Promotion Effects of Ultra-High Molecular Weight Poly-γ-Glutamic Acid on Wound Healing

Abstract

We examined the in vivo efficacy of ultra-high molecular weight poly-γ-glutamic acid (UHMW γ-PGA) for wound healing. The wound area was measured by a ruler and documented by digital photography before the animals were sacrificed at days 8 and 16 post wounding. The areas of wounds treated with UHMW γ-PGA were significantly decreased on days 8 and 16, as compared with those receiving a control treatment, and more than 70% of the UHMW γ-PGAtreated area was repaired by day 8. Hematoxylin and eosin staining confirmed that the epidermis had regenerated in the UHMW γ-PGA-treated wounds. At 16 days post wounding, collagen pigmentation and cross-linking were increased as compared with the control groups, and greater regeneration of blood vessels had occurred in UHMW γ-PGA-treated groups. Increased levels of transforming growth factor-beta and β-catenin were also observed in skin samples collected from UHMW γ-PGA-treated animals on days 8 and 16 post incision. Taken together, these findings suggest that UHMW γ-PGA promotes wound healing in vivo.

Introduction

A wound is a breakdown in the protective function of the skin. Skin provides physical and chemical protection against invasion by toxins and microorganisms and plays an important role in the prevention of dehydration, which can result from a loss of barrier function [2]. Effective wound-care is important because it can accelerate wound repair, prevent microbial infection, and reduce transepidermal water loss [6]. Wound healing is a complex process involving multiple cellular events, including cell proliferation, migration, and tissue remodeling [20,22]. The characteristic features of wound healing, which include the proliferation and migration of keratinocytes and fibroblasts and matrix deposition, are regulated by multiple signaling mechanisms. These involve growth factors, such as transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), transforming growth factor- alpha (TGF-α), and keratinocyte growth factor (KGF); and cytokines, such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (INF-γ) [4,5].

Ultra-high molecular weight poly-γ-glutamic acid (UHMW γ-PGA) is a safe, edible biomaterial that is naturally synthesized by Bacillus subtilis subsp. Chungkookjang (KCTC 0697BP) and characterized as ultra-high molecular weight, being over 2,000 kDa [18]. Development of γ-PGA has been pursued for many applications, including cosmetics, skin care, bone care, nanoparticulate drug delivery systems, and hydrogels. More recently, UMHW γ-PGA has been produced industrially and used for new functional applications in foods, cosmetics, and pharmaceuticals [10,13,18]. Previous studies showed that UHMW γ-PGA successfully stimulated corneal wound healing by inducing an inflammatory effect, enhancing cell migration and cell proliferation [3]. In the present study, we investigated the effects of UHMW γ-PGA on skin wound healing.

 

Materials and Methods

Preparation of UHMW γ-PGA

Bacillus subtilis subsp. Chungkookjang (KCTC 0697BP) was inoculated into 30 L of preparative Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) and incubated at 37℃ for 8 h. The resulting seed culture was transferred to a 500 L jar fermenter containing 300 L of medium composed of 5% L -glutamic acid, 5% glucose, 1% (NH4)2SO4, 0.27% KH2PO4, 0.42% Na2HPO4·12H2O, 0.05% NaCl, 0.3% MgSO4·7H2O, and 1 ml of vitamin solution/L; pH 6.8), and incubated at 37℃ for 72 h. The cells were then separated from the culture broth by filtration. The supernatant was precipitated after adding 5 N HCl for 12 h. The resulting precipitated UHMW γ-PGA was freeze-dried to obtain pure UHMW γ-PGA.

The molecular weight was measured by gel permeation chromatography, using a GMPWXL column (7.8 mm × 30 cm; Viscotek, USA) and a LR125 Laser Refractometer (Viscotek); polyacrylamide was used as a standard material. The molecular weight of the UHMW γ-PGA in the present study was 2,000-15,000 kDa.

Experimental Design

Ten-week-old male Sprague-Dawley rats were kept in cages in a temperature-controlled environment with a 12-h light-dark cycle. The animals were randomized equally into control and experimental groups. All rats were fed on a basal diet and allowed to drink distilled water. The animals were anesthetized by intramuscular injection. After anesthesia, the dorsal hair was carefully shaved. Four sites of full-thickness skin wounds above the fascia on the dorsal side of the rats were surgically created using a biopsy punch with a 6 mm diameter. The incision was not sutured. The wounds were treated daily with phosphate-buffered saline (PBS) alone or with 0.25% UHMW γ-PGA in PBS. Wound closure was measured by a ruler and documented by digital photography before the animals were sacrificed on days 8 or 16 post wounding.

Histological Studies

One part of the skin around the wound area was stained using hematoxylin and eosin (H&E) in accordance with the method described by Adam et al. [1]. The other part was lysed and used for western blot analysis. The fixed tissues were dehydrated in alcohol, cleared in xylene, infiltrated, and embedded in paraffin. Paraffin blocks of each sample were then cut into 5 µm sections and stained with H&E. Each section was visualized using a microscope equipped with a digital camera at 40× magnification. Van Gieson’s staining method was used to identify type 1 collagen fibers (red color) and to score collagen fiber deposition and epithelialization [8].

Western Blot Analysis

The other part of the wound tissue samples collected on days 8 and 16 post incision was homogenized in lysis buffer and incubated on ice for 3 0 min. The lysates were centrifuged at 1,500 rpm for 20 min at 4℃ prior to western blot analysis [17]. The samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Millipore Corp., USA). The membrane was blocked for 1 h in PBS containing 5% casein. The membrane was then incubated overnight with a primary antibody raised against TGF-β (Abcam, USA) and then for 1 h with a secondary antibody (alkaline phosphataseconjugated goat anti-rabbit IgG, 1:5,000; Abcam).

Immunohistochemical Analysis

The paraffin-embedded samples described above were incubated with anti-KGF (Abcam, UK). After washing in PBS, the slides were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, USA), followed by a streptavidin-alkaline phosphatase conjugate (Zymed Laboratories, USA). The Sigma Fast substrate reaction mixture (Sigma Immunochemicals, USA) was then added. After counterstaining with H&E, the localization of KGF was examined under a light microscope (400× magnification) [14].

 

Results

Effect of UHMW γ-PGA on Skin Wound Healing

In the present study, the wound healing effect of UHMW γ-PGA purified in accordance with the purification method using acid precipitation was examined [18]. As shown in Fig. 1, the sizes of wounds treated with UHMW γ-PGA were significantly decreased on days 8 and 16, as compared with those receiving the control treatment, and more than 70% of the area treated with UHMW γ-PGA was repaired on day 8. Acceleration of wound healing was obvious in wounds treated with UHMW γ-PGA, especially during the early phase of the healing process.

Fig. 1.Macroscopic appearance of punch wounds on the control (con.) and ultra-high molecular weight poly-γglutamic acid (UHMW γ-PGA)-treated rats at the indicated time-points post wounding.

Histological Results

The results of the histological investigation of the wounds on days 8 and 16 post wounding are shown in Fig. 2. H&E staining showed that the epithelium at the edges of all wounds migrated toward the center. At 8 days post wounding, the epidermis had regenerated in the wounds of the rats treated with UHMW γ-PGA. The extent of reepithelialization of wounds treated with UHMW γ-PGA was higher than that observed in the control group. In addition, the length of the wound was significantly decreased by treatment with UHMW γ-PGA, as compared with control. At 16 days post wounding, greater pigmentation of collagen, cross-linking of collagen, regeneration of blood vessels, and formation of hair follicles were observed in the wounds of animals treated with UHMW γ-PGA, as compared with the control group. Collagen fiber pigmentation and cross-linking were analyzed using Van Gieson’s staining (Fig. 3).

Fig. 2.Hematoxylin and eosin (H&E) staining of the control (con.) and ultra-high molecular weight poly-γ-glutamic acid (UHMW γ-PGA)-treated wounds at the indicated time-points post wounding. C, collagen; Der, dermis; Epi, epidermis; F, fibroblast; HF, hair follicle; NS, normal skin; WS, wound skin; arrow, wound edge. Original magnification, 40×.

Fig. 3.Photomicrographs showing a marked increase in granulation tissue and collagen content in the control (con.) and ultrahigh molecular weight poly-γ-glutamic acid (UHMW γ-PGA)-treated group at the indicated time-points post wounding. The red color indicates collagen fibers (40× magnification).

Identification of KGF using Immunohistochemical Staining

At 8 days post wounding, the dermal expression of KGF in the UHMW γ-PGA-treated group was increased, as compared with the control group, as shown in Fig. 4. However, KGF expression showed no significant increase in the UHMW γ-PGA-treated group at 16 days post wounding, as compared with the control group.

Fig. 4.Immunohistochemical analysis of keratinocyte growth factor (KGF) levels in the control (con.) and ultra-high molecular weight poly-γ-glutamic acid (UHMW γ-PGA)-treated wounds at the indicated time-points post wounding. The brown color indicates keratinocyte growth factor.

Expression of TGF-β and β-catenin in Wounded Tissue

Western blot analysis of wound tissue sections from the UHMW γ-PGA-treated group revealed a higher level of TGF-β and β-catenin proteins on days 8 and 16 post incision (Fig. 5). The expression of β-catenin was particularly elevated on day 16, as compared with 8 days post wounding.

Fig. 5.Expression of TGF-β and β-catenin in wounded tissue.

 

Discussion

An altered inflammatory response, which can include collagen synthesis, delayed angiogenesis, and slower reepithelialization, are observed during the wound healing process [9,11]. Wound healing is a complex process involving the integration of a variety of tissue and cell types. Re-epithelialization, an important process during the early phase of wound healing, involves the migration of keratinocytes from the edges of the wound [12]. Rapid re-epithelialization is crucial because it provides an appropriate environment for wound healing, which includes the regeneration of cells and increased levels of growth factors; these are indispensable for the wound healing process [16].

Many studies have identified a correlation between wound healing and stimulation of angiogenesis [23]. In this study, we observed an increased KGF level on day 8 post incision in the UHMW γ-PGA-treated rats. KGF is a growth factor that induces angiogenesis and is present in the epithelialization phase of wound healing.

TGF-β is a member of a family of growth factors involved in a number of essential cellular functions. TGF-β is involved in a number of processes during wound healing, including inflammation, stimulation of angiogenesis, fibroblast proliferation, collagen synthesis and deposition, and remodeling of the new extracellular matrix [7,15]. Interestingly, chronic (non-healing) wounds often show a loss of TGF-β1 signaling [19]. β-Catenin plays a crucial role as a mediator in the canonical Wnt signaling pathway. βCatenin protein levels are elevated during the proliferative phase of wound healing. β-Catenin stabilization increases the proliferation rate, motility, and invasiveness of fibroblasts [21]. The increased expression of TGF-β and β-catenin observed in the experimental group on days 8 and 16 post incision may be crucial for the positive effects of UHMW γPGA on wound healing.

The properties of UHMW γ-PGA will be studied more thoroughly in our future investigations, particularly with respect to the molecular mechanisms involved in the regulation of wound angiogenesis and the interactions between the extracellular matrix and angiogenesis during wound healing.