Anisotropic Biomimetic Silk Scaffolds for Improved Cell Migration and

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Biological and Medical Applications of Materials and Interfaces

Anisotropic Biomimetic Silk Scaffolds for Improved Cell Migration and Healing of Skin Wounds Guozhong Lu, Zhaozhao Ding, Yuanyuan Wei, Xiaohong Lu, Qiang Lu, and David L Kaplan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18626 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 9, 2018

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ACS Applied Materials & Interfaces

Anisotropic Biomimetic Silk Scaffolds for Improved Cell Migration and Healing of Skin Wounds Guozhong Lua,#, ZhaoZhao Dingb,#, Yuanyuan Weic,#, Xiaohong Lub, Qiang Lu b,*, David L. Kapland

aDepartment

of Burns and Plastic Surgery, The Third Affiliated Hospital of Nantong University, Wuxi 214041, People's Republic of China

bNational

Engineering Laboratory for Modern Silk & Collaborative Innovation Center

of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China cDepartment

of Maternity and Child Care Hospital, Gansu Province, Lanzhou730050, People's Republic of China

dDepartment

#The

of Biomedical Engineering, Tufts University, Medford, MA 02155, USA

authors have the same contribution.

Corresponding author: *Qiang Lu, Tel: (+86)-512-67061649; E-mail: [email protected]

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ABSTRACT Improved and more rapid healing of full-thickness skin wounds remains a major clinical need. Silk fibroin (SF) is a natural protein biomaterial that has been used in skin repair. However, there has been little effort aimed at improving skin healing through tuning the hierarchical microstructure of SF-based matrices and introducing multiple physical cues. Recently, enhanced vascularization was achieved with SF scaffolds with nanofibrous structures and tunable secondary conformation of the matrices. We hypothesized that anisotropic features in nanofibrous SF scaffolds would promote cell migration, neovascularization and tissue regeneration in wounds. To address this hypothesis, SF nanofibers were aligned in an electric field to form anisotropic porous scaffolds after lyophilization. In vitro and in vivo studies indicated good cytocompatibility, and improved cell migration and vascularization than nanofibrous scaffolds without these anisotropic features. These improvements resulted in more rapid wound closure, tissue ingrowth and the formation of new epidermis, as well as higher collagen deposition with structure similar to the surrounding native tissue. The new epidermal layers and neovascularization were achieved by day 7, with wound healing complete by day 28. It was concluded that anisotropic SF scaffolds alone, without a need for growth factors and cells, promoted significant cell migration, vascularization and skin regeneration and may have the potential to effectively treat dermal wounds. KEYWORDS: Silk, anisotropic, wound healing, vascularization, cell migration


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1. INTRODUCTION Millions of patients worldwide suffer severe skin loss each year. Many of these full-thickness skin wounds resulting from trauma, burns or chronic disease cause physiological and functional problems.1,2 Would healing is a complex physiological process which involves a series of interactions between multiple cell types, soluble factors and extracellular matrix (ECM) components.3-6 Recovery from such wounds, if such healing is to happen, typically occurs within 10 weeks with an increased risk of scar contraction with longer healing duration.7,8 Autologous skin grafting can be effective for patients with extensive burn injuries or traumatic skin loss, however, sufficient donor sites for skin on the patient and the formation of new wounds at the donor sites remain serious clinical challenges.9-11 Skin grafts have been developed to accelerate wound healing and address contraction. However, the deficiency of early blood vessel and neodermis formation for these grafts hampers high-quality tissue regeneration.5,12,13 There is a unmet need for developing new skin substitutes that provide favorable microenvironments for improved vascularization, more rapid host cellular infiltration and progressive remodeling to form neodermis and epidermis.

Silk fibroin (SF) has been used in scaffolds for tissue regeneration due to its biocompatibility, unique mechanical properties, tunable biodegradability and minimal inflammatory reactions.14-21 Improved skin regeneration on SF scaffolds through tuning microstructure, mechanical properties, retention of volume with minimal contraction, as well as degradation behavior has been reported, suggesting a



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promising future for these types of scaffolds as skin substitutes.22,23 However, few studies were successfully introduced multiple physical cues such as tunable stiffness, nanofibrous structures and aligned morphologies simultaneously into same SF scaffolds for better wound healing capacity.

Recently, a self-assembly mechanism to induce SF nanofiber formation in aqueous solution was developed and silk scaffolds composed of ECM-like nanofibers were prepared.24

The

scaffolds

with

biomimetic

nanostructures

showed

better

biocompatibility and achieved vascularization and enhanced fibroblast penetration when implanted subcutaneously in mice through tuning the secondary structure of the scaffolds. Meanwhile, silk nanofiber hydrogels with hierarchical structural alignment were also developed and demonstrated potential to enhance the alignment and migration of cells.25-27 All of these developments supported our hypothesis that scaffolds forming suitable microenvironments with multiple physical cues for skin regeneration through the careful fabrication of SF structures would promote enhanced wound healing outcomes.

Based on our recent developments, we further developed SF nanofibrous scaffolds with aligned porous structures and evaluated their efficacy as substitutes for skin regeneration (Scheme 1). The hierarchical ECM-like morphology promoted cell attachment, proliferation, and organization in the scaffolds, while the aligned structures favored the penetration of fibroblasts and endothelial cells, which promoted


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vascularization and would closure.

Scheme 1. The preparation of the scaffolds.

2. EXPERIMENTAL SECTION 2.1 Preparation of Aqueous Silk Solutions and Silk Nanofiber Solutions. Aqueous silk solution was obtained according to typical lithium bromide solution system.25 After

the

dissolution-dialysis-centrifugation

processes,

silk

solution

with

concentration of about 6 wt% was prepared and stored at 4oC for further use. Silk nanofiber solution was then assembled through concentration-dilution-thermal culture method reported previously.24 Silk assembled to metastable particles with size of about 100 nm in the concentrating process and then disassembled to form nanofibers in the diluted solutions with concentration of 0.7wt% at 60oC.

2.2 Preparation of Aligned Silk Nanofiber Scaffolds. Silk nanofiber solution (0.7 wt%) was treated with electrical field based on previous protocol. 25,28-30 After treated under 50 VDC for 20 min, aligned silk nanofiber hydrogels formed near the positive electrode. The hydrogels were frozen at -20oC for 24 h and lyophilized for above 48 h to obtain aligned silk nanofiber scaffolds. The aligned scaffolds were termed AS. Porous silk nanofiber scaffolds without aligned structure were also prepared through



freeze-drying the silk nanofiber solutions, but failed to be used in our present study due to their fragile property. Therefore, as a control, porous silk scaffolds with vascularization capacity were prepared through our acid-assisted lyophilization processes. 31 The porous scaffolds were termed PS.

2.3 Characterization. The morphology of silk scaffolds was observed via a Hitachi S-4800 microscope (Hitachi, Tokyo, Japan) at 3 kV. Before investigation, the scaffold samples were sprayed with gold according to the protocol used in our previous study.32 The secondary structure of the different scaffolds were estimated by Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectrometry. The specific parameters keep same with previous works in our group.32-34

2.4 Scaffolds Degradation Analysis. All samples (30 mg) were soaked in protease XIV solutions with the concentration of 5U ml-1 in phosphate buffers, and kept at 37oC in a shaking water bath.31 At designed time points, the samples were washed several times with distilled water and dried at 60oC for above 12 h. The degradation rate was then calculated through the following formula:  (%)  m0  m1 / m0 100 Where ε expressed the degraded rate of the scaffold, m0 represented the original mass of the scaffold, while m1 was the mass of degraded scaffold. Five samples were measured for each scaffold.

2.5 Mechanical Properties. The compressive moduli of the scaffolds in wet states


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were analyzed using a Food Texture Analyzers (TMS-Pro, FTC, USA) with a 25 N loading cell.35 Samples were cut into 12mm in diameter and 13mm in height, and immersed in distilled water for above 4 h to maintain hydrated state and then compressed to above 30% of their primary length at rate of 2 mm/min. Each scaffold was measured five times to get the mean value of the stiffness.

2.6 Cells Culture and Characterization of Cells Behavior on Different Scaffolds. Rat bone marrow cells (BMSCs) were isolated from 3 to 4 week-old male Sparague-Dawley rats by flushing femurs with the Dulbecco's modified Eagle medium (DMEM, low glucose), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, cultured in cell incubator at 37 oC, 5% CO2.

Human

umbilical vein endothelial cell (HUVECs, obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China ) were cultured in Dulbecco's modified Eagle medium (DMEM, high glucose). Other culture conditions remain same as BMSCs.

The BMSCs and HUVECs morphology on the tested different silk scaffolds were examined by laser confocal scanning microscopy (CLSM, Olympus FV10 inverted microscope, Nagano, Japan) and Hitachi S-4800 microscope (Hitachi, Tokyo, Japan). At specified time points, CLSM samples were fixed with 4% paraformaldehyde at room temperature for 30 min, then washed with PBS and penetrated by 0.1% Triton X-100. Finally stained the cytoskeleton and nuclear by FITC-Phallodin and DAPI at room temperature, respectively. SEM samples were fixed with 2.5% glutaradehyde at



4oC for 2 hours, washed with PBS and dehydrated by gradient of alcohol from low to high concentration, then samples were dried and sprayed gold before observed, as described in our previously studies.36,37 To analyze the cell proliferation on the different scaffolds at the indicated time point, the DNA content was detected by Quanti-iTTM PicoGreenTM dsDNA assay kit according to the reported protocol.31

2.7 In Vivo Study. 6 to 8 week-old male Sparague-Dawley rats were used in the in vivo study. The use was approved and granted by animal ethnic committee of Soochow University. Two animal models including subcutaneous implantation and full-thickness skin defects were provided to evaluate the vascularization and tissue ingrowth. Both the animal models were established according to previous studies.35,38,39 For the subcutaneous implantation experiment, the rats were anesthetized with 4% chloraldurate through intraperitoneal injection, then cut four incisions with size of 1 cm on the dorsal of rats. Sterile scaffolds were implanted into the subcutaneous pockets. For full-thickness skin defects model, dermal defects wound with a diameter of 1.2 cm were constructed on the dorsal. Each group of the scaffolds was covered on the wound, and wrapped with sterile gauze. At the indicated time points of post operation, animals were euthanized for sample collection. Histology analysis (hematoxylin and eosin staining (H&E), Masson Trichrome Staining, immunohistochemistry for CD34 staining (1:100 dilution, Abcam) and immunofluorescence staining for CD31(1:100 dilution, Ab28364, Abcam) ) were used to evaluate the blood vessel formation and tissue ingrowth according to previous


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studies.29,40

2.8 Statistical Methods: All results were statistically analyzed by SPSS v.16.0 software. One-way AVOVA was used to compare the mean values of the data. Values are presented as means ± standard deviations. A value of P < 0.05 was supposed to be statistical significant.

3. RESULTS AND DISCUSSION 3.1 Formation and Characterization of the Anisotropic Scaffolds. Insight into silk self-assembly provided the capacity to design hierarchical microstructures and conformations of silk-based materials, resulting in the introduction of multiple physical cues.24,41 Stable silk nanofibers with high beta-sheet (crystalline) structure were assembled in aqueous solutions without salt and then aligned under an electric field, forming silk nanofiber hydrogels with anisotropic features.25,28 These hydrogels had good biocompatibility because of the ECM-mimetic nanofibrous structure and composition, and also regulated cell migration through the aligned structures.25 However, it was difficult to entrap the cells inside the hydrogels since the existence of salts in SF solution could result in high electrical current and then quick temperature increase and even boiling of the solution. Therefore, anisotropic hydrogels were freeze-dried to transform the scaffolds without structural damage. The scaffolds maintained aligned porous morphology (Figure 1A a and c) with high magnification images indicating the porous walls composed of silk nanofibers and confirming



hierarchically aligned structures. The spacing of the aligned nanofiber layers was about 100-200 um, suitable for penetration of fibroblasts and endothelial cells. Both FTIR and Raman spectra (Figure 1B and C) revealed the existence of silk II structure, which endowed the scaffolds with sufficient hydrophobic properties and insolubility in water. 29,42 Since homogeneous hydrogels composed of the same nanofibers could not form stable scaffolds after lyophilization, silk nanofibrous scaffolds with vascularization capacity previously developed in our group were used as controls (Figure 1A b and d).43 The two scaffolds were termed PS (porous scaffolds) and AS (Aligned scaffolds), respectively. Unlike the AS scaffolds, the PS scaffolds are mainly composed of amorphous state (Figure 1B and C), resulting in softer mechanical properties with stiffness of about 6 kPa (Figure 1D). Based on plenty of previous studies,

43-45

the stiffness within 1-7 kPa could provide suitable mechanical cues to

induce endothelial differentiation and better blood vessel formation.31,46 Similar to anisotropic hydrogels reported previously, the anisotropic structure of the AS scaffolds derived from aligned layers and nanofibers (Figure 1A) also lead to anisotropic stiffness. Compared to that measured when the compressive force was parallel to the aligned silk layers, significantly lower compressive moduli appeared after the compressive force was orthogonal to the layers. Similar to previous studies,29,32,43 the different β-sheet content had significant influence on the degradation behaviors after exposure to protease XIV solution for 24h where the PS scaffolds mainly composed of amorphous state lost above 70% of their original weight while the AS scaffolds containing higher β-sheet content maintained about


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50% of their original weight (Figure 1E). Therefore, besides aligned micro-nano hierarchical structures, the AS scaffolds also achieved various mechanical properties and degradation behaviors that would further influence cell behaviors and wound healing capacity.

Figure 1. Characterization of silk scaffolds: (A) SEM images of the silk scaffolds structures. a and b are the macroscopic of aligned silk scaffold and porous silk scaffold respectively; c and d are the high magnification of the porous walls of aligned and porous silk scaffold respectively. The red arrow indicates the oriented direction inside the AS scaffolds. (B, C) FTIR and Raman spectra of silk scaffolds. (D) The mechanical of silk scaffolds. (E) The enzyme degradation behaviors of silk scaffolds. Statistically significant*P≤0.05.



3.2 In Vitro Cytocompatibility of the Scaffolds. BMSCs adhesion and proliferation were carried out to assess cell compatibility. Different cell proliferation behavior was found in Figure 2, where cell numbers continued to increase without reaching a plateau until 12 days on the two scaffolds (PS and AS). Confocal microscopy and SEM revealed the morphology and distribution of the cells on the scaffolds. Although the cells adhered well on the PS and AS scaffolds, the cytoskeleton showed aligned and elongated morphology on the AS scaffolds, implying the influence of the anisotropic features. DNA content revealed higher cell numbers on the aligned scaffolds, suggesting better BMSC cell proliferation possibly due to the ECM-biomimetic nanofibrous morphology and the convenience of nutrient transportation inside aligned structures.

Figure 2. In vitro cytocompatibility of the different scaffolds: (A) Laser confocal scanning microscopy images of BMSCs fostered on porous silk scaffolds and aligned


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silk scaffolds at day 1(a-d), day 6 (e-h) and day 12 (i-l). The red arrows indicate cell orientation. The scale bar was 100μm. (B) SEM results of BMSCs cultivated on porous silk scaffolds (a-b) and aligned silk scaffolds (c-d) at day 12. (C) BMSC proliferation on different silk scaffolds on days 1, 3, 6, 9 and 12. Statistically significant ***P≤0.001.

Enough vascularization is a critical step in wound healing process. Our previous study revealed that suitable stiffness of the PS scaffolds endowed the scaffolds vascularization capacity to facilitate tissue regeneration.29 Here, human umbilical vein endothelial cell (HUVECs) was cultured on the AS and PS scaffolds to evaluate their angiogenic ability in vitro (Figure 3). Although the HUVECs proliferated better on the PS scaffolds than on the AS scaffolds due to more appropriate mechanical cues of the PS scaffolds (Figure 3A and C), the cells migrated faster and deeper on the AS scaffolds because of the aligned hierarchical structures (Figure 3B and D). After cultured for 3, 6 and 12 days, the migration depth of the endothelial cells inside the AS scaffolds was 184μm, 282μm and 343μm which was significantly deeper than that cultured in the PS scaffolds (121μm, 181μm and 244μm). The results suggested that various physical cues such as stiffness and aligned structures could regulate the vascularization capacity of the scaffolds differently. The final vascularization capacity of the scaffolds should be confirmed via animal studies in vivo. These in vitro cell culture studies also imply the possibility of further optimizing angiogenic ability of the scaffolds through tuning the stiffness and aligned structure simultaneously in



future.

Figure 3. (A) Human umbilical vein endothelial cells seeded on PS and AS scaffolds and examined by laser confocal scanning microscopy at 1, 6 and 12 days. The blue color (DAPI) indicates silk scaffolds and cell nuclear, while the red color (rhodamine labeled phalloidin) indicates actin cytoskeleton. The scale bar was 100μm. (B) Human umbilical vein endothelial cells migration on different scaffolds at days 3, 6 and 12 were observed via laser confocal scanning microscopy. The scale bar was 100μm. (C and D) Quantification analysis of DNA and the human umbilical vein endothelial cells migration distance. Statistically significant *P≤0.05, **P≤0.01.

3.3 In Vivo Compatibility of the Scaffolds in Rat. We next evaluated the in vivo compatibility of the scaffolds after a 3-week subcutaneous implantation in rats. PS and AS scaffolds were implanted in the same location of the same rat. There was no redness or swelling at the implant site over the course of the study. Macroscopic images of the implanted scaffolds showed integration with host tissue without obvious mass loss or evidence of infection of the implants. Blood vessels moved into the


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scaffolds after 1 week of implantation, suggesting good in vivo compatibility of the scaffolds (Figure 4A).

The explants were further evaluated by H&E and Masson's trichrome staining (Figure 4 B and C). Few macrophages or lymphocytic infiltration appeared around both the PS and AS parts, confirming a limited inflammatory response at this 3-week time point. Tissue infiltration into PS and AS scaffolds was also detected. The surrounding tissue was completely infiltrated 7 days after implantation, and occupied almost all the space of the PS and AS scaffolds after 21 days. Improved tissue infiltration was achieved on the AS scaffolds. After 2-weeks of implantation, the AS scaffolds had been almost overtaken by tissue ingrowth while only about 60% of the PS scaffolds was occupied by tissue ingrowth. Higher magnification of H&E staining slices indicated various morphology of the cells insides the scaffolds (Figure 4B b, e, h and c, f, i). Similar to the in vitro results, the cells migrated along the aligned pores of the AS scaffolds and formed elongated shapes, which would be helpful for forming mature fibrous granulation tissue.25,43,44

Since the PS scaffolds degraded more

quickly than the AS scaffolds, the better tissue ingrowth inside the AS scaffolds should be attributed to the aligned structures rather than the cell penetration following scaffold degradation. Masson staining further revealed better tissue ingrowth and collagen deposition on the AS scaffolds (Figure 4C). For example, above 73% of the area inside the AS scaffolds was deposited with collagen after 21 days, while only 57% of the PS scaffolds exhibited collagen formation (Figure 4D). Unlike that on the



PS scaffolds without regular morphology, the deposited collagen ECM had aligned structures that were similar with the surrounding native tissue, indicating the formation of granulating tissue inside the AS scaffolds.7,46

Figure 4. In vivo compatibility of the two scaffolds: (A) Macroscopic observation of scaffolds embedded subcutaneously in rats for 7, 14 and 21 days; (B) Haematoxylin-eosin (H ＆ E) staining of sections of the scaffolds implanted subcutaneously in rats for 7, 14 and 21 days; (C) Masson’s trichrome staining of sections of the scaffolds implanted subcutaneously in rats for 7, 14 and 21 days; (D) Quantification of the collagen deposition in PS and AS scaffolds for 7, 14 and 21 days by Image J software. Statistically significant **P ≤ 0.01, ***P ≤ 0.001. (E) CD34 immunohistochemical staining of sections of the scaffolds implanted subcutaneously in rats for 7, 14 and 21 days, and (F) vessel density within the scaffolds after


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subcutaneous implantation on the dosal region. For B, C and E images, the samples (a, d and g) were the overall morphology of the PS and AS scaffolds after implantation for 7, 14 and 21 days, the area above the black dashed line was the PS, and under the black dashed line was the AS, while the samples (b, e, h) and the samples (c, f, i) were the magnified images of the PS and AS scaffolds after implantation for 7, 14 and 21 days, respectively. The red double arrows point to the aligned cells inside the PS scaffolds. The yellow arrows indicate the blood vessels inside the scaffolds. The scale bar was 100 μm. Statistically significant *P≤0.05.

CD34 immunostaining was performed to evaluate angiogenesis after the implantation of the scaffolds (Figure 4E). Endothelial cells and the formation of new blood vessels was identified after 1 week of implantation, and gradually increased after 2 and 3 weeks inside both of the scaffolds. Significantly higher blood vessel numbers formed inside the AS scaffolds than inside the PS scaffolds, implying improved angiogenesis capacity with aligned structure (Figure 4E). Our recent study revealed that PS scaffolds achieved enhanced neovascularization and tissue ingrowth in vivo than previously reported non aligned silk materials through tuning the self-assembly of silk.29 The in vitro HUVECs cell culture also exhibited better proliferation on the PS scaffolds. Here, further improvements in angiogenesis and tissue ingrowth were achieved on the AS scaffolds, suggesting more significant role of the anisotropic and hierarchical microstructures than the mechanical cues. Although AS and PS scaffolds were implanted in the same location of the same rats, the in vivo microenvironments



for the two scaffolds might remain slightly different, which would result in the bias of the tissue ingrowth. Therefore, according to previous studies,38,39 rodent full-thickness wound model was also used to evaluate the influence of the scaffolds on the tissue regeneration.

3.4 In Vivo Wound Healing Study. The rat full-thickness skin defect model was used to assess whether the AS scaffolds would allow for quicker wound healing when compared to the PS scaffolds.47,48 A splint ring was affixed around the skin wound tightly to prevent local skin contraction.49,50 Therefore, the wound healing was promoted through granulation and re-epithelialization rather than contraction, which is similar to that healing mechanism occurring in humans.51-53

Figure 5A presents the overall observation photos of wounds at days 0, 7, 14, 21 and 28 after implantation of the AS and PS scaffolds where scaffold free treated samples were used as the control group. All the wound sizes were gradually reduced after the implantation and significantly faster wound reduction was achieved for the AS scaffolds. Tracing the wound edges and calculating the wound area by using NIH Image J software. As shown in Figure 5B, the wound area treated with the AS scaffold started to reduce at the first 7 days due to quicker new vessel formation. Unlike the control group where only 71% of the wound was closed, the closed areas of the wounds treated with the PS scaffold reached to above 77% and then further increased to 93% for the wounds treated with the AS scaffolds after 14 days. After the


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28 days, the wounds were nearly completely closed. The results suggested that the AS scaffolds with aligned structures provided better microenvironments for the wound closure than the PS scaffolds.

Figure 5. The skin regeneration capacity of the porous and aligned scaffolds in a full-thickness skin defect model: (A) Macro-observation of wound healing process of full-thickness skin defect. (B) Quantification of the wound area through the Image J software. Statistically significant *P≤0.05, **P≤0.01, ***P≤0.001.

Histological analysis of full-thickness wound repairing process was investigated to further reveal the effect of the two different scaffolds. Angiogenesis is a prerequisite for tissue repair. Endothelial cells usually migrated from the surrounding tissues into the wound area to accelerate the healing. Compared to the control without the scaffolds, significantly higher expression of CD 31 and vascular hole area with CD 31 staining were observed at the first 7 days for the wounds treated with PS and AS



scaffolds, following a ascending in the CD 31 positive area at 14 day for both of the scaffolds. Subsequently, the positive area of CD 31 began to decline (Figure 6A and B). These results indicated that PS and AS scaffolds facilitated rapid induction of infant vascular endothelial cells and faster formation of immature blood vessels inside the scaffolds since CD 31 is a marker of immature cells. Similar to plenty of previous studies,54,55 various vasculization processes appeared in subcutaneous implantations and skin wound model due to the different tissue ingrowth behaviors in the two models. However, significantly higher density of new vessels appeared in wound sites treated with the AS scaffolds than those treated with the PS scaffolds, consistent with subcutaneous implantation experiments. The results further confirmed the critical influence of the aligned microstructures on cell migration and blood vessel formation.

Figure 6. (A) The wound bed on 7, 14, 21 and 28 days were immunofluorescence


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stained with CD31. The white arrows indicated red cavity of the new vessels. (B) Quantification of the microvessel area by using Image J software. Statistically significant *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 7. (A) H＆E stained wound bed on 7, 14, 21 and 28 days. The black triangle reflects the original cuticle and the green triangle reflects the new epithelium. Scale bars were 500μm. (B) Quantification of the length of the new epidermis by Image J software. Statistically significant *P≤0.05, **P≤0.01, ***P≤0.001.

The regeneration of the epidermis was crucial as it acts as a barrier to prevent moisture loss and infection.9,56,57 The extension distance of new epidermis at different



time points were measured among the three groups (Figure 7A). The AS scaffold-dressed wound was predominantly covered by new epidermis layer after one week of implantation and the thickness of new epidermis was almost the same as that of healthy skin after three weeks. Unlike the AS scaffold treatment, the epidermal tissue in the wounds treated with the PS scaffolds remained fragmentary and fragile even after three weeks (Figure 7A). Considering that substantial spaces still existed inside the AS scaffolds at one week, the epidermal cells should migrate from the surround normal tissues. The aligned structure of the AS scaffolds provided positive cues for migration, as shown in the vitro cell results and previous studies.19 Therefore, through the fabrication of hierarchical microstructures and different physical cues, the AS scaffolds provided a better microenvironment for cell migration, vascularization and tissue ingrowth, achieving faster and higher quality skin regeneration in vivo. Quantitative measurement showed quicker new epidermal increase from 17mm to 73mm for AS-treated rats after 1 and 3 weeks while the length of the new epidermal tissue was below 62 mm for PS-treated rats after 3 weeks (Figure 7B).

Neovascularization that could facilitate cell and nutrient transport is critical for skin regeneration.58-62 Masson's trichrome stained sections confirmed better ingrowth and regeneration of new skin tissues inside the AS scaffolds (Figure 8A). Strikingly new granulation tissue with thicker and better structure appeared on the AS scaffold treated wounds, indicating the improved tissue regeneration process than that treated with the PS scaffolds. Collagen deposition is a key process among wound healing


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process.21Although both the scaffolds had been almost replaced by tissue ingrowth after 3 weeks, significantly higher collagen deposition was observed in the wounds treated with the AS scaffolds, and the collagen deposited inside the AS scaffolds was arranged in an orderly fashion which was similar to that of the native surrounding tissue, reflecting better repair with the AS scaffolds. The collagen deposition was also quantified and showed in Figure 8B. Although the collagen deposition inside AS and PS scaffolds was similar after 28 days, significantly higher collagen deposition was observed in the wounds treated with the AS scaffolds where the deposited collagen was 69% inside the AS scaffolds, and decreased to 63% in the PS scaffolds after 14 days.

Figure 8. (A) Masson’s trichrome staining of wound bed on different points. The blue



stained the new-formed collagen tissue. The scale bars were 100 μm. (B) Quantification of the collagen deposition in wound sites by Image J software. Statistically significant **P≤0.01, ***P≤0.001.

It remains a great challenge to introduce and optimize various physical cues into same scaffolds since few feasible techniques could effectively regulate various physical cues simultaneously. Here, silk nanofibers with stable conformations and microstructures were used to design silk scaffolds and introduce multiple physical cues via sequential fabrication processes (electrical treatment and lyophilization). Both the in vitro and in vivo results indicated significantly better wound healing capacity for these scaffolds containing multiple physical cues, suggesting a promising strategy of developing bioactive silk-based biomaterials through integrating silk nanofiber elements with various fabricating processes.

4. CONCLUSIONS Fast and effective wound repair requires the promotion of processes including angiogenesis and tissue migration. Through tuning the secondary conformation and inducing ECM-biomimetic nanofiber formation, silk scaffolds were developed with vascularization capacity to facilitate tissue regeneration. The microstructures of the scaffolds were further fabricated to achieve aligned features, providing positive signals for cell migration. Thus, unlike previously used silk scaffolds, the anisotropic silk nanofibrous scaffolds accelerated cell migration to wounds, enabling more rapid


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neovascularization, tissue infiltration and epidermis formation. Finally, the wound treated with these scaffolds resulted in faster and higher quality skin regeneration. Overall, the study demonstrates that silk scaffolds alone, without the addition of growth factors, could promote skin regeneration through tuning the hierarchical microstructures, offering potential for treating skin wounds.

ACKNOWLEDGMENTS The authors thank the National Key R&D Program of China (2016YFE0204400), NSFC (81671912), NIH (R01NS094218, R01AR070975) and the AFOSR. We also thank the Social Development Program of Jiangsu Province (BE2018626) for support of this work.



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TABLE OF GRAPHIC


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Anisotropic Biomimetic Silk Scaffolds for Improved Cell Migration and

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