The potential of cellulose nanocrystals in tissue engineering strategies.

Subscriber access provided by UNIV OF CONNECTICUT

Review

The potential of Cellulose Nanocrystals in Tissue Engineering strategies Rui M. A. Domingues, Manuela E. Gomes, and Rui L. Reis Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 10 Jun 2014 Downloaded from http://pubs.acs.org on June 24, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The potential of Cellulose Nanocrystals in Tissue Engineering strategies AUTHOR NAMES: Rui M. A. Domingues, Manuela E. Gomes*, Rui L. Reis AUTHOR ADDRESS: 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Indústrial da Gandra, 4806-909 Caldas das Taipas, Guimarães, Portugal ICVS/3B's—PT Government Associate Laboratory, Braga/Guimarães, Portugal

ABSTRACT Cellulose nanocrystals (CNCs) are a renewable nanosized raw material which is drawing a tremendous level of attention from the materials community. These rod-shaped nanocrystals that can be produced from a variety of highly available and renewable cellulose-rich sources are endowed with exceptional physicochemical properties which have promoted their intensive exploration as building blocks for the design of a broad range of new materials in the past few decades. However, only recently these nanosized substrates are being considered for bioapplications following the knowledge on their low toxicity and ecotoxicological risk. This review provides an overview on the recent developments on CNCs-based functional biomaterials with potential for tissue engineering (TE) applications, focusing on 1 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 60

nanocomposites obtained through different processing technologies usually employed in the fabrication of TE scaffolds into various formats, namely, dense films and membranes, hierarchical three-dimensional (3D) porous constructs (micro/nanofibers mats, foams and sponges), and hydrogels. Finally, while highlighting the major achievements and potential of the reviewed work on cellulose nanocrystals, alternative applications for some of the developed materials are provided and topics for future research to extend the use of CNCs-based materials in the scope of the TE field are identified.

KEYWORDS Cellulose nanocrystals, nanowhiskers, tissue engineering, nanocomposites, polymers

1. Introduction Cellulose is one of the most ubiquitous and abundant biopolymers produced in the biosphere and it is considered as a virtual inexhaustible source of raw material meeting the increasing demand for green and biocompatible products 1. It has been traditionally used in the form of wood and plant fibers as an energy source, for building materials, paper, textiles and clothing 2. Wood, the main raw material of pulp and paper industry, has been the most commercially exploited source of cellulose. However, other plants also contain a large amount of this polysaccharide, including hemp, flax, jute, ramie and cotton 3. Besides plants, certain bacteria, algae, fungi and several marine animals (e.g., tunicates) also produce cellulose 3. Cellulose is a long-chain polysaccharide composed of D-glucose with a disaccharide repeat unit called cellobiose, which consists of two glucose residues linked via a β-1,4 glycosidic bond 4. In the plant cell wall, cellulose molecule chains connect with each other through hydrogen bonds and van der Waals

2 ACS Paragon Plus Environment

Page 3 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

forces, thus forming elementary fibrils, which are packed into larger microfibrils 5-50 nm in 2

diameter and several micrometers in length, which are in turn assembled into fibers

. These

microfibrils presents highly ordered (crystalline) regions alternating with disordered (amorphous) regions 5

. When subjected to the proper combination of mechanical, chemical and/or enzymatic treatments 1, these

highly crystalline regions contained within the cellulose microfibrils can be extracted, resulting in socalled cellulose nanocrystals (CNCs) 5. CNCs are also often referred to as whiskers, nanowhiskers, nanoparticles, nanofibers, microcrystals or microcrystallites, despite their nanoscale dimensions (at least one dimension). However, in this review they are all termed cellulose nanocrystals provided that they are obtained through a “top down” deconstruction of cellulose fibers. For a more comprehensive and detailed cellulose particles classification, the readers are referred to the review by Moon et al. 2. The most simple and usually employed process to produce these CNCs involves an acid hydrolysis of cellulose with concentrated sulfuric acid, resulting in the characteristic rod-shaped nanofibers bearing anionic sulfate ester groups at their surface (Figure 1A) 1. They have been isolated from a vast number of different cellulose sources (Figure 1B) which include, for example, wood pulp wheat

12

and rice

13

straw, microcrystalline cellulose (MCC)

14, 15

6, 7

, cotton

, bacterial cellulose

16, 17

8-10

, ramie 11,

, algae

18, 19

and

tunicates 20-23.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 60

Figure 1. A) Schematics of idealized cellulose fibers showing one of the suggested configurations of the crystalline and amorphous regions, and cellulose nanocrystals after sulfuric acid hydrolysis of the disordered regions, exhibiting the characteristic sulfate half ester surface groups formed as a side reaction. (Adapted from ref. 2. Copyright 2011, with permission of The Royal Society of Chemistry. http://dx.doi.org/10.1039/c0cs00108b); B) TEM micrographs of dispersion of cellulose nanocrystals derived from different sources: b1) microcrystalline cellulose (Avicel), b2) tunicate

23

, b3) green algae

(Cladophora sp.) 18, and b4) ramie 11. Reproduced from ref. 11. Copyright 2008, with permission of The Royal Society of Chemistry. http://dx.doi.org/10.1039/b809212e. Reprinted with permission from ref. 18 and 23. Copyrights 2012 and 2008 American Chemical Society.


Page 5 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Due to their unique features, CNCs have garnered a tremendous level of attention in the material community, which can be confirmed by the increasing number of scientific publications in the field over the past decades. Besides their nanoscale dimensions, typically of 2–50 nm in width and 100–2000 nm in length

5, 24

, this cellulose elementary “building blocks” have excellent mechanical properties: the axial

elastic modulus of native CNCs ranges from 110 to 220 GPa, while its tensile strength is in range of 7.5 7.7 GPa

2, 25-28

. These outstanding mechanical properties are within the range of other reinforcement

nanomaterials, such as clay nanoplatelets or carbon nanotubes 2. Its reactive surface covered with numerous hydroxyl groups, enables different chemical modifications of CNCs, such as oxidation, esterification, etherification, silylation or polymer grafting

5, 25, 29

. This vast panoply of possible

modifications also allows their effective incorporation and dispersion into different polymer matrices (both water-soluble and water-insoluble)

5, 25, 29, 30

rendering CNCs the status of an ideal candidate as a

reinforcement nanofiller material. CNCs also have other key properties such as low density, high aspect ratio and high surface area which greatly contribute to their attractiveness in the development of promising novel functional nanomaterials. Moreover, CNCs have proven excellent biodegradability (cellulose nanoparticles were found to degrade faster than their macroscopic counterpart)

31

and have low ecotoxicological risk 32. From a toxicological

point of view, cellulose-based materials have been generally showing excellent biocompatibility on a bulk basis 33, 34 and, CNCs in particular, exhibit low cytotoxicity to a range of animal and human cell types 32, 35-39

. Although some studies have recently suggested that cytotoxic responses can occur at high CNCs

concentration

40, 41

, those are still significantly lower than the ones found for other nanofibers such as

multiwalled carbon nanotubes and crocidolite asbestos

41

, providing impetus for their use in

bioapplications. This set of characteristics and the growing interest in the bioconversion of renewable lignocellulosic biomass rendered to CNCs a substantial interest directed toward their application in various fields such as high performance materials, electronics, catalysis, biomedical, and energy

1, 4, 5, 30, 42-44

. Several reviews 5

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 60

have been published in the last decade covering the various aspects related to CNCs, including processing, chemical modification of surfaces, CNCs-containing nanocomposites and self-assembly of suspensions 1-5, 24, 25, 29, 30, 43, 45-52. One of the main drawbacks concerning the use of CNCs in commercial applications is related to their efficient production at affordable quantity and quality

44, 53

. However, the first demonstration plants for

the production of CNCs at a commercial scale are currently under operation in Canada and U.S.A., and its mass production is expected to rise to multiple tons per day in the near future 54, giving a strong boost for application in composites at industrial scale. Although research on CNCs have experienced a tremendous increase on a variety of fields over the last two decades 5, only recently it has attracted the interest of researchers in the field of tissue engineering (TE) and regenerative medicine, both as scaffolds or as drug-delivery systems. In fact, most of the studies addressing this topic have been published during the last 5 years, indicating that within the TE field, CNCs have a large unexploited potential despite their many well-known attractive properties and possible applications in this context, as will be discussed later in this review. Recently, Ning et al.

25

included in their review on polysaccharides nanocrystals for advanced functional

nanomaterials an overview of some works dealing with the application of CNCs in the biomedical field, as drug carriers and nanoscaffolds. Dugan et al. 55 reviewed the literature on bacterial cellulose scaffolds, which is probably the most studied cellulose nanomaterials for TE, and discussed in detail the studies on the biocompatibility of CNCs, addressing its potential in this field. However, they restricted their discussion to studies that demonstrated proof of concept using specific biological in vitro or in vivo models. More recently, an extensive review from our research group on bionanocomposites from lignocellulosic resources 53, discussing the properties, applications and future trends for their use in the biomedical field, also addressed, to some extent, the theme of CNCs on the scope of TE.


Page 7 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

In the light of the latest advances and the growing number of studies on the use of CNCs alone or in nanocomposites aiming at potential applications in TE strategies, here we comprehensively review the recent research developments employing these outstanding natural nanoparticles for the fabrication of scaffolds and drug-delivery systems in the scope of TE. A concise introduction to the principles and strategies of TE is provided in order to highlight the potential of CNCs within this area. The literature on nanocomposites based on both natural and synthetic origins polymer matrices relevant for this field is reviewed. Particular emphasis is given to functional materials obtained through different fabrication strategies usually employed to obtain scaffolds with various formats, namely, dense films and membranes (mostly considered as a prequel for three-dimensional (3D) scaffolds development), porous constructs (micro/nanofibers mats, foams and sponges), and hydrogels (Figure 2). Finally, in the last section we highlight the major achievements of the published work and envisage potential future perspectives for the use of CNCs within the field of TE.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 60

Figure 2: Summary of the current utilization of CNCs on nanocomposites processing for tissue

engineering applications.

2. Introduction to principles and strategies of Regenerative Medicine and Tissue Engineering In vivo, cells interact with biochemical and biophysical cues within their surrounding microenvironment and such interactions collectively regulate cell behavior, function, and fate 56. The cell microenvironment comprises the extracellular matrix (ECM) proteins, soluble and sequestered bioactive factors, and neighboring cells (Figure 3). Understanding and interrelating this bidirectional cross talk between the microenvironment and resident cells is vital for developing strategies to regenerate tissues 57. 8 ACS Paragon Plus Environment

Page 9 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 3: Schematic of the extracellular matrix. Fibrous matrix proteins (e.g., collagen, fibrin, elastin) provide structural and mechanical cues to direct cell behavior; soluble signals are sequestered by proteoglycans (proteins with polysaccharide moieties) and interact with cell surface receptors to direct cell migration, proliferation, and differentiation; integrins (transmembrane receptors) bind to matrix proteins for cell adhesion; ECM degradation enzymes (e.g., matrix metalloproteinases, serine proteinases, plasmin) cleave matrix components during cell motility and matrix remodeling 58. Reprinted from ref. 58, Copyright 2012, with permission from Elsevier.

One of the simplest acellular approaches in Regenerative Medicine consists on the implantation of a scaffold device that has the intrinsic ability to actively promote the body's inherent capacity of healing and self-repair. The scaffold materials should have the ability to mimic the function of the ECM in order to stimulate cellular invasion, attachment and proliferation, thus rendering the lost tissue regeneration 59. In the current standard Tissue Engineering approach, the cells (particularly stem cells, either autologous or allogeneic) are cultured onto biodegradable and bioactive scaffolds that recapitulate ECM in order to guide their attachment, migration, proliferation and differentiation to produce 3D tissues substitutes for implantation 59. 9 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 60

The ECM generally exhibits a hierarchical organization from nano to macro scale structures 60. It differs in composition and spatial organization of their components (collagens, elastins, proteoglycans and adhesion molecules) in the various tissues of the body, naturally assembled to maintain specific tissue morphologies and to provide specific instructive cues for the cells of the different organs 60. TE scaffolds present several features that can affect their performance. Therefore, the considerations behind the design of a scaffold should be adapted to the targeted tissue. It is generally acknowledged that scaffolds’ surface properties such as roughness, topography and chemistry all play a pivotal role in specific cells behavior

57, 61-63

. Biomimetic features can also be incorporated into advanced constructs

design by the modification of 3D morphological properties. It has been suggested that scaffolds’ porosity, pore size, pore interconnectivity and surface area to volume ratio are key aspects that should be considered in their design as these have a known impact over, e.g. tissue in-growth, vascularization and nutrient supply

64-67

. Furthermore, besides providing the physical and biochemical cues recapitulating

those found in the native tissue environment, all these features should be combined into the scaffolds design based on materials that are absorbable and are subjected to degradation rates matching those of tissue formation, until the injured tissue is completely replaced by healthy tissue and its functionality is restored 64, 68. Based on these considerations, the selection of the material and the individual preparation technology depends on the requirements of the desired application and confers unique properties to the scaffold 68. One of the most critical issues in TE is thus the fabrication of scaffolds with specific physical, mechanical and biological properties. In terms of mechanical properties, the scaffolds should present similar performance to the tissue to be repaired, since in such case they would enable a better mechanical integration of the constructs in the biological tissue 69. Moreover, mechanical signals are well-known to influence cells’ fate 70, and growing importance is being given to the role of mechanotransduction during cell expansion and differentiation 71,


Page 11 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

72

. Thus, the accurate modulation of these properties is a very challenging and a desired goal because

biological tissues show a large spectrum of mechanical properties, depending on their anatomical function: many tissues are viscoelastic with non-linear, anisotropic, and heterogeneous mechanical properties 73. Numerous materials (synthetic and/or from natural origin) and fabrication methods have been explored enabling architectural scaffold designs with defined macro-, micro- and nanostructure length scales for different TE applications 74, 75. Many of the fabrication strategies have been based on biodegradable polymer matrices, including both natural-based and the synthetic polymers 67. In the natural-based category, polysaccharides (e.g., starch, carrageenan, alginate, chitin/chitosan, gellan gum, hyaluronic acid and chondroitin sulfate), proteins (e.g., collagen, gelatin, silk fibroin, elastin and fibrin), natural biopolyesters such as polyhydoxyalkanoates (PHA) and, as reinforcement, a variety of biofibers such as lignocellulosic natural fibers, have been applied

53, 75, 76

. Several chemically modified cellulose derivatives have been also proposed for TE

applications, such as, for example, cellulose acetate (CA)

33

and cellulose acetate propionate (CAP)

hydroxypropylcellulose (HPC) 78 or carboxymethyl cellulose (CMC)

79

77

,

. These naturally derived

components are commonly employed in the synthesis of materials for cell culture and scaffolds design owing to their inherent bioactivity, biocompatibility, and general biodegradability

75

.

Concerning

synthetic polymers, the aliphatic polyesters poly(L-lactide) (PLLA), poly(ɛ-caprolactone) (PCL), poly(glycolide) (PGA), poly[D,L-(lactide-co-glycolide)] (PLGA) have been widely used materials for TE scaffolds due to their excellent biocompatibility, biodegradability and reasonable mechanical properties 67, 80

. Other synthetic biocompatible polymers such as, for example, poly(ethylene glycol) (PEG), poly(vinyl

alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAm), poly(acrylic acid) (PAA) and its derivatives (such as poly(2-hydroxyethyl methacrylate) (PHEMA)) are also extensively used in TE 81.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 60

In terms of processing, a variety of techniques have been reported in the literature for scaffold fabrication. 3D structures or particulate systems are routinely obtained by conventional processes such as foaming, freeze-casting, freeze-drying-particle leaching, solvent casting-particle leaching, thermally induced phase separation, compression molding, injection molding, rapid prototyping, extrusion, wet-spinning and electrospinning, self-assembly, among others 82-84. Although considerable advances have been achieved along the years, it is hardly conceivable that conventional single-component polymer materials can meet all the requirements for TE scaffolds 80. In order to better mimic the ECM hierarchical structure, during the last few decades many of the fabrication strategies have been directed toward nanocomposites and nanotopography-guided approaches due to the matching of the length scales of their structure and the components of the ECM. Several excellent reviews covering these topics can be found in recent literature 60, 80, 85-88. From a conceptual point of view, the rationale behind the incorporation of nanostructures in the scaffold matrix is to compensate for other limitations such as, for instance, weak mechanical properties, lack of electrical conductivity, the absence of adhesive and microenvironment-defining moieties, or the inability to enable cells to self-assemble to 3D tissues 86. Several inorganic and organic nanofillers have been used in combination with polymer matrices for the fabrication of nanocomposite scaffolds aimed at regeneration of different tissues. For example, hydroxyapatite (HA) has been widely used as a biocompatible ceramic material for contact with bone tissue 89; carbon nanotubes have been used for the enhancement of weak mechanical properties and the increase in conductivity of polymers matrices; metal nanoparticles (e.g., silver, gold and iron oxide) have been applied for materials endowed with antibacterial properties , for the controlled localization of biomolecules or to engineer complex tissues composed of several cell types 80, 86, 90. Most of the nanofillers used to prepare nanocomposites are inorganic or carbon based (e.g. metal nanoparticles, carbon nanotubes or nanoceramic materials such as bioactive glasses, hydroxyapatite or β12 ACS Paragon Plus Environment

Page 13 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

tricalcium phosphate

91, 92

). However, despite their enormous potential in biomedicine, there are several

important challenges and issues that remain to be addressed such as their processability, biocompatibility and biodegradability. Such features are referred to be much more limited than those of organic nature 80. Although this is a very controversial theme, just to mention an example, in recent years several authors have reported possible negative effects of carbon nanotubes on cells and their potential to induce oxidative stress, inflammation, genetic damage and long-term pathological effects

93

. These

considerations are not intended to generalize safety concerns for all inorganic or carbon based nanomaterial, neither exclude organic nanoparticles from possible adverse effects in living organisms. It rather emphasizes the need for a continued work in the field of nanotoxicology in order to better understand how the physicochemical properties of nanoparticles affect biological systems and thus better evaluate their safety in potential biomedical applications. As an alternative among potential nanofillers to prepare nanostructured polymeric materials, CNCs are emerging as reasonable candidates for TE applications. Their referred biocompatibility, potential low cost, renewable raw material nature and hydrophilicity suggest that CNCs could be an ideal material for the fabrication of TE scaffolds. The issues of biodegradability and cytotoxicity of CNCs are particularly relevant for their potential use in this field and it has been recently reviewed be Dugan et al 55. Although recognizing that the number of in vitro or in vivo studies available for evaluating the potential hazards of CNCs when in contact with living cells is limited, these nanocrystals are generally being considered nontoxic or to elicit only low cytotoxicity against various cell lines in concentration up to 250 µg/mL 35, 36, 38, 94

. This behavior is also found to vary with their dimensions and surface chemistry properties

(particularly surface charge density) which depend on the cellulose source and preparation method. In a recent study however, high CNCs concentrations (2000 and 5000 µg/ml) caused decreased cell viability and affected the expression of stress- and apoptosis-associated molecular markers of fibroblasts cultured in vitro

40

. Also, while in a 3D in vitro model simulating the human epithelial airway barrier,

CNCs exhibited lower cytotoxicity and (pro-)inflammatory response than multiwalled carbon nanotubes


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 60

and crocidolite asbestos fibers 41, a recent in vivo study demonstrated that different forms of CNCs elicit dose-dependent oxidative stress, tissue damage, and robust inflammatory responses in mice lungs, which was found more prominent than those triggered by crocidolite asbestos 95. The data available suggest that, like for any other nanomaterial, the current understanding of how CNCs physicochemical and morphological properties affect living cells is very limited, thus, despite some encouraging result on their low toxicological effects, research on this field should continue along with the development of new biomaterials incorporating these nanoparticles. The in vivo degradation and fate of CNCs and its derivatives is also currently unknown, being an issue to consider in developing TE scaffolds. In general, cellulose does not degrade or undergo slow degradation in vivo although the form of cellulose may affect these mechanisms and the introduction of modifications such as aldehyde groups on the cellulose chain may improve its decomposition (for more details consult 44, 55). Notwithstanding, CNCs have low extension to break, high aspect ratios, high surface areas and high crystallinity, which make them excellent candidates as load-bearing components for biomedical applications comparatively to the other non-biocompatible reinforcing nanofillers

96, 97

. Furthermore, the

high surface area-to-volume ratio of the CNCs combined with their susceptibility to relative orientation by mechanical forces (shearing)

98

or when exposed to external magnetic

99

or electric

100

fields, constitute very important properties not only for the improvement and anisotropic modulation of the nanocomposites mechanical performances, but can also provide nanostructured cues that can have an impact over cells behavior, namely in the mechanisms of adhesion, proliferation, migration, and differentiation, which are highly relevant in the context of TE strategies. In fact, Dugan et al. have firstly demonstrated the potential of CNCs to provide nanoscale structural cues for TE in 2010 101. In this pioneering study, the authors prepared radially oriented submonolayer surfaces of high aspect ratio CNCs from tunicate by spin-coating onto flat glass substrates. It was shown that, despite the small size of CNCs, the myoblasts oriented along the CNCs surfaces and fused to form highlyoriented multinuclear myotubes, disclosing how this straightforward nanopatterning method may be 14 ACS Paragon Plus Environment

Page 15 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

applied for the oriented growth of tissues 101. More recently, the same group further explored this concept using CNCs extracted from Ascidiella aspersa, which have particularly small diameters of only a few nanometres (mean height of only 5–6 nm) 102. The CNCs nanopatterned surfaces induced the adoption of similar oriented morphologies by C2C12 myoblasts, enhancing the degree of cellular fusion and serving as template for the deposition of an oriented fibrillar ECM (Figure 4) 102. According to the authors, these CNCs surfaces present some of the smallest features ever demonstrated to induce contact guidance in mammalian cells (several orders of magnitude smaller than myoblast cells)

102

, opening the way for the

potential application of these bionanoparticles for engineering highly ordered tissues such as, e.g. skeletal muscle (as tested) or tendons.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 60

Figure 4. Fluorescence micrographs of C2C12s stained for MHC (green), fibronectin (red) and nuclei (blue) on C-500-12 and C-6000-12 surfaces, 1 and 4 days after seeding. The arrow indicates the approximate radial axis. (a) C-500-12, 1 day; (b) C-6000-12, 1 day; (c) C-500-12, 4 days; (d) C-6000-12, 4 days; (e) C-500-12, 4 days, high-magnification zoom of boxed region in (c); (f) C-6000-12, 4 days, high-magnification zoom of boxed region in (d). Scale bars = 50 μm (a-d) or 20 μm (e and f)

102

.

Reprinted from ref. 102, Copyright 2012, with permission from Elsevier.

In the following sections we will explore recent literature dealing with the use of CNCs in combination with other polymer matrices, thus resulting in nanocomposite materials with promising applications in the TE field.

3. CNCs nanocomposites for TE 3.1 Dense films and membranes When developing a scaffold material as substrate for cell culture its properties must be properly characterized and fully-optimized. Usually, the first approach involves the preparation of dense films as a prequel to 3D scaffold development, since it facilitates the introduction of single variables with the purpose of unveiling their impact on cell behavior

80

. These 2D structures are also relevant in some

particular applications, e.g. in skin TE where films can present some advantages over highly porous materials

103

, as complements of some specific TE scaffolds or as films that prevent tissue adhesions in

surgeries involving soft tissue and regenerative medicine, for example of blood vessels 104, 105. A variety of processing techniques have been studied to fabricate polymer nanocomposite films and membranes containing CNCs as nanofillers, solvent casting being the preferred strategy. Nonetheless, layer-by-layer (LBL) assembly and in situ polymerization have also been used. 16 ACS Paragon Plus Environment

Page 17 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Generally, the interfacial adhesion between CNCs and a polymer matrix is one of the most important factors affecting nanocomposite properties. In order to enhance the dispersion of CNCs and increase the interfacial strength between the two phases, various methods have been attempted, including magnetic field alignment within the matrix modifications

110, 111

106

, use of plasticizers

107

and surfactants

108, 109

, or through surface

. A quite extensive range of polymer matrices, both synthetic and natural based, has

been used to produce nanocomposite films and membranes containing CNCs, which will be described in the following sections.

3.1.1 Natural-based polymeric systems Taking advantage of the excellent properties of cellulose and its derivatives, i.e. good biocompatibility, controlled biodegradability and non-acidic byproducts 53, Pooyan et al. have designed a fully cellulosebased nanohybrid material in which CNCs were dispersed within a CAP matrix to form a 3D rigid percolating network, aimed at fabricating a potential scaffold candidate in TE of small diameter vascular grafts

112

. CNCs extracted from microcrystalline cellulose were suspended in acetone prior to the

composite fabrication in order to ensure a uniform distribution. Thin films were produced by solvent casting and, according to the authors, the obtained bionanocomposites exhibited excellent mechanical performance at body temperature, claiming that CNCs impart significant strength and directional rigidity even at 0.2 wt.% (about twofold higher compared to CAP) and almost double that at only 3.0 wt.%. In a following study, these authors explored the same concept but aligned the CNCs embedded within the CAP matrix by applying a relatively weak external magnetic field (0.3 T) 106. The alignment of the CNCs substantially enhanced the mechanical and thermal properties of the nanocomposites up to 3 wt.% nanofiller concentration. The authors hypothesized that the magnetic alignment of the nanoparticles could be advantageous in different aspects, such as in promoting favorable filler/filler interactions, enhancing their dispersion within the matrix and providing a better filler/matrix interfacial contact. From the


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 60

microstructural point of view, the composites exhibited a relatively controlled porous structure (average pore diameter of 3 µm and a void fraction of ∼13%, for a 0.2 wt.% CNCs loading), which could be potentially helpful for cell seeding and proliferation because it would allow diffusion of fluids and gases deep into the material

106

. Furthermore, the alignment of the CNCs also confers an oriented microporous

nanostructure to the system that could potentially induce contact guidance to cells in the direction of the aligned CNCs 106. Although not specifically aimed at TE applications, other works also thoroughly investigated the influence of CNCs alignment on the mechanical properties of all-cellulose nanocomposites prepared by solvent casting, either through the application of a magnetic field

113

or using a different wet-stretching

approach 114. In terms of processing, the wet-stretching approach may be an interesting route of achieving significant levels of CNCs orientation within all-cellulose nanocomposites processed in water based systems. Besides CNCs alignment, the cellulose molecular chains from the matrix phase also showed high levels of orientation after stretching, thus significantly contributing for the enhancement of the mechanical properties of the resulting nanocomposites 114. However, it was also proven that the Young’s modulus and strength of the composites decrease dramatically when the material is wetted 114, something that should be considered when developing all-cellulose nanocomposites for potential TE applications. Proteins, such as collagen and silk fibroin, are widely used in the fabrication of TE scaffolds because they can closely mimic the composition of ECM

76, 115

. However, the poor mechanical properties of collagen

and the brittleness and reduced flexibility of as-prepared silk fibroin films from regenerated silk fibroin solutions are often referred as limiting properties for their application in TE strategies 76, 116. In order the improve the mechanical properties of collagen, Li et al. have designed collagen-based composite films reinforced with CNCs 117, for potential applications in skin TE. The films were prepared by a solvent casting method and CNCs concentration varied up to 10 wt. % of collagen mass. No aggregates were observed up to 7 wt. % CNCs, thus indicating good dispersion and compatibility between


Page 19 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

the CNCs and collagen matrix. The incorporation of CNCs within collagen-based composite films improved its bulk ordering and stability and led to an increase in the swelling capacity, (twofold improvement over pure collagen films for 7 wt. % CNCs), which is highly desirable for the proposed application, as it enables the absorption of large amount of exudates in the early stages of wound healing. The mechanical properties of the composite films were also significantly improved when compared with those of the pure collagen film. The ultimate tensile strength of the 7 wt. % CNCs film was almost 2.4 fold larger than that of the pure collagen film and was explained by the interaction between the collagen matrix and the CNCs through hydrogen bonding, which permitted stress transfer at the interface between the collagen and the CNCs. Results from in vitro assays with 3T3 fibroblasts cell culture indicated that the composite films showed no obvious cytotoxicity and facilitated cell adhesion, thus displaying excellent biocompatibility 117. LBL assembly was proposed for the preparation of collagen/CNCs multilayered films as an effective method to maximize the interaction between the two structural materials 118. The LBL technique consists in the sequential deposition of complimentary multivalent molecules on a substrate via electrostatic and non-electrostatic interactions 119. This technique has gained considerable interest in TE research because it can be performed under mild conditions with inexpensive materials requiring no exposure to harmful solvents. Moreover it allows a precise nanometer-scale control over the thickness and composition of thin films, which opens the possibility of controlling (at the molecular level) some characteristics of the further attached cells, such as adhesion and proliferation

120

. In the referred work, LBL thin films were

built up by alternate deposition of positively charged collagen and negatively charged CNCs obtained from eucalyptus wood pulp (145 ± 25 nm length and 4.6 ± 0.5 nm height). The average thickness of a single bilayer was about 9.0 nm, formed by thickness increments of 2.7 ± 0.5 nm for the collagen layers and 6.3 ± 1 nm for the CNCs layers. According to the authors, hydrogen bonding between amide groups from collagen and OH groups from CNCs was more important in the LBL construction than the contribution of electrostatic interactions. Relatively smooth surfaces were obtained when ending the LBL


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 60

process with CNCs (roughness ∼ 9.5 nm), showing high density and uniform distribution, indicative of a good interaction between polymers and nanocrystals

118

. This research group had previously reported a

similar strategy to fabricate multilayered films composed of CNCs/chitosan, a biopolymer with tradition in drug delivery, TE and wound healing applications 121, and observed the same morphology in the layers of CNCs

122

. This characteristic is important to improve the mechanical properties of nanocomposites

because it allows maximizing the interaction and the load transfer between the biopolymers and the nanofibers while avoiding nanofillers aggregation in the polymer matrix, thus enabling to obtain materials with high loading of nanofillers 118, 122. Noishiki et al. firstly studied the reinforcement effect of CNCs from tunicate on silk fibroin composites films and evaluated their influence on the higher-order structure of this bioplolymer

123

. The tensile

strength (∼ 160 MPa) and ultimate strain (∼ 4%) of the casted composite films showed a maximum at 70– 80% cellulose content, reaching five times those of fibroin-alone or cellulose-alone films. This effect was considered to arise from the β-structure formation of fibroin induced by contact with the highly ordered surface of CNCs which probably serves as a template in this process. This is a particularly interesting result because the assembly of silk fibroin β-sheets usually requires shearing and elongation stress for proper molecular arrangement

124

. In order to improve the flexibility of the composite films, Li et al.

proposed to use PEG (30 wt. %) as plasticizer in combination with silk fibroin and CNCs (up to 15 wt. %) obtained from mulberry branch barks

107

. The CNCs with diameter of 20 to 40 nm and length of ∼ 500

nm showed a uniform distribution in the matrix for composites with contents as high as 12 wt. %. Flexible and transparent composite films were obtained, with a maximum tensile strength of 36 MPa and strain at break of 10 % for films with 12 wt. % CNCs. This results may be significant in order to overcome some mechanical limitations of silk fibroin films and extend potential applications in TE as cell culture substrates or implantable materials 107, 123. Polyhydoxyalkanoates (PHA) are natural biopolyesters produced by microorganisms under unbalanced growth conditions. Due to their general good biodegradability and biocompatibility, these biomaterials are 20 ACS Paragon Plus Environment

Page 21 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

very attractive for TE, particularly poly 3-hydroxybutyrate (PHB) and its copolymers used in several TE strategies, particularly in hard tissue regeneration

126-128

125

. PHB has been

. However, its use has been

limited mainly due to its brittleness and the narrow processing window. The incorporation of CNCs in a PHB matrix was proposed as a way to increase its elongation at break as well as its thermal properties 109. These bionanocomposites were produced by solvent casting, using chloroform as solvent and low concentrations of CNCs (between 0 and 0.75 wt.%) dispersed in PEG plasticizer to increase the compatibility between CNCs and a PHB matrix. The films showed an effective dispersion of the CNCs within the matrix and an enlargement of the thermal processing window of the nanocomposites, when compared to the neat PHB. In terms of mechanical performance, the nanocomposites showed a 50 fold increase in the strain level without a significant loss of the tensile strength with the incorporation of CNCs up to 0.45 wt.%. The authors hypothesized that this may be due to considerable chain orientation promoted by the presence of CNCs in the same direction of the applied load, which activated shear flow of the polymer matrix 109. Wolcott’s research group has systematically studied the preparation and properties of poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/CNCs composites

129-133

. These studies showed that

PHBV reinforced with homogeneously dispersed CNCs (1-5 wt.% ) could be prepared by solvent casting and exhibited significant improvements in tensile strength, Young's modulus, toughness and dynamic modulus. It has been demonstrated that strong hydrogen bonding interactions occur between carbonyl groups of PHBV and hydroxyl groups in CNCs PHBV crystallization

129

131

. Furthermore, CNCs also played an important role in

. The solvent exchange approach has been followed by others to obtain well-

dispersed CNCs in a good solvent for PHBV and prepare nanocomposites with higher amounts of CNCs (up to 10 wt.%), resulting in simultaneous enhancements of the mechanical properties and thermal stability of this polymer

134

. Different types of CNCs obtained by either sulfuric and hydrochloric acid

hydrolysis also proved to have distinct reinforcing effects on PHBV that was found stronger when using CNCs obtained with hydrochloric acid

135

. This behavior was attributed to a combination of a larger


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 60

aspect ratio, higher crystallinity and no residual acid groups of hydrochloric acid CNCs, with a stronger heterogeneous nucleation effect and the formation of more intermolecular hydrogen bonding interactions arising from a more uniform dispersion within the PHBV matrix, comparatively to CNCs obtained with sulfuric acid 135. CNCs were also aligned in PHBV using an external electric field, which was effective in aligning CNCs up to 4 wt.% loading

133

. The aligned PHBV/CNCs composites showed considerable

mechanical anisotropy, highlighting the potential of this method to prepare nanocomposites with directional reinforcement

133

. More recently, CNCs were grafted with PHBV side chains in order to

improve the dispersion and compatibility of CNCs within PHBV matrix

136

. This approach allowed an

effective increase in the loading levels of well dispersed CNCs up to 20 wt.% which, combined with a good interfacial adhesion between filler and matrix by chain entanglements, cocrystallization, and hydrogen bonding interactions, resulted in superior mechanical performance and thermal stability, comparatively to neat PHBV. These materials exhibited even better cytocompatibility, evaluated with human MG-63 cells, than neat PHBV

136

. In general, these works demonstrate that CNCs can play a

remarkable role in enhancing the performance of these natural polyesters, widening their potential application in TE strategies.

3.1.2 Synthetic-based polymeric systems Besides natural origin polymers, some studies have been reporting the use of CNCs as reinforcements of synthetic polymer matrices, such as PLA 111

137

, poly(vinyl alcohol) (PVA)

138

, poly(vinyl acetate) (PVAc)

, and polyurethane (PU) 110, in the fabrication of nanocomposite films that may find applications in TE.

The preparation of cotton-based CNCs reinforced PLA nanocomposites was carried out by solvent casting in chloroform 137. The incorporation of CNCs (1 to 5 wt.%) within a PLA matrix significantly improved the thermomechanical, crystallisation and degradation properties of the PLA, particularly in the case of nanocomposites containing 1 wt.% CNCs which showed a 34 and 31% increase in tensile strength and 22 ACS Paragon Plus Environment

Page 23 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

modulus, respectively, when compared to pure PLA. The dispersion of the rod-like nanocrystals within the PLA matrix was however limited above 1 wt.% CNCs and seen to be largely aggregated leading to a less pronounced enhancement of the mechanical properties. This is a rather typical observation because the hydrophilic nature of cellulose promotes the irreversible agglomeration and aggregation in nonpolar matrices 53. Attempts to use surfactants to promote the dispersion of CNCs within the PLA matrix have been reported, showing improved dispersion of CNCs in the polymer matrix 108. The surface modification of CNCs, for instance, through silylation 139 or PLA grafting

140, 141

, has also shown to improve their

dispersion on PLA, having an important influence over their nucleation effect and consequently enhancing the nanocomposite mechanical properties. Ternary multifunctional bionanocomposites have been proposed by combining the good biocompatibility and the high mechanical response of PVA/CNC systems with the protein controlled release by polymeric PLGA nanoparticles (NPs)

138

. CNCs were obtained from microcrystalline cellulose and PLGA

nanoparticles were prepared by a double emulsion with subsequent solvent evaporation method. The NPs were loaded with bovine serum albumin fluorescein isothiocynate conjugate (FITC-BSA). In the optimized conditions (0.5 wt. % of CNCs and 0.5 wt.% of PLGA NPs), the thin films prepared by solvent casting in water had a thickness ranging from 200 and 300 µm. CNCs induced an increase of the Young's modulus and of the elongation at break (15% and 57%, respectively, comparatively to PVA matrix), underlining the capability of the lower content of CNCs (0.5 wt.%) to induce a reinforcement effect in both the elastic and plastic regions. Similar results had been previously reported on the reinforcements of PVA films with CNCs obtained from various sources, which were proposed for other applications 142, 143. The effectiveness of the ternary system as suitable vehicle for biopolymeric nanoparticles was tested with adult bone marrow mesenchymal stem cells (BM-MSCs) (Figure 5). The results didn’t show any signs of toxicity in cell cultures, indicating that these nanocomposites do not affect the cellular viability and thus could represent a good device to be used for drug delivery strategies in TE applications 138.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 60

Figure 5. a) XTT assay shows the biocompatibility of PVA, PVA/0.5NPs and PVA/0.5CNC/0.5NPs binary and ternary systems towards BM-MSCs. b) Up-take of NPs released by PVA/0.5NPs binary (a) and PVA/0.5CNC/0.5NPs (b) ternary systems

138

. Reprinted from ref. 138, Copyright 2013, with

permission from Elsevier.


Page 25 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

In a different ternary nanocomposite systems based on PVA matrix, George et al. have combined bacterial CNCs (up to 4 wt. %) and Ag nanoparticles (1 wt. %) as nanofillers

144

. In addition to the

antimicrobial properties imparted by the Ag nanoparticles, a synergistic effect on the improvement of the mechanical properties of the PVA films was obtained by combining these two nanomaterials: while CNCs increased the modulus and tensile strength of the films, the Ag nanoparticles decreased its brittleness. These effects could be useful in the development of multifunctional hybrid materials for TE applications, although the toxicity issues associated to Ag nanoparticles are still an open debate 145. Polyurethanes (PU) are one of the most popular groups of biomaterials applied for medical devices

146

.

Several studies on PU/CNCs nanocomposites have demonstrated the positive effect of these nanofillers on the thermal, mechanical and morphological properties of these systems

110, 147-149

outperforming PU nanocomposites reinforced with other nonofillers, such as nanoclays

147

, in some cases

carbon nanotubes or

. Recently, Rueda et al. synthesized PU/CNC nanocomposites (0.25 to 1 wt.% CNCs) by

means of in situ polymerization and evaluated their biocompatibility

110

. The incorporation of low CNC

contents (up to 0.5 wt.%) led to an increase of both strength and ductility, therefore greatly improving the toughness. The PU/CNC nanocomposites displayed no toxicity toward L-929 fibroblasts, which adhered and massively proliferated on the films surface, thus highlighting the potential of these materials for biomedical applications. In a creative approach, nanocomposite films with water-enhanced mechanical gradient properties mimicking the squid beak have been developed by controlling the degree of cross-linking along the length of the film

111

. Tunicate CNCs functionalized with allyl moieties were embedded within a poly(vinyl

acetate) (PVAc) matrix through a photoinduced crosslinking procedure using a tetrathiol as crosslinker. By controlling the photoirradiation exposure time of different parts of the film, a gradient of the degree of covalent cross-linking was created. As result, a significant mechanical contrast was observed along the film up on exposure to water due to the “switches off” of the noncovalent CNCs interactions. The authors hypothesize that these materials could find applications in soft-to-hard interface TE 150. 25 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 60

3.2 Porous scaffolds 3.2.1 Nano and microfibers mats Electrospinning is a simple and versatile method of preparing ultrathin fibers with diameters ranging from a few micrometers down to a few nanometers through highly-charged polymeric solutions or melts 151, 152. The enormous interest in nonwovens nanofibrous mats produced by electrospinning for TE scaffolds manufacture is related with the ability of these fibrilar systems to better mimic ECM dimensions and structure as compared to other conventional techniques 151. Among the several advantages offered by this technique, one can include the ability to manipulate nanofiber composition, while allowing to tailor scaffolds parameters such as fiber diameter, orientation, and density and to form scaffolds with high porosity as well as high surface area-to-volume ratio 151, 153-155. As the diameters of electrospun nanofibers are generally orders of magnitude smaller than the cell sizes, cells are able to organize over the scaffold fibers or spread and attach to adsorbed proteins at multiple focal points

156

. However, despite the

numerous attractive features of electrospun nanofibers, it has been difficult to design macroscopically porous 3D architectures based on these materials, which are characterized by entangled fibers and densely packed membranous structure which poses obvious limitations to cellular infiltration. In order to overcome these limitations a plethora of different fabrication approaches have been proposed for enlarging the pore size of electrospun nanofibrous scaffolds and promote cellular infiltration. These include the combination with salt/polymer leaching using of ice crystals as a collection device

160

157, 158

, wet electrospinning using bath collector

, laser/UV photolithography

161, 162

159

,

, or combination of

nanofibers and microfibers 163, among others. For further details on this topic, the readers are directed to the review by Zhong et al. 164. Several studies have reported the performance of nanofibrous materials applied to TE strategies targeting skin, blood vessel, cartilage, muscle, adipose, nerve and bone regeneration 156, 165, 166. 26 ACS Paragon Plus Environment

Page 27 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Notwithstanding the works published on the performance of nanofibrous materials aiming for TE applications, electrospun nanofibrous mats are not strong enough for many other TE applications. Incorporation of stiff nano reinforcements, including CNCs with high aspect ratios, has been studied as an effective approach to enhance the mechanical properties of electrospun polymer matrices and also to add new functionalities to the electrospun nanofibres. Electrospinning has been used to orient CNCs along the fiber axis, thus providing unidirectional fiber-reinforced composites for applications requiring high mechanical performance

167-170

. Generally, as will be discussed, the addition of the CNCs has a very

positive impact on the fiber stiffness, morphology and has reduced the fiber diameter. The potential application of microcrystalline cellulose and CNCs as fillers in electrospun CA for vascular tissue scaffolds has been investigated

171

. In fact, various electrospun nanocomposite materials have

demonstrated a reduction in thrombogenicity while also improving mechanical properties

172

. The

introduction the micro- and nanoscaled cellulose particulates into the fibers was considered to fabricate composite scaffolds with multiscaled features which would better mimic the natural hierarchical organization of ECM. The diameters of the obtained electrospun fibers were at submicro-range and the blending of microcrystalline cellulose and CNC (especially microcrystalline cellulose) increased the porosity of the scaffolds (e.g. 1016 ± 572 nm of mean diameter and 79.7 ± 8.8 % porosity at 10 wt.% microcrystalline cellulose/CNC 1:1 content). The effect of microcrystalline cellulose and CNC over the viability of rat aortic vascular smooth muscle cells (VSMC) within the electrospun scaffolds was tested in vitro. The results suggested that cell viability, adhesion and proliferation within the electrospun scaffolds containing both microcrystalline cellulose and CNC was considerably improved when compared to CA alone or to composites with only microcrystalline cellulose or CNC, proving the synergistic enhancement provided by the micro- and nanoscaled features within the 3D fibrous mesh structure. Recently, He et al. prepared electrospun all-cellulose nanocomposite nanofibers reinforced with CNCs (up to 20%), which were uniaxially aligned using a rotating drum as collector

170

. The resulting

electrospun nanofibers exhibit well dispersed and considerable orientated CNCs along the fiber axis 27 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 60

direction, having a positive effect on creating a more uniform morphology (diameters ranging from 212 nm to 221 nm). This ordered microstructure combined with the strong interfacial bonding between CNCs and the regenerated cellulose matrix, remarkably improved the tensile properties of non-woven mats (tensile strength and elastic modulus increased by 101.7 % and 171.6 %, respectively, in the fiber alignment direction, with 20 % loading of CNCs). In terms of potential as scaffold materials for TE, the fiber mats were non-toxic for human dental follicle cells (hDFCs), demonstrating that hDFCs could attach and proliferate in the entire scaffold (not only on the surface but also deep inside the fiber mats) and induce ordered cellular organization toward the fiber alignment direction (Figure 6). Considering these features, the authors proposed that these scaffolds can find applications in TE strategies where mechanical performance and cell orientation are critical issues (e.g. blood vessel, tendon or nerve) 170.

Figure 6. Confocal laser scanning microscopy images of hDFCs loaded in electrospun cellulose/CNCs nanocomposite nanofibers: (a) cultured for 3 days, (b) cultured for 7 days, (c) the 3D view of electrospun


Page 29 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

nanofibers with cells. Scale bar: 50 µm

170

. Reprinted with permission from ref. 170. Copyright 2014

American Chemical Society.

Silk fibroin/CNCs nanocomposites have also been prepared by electrospinning

168, 173

. Huang et al.

prepared electrospun silk fibroin nanofiber mats reinforced by CNCs extracted from Morus alba L. branch bark (20-40 nm in diameter and 400-500 nm in length)

168

. They reported that CNCs were

homogeneously dispersed and aligned along the fiber axis in the silk fibroin matrix. The tensile strength and Young’s modulus of the reinforced silk fibroin nanofiber mats were almost twice those of unreinforced silk fibroin mats when the CNCs content was 2 wt. %. Similar enhanced mechanical properties were achieved with bacterial cellulose nanocrystal as nanofillers in silk fibroin nanofibers 173. Although natural origin polymers are highly attractive for TE applications, electrospinning of biological materials is less versatile because a suitable solvent that does not compromise its integrity has to be used or it requires blending with synthetic polymers or salts to increase the solution viscosity and consistency in electrospinning 156, 174. Thus, synthetic polymers, particularly the bio-based PLA 175-178, have been at the research focus for the fabrication of electrospun nanocomposites fibers using CNCs as nanofiller. Xiang el al. firstly incorporated CNCs prepared from microcrystalline cellulose into electrospun PLA fibers, reporting that the strength of the electrospun non-woven fabrics was improved by 30 % with 1 wt. % loading of CNCs, which acted as nucleating agent of PLA crystallization and hence increased the crystallinity of PLA in the resulting nanocomposite fibers

179

. Shi et al. prepared PLA/CNCs fiber mats

electrospun from a solvent mixture consisting of N,N’-dimethylformamide and chloroform at room temperature and investigated their in vitro degradation

177

. At a CNCs content of 5 wt. %, the maximum

tensile stress and Young's modulus of the nanocomposite mats increased by 5 and 22 fold respectively, and the nanocomposite mats were also found to degrade faster in phosphate-buffered saline solution, compared with neat PLA mats. It was hypothesized that the rapid in vitro biodegradability combined with 29 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 60

impressive mechanical properties make PLA/CNCs mats particularly suitable for TE scaffolds aimed at short-term applications. Due to the high polarity of CNCs and known tendency for the formation of strong hydrogen bonding promoting aggregation, alternative strategies were proposed to overcome the obstacles of dispersing CNCs in PLA matrices, which are not water-based and nonpolar. Li et al. proposed a water-in-oil (W/O) emulsion system consisting of two immiscible phases: a dispersed phase of CNCs aqueous suspension and an immiscible continuous phase of PLA solution 178

. Interestingly, depending on the emulsion droplet size, the electrospun composite fiber assumed a

core–shell or hollow structure, in which CNCs were aligned along the core or on the wall of the hollow cylinder, respectively (Figure 7). The addition of CNCs (5 wt. %) and the alignment of the fibers effectively improved the strength and stiffness of electrospun composite fibers mats (increased the Young’s modulus by 549 % and maximum tensile strength by 90 %). Besides the reinforcement effect provided by the CNCs, these hollow nanofibers could potentially be used as multifunctional systems in TE applications. Bioactive molecules/factors could be loaded in the hollow nanofibers and be used simultaneously as scaffolds and controlled delivery systems, following similar strategies proposed for other fibrous materials 180.


Page 31 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 7: Correlations between emulsion droplet size (a ∼ 6 µm, b ∼ 3 µm, and c < 1 µm) and electrospun fiber structure (d and g represent core–shell, e and f hollow cylinder, and h CNC aggregation) 178

. Reprinted with permission from ref. 178. Copyright 2013 American Chemical Society.

In another study, PLA was grafted with maleic anhydride (MPLA) as a strategy to improve the interfacial adhesion between the hydrophobic PLA matrix and the hydrophilic CNCs

176

. The diameter and

polydispersity of electrospun MPLA/CNCs nanofibers were reduced with increased CNCs content and the fibrous scaffolds showed improved thermal and mechanical properties (tensile strength of more than 10 MPa at 5 wt. % CNCs). In vitro degradation and cytocompatibility was evaluated using human adult adipose derived mesenchymal stem cells (hASCs), and the results showed that these MPLA/CNC scaffolds are biodegradable and cytocompatible, indicating that they could be suitable for bone TE 176.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 60

CNCs obtained from ramie cellulose fibers were used to reinforce PCL electrospun nanofibers

181

. The

surface of CNCs was modified by grafting with low-molecular-weight PCL diol in an attempt to improve the interfacial adhesion with the polymer matrix. This strategy, however, resulted in a negative effect on the morphology of non-woven mats in which individual nanofibers became annealed during the electrospinning process. Nevertheless, a significant improvement was achieved in the mechanical properties of the nanofibers after reinforcement with unmodified CNCs (2.5 wt. %), resulting in a 1.5-fold increase in Young’s modulus and ultimate strength compared to PCL mats. Due to the good dispersion of CNCs in water, electrospun nanocomposite fibers based in water soluble polymer matrices relevant for TE applications, such as PEO

182, 183

and PVA

169, 184

, have been

investigated. In this case, a direct mixing method can be used to disperse CNCs into polymer aqueous solutions. CNCs demonstrated a more significant reinforcement effect on the aligned electrospun PVA fiber mats compared with the isotropic ones 169. The modulus and tensile strength of the aligned mats increased 35% and 45%, respectively, compared to the isotropic mats. Aligned PVA/CNCs fibers presented lower diameter when compared with the fibers produced with PVA alone. The addition of 15% of CNCs to aligned PVA electrospun fiber mats induced a 95% higher tensile strength and an increase of 118% in the modulus. Moreover, Peresin et al. studied CNCs reinforcement effect on electrospun PVA with different concentrations of acetyl groups

184

. It was shown that the higher the hydrolysis degree of PVA, the

stronger the PVA-CNC interaction, which resulted in an effective reinforcement of the fiber mats. This observation was ascribed to the reinforcing effect of CNCs via the percolation network held by hydrogen bonds. Similar results were obtained for PEO/CNC electrospun nanocomposite fiber mats with different woodbased CNCs loadings (up to 20 wt. %)

182

. Nanofibers also became more uniform and finer while

increasing of CNCs content due to the enhanced electric conductivity of the electrospinning solution. The


Page 33 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

mechanical properties of the mats were effectively improved by CNCs (152% and 180% in the modulus and tensile strength, respectively, with 20 wt. % CNCs) due to the efficient stress transfer from PEO to CNCs originating from their strong interactions through hydrogen bonding and the uniform dispersion and high alignment of CNCs in the electrospun fibers. Considering that cellular infiltration is hindered by decreasing the scaffolds’ pore size, the general decrease of fiber diameter resulting from the incorporation of CNCs into electrospun polymer mats may be a detrimental feature of these systems in TE applications. Although the data on pore sizes of nanofiber mats are not available from the cited works, a significant decrease of fiber diameter has been observed with increasing amount of CNCs incorporation, e.g. 24% decrease for electrospun PEO with 20 wt. % CNCs

182

and 19% decrease for MPLA with 5 wt. % CNCs 176, compared with neat polymer nanofibres.

However, this issue can be mitigated if fabrication techniques to increase the pore size of electrospun nanofibrous mats are adopted, as previously referred

164

. Furthermore, it is known that the mechanical

strength of scaffolds is impaired with increasing pore size and porosity

164

, thus the incorporation of

CNCs may be a useful strategy in order to preserve or improve the mechanical competence of nanofibrous scaffolds fabricated through these techniques, while providing an effective 3D nanofibrous structure to accommodate sufficient cell infiltration. Wet-spun microfibers (typical diameters of 30–200 µm) is another class of materials which has been deserving considerable interest as 3D scaffolding substrates for several TE strategies over the past decade 185, 186

. Some studies on using the wet-spinning technique for the preparation of alginate microfibers

containing CNCs that could find application in TE have also been reported

187-189

. It was demonstrated

that by controlling the CNC weight fraction loading and the fiber wet-spinning properties, it was possible to tailor in a very effective way its mechanical properties, which correlate directly with the CNC orientation within alginate fibers

187, 189

. A reinforcement of 38% in tenacity and 123% in modulus was

obtained in wet-spun fibers at an apparent jet stretch of 4.2 and a CNCs load of 10 wt. % 188.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 60

Recently, in a different strategy, Hossain et al. used a melt-drawing process to produce PLA microfibers (c.a. 11 µm average diameter) which were further coated with blends of CNCs (65 to 95 wt %) and PVAc (used as a binding agent)

190

. The rational of this approach was to obtain a more hydrophilic and

roughened fiber surface that could positively impact cells/fiber interactions. Besides some improvement of the fibers mechanical properties (45% increase in tensile modulus for PLA fibers coated with 75 wt % CNCs-PVAc blend), a significant increase on moisture absorption was achieved, proving the increased hydrophobicity of the coated microfibers, while imparting surface roughness to the otherwise smooth PLA fibers. The cytocompatibility of CNCs coated PLA fibers was tested using NIH-3T3 mouse fibroblast cells, which revealed a better cell adhesion compared with the PLA control. In fact, cells completely covered the fibers within 2 days in culture, suggesting the enhanced potential of this system for TE application 190.

3.2.2 Foams and sponges As discussed previously in section 2, the preparation of 3D scaffolds must result in structures with adequate porosity, interconnectivity, pore size distribution and mechanical properties which make then suitable for the tissue to be engineered 74, 75, 191. In this perspective, polymer foams and sponges obtained through various processing techniques have been among the most studied porous materials aimed at a variety of TE strategies 75, 191-193. Regarding the incorporation of CNCs within composite foams and sponges, some studies have been exploring various fabrication routes in order to produce porous nanostructured materials based on several natural

194-196

and synthetic

197, 198

polymer matrices. Besides improving the foams and sponges stability

and mechanical properties, in some cases, nanotopography was added to the pore walls, which ultimately would result in a positive effect on the cells/scaffold interaction.


Page 35 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Starch/PVA nanocomposite sponges reinforced with CNCs (0-30 wt. %) were fabricated through repeated cycles of freezing and thawing

195

. The obtained porous materials exhibited a honeycomb like structure

with interconnected pores (Figure 8A), and improved dimensional stability and strength (nearly an order of magnitude higher, with 30 wt. % CNCs). The enhancement of the mechanical properties was ascribed to the formation of the honeycomb like structures and strong hydrogen-bonding interactions between the CNCs and the starch matrix. The size and shape of the pores were controlled by the addition of CNCs, and sponges with higher content of CNCs exhibited a more homogeneous porous architecture. Biocompatibility of the nanocomposite sponges was evaluated with COS-7 cells (fibroblast-like cells). The seeded cells adhered onto the surface of the sponge and spread through the pores, indicating that this nanocomposite was cytocompatible and thus could be considered as a promising material for wound dressing and TE applications 195. Ionic cross-linked alginate/CNCs nanocomposite sponges, using TEMPO-mediated oxidized cellulose nanocrystals (OCNCs), were prepared by freeze-drying in order to enhance the mechanical performance and degradation properties of alginate sponges

196

. In theory, the carboxyl groups of OCNCs could

participate in the construction of the cross-linked network from alginate-based sponge, thus playing a fundamental role on the structural and mechanical stability of the material. Although, comparatively, the use of oxidized microfibrillated cellulose resulted in a superior improvement of the sponge’s porosity and water absorption, OCNCs significantly enhanced its mechanical strength by participating in the crosslinking and serving as the coupling points with alginate. The highest mechanical performance was obtained with 10 wt. % OCNCs, resulting in a compressive strength increased by factors of 2.78, 2.80, and 2.94 at compressive strain of 30%, 50%, and 70%, respectively, in comparison to neat alginate sponge 196. Ragauskas’ research group has been reporting a number of highly structured foams prepared by an icetemplating technique, also known as freeze-casting

194, 198, 199

. Freeze-casting consists of directional

freezing a liquid suspension/solution followed by sublimation of the solvent under reduced pressure

200

.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 60

During the freezing process, the suspended particles are ejected from the moving solidification front, concentrated and finally entrapped between the growing solvent crystals, which results in an ordered structure after sublimation. In one of their studies they prepared highly structured biofoams from xylans and CNCs suspensions

194

.

By employing unidirectional solidification, that caused the ice-segregation-induced self-assembly, it was possible to design low density xylan biofoams which possessed a highly ordered lamellar structure (Figure 8B), a porosity of 92–95% and a very high degree of pore interconnectivity (>99%). Addition of 25 wt. % CNCs also increased the stress at 50% strain by about 250% and the modulus by about 400% of the fabricated nanocomposite foams. A similar approach has been followed for the preparation of these ordered porous CNCs structures with PVA as a support material

198

or as a binder

199

. Furthermore, the

pore morphology of the foams could be controlled and its mechanical properties improved by adding small amounts of dimethylsulfoxide (DMSO) to the water solvent 199. These structured foams prepared by freeze-casting show potential for TE applications as they can provide hierarchical composite features that can better emulate the naturally-occurring tissues exhibiting a preferential alignment and a well-ordered structure 201.


Page 37 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 8. SEM image of the A) cross-sections of starch/PVA nanocomposite sponges at low magnification (top) and at high magnification (bottom) obtained with different amounts of CNCs (0-30 wt.%) 195; B) freeze-cast glucuronoarabinoxylan/CNCs foams unidirectionally frozen in liquid nitrogen 194 C) PLA foams containing 30 wt.% bacterial CNCs and 20 vol.% ice microspheres as a template

197

.

Reproduced from ref. 194. Copyright 2012, with permission of The Royal Society of Chemistry. http://dx.doi.org/10.1039/c2gc35413f. Reprinted from ref. 195. . Copyright 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. With permission from John Wiley and Sons. Reprinted from ref. 197, Copyright 2010, with permission from Elsevier.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 60

A common method which provides great control over the microscale structure within low-density porous foams is to template sintered spheres and then remove the template. Blaker et al. proposed the use of CNCs from bacterial cellulose to produced PLA/CNCs scaffolds using a thermally induced phase separation route combined with an ice-microsphere templating

197

. Through this process, the scaffolds

pore structure, interconnects and surface area could be controlled, and exhibited nanotopography on the pore walls of the 3D porous forms (Figure 8C). The scaffolds exhibited porosities up to 97% with spherical interconnected pores which, by exploiting interactions between the hydrophilic and hydrophobic phases, were lined by CNCs where they are required for direct interaction with cells.

3.3 Hydrogels Polymeric hydrogels are 3D networks of cross-linked (physically and/or chemically) macromolecules that can entrap substantial amounts of water, typically through surface tension and capillary forces

202

.

Hydrogels are highly attractive for TE applications because, within this context, they are typically based on biodegradable materials, can be processed under relatively mild conditions, have mechanical and structural properties similar to many tissues and the ECM, and in some cases they can be delivered in a minimally invasive manner

202, 203

. Furthermore, their high tissue-like water content closely mimic the

natural environment in the body, enabling efficient transport of nutrients and waste

204

. The

biofunctionality of these highly versatile materials can be engineered by tailoring their chemical and mechanical properties, each of which directly influences cell fate 72. Besides acting as porous scaffolds for neo-tissue ingrowth, hydrogel based systems have been proposed as carriers for cells, growth factors and bioactive molecules in many TE strategies 205-207. Although most hydrogels are similar to biological tissues, they are generally brittle and prone to fracture at low deformation, which hinders their applications where high stress is required

208

. The incorporation

of nanoparticles into the hydrogels 3D matrix, producing a class of materials known as nanocomposite 38 ACS Paragon Plus Environment

Page 39 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

hydrogels (NHPs), has been a widely investigated strategy to improve some existing physical properties or to provide them new physical or chemical features 208, 209. Considering the excellent dispersion of CNCs in water, they are obvious candidates to prepare NHPs, having many advantages compared to other polymeric or metal nanoparticles

210

. CNCs have been

incorporated in several polymeric hydrogel matrices, either as fillers or chemically cross-linked within the host polymer. The general work published on this topic has been included in some review articles 210

25,

. Here, the main focus is given to the recent works on NHPs containing CNCs with potential

applications in TE. Generally, the introduction of rigid CNCs in polymeric matrices to prepare gel-based nanomaterials improves the overall gelation process, the structural stability of the NHPs framework, and enhances their mechanical strength 25. In terms of swelling properties, the incorporation of CNCs has been referred to impart both the increasing 211-213 and decreasing 214-220 effects on the equilibrium swelling ratio of NHPs, depending mostly on the nature of polymer matrix and nanocomposite preparation strategy. However, as CNCs generally act as a multifunctional cross-linkers resulting in increased hydrogels cross-link density, the swelling ratio is most often inversely proportional to CNCs concentration in the hydrogels 216, 218-220. As fillers (i.e., with no covalent attachment to the hydrogel), CNCs have been incorporated to reinforce polymer hydrogels based on PVA 212

213, 221

, α-cyclodextrin

222

, PEG

223

, PNIPAm

211

, hemicellulose (xylan)

, regenerated cellulose 216, and carboxymethyl cellulose/hydroxyethyl cellulose 214.

Wang et al. studied the impact of CNCs on the formation kinetics and properties of all-cellulose composite gels using CNCs as the reinforcing phase (0 and 50 wt. %) and regenerated cellulose as the matrix

216

. In this study, all-cellulose hydrogels were developed by a rapid thermal-induced phase

separation followed by a regenerating process

216

. In the resulting physically cross-linked composite

hydrogels, CNCs acted as bridges that facilitate the cross-linking of cellulose chains, providing a good support to the gel network to maintain a homogeneous shrinkage during the regeneration process. 39 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 60

Moreover, CNCs significantly improved the dimensional stability and mechanical strength of the regenerated gels: the shear modulus of the neat cellulose gel increased from 81 to 160 kPa at 50 wt. % of CNCs content. According to the authors, the composites exhibited similar modulus compared to those of PLA-PEO-PLA hydrogels and PLA-fibrin gels which have been proposed as cartilage TE materials 225

224,

.

Karaaslan et al. designed hydrogels based on aspen wood hemicellulose (xylan) and CNCs

212

.

Hemicellulose was first chemically modified with 2-hydroxyethylmethacrylate (HEMA) and then adsorbed onto the CNCs surface, making use of its inherent affinity. NPHs were prepared through in situ free radical polymerization of HEMA, a common biocompatible monomer

202

.

The resulting

hydrogels had enhanced toughness, increased viscoelasticity, and improved recovery behavior, when compared to those prepared using conventional cross-linking agents. Considering the characteristics of the produced hydrogels, it was hypothesized that these materials have potential for use in load-bearing biomedical applications such as articular cartilage replacement 212. A few recent works have investigated the incorporation of CNCs with particular surface modifications in the preparation of NPHs, thus acting as both filler and cross-linker to reinforce hydrogel systems. The polymer matrices include polyacrylamide (PAAm)

217, 218

, PAA

219

, gelatin

215

, cyclodextrin

226

, and

CMC/dextran systems 220. The in situ free-radical polymerization route was proposed for the preparation of NPHs reinforced with CNCs based on PAAm

217, 218

, a polymer with wide variety of applications in TE

207

. Zhou et al.

demonstrated that the CNCs acted as reinforcing agents and also as multifunctional cross-linkers, accelerating the formation of hydrogels and increasing their effective cross-linking density 218. Compared to neat PAAm hydrogels, the NPHs exhibited a significant improvement in the shear storage modulus and compression strength (4.6 and 2.5-fold, respectively, loaded with 6.7 wt. % CNCs). The improvements were assigned to the good dispersion of CNCs in the PAAm matrix as well as to the PAAm grafting on


Page 41 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

the surface of CNCs, thus allowing enhanced interfacial interaction between the two components. The authors suggested that such NPHs could be applied in traditional bone-defect repair and bone TE 218. Chemically cross-linked gelatin/CNCs hydrogels were prepared using oxidized CNCs as cross-linkers 215. In this study, the dialdehyde groups of oxidized CNCs reacted with the free amine groups of gelatin to cross-link the hydrogel framework. The properties of these materials were dependent on the amounts of CNCs’ aldehyde groups (i.e., degree of oxidation). It was shown that an increase in aldehyde groups resulted in an increase of the cross-linking degree, leading to the formation of a rigid dense network, which relatively reduced the water uptake ability of the hydrogels. Conversely, the increase in crosslinking degree improved the storage modulus of hydrogels by 150% and also their thermal stability up to 50 °C. This could be a suitable strategy to overcome the poor thermal and mechanical properties of neat gelatin gels, thus broadening the application of chemically cross-linked gelatin hydrogels in TE, among other biomedical applications 215. Recently, Yang et al. arguably reported the first injectable hydrogels reinforced both physically and covalently with CNCs, based on a CMC/dextran system

220

. Their approach was based on coextruding

aldehyde functionalized CNCs with dihydrazide-modified CMC and aldehyde-modified dextran solutions through a double-barrel syringe (Figure 9A). Hydrazone cross-links are formed when the functionalized hydrogel components come into contact under normal physiological conditions, without the need of additional chemicals, changes in temperature, or processing for gelation to occur. The resulting CNC cross-linked hydrogels were more elastic (>140% increase of elastic modulus at peak strength by adding 0.375 wt.% of functionalized CNCs ) and more dimensionally stable over an extended period of time (60 days) compared to unfilled hydrogels (Figure 9B), without significantly impacting the pore structure of the hydrogels. Furthermore, these NPHs and their components (including CNCs and aldehyde functionalized CNCs in a range of 100 to 1000 µg/mL) revealed no evident cytotoxicity to NIH 3T3 fibroblast cells. The authors hypothesize that these CNC-reinforced injectable polysaccharide hydrogels


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 60

are of potential interest for TE applications where longer term dimensional stability and enhanced mechanical strength are desirable 220.

A

B

Figure 9. A) Schematic representation of injectable hydrogels reinforced with cellulose nanocrystals (CNCs), prepared using a double-barrel syringe. The cross-linking hydrogel components include hydrazide-functionalized carboxymethyl cellulose (CMC-NHNH2), aldehyde-functionalized dextran (dextran-CHO), and either unmodified CNCs or aldehyde-modified CNCs (CHO–CNCs); B) digital photographs of hydrogels with no CNCs (squares), CNCs (circles), and aldehyde-modified CNCs (triangles) after swelling for both 6 h and 60 days (background mat has 1 cm grid spacings and degraded hydrogel fragments are outlined with boxes) 220. Reprinted with permission from ref. 220. Copyright 2013 American Chemical Society.

In another recent work, aldehyde-functionalized CNCs were also used to reinforce the biocompatible fibrin hydrogel sheets for vascular graft replacement applications

227

. The rationale behind this approach

was to produce a nanocomposite that resembles the collagen-elastin makeup of the native blood vessels, where CNCs and fibrin could mimic the role of collagen and elastin, respectively. It was demonstrated


Page 43 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

that the molecular interaction between oxidized CNCs and fibrin induced by the carbonyl group is the key to obtain an effective reinforcement, although an excessive covalent binding could impair the elongation of nanocomposites and high oxidized CNCs loadings could hamper fibrin formation (Figure 10). Nanocomposite synthesized by combining fibrin and CNCs at a 1:1 ratio and with an optimized degree of oxidation yielded both comparable strength and elasticity to the porcine coronary artery, used as a native blood vessel control 227.

Figure 10. Postulated molecular interactions between oxidized CNCs and fibrin in nanocomposites

227

.

Reprinted with permission from ref. 227. Copyright 2013 American Chemical Society.

4. Conclusions and future perspectives Over the last few years, a growing interest has emerged on applying CNCs as biomaterials for the development of advanced functional bionanocomposites which could find a wide range of potential applications in TE. The current review has attempted to provide a general overview of the potential of CNCs in the design of these functional nanomaterials, through various examples involving different


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 60

approaches and processes. Recent studies demonstrated that different types of nanomaterials presenting desired properties and functions could be produced from CNCs. Research on nanocomposite films and membranes with potential application in TE strategies was summarized. The development of well-defined hierarchical 3D nanocomposite biomaterials fabricated through different techniques usually adopted when dealing with porous scaffolds in TE strategies were presented such as electrospun nanofiber mats, wetspun fibers, foams, sponges, and hydrogels. The main research focus has been directed to the reinforcement effect provided by CNCs to nanocomposites which would enable their application as higher performance materials. However, despite the considerable advances achieved to date, properly tailoring their surface functionalization and reach homogeneous dispersion within polymer matrices (particularly in the case of nonpolar ones) are still considered among the major challenges in the development of these materials. A considerable number of works have been reported on electrospun nanocomposites fiber mats containing CNCs, showing the great potential of combining this fabrication route with the incorporation of CNCs to achieve improved dispersion and effective reinforcement of the nanofibers. The reinforcement effect provided by CNCs has also shown to be anisotropic. Strategies to produce hollow fibers or to line CNCs on the surface of the nanofibers have been developed. These features could be relevant for the microenvironment that regulates cell behavior in the case of TE and regenerative medicine applications. In fact, the mechanical properties of many native tissues are also anisotropic, requiring that scaffolds have embedded microstructures that exhibit preferred orientations 73. Moreover, materials’ nanotopography is well-known to regulate the cell–material interaction due to the involvement of nanoscale topography and integrins

60, 88

. From another perspective, hollow nanofibers could potentially be used as multifunctional

systems acting simultaneously as scaffolds and delivery vehicles for drugs or other bioactive factors in TE applications 180.


Page 45 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Hydrogel systems containing CNCs have been particularly investigated, taking advantage of the good dispersion of these nanocrystals in aqueous media. Generally, CNCs imparted a positive effect on the gelation mechanism, improved the overall dimensional stability and enhanced the mechanical strength of the materials. These improvements were shown to be depending on the CNCs’ concentration and aspect ratio, but also on their surface modification when used within certain polymer matrices. There is a current trend in developing hydrogel systems for 3D cell culture and TE applications. Within this context, a growing interest has emerged in the role of mechanotransduction during cell expansion and differentiation 72

. CNCs could play an important role in the design of hydrogel nanocomposites, enabling user-defined

tailorability of the physical cues within the surrounding ECM, which would potentially promote lineagespecific differentiation of stem cells. Furthermore, various stimuli responsive (e.g., pH, temperature, ionic strength, biomolecules) nanocomposite hydrogels reinforced with CNCs can be designed in a very controlled fashion and are expected to find applications in TE strategies. The combination of stem cells therapy and nanotechnology allows the engineering of scaffolds with various features to control cells fate decisions. How to design and fabricate functionalized scaffolds in micro- and nanotechnologic length scales for each specific application and improving cellular responses have become important trends in TE and regenerative medicine. However, regardless of the considerable amount of different materials fabricated with CNCs having a plethora of physical cues that could potentially be advantageous and adapted for specific TE strategies, very few in vitro and no in vivo biological tests have been carried out in order to assess the performance of the proposed platforms for practical applications, making this topic a vast field to be explored. Another interesting issue expected to be addressed in the upcoming years is the effect of CNCs on the self-assembling of peptide-based biomaterials. The development of self-assembled peptide biomaterials for 3D tissue engineering scaffolds has emerged as a very active research field in the last few years

228

.

Recently, Haghpanah et al. reported a study exploring the ways in which protein diblock copolymers interact with CNCs, depicting that different protein copolymers possess different modes of assembly in 45 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the presence of CNCs and thus leading to dissimilar physicochemical properties

Page 46 of 60

229

. This work may

provide some insights into the design of future bionanocomposites involving peptide-based polymers and CNCs. Finally, a topic that has been previously identified by Dugan et al. and still remains to be realized

55

,

concerns the development of CNCs-based systems as therapeutic agent delivery vehicles or their modification with specific chemical signaling motifs (e.g., peptide sequences) to guide or enhance in vitro or in vivo tissue formation. With this overview we expect to provide an impetus for the development of novel functional biomaterials with tailored properties for TE applications, while hopefully contribute for highlighting the various interesting properties of CNCs as bio-based building blocks and their potential in this field.

AUTHOR INFORMATION *Corresponding Author Mailing address: 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics,

Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Indústrial da Gandra, 4806-909 Caldas das Taipas, Guimarães, Portugal E-mail address: [email protected], Tel.: +351253510906

Notes The authors declare no competing financial interest.


Page 47 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

ACKNOWLEDGMENT The authors acknowledge the financial support from the Project RL1 - ABMR - NORTE-01-0124FEDER-000016 cofinanced by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

References (1) Brinchi, L.; Cotana, F.; Fortunati, E.; Kenny, J. M. Carbohydr. Polym. 2013, 94, 154-169. (2) Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chem. Soc. Rev. 2011, 40, 39413994. (3) Klemm, D.; Heublein, B.; Fink, H. P.; Bohn, A. Angew. Chem. Int. Ed. 2005, 44, 3358-3393. (4) Azizi Samir, M. A. S.; Alloin, F.; Dufresne, A. Biomacromolecules 2005, 6, 612-626. (5) Habibi, Y.; Lucia, L. A.; Rojas, O. J. Chem. Rev. 2010, 110, 3479-3500. (6) Beck-Candanedo, S.; Roman, M.; Gray, D. G. Biomacromolecules 2005, 6, 1048-1054. (7) Araki, J.; Wada, M.; Kuga, S.; Okano, T. J. Wood Sci. 1999, 45, 258-261. (8) De Souza Lima, M. M.; Wong, J. T.; Paillet, M.; Borsali, R.; Pecora, R. Langmuir 2003, 19, 24-29. (9) Araki, J.; Wada, M.; Kuga, S.; Okano, T. Langmuir 2000, 16, 2413-2415. (10) Dong, X. M.; Revol, J. F.; Gray, D. G. Cellulose 1998, 5, 19-32. (11) Habibi, Y.; Goffin, A.-L.; Schiltz, N.; Duquesne, E.; Dubois, P.; Dufresne, A. J. Mater. Chem. 2008, 18, 5002-5010. (12) Dufresne, A.; Cavaillé, J. Y.; Helbert, W. Polym. Compos. 1997, 18, 198-210. (13) Lu, P.; Hsieh, Y.-L. Carbohydr. Polym. 2012, 87, 564-573.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 60

(14) Capadona, J. R.; Shanmuganathan, K.; Trittschuh, S.; Seidel, S.; Rowan, S. J.; Weder, C. Biomacromolecules 2009, 10, 712-716. (15) Bondeson, D.; Mathew, A.; Oksman, K. Cellulose 2006, 13, 171-180. (16) Araki, J.; Kuga, S. Langmuir 2001, 17, 4493-4496. (17) Hirai, A.; Inui, O.; Horii, F.; Tsuji, M. Langmuir 2009, 25, 497-502. (18) Osorio-Madrazo, A.; Eder, M.; Rueggeberg, M.; Pandey, J. K.; Harrington, M. J.; Nishiyama, Y.; Putaux, J.-L.; Rochas, C.; Burgert, I. Biomacromolecules 2012, 13, 850-856. (19) Revol, J. F. Carbohydr. Polym. 1982, 2, 123-134. (20) Anglès, M. N.; Dufresne, A. Macromolecules 2001, 34, 2921-2931. (21) Anglès, M. N.; Dufresne, A. Macromolecules 2000, 33, 8344-8353. (22) Kimura, F.; Kimura, T.; Tamura, M.; Hirai, A.; Ikuno, M.; Horii, F. Langmuir 2005, 21, 2034-2037. (23) Elazzouzi-Hafraoui, S.; Nishiyama, Y.; Putaux, J.-L.; Heux, L.; Dubreuil, F.; Rochas, C. Biomacromolecules 2008, 9, 57-65. (24) Hamad, W. Can. J. Chem. Eng. 2006, 84, 513-519. (25) Lin, N.; Huang, J.; Dufresne, A. Nanoscale 2012, 4, 3274-3294. (26) Dri, F.; Hector, L., Jr.; Moon, R.; Zavattieri, P. Cellulose 2013, 20, 2703-2718. (27) Rusli, R.; Eichhorn, S. J. Appl. Phys. Lett. 2008, 93, 033111. (28) Šturcová, A.; Davies, G. R.; Eichhorn, S. J. Biomacromolecules 2005, 6, 1055-1061. (29) Habibi, Y. Chem. Soc. Rev. 2014, 43, 1519-1542. (30) Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Angew. Chem. Int. Ed. 2011, 50, 5438-5466. (31) Kümmerer, K.; Menz, J.; Schubert, T.; Thielemans, W. Chemosphere 2011, 82, 1387-1392. (32) Kovacs, T.; Naish, V.; O'Connor, B.; Blaise, C.; Gagné, F.; Hall, L.; Trudeau, V.; Martel, P. Nanotoxicology 2010, 4, 255-270. (33) Entcheva, E.; Bien, H.; Yin, L.; Chung, C.-Y.; Farrell, M.; Kostov, Y. Biomaterials 2004, 25, 57535762. 48 ACS Paragon Plus Environment

Page 49 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(34) Märtson, M.; Viljanto, J.; Hurme, T.; Laippala, P.; Saukko, P. Biomaterials 1999, 20, 1989-1995. (35) Roman, M.; Dong, S.; Hirani, A.; Lee, Y. W., In Polysaccharide Materials: Performance by Design, Edgar, K. J.; Heinze, T.; Buchanan, C. M., Eds. American Chemical Society: Washington DC, 2009, pp 81-91. (36) Dong, S.; Hirani, A. A.; Colacino, K. R.; Lee, Y. W.; Roman, M. Nano LIFE 2012, 02, 1241006. (37) Mahmoud, K. A.; Mena, J. A.; Male, K. B.; Hrapovic, S.; Kamen, A.; Luong, J. H. T. ACS Appl. Mater. Interfaces 2010, 2, 2924-2932. (38) Male, K. B.; Leung, A. C. W.; Montes, J.; Kamen, A.; Luong, J. H. T. Nanoscale 2012, 4, 13731379. (39) Jackson, J. K.; Letchford, K.; Wasserman, B. Z.; Ye, L.; Hamad, W. Y.; Burt, H. M. Int. J. Nanomedicine 2011, 6, 321-330. (40) Pereira, M. M.; Raposo, N. R. B.; Brayner, R.; Teixeira, E. M.; Oliveira, V.; Quintão, C. C. R.; Camargo, L. S. A.; Mattoso, L. H. C.; Brandão, H. M. Nanotechnology 2013, 24, 075103. (41) Clift, M. J. D.; Foster, E. J.; Vanhecke, D.; Studer, D.; Wick, P.; Gehr, P.; Rothen-Rutishauser, B.; Weder, C. Biomacromolecules 2011, 12, 3666-3673. (42) Dufresne, A. Can. J. Chem. 2008, 86, 484-494. (43) Eichhorn, S. J. Soft Matter 2011, 7, 303-315. (44) Lam, E.; Male, K. B.; Chong, J. H.; Leung, A. C. W.; Luong, J. H. T. Trends Biotechnol. 2012, 30, 283-290. (45) Dufresne, A. Molecules 2010, 15, 4111-4128. (46) Durán, N.; Lemes, A. P.; Seabra, A. B. Recent Pat. Nanotechnol. 2012, 6, 16-28. (47) Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.; Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.; Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T. J. Mater. Sci. 2010, 45, 1-33. (48) Holt, B. L.; Stoyanov, S. D.; Pelan, E.; Paunov, V. N. J. Mater. Chem. 2010, 20, 10058-10070. 49 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 60

(49) Hubbe, M. A.; Rojas, O. J.; Lucia, L. A.; Sain, M. BioResources 2008, 3, 929-980. (50) Ramires, E. C.; Dufresne, A. Tappi J. 2011, 10, 9-16. (51) Siqueira, G.; Bras, J.; Dufresne, A. Polymers 2010, 2, 728-765. (52) Visakh, P. M.; Thomas, S. Waste and Biomass Valorization 2010, 1, 121-134. (53) Fernandes, E. M.; Pires, R. A.; Mano, J. F.; Reis, R. L. Prog. Polym. Sci. 2013, 38, 1415-1441. (54) The global market for nanocellulose to 2017; Futures Markets Inc.: Canada, 2013, pp 1-66. (55) Dugan, J. M.; Gough, J. E.; Eichhorn, S. J. Nanomedicine 2013, 8, 287-298. (56) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47-55. (57) Keung, A. J.; Kumar, S.; Schaffer, D. V. Annu. Rev. Cell. Dev. Biol. 2010, 26, 533-556. (58) Wade, R. J.; Burdick, J. A. Mater. Today 2012, 15, 454-459. (59) Place, E. S.; Evans, N. D.; Stevens, M. M. Nature Mater. 2009, 8, 457-470. (60) Kim, H. N.; Jiao, A.; Hwang, N. S.; Kim, M. S.; Kang, D. H.; Kim, D.-H.; Suh, K.-Y. Adv. Drug Del. Rev. 2013, 65, 536-558. (61) Bauer, A. L.; Jackson, T. L.; Jiang, Y. PLoS Comput Biol 2009, 5, e1000445. (62) Edalat, F.; Bae, H.; Manoucheri, S.; Cha, J.; Khademhosseini, A. Ann. Biomed. Eng. 2012, 40, 13011315. (63) Stevens, M. M.; George, J. H. Science 2005, 310, 1135-1138. (64) Hutmacher, D. W. Biomaterials 2000, 21, 2529-2543. (65) Hutmacher, D. W.; Schantz, J. T.; Lam, C. X. F.; Tan, K. C.; Lim, T. C. J. Tissue Eng. Regen. Med. 2007, 1, 245-260. (66) Karageorgiou, V.; Kaplan, D. Biomaterials 2005, 26, 5474-5491. (67) Rezwan, K.; Chen, Q. Z.; Blaker, J. J.; Boccaccini, A. R. Biomaterials 2006, 27, 3413-3431. (68) Holzapfel, B. M.; Reichert, J. C.; Schantz, J.-T.; Gbureck, U.; Rackwitz, L.; Nöth, U.; Jakob, F.; Rudert, M.; Groll, J.; Hutmacher, D. W. Adv. Drug Del. Rev. 2013, 65, 581-603.


Page 51 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(69) Luo, Y.; Engelmayr, G.; Auguste, D. T.; Ferreira, L. d. S.; Karp, J. M.; Saigal, R.; Langer, R., In Principles of Tissue Engineering 3rd ed.; Lanza, R.; Langer, R.; Vacanti, J., Eds. Academic Press: Burlington, 2007, pp 359-373. (70) Discher, D. E.; Janmey, P.; Wang, Y.-l. Science 2005, 310, 1139-1143. (71) Chen, C. S. J. Cell Sci. 2008, 121, 3285-3292. (72) DeForest, C. A.; Anseth, K. S. Annu. Rev. Chem. Biomol. Eng. 2012, 3, 421-444. (73) Hollister, S. J. Adv. Mater. 2009, 21, 3330-3342. (74) Tian, H.; Tang, Z.; Zhuang, X.; Chen, X.; Jing, X. Prog. Polym. Sci. 2012, 37, 237-280. (75) Mano, J.; Silva, G.; Azevedo, H.; Malafaya, P.; Sousa, R.; Silva, S.; Boesel, L.; Oliveira, J.; Santos, T.; Marques, A.; Neves, N.; Reis, R. J. R. Soc. Interface 2007, 4, 999-1030. (76) Gomes, S.; Leonor, I. B.; Mano, J. F.; Reis, R. L.; Kaplan, D. L. Prog. Polym. Sci. 2012, 37, 1-17. (77) Gomes, G. d. S.; Almeida, A. T. d.; Kosaka, P. M.; Rogero, S. O.; Cruz, Á. S.; Ikeda, T. I.; Petri, D. F. S. Mat. Res. 2007, 10, 469-474. (78) Kitamura, M.; Ohtsuki, C.; Iwasaki, H.; Ogata, S. I.; Tanihara, M.; Miyazaki, T. J. Mater. Sci. Mater. Med. 2004, 15, 1153-1158. (79) Barbieri, D.; Yuan, H.; de Groot, F.; Walsh, W. R.; de Bruijn, J. D. Acta Biomater. 2011, 7, 20072014. (80) Armentano, I.; Dottori, M.; Fortunati, E.; Mattioli, S.; Kenny, J. M. Polym. Degradation Stab. 2010, 95, 2126-2146. (81) Place, E. S.; George, J. H.; Williams, C. K.; Stevens, M. M. Chem. Soc. Rev. 2009, 38, 1139-1151. (82) Gomes, M. E.; Malafaya, P. B.; Reis, R. L., In Biopolymer Methods in Tissue Engineering - Methods in Molecular Biology Series, Hollander, A., Ed. Humana Press Inc.: Totowa, 2003, pp 65-67. (83) Ma, P. X. Adv. Drug Del. Rev. 2008, 60, 184-198. (84) Ma, P. X. Mater. Today 2004, 7, 30-40. (85) Chua, C. K.; Tan, L. P.; An, J. Nanomedicine 2013, 8, 501-503. (86) Dvir, T.; Timko, B. P.; Kohane, D. S.; Langer, R. Nature Nanotech. 2011, 6, 13-22. 51 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 60

(87) McMurray, R. J.; Gadegaard, N.; Tsimbouri, P. M.; Burgess, K. V.; McNamara, L. E.; Tare, R.; Murawski, K.; Kingham, E.; Oreffo, R. O. C.; Dalby, M. J. Nature Mater. 2011, 10, 637-644. (88) King-Chuen, W.; Ching-Li, T.; Chi-Chang, W.; Feng-Chen, K.; Yuan-Kun, T.; Edmund, C. S.; Yang-Kao, W. Sci. Tech. Adv. Mater. 2013, 14, 054401. (89) Wei, G.; Ma, P. X. Biomaterials 2004, 25, 4749-4757. (90) Okamoto, M.; John, B. Prog. Polym. Sci. 2013, 38, 1487-1503. (91) Boccaccini, A. R.; Erol, M.; Stark, W. J.; Mohn, D.; Hong, Z.; Mano, J. F. Composites Sci. Technol. 2010, 70, 1764-1776. (92) Harrison, B. S.; Atala, A. Biomaterials 2007, 28, 344-353. (93) Kunzmann, A.; Andersson, B.; Thurnherr, T.; Krug, H.; Scheynius, A.; Fadeel, B. Biochim. Biophys. Acta Gen. Subj. 2011, 1810, 361-373. (94) Hanif, Z.; Ahmed, F. R.; Shin, S. W.; Kim, Y.-K.; Um, S. H. Colloids Surf. B. Biointerfaces, http://dx.doi.org/10.1016/j.colsurfb.2014.04.018. (95) Yanamala, N.; Farcas, M. T.; Hatfield, M. K.; Kisin, E. R.; Kagan, V. E.; Geraci, C. L.; Shvedova, A. A. ACS Sustainable Chem. Eng. 2014. (96) Dreher, K. L. Toxicol. Sci. 2004, 77, 3-5. (97) El-Ansary, A.; Al-Daihan, S.; Bacha, A.; Kotb, M., In Oxidative Stress and Nanotechnology, Armstrong, D.; Bharali, D. J., Eds. Humana Press: New York, 2013, pp 47-74. (98) Hoeger, I.; Rojas, O. J.; Efimenko, K.; Velev, O. D.; Kelley, S. S. Soft Matter 2011, 7, 1957-1967. (99) Sugiyama, J.; Chanzy, H.; Maret, G. Macromolecules 1992, 25, 4232-4234. (100) Habibi, Y.; Heim, T.; Douillard, R. J. Polymer Sci. B Polymer Phys. 2008, 46, 1430-1436. (101) Dugan, J. M.; Gough, J. E.; Eichhorn, S. J. Biomacromolecules 2010, 11, 2498-2504. (102) Dugan, J. M.; Collins, R. F.; Gough, J. E.; Eichhorn, S. J. Acta Biomater. 2013, 9, 4707-4715. (103) Faraj, K. A.; Van Kuppevelt, T. H.; Daamen, W. F. Tissue Eng. 2007, 13, 2387-2394. (104) Shunqing, T.; Wei, Y.; Xuan, M. Biomed. Mater. 2007, 2, S129.


Page 53 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(105) Steele, T. J.; Huang, C.; Nguyen, E.; Sarig, U.; Kumar, S.; Widjaja, E.; Loo, J. C.; Machluf, M.; Boey, F.; Vukadinovic, Z.; Hilfiker, A.; Venkatraman, S. J. Mater. Sci. Mater. Med. 2013, 24, 2013-2027. (106) Pooyan, P.; Kim, I. T.; Jacob, K. I.; Tannenbaum, R.; Garmestani, H. Polymer 2013, 54, 21052114. (107) Li, R.; Zhang, Y.; Zhu, L.; Yao, J. J. Appl. Polym. Sci. 2012, 124, 2080-2086. (108) Fortunati, E.; Peltzer, M.; Armentano, I.; Torre, L.; Jiménez, A.; Kenny, J. M. Carbohydr. Polym. 2012, 90, 948-956. (109) Patrício, P. S. d. O.; Pereira, F. V.; dos Santos, M. C.; de Souza, P. P.; Roa, J. P. B.; Orefice, R. L. J. Appl. Polym. Sci. 2013, 127, 3613-3621. (110) Rueda, L.; Saralegi, A.; Fernández-d’Arlas, B.; Zhou, Q.; Alonso-Varona, A.; Berglund, L. A.; Mondragon, I.; Corcuera, M. A.; Eceiza, A. Cellulose 2013, 20, 1819-1828. (111) Fox, J. D.; Capadona, J. R.; Marasco, P. D.; Rowan, S. J. J. Am. Chem. Soc. 2013, 135, 5167-5174. (112) Pooyan, P.; Tannenbaum, R.; Garmestani, H. J. Mech. Behav. Biomed. Mater. 2012, 7, 50-59. (113) Pullawan, T.; Wilkinson, A. N.; Eichhorn, S. J. Biomacromolecules 2012, 13, 2528-2536. (114) Pullawan, T.; Wilkinson, A.; Eichhorn, S. J. Mater. Sci. 2013, 48, 7847-7855. (115) Chan, G.; Mooney, D. J. Trends Biotechnol. 2008, 26, 382-392. (116) Kundu, B.; Rajkhowa, R.; Kundu, S. C.; Wang, X. Adv. Drug Del. Rev. 2013, 65, 457-470. (117) Li, W.; Guo, R.; Lan, Y.; Zhang, Y.; Xue, W.; Zhang, Y. J. Biomed. Mater. Res., Part A 2014, 102A, 1131-1139. (118) de Mesquita, J. P.; Patricio, P. S.; Donnici, C. L.; Petri, D. F. S.; de Oliveira, L. C. A.; Pereira, F. V. Soft Matter 2011, 7, 4405-4413. (119) Decher, G. Science 1997, 277, 1232-1237. (120) Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203-3224. (121) Di Martino, A.; Sittinger, M.; Risbud, M. V. Biomaterials 2005, 26, 5983-5990. (122) de Mesquita, J. o. P.; Donnici, C. L.; Pereira, F. V. Biomacromolecules 2010, 11, 473-480.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 60

(123) Noishiki, Y.; Nishiyama, Y.; Wada, M.; Kuga, S.; Magoshi, J. J. Appl. Polym. Sci. 2002, 86, 34253429. (124) Lapidot, S.; Meirovitch, S.; Sharon, S.; Heyman, A.; Kaplan, D. L.; Shoseyov, O. Nanomedicine 2012, 7, 1409-1423. (125) Chen, G.-q.; Wang, Y. Chin. J. Polym. Sci. 2013, 31, 719-736. (126) Misra, S. K.; Ansari, T. I.; Valappil, S. P.; Mohn, D.; Philip, S. E.; Stark, W. J.; Roy, I.; Knowles, J. C.; Salih, V.; Boccaccini, A. R. Biomaterials 2010, 31, 2806-2815. (127) Misra, S. K.; Mohn, D.; Brunner, T. J.; Stark, W. J.; Philip, S. E.; Roy, I.; Salih, V.; Knowles, J. C.; Boccaccini, A. R. Biomaterials 2008, 29, 1750-1761. (128) Misra, S. K.; Ohashi, F.; Valappil, S. P.; Knowles, J. C.; Roy, I.; Silva, S. R. P.; Salih, V.; Boccaccini, A. R. Acta Biomater. 2010, 6, 735-742. (129) Ten, E.; Jiang, L.; Wolcott, M. P. Carbohydr. Polym. 2012, 90, 541-550. (130) Ten, E.; Bahr, D. F.; Li, B.; Jiang, L.; Wolcott, M. P. Ind. Eng. Chem. Res. 2012, 51, 2941-2951. (131) Ten, E.; Turtle, J.; Bahr, D.; Jiang, L.; Wolcott, M. Polymer 2010, 51, 2652-2660. (132) Jiang, L.; Morelius, E.; Zhang, J.; Wolcott, M.; Holbery, J. J. Compos. Mater. 2008, 42, 2629-2645. (133) Ten, E.; Jiang, L.; Wolcott, M. P. Carbohydrate Polymers 2013, 92, 206-213. (134) Yu, H.-Y.; Qin, Z.-Y.; Liu, Y.-N.; Chen, L.; Liu, N.; Zhou, Z. Carbohydr. Polym. 2012, 89, 971978. (135) Yu, H.-Y.; Qin, Z.-Y.; Liu, L.; Yang, X.-G.; Zhou, Y.; Yao, J.-M. Composites Sci. Technol. 2013, 87, 22-28. (136) Yu, H.-Y.; Qin, Z.-Y.; Yan, C.-F.; Yao, J.-M. ACS Sustainable Chem. Eng. 2014, 2, 875-886. (137) Hossain, K. Z.; Ahmed, I.; Parsons, A.; Scotchford, C.; Walker, G.; Thielemans, W.; Rudd, C. J. Mater. Sci. 2012, 47, 2675-2686. (138) Rescignano, N.; Fortunati, E.; Montesano, S.; Emiliani, C.; Kenny, J. M.; Martino, S.; Armentano, I. Carbohydr. Polym. 2014, 99, 47-58. (139) Pei, A.; Zhou, Q.; Berglund, L. A. Composites Sci. Technol. 2010, 70, 815-821. 54 ACS Paragon Plus Environment

Page 55 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(140) Goffin, A.-L.; Raquez, J.-M.; Duquesne, E.; Siqueira, G.; Habibi, Y.; Dufresne, A.; Dubois, P. Biomacromolecules 2011, 12, 2456-2465. (141) Braun, B.; Dorgan, J. R.; Hollingsworth, L. O. Biomacromolecules 2012, 13, 2013-2019. (142) Fortunati, E.; Puglia, D.; Luzi, F.; Santulli, C.; Kenny, J. M.; Torre, L. Carbohydr. Polym. 2013, 97, 825-836. (143) Fortunati, E.; Puglia, D.; Monti, M.; Santulli, C.; Maniruzzaman, M.; Kenny, J. M. J. Appl. Polym. Sci. 2013, 128, 3220-3230. (144) George, J.; Sajeevkumar, V. A.; Ramana, K. V.; Sabapathy, S. N.; Siddaramaiah J. Mater. Chem. 2012, 22, 22433-22439. (145) Cronholm, P.; Karlsson, H. L.; Hedberg, J.; Lowe, T. A.; Winnberg, L.; Elihn, K.; Wallinder, I. O.; Möller, L. Small 2013, 9, 970-982. (146) Santerre, J. P.; Woodhouse, K.; Laroche, G.; Labow, R. S. Biomaterials 2005, 26, 7457-7470. (147) Pei, A.; Malho, J.-M.; Ruokolainen, J.; Zhou, Q.; Berglund, L. A. Macromolecules 2011, 44, 44224427. (148) Rueda, L.; Fernández d’Arlas, B.; Zhou, Q.; Berglund, L. A.; Corcuera, M. A.; Mondragon, I.; Eceiza, A. Composites Sci. Technol. 2011, 71, 1953-1960. (149) Rueda, L.; Saralegui, A.; Fernández d’Arlas, B.; Zhou, Q.; Berglund, L. A.; Corcuera, M. A.; Mondragon, I.; Eceiza, A. Carbohydr. Polym. 2013, 92, 751-757. (150) Seidi, A.; Ramalingam, M.; Elloumi-Hannachi, I.; Ostrovidov, S.; Khademhosseini, A. Acta Biomater. 2011, 7, 1441-1451. (151) Bhardwaj, N.; Kundu, S. C. Biotechnol. Adv. 2010, 28, 325-347. (152) Greiner, A.; Wendorff, J. H. Angew. Chem. Int. Ed. 2007, 46, 5670-5703. (153) Li, D.; Xia, Y. Adv. Mater. 2004, 16, 1151-1170. (154) Roh, J. D.; Nelson, G. N.; Brennan, M. P.; Mirensky, T. L.; Yi, T.; Hazlett, T. F.; Tellides, G.; Sinusas, A. J.; Pober, J. S.; Saltzman, W. M.; Kyriakides, T. R.; Breuer, C. K. Biomaterials 2008, 29, 1454-1463. 55 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 60

(155) Cleary, M. A.; Geiger, E.; Grady, C.; Best, C.; Naito, Y.; Breuer, C. Trends Mol. Med. 2012, 18, 394-404. (156) Pham, Q. P.; Sharma, U.; Mikos, A. G. Tissue Engineering 2006, 12, 1197-1211. (157) Nam, J.; Huang, Y.; Agarwal, S.; Lannutti, J. Tissue Eng. 2007, 13, 2249-2257. (158) Ekaputra, A. K.; Prestwich, G. D.; Cool, S. M.; Hutmacher, D. W. Biomacromolecules 2008, 9, 2097-2103. (159) Ki, C. S.; Kim, J. W.; Hyun, J. H.; Lee, K. H.; Hattori, M.; Rah, D. K.; Park, Y. H. J. Appl. Polym. Sci. 2007, 106, 3922-3928. (160) Leong, M. F.; Rasheed, M. Z.; Lim, T. C.; Chian, K. S. J. Biomed. Mater. Res., Part A 2009, 91A, 231-240. (161) Yixiang, D.; Yong, T.; Liao, S.; Chan, C. K.; Ramakrishna, S. Tissue Eng. Part A 2008, 14, 13211329. (162) Sundararaghavan, H. G.; Metter, R. B.; Burdick, J. A. Macromol. Biosci. 2010, 10, 265-270. (163) Pham, Q. P.; Sharma, U.; Mikos, A. G. Biomacromolecules 2006, 7, 2796-2805. (164) Zhong, S.; Zhang, Y.; Lim, C. T. Tissue Engineering - Part B: Reviews 2012, 18, 77-87. (165) Liang, D.; Hsiao, B. S.; Chu, B. Adv. Drug Del. Rev. 2007, 59, 1392-1412. (166) Barnes, C. P.; Sell, S. A.; Boland, E. D.; Simpson, D. G.; Bowlin, G. L. Adv. Drug Del. Rev. 2007, 59, 1413-1433. (167) Changsarn, S.; Mendez, J. D.; Shanmuganathan, K.; Foster, E. J.; Weder, C.; Supaphol, P. Macromol. Rapid Commun. 2011, 32, 1367-1372. (168) Huang, J.; Liu, L.; Yao, J. Fiber. Polym. 2011, 12, 1002-1006. (169) Lee, J.; Deng, Y. Macromol. Res. 2012, 20, 76-83. (170) He, X.; Xiao, Q.; Lu, C.; Wang, Y.; Zhang, X.; Zhao, J.; Zhang, W.; Zhang, X.; Deng, Y. Biomacromolecules 2014, 15, 618-627. (171) Jia, B.; Li, Y.; Yang, B.; Xiao, D.; Zhang, S.; Rajulu, A. V.; Kondo, T.; Zhang, L.; Zhou, J. Cellulose 2013, 20, 1911-1923. 56 ACS Paragon Plus Environment

Page 57 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(172) Ravi, S.; Chaikof, E. L. Regen. Med. 2009, 5, 107-120. (173) Park, D. J.; Choi, Y.; Heo, S.; Cho, S. Y.; Jin, H.-J. J. Nanosci. Nanotechnol. 2012, 12, 6139-6144. (174) Dahlin, R. L.; Kasper, F. K.; Mikos, A. G. Tissue Eng. Part B Rev. 2011, 17, 349-364. (175) Liu, D.; Yuan, X.; Bhattacharyya, D. J. Mater. Sci. 2012, 47, 3159-3165. (176) Zhou, C.; Shi, Q.; Guo, W.; Terrell, L.; Qureshi, A. T.; Hayes, D. J.; Wu, Q. ACS Appl. Mater. Interfaces 2013, 5, 3847-3854. (177) Shi, Q.; Zhou, C.; Yue, Y.; Guo, W.; Wu, Y.; Wu, Q. Carbohydr. Polym. 2012, 90, 301-308. (178) Li, Y.; Ko, F. K.; Hamad, W. Y. Biomacromolecules 2013, 14, 3801-3807. (179) Xiang, C.; Joo, Y. L.; Frey, M. W. J. Biobased Mater. Bioenergy 2009, 3, 147-155. (180) Tamayol, A.; Akbari, M.; Annabi, N.; Paul, A.; Khademhosseini, A.; Juncker, D. Biotechnol. Adv. 2013, 31, 669-687. (181) Zoppe, J. O.; Peresin, M. S.; Habibi, Y.; Venditti, R. A.; Rojas, O. J. ACS Appl. Mater. Interfaces 2009, 1, 1996-2004. (182) Zhou, C.; Chu, R.; Wu, R.; Wu, Q. Biomacromolecules 2011, 12, 2617-2625. (183) Park, W.-I.; Kang, M.; Kim, H.-S.; Jin, H.-J. Macromol. Symp. 2007, 249-250, 289-294. (184) Peresin, M. S.; Habibi, Y.; Zoppe, J. O.; Pawlak, J. J.; Rojas, O. J. Biomacromolecules 2010, 11, 674-681. (185) Mathiowitz, E.; Lavin, D. M.; Hopkins, R. A. Ther. Deliv. 2013, 4, 1075-1077. (186) Tuzlakoglu, K.; Reis, R. L. Tissue Eng. Part B Rev. 2009, 15, 17-27. (187) Ureña-Benavides, E. E.; Kitchens, C. L. Mol. Cryst. Liquid Cryst. 2012, 556, 275-287. (188) Ureña-Benavides, E. E.; Brown, P. J.; Kitchens, C. L. Langmuir 2010, 26, 14263-14270. (189) Ureña-Benavides, E. E.; Kitchens, C. L. Macromolecules 2011, 44, 3478-3484. (190) Hossain, K. M. Z.; Hasan, M. S.; Boyd, D.; Rudd, C. D.; Ahmed, I.; Thielemans, W. Biomacromolecules 2014, 15, 1498-1506. (191) Duarte, A. R. C.; Santo, V. E.; Alves, A.; Silva, S. S.; Moreira-Silva, J.; Silva, T. H.; Marques, A. P.; Sousa, R. A.; Gomes, M. E.; Mano, J. F.; Reis, R. L. J. Supercrit. Fluids 2013, 79, 177-185. 57 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 58 of 60

(192) Mikos, A. G.; Sarakinos, G.; Leite, S. M.; Vacant, J. P.; Langer, R. Biomaterials 1993, 14, 323-330. (193) Wegst, U. G. K.; Schecter, M.; Donius, A. E.; Hunger, P. M. Phil. Trans. R. Soc. A 2010, 368, 2099-2121. (194) Kohnke, T.; Lin, A.; Elder, T.; Theliander, H.; Ragauskas, A. J. Green Chem. 2012, 14, 1864-1869. (195) Yixiang, W.; Chunyu, C.; Lina, Z. Macromol. Mater. Eng. 2010, 295, 137-145. (196) Lin, N.; Bruzzese, C.; Dufresne, A. ACS Appl. Mater. Interfaces 2012, 4, 4948-4959. (197) Blaker, J. J.; Lee, K. Y.; Mantalaris, A.; Bismarck, A. Composites Sci. Technol. 2010, 70, 18791888. (198) Dash, R.; Li, Y.; Ragauskas, A. J. Carbohydr. Polym. 2012, 88, 789-792. (199) Zhou, Y.; Fu, S.; Pu, Y.; Pan, S.; Levit, M. V.; Ragauskas, A. J. RSC Adv. 2013, 3, 19272-19277. (200) Zhang, H.; Hussain, I.; Brust, M.; Butler, M. F.; Rannard, S. P.; Cooper, A. I. Nature Mater. 2005, 4, 787-793. (201) Murugan, R.; Ramakrishna, S. Tissue Eng. 2007, 13, 1845-1866. (202) Lee, K. Y.; Mooney, D. J. Chemical Reviews 2001, 101, 1869-1880. (203) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337-4351. (204) Risbud, M. V.; Sittinger, M. Trends Biotechnol. 2002, 20, 351-356. (205) Vermonden, T.; Censi, R.; Hennink, W. E. Chem. Rev. 2012, 112, 2853-2888. (206) Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biomacromolecules 2011, 12, 1387-1408. (207) Calvert, P. Adv. Mater. 2009, 21, 743-756. (208) Schexnailder, P.; Schmidt, G. Colloid. Polym. Sci. 2009, 287, 1-11. (209) Das, D.; Kar, T.; Das, P. K. Soft Matter 2012, 8, 2348-2365. (210) Rodrigues, F. H. A.; Spagnol, C.; Pereira, A. G. B.; Martins, A. F.; Fajardo, A. R.; Rubira, A. F.; Muniz, E. C. J. Appl. Polym. Sci. 2014, 131, 39725. (211) Cha, R.; He, Z.; Ni, Y. Carbohydr. Polym. 2012, 88, 713-718. (212) Karaaslan, M. A.; Tshabalala, M. A.; Yelle, D. J.; Buschle-Diller, G. Carbohydr. Polym. 2011, 86, 192-201. 58 ACS Paragon Plus Environment

Page 59 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(213) Abitbol, T.; Johnstone, T.; Quinn, T. M.; Gray, D. G. Soft Matter 2011, 7, 2373-2379. (214) Dai, Q.; Kadla, J. F. J. Appl. Polym. Sci. 2009, 114, 1664-1669. (215) Dash, R.; Foston, M.; Ragauskas, A. J. Carbohydr. Polym. 2013, 91, 638-645. (216) Wang, Y.; Chen, L. Carbohydr. Polym. 2011, 83, 1937-1946. (217) Yang, J.; Han, C.-R.; Duan, J.-F.; Ma, M.-G.; Zhang, X.-M.; Xu, F.; Sun, R.-C. Cellulose 2013, 20, 227-237. (218) Zhou, C.; Wu, Q.; Yue, Y.; Zhang, Q. J. Colloid Interface Sci. 2011, 353, 116-123. (219) Yang, J.; Han, C.-R.; Duan, J.-F.; Ma, M.-G.; Zhang, X.-M.; Xu, F.; Sun, R.-C.; Xie, X.-M. J. Mater. Chem. 2012, 22, 22467-22480. (220) Yang, X.; Bakaic, E.; Hoare, T.; Cranston, E. D. Biomacromolecules 2013, 14, 4447-4455. (221) Han, J.; Lei, T.; Wu, Q. Carbohydr. Polym. 2014, 102, 306-316. (222) Zhang, X.; Huang, J.; Chang, P. R.; Li, J.; Chen, Y.; Wang, D.; Yu, J.; Chen, J. Polymer 2010, 51, 4398-4407. (223) Yang, J.; Han, C.-R.; Duan, J.-F.; Xu, F.; Sun, R.-C. ACS Appl. Mater. Interfaces 2013, 5, 31993207. (224) Sanabria-DeLong, N.; Crosby, A. J.; Tew, G. N. Biomacromolecules 2008, 9, 2784-2791. (225) Zhao, H.; Ma, L.; Gong, Y.; Gao, C.; Shen, J. J. Mater. Sci. Mater. Med. 2009, 20, 135-143. (226) Lin, N.; Dufresne, A. Biomacromolecules 2013, 14, 871-880. (227) Brown, E. E.; Hu, D.; Abu Lail, N.; Zhang, X. Biomacromolecules 2013, 14, 1063-1071. (228) Kyle, S.; Aggeli, A.; Ingham, E.; McPherson, M. J. Trends Biotechnol. 2009, 27, 423-433. (229) Haghpanah, J. S.; Tu, R.; Da Silva, S.; Yan, D.; Mueller, S.; Weder, C.; Foster, E. J.; Sacui, I.; Gilman, J. W.; Montclare, J. K. Biomacromolecules 2013, 14, 4360-4367.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 60 of 60

TABLE OF CONTENTS GRAPHIC For Table of Contents Only

The potential of Cellulose Nanocrystals in Tissue Engineering strategies Rui M. A. Domingues, Manuela E. Gomes*, Rui L. Reis


Uniaxially aligned electrospun all-cellulose nanocomposite nanofibers reinforced with cellulose nanocrystals: scaffold for tissue engineering.

Cellulose Nanocrystals Bionanocomposite Hydrogels for Tissue Engineering Applications.

Incorporation of poly(ethylene glycol) grafted cellulose nanocrystals in poly(lactic acid) electrospun nanocomposite fibers as potential scaffolds for bone tissue engineering.

Tissue-engineering-based strategies for regenerative endodontics.

Cell-Based Strategies for Meniscus Tissue Engineering.

Imaging strategies for tissue engineering applications.

The potential impact of bone tissue engineering in the clinic.

Nanostructured materials from hydroxyethyl cellulose for skin tissue engineering.

Potential of graphene for tissue engineering applications.

Control of scar tissue formation in the cornea: strategies in clinical and corneal tissue engineering.

New strategies in kidney regeneration and tissue engineering.

Tissue engineering strategies for fetal myelomeningocele repair in animal models.

Fibroblast-endothelial partners for vascularization strategies in tissue engineering.

Preparation and characterization of functionalized cellulose nanocrystals.

The feasibility of using irreversible electroporation to introduce pores in bacterial cellulose scaffolds for tissue engineering.

Kinetic aspects of the adsorption of xyloglucan onto cellulose nanocrystals.

Individually Dispersed Wood-Based Cellulose Nanocrystals.

Cellulose nanocrystals: synthesis, functional properties, and applications.

Periodontal tissue engineering strategies based on nonoral stem cells.

Tissue engineering strategies for promoting vascularized bone regeneration.

Skeletal muscle tissue engineering: strategies for volumetric constructs.

Cellular and developmental strategies aimed at kidney tissue engineering.

3D biofabrication strategies for tissue engineering and regenerative medicine.

Tissue Engineering Strategies as Tools for Personalized Meningioma Treatment.