www.ietdl.org Published in IET Nanobiotechnology Received on 25th March 2014 Revised on 12th June 2014 Accepted on 13th June 2014 doi: 10.1049/iet-nbt.2014.0006

ISSN 1751-8741

Electrospun scaffold containing TGF-β1 promotes human mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype under hypoxia Xiang Cui1, Minghan Liu1, Jiaxu Wang1, Yue Zhou1, Qiang Xiang2 1

Department of Orthopedics, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, People’s Republic of China 2 Department of Emergency, Southwest Hospital, Third Military Medical University, Chongqing 400038, People’s Republic of China E-mail: [email protected]

Abstract: The study was aimed at evaluating the effect of electrospun scaffold containing TGF-β1 on promoting human mesenchymal stem cells (MSCs) differentiation towards a nucleus pulposus-like phenotype under hypoxia. Two kinds of nanofibrous scaffolds containing TGF-β1 were fabricated using uniaxial electrospinning (Group I) and coaxial electrospinning (Group II). Human MSCs were seeded on both kinds of scaffolds and cultured in a hypoxia chamber (2% O2), and then the scaffolds were characterised. Cell proliferation and differentiation were also evaluated after 3 weeks of cell culture. Results showed that both kinds of scaffolds shared similar diameter distributions and protein release. However, Group I scaffolds were more hydrophilic than that of Group II. Both kinds of scaffolds induced the MSCs to differentiate towards the nucleus pulposus-type phenotype in vitro. In addition, the expression of nucleus pulposus-associated genes (aggrecan, type II collagen, HIF-1α and Sox-9) in Group I increased more than that of Group II. These results indicate that electrospinning nanofibrous scaffolds containing TGF-β1 supports the differentiation of MSCs towards the pulposus-like phenotype in a hypoxia chamber, which would be a more appropriate choice for nucleus pulposus regeneration.

1

Introduction

Lower back pain is one of the common diseases in clinic which affects about 75–85% of all people [1, 2]. Intervertebral disc (IVD) degeneration is considered to play the main role [3, 4]. Conservative treatment could only relieve symptoms. Although surgical treatments including discectomy and spinal fusion might provide short-term pain relief, they would result in complications and impairment such as reduction of spinal mobility, degenerative post-discectomy spondylosis and disc herniation recurrence [5]. In recent years, IVD nucleus pulposus (NP) tissue engineering exhibits extensive prospects in repairing and reconstructing IVD [6]. Generation of a biological NP replacement by tissue engineering appears to be a promising approach for the therapy of early stages of IVD degeneration. However, there are still many problems such as the lack of perfect cell type and appropriate scaffolds. Among the mainly used engineering cells like stem/ progenitor cells and embryonic stem cells, mesenchymal stem cells (MSCs) were the most widely used [7, 8]. Various growth factors have been proven to promote MSCs’ proliferation and differentiation. However, high price and short lives in vivo limit the application of growth 76

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factors. Thus, a controlled-release scaffold would help sustain the existence and function of biologically active growth factors [9]. Electrospinning has emerged as a new technique to produce various functional polymer nanofibres, which have been developed as a tool to deliver the bioactive reagents [10–12]. The surface pores of the nanofibres are used to release the drugs that are contained in the fibres. Compared with traditional drug delivery systems, this method greatly increases the action time. This method also offers the advantages of low mass and high wound closure, effectively facilitating wound recovery. Notably, fabrication of nanofibres containing diverse growth factors, such as platelet-derived growth factor (PDGF) [13], vascular endothelial growth factor and PDGF [14] and transforming growth factor-beta (TGF-β) [15], has been reported. In addition to uniaxial electospinning, co-axial electospinning technology has also been widely applied [16]. It would provide new access to biologic treatment by fabricating electrospinning scaffolds combining with growth factors which promote MSCs to differentiate towards a NP-like phenotype. Autologous MSCs represent an attractive cell source compared with NP cells that are already altered in their IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

www.ietdl.org phenotype because of degenerative processes [17]. MSCs can be induced to differentiate into a NP-like phenotype following direct co-culture with degenerate NP cells [18]. Vadala et al. [19] reported that co-culture of MSC and NP cells induced a change in MSCs towards a more chondrogenic gene expression profile, suggesting MSC differentiation. These investigations indicate that MSC-based therapies with degenerate NP cells may enhance matrix synthesis for self-repair of IVD. Risbud et al. [20] confirmed for the first time that bone marrow MSC (BMSC) could direct the differentiation of BMSCs towards a NP-like phenotype under the effect of hypoxia and TGF-β1 in vitro. Sakai et al. [21] found that autologous MSCs were embedded in Atelocollagen® gel and transplanted them into the discs of rabbits which resulting in a retardation of degeneration. Furthermore, TGF-β has been demonstrated to promote chondrogenesis of mesenchymal progenitor cells and immortalised human MSCs; TGF-β at 1 ng/ml effectively promotes NP cells proliferation. Furthermore, hypoxia has been suggested to be suitable for directing MSCs towards a NP-like phenotype [22]. In this study, uniaxial and coaxial electrospinning technologies were utilised to fabricate nanofibre scaffolds with or without TGF-β. The effects of those nanofibre scaffolds on proliferation, adhesion and gene expression of MSCs upon hypoxia were also investigated.

2 2.1

Materials and methods Materials and reagents

Poly (lactic-co-glycolicacid) (PLGA, LA/GA = 75:25, M W = 66 − 107 kDa) was obtained from Daigang company (Shandong, China). Polyvinyl alcohol (PVA, M W = 89 − 98 kDa, 99+% hydrolysed) and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Recombinant Human TGF-β1 was supplied from ProSpec Company. Other reagents and culture supplements were commercially available and used as received unless otherwise stated.

2.2

Fabrication of electrospinning scaffolds

PLGA solution of 15% w/v was prepared by adding 1.5 g PLGA to 10 ml hexafluoroisopropanol (HFIP, Fluka Chemie GmBH, Germany), while PVA solution was at a concentration of 15% w/v by dissolving 1.5 g PVA in 10 ml of double distilled water. TGF-β1 was dissolved in 0.1% BSA at a concentration of 1 ng/μl. For Group I, the blend solution of 1 ml PLGA, 1 ml PVA and 3 μl TGF-β1 was loaded into a syringe after mixing and stirring for 10 min. Then the syringe was fixed on a syringe pump (LongerPump, LSP02-1B, China) to maintain a constant flow of 0.4 ml/h. The group I ( + ) scaffolds were fabricated with the high-voltage RXZGF (Rixing Electronics, Co. Ltd., Shanghai, China) set at 16–18 kV and collected on a grounded plate placed 15 cm from the needle tip. For group II ( + ), 1 ml PLGA solution made up the outer channel, while the mixed solution of 1 ml PVA and 3 μl TGF-β1 formed the inner channel. In addition, control scaffolds (without TGF-β1) of group I ( − ) and group II ( − ) were fabricated as well. Scaffolds were preserved under −20°C before further use. IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

2.3

Characterisation

2.3.1 Scanning electron microscopy (SEM): The morphology of nanofibres was observed with SEM (Agilent Technologies Inc, Model s-3400II, Japan) after samples were sputter-coated with gold [10 mA, 10 psi for 50–60 s (JFC-1200 Fine Coater, JEOL)]. SEM images were analysed by Image-J 1.34 software (National Institutes of Health, USA) to determine the fibres diameter distribution of uniaxial and coaxial electrospun scaffolds. 2.3.2 Transmission electron microscopy (TEM): TEM (Libra200, Carl Zeiss AG Germany) was employed to examine the core-shell structure of electrospun nanofibres. TEM samples were prepared by placing copper grids on the collector and directly depositing a very-thin layer of electrospun nanofibres onto the copper grids. TEM images were subsequently obtained to observe the core/sheath structure of nanofibres. 2.3.3 Water contact angles measurement: Water contact angles of nanofibrous membranes were measured by a water contact angle analyser (SCA; VCA-Optima, AST Products, MA) to evaluate the hydrophilicity of each sample. Samples of 1 cm by 3 cm were cut from the membranes and placed on the testing plate, after the distilled water was dropped on the surface of samples carefully, then the contact angles were measured. 2.3.4 In vitro TGF-β1 release study: The uniaxial and coaxial electrospinning scaffolds were immersed in dialysis bag containing PBS buffer in 25 ml tubes. Then the samples were incubated at 37°C and 50 rpm in a shaking incubator. At specified time points, 5 ml PBS was taken out, and fresh PBS was added at the same time. The amount of TGF-β1 released was calculated based on HPLC/ MS (Agilent, USA). 2.3.5 Attenuated total reflectance-fourier transform infrared (ATR-FTIR): To determine the protein contained in scaffolds, ATR-IR spectra of the nanofibres films were carried out with FTIR spectrometer (Lambda-900, Lambda35 PerkineElmer) to observe the groups difference of group I and group II. The uniaxial electrospinning scaffolds only containing PLGA and PVA were also used as controls. 2.4

Cell culture and seeding

Human MSCs (hMSCs, Cyagen, Chicago) are derived from the healthy adult bone marrow. hMSCs were incubated in a humidified incubator at 37°C with 5% CO2 using hMSCs growth medium (Cyagen) containing 10% foetal bovine serum (FBS, Cyagen). The medium was changed every other day. Cells at passage 6–8 were used. According to their size, the cut nanofibre mats were inserted into 6-well and 24-well culture plate, respectively. Then these culture plates, sealed by hermetic bag, were sterilised in ethylene oxide steriliser (Shenyang Longteng Company, YWM30). To promote cell attachment, the scaffolds were immersed in hMSCs growth medium for 6 h prior to cell seeding. hMSCs were pipetted directly onto the scaffolds at the density of 1.5 × 104 cells/well and incubated at 37°C with 5% CO2 for 24 h. Then the nanofibrous matrix was transferred to the hypoxia chamber and the media was replaced with a chondrogenic differentiation media (Cyagen) in a hypoxia chamber at 2% O2. Controls were 77

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www.ietdl.org maintained in the same medium but at 20% O2. The medium was changed twice a week. 2.5

MTT

2.5.1 Cell proliferation: Cells were cultured in hMSCs growth medium at the density of 5 × 104 cells/well (24 well). Cell proliferation was determined using MTT Kit (Vybrant® MTT Cell Proliferation Assay Kit, Life Technologies, USA) according to the manufacturer’s instructions. Briefly, after each time period (2, 4, 6 and 8 days), the medium was sucked, and then 24 μl MTT solution (5 mg/ml) and 600 μl fresh hMSCs growth medium were added to each well, incubated at 37°C and 5% CO2 for 4 h. After the medium was discarded, 450 μl DMSO (Sigma) was added to each well and the plate was incubated for 30 min. Absorbance was measured at 570 nm by microplate reader (MK3, Thermo, USA). Each assay was carried out in triplicate. 2.5.2 Cell attachment: Cells were cultured in hMSCs growth medium at the density of 1 × 105 cells/well (24 well). Adherent cells were quantified using MTT Kit according to the manufacturer’s instructions. Briefly, after each time period (30 min, 1 h and 2 h), the medium was discarded, and the non-adherent cells were removed by washing with PBS (pH 7.4). Then these wells were added to 24 μl MTT solution and 600 μl fresh hMSCs growth medium, incubated at 37°C and 5% CO2 for 4 h. After the medium was removed, 450 μl DMSO was added to each well and the plate was incubated for 30 min. Absorbance was measured at 570 nm by microplate reader. Each assay was carried out in triplicate. 2.5.3 SEM: On day 21, cell-scaffold constructs, cultured in normal oxygen and hypoxia, respectively, were rinsed in PBS, fixed with 3% glutaraldehyde for 2 h and dehydrated in increasing concentrations of ethanol and tert butyl-alcohol, and vacuum-drying. The specimens were then sputter-coated with gold and observed under SEM. 2.6 Reverse transcriptase polymerase chain reaction (qRT-PCR) Total RNA was extracted with TRIZOL (Invitrogen) according to the manufacturer’s instructions. The RNA samples were reverse transcribed into first-strand cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Company, Dalian, China). Gene-specific amplimer from the cDNA of each sample were amplified and quantitatively measured by real-time PCR using SYBR® Premix Ex TaqTM II kit (Takara Company, Dalian, China). After PCR (ABI 7500 PCR instrument, Applied Biosystems), the expression of Sox-9, Collagen type II, Aggrecan and hypoxia inducible factor-1 α (HIF-1α) were

analysed. β-actin was used as an internal standard. PCR conditions were at 95°C for 2 min, 40 cycles at 95°C for 30 s, 60°C for 30 s. These gene primers were shown in the Table 1. 2.7

Statistical analysis

Results are presented as mean ± standard deviation. Significance between the mean values was calculated using analysis of variance (ANOVA) one-way analysis (Origin8.1 SRO, Northampton, MA, USA). Probability values p < 0.05 were considered significant (n = 8).

3 3.1

Results Morphology of the nanofibres

The surface morphology of Groups I and II nanofibres was revealed using SEM (Figs. 1a and c), and the fibres’ diameter of uniaxial and coaxial electrospun scaffolds were analysed. The diameters of nanofibres were between 100 and 600 nm (Figs. 1b and d ). The average diameter of Group I and Group II nanofibres was 418.31 ± 130.03 and 410.84 ± 114.95 nm, respectively. There was no significant difference between the two groups’ nanofibres ( p > 0.05). 3.2

TEM characterisation

The core-shell structure of electrospun nanofibres was observed using TEM. In Fig. 2, the images display the internal structure of coaxial electrospinning prepared nanofibres. The dark region in the images is core, and the bright region is shell. The contrast in the TEM images demonstrated that core-shell double-layer structure of nanofibres was prepared successfully. 3.3

Water contact angle measurements

On hydrophilic surfaces of nanofibres, the water droplets were spread quickly, pulled continuously and moved towards the loose fibres, accelerating wetting and reducing the contact angle. Therefore, the contact angle was measured at different time points to prove the point. According to the results, the average water contact angles of Group I ( + ) were 51.2°, 50.1°, 47.6° and 44.7° at 3, 6, 12 and 18 s, respectively. In the Group II ( + ), the average water contact angles were 65.8°, 63.7°, 62.5° and 60.8° at 3, 6, 12 and 18 s, respectively (Table 2). Therefore the hydrophilicity of nanofibres fabricated by uniaxial electrospinning was better than that of coaxial electrospinning. In addition, the water contact angles of Group I ( − ) and Group II ( − ) were also determined. In the Group I ( − ), the average water contact angles were 57.1°, 56.8°, 54.6° and 53.8° at 3, 6, 12 and 18 s, respectively. In the Group II ( − ), the average water

Table 1 RT-PCR primers for Sox-9, Collagen Type II, Aggrecan, HIF-1α and β-Actin genes Genes Sox-9 collagen type II Aggrecan HIF-1α β-Actin

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Forward primer

Reverse primer

5-GCGGAGGAAGTCGGTGAAGA-3 5-GGAGCAGCAAGAGCAAGGAGA-3 5-ACTCTGGGTTTTCGTGACTCT-3 5-GTCGGACAGCCTCACCAAACAGAG-3 5-GTCCTCTCCCAAGTCCACAC-3

5-GAAGATGGCGTTGGGGGAGA-3 5-GTGGACAGCAGGCGTAGGAAG-3 5-ACACTCAGCGAGTTGTCATGG-3 5-GTTAACTTGATCCAAAGCTCTGAG-3 5-GGGAGACCAAAAGCCTTCAT-3

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Fig. 1 Comparison of uniaxial and coaxial electrospun nanofibres a SEM images of uniaxial electrospun nanofibres b Diameters distribution of uniaxial electrospun nanofibres c SEM images of coaxial electrospun nanofibres d Diameters distribution of coaxial electrospun nanofibres

contact angles were 78.2°, 75.9°, 73.2° and 70.1° at 3, 6, 12 and 18 s, respectively. 3.4

In vitro release of TGF-β1

As shown in Fig. 3, no obvious differences were observed in the release characteristics between the uniaxial and coaxial electrospun nanofibres. In the Group I ( + ), TGF-β1 released faster, and almost completely released in about 7 days with encapsulation efficiency of 86.3 ± 3.8%. However, sustained release of TGF-β1 from nanofibres in the Group II ( + ) was observed over a period of 14 days, indicating high efficient encapsulation of TGF-β1 (92.7 ± 2.8%) in core/sheath nanofibres. 3.5

contained protein in Group I ( + ) and Group II ( + ). These results indicate that nanofibres containing TGF-β1 were fabricated from uniaxial and coaxial electrospinning. 3.6

MTT

3.6.1 Cells proliferation and adhesion: In Group I ( + ), nanofibrous mats showed the best ability to encourage the

ATR-FTIR spectra analysis

In Fig. 4, curve I corresponds to electrospun scaffolds only including PLGA and PVA, and curves II and III correspond to Group I ( + ) and Group II ( + ), respectively. The characteristic absorption band observed at 1760 cm−1 is ascribed to C = O characteristic vibration of PLGA, the band at about 1640 cm−1 corresponds to peptides amide peak and the band at about 1428 cm−1 is assigned to CH2 bending vibration. The band at about 1089 cm−1 corresponds to C–O peak and the band at about 871 cm−1 is assigned to CH2 bending vibration of PVA. Compared with curve I, curves II and III exhibit characteristic absorption bands at 1640 cm−1, which represent the amide bonds of protein, demonstrating that the nanofibres IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

Fig. 2 TEM image of the internal structure of coaxial electrospinning nanofibres 79

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www.ietdl.org Table 2 The water contact angle of Group I ( − ), I ( + ), II ( − ) and II ( + ) at 3, 6, 12 and 18 s, respectively Water contact angle (mean ± SD, °)

Group I ( − ) Group I ( + ) Group II ( − ) Group II ( + )

3s

6s

12 s

18 s

57.1 ± 1.92 51.2 ± 1.56a 78.2 ± 3.47 65.8 ± 2.46b

56.8 ± 1.84 50.1 ± 1.35a 75.9 ± 2.93 63.7 ± 3.04b

54.6 ± 1.63 47.6 ± 1.19a 73.2 ± 2.86 62.5 ± 2.83b

53.8 ± 1.76 44.7 ± 1.27a 70.1 ± 3.17 60.8 ± 2.79b

Compared with uniaxial electrospun nanofibres without TGF-β1, P < 0.05. Compared with coaxial electrospun nanofibres without TGF-β1, P < 0.05.

improvement between the uniaxial and coaxial electrospun nanofibres when added TGF-β1.

Fig. 3 Release rate of TGF-β1in the uniaxial and coaxial electrospun nanofibres a TGF-β1 release of uniaxial electrospun nanofibres b TGF-β1 release of coaxial electrospun nanofibres

cells’ proliferation (Fig. 5a). Compared with cell proliferation on nanofibres scaffolds contained TGF-β1 or not, the cell population increased obviously on nanofibrous mats with TGF-β1. Furthermore, the uniaxial electrospun nanofibres enhanced cell proliferation compared with coaxial electrospun nanofibres. Cells’ attachment on nanofibres after cell culture was shown in Fig. 5b. The cells attached well to porous uniaxial electrospun nanofibres. However, there was no obvious

3.6.2 Morphology of the cells grown observed by SEM: After 21 days of culture, surface morphology of cell-scaffold constructs among Group I ( − ), I ( + ), II ( − ) and II ( + ), cultured under hypoxia and normoxia, respectively, was observed by SEM (Fig. 6). Compared to cell-scaffold constructs cultured under normoxia, there was a great deal of extracellular matrices (ECM) on scaffolds cultured under hypoxia. Compared with the scaffolds without TGF-β1, the produced ECM on TGF-β1 nanofibres was a little more, but had no significant difference when cultured under hypoxia and normoxia. Furthermore, the amount of generated ECM on uniaxial electrospun nanofibres was more than that of coaxial electrospun nanofibres. However, the cell morphology was indistinct because of covering by ECM. 3.7

qRT-PCR analysis

The mRNA expression differences of Sox-9, Type II collagen, aggrecan and HIF-1α of each group under hypoxia and normoxia were tested. On Day 21, compared with the normoxia-induced group, Sox-9, Type II collagen and aggrecan mRNA expression was significantly greater in the hypoxia-induced group (P < 0.05) (Figs. 7a–c). Compared to groups without TGF-β1, there was a significant difference in the expression of these three genes in groups containing TGF-β1 (P < 0.05) (Figs. 7a–c). The relative expression of HIF-1α was higher in the hypoxic group compared with the normoxia group at 21 days

Fig. 4 ATR-FTIR spectra analysis of nanofibres Curve I: only including PLGA and PVA; curve II: Group I ( + ); and curve III: Group II ( + ) 80

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Fig. 5 The MTT absorbance values of Group I ( − ) (namely D( − )), I ( + ) (D( + )), II ( − ) (T( − )) and II ( + ) (T( + )) a Cells proliferation on nanofibres b Cells adherent on nanofibres

(Fig. 7d ). Especially when TGF-β1 was added, the HIF-1α mRNA expression was significantly higher in the hypoxic group compared to the normoxia group (P < 0.05) (Fig. 7d ). Additionally, the inducing differentiation of the uniaxial electrospun nanofibres was best under hypoxia condition.

4

Discussion

Tissue engineering is considered a promising approach for restoring, repairing and regenerating IVD, but current works are pre-clinical studies. This new approach involves the replacement of the damaged tissue by a biomaterial alone (scaffold) or by the scaffold associated with the

appropriated cells and/or biochemical factors (e.g. growth factors, drugs and anti-angiogenic peptides) [23]. Clinically, such a scaffold might be used after discectomy to cork the hole in annulus fibrosus (AF) left after disc herniation and surgical procedure. The scaffold might either be sutured or fixed with fibrin glue or other biological glue materials. This bioactive scaffold might also be associated with NP tissue engineering strategies for the treatment of IVD degeneration. Recently, these cell-seeded engineered discs have been placed in situ between rat lumbar and caudal vertebrae [24, 25]. However, in the field of biomaterials for IVD regeneration, namely the development of adequate materials for closure of the AF and the avoidance extrusion of NP, this still remains a huge challenge.

Fig. 6 Surface morphology of cell-scaffold constructs cultured after 21 days a Normoxia, Group I ( − ) b Normoxia, Group I ( + ) c Normoxia, Group II ( − ) d Normoxia, Group II ( + ) e Hypoxia, Group I ( − ) f Hypoxia, Group I ( + ) g Hypoxia, Group II ( − ) h Hypoxia, Group II ( + ) IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

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Fig. 7 Analysis of mRNA expression a mRNA expression of Sox-9 of Group I ( − ), I ( + ), II ( − ) and II ( + ) under hypoxia and normoxia b mRNA expression of Type II collagen of Group I ( − ), I ( + ), II ( − ) and II ( + ) under hypoxia and normoxia c mRNA expression of Aggrecan of Group I ( − ), I ( + ), II ( − ) and II ( + ) under hypoxia and normoxia *Indicates significant variation in mRNA expression at P < 0.05 level by ANOVA one-way analysis

In our investigation, the uniaxial and coaxial electrospun nanofibres containing TGF-β1 were fabricated. The similar diameter between these two scaffolds was found by SEM and TEM, and the core-shell structure of coaxial electrospinning scaffolds was demonstrated by TEM. In addition, nanofibres containing TGF-β1, tested by ATR-FTIR, were fabricated from uniaxial and coaxial electrospinning. Previous studies have investigated the release properties of protein in electrospinning nanofibres. In the present study, pores appeared in nanofibres’ surface during nanofibres’ degradation. The release of protein was mainly diffused through these pores into the surrounding solution [26, 27]. Previously, DeFail et al. [28] reported bioactivity of TGF-β1 from PLGA microspheres embedded in poly (ethylene glycol) hydrogels. Their work showed that TGF-β1 was stable, released over 21 days from release study. Moreover, Jaklenec et al. [29] found that TGF-β1, encapsulated in PLGA microspheres, was released for up to 70 days in bioactive form, determined by HT-2 inhibition assay. Similarly, Vadala et al. [15] also reported that there was a slightly diminishing value at day 21 which could be ascribed to TGF-β1 denaturation. In the present study, there was an initial burst release within the first 24 h, followed by a much slower and sustained release of TGF-β1 in uniaxial and coaxial electrospun nanofibres. This release behaviour is in agreement with the previous study [15]. TGF-β1 from nanofibres in the Group I ( + ) released faster, and almost completely released in about 1 week. However, sustained release of TGF-β1 from nanofibres in 82

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the Group II ( + ) was observed over a period of 2 weeks. This may be because of the effect of the surface morphology and core-sheath structure, indicating that TGF-β1 is restricted to the core layer. The cell adhesion, migration and proliferation on a material surface or a scaffold are strongly influenced by the nanofibres’ hydrophilicity [30, 31]. Results from our contact angle tests showed that compared with coaxial electrospun nanofibres, uniaxial electrospun nanofibres with higher hydrophilicity are better in terms of promoting cell proliferation and adhesion. Moreover, water contact angle results revealed that coaxial electrospun nanofibres were less hydrophilic and the hydrophilicity of nanofibres was increased by adding TGF-β1. Wang et al. [32] reported that electrospun fibrous scaffolds had much larger water contact angles than the non-porous films fabricated by solvent-casting method owing to different surface roughness. However, another study has suggested that emulsion electrospun nanofibres had a smaller water contact angle [33]. In our study, uniaxial electrospinning scaffolds electrospun from emulsions because of liposolubility of PLGA and water-solubility of PVA. The following contact angle tests showed that uniaxial electrospun nanofibres had a smaller water contact angle, and this was similarly observed in previous investigation [33]. It is also in agreement with a report that hydrophilicity of scaffolds was improved by joining hydrophilic proteins [34]. The aim of the current study was to develop efficient methods to generate NP grafts with phenotypic stability. IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

www.ietdl.org The NP is a highly avascular tissue with no independent blood supply. It has been reported that low oxygen environment plays a key role in maintaining IVD physiological function or promoting the ideal NP phenotype [35–37]. Mwale et al. [38] found that the best NP phenotype for bovine IVD cells could be promoted at a physiologic O2 (1%) in alginate. Feng et al. [39] also found that hypoxia markedly enhanced NP cells phenotype, which resulted in greater production of collagen type II and sulphated glycosaminoglycan (GAG) in the nanofibrous scaffolds. In this study, low oxygen physiological environment of NP was mimicked. The current investigation has demonstrated that when combined with hypoxia and TGF-β1, the nanofibrous scaffold was able to direct the differentiation of hMSCs to a NP-like and stable phenotype. Our results showed that the nanofibres cultured under hypoxia could facilitate differentiation of hMSCs persistently. Under hypoxia, scaffolds with TGF-β1 could also promote differentiation of hMSCs compared with scaffolds without TGF-β1, although this enhancement was not obvious. This may be because hypoxia condition plays a dominant role in accelerating differentiation of hMSCs. In NP cells and chondrocytes, characteristic markers of type II collagen, aggrecan, Sox-9 and HIF-1α are expressed. HIF-1α is a key transcription factor that can be used as a phenotypic marker to distinguish NP cells from chondrocytes [36]. In our study, the gene expression of Sox-9, Type II collagen, aggrecan and HIF-1α was also examined by RT-PCR. These results clearly demonstrated that a hypoxic environment led to significantly increased expression of Sox-9, Type II collagen, aggrecan and HIF-1α at 21 days when compared with normal oxygen conditions, which indicates that cells grown in a hypoxic environment more closely resemble NP-like cells. The results of this study support previous results by Risbud et al. [20], Feng et al. [39] and Ni et al. [40] in that hypoxic conditions lead to a higher gene expression of NP-like cell markers (type II collagen, Sox-9, aggrecan and HIF-1α) than normoxia conditions. Rutges et al. [4] reported that calcification of IVD is associated with disease and aging, and inflammatory response and angiogenesis are caused during disease and aging, which leading to abnormal calcification. Thus, this knowledge contributes to put forward ideas, namely the potential clinical application of MSCs for NP repair. Araldi et al. [41] found that endochondral bone formation could be regulated under hypoxia, and HIF-1α may have effects of regulation on preventing mineralisation in the IVD. A similar observation was also made by Skubutyte et al. [42]. These investigations implied that HIF-1α is a survival factor in NP cells and that hypoxia-induced matrix gene expression requires the presence of HIF-1α. There were several limitations in the present study. The fabricated nanofibres were two-dimensional, so the promise of three-dimensional nanofibrous scaffolds should be made to engineer NP tissue. Furthermore, the implanted experiment in animals also should be conducted to confirm that hypoxic induction could contribute to maintain their phenotype and resist calcification in vivo.

5

Conclusions

In this work, the loaded TGF-β1 nanofibres were successfully fabricated by uniaxial and coaxial electrospinning. The IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

uniaxial electrospun nanofibres had similar diameter to coaxial electrospun nanofibres, and higher hydrophilicity, possibly facilitating cell adhesion and proliferation. The investigation for inducing differentiation of MSCs has demonstrated that nanofibrous scaffolds were able to promote the differentiation of hMSCs towards a NP-like phenotype under hypoxia, and the uniaxial electrospun nanofibrous matrices could bring better results. Hypoxic environment enhanced expression of the NP-like cell markers, indicating increased differentiation and proliferation of NP-like cells. The experimental data presented above suggest that the porous nanofibrous scaffolds, combined with TGF-β1 and hypoxic induction, may serve as an efficient way to achieve a stable and functional NP graft for IVD regeneration.

6

Acknowledgment

This research was supported by National Nature Science Foundation of China (project No. 81171691).

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IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 76–84 doi: 10.1049/iet-nbt.2014.0006

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