Name: tissue characterization after a new disaggregation method for skin micro-grafts generation
Uploaded: 2016-03-04T13:00:00
Description: Watch this Scientific Journal Video about Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation at JoVE.com

The authors wish to thanks Dr. Federica Zanzottera for contributing to the study performing flow cytometry analysis.

Ethics statement: since the clinical application of the protocol requires the use of cutaneous autologous tissue of the patient, its characterization in vitro was performed before clinical use on homologous cutaneous tissue at Emilia Romagna Regional Skin Bank following the guidelines of National Rules on harvesting, processing and distributing tissues for transplantation (CNT 2013).
1. Bio-complex Building for Clinical Application
NOTE: This protocol is clin...

<ol>
	<li>Sen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair Regen. 17, 763-771 (2009).</li><li>Ellis, E., Sinn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+homologous+bone+grafts+in+maxillofacial+surgery.">Use of homologous bone grafts in maxillofacial surgery.</a> J Oral Maxillofac Surg. 51, 1181-1193 (1993).</li><li>Nguyen, A., Pasyk, K. A., Bouvier, T. N., Hassett, C. A., Argenta, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+survival+of+autologous+adipose+tissue+taken+and+transplanted+by+different+techniques.">Comparative study of survival of autologous adipose tissue taken and transplanted by different techniques.</a> Plast Reconstr Sur. 85, 378-389 (1990).</li><li>Abuzeni, P. Z., Alexander, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+Autologous+Fat+Transplantation+With+Platelet+Rich+Plasma.">Enhancement of Autologous Fat Transplantation With Platelet Rich Plasma.</a> AJCS. 18 (2), 59-70 (2001).</li><li>Trovato, L., 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+New+Medical+Device+Rigeneracons+Allows+to+Obtain+Viable+Micro-Grafts+from+Mechanical+Disaggregation+of+Human+Tissues.">A New Medical Device Rigeneracons Allows to Obtain Viable Micro-Grafts from Mechanical Disaggregation of Human Tissues.</a> J Cell Physiol. , (2015).</li><li>Zanzottera, F., Lavezzari, E., Trovato, L., Icardi, A., Graziano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Derived+Stem+Cells+and+Growth+Factors+Applied+on+Hair+Transplantation+Follow-Up+of+Clinical+Outcome.">Adipose Derived Stem Cells and Growth Factors Applied on Hair Transplantation Follow-Up of Clinical Outcome.</a> JCDSA. 4 (4), 268-274 (2014).</li><li>Giaccone, M., Brunetti, M., Camandona, M., Trovato, L., Graziano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Medical+Device,+Based+on+Rigenera+Protocol+in+the+Management+of+Complex+Wounds.">A New Medical Device, Based on Rigenera Protocol in the Management of Complex Wounds.</a> J Stem Cells Res, Rev &amp; Rep. 1 (3), 3 (2014).</li><li>Aimetti, M., Ferrarotti, F., Cricenti, L., Mariani, G. M., Romano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+dental+pulp+stem+cells+in+periodontal+regeneration:+a+case+report.">Autologous dental pulp stem cells in periodontal regeneration: a case report.</a> Int J Periodontics Restorative Dent. 34 (3), s27-s33 (2014).</li><li>Graziano, A., Carinci, F., Scolaro, S., D'Aquino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+tissue+generation+using+autologous+dental+ligament+micro-grafts:+case+report+with+6+months+follow-up.">Periodontal tissue generation using autologous dental ligament micro-grafts: case report with 6 months follow-up.</a> AOMS. 1 (2), 20 (2013).</li><li>Brunelli, G., Motroni, A., Graziano, A., D'Aquino, R., 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=Sinus+lift+tissue+engineering+using+autologous+pulp+micro-grafts:+A+case+report+of+bone+density+evaluation.">Sinus lift tissue engineering using autologous pulp micro-grafts: A case report of bone density evaluation.</a> J Indian Soc Periodontol. 17 (5), 644-647 (2013).</li><li>Ellner, P. D., Stoessel, C. J., Drakeford, E., Vasi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+culture+medium+for+medical+bacteriology.">A new culture medium for medical bacteriology.</a> Am J Clin Pathol. 45, 502-504 (1966).</li></ol>

This preliminary study showed that micro-grafts obtained by this protocol can be combined with collagen sponges, as already reported in other clinical applications, to optimize the efficacy of micro-grafts implants9, 10. In particular, this study reported the capacity of bio-complexes, constituted by micro-grafts and collagen sponges, to adjuvant the wound healing of a leg lesion after 30 days from clinical application. Furthermore, in vitro results provide evidence about the effectiveness of this pro...

In the last years, several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery in order to provide one-step treatments for acute and chronic skin lesions. Moreover, the management of acute but mainly chronic wounds resulting from trauma, diabetes, infections, and other causes, remains challenging. There is mounting evidence that chronic wounds have become a serious global health issue, causing an enormous financial burden on healthcare systems worldwide1.

To increase the rate of success in the treatment of skin lesions, the absence of extensive manipulation (including cellular enhancement) and the maintenance of sterile conditions are essential, in order to create a cellular suspension that can be immediately applied on the damaged area of the patients, thereby avoiding a longer processing in cleanrooms such as Cell Factories. Starting from small skin biopsies, grinding, centrifugation and other separation methods (e.g., enzymatic or mechanical), are frequently used to obtain a cellular suspension, which can be cultured in a growth medium. All of these methods generally require a long time of execution, stressing the cell structures, and leading to a reduction of cell viability. Another significant aspect is to obtain an autologous cellular suspension ready to be used by clinicians, for example, to repair damaged areas. Furthermore, it is well established that autologous tissue grafts survive the transfer procedures to eventually survive in the recipient site by the principles of induction and conduction 2, 3. The ideal graft tissue should be readily available and have low antigenicity and donor site morbidity 4.

On the basis of these evidences, the first aim of this study was to create autologous bio-complexes suitable for clinical application in the tissue repair. For this purpose, we describe a new method to obtain autologous micro-grafts starting from cutaneous tissues which were disaggregated by this protocol. A case presentation is also herein described as a clinical application of autologous micro-grafts obtained by this protocol in combination with collagen sponges. This approach has already been reported to be efficient in the mechanical disaggregation of human tissues5 and it has been used clinically for grafts and regeneration of dermal tissues 6,7 as well as for regenerative therapies of connective tissues in oral-maxillofacial surgery 8-10.

In addition, the second aim of this study was the biological characterization of the cutaneous tissues after their disaggregation by this protocol. To this purpose, different homologous samples of cutaneous tissue derived from the trunk area of different multi-organ and/or multi-tissue donors were processed following National Rules on harvesting, processing and distributing tissues for transplantation (CNT 2013) at Emilia Romagna Regional Skin Bank.
CASE PRESENTATION:&#160; 
A 35-year-old female patient showing a complex trauma due to car accident was admitted to the Intensive Care Unit of Ancona Hospital. The patient showed an infection on the leg due to an open wound and a compound fracture stabilized with external fixation. Two radical debridement were performed and when the wound became clean after negative pressure therapy (V.A.C. therapy) and the periosteum appeared healthy, we applied the protocol after two months from recovery. After disaggregation with this system, the micro-grafts obtained were used to create bio-complexes with a collagen sponge which were subsequently implanted in order to investigate their efficacy on the lesion repair.

<table border="0" cellpadding="0" cellspacing="0" width="978">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Rigenera Machine</td>
			<td style="width:257px;">Human Brain Wave</td>
			<td style="width:217px;">79210R</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Rigeneracons</td>
			<td style="width:257px;">Human Brain Wave</td>
			<td style="width:217px;">79450S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MTT</td>
			<td style="width:257px;">Roche Diagnostic GmbH</td>
			<td style="width:217px;">11465007001</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:245px;">RPMI medium</td>
			<td style="width:257px;">PBI International</td>
			<td style="width:217px;">733-2292</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:245px;">DMEM medium</td>
			<td style="width:257px;">PBI International</td>
			<td style="width:217px;">F 0415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Antibiotics</td>
			<td style="width:257px;">Biological Industries PBI</td>
			<td style="width:217px;">03-038-1 &#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:245px;">Antibodies for FACS Analysis</td>
			<td style="width:257px;">eBiosciences</td>
			<td style="width:217px;">code 12-1469 for CD146-P; code 17-0349&#160; for CD34-APC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Columbia agar</td>
			<td style="width:257px;">BioMerieux Company</td>
			<td style="width:217px;">43041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Condress&#174;</td>
			<td style="width:257px;">Abiogen Pharma</td>
			<td style="width:217px;"></td>
			<td>collagen sponge used for bio-complexes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">DMSO&#160;</td>
			<td style="width:257px;">Bioniche Pharma USA LLC, Lake Forest, IL</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl solution</td>
			<td>Fresenius Kabi, Bad Homburg, Germany</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene&#160;</td>
			<td style="width:257px;">Carlo Erba</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue characterization after a new disaggregation method for skin micro-grafts generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

In this preliminary study, the first aim was to investigate the ability of human autologous micro-grafts combined with a biological support, such as collagen, to produce bio-complexes ready to use. These bio-complexes were implanted in a patient with a leg lesion caused by a car accident (Figure 2A) and a complete re-epithelialization associated with tissue repair after 30 days (Figure 2B) was observed. Moreover, the clin...

Watch this Scientific Journal Video about Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation at JoVE.com

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Research

JoVE Journal

Medicine

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1ÃƒÅ½Ã‚Â² using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...