Article pubs.acs.org/molecularpharmaceutics
Epidermal Stem Cells Manipulated by pDNA-VEGF165/CYD-PEI Nanoparticles Loaded Gelatin/β-TCP Matrix as a Therapeutic Agent and Gene Delivery Vehicle for Wound Healing Li-Hua Peng,† Wei Wei,†,‡ Xiao-Tian Qi,† Ying-Hui Shan,† Fang-Jun Zhang,† Xi Chen,† Qian-Ying Zhu,† Lian Yu,‡ Wen-Quan Liang,† and Jian-Qing Gao*,† †
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, P. R. China Department of Pharmaceutics, College of Pharmaceutical Sciences, Jiamusi University, Jiamusi, Heilongjiang, 154007, P. R. China
‡
ABSTRACT: The success of gene therapy largely relies on a safe and effective gene delivery system. The objective of this study is to design a highly efficient system for the transfection of epidermal stem cells (ESCs) and investigate the transfected ESCs (TESCs) as a therapeutic agent and gene delivery reservoir for wound treatment. As a nonviral vector, β-cyclodextrin-linked polyethylenimines (CYDPEI) was synthesized by linking β-cyclodextrin with polyethylenimines (600 Da). Gelatin scaffold incorporating β-tricalcium phosphate (β-TCP) was utilized as a substrate for the culture and transfection of ESCs. With the CYD-PEI/pDNAVEGF165 polyplexes incorporated gelatin/β-TCP scaffold based 3D transfection system, prolonged VEGF expression with a higher level was obtained at day 7 in ESCs than those in two-dimensional plates. Topical application of the TESCs significantly accelerated the skin re-epithelization, dermal collagen synthesis, and hair follicle regeneration. It also exhibited a potential in scar inhibition by regulating the distribution of different types of collagen. In contrast to ESCs, an additive capacity in stimulating angiogenesis at the wound site was observed in the TESCs. The present study provides a basis for the TESCs as a promising therapeutic agent and gene delivery reservoir for wound therapy. KEYWORDS: β-cyclodextrin-linked polyethylenimine, gelatin/β-tricalcium phosphate scaffold, epidermal stem cells, nonviral gene delivery, wound repair and regeneration
1. INTRODUCTION Advances in understanding the molecular and cellular responses involved in wound repair and regeneration has led to the extensive development of stem cells based tissue engineering and gene therapy for wound therapy. One of the exciting approaches is likely to be the development of recombinant stem cell-based skin grafts that are then able to develop into new skin layers with normal functions.1 As the important progenitor cells for skin, the self-renewal ability and multilineage differentiation potential of epidermal stem cells (ESCs) suggest their great potential in wound treatment.2 However, in our recent study, compared with the stimulating effect expressed by BMSCs in blood vessels formation at the wound site, ESCs showed no angiogenic effect.3 Up to now, ESCs have not been reported for their capacity in activating blood vessels formation. Since angiogenesis is essential for wound healing, impaired angiogenesis causes delayed or nonhealing wounds.4 Vascular endothelial growth factor (VEGF), a glycoprotein produced by skin keratinocytes, fibroblasts, and macrophages, is one of the main promoters of angiogenesis.5 But the high concentrations of VEGF protein required for a biological effect and its rapid degradation after introduction into the body has rendered its use as cost ineffective. The delivery of VEGF has been widely employed to stimulate an angiogenic response for © 2013 American Chemical Society
clinical therapy. Therefore, the hypothesis of this study is that genetic manipulation of ESCs with the VEGF gene may be a promising strategy to enhance the ESCs angiogenic capacity and overcome the limitations associated with the conventional delivery of VEGF protein. However, the data available on gene delivery to ESCs have been scarce until recently. The efficacy and safety of gene therapy depend not only on gene construct being delivered, but more importantly on the delivery vehicle itself. The viral infection-associated toxicity, immunologic compromise, and possible mutagenic or carcinogenic effects make viral vectors potentially dangerous. Nonviral vectors have advantages, such as ease of synthesis, cell/tissue targeting, low immune response, and unrestricted plasmid size; however, they expressed quite low transfection efficiency, nor do they allow long-term transfection when used in stem cells.6 This study primarily aims to develop an efficient nonviral system to enhance the level and prolong the duration of gene expression for recombinant ESCs. Especially, in conventional transfection, cells are seeded on the culture plates for complete Received: Revised: Accepted: Published: 3090
March 16, 2013 June 7, 2013 June 12, 2013 June 12, 2013 dx.doi.org/10.1021/mp400162k | Mol. Pharmaceutics 2013, 10, 3090−3102
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with keratinocyte serum-free medium (Gibcol, USA) containing 10% FBS, the cells were suspended. The cell suspension was filtered through a stainless steel mesh to remove residual tissues. The cells were collected by centrifugation for 5 min at 1200 rpm and then plated onto 0.01% (g/g) collagen type IVcoated dishes at a cell density of 1 × 106/mL for 10 min at room temperature. The unattached cells were removed, and the rapidly adherent epidermal cells were cultured in KSFM supplemented with 100 IU/mL penicillin at 37 °C in a humidified 5% CO2 atmosphere for 3 d before replacing the medium. The medium was changed every other day. After the cells reached 80− 90% confluence, they were subcultured by detaching them with trypsin-EDTA solution. 2.3. In Vitro Transfection of ESCs by CYD-PEI Nonviral Vector. 2.3.1. Preparation and Characterization of CYD-PEI and CYD-PEI/pDNA Complexes. The nonviral vector CYD-PEI was prepared according to the method described previously.11 CYD-CDI was conjugated to PEI600 to obtain the CYD-PEI copolymer via a polycondensation reaction. The chemical structure of the prepared CYD-PEI copolymer was characterized by measuring 1H nuclear magnetic resonance (1H NMR) (AvanceTM 600, Bruker, Germany). CYD-PEI/pDNA complexes were prepared by mixing the aqueous solution of CYD-PEI with that of plasmid DNA coding for luciferase/ eGFP genes. Briefly, CYD-PEI and pDNA which have been dissolved with phosphate-buffered saline solution (PBS, pH 7.4) were mixed together and incubated for 20 min at room temperature and then diluted with PBS with the different concentrations of pDNA for test. The particle size and surface charge of the synthesized CYD-PEI/pDNA complexes with different N/P ratios (the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of DNA) were measured using laser diffraction spectrometry (Malvern Zetasizer 3000HS, Malvern, U.K.). The volume of the samples was 1 mL containing a final DNA concentration of 50 μg/mL. The cytotoxicity of CYD-PEI/pDNA complexes to seeded ESCs at day 3 was tested with MTT assay with a standardized protocol. Optical density of the solution was recorded using a Microplate Reader (Bio-Rad, model 550) at a wavelength of 570 nm. 2.3.2. Cellular Uptake of the CYD-PEI/pDNA Complexes by ESCs. ESCs were seeded on a 24-well plate precoated with 0.01% (w/w) human collagen I and then incubated overnight to reach 30−40% confluence. After replacing the medium with Opti-MEM, the CYD-PEI/FITC-DNA complexes was added, followed by incubation for 1, 3, 6, and 9 h. The cells were washed thrice with PBS, and the cellular uptake of the complexes by ESCs was observed under a microscope (Leica, Germany). 2.3.3. EGFP Expression by CYD-PEI Transfected ESCs. A widely used nonviral vector, lipofectamine 2000 (Lipo), was selected as a reference to evaluate the transfection efficiency of CYD-PEI in ESCs. ESCs were seeded in a 24-well plate at a density of 1 × 105 cells/well and then cultured overnight. The culture medium was removed before transfection, and the cells were rinsed with PBS. Each well received 1 μg of eGFP-DNA, in exclusion from the vector. The CYD-PEI/eGFP-DNA complexes were diluted in 500 μL of Opti-MEM medium. After 6 h of incubation at 37 °C, the Opti-MEM medium was replaced with KSFM medium. After 48 h of incubation, green fluorescence was observed under fluorescent micrographs, and the transfection efficiency was quantified by flow cytometry (FACS Calibur, BD Biosciences, San Jose, CA).
attachment prior to the addition of gene complexes. When the gene complexes are fixed to the substrate, 2D culture plates, or 3D scaffold, before the cells are seeded, such transfection is referred to as reverse transfection (substrate-mediated transfection).7 Gelatin is a denatured collagen highly used as a component for tissue engineering.8 In our previous study, gelatin based hydrogel exhibited a promoting effect in wound healing.9 β-TCP incorporated in the gelatin scaffold has been demonstrated to improve its cellular responses.10 A biodegradable nonviral vector, β-cyclodextrin-linked polyethylenimines (CYD-PEI), was prepared through using a cationic polymer composed of low-molecular-weight PEI (600 Da) cross-linked by β-cyclodextrin to decrease the cytotoxicity and enhance the transfection efficiency of PEI. Based on the CYD-PEI vector and gelatin/β-TCP scaffold, a 3D transfection system was constructed, and the ESCs were recombinant by the 3D system with reverse transfection method. The gelatin/β-TCP scaffold and CYD-PEI mediated 3D transfection system were compared for its in vitro transfection efficiency in ESCs with the 2D conventional and reverse transfection methods. Upon that, the ESCs recombination by the 3D transfection system was topically applied to the wound site, and their therapeutic potential in wound healing and angiogenesis was investigated.
2. EXPERIMENTAL SECTION 2.1. Materials. Gelatin/β-TCP scaffold and Nonwoven PET fabric were provided by Department of Biomaterials, Kyoto University, Japan. CYD-PEI vectors were supplied by Department of Chemistry, ZheJiang University. Collagen sponges were purchased from Qisheng Co. (Qisheng, China). Plasmid DNAs encoding luciferase or eGFP were provided by the Institute of Infectious Diseases, Zhejiang University, China. pDNA-VEGF165 were obtained from Department of Polymer Science and Engineering, Zhejiang University, China. The luciferase assay and BCA protein assay kit were purchased from Beyontime Co. (Beyontime, China). Pronectin was purchased from Arysta Health & Nutrition Science Corporation (Japan). Human collagen type IV was purchased from Sigma (Sigma, St. Louis, MO, USA). Defined keratinocyte serum-free medium (KSFM), penicillin, streptomycin, and trypsin were obtained from Gibco BRL (Gaithersburg, MD, USA). VEGF ELISA kit was obtained from Ming Rui Biotech Company (Shanghai, China). Mouse antirat CD31 monoclonal antibody was purchased from BD (New Jersey, U.S.A.). Mouse monoclonal antibodies, p63-FITC, CD34-FITC, and goat antirabbit IgGFITC were purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidase (HRP)-labeled goat antimouse IgG1 antibody was purchased from Boster, Inc. (Wuhan, China). Masson’s trichrome staining kit was purchased from Nanjing Keygen, Inc. (Nanjing, China). Buffered paraformaldehyde (4%) was purchased from Boster, Inc. (Wuhan, China). All other reagents were of analytical grade and used as received. 2.2. Isolation and Culture of ESCs. ESCs were prepared and identified with a reported protocol.3 Three-day-old rats (18−20 g) used for the ESCs extraction were supplied by Zhejiang Academy of Medical Sciences, China. Briefly, skin tissue biopsy was obtained from the back of adult rats via plastic surgical procedures. The skin sample was sterilized with 75% ethanol, rinsed in phosphate-buffered saline (PBS), and then minced into 2 mm wide strips treated with 0.25% Dispase II overnight. The epidermis was mechanically separated from the dermis and then incubated in trypsin-EDTA (0.05%) at 37 °C for 10 min to dissociate cells. After enzyme activity was blocked 3091
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2.3.4. Luciferase Activity Expressed by CYD-PEI Transfected ESCs. The plasmid PGL3 was also used to examine the transfection efficiency of both the CYD-PEI and Lipo vectors on ESCs. ESCs were seeded in a 24-well plate at a density of 1 × 105 cells/well and then cultured overnight before transfection. CYD-PEI/pDNA-PGL3 or Lipo/pDNA-PGL3 complexes were prepared by adding a copolymer solution with equal volumes of PGL3 solution with gentle vortex mixing and then incubated at room temperature for 20 min. The original cell culture medium was replaced with the solution containing the complexes and an additional 500 μL of Opti-MEM in each well. After incubation for 6 h at 37 °C, the transfection medium was replaced with fresh KSFM, and the cells were incubated for 48 h. The luciferase assay was carried out according to the manufacturer’s instructions (Promega, United States). Light units due to luciferase activity were measured with a chemiluminometer (Autolumat LB953, EG&G Derthold, Germany). All experiments were carried out in triplicate to ascertain reproducibility. 2.4. Fabrication and Characterization of CYD-PEI and Scaffold Based 3D Transfection System. 2.4.1. Biocompatibility Tests of Different Scaffolds to ESCs. The gelatin/β-TCP scaffold was prepared and characterized according to the method described previously.12 The collagen, gelatin/β-TCP scaffolds, and nonwoven PET fabric were compared for their biocompatibility as a substrate for ESC growth/attachment. Briefly, blank scaffold and scaffold with the ESCs were rinsed with PBS and then incubated in 10% formalin at 4 °C overnight. The samples were rinsed in PBS, immersed in 1% OsO4 solution for 1 h, and then dehydrated in 50%, 70%, 95%, and 100% ethanol for 20 min for each respective ethanol change. The samples were mounted on specimen holders and dried from CO2. Morphology of the samples was finally sputter coated with gold for scanning electron microscopy (SEM) observation (Hitachi, S-3000N) with an accelerating voltage of 25 kV after the samples were sputter-coated with a thin gold layer. 2.4.2. Fabrication and Characterization of CYD-PEI and Gelatin/β-TCP Scaffold Based 3D Transfection System. Based on the biocompatibility tests of different scaffolds to ESCs, the gelatin/β-TCP scaffold was selected for the construction of 3D transfection system. The gelatin/β-TCP scaffold was cut into sheets of 10 mm × 10 mm, with a thickness of 3 mm. The scaffold sheets were presterilized with 75% (v/v) ethanol and then washed repeatedly with PBS to remove any residual alcohol. Sequentially, the scaffolds were coated with anionic gelatin, pronectin solution, and the CYD-PEI/pDNA-PGL3 complexes. Morphology of the cross sections of the scaffolds with or without the CYD-PEI/pDNA-PGL3 complexes were examined by SEM (Hitachi, S-3000N). 2.5. In Vitro Transfection Efficiency of the 2D Conventional, Reverse and 3D Systems on ESCs. Anionic gelatin was prepared and characterized with a previous reported method.13 For 2D conventional transfection, ESCs were seeded in a 24-well plate at a density of 1 × 105 cells/well and then cultured overnight before transfection. CYD-PEI/pDNAVEGF165 complexes were prepared by adding a copolymer solution with equal volumes of pDNA-VEGF165 solution with gentle vortex mixing and then incubated at room temperature for 20 min. The original cell culture medium was replaced with the solution containing the 40 μL of CYD-PEI/pDNAVEGF165 complexes and an additional 500 μL of Opti-MEM in each well. After incubation for 6 h at 37 °C, the transfection
medium was replaced with 2 mL of fresh KSFM, and the cells were incubated for 3 or 7 days. For 2D reverse transfection, the 24-well plate was precoated with aqueous solutions of 100 μg/ mL anionic gelatin and 200 μg/mL pronectin in sequence at 37 °C. Each well was rinsed with PBS and then coated with 40 μL CYD-PEI/pDNA-VEGF165 complexes for 1 h. Subsequently, the well was rinsed with PBS and seeded with cells (1 × 105/ well) in 2 mL of KSFM. The cells were cultured for 3 or 7 days for test. For the 3D reverse transfection, the gelatin/β-TCP scaffold was cut into sheets of 1 mm × 1 mm, with a thickness of 3 mm. The 3D matrix was presterilized with 75% (v/v) ethanol and then washed repeatedly with PBS to remove any residual alcohol. Sequentially, the matrix was coated with anionic gelatin, pronectin, and pDNA-VEGF165/CYD-PEI complex. Afterward, ESCs (1 × 105/well) were seeded to the scaffold and incubated for 2 h for cell attachment. Approximately 2 mL of the KSFM was added slowly to the wells, and the seeded scaffold was transferred to fresh 24-well culture plates containing 2 mL of KSFM in the next day for 3 or 7 days of culture. The amount of VEGF protein secreted by the transfected ESCs was determined by a rat VEGF enzyme-linked immunoabsorbance assay (ELISA) kit (Ming Rui Biotech Company, Shanghai, China) according to the manufacturer’s instructions. All experiments were carried out in triplicate to ascertain reproducibility. 2.6. In Vivo Study: Therapeutic Effects of the Recombinant ESCs by 3D Transfection System in Rat Skin Wounds. Six-week-old (170−200 g) Sprague−Dawley rats used for the in vivo wound healing test were supplied by Zhejiang Academy of Medical Sciences, China. All experimental procedures were in accordance with the Zhejiang University guidelines for the welfare of experimental animals (Animal Experimentation Ethics Approval No: Zju2010-1-02-015). The healing properties of the transfected ESCs were studied in rat skin full-thickness excision model.14 A skin excision of 10 mm × 10 mm was made by excising the skin within the confines of the square down to the level of subcutaneous panniculus carnosus. The animals were randomly divided into four groups of six: (1) blank control group (BC), received no treatment; (2) matrix control group (MC), received topical application of gelatin/β-TCP scaffold alone with medium; (3) ESC-treated group (ESC), received topical application of the scaffold loaded with ESC suspension; and (4) transfected ESC-treated group (TESC), received topical application of the scaffold loaded with CYD-PEI/pDNA-VEGF and ESCs suspension. Scaffold with or without cell suspension was pasted over the wound bed; the wounds were further covered with sterile adhesive tegaderm dressings punctured with sterile needles to allow air exposure. After surgery, each animal was placed in one cage. Fresh medium was added into the scaffold, and newly tegaderm dressings were attached every 3 d. All animals were maintained under constant conditions and had free access to standard diet and drinking water. Any postsurgery pain, distress, or complications were checked daily. On postoperative days 14 and 34, the rats were euthanized, and their reconstituted skins were harvested for assays. 2.6.1. Wound Closure Test. The progressive healing in various groups on days 3, 5, 7, 9, and 14 was recorded by macroscopic observation. The wound closure percentages at different time points at postwounding were measured every 2 days since day 3 by copying the wounds with filter papers and calculating the weight percentage of the filter papers. Wound 3092
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Figure 1. Characterization, cytotoxicity, and in vitro transfection of ESCs by nonviral vectors. (A) Chemical structure of CYD-PEI. (B) Cytotoxicity of the CYD-PEI/pDNA polyplexes to ESCs. (C) Characterization (particle size and zeta potential) of the CYD-PEI/pDNA polyplexes. (D) Cellular uptake of the CYD-PEI/pDNA polyplexes by ESCs at 1, 4, 6, and 9 h. (E) eGFP expression in ESCs transfected by CYD-PEI and Lipo (control) vectors observed under a fluorescence microscope. (F) Luciferase activities of ESCs transfected by CYD-PEI and Lipo (control) vectors.
closure percentage (%) = (area on day 0 − open area on day n)/area on day 0 × 100. 2.6.2. Scanning Electronic Microscopy Characterization of the Dermal Collagens. The wounded specimens (hypodermis removed) were fixed in 4% buffered paraformaldehyde for 24 h at 4 °C and then washed well in PBS to remove excess fixatives. The tissues were sequentially dehydrated in 75%, 85%, 95%, and 100% ethanol. Afterward, the samples were mounted on specimen holders and dried from CO2. Finally, the samples were sputter-coated with gold for SEM observation (Hitachi X650, Tokyo, Japan). The imaging of the collagen deposition was performed using SEM. 2.6.3. Sirius Red Staining of the Collagens of Type I and Type III. The wounded specimens including the full-thickness skin layers (epidermis and dermis) on day 14 postwounding were fixed in 4% buffered paraformaldehyde solution at 4 °C, dehydrated with a graded series of ethanol, and embedded into paraffin according to routine light microscope tissue processing methods. The sectioned samples with a thickness of 5 μm were then stained with sirius red and visualized by a polarizing
microscope (Nikon E200, Japan). Three regions with the same area were selected randomly from each skin specimen to calculate the distribution of collagen types I and III (percentage of collagen I or III in the sum of collagens I and III) with Image Pro-plus 5.1 (Nikon). 2.6.4. Masson’s Trichrome Staining. The sectioned samples with a thickness of 5 μm was stained with Masson’s trichrome and visualized with an optical microscope image system (Leica). 2.6.5. FITC-p63 and FITC-CD34 Molecular Immune Fluorescence Staining of the TESCs/ESCs Engrafted in the Wounded Skins. The wounded skins were stained with two molecular surface antigen markers of ESCs, FITC-p63, and FITC-CD34 to analyze the engraftment of the topically applied ESCs/TESCs into the wound sites. Paraffin sections (5 μm) were deparaffinized in xylol and then rehydrated in graded alcohol series. Endogenous peroxidase was inhibited using 3% H2O2 in methanol for 10 min. The sections were washed with distilled water and then soaked in 0.01 M citrate buffer (pH 6.0) for epitope retrieval. The slices were washed thrice with 1 3093
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Figure 2. (A−F) Biocompatibility test of the collagen, gelatin/β-TCP, and nonwoven PET fabric scaffolds. SEM images of the collagen sponge at 100 μm (A), gelatin/β-TCP matrix at 100 μm (B), and nonwoven PET fabric at 100 μm (C) without ESCs. SEM images of the collagen sponge at 10 μm (D), gelatin/β-TCP matrix at 50 μm (E), and nonwoven PET fabric (F) seeded with ESCs at 50 μm. Red arrows indicate the attached ESCs. (G−L) Characterization of the gelatin/β-TCP matrix loaded with the CYD-PEI/pDNA polyplexes. (G and J) SEM images of the blank gelatin/βTCP matrix at 5 μm and 500 nm, respectively (blue arrows indicate the matrix material particle); (H and K) SEM images of the gelatin/β-TCP matrix loaded with ESCs at 1 μm and 500 nm, respectively (yellow arrows indicate the attached ESCs); (I and L) SEM images of the gelatin/β-TCP matrix loaded with the pDNA/CYD-PEI complex at 1 μm and 500 nm, respectively (green arrows indicate the incorporated complex). (M−O) In vitro VEGF expression by ESCs upon 2D conventional, reverse, and 3D transfection systems on day 3 (M) and day 7 (N). (O) VEGF expression by ESCs upon 3D transfection on day 3 and day 7 (n = 3; *denotes statistically significant difference, p < 0.05).
M PBS (pH 7.2−7.6) and then incubated with 5% bovine serum albumin at room temperature for 20 min. The tissue
sections were incubated with diluted FITC-p63 or FITC-CD34 primary antibody at 4 °C overnight and then washed thrice 3094
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3.1.2. Cellular Uptake of the CYD-PEI/pDNA Complexes by ESCs. Figure 1D shows the cellular internalization procedure of the FITC-DNA/CYD-PEI complexes; the complexes were internalized for 2 h. The complexes entered the nucleus and reached the peak within approximately 4 h, and fluorescence decreased apparently within approximately 9 h. Therefore, the ability of nonviral vectors to escape quickly from endosomes and enter the nucleus is essential for their transfection efficiency and cytotoxicity. The cellular uptake studies showed that the CYD-PEI complexes can be absorbed within 6 h and can escape from endosomes at a proper rate within approximately 9 h. 3.1.3. In Vitro Transfection Efficiency of CYD-PEI in ESCs. It was seen that pEGFP of ESCs transfected by CYD-PEI showed a relatively stronger fluorescence signal (Figure 1E-I) than those transfected by Lipo (Figure 1E-II). As displayed in Figure 1E and F, the percentages of the ESC-expressed eGFP after transfection by CYD-PEI were relatively 3-fold higher (5.43%, Figure 1E-III) than those after transfection by Lipo (1.84%, Figure 1E-IV). Similar results were obtained in the delivery of pDNA-PGL3, in which the PGL3 expressions of 6 × 10 × 104RUL/mg and 2 × 10 × 104RUL/mg were obtained in the groups transfected by CYD-PEI and Lipo, respectively (Figure 1F). 3.2. Fabrication and Characterization of CYD-PEI and Scaffold Based Three-Dimensional (3D) Transfection System. 3.2.1. Biocompatibility of Different Scaffolds to ESCs. With the commercially available collagen sponge and PET nonwoven fabric as a reference, the biocompatibility of gelatin/β-TCP as substrate for ESC growth/attachment was evaluated. Figure 2A/D, B/E, and C/F shows the SEM images of the collagen, gelatin/β-TCP, and PET fabric scaffolds without/with ESCs after 3 d of incubation, respectively. Several ESCs with characteristic morphology were distributed homogeneously inside the gelatin/β-TCP scaffold (Figure 2D). By contrast, the number of cells attached to the collagen scaffold (Figure 2E) or PET nonwoven fabrics (Figure 2F) were much less. In addition, the poor mechanical strength of collagen scaffold and the nondegradability of PET fabric limit their application. The gelatin incorporated with 50% β-TCP was prepared with an optimized protocol; this 3D scaffold presented a highly interconnected structure with a pore size distribution of 179.1 μm ± 27.8 μm and a porosity of 96%. The rough surface of this scaffold is helpful in promoting the attachment of cells and nanoparticles.17 As previously reported, the gelatin/β-TCP scaffold was also demonstrated for its superiority for ESC growth/attachment in present study.3 Therefore, it was selected as a scaffold reservoir for the gene transfection and topical delivery of ESCs. 3.2.2. Characterization of CYD-PEI and Gelatin/β-TCP Scaffold Based 3D Transfection System. The incorporation of the CYD-PEI/pDNA complexes in the gelatin/β-TCP scaffold was characterized via SEM. Scaffold material particles, cells, and pDNA-vector complexes with different sizes contained in the scaffold were identified. Only scaffold material particles >1 μm in size were identified in the blank gelatin/β-TCP scaffold (Figure 2G and J, indicated by blue arrows). By contrast, ESCs approximately 500−700 nm in size (indicated by yellow arrows, Figure 2H and K) and the pDNA-vector complexes approximately 200 nm in size (indicated by green arrows, Figure 2I and L) with spherical shapes were identified in the scaffold. 3.3. In Vitro Transfection Efficiency of the 2D Conventional, Reverse and 3D Systems on ESCs. The
with PBS for 15 min each. Afterward, the slices were incubated with goat antirabbit IgG-FITC secondary antibody at 37 °C for 30 min. The tissue sections were washed thrice with 1 M PBS (pH 7.2 to 7.6) for 15 min each. After drying, the sections were kept in resin and observed using the Leica image system. 2.6.6. Observation of Blood Vessel Formation in Wounded Skins. The wounded skin specimens (included epidemis and dermis) on days 14 and 34 postwounding were observed for the blood vessels formation under anatomical lens. 2.6.7. CD31 Molecular Immunohistochemical Staining of the Wounded Skin. To study the angiogenesis during the healing process, one of the specific molecular markers, CD31 was detected by immunohistochemistry. Paraffin sections (5 μm) were deparaffinized in xylol and then rehydrated in graded alcohol series. Endogenous peroxidase was inhibited using 3% H2O2 in methanol for 10 min. The sections were washed with distilled water and then soaked in 0.01 M citrate buffer (pH 6.0) for epitope retrieval. The slices were washed thrice with 1 M PBS (pH 7.2 to 7.6) and then incubated with 5% bovine serum albumin at room temperature for 20 min. The tissue sections were incubated with diluted CD31 primary antibody at 4 °C overnight and then washed thrice with 1 M PBS for 15 min each. Subsequently, the slices were incubated with HRPlabeled secondary antibody at 37 °C for 30 min. The tissue sections were washed thrice with 1 M PBS for 15 min each, stained with 3,3′-diaminobenzidine chromogenic kit, and then rinsed with distilled water. After drying, the sections were kept in resin and observed using the Leica image analyzing system. 2.6.8. ELISA Determination of the VEGF Expression in Wounded Skin Homogenate. VEGF protein levels in the wounded skin homogenate, another key factor of angiogenesis, were also detected. Briefly, the wounded skins were homogenized in lysis buffer (0.1 M Tris-HCl, 2 mM EDTA, and 0.1% Triton X-100). The lysate (2 mL) was centrifuged at 12 000 rpm at 4 °C for 5 min to collect the supernatant. The amount of VEGF protein was determined using a human VEGF ELISA kit following the manufacturer’s instructions. 2.7. Statistical Analysis. All values were expressed as mean ± standard deviation (SD). Two-tailed Student’s t tests was performed for data comparison of paired samples. One-way ANOVA analysis was used for multiple group comparisons. A probability (p) value of
