Asiaticoside-laden Silk Nanofiber Hydrogels to Regulate Inflammation and Angiogenesis for Scarless Skin Regeneration

Abstract

Scarless skin regeneration remains a challenge due to the complicated microenvironment involved in wound healing. Here, the hydrophobic drug, asiaticoside (AC), was loaded inside silk nanofiber hydrogels to achieve bioactive and injectable matrices for skin regeneration. AC was dispersed in aqueous silk nanofiber hydrogels with retention of biological functions that regulated inflammatory reactions and vascularization in vitro. After implantation in full-thickness wound defects, these AC-laden hydrogel matrices achieved scarless wound repair. Inflammatory reactions and angiogenesis were regulated during inflammation and remodeling, which was responsible for wound regeneration similar to normal skin. Both in vitro and in vivo studies demonstrated promising applications of these AC-laden silk hydrogels towards scarless tissue regeneration.

1. Introduction

Major dermal injuries caused by burn, trauma and surgery often result in hypertrophic scars during healing, resulting in reduced functions of normal skin, such as restricted movement as well as aesthetic and psychological trauma.¹ Multiple tissue engineering strategies, as well as bioactive wound matrices, have been developed to accelerate wound healing,^1,2 however, scarring remains a challenge. Different clinical treatments and non-surgical approaches have been pursued to address this problem, including laser therapy, radiotherapy, cryosurgery, silicon dressings and topical medicines,^1,3–7 but failed to achieve functional recovery.

Skin healing is a complex and dynamic process characterized by a harmony of various cells, growth factors, cytokines, pathways and extracellular matrix (ECM) synthesis.⁸ An imbalance of these factors induce scar formation.^9,10 The simultaneous introduction of multiple growth factors, cytokines and cells to wound matrices has resulted in improved wound healing with less scar tissue, however, scarless outcomes remain to be solved.^11–13 The wound healing process involves cascades of inflammation, granulation, and remodeling where inflammatory reactions and angiogenesis undergo dynamic changes at various stages of the process to determine ECM production and ultimately, scar formation.¹⁴ The complex factors (growth factors, cytokines, cells, ECM) determine scar outcomes during healing by tuning inflammation and angiogenesis at different stages.^15,16 Different bioactive biomaterials, including cell-laden matrices have been designed to actively regulate inflammation and angiogenesis to stimulate scarless wound healing.^17,18 Most often, a complex of multiple ingredients is used in these matrices, which can also complicate outcomes.^19–20

Asiaticoside (AC), an extract from the plant (Centella asiatica), has been used clinically to stimulate wound healing and reduce scar formation.^21–23 AC displays versatile biological functions such as anti-inflammation, antioxidant activity, stimulation of collagen synthesis, and promotion of angiogenesis.^21–23 However, the insolubility of AC in water limits clinical applications as a topical drug or as a bioactive additive in wound dressings and matrices.²⁴ Some studies revealed successful loading of AC into wound dressings and the use of liposome carriers,^25–27 however, the regulation of angiogenesis and inflammation during wound healing to control the outcomes remains a challenge. Thus, we hypothesized that improved control of loading and release of AC would empower improved outcomes towards scarless wound healing.

Silk fibroin (hereafter referred to as silk) has been used in wound healing as a promising scaffold because of its biocompatibility, low immune reactions, and tunable mechanical properties and degradation behavior.^28,29 Recently, beta-sheet rich silk nanofibers were assembled to form injectable hydrogels that accelerated skin regeneration as wound matrices.^17,30 These silk nanofibers were able to transport hydrophobic cargos into aqueous solutions with retention of bioactivity.³¹ Here, AC was loaded onto these silk nanofibers and then formed aqueous silk hydrogels. Sufficient AC was loaded inside the nanofiber hydrogels compared to the loading capacity of liposome systems.^32,33 These AC-laden silk nanofiber hydrogels retained shear-thinning behavior and injectability, to support their use towards scarless wound healing applications.

2. Materials and methods

2.1. Preparation of Silk Nanofiber Hydrogels

The silk nanofiber hydrogels were prepared via a concentration-dilution-thermal incubation process.³⁴ Fresh silk solution (6 wt%) were concentrated to above 20 wt% for above 24 h at 60°C, and then diluted to 2 wt% with distilled water. The diluted solution was cultured at 60°C until the formation of silk nanofiber hydrogels. The silk nanofiber hydrogels were termed SNF.

2.2. Preparation of AC-laden SNF Hydrogels

The AC-laden SNF hydrogels were prepared using silk nanofibers as drug shuttles.³¹ Hydrophobic AC (MCE, New Nishizawa, USA) dissolved in methanol was blended with silk nanofiber hydrogels (2 wt%) at a volume ratio of 1:1. After stirring for 24 h at 200 rpm, the mixed solution was centrifuged at 10,000 rpm for 30 min to separate the AC-laden silk nanofibers and methanol, obtaining AC-laden hydrogels. The hydrogels were washed with distilled water three times to remove the residual methanol, and termed SNF-AC.³¹ The centrifuged supernatant was collected to calculate the content of unloaded AC. The content of AC in solution was determined with high-performance liquid chromatography (HPLC, Thermo Fisher, USA) with a C18 column (5μm, 250 mm×4.6 mm, Thermo Fisher, USA). The mobile phase consisted of acetonitrile/water (25:75 up to 2 min; 90:10 up to 8 min; 25:75 up to 10min) and the flow rate was 1.0 ml/min. The detection was 205 nm. The loading efficiency (LE) and loading capacity (LC) of AC were calculated by the following formulas:

$$\text{LE}\left(\text{\%}\right)=\frac{W_T-W_F}{W_T}\times 100\text{\%}.$$

$$\text{LC}\left(\text{\%}\right)=\frac{W_T-W_F}{W_S}\times 100\text{\%}.$$

Where W_T is the total weight of AC, W_F is the weight of unloaded AC in the supernatant, W_S is the total weight of silk nanofibers.

2.3. Characterization of AC-laden SNF Hydrogels

AFM (Nanoscope V, Veeco, NY, USA) was used to measure the morphology of AC-laden silk nanofibers. Confocal Raman spectrometer (JY HR 800, Renishaw, 532 nm diode laser) was used to evaluate the loading of AC in the SNF hydrogels.³⁴ Rheological properties of all hydrogels were measured with a rheometer (AR2000, TA Instruments, New Castle, DE) fitted with a 20 mm cone plate. The used parameters were consistent with our previous work.³⁴

2.4. In Vitro Release of AC from SNF Hydrogels

The AC-laden SNF hydrogels (2 ml) were transferred to dialysis tubing (MWCO 2,000, Thermal Scientific, USA) and immersed in 10 ml of release medium at 37°C in an oscillating water bath. Both phosphate buffered saline (PBS) and PBS/methanol blend solution (methanol 10 vol%) were used as the release medium to evaluate the release behavior of AC where PBS solution simulated the in vivo aqueous situation while PBS/methanol blend solution could reveal the interaction of AC and SNF due to the solubility of AC in the blend solution.³⁵ At designed time points, 1 ml of solution was collected and the contents refilled with an equal amount of release medium. The amount of released AC was determined with HPLC. The samples were measured in triplicate at each time points.

2.5. In Vitro Cytocompatibility of AC-laden SNF Hydrogels

Adult human dermal fibroblasts (HDF-a) and mouse RAW 246.7 macrophages (RAW 246.7) were obtained from Beina Chuanglian Biotechnology Co. (LTD, Beijing, China). Human umbilical vein endothelial cells (HUVECs) were supplied by the Chinese Academy of Sciences (Shanghai, China). AC-laden silk nanofiber hydrogels with AC concentrations of 0.625 mg/ml, 1.25 mg/ml and 2.5 mg/ml were blended with the media, tuning the concentration of AC to 125 μg/ml, 250 μg/ml and 500 μg/ml, respectively. Cells were cultured on these composite media with AC-laden SNF at a cell density of 2×10⁴ per well in 24-well plates, and supplied with high glucose (DMEM) containing 10% Fetal Bovine Serum (FBS) and 100 units/ml penicillin streptomycin. These groups were termed SNF-AC125, SNF-AC250 and SNF-AC500. As controls, AC was dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium at a volume ratio of 1:200, forming the modified media containing free AC with concentrations of 125, 250 and 500 μg/ml. Different cells were also cultured in these modified media and termed AC125, AC250 and AC500. The same amount of DMSO was also added to the medium directly and cultured the cells to evaluate the influence of DMSO. The group was termed DMSO. Cell proliferation was evaluated with a cell counting kit 8 (CCK-8) (Beyotime, Shanghai, China). Briefly, at designed time points, DMEM contained 10% CCK-8 solution was added to each well and incubated for 3 h. Then the solutions were transferred to new 96-well plates and measured at 450 nm with multiscan spectra (Biotek Synergy 4, Winooski, VT, USA).

2.6. Tube Formation Assay

To estimate the effect of AC-laden SNF on vascularization in vitro, matrigel (200 μl/well, BD Biosciences, USA) was coated on the plates at 37°C for 30 min and seeded with HUVECs at a density of 8×10⁴ cell/well in a 24-well plate. The cells were then cultured in the media containing AC-laden SNFs for 12 h, and observed with an inverted microscope (AxioVert A1, Carl zeiss, Germany). Each samples were repeated three times for statistical analysis.

2.7. Enzyme-linked Immunosorbent Assays (ELISA)

To estimate the influence of SNF, free AC (125, 250, 500 μg/ml) and AC-laden SNF (125, 250, 500 μg/ml) on inflammation, the secretion of TNF-α and IL-6 from Raw 246.7 cells was measured by ELISA. Raw 246.7 cells (1×10⁵ cells/well) were seeded in 24-well plates and cultured for 6 h. Then the media containing free SNF, free AC and AC-laden SNF were used to culture the cells for 6 h. Then 100 ng/ml lipopolysaccharides (LPS) was added to the media and the cells were cultured for 24 h, the supernatants were collected and measured with a mouse IL-6 ELISA kit (ab222503, Abcam, USA) and a mouse TNF-α ELISA kit (ab208348, Abcam, USA) according to the manufacturer’s protocols.

2.8. In Vivo Wound Healing

Based on the in vitro results, the concentration of AC in the silk nanofiber hydrogels used in wound healing was tuned to 250 and 500 μg/ml, respectively. All animal procedures were performed in accordance with the guidelines for Care and Use of Laboratory Animals of Soochow University and approved by the Animal Ethics Committee of the Soochow University. Eighteen Sprague-Dawley (SD) adult male rats weighting 250 g were used in the in vivo wound experiments. Each group has six samples. Six full-thickness wounds with a diameter of 1.5 cm were generated on each rat.^30,36,37 The same volume (500μl) of 0.9% NaCl solution, pure SNF hydrogel (2 wt%), AC solutions with concentrations of 250 μg/ml and 500 μg/ml and SNF hydrogels (2 wt%) with AC concentrations of 250 μg/ml and 500 μg/ml were injected with needles (25G) into the full-thickness wounds and divided into six groups: Control, SNF2, AC250, AC500, SNF2-AC250, and SNF2-AC500. Donut-shaped silicone splints were sutured into the sites to prevent wound shrinkage.³⁸ After 7, 14, and 21 days post-operation, the digital images of the wound areas were obtained using ImageJ software to calculate the healing rate and draw the wound healing simulated diagram.

2.9. Histology Analysis and Immunofluorescence Staining

On days 7, 14, and 21 after the operation, the wound and adjacent skin tissues were collected, fixed with 4% paraformaldehyde and embedded in paraffin. The embedded samples were sectioned (5 μm thickness) and stained with hematoxylin-eosin (H&E), Masson’s trichrome and Picrosirius Red (Sigma-Aldrich, USA). H&E staining was used to evaluate granulation tissue formation and re-epithelialization. Granulation tissue and newborn epidermis were calculated with ImageJ software. Masson’s trichrome and Picrosirius Red staining were used to analyze collagen deposition and collagen type I/III ratios. In order to evaluate angiogenesis at days 7 and 21 after surgery, CD31 (1:200, Abcam, USA) antibody, α-SMA (1:200, Abcam, USA) antibody and VEGF (1:200, Abcam, USA) were used for immunofluorescence (IF) staining and immunohistochemical (IHC) staining. To evaluate inflammation at days 7 and 21 post-operation, the Pan macrophage marker CD68 (ab201340, Abcam) antibody and M2 macrophage marker CD206 (Ab64693, Abcam) antibody were used for immunofluorescence staining. The nucleus was stained by DAPI. Immunofluorescence and immunohistochemistry stained images were obtained with a confocal microscope (Olympus FV10 inverted microscope, Japan) and an inverted microscope (Axio Vert A1, Carl Zeiss, Germany), respectively. Image J software was also used to analyze the results.

2.10. Statistical analyses

Comparisons between groups were evaluated by one-way or two-way ANOVA. Comparisons between two groups were evaluated with t-test. Values were presented as mean ± standard deviations. A p-value of less than 0.05 was considered as significant. All the results were statistically analyzed by SPSS v.16.0 software (IBM SPSS Statistics, US).

3. Results and Discussion

3.1. Characterization of AC-laden silk nanofiber hydrogels

AC accelerated skin regeneration and reduced scar formation by modulating inflammatory reactions and angiogenesis.^21,22,24 However, the hydrophobic features of AC limited delivery in aqueous environments. Although aqueous-organic composite solvents and liposome carriers were used to improve the dispersion of AC in aqueous systems,^32,33,39 therapeutic efficacy remained a challenge. Recently, a relatively simple blending-centrifugation process was developed to directly transport hydrophobic cargos into aqueous solutions using silk nanofibers as shuttles.³¹ This transport method was used to load AC into aqueous silk hydrogels as a route to address therapeutic impact. AC-laden silk nanofiber hydrogels with an AC concentration above 2.8 mg/ml were prepared using this approach, a 7-fold higher drug loading than liposome systems (Table S1).³² Due to the hydrophilic/hydrophobic properties of the silk nanofibers, the loading efficiency for AC was above 65% at different concentrations (1 mg/ml, 2 mg/ml and 4 mg/ml), also superior to liposome carriers.³⁹ Similar to our previous cargo-laden silk nanofibers,³¹ AFM images of these AC-laden silk nanofibers revealed gradually increased diameters from 15 nm (free silk nanofibers) to 19 nm, 22 nm and 28 nm, respectively (loaded AC 0.7 mg/ml, 1.4 mg/ml and 2.8mg/ml, respectively) (Figure 1a), which revealed that the AC adhered on the nanofibers. Similar to our previous study,³¹ hydrophobic AC loaded on silk nanofibers through hydrophobic interaction. Raman spectra of the AC-laden silk nanofiber hydrogels exhibited typical peaks for AC at 450–650 cm⁻¹ and for silk at 1669 cm⁻¹ (β-sheet structure of silk nanofibers), confirming the successful loading of AC (Figure 1b). After the AC loading, the negative charge repulsion of silk nanofibers was altered, reflected in a change in zeta potential from −39.50±2.13 mV to −36.74±0.38 mV (Table S1). Similar to previous hydrophobic cargo-laden hydrogels,³¹ AC loaded on these nanofibers supported dispersion in aqueous hydrogels due to the charge repulsion.

Figure 1.

Characterization of silk nanofiber hydrogels and AC-laden silk nanofiber hydrogels: (a) AFM images of AC-laden silk nanofiber hydrogels with different AC concentrations (0, 0.7, 1.4, 2.8 mg/ml). Scale bars 500 nm; (b) Raman spectra; (c) Modulus of AC-laden silk nanofiber hydrogels with different AC concentrations (0, 0.7, 1.4, 2.8 mg/ml); (d) Viscosity of AC-laden silk nanofiber hydrogels with different AC concentrations (0, 0.7, 1.4, 2.8 mg/ml); (e) AC release behavior from AC-laden silk nanofiber hydrogels (SNF-AC-2.8 mg/ml). Data presented as mean ± SD, n=3. SNF, silk nanofiber hydrogels; SNF-AC-0.7 mg/ml, AC-laden silk nanofiber hydrogel with AC concentration of 0.7 mg/ml; SNF-AC-1.4 mg/ml, AC-laden silk nanofiber hydrogel with AC concentration of 1.4 mg/ml; SNF-AC-2.8 mg/ml, AC-laden silk nanofiber hydrogel with AC concentration of 2.8 mg/ml. Silk concentration at 2 wt% for all hydrogels.

After the introduction of AC, the silk nanofiber hydrogels continued to exhibit similar shear-thinning behavior as the materials without AC, and were injectable through needles (25G) (Figure S1). The modulus increased slightly from 1.5 kPa to 1.9 kPa when different amounts of AC were loaded into the hydrogels (Figure 1c). The loading and release of AC could facilitate improvements in skin regeneration.^24,40 AC easily dissolved in methanol-PBS solutions, and when the AC-laden silk nanofiber hydrogels were incubated in methanol-PBS solution, the AC was gradually released over 72 h (Figure 1e). Little AC was released from the hydrogels in PBS, suggesting that the loaded AC would persist in wound healing processes in vivo based on the local environment.

3.2. Cytocompatibility and biological activity of AC-laden hydrogels

Based on previous studies,^24,40 AC concentrations in cell culture were adjusted to 125 μg/ml, 250 μg/ml and 500 μg/ml by blending AC-laden hydrogels with the culture medium to form the composite solutions. To evaluate the cytocompatibility of the AC-laden hydrogels in vitro, human dermal fibroblasts (HDF-α), human umbilical vein endothelial cells (HUVECs) and mouse RAW 264.7 macrophages (RAW 264.7) were cultured in the composite media. All cells exhibited sustained proliferation in the samples, suggesting the cytocompatibility of AC and silk nanofibers. Both fibroblast and endothelial cells had similar proliferation behavior with the various amounts of AC (free AC and AC when loaded in the silk nanofiber hydrogels), indicating that AC had no negative influence on cell viability (Figure 2a, b). Previous studies revealed the influence of AC on inflammatory reactions.^41–43 The growth of macrophages was dose dependent, where higher amounts of AC in solution resulted in slower growth (Figure 2c). Similar trends occurred, but significantly faster growth was achieved, when the macrophages were exposed to the same amount of AC loaded in silk nanofiber hydrogels. Except the SNF-AC500 group, all the AC-laden nanofiber groups exhibited similar proliferation with the control group. Previous studies revealed that silk as drug carriers and matrices could alleviate the inhibition effect of drugs on cell proliferation through controlling the release behaviors of drugs from the carriers.³⁰ Similar results here was achieved, which suggested that AC-laden silk nanofibers also affected macrophage behavior. At early stages of inflammation during wound healing, macrophages secreted VEGF to induce angiogenesis.⁴⁴ Several studies have indicated that silk nanofibers promoted vascularization to accelerate skin regeneration.^17,30 The faster growth of macrophages on silk nanofiber hydrogels might be beneficial to early angiogenesis activity during wound healing. The macrophages were cultured in medium containing AC-laden silk nanofibers and then polarized with lipopolysaccharides (LPS). The macrophages were also cultured in media containing free AC and then polarized as controls. As expected, the secretion of IL-6 and TNF-α was reduced when free AC was introduced to the media and a dose-dependent response was evident (Figure 3a, b). Compared to the free AC control, while the anti-inflammatory capacity of AC when loaded on the silk nanofibers was slightly decreased since most of AC was loaded on the nanofibers rather than dispersed in solutions directly,³⁰ the expression of IL-6 and TNF-α was inhibited for the polarized macrophages. Considering the similar proliferation of macrophages between AC-laden silk nanofiber groups (SNF-AC125 and SNF-AC250) and the control group, the inhibition of IL-6 and TNF--α secretion in these groups should be attributed to the bioactivity of AC. Similar inflammatory inhibition was achieved for free AC and the AC-laden nanofiber groups at high dose (500 μg/ml), also suggesting the preservation of biological activity for the loaded AC. The anti-inflammation at early stages is considered beneficial to angiogenesis and granulation processes during wound healing.³² Multiple studies have revealed that anti-inflammation of AC stimulated the secretion of VEGF, accelerating wound healing.^45,46 Inhibition of inflammation is critical for scarless tissue formation at the remodeling stage.¹⁴ However, a contradiction remained since anti-inflammation behavior stimulated angiogenesis while higher angiogenesis during remodeling stage results in scar formation.^47–49 Although the clinical effects of AC have been confirmed in recent studies,^24,45 a deeper understanding is necessary to clarify the contradictory effects of AC (stimulating angiogenesis and reducing scar formation during remodeling). To reveal the impact of AC in wound healing, a tube-formation assay was used to assess vascularization of the AC-laden hydrogels. Interestingly, tube formation was reduced on AC-laden hydrogels where higher amounts of AC were presented (Figure 3c, d). The results suggested that AC inhibited the assembly of HUVECs. Therefore, it is possible that AC regulates angiogenesis in a dynamic fashion, by impacting inflammation (down-regulation) and HUVECs tube assembly.

Figure 2.

Cell proliferation with different amounts of AC and AC-laden silk nanofibers: (a) proliferation behavior of HDF-a; (b) proliferation behavior of HUVECs; (c) proliferation behavior of RAW264.7. AC-laden silk nanofiber hydrogels containing 0.625, 1.25 and 2.5mg/ml of AC added to media at a volume ratio of 1:4, for concentrations of 125 μg/ml, 250 μg/ml and 500 μg/ml, respectively. Free AC with concentrations of 25, 50 and 100 mg/ml in DMSO added to the media at a volume ratio of 1:200, for final concentrations of 125 μg/ml, 250 μg/ml and 500 μg/ml, respectively. The same amount of silk nanofiber hydrogels and DMSO without AC added to the media at the same ratios, respectively. The samples were as follows: Controls, media without SNF, DMSO and AC; SNF, media containing SNF without DMSO and AC; DMSO, media containing DMSO without SNF and AC; AC125, AC250 and AC500 represent media containing free AC and the concentrations of free AC in the media were 125 μg/ml, 250 μg/ml and 500 μg/ml, respectively; SNF-AC125, SNF-AC250 and SNF-AC500 represent the media containing AC-laden silk nanofiber hydrogels and the AC concentrations were 125 μg/ml, 250 μg/ml and 500 μg/ml, respectively. Data presented as mean ± SD, n=3. Statistically significant *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 3.

Inflammatory behavior and tube formation In vitro: (a, b) secretion of IL-6 and TNF-α from RAW 264.7 cells. Before supernatant collection, macrophages were cultured in media containing different amounts of AC and AC-laden silk nanofibers for 6 h, then 100 ng/ml of lipopolysaccharides (LPS) added to the media and cultured for 24 h. As controls, cells were cultured in media for 6 h, then further cultured for 24 h with and without the introduction of LPS. Control samples were Control and LPS, respectively; (c, d) In vitro tube formation from HUVECs when cultured in media containing different amounts of AC-laden silk nanofibers for 12h. Scale bars 100 μm. Samples were as follows: SNF, the medium containing SNF without AC; SNF-AC125, SNF-AC250 and SNF-AC500 represent media containing AC-laden silk nanofiber hydrogels and the AC concentrations were 125 μg/ml, 250 μg/ml and 500 μg/ml, respectively. Data presented as mean ± SD, n=3. Statistically significant *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

3.3. Scarless Wound Healing

Injectable silk nanofiber hydrogels accelerated wound healing, while free AC has been used clinically to reduce scar formation.^17,24,30 The in vitro cell results indicated that AC-laden silk nanofibers with AC concentrations of 250 and 500 μg/ml regulated inflammatory reactions using macrophages. Silk nanofiber hydrogels with SNF concentrations of 2 wt% were directly loaded with AC and the concentrations of AC in the hydrogels were 250 and 500 μg/ml, with treatment groups termed SNF2-AC250 and SNF2-AC500. Pure silk nanofiber hydrogels and free AC solutions with different AC amounts as controls were also used to treat full-thickness wounds and termed SNF2, AC250 and AC500. Based on previous reported wound models,^30,36,37 six wounds with diameter of 15 mm were prepared and treated with different groups. The donut-shaped silicone splints were sutured into the wounds to prevent the unwanted contraction healing.³⁸ Defects were also filled with 0.9% NaCl solution and used as blank controls. Except for the blank controls, all wounds had almost healed within 21 days (Figure 4a–c), suggesting a positive influence for the silk nanofibers and AC. Granulation tissue plays a vital role in the process of wound repair. The thicker granulation tissue during wound healing is an important indicator for evaluating wound healing.^50,51 Higher amounts of AC in solution induced slightly faster wound healing and granulation ingrowth than solutions with lower AC concentrations (Figure 4d, e), while the AC solutions were inferior to pure silk nanofiber hydrogels. The AC-laden hydrogels stabilized AC at the wound sites and also facilitated cell/tissue ingrowth, which resulted in significantly faster wound healing. The hydrogels loaded with 500 μg/ml of AC (SNF2-AC500) showed the fastest healing rate. The results indicated the synergistic effects of AC and silk nanofiber hydrogels. The thickness of regenerated epidermis is considered a key indicator of scar tissue,^12,16 with thicker epidermis than normal skin implicating scar formation.⁵² The AC reduced scar formation.²⁴ Compared to the control group, a thinner epidermis appeared in the AC250 and AC500 groups. However, the epidermis was still significantly thicker than that of normal skin, which implied scar tissue formation in the free AC groups (Figure 4f). The AC-laden silk nanofiber hydrogels showed better scar inhibition than the AC solutions and the pure silk nanofiber hydrogels. Compared to the control, the thickness of epidermis decreased from 101.4±11.0 to 64.4±7.6 μm and 52.4±3.7 μm for the SNF2-AC250 and SNF2-AC500 groups, respectively (Figure 4f). Although the thickness of epidermis was still slightly higher than that of normal skin (52.4±3.7 μm vs 41.4±7.4 μm), the epidermis in the AC-laden silk hydrogel groups were significantly thinner than in previously reported biomaterial systems containing AC.²⁴

Figure 4.

Wound regeneration: (a) Digital photographs of wounds at 0, 7, 14 and 21 days post-operation; (b) Simulated diagram of wound healing at 0, 7, 14 and 21 days after surgery. The green, blue, yellow and red areas represent the unhealed areas at 0, 7, 14, and 21 days, respectively; (c) Wound closure rate analyzed via ImageJ; (d) H&E stained wound bed at 7, 14 and 21 days. Scale bars are 200 μm. The black arrow represents the granulation tissue and the black solid line represents the newborn epidermis; (e) Quantitative analysis of granulation tissue thickness at 7 and 14 days with ImageJ; (f) Quantitative analysis of regenerated epidermis thickness at 21 days with ImageJ. Control, 0.9 % NaCl solution; SNF2, 2 wt% silk nanofiber hydrogels; AC250, AC solution with concentration of 250 μg/ml; AC500, AC solution with concentration of 500 μg/ml; SNF2-AC250, AC-laden silk nanofiber hydrogel (2 wt%) with AC concentration of 250 μg/ml; SNF2-AC500, AC-laden silk nanofiber hydrogel (2 wt%) with AC concentration of 500 μg/ml. Data presented as mean ± SD, n=6. Statistically significant *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

The in vitro results revealed that AC regulated angiogenesis and inflammatory reactions. Therefore, these reactions were investigated after implantation of the hydrogels for 7 and 21 days. At day 7 after surgery, higher M2 macrophages appeared in the AC250, AC500 and SNF groups (Figure 5a, c, d), suggesting the anticipated anti-inflammatory regulation. The synergistic action of the silk nanofibers and AC further strengthened the anti-inflammation, with the highest ratio of M2/M1 macrophages in the SNF2-AC500 group. The regulation also resulted in higher expression of VEGF. Gradually higher amounts of VEGF were found in the AC250, AC500, SNF2, SNF2-AC250 and SNF2-AC500 groups (Figure 6a, b). The highest VEGF expression appeared in the SNF2-AC500 group, which was beneficial to angiogenesis and promote wound repair in the early stage. After implantation of the hydrogels for 21 days, the reversed macrophage density appeared in the different groups where high macrophage density was maintained in the control group while the fewest macrophages were present in the AC-loaden hydrogel groups (SNF2–250, SNF2–500). Although the highest ratio of M2/M1 macrophages remained in the AC-laden hydrogel groups (Figure 5b, d), the lower amounts of macrophages resulted in lower secretion of VEGF (Figure 6), which is beneficial to prevent scar formation during the remodeling stages.^47–49 The highest VEGF secretion was found in the control group while the wounds treated with the SNF2–250, SNF2–500 groups exhibited the lowest VEGF secretion. Both the lower secretion of VEGF and the inhibition of AC on HUVEC assembly reduced the angiogenesis in the AC-laden hydrogel groups during remodeling. Similar angiogenesis changes happened for the SNF2–250 and SNF2–500 groups in vivo, which might be due to the saturation effect of AC at dose of above 250 μg/ml. Further study is necessary to clarify the optimized AC dose in vivo in our future study. The results indicated that inflammatory reactions and vascularization were dynamically influence in the inflammation and remodeling stages of wound healing to achieve the best and weakest angiogenesis in the inflammation and remodeling stages for the AC-laden hydrogel groups (Figure 6c, d, Figure S2), which would be beneficial towards scarless wound healing.

Figure 5.

Inflammation in wounds: (a) Dual immunofluorescence staining for CD 68 (green) and CD 206 (red) performed in wound tissues at day 7. Nuclei stained with DAPI (blue). Scale bars 50 μm; (b) Dual immunofluorescence staining for CD 68 (green) and CD 206 (red) performed in the wound tissues at day 21. Nuclei stained with DAPI (blue). Scale bars 50 μm; (c) CD68⁺macrophages per mm² at wound sites; (d) Quantitative analysis of M2 macrophage ratio in immunofluorescence images. Control, 0.9% NaCl solution; SNF2, 2 wt% of silk nanofiber hydrogels; AC250, AC solution with 250 μg/ml; AC500, AC solution with 500 μg/ml; SNF2-AC250, AC-laden silk nanofiber hydrogel (2 wt%) with AC 250 μg/ml; SNF2-AC500, AC-laden silk nanofiber hydrogel (2 wt%) with AC 500 μg/ml. Data presented as mean ± SD, n=6. Statistically significant *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 6.

Angiogenesis at wound sites at days 7 and 21 after the surgery: (a) Immunohistochemical staining for VEGF performed in wound tissues at days 7 and 21 after implantation. Scale bars 100 μm; (b) Quantitative analysis of VEGF expression; (c) Representative immunofluorescence images of DAPI (blue), CD31 (red) and α-SMA (green) stained tissue samples collected from the wounds at 7 and 21 day. Scale bars 200 μm; (d) Blood vessel density in the wound sites at day 7 and 21 post-implantation. Control, 0.9% NaCl solution, SNF2, 2 wt% of silk nanofiber hydrogels; AC250, AC solution 250 μg/ml; AC500, AC solution 500 μg/ml; SNF2-AC250, AC-laden silk nanofiber hydrogel (2 wt%) with AC 250 μg/ml; SNF2-AC500, AC-laden silk nanofiber hydrogel (2 wt%) with AC 500 μg/ml. Data presented as mean ± SD, n=6. Statistically significant *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

At day 14 after implantation, significantly higher amounts of collagen were deposited in the wounds treated with the AC-laden hydrogel groups (Figure 7a, b). The ratio of type I versus type III collagen was used to evaluate scarless wound healing.⁵³ As shown in Figure 7c, a lower ratio of type I versus type III was achieved for the AC-laden hydrogel groups. Compared to the blank group, the ratio decreased from 51.1±1.2 to 42.0±2.3, 23.8±3.7, 16.2±1.1, 11.8±0.4 and 10.0±0.4 for the AC250, AC500, SNF2, SNF2-AC250 and SNF2-AC500 groups, respectively. Similar to previous studies,^17,24 both silk hydrogels and AC reduced scar formation. The AC-laden silk hydrogels provided an optimal microenvironment for scarless wound repair. The ratio of type I and type III collagen in the SNF2-AC500 group was similar to that found in normal skin (Figure 7c), which further confirmed scarless tissue recovery. The deposited collagen microstructure also indicated high-quality wound healing in the AC-laden hydrogel groups. The deposited collagen in the blank and AC250 groups was composed of anisotropic collagen fiber bundles (Figure 7d, Figure S3), indicating scar tissue. Similar to that of normal skin, the deposited collagen in the SNF2-AC500 group exhibited a homogeneous network structure (Figure 7d, Figure S3). Similar wound regeneration quality was achieved for the SNF2-AC250 and SNF2-AC500 groups possible due to the similar angiogenesis and inflammation behaviors at day 21.

Figure 7.

Collagen deposition in the wound beds: (a) Representative images of wounds stained with Masson’s trichrome at days 14 and 21 after surgery. Scale bars 100 μm; (b) Quantitative analysis of collagen deposition; (c) Ratios of collagen types I/III; (d) Picrosirius red stained images of wounds at day 21 after surgery. Scale bars 100 μm. Control, 0.9% NaCl solution; SNF2, 2 wt% of silk nanofiber hydrogels; AC250, AC solution 250 μg/ml; AC500, AC solution 500 μg/ml; SNF2-AC250, AC-laden silk nanofiber hydrogel (2 wt%) with AC 250 μg/ml; SNF2-AC500, AC-laden silk nanofiber hydrogel (2 wt%) with AC 500 μg/ml. Data presented as mean ± SD, n=6. Statistically significant *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Various strategies have been developed to restrain the scar formation and stimulate the regeneration of the appendages.^{15–18,54,55} Hydrophobic AC has been used to alleviate the scar tissues in clinic applications.^24,45 Our present study successfully transport AC in aqueous silk nanofiber hydrogels without the compromise of AC bioactivity. The regenerated wounds treated with the AC-laden hydrogels still lacked the appendages (Figure 4d), indicating the need for further optimization of the AC-laden hydrogel systems. Our recent study also revealed that stem cells could be loaded inside the silk nanofiber hydrogels and stimulate the appendage regeneration.¹⁷ It is possible to develop the stem cell and AC-laden silk nanofiber hydrogels for better scarless wound repair, which would be realized in our future study. Although more studies such as the mechanical properties of the regenerated wounds and the remodeling dermal quality after longer times are necessary to further evaluate the scarless wound healing, our present results revealed that AC-laden silk nanofiber hydrogels restrained the scar formation effectively through tuning the inflammation and angiogenesis, suggesting its potential in clinical applications.

4. Conclusions

Hydrophobic AC was loaded into silk nanofiber hydrogels through a simple blending-centrifugation transport process, achieving better loading efficiency than previous liposome systems. The loaded AC remained bioactive and regulated inflammation and vascularization in a dynamic fashion based on the stage of wound healing in vivo. Scarless wound healing was achieved with the AC-laden hydrogels, superior to previous AC-loaded systems.