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Advances in Wound Care logoLink to Advances in Wound Care
. 2020 Jul 23;9(8):441–452. doi: 10.1089/wound.2018.0879

Electrospinning Nanofiber-Reinforced Aerogels for the Treatment of Bone Defects

Yishan Zhang 1,†, Chengcheng Yin 1,†, Yuet Cheng 1, Xiangyu Huang 2, Kai Liu 2,‡, Gu Cheng 1,*,‡, Zubing Li 1,*,‡
PMCID: PMC7382394  PMID: 32857019

Abstract

Objective: Application of aerogels in bone tissue engineering is an emerging field, while the reports of electrospinning nanofiber-reinforced aerogels are limited. This research aimed at fabricating the nanofiber-reinforced aerogels and evaluating their physiochemical and biological properties.

Approach: The chitosan (CS) aerogels incorporated with cellulose acetate (CA) and poly (ɛ-caprolactone) (PCL) nanofibers were fabricated via ball milling and freeze-drying techniques. Scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectrum, X-ray photoelectron spectroscopy (XPS), compressive experiment, and in vitro experiment were conducted to assess their physiochemical properties and biological behavior.

Results: The SEM examination showed that satisfying morphology was attained in the CA/PCL/CS aerogels with incorporation of CA/PCL nanofibers and CS solution. The results of FT-IR and XPS indicated the perfect incorporation of CA, PCL, and CS. A compressive experiment confirmed that the CA/PCL/CS aerogels enhanced the compressive modulus of the pure CS aerogel. For in vitro experiment, the CA/PCL/CS composite scaffolds were proven to possess better cytocompatibility compared with the pure CS. Also, cells on the CA/PCL/CS showed well-extended morphology and could infiltrate into a porous scaffold. Furthermore, confocal experiment revealed that the CA/PCL/CS could also promote the osteogenic differentiation of MC3T3-E1 cells.

Innovation: This study fabricated the nanofiber-reinforced aerogels mainly to optimize the cell/material interaction of the pure CS scaffold.

Conclusion: The CA/PCL nanofibers not only improved the mechanical property of the CS aerogel to some extent but also facilitated cell adhesion and osteogenic differentiation. Thus, it could be considered a promising candidate for bone tissue engineering.

Keywords: aerogel, bone tissue engineering, electrospinning, nanofibers

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Zubing Li, PhD

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Gu Cheng

Introduction

Autograft is defined as the “gold standard” for bone defect reconstructing, since it obtains superlative osteogenic competence. However, patients may be subjected to supplement limitation, donor-site morbidity, and harvest-site surgery complications such as vessel injury or visceral hemorrhage. To address the limited treatment for bone defects, tissue engineering has proven to be an alternative treatment plan instead of bone autograft because of reducing the above complications, as well as diminishing antigenicity compared with allograft.1

The ideal scaffold for bone tissue engineering should have suitable porosity and pore size with interconnected structures (for instance, pore sizes about 300 μm favor osteogenesis in vivo), similar mechanical property to human bone (the compressive strength of cortical bone is 100–230 MPa and of trabecular bone is 1–100 MPa), and similar biodegradation rate to native bone and biocompatibility.1,2 Almost no scaffolds used in the previous studies for bone tissue engineering fit all the features.

Many researches have reported that aerogels obtain qualified morphology and good biocompatibility, as well as possessing structures with remarkably high surface area, low density, and high porosity.3–6 As a result, they could be treated as a suitable scaffold for bone tissue engineering. Novel types of aerogels are manufactured every year, indicating the spurring demands of aerogel materials. Drying processes such as freeze-drying (or lyophilization), supercritical drying, and ambient pressure drying are undertaken to transform the wet gels into aerogels by replacing liquid inside the pores with gas.6

Cellulose acetate (CA), poly(ɛ-caprolactone) (PCL), and chitosan (CS) have been extensively utilized in tissue engineering. The polymer composites possess the integrated properties of CA, PCL, and CS, including good biodegradability, hydrophilicity, biocompatibility, and mechanical constancy.7,8 Although the CS scaffolds obtain excellent biocompatibility, their instable porous structure, insufficient mechanical strength, and biological property inhibit wide application for bone tissue engineering.9,10 Numerous modification methods have been reported to modify the inadequate properties of the CS scaffolds.11 Liu et al. prepared CS-graphene oxide scaffolds via directional freezing and improved the pore sizes and porous structure.10 Thein-Han and Misra introduced hydroxyapatite into CS scaffolds, and the porosity, degradation rate, and cytocompatibility of the composite scaffolds were optimized.9 However, the cell/material interconnection of these CS-base scaffolds also needs to be further improved. In this experiment, the pure CS aerogel was incorporated with CA/PCL nanofibers mainly aiming at optimizing its cell/material interconnection for bone tissue engineering. We hypothesized that the CA/PCL/CS hybrid aerogels with an optimized interconnected structure would promote proliferation, adhesion, and osteogenic differentiation of cells attached to the pure CS aerogel.

The CA/PCL composite nanofibers with a rational ratio of CA and PCL were fabricated via the electrospinning technique, and subsequently added into pure CS aerogel via ball milling and freeze-drying techniques.

Accordingly, experiments were conducted in the aspects of composite morphology, chemical constitution, mechanical property, and biocompatibility.

Clinical Problem Addressed

Bone tissue engineering is considered a promising alternative treatment to bone grafts for bone defect, while the ideal scaffold has not been developed yet. The CS scaffolds were inappropriate for bone tissue engineering with respect to their three-dimensional (3D) porous structure, mechanical property, and biological property. Polymers such as collagen12 and cellulose,13 bioceramic such as hydroxyapatite,9 and inorganic material such as metal14 have been introduced into the pure CS to enhance the multiple properties, while limited literatures referred to the electrospinning nanofiber-reinforced CS-base scaffold. This study manufactured the novel nanofiber-reinforced CS aerogels to improve the capability of cell/material interconnection.

Materials and Methods

Fabrication of CA/PCL electrospun nanofibrous membranes

To fabricate CA/PCL electrospun nanofibrous membranes, the electrospinning machine (Shanghai Oriental Flying Nanotechnology Co., Ltd.) was operated. CA (average Mn∼30,000; Sigma-Aldrich Co.) and PCL (average Mn = 80,000; Sigma-Aldrich Co.) were dissolved in hexafluoroisopropanol (DuPont Chemical Co.) separately and magnetically stirred for 12 h. Then, the CA and PCL solutions at various weight ratios of 8:2, 7:3 and 6:4 were blended thoroughly. The concentrations of all the mixed solutions were constantly 13%. The prepared solutions were poured into several 10 mL injectors equally. The fluid flow rate was 1 mL/h−1. The distance between the syringe tips and cylindrical collector was 15 cm and the direct current voltage was set up at 15 kV. The whole process of electrospinning was operated in an atmosphere of 25°C and 65% humidity. The collected membranes were dried in a vacuum drying oven (DZF-6050; Shanghai CIMO Medical Instrument Manufacturing Co., Ltd.) for at least 3 days.

Characteristics of the fabricated CA/PCL electrospun membranes

Dry membranes were collected and scissored into 1 × 1 cm samples, which were then sputter coated with gold. The morphology of the CA/PCL electrospun nanofibers was examined by scanning electron microscopy [SEM] (FE-SEM; ZEISS SIGMA, Germany). Nano-measurer 1.2 software was used to determine the diameters of the fibers in each electrospun mat derived from SEM images. Mean diameters and standard deviations were calculated by measuring at least 100 different fibers per image.

Fabrication of the CA/PCL/CS composite aerogels

After selecting the most uniform electrospun mat with the ideal ratio of CA and PCL, the CA/PCL/CS aerogels were then fabricated. The pure CS aerogel without CA/PCL nanofiber incorporation was used for the control group. The chosen electrospun mat was crosslinked in ethanol with concentration gradients to promote the tenacity of the nanofibers, dried thoroughly in the vacuum oven, and chopped into pieces. CS (1% w/v) (Sinopharm Chemical Reagent Co., Ltd.) was dissolved in 1% (v/v) acetic acid solution. For every 0.3 g dry mat, 13, 15, and 17 mL of CS solution was added and the ultimate fabricated aerogels were marked as the (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17, respectively. The ball milling machine (Retsch, Germany) equipped with two ZrO2-linning pots was utilized to grind and disperse the nanofibers within the CS solution. The mixture of chopped nanofibers and CS solution was put into each pot containing ZrO2 milling balls. The ball milling machine was operated at a speed of 1,000 rpm for 20 min. The obtained homogeneous solutions were injected into a 48-well culture plate (Nest Biotechnology Co., Ltd., China), frozen in liquid nitrogen, and subsequently dehydrated to the 3D scaffolds by a lyophilizer (SCIENTZ-10ND; Ningbo Scientz Biotechnology Co., Ltd). The pure CS aerogel was fabricated according to the above procedures but without incorporation of the (CA/PCL)6:4 nanofibers.

Characteristics of the CA/PCL/CS aerogels

The pure CS, (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 aerogels were dried in the vacuum drying chamber for 24 h and sputter coated with gold. The morphology of aerogels was examined by scanning electron micrographs. Besides, the ball milling (CA/PCL)6:4 nanofibers alone were examined by SEM to precisely reveal the result of ball milling procedure. Nicolet 170-SX spectrophotometer (Thermo Nicolet Ltd.) was used for Fourier transform infrared (FT-IR) analysis to examine the chemical composition and structures of the aerogels. To further get light of the chemical elements and structure changes during the process of synthesizing, X-ray photoelectron spectroscopy (XPS) examination was performed on an Axis Ultra DLD apparatus (Kratos, UK). XPS peak software was used to divide and fit the peaks of the narrow scan spectrums. The mechanical experiment was operated on four samples of each group, which were cut into 10 mm height and immersed in 75% ethanol for over 48 h. An ETM502A tester (Shenzhen Wance Instrument Co., Ltd., China) was used for compressive experiment at a pushing speed of 1 mm/min with a 100 N load, whereas samples were still not fractured. Subsequently, the compressive modulus was calculated by analyzing the obtained stress/strain curve.

Cell seeding procedure

The CA/PCL/CS composite scaffolds were cut into a 5-mm-high cylinder and soaked in 1 M NaOH solution overnight to neutralize the acetic acid. Then, they were rinsed thoroughly with phosphate-buffered saline (PBS) and exposed to ultraviolet for 30 min on each side before usage. Finally, the aerogels were incubated in alpha-modified Eagle's medium (α-MEM; Hyclone Life Technology Co.) for at least 48 h before use. MC3T3-E1 cells were seeded on both sides of the cylindrical aerogels according to our previous article.15 The culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS, Hyclone Life Technology Co.) and 1% antibiotics (100 U/mL streptomycin and 100 U/mL penicillin; Hyclone Life Technology Co.). The osteogenic medium was α-MEM containing 10% FBS, 1% antibiotics, 10 mM β-glycerophosphate, 25 μg/mL ascorbic acid, and 10 nM dexamethasone (Sigma). For cell viability assay and cell adhesion, cells/scaffolds were incubated in a culture medium. To assess osteogenic differentiation, cells/scaffolds were cultured in the culture medium on the first 2 days and then replaced with the osteogenic medium. The medium was replaced with a fresh one every other day. The cell/scaffold compounds were cultivated at 37°C with 5% CO2 surrounding.

Cell toxicity test and morphological examination

Cell toxicity of scaffolds was evaluated by the Cell Counting Kit-8 (CCK-8; Dojindo, Japan). Cells were seeded into scaffolds at a cell density of 5 × 104 cells per well in a 48-well culture plate (Nest Biotechnology Co., Ltd.). After 3, 5, and 7 days of culturing, the culture medium containing 10% CCK-8 solution was added into each well. After incubating at 37°C with 5% CO2 for 1 h, optical density (OD) values at 450 nm were measured using a microplate reader (Hyclone Life Technology Co.). Subsequently, cell viability was calculated according to OD values. The CS aerogel served as the control group.

Cell morphology and initial adhesion on the surface of cell-seeded scaffolds were detected. After 1 day of culturing, the aerogels were washed with PBS solution twice, subsequently fixed with 4% paraformaldehyde at 4°C for 15 min, and rinsed with distilled water thoroughly. After dehydrated with graded ethanol, the scaffolds were freeze-dried and sputter coated with gold and observed via SEM.

Immunofluorescence staining

F-actin, alkaline phosphatase (ALP), and collagen type-I were detected through immunofluorescence staining. Cells were cultured at a density of 3 × 105 per well. The cytoskeleton was staining by fluorescein isothiocyanate (FITC) phalloidin (1:200 dilution in PBS; Yeason, China) after incubating in culture medium for 5 days to assess cell adhesion and infiltration. First, scaffolds were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.5% Triton X-100 for 10 min. Then, scaffolds were washed again with PBS and treated with FITC phalloidin for 30 min. After being washed with PBS and stained with 4′,6′-diamidino-2-phenylindole (DAPI; Beyotime, China) for 15 min, the samples were washed with PBS and observed by a confocal microscope (OLYMPUS, Japan). The images were derived from the inside of the scaffolds.

To further confirm the osteogenic differentiation, ALP and collagen type-I staining was conducted after being cultured in osteogenic medium for 7 days. Briefly, scaffolds were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% bovine serum albumin (BSA; BioFroxx, Germany) in PBS. Cells were incubated with the ALP antibody (1:200 dilution in PBS; BA0632, Boster Biological Technology, China) or rabbit collagen type-I antibody (1:100 dilution in 5% BSA; A16891, ABclonal, China) overnight at 4°C and stained with a secondary antibody, DyLight 594, goat anti-rabbit IgG (H + L) (1:200 dilution in PBS; Abbkine Scientific Co., Ltd.) for 60 min. Finally, cell nuclei were stained with DAPI and examined by a confocal microscope.

Statistical analyses

All data are expressed as means ± standard deviations. One-way analysis of variance (ANOVA) was applied using GraphPad Prism 7 software for statistical analyses, and p < 0.05 was considered statistically significant.

Results

Characteristics of the electrospun mat with different ratios of CA and PCL

The morphological characteristics of the electrospun mats with different ratios of CA and PCL were examined by SEM examination (Fig. 1). The ratio of PCL in the CA/PCL nanofibrous mat significantly influenced the morphology of the composite mats.7 The pure CA electrospun mat exhibited some bended nanofibers and slim branches deviating from the core fibers. Furthermore, the diameter of the pure CA nanofibers (1.84 ± 0.40 μm) was not uniform (Fig. 1A). The high-magnification image also showed that some dispersed nodules formed on the rough surface of the CA nanofibrous mats. The incorporation of PCL into the CA/PCL composite mats presented smoother surfaces and more uniform diameter of nanofibers when compared with the pure CA nanofibers. The mean diameter of the CA/PCL8:2, CA/PCL7:3, and CA/PCL6:4 nanofibers was 1.36 ± 0.17, 1.55 ± 0.15, and 1.57 ± 0.12 μm, respectively (Fig. 1B–D), which revealed that all the CA/PCL composite nanofibrous mats were slender than that of the pure CA mat and the average diameter of the composite nanofibers also widened with the increasing content of PCL. Furthermore, the polymer PCL improved mechanical constancy in the blended fibers.7 Compared with other groups, the CA/PCL6:4 nanofibrous mat exhibited the most uniform diameter distribution and was incorporated into the CS solution in the following experiments to construct the CA/PCL/CS composite aerogels.

Figure 1.

Figure 1.

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SEM images of the electrospun membranes with different ratios of CA and PCL: pure CA (A); CA/PCL8:2 (B); CA/PCL7:3 (C); CA/PCL6:4 (D). The inserted histograms exhibited the frequency distributions of CA/PCL fiber diameters (E–H). CA, cellulose acetate; PCL, poly (ɛ-caprolactone); SEM, scanning electron microscopy.

Morphology of the CA/PCL/CS aerogels

The SEM of ball milling nanofibers revealed that the chopped fibers were generally dispersed and some were bundled (Fig. 2A). Most fibers presented long and bended structure, still few short or even granule-like fibers were seen. As shown in the high-magnitude image, the fibers underwent slight deformity such as swelling and bending. Generally, the state of nanofibers maintained original morphology after the ball milling process. The SEM images of the aerogels showed that the pure CS aerogel without nanofibers imbedded presented a smooth surface (Fig. 2B). However, the composite CA/PCL/CS aerogels revealed an interconnective network of sheet-like structures and well-distributed nanofibers with rougher surface (Fig. 2C–E). With the increasing content of CS, the mean pore size and porosity of the composite aerogels gradually expanded.

Figure 2.

Figure 2.

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The SEM images of chopped fibers (A), the pure CS aerogel (B), (CA/PCL)6:4/CS13 (C), (CA/PCL)6:4/CS15 (D), and (CA/PCL)6:4/CS17 (E), and the respective high-magnification images were presented. The crosslinking structures in composite aerogels were circumscribed by frameworks, and sheet-like structures were marked with asterisk (*). CS, chitosan.

FT-IR examination

FT-IR analysis was performed to confirm the chemical bonds of the hybrid aerogels. Typical peaks of CA, PCL, and CS were observed in the transmittance spectrum of the aerogel (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 (Fig. 3A). The characteristic transmittance peaks of the aerogel CA/PCL/CS at 2,870 cm−1 aligned to symmetric -CH2 stretching vibrations from PCL,14 which demonstrated that PCL might be incorporated into the CA/PCL/CS composite aerogels; The characteristic peaks of composite aerogels at 1,730, 1,240, and 1,170 cm−1 were attributed to carbonyl stretching, C-C-O stretching, and C-O stretching respectively, corresponding with CA transmittance spectra.16 The main peak of CS located at 1,630 and 1,380 cm−1 represented C-N stretching and CH3 stretching. However, the peak at 1,630 and 1,380 cm−1 also presented in CA, which could not fully support the successful incorporation of CS. Therefore, XPS analysis was performed in the following experiments. There was no new peak generated according to FT-IR spectra and this result demonstrated that PCL and CA were integrated into aerogels successfully and no chemical reaction occurred during the process of electrospinning.

Figure 3.

Figure 3.

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(A) FT-IR analysis of the aerogel (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, (CA/PCL)6:4/CS17, the pure CA, pure CS, and pure PCL. (B) XPS wide scans of CA, PCL, CS, and the (CA/PCL)6:4/CS13 composite aerogel. (C) XPS narrow scan spectrum of CA, PCL, CS, the composite aerogels (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 illustrating the curves of C, N, and O. (D) Typical strain/stress curve was obtained from the compression experiment of the aerogel CS, (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17. It revealed that the aerogel (CA/PCL)6:4/CS13 exhibited the highest compressive modulus. FT-IR, Fourier transform infrared; XPS, X-ray photoelectron spectroscopy.

XPS examination

XPS was used to further validate chemical elements on the surface of the fabricated aerogels and verify the successful incorporation of CA, PCL, and CS. Among all the fabricated aerogels, the XPS wide scan spectrums clearly evidenced the existence of C, N, and O ions, which presented dominant peaks at 292, 524, and 402 eV, respectively (Fig. 3B). The N1s peak of aerogels locating at 399.1 eV was attributed to imine nitrogen corresponding with that of CS (Fig. 3C), which indicated that CS had been successfully incorporated into the composite aerogels.12 Furthermore, the intensity value of N1s gradually increased in the aerogel (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 aerogels, which indicated that the content of CS was increasing.

The details of C and O elements of the aerogels were also exhibited (Fig. 3C). The C1s spectra of PCL was deconvoluted and three peaks were revealed as follows: 284.8 eV (hydrocarbons C-H and C-C), 286.3 eV (ether group C-O), and 288.8 eV (ester group O-C = O). The O1s spectra of PCL illustrated three characteristic peaks. The peak at 531.9 and 533.2 eV assigned to C = O and C-O, respectively. The third peak with low intensity at 531 eV might be attributed to -OH.17,18 Three characteristic peaks at 284.7, 286.2, and 287.8 eV were detected in the C1s spectra of CS, which represented C-C/C-H, C-N/C-O, and O-C-O, respectively.19,20 In the O1s narrow spectra of CS, the peak at 532.4 was attributed to O-C.21 As for the C1s spectra of CA, four characteristic signals were noted. They were C-C/C-H at 284.6 eV, C-O at 286.1 eV, C = O/O-C-O at 287.0 eV with fairly low intensity, and O-C = O at 288.7 eV.16,22 The peak in O1s spectra of CA located at 532.3 was assigned to O-C.23

Four binding energy intensities were presented in C1s narrow spectra of the (CA/PCL)6:4/CS(13, 15, 17). The peak at 287.8 eV was assigned to -O-C-O in CS; the peak with high intensity at 286.2 eV was assigned to C-O (in PCL, CA, and CS) or C-N (in CS); The peak at 284.6 eV was derived from C-H/C-C (in PCL, CS, and CA).24,25 The peak assigning to O-C = O in PCL and CA observed an upshift to 289.4 eV, which was attributed to the hydrogen bonding among PCL, CA, and CS.26 Three characteristic peaks at 535.4, 532.4, and 531.1 eV were revealed in O1s narrow scan spectrums of the CA/PCL/CS aerogel. The peak at 531.1 eV revealed a decrease from the binding energy of C = O in PCL. This phenomenon accounted for the crosslinking of CA, CS, and PCL.27 The dominant peak at 532.4 eV corresponding to C-O of CS and CA was unchanged. The new signal located at 535.4 with less intensity could be explained by hydrogen bonding.17 The XPS analysis results confirmed that CA, PCL, and CS were successfully incorporated into the composite aerogels.

Compressive examination

A compressive examination was performed to validate that the CA/PCL/CS aerogels modified with nanofibers could improve the mechanical property of pure CS aerogels. The typical stress/strain curves of the aerogel CS, (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 are shown in Fig. 3D. The mean compressive modulus of the pure CS aerogel was 12.5 ± 5 kPa. As for the aerogels (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17, the mean compressive moduli were 45 ± 6, 35 ± 10, and 22.5 ± 5 KPa, respectively, much higher than that of the pure CS aerogel. These results demonstrated that incorporation of nanofibers enhanced the stiffness of the CS aerogel. The pure CS aerogel had the lowest compressive modulus, and thus, it might not bolster certain compressive stress compared with the composite aerogels.

Cell viability, morphology, and adhesion

The results of CCK-8 test (Fig. 4A) revealed that cell viability of the CA/PCL/CS aerogels was similar or higher than that of the pure CS aerogel although there were no significant differences. The (CA/PCL)6:4/CS15 obtained the best cell viability among all the scaffolds. Therefore, incorporating nanofibers would not diminish the cytocompatibility of the CS scaffold and to some extent could enhance cell proliferation.

Figure 4.

Figure 4.

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Cell viability of MC3T3-E1 cells seeded on the aerogels (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 was compared with that of the control group on days 3, 5, and 7 of culture. There were no significant differences between the composite aerogels and the CS aerogel (A). One day after culturing, cell attachment condition on the surface of the pure CS, (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 was examined by SEM (B). Arrows marked the attached cells and frameworks labeled cell/material connection.

Cell morphology of MC3T3-E1 and cell/material interaction on the surface of scaffolds were observed via SEM after 1-day culturing (Fig. 4B). MC3T3-E1 cells on all the scaffolds showed spread morphology with polygonal shape. Cells on the pure CS aerogel presented small morphology with rather thin filopodia. On the contrary, the filopodia of cells on the CA/PCL/CS were long and remarkable, which indicated that the CA/PCL/CS provided a preferable environment for the cell's initial adhesion. Furthermore, the high magnitude of SEM images exhibited detailed cell/material interaction that cells were majorly attached to the fibrous structure on the CA/PCL/CS, which denoted well the cell/material interaction on the nanofiber-reinforced scaffolds. F-actin staining distinctively displayed the cytoskeleton of cells inside the scaffolds after 5 days of culturing and it further confirmed that cells stretched more extensively on the CA/PCL/CS (Fig. 5) than on the CS aerogel. These results demonstrated that the CA/PCL/CS aerogel facilitated cell adhesion and permitted cell infiltration.

Figure 5.

Figure 5.

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Confocal micrographs of cell-seeded scaffolds to visualize F-actin (green) after 5 days of culturing, which were extracted inside the scaffolds. ALP (red) and collagen type I (red) staining was conducted after 7 days of cell culturing. Cell nuclei were stained with DAPI (blue). ALP, alkaline phosphatase; DAPI, 4′,6′-diamidino-2-phenylindole.

Osteogenic differentiation

ALP is an early-stage marker of osteogenic differentiation. Confocal microscope images of ALP immunohistochemistry illustrated that a more abundant expression of ALP was observed in the CA/PCL/CS aerogels when compared with that of other groups (Fig. 5). Collagen type I was produced by mature osteoblasts before the mineralization of extracellular matrix. After culturing in osteogenic medium for 7 days, the deposition of collagen type I could be detected in both the CS and CA/PCL/CS scaffolds (Fig. 5). Cells gathered in clusters in the CA/PCL/CS scaffolds and considerable amount of collagen type I was expressed. The results of the confocal microscope experiment indicated that the CA/PCL/CS scaffolds could promote ALP activity and collagen type I production and enhance osteogenic differentiation.

Discussion

Aerogel is characterized by a remarkably high surface area, low density, and high porosity. Since this scaffold obtains qualified morphology and good biocompatibility, the application in bone tissue engineering is a prospective field.4

The CS aerogel with well biocompatibility should be modified by different methods and materials to improve the porous structure and mechanical and biological properties. Other kinds of biomaterials could be added into aerogels during the manufacturing process to optimize the mechanical property, degradation rate, and biocompatibility. Nissilä et al. incorporated bioepoxy resin into cellulose nanofibrous aerogel and the mechanical properties of composite material improved dramatically compared with pure cellulose.13 Takahashi et al. added β-tricalcium phosphate into gelatin sponges to specifically control the degradation rate, as well as to enhance mechanical properties and biocompatibility.28 It was also reported that hybrid aerogels reinforced with hydroxyapatite obtained well porous structure and enhanced cell proliferation.9

In our experiment, we incorporated the CA/PCL nanofibers into the pure CS aerogel, aimed at promoting the cell/material interconnection of the scaffold for application. Our study was based on the hypothesis that the nanofiber-reinforced CS aerogels could facilitate the mechanical and biological properties of the pure CS aerogel. The electrospun CA/PCL(6:4) nanofibers were added into pure CS aerogel via ball milling and freeze-drying techniques.

The ideal scaffold for bone tissue engineering should mimic the extracellular matrix of native bone structure as the characteristics of materials have a great impact on cell phenotype.29,30 As an important characteristic of biomaterials, the surface morphology of composite aerogels should be taken into account. The incorporation of the (CA/PCL)6:4 nanofibers into the composite aerogels can optimize the porosity structure and built 3D networks of the composite aerogels.31,32 According to the results of the previous studies, the content of nanofibers plays a critical part in bone formation.33 Correspondingly, the pore size and porosity of the composite aerogels decreased with the increasing content of the (CA/PCL)6:4 nanofibers.

The results of compressive test presented that the composite CA/PCL/CS aerogels exhibited a higher compressive modulus compared with that of the pure CS aerogels, which might account for the denser 3D network structures due to the incorporation of the (CA/PCL)6:4 nanofibers.33 The introduction of the CA/PCL composite nanofibers could to some extent enhance the stiffness of the CS aerogel, although the mechanical property of the scaffold for bone tissue engineering is yet to reach the requirement. With the increase in volume of CS solution, the compressive modulus of the (CA/PCL)6:4/CS13, (CA/PCL)6:4/CS15, and (CA/PCL)6:4/CS17 aerogels gradually decreased, which might be because the concentration of nanofibers could affect the density of the CA/PCL/CS composite aerogels.

Cell response to the scaffolds was of vital importance, and so, the biological characteristics were also evaluated in in vitro experiments. Cell viability assay demonstrated that the CA/PCL/CS scaffolds possessed similar or better cytocompatibility compared with the CS scaffold. Some researchers claimed that the higher mechanical property might supply a favorable environment for cell proliferation.34 Accordingly, the advanced mechanical stiffness of the CA/PCL/CS scaffolds might attribute to their better cell viability. The results of SEM and F-actin staining confocal microscope revealed that the CA/PCL/CS aerogels could promote cell adhesion. It is commonly recognized that cell adhesion acts as a pivot premise of cell proliferation and differentiation. In our study, cells spread extensively on the CA/PCL/CS aerogels, which might contribute to the excellent behavior of cell growth and osteogenic differentiation.35 It has been reported that the roughness of the scaffold could benefit effective cell attachment.36 Thus, we believed that the elevated stiffness of nanofiber-modified CS aerogel with rough surface induced better cell adhesion. Intriguingly, the cells were interconnected with adding fibers, which might provide the suitable sites for cell adhesion. The results of ALP activity and collagen type I production demonstrated that the CA/PCL/CS composite aerogels could enhance osteogenic differentiation of MC3T3-E1 cells, which confirmed biocompatibility for bone tissue engineering.

Above all, we could safely conclude that all the nanofiber-reinforced aerogels, with promoted stiffness, were beneficial for cell attachment and osteogenic differentiation. As a result, they could be regarded as the biocompatible materials for bone regeneration. In the future, the mechanical stiffness should be further elevated and in vivo experiment should be performed for CA/PCL/CS aerogels.

Innovation

The CS aerogel requires improved mechanical and biological properties for bone tissue engineering. The present study researched in incorporating nanofibers into the pure CS aerogel mainly to enhance cell/material interconnection. The results of in vitro experiment demonstrated that the composite CA/PCL/CS aerogel, with an interconnected porous structure and rough surface, could improve cell adhesion, infiltration, and osteogenic differentiation. Moreover, the stiffness of the composite aerogel was increased to some extent. Hence, it can be regarded as a suitable candidate for bone tissue engineering and may serve as a treatment for bone defects.

Key Findings

  • After incorporating (CA/PCL)6:4 nanofibers into the pure CS aerogel, the CA/PCL/CS composite aerogels were observed with a nanofibrous network, which elevated the roughness and stiffness of the CS aerogel. With the increasing content of CS solution in the composites, the mean pore sizes and porosity of the composite aerogels amplified simultaneously, while the mechanical stiffness descended.

  • The seeded cells on the CA/PCL/CS scaffold could interconnect with the nanofiber structure and exhibited sufficient spread.

  • The CA/PCL/CS attained better cytocompatibility, cell attachment, and osteogenic differentiation than the CS aerogel.

Abbreviations and Acronyms

3D

three dimensional

ALP

alkaline phosphatase

CA

cellulose acetate

CCK-8

Cell Counting Kit 8

CS

chitosan

DAPI

4′,6′-diamidino-2-phenylindole

FT-IR

Fourier transform infrared

PBS

phosphate-buffered saline

PCL

poly(ɛ-caprolactone)

SEM

scanning electron microscopy

XPS

X-ray photoelectron spectroscopy

Acknowledgments and Funding Sources

This work was supported by the Natural Science Foundation of China (Nos. 81771051 and 81800943), the Natural Science Foundation of Hubei Province (No. 2018CFB497), and the Natural Science Foundation of Zhejiang Province (No. LY17H140002).

Author Disclosure and Ghostwriting

No competing financial interests exist. The content of this article was expressly written by the authors listed. No ghostwriters were used to write this article.

About the Authors

Yishan Zhang, MD, and Yuet Cheng, PhD, are both undergraduates from Wuhan University Stomatological Hospital. Chengcheng Yin, MS, received her Master's degree at Jilin University Stomatological Hospital and currently studies at Wuhan University Stomatological Hospital. Xiangyu Huang, PhD, and Kai Liu, PhD, now work at Lishui University, Department of Oral and Maxillofacial Surgery. Gu Cheng, PhD, received his PhD at Wuhan University Stomatological Hospital. He works at Wuhan University Stomatological Hospital and researches osteogenic scaffolds for bone tissue engineering. Zubing Li, PhD, is a professor at Wuhan University Stomatological Hospital. He researches in oral and maxillofacial post-trauma therapy and plastic surgical treatment.

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