methodology of theses!

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1- preparation and characterization of axolotl skin ECM scaffolds and Commercial scaffolds (SYMBIOS PeriDerm)

Axolotls used in experiments were purchased from the Ambystoma Genetic Stock Center (AGSC) and breed in Montreal University animal care facility. Animals were lodged in aquaria at ~20 °C

To anesthesia , axolotl (female,blank wind 3 years old) placed in into a plastic clear container filled of the 0.2% MS222 a 20-minute and than euthanized it by decapitation All procedures were conducted in accordance with, and approved by, the University of Animal Care

The skin will be removed from freshly euthanized adult axolotls (female,blank wind 3 years old). Once the skin has been removed any underlying tissue that is not from the dermis (e.g. muscles and excess fat) will be removed manually by scalpel and then the skin will be washed extensively in 1X PBS

The skin will then be processed for decellularization of the ECM, this is very important in order to reduce the risks of rejection. One of the first steps is to break the cells apart without destroying the ECM scaffold. This is achieved by three cycles of freeze (-80˚) and thaw (37˚) of the skin explants. These freeze thaw cycles break up the cellular membrane as well and the nuclei of cells making it easier to wash them out of the collagen scaffold of the ECM27,28. Method #1 uses a hypotonic/hypertonic shock to further break cells and their organelles followed by treatments with detergents to eliminate the cellular debris.

Preparation of ECM scaffolds from axolotl skin has been initiated using the first method (see table-1)

Table-1: different methods for the preparation of ECM scaffold Method #1(mild on proteins; minimal damage expected) -Freeze thaw 3X (-80˚ and 37˚) -rinse dH2O -Rinse in 1X PBS 5h at room T˚

-Treat with 1M KCL (can be 1M NaCL) 24h at 37˚ with shaking -rinse dH2O -Treat with 1% SDS 48h at 37˚ with shaking (change solution after 24h) -rinse dH2O (3X 1h each time with shaking to remove SDS). -Treat with detergent 1% Triton X-100 24h at 37˚ -rinse dH2O (3X 1h each time with shaking to remove Triton-X100). -rinse in 1X PBS 5h at room T˚ -rinse dH2O

-Lyophilize overnight -Ready to use (store at -80˚)

Commercial scaffolds (SYMBIOS PeriDerm)

Symbios PerioDerm (derived from donated human skin , Thick 1 x 2 cm) is an acellular dermal allograft designed to replace damaged or inadequate tissue in the repair, reinforcement or supplemental support of soft tissue defects. Advantages of PerioDerm are that it has excellent, consistent handling characteristics which are similar to autogenous connective tissue grafts. The rehydration time is very quick, reducing surgery and procedural costs. The graft is freeze-dried and requires no refrigeration. Maintains over 90% thickness consistency for uniform revascularization and aesthetic results.

2-DNA isolation and RT-PCR

Axolotl ECM/ intact skin, and DAPI Staining to detect if there are any cells to avoid immune system rejection.

Pisces of axolotl decellularized scaffold (ECM) and pisces of skin as positive control were frozen with liquid nitrogen and broken/ground the sample into powder by using mortar and pestle. Collect powder put tube with 2ml BPS X 1 an homogenized sample by high shear homogenizers.

PCR reactions were carried out using rTaq (Takara) for 32 cycles with the following conditions: 55 ° for 20 PCR products were loaded and resolved in 2% agarose gels. using with the following primers: Hdm2 forward CAGCTTCG- GAACAAGAGACC, reverse GAAGCCAATTCTCAC-

GAAGG;

and another axolotl decellularized scaffold and pisces of skin as positive control were fixed in 4% formaldehyde and then sample were then rinsed, dehydrated, and put in paraffin the next day and Sectioning was performed at a thickness of 10 mm. Saining with DAPI and Mounting was done in Permount TM Mounting medium (Fisher Scientific, Sp15-100)

3-Proteomics Analysis for Decellularized Axolotl ECM and Intake Skin Axolotl to measure proteins proteome analysis was performed using multidimensional gel electrophoresis to identify differentially expressed protein Decellularized Axolotl ECM proteome analysis AND intack skin.

Immunofluorescence enhanced with tyramide for Col IV

Removal of paraffin from slides by 3 consecutive incubations in xylene followed by rehydration to water. Epitope retrieval was performed ( citric acid for 20 min at 95°C). Slides were blocked using 2% BSA in TBS-T (Tris-buffered saline with 0.1%Tween 20) for 1 h at room temperature. Primary antibodies anti-Col IV (ab6586, Abcam; 1/500) in PBS and incubated overnight at 4°C. Anti-rabbit-HRP and anti-mouse-HRP (170-6515 and 170-6516, Bio-Rad; 1/400) secondary antibodies were diluted in blocking solution and incubated at room temperature for 45 min. Tyramide (Biotium, 92175) was diluted in TBS with 0.0015% H2O2 to 11.6 μM then incubated at room temperature for 8 min. All slides were mounted with ProLong Gold antifade reagent containing DAPI (Invitrogen, 36931). Slides were visualized with a Zeiss Axio Imager M2 optical microscope. The images were saved as tif files

5-Animal experimentation and Ethical statement

All experiments were performed in accordance with tthe University animal ethics committee which is overseen by the Canadian Council of Animal Care. Animal protocols were approved by University ethics committee (authorization number 38828770-E.2305). Animals were caged with free access ad libitum to standard pellet food and water, and following conditions: at room temperature (at around 24°C), with 12h dark: 12h light cycle.

18 immunocompetent furred mice models ( aged 6–7 weeks) were purchased from University and housed at the animal facility in controlled conditions. 18 mice were divided into two major groups (n=18, 2 groups consisted of 9 animals each) aged 4-5 months and weighing between

25-30 g were selected and grouped randomly. The first group is going to 7 days and the second is going to 30 days.

The first group (consisted of 9 animals mice ) were divided 3 group (3 Axolotl groups, Commercial Groups,3 control groups). The seconed group (consisted of 9 animals mice ) were divided 3 group (3 Axolotl groups, Commercial Groups,3 control groups) as well

Axolotl groups will be treated with axolotl ECM scaffolds and Commercial Groups will be treated with Commercial scaffolds(SYMBIOS PeriDerm), and control groups will be left without treatment as positive control

All surgery was performed under Using inhalative anesthesia (Isoflurane), and all efforts were made to minimize suffering Before anesthesia, each group of mice will be injected with (Buprenorphine) subcutaneously that will provide pain relief after surgery. (Dose - 0.05-0.1 mg/kg).

excision circles by biopsy punches (8 mm).

Surgical tools and nonsurgical materials

Isoflurane was purchased from university . needle holders and Adison forceps , Suture scissors, were obtained from our lab (Montreal, Canada). Other material that were used in this study include: Tegaderm Film dressing (3M, St. Paul, MN), Opsite spray dressing (Smith and Nephew, MA) Sterile gauze pads, 10% povidone/iodine, Vaseline infused gauze, and Co-Flex self-adherent wrapping (Andover Healthcare, Salisbury, MA). 7-0 Prolene and 8-0 Nylon sutures were purchased from S&T (Neuhausen, Switzerland).

biopsy punches dimeter (8 mm) and to make excision circles of the dorsal skin of mice

biopsy punches dimeter (8 mm) and to make circle pieces of Titanized Mesh

suture needles (Nylon Suture) nonabsorbable surgical suture

Suture scissors

Needle holder

Adison forceps

Other material that were used in this study include were purchased Montreal University ( montreal, Canada) Canada

Needle and syringe

normal saline

wound dressing

Sterile gauze pads

samples of Axolotl ECM scaffolds (8 mm) , stratus

samples of Commercial scaffolds (SYMBIOS PeriDerm) (8 mm)

Generate 18 samples of the Titanized Mesh by using 10 mm diameter that will be placed under all scaffold and control group to prevent contraction muscles ( panniculus carnosus)

wound dressing ( band /Tegaderm (Nexcare 3M) + cushion

In vivo excisional wound model Full-thickness wounds were created in the dorsal skin and Scaffold implantation

Lying mouse on operating table and stretching skin out and then making excision circles by biopsy punches (8 mm).

2 full-thickness 8 mm excisional circle wounds will be created by a sterile punch biopsy (8 mm).

on the dorsal surface for each group (axolotl group, commercial scaffolds (SYMBIOS PeriDerm), and control groups) . .

Placing the titanized mesh (10 mm) into wound bed and under wound edges for each group

axolotl group/ commercial (SYMBIOS PeriDerm) scaffolds will be placed directly in the wound bed immediately after wounding

The control excisional wounds will be left untreated just placing by Titanized Mesh.. Suture the scaffold to wound edges with 8 stitches to secure the scaffold in place.

Taking picture 0 day by Canon EOS M50 Mirrorless Camera

Placing cushion by suture and Covering Tegaderm (Nexcare 3M) after that The scaffolds will be covered with a non-sticky dressing.

Postoperative Care

Postoperative monitoring was provided for approximately 30 minutes following the completion of anesthesia. According to our approved protocol

To avoid dehydration, mice will be injected with normal saline subcutaneously (Dose- 0.5 ml)

Keep one mouse in each cage to avoid any errors (each cage there will be name of mouse and which a scaffold and group is)

Observing motor activity, signs of pain and wound area for bleeding or any sign of infection at the position of dressing.

To reduce pain if necessary, it will inject Buprenorphine (Dose 0.05-0.1 mg/kg)and were monitored weekly for graft outcomes

If the wound dressing taking off or damage, it will replace or fix it under anaesthesia

Photographs were taken at day 0, 7, 22, 30 for each wound with a ruler by using a Canon model digital camera to calculate the wound closure rate. All pictures were taken from the top view of the wounds with an angle wound healing assessment

12-Sacrificing mice and Sample preparation

First group (consist of 9 Animals , Axolotl groups,3 Commercial Groups,3 control groups) were sacrificed by CO2 exposure and then decapitate them on day 7 and second group (consist of 9 Animals, Axolotl groups,3 Commercial Groups,3 control groups) were sacrificed on day 30 , and skin covering the wound site including the original wound margin was removed by using surgical scissor and scalpel for further analysis for all group

Transillumination to detect and quantify a vascular network

Place Petri dish on a strong of white light bulb and take a picture by Canon EOS M50 Mirrorless Camera.

Save picture in a TIFF for digital analysis by VesSeg- Tool software

extracted tow samples were extracted from Each mice , One Sample extracted were fixed in 4% formaldehyde and then sample were then rinsed, dehydrated, and put in paraffin the next day and Sectioning was performed at a thickness of 10 mm of all samples Axolotl ECM scaffold, Commercial scaffolds (SYMBIOS PeriDerm) and no grafted group (positive control)

14 -Histology analysis

Before staining, slides were deparaffinized through three baths of xylene for 5 min each.

Slides were then rehydrated in a graded series of 100, 90, 70, and 50% ethanol and then with distilled water for 5min each

Slides were successively put in Mayer’s hematoxylin (Dako Cytomation, S3309) for 75 sec, thor- oughly rinsed in water, put in 0.08% NH4OH for 20 sec, in 80% ethanol for 1 min, in Eosin for 30 sec and rinsed in 80% ethanol for 30 sec. Samples were dehydrated in 95% ethanol two times for 1 min, 100% twice for 1 minute and two times in xylene for 1 minute. Mounting was done in Permount TM Mounting medium (Fisher Scientific, Sp15-100). Slides were visualised using Imager Optical Microscope

Collagen deposition (indicator of scarring) and wound healing maturation will be analyzed by Masson’s trichrome and Sirius Red staining 33,34

Masson’s Trichrome Stain:

Using Masson’s Trichrome Skin samples were embedded in paraffin and cut in sections of 5 μm, stained with Masson’s Trichrome for analysis of collagen-rich and fibrotic areas. Masson’s trichrome staining method was used to stain cell cytoplasm in red, nuclei in black, and collagen in blue (Luna et al., ’68). Sirius red staining was used to specifically stain collagen fibers (protocol of John A. Kiernan on www.ihcworld.com). To permit the visualization of collagen- specific staining, slides stained with Sirius red were visualized using a microscope equipped with a polarized light. After staining, slides were serially dehydrated for 2 x 2min each in 90% EtOH, 100% EtOH, and 100% Xylene. Slides were mounted with Permount (Fisher scientific, Ottawa,

Sirius Red Chemical Stain:

Removal of paraffin by incubations of slides in 3 changes of xylene followed by rehydration to water and staining for 10min in Weigerts Hematoxlylin occurred prior to the incubation of 1 hour in the Sirius Red solution composed of Direct Red 80(Alfa Aesar, B21693) in a saturated solution of Picric Acid). Two 5min rinses in 0.5% v/v Acetic Acid and rapid dehydration in 90 and 100% EtOH followed by clearing in xylene and coverslip with organic mounting media.

Sirius red staining was used to specifically stain collagen fibers (protocol of John A. Kiernan on www.ihcworld.com). To permit the visualization of collagen- specific staining, slides stained with Sirius red were visualized using a microscope equipped with a polarized light

Statistical analysis

All statistical analyses were performed with SPSS 18.0 software program. The closure rate for each treatment is calculated as means ± Standard deviation. The significance of differences between groups was analyzed by one-way ANOVA for the macroscopic assessment of wound healing. Multiple comparisons between groups were carried out by using Dunnett’s test. According to Post-hoc tests; p<0.05 was considered as statistically significant

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