Significantly Accelerated Wound Healing of Full-Thickness Skin Using a Novel Composite Gel of Porcine Acellular Dermal Matrix and Human Peripheral Blood Cells

Abstract

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls (p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

Keywords

Scaffolds Tissue engineering Regenerative medicine Porcine skin Decellularization Wound healing

Introduction

Wound healing is the process in which cells in the body regenerate and repair to reduce size of damaged or necrotic areas (22). Wound healing involves a set of phases that include inflammation surrounding a region of injury, cell migration and mitosis, angiogenesis and the development of granulation tissue, repair of the connective tissue, regeneration of the extracellular matrix (ECM), and remodeling that leads to a healed wound (30). Each phase has its distinctive mechanism and is also interconnected with the other phases. The duration of these individual phases varies depending on the intensity and depth of the wound. Most wound-dressing treatments aim to facilitate these stages of wound healing by providing a moist environment, controlling excessive exudate build up, and protecting against infection that would perturb normal healing.

Normally, skin injuries caused by various physical factors or chemical agents can heal spontaneously (9,13). However, in the therapy of extensively burned patients, the limited amount of donor sites for skin autotransplantation is a constant problem. While full-thickness wounds are resurfaced with split-thickness skin autografts, deep dermal burns are usually covered with biological or synthetic covers (dressings). Wound covers work as temporary substitutes. If the wound does not heal spontaneously, they have to be replaced with the patient's own skin.

Today, several modern skin substitutes are available and commonly used in clinical practice. They include autografts, allografts, xenografts, and tissue-engineered skin products. However, the properties and applicability of the skin substitutes differ significantly, depending on the materials used, cost effectiveness, and the manufacturing process of the substitute. Therefore, there is a clinical need for a wound treatment system that is efficient, easily applicable, and available on demand.

ECM is composed of collagens, proteoglycans, structural proteins, and basement membrane and is the largest component of normal skin, which provides the unique properties of elasticity, tensile strength, and compressibility of the skin (14,31). An important step in the regenerating process is synthesizing the same ECM, resulting in tissue remodeling and less scar formation (20,29). In order to augment and support this natural procedure, introduction of a similar template to the wound area may induce efficient cell migration, proliferation, and differentiation and generate natural ECM (24). Pig skin possesses a comparable structure to natural human ECM, and the presence of several similar human skin ECM components has been verified in pig skin (4,27). We therefore reasoned that decellularized pig skin would be an ideal template for preparation of a gel to be used as a dermal substitute for the reconstitution of full-thickness wounds.

Decellularization technology can be used to reduce the antigenicity of xenogeneic tissues and organs and at the same time retain many of the main components of the ECM (12). Here we employed this technology to decellularize pig skin, which was further processed to produce a gel. We then speculated that embedding human peripheral blood mononuclear cells (hPBMCs) in the gel would further accelerate formation and maturation of well-organized wound tissue in the setting of acute skin wounds. The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-human tissue and organ xenotransplantation (7). Since the process of decellularization does not result in complete removal of all cellular material (11), we decided to decellularize skin grafts taken from galactose-α1,3-galactose (Gal/gal) knockout (KO) pigs. In addition, since normal mice will reject human cells, immunocompromised mice were used in all animal experiments. So we aimed to test the feasibility and effectiveness of ECM gel derived from decellularized galactose-α1,3-galactose KO porcine skin in wound healing using a model of nude mouse with full-thicknvess cutaneous wound.

Materials and Methods

Preparation of Porcine Skin Scaffolds

Gal/gal porcine skin (n = 1) was purchased from Avantea (Cremona, Italy). A piece of skin measuring 20 × 15 cm² was dissected from a Gal/gal KO pig and was vacuum sealed in a plastic bag and stored frozen at −20°C until use. The skin was thawed at room temperature (RT) and cleaned by excision of the subdermal fat tissue. The hair was removed and the skin was washed with distilled water. The skin was then placed in a 5-L plastic container and agitated at 200 rpm with 0.5% sodium dodecyl sulfate (SDS; Sigma-Aldrich, Steinheim, Germany) containing 0.02% sodium azide (Sigma-Aldrich) and 1.86% ethylenediaminetetraacetic acid (EDTA; Alfa Aesar, Karlsruhe, Germany) at 37°C for 9 days continuously. SDS was changed first after 24 h and then every 48 h. During every change of SDS, the skin was washed for 1 h with distilled water.

Verification of Decellularization and Characterization of the Extracellular Matrix

The decellularization was verified by histology and DNA quantification using the following procedures. Two pieces from normal and decellularized skin were fixed in formalin (Histolab, Gothenburg, Sweden) for 48 h and embedded in paraffin (Histolab). The sections were cut at 5-μm thickness using a microtome and stained by hematoxylin and eosin (H&E), Masson's trichrome (MT) (25088; Polysciences, Warrington, PA, USA), and Verhoeff Van Gieson (VVG) (25089; Polysciences) methods to identify the presence of nuclei, collagen, and elastin. Pieces of tissue (25 mg) were cut from normal and decellularized skin, and total DNA was isolated using Qiagen Blood and Tissue DNA Kit (Qiagen, Hilden, Germany) following the manufacturer's instruction and quantified using NanoDrop (Implen, München, Germany) at 260 nm wavelength.

Preparation and Characterization of Decellularized Pig Skin Powder

After decellularization, the skin was washed for 5 days in distilled water. The water was changed every 12 h. The decellularized skin was cut into pieces of 3 × 3 cm² and freeze dried (lyophilized) for 72 h. The lyophilized pieces were pulverized in a cryomill with a 0.75-μm sieve at 14,000 rpm. The filamentous powder obtained was sterilized by g irradiation at 25 kGy for 3 min and 25 s.

ECM quantification in the pig skin powder for collagen, elastin, and glycosaminoglycans (GAGs) was performed as we previously described (21). The powder structure and morphology were investigated by optical and scanning electron microscopy (SEM). Optical imaging was performed by an Olympus SZX16 stereo microscope (Olympus Europa SE & Co.KG, Hamburg, Germany) equipped with ColorView IIIu CCD camera (Soft Imaging System GmbH, Münster, Germany) and Olympus Cell∗∗∗^D image analysis software. SEM analysis was performed by a Zeiss Supra 40VP instrument (Carl Zeiss NTS GmbH, Oberkochen, Germany) in secondary electron detection mode. To reduce sample charging during SEM imaging, powder samples were deposited on carbon pads and sputter coated by 15-nm-thick Au/Pd film in a Gatan PECS Mod 682 instrument (Gatan Inc., Pleasanton, CA, USA).

Preparation of Pig Skin Gel (PSG)

A gel was prepared by mixing 50 mg of decellularized skin powder and 250 μl of hyaluronic acid (HA) (Sigma-Aldrich) to get a gel-like consistency. The HA was constituted at 1 mg/ml in keratinocyte medium (Lonza, Verviers, Belgium).

Sterility

Three random samples of 1 mg of irradiated powder were added into thioglycolate broth (Fluka, Steinheim, Germany) and cultured for 2 weeks in an incubator at 37°C. Every other day, 200 μl of broth was collected and verified for turbidity by measuring optical density in a spectrophotometer (Synergy 2; BioTek, Winooski, VT, USA) at 600 nm wavelength. An increase in turbidity indicates contamination.

Animal Experiments

All animal experiments were performed after prior approval from the local ethics committee for animal studies at the administrative court of appeals in Gothenburg, Sweden. In total, 72 BALB/c nude female mice (7–8 weeks of age and weighing 17–18 g; Taconic, Silkeborg, Denmark) were used to study wound healing rate (WHR). The mice were divided into four groups after induction of full-thickness skin wound: (i) untreated; (ii) treated with HA; (iii) treated with PSG only; and (iv) treated with PSG + hPBMCs (10⁶ cells). All groups except the untreated group (i) contained HA reconstituted in keratinocyte medium. A total of 12 mice were included in the untreated and HA-treated groups, and 3 mice per group were sacrificed at each time point. A total of 24 mice were included in the PSG- and PSG + hPBMC-treated groups, and 6 mice per group were sacrificed at each time point. The mice within untreated and treated with HA groups served as controls. The mice were anesthetized using isofluorane, and carprofen (Rimadyl) (Pfizer, New York, NY, USA) was given by intraperitoneal (IP) injection preoperatively at 10 mg/kg for postoperative pain relief as well as once daily for 2 days after surgery. The skin was wiped with a few drops of chemical gasoline, and a full-thickness 1 × 1-cm² excision wound was created in the upper back area of each animal. Gel with or without cells was applied using a sterile wooden stick. Tegaderm (3M^TM, Neuss, Germany) was then placed on the wounds and sutured to the skin using a 4-0 nonabsorbable monofilament suture (Ethicon, San Lorenzo, Puerto Rico, USA) for protection and to keep the gel in place. The Tegaderm was further covered with Hydrofilm (Hartmann ScandiCare AB, Anderstorp, Sweden) to prevent removal of the Tegaderm by the animals. Both Tegaderm and Hydrofilm are thin polyurethane membranes permeable to oxygen and water vapor but protect the wound from contamination and infection. On the day of surgery, peripheral blood was collected from a healthy human donor, and mononuclear cells were separated on lymphoprep (Axis-Shield PoC, Oslo, Norway), and the isolated cells were washed in phosphate-buffered saline (PBS) thrice and suspended in keratinocyte medium. The cells were counted using Bio-Rad (Singapore) automated cell counter. At 5, 10, 15, and 25 days after treatment, animals were sacrificed, and the skins, including the wounds, were excised for histological examination. The cutaneous wounds were also photographed as described in the Measurement of Wound Area section.

Histology and Immunohistochemistry of Wounds

Paraffin blocks of the skin wounds from all groups were sectioned at 5-μm thickness using a microtome, and five sections, covering the whole region of the wound, were stained with H&E and MT following the standard procedure of the staining kit producer (Polysciences). The slides were scanned using a Leica SCN400 microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Skin sections were stained with human-specific anti-mitochondrial antibody (1:100; Merck Millipore, Stockholm, Sweden) for detection of human cells. Secondary antibody used was peroxidase-conjugated affinipure (Fab)₂ fragment goat anti-mouse IgG, F(ab)₂ fragment specific (1:500; Jackson Immunoresearch, West Grove, PA, USA).

Immunofluorescence

Cryosections were cut at 5-μm thickness using a cryotome. Antibodies specific for human CD31 (1:25; 10148-MM13; Sino Biologicals, North Wales, PA, USA) and mouse-specific CD31 (1:400; LS-C348704; LSBio, Seattle, WA, USA) and the secondary antibodies Alexa GaM 568 (1:100; A11031; Life Technologies, Eugene, OR, USA) and GaR fluorescein isothiocyanate (FITC) (1:100; SC3825; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. Briefly, cryoslides were fixed in acetone/methanol (4:6) for 10 min at −20°C and washed in PBS for 5 min. The slides were blocked with serum of secondary antibody host and incubated with primary antibody overnight at +4°C. The slides were washed in PBS thrice and incubated with secondary antibody in the dark at +4°C for 45 min. The slides were washed thrice in PBS and coverslipped with a drop of mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (H1500; Vector Laboratories, Burlingame, CA, USA).

Quantification of Collagen

The amount of collagen in all animal groups on day 25 (newly formed skin) was quantified based on the intensity of blue-stained collagen in MT staining using the principle of densitometry. Five representative images were taken from each animal, and the total intensity of blue color per image was quantified with the BioPix iQ 2.1.8 software (Gothenburg, Sweden). The collagen intensity was read as arbitrary units (a.u.), and the average for each animal was calculated.

Measurement of Wound Area

To calculate the wound size, a measuring scale was placed next to the wound, and the whole mouse was photographed using a Sony a 33 digital camera (Thailand). The area of unclosed wound (not scars) per animal was then quantified using ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA]. Each image was calibrated using the photographed scale, and the contours were marked using a manual drawing tool. The average of unclosed wound area per animal in each group was then calculated. Furthermore, the WHR for each group was calculated at the following time intervals: 0–5, 5–10, 10–15, and 15–25 days. WHR was calculated as (A_i - A_f)/A_i, where A_i is the initial wound area at the starting time point of the interval, and A_f is the final wound area at the final time point of the interval. WHR greater than 0 indicates healing, while WHR less than 0 indicates an increase in wound area (3,18).

Gene Analysis

RNA was extracted by adding 1 ml of ice-cold Qiazol (Qiagen GmbH) to the frozen skin samples. A steel bead was added to each sample, and samples were homogenized in a Qiagen Tissue Lyser at 25 Hz for 2 × 5 min, 200 μl of chloroform was added, and the samples were shaken vigorously for 15 s, left at RT for 3 min, and centrifuged at 12,000 × g for 15 min at 4°C. Following centrifugation, 350 μl of the supernatant was transferred to a new tube and mixed with 350 μl of 70% ethanol and mixed carefully by pipetting and added to the MiniElute column of an RNeasy Mini Kit (Qiagen GmbH). The rest of the procedure was done according to the manufacturer's instructions.

Extracted RNA was reverse transcribed using TATAA GrandScript cDNA Synthesis Kit (Tataa Biocenter, Gothenburg, Sweden) by adding 2 μg of total RNA in a final reaction volume of 20 μl in a Bio-Rad CFX96 instrument according to the manufacturer's instruction.

The quantitative polymerase chain reaction (qPCR) was performed using TATAA SYBR Grandmaster Mix (Tataa Biocenter) in a Bio-Rad CFX384 instrument with a temperature protocol of 95°C for 60 s, followed by 45 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 10 s, followed by a melt curve from 55°C to 95°C in 0.5°C steps. For each reaction, 4 μl of cDNA was used in a total reaction volume of 20 μl. For the qPCR, two assays were done, one directed against human cytochrome B (hCytB) and the other against mouse cytochrome B (mCytB). The qPCRs were performed in duplicates for the hCytB assay and in single measurements for the mCytB assay.

Statistics

All values represent the average of that experiment, the graphs plotted are the mean of that group, and the error bars represent the standard error of the mean. The graphs were plotted using GraphPad Prism software version 6.0 (La Jolla, CA, USA). Mann–Whitney U-test was used to calculate significant differences between groups. A value of p < 0.05 was considered significant.

Results

Decellularized Pig Skin

We found that the Gal/gal KO pig skin was completely decellularized in 9 days after continuous treatment with 0.5% SDS. The gross morphology and histology of the pig skin before (Fig. 1A–C) and after decellularization (Fig. 1D–F) are shown in Figure 1. No presence of nuclei (blue) or cellular remnants was observed in the decellularized pig skin (Fig. 1F). DNA quantification showed a rapid decrease in DNA amount to 13.9 ng/mg wet tissue weight in decellularized skin from 245 ng/mg wet tissue in normal skin. The pig skin gel prepared using HA and keratinocyte medium was white in color (Fig. 1G) and was used for all wound skin healing experiments.

Figure 1

Gross morphology and histology of pig skin. Gal/gal knockout native pig skin with (A) epidermal side up and (B) dermal side up demonstrating a pinkish color, presence of hair, and subcutaneous fat. (C) Hematoxylin and eosin staining of native pig skin showing presence of nuclei (blue) and cellular material. (D, E) Decellularized pig skin after 9 days of decellularization. The skin looked pale and white. (F) Hematoxylin and eosin staining of decellularized pig skin showing complete absence of nuclei and cellular material. (G) Picture showing the gel prepared using decellularized pig skin containing hyaluronic acid and keratinocyte medium. Scale bar: 50 μm.

The decellularized pig skin powder retained collagen (66.6 μg/mg), which is one of the major ECM components required for regeneration of skin as well as some elastin (3.5 μg/mg) and sulfated GAGs (4.6 μg/mg) (Fig. 2A). MT and VVG stainings also confirmed that collagen and elastin were preserved after decellularization (Fig. 2B and C). Furthermore, characterization of the powdered pig skin showed that the decellularized pig skin powder had a filamentous, white macroscopic appearance and was transparent to light when investigated by optical microscopy (Fig. 2D). SEM showed that it consisted of ribbon-like fibers that varied in shape and size. The predominant fiber dimensions were 20–30 μm in width, 1–3 μm in thickness, and up to 2 mm in length. The fibers were irregularly wrinkled and twisted, forming easily dispersible bundles (Fig. 2D–F). The sterility testing of the pig skin powder also showed no increase in optical density measured for 2 weeks during culture, showing that gamma irradiation can be used for sterilization of pig skin powder.

Figure 2

Characterization of decellularized pig skin. (A) Graph showing presence of residual extracellular matrix components in decellularized pig skin powder despite removal of cells. (B) Masson's trichrome (MT) staining pictures of native (upper panel) and decellularized (lower panel) pig skin showing collagen (blue) and (C) Verhoeff Van Gieson (VVG) staining for elastin (black) in (upper panel) native and (lower panel) decellularized pig skin. (D) Optical microscopy picture of powdered pig skin showing a filamentous, white macroscopic appearance that was transparent to light. (E, F) Scanning electron micrographs showing that the pig skin powder consisted of ribbon-like fibers that varied in shape and size. The fibers were irregularly wrinkled and twisted, forming easily dispersible bundles. Scale bars: 200 μm.

Effect of the Pig Skin Gel and Human Peripheral Blood Mononuclear Cells on Full-Thickness Wound Healing

To determine the wound healing capacity of the PSG, acute full-thickness excision wounds created on the back of nude mice were either untreated or treated with HA, PSG only, or PSG + hPBMCs. Figure 3 shows the wound healing process in all animals over time. At 5 and 10 days postoperation, scabs were observed in all groups.

Figure 3

Gross morphology of wound healing of a full-thickness cutaneous wound in nude mice. Healing was found to be significantly accelerated in mice treated with pig skin gel (PSG) only or PSG + human peripheral blood mononuclear cells (PSG + hPBMCs) already at day 15 compared to untreated or those treated with hyaluronic acid (HA). In addition, a dark pink-colored scar was still observed in the healed skin of the untreated group due to excessive contraction at day 25, and an elongated scar was also observed in the animals treated with HA and keratinocyte medium, while a remarkably clear and almost completely healed skin with a slight or no scar was seen in animals treated with PSG or PSG + hPBMCs. The mouse pictures used are not equally scaled but illustrate wound healing.

However, by day 15, the scabs were peeling off in all PSG-treated groups, and the wounds were repleted with regenerated skin. As seen, clear differences were observed with regard to wound closure in animals treated with PSG only and PSG + hPBMCs. By day 15, the wounds in these animals represented the most significant difference in wound closure. Already on day 15 complete wound closure was observed in five of six (83%; p < 0.05) mice treated with PSG + hPBMCs and in four of six (66%, p < 0.05) mice treated with PSG only, compared to zero of three (0%) in untreated and mice treated with HA. On day 25, complete wound healing was observed in all groups. The healed skin in animals treated with PSG only or PSG + hPBMCs was similar to normal skin, while an elongated scar was still observed in the healed skin of the control groups, which may be due to excessive contraction. As shown in Figure 3, the difference between the control groups and both PSG-treated groups was remarkable at 15 days after the operation but was not significant at 25 days.

Measurements of wound sizes on day 15 showed that the animals treated with PSG (p < 0.05) or PSG + hPBMCs (p < 0.05) had significantly smaller wound areas compared to untreated animals (Table 1). Although not significant, the wound areas in animals treated with PSG or PSG + hPBMCs were also lower when compared to animals treated with only HA (Table 1). Furthermore, the WHR was also increased in both PSG-treated groups in a period of 10–15 days (Table 2) in comparison to both control groups. The observed decrease in wound area in the untreated group on day 5 could be due to excessive skin edge contraction, which might not be the case for the wounds filled with gel.

Table 1

Measurement of Wound Sizes at Various Time Points in Untreated and Animals Treated With Hyaluronic Acid, Pig Skin Gel, or Pig Skin Gel and Human Peripheral Blood Cells

	0 Day	5 Days	10 Days	15 Days	25 Days
	Wound Size in mm²
Untreated (n = 3)		62 ± 1	70 ± 7	30 ± 5	2 ± 2
H A (n = 3)	109 ± 10 (n = 5)	100 ± 19	50 ± 3	12 ± 4 (p = ns)	0
PSG (n = 6)		121 ± 8	78 ± 12	9 ± 2^∗ (p = 0.01)	0
PSG + hPBMCs (n = 6)		85 ± 7	82 ± 6	4 ± 3^∗ (p = 0.02)	0

∗

Compared to untreated animals on day 15. ns, not significant.

Table 2

Calculation of Wound Healing Rate at Various Time Points in Untreated, HA-Treated, PSG-Only, and PSG + hPBMC Groups

	Wound Healing Rate
Sample	0–5 Days	5–10 Days	10–15 Days	15–25 Days
Untreated	0.43 ± 0.04	-0.14 ± 0.01	0.57 ± 0.11	0.92 ± 0.93
HA	0.08 ± 0.02	0.50 ± 0.10	0.76 ± 0.28	1.00 ± 0.36
PSG	-0.12 ± 0.01	0.36 ± 0.06	0.88 ± 0.24	0.97 ± 1.00
PSG + hPBMC	0.22 ± 0.03	0.04 ± 0.0	0.95 ± 0.58	1.00 ± 0.61

Histological stainings were performed to assess the wound-healing processes and the structure of the restored tissues (Fig. 4). Five days after the operation, all the groups exhibited abundant inflammatory cells. Ten days later, wounds in the untreated and HA-treated groups (control groups) were discernable from adjacent tissues, and no distinguishable keratin layer was observed. In the PSG- and PSG + hPBMC-treated groups, new epidermal cells had migrated around the wound edge, and a keratin layer was distinctly noticeable. On days 15 and 25, H&E staining of cross sections of the healed wounds revealed that the newly formed epidermal layer displayed great similarity to surrounding epidermis with regard to organization and morphology in PSG- and PSG + hPBMC-treated wounds compared to the untreated and HA-treated wounds. Additionally, in the untreated and HA-treated groups, the migration rate was limited and could not bridge the entire wound surface by 25 days, showing a nonkeratinized center (Fig. 4).

Figure 4

Hematoxylin and eosin staining of skin biopsies showing wound healing progression in nude mice. Histology images of the progression in healing of a full-thickness cutaneous untreated wound or treated with hyaluronic acid (HA), PSG only, or PSG + hPBMCs. As seen on day 5 after the operation, animals in all groups exhibited abundant inflammatory cells. The wounds in the control groups (untreated and HA) were distinguishable from adjacent tissues even at day 25, and no clear dermis layer was observed, while in the PSG-only and PSG + hPBMC groups keratin + dermal layers were clearly observed. While the restored skin of these animals showed a similar structure to that of normal skin, the dermal layer in the control group was still incomplete. The arrows indicate the wound area in all pictures, and host skin is seen to the right of this region. Scale bars: 200 μm.

As increased, organized collagen deposition is associated with improved wound bed maturation, the degree of collagen deposition within the wound bed was assessed using MT staining. MT staining showed a markedly increased presence of collagen in the images from skin biopsies of PSG- and PSG + hPBMC-treated animals compared to controls (Fig. 5A–D). In order to confirm the histopathological analysis, quantification of collagen by densitometry was performed in the newly regenerated skin (day 25 after operation). We found that MT images in the groups treated with PSG only or with PSG + hPBMCs had higher collagen intensity (252,314 ± 70,005 and 245,011 ± 35,832 a.u., respectively) compared to the untreated group (127,001 ± 25,429 a.u.), although the difference was not statistically significant. The HA-treated group had 177,239 ± 31,097 a.u. density and was also not significantly different compared to the untreated group.

Figure 5

Collagen staining of wounds in untreated and PSG-treated nude mice. MT staining of the wounds in (A) untreated and (B) HA-treated animals showed weaker staining of collagen (blue), while animals treated with (C) PSG only and (D) PSG + hPBMCs showed stronger staining for collagen at day 25 of wound healing, although this was not statistically significant. Scale bars: 100 μm.

To determine whether human cells in PSG + hPBMCs survived after application in wounds, paraffin sections of partially healed wounds at day 5 and day 10 were immunostained with human-specific anti-mitochondrial antibodies. The results demonstrated absence of positively stained cells in the controls (Fig. 6A and B), but presence of human mitochondrial-positive cells in human liver tissue (positive control) (Fig. 6C) as well as in both dermal and epidermal layers of animals treated with PSG + hPBMCs (Fig. 6D–F). These results indicate that human cells may accelerate the reepithelialization of wounded skin by facilitating neovascularization.

Figure 6

Detection of human cells in the wounds of animals treated with PSG and hPBMCs. Presence of human cells in the epidermis and dermis of animals treated with PSG and hPBMCs was detected using a human-specific mitochondrial antibody. No positive cells were found in (A) the PSG-treated animals, and no background staining was observed in (B) the negative control. The specificity of the antibody is demonstrated in a positive control using a human liver tissue (C). (D, E) Presence of positively stained cells in the epidermis and dermis of skin biopsies (black arrows) taken from animals treated with PSG + hPBMCs demonstrating the presence of human cells on day 5 of wound healing. (F) Skin biopsies taken from the same animals showing the presence of human cells in new blood vessels formed during wound healing. Scale bars: 50 μm.

Human and Host Blood Vessel Formation in the Wounds

CD31 staining showed vascularization in each experimental group at 5 and 10 days after surgery (Fig. 7). As seen, large numbers of host (mouse) microblood vessels (green) were observed in the PSG + hPBMC-treated group on day 5 and day 10, and the microblood vessel density was much higher compared with the control untreated group on day 5 and day 10 (Fig. 7A, D and E, H, respectively). Importantly, when stained with human-specific anti-CD31 antibodies, several small blood vessels were positive (red) in the PSG + hPBMC-treated group at day 5 (Fig. 7B). However, the number of blood vessels positive for human CD31 decreased on day 10 (Fig. 7F) and was negligible on day 15 (data not shown). The control untreated group on days 5 and 10 was negative for human CD31 staining (Fig. 7D and F). This finding suggests that the human PBMCs participate in acceleration of wound healing by facilitating neovascularization.

Figure 7

Detection of human blood vessels in the wounds of animals treated with PSG and human blood cells. Distribution and density of newly formed blood vessels were assessed using immunofluorescence. (A, E) A mouse-specific CD31 antibody (green) for mouse endothelial cells was used to detect host blood vessels on days 5 and 10. (B, F) To determine whether human cells also contributed to neovascularization on days 5 and 10, we used a human-specific CD31 antibody (red). (C, G) Merged pictures of (A) and (B) and (E) and (F), respectively. Compared to (D, H) control untreated animals, markedly increased numbers of host blood vessels staining for mouse CD31 (green) and presence of human CD31 (red), respectively, were observed on day 5 in animals treated with PSG and human cells (top). On day 10, however, a lower number of human blood vessels was observed (bottom). Scale bars: 75 μm.

Detection of Human RNA in Newly Formed Skin

A total of 38 skin samples taken at days 5, 10, 15, and 25 were analyzed with qPCR for detection of human cells. Twenty-three of the samples were taken from mice treated with hPBMCs, and 15 samples were from mice not treated with human cells. The Cq values for the mouse qPCR assay were low in all cases ranging between 10 and 14, while the Cq values for the human assay were 20 cycles higher, indicating, as expected, a low number of human cells present in the mouse tissue. Human RNA could be identified in 20 out of the 23 samples treated with human cells, while no human RNA was identified in three of the samples. For the samples not treated with human cells, a positive qPCR result with Cq value in similar range was obtained in 4 of the 15 samples.

Discussion

The present study was conducted to offer an “off-to-the-shelf” scaffold-based gel for skin tissue engineering applications. A gel prepared from decellularized skin of Gal/gal KO pig was directly applied on excision wounds of nude mice in vivo with or without the addition of human cells. We found that the ECM gel significantly contributed to the WHRs within 15 days. The ECM gel interacted effectively and protected the wound in the nude mouse model, providing good adherence of cells and a moist healing environment. Interestingly, the addition of human cells to the gel prior to wound application markedly improved host blood vessel formation in the wounds and significantly accelerated the wound healing process. Importantly, in the same group of animals, we also found human blood vessels in the wounds at days 5 and 10 as evidenced by the presence of human CD31 and positive staining with the human-specific anti-mitochondrial antibody. Furthermore, qPCR detection also confirmed the presence of low numbers of human cells. The low number of human cells might be the reason for not finding human RNA in three mice treated with hPBMCs. However, it is important to state that identification of the few human-specific cells in a high background of other host (mouse) cells is difficult. The reason for the detection of human RNA in four mice samples not treated with hPBMCs may be a possible contamination while collecting and processing the samples, since even a minute amount of contamination could cause a positive signal in qPCR. Nevertheless, our results clearly indicate that the human cells survived and participated initially in neovascularization leading to accelerated wound healing.

Various cell-based therapies and tissue engineering strategies have been developed to provide the most adequate treatment options for patients suffering from chronic wounds or acute skin trauma (17,26). Biological products currently commercially available are mainly engineered using dermal fibroblasts such as Dermagraft® (15), Apligraft® (5), and ICX-SKN® (2). However, a major clinical demand is the use of an effective approach to rapidly cover wounds and shorten the skin substitute preparation time to avoid patient complications. Therefore, instead of a full 3D skin biomimetic model, we prepared an ECM gel using HA. HA was chosen based both on its importance as a main skin ECM component, with an active role in skin remodeling (23), and on angiogenesis (10). It has been used in wound dressings, skin substitute products, and other regenerative medicine applications (28). We hydrated the HA, which is commercially available as a powder, with keratinocyte medium supplemented with growth factors in order to further improve wound healing. Moreover, the gel was prepared from the hydration of off-the-shelf dried pig skin powder at the time of application, which opens up the possibility of a ready-to-use application. In addition, we found that the gel could be easily manipulated upon application on the wounds and to possess a high ability to shape adaptability, but more importantly, it presented attractive cell adhesive features that distinguish it from traditional skin substitutes.

To achieve durable wound closure, the formation of a functional vascular network is very important for the regeneration of wound beds (8). Recently, mesenchymal stem cell-based therapies have emerged as promising strategies to promote skin wound healing in light of the therapeutic effects associated with their important secretion of potent soluble mediators. In addition, a number of studies have demonstrated the capacity of adipose-derived regenerative cells to promote angiogenesis and modulate inflammation (16,25). In the course of translating a skin gel for extensive wound treatments in clinical application, sufficient autologous cells are needed. We were interested in finding a simple source of cells. To this end, we used autologous PBMCs as a viable strategy. Given the pro angiogenic and regenerative properties of hPBMCs (1,19), we hypothesized that loading the gel with these cells would overcome the delayed angiogenesis sometimes reported with several skin substitutes and thereby accelerating the formation of a mature, well-vascularized wound bed. Adding PBMCs to the gel appears to be both safe and feasible with no significant adverse systemic health effects. Our data suggest that seeding uncultured hPBMCs into this dermal gel enhances tissue integration by increasing blood vessel formation, maturation, and matrix remodeling. These results provide new insights for novel strategies in skin regeneration after thermal burns, consisting of the combination of PBMCs with engineered biomaterials. Utilizing uncultured population of cells may facilitate potential clinical application. Further development of this matrix scaffold gel engineered with PBMCs may lead to improved healing in future clinical trials. With successful demonstration of the skin gel in nude mice, further evaluation on the efficiency of such a gel in large animals and humans will be the next step toward clinical translation.

Collagen is one of the most important components of regenerated skin. In fact, increased, organized collagen deposition is associated with improved wound bed maturation (6). In our study, we observed an abundant collagen in the MT staining of animals treated with PSG as well as those treated with PSG + hPBMCs in comparison to the controls. This finding is not surprising since the decellularized pig skin had retained considerable amounts of the ECM proteins such as collagen and elastin, both of which are important components of the normal skin. It is therefore likely that the higher amounts of collagen in the PSG may promote rapid infiltration of host cells to the wound and improved wound stabilization.

Conclusions

In this study, pig skin-derived ECM gel composed of various ECM components was developed and tested as an efficient filler for wound treatment. Our findings suggest that this gel together with human peripheral blood cells can effectively promote the neovascularization and collagen deposition due to compositional properties, leading to improved wound healing.

Footnotes

Acknowledgments

This study was financed by the Swedish Government LUA ALF grant, The Inga Britt and Arne Lundbergs Foundation, and the Swedish Medical Council (K2013-65X-22347-01-3) to S.S.H. The authors also wish to thank Murali K. Akula for help with the quantification of collagen intensity. S.S.H. holds shares in NovaHep AB, a company developing tissue engineered blood vessels for diagnostic and therapeutic purposes. The other authors declare no conflicts of interest.

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