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.

All animal protocols were pre-approved by the Stockholm County Committee on Animals and all procedures conformed to the regulations for animal use, as well as relevant federal statutes.
1. Animal Procedures
<ol>
	<li>Preparing the Animal for Surgery
	<ol>
		<li>Prepare the surgical table with all the materials and instruments needed for the operation under sterile conditions. Perform the surgery exclusively under sterile conditions to reduce the risk of infection and optimize the conditions ...

<ol>
	<li>Rheinwald, J. G., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+cultivation+of+strains+of+human+epidermal+keratinocytes:+the+formation+of+keratinizing+colonies+from+single+cells.">Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.</a> Cell. 6, 331-343 (1975).</li><li>Fossum, M., Nordenskjold, A., Kratz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+multilayered+urinary+tissue+in+vitro.">Engineering of multilayered urinary tissue in vitro.</a> Tissue Engineering. 10, 175-180 (2004).</li><li>Salmikangas, 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=Manufacturing,+characterization+and+control+of+cell-based+medicinal+products:+challenging+paradigms+toward+commercial+use.">Manufacturing, characterization and control of cell-based medicinal products: challenging paradigms toward commercial use.</a> Regen Med. 10, 65-78 (2015).</li><li>Fossum, 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=Minced+skin+for+tissue+engineering+of+epithelialized+subcutaneous+tunnels.">Minced skin for tissue engineering of epithelialized subcutaneous tunnels.</a> Tissue Engineering. Part A. 15, 2085-2092 (2009).</li><li>Fossum, 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=Minced+urothelium+to+create+epithelialized+subcutaneous+conduits.">Minced urothelium to create epithelialized subcutaneous conduits.</a> The Journal of Urology. 184, 757-761 (2010).</li><li>Reinfeldt Engberg, G., Lundberg, J., Chamorro, C. L., Nordenskjold, A., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+autologous+minced+bladder+mucosa+for+a+one-step+reconstruction+of+a+tissue+engineered+bladder+conduit.">Transplantation of autologous minced bladder mucosa for a one-step reconstruction of a tissue engineered bladder conduit.</a> BioMed Research International. 2013, 212734 (2013).</li><li>Meek, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+microdermagrafting+using+the+Meek-Wall+microdermatome.">Successful microdermagrafting using the Meek-Wall microdermatome.</a> Am J Surg. 96, 557-558 (1958).</li><li>Tanner, J. C., Vandeput, J., Olley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mesh+skin+graft.">The Mesh skin graft.</a> Plastic and Reconstructive Surgery. 34, 287-292 (1964).</li><li>Svensjo, T., 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=Autologous+skin+transplantation:+comparison+of+minced+skin+to+other+techniques.">Autologous skin transplantation: comparison of minced skin to other techniques.</a> The Journal of Surgical Research. 103, 19-29 (2002).</li><li>Ajalloueian, F., Zeiai, S., Rojas, R., Fossum, M., Hilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-stage+tissue+engineering+of+bladder+wall+patches+for+an+easy-to-use+approach+at+the+surgical+table.">One-stage tissue engineering of bladder wall patches for an easy-to-use approach at the surgical table.</a> Tissue Engineering. Part C, Methods. 19, 688-696 (2013).</li><li>Engelhardt, E. 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=A+collagen-poly(lactic+acid-co-varepsilon-caprolactone)+hybrid+scaffold+for+bladder+tissue+regeneration.">A collagen-poly(lactic acid-co-varepsilon-caprolactone) hybrid scaffold for bladder tissue regeneration.</a> Biomaterials. 32, 3969-3976 (2011).</li><li>Brown, R. A., Wiseman, M., Chuo, C. B., Cheema, U., Nazhat, S. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructs+of+electrospun+PLGA,+compressed+collagen+and+minced+urothelium+for+minimally+manipulated+autologous+bladder+tissue+expansion.">Constructs of electrospun PLGA, compressed collagen and minced urothelium for minimally manipulated autologous bladder tissue expansion.</a> Biomaterials. 35, 5741-5748 (2014).</li><li>Orabi, H., AbouShwareb, T., Zhang, Y., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-seeded+tubularized+scaffolds+for+reconstruction+of+long+urethral+defects:+a+preclinical+study.">Cell-seeded tubularized scaffolds for reconstruction of long urethral defects: a preclinical study.</a> Eur Urol. 63, 531-538 (2013).</li><li>Blais, M., Parenteau-Bareil, R., Cadau, S., Berthod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+tissue-engineered+skin+and+nerve+regeneration+in+burn+treatment.">Concise review: tissue-engineered skin and nerve regeneration in burn treatment.</a> Stem Cells Transl Med. 2, 545-551 (2013).</li><li>Serpooshan, V., Muja, N., Marelli, B., Nazhat, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+contractility+and+growth+in+plastic+compressed+collagen+gel+scaffolds+with+microstructures+correlated+with+hydraulic+permeability.">Fibroblast contractility and growth in plastic compressed collagen gel scaffolds with microstructures correlated with hydraulic permeability.</a> J Biomed Mater Res A. 96, 609-620 (2011).</li></ol>

This study presents an easy-to-use approach to produce bladder wall patches with autologous tissue for transplantation at the surgical table. The patches are formed by the combination of a biodegradable polymer knitting in the middle and collagen with and without minced tissue in the outer surfaces in combination with plastic compression. Plastic compression is a method previously described by other authors and can be defined as a rapid expulsion of fluid from collagen gels12,13. Minced tissue of bladder mucos...

Most tissue engineering studies on transplantation to the skin and urogenital tract include autologous cell harvests from healthy tissue and cell expansion in specially equipped cell-culturing facilities1,2.
After cell expansion, cells are usually stored for later use when the patient is prepared to receive the autograft. Nitrogen freezers allow long-term storage at low temperatures of -150 &#176;C or lower. The process of freezing must be careful and controlled in order not to lose the cells. One risk of cell death is crystallization of intracellular water during the thawing process, which can lead to rupture of the cell membranes. Cell freezing is usually performed by slow and controlled cooling (-1 &#176;C per min), using a high concentration of cells, fetal bovine serum, and dimethyl sulfoxide. After thawing, the cells need to be processed again by removing the freezing medium and culturing on cell culture plastic or a biomaterial before transplantation back to the patient.
All the above-mentioned steps are time-consuming, laborious, and costly3. In addition, all in vitro processing of cells intended for patient transplantation are highly regulated and requires well-trained and accredited personnel and laboratories4. All in all, to procure a safe and reliable manufacturing process, the technique could only be established in a very small number of technically advanced centers and a wider use in common surgical disorders is doubtful.
In order to overcome the limitations of cell culturing in the laboratory environment, the concept of transplanting minced tissue for cell expansion in vivo is introduced by using the body itself as a bioreactor. For these purposes, the autografts would preferentially be transplanted on a 3D mold according to the shape that is needed for the final reconstruction of the organ of interest5-7.
Originally, the idea of transplanting minced epithelium was presented by Meek in 1958&#160;when he described how epithelium grows from the edges of a wound. He demonstrated that a small piece of skin would increase its margins and thereby its potential for cell expansion by 100% by cutting the piece twice in perpendicular directions (Figure 1)8. The theory has been supported by the use of meshed partial thickness skin grafts for skin transplantation9 and in skin wound healing models10.
<img alt="Figure 1" src="/files/ftp_upload/53061/53061fig1.jpg" width="600" /> 
Figure 1: Meek theory. According to Meek&#8217;s theory, epithelium grows from the edges of a wound. By increasing the area exposed by the mincing technology, minced tissue epithelializes wounds from many spots.
The present study is based on the hypothesis that the same principle could be applied to the subcutaneous tissue by placing minced epithelium around a mold. The epithelial cells would mobilize from minced transplants (reorganize), cover the wound areas (migrate) and divide (expand) in order to form a continuous neoepithelium that covers the wound area and separates the foreign body (the mold) from the inner body (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/53061/53061fig2.jpg" /> 
Figure 2: Cartoon of a 3D mold with minced epithelium for in vivo intracorporal tissue expansion according to the theory of Meek. By using minced tissue placed on a mold and then transplanted to the subcutaneous tissue, the hypothesis is that the epithelial cells migrate from the edges of the minced tissue, reorganize, and expand so as to form a continuous neoepithelium that covers the wound area and separates the foreign body (the mold) from the inner body.
Although previous in vivo studies show promising results, further improvements could be achieved by reinforcing the autografts so that the regenerated epithelium could withstand mechanical trauma better7. For these purposes, important prerequisites for a successful biomaterial were identified, such as: easy diffusion of nutrients and waste products, possibility to mold in a 3D manner and easiness of surgical handling. Conclusions were made that these needs could be met by adding a composite biomaterial to the minced tissue.
The current study aimed at developing a scaffold composed of minced tissue in plastic-compressed collagen containing a reinforcing core of a biodegradable fabric. By these means, viable cells could migrate from the minced tissue particles and proliferate with morphological features characteristic of the original epithelium (skin or urothelium). Using plastic compression, the scaffold was reduced in size from 1 cm to about 420&#8201;&#181;m as the minced particles were encased in the upper layer collagen. The core fabric could be any polymer but needs to be modified with a hydrophilic surface in order to interlink with the covering collagen layers11.
The method provided an enhanced scaffold integrity by incorporating a knitted mesh consisting of poly(&#949;-caprolactone) (PCL) within two plastic compressed collagen gels using it as a scaffold for culturing minced bladder mucosa or minced skin from pigs. The construct was maintained in cell culture conditions for up to 6 weeks in vitro, demonstrating successful formation of a stratified, multilayered urothelium or squamous skin epithelium on the top of a well-consolidated hybrid construct. The construct was easy to handle and could be sutured in place for bladder augmentation purposes or covering of skin defects. All parts of the tissue scaffold are FDA-approved and the technique could be used for single-stage procedures by tissue harvesting, mincing, plastic compression, and transplanting back to the patient as a single-staged intervention. The procedure could be performed for tissue expansion and reconstruction under sterile conditions in any general surgery unit.

<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.

This study presents a method that shows how to produce a biomaterial for transplantation using plastic compression of collagen and minced tissue.
Bladder mucosa and skin can be harvested and then mechanically minced into small particles (Figure 3). By plastic compression, the minced particles are incorporated within the composite scaffold composed of a centrally placed biodegradable polymer that is mechanically strong within outer layers of a collagen gel (Figure 4</st...

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

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

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

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

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

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

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

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

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

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

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

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

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