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Effects of Dimethyloxalylglycine-Embedded Poly(#caprolactone) Fiber Meshes on Wound Healing in Diabetic Rats Qiankun Zhang, Joung-Hwan Oh, Chan Ho Park, Jeong-Hwa Baek, Hyun-Mo Ryoo, and Kyung Mi Woo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15815 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 19, 2017
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Effects of Dimethyloxalylglycine-Embedded Poly(ε-caprolactone) Fiber Meshes on Wound Healing in Diabetic Rats
Qiankun Zhang †, Joung-Hwan Oh †, Chan Ho Park †, Jeong-Hwa Baek †‡, Hyun-Mo Ryoo †‡, and Kyung Mi Woo †‡*
† Department of Molecular Genetics, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea ‡ Department of Pharmacology & Dental Therapeutics, School of Dentistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
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ABSTRACT Impaired wound healing in diabetic patients is associated with altered inflammatory responses, poor angiogenesis, deficient extracellular matrix (ECM) component, and peripheral neuropathy. To develop a wound dressing that is capable of the controlled delivery of bioactive small molecules that can improve diabetic wound healing, dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) (PCL) fiber (PCLF/DMOG) meshes are fabricated by electrospinning, and the effects of the PCLF/DMOG meshes on wound healing in diabetic rats are evaluated. Electrospun PCLF/DMOG meshes increase not only the wound closure, re-epithelialization ratio, epithelial maturation (K-10positive epidermis), and collagen-positive area but also the numbers of angiogenic marker (CD-31)positive and neuronal marker (neurofilament)-positive cells compared to PCLF (p 300 mg dl-1 or hemoglobin A1c (HbA1c) levels > 6.5% were considered DM. Wound healing assay. For the wound healing studies, a silicone-splinted full-thickness wound model was adapted to be compatible with the application of fibers. The use of silicone rings reduces contraction upon wounding, thus allowing loose-skinned mammals to heal by secondary intention processes more reminiscent of human wound healing. Silicone rings with an internal diameter of 8 mm were cut from 0.5 mm-thick silicone sheets (Grace Bio-Labs, Bend, OR, USA) and then sterilized. Normal rats and DM rats were anesthetized with isoflurane (HAHA PHARM, Seoul, Korea). Their dorsal hairs were shaved, and two 6-mm-diameter, full-thickness wounds were created with a biopsy punch at either side of the dorsal central line in experimental animals (normal rats, n rat = 12; DM rats, 8
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n rat = 12). To avoid the differences between individuals, all wounds in each rat were randomly treated by control, PCLF, and PCLF/DMOG meshes. In all groups, the wounds were wrapped with a surgical film (Tegaderm, 3M Health Care, St. Paul, MN, USA). Various healing analyses were performed at 3 (n rat = 3), 9 (n rat = 6) and 14 (n rat = 3) post-surgery. Wounds were photographed at days 0, 3, 9, and 14 post-surgery using a Canon digital camera. Each image included a ruler to allow standard calibration of measurements. Surface wound area was quantified at each time-point using the ImageJ software. The rate of wound closure was determined by calculating the wound area (wound closure rate = (wound area day 0 − wound area day #)/wound area day 0). In vitro cell migration (scratch) assay. To evaluate re-epithelialization in vitro, a keratinocyte migration assay was performed. HaCaT cells were seeded at a density of 2.5x104 cells cm-2 into 12well plates, allowed to form a confluent monolayer, and then starved with FBS-free media for 2-4 h. The cells were carefully scratched with sterile 200 µL pipette tips and washed with cold PBS, and then the medium was replaced with FBS-free media containing 0, 0.25, 0.5, and 1 mM of DMOG or collected CMs. Twenty-four hours after incubation, the cells were washed with PBS, and the scratched wounds were photographed. Keratinocyte migration areas were determined by calculating the scratched wound areas (migration areas = [scratched wound areas
0h
- scratched wound areas
24 h]).
Scratched
wound area was quantified at each time-point using ImageJ software. Histology. Normal and DM rats for control, PCLF, and PCLF/DMOG treatments were sacrificed at day 9 post-surgery. Square-shaped skin samples including the wounded and unwounded area were collected, and cut into 2 pieces, one half for hematoxylin and eosin staining (H&E staining) and the other half for immune-fluorescence staining. For H&E staining, skin samples were fixed with 10% formaldehyde solution, embedded in paraffin, sectioned at 5-µm intervals, and stained with H&E. The ratio of re-epithelialization was determined by calculating the wound length between wound edges (ratio of re-epithelialization = (wound length day 0 − wound length day #)/wound length day 0).31 For tissue 9
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collagen staining, the sections were deparaffinized, washed in PBS, and stained with a collagen staining kit (Chondrex, Redmond, WA, USA). The ratio of the collagen-positive area was determined by calculating the collagen-stained area in the field of the wound bed. Another half of the skin sample was quickly frozen by liquid nitrogen, embedded in Optimal Cutting Temperature (OCT) compound (Sukura, Tokyo, Japan), cryo-sectioned at 10-µm intervals, fixed in cold acetone, and stored at – 80 °C. Immunostaining was performed for cluster of differentiation 31 (CD-31), Keratin-14 (K-14), Keratin10 (K-10), and Neurofilament heavy chain (NEFH). Briefly, the cryo-sections were warmed and airdried at room temperature (RT) for 30 min. After washing with PBS, the cryo-sections were blocked with 5% anti-donkey serum in PBS for 30 min at RT and then incubated with primary anti-CD-31 antibody (BD Biosciences, San Jose, CA, USA), anti-K-14 antibody (Biolegend, Dedham, MA, USA), anti-K-10 (BD Biosciences), or anti-NEFH (Sigma) at RT for 1 h. After washing with PBST (0.05% tween 20 in PBS), the cryo-sections were incubated with Alexa Fluor Cy3 & 488-conjugated donkey anti-rabbit or mouse IgG (Santa Cruz Biotechnology) at RT for 1 h. After washing with PBST, the cryosections were mounted with DAPI Fluoromount-G solution (Southern Biotech, Birmingham, AL, USA). The number of CD-31-positive cells was determined by calculating CD-31-stained cells in the fields of the wound bed. The elongation of K-10-positive epidermis was determined by calculating K-10 and K14-stained epithelial length between the first hair follicle on both sides of the wound beds (elongation of K-10-positive epidermis = K-10-stained length/ K-14-stained length). Western blot analysis. Whole NIH/3T3 cell lysates in each well from the six well plates were prepared using a buffer containing 12.5 mM Tris-Cl (pH6.8, Tris-base, Biosesang, ≥99.9%, Suwon, Korea), 20% glycerol (sigma, ≥99%), 4% SDS (sigma, ≥99%), 10% mercaptoethanol (Biosesang, ≥99.9%), and a complete protease inhibitor cocktail solution. The samples were subjected to 8% SDS– PAGE and then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked by 5% non-fat dried milk and incubated with primary anti-HIF-1α antibody (Abcam, Cambridge, MA, 10
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USA). This procedure was followed by incubation with an HRP-conjugated secondary antibody. Luminescence was detected using a Bio-Image Analyzer (Bio-Rad, Hercules, CA, USA). β-actin was used as a loading control. Extraction of total RNAs and qPCRs. The qPCR was used to evaluate the transcript expression. Total RNAs were extracted from a well in the 6-well plate using the RNAiso Plus reagent (Takara, Tokyo, Japan). cDNA was synthesized using the PrimeScriptTM RT reagent kit (Takara) according to the manufacturer’s instructions. qRT-PCR was performed on a Real-time PCR system using SYBR® Premix Ex TaqTM according to the manufacturer’s instructions. Table S1 (Supporting Information) lists the rat, mouse and human primer sequences that were used for qPCR analysis in this study. The relative levels of the target gene mRNAs were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each experiment was duplicated at least three times. Statistical analysis. Data from the repeated experiments are presented as the mean and standard deviation (mean ± SD). Statistical analyses were performed using the analysis of variance (ANOVA) method. Differences were considered significant if the p value was
