3D Electrospun Fibrous Structures from Biopolymers - ACS Publications

Chapter 7

3D Electrospun Fibrous Structures from Biopolymers Downloaded by MONASH UNIV on December 6, 2014 | http://pubs.acs.org Publication Date (Web): October 21, 2014 | doi: 10.1021/bk-2014-1175.ch007

Helan Xu1 and Yiqi Yang*,1,2,3 1Department

of Textiles, Merchandising and Fashion Design, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802, United States 2Department of Biological Systems Engineering, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802, United States 3Nebraska Center for Materials and Nanoscience, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802, United States *Tel: +001 402 472 5197. Fax: +001 402 472 0640. E-mail: [email protected].

Electrospun three-dimensional (3D) fibrous biopolymers are receiving increasing attention as tissue engineering scaffolds. 3D structures could more closely resemble the stereoscopic architectures of native extracellular matrices (ECMs), and thus could provide similar guidance to signaling and migration of cells. Furthermore, fibrous structures could provide larger surface area than non-fibrous ones to facilitate cell attachment and growth. Due to the high efficiency and broad applicability, electrospinning has become the most widely accepted method in developing ultrafine fibers from biopolymers. However, since last decade, researchers started applying electrospinning technology to produce 3D ultrafine fibrous scaffolds. Via incorporating porogens or microfibrous frames, using coagulation bath as receptors, and changing electrical properties of spinning dopes, 3D fibrous structures have been developed from natural biopolymers, including proteins (collagen, gelatin, silk fibroin, zein, soyprotein, wheat gluten, etc.), polysaccharides (chitosan, alginate, hyaluronic acid, etc.), and bio-derived synthetic polymers, mainly polylactic acid © 2014 American Chemical Society In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

(PLA) and polycaprolactone (PCL). These 3D fibrous scaffolds from biopolymers played increasingly important roles in tissue engineering and medical applications.

Introduction

Downloaded by MONASH UNIV on December 6, 2014 | http://pubs.acs.org Publication Date (Web): October 21, 2014 | doi: 10.1021/bk-2014-1175.ch007

Tissue Engineering The ultimate target of scaffold design in tissue engineering is to produce substrates that can function as artificial extracellular matrices (ECMs) until new ECMs are resynthesized by cells cultured. To achieve this goal, the substrates should have architectures similar to the natural ECMs. Typical natural 3D ECM is shown in Figure 1 (1). The cell attaches on the fibrils in the ECM and spread into a stereo shape. As dynamic, mobile and flexible three dimensional (3D) sub-micron fibrous networks, natural ECMs not only provide physical support to cells, but also define cellular behaviors and final tissue functions. In natural ECMs, highly organized ultrafine collagen fibrils in 3D architectures play dominant roles in maintaining the biological and structural integrity of the tissues or organs (2). The 3D structures could provide better connection among cells than conventional 2D cultures, by providing another dimension for interaction, migration and morphogenesis of cells. Figure 2 showed the cross-sections of 2D fibrous and 3D fibrous scaffolds after culturing with cells for 15 days (3). The red color represented stained cells, indicating that more cells could be found in the interior of 3D fibrous scaffolds. Moreover, the collagen ECMs are highly dynamic because they undergo constant remodeling to maintain proper physiologic functions. Hence, optimal tissue engineering scaffolds should be able to restore both structural integrity and physiological functions of native ECMs.

Figure 1. Scheme of a cell in 3D natural extracellular matrix. Reproduced with permission from reference (1). Copyright (2013) Nature. 104 In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Ideally, the basic building blocks of tissue engineering scaffolds should be ultrafine fibers oriented in multiple dimensional scales. Comparing to films, sponges and other types of scaffolds, fibrous architectures could more closely emulate the structures of the native ECMs, provide better guidance for cell migration and proliferation, and ultimately, determine the functions of neo-tissues. Fibers also can provide much larger surface area to facilitate adsorption of receptor proteins, such as integrin, fibronectin and vitronectin, and thus enhance cell adhesion. In general, properties and processing techniques are the two major concerns in developing 3D ultrafine fibrous scaffolds.

Figure 2. Histological analysis of the Oil Red O stained scaffolds with adipose derived mesenchymal stem cells 15 days after induction of adipogenic differentiation. Left, 2D wheat glutenin (WG) fibrous scaffolds; right, 3D WG fibrous scaffolds. Scale bar represents 100 µm. Reproduced with permission from reference (3). Copyright (2014) Elsevier. Electrospinning Electrospinning is one common method to fabricate nano- to micro-scale fibers from various biopolymers because it is applicable to a variety of biopolymers and efficient (4). During electrospinning, polymer solution is forced through a capillary, forming a drop of polymer solution at the tip. By applying a high voltage between the tip and a collecting surface, the polymer solution carries charges and is attracted by the collecting surface with opposite charges. As soon as the electric field strength overcomes surface tension of the droplet, the droplet travels to the collector. During flying, the polymer solution is drawn in the electric field and the solvent evaporates, subsequently, a nonwoven fibrous mat is formed on the targeted surface. Most tissue engineering applications required for three-dimensional (3D) scaffolds with sufficient interconnected pores to allow cells to penetrate, facilitate formation of uniform tissue and transport mass. The goal could be achieved via random arrangement of fibers. Conventional electrospinning technologies generated flat fibrous composed of nanofibers randomly oriented in 105 In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


two dimensions (2D) (5). Without allowing penetration of cells into interior of the scaffold, the 2D electrospinning mats could only be developed into flat shapes instead of stereo spheroid shapes as in most native ECMs. To fabricate ultrafine fibrous structures with 3D organization, conventional electrospinning methods have been modified via multiple approaches. Porogens, such as ice and salt crystals, have been incorporated to increase bulkiness of electrospun ultrafine fibrous scaffolds (6). Wet electrospinning was developed using a coagulation bath instead of a collecting board to receive fluffy fibers (7). Using porous structures as receptors for electrospinning was a facile approach to obtain 3D ultrafine fibrous scaffolds. Micro-fibrous scaffolds were usually used to collect electrospun fibers from various materials. In addition, to fabricate special tubular scaffolds, such as vascular prostheses, rotating drums with desired diameter were used instead of collecting boards (8). All the methods have their own merits and drawbacks. These methods shared a disadvantage of retained the 2D alignment of the conventional 2D electrospinning. Presence of coarse fiber frames, porogens and wet bath only enlarged the distance among deposited fibers, but did not change the alignment of the fibers. With higher volume of voids inside, the cells could penetrate more deeply. However, orientation of attached cells determined by the alignment of the fibers would not be changed. Stacking of cells oriented in planar directions could be a potential issue in regeneration of thick 3D tissues or organs, such as liver. Adjusting the surface charge of spinning solution was another newly emerging approach to develop scaffolds composed of 3D randomly oriented ultrafine fibers (9, 10). Figure 3 shows that plant protein, zein and synthetic polymer polyethylene glycol could be electrospun 3D cotton ball-like structures on the left sides of Figure 3(a) and 3(b). The conventional 2D electrospun scaffolds with the same mass are shown on the right sides of Figure 3(a) and 3(b). Therefore, porosity and accessible area for cells in the 3D scaffolds could be remarkably increased.

Figure 3. (a) Electrospun zein fibrous scaffolds, left: 3D; right: 2D; (b) electrospun Poly ethylene glycol, left: 3D; right: 2D. Reproduced with permission from reference (9). Copyright (2013) The American Chemical Society. 106 In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Biopolymers for Tissue Engineering Materials for development of tissue engineering scaffolds should be biocompatible and biodegradable, as they usually perform roles as temporary therapeutic substrates to shelter cells. In long term, the neo-tissues are expected to restore physiological functions after being synthesized by the cells cultured on scaffolds. During neo-tissue regeneration, scaffolds, the artificial tissues, should degrade gradually to give room to the newly formed tissues. Proteins, polysaccharides and degradable synthesized polymers are the major biomaterials for fabrication of tissue engineering scaffolds. Proteins have been employed to develop tissue engineering scaffolds for their biocompatibility, biodegradability, versatility in loading therapeutic agents and provide unique affinity to cells. The basic units of proteins are similar to that of collagen, the major component in natural ECMs. Proteins could be easily degraded into short peptides or amino acids, which are building blocks for native proteins in body and could be reused or metabolized in physiological environments. Hydrophilic protein scaffolds are preferred to hydrophobic ones for cell attachment (11). In addition, with tunable surface charges under different pHs, proteins could load substrates with different charges. Meanwhile, the hydrophobic domains in proteins could attract water insoluble substrates via hydrophobic interactions. Some animal proteins, such as collagen and keratin have bio-signaling moieties, such as tripeptide, Arg-Gly-Asp (RGD) in the molecules to promote cell adhesion (12–14). Proteins have been extensively electrospun into 2D fibrous structures for tissue engineering (15), and are attracting attention in 3D electrospinning (10). Polysaccharides, polymers of monosaccharides in multiple combinations, have several merits, i.e. hygroscopicity, biocompatibility and non-toxicity for biomaterial applications. Polysaccharides have diverse origins, chemical structures, molecular weights and ionic characters, leading to different processability, stability and degradability. In addition, some polysaccharides, like hyaluronic acid existed in special tissues, like cartilages, and therefore showed intrinsic advantages in developing engineering scaffolds for these tissues. The widely used polysaccharides, i.e., chitosan, hyaluronic acid and alginate will be introduced in terms of processing methods and properties of products in this chapter. A plethora of synthetic biopolymers, including polycaprolactone (PCL), polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA), have been investigated and used as scaffold matrices (16). These polymers are generally biodegradable and biocompatible. Featured with highly repeated molecular compositions and structures, scaffolds from these bio-based synthetic polymers usually have highly predictable and reproducible chemical, physical and biological properties. Easy processability makes these synthetic biopolymers favorable in scaffold design. With mechanical properties superior to most natural polymers, synthetic biopolymers are especially advantageous in constructing scaffolds to repair high load-bearing defects. Degradation rates of synthetic polymers can be regulated to match tissue growth into neo-ECMs. Moreover, a variety 107 In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

of copolymers, polymer blends, biologically and chemically functionalized polymers significantly promoted broader applications of these synthetic polymers.

Proteins Animal Proteins


Collagen Collagen is a category of fibrous proteins existing in connective tissues, providing structural integrity and mechanical support to many tissues, such as skin, cartilage, tendons and blood vessels. As biomaterial, collagen has its unique advantages. Collagen has biological cues in the molecules to facilitate cell adhesion (13). Collagen can be degraded by enzymes in body, such as collagenases and serine proteases. However, collagen has issues of adverse immune response and risks of transmitting pathogens even after purification. Furthermore, collagen-based scaffolds usually suffer from poor mechanical properties, difficulty in spinning and fast degradation. Collagen has been extensively electrospun into nanofibers (17), and shown compatibility with a number of cell lines. In Table 1, publications regarding electrospun 3D ultrafine fibers from collagen are summarized. There have been no examples of using sole collagen as scaffolds in Table 1. As shown in Table 1, all the collagen based scaffolds combined two or more materials, usually one biodegradable synthetic polymer, such as polycaprolactone (PCL). Collagen was added to promote the initial attachment of cells. However, as collagen degraded or dissolved, the affinity to cells of the whole scaffolds might be reduced. The poor water stability and mechanical properties of collagen-based materials could be the critical reason. Solvent for collagen dissolution could be one cause, and usually crosslinking was used to compensate. Careful selection of solvent could be critical for collagen regeneration. Native collagen fibrils in physiological conditions are water stable and mechanically robust. However, regenerated collagen fibers usually were not water stable probably due to the destroyed triple helical configuration during dissolution. 1,1,1,3,3,3-hexaflouro-2-propanol (HFIP) was the most widely used solvent for collagen. However, due to its corrosive nature, HFIP introduced apparent loss of the natural triple helical configuration, increased the solubility of regenerated collagen in physiological environments, and thus decreased the water stability of collagen based fibers (18). To substitute HFIP, acetic acid has been used to dissolve collagens, but still caused 70% decrease in the percentage of triple helical configuration of collagen. In addition, a water/alcohol/salt solvent system has been developed to dissolve collagen (19). But the salt concentration as high as 50% impaired the protein fibers, indicated by decrease in strength and water stability. Aqueous ethanol solution was found to be a more non-corrosive and non-toxic solvent system that could more efficiently preserve the triple helical configuration of collagen (20). 108 In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

Table 1. 3D fibrous scaffolds from collagen

109


Material

Solvent

Crosslinker

Diameter (nm)

Method

Cell culture

Reference

Collagen I

Acetic acid

EDC/NHS

1200 ± 210

Coarse fiber incorporation

BMSCs

(23)

Collagen I

HFIP

EDC

160


Osteoblasts

(24)

Collagen II

HFIP

GTA

5200 ± 700


BMSCs

(25)

Collagen I

HFIP

N/A

350 ± 85


Osteoblasts

(26)

Collagen I/PCL/HA/PEO

HFIP

N/A

1000

PEO removed as sacrificial substrates

BMSCs

(27)

Collagen I/PCL/HA

Acetic acid

N/A

N/A

NaCl as porogens

Osteoblasts

(28)

Collagen I/PCL/PEO

HFIP

N/A

250 ± 73

PEO removed as sacrificial substrates

In vivo study using rats

(29)

EDC: 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide; NHS: N-hydroxysuccinimide; BMSCs: Bone marrow-derived mesenchymal stem cells; HFIP: 1,1,1,3,3,3 hexafluoro-2-propanol; GTA: Glutaraldehyde; N/A: Not Applicable; PCL: polycaprolactone; PEO: Polyethylene oxide.

In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Crosslinking was usually needed to improve water stability and performance properties of collagen fibers. Glutaraldehyde (GTA) was the primary crosslinking agent for proteins to enhance the mechanical properties and water stability (21). However, the cytotoxicity and calcification effect on protein materials rendered GTA not an optimal crosslinker for collagen targeting biomedical applications. Recently, non-toxic crosslinkers, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and genipin, have been applied to crosslink protein biomaterials (22). However, the crosslinking efficiency of these crosslinkers could not endow adequate water stability, as the crosslinked proteins still easily collapsed into films in high humidity or aqueous conditions. Citric acid with glycerol as extender has also been used to crosslink electrospun collagen nanofibers (20).

Silk Fibroin Silk fibroin is the insoluble protein secreted by silk worms. It has been used as surgical sutures for decades due to its outstanding mechanical properties and water stability. Comparing with many other proteins, silk fibroin has higher percentage of crystalline formed by anti-parallel sheets of hydrophobic peptide chains (30). The highly stable configuration of silk fibroin is due to the strong hydrophobic interaction among the polypeptides, which primarily consist of glycine (Gly) (43%), alanine (Ala) (30%) and serine (Ser) (12%) (31). All the three amino acids have small side groups, facilitating tight packing of polypeptides to form crystals. Most regenerated fibroin fibers in nano- or micro-scales, showed good mechanical behaviors due to easy formation of beta sheet of polypeptides (32). Silk fibroin showed promising potential to support cell adhesion, proliferation, growth and differentiation to promoting neo-tissue formation. Moreover, silk fibroin scaffolds were preferred in regenerating load-bearing tissues, such as ligament (33), cartilage (34) and bone (35) tissue engineering due to its superb mechanical properties and slow degradation rate. Table 2 demonstrates a few examples of nano- and micro-scale silk fibroin fibers in 3D dimensions produced via electrospinning methods. Strong solvents, such as HFIP and trifluoro acetic acid (TFA) were used to dissolve silk fibroin. One example of using water as solvent for electrospinning produced micro-scale fibers, indicating worse spinnability of fibroin in water than in HFIP and TFA (36). Fibroin fibers with diameter on micro-scale were also generated by electrospinning fibroin dissolved in 9.3 M LiBr (37). The precipitation of salts and poor solubility of fibroin in water could also be the cause. Methanol bath, microfiber template and small-diameter mandrel were used to induce formation of 3D structures.

110 In Lightweight Materials from Biopolymers and Biofibers; Yu, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

Table 2. 3D Fibrous scaffolds from silk fibroin

111


Material

Solvent

Diameter (nm)

Method

Cell culture

Reference

Silk fibroin

CaCl2 /water/ethanol

100-400

Methanol bath

N/A

(38)

Silk fibroin/PEO

9.3 M LiBr

3D Electrospun Fibrous Structures from Biopolymers - ACS Publications

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