This study aimed to investigate the potential of cultured dermal substitute (CDS) to release angiogenic growth factors when laminated with a membrane containing epidermal growth factor (EGF) as a top dressing. Membranes were prepared by air-drying a solution of hyaluronic acid (HA) and collagen (Col) with or without EGF. Membranes were designed to contain EGF at concentrations of 0, 0.1, 0.2 or 0.5 μg/cm2. CDS was prepared by incorporating fibroblasts into a collagen gel combined with a cross-linked HA spongy matrix, followed by culturing for 5 days. CDS was designed to contain fibroblasts at a density of 2 × 105 (Group I) or 4 × 105 cells/cm2> (Group II). CDS was elevated at the interface between air and culture medium, on the top of which each membrane was placed. This culture system was employed as a wound surface model. Metabolic activity of the fibroblasts in the CDS cultured for 7 days on a wound surface model was measured by MTT assay. The amounts of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) after 7 days of cultivation were measured by using ELISA. Membranes containing EGF ranging from 0.1 to 0.5 μg/cm2> facilitated production of both VEGF and HGF, as compared with control membranes without EGF. However, a membrane containing EGF at a concentration of 0.5 μg/cm2> failed to facilitate fibroblast cytokine production in Group I. These results demonstrated that the EGF-incorporating membrane was able to stimulate fibroblasts in the CDS to synthesize an increased amount of VEGF and HGF in a dose-dependent manner.
Many types of injury result in damage to the skin. The body is typically capable of closing these wounds spontaneously to restore the original functions of its protective covering as quickly as possible. This process involves various repair mechanisms in the individual layers of skin as well as the growth and differentiation of numerous cells. Understanding these processes provides valuable information for designing wound dressings and cultured skin substitutes. We have developed various wound dressings and cultured skin substitutes. The wound dressing is composed of hyaluronic acid (HA) and collagen (Col) spongy sheet containing epidermal growth factor (EGF) [
In our clinical study, the allogeneic CDS is covered with conventional ointment-coated gauze and secured with a tie-over dressing. To improve the conventional covering procedure, we reported the combined use of an allogeneic CDS and EGF-incorporating sponge-type wound dressing [
HA powder (Bio Sodium Hyaluronate HA 20; molecular weight, 2000 kDa; Shiseido, Tokyo, Japan), Col powder (NMP Collagen PS; Nippon Meat Packers, Osaka, Japan), and freeze-dried EGF (rh-HGF; Shanghai Haohai Biological Technology Co., Ltd., Shanghai, China) were used to prepare the matrices for CDS and EGFincorporating membrane. This recombinant human (rh-) EGF has been commercially available as a medical drug for the treatment of intractable skin ulcer. Ethylene glycol diglycidyl ether (EX810; Nagasekasei Co. Ltd., Osaka, Japan) was used as a cross-linking agent. Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St.Louis, USA) and fetal bovine serum (FBS; Gibco, California, USA) were used for fibroblast culture.
HA powder (4 g) was dissolved in distilled water (400 mL) in order to prepare a high molecular weight (HMW)- HA solution. Another amount of HA powder (4 g) was dissolved in distilled water (400 mL), and was then placed in an autoclave at 120˚C for 1 h to obtain a partially hydrolyzed low molecular weight (LMW)-HA solution (molecular weight, 150 kDa). Col powder (4 g) was dissolved in distilled water (400 mL), and was then warmed at 50˚C for 10 min to yield heat-denatured Col solution. These three solutions were mixed and adjusted to pH 7.0 using 1 N-NaOH, and then divided into 4 portions (each 300 mL), in which a given amount of EGF dissolved in distilled water (15 mL) or only distilled water (15 ml) was added. Each mixture (21 mL) was poured into a plastic tray (5 cm × 8 cm), and was then placed in a clean bench for 5 days in order to prepare a membrane containing EGF. The EGF concentration in each membrane is 0, 0.1, 0.2, or 0.5 µg/cm2. The EGF concentration was expressed for convenience as a unit of µg/cm2, because the thickness of membrane was very thin (10 μm).
HA powder (30 g) was dissolved in distilled water (2000 mL). This HA solution was adjusted to pH 3.5 with 10% diluted HCl. EX810 (6 g) was mixed with distilled water (14 mL). The aqueous solution of EX810 was then added drop-wise to the HA solution under vigorous stirring. The weight ratio of HA to EX810 was adjusted to 5:1. This mixed solution (40 mL) was poured into a tray (5 cm × 8 cm) and kept at 50˚C for 5 h in order to promote cross-linking. During this procedure, the volume of HA solution was reduced by about 50%. The tray was then kept at 4˚C for 2 h, and was quickly frozen in a freezer at −85˚C, followed by freeze-drying to obtain the HA spongy sheet. The spongy sheet was rinsed with distilled water in order to remove free cross-linking agent. After rinsing, the hydrated HA spongy sheet was again placed in a tray and kept at 4˚C for 2 h, followed by quick freezing at −85˚C and freeze-drying to yield purified HA spongy sheets. These HA spongy sheets were punched to produce numerous holes (diameter 0.5 mm) separated by a distance of 4 mm. Each spongy sheet was packed into a bag and kept in a dry sterilizer at 110˚C for 1 h.
Col powder was sterilized in a vacuum drying oven at 121˚C for 2 h and dissolved in sterilized distilled water at a concentration of 1%. The DMEM used for mixing with Col solution was prepared at twice the standard concentration. Fibroblasts were suspended in double concentrated DMEM supplemented with 20% FBS. Cell suspension (10 mL) and Col solution (10 mL) were mixed at less than 10˚C, and were then poured into a dish measuring 5 cm × 8 cm, in which a sterilized HA spongy sheet of the same size was immersed, followed by overnight incubation under 5% CO2 at 37˚C to allow jellification. The resulting product was cultured for 5 days in a dish containing additional 30 ml of culture medium (DMEM supplemented with 10% FBS) in order to give cultured dermal substitute (CDS). Cell seeding density was 2 × 105 cells/cm2 (Group I) and 4 × 105 cells/cm2 (Group II).
Cultivation on the wound surface model was conducted according to the method described in our previous article [
The procedure was described previously [
Laboratories Inc., California, USA) at 570 nm.
The procedure was described previously [
Data are expressed as mean ± standard error. Statistical analysis was performed using Tukey Kramer test.
This membrane was composed of HMW-HA, LMW-HA, and heat-denatured Col. The heat-denatured Col aqueous solution was not precipitated even at a neutral pH condition. Therefore, it is possible to prepare a clear mixed solution at neutral pH condition. The resulting clear mixture had an appropriate viscosity, and thereby could prepare a membrane with thickness as thin as 10 μm.
Scanning electron microphotograph of a cross-section of the cross-linked HMW-HA spongy matrix is shown in
In general, the size of collagen gel sheet including fibroblasts decreases easily, because fibroblasts contract the collagen microfibers in gel. To improve the size contraction, a unique structure was designed. The CDS is composed of a fibroblasts-incorporating collagen gel combined with a cross-linked HMW-HA spongy matrix. This HA spongy sheet was punched to produce numerous holes (diameter 0.5 mm), thereby a fibroblasts-incorporating collagen gel was found within a HA spongy structure (
The optical density (OD) value on MTT assay corresponds to the metabolic activity of fibroblasts in CDS under each culture condition. The data after 7 days of cultivation at the interface between air and culture medium are shown in
but slightly decreased at EGF concentration of 0.5 µg/cm2. A membranes containing EGF at 0.2 µg/cm2 stimulated fibroblasts in CDS to release 2.3 times more VEGF after 7 days of cultivation, as compared with a control. The amount of VEGF in Group II increased sufficiently when CDS was laminated with membranes containing EGF ranging from 0.1 to 0.5 µg/cm2. A membrane containing EGF at 0.5 µg/cm2 stimulated fibroblasts in CDS to release 2.7 times more VEGF after 7 days of cultivation, as compared with a control.
cells/cm2) and Group II (4 × 105 cells/cm2). The amount of HGF in Group II was double of those in Group I, corresponding to the double cell number. The amount of HGF in Group I increased sufficiently when CDS was laminated with membranes containing EGF ranging from 0.1 to 0.2 µg/cm2, in spite of the dose-dependence in OD values, but decreased at EGF concentration of 0.5 µg/cm2. A membranes containing EGF at 0.2 µg/cm2 stimulated fibroblasts in CDS to release 5.8 times more HGF after 7 days of cultivation, as compared with a control. The amount of HGF in Group II increased sufficiently when CDS was laminated with membranes containing EGF ranging from 0.1 to 0.5 µg/cm2. A membrane containing EGF at 0.5 µg/cm2 stimulated fibroblasts in CDS to release 6.0 times more HGF after 7 days of cultivation, as compared with a control.
HA is a particularly promising biomaterial [
CDS was designed to combine the wound healing effect via growth factors released from fibroblasts and the wound healing effect via biomaterials, i.e., HA and Col used as matrix. This type of CDS was employed as an autologous cultured gingival dermal substitute in clinical study. The excellent clinical result was reported in our previous article [
It is known that growth factors accelerate wound healing by regulating various cell functions such as proliferation, differentiation, migration and morphogenesis [
In the present study, we aimed to determine the appropriate concentration of EGF in a membrane by altering the EGF concentration in the membrane, and by altering the seeding density of fibroblast in the matrix. The practical issue is to find out the appropriate EGF concentration in the membrane under the practical condition in which EGF-incorporating membrane was placed on the CDS for one week.
The effects of growth factor on cell proliferation are generally dependent on its concentration. In a previous study [
The EGF-incorporating membrane is capable of enhancing angiogenic cytokine production by fibroblasts in CDS, as well as serving as a top covering to protect CDS. In practice, the appropriate concentration of EGF in the membrane was found to be between 0.1 and 0.2 µg/cm2. EGF released from the membrane is able to stimulate fibroblasts in CDS to release an increased amount of VEGF and HGF that are essential for angiogenesis.