Angiogenesis and Full-Thickness Wound Healing Efficiency of a

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Biological and Medical Applications of Materials and Interfaces

Angiogenesis and full-thickness wound healing efficiency of copper doped borate bioactive glass/poly(lactic-co-glycolic acid) dressing loaded with vitamin E in vivo and in vitro Haoran Hu, Yue Tang, Libin Pang, Cunlong Lin, Wenhai Huang, Deping Wang, and Weitao Jia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04903 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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ACS Applied Materials & Interfaces

Angiogenesis and full-thickness wound healing efficiency of copper-doped borate bioactive glass/poly(lactic-co-glycolic acid) dressing loaded with vitamin E in vivo and in vitro

Haoran Hua,1, Yue Tangb,1, Libin Pangb, Cunlong Linb, Wenhai Huangb, Deping Wangb,*, Weitao Jiaa,* a

Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China b School of material science and engineering, Tongji University, Rd Caoan, Shanghai 201800, China

ABSTRACT: There is an urgent demand for wound healing biomaterials because of the increasing frequency of traffic accidents, industrial contingencies and natural disasters. Borate bioactive glass has potential applications in bone tissue engineering and wound healing; however, its uncontrolled release runs a high risk of rapid degradation and transient biotoxicity. In this study, a novel organic-inorganic dressing of copper-doped borate bioactive glass/poly(lactic-co-glycolic acid) loaded with vitamin E (0-3.0 wt.% vitamin E) was fabricated to evaluate its efficiency for angiogenesis in cells and full-thickness skin wounds healing in rodents. In vitro results showed the dressing was an ideal interface for the organic-inorganic mixture and a controlled release system for Cu2+ and vitamin E. Cell culture suggested the ionic dissolution product of the copper-doped and vitamin E-loaded dressing showed the best migration, tubule formation and vascular endothelial growth factor (VEGF) secretion in human umbilical vein endothelial cells (HUVECs) and higher expression levels of angiogenesis-related genes in fibroblasts in vitro. Furthermore, this dressing also suggested a significant improvement in the epithelialization of wound closure and an obvious enhancement in vessel sprouting and collagen remodeling in vivo. These results indicate that the copper-doped borate bioactive glass/poly(lactic-co-glycolic acid) dressing loaded with vitamin E is effective in stimulating angiogenesis and healing full-thickness skin defects and is a promising wound dressing in the reconstruction of full-thickness skin injury. KEYWORDS: Wound healing, Borate bioactive glass, Poly(lactic-co-glycolic acid), Vitamin E, Angiogenesis 1. INTRODUCTION Skin, which is the largest organ of the body, refers to an organized wrapping outside the muscles that has significant functions to protect of the body, perspire and perceive temperature and pressure1. In recent years, the increasing frequency of traffic accidents, industrial contingencies and natural disasters has led to a greater demand for wound healing biomaterials for serious defects that threaten a patient’s life if medical intervention is not successful2-3. Therefore, the study of ideal wound dressings of skin substitutes that can heal wounds and full-thickness skin injury is urgent and necessary4-5. Bioglass fibers, among the various wound dressings being researched, have received lots of attention due to their large specific surface area, excellent mechanical properties and high porosity.

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However, wound healing is a complex and interactive process involving acute inflammation, re-epithelialization, granulation tissue formation, and tissue remodeling, which involves the interaction between cells, extracellular matrix (ECM)4 and angiogenesis. Thus, bioglass fibers are a reasonable option for a wound dressing because they accelerate the healing and regeneration process6-9. Borate bioactive glass (BG) is a material with good chemical reactivity, degradability and biocompatibility10, and its potential application in bone tissue engineering and wound healing has been reported11-13. In our previous study, Cu-doped BG microfibers have been proven to stimulate the proliferation of endothelial cells and upregulate VEGF gene expression14-15. Copper ions have also been reported16-17 to enhance angiogenesis by stabilizing the expression of hypoxia-inducible factor (HIF-1a), thus mimicking hypoxia, which plays a critical role in the recruitment and differentiation of cells and the formation of blood vessels. However, the release of these effective ions are uncontrolled in the degradation process of BG, leading to initially burst release, which is reported to be toxic potentially to the surrounding cells18. To solve the problem mentioned above, poly (lactic-co-glycolic acid) (PLGA), a biodegradable polyester copolymer composed of two monomers, lactic acid and glycolic acid19, is adopted as a substitute matrix material for the Cu-doped BG micro-fibers in the present study20-21. The PLGA solution is viscous and can be used as a binder for BG micro-fibers in wound dressings fabrication22. Meanwhile, liposoluble drugs can be easily incorporated into this system due to the properties of PLGA. Vitamin E (VE) was chosen to be loaded because of its antioxidant23-25 and anti-inflammatory properties26-27. As a naturally occurring lipophilic antioxidant, VE has been used in the prevention and therapy of diseases of inflammation and aging due to its antioxidant properties and of oxidative stress and inflammation28 due to its protective effects. In the current study, Cu-doped BG/PLGA with various concentrations of VE (VE-Cu BG/PLGA) was fabricated into a dressing to study wound healing. To make a comprehensive understanding of this novel organic-inorganic composite in vivo and in vitro, an investigation of the properties, characteristics, cell bioactivity and full-thickness wound healing of VE-Cu BG/PLGA dressing was conducted. 2. MATERIALS AND METHODS 2.1. Preparation of copper-doped BG Fibers and VE-Cu BG/PLGA Dressing The copper-doped BG fibers used in the present study were prepared by melt-derived glasses with composition 6Na2O-8K2O-8MgO-22CaO-18SiO2-36B2O3-2P2O5-6SrO (mol%) and 3.0 wt% CuO according to previous work14-15, 29. Briefly, the requisite amounts of analytical grade raw materials, Na2CO3, K2CO3, CaCO3, H3BO3, SiO2, (MgCO3)4·Mg(OH)·25H2O, NaH2PO4·2H2O and CuSO4·5H2O (Sinopharm Chemical Reagent Co., Ltd. China), were mixed and heated for 2 h at 1150 °C, and molten glass fluid was obtained. Then, the fluid was poured from a crucible at 0.10 L/s, blown by high-pressure (0.35 MPa) cold air and cooled to room temperature (RT). Finally, the cotton-like glass fibers were obtained, and the micro-fibers were mixed with alcohol and ultrasonically dispersed. To obtain the VE-Cu BG/PLGA dressing, PLGA (50:50) (Dai Gang Biology, China) was dissolved in dichloromethane, and the dispersed Cu-doped BG fibers were mixed with PLGA with a weight ratio of 1/1 (w/w), VE (0, 0.5, 1 and 3 wt%) was added, and the mixture was reacted at 40 °C for 1 h.


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2.2. Characterization and Conversion Reaction of VE-Cu BG/PLGA Dressing in vitro Scanning electron microscopy (SEM; Model Quanta 200 FEG, FEI Co.) was used to observe the morphology of the composite after coating with Au/Pd. Simulated body fluid (SBF), the immersion solution, was prepared according to a previously published method30-33. The ratio of the composite/SBF was 1/50 (g/mL). The weight loss of the VE-Cu BG/PLGA sample and the pH value of the solution were measured. The release of ions was detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optima 2100DV), and the concentration of VE was investigated using an enzyme-linked immunosorbent (ELISA) kit (Jiancheng Bioengineering Institute). The fresh immersion solution was changed at each point after the test. Attenuated total reflection flourier transformed infrared spectroscopy (ATR-FTIR; Model Equinoxes/Hyperion 2000, TA CO.) was used for phase analysis before and after immersion in the SBF to assess the reaction of concerting to hydroxyapatite (HA) and the bioactivity. 2.3. In vitro Biocompatibility and Angiogenesis 2.3.1. Cells and cell culture To examine the in vitro cytotoxicity and angiogenesis of the VE-Cu BG/PLGA dressing, human fibroblasts and umbilical vein endothelial cells (HUVECs) were cultured in minimum essential medium α (α-MEM, Gibco) with 10% fetal bovine serum (Gibco) in an incubator with 5% CO2 at 37 °C. The usage of these two cell types was approved by the ethical committee of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. The cells were isolated from human skin and an umbilical cord vein with the approval of the donors. Extracts of the ionic dissolution product of the VE-Cu BG/PLGA composite were prepared by immersing the composite into α-MEM containing 10% fetal bovine serum with a concentration of dressing in DMEM of 100 mg/mL in accordance with International Standard Organization (ISO/EN) 10993-534 at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. 2.3.2. Cell proliferation The effect of the ionic dissolution product of the VE-Cu BG/PLGA wound dressing on the proliferation of fibroblasts and HUVECs was assessed using the CCK-8 method (Dojindo, Japan). The two cell types were seeded in 96-well plates at an initial density of 5×103 cells/well with different concentrations of the ionic extracts. At days 1, 3 and 7, 20 µL of CCK-8 solution and 180 µL of culture medium were added to each well. After another 4 h incubation at 37 °C, 100-µL aliquots were taken from each well and transferred to another 96-well plate. The absorbance values at 450 nm were recorded to measure the cell viability. 2.3.3. Migration and tubule formation activities of HUVECs To measure the migration potential of HUVECs in the ionic dissolution product, a transwell assay was used by seeding 5.0×104 cells/mL in the upper chambers of a 24-well transwell plate (3422, Corning) and adding 600 µL ionic dissolution product of each group to the lower chambers. After 24 h of incubation, the cells on the upper surface of the transwell membrane were gently wiped with a cotton swab, and cells on the lower surface were fixed with 4% paraformaldehyde and stained for 10 min with 0.5% crystal violet. Finally, 6 random lower surfaces of each filter were chosen and counted twice in a blinded manner by two independent evaluators. Meanwhile, the tubule formation ability of HUVECs in the ionic dissolution product was



tested by seeding the cells on MatrigelTM (BD Bioscience). In brief, a HUVEC suspension of 2.0×104 cells/mL was seeded on the matrix after 500 mL of cold Matrigel per well was spread in each hole of a 24-well plate and gelatinized at 37 °C for 30 min. After 12 h of incubation, the number of complete capillaries connecting individual points of each hole was counted to detect the tubule formation ability. 2.3.4. HUVEC morphology and VEGF secretion To investigate the morphology and VEGF secretion of HUVECs, the cells were cultured in the ionic dissolution product of each group in an incubator with 5% CO2 at 37 °C at a density of 2.0×104 cells/mL in a 24-well plate. After 3 days of incubation, the cells grown on the plate were washed with PBS and fixed with 4% polyoxymethylene for 15 min at RT. After being washed with PBS, cells were incubated with primary antibodies against VEGF (1:200, Proteintech) overnight at 4 °C followed by incubation with Alexa Fluor 488-conjugated secondary antibody (1:200, Proteintech) for 1 h, rhodamine phalloidin (1:200, Sigma) for 40 min and 4',6-diamidino-2-phenylindole (1:200, DAPI, Solarbio) for 10 min at RT. Finally, the samples were observed using an imaging fluorescence microscope (Leica). 2.3.5. Angiogenesis-related gene expression of fibroblasts Fibroblasts cultured in the ionic dissolution were collected on days 3 and 7 to estimate the angiogenesis-related gene expression of VEGF, b-FGF and PDGF via real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR). To extract the total RNA of each specimen and synthesize the complementary DNA (cDNA), TRIzol reagent (Invitrogen) and Reverse Transcriptase M-MLV (Takara) were used in turn. Quantification of the chosen genes was performed using real-time PCR with SYBR Premix Ex Taq (Takara). The primers used in this experiment are listed in Table I, and GAPDH was used as the internal control gene. 2.4. In vivo Evaluation of Healing of Full-thickness Skin Defects 2.4.1. Animal experiments Thirty adult Sprague-Dawley (S-D) rats (2 months old; 250 ± 15 g) were used in this study. The animal experimental protocol was approved by the Animal Care and Experiment Committee of Shanghai Jiao Tong University Affiliated No. 6 Hospital, and all procedures strictly followed the description. The VE-Cu BG/PLGA dressing was sterilized using 60Co irradiation prior to transplantation, and the animals were anesthetized with chloral hydrate sodium (250 mg/kg) via intraperitoneal injection. The wound areas were marked and then sterilized with iodine prior to incision. To create a full-thickness skin wound, a standardized defect (round, diameter＝20 mm) was incised on the dorsum by an experienced surgeon. Then, 0VE-Cu or 3VE-Cu dressing was placed on each skin defect, and a Band-Aid was placed over the wound to cover and fix the dressing. Meanwhile, the control group received a Band-Aid with no dressing after the standardized defect was created. 2.4.2. Wound closure measurement


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On days 7 and 14 post-surgery, the Band-Aids on the wounds were moved, and each defect was photographed by a digital camera. The size of the wound was measured using image analysis software (NIH Image) and calculated by the following formula: Percent wound size reduction ＝ [(A0- At)/A0] × 100

Equation (1)

where A0 refers to wound area (t＝0), and At refers to wound area at appointed time.

2.4.3. Vascular perfusion To observe the new-born vasculature, the rats were perfused with Microfil (MV-122, Flow Tech) 14 days after surgery. After the animals were euthanized, 100 mL heparinized saline and 20 mL Microfil were successively perfused into the left ventricles at 2 mL/min according to a previous description15. The wound samples were scanned to measure the newly formed vasculature by an NRecon micro-CT scanner (Skyscan Company) with a 100-kV/100-µA X-ray source at an isotropic voxel size of 18 µm after polymerizing the contrast agent at 4 °C overnight. 2.4.4. 3D reconstruction of vasculature After micro-CT scan, a 3D vascular enhancement image of the wound was reconstructed by Skyscan software (Skyscan Company). Meanwhile, the blood vessel area and blood vessel number were also calculated by the software. 2.4.5. Histological, immunofluorescence and immunohistochemical observation The wounds were removed along with the surrounding healthy skin after the rats were sacrificed. The samples were fixed in 10% formalin for 2 days, dehydrated with a graded series of ethanol and embedded in paraffin. After that, sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome according to the previous procedure and examined with an optical microscope. For immunofluorescence staining, sections were treated with antigen retrieval and then incubated with α-SMA (1:200, Abcam) and CD31(1:200, Abcam) primary antibody at 4 °C overnight. Next, the Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary antibodies (1:200, Abcam) were applied for 1 h at RT and 4',6-diamidino-2-phenylindole (1:100, DAPI, Solarbio) for 10 min at RT. The images were examined under a confocal laser scanning microscope (CLSM; SP8, Leica). For immunohistochemical staining, sections incubated with CD34 primary antibody at 4 °C overnight and incubated with secondary antibody for 1 h. Subsequently, diaminobenzidine (Dako) was used to develop the color reaction. Finally, the sections were stained with hematoxylin and examined under a light microscope (Leica). To count the number of new vessels, 6 random sections chosen from different specimens of each group were used for the pathologist’s evaluation. The evaluation was standardized by choosing three high-power fields containing the entire portion of the defects at random. 2.5. Statistical Analysis All statistical analyses were conducted using SPSS 20.0 software. The count data were presented as the mean ± standard deviation. Differences among groups were analyzed using one-way



ANOVA followed by the SNK test. The differences were considered to be statistically significant if P

Angiogenesis and Full-Thickness Wound Healing Efficiency of a

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