Fabrication of Aligned Nanofiber Polymer Yarn Networks for

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Fabrication of Aligned Nanofiber Polymer Yarn Networks for Anisotropic Soft Tissue Scaffolds Shaohua Wu, Bin Duan, Penghong Liu, Caidan Zhang, Xiao-Hong Qin, and Jonathan T. Butcher ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05199 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 16, 2016

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ACS Applied Materials & Interfaces

Fabrication of Aligned Nanofiber Polymer Yarn Networks for Anisotropic Soft Tissue Scaffolds Shaohua Wu, Butcher* †

†§‡

Bin Duan,

§‡

†

†

Penghong Liu, Caidan Zhang, Xiaohong Qin,*

†⊥

Jonathan T.

§

Key Laboratory of Textile Science & Technology, Ministry of Education, College of Textiles,

Donghua University, No.2999 North Renmin Road, Songjiang, Shanghai, 201620, China §

Department of Biomedical Engineering, Cornell University, Ithaca, NY, 14850, USA

⊥

Key Laboratory of Shanghai Micro & Nano Technology, Shanghai, 201620, China

KEYWORDS: heterogeneous, biomechanics, adipose derived stem cells, valve interstitial cells, hydrogel/woven fabric composite scaffolds

ABSTRACT: Nanofibrous scaffolds with defined architectures and anisotropic mechanical properties are attractive for many tissue engineering and regenerative medicine applications. Here, a novel electrospinning system is developed and implemented to fabricate continuous processable uniaxially aligned nanofiber yarns (UANY). UANY were processed into fibrous tissue scaffolds with defined anisotropic material properties using various textile-forming technologies, i.e., braiding, weaving, and knitting techniques. UANY braiding dramatically

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increased overall stiffness and strength compared to the same number of UANY unbraided. Human adipose derived stem cells (HADSC) cultured on UANY or woven and knitted 3D scaffolds aligned along local fiber direction and were >90% viable throughout 21 days. Importantly, UANY supported biochemical induction of HADSC differentiation towards smooth muscle and osteogenic lineages. Moreover, we integrated an anisotropic woven fiber mesh within a bioactive hydrogel to mimic the complex microstructure and mechanical behavior of valve tissues. Human aortic valve interstitial cells (HAVIC) and human aortic root smooth muscle cells (HASMC) were separately encapsulated within hydrogel/woven fabric composite scaffolds for generating scaffolds with anisotropic biomechanics and valve ECM like microenvironment for heart valve tissue engineering. UANY have great potential as building blocks for generating fiber-shaped tissues or tissue microstructures with complex architectures.

1. INTRODUCTION Electrospinning has been recognized as an economical and versatile method to continuously produce polymer fibers with diameters in the nanoscale range of 50-1000 nm, which is on the same order of biological fibers contained in the extracellular matrix (ECM) of soft tissues, including collagens, fibrin, and fibronectin.1-3 Moreover, many studies have shown that the fiber size control and high surface-to-volume ratio of electrospun nanofibrous scaffolds could improve cell adhesion, proliferation as well as differentiation in various tissue engineering applications.4-6 Typical electrospinning processes create sheet-like nanofiber mats that are poorly aligned, and tightly packed.7 The relatively chaotic architecture and weak mechanical properties restrict their applications in tissue engineering, in particular where tight control of fiber organization is required.8,9 Aligned nanofiber scaffolds can mimic anisotropic feature of many native tissues,


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including blood vessel, nerve, muscle heart valve leaflets and improve cell growth and differentiation and guided tissue formation.10-14 Although nanofiber mats with aligned fibers can also be fabricated via modified electrospinning process,15,16 the obtained mats cannot be tailored into different architectures to satisfy the requirements of shape and structure of various tissueengineered scaffolds. In addition, the tightly packed fibrous structure, small pore sizes existed between fibers, and poor inferior controllability may limit cell growth and migration only on the superficial surface, rather than infiltration into the inner layers, due to the inefficiency of nutrient exchange and metabolic product removal.17,18 Textile techniques, including braiding, weaving, and knitting, are an attractive technology to produce excellent 2D and 3D fibrous yarn based architectures with precise control of size, shape, porosity, and mechanical properties.19 Textile based scaffolds have replicated anisotropic mechanical properties of some tissues, such as tendon,20 blood vessel,21 and cartilage,22 which are known to effectively control behaviors and activities of the cells. However, these scaffolds were formed from microfiber yarns with fiber diameter larger than 10 µm. This fiber size is too large relative native cellular ECM, contributing to reduced cell attachment and proliferation.19, 23 The production of nanofiber yarns and further nanofiber fabrics through textile braiding, weaving or knitting technologies would be of substantial interest to merge excellent control of biomechanical anisotropy with excellent cellular integration. Several researchers have attempted to modify eletrospinning technology to obtain nanofiber yarns.24 Barber et al. and Czaplewski et al. employed a modified electrospinning collector to fabricate nanofiber bundles that were braided into nanofibrous scaffolds. These scaffolds possessed uniaxial biomechanical performance similar to that of native tendons and supported expression of tendon fibroblast cell phenotype and elongated morphology.25,26 Nanoyarn-


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reinforced fibrous scaffolds showed significantly increased mechanical properties in the nanoyarn-enhanced direction and possessed higher cell infiltration than random electrospun nanofibrous mats.27 However, these nanofiber bundles and nanoyarns were very short in length and biomechanically weak. These features prevent interior processing continuity, and are thus not amenable to textile processing. Alternatively, electrospun nanofiber membranes can cut into thin strips and then further processed, but still suffer from lack of fiber continuity and property control, which limit their potential for textile processing.28,29 In this study, we design and implement a novel electrospinning system to efficiently manufacture continuous uniaxially aligned, nanofiber yarns (UANY). We generate and characterize UANY created from degradable and non-degradable biopolymers. We also evaluate the cellular behaviors of human adipose derived stem cells (HADSC) on UANY and investigate the differentiation capacity of HADSC towards smooth muscle and osteogenic lineage on UANY. Subsequently, the UANY are fabricated into 3D heterogeneous constructs using textile techniques, i.e. braiding, weaving, and knitting, demonstrating the feasibility and versatility for designing 3D nanofibrous scaffolds with tunable multi-scale structure and material properties. We also integrate woven fabric with heart valve cell laden bioactive hydrogel to generate composite scaffolds for heart valve engineering. 2. RESULTS AND DISCUSSION 2.1. Fabrication of Uniaxially Aligned Nanofiber yarns (UANY) The electrospinning system for UANY is shown in Figure 1. We applied opposing voltages to two needles (Figure 1A), to attract oppositely charged jets of polymer solution (each pulled from a needle) towards the space between a neutral hollow metal rod (NHMR) and a neutral metal disc (NMD). Contact between the oppositely charged nanofibers causes bonding and charge


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neutralization, and the nanofibers ware aligned uniaxially across the gap between the sharptapered point of NHMR and the NMD. By anchoring the NHMR and rotating the NMD , a stable spinning triangle cone formed between the NHMR andNMD , enabling the highly aligned nanofibers to be gathered and twisted into UANY. The obtained UANY then pass through the inner part of NHMR by the guidance of metal wire and are continuously collected on the rotating take-up roll. Figure S1 outlines the mechanism of nanofiber alignment. The needle with positive charges induces negative charges on the positively charged needle-facing side of the sharptapered point of NHMR and the NMD, while the needle with negative charges brings positive charges on the negatively charged needle-facing side (Fig. S1A). Due to the air gap between NHMR and NMD, the electric field lines near the gap are split into two parts and pointing separately to the sharp-tapered point of NHMR and the NMD (Fig. S1C). The joint action of the external electrostatic force and attractive force eventually aligned the nanofibers uniaxially across the space between the the sharp-tapered point of NHMR and NMD. This setup significantly surpass previous designs by improving the alignment and length of nanofiber yarn, and fabrication continuity and productivity . We employed a 10% (w/w) polyacrylonitrile (PAN)/N, N-Dimethylformamide (DMF) solution as a model to demonstrate the capacity of our setup for continuously fabricating UANY for about 6 hours (Figure 1B). A video (Video S1) shows the continuous fabrication of PAN UANY. With this system, we can easily produce PAN UANY with near limitless length (>100 m within 2 hours). Figure 1C and D show the typical FESEM images of PAN UANY. FESEM images of PAN nanofibers in the yarn demonstrated that they were free from beading and exhibited generally circular cross section. The diameters of the PAN UANY and nanofibers were 136.59 ± 28.55 µm and 550.32 ± 66.91 nm, respectively. More than 70% of nanofibers were highly


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aligned along the yarn longitudinal direction (within ±10°, Figure 1E). Other polymers, such as polycaprolactone (PCL) and polyurethane (PU) can also be fabricated into UANY (Figure S2), which supports the versatility and feasibility of the designed electrospinning system.

Figure 1. Continuous fabrication of uniaxially aligned nanofiber yarns (UANY). (A) Schematic illustration of our novel electrospinning setup and its use to continuously fabricate UANY. (B) Photograph of a PAN UANY package produced about 6 hours using the novel electrospinning setup. (C, D) FESEM images of PAN UANY generated with a concentration of 10% PAN/DMF


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solution. The arrows indicate the direction of the long axis of UANY. Scale bars are 200 µm for (C) and 20 µm for (D), respectively. (E) Nanofiber alignment quantification of PAN UANY. 2.2. UANY Support HADSC Viability and Alignment We first evaluated cell viability, adhesion, proliferation and alignment of HADSC on our PAN UANY. After 7-day culture, cells cultured on PAN UANY were viable (>90%, Figure 2A). The cell numbers increased apparently through 21 days of culture (Figure 2B). FESEM imaging determined that HADSC adhered, elongated and aligned along the nanofiber-oriented direction in the UANY (Figure 2C, indicated by the arrow). Moreover, after 21-day culture, the cells became confluent on the surfaces of the UANY and more aligned cells were found comparing to those on UANY after 7-day culture (Figure 2D). PAN is not a widely used biodegradable material, but it is a recognized biocompatible material. Many researchers have been using PAN for bone regeneration,30 for treatment of skin cancer (with holmium-166 iron garnet nanoparticles,31 myocardium regeneration32 and for hemodialysis applications.33 All these studies have demonstrated that PAN has good cytocompatibility and hemocompatibility. PAN membranes and their derivatives are also widely used as dialysis membranes (Evodial® by Gambro) in clinical application. Our results also demonstrate that UANY are able to support cell adhesion, proliferation, as well as alignment, thereby useful candidates for fabricating fibrous tissue scaffolds.


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Figure 2. UANY promote high cell viability and induce cell alignment by the aligned nanofibrous structure. (A, B) Fluorescent images of dead cells (red) and living cells (green) on cultured on PAN UANY for 7 days and 21 days. The dashed lines indicate the border edges of different PAN UANY. Scale bars = 100 µm. (C, D) FESEM images demonstating HADSC orientation and colonization alignment along the direction of underlying nanofibers in the UANY. The arrows indicate the direction of the long axis of UANY. Scale bars = 50 µm. 2.3. UANY Support Differentiation of HADSC towards Smooth Muscle or Osteogenic Differentiations in Culture


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HADSC were seeded on PAN UANY for 21 days in either control growth medium (GM) or SMC-differentiation medium (SMC-DM) to assess the SMC phenotype differentiation. HADSC expressed α-SMA and calponin sparsely, with no expression of SM-MHC, when cultured in GM (Fig 3A-C). SMC-DM culture however induced robust expression of α-SMA, calponin and SMMHC, indicating mature SMC phenotype (Figure 3D-F). Smooth muscle-specific gene expression was also quantified by qPCR. HADSC significantly upregulated α-SMA (4.08±0.53fold increase; p

Fabrication of Aligned Nanofiber Polymer Yarn Networks for

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