Nanostructured surface of electrospun PCL/dECM fibres treated with oxygen plasma for tissue engineering

HoJun Jeon, JaeYoon Lee, Hyeongjin Lee and Geun Hyung Kim*
Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, South Korea. E-mail: gkimbme@skku.edu; Fax: +82-31-290-7870; Tel: +82-31-290-7828

Received 11th February 2016 , Accepted 22nd March 2016

First published on 24th March 2016


Recently, various biomimetic scaffolds using a decellularised extracellular matrix (dECM) have been applied in tissue regeneration applications because dECM consists of highly bioactive fibrous proteins and polysaccharides. Here, to enhance the biofunctionality of the dECM surface, homogeneously roughened nanoscale patterns (Ra = 524 ± 66 nm) on the surface of poly(ε-caprolactone) (PCL)-based dECM were developed by selecting optimal plasma treatment conditions (power: 15 W, oxygen flow rate: 5 standard cubic cm per min, and total plasma exposure time: 4 h). To determine its efficiency as a biomedical scaffold, the plasma-treated PCL/dECM was evaluated for various cellular activities, metabolic activity and osteogenic differentiation, and compared with two controls: plasma-treated pure PCL fibres and an untreated PCL/dECM fibrous structure. Significant increases in cell viability (>1.5-fold) and calcium mineralisation (>1.8-fold) were observed for the nanoscale-roughened PCL/dECM fibrous structure compared with the untreated PCL/dECM fibrous mat. Based on the results, we propose a new surface model of a surface-modified biomedical scaffold, which can significantly promote cell activities at the interface between cells and the surface to regenerate hard tissues.


Introduction

It has been clarified that cellular behaviour corresponds to the topographical conditions at the cell's surface. Recently, to observe the relation between topographical surface patterns and cell responses, a variety of micro/nanoscale pattern models have been investigated.1–4 Due to this, physical and chemical surface modifications of biomedical substitutes have been widely investigated for tissue engineering applications.5,6 In particular, it has been shown that microscale and nanoscale topographical features strongly affect cell behaviours including adhesion,7–9 proliferation,10,11 migration12,13 and even differentiation.10,14 Micropatterned surfaces can affect cell morphology and cytoskeletal structure, while nanosized patterns can influence cell functions, including proliferation and differentiation.15–17 In some cases, microsized ridge patterns have shown a marked effect on MG63 cell differentiation, including increased activities of alkaline phosphatase (ALP) and Runx2, which dramatically improve bone tissue regeneration.18 Furthermore, micro/nanostructured combinational patterns (i.e. hierarchical structure) have been investigated for their effects on cellular activities. According to Martins et al., human osteosarcoma-derived cells, which were cultured on hierarchical poly(ε-caprolactone) (PCL) scaffolds, showed significantly higher levels of proliferation and bone maturation.19,20

For these reasons, physical and chemical treatments of biomedical substitutes, such as laser sintering,21 ion beams22 and plasma treatment,23–25 are widely used. Among these techniques, plasma treatment has been used recently to modify the surfaces of polymeric biomaterials because this method induces the desired chemical groups without damaging the bulk properties of materials, and can change a surface from being hydrophobic to hydrophilic.26–28 Moreover, plasma treatment does not employ harmful toxic solvents, which can remain on the surface and cause damage to seeded cells.23 According to Yang et al., an oxygen-plasma-treated polydimethylsiloxane substrate showed significantly enhanced focal adhesion of human mesenchymal stem cells (hMSCs), resulting in enhanced focal adhesions favouring cell spreading, cytoskeletal organisation and osteogenesis of hMSCs.23 Furthermore, polystyrene films with micropatterned grooves and nanostructured roughness were developed using an oxygen plasma treatment by Mattioli et al., and were found to exhibit outstanding alignment and elongation of human bone-marrow-derived mesenchymal stem cells.29 Savoji et al. also demonstrated the advantages of an etching process including minimal apparent damage to fibre surfaces and insignificant changes in mechanical properties by applying oxygen plasma to poly(ethylene terephthalate) nanofibrous mats.30

Recently, scaffolds composed of decellularised extracellular matrix (dECM) have been widely used in regenerative medicine approaches for various tissues and organs.31,32 In general, ECM helps cells interact in tissues, regulates dynamic cellular activities, and performs protective and supportive functions. The structural and functional molecules of ECM have not been fully characterised; however, its individual components, such as collagen, elastin, laminin, fibronectin and glycosaminoglycans, have been isolated and used for many applications.33 ECM-based scaffolds have been achieved for various tissues including small intestinal submucosa,34 trachea,35 heart valves,36 tendon,37 muscle38 and abdominal walls.39 Recent investigations have demonstrated that efficient improvements for specific bone regeneration can be achieved by the combined use of MSCs with ECM-based scaffolds.40,41 However, unfortunately, the processability of ECM and dECM has been limited by their insoluble components, such as fibril collagen, proteoglycans and fibronectin.

In recent, we employed an electric-field-aided casting technique to mimic a typical natural hierarchical structure (a lotus leaf) on a PCL film. The unique micro/nano-structure induced high cell responses (cell proliferation and calcium deposition) compared to the smooth PCL film.42 Also, to obtain nanoscale roughened surface consisting of electrospun PCL micro/nanofibers coated with type-I collagen, a selective oxygen plasma-etching process was performed.43 Here, we propose another new scaffold with unique nanoscale-surface-patterned dECM laden on an electrospun PCL fibrous mat. Although we use the same fibrous PCL as a matrix material, the proposed scaffold is completely different with those of our previous works, because we used decellularised ECM and, to achieve nanoscale roughened dECM, a low temperature plasma-processing was applied.

To achieve the surface-patterned fibrous mat, preosteoblasts (MC3T3-E1) were cultured on the electrospun PCL fibres and then, using a decellularisation process (freeze–thaw cycles), we obtained decellularised ECM. The PCL/dECM fibrous mat was treated with oxygen plasma. To avoid denaturalisation of the ECM components, we applied the plasma treatment with a low working temperature (below 30 °C). By using the selected conditions of the plasma treatment, we can achieve a nanoscale-roughened surface of the dECM as well as of the PCL micro/nanofibres. To determine the feasibility of the biomedical scaffold having unique topological and biochemical components, various in vitro cellular activities were examined using the preosteoblasts. Our results suggest that the newly/topologically designed fibrous structure is a highly promising dECM-based scaffold because of the synergistic effect of the hierarchical topological structure (microscale of PCL fibres and plasma-etched nanoscale on the surface of both dECM and PCL fibres) and the biochemical components (dECM).

Experimental

Fabrication of electrospun PCL mats

We used PCL (density = 1.135 g cm−3, molecular weight = 90[thin space (1/6-em)]000 g mol−1) purchased from Sigma-Aldrich (St. Louis, MO, USA). The fabrication of the electrospun PCL fibrous mat was performed with a 20 mL glass syringe, 16 G electrospinning nozzle and 10 wt% PCL in an 8[thin space (1/6-em)]:[thin space (1/6-em)]2 solvent mixture of dimethylformamide and methylene chloride (Junsei Chemical Co., Tokyo, Japan). The PCL solution was consistently supplied using a syringe pump at a flow rate of 0.5 mL h−1. The voltage applied to the needle was 15 kV and the distance between the needle and the target was 10 cm.

Decellularisation

To obtain decellularised PCL/ECM, PCL 12 × 12 mm2 fibrous mats were prepared and sterilised in 70% ethanol for 1 h while ultraviolet light was applied for 2 h. The sterilised PCL fibre mats were then placed in culture medium overnight. Mouse preosteoblast cells (MC3T3-E1; ATCC, Manassas, VA, USA) were cultured in the fibrous mats and maintained in alpha-Minimum Essential Medium (α-MEM) (Life Sciences Advanced Technologies Inc., St Petersburg, FL, USA) containing 10% foetal bovine serum (Gemini Bio-Products, Calabasas, CA, USA) and 1% antibiotic–antimycotic (Cellgro, Herndon, VA, USA). The cells were seeded onto the mats at a density of 1 × 105 for each specimen and incubated in an atmosphere of 5% CO2 at 37 °C, with medium exchange every second day. After culturing the cells for 4 weeks, fibrous mats were decellularised to obtain an ECM-based PCL scaffold, in accordance with previous protocols.44 In brief, three cycles of freeze–thaw were applied to samples in liquid nitrogen and a 37 °C water bath (10 min each), respectively. After each thaw step in deionised water, the remaining cells were hypotonically lysed by rinsing the samples in sterile phosphate-buffered saline (PBS). A decellularisation process was performed to maximise the removal of cellular debris, while minimising the disruption of ECM components.

Plasma treatment

Oxygen plasma was applied on pure PCL and PCL/dECM fibrous mats using LF plasma (CUTE-MP/R; Femto-Science, Inc., Gyeonggi, South Korea). A low frequency of 50 kHz, power of 10–30 W, pressure of 5.41 × 10−1 Torr and an oxygen flow rate of 5–50 standard cubic cm per min (sccm) were used. Before the plasma etching process, to remove any impurities, the plasma chamber was cleaned by performing a cycle without samples for 30 min. The fibrous mats were placed in the chamber and subjected to plasma treatment for various cycles (1 cycle = 1 h). As controls, untreated PCL/dECM fibres and plasma-treated PCL fibres were used.

Characterisation of plasma-treated PCL/dECM fibres

A scanning electron microscope (SEM) (SNE-3000M; SEC Inc., Suwon, South Korea) and an optical microscope (BX FM-32; Olympus, Tokyo, Japan) connected to a digital camera were used to observe the surface morphology of the fibrous mats. To measure the fibrous surface morphology and roughness, an atomic force microscope (AFM) (Nanowizard AFM; JPK Instruments, Berlin, Germany) and a surface optical topographical tester (Nanoview-m4151p; Nanoview, Gyeonggi, South Korea) were used. To determine the roughness of the specimens, measurements were performed at 30 randomly chosen points on the similar diameter (1.5 ± 0.68 μm) of fibre surface (measured area = 0.25 μm2) and the average was calculated.

To analyse the stress–strain curves, the specimens were cut into small strips (5 × 20 mm2). Uniaxial tests were performed using a tensile machine (Top-tech 2000; Chemilab, Kyunggi, South Korea). The stress–strain curves for the fibrous mats were recorded at a stretching speed of 0.5 mm s−1. All values are expressed as means ± standard deviation (SD; n = 5).

In vitro cell culture on the fibrous mats

After plasma treatment, MC3T3-E1 cells were reseeded on the plasma-treated pure PCL and PCL/dECM fibrous mat. In addition, a PCL/dECM fibrous mat, which was not treated with the plasma, was also cultured. The cell culture procedure was identical to the process used in the Decellularisation section.

Live/dead and cell proliferation assessment

After 24 h of cell culture, to obtain images of live and dead cells, the fibrous mats were exposed to 0.15 mM calcein AM and 2 mM ethidium homodimer-1 for 45 min in an incubator. Stained specimens were analysed using a fluorescence microscope (CKX41; Olympus, Tokyo, Japan). In the captured images, green and red indicate live and dead cells, respectively.

The proliferation of viable cells was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Cell Proliferation Kit I; Boehringer Mannheim, Mannheim, Germany). This assay is based on the cleavage of the yellow tetrazolium salt MTT by mitochondrial dehydrogenases in viable cells to produce purple formazan crystals. Cells on the surface were incubated with 0.5 mg mL−1 MTT for 4 h at 37 °C. The absorbance at 570 nm was measured using a microplate reader (EL800; Bio-Tek Instruments, Winooski, VT, USA). Six samples were used for each incubation period.

Immunohistochemistry

To observe the ECM components of PCL/dECM fibrous mats, the specimens were fixed in 4% paraformaldehyde for 45 min and permeabilised with 0.25% Triton-X for 15 min at room temperature. Then, the samples were immersed in PBS supplemented with 3.5% bovine serum albumin (BSA) overnight at 4 °C. The samples were then incubated with primary antibodies, including against fibronectin (ab6328, 1:100; Abcam, Cambridge, MA, USA) and collagen I (SC-25974; Santa Cruz Biotechnology, Santa Cruz, CA, USA), at the manufacturers' recommended dilutions in PBS and 0.35% BSA, at 4 °C overnight. The next day, the samples blocked with fluorescent secondary antibodies (AlexaFluor 488/594, Pacific Orange; Invitrogen, Carlsbad, CA, USA) were incubated at 4 °C overnight, washed and observed with a confocal microscope (LSM 800; Zeiss, Oberkochen, Germany).

To characterise the cell nuclei, the samples were subjected to 4′,6-diamidino-2-phenylindole (DAPI) fluorescent staining after 3 days of cell culture. Phalloidin was also applied to visualise the F-actin cytoskeleton. From fluorescence images of the surface of the fibrous mats, the cell number and area fraction and aspect ratio (i.e. stretching degree) of F-actin identified by DAPI and phalloidin staining, respectively, were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

ALP activities and Alizarin Red S staining

After 7 and 14 days of cell culture, the ALP activity of the cultured samples was determined by measuring the release of p-nitrophenol from p-nitrophenyl phosphate (p-NPP). The specimens were washed gently with PBS and incubated in Tris buffer (10 mM, pH 7.5) containing 0.1% Triton X-100 for 10 min. Aliquots of the lysate (100 μL) were added to 96 well tissue culture plates containing 100 μL of p-NPP solution, prepared using an ALP kit (procedure no. ALP-10; Sigma-Aldrich). In the presence of ALP, p-NPP was transformed to p-nitrophenol and inorganic phosphate. The ALP activity was determined by measuring the absorbance at 405 nm (λ405) using a microplate reader (Spectra III; SLT-Lab Instruments, Salzburg, Austria).

The calcium mineralisation of the cells in 24 well plates was assayed by Alizarin Red S staining. The cells were cultured in α-MEM containing 50 μg mL−1 vitamin C and 10 mM β-glycerophosphate. Then, the cells were washed three times with PBS, fixed in 70% (v/v) cold ethanol (4 °C) for 1 h and dried in the air. The ethanol-fixed specimens were stained with 40 mM Alizarin Red S (pH 4.2) for 1 h and washed three times with purified water. Then, the specimens were destained with 10% cetylpyridinium chloride in 10 mM sodium phosphate buffer (pH 7.0) for 15 min. To observe the extent of staining, an optical microscope was used and the optical density (OD) was measured at 562 nm using the Spectra III UV microplate reader. The OD of calcium deposition was normalised to the total protein content. All values are expressed as means ± SD (n = 6).

Total protein content

The bicinchoninic acid (BCA) protein assay (Pierce Kit; Thermo Scientific, Waltham, MA, USA) was used to measure the total protein content. After 7 and 14 days of cell culture, the scaffold was assayed. Samples were washed and lysed using PBS and 1 mL of 0.1% Triton X-100, respectively. A mixture of 200 μL of BCA working reagent and a 25 μL aliquot of the lysate was prepared and placed in an incubator for 30 min at 37 °C. A plate reader measured the protein concentration from the absorbance at 562 nm.

Statistical analysis

The data obtained in this study are presented as means ± SD. SPSS for Windows software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis, and single-factor analysis of variance was applied. A value of p < 0.05 was considered to indicate statistical significance.

Results and discussion

Decellularisation and plasma process to obtain nanopatterned roughened PCL/dECM microfibres

A multistep approach to fabricate physically roughened PCL/dECM fibres is shown in Fig. 1(a)–(d). Firstly, to obtain the decellularised ECM, the preosteoblasts (MC3T3-E1) were cultured on electrospun PCL fibres for 4 weeks. After culturing the cells on the fibres, the decellularisation process, namely, freeze–thaw cycles, was conducted to obtain the decellularised ECM. The PCL/dECM fibrous structure was treated with oxygen plasma. Generally, various plasma treatment conditions, such as applied power, exposure time of plasma and gas flow rate, should be appropriately controlled to obtain high etching efficiency for polymeric substrates.25,45 To assess the effect of the processing conditions on the etching efficiency of the PCL/dECM fibres, various powers (10–30 W), total exposure times (0–5 h) and gas flow rates (5–50 sccm) were used. In addition, as an important processing criterion in the plasma process, we set the plasma processing temperature to below 30 °C because a high processing temperature can denaturalise the fibrous proteins within the ECM.
image file: c6ra03840a-f1.tif
Fig. 1 Schematic of plasma-treated poly(ε-caprolactone) (PCL)/decellularised extracellular matrix (dECM) fibrous structure. (a) Electrospinning and (b) cell culturing of preosteoblasts on the micro/nanofibres for 4 weeks. (c) Decellularisation of the cultured cells to obtain dECM and (d) plasma treatment of the PCL/dECM fibrous structure.

To achieve the appropriate processing conditions, we measured the temperature of the plasma chamber for various plasma powers (10–30 W). Fig. 2(a) shows the measured temperature of the chamber under a fixed oxygen flow rate (5 sccm); it is shown that 15 W is reasonable power to achieve a low temperature (28.4 °C). After setting the power, the gas flow rate was varied and the temperature of the plasma chamber was measured [Fig. 2(b)]. Below a flow rate of 10 sccm, the temperature did not exceed 30 °C. However, since the processing temperature in the plasma chamber can be highly dependent on the plasma processing time, we measured the processing temperature for 1 h with the plasma conditions set as 15 W and 5 sccm. Fig. 2(c) shows the temperature change for various numbers of plasma exposure cycles (1 cycle = 1 h) under the plasma conditions (15 W and 5 sccm for 1 h). As shown in the results, for plasma cycles with an intermediate time (10 min), the plasma chamber temperature did not increase. Based on these results, to achieve appropriate etching efficiency of the PCL/dECM fibrous structure, we used various numbers of plasma cycles.


image file: c6ra03840a-f2.tif
Fig. 2 The effects of various plasma treatment conditions on the processing temperature in a plasma chamber. (a) Applied plasma power, (b) oxygen flow rate and (c) plasma exposure time (n = 3).

Fig. 3(a) and (b) shows the SEM and 3D topological images of roughened PCL/dECM fibres subjected to various numbers of plasma cycles (n = 1–5). In the plasma cycles, the plasma power and oxygen flow rate were set to 15 W and 5 sccm, respectively. As shown in the SEM and topological images, the roughened pattern on the surface of the fibrous PCL/dECM structure gradually became more prominent, but at cycle 5, its fibrous structure collapsed due to excessive exposure to plasma ions. To characterise the roughened surface of the PCL/dECM quantitatively, we measured the roughness (Ra) of the fibrous structures [Fig. 3(c)]. As expected, upon increasing the number of cycles of plasma exposure, the roughness gradually increased. However, as shown in the SEM image for cycle 5 (total plasma exposure time = 5 h), the fibrous structure was significantly degraded.


image file: c6ra03840a-f3.tif
Fig. 3 The effect of plasma treatment on the surface roughness of the PCL/dECM fibrous structure. (a) Scanning electron microscope (SEM) and (b) optical topological images showing the roughened surface of fibrous PCL and dECM upon exposure to various numbers of plasma cycles (1 cycle = 1 h). (c) Measured surface roughness and (d) Young's modulus of the PCL/dECM upon exposure to various numbers of cycles.

In addition, it has been shown that the mechanical stiffness of a biomedical scaffold can be strongly related to the mechanical sustainability during tissue growth, as well as cellular activities.46 It is well known that the stiffness of a scaffold can affect cytoskeletal organisation, which can finally influence cell morphology (high stiffness encourages wider cell spreading, while low stiffness can result in rounded cell shapes) and even differentiation.47 For example, Discher et al. demonstrated that contractile myocytes differentiated into myotubes on a substrate with stiffness similar to that of muscle tissue.48 It is also known that plasma treatment can induce a significant decrease of the mechanical strength of the exposed fibrous structure.45 In our previous work, Young's modulus of plasma-etched PCL micro/nanofibres was significantly decreased from 9.1 ± 0.8 MPa for an untreated PCL surface to 8.2 ± 0.8 MPa for a plasma-treated surface due to the highly roughened surface of the micro/nanofibres. From this reason, we measured the mechanical change before and after plasma treatment. Fig. 3(d) shows the change of Young's modulus for various numbers of plasma treatment cycles. Significant deterioration of the mechanical properties of the plasma-treated PCL/dECM was observed after the treatment (five cycles). However, before cycle 2, the mechanical loss was not significant and a homogeneous nanoscale-roughened surface did not develop. From this reason, although a small amount of mechanical loss occurred for the PCL/dECM treated up to cycle 4, radical mechanical loss was not attained compared with that of the PCL/dECM treated up to cycle 5. This phenomenon was because the mechanically reinforcing proteins (e.g. collagen or elastin fibril) of the ECM were not critically damaged during the plasma treatment.

Based on the features of the surface roughness, fibrous morphology and mechanical stiffness of PCL/dECM, we can select appropriate processing conditions (applied power = 15 W, gas flow rate = 5 sccm, number of cycles of exposure to plasma = 4).

In this work, we compared the plasma-treated PCL/dECM (P-PCL/dECM) with two controls: plasma-treated PCL (P-PCL) and PCL/dECM, which was not treated with plasma. Specifically, the P-PCL (Ra = 644.8 nm ± 75) was subjected to plasma conditions as set in our previous work.25 Fig. 4(a)–(c) shows the SEM, AFM and topological images of the modified surfaces (P-PCL, PCL/dECM and P-PCL/dECM), respectively. The images of plasma-treated P-PCL and P-PCL/dECM surfaces clearly show the nanoscale-roughened shape, while PCL/dECM, which was not treated with the plasma process, showed a smooth surface of the PCL fibres and dECM region. In addition, to show the dECM components (e.g. fibronectin and collagen type I) in the fibrous mats, immunofluorescence images were obtained, as shown in Fig. 4(d) and (e). As expected, the biochemical components of ECM were well distributed in the PCL/dECM fibrous mat, regardless of the plasma treatment.


image file: c6ra03840a-f4.tif
Fig. 4 Topological views and biochemical components of the plasma-treated P-PCL, PCL/dECM and P-PCL/dECM surfaces. (a) Scanning electron microscope (SEM), (b) atomic force microscope and (c) optical images showing the roughened surfaces of the P-PCL, PCL/dECM and P-PCL/dECM. (d) Fibronectin (green) and (e) collagen I (red) on the surfaces.

Surface roughness and tensile properties of P-PCL, PCL/dECM and P-PCL/dECM

It has been shown that a microstructured surface can directly interact with the cell morphology and cytoskeletal shape, while a nanostructured surface can affect various cellular activities, including cell adhesion, growth, alignment and even differentiation, although the results differ depending on the surface-pattern shape and cell type.49 In particular, nanosized patterns were shown to markedly influence cell proliferation and differentiation, resulting in significant bone tissue regeneration.50 Based on these findings, physical treatments of surfaces can provide topological cues that are among the most important parameters for determining how successful scaffolds are for tissue regeneration, regarding various cell activities.

To characterise the nanopatterned features on the surfaces quantitatively, AFM and topological optical images were obtained, as shown in Fig. 4(b) and (c). Fig. 5(a) and (b) shows the roughness results for the four surfaces: pure PCL, P-PCL, PCL/dECM and P-PCL/dECM. The results show that the plasma-treated surfaces (P-PCL and P-PCL/dECM) were significantly roughened, while the pure PCL and PCL/dECM had low values of Ra and RMS.


image file: c6ra03840a-f5.tif
Fig. 5 Characterisations of the surface and mechanical properties of the fibrous structures. (a) Roughness distribution and (b) roughness on a single fibre for pure PCL, P-PCL, PCL/dECM and P-PCL/dECM fibrous structures. (c) Tensile stress–strain curves and (d) Young's modulus and maximum stress for the fibrous structures (n = 5).

Because of its simplicity, plasma treatment has been widely used for modifying the surface properties in tissue engineering applications, although it has been reported that this may be associated with deterioration of the mechanical properties. To evaluate the mechanical properties of fibrous mats, a tensile test was performed. Fig. 5(c) and (d) shows the stress–strain curves of the fibrous mats and the measured Young's modulus and maximum tensile stress. The modulus was obtained using a tensile mode at a constant stretching velocity of 0.5 mm s−1.

As expected, for P-PCL and P-PCL/dECM, Young's modulus and maximum stress decreased after the plasma treatment due to the roughened surface of the substrates. However, the modulus and stress of P-PCL/dECM did not differ much from those of the pure PCL because the dECM components embedded in the fibrous PCL mat can reinforce the PCL fibres.

Comparisons of in vitro cellular activities for P-PCL/dECM

In previous studies, we showed that the physically nanoscale-patterned PCL fibrous surface had significant increases of initial cell attachment and proliferation compared with the unpatterned fibrous surface.42,43 To evaluate initial cellular activities using the preosteoblasts, live/dead images and SEM images at day 1 were obtained for the surfaces (pure PCL, P-PCL, PCL/dECM and P-PCL/dECM) [Fig. 6(a)]. In the live/dead images, green and red indicate live and dead cells, respectively. As shown in the live/dead and SEM images, the attached cells on the P-PCL/dECM were much larger than on the P-PCL and PCL/dECM fibrous structures. In addition, for the plasma-treated groups (P-PCL and P-PCL/dECM), the attached cells displayed significant spreading (like focal adhesion) compared with those of the PCL/dECM. After culture for 3 days, the morphological features and nucleus/cytoskeleton distribution on the fibrous structures were observed using SEM and DAPI/phalloidin staining. The SEM images showed that the cells were well proliferated and, even in the P-PCL/dECM, they entirely covered the fibrous surface. To quantitatively analyse the images, the number of nuclei, the area of F-actin and the aspect ratio of F-actin were analysed for the cultured cells [Fig. 6(d)–(f)]. As shown in the results, there were significant increases in the number of nuclei and F-actin spreading, and in the area of the P-PCL/dECM surface compared with the other surfaces. These results indicate that the P-PCL/ECM scaffold provided more favourable microenvironmental conditions, which developed via the synergistic effects of the biochemical components of dECM and physically patterned nanoscale-topological surfaces, for the cultured cells.
image file: c6ra03840a-f6.tif
Fig. 6 Surface characterisations of the cell-cultured fibrous structures (pure PCL, P-PCL, PCL/dECM, P-PCL/dECM). (a) Live (green)/dead (red) images after cell culture for 1 day. (b) SEM images of the fibrous structures after cell culture for 1 and 3 days. (c) DAPI images after cell culture for 3 days. MC3T3-E1 cell morphology during culture on the fibrous structures was analysed by focusing on the nucleus and F-actin after 3 days of culture, for (d) number of nuclei, (e) F-actin area and (f) aspect ratio. Asterisks indicate significant differences.

An analysis of the proliferation of viable cells was conducted using the MTT assay after culturing for 1, 3 and 7 days [Fig. 7(a)]. The P-PCL/dECM structure showed the highest proliferation among the surfaces, indicating that the plasma-treated P-PCL/dECM surface can provide a platform for outstanding metabolic activity.


image file: c6ra03840a-f7.tif
Fig. 7 Cell proliferation and osteogenic activities of the fibrous structures. (a) MTT assay of viable cells (optical density) on the surfaces after 1, 3 and 7 days. (b) Relative alkaline phosphatase (ALP) activity and (c) calcium mineralisation for 7 and 14 days. (d and e) Optical images of Alizarin Red S staining and ALP staining for P-PCL, PCL/dECM and P-PCL/dECM after 7 days of cell culture.

ALP activity and calcium deposition in the fibrous scaffolds were quantitatively assessed [Fig. 7(b) and (c)]. The measured values were normalised with total protein content (Table 1), and the value of the P-PCL fibrous structure was set to 100%. As shown in the results, the mineralisation of the P-PCL/dECM structure on days 7 and 14 was significantly higher than for the others. To qualitatively compare the mineralisation of the fibrous structures, we obtained optical images of ALP activity and Alizarin Red S staining of the fibrous structures cultured for 7 days [Fig. 7(d) and (e)]. The stained surface showing the mineralisation for P-PCL/dECM was much denser than for the other fibrous structures. Our results indicate that cellular activities including proliferation, and osteogenic abilities of the preosteoblasts, were significantly affected by a physically patterned nanoscale topology and the biological environment provided by the plasma treatment and dECM.

Table 1 Total protein content of P-PCL, PCL/dECM and P-PCL/dECM scaffolds. (n = 3)
Samples P-PCL (mg) PCL/dECM (mg) P-PCL/dECM (mg)
7 days 192.5 ± 14.7 256.7 ± 17.6 278.2 ± 5.5
14 days 278.5 ± 6.4 295.8 ± 6.7 309.5 ± 16.8


Conclusion

Here, a versatile plasma treatment was used to develop nanoscale-roughened electrospun PCL/dECM, which was obtained via an electrospinning and decellularisation process. This method was used to obtain a nanoscale pattern under appropriate plasma conditions without significant loss of mechanical properties. To verify the cellular activities, including osteogenic activity using preosteoblasts, the plasma-etched P-PCL/dECM was compared with two controls (plasma-etched P-PCL and an untreated PCL/dECM fibrous structure). In vitro cellular activities demonstrated that the combination of dECM and the nanoscale-roughened surface of the fibrous structure provides meaningful effects, such as significantly high initial cell adhesion, proliferation and even differentiation of the preosteoblasts. These results confirm the potential of surface-roughened P-PCL/dECM as a biomaterial for hard-tissue regenerative applications and also demonstrate that the fabrication technique using plasma treatment is effective to obtain fibrous structures with a nanosized surface pattern.

Acknowledgements

This study was partially supported by a grant from the National Research Foundation of Korea grant funded by the Ministry of Education, Science, and Technology (MEST) (Grant no. NRF-2015R1A2A1A15055305) and also supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (Grant no. HI15C3000).

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03840a

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