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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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: 

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.

Protokół

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 clinically based on the use of Rigeneracons (tissue disruptor) and the Rigenera Machine (tissue disruptor system) (Figure 1A). The tissue disruptor is a biological medical disruptor of human tissues able to disrupt small pieces of tissues using a grid provided by hexagonal blades and filtering cells and components of extracellular matrix with a cut-off of about 50 microns.

1. Collect skinsamples of the patient through a biopsy punch (Figure 1B) and disaggregate adding 1 ml of saline solution for each piece to obtain autologous micro-grafts ^6,7,9 (or see step 2.1).
2. Place 1 ml of micro-grafts on collagen sponges (Figure 1C) toform bio-complexes to use for clinical application.
3. Culture another 1 ml of micro-grafts on collagen sponges in the presence of 6 ml of DMEM medium supplemented with 10% Fetal Bovine Serum at 37 °C in a 5% CO₂ humidified atmosphere.
4. Following 3 days of culture, fix the bio-complexes with 0.3% Paraformaldehyde for 10 min at RT. Pour the paraffin with a specific dispenser directly onto the sample. Obtain slices with a microtome with a thickness of 5 µm and put directly in a glass-slide.
5. Immerse paraffin slices of 5 µm in a glass for histological analyses, containing 15 - 20 ml Xylene (commercially available - a mix of m-xylene (40 - 65%), p-xylene (20%), o-xylene (20%) and ethyl benzene (6 - 20%) and traces of toluene, trimethyl benzene, phenol, thiophene, pyridine and hydrogen sulfide) for 3 min each.
6. Immerse the slices in decreasing grades (100% to 70%) of ethanol (100% ethanol for 1hr, 95% ethanol for 1 hr, 80 % ethanol for 1hr, 70% ethanol for 1 hr) and then deionized water for de-paraffinizing and rehydrating the sections.
7. Stain the sections with 1 - 2 ml of 1g/L Ematoxylin for 1 - 2 min and subsequently rinse in water to remove any Ematoxylin surplus.
8. Stain the sections for 4 - 5 min with Eosin Y alcoholic solution at 1% of concentration mixed with ethanol 70% and diluted in water.
9. Use 1 - 2 ml of Eosin Y for each slide section and rinse under running tap water.
10. Immerse sections in increasing grades of ethanol (see step 1.6) and finally, after a passage in Xylene for 1h, coverslip with a based-mounting mediumand observe under a light microscope at 100X magnification (Figure 1D).

2. Collection, Disaggregation and In Vitro Analysis of the Tissues

1. Using a dermatome, take independent skin tissue, papillary de-epidermized dermis (Ded) or reticular dermis (Dermis) respectively of 0.6 mm, 1 mm or 2 mm in thickness from the trunk area of 4 different multi-organ and/or multi-tissue donors in a range from 40 to 55 years, following National Rules on harvesting, processing and distributing tissues for transplantation (CNT 2013).
   1. Gently rinse all samples in 0.9% NaCl solution putting them in a dish on an orbital shaker for 5 min.
   2. Using the 5-mm biopsy punch, create samples which are uniform in diameter from the skin tissue, Ded and Dermis and weigh all tissue specimens before the disaggregation.
   3. Insert eight, three or four uniform samples of skin tissue, Ded or Dermis respectively, in the tissue disruptor, adding 1.5 ml of saline solution for the disaggregation.
   4. Perform different times of disaggregation for all tissue samples as indicated in Table 1.
   5. Use a correspondent number of punch biopsies derived from intact tissue samples as controls.
   6. After mechanical disaggregation, aspirate the saline solution containing micro-grafts and place separately each sample in a single well of a 12-well plate. Perform the same protocol for intact control punch biopsies.
   7. Add 1ml of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics to each sample.
   8. Evaluate the cell viability immediately. To each well containing a micro-graft (obtained by the simultaneous disaggregation of eight, three or four uniform samples of skin tissue, Ded or Dermis respectively) add 1ml of medium containing 0.5 mg/ml of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) solution and incubate for 3 hr at 37 °C in an atmosphere of 5% CO₂/air. Perform the same protocol for intact control punch biopsies.
   9. After incubation, remove all medium containing MTT and add to each sample 1 ml dimethyl sulfoxide (DMSO) for 10 min.
   10. Transfer each sample and DMSO in a cuvette and read at optical density (OD) at 570 nm using a spectrophotometer. Calculate cell viability as the ratio of absorbance at 570 nm and the weight in grams (gr) of tissue used before disaggregation. Perform the same protocol for intact control punch biopsies.
2. After mechanical disaggregation, aspirate the saline solution containing micro-graft derived from skin tissue, Ded and Dermis samples of a single donor.
   1. Place separately each sample in a single well of a 12-well plate or in a culture flask for cell viability test and morphological analysis respectively.
   2. Culture micro-grafts adding 1 ml (12-well plate) or 5 ml (culture flask) of RPMI 1640 medium supplemented with 10% FBS and antibiotics at 37 °C in an atmosphere of 5% CO₂/air for 24 hr or 7 days.
   3. Evaluate the cell viability after 24 hr or 7 days (repeat steps 2.1.8-2.1.10).
   4. Perform morphological analysis evaluating the presence of cell suspension by light microscopy after 24 hr and 7 days of culture in flask.
   5. Analyze the samples of Dermis for positivity to the mesenchymal and hematopoietic cell markers, including CD146, CD34 and CD45 antigens by FACS analysis ⁶.
   6. Under laminar flow hood seed each micro-graft (obtained by the simultaneous disaggregation of eight, three or four uniform samples of skin tissue, Ded or Dermis respectively) and a correspondent small fragment of each tissue sample not totally disaggregated (>50 micron in size after disaggregation process) on Columbia agar plate containing 5% sheep blood broth 100 µl.
   7. Incubate the plate at 37 °C for three days and perform microbiological analysis on Columbia agar plate in order to assess the sterility¹¹.

Wyniki

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...

Dyskusje

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 implants^{9, 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...

Materiały

Name	Company	Catalog Number	Comments
Rigenera Machine	Human Brain Wave	79210R
Rigeneracons	Human Brain Wave	79450S
MTT	Roche Diagnostic GmbH	11465007001
RPMI medium	PBI International	733-2292
DMEM medium	PBI International	F 0415
Antibiotics	Biological Industries PBI	03-038-1
Antibodies for FACS Analysis	eBiosciences	code 12-1469 for CD146-P; code 17-0349  for CD34-APC
Columbia agar	BioMerieux Company	43041
Condress®	Abiogen Pharma		collagen sponge used for bio-complexes
DMSO	Bioniche Pharma USA LLC, Lake Forest, IL
NaCl solution	Fresenius Kabi, Bad Homburg, Germany
Xylene	Carlo Erba