Applications of Nanotechnology for Regenerative Medicine

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Applications of Nanotechnology for Regenerative Medicine Benjamin S. Harrison, Sirinrath Sirivisoot Wake Forest Institute for Regenerative Medicine, Wake Forest University, Medical Center BLVD, Winston-Salem, NC, USA

INTRODUCTION While organ transplants have provided renewed life in individuals with failed organs, the reality is that the demand of organs far exceeds the available supply. The potential ability to build organs is therefore an attractive option to fill the deficit in the supply of organs available. In constructing regenerative therapies, there will be a need to develop new tools to aid in the engineering of neo-organs. From chemistry, physics, and biology disciplines has emerged the field of nanotechnology, which has the potential to provide the tools needed to accelerate the engineering of organs. Nanotechnology is a bottom-up approach that focuses on assembling simple elements to form complex structures. According to the United States National Nanotechnology Initiative, nanotechnology is broadly defined as “the understanding and control of matter at dimensions of roughly 1 to 100 nanometers, where unique phenomena enable novel applications.” Nanomaterials are those with at least one dimension in nanometer scale. Nanotechnology can be understood as a technology of design, fabrication, and applications of nanostructures and nanomaterials, as well as the fundamental understanding of its physical properties and phenomena (Cao, 2004). At the nanometer scale, where many biological processes operate, nanotechnology can provide the tools to probe and even direct these biological processes. Thus, nanotechnology could potentially repair damaged parts, cure diseases, and even actively monitor and respond to the needs of the body. The broad potential of nanotechnology is owed to the fact that cells and the extracellular matrix possess a multitude of nanodimensionality, which affects cell behaviors (e.g. adhesion, proliferation, differentiation). Cells, typically microns in diameter, are composed of numerous nanosized components all working together to create a highly organized, self-regulating machine. For example, the cell surface is composed of ion channels that regulate the coming and going of ions such as calcium and potassium in and out of the cell. Enzyme reactions, protein dynamics, and DNA all possess some aspect of nanodimensionality. These nanodimensional components control how cells produce the extracellular matrix (ECM) including its composition and architecture. The extracellular matrix that cells interact with also abounds with nanosized features that influence the behaviors of other cells and tissues. These nanosized features, such as fiber diameter and pores, in concert with the intrinsic properties of the matrix itself, control the mechanical strength, the adhesiveness of the cells to the matrix, cell proliferation, and the shape of the ECM. Principles of Regenerative Medicine. DOI: 10.1016/B978-0-12-381422-7.10030-6 Copyright Ó 2011 Elsevier Inc., All rights reserved.

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Nanomaterials allow for different functional components to be contained together in a single unit. For example, therapeutic, targeting, contrast, and/or bio-compatibilizing components can be combined. A description of the different components of a nanocarrier can be found in Table 30.1. These components can be added or removed to create the desired effect without necessarily compromising the overall function of the particle. This is inherently different from the high costs approach to drug development, where a small change in molecular structure can dramatically influence the pharmokinetics and even potency of the drug. The ability to readily combine different components into a small physical space is not the only advantage of nanoscale materials. For example, quantum effects become more prominent at the nanoscale, which can result in high optical absorptivities, large photostabilities, or unusual magnetic properties that can be used to enhance cellular imaging (Zhang et al., 2002; Medintz et al., 2005). Besides imaging, these quantum effects can allow for novel methods of drug delivery-triggered light, electric, or magnetic fields (Yuan et al.). Therefore, there is great potential for using quantum dots in imaging, diagnostics, therapy, bioconjugation, and drug delivery for regenerative medicine. Nanotechnology’s impact on regenerative medicine will be through development of multifunctional tools to enhance effectiveness of implants, cell therapies, and tissue engineering. Since nanotechnology is at the interface of modern physical science and medicine, new and unconventional ideas will be developed, capable of bringing about major revolutions in science and medicine. Therapies developed using nanotechnology could someday minimize or eliminate the side-effects of drugs through targeted delivery and will provide real-time, and TABLE 30.1 A typical Nanocarrier of Image Contrast and/or Therapeutic Agents is Composed of Six Components 530

Binder

Biocompatibilization

Imaging contrast

Sensor

Targeting

Therapeutics

All the different components are held together using a binder. The binder may be an inert piece of the nanocarrier; however, it also often serves another purpose. The binder may also be the image contrasting agents. For example, iron nanoparticles and quantum dots serve as the core for the attachment of the other components. Polymers such as polyglycolic acid may serve as the binder of the therapeutic but also the biocompatiblizing agent. This component makes the nanocarrier compatible with the biological environment. It does this by minimizing aggregation of the nanocarrier and increases the lifetime of the carrier by avoiding the defense mechanisms of the biological systems such as the reticuloendothelial system. This component provides the means for imaging modalities to observe the nanocarrier. These contrasting agents may be observed using optical, magnetic, ultrasound, and scintillating methods. The sensor or trigger is used to alter the behavior of the nanocarrier once it has been deployed. For example, near-infrared light or electromagnetic radiation may be used to accelerate the release of a therapeutic or cause rapid localized heating as part of a therapy. Chemical sensors such as polymers that are pH or ion sensitive may also provide feedback to the nanocarrier in delivery of its payload. This component provides the means of driving the nanocarrier to its desired location. There are two types of targeting: passive and active. Passive targeting incorporates only nonspecific targeting agents, which may be useful for determining microenvironment permeability or areas of increased angiogenesis. Active targeting uses ligands or antibodies that bind to specific receptors at the target site. Active targeting aids in obtaining higher concentrations of therapeutics and contrasting agents at the desired site. Also, multiple targeting agents can be bound to the nanocarrier, allowing lower binding affinity molecules to be used to increase binding probabilities. Bioactive agents such as drugs or DNA are typical payloads of the nanocarrier. Drugs that are incapable of penetrating cellular membranes or hydrophobic drugs that cannot be administered systemically by themselves can be contained within the nanocarrier awaiting release in a controlled manner. Other novel properties of nanoparticles have also shown promise as hyperthermic agents.

CHAPTER 30 Applications of Nanotechnology for Regenerative Medicine

even non-invasive, monitoring of the disease and tissue repair. In this chapter we will examine the impact nanotechnology will have on regenerative medicine related to cellular therapies and biomaterial control, which play an important role for implant design and tissue engineering.

NANOTECHNOLOGY AS A MULTI-FUNCTIONAL TOOL FOR CELLBASED THERAPIES There is much excitement driving research into cell-based therapies to regenerate tissue function. However, many questions still remain as to how the cells behave once placed in vivo. One way to better understand how cells behave in vivo is through the use of nanoparticles as a multi-functional tool to improve monitoring or even potentially modify cell behavior. For example, with the enormous self-repair potential of stem cells, it is important to be able to locate, recruit, and signal these cells to begin the regeneration process. Improving non-invasive monitoring methods is particularly desirable since current methods of evaluating cell treatments typically involve destructive or invasive techniques such as tissue biopsies. Traditional non-invasive methods such as MRI and PET, which rely heavily on contrast agents, lack the specificity or resident time to be a viable option for cell tracking. However, in vitro and in vivo visualization of nanoscale systems can be carried out using a variety of clinically relevant modalities such as fluorescence microscopy, single photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), four-photon microscopy, near-infrared surface-enhanced Raman scattering, X-ray fluorescence micro- and nano-probe imaging, coherent X-ray diffraction imaging, ultrasound, and radiotracing such as gamma scintigraphy (Hong et al., 2009). Nanoparticulate imaging probes include semiconductor quantum dots, magnetic and magnetofluorescent nanoparticles, gold nanoparticles, and nanoshells, among others. Nanoparticles that are novel intravascular or cellular probes are being developed for diagnostic (imaging) and therapeutic (drug and gene delivery) purposes (Fig. 30.1) (Heller et al., 2005). There are a growing number of nanomaterials being used to probe aspects of the tissue regeneration process, such as monitoring angiogenesis (Winter et al., 2003), apoptosis (Jung et al., 2004; Sosnovik et al., 2009), and tissue viability (Sosnovik et al., 2009). These nanoparticles can play a critical role in future regenerative medicine, especially in the areas of targetspecific drug and gene delivery. Quantum dots (QDs) are one class of nanomaterial that is receiving special attention. QD are inorganic nanocrystals that possess physical dimensions between 2 and 10 nanometers, composed of a core of a semiconductor material. Quantum dots are tunable in a broad spectrum of colors by varying particle size or composition. They also possess strong and narrow symmetrical emission spectra and usually have high photochemical stability. The emission wavelength is controlled by the size of the nanocrystal and can be tuned throughout the visible spectrum to the near-infrared region (>670 nm). Early live cell experiments using fluorescent quantum dots sparked interest in using nanoparticles for immunocytochemical and immunohistochemical assays as well as for cell tracking (Akerman et al., 2002; Tokumasu and Dvorak, 2003; Sukhanova et al., 2004). A significant advantage of quantum dots is their increased photostability (typically 10e1,000 times more stable) compared to organic dyes. This allows quantum dots and the cells or proteins attached to them to be tracked over longer periods of time. Tumor cells labeled with QDs have been intravenously injected into mice and successfully followed using fluorescence microscopy (Gao et al., 2004; Voura et al., 2004). As passive imaging agents, quantum dots can be used for imaging microvascularity in animals since PEG-coated quantum dots injected into mice have shown good tissue perfusion and appear to be biocompatible (Ballou et al., 2004). Moreover, the capability to modify both surface chemistry and size of quantum dots allows for multiple targeting analyses within the same cell. Quantum dots can be functionalized with biomolecules, which interact with

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Nanomedicine

(A) Nano-vehicles travel in bloodstream to the targeted tissues or organs.

Blood vessels

(B)

Gold shell

Gold or gold-shell nanoparticles accumulated in mice as a contrast agent or targeted-drug delivery.

Core

Gold nanoparticle

(C) Light or laser

Quantum dots accumulate in targeted tissue or organ, and become fluorescent after exposure to light or laser.

Antibody coating Core Shell

FIGURE 30.1

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Nanomedicine overview. (A) Nanostructured vehicles reach targeted tissues through a bloodstream. (B) Nanoshells or gold nanoparticles can be used as contrast agents for medical imaging and as vehicles for drug and gene delivery. (C) Quantum dot with a semiconductor nanocrystal core emits fluorescent light. (D) Polyethylene glycol-nanoparticles functionalized with targeting molecules deliver therapeutics via receptors and membranes. Modified from Kateb et al. (2007).

(D)

Targeting molecule Polyethylene glycol stalk

Nanoparticles deliver therapeutic agents to a targeted tissue.

Receptor

Therapeutic core

a biological entity through electrostatic or hydrogen bonding, to suit their targets. For example, when quantum dots are coated with trimethoxysilylpropyl urea and acetate groups, they have shown their ability to bind with the nuclear membrane (Bruchez et al., 1998). Wu et al. used quantum dots to image cell surface markers (Her2), cytoplasmic proteins (actin and microtubules), and nuclear antigens (Wu et al., 2003). CdSe-CdS core-shell nanocrystals linked covalently with biotin have been used as the secondary antibody, binding F-actin filaments in 3T3 mouse fibroblasts that were labeled with phalloidin-biotin and streptavidin (Bruchez et al., 1998). Quantum dots represent just one novel class of nanomaterials whose ability to aid in imaging cells could help develop better regenerative therapies. Other nanoparticles are showing promise for optical cell tracking and imaging. For instance, nanosized tubes of carbon known as carbon nanotubes possess optical transitions in the nearinfrared that can be used for tracking cells. However, unlike quantum dots, which are typically composed of heavy metals such as cadmium, carbon nanotubes are made of carbon, an abundant element in nature. Carbon nanotubes possess large aspect ratios with nanometer diameters and lengths ranging from submicrons to millimeters. These tubes can contain a single wall of carbon or multiple walls (typically 3e10) of carbon, commonly called single wall carbon nanotubes (SWNTs) or multi-wall carbon nanotubes (MWNTs), respectively. The versatile chemistry of carbon nanotubes in combination with their intrinsic optical properties can lead to a multifunctional nanoplatform for multimodality molecular imaging and therapy (Fig. 30.2) (Hong et al., 2009).

CHAPTER 30 Applications of Nanotechnology for Regenerative Medicine

Immunoglobin

Functionalized

Biocompatible polymer coating (polyethylene glycol) with a targeting ligand

Gene

FIGURE 30.2 Intrinsic properties of carbon nanotube for imaging are infrared radiation and Raman scattering

Drug Radioisotope

Multifunctional carbon nanotube-based platform functionalized with antibody, polymer coating, ligand, drug, gene, and radioisotope for a multimodality imaging and multiple therapeutic delivery.

The infrared spectrum between 900 and 1300 nm is an important optical window for biomedical applications because of the lower optical absorption (greater penetration depth of light) and small auto-fluorescent background. Like quantum dots, carbon nanotubes possess good photostability and can be imaged over long periods of time using Raman scattering and fluorescence microscopy. Single-wall carbon nanotubes dispersed in a Pluorinc surfactant can be readily imaged through fluorescence microscopy after being ingested by mouse peritoneal macrophage-like cells. The small size of the SWNT makes it possible for 70,000 nanotubes to be ingested, where they can remain stable for weeks inside 3T3 fibroblasts and murine myoblast stem cells (Cherukuri et al., 2004; Heller et al., 2005). Having such a high concentration of carbon nanotubes within a cell without distributing the cell behavior means such probes could be used for studying cell proliferation and stem cell differentiation, even through repeated cells. While such nanomaterials have yet to reach clinical applications, it does show the potential for non-invasive optical imaging. Still, there is much to learn about how carbon nanotubes interact with cells. For example, it has been shown that SWNTs and double-walled carbon nanotubes can trigger immunological responses (Salvador-Morales et al., 2006). However, MWNTs reportedly do not result in proliferative or cytokine changes in vitro. Some studies have shown that the size and composition of carbon nanotubes must be carefully controlled to promote their biocompatibility and to prevent body immune reaction (Kateb et al., 2007). Brown et al. suggested that MWNTs enter the cell by either mechanically translocating through the lipid bilayer or by incomplete or frustrated phagocytosis, which occurs when MWNTs are too large for the cell to phagocytose (Brown et al., 2007). It has been suggested that if MWNTs penetrate into cell membranes they can promote reactive oxygen species (ROS) generation and cause cell death (Nel et al., 2006). Others have shown that, if MWNTs enter via incomplete phagocytosis, the cell can release digestive enzymes from the phagosome into extracellular regions and cause chronic inflammation (Zeidler-Erdely et al., 2006). It is unclear with certainty what properties of carbon nanotubes will have the most impact on biological systems whether it is their chemical structure, length and aspect ratio, surface area, degree of aggregation, extent of oxidation, surface topology, bound functional groups, and catalyst residues/produced impurities (Foldvari and Bagonluri, 2008). For instance, heparinizing CNT reduced or eliminated complement activation (Murugesan et al., 2006). Other reports imply that the accumulation of carbon nanotubes within cells depends on the functional groups and the functionalization degree (Wang et al., 2004; Lacerda et al., 2008; Schipper et al., 2008; Yang et al., 2008). Even though understanding how carbon nanotubes interact with cells is still in its infancy, these examples illustrate that nanomaterials have the potential for large impact in cell biology and by extension regenerative medicine.

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Along with optical contrast agents, magnetic nanoparticles have been used to track cells and report on cell behavior. Many nanoparticle contrasting agents are based on superparamagnetic iron oxide nanoparticles and some have already been approved as clinical MRI contrast agents. When placed into a magnetic field, magnetic nanoparticles create perturbations of the external field that significantly reduce the spin-spin relaxation time (T2) of the nearby environment generating MR contrast. Typically, these probes consist of a magnetic iron oxide core surface functionalized with an agent, such as dextran or other polymers, to prevent aggregation and to enhance stability and solubility. Sizes of these particles can range from one nanometer to hundreds of nanometers in diameter. Magnetic iron oxide nanoparticles and their composites are emerging as novel contrast agents for MRI and are much more sensitive than conventional gadolinium-based contrast agents (Chemaly et al., 2005). When used in conjunction with HIV-Tat and polyArginine peptides, these particles are readily taken up by many cell types (Dodd et al., 2001; Zhao et al., 2002). For example, superparamagnetic iron oxide (SPIO)-labeled rat mesenchymal stem cells injected into rats could be imaged and tracked to the liver and kidneys (Bos et al., 2004). Another example of a composite nanoparticle is the triple-labeled (magnetic, fluorescent, and isotope) SPIO that can be readily internalized by hematopoietic stem and neural progenitor cells and not affect their potential for viability, proliferation, or differentiation (Lewin et al., 2000). A third example of functionalized nanoparticles for imaging includes using an antitransferrin receptor monoclonal antibody-functionalized nanoparticle to label oligodendrocyte progenitor cells by targeting the transferring receptors on the cells (Bulte et al., 1999). The oligodendrocyte progenitor cells, which as shown previously significantly myelinate a large area in the central nervous system (Duncan and Milward, 1995), were transplanted into the spinal cord of myelin-deficient rats. Since they were labeled with nanoparticles, they could be tracked easily using MRI and the extent of myelination could be determined. 534

Apoptosis is commonly detected by using the binding of annexin V to externalized phosphatidylserine. This binding event is the basis of optical and radiolabel methods for detecting apoptotic cells and can be bound to iron nanoparticles for sensing using MRI. It has been demonstrated that tumor-bearing mice injected with SPIO particles bearing apoptotic sensing proteins showed a sharp decrease in the T2) weight image corresponding to the location of the tumor (Zhao et al., 2001). This demonstrated that nanomaterials can be used to create highspecificity MRI contrast agents for apoptotic cells. Such results are encouraging because they show that nanomaterials can be used not only for imaging the physical location of cells but also to provide information on the biological state of cells. While MRI has revolutionized our way of visualization in vivo, allowing cells to be tracked noninvasively, it is difficult to quantify the MRI signals and provide real quantification of cell numbers. The difficulty arises because MRI contrasting agents that are based on paramagnetic gadolinium and iron metals are not directly detected by the scanner but are indirectly detected by their influence on surrounding water molecules. However, the use of perfluoronated nanoparticles has recently been shown to be a new way to provide quantitative numbers to MRI since the fluorine nuclei (19F) can be directly detected (Morawski et al., 2004; Ahrens et al., 2005). Since endogenous fluorine is negligible in the body, 19FMRI is capable of directly detecting fluorine against a dark background similarly to radiotracers and fluorescent dyes. While this has been demonstrated with dendritic cells, similar results should be obtainable using other cell types. Besides imaging enhancements, nanotechnology can produce carriers for delivery of therapeutics for aiding the regeneration process. For example, biodegradable nanoparticles can deliver drugs, growth factors, and other bioactive agents to cells and tissue (Panyam and Labhasetwar, 2003). Nanomaterials can be used as immensely powerful tools for gene delivery in specific differentiation of stem cells. Gold nanoparticles (20 nm in diameter) conjugated with a DNA-poly-ethylenimine complex were patterned on a solid surface (glass) and used as

CHAPTER 30 Applications of Nanotechnology for Regenerative Medicine

nanoscaffolds for the delivery of DNA into hMSCs through reverse transfection (Uchimura et al., 2007). The development of safe and efficient gene delivery systems, which can lead to high levels of gene expression within stem cells, is a strong indicator for the effective implementation of regenerative therapies (Solanki et al., 2008). Nanodelivery vehicles possess three distinct advantages over conventional drug delivery methods. First, nanoparticles, due to their small size, are able to bypass biological barriers such as cell membranes and the blood brain barrier (BBB), allowing greater concentrations of therapeutics to be delivered. Second, nanocarriers can be functionalized with active targeting agents to allow selective delivery of bioactive agents. Third, drug delivery systems can incorporate nanotriggers for non-invasive delivery of therapeutic agents. These sensitive triggers can be activated using in vivo signals such as pH, ion concentration, and temperature or external sources such as near-infrared light, ultrasound, and magnetic fields. Nanotechnology can provide powerful new tools for non-invasive tracking of cells in engineered tissues. As was also mentioned at the outset, the real benefits of nanotechnology are the multifunctional tools that it can bring. As nanotechnology progresses, new nanomaterials and techniques are being developed regarding cellular imaging and drug delivery that will better equip those practicing regenerative medicine to reach their goals. Cellular therapies for regenerative medicine would benefit from nanotechnology since tracking of implanted cells would provide the means to better evaluate the viability of engineered tissues and help in understanding the biodistribution and migration pathways of transplanted cells. Nanotechnology would also allow better and more intelligent control of the bioactive factors which can influence cellular therapies. The potential of nanotechnology for impacting regenerative medicine is great, creating the hope of individualized and targeted therapies.

NANOTECHNOLOGY AS A MULTI-FUNCTIONAL TOOL FOR BIOMATERIAL CONTROL Biomaterials play an important role in regenerative medicine because they make up a large component of implants and tissue scaffolds. Biocompatible scaffolds can provide temporary structural support guiding cell growth, assist the transportation of essential nutrients, and facilitate the formation of functional tissues and organs. Increasing evidence shows that the nature of the biomaterial greatly affects long-term success of biomedical implants and shortterm wound healing response. Substrate features such as the chemical composition and surface morphology affect the viability, adhesion, morphology, and motility of cells. Therefore, controlling the three-dimensional structure and surface composition of a biomaterial is important to promoting normal tissue growth or minimizing foreign body response. To illustrate the importance of controlling the biomaterial surface, one can examine the use of implants to repair bone defects. Currently, there are several strategies for repairing large bone defects including using implants made of metal, plastic, ceramics, and or graphing of tissue. However, there are limitations to these biomaterials. Autographs can be expensive and difficult to handle, and may have physical limitations in their use. Allographs are also expensive and carry additional risks of an autoimmune response and disease transmission. Bone tissue engineering seeks to develop strategies to heal bone loss due to trauma or disease without the limitations and drawbacks of current clinical autografting and allografting treatments (Langer and Vacanti, 1993; Mistry and Mikos, 2005). While metal and plastics mitigate many of the aforementioned risks, implants made from these materials, instead of integrating with bone, often form soft undesirable fibrous tissue. This is especially true with surfaces that are uniform and non-porous. This mechanical mismatch between tissue leads to wear of the implant that either aggravates or in some cases leads to cell death in nearby tissue, causing implant failure. However, inclusion of nanosized particles into implant materials, for example, has been shown to increase osteoblast adhesion (Kay et al., 2002). While this may be partially due to increased surface area, other factors may

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be involved, such as controlling protein adsorption. For instance, on carbon nanofiber surfaces, osteoblast adhesion was greater than other competitive cell types, possibly due to the fact that the aspect ratio and physical shape of these fibers mimic the crystalline hydroxyapatite structures of natural of nature bone (i.e. hydroxyapatite crystal dimensions from 50 to 100 nm in length and 1 to 10 nm in diameter) (Price et al., 2003). Sitharaman et al. demonstrated after 12 weeks that bone formation in defects (4 mm in diameter and 8 mm in depth) containing ultrashort-SWNT/poly(propylene fumarate) scaffolds had significantly higher (about 200% increase) bone volumes than poly(propylene fumarate) (PPF) scaffolds alone (Sitharaman et al., 2008). The histological sections of the ultrashort-SWNT/PPF implants showed increased collagen matrix production along with decreased foreign body giant cell density when compared to PPF scaffolds. Taking advantage of the electroactive properties of carbon nanotubes, scaffolds could be formed that could be electrically conductive and thus stimulate cells contained on the scaffolds. For example, applying an alternating current to a nanocomposite of polylactic acid and multi-walled carbon nanotubes resulted in an increase in osteoblast proliferation by 46% and a greater than 300% increase in calcium production (Supronowicz et al., 2002). Also, upregulation of collagen I (a major component in organic bone formation), osteonectin, and osteocalcin was observed. Such results suggest that nanocomposites could accelerate the bone regeneration process.

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For neuronal regeneration, carbon nanotube scaffolds could guide neurite growth into a specific neural bundle or network. Functionalized carbon nanotubes (f-CNT) have been able to guide neurite growth by providing a platform for the growth cone to grasp onto, instead of relying on, physiosorption alone (Hu et al., 2004). Zhang et al. suggested that positively charged carbon nanotubes are suitable to use as a template and patterned guide to grow an elaborate and controlled neuronal network (Zhang et al., 2005). The number of neurite growth cones, length of neurite outgrowths, and the degree of branching on positively charged polyethyleneimine f-CNT templates were significantly higher than on neutral or negatively charged CNT substrates (Hu et al., 2005). Lovat et al. demonstrated an increase in spontaneous post-synaptic currents in hippocampal neurons grown on a CNT substrate even when the neurons were randomly spaced apart from the substrate, suggesting that electric coupling had occurred between neurons and the CNT (Lovat et al., 2005). Such examples demonstrate that carbon nanotubes are potentially useful materials that can serve as both a supportive matrix and a conduit for delivering electrical signals. Nanomaterials, like carbon nanotubes, are part of a growing new class of multifunctional biomaterial e smart biomaterials. Unlike passive structural biomaterials, smart biomaterials are designed to interact with their environment either by responding to changes in their surroundings or by stimulating or surpressing specific cellular behavior. They can change their shape, porosity, or hydrophilicity based on changes in temperature (Gan et al., 2005), pH (Bulmus et al., 2003), or external stimuli such as electric (Lahann et al., 2003) or magnetic (Jordan et al., 1999) fields. Such control of the biomaterial behavior through nanotechnology could create a major shift in the way biomaterials are used. Examples of some techniques used for creating nanostructured surfaces for tissue engineering are shown in Table 30.2. The current paradigm to tissue regeneration is to isolate a patient’s cells and then expand the cell population outside the body and finally place or seed the cells onto scaffold-like biomaterials before implantation. This method of engineered tissue using two different cell types has met with great success (Atala et al., 2006). Ideally, one would want to directly implant a biomaterial into the patient that would then selectively recruit the correct cell types. This approach would be especially important for engineering organs with very elaborate structures. Another area where nanotechnology can impact the effectiveness of biomaterial surfaces is affecting stem cell differentiation within the engineered tissue. The unique properties of

CHAPTER 30 Applications of Nanotechnology for Regenerative Medicine

TABLE 30.2 Examples of Tissue Scaffolds Created using Nanofabrication Techniques Technique Lithography Electrospinning Self-assembly Polymer demixing Solvent casting Salt leaching

Tissue scaffold prepared Nerve (Gabay et al., 2005) Heart (Zong et al., 2005), nerve (Yang et al., 2005), bone (Fujihara et al., 2005) Nerve (Ellis-Behnke et al., 2006) Bone (Kim et al., 2005; Liao et al., 2004; Kikuchi et al., 2001; Du et al., 1999) Bladder (Pattison et al., 2005; Thapa et al., 2003a,b) Bladder (Pattison et al., 2005; Thapa et al., 2003a,b)

Stem/Progenitor Cell Responses:

Nanotechnology Approaches: - Drug delivery - Molecular imaging - Biodetection - Cell arrays - Biocompatible scaffolds and grafts - Nanostructured biomaterials - Extracelluar matrix patterning

Signals or Cues:

(Based on gene expression) - Self-renewal - Differentiation - Apoptosis - Migration

Soluble signal: - Growth factors - Cytokines - Chemokines Cell-cell interactions: - Cadherins Insoluble or physical signals: - Laminin - Fibronectin - Mechanical forces

FIGURE 30.3 Regulation of stem cell fate corresponding to applications of nanotechnology and environmental signals (modified from Solanki et al., 2008).

nanomaterials and nanostructures can be particularly useful in controlling intrinsic stem cell signals and in dissecting the mechanisms underlying embryonic and adult stem cell behavior (Fig. 30.3) (Solanki et al., 2008). Currently, blends of expensive growth factors are used to guide the differentiation of stem cells. With the ability to control the surface morphology and chemistry at the nanoscale, nanobiomaterials may eliminate the need to culture different cell types for reassembly into an engineered tissue as they can recruit the body’s own stem cells and differentiate them into the correct phenotype (Silva et al., 2004). Biomaterials play an important role in regenerative medicine through their use in implants and tissue scaffolds. Nanotechnology is poised to provide the tools for rapidly increasing the pace of biomaterials development. Through the ability to control the nanostructure of a biomaterial, better understanding and control of cell behaviors will result, creating better regenerative therapies. The timeline of the impact of nanotechnology on biomaterial development as it relates to regenerative medicine will first be felt through better-performing, longer-lasting implants, and will eventually give way to smart biomaterials that can be implanted and direct the regenerative process at the cellular level.

CONCLUSION As nanotechnology continues to grow, it will provide new and powerful tools that will revolutionize regenerative medicine. The most significant impact nanotechnology will have on regenerative medicine is that it will help in providing a detailed understanding and control of

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biology. Already the young field has demonstrated significant advances over traditional imaging, sensing, and structural technologies. Many of these advantages stem from the capability of nanomaterials to be multifunctional. These advances help in tackling one of most significant challenges faced in designing new biomedical technologies e targeting biological functions while at the same time avoiding non-specific effects. While there have been challenges for some time, nanotechnology provides us with the means to successfully negotiate these challenges and create new innovations in regenerative medicine.

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CHAPTER 30 Applications of Nanotechnology for Regenerative Medicine

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PART 2 Cells and Tissue Development

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