Hyaluronic acid (Shisheido, Shizuoka, Japan), Collagen (Koken, Tokyo, Japan) and Pluronic F68 (Daebong LS, Incheon, Korea) are base materials for production of hyaluronate- collagen dressing (HCD) matrix. For the production of HCD matrix, we first dissolved the 0.1 % of collagen in refined water (pH3 ~ 4) and raised pH to 7 ~ 8 then dissolved 0.8 % of HA. The 0.1 % of F68 was added to help blending collagen and HA then evenly mixed them with homogenizer. Subsequently, we added stabilized growth factors with concentrations of 0.1, 0.3, 1, and 2.5ug/cm2 and aliquot them into the mold for the lyophilization (Additional file 1: Figure S1). For selection of optimal sterilization method of S-EGF and S-bFGF, we exerted various kinds of methods following ethylene oxide (EO) gas, gamma irradiation (25 kGy) and electronic irradiation. The stabilized epidermal growth factor (S-EGF) and basic fibroblast growth factor (S-bFGF) became more thermostable through structural modification when compared to existing growth factors. The stabilized growth factor loaded HCD matrixes were received from GENEWEL (Seongnam, Gyeonggi-do, Korea).
MTT Assay and cell proliferation assay
L929 cells (Sigma-Aldrich, St Louis, MO, USA) were cultured in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S). The NIH/3T3 fibroblast cells (Sigma-Aldrich) and Balb/3T3 fibroblast cells (Sigma-Aldrich) were cultured in DMEM (Gibco) supplemented with 0.5 % bovine calf serum (BCS) and 1 % P/S. These cells were incubated at 37 °C incubator with 5 % CO2 and medium was replenished every two days. Each of cells were seeded at density of 1X105cells/well on 96 well plates and incubated for 24 h. We classified the groups as negative control (no treatment), positive control (latex glove extracted solution; ISO-10993-5) , S-EGF, S-bFGF, and HCD matrix containing S-EGF and S-bFGF and added them in medium and cultured for 48 h. The S-EGF and S-bFGF are loaded on HCD matrix with the concentration of 0.1, 0.3, 1, and 2.5 μg/cm2. Then thyiazolyl blue tetrazolium bormide (MTT, M2128-100MG, Sigma-Aldrich) was added and incubated for 3 h. At the indicated time, DMSO was added and absorbance was read at 570 nm.
L929 cells were seeded 4X105cells/well on 6well plate. After 24 h of cultivation, medium were removed and 1.5 % agar (BD, Franklin Lakes, NJ, USA) diluted in distilled water was overlaid on the cells. After solidification of agar, 0.01 % neutral red vital dye (Sigma-Aldrich) was treated on medium and placed in dark 37 °C incubator for 90 min. The sterilized paper discs (6 mm) containing HA, were laid on agar and incubated in dark for 24 h. After incubation, cytotoxicity was rated with decolorized zone around the paper discs .
Enzyme-Linked Immunosorbent Assay (ELISA)
Each of disk-shaped HCD matrix containing 1 μg/cm2 of S-EGF and S-bFGF (diameter of 10 mm) was immersed in 10 ml of 0.1 % BSA (Sigma-Aldrich) dissolved in PBS and incubated in shaking incubator at 37 °C with 15 rpm for 7 days. Thereafter, to determine the amount of growth factors, these mixtures were diluted to 1/1000 and analyzed by ELISA kit (R&D system, St Cloud, MN, USA). Also, to confirm concentration of released S-EGF and S-bFGF from HCD matrix, we installed matrices (diameter of 30 mm) in the Franz Cell and incubated in shaking incubator at 37 °C with 50 rpm. Then, collected samples on day 1, 3, 7, 10, 14, 21 and confirmed quantity of S-EGF and S-bFGF with ELISA kit (R&D system).
Preparation of streptozotocin (STZ)-induced type1 diabetic mouse model
ICR mice (Male, aged 7 weeks) were purchased from Orient Bio (Seongnam, Gyeonggi-do, Korea) and housed in wire cage at 20–22 °C at a relative humidity of 40–50 %. We used type1 diabetic mice, induced by intraperitoneal (I.P.) injection of streptozotocin (STZ) (200 mg/kg body weight; Sigma-Aldrich) dissolved in 0.05 M citrate buffer (pH4.5). One week after induction of diabetes, blood glucose levels were measured using OneTouch Select meter (Johnson&Johnson, New Brunswick, NJ, USA). The diabetic phenotype in animals was confirmed by blood glucose levels over 300 mg/dL and maintained for three weeks. The animal experiments were carried out in accordance with guidelines set by the Department of Laboratory Animal Resources, Yonsei University College of Medicine and Seoul, Korea (Permit number = 2015-0190).
Biocompatibility test of S-EGF and S-bFGF loaded on HCD matrix in type1 diabetic mouse model
STZ-induced type1 diabetic mice (Male, aged 9 weeks) were anesthetized with I.P. injection of Zoletile (30 mg/kg body weight) and Rumpon (10 mg/kg body weight). The hair on the back of mouse was shaved and subsequently wiped with 70 % ethanol. A 10 mm diameter full-thickness of skin wounds were created on the backs of STZ-induced diabetic mice and fixed with a silicone ring to prevent wound contraction. The groups are classified as Defect control, HCD only, HCD+S-EGF 0.3 μg/cm2, HCD+S-EGF 1 μg/cm2, HCD+S-EGF 2.5 μg/cm2, HCD+S-bFGF 0.3 μg/cm2, HCD+S-bFGF 1 μg/cm2, HCD+S-bFGF 2.5 μg/cm2. After application of matrix on wound site, Vaseline gauze (Covidien, St. Louis, MO, USA) and Neo dressing (Everaid, Gangnam, Seoul, Korea) were placed on matrix for the minimal dehydration of wound sites. In each groups, twelve animals were used.
Statistical analysis was performed via Student’s T-TEST and ANOVA. The data was expressed as the mean ± standard deviation (SD). Values of *p < 0.05, **p < 0.01 were considered statistically significant.