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Electrospun Core–Shell Fibrous 2D Scaffold with Biocompatible Poly(Glycerol Sebacate) and Poly-l-Lactic Acid for Wound Healing

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Abstract

Biomimetic scaffolds made by synthetic materials are usually used to replace the natural tissues aimed at speeding up the skin regeneration. In this study, a flexible and cytocompatible poly(glycerol sebacate)@poly-l-lactic acid (PGS@PLLA)  fibrous scaffold with a core–shell structure was fabricated by coaxial electrospinning, where the shell PLLA was used to be a skeleton with pores on the fibrous surface. The fibrous morphology with pores on the surface of the prepared fibers was observed by SEM. The core–shell microstructure of PGS@PLLA fibers was confirmed by TEM and Laser Scanning Confocal Microscopy (LSCM). In addition, the prepared fibers exhibited a strong ability to repair tissues of the skin wound, where the stability of cell security and proliferation, and the lower inflammatory response were all superior to those of pure PLLA scaffold. It’s worth noting that the percentage of skin tissue was regenerated by 95% within 14 days, which suggests the potential application for electrospun-based synthetic fibrous scaffolds on wound healing.

Introduction

Nowadays, the death rate caused by the infection of hard-to-healing wounds is high [1], especially in the cases of the necrosis of tissues [2] or the organs grafting [3] in the war and the poor countries [4, 5]. The tissues regeneration and engineering could alleviate the healthcare burden worldwide. Recently, the understanding of the interactions between cells and tissues, tissue engineering scaffolds prepared by biocompatible materials to mimic the extracellular matrix (ECM) mainly have been used in those regards by combing material property and tissue engineering principle [6,7,8]. Biocompatible materials play a more and more important role in tissue engineering [9], medicine delivery [10], antibacterial application [11], and wound healing [12]. Among them, synthetic polymer materials are very popular in the skin regeneration [13, 14]. As one of the synthetic polymers, Poly-l-lactic acid (PLLA) with controlled degradation has been studied in clinic medicine for a long time [15]. PLLA has advantages of high recyclability, good stability and spinnability [16]. More and more natural materials are appeared to apply in bioengineering lately [17]. However, most natural materials can’t be electrospun to hold stable fibrous morphology. The chain character of natural materials won’t provide the effect of framework in the fibers. What’s more, during the electrospinning process, pores could appear on the surface of the PLLA fibers due to high evaporation of solvent, which was named as “breath figure” [18, 19]. However, PLLA has properties of high hydrophobicity and slow degradation, which hinders its development in the regeneration of soft tissues or wounds [20, 21]. Thus, other different synthetic polymers with good biocompatibility and fast degradative property were studied. Among them, poly(glycerol sebacate) (PGS) has been attracted attention as it was formed by non-toxic and tough materials—glycerol and sebacic acids, which have been approved by the US Food and Drug Administration [22, 23]. PGS can be degraded under human condition, and the degradative intermediates can be eliminated from body by metabolism [24]. It has been identified to be well biocompatible, flexible and degradable, which are widely used as kinds of patch of organs and wounds [25]. However, PGS has low specific surface area and low spinnablility, which cannot support the formation of fibrous morphology, and further restrict cells attachment and migration for tissues or wounds regeneration [26]. Thus, using tissue scaffolds with micro-nanofibers can provide more space to go deep into scaffolds for cells attachment and migration [27].

Electrospinning technology has been widely used to fabricate fibrous scaffold [28, 29]. Owing to the similar properties of the dermal extracellular matrix (ECM), it makes a wide range of applications in tissues regeneration [30]. Both porous network and high surface-to-volume ratio are in favour of cellular attachment, migration and proliferation. In addition, good permeability of oxygen, carbon dioxide and water, outputting of metabolic waste and effective inhibiting pathogen invasion are the additional advantages of the fibrous membrane matrix [7, 31,32,33]. Coaxial electrospinning is a novel method of the fabrication of micro-/nanofibers, and it is facile and high effective to produce fibrous membranes with a core–shell structure, which can integrate more than two different materials with synergistic effect [26, 34]. The prepared core–shell fibers were normally applied to tissue scaffolds [35, 36], drugs controlled release [9, 18, 37], and antibacterial application [38].

In our research, the PGS@PLLA fibers scaffold with PGS core and PLLA shell was fabricated by coaxial electrospinning. During the electrospinning process, the inner core pre-PGS was wrapped by the outer layer PLLA, which formed a Taylor cone on the tip of needle under the high voltage. The pre-PGS core of PGS@PLLA fibers scaffold was cross-linked with the efficient covalent and hydrogen bonds[22] to form PGS by thermocuring (depicted in Fig. 1a). The PLLA acting as the skeleton to form the PGS@PLLA fibers scaffold with a core–shell structure rarely appears in tissue regeneration of wounds. The PGS fibers can be degraded by the infiltration of water and exposure after the degradation of thin PLLA shell, which accelerates the degradation of scaffolds and it is beneficial to the wettability of the PGS@PLLA fibers scaffold, which the shell layer was composed by PLLA and PGS. It means the shell layer is not the strict PLLA layer, so the surface of PGS@PLLA fibers scaffold was moist as shown in Figure S1 [6]. The flexible PGS@PLLA fibers scaffold was applied and covered on the wound of rat back (shown in Fig. 1b). The scaffold provides a compatible independent condition for cells attachment, migration, proliferation, and the accesses for metabolic gases, nutrition and wastes. In addition, it also provides a barrier against the external bacteria as described in Fig. 1b, which shows a lower inflammatory response. Therefore, our prepared PGS@PLLA fibers scaffold exhibited an excellent prospect for the regeneration of hard-to-healing wounds.

Fig. 1
figure1

Schematic captions of the preparation, structure and application of PGS@PLLA fibers scaffold. a The fabrication of PGS@PLLA fibers scaffold from coaxial electrospinning. b Illustration of PGS@PLLA fibers scaffold applied to back wounds in mice

Experimental Section

Materials

Sebacic acid (99%, MW = 202.25, C10H18O4) was purchased from Aladdin, glycerol (A.R., MW = 92.09, C3H8O3) was purchased from Sigma and poly-l-lactic acid (PLLA, Mn = 200 KDa) were purchased from the company named Jinan Daigang Biomaterial Co. Ltd. The fluorochromes including coumarin and perylene were all obtained from Aladdin. Dichloromethane (DCM), trichloromethane and N,N-dimethylformamide (DMF) were of reagent grade and purchased from Sigma. PBS was purchased from HyClone and deionized water was prepared by water purification machine (UTP-1-10T). All solvents were used without further purification.

Fabrication of the PGS@PLLA Fibers Scaffold

Sebacic acid was recrystalled from absolute ethyl alcohol before being used. Equimolar ratios of glycerol and sebacic acid were mixed in the flack at 140 ℃ to melt the sebacic acid, then heat up to 160 ℃ for 8 h to formed the PGS pre-polymer. And the nitrogen flow was blown at a velocity of 0.2 m3 h−1 during the reaction.

During fabrication of the PGS@PLA fibers scaffold with core–shell structure, Pre-PGS was dissolved in the mixed solvent of DCM and DMF (v:v = 3:1) to achieve concentrations of 35, and 40 wt%. The concentration of PLLA/trichloromathane was 10 wt%. The as-prepared solutions were loaded into two 5 ml syringe with a coaxial needle and positioned on two syringe pumps, respectively. The inner and outer needles were 22G and 17G. And the flow rate of accesses was 0.4 mL h−1 and 1.2 mL h−1, respectively. In addition, the electrospinning voltage was 20 kV and the distance between the needle and collector was set at 15 cm. The obtained PGS@PLLA fibers scaffolds were exposed on room temperature to evaporate any residual solvent. Furthermore, the membrane was thermo-cured in 120 ℃ under vacuum for 72 h to cure PGS fibers.

Characterizations of Pre-PGS and the PGS@PLLA Fibers Scaffold

The pre-PGS was smeared on the KBr salt tablet for Fourier-transform infrared (FTIR) characterization using an FT-IR spectrometer (Nicolet 5700, Thermo Company, USA). 1H NMR (AV400 NMR, Bruker Company, Germany) spectra was used to evidence the structure of pre-PGS as well. It was tested at 600 MHz using DCl3 as solvent. The morphology of the PGS@PLLA fibers scaffold was examined by SEM (FEI Quanta 250, the Netherlands). The dry scaffold sample ensuring they are completely free of moisture was pasted on the sample table and then were sputter coated with gold (Model 550; Electron Microscope Sciences) in preparation for SEM. The SEM image were taken on 500× and 2000× . At least 60 different fibers and 100 segments were randomly counted to measure average diameter using Nano Measurer. The thermal properties for the PGS@PLLA fibers scaffold, PGS and PLLA were measured by Differential Scanning Calorimetry (DSC, Q1600, TA Instruments, USA). They underwent two cycle of heating to 250 ℃ and cooling to – 50 ℃. The rate of heating and cooling was 5 ℃ min−1. The crystal structure was further studied by Wide-angle X-ray powder diffraction (WXRD, D8 Advance, Bruker, Germany) after curing progress with an angle range of 5°–90°. The micro-structure image of the PGS@PLLA fibers encapsulated outer PLLA was recorded by the Transmission Electron Microscopy (TEM, Tecnai G2 T20, FEI, USA). The fluorescent image of the PGS@PLLA fibers scaffold was obtained from the Laser Scanning Confocal Microscopy (LSCM, Leica TCS SP8, Germany), which coumarin and perylene as fluorescent dye were added in outer and inner resolutions, respectively. The concentration of the coumarin dye was 0.01 mg per 3 mL pre-PGS solution and 5 mL PLLA solution had 0.01 mg perylene dye.

Characterization of Physical Properties

The degradation property of the PGS@PLLA fibers scaffold and PLLA fibrous scaffold were evaluated by the ratio of mass loss at different time, which were immersed in the phosphate buffer (PBS) at 37 ℃. Morphology of scaffolds after degradation at different time were characterized by SEM. The ratio of mass loss (D%) was determined using equation [39].

$$D\% = \frac{{W_{0} - W_{t} }}{{W_{0} }} \times 100\% ,$$
(1)

where W0 is the initial scaffolds dry weight and Wt is the residual scaffolds dry weight after degradation at time t.

The strips of scaffolds (30.0 × 10.0 mm) were tested by a mechanical tester (UTM5205XHD, SUNS, China) in Uniaxial Tensile Tests. The effective length of samples was 10 mm and then the strips were stretched to failure at a speed of 5 mm min−1. Young’s modulus of the samples was obtained from the stress–strain curves [39].

Cytotoxicity Tests of the PGS@PLLA Fibers Scaffold

The L929 cells were used to measure the biosecurity of the PGS@PLLA fibers scaffold, which cultured in DMEM with the FBS. The PGS@PLLA fibers scaffold with core–shell structure and PLLA fibrous scaffold that were similar with weight were added to 96-wells cell culture dishes that each well containing 1 ml culture medium and incubated for 24 h, and then sterilized with exposure to an ultraviolet ray light lamp in a laminar flow hood for 30 min per side. The L929 cells were adjusted to different concentration after digesting and rehung. Then the L929 cells were seeded in 96-wells cell dishes with a density of 8000 cells per well (100 μL) for 24 h in order to allow attachment before the test. The cells were rinsed by PBS after the media in the 96-wells dish was regularly removed. Afterwards, 24 h aged extract material solutions were added to the wells and the cells were incubated at 37 ℃ with the extract solutions for another 24 h. Every concentration was set up ten groups. The MTT was used to evaluate the cell metabolic and viability. 50 μL MTT reagent was added to the solution from where the culture medium was removed under dark environment. The cells were incubated for another 4 h. Furthermore, the solution was added 150 μL DMSO and cultured for 15 min in a shaker after the liquid supernatant was removed. The absorbance intensity of each sample and control well were measured at a wavelength of 490 nm using a microplate reader (Thermo Labsystem, USA). In addition, an equivalent number of cells on tissue culture plate (TCP) which served as control. The experiment condition of control was the same as the PGS@PLLA fibers scaffold and the PLLA fibrous scaffold.

The cell viability expressed by the percentage of living/dead cells with respect to the control group and the change of the cell viability from day 1 to day 3. The L929 cells were seeded in 6-wells cell dishes with a density of 20,000 cells per well (2 mL) for 24 h to guarantee the attachment. The cells were transferred to the 24 h aged extracted material solutions and incubated for 24 h and 72 h. Every wells were added to the staining solution and moved to incubator for 30 min. Afterwards, the media in the 6-well dish were removed, and the cells were washed by PBS. All experiments were repeated for three times. Finally, the fluorescent images were obtained by using the Inverted Fluorescence Microscopy.

Chronic Wound Healing Study of the PGS@PLLA Fibers Scaffold

Twenty-four 6-week adult female mice (20 ± 5 g) were used in our experiment after breeding 1 week in order to adapt the condition. We set up two groups (blank and pure PLLA fibrous scaffold) as the control to compare with the PGS@PLLA fibers scaffold. The animals were divided into three groups randomly, the PGS@PLLA fibers scaffold, the PLLA fibrous scaffold and control. The rats were anesthetized by using an intraperitoneal injection of 1% chloral hydrate (20 mg kg−1) and their backs were shaved under sterile condition. A 1 cm2 area wound which was deep to the fascia was cut out on the back of each mouse. 100 μL E. coli and 100 μL S. aureus were dropwise added on the wound bed. The wounds were closed by film of indwelling needle to wait for inflection. Afterwards, the sterilized wound dressings containing saline solution were applied to the inflect wound and closed to avoid multiple inflection from other bacteria. The rats were housed in cage and allowed to heal for 14 days with changing wound dressing every 2 days, respectively. On 0th, 3th, 7th, 10th, 14th day, the wound regeneration of each group should be recorded and photos were taken by digital camera. All mice were euthanized after 14 days and granulation tissues over the surface of wounds was cut out for the immunohistochemistry analysis.

Inflammation and Histology

The wound tissue samples were isolated from sacrificed mice that healed for 2 days and 14 days and fixed by 4% paraformaldehyde overnight. Then the samples were dehydrated by gradient ethanol solution from 50 to 100%. The dehydrated tissues were soaked in the mixed ethanol/xylene (v:v  = 1:1) solution and xylene reagent in turn until the tissues appeared to be transparent. Afterwards, as addressed tissues were embedded in I, II paraffin at 40 °C for 1 h to eliminate transparent reagent. Several 5 μm-thick sections of the addressed tissue were gained by microtome and prepared to hematoxylin–eosin staining. The tissue slices were closed by neutral resin and viewed under the optical microscopy for further analysis.

Statistical Analysis

Data were performed by SPSS 17.0 statistical analysis software. Statistical comparisons were conducted by Student’s t test or one-way ANOVA followed by post hoc Student–Newman–Keuls test. p < 0.05 was considered statistically significant.

Ethical Statements

L929 cells were obtained from Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, (Jiangsu, China). These animal protocols were approved by the Xuzhou Medical University Laboratory Animal Center.

Results and Discussion

Preparation of the PGS@PLLA Fibers Scaffold

The pre-PGS was synthesized by the condensation reaction, the FTIR spectrum of pre-PGS in Figure S2 shows the peaks for –C=O at 1740 cm−1 and the peaks for –OH at 3438 cm−1, which is completely consistent with the previous report [40]. As depicted in 1H NMR spectrum of Figure S3, it shows –CH2 groups in the backbone supported by sebacic acid at 1.3, 1.6 and 2.34 ppm, and –CH2, –CH– at 4.18 and 3.7 ppm from glycerol, which were influenced by the distance from ester groups. It also confirmed the formation of PGS pre-polymer. The PGS elastomer shown in Figure S4 was fabricated after thermo-curing according to the experiment section. In the research, 35 wt% concentration of PGS as core solution was chosen to prepare the desired PGS@PLLA fibers scaffold that could retain the fibrous morphology, while the membrane spun with 40 wt% core concentration of PGS presented irregular fibers with beads (Figure S5). As known to all, the main factors of beaded nanofibers’ formation are the surface tension of solution, charge density carried by the jet and the viscoelasticity of solution, and these factors are influenced by the characters of the solution, electrospinning condition and the environmental condition, for example the solvent volatilization, temperature and humidity, solubility of materials, voltage and so on. Pre-PGS with low molecular weight is a kind of material that can’t be electrospun, and it will form droplets with an electrically driven jet [41]. PLLA was used to electrospinning in our experiment with a suitable concentration that reported [42]. While the concentration of core solution increased, the shell PLLA solution will not wrap the core solution because of the jam with the solvent volatilization. The beaded fibers were formed by the low surface tension of solution with the leakage of core pre-PGS solution.

The PGS@PLLA fibers scaffold as a kind of electrospun membrane could be folded, exhibiting an excellent toughness (shown in Figure S6). It is clear that the PGS@PLLA fibers scaffold still remain pores because of the outer solvent flash evaporation (shown in Fig. 2a). The fiber diameter is from 0.75 to 2.75 μm (Fig. 2b). The porous microstructure and rough surface of fibers are beneficial to cell attachment [14]. In addition, the partly crosslinked fibers formed from the phase separation of low molecular pre-PGS may be worked to biological wettability and mechanical strength [43, 44].

Fig. 2
figure2

The characterization of PGS@PLLA fibers scaffold. a SEM images of PGS@PLLA fibers scaffold with pores on fibers. b The fiber diameter profile of PGS@PLLA fibers scaffold. c DSC curves of PGS, PLLA and PGS@PLLA fibers scaffold for verifying non-reactions between PGS and PLLA. d X-ray diffraction pattern of PLLA and the PGS@PLLA fibers scaffold

In our experiment, the PGS@PLLA fibers scaffold shows two groups of melting temperature (Tm) where Tm of PGS at about 23 ℃ and 8 ℃, and Tm of PLLA at 168 ℃. PGS is amorphous at room temperature as shown in Fig. 2c, while the glass temperature (Tg) of PLLA is 61 ℃ and the crystal peaks are shown in Fig. 2d. The peaks of two independent groups offset from the peaks of PGS (21 ℃ and 6 ℃) and PLLA (180 ℃) because of the mixture of two materials, as shown in Fig. 2c in DSC curve of the PGS@PLLA fibers scaffold. It confirms that the cured progress of pre-PGS had no influence on the PLLA that used to support the core of PGS. The WXRD pattern shows three peaks of the PGS@PLLA fibers scaffold at 2θ = 16.5, 19.1 and 23.2°, which are a bit wider than the PLLA lattice parameters impacted by the mixture of amorphous and semi-crystal polymers (Fig. 2d). Based on the XRD analysis, it can be confirmed that the semi-crystal structure was not destroyed by the condition of thermal cross-linking of PGS and there not have been any interaction between the PGS and PLLA chains.

The core–shell structure of the PGS@PLLA fibers scaffold were evaluated by Laser Scanning Confocal Microscopy (LSCM). The view of the PGS@PLLA fibers scaffold in LSCM is shown in Fig. 3a, exhibiting two different fluorescent colors. The PGS that reflected red fluorescence (Fig. 3c) was encapsulated into the fiber core and fluorochrome coumarin was mixed in the PLLA solution, which shows the green fluorescence (Fig. 3b). Figure 3d was obtained by overlapping the images of Fig. 3b, c, showing the obvious distribution of the external of PLLA and the core PGS within the nanofibers. It is indicated that the outer PLLA layer covered on the PGS fiber through the fluorescent image of cross section of fiber shown in Fig. 3d, e. The TEM image of the core–shell PGS@PLLA fibers scaffold, as shown in Fig. 3f, presents a clear interface between the core and shell of the fiber. In addition, the overall diameter of the fiber is around 1000 nm and the core diameter is around 700 nm. The fibers with the core–shell structure can be adequately developed by controlling the core and shell layers [45].

Fig. 3
figure3

The structural characterization of the fiber. a White light image of the PGS@PLLA fibers scaffold. b LSCM image distribution of PLLA shell (green) and c PGS core (red). d The coincident drawing of b and c. e The enlarged image of part of d. f TEM images of the core–shell structure

Mechanical Testing and Biodegradation of the PGS@PLLA Fibers Scaffold

Previous studies have been reported that soft matrices can improve the regeneration of impaired tissues [46]. In our study, three kinds of samples displayed typical stress–strain curves (shown in Fig. 4e). It can be seen that the PGS@PLLA fibers scaffold with cured (~ 67%) had a lower tensile strength compared with the pure PLLA fibrous scaffolds (~ 71%). However, it was higher than that of the uncured PGS@PLLA fibers scaffold which accounted for only 43% of the cured PGS@PLLA fibers scaffold. For a quantitative comparison, Young’s modulus determined from the initial slope at small strains [40]. Figure 4f reveales Young’s modulus of the PGS@PLLA fibers scaffold (19.99 MPa) was higher than that of uncured PGS@PLLA fibers scaffold (12.01 MPa). Due to a little pre-PGS emigrated to attach to the surface of fibers while electrospinning, it made the cured PGS become the crosslinker among the fibers of the scaffold. Although the stress–strain curves results showed that mechanical strength of the PGS@PLLA fibers scaffold as well as the PLLA fibrous scaffold could meet the requirement of tissue engineering materials, the PGS@PLLA fibers scaffold performed softer than the PLLA fibrous scaffold. As a result, the PGS@PLLA fibers scaffold is suitable for skin wound regeneration [20].

Fig. 4
figure4

Degradative morphologies of PGS@PLLA fibers for a 0 day, b 28 days, c 60 days and d the ratio mass loss of different degradation time. The mechanical characterization of PGS@PLLA fibers scaffold that cured and uncured, PLLA fibrous scaffold. e Stress–stain curves and f bar graphs of Young’s modulus

SEM images (Fig. 4a) were used to show the fibrous morphology at degradation time of 0 day, 28 days and 60 days, it can be found that the fibers of the PGS@PLLA fibers scaffold was close-knit before degradation. After 28 days of degradation (shown in Fig. 4b.), the structure tent to be loose but still held the fibrous structure. However, in the 60th day (as shown in Fig. 4c), the whole scaffold was corroded to lead fibers broken and damaged gaps, and it can not keep structure of micro-nanofiber network. In addition, Fig. 4d shows that the mass loss percentage is almost 14% for the PGS@PLLA fibers scaffold, and 7% for the PLLA fibrous scaffold over 28 days, following the two samples more loss of 32% and 8% after 60 days. At first, PGS was degraded along with water infiltrating, as the degradative time passed, the PGS core was more exposed after the PLLA shell was degraded. Thus, the degradation rate of PGS@PLLA fibers scaffolds exhibited faster than that of PLLA fibrous scaffold. The ester groups of PGS can be accounted for the run-up of degradation rate of the PGS@PLLA fibers scaffold. Besides, the PGS can be degraded to no-toxic intermediate products in body condition and the degradation products can be eliminated by human metabolism [22].

Biomedical Properties of the PGS@PLLA Fibers Scaffold

The cytotoxicity of material is important for the biomedical potential of materials [47]. To investigate the biocompatibility of the PGS@PLLA fibers scaffold with L929 cells, firstly, the cells viability were assessed by the cell counting kit-8 (CCK-8) assay and Live/dead analysis, and gradient cells concentrations (from 0.01 to 0.2 μg mL−1) were set up to evaluate the survive and grow ability of cells. CCK-8 assay demonstrated that the L929 cells were viable on the PGS@PLLA fibers scaffold with over 90% viability in all concentration gradient (in Fig. 5a). Compared to the PLLA fibrous scaffold, the cells viability of the PGS@PLLA fibers scaffold was lower than that of the PLLA fibrous scaffold in the low cell concentration (0.01 μg mL−1). When the cell concentration raised as 0.05, 0.1, 0.15 and 0.2 μg mL−1, as contrasted with the viability of cells on the PLLA fibrous scaffold that changed severally at 92%, 92%, 91%, 82%, the viability of cells cultured on the PGS@PLLA fibers scaffold was changed at 98%, 96%, 92% and 91%, respectively. It confirms that the results of PGS@PLLA fibers scaffold were stable when the cell concentration increased, which reveals the stead biosecurity and excellent cells metabolism of the PGS@PLLA fibers scaffold.

Fig. 5
figure5

Viability of cells growth on the electrospun fibrous scaffolds. a L929 cells viability with different cell gradient concentrations cultured on PGS@PLLA fibers scaffolds and PLLA fibrous scaffolds. b Live/dead staining of L929 cells on PGS@PLLA fibers scaffolds and PLLA fibrous scaffolds on 1 day and 3 day. The statistical number of living cells (c) and dead cells (d) were counted from fluorescent images. Cells shown green and red fluorescence are alive and dead cells, respectively. *p < 0.05 compared with the corresponding groups

Live/dead analysis was used to express and calculate the situation of live and dead of L929 cells adhered on the sample scaffold (Fig. 5c, d). At 1 day, there was no obvious quantitative difference between both kinds of scaffold, but at the 3th day, compared to the PLLA fibrous scaffold, the quantity of live cells (dyed green) grown on the PGS@PLLA fibers scaffold was more abundant and the dead cells (dyed red) were lower (in Fig. 5b). As the quantity of viable cells seeded on both kinds of scaffold for 3 days, the statistic number of living cells of the PGS@PLLA fibers scaffold is 1.4 times higher than that of PLLA fibrous scaffold (Fig. 5c). In addition, the PGS@PLLA fibers scaffold presented a low quantity of dead cells after 3 days culturing, which is just account for 35% of dead cells on the PLLA fibrous scaffold (Fig. 5d). Thus, being the cellular compatibility and advanced metabolism, the PGS@PLLA fibers scaffold is better than the PLLA fibrous scaffold in vitro, which provides a gentle and suitable condition for cells attachment, growth and proliferation.

The PGS@PLLA Fibers Scaffold for Wound Healing

For the vivo experiment, the wound healing capacity of the PGS@PLLA fibers scaffold was investigated by constructing the E. coli infected full-thickness skin defect model with a area of 1 cm2 on the back of the mouse. The testing samples were divided into three groups, in which the control group and the PLLA fibrous scaffold were treated as comparison. The wounds closure process and potential were all recorded to analyze the difference of the testing samples in detail. The decease of the wound area was depended on the time and was not obvious at day 4 in all groups. By day 7, the reduction in wound area started to present slightly and the decrease of wounds closure could be observed prominently at day 10. The wound covered with the PGS@PLLA fibers scaffold was almost healed after 14 days compared to the other two samples (shown in Fig. 6a). A kind of PCL-Chitosan core–shell scaffold that reported recently was compared with PGS@PLLA fibers scaffold, which the area of healing wound was 80% approximately after 14 days [7]. While Fig. 6c estimated the quantitative wound-healing datas about our scaffold, which was upon 95% by 14 days for the PGS@PLLA fibers scaffold. From the results, PGS@PLLA fibers scaffold has a superior repair effect. It also evidenced by the growth of epidermal in the histology experiment as shown in Fig. 6d. The epidermal thickness for the PGS@PLLA fibers scaffold was approximately two times thicker than that of the PLLA fibrous scaffold and about nine times thicker than that of the control one. It also evaluated that the wound treated with the PGS@PLLA fibers scaffold experienced a shorter healing period than that of the other two samples.

Fig. 6
figure6

Analysis of healing progress and inflammatory response of the wound. a Digital photograph of covering electrospun fibrous scaffolds from 0 to 14 day. b Images of histological section for the control, PLLA fibrous scaffold and PGS@PLLA fibers scaffold 2 and 14 days postoperation (the black, blue and red arrows point to the broken skin, plasmocytes and new vessels). c The statistical areas of wound healing for electrospun fibrous scaffolds. d Quantitative analysis of the dermal thickness of wound healing. *p < 0.05 compared with the corresponding groups

The re-epithelialization processes of the testing samples were represented by histological sections shown in Fig. 6b. The tissues were stained by hematoxylin & eosin in our study. At the second day after wound presence, it can be seen the broken skin tissue indicated by the black arrows in Fig. 6b(A–C). The epidermal of the wounds were represented, vessues and other cells at the derma also recovered at 14th day in Fig. 6b(D–F), while the group for the PGS@PLLA fibers scaffold put up the outstanding effectiveness. In addition, the histological sections can be used to assess the inflammatory response in the wound area as well. There were a number of plasmacyte appeared in the wound sections indicated by blue arrows at day 2 for the control and the PLLA fibrous scaffold groups. The PGS@PLLA fibers scaffold presented the slight inflammatory response due to its good biocompatibility.

Conclusion

We successfully fabricated the PGS@PLLA fibers scaffold with a core–shell structure by the coaxial electrospinning technology. The PLLA shell layer provides a holder to support the fibrous morphology of PGS as a core part of fibers, exhibiting the good cytocompatibility, flexibility and degradability. This electrospun-based engineering scaffolds consisted of synthetic materials and core–shell structure makes it possible to facilitate a wide range of biomedical and clinical applications. In our study, it was evaluated that the PGS@PLLA fibers scaffold presented more flexibility and higher degradable ability than that of the PLLA fibrous scaffold. The cells viability of the PGS@PLLA fibers scaffold is all above 90% at different cell concentrations in vitro, the inflammation on the skin tissue is lower than the other two samples and the area of wound regeneration is 95% in vivo, which is due to the composition and construction of PGS. The porosity, architecture and composition of the PGS@PLLA fibers scaffold contributed to the scaffold, tissue attachment and healing process. Therefore, we believe that the PGS@PLLA fibers scaffold with the core–shell structure is highly potential for the applications of wound healing. We will probe its application into the necrotic tissues and explore its role as a kind of organs patch in the clinical field.

References

  1. 1.

    Chen G, Yu Y, Wu X, Wang G, Gu G, Wang F, Ren J, Zhang H, Zhao Y. Microfluidic electrospray niacin metal-organic frameworks encapsulated microcapsules for wound healing. Research.2019;2019:1.

  2. 2.

    Sun X, Zheng R, Cheng L, Zhao X, Jin R, Zhang L, Zhang Y, Zhang Y, Cui W. Two-dimensional electrospun nanofibrous membranes for promoting random skin flap survival. RSC Adv.2016;6:9360.

  3. 3.

    Ravichandran R, Venugopal JR, Sundarrajan S, Mukherjee S, Sridhar R, Ramakrishna S. Expression of cardiac proteins in neonatal cardiomyocytes on PGS/fibrinogen core/shell substrate for cardiac tissue engineering. Int J Cardiol.2013;167:1461.

  4. 4.

    Griffin DR, Weaver WM, Scumpia PO, Di Carlo D, Segura T. Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat Mater.2015;14:737.

  5. 5.

    Chen G, Yu Y, Wu X, Wang G, Ren J, Zhao Y. Bioinspired multifunctional hybrid hydrogel promotes wound healing. Adv Funct Mater.2018;28:1801386.

  6. 6.

    Ifkovits JL, Devlin JJ, Eng G, Martens TP, Vunjak-Novakovic G, Burdick JA. Biodegradable fibrous scaffolds with tunable properties formed from photo-cross-linkable poly(glycerol sebacate). ACS Appl Mater Interfaces.2009;1:1878.

  7. 7.

    Pal P, Srivas PK, Dadhich P, Das B, Maulik D, Dhara S. Nano-/microfibrous cotton-wool-like 3d scaffold with core-shell architecture by emulsion electrospinning for skin tissue regeneration. ACS Biomater Sci Eng.2017;3:3563.

  8. 8.

    Baker BM, Trappmann B, Wang WY, Sakar MS, Kim IL, Shenoy VB, Burdick JA, Chen CS. Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments. Nat Mater.2015;14:1262.

  9. 9.

    Sperling LE, Reis KP, Pranke P, Wendorff JH. Advantages and challenges offered by biofunctional core-shell fiber systems for tissue engineering and drug delivery. Drug Discov Today.2016;21:1243.

  10. 10.

    Thakur N, Sargur Ranganath A, Sopiha K, Baji A. Thermoresponsive cellulose acetate-poly(N-isopropylacrylamide) core-shell fibers for controlled capture and release of moisture. ACS Appl Mater Interfaces.2017;9:29224.

  11. 11.

    Lv D, Wang R, Tang G, Mou Z, Lei J, Han J, De Smedt S, Xiong R, Huang C. Ecofriendly electrospun membranes loaded with visible-light-responding nanoparticles for multifunctional usages: highly efficient air filtration, dye scavenging, and bactericidal activity. ACS Appl Mater Interfaces.2019;11:12880.

  12. 12.

    Sun W, Chen G, Wang F, Qin Y, Wang Z, Nie J, Ma G. Polyelectrolyte-complex multilayer membrane with gradient porous structure based on natural polymers for wound care. Carbohydr Polym.2018;181:183.

  13. 13.

    Liu Y, Liang X, Zhang R, Lan W, Qin W. Fabrication of electrospun polylactic acid/cinnamaldehyde/beta-cyclodextrin fibers as an antimicrobial wound dressing. Polymers (Basel).2017;9:464.

  14. 14.

    Wei DX, Dao JW, Chen GQ. A micro-ark for cells: highly open porous polyhydroxyalkanoate microspheres as injectable scaffolds for tissue regeneration. Adv Mater.2018;30:1802273.

  15. 15.

    Huan S, Liu G, Cheng W, Han G, Bai L. Electrospun poly(lactic acid)-based fibrous nanocomposite reinforced by cellulose nanocrystals: impact of fiber uniaxial alignment on microstructure and mechanical properties. Biomacromolecules.2018;19:1037.

  16. 16.

    Rezabeigi E, Wood-Adams PM, Demarquette NR. Complex morphology formation in electrospinning of binary and ternary poly(lactic acid) solutions. Macromolecules.2018;51:4094.

  17. 17.

    Omenetto FG, Kaplan DL. New opportunities for an ancient material. Science.2010;329:528.

  18. 18.

    Fernandes JG, Correia DM, Botelho G, Padrão J, Dourado F, Ribeiro C, Lanceros-Méndez S, Sencadas V. PHB-PEO electrospun fiber membranes containing chlorhexidine for drug delivery applications. Polym Test.2014;34:64.

  19. 19.

    Gupta K, Kumar MR. Preparation, characterization and release profiles of pH-sensitive chitosan beads. Polym Int.2000;49:141.

  20. 20.

    Sun X, Lang Q, Zhang H, Cheng L, Zhang Y, Pan G, Zhao X, Yang H, Zhang Y, Santos HA, Cui W. Electrospun photocrosslinkable hydrogel fibrous scaffolds for rapid in vivo vascularized skin flap regeneration. Adv Funct Mater.2017;27:1604617.

  21. 21.

    Valerio O, Pin JM, Misra M, Mohanty AK. Synthesis of glycerol-based biopolyesters as toughness enhancers for polylactic acid bioplastic through reactive extrusion. ACS Omega.2016;1:1284.

  22. 22.

    Wang Y, Ameer GA, Sheppard BJ, Langer R. A tough biodegradable elastomer. Nat Biotechnol.2002;20:602.

  23. 23.

    Jeffries EM, Allen RA, Gao J, Pesce M, Wang Y. Highly elastic and suturable electrospun poly(glycerol sebacate) fibrous scaffolds. Acta Biomater.2015;18:30.

  24. 24.

    Liu G, Hinch B, Beavis AD. Mechanisms for the transport of α, ω-dicarboxylates through the mitochondrial inner membrane. J Biol Chem.1996;271:25338.

  25. 25.

    Loh XJ, Abdul Karim A, Owh C. Poly(glycerol sebacate) biomaterial: synthesis and biomedical applications. J Mater Chem B.2015;3:7641.

  26. 26.

    You ZR, Hu MH, Tuan-Mu HY, Hu JJ. Fabrication of poly(glycerol sebacate) fibrous membranes by coaxial electrospinning: Influence of shell and core solutions. J Mech Behav Biomed Mater.2016;63:220.

  27. 27.

    Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater.2005;4:518.

  28. 28.

    Chen Y, Sui L, Fang H, Ding C, Li Z, Jiang S, Hou H. Superior mechanical enhancement of epoxy composites reinforced by polyimide nanofibers via a vacuum-assisted hot-pressing. Compos Sci Technol.2019;174:20.

  29. 29.

    Yang D, Li L, Chen B, Shi S, Nie J, Ma G. Functionalized chitosan electrospun nanofiber membranes for heavy-metal removal. Polymer.2019;163:74.

  30. 30.

    Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment. J Biomed Mater Res Part A.2003;67A:531.

  31. 31.

    Dai Z, Deng J, Yu Q, Helberg RML, Janakiram S, Ansaloni L, Deng L. Fabrication and evaluation of bio-based nanocomposite TFC hollow fiber membranes for enhanced CO2 capture. ACS Appl Mater Interfaces.2019;11:10874.

  32. 32.

    Liu Y, Zhou G, Liu Z, Guo M, Jiang X, Taskin MB, Zhang Z, Liu J, Tang J, Bai R, Besenbacher F, Chen M, Chen C. Mussel inspired polynorepinephrine functionalized electrospun polycaprolactone microfibers for muscle regeneration. Sci Rep.2017;7:8197.

  33. 33.

    Miao D, Huang Z, Wang X, Yu J, Ding B. Continuous, spontaneous, and directional water transport in the trilayered fibrous membranes for functional moisture wicking textiles. Small.2018;14:e1801527.

  34. 34.

    Nie G, Lu X, Chi M, Jiang Y, Wang C. CoOx nanoparticles embedded in porous graphite carbon nanofibers derived from electrospun polyacrylonitrile@polypyrrole core–shell nanostructures for high-performance supercapacitors. RSC Adv.2016;6:54693.

  35. 35.

    Sedghi R, Sayyari N, Shaabani A, Niknejad H, Tayebi T. Novel biocompatible zinc-curcumin loaded coaxial nanofibers for bone tissue engineering application. Polymer.2018;142:244.

  36. 36.

    Hou L, Zhang X, Mikael PE, Lin L, Dong W, Zheng Y, Simmons TJ, Zhang F, Linhardt RJ. Biodegradable and bioactive PCL-PGS core-shell fibers for tissue engineering. ACS Omega.2017;2:6321.

  37. 37.

    Cheng G, Yin C, Tu H, Jiang S, Wang Q, Zhou X, Xing X, Xie C, Shi X, Du Y, Deng H, Li Z. Controlled co-delivery of growth factors through layer-by-layer assembly of core-shell nanofibers for improving bone regeneration. ACS Nano.2019;13:6372.

  38. 38.

    Memic A, Abudula T, Mohammed HS, Joshi Navare K, Colombani T, Bencherif SA. Latest progress in electrospun nanofibers for wound healing applications. ACS Appl Biol Mater.2019;2:952.

  39. 39.

    Yang X, Yang D, Zhu X, Nie J, Ma G. Electrospun and photocrosslinked gelatin/dextran–maleic anhydride composite fibers for tissue engineering. Eur Polym J.2019;113:142.

  40. 40.

    Yan Y, Sencadas V, Jin T, Huang X, Chen J, Wei D, Jiang Z. Tailoring the wettability and mechanical properties of electrospun poly(l-lactic acid)-poly(glycerol sebacate) core-shell membranes for biomedical applications. J Colloid Interface Sci.2017;508:87.

  41. 41.

    Fong H, Chun I, Reneker DH. Beaded nanofibers formed during electrospinning. Polymer.1999;40:4585.

  42. 42.

    Michael B, Wolfgang C, Thomas F, et al. Nanostructured fibers via electrospinning. Adv Mater.2001;1(13):70.

  43. 43.

    Yan Y, Sencadas V, Zhang J, Wei D, Jiang Z. Superomniphilic poly(glycerol sebacate)-poly(l-lactic acid) electrospun membranes for oil spill remediation. Adv Mater Interfaces.2017;4:1700484.

  44. 44.

    Huang W, Restrepo D, Jung JY, Su FY, Liu Z, Ritchie RO, McKittrick J, Zavattieri P, Kisailus D. Multiscale toughening mechanisms in biological materials and bioinspired designs. Adv Mater.2019;1901561:1.

  45. 45.

    Qin Z, Zhang P, Wu Z, Yin M, Geng Y, Pan K. Coaxial electrospinning for flexible uniform white-light-emitting porous fibrous membrane. Mater Des.2018;147:175.

  46. 46.

    Lee J, Song B, Subbiah R, Chung JJ, Choi UH, Park K, Kim SH, Oh SJ. Effect of chain flexibility on cell adhesion: semi-flexible model-based analysis of cell adhesion to hydrogels. Sci Rep.2019;9:2463.

  47. 47.

    Schoen B, Avrahami R, Baruch L, Efraim Y, Goldfracht I, Elul O, Davidov T, Gepstein L, Zussman E, Machluf M. Electrospun extracellular matrix: paving the way to tailor-made natural scaffolds for cardiac tissue regeneration. Adv Funct Mater.2017;27:1700427.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant no. 51973009) and Xuzhou Natural Science Foundation in China (KC18201 and KC18108).

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Correspondence to Dongzhi Yang or Guiping Ma.

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Yang, X., Li, L., Yang, D. et al. Electrospun Core–Shell Fibrous 2D Scaffold with Biocompatible Poly(Glycerol Sebacate) and Poly-l-Lactic Acid for Wound Healing. Adv. Fiber Mater. (2020). https://doi.org/10.1007/s42765-020-00027-x

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Keywords

  • Coaxial electrospinning
  • Core–shell structure
  • Porous
  • PGS
  • PLLA
  • Wound healing