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. 2016 May;22(9-10):754-64.
doi: 10.1089/ten.TEA.2015.0536.

Cellularized Bilayer Pullulan-Gelatin Hydrogel for Skin Regeneration

Mathew N Nicholas  1 , Marc G Jeschke  1 , Saeid Amini-Nik  1
Affiliations

Cellularized Bilayer Pullulan-Gelatin Hydrogel for Skin Regeneration

Mathew N Nicholas et al. Tissue Eng Part A. 2016 May.

Abstract

Skin substitutes significantly reduce the morbidity and mortality of patients with burn injuries and chronic wounds. However, current skin substitutes have disadvantages related to high costs and inadequate skin regeneration due to highly inflammatory wounds. Thus, new skin substitutes are needed. By combining two polymers, pullulan, an inexpensive polysaccharide with antioxidant properties, and gelatin, a derivative of collagen with high water absorbency, we created a novel inexpensive hydrogel-named PG-1 for "pullulan-gelatin first generation hydrogel"-suitable for skin substitutes. After incorporating human fibroblasts and keratinocytes onto PG-1 using centrifugation over 5 days, we created a cellularized bilayer skin substitute. Cellularized PG-1 was compared to acellular PG-1 and no hydrogel (control) in vivo in a mouse excisional skin biopsy model using newly developed dome inserts to house the skin substitutes and prevent mouse skin contraction during wound healing. PG-1 had an average pore size of 61.69 μm with an ideal elastic modulus, swelling behavior, and biodegradability for use as a hydrogel for skin substitutes. Excellent skin cell viability, proliferation, differentiation, and morphology were visualized through live/dead assays, 5-bromo-2'-deoxyuridine proliferation assays, and confocal microscopy. Trichrome and immunohistochemical staining of excisional wounds treated with the cellularized skin substitute revealed thicker newly formed skin with a higher proportion of actively proliferating cells and incorporation of human cells compared to acellular PG-1 or control. Excisional wounds treated with acellular or cellularized hydrogels showed significantly less macrophage infiltration and increased angiogenesis 14 days post skin biopsy compared to control. These results show that PG-1 has ideal mechanical characteristics and allows ideal cellular characteristics. In vivo evidence suggests that cellularized PG-1 promotes skin regeneration and may help promote wound healing in highly inflammatory wounds, such as burns and chronic wounds.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Surgical model using bilayer cellularized PG-1. Schematic drawing (A) and representative image (B) of mouse model where two 6 mm diameter mouse skin punch biopsies were created on the back of immunosuppressed mice and skin substitutes were held in place on the wound using newly developed plastic domes. Animal wounds were randomly divided into three groups: control (no hydrogel), acellular hydrogel (hydrogel without cells), and cellularized hydrogel (bilayer hydrogel). The figure above shows the dome held in place using tissue glue (left) and nylon suture (right). In this experiment, domes were only secured with tissue glue. Color images available online at www.liebertpub.com/tea
<b>FIG. 2.</b>
FIG. 2.
Mechanical characteristics of PG-1. (A) Scanning electron microscopic images of pullulan-gelatin hydrogel at 27× magnification (left panel) and 75× magnification (center panel). Images at 75× magnification were used to quantify pore sizes. Pore size distribution was plotted (right panel). Average pore size was 61.69 ± 2.76 μm (mean ± SEM) with a range of pore sizes from 20 to 200 μm. (B) The mass of PG-1 over time when submerged in pullulanase microbial is shown. Hydrogels could no longer be placed on a scale after 20 min due to significant degradation. (C) Image shows increase in hydrogel size after being incubated in PBS. Swelling percentage of pullulan-gelatin hydrogel ranged from 1812% ± 25.46%, 1758% ± 31.74%, and 1801% ± 43.49% (mean ± SEM) when incubated in PBS, fibroblast media, and keratinocyte media respectively. (D) Sample stress-strain curve for PG-1 is shown. Elastic modulus of the pullulan-gelatin material was 22.43 ± 4.54 kPa (mean ± SEM). PBS, phosphate-buffered saline; SEM, standard error of the mean. Color images available online at www.liebertpub.com/tea
<b>FIG. 3.</b>
FIG. 3.
Cellular characteristics of cellularized PG-1. Images of epidermal component (keratinocytes) and dermal component (fibroblasts) of skin substitute are shown on the left side and right side respectively. Confocal microscopic images (20× magnification) of (A) top and (F) bottom of cellularized hydrogels (green—phalloidin, red—K14, blue—DAPI). Images taken 5 days post fibroblast seed and 3 days post keratinocyte seed show clusters of keratinocytes on the top surface of the hydrogel while elongated fibroblasts without keratinocytes are seen on the bottom of the hydrogel. Scale bar represents 50 μm. Live/dead viability (green—calcein AM [live cells], red—ethidium homodimer-1 [dead cells]) of (B) keratinocytes and (G) fibroblasts show 92.46% ± 2.26% (mean ± SEM) viable keratinocytes 3 days post seed and 97.74% ± 1.17% (mean ± SEM) viable fibroblasts 5 days post seed. Arrows indicate live cells while arrowheads indicate dead cells. Images taken at 20× magnification and scale bar represents 50 μm. BrdU proliferation assay (green—BrdU, blue—DAPI) of (C) keratinocytes and (H) fibroblasts show 18.27% ± 3.45% (mean ± SEM) BrdU-positive keratinocyte and 3.86% ± 1.42% BrdU-positive fibroblasts. Arrows indicate BrdU-positive cells while arrowheads indicate BrdU-negative cells. Images taken at 20× magnification and scale bars represent 50 μm. (D) Confocal microscopic image of different keratin expression of keratinocytes adhered to pullulan-gelatin hydrogel (green—phallodin, red—K10, blue—DAPI). Arrows indicate K14-positive keratinocytes while arrowheads indicate K10-positive keratinocytes. Images taken at 20× magnification and scale bars represent 50 μm. (E) Confocal microscopic image shows tight clustering of keratinocytes with adherens junction formation (green–phalloidin, red–e-cadherin, blue–DAPI). Image taken at 63× magnification and scale bar represents 20 μm. (I) Schematic showing total thickness of hydrogel and areas from which epidermal and dermal component images were taken. BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/tea
<b>FIG. 4.</b>
FIG. 4.
Cellularized PG-1 significantly increased new skin formation 14 days post punch biopsy compared to acellular hydrogels and control. Representative trichrome-stained images (10× magnification) show new dermal formation 14 days post punch biopsy. Capped bars on images represent dermis thickness (wound depth) in (A) control, (B) acellular hydrogels, and (C) cellular hydrogels. Scale bars represent 200 μm. (D) Wound depth was 123.77 ± 21.37 μm, 115.63 ± 9.83 μm, and 204.00 ± 19.65 μm, (mean ± SEM) for control, acellular hydrogels, and cellular hydrogels respectively (*p < 0.05). Color images available online at www.liebertpub.com/tea
<b>FIG. 5.</b>
FIG. 5.
Human cells were incorporated into newly formed murine dermis 14 days post punch biopsy. Immunohistochemistry staining of HLA showed 4.02% ± 0.78% (mean ± SEM) of skin cells in the murine dermis were HLA-positive cells. (B) 40× magnification image of rectangle outlined in (A) 10× magnification image. Arrow indicates HLA-positive cell. Scale bars represent (A) 200 μm and (B) 50 μm. HLA, human leukocyte antigen. Color images available online at www.liebertpub.com/tea
<b>FIG. 6.</b>
FIG. 6.
Cellularized PG-1 increased skin cell proliferation, suppressed excessive immune response, and increased neovascularization. Representative images of immunohistochemistry stained images for (A–C) PCNA-positive cells, (E–G) F4/80-positive cells, and (I–K) ASM-positive cells. Arrows indicate cells positive for their respective stains. Scale bars represent 50 μm (40× magnification) for PCNA and F4/80 images and 100 μm (20× magnification) for ASM images. (D) Percentage of PCNA-positive cells were 9.93% ± 1.35%, 7.97% ± 2.54%, and 19.03% ± 2.40% (mean ± SEM) for control, acellular hydrogels, and cellular hydrogels respectively. (H) Percentage of F4/80-positive cells were 27.94% ± 4.19%, 1.22% ± 0.19%, and 2.84% ± 0.36% (mean ± SEM) for control, acellular hydrogels, and cellular hydrogels respectively. (L) Relative proportion of increased number of capillaries found in newly formed dermis was 5.81 ± 0.65 and 2.61 ± 0.34 for cellular hydrogels and acellular hydrogels respectively compared to control (1 ± 0.22) (mean ± SEM) (*p < 0.05, ***p < 0.001). ASM, alpha-smooth muscle antibody; PCNA, proliferating cell nuclear antigen. Color images available online at www.liebertpub.com/tea
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