Fisher John P. e.a. (ed.) Tissue Engineering

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11-2 Tissue Engineering

Macroscale

Microscale

Nanoscale

Hydroxyapatite

nanocrystal

Collagen

Osteoblast

FIGURE 11.1 Nanocomposite structure of bone. The interaction between collagen ﬁbers and HA nanocrystals in the

nanoscale gives rise to the complex mechanical properties of bone tissue observed in the macroscale. Cells operating

on this collagen/hydroxyapatite nanocomposite continually remodel bone on the microscale.

properties. More speciﬁcally, nanoﬁllers have been shown to improve a composite material’s ﬂexural mod-

ulus [4], tensile strength, stiffness, toughness [5–8], fatigue resistance [9], wear resistance [10], thermal

stability [11,12], and gas permeability properties [13]. Nanophase ceramic materials have been shown

to also improve bone cell functions [14] and can impart osteoconductivity and improved biocompat-

ibility to synthetic polymers [15,16]. Alternatively, HA nanocrystals can improve the osteoconductivity

and biocompatibility of natural polymers, such as collagen, by mimicking the natural composition of

bone [17–19]. The present chapter highlights current efforts toward nanocomposite scaffolds for tissue

engineering applications.

11.2 Nanocomposite Materials

11.2.1 Nanomaterials Overview

Nanomaterials, as described here, are deﬁned as materials with at least one of three dimensions <100 nm.

Spherical nanoparticles, such as alumoxanes or silica nanoparticles, are nano-sized in all three dimen-

sions. Nanotubes (carbon nanotubes), rods, or needles (HA) have two nanometer-sized dimensions.

Nanosheets, such as layered silicates, have only one dimension in the nanoscale. Each of these nanoma-

terials offers mechanical reinforcement or osteoconductivity by dispersing into a matrix and chemically

interacting with the macroscopic material. However, these particles are typically hydrophilic while the

macroscopic material into which they are dispersed is usually hydrophobic. Thus, nanomaterial dispersion

and promotion of interactions between the nanoﬁllers and the macroscopic material are the two primary

challenges for nanocomposite development.

Table 11.1 describes some of the many different nanomaterials currently being investigated for biomed-

ical scaffolds. Each section of this chapter discusses the synthesis of a nanomaterial as well as the fabrication

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Nanocomposite Scaffolds for Tissue Engineering 11-3

TABLE 11.1 Nanomaterials for Biomedical Composites

Composite materials for

Nanomaterial Chemical formula biomedical applications References

Carboxylate [Al(O)

(OH)

CR)

]

PPF Horch et al. [4]

Alumoxane

Montmorillonite M

(Al

4−x

)Si

(OH)

PLLA Lee et al. [11]

Polyurethanes Xu et al. [26]

Hydroxyapatite Ca

(PO

)

(OH)

PMMA Fang et al. [39]

PEG, PMMA, PBMA, Liu et al. [40–42]

PHEMA, PEG/PBT

Chitosan Hu et al. [43]

PPF Lewandrowskiet al. [44]

Collagen Zhang et al. [45]

Silica SiO

PMMA, PCL Rhee et al. [16, 46–47]

Yoo et al. [48]

Alumina Al

Ce-TZP Nawa et al. [8,49]

Tanaka et al. [10]

Uchida et al. [50]

Carbon nanotube C PMMA Cooper et al. [65]

PLA Supronowicz et al. [67]

of nanocomposites from this material. A brief description of relevant issues and results of physical and

biological testing is also included for each material.

11.2.2 Functionalized Alumoxane Nanocomposites

Carboxylate-alumoxanes are alumina-based nanoparticles developed as inorganic ceramic ﬁllers for a

variety of engineering applications. Alumoxanes are prepared directly from boehmite mineral in a “top-

down” synthesis involving acid hydrolysis [20]. Nanoparticle size is controlled by conditions during

synthesis and particles are easily functionalized based on the type of carboxylic acid used during synthesis

[21]. Certain functional groups can be added to the hydrophilic alumoxane nanoparticles to aid in

dispersion and covalent interaction with the composite medium. Vogelson et al. [6] modiﬁed alumoxanes

with lysine and p-hydroxybenzoic acid to reinforce organic epoxy resins and yield sizable increases in

thermal stability and tensile strength over blank resin [6].

In our laboratory, we have studied the effects of various surface-modiﬁed alumoxane nanoparticles

on the mechanical properties of a biodegradable polymer for load-bearing bone tissue applications [4].

Alumoxane nanoparticles with three different surface modiﬁcations were tested — “activated” alumox-

anes possessing two reactive double bonds available for interaction with the cross-link network of the

polymer; “surfactant” alumoxanes modiﬁed with long fatty acid chains to aid in dispersion within the

hydrophobic polymer; and “hybrid” alumoxanes modiﬁed with a surfactant chain and a reactive double

bond within the same substituent (Figure 11.2). These nanoparticles were incorporated into a biodegrad-

able poly(propylene fumarate)-based (PPF) system and the nanocomposites were tested for ﬂexural and

compressive mechanical properties.

Unmodiﬁed boehmite particle composites showed no signiﬁcant improvement in mechanical proper-

ties compared to polymer resin alone and demonstrated a signiﬁcant decrease in ﬂexural fracture strength

with increased loading. This is explained by the formation of large aggregates within the hydrophobic

polymer, which promote crack formation (Figure 11.3a). The activated alumoxane nanocomposites were

expected to covalently interact with the PPF matrix, but instead tended to aggregate into micron-sized

clusters, which decreased ﬂexural fracture strength with increased loading. Surfactant alumoxane nano-

composites demonstrated signiﬁcant improvements in ﬂexural modulus over blank polymer resin due

to the ﬁne dispersion of nanoparticles within the polymer matrix as determined by scanning electron

microscopy (SEM). The hybrid alumoxane nanocomposites performed the best out of all materials by

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11-4 Tissue Engineering

(a) (b) (c)

O OH

OH OH

HO O OH

FIGURE 11.2 Chemical structures of modiﬁed alumoxanes: (a) diacryloyl lysine–alumoxane (activated), (b) stearic

acid–alumoxane (surfactant), and (c) acryloyl undecanoic amino acid–alumoxane (hybrid).

(a) (b)

FIGURE 11.3 SEM images of fracture planes of nanocomposite samples after mechanical testing: (a) unmodiﬁed

boehmite crystals in polymer, bar is 1 µm; (b) hybrid alumoxane nanocomposite, bar is 1 µm.

improving the ﬂexural modulus of PPF at loading concentrations between 0.5 and 5 wt.%. Ata1wt.%

loading, the ﬂexural modulus reached 5410 ± 460 MPa, a factor of 3.5 greater than polymer alone

(Figure 11.4a). Additionally, hybrid nanocomposites caused no signiﬁcant loss of ﬂexural or compressive

fracture strength up to 5 wt. % loading (Figure 11.4b). SEM images revealed that the surfactant chain

within the functional group of hybrid alumoxanes aided in dispersion within the polymer (Figure 11.3b)

while the signiﬁcant increase in mechanical properties may be explained by covalent interaction between

PPF polymer and alumoxane nanoparticles. Thus, surface modiﬁcation of alumoxane nanoparticles sig-

niﬁcantly increased the ﬂexural modulus of polymer nanocomposites without a detrimental effect on

fracture strength.

11.2.3 Polymer-Layered Silicate Nanocomposites

Layered silicates, derived from smectite clays, are commonly used as ﬁllers for polymeric materials for

manydifferent applications as their chemistries have been extensively studied [22]. Polymer-layered silicate

nanocomposites have shown improvements in mechanical, thermal, optical, physicochemical, and barrier

properties as well as ﬁre resistance compared to pure polymers or conventional composites (composed of

micron-sized particles) [5,12].

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Nanocomposite Scaffolds for Tissue Engineering 11-5

1000

2000

3000

4000

5000

6000

7000

0.0 0.5 1.0 2.5 5.0

Wt.% loading

0.0 0.5 1.0 2.5 5.0

Wt.% loading

Flexural modulus (MPa)

(a)

Flexural fracture strength (MPa)

(b)

FIGURE 11.4 Flexural testing of hybrid alumoxanes. Flexural modulus (a) and ﬂexural fracture strength (b) of

hybrid alumoxane nanocomposites as a function of nanoparticle loading weight percentage. Error bars represent

mean ± standard deviation for n = 5. The symbol “*” indicates a statistically signiﬁcant difference compared to the

pure polymer resin (p < .05).

These materials, unlike the other nanophase materials described in this chapter, are nano-sized in

only one dimension and thereby act as nanoplatelets that sandwich polymer chains in composites. Mont-

morillonite (MMT) is a well-characterized layered silicate that can be made hydrophobic through either

ionic exchange or modiﬁcation with organic surfactant molecules to aid in dispersion [5,23]. Polymer-

layered silicates may be synthesized by exfoliation adsorption, in situ intercalative polymerization, and

melt intercalation to yield three general types of polymer/clay nanocomposites. Intercalated structures

are characterized as alternating polymer and silicate layers in an ordered pattern with a periodic space

between layers of a few nanometers [13]. Exfoliated or delaminated structure occurs when silicate layers

are uniformly distributed throughout the polymer matrix. In some cases, the polymer does not intercalate

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11-6 Tissue Engineering

the layers of silicate, resulting in a phase-separated structure containing micron-sized clusters of multiple

silicate layers [5]. Typically, the greatest improvement of properties is observed in exfoliated structures

based on the degree of layered-silicate dispersion within the polymer [13,24,25], though intercalated

systems show more signiﬁcant improvements in certain properties such as fracture behavior [23].

For biomedical applications, layered silicates have been incorporated into biodegradable lactide-based

polymers to improve mechanical properties for hard-tissue applications. Lee et al. [11] incorporated

MMT nanoplatelets into poly(l-lactic acid) (PLLA) with the aim of improving the intrinsic stiffness of

porous polymer scaffolds. Exfoliated composites were prepared by the exfoliation-adsorption process and

thermal and tensile mechanical properties were examined. The authors observed decreased glass transition

temperatures with the addition of MMT to PLLA along with a larger amorphous region, which may have

a positive effect on the biodegradation behavior of the composite. The tensile modulus of PLLA loaded

with 5.79 wt.% MMT increased approximately 40% compared to pristine PLLA while maintaining more

than 90% porosity.

Biomedical polyurethanes have also been modiﬁed with organically modiﬁed layered silicates (OLS) to

improve mechanical properties and reduce gas permeability. Xu et al. [26] demonstrated an increase in

tensile modulus with increased OLS concentration without the loss of strength and ductility that is typical

for ﬁller systems. Additionally, they observed a ﬁvefold decrease in water vapor permeability, which is a

major advantage for blood-contacting biomedical devices.

11.2.4 Hydroxyapatite Nanocomposites

As the nanostructure of bone was revealed, many researchers started to synthesize nanoscale HA and

investigate its properties. Among various methods to prepare HA, the wet chemical method is most

commonly used because it is well developed and easily adjusted for mass production [27]. Brieﬂy, solutions

of either calcium hydroxide and orthophosphoric acid or calcium salts and phosphate salts are mixed in

a Ca/P ratio of 5 : 3. Under these conditions, HA will precipitate from the solution [28]. Researchers can

ﬁnely tune this simple method to make HA nanocrystals in various shapes such as spheres, rods, needles,

and plates [28–33].

Due to its low ﬂexural strength and toughness, commercial HA powders (of micron-sized particles)

are usually limited to use as non-load-bearing implants or bioactive coatings on stronger materials, such

as titanium alloys, to promote bone ingrowth [34]. Nanocrystalline HA with an average grain size of

100 nm possesses superior bending strength (182 MPa) and compressive strength (879 MPa) compared

to conventional HA (in the range of 38–113 MPa in bending and 120–800 MPa in compression) [35].

Improved osteoblast adhesion, proliferation, and mineralization were also observed on the surface of

nanoscale HA [14,36–38].

It is very attractive to introducenanoscale HA as a ﬁller for widely used biopolymers to improve bioactive

and mechanical properties. As mentioned previously, however, the major challenge is achieving uniform

dispersion of hydrophilic HA nanoparticles throughout hydrophobic polymer matrixes. Surfactants, such

as lecithin, can be used to prevent aggregation of HA nanocrystals and homogeneously distribute them

into poly(methylmethacrylate) (PMMA) polymer [39]. Liu et al. [40,41] successfully modiﬁed the surface

of HA nanocrystals with poly(ethylene glycol) (PEG), PMMA, poly(butyl methacrylate) (PBMA), and

poly(hydroxyethyl methacrylate) (PHEMA) to produce chemical bonding between the ﬁller and matrix

[40,41]. In another study, tensile strength and modulus of composites were signiﬁcantly enhanced by

the improved interface of HA nanocrystals and PEG/poly(butylenes terephthalate) (PEG/PBT) block

copolymer [42].

Natural polymers have also been investigated for nanocomposites. In fact, a biodegradable chitosan/HA

nanocomposite made by in situ hybridization exhibited higher bending strength and modulus than

PMMA [43]. Moreover, nanoscale HA ﬁllers can reduce water absorption, thus retaining material mech-

anical properties under moisture conditions for the potential application of internal ﬁxation of bone

fractures [43].

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Nanocomposite Scaffolds for Tissue Engineering 11-7

In an in vivo study, the biocompatibility and osteointegration of biodegradable PPF polymer grafts

were improved when nanoscale HA was employed as opposed to micron-sized particles [44]. In another

study, HA nanocrystals were reported to grow on the surface of self-assembled collagen triple helices

along the longitudinal axes of their ﬁbrils [45]. This designed hierarchical structure is very close to the

actual nanostructure of bone. Thus, HA nanocomposites provide signiﬁcant advantages for successful

orthopaedic and dental applications in that they closely mimic natural bones and improve the bioactivity

of many materials.

11.2.5 Other Ceramic Nanocomposites

In an effort to enhance the osteoconductivity of PMMA bone cement, Rhee and Choi [16,46] incorporated

silica nanoparticles into the polymer. This composite was synthesized by sol–gel processing with the goal

of improving binding at the bone-implant interface. The authors observed high mechanical properties in

addition to crystalline apatite formation on implants in simulated body ﬂuid.

While PMMA is useful as a bone cement, it is not ideal for tissue engineering applications as it is

nondegradable. Researchers incorporated the same type of silica nanoparticles into the biodegradable

poly(ε-caprolactone) (PCL) and also observed apatite formation and favorable mechanical properties

[47,48].

Another noteworthy effort at nanocomposite fabrication applied ceramic nanoparticles to a ceramic

material to enhance osteoconductivity and mechanical performance. Nawa et al. [49] developed a

ceria-stabilized tetragonal zirconia polycrystal (Ce-TZP) ceramic and incorporated alumina (Al

)

nanocrystals into it via wet chemistry methods for load-bearing bone applications. Further studies of this

material investigated its ability to induce apatite formation [50], in vivo biocompatibility, and resistance

to wear [10] with favorable results.

11.2.6 Carbon Nanotube Nanocomposites

Carbon nanotubes are among the strongest materials known because of their almost defect-free graphite

architecture with the sp

type of carbon–carbon covalent bond, which is one of the strongest chem-

ical bonds in nature [51,52]. There are two types of carbon nanotubes: multiwalled carbon nanotubes

(MWNTs), ﬁrst discovered in 1991 [53], and single-walled carbon nanotubes (SWNTs), ﬁrst reported in

1993 [54,55]. Depending on the quality of nanotubes, elastic moduli can be as high as 1 TPa for SWNTs

and 0.3 to 1 TPa for MWNTs, while strength ranges from 50 to 500 GPa for SWNTs and 10 to 60 GPa for

MWNTs [56]. Owing to their very small diameters (ranging from 0.42 nm to dozens of nanometers) and

lengths of more than several micrometers, the aspect ratio (length-to-diameter ratio) of carbon nanotubes

can be more than 1000, while those of conventional carbon ﬁbers are only about 100 [57]. Therefore,

carbon nanotubes could become the best reinforcing ﬁber for composite materials.

Both types of carbon nanotubes are synthesized by three methods involving gas phase processing,

namely, arc-discharge, laser ablation, and chemical vapor deposition (CVD) [58]. Subsequent puriﬁc-

ation procedures are required to remove impurities, such as catalyst particles, amorphous carbon, and

nontubular fullerenes, from the nanotubes [59].

One of the greatest challenges for developing carbon nanotube nanocomposites is separating nanotube

bundles, which aggregate into ropes due to strong inter-tube van der Waals attractions. This makes it

quite difﬁcult to obtain a uniform dispersion of individual nanotubes into a matrix material. Another

signiﬁcant challenge is effectively transferring load from a matrix to nanotubes, which have atomically

smooth surfaces. The main dispersion methods include mechanical procedures, sonication of nanotubes

in solvents and surfactants, and surface functionalization [60]. Among them, surface functionalization

seems superior in that functional groups on the surfaces can not only isolate individual nanotubes from

each other and therefore achieve uniform distribution throughout a matrix, but can also provide possible

sites for covalent bonding between nanotubes and matrix to facilitate load transfer. Dyke and Tour [61]

added various functional moieties to the surfaces of SWNTs by diazonium-based reactions and were able

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11-8 Tissue Engineering

to separate bundles into individual nanotubes. Mitchell et al. [62] showed that such functionalized SWNTs

demonstrated much better dispersion in polymer than nonfunctionalized ones.

Many researchers have reported signiﬁcant improvements of mechanical properties in thermoplastics

and epoxy resins by the addition of MWNTs or SWNTs [63,64]. Cooper et al. [65] found that impact

strengths of both types of carbon nanotubes in PMMA were signiﬁcantly improved compared to pure

polymer. Carbon nanotubes provide another opportunity for creating dense ceramic composites with

enhanced mechanical properties by absorbing energy through their highly ﬂexible elastic behavior during

deformation [66]. With the addition of 5 or 10% well-dispersed MWNTs, both the strength and fracture

toughness of alumina were greatly increased [56]. In addition to their exceptional mechanical properties,

carbon nanotubes also possess superior electric properties [58]. In an in vitro study, current-conducting

composites of polylactic acid (PLA) and multiwalled carbon nanotubes were effectively used as substrates

to expose osteoblasts to electrical stimulation, which promotes cellular functions for new bone formation

[67]. Though carbon nanotubes are a relatively new material for biomedical applications, they show great

potential for engineering biomaterials for hard tissue scaffolds.

11.3 Conclusions

As is evident by the described studies, a great deal of progress has been made toward improving bio-

materials for tissue engineering through nanotechnology. Nanocomposite scaffolds have demonstrated

enhanced mechanical properties and improved osteoconductivity of polymers as well as other materials.

The challenge remains to design a nanocomposite scaffold with mechanical properties suitable for hard,

cortical bone regeneration therapies. Future studies of nanocomposites should focus on answering an

important question: How do these novel materials perform in vivo?Thein vivo biocompatibility and

osteoconductivity must be well characterized before the high potential of nanocomposite scaffolds for

tissue engineering can be achieved.

Acknowledgments

This work was supported by the National Institutes of Health (R01 AR48756) (AGM) and the Nanoscale

Science and Engineering Initiative of the National Science Foundation (EEC-0118001). ASM acknowledges

the support of the NIH Biotechnology Training Grant (5 T32 GMO 08362).

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