Introduction

Skin is the outer covering of the body and it is the largest organ of the integumentary system, guards the underlying muscles, bones, ligaments and internal organs. Wound or injury to skin can be due to burn, accidental trauma or chronic ulcerations (venous stasis, diabetic wounds and pressure ulcers) [1]. Wound healing is a complex and dynamic process comprising four distinct phases namely hemostasis, inflammation, proliferation and remodeling, requiring cellular and biochemical interaction of various cells such as keratinocytes, fibroblasts and endothelial cells of which fibroblast cells play vital role in initiating angiogenesis, epithelialization and collagen formation [2–4]. The ability of the skin to regenerate itself following minor epidermal injury is remarkable; however, during bacterial infection or when the injury is severe, such as loss of epidermis and dermis in full-thickness skin wounds, the damaged skin cannot initiate the healing process leading to life threatening complications. Use of antiseptics and antibiotics can only prevent or treat infections but fails to induce healing process. Hence, developments of therapeutic approaches focusing on wound healing are of prime importance and need to be explored. Natural medicines, especially pharmaceutical herbs, have been considered the principal cure for several years. These have been used in the treatment of a variety of disorders including wounds and burns since ancient times [5].

Aloe vera (AV) is one of the pharmaceutical herbs belonging to the Liliaceae family. It has been used in the treatment of a variety of skin disorders including burns, infections and dermatologic conditions since ancient times [6, 7]. The gel inside the leaf consists of 99 % water with long chain polysaccharide of acetylated glucomannan and other carbohydrates. It also contains the complex of amino acids, salicylic acid, ascorbic acid, vitamin A and vitamin E with antioxidant properties [8]. Aloe gel prevents skin dryness due to the high volume of water. The high percentage of glucose present in gel prevents bacterial growth due to the high osmotic virtue [9]. Prostaglandin and bradykinin hydrolyzing enzymes in AV reduce pain and inflammation. The existence of amylase enzyme in aloe annihilates the necrosal tissue, aloctin-A, which has a cell division and mitosis effect, and causes acceleration in the healing and stimulating macrophage to excrete the dead tissue. Amino acids present in plant gel are used to produce protein, causing tissue growth and healing. Vitamins including β-carotene, vitamin E, vitamin C and B complex, used in cell reaction, are consumed as antioxidants in strengthening the body immune system. Plant juice, antrakinons with antimicrobial, antiviral, antifungal and antiinflammatory properties and the saponins with antiseptic properties are effective in preventing infections [10, 11]. The glycoprotein fraction present in AV was reported to enhance cell proliferation and migration of keratinocytes and fibroblast cells thereby act as an effective wound healing agent [12].

The above information proves AV would be a promising agent for preventing infection and brings effective wound healing. However, some of the key challenges particularly issues of solubility, targeting, drug degradation and cellular interaction are undesirable in drug delivery applications and have led to a renewed search for alternative approaches of delivery system that prevent and combat skin wound infections and healing effectively.

Tissue engineering has emerged as a promising approach to overcome these drawbacks with the use of scaffolds that act as a drug carrier as well mimic extracellular matrix [13]. Scaffolds are highly porous, typically macromolecular, solids that are used to synthesize tissue or organ in vitro or in vivo [14]. Several methods are available for the preparation of an ideal scaffold, but the electrospinning process has attracted a great deal of attention due to its cost effective and easiest way to produce ultrafine fibers from polymer solutions with diameters in the range of nanometer to sub-micrometers that exhibit high surface-area-to-volume using electrostatic forces [15–17]. The topological structure of electrospun products can mimic the ECM and enhance both cell migration and proliferation [18, 19]. Nanotopographical features such as pores, ridges, groves, fibers, nodes or a combination of these have been reported to influence the cell behavior [20]. The possibility to immobilize antibiotics, enzymes, bioactive antimicrobial peptides and growth hormones to nanofibers, or encapsulation into fiber matrices, opens a new field in biomedical engineering [21].

The current study focuses on the fabrication of nanofibers using PCL and AV and their potential use as scaffolds for the culture of fibroblasts. PCL is an aliphatic, biodegradable and biocompatible polyester with good tensile properties [22]. However, its poor hydrophilicity, slow degradation kinetics and lack of natural cell recognition sites greatly limit its application in biomedical field.

Blending of AV with PCL provides an attractive option in improving its biological, mechanical and degradation properties in comparison to individual components. Previous research has proved collagen as a substrate for skin tissue engineering as it is easily degraded and resorbed by the body [23]. However, the use of collagen is expensive and the availability is limited when used as skin biomaterials [24]. Use of AV overcomes these problems, and in combination with a synthetic polymer PCL, gives a ‘bioartificial polymer’ with enhanced biocompatibility and chemical properties. This study involves the characterization of these nanofibers and analysis of cell growth and proliferation to determine the efficiency of skin tissue regeneration on these biocomposite nanofibrous scaffolds.

Experimental

Materials

Mice fibroblast cells (ATCC) obtained from Arlington, Virginia. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics, trypsin–EDTA and tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin were purchased from GIBCO Invitrogen USA. CellTiter 96® Aqueous one solution was purchased from Promega, Madison, WI, USA. PCL, chloroform, methanol, 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP), bovine serum albumin (BSA), Sirius red, collagen, Harris hematoxylin and formaldehyde were purchased from Sigma-Aldrich, St. Louis, USA. Lyophilized AV powder was purchased from Xi`an Yuensun Biological Technology Co., Ltd, China.

Fabrication of PCL, PCL-AV and PCL/Collagen scaffolds

PCL was dissolved in 3:1 (v/v) chloroform: methanol solvent mixture to form 10 % solution and kept in stirring overnight. PCL-AV 5 % represents AV 5 % (w/w) loaded in PCL, prepared by dissolving 10 % PCL and 5 % lyophilized AV powder in 3:1 (v/v) chloroform: methanol solvent mixture. Similarly, PCL-AV 10 % represents AV 10 % (w/w) loaded in PCL and prepared by dissolving 10 % PCL and 10 % AV powder in 3:1 (v/v) chloroform: methanol solvent mixture. PCL/Collagen solution was prepared by dissolving 10 % PCL and 2 % collagen in 10 mL HFP. The solutions were fed into a 5 mL standard syringe attached to a 21G blunted stainless steel needle, respectively, using a syringe pump (KDS 100, KD Scientific, Holliston, MA) at a flow rate of 1 mL/h with an applied voltage of 12 kV for all the solutions (Gamma High Voltage Research, USA). Random fibers were collected on a flat collector plate wrapped with aluminum foil that was kept at a distance of 12 cm from the needle tip. On application of high voltage the polymer solution was drawn into fibers. These nanofibers were collected on 15 mm cover slips by spreading them on the collector plate and used for cell culture studies.

Characterization of nanofibrous scaffolds

The electrospun nanofibers were sputter coated with gold (JEOL JFC-1200 Fine Coater, Japan) and visualized using a field emission scanning electron microscope (FESEM) (JEOL JSM 6700, Japan). Diameters of the electrospun fibers were analyzed from the FESEM images using image analysis software (Image J, National Institutes of Health, USA). Tensile properties of electrospun nanofibrous scaffolds were determined with a tabletop tensile tester (Instron 3345, USA) using load cell of 10 N capacities. Rectangular specimens of dimensions 10 × 20 mm were used for testing, at a crosshead speed of 10 mm/min and the data was recorded for every 50 μs.

Tensile stress, strain and elastic modulus were calculated based on the generated tensile stress–strain curve. Hydrophilic nature of the electrospun nanofibrous scaffolds was measured by sessile drop water contact angle measurement using a VCA optima surface analysis system (AST products, Billerica, MA). Distilled water was used for drop formation. The measured contact angle value reflected the hydrophilicity of the scaffolds. FTIR spectroscopic analysis of electrospun nanofibrous scaffolds was performed on Avatar 380 (Thermo Nicolet, USA) over a range of 500–4,000 cm−1 at a resolution of 2 cm−1. The thermal behavior of the electrospun nanofibrous scaffolds was examined by thermo gravimetric analyzer (TGA) TA Q500 (TA Instruments, USA). Measurements were conducted over a temperature range of 0–700 °C, at a heating rate of 20 °C/min under nitrogen purge.

Culture of fibroblasts

Mice fibroblasts were cultured in DMEM supplemented with 10 % FBS and 1 % antibiotic/antimycotic solutions in a 75 cm2 tissue-culture flask and incubated at 37 °C in a humidified atmosphere with 5 % CO2. The populations of fibroblast passage 3 were used for this experiment. The electrospun scaffolds on coverslips were UV-sterilized, rinsed in phosphate-buffered saline (PBS) and soaked in cell culture medium overnight prior to cell seeding to facilitate protein adsorption and cell attachment. The fibroblasts were separated by trypsinization, centrifuged, counted using a hemocytometer and seeded on the scaffolds at a cell density of 7.5 × 103 cells/well and incubated at conditions suitable for cell growth. Tissue culture polystyrene (TCP) was used as the control for cell culture studies.

Proliferation of fibroblasts

The proliferation of cultured fibroblasts was monitored on the third, sixth and ninth day of culture using the colorimetric MTS assay. MTS [3-(4, 5-dimethylthiazol–2-yl)-5-(3-carboxymethoxyphenyl)-2(4 sulfophenyl)-2H tetrazolium], inner salt has a yellow tetrazolium salt which was reduced by the dehydrogenase enzymes secreted by metabolically active cells to form purple formazan crystals. The amount of formazan crystals formed varies proportionally with the number of cells. The samples were prepared for MTS assay by rinsing with PBS to remove non-adherent cells and then incubated in a serum-free medium containing 20 % MTS reagent for 3 h at 37 °C. After incubation, the samples were transferred to a 96-well plate and their absorbance was read in a spectrophotometric plate reader (FLUOstar OPTIMA, BMG Lab Technologies, Germany) at 492 nm.

Morphology of fibroblasts on scaffolds

The cell morphology of in vitro-cultured fibroblasts was analyzed for sixth and ninth day of culture by processing them for SEM studies. After removal of non-adherent cells by repeated PBS wash, the scaffolds with cells were fixed in 3 % glutaraldehyde for 3 h followed by rinsing with deionized water. The cell–scaffold samples were dehydrated using increasing concentrations of ethanol (30, 50, 70, 90 and 100 %), finally treated with hexamethyldisilazan and air-dried in a fume hood overnight to maintain normal cell morphology. The electrospun nanofibrous membranes were sputter coated with gold up to 90 s, and their morphologies were observed by FESEM at an accelerating voltage of 15 kV.

Expression of CMFDA dye

Fluorescent dye expression was observed in sixth day of fibroblasts culture using CMFDA (5-chloromethylfluorescein diacetate), which on cleavage of its acetates by cytosolic esterases produces a brightly fluorescent CMFDA derivative. The cell culture medium was removed, followed by the addition of 180 μL of DMEM and 20 μL CMFDA (25 μM) to the cells adhered on the scaffolds and incubation at 37 °C for 2 h. The CMFDA medium was replaced by adding complete medium and cells were incubated overnight in the incubator. Then the culture medium was removed and the cells were washed with PBS and, after addition of serum-free medium, observed under an inverted Leica DM IRB laser scanning microscope (Leica DC 300F, Germany) at 488 nm.

Expression of collagen

Sirius red staining method was used for analyzing the presence of collagen in the cell matrix. It is a strong anionic dye whose sulfonic acid groups interact with the basic groups of collagen staining it red. This helped us to determine the secretion of collagen-containing ECM by fibroblasts in culture. The cells were first fixed with 10 % formaldehyde, stained with Harris hematoxylin to distinguish the nucleus of the cells and washed three times with deionized water. This was followed by staining with Sirius red stain consisting of 0.1 % Sirius red F3B in a saturated aqueous solution of picric acid for 1 h. The cells were washed with mild acidified water followed by 100 % ethanol and viewed under Leica DM IRB microscope. Collagen is stained red on a yellow background in the nanofibrous scaffolds.

Expression of F-actin

Fibroblast cells on the scaffolds were stained for F-actin on the sixth day of cell culture. The cells were first fixed in 100 % ice-cold methanol for 15 min. The samples were washed with PBS for 15 min and incubated in Triton-X100 solution (0.5 %) for 5 min to permeabilize the cell membrane. Non-specific sites were blocked by incubating the cells in 3 % BSA for 1 h. The samples were then incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin in the dilution of 1:200 for 90 min at room temperature. The samples were washed with PBS thrice to remove the excess staining and then incubated with DAPI in the dilution of 1:3,000 for 30 min at room temperature. The samples were then removed and mounted over a glass slide using Vectashield mounting medium and examined under Olympus FV1000 (USA) fluorescent microscope.

Statistical analysis

The data presented are expressed as mean ± standard deviation. Statistical analysis was done using Student’s t test and the significance level of the data was obtained. The p value ≤ 0.05 was considered to be statistically significant.

Results and discussion

Characterization of nanofibrous scaffolds

The morphology of nanofibrous membranes was analyzed by FESEM. Fig. 1a–d shows the FESEM micrographs of the scaffolds PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen. Evidently fibers with smooth surface without any aggregation were obtained indicating that the AV and collagen were incorporated well within the fibers. Fiber diameters were calculated from the FESEM pictures using the ImageJ analysis software. The average fiber diameters of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofibers were in the range of 519 ± 28, 264 ± 46, 215 ± 63 and 249 ± 52 nm, respectively, and results were tabulated in Table 1.

Fig. 1
figure 1

FESEM micrographs of: a PCL, b PCL-AV 5 %, c PCL-AV 10 % and d PCL/Collagen nanofiber scaffolds. (The average diameter of the composite fibers decreased and the morphology of fibers became finer with the increasing content of aloe vera)

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Table 1 Characterization of electrospun nanofibers for fiber diameter, water contact angle and tensile properties
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Similar to the PCL nanofibers (Fig. 1a) AV and collagen loaded PCL nanofibers (Fig. 1b–d) showed bead-free morphology. However, the loading of AV and collagen significantly decreased the fiber diameter due to increased concentration and solution conductivity. Fibroblasts adhered to nanofibers with smaller diameters compared to fibers with higher diameters [25]. The SEM morphology of PCL-AV 10 % is finer when compared to other scaffolds and hence will be more suitable for fibroblast adhesion and proliferation.

The water contact angle parameter was also analyzed for all the nanofibrous scaffolds as shown in Table 1. It was found that PCL nanofibers were hydrophobic having a contact angle of 133.30°. Upon the incorporation of AV and collagen the scaffolds became hydrophilic. The contact angles obtained for PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofiber scaffolds were 76.40°, 63.90° and 66.30°, respectively. The hydrophilic nature of the scaffolds is very important since it is important for cell adhesion and growth [26]. Blending of natural extracts imparts functional groups such as hydroxyl and carboxyl groups to the scaffolds thereby improving their hydrophilic properties. The PCL-AV 10 % scaffold has better hydrophilic properties when compared to other scaffolds.

To maintain the scaffold when used as a skin substitute, the scaffold has to be strong enough in order to support extensive vasculatures, the lymphatic system, nerve bundles and other structure in the skin. Therefore, the scaffold should have appropriate mechanical properties to absorb forces when they are implanted into the wounds [27]. Fig. 2 shows the nonlinear stress–strain graphs of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen samples. The tensile stress, strain and elastic modulus of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofiber scaffolds are illustrated in Table 1. Tensile stress for PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen samples were 2.43, 2.96, 6.28, and 6.16 MPa and can bear a strain of 84, 78, 113, and 68 %, respectively.

Fig. 2
figure 2

Tensile stress–strain curves of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofiber scaffolds. (The incorporation of aloe vera (10 %) to PCL increased mechanical properties with a higher tensile strength of 6.28 MPa with tensile strain of 113 % desirable for skin tissue engineering)

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The Young’s modulus value for PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofiber scaffold samples were 11.61, 10.03, 16.11 and 28.32 MPa. The data shows higher tensile stress−strain values obtained for PCL-AV 10 % than other nanofiber samples owing to increased mechanical properties. The results implied that blending PCL with AV 10 % gives favorable mechanical properties to the nanofibrous scaffolds. Very high tensile strength may result in the scaffold staying with the wound bed longer until its regeneration.

The FTIR spectrum of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen scaffolds is shown in Fig. 3 (spectra a–d, respectively). The C = O vibration of ester occurs at 1,728 cm−1 in PCL nanofibers (Fig. 3, spectrum a). The CH2 band vibrations of the polymer are presented at 1,362, 1,407 and 1,465 cm−1 and the ester COO vibrations occur at 1,181 and 1,238 cm−1. The peaks at 1,099, 1,047 and 961 cm−1 are due to O–C vibrations with CH2 rocking vibration occurring at 729 cm−1.

Fig. 3
figure 3

FTIR spectra of: a PCL, b PCL-AV 5 %, c PCL-AV 10 % and d PCL/Collagen nanofiber scaffolds. (The spectra confirm the successful incorporation of aloe and collagen to PCL backbone by detection of their characteristic functional groups)

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The FTIR spectrum of AV (5 %) incorporated PCL scaffolds is shown in spectrum b in Fig. 3, the peaks corresponding to hydroxyl groups are observed at 3,012 cm−1. Specifically, the peaks at 1,736 and 1,248 cm−1 clearly indicated the presence of O-acetyl esters. Furthermore, the absorption band of carboxyl groups at around 1,646 cm−1 (probably an asymmetrical COO stretching vibration or O–H deformation vibration of H2O) was found. A peak at 1,032 cm−1 might be due to the glucan units. The pyranoside ring absorption band at 879 cm−1 (C–H ring vibration) and mannose absorption peak at 811 cm−1 were also detected which confirms the successful incorporation of AV in the scaffolds.

The spectrum c in Fig. 3 of PCL-AV 10 % shows all the features of spectrum d (Fig. 3) with increased intensity of the characteristic peaks due to increased concentration of AV. The FTIR spectrum of PCL/Collagen scaffold (Fig. 3, spectrum d) shows the characteristic peaks of collagen along with PCL. N–H stretching around 3,000 cm−1 for amide A, C–H stretching at 3,068 cm−1 for amide B, C = O stretching at 1,600–1,700 cm−1 for amide I, N–H deformation at 1,500–1,550 cm−1 for amide II and N–H deformation at 1,200–1,300 cm−1 for amide III band. The amide I, II and III band regions of the spectrum are directly related to polypeptide conformation confirming the presence of collagen. The spectral result shows the characteristic peaks of aloe and collagen which confirm the successful incorporation of these materials into the polymeric backbone.

The thermal degradation of nanofibrous scaffolds was analyzed by thermo gravimetric analyzer (TGA) and is presented in Fig. 4. Samples (1.5 mg) of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofibrous scaffolds were heated from 0 to 700 °C at a rate of 20 °C per min under nitrogen purge 200 mL per min. PCL degradation curve gives a first weight loss, starting from 385 to 413 °C whereas the PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen samples give first weight loss at 160, 220 and 168 °C, respectively. It shows that the interaction between PCL and the incorporated aloe and collagen which results in decrease of its thermal stability. Hence it is a clear evidence for the successful blend of aloe and collagen with PCL.

Fig. 4
figure 4

The TGA curves of PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofiber scaffolds. (Incorporation of aloe and collagen altered the thermal property of PCL nanofibrous scaffold)

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Cell-scaffold interaction and proliferation

The proliferation of fibroblasts on TCP, PCL, PCL-AV5 %, PCL-AV10 %, PCL/Collagen was analyzed for three different time periods 3, 6 and 9 using MTS assay (Fig. 5). Initially, proliferation on PCL/Collagen scaffold was significantly higher (p ≤ 0.05) than PCL–AV 10 % nanofiber scaffolds and percentage level increases up to 12.55 on day three. However, the rate of proliferation was significantly increased in PCL-AV 10 % when compared to all other scaffolds on days six and nine. It was found that PCL/AV 10 % nanofiber matrix favored cell proliferation which almost increased linearly by (p ≤ 0.01) 17.79 % (6 days) and (p ≤ 0.001) 21.28 % (9 days) compared to PCL. Aloe vera contains significant growth factors which aid in cell proliferation and differentiation. Cell growth was higher on PCL-AV 10 % than other scaffolds and TCP. Suitable mechanical properties, molecular signals from the nanofibers may guide cells entering the cell substrate by their ameboid movement [28].

Fig. 5
figure 5

MTS assay for mice fibroblast proliferation on TCP, PCL, PCL-AV 5 %, PCL-AV 10 % and PCL/Collagen nanofibrous scaffolds on days three, six and nine. (Fibroblast proliferation significantly increased up to 12.55 % in PCL/Collagen scaffold compared to PCL-AV 10 % on day three. However, the rate of proliferation was significantly increased linearly in PCL-AV 10 % by 17.79 % (6 days), and 21.28 % (9 days) compared to PCL. *Indicates significant difference of p ≤ 0.05; **indicates significant difference of p ≤ 0.01; ***indicates significant difference of p ≤ 0.001)

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The hydrophilic nature of the PCL-AV 10 % scaffolds is another reason for better adhesion and proliferation of fibroblasts. Scaffold properties play a vital role in controlling the cell growth and morphology, and impose a direct influence on intracellular responses. Cell behaviors such as adhesion, spreading and proliferation represent the initial phase of cell-scaffold communication.

Figures 6 (a–e) and 7 (a–e) show FESEM micrographs of cell cultured on TCP, PCL, PCL-AV5 % and PCL-AV10 % on days six and nine, respectively. PCL-AV 10 % scaffolds showed the normal morphology and higher cell growth on both days six and nine when compared to other scaffolds and favored the cells to migrate slowly into the fibers and indulge in cell-to-cell interaction via extension of filopodia. Evidence shows that certain aloe derived Glucomannan, a mannose-rich polysaccharide bind specifically to carbohydrate-binding, sites on two β2-integrins called LFA-01 and Mac-1 on the fibroblast and promotes cell adhesion, migration and proliferation. Gibberellins, the growth hormone in aloe stimulates fibroblast proliferation and protein synthesis [29]. The hydrophilic nature of PCL-AV 10 % scaffolds was another reason for better adhesion, migration and proliferation of fibroblasts for skin tissue regeneration.

Fig. 6
figure 6

FESEM micrographs of fibroblast interaction with nanofibrous scaffolds after 6 days of culture on: a TCP, b PCL, c PCL-AV 5 %, d PCL-AV 10 % and e PCL/Collagen nanofiber scaffolds at ×500 magnification

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Fig. 7
figure 7

FESEM micrographs of fibroblast interaction with nanofibrous scaffolds after 9 days of culture on: a TCP, b PCL, c PCL-AV 5 %, d PCL-AV 10 % and e PCL/Collagen nanofiber scaffolds at ×500 magnification

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Expression of CMFDA, collagen and F-actin

The compound CMFDA belongs to a group of chloromethyl derivatives developed for labeling the living cells in vitro. The CMFDA compound is metabolized intracellular in viable cells and converted to a cell-impermeant and fluorescent state within 1 h. The addition of CMFDA to the cell culture medium caused the compound to freely permeate the cell membrane and acted upon by cytosolic esterase, producing a CMFDA derivative that was brightly fluorescent. CMFDA dye expression was observed on the sixth day of fibroblast culture using Leica fluorescence microscope.

The fibroblast density and morphology were observed to be increased in PCL-AV 10 % treated scaffolds compared to other nanofibrous scaffolds. Figure 8 (a–e) shows the CMFDA stained fibroblast cells on different scaffolds. Collagen is the major protein of the extracellular matrix, and is the component which ultimately contributes for skin strength and elasticity. Collagen staining with Picro-sirius red confirmed the secretion of the ECM by the cells in culture.

Fig. 8
figure 8

The detection of CMFDA-labeled fibroblasts on day six of cell culture on: a TCP, b PCL, c PCL-AV 5 %, d PCL-AV 10 % and e PCL/Collagen nanofiber scaffolds at ×10 magnification. (Fibroblast cell density and morphology were better in PCL-AV 10 % nanofiber scaffold compared to TCP and other nanofibrous scaffolds)

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Figures 9 (a–e) and 10 (a–e) show the secretion of collagen on fibroblasts grown on TCP, PCL, PCL-AV5 %, PCL-AV10 % and PCL/Collagen nanofibrous scaffolds on days six and nine, respectively. The results showed the increased secretion of collagen on PCL-AV 10 % than all other scaffolds. Previous studies showed aloe derived Glucomannan, a mannose-rich polysaccharide, and gibberellin, a growth hormone, interacted with growth factor receptors on the fibroblast, thereby stimulating its activity and proliferation, which in turn significantly increased the collagen synthesis after topical and oral applications. Aloe not only increased collagen content of the wound but also changed collagen composition (type III) and increased the degree of collagen cross-linking. Therefore, it accelerated wound contraction and increased the breaking strength of resulting scar tissue [30].

Fig. 9
figure 9

Optical microscope images showing the secretion of collagen by fibroblast cells on day six using picro-sirius red staining on: a TCP, b PCL, c PCL-AV 5 %, d PCL-AV 10 % and e PCL/Collagen nanofiber scaffolds at ×10 magnification. (PCL-AV 10 % nanofiber scaffold showing significant increase in collagen secretion compared to TCP and other nanofibrous scaffolds)

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Fig. 10
figure 10

Optical microscope images showing the secretion of collagen by fibroblast cells on day nine using picro-sirius red staining on: a TCP, b PCL, c PCL-AV 5 %, d PCL-AV 10 % and e PCL/Collagen nanofiber scaffolds at ×10 magnification. (PCL-AV 10 %, nanofiber scaffold showing significant increase in collagen secretion compared to TCP and other nanofibrous scaffolds)

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The increased secretion of collagen proved that the biocomposite nanofibers PCL-AV 10 % scaffold have potential for wound healing through skin tissue regeneration. Staining for cytoskeleton protein, F-actin of mice primary fibroblasts cultured on the TCP and scaffolds are shown in Fig. 11 (a–e). The cell cytoskeleton was labeled with TRITC conjugated phalloidin (red) and nucleus with DAPI (blue) and visualized by confocal microscopy. The PCL fibers were autofluorescent; however, their fluorescence was not as clear as the stained cell. Cells generally exhibited bipolar and elongated morphology, which is the characteristic of cultured mice dermal fibroblast, but it is distinctly different for their fully spread morphology on different materials. Fibroblast growing on the PCL-AV 10 % nanofibers showed a large number of microfilaments and highly oriented morphology.

Fig. 11
figure 11

F-actin expression of fibroblast cells on: a TCP, b PCL, c PCL-AV 5 %, d PCL-AV 10 % and e PCL/Collagen nanofiber scaffolds at ×60 magnification. (The cells’ cytoskeleton were labeled (red) with TRITC conjugated phalloidin and nucleus (blue) with DAPI)

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The stained actin showed well-defined stress fibers, which appeared oriented in a parallel direction as long linear arrays following the main cellular axis throughout the entire cytoplasm (Fig. 11d) while cells on the other nanofibers have vague microfilaments, and they were dispersed randomly without noticeable structural organization (Fig. 11a–c and e). Cytoskeletal organization is related to cell adhesion, spreading and functions on the substrates. These results showed that fibroblasts can have better and faster attachment, proliferation and guided growth on the PCL-AV 10 % nanofibers for skin tissue engineering.

Conclusion

The PCL-AV 10 % electrospun nanofibrous scaffold showed good potential for supporting cell attachment and proliferation for skin tissue engineering. Cells seeded on such scaffold tend to maintain phenotypic morphology and guided growth according to nanofiber orientation. It is probably correlated to the different components present in AV that are able to promote the regenerative process of the skin as a function of the dose-dependent concentrations. The obtained results showed that AV offers various opportunities for the development of natural therapeutic strategies through the topical route. This novel herbal biodegradable scaffold has shown that the plant may be used as natural agent for the treatment of different skin disorders or damage with potential applications for tissue engineering. The PCL-AV 10 % scaffold in comparison with the previous scaffolds of PCL/Collagen will serve as a better tissue-engineering scaffold in the longer run because of the relatively low cost and natural origin in addition to its water retention properties, which are typically required for light to moderate exudate wounds. In future, better approaches would be available to devise nanofibrous scaffolds which are capable of supporting the tissues in their natural environment and possess controlled surface topography as well as structural morphology. One such approach could be to formulate nonwoven 3D scaffolds for wound dressing and skin tissue regeneration.