| Background: Human skin is composed of the epidermis, dermis and hypodermis. The thickness of skin varies from 1 to 4 mm, accounting for approximately 15% of the total body weight. The stratum corneum, with a thickness of 10 to 15 μm, forms the outermost region of the epidermis and prevents excessive water loss and unwanted materials from entering the body. Hair follicles, sebaceous glands and sweat glands are specialized epithelial structures that are primarily located within the dermis and hypodermis and connected to the surface epidermis. The dermis consists of cells, fibrous molecules(mainly collagen and elastic fibers) and a ground substance; the dermis is separated from the epidermis by the dermal-epidermal junction. Both the components and the morphological structures of skin are crucial for the skin’s normal functions. Dermal structures vary somewhat depending on depth(papillary or superficial dermis; reticular or deep dermis) and dermal injuries(normal skin or scars). Collagen and elastic fibers are the main components of dermal fibrous connective tissue and are closely related to wound healing, scar formation and the biomechanical properties of the skin. The thickness and orientation of collagen bundles vary between the superficial and deep dermis, longitudinal and transverse dermis sections, and scarred and normal skin. For example, the diameters of the elastic fibers in scars are reduced compared with normal skin, both in superficial(0.54 ± 0.04 μm vs. 1.08 ± 0.04 μm) or deep dermis(1.16 ± 0.02 μm vs. 1.99 ± 0.07 μm). Dermal morphological structures also affect the skin’s biomechanics. However, to our knowledge, whether the dermal structures are different close to or far away from hair follicles has never been reported. Tissue-engineered(TE) skin is one of the most promising methods for extensive skin defects and enhanced cosmetic outcomes. The physical properties of engineering scaffolds, such as their constituent elements, porosity, pore diameter and wall thickness, are directly related to the fabrication methods used and greatly affect nutrient penetration, biomechanics, biodegradability, cell proliferation and conformation. Engineered skin that contains components and micro-structures similar to normal skin may be more effective in wound healing. Therefore, gaining a better understanding of skin micro-structures should be helpful for the next generation of skin engineering. In the first part of our study, we focused on the three-dimensional(3D) reconstructionof human dermal collagen and elastic fibers using serially sectioned specimens and quantitative analysis of dermal fibers based on subregions defined by their distance from hair follicles and subregions defined by dermal depth. The collagen fibers referred to in our manuscript are collagen type I. The porosity, pore diameters and wall thickness of human acellular dermal matrix(ADM), which was made from the same skin samples, were acquired by micro-computed tomography(micro-CT). The structure of dermal scaffolds greatly affects the engineered tissue’s functions and the activities of seeded cells.Current strategies of dermal scaffold design tend to yield a homogeneous architecture with a uniform pore size. However, based on the results of the first part of our study, the structures of the human dermis are not homogeneous in terms of either interstitial spaces or architecture at different dermal depths. In the second part of our study, a biomimetic fibroblasts-loaded artificial dermis composed of three-layer scaffolds with different pore sizes was prepared. The three-layer scaffolds, which look similar to a sandwich, mimic the natural structures of the human dermis, which has comparatively larger pores in the outer layers and smaller pores in the middle layer. The fibroblasts-loaded artificial dermis were shown to favor wound healing by promoting granulation tissue formation and wound re-epithelialization, as determined by a histological study and Western blotting. Our data indicated that the biomimetic fibroblasts-loaded artificial dermis with "Sandwich" structure and designed gradient pore sizes may hold promise as tissue-engineered dermis. In the third part of our study, we aimed to fabricate another artificial dermis, which was able to promote angiogenesis, because the "Sandwich" artificial dermis was not found to benefit vascular formation. In order to solve this problem, we used a convenient and novel method to produce VIP loaded microspheres in polycaprolactone(PCL) nanofibrous membrane without complicated processes. Vasoactive intestinal peptide(VIP) was reported to promote angiogenesis. Electrospun nanofibers lead to idea wound dressing substrates. We first coated mussel-inspired dopamine(DA) to nanofibers, then used strong adhesive DA to absorb the functional peptide. PCL membrane was then immersed into acetone to generate microspheres with VIP loading. We employed high pressure liquid chromatography to record encapsulation efficiency of(31.8 ± 2.2) % and loading capacity of(1.71 ± 0.16) %. The release profile of VIP from nanosheets showed a prolonged release. The results of laser scanning confocal microscope, scanning electron microscope and cell counting kit-8 proliferation assays showed that cell adhesion and proliferation were promoted. The resulting VIP-DA-coated PCL(PCL-DA-VIP) nanosheets with spatiotemporal delivery of VIP could be a potential application in wound treatment and vascular tissue engineering.Objective: One aim of the present study is to acquire the information of the natural histological structures of the human dermis. Another aim of our study is to fabricate the biomimetic artificial dermis based on the previous information.Part One Three-dimensional histological structures of the human dermisMethods: 1. Acquiring the 3D reconstructed histological images of human dermis(Image registration, image segmentation, 3D imaging and visualization). 2. Evaluation of the fiber distribution in subregions. Subregions defined by the distance from hair follicles: Close to hair follicles(C-HF) and far away from hair follicles(F-HF) Subregions defined by depth: from upper to lower dermis(from papilla layer to reticular layer) 3. Porosity, pore diameters and wall thickness of human ADM was determined by micro-CTResults: 1. We successfullyacquired the 3D reconstructed histological images of human dermis. 2. Fiber distribution in subregions defined by distance from hair follicles 3. Fiber distribution in subregions defined by depth from the upper to lower dermis 4. Porosity, pore diameter and wall thickness of human ADM as determined by micro-CTPart Two Biomimetic fibroblasts-loaded artificial dermis with "Sandwich" structure and designed gradient pore sizes promoted wound healing via favoring granulation tissue formation and wound re-epithelializationMethods: 1. Preparation of collagen scaffolds with different pore sizes Determination of scaffold pore sizes and microstructures Measurement of scaffold biodegradation in vitro 2. One-layer artificial dermis in vitro: Culture and seeding of mouse fibroblasts Evaluation of the cell distribution in the scaffolds Determination of cell proliferation In vivo: Implantation of fibroblast-loaded one-layer artificial dermis in vivo 3. Three-layer artificial dermis(“Sandwich†and “Homogeneous†structures) In vitro: Culture and seeding of mouse fibroblasts Evaluation of the cell distribution in the scaffolds Determination of cell proliferation In vivo: Fabrication of three-layer artificial dermis(“Sandwich†and “Homogeneous†structures) Implantation of fibroblast-loaded three-layer artificial dermis Measurement of wound healing Hematoxylin-eosin and Masson trichrome staining Immunohistochemistry Western blottingResults: 1. One-layer artificial dermis The effect of pore size on the biodegradation of collagen scaffolds in vitro The effect of pore size on the adhesion and proliferation of seeded fibroblasts in vitro The effect of pore size on wound healing in vivo 2. Three-layer scaffolds The effect of three-layer scaffolds on wound healing in vivo Mechanism:The 166.9/87.7/166.9 μm-pore-size “Sandwich†artificial dermis favored cell proliferation in vivo.Part Three In-situ Generated Vasoactive Intestinal Peptide Loaded Microspheres in Mussel-inspired Polycaprolactone Nanosheets Creating Spatiotemporal Releasing Microenvironment to Promote Cell Proliferation in vitro.Methods: 1. Preparation and characteristics of PCL-DA-VIP nanosheets. Measurement of the VIP-release behavior in vitro 2. In vitro Evaluation of the cell proliferation in vitroResults: 1. Preparation and characteristics Microspheres were in-situ generated in nanosheets after the treatment of acetone The loading capacity(LC) and the encapsulation efficiency(EE) Microspheres prolonged the releasing of VIP 2. In vitro Spatiotemporal delivery of VIP significantly promoted cell proliferation in vitroConclusion: In this study, we acquired the 3D reconstructed histological images of human dermis and fabricated two artificial dermis( "Sandwich" collagen scaffolds and PCL-DA-VIP nanosheets).We observed the following information: 1. Fiber distribution in natural human dermis and parameters for fabricating biomimetic scaffolds. Fewer collagen and elastic fibers in the layers either close to the epidermis or close to the hypodermis. The pixels pertaining to collagen fibers and elastic fibers constituted(28.96 ± 14.63) % and(8.06 ± 3.75)% of all dermal pixels. The porosity of human ADM was(68.3 ± 5.8)%; The mean pore diameter of human ADM was 131.2 ± 96.8 μm and the median value was 95 μm; The mean wall thickness of ADM was 207.2 ± 251.7 μm as assessed by micro-CT. 2. We studied the effect of one-layer scaffolds with different pore sizes on cellular behavior and wound healing. We studied the effect of three-layer scaffolds with “Sandwich†and “Homogeneous†structures on cellular behavior and wound healing. The 166.9/87.7/166.9 μm-pore-size “Sandwich†artificial dermis significantly promoted wound healing. 3. We report a convenient and novel method to produce VIP loaded microspheres in polycaprolactone(PCL) nanofibrous membrane without complicated processes. The spatiotemporal delivery of VIP significantly promoted cell proliferation in vitro In summary, we acquired the 3D reconstructed histological images of human dermis and fabricated two artificial dermis( "Sandwich" collagen scaffolds and PCL-DA-VIP nanosheets). Firstly, this study represents the first time that 3D reconstructed digital images of collagen and elastic fibers in the human dermis have been examined. The results of this study should not only provide insight into the micro-structures of the skin but should also serve as a guide for fabricating bionic skin. Secondly, pore size effect on wound healing had been extensively studied. It is still not well understood whether dermal scaffolds with a uniform pore size are better than that with varied pore sizes, which are similar to human dermis as determined by our previous work. In our study, we demonstrated that the ‘‘sandwich†collagen scaffolds mimicking the natural structures of the human dermis significantly promoted wound healing compared with the ‘‘Homogeneous†scaffolds with a uniform pore size. These results may be helpful in the design of dermal scaffolds. Thirdly, we proposed a convenient and novel method to in-situ produce the peptide-loaded microspheres to prolong the spatiotemporal releasing of peptides, instead of using traditional methods that microspheres had to be pre-made and then transferred. The prepared nanosheets with spatiotemporal delivery of VIP were in vitro showed to significantly promote cell proliferation... |
