main-tain normal tissue or organ functions. To these ends, one or more of the following three components:
progenitor cells, signaling molecules, and engineered biomaterials or scaffolds are applied (Langer and Vacanti, 1993). The advancement of TE requires new biomaterials to deliver important biochemical moi-eties (e.g., growth factors) to the engineered tissues, to direct tissue growth, and to improve monitoring and evaluating of the regenerating tissue.
Nanotechnology-based approaches are currently being pursued for a variety of biomedical applications. Nanotechnology is a relatively new field of science broadly defined as research and tech-nology development at length scales between 1 and 100 nm to create materials, gain fundamental insights into their properties, and to use the nanoscale materials as components or building blocks to create novel structures, or devices (Rodgers et al., 2006). At these length scales, materials show unique properties and functions. However, in certain cases, the length scales for these novel properties maybe under 1 nm (down to 0.1 nm for atomic and molecular manipulation) or over 100 nm (up to 300 nm in case of nanopolymers and nanocomposites). Nanotechnology is a convergent technology in which, the boundaries separating discrete disciplines become blurred. Biochemists, materials scientists, electrical engineers, and molecular biologists may all be considered experts in the field if they are involved in the development of nanosized structures.
Specifically for TE, nanotechnology-based approaches allow the synthesis of unique nanobiomaterials having nanoscale features that can mimic the natural extracellular matrix to affect the cellular functions (e.g., adhesion, mobility, and differentiation) (Harrison and Atala, 2007; Kim and Fisher, 2007). Further, these nanobiomaterials could be developed with multifunctional capabilities as delivery agents of sig-naling molecules and genes as well as efficient imaging probes to noninvasively monitor implanted cells, and the process of tissue regeneration in tissue-engineered constructs. This chapter provides the reader
11
Nanobiomaterials for Tissue Engineering
Pramod K. Avti
Stony Brook University
Sunny C. Patel
Stony Brook University
Pushpinder Uppal
Stony Brook University
Grace O’Malley
Stony Brook University
Joseph Garlow
Stony Brook University
Balaji Sitharaman
Stony Brook University
11.1 Introduction ... 11-1 11.2 Nanobiomaterials to Improve Bulk and Surface Properties
of Tissue Engineering Scaffolds ... 11-2 Nanofibrous Scaffolds • Nanobiomaterial-Incorporated Polymer
Scaffolds
11.3 Nanobiomaterials for Therapeutic Delivery ... 11-5 11.4 Nanobiomaterials to Image the Process of Tissue Formation ....11-9 11.5 Continuing and Future Developments... 11-13 Abbreviations ... 11-13 References ... 11-15
11-2 Tissue Engineering a perspective of nanobiomaterials for TE. It discusses the various nanobiomaterials been developed to (a) improve bulk and surface properties of TE scaffolds; (b) deliver biochemical moieties (e.g., genes, growth factors); and (c) image the process of tissue formation.
11.2 Nanobiomaterials to Improve Bulk and Surface Properties of Tissue Engineering Scaffolds
Nanobiomaterial-based approaches have been used in the developments of two types of TE structures:
(1) Nanofibrous scaffolds and (2) Nanobiomaterial-incorporated polymer composite scaffolds.
11.2.1 Nanofibrous Scaffolds
Scaffolds are porous biomaterials and play a pivotal role in the TE paradigm by providing temporary structural support, guiding cells to grow, assisting the transport of essential nutrients and waste products, and facilitating the formation of functional tissues and organs. Nanofiber scaffolds are TE scaffolds with nanoscopic structure and morphologies fabricated using natural and synthetic materials. These materi-als are biodegradable or nonbiodegradable polymers and generally biocompatible. Some examples of the natural materials used as starting materials for the development of nanofiber scaffolds are self-assembling polypeptides, DNA, RNA, carbohydrates, peptides, collagen, fibrin, glycosaminoglycans, fibrinogen, gel-atin, elastin, silk, hyaluronan, and chitosan (Matthews et al., 2002; Min et al., 2004; Silva et al., 2004;
Bhattarai et al., 2005; Li et al., 2005; Hamdi et al., 2009; Carneiro et al., 2010). Examples of synthetic mate-rials include poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(hydroxyethyl methacrylate), poly(lactic acid) (PLA), poly(glycolic acid), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), poly(methyl methacrylate) (PMMA), poly(propylene fumarate) (PPF) (Ding et al., 2002; Kenawy et al., 2003; Gupta et al., 2005; Kim et al., 2005; Chen et al., 2008; Choi et al., 2008; Corey et al., 2008; Powell and Boyce, 2008). Nanofiber scaffolds have special characteristics such as high surface area to volume ratio, functional groups for high density functionalization, and high porosities. The nanofibers can also mimic natural extracellular matrix (ECM). The fabrication techniques used in synthesizing these scaffolds are phase separation (Smith and Ma, 2004), melt-blowing, template synthesis, electrospinning (Li et al., 2002), and self-assembly (Whitesides and Boncheva, 2002). Among these methods, nanofibers obtained from electrospinning and self-assembly have widespread applications in bone, skin, neural, cartilage, vascular heart, and lung TE (Pham et al., 2006; Rubenstein et al., 2007; Chew et al., 2008; Venugopal et al., 2008).
The electrospinning method allows the fabrication of solid, hollow, or core−shell nanofiber scaffolds, where the nanofibers can be randomly arranged or aligned along a particular direction (Bini et al., 2004;
Corey et al., 2007). The hollow fiber scaffolds (Figure 11.1) are suitable for loading drugs, and enzymes to improve tissue regeneration (Bini et al., 2004).
The core−shell fibers are also well suited for drug-delivery applications as the core helps in loading the drug and the shell controls release kinetics. Synthetic and overexpressed peptide and protein pre-cursors have been widely used as starting materials for the development of self-assembled nanofiber scaffolds (Koide et al., 2005; Kotch and Raines, 2006; Paramonov et al., 2006; Woolfson and Ryadnov, 2006; Gauba and Hartgerink, 2007). For instance, elastin is a self-assembling polymeric protein with good mechanical strength properties with potential applications for vascular TE (Bellingham et al., 2003; Miao et al., 2005; Daamen et al., 2007). Recently, recombinant (synthetic) polypeptides of elastin were self-assembled to form novel macromolecular nanostructures (Bellingham et al., 2003; Vieth et al., 2007). Self-assembled nanofiber scaffolds are well-suited to incorporate cell signaling molecules that can affect progenitor cell behavior such as their attachment, differentiation, and proliferation (Hofmann et al., 2006; Meinel et al., 2006, 2009). The complexity of the self-assembly techniques leads to low yields and relatively high cost; its main limitations compared to the electrospinning method. Table 11.1 lists some of the recent advances in the development of nanofibrous scaffolds for TE.
10kV
(a) (b)
×65 200 μm 10kV ×9,000 2 μm
FIGURE 11.1 SEM imaging of micro- and nanofiber electrospun poly(1-caprolactone)/poly(d,l-lactic-co-glycolic acid) tubular scaffolds designed for regenerating sciatic nerve transections. (a) Tube lumen and (b) zoomed details of the tube wall. Both nano- and microfibers are visible. Fiber links are obtained via partial solvent evaporation and polymer annealing subsequent to electrospinning in order to increase the overall prosthesis mechanical properties.
(Reprinted from Biomaterials 26(31), Bhattarai N. et al. Electrospun chitosan based nanofibers and their cellular compatibility, 6176–84. Copyright 2005, with permission from Elsevier.)
TABLE 11.1 Nanofibrous Scaffolds Nanofiber Material Intended
Application Functions and Biological Response/Improvements Reference
PLGA Neural TE Neurite formation and elongation Lee et al. (2009)
Bladder tissue
engineering Increased bladder cell adhesion and growth, increased production of elastin, and collagen proteins enhance in urinary bladder wall replacement
Pattison et al. (2005)
PGS Retinal
transplantation Improved the growth of graft-host cells without signs
of inflammation Pritchard et al. (2010)
Vascular
regeneration Decreased thrombogenecity (platelet adhesion and aggregation) and inflammatory response when used as blood contacting surface
Motlagh et al. (2006)
Cardiac tissue
engineering Bioinert, biocompatible, wide degradation properties, good mechanical properties matching the physical characteristics of heart tissue
Chen et al. (2008)
PEG Cartilage
regeneration Improved cellularity, collagen and glycosaminoglycan
content Mahmood et al. (2006)
Wound healing Fibronectin-coupled PEG are cytocompatible, improves proliferation and migration of fibroblasts both in vitro and in vivo
Ghosh et al. (2006)
PLLA Neural tissue
engineering Improves neurite growth, elongation, and
differentiation Corey et al. (2007);
Yang et al. (2004, 2005)
Cardiac tissue
engineering Increased contractile machinery (sarcomeres) in
cardiomyocytes Zong et al. (2005)
Bone tissue
engineering Increased osteoblast adhesion, proliferation,
mineralization, and protein marker expression Woo et al. (2003, 2006)
CNFs Neural tissue
engineering Increased elastic modulus, neuronal cell adhesion,
neurite extension, and decreased astrocyte adhesion McKenzie et al. (2004) Vascular tissue
engineering Supports the aggregation and enhances the migration
ability of endothelial cells Han et al. (2009) continued
11-4 Tissue Engineering
11.2.2 Nanobiomaterial-Incorporated Polymer Scaffolds
Nanoparticles have also been incorporated into porous scaffolds to improve their bulk and surface properties. A large number of porous scaffolds do not possess mechanical properties (a bulk property) necessary for in vivo applications (Mistry and Mikos, 2005). Thus, nanobiomaterials are incorporated into these scaffolds to improve their mechanical properties. For instance, carbon nanotubes have high Young’s modulus (~1 TPa), and therefore, have been incorporated as reinforcing agents into porous poly-mer scaffolds to improve their mechanical properties (Lukic et al., 2005; Shi et al., 2005) (Figure 11.2).
Further, organic or inorganic nanomaterials have also been incorporated to induce bioactive properties (a surface property) into the scaffolds. The rationale here is that the physical interface between biological systems (e.g., proteins, DNA) and nanobiomaterials share a number of common (e.g., similar size scales) as well as complementary (e.g., inorganic/organic versus biological composition) attributes. Since, the TABLE 11.1 (continued) Nanofibrous Scaffold
Nanofiber Material Intended
Application Functions and Biological Response/Improvements Reference Bone tissue
engineering Increased osteoblast adhesion, enhanced mineral
deposition, decreased fibroblast adhesion Khang et al. (2006);
Price et al. (2003) Peptide nanofibers Bladder tissue
regeneration Promotes bladder smooth muscle cells attachment,
matrix production and spindled morphology Harrington et al.
(2006) Neural
regeneration IKVAV-Peptide nanofibers increased the neural progenitor cells attachment, migration, neurite outgrowth and undergo selective and rapid differentiation.
Silva et al. (2004)
Vascular tissue
regeneration Heparin binding-peptide nanofibers influenced the
tube formation in endothelial cells Rajangam et al. (2006, 2008)
Cardiac tissue
engineering Heparin binding-peptide nanofibers restored the hemodynamic functions in acute myocardial infarction
Rajangam et al. (2006, 2008)
PCL Skin grafting Improved growth, longevity of keratinocytes and
fibroblasts during wound healing Reed et al. (2009) Vascular tissue
engineering Modulated smooth muscle cells behavior to express contractile phenotype, attained spindle shape, oriented and directional migration
Xu et al. (2004)
Cardiac tissue
engineering Enhanced cardiomyocytes attachment, growth and proliferation. Increased synchronized contraction and contractile machinery (actin, tropomyosin, cardiac troponin)
Ishii et al. (2005); Shin et al. (2004)
Bone tissue
engineering Improved cellular adhesion, penetration into the scaffold thereby releasing ECM and helps in differentiation
Yoshimoto et al.
(2003); Shin et al.
(2004); Li et al.
(2005a,b) Polyurethane Skin grafting Increased rate of epithelialization, well organized
dermis formation Khil et al. (2003)
Chitin nanofiber Skin tissue
engineering Promoted keratinocyte and fibroblast cellular
attachment and proliferation Noh et al. (2006)
SF Skin tissue
engineering Highly porous, high surface area and improved mechanical properties of SFs responsible for use in wound dressing material and skin regeneration application
Kim et al. (2003)
PET Vascular tissue
engineering Increased hydrophobicity of scaffold enables endothelial cells to attain polygonal morphology and express cell adhesion markers PECAM, ICAM, VCAM responsible for vascularization
Ma et al. (2005)
nanoscale interactions in tissues (e.g., protein–protein interaction) are crucial for controlling many cellular functions such as cell–cell interactions, migration, proliferation, and ECM production (Benoit and Anseth, 2005), the nanomaterials could affect these interactions to achieve the desired result. For instance, incorpo-rating ceramic nanoparticles into polymer scaffolds has been shown to induce bioactive properties into the scaffolds (Liu et al., 2006). The bioactive properties are induced by the ceramic nanoparticles by improving the adsorption characteristics of proteins such as fibronectin, vitronectin, laminin, and collagen involved in osteoblast functions (Webster et al., 1999, 2000, 2001). Some of the common fabrication techniques to incorporate nanoparticles into scaffolds include solvent casting, salt leaching, and freeze drying. Using all these methods, the individual nanoparticles are randomly distributed into the scaffolds. More examples of the nanoparticle-incorporated scaffolds examples and their applications are listed in Table 11.2.
11.3 Nanobiomaterials for Therapeutic Delivery
Controlled production and/or delivery of tissue-inducing macromolecules such as cytokines and growth factors are widely applied strategies in regenerative medicine. The physical and chemical properties of a large number of nanobiomaterials make them suitable for a variety of therapeutic and drug-delivery
Acc. V
20.0 kV 3.0 25000 x SE 10.1 HivacSpot magn Det WD 1 μm
Acc. V
15.0 kV 3.0Spot magn20000 x SEDet WD14.5 Hivac 1 μm
(a)
(b)
FIGURE 11.2 SEM images of single-walled carbon nanotubes (SWCNTs) incorporated polymer scaffolds.
(a) SWCNT bundles pulled out of the fracture surface from SWCNT polymer scaffold (0.05% by weight of SWCNT was dispersed in the scaffold). (b) Crack region propagation is prevented by spanning the SWCNT bundles. (Shi, X. et al. Rheological behaviour and mechanical characterization of injectable poly(propylene fumarate)/single-walled carbon nanotube composites for bone tissue engineering. Nanotechnology. 16: S531–8. Copyright 2005 IOP Science.)
11-6 Tissue Engineering applications in TE (Figure 11.3). The external surfaces of the nanobiomaterials can be covalently or noncovalently functionalized with biological moieties that target specific cell or tissues types and/or pharmaceutical agents. Here, the nanobiomaterials target a specific cell or tissue type, and act as biologi-cal cargo vehicles to transport, and deliver therapeutic agents via a biochemibiologi-cal or biophysibiologi-cal stimulus (Langer and Tirrell, 2004).
Furthermore, the nanobiomaterial themselves can be used as a therapeutic agent by exploiting their unique physical properties (Shi et al., 2010). For example, the strong optical absorption properties of SWCNTs and gold nanoparticles render them capable of generating acoustic waves upon irradiation.
These waves have shown to affect the process of osteoinduction (Green et al., 2009). Other advantages of using nanobiomaterials for therapeutic purposes, and as delivery vehicles include their nanoscale dimensions, which enhance their retention and permeability in the regenerating tissues (Gannon et al., TABLE 11.2 Nanobiomaterial-Based Polymer/Composite Scaffolds
Nanoparticle Material Intended
Applications Function and Biological Improvements Reference n-HA/PA, n-HA/PA/
MSC Bone Tissue
engineering Enhanced osteogenesis than pure n-HA/PA scaffolds Wang et al. (2007) Bioactive-glass
ceramic nanoparticles Bone tissue
engineering Higher amount of mineral deposited on the composite scaffold, which increased with increasing time of incubation
Peter et al. (2010)
Bioglass-based
glass-ceramic pellets General
applications Bioactive and resorbable nanofibrous coatings can be used to tailor the surface topography of bioactive glass-ceramics
Bretcanu et al.
(2009) Mesoporous bioactive
glasses Bone tissue
engineering In vitro bioactivity of these MBGs scaffolds was
dependent on the chemical composition Zhu et al. (2008), Yan et al. (2006) Forsterite Bone tissue
engineering Significantly promoted cell proliferations, cell adhesion, spread, and growth on the surface of the nanostructured forsterite ceramic cytocompatibility seen in chitosan–nHA porous scaffolds
Thein-Han and Misra (2009)
HA and PEG/PBT Bone tissue
engineering Increased Young’s modulus, tensile strength, and
elongation at break of composite scaffold Liu et al. (1998)
HA/PLLA Bone tissue
engineering Increased compressive modulus and protein
adsorption Wei and Ma (2004)
HA/PLGA Bone tissue
engineering Stimulated cell proliferation and osteogenic
differentiation Kim et al. (2006)
HA/PLGA Bone tissue
engineering In vivo bone formation after 8 weeks of implantation
to critical size defects in rat skulls Kim et al. (2007)
POC Cardiac tissue
engineering Decreased porosity caused a rise in the elastic modulus, ECM proteins promoted cell adhesion in a protein-type- and concentration-dependent manner
Hidalgo-Bastida et al. (2007) Cellulose acetate,
regenerated cellulose Cardiac tissue
engineering Cellulose acetate and regenerated cellulose surfaces promoted cardiac cell growth, enhanced cell connectivity (gap junctions) and electrical functionality
Entcheva et al.
(2004)
Fibrin Cardiac tissue
engineering Dense fibrin scaffolds had mechanical properties
closer to native myocardium than fibrin gels Robinson et al.
(2008), Thomson et al. (2010)
PF Cardiac tissue
engineering PF hydrogel biomaterial can be used as an in situ polymerizable biomaterial for stem cells and their cardiomyocyte derivatives
Shapira-Schweitzer et al. (2009)