚や尿路上皮）の特徴的な形態学的特徴で増殖します。塑性圧縮を使用して、足場を減らすました約420μmのみじん切り粒子として、1 CMからサイズのdは上位層のコラーゲンに包まれました。コア・ファブリックは、任意のポリマーであるが、被覆コラーゲン層11とインターリンクするために、親水性表面で修飾することが必要である可能性があります。 この方法は、ブタからミンチ膀胱粘膜やみじん切り皮膚を培養するための足場としてそれを使用して、2つのプラスチック製の圧縮コラーゲンゲル内にポリ（εカプロラクトン）からなるニットメッシュ（PCL）を組み込むことによって強化された足場の整合性を提供します。構築物は、よく連結ハイブリッド構築物の上に層状、多層尿路上皮または扁平皮膚上皮の成功形成を実証し、in vitroで 6週間まで細胞培養条件下で維持しました。構築物は、取り扱いが容易であったと膀胱増強の目的や皮膚の欠陥の被覆のための場所に縫合することができました。すべての組織スキャフォールドの部分がFDA承認されており、技術組織細断収穫、プラスチック圧縮、および単一段階介入として患者に戻し移植することにより、一段階手順を使用することができます。手順は、任意の一般外科ユニット内の無菌条件下で組織拡大と再構築のために行うことができました。

<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.

この研究は、コラーゲンやミンチ組織の塑性圧縮を使用して、移植のための生体材料を製造する方法を示しています方法を提示します。 膀胱粘膜や皮膚を採取した後、機械的に小さい粒子（ 図3）にみじん切りすることができます。プラスチック圧縮することにより、みじん切り粒子は、コラーゲンゲル（ 図4）の外側層内に機械的に強固である...

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

組織工学は、多くの場合、組織再生のための自家移植を作成するためにインビトロで拡張に含まれます。この研究では、組織拡張、再生、およびin vivoでの再構成のための方法は、体外細胞および生体物質の処理を最小限にするために開発されました。

圧縮されたコラーゲンで細分化した組織：シングル段再建修復のための細胞含有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と3D環境での神経前駆細胞の電界制御監督移行

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

生物工学

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

レーザー組織修復にローズベンガル-キトサン膜の作製と応用

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

皮膚からのI型コラーゲンの調製のための改良された方法

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

変性コラーゲンストランドを画像化する<em&gt;インビボ</em&gt;と<em&gt;生体外</em&gt;リテーナ入りコラーゲン模倣ペプチド写真·トリガブリダイゼーション経由

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

三次元コラーゲンマトリックス内の細胞移動の解析

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

三次元の全層皮膚等価物と自動傷つけ処理の発生

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

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

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

偏神経組織のエンジニア3Dシルクコラーゲンベースモデル

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安定化のためのマイクロ流体デバイスの層ごとのコラーゲン沈着

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

の応用<em&gt;インビボ</em組織工学骨格筋の修復を評価するためのラット前脛骨の&gt;機能テスト

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