Release Of Epidermal Growth Factor Research Articles

Effects of epidermal growth factor-loaded mucoadhesive films on wounded oral tissue rafts

Current treatments for traumatic oral mucosal wounds include the gold standard of autologous tissue and alternative tissue-engineered grafts. While use of autografts has disadvantages of minimal availability of oral keratinized tissue, second surgery, and donor site discomfort, tissue-engineered grafts are limited by their unavailability as off-the-shelf products owing to their fabrication time of 4–8 weeks. Hence, the current work aimed to develop a potentially cost-effective, readily available device capable of enhancing native mucosal regeneration. Considering the key role of epidermal growth factor (EGF) in promoting mucosal wound regeneration and the advantages of mucoadhesive delivery systems, mucoadhesive films composed of polyvinylpyrrolidone and carboxymethylcellulose were developed to provide sustained release of EGF for a minimum of 6 h. Bioactivity of released EGF supernatants was then confirmed by its ability to promote proliferation of BALB/3T3 fibroblasts. Efficacy of the developed system was then investigated in vitro using buccal tissues (ORL 300-FT) as a potential replacement for small animal studies. Although the mucoadhesive films achieved their desired role of delivering bioactive EGF in a sustained manner, treatment with EGF, irrespective of its release from the films or solubilized in medium, caused a hyperparakeratotic response from in vitro tissues with distinguishable histological features including thickening of the spinous layer, intra- and intercellular edema, and pyknotic nuclei. These significant morphological changes were associated with no improvements in wound closure. These observations raise questions about the potential of using in vitro tissues as a wound healing model and substitute for small animal studies. The mucoadhesive delivery system developed, however, with its potential for sustained release of bioactive growth factors and small molecules, may be loaded with other desired compounds, with or without EGF, to accelerate the process of wound healing.

Decellularized scaffolds containing hyaluronic acid and EGF for promoting the recovery of skin wounds.

There is no effective therapy for the treatment of deep and large area skin wounds. Decellularized scaffolds can be prepared from animal tissues and represent a promising biomaterial for investigation in tissue regeneration studies. In this study, MTT assay showed that epidermal growth factor (EGF) increased NIH3T3 cell proliferation in a bell-shaped dose response, and the maximum cell proliferation was achieved at a concentration of 25ng/ml. Decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments. Hyaluronic acid (HA) increased EGF adsorption to the scaffolds. Decellularized scaffolds containing HA sustained the release of EGF compared to no HA. Rabbits contain relatively large skin surface and are less expensive and easy to be taken care, so that a rabbit wound healing model was use in this study. Four full-thickness skin wounds were created in each rabbit for evaluation of the effects of the scaffolds on the skin regeneration. Wounds covered with scaffolds containing either 1 or 3μg/ml EGF were significantly smaller than with vaseline oil gauzes or with scaffolds alone, and the wounds covered with scaffolds containing 1μg/ml EGF recovered best among all four wounds. Hematoxylin-Eosin staining confirmed these results by demonstrating that significantly thicker dermis layers were also observed in the wounds covered by the decellularized scaffolds containing HA and either 1 or 3μg/ml EGF than with vaseline oil gauzes or with scaffolds alone. In addition, the scaffolds containing HA and 1μg/ml EGF gave thicker dermis layers than HA and 3μg/ml EGF and showed the regeneration of skin appendages on day 28 post-transplantation. These results demonstrated that decellularized scaffolds containing HA and EGF could provide a promising way for the treatment of human skin injuries.

Lysozyme-Triggered Epidermal Growth Factor Release from Bacterial Cellulose Membranes Controlled by Smart Nanostructured Films

A novel wound-dressing biodevice, sensitive to lysozyme, an enzyme commonly found at infected skin wounds, was assembled by the layer-by-layer deposition of nanopolymeric chitosan and alginate films onto oxidized bacterial cellulose membranes incorporated with epidermal growth factor (EGF). Distinct EGF release profiles were obtained according to specific stimuli caused by infection. In in vitro conditions simulating noninfected wounds, the EGF rate and burst release effect were reduced by three deposited layers (Mt /M∞ of 0.25 at 3 h) in a process dependent on the porosity of the compact chitosan-alginate complex. The importance of the organized structure was revealed when an infected wound was simulated by adding lysozyme to the release medium, thus inducing the formation of a loosely polyelectrolyte architecture that caused rapid EGF diffusion (Mt /M∞ of 0.75 at 30 min). The results indicate that the nanopolymeric layers were capable of slowly releasing EGF as required for normal wound repair and rapidly undergoing architectural transitions that allow the diffusion of massive amounts of drug to enhance the process of re-epithelialization. In summary, the proposed system comprises the roles of both wound dressing and local delivery mechanism to recognize infections and respond with a burst of EGF release.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor

Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin–Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing.

Layer-by-layer assembly of epidermal growth factors on polyurethane films for wound closure

To facilitate the healing of chronic wounds, growth factors such as epidermal growth factor need to be safely encapsulated for their sustained and effective delivery to the wound bed. Using a layer-by-layer assembly technique, epidermal growth factor is successfully encapsulated on the surface of poly(acrylic acid)-modified polyurethane film. The amount of encapsulated epidermal growth factor is controlled by adjusting the number of chitosan/epidermal growth factor bilayers. A controlled release of epidermal growth factor from the surface of polyurethane film for a period of five days is achieved with well-retained bioactivity (over 90%) as evidenced by a cell proliferation assay. In an invitro cellular wounding assay, the cell gap covered with the epidermal growth factor-loaded polyurethane film closes at a rate more than twice as fast as that covered with a control polyurethane film. Fluorescent staining of F-actin reveals that the released epidermal growth factor induces differences in cytoskeletal organization, suggesting that stimulated cell migration also contributes to the close of the cell gap.

Influence of Terminal Differentiation and PACAP on the Cytokine, Chemokine, and Growth Factor Secretion of Mammary Epithelial Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with trophic and cytoprotective effects, has been shown to affect cell survival, proliferation, and also differentiation of various cell types. The high PACAP level in the milk and its changes during lactation suggest a possible effect of PACAP on the differentiation of mammary epithelial cells. Mammary cell differentiation is regulated by hormones, growth factors, cytokines/chemokines, and angiogenic proteins. In this study, differentiation was hormonally induced by lactogenic hormones in confluent cultures of HC11 mouse mammary epithelial cells. We investigated the effect of PACAP on mammary cell differentiation as well as release of cytokines, chemokines, and growth factors. Differentiation was assessed by expression analysis of the milk protein β-casein. Differentiation significantly decreased the secretion of interferon gammainduced protein (IP)-10, "regulated upon activation normal T cell expressed and presumably secreted" (RANTES), insulin-like growth factor-binding protein (IGFBP)-3 and the epidermal growth factor receptor (EGFR) ligands, such as epidermal growth factor (EGF) and amphiregulin (AREG). The changes in the levels of IP-10 and RANTES may be relevant for the alterations in homing of T cells and B cells at different stages of mammary gland development, while the changes of the EGFR ligands may facilitate the switch from proliferative to lactating stage. PACAP did not modulate the expression of β-casein or the activity of hormone-induced pathways as determined by the analysis of phosphorylation of Akt, STAT5, and p38 MAPK. However, PACAP decreased the release of EGF and AREG from non-differentiated cells. This may influence the extracellular signal-related transactivation of EGFR in the non-differentiated mammary epithelium and is considered to have an impact on the modulation of oncogenic EGFR signaling in breast cancer.

Regeneration effect of visible light‐curing furfuryl alginate compound by release of epidermal growth factor for wound healing application

ABSTRACTA visible light‐curable polysaccharide conjugate was prepared and was included with epidermal growth factor (EGF) for wound healing application. Alginate was modified with furfurylamine, and the furfuryl alginate (F‐alginate) was mixed with Rose Bengal for visible light reactivity. The introduction of furfuryl group was confirmed by 1H‐NMR and UV/vis spectroscopy. Visible light‐induced crosslinking was evaluated by micropatterning and weight measurement. Swelling ratio was also determined. To verify its applicability in wound healing applications, EGF was included in the system of F‐alginate and Rose Bengal. After the confirmation of no cytotoxicity of F‐alginate and murine EGF release from the system, cell proliferation, antibacterial activity, and animal test were performed on the system. In conclusion, it was demonstrated that the system was useful for wound healing. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40113.

The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

Wiskott-Aldrich Syndrome Protein Regulates Leukocyte-Dependent Breast Cancer Metastasis

A paracrine interaction between epidermal growth factor (EGF)-secreting tumor-associated macrophages (TAMs) and colony-stimulating factor 1 (CSF-1)-secreting breast carcinoma cells promotes invasion and metastasis. Here, we show that mice deficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable to support TAM-dependent carcinoma cell invasion and metastasis in both orthotopic and transgenic models of mammary tumorigenesis. Motility and invasion defects of tumor cells were recapitulated ex vivo upon coculture with WASp(-/-) macrophages. Mechanistically, WASp is required for macrophages to migrate toward CSF-1-producing carcinoma cells, as well as for the release of EGF through metalloprotease-dependent shedding of EGF from the cell surface of macrophages. Our findings suggest that WASp acts to support both the migration of TAMs and the production of EGF, which in concert promote breast tumor metastasis.

Effect of Contact Lens Wear on the Release of Tear Mediators in Keratoconus

The release of different cytokines and mediators in tears of patients with keratoconus (KC) wearing contact lenses (CLs) may contribute to the pathology of KC. Cohort study was established in patients with KC wearing rigid gas permeable (RGP) CL (group I), patients with ametropia wearing silicone hydrogel (Si-Hi) CL (group II) and ametropic patients wearing RGP CL (group III). Our findings indicate that before CL wear, the release of epidermal growth factor (EGF) and tissue-type plasminogen activator (t-PA) was attenuated, whereas matrix metalloproteinase (MMP)-9, interleukin (IL)-6, chemokine (C-C motif) ligand 5 (CCL5), IL-13, and plasminogen activator inhibitor (PAI)-1 were enhanced in KC compared with ametropes. An increasing linear trend over time was found for MMP-9, EGF, and CXCL8 in KC and MMP-9, MMP-13, IL-6, and CXCL8 in group III. Significant differences were observed in the linear trend over time between groups I and III for MMP-13 and tissue inhibitor of metalloproteinases (TIMP)-1; between groups I and II for MMP-9 and CXCL8; and between groups III and II for MMP-9, CXCL8, and MMP-13. In KC, the release of MMP-9 at week 6 and nerve growth factor (NGF) at 10 min was higher, but NGF at week 2 was lower than that in group II. The release of MMP-13 and NGF at week 2 and 6 were lower in the KC group as compared with group III, and similarly, with IL-6 and CXCL8 at week 2 and PAI at all time points. Contact lens wear can influence the levels and dynamics of various mediators in the tears of patients with KC that might have an impact on the progression of the disease.

GnRH-(1–5) Transactivates EGFR in Ishikawa Human Endometrial Cells via an Orphan G Protein-Coupled Receptor

The decapeptide GnRH is known for its central role in the regulation of the hypothalamo-pituitary-gonadal axis. In addition, it is also known to have local effects within peripheral tissues. The zinc metalloendopeptidase, EC 3.4.24.15 (EP24.15), can cleave GnRH at the Tyr(5)-Gly(6) bond to form the pentapeptide, GnRH-(1-5). The central and peripheral effect of GnRH-(1-5) is different from its parent peptide, GnRH. In the current study, we examined the effect of GnRH-(1-5) on epidermal growth factor receptor (EGFR) phosphorylation and cellular migration. Using the Ishikawa cell line as a model of endometrial cancer, we demonstrate that GnRH-(1-5) stimulates epidermal growth factor release, increases the phosphorylation of EGFR (P < .05) at three tyrosine sites (992, 1045, 1068), and promotes cellular migration. In addition, we also demonstrate that these actions of GnRH-(1-5) are mediated by the orphan G protein-coupled receptor 101 (GPR101). Down-regulation of GPR101 expression blocked the GnRH-(1-5)-mediated release of epidermal growth factor and the subsequent phosphorylation of EGFR and cellular migration. These results suggest that GPR101 is a critical requirement for GnRH-(1-5) transactivation of EGFR in Ishikawa cells.

Estradiol Protects Dermal Hyaluronan/Versican Matrix during Photoaging by Release of Epidermal Growth Factor from Keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.

Controlled Release of Epidermal Growth Factor from Hydrogels Accelerates Wound Healing in Diabetic Rats

We sought to develop new recombinant human epidermal growth factor (rhEGF)-containing hydrogels and to investigate their biological activity and therapeutic effects on wound healing in diabetic rats. Levels of rhEGF released from hydrogels were measured by enzyme-linked immunosorbent assay. The cellular proliferating activity of released rhEGF was evaluated by MTT assay. Fifty-six wounded diabetic rats were randomly divided into four groups with different topical treatment daily. The therapeutic effects were evaluated by wound area measurement, histologic analysis, immunohistochemical assessment of proliferating cell nuclear antigen and B-cell lymphoma/leukemia-2, and Western blotting of EGF receptor. The rhEGF released from the hydrogel matrix kept its bioactivity on stimulating proliferation of the BALB/c3T3 cell line. Wound closure rates on postoperative day 14 were 75.8% in the negative control group, 82.83% in the group treated with hydrogel matrix, 85.87% in the group treated with rhEGF-containing hydrogel, and 81.18% in the group treated with rhEGF solution. Compared with hydrogel matrix, rhEGF-containing hydrogel had an additional effect on induction of EGF receptor expression (P < .05). Compared with negative controls, protein expression of B-cell lymphoma/leukemia-2 was higher in the rhEGF-containing groups (P < .05). Proliferating cell nuclear antigen was induced at its highest level on day 7 in the rhEGF-containing hydrogel-treated group (P < .05). These data from in vitro release and diabetic animal models highlight the efficacy of hydrogels as a controlled releasing system for topical application of EGFs. The rhEGF-containing hydrogel we developed holds the merits of prolonged and sustained releasing of bioactive rhEGF and therapeutic potential in enhancing diabetic wound healing.

Dopamine stimulates epidermal growth factor release from adult neural precursor cells derived from the subventricular zone by a disintegrin and metalloprotease

Dopamine plays a key role in the regulation of stem cell turnover and neurogenesis in the subventricular zone. This effect is mediated by dopamine-induced release of epidermal growth factor (EGF), to promote stem cell proliferation in this area. We, therefore, sought to investigate whether a disintegrin and metalloprotease (ADAMs) are implicated in this process, as they have previously been shown to play a role in transactivation of the EGF receptor after stimulation of G protein-coupled receptors. We found that dopamine stimulation of stem cells caused the release of EGF, in agreement with our previous findings. However, the inhibition of ADAMs reversed this effect. Our results support a role for ADAMs in dopamine-induced release of EGF from stem cells in the subventricular zone.

Dopamine‐dependent ectodomain shedding and release of epidermal growth factor in developing striatum: target‐derived neurotrophic signaling (Part 2)

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D(1) -like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D(2) -like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D(1) -like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.

Contact lenses as a drug delivery device for epidermal growth factor in the treatment of ocular wounds

Background: This work was conducted to investigate the uptake and release of epidermal growth factor (EGF) from hydrogel contact lenses and to determine whether the released protein would be therapeutically active in a rabbit corneal epithelial defect model of ocular trauma, prior to use in humans.Methods: The uptake and release of EGF from hydrogel contact lens materials were determined by high‐pressure liquid chromatography. Contact lenses composed of vasurfilcon A or lotrafilcon A (containing silicone) were incubated in a source solution containing 0.4-ppm EGF for seven hours. To determine the kinetics of drug uptake into the contact lens matrix, drug concentration in the source solution was measured at zero, one, 60, 240 and 420 minutes. To determine the kinetics of release, loaded contact lenses were immersed in a recipient solution of phosphate‐buffered saline. Therapeutic activity in vivo was investigated by placing prepared lenses on the surface of abraded corneas of New Zealand White rabbits, with abraded corneas of contralateral eyes used as controls. Control eyes were treated with contact lenses placed in saline for injection. Wound closure was assessed hourly.Results: Uptake and release of EGF were demonstrated for vasurfilcon A but not lotrafilcon A contact lens materials. The retention time of EGF released from vasurfilcon A contact lenses was similar to control EGF not exposed to contact lens polymers. The greatest adsorption of EGF into the lens material occurred within approximately 120 minutes, with a flattening of the rate of uptake thereafter. Abraded eyes in rabbits showed a significantly higher overall healing rate for EGF‐treated contact lenses compared with control eyes (p < 0.0001).Conclusions: EGF can be delivered from some but not all hydrogel materials. Lens materials composed of silicone may not be useful for delivering EGF to the eye. EGF‐treated contact lenses may be a useful device to facilitate healing of ocular wounds.

Development of a Wound Dressing Composed of Hyaluronic Acid Sponge Containing Arginine and Epidermal Growth Factor

Hyaluronic acid (HA) has the ability to promote wound healing. Epidermal growth factor (EGF) is able to promote the proliferation of various cell types, in addition to epidermal cells. A novel wound dressing was designed using high-molecular-weight hyaluronic acid (HMW-HA) and low-molecular-weight hyaluronic acid (LMW-HA). Spongy sheets composed of cross-linked high-molecular-weight hyaluronic acid (c-HMW-HA) were prepared by freeze-drying an aqueous solution of HMW-HA containing a crosslinking agent. Each spongy sheet was immersed into an aqueous solution of LMW-HA containing arginine (Arg) alone or both Arg and epidermal growth factor (EGF), and were then freeze-dried to prepare two types of product. One was a wound dressing composed of c-HMW-HA sponge containing LMW-HA and Arg (c-HMW-HA/LMW-HA + Arg; Group I). The other was a wound dressing composed of c-HMW-HA sponge containing LMW-HA, Arg and EGF (c-HMW-HA/LMW-HA + Arg + EGF; Group II). The efficacy of these products was evaluated in animal tests using rats. In the first experiment, each wound dressing was applied to a full-thickness skin defect with a diameter of 35 mm in the abdominal region of Sprague–Dawley (SD) rats, leaving an intact skin island measuring 15 mm in diameter in the central area of this skin defect. Commercially available polyurethane film dressing was then applied to each wound dressing as a covering material. In the control group, the wound surface was covered with polyurethane film dressing alone. Both wound dressings (Group I and Group II) potently decreased the size of the full-thickness skin defect and increased the size of the intact skin island, when compared with the control group. The wound dressing in Group II showed particularly potent activity in increasing the distance of epithelization from the intact skin island. This suggests that EGF release from the spongy sheet serves to promote epithelization. The wound dressing in Group II enhanced early-stage inflammation after 1 week, as compared with the other two groups. In the second experiment, each wound dressing was applied to a full-thickness skin defect measuring 35 mm in diameter in the abdominal region of SD rats, after removing necrotic skin caused by dermal burns. Polyurethane film dressing was applied to each wound dressing as a covering material. In the control group, the wound surface was covered with polyurethane film dressing alone. Both wound dressings (Group I and Group II) potently decreased the size of the full-thickness skin defect and increased epithelization from the wound margin, as compared with the control group. The wound dressing in Group II was found to enhance early-stage inflammation after 1 week, as compared with the other two groups. The findings in both experiments indicate that the wound dressing composed of HA-based spongy sheets containing Arg and EGF potently promotes wound healing by inducing moderate inflammation. The release of EGF in the early stages of wound healing induces moderate inflammation. This suggests that wound healing is facilitated directly by topical application of EGF, and indirectly by cytokines derived from inflammatory cells stimulated by EGF.

Collagen-Binding Human Epidermal Growth Factor Promotes Cellularization of Collagen Scaffolds

Previous studies have demonstrated that epidermal growth factor (EGF) can promote tissue regeneration effectively. The aim of the present study was to combine a collagen-binding domain (CBD) with native EGF (NAT-EGF) to form a fusion protein, aiming at that it could bind to collagen scaffolds specifically. This approach could restrict the diffusion of EGF despite of extracellular fluids and limit the release of EGF to surrounding tissues. Thus, the retained CBD-EGF would render a continual function during the tissue regeneration. In our study, in vitro bioactivity assay showed that there was no significant difference between CBD-EGF and NAT-EGF to stimulate human fibroblasts to proliferate. In the collagen-binding assay, CBD-EGF exhibited a specific binding ability to collagen compared to NAT-EGF. Finally, in vivo experiment demonstrated that CBD-EGF-loaded collagen membranes uniformly possess a high capability to promote cellularization of scaffolds as compared with that of NAT-EGF. All results aforementioned suggested that collagen loaded with CBD-EGF is a functional biomaterial with potential clinical applications.

Functional Bilayered Skin Substitute Constructed by Tissue-Engineered Extracellular Matrix and Microsphere-Incorporated Gelatin Hydrogel for Wound Repair

Our aim in this study was to develop a new methodology for constructing a bilayer skin equivalent to create a clinically compliant skin graft composite for the treatment of various skin defects. We utilized tissue-engineered extracellular matrix (TEECM) that maintains the native dermal components, as analogical dermal layer, and gelatin hydrogel containing epidermal growth factor (EGF)-loaded microspheres, as epidermis layer upon TEECM. The bilayer skin equivalent (GG-EGF/TEECM) could be synthesized appropriately and has been hypothesized to provide an enhanced effect on re-epithelization, apart from dermal reconstruction improved by TEECM. Morphological properties, EGF release efficiency, cytotoxicity, and effects on wound repair of this composite were investigated in this study. It was shown that the two layers adhered firmly to each other, and the percentage of cumulative release was approximately 80% by day 14. Moreover, as a biocompatible equivalent, GG-EGF/TEECM improved the healing of the wound area as indicated by significantly earlier re-epithelialization and dermal maturation. These results suggest that GG-EGF/TEECM can provide an optimal microenvironment and proper template for skin tissue engineering.