Nanocomposites for Bone Tissue Regeneration

Nanda Gopal Sahoo; Yong Zheng Pan; Lin Li; Chao Bin He

Disclosures

Nanomedicine. 2013;8(4):639-653. 

In This Article

Materials for the Composites Used in Bone Regeneration

Bone tissue is the major structural and supportive connective tissue of the body. It is a mineralized connective tissue formed by osteoblasts. Osteoblasts deposit a matrix of collagen and also release calcium, magnesium and phosphate ions that chemically combine within the collagenous matrix into a crystalline mineral, known as bone mineral, in the form of HA. Therefore, bone tissue is a natural composite consisting of a collagenous matrix and a mineral phase. Therefore, it is necessary to develop new therapeutic materials with the same hybrid structure that combine the strength, stiffness and osteoconductivity of an inorganic component with the flexibility, toughness and resorbability of an organic phase.[13,14] As such, in recent years, researchers have concentrated on developing the polymer/ceramic nanocomposites, which have the advantages of both polymers (structural stability, strength, biocompatibility and desired shape) and ceramics, and are more similar to natural bones.

Bioceramic/Synthetic Polymer Nanocomposites

Bioceramic/Biodegradable Polymer Nanocomposites Bioceramics such as calcium phosphate, calcium sulfate, β-TCP and HA are clinically used as implant coatings or bone-void fillers because of their attractive biodegradable, bioactive and osteoconductive properties, and also because they have a notable ability to bond directly to bone.[15,16] However, the main drawback of these materials is their brittleness, which makes them susceptible to catastrophic failure. On the other hand, because synthetic biodegradable polymers are biocompatible, biodegradable, nontoxic and easier to process, they have extensive applications in the biomedical field, for example temporary scaffolds (e.g., sutures, bone fixation devices and artificial skin) for tissue repair, drug delivery and release devices, and other surgical implants. The polymers commonly used in these applications are poly(glycolic acid), poly(lactic acid) (PLA) and their copolymer poly(lactide-co-glycolide) (PLGA), polydioxanone, poly(ethylene oxide) and poly(trimethylene carbonate).[2,17] In addition, poly(ε-caprolactone) (PCL), polyanhydrides, poly(vinyl alcohol) (PVA) and polyurethanes have also been investigated for bone regeneration.[17] However, out of these polymers, PLA, poly(glycolic acid) and PLGA have received the highest interest because the architectures and properties of these polymers can be easily controlled.

Ceramic–polymer composites are widely studied alongside their application in bone/tissue regeneration because of the excellent combination of bioactivity and osteoconductivity of the ceramics, and the flexibility and shape controllability of the polymers. For example, HA and TCP powders were mechanically mixed with different polymers, such as poly(glycolic acid), PCL and their copolymers, and their biomedical applications were studied.[18–20] The addition of bioactive ceramic particles, fibers or whiskers to polymers improves not only the biological properties, but also the mechanical properties of polymeric scaffolds.[9,21] The mechanical properties of the nanocomposites play a significant role in bone tissue engineering.

Laurencin's group developed a nanocomposite using PLGA microspheres as a matrix and amorphous calcium phosphate nanoparticles as a reinforcement phase to optimize the structure of the sintered microsphere matrix with various microsphere diameters. They studied the osteoconductivity of the optimal structure with respect to human osteoblasts.[22,23] The cell studies demonstrated that the primary culture human cells proliferated through the pores of the matrix and also continued to express their osteoblast phenotype. The compressive modulus of 64.7 MPa was obtained by heating the microspheres at 4°C for 90 min, with a high polymer:ceramic ratio.[23] This modulus is within the range of trabecular bone.

Since bone is composed of HA and collagen, many attempts have been made to introduce nano-HA (n-HA) particles into a polymer matrix to improve the mechanical as well as biological properties of the scaffolds, and mimic the synthetic structure of bone.[24–28] Sharifi et al. developed a new biodegradable nanocomposite based on poly(hexamethylene carbonate fumarate) and n-HA.[29] The storage modulus of the nanocomposite increased with increasing n-HA content due to the enhanced interaction between HA particles and poly(hexamethylene carbonate fumarate) at their interface. The cell proliferation of the poly(hexamethylene carbonate fumarate)–HA composites was significantly increased by the addition of n-HA particles into the composites. Therefore, this composite scaffold can be used for bone tissue engineering applications. Kim developed a nanocomposite consisting of HA and PCL through the mediation of a surfactant oleic acid, where the HA nanoparticles were uniformly dispersed within the PCL matrix.[25] This nanocomposite showed significantly higher mechanical strength (22 MPa) compared with the conventional nanocomposite and the pure PCL. The nanocomposites also showed significant improvement of osteoblastic cell proliferation compared with the conventional nanocomposites. This nanocomposite may be useful in bone tissue regeneration.

Cui et al. investigated the in vitro and in vivo biodegradation of a biomimetic bone scaffold composite, n-HA/collagen/PLA, which can be used for bone tissue engineering.[30,31] The compressive strengths increased with increasing PLA content in the composites and reached the lower limit (1 MPa) of natural cancellous bone. The elastic modulus was at the maximum value of 47.3 MPa in the case of 10% PLA-containing composites, which is comparable with the compressive modulus of trabecular bone (50 MPa). Within a week, osteoblasts adhered, spread and proliferated throughout the pores of the scaffold composite materials. In vivo, a rabbit segmental defect model was used to evaluate these nanocomposites. It was observed that the segmental defect was integrated 12 weeks after surgery, and the implanted composite was partially substituted by new bone tissue. This composite scaffold was shown to be a promising material for bone tissue engineering.

However, the interfacial compatibility and interaction between the n-HA filler and the polymer matrix are vital factors to determine the dispersion of n-HA in a polymer matrix and the mechanical properties of the nanocomposite. The poor interaction between n-HA and a polymer matrix will result in an early failure at the interface and, thus, weakening the mechanical property and structural stability of the scaffold.

Zhang et al. first prepared n-HA surface-grafted with PLA (g-HA) and mixed with PLGA using a solvent casting (SC)/particulate leaching (PL) method to investigate its applications in bone replacement.[32] Composites of ungrafted HA/PLGA and pure PLGA were used for comparison. They investigated in vivo mineralization and osteogenesis after intramuscular implantation for repairing radius defects in rabbits. Figure 1 represents the radiographic results of rabbit radius defects (2.0 cm in length) repaired with the implant of BMP-2/g-HA/PLGA, g-HA/PLGA, HA/PLGA and PLGA at 0, 2, 4 and 8 weeks postsurgery. At 2 weeks postsurgery, the radiographs in the repaired areas of BMP-2/g-HA/PLGA, g-HA/PLGA and HA/PLGA groups appeared similar and the implants exhibited distinct mineralization (Figure 1Aii, Bii & Cii). There was more new bone formation in the group of BMP-2/g-HA/PLGA compared with other groups. The densities of the repaired areas for all groups increased gradually at 4 and 8 weeks postsurgery. However, the scaffold of g-HA/PLGA exhibited rapid and strong mineralization and osteoconductivity, and the incorporation of BMP-2 promoted the osteogenic process of the composite implant.

Figure 1.

Representative computer radiographs of rabbit radius defects implanted with porous scaffolds.
Scaffolds of (A) BMP-2/nano-hydroxyapatite surface grafted with poly(lactic acid) (g-HA)/poly(lactide-co-glycolide) (PLGA), (B) g-HA/PLGA, (C) hydroxyapatite/PLGA and (D) PLGA at 0, 2, 4 and 8 weeks postsurgery. There was more mineral deposit and new bone formation in the groups of BMP-2/g-HA/PLGA, g-HA/PLGA and hydroxyapatite/PLGA than in the group of PLGA. (Ai, Bi, Ci & Di) 0 weeks (the first day after surgery); (Aii, Bii, Cii & Dii) at 2 weeks; (Aiii, Biii, Ciii & Diii) at 4 weeks; and (Aiv, Biv, Civ & Div) at 8 weeks.
Reproduced with permission from [32].

The addition of BMP-2 and HA into polymer scaffolds is a very helpful way to produce high performance bone regenerative materials.[33] Recently, Jeon et al. developed a novel bone-regenerative scaffold based on PLGA microspheres, n-HA and BMP-2.[34] Jeon et al. functionalized HA and PLGA with polyphosphate and amine, respectively, and this polyphosphate-functionalized HA (PP-n-HA) was immobilized on the pore surface of the PLGA and amino-functionalized PLGA microspheres to control the addition and release of BMP-2. BMP-2 was controllably released for approximately 1 month, as the positive charge of BMP-2 was attached with high efficiency to the anionic phosphates of surface-immobilized PP-n-HA. The dynamic 3D cell culture system was used for determining the osteogenic differentiation and proliferation of human adipose-derived stem cells within the HA/BMP-2/PLGA microspheres. The results showed that the PP-n-HA-immobilized surface supported cell adhesion, proliferation and osteoconduction. In addition, the BMP-2 further improved the bone tissue regenerative activity of the porous microspheres with respect to osteoinductive property.

Methods such as SC/PL or phase separation methods are commonly used for the fabrication of polymer/bioceramic composite scaffolds.[32,35] However, residual solvents in the scaffolds may not be safe for cells or tissues[36] and the ceramics coated by polymer may hinder the contact of the ceramics to the scaffold surfaces, which may decrease the ability of osteogenic cells to make contact with the bioactive ceramics. Kim et al. fabricated PLGA/n-HA nanocomposite scaffolds by the gas forming (GF)/PL method without the use of organic solvents.[37] Compared with the conventional SC/PL method, this method allows exposure of significantly more HA nanoparticles at the scaffold surface, and scaffolds prepared by this method showed interconnected porous structures without a skin layer and exhibited superior mechanical properties. The compression and tensile moduli of the GF/PL scaffolds increased by 99 and 1331%, respectively, compared with the SC/PL scaffolds. From the in vitro study, it was observed that the GF/PL scaffolds exhibited the higher cell growth, alkaline phosphatase activity and mineralization than the SC/PL scaffolds. At 5 and 8 weeks after in vivo implantation, the GF/PL scaffolds showed improved bone formation compared with the SC/PL scaffolds and the PLGA scaffolds without HA. This is likely to be due to the higher exposure of HA nanoparticles at the GF/PL scaffold surface, which allowed direct contact with the transplanted cells and stimulated cell proliferation and osteogenic differentiation. Based on these results it can be concluded that the polymer nanocomposite scaffolds prepared by the GF/PL method are able to increase bone regeneration more significantly compared with those fabricated by the conventional SC/PL method.

Duan et al. prepared 3D nanocomposite scaffolds based on calcium phosphate/poly(hydroxybutyrate-co-hydroxyvalerate) and carbonated HA/poly(L-lactic acid) (PLLA) nanocomposite microspheres using selective laser sintering.[3] The in vitro study suggested that cell proliferation and alkaline phosphatase activity for calcium phosphate/poly(hydroxybutyrate-co-hydroxyvalerate) scaffolds were significantly improved after the incorporation of calcium phosphate nanoparticles, whereas a similar cell response was observed for both carbonated HA/PLLA nanocomposite and PLLA polymer scaffolds. These nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue regeneration applications.

The bioactivity and mechanical properties of the nanocomposite scaffolds depend on the shape and size of HA particles on the nanocomposites. Roohani-Esfahani et al. prepared a nanocomposite biphasic calcium phosphate scaffold by coating a nanocomposite layer consisting of HA nanoparticles and PCL, and found that the compressive strength of the HA nanoparticle composite-coated scaffolds was 2.1 ± 0.17 MPa, which was significantly higher than that of pure HA/β-TCP (0.1 ± 0.05 MPa) and micron HA composite-coated scaffolds (0.29 ± 0.07 MPa).[38] The use of nanoparticles instead of the micron-sized particles resulted in enhancement of the interfacial bonding between the nanosized particles and the polymer matrix, due to a higher surface area and better wettability. It can also be concluded that the needle-shape particles had significant capability to increase mechanical properties of PCL compared with rod and spherical shapes. These needle-shaped scaffolds also showed the strongest osteoblast differentiation profile compared with other groups, suggesting their potential application in bone tissue regeneration.

In addition to the aforementioned biodegradable synthetic polymers, PVA is also commonly used in biomedical fields because of its nontoxic, highly hydrophilic and biocompatible nature. PVA has a high tensile strength and flexibility. Poursamar et al. synthesized HA/PVA nanocomposite scaffolds using colloidal HA nanoparticles in a PVA solution and freeze drying.[39] The elastic modulus of the nanocomposites decreased from 254.7 to 226.6 MPa, whereas the compression yield strength (~113 MPa) was almost constant and the toughness increased from 3.27 to 3.72 J/mm3 with the addition of 40–60% PVA in the composites. The SaOS-2 cell line was used for the in vitro biocompatibility study of the HA/PVA scaffolds. The cells were well spread on the scaffolds, which indicated good attachment and penetration to the pores of the scaffolds. Cell aggregation on the scaffolds increased continuously during 3 days of incubation. These results proved that the HA/PVA scaffold had not only an excellent biocompatibility but also an ability to enhance cell adhesion and proliferation. Zeng et al. synthesized HA/PVA nanocomposite membranes by a solvent casting and evaporation method; they proved that the surface of composite membranes was suitable for the adhesion and proliferation of osteogenic cells and that the composite membranes had a good biocompatibility.[40] Therefore, the HA/PVA nanocomposite scaffolds could be used for guided bone regeneration.

As natural bone is composed of HA nanocrystallites and collagen, it is important to design composites containing either HA nanocrystals or a component that induces the formation of HA nanocrystals, to mimic the bone composition and structure. Researchers have considered SiO2 as an ideal candidate material because silanolan groups interact with calcium and phosphate ions forming an amorphous calcium phosphate that can also be found in the natural bone. Stodolak-Zych et al. fabricated nanocomposite scaffolds based on modified poly-(L/DL)-lactide and SiO2.[41] The in vitro study showed that the silica nanoparticles present in the nanocomposite foils acted as nucleating centers of apatite and accelerated its crystallization. The chemically active bonds created on the scaffold surface, in other words Si–O on poly-(L/DL)-lactide/2% SiO2, constituted the biomimetic structure of an apatite. In other studies, bio-nanocomposites were fabricated for bone regeneration based on PCL and silica nanoparticles, where both PCL and silica nanoparticle surface were modified to improve the interaction between the nanoparticles and the polymer matrix.[42] The authors investigated the in vitro biocompatibility of the biocomposites as well as their capacity to induce the osteogenic differentiation of human bone marrow stromal cells. In particular, they analyzed the adhesion and the growth of these cells. The results suggested that the cells adhered and grew onto all the materials but to different extents. These nanocomposite seem to have promise in regenerative medicine applications.

Mg2SiO4 is a new bioceramic that exhibits good bioactivity for nanoscale structures and also shows mechanical properties superior to those of HA and bioglass.[43,44] Diba et al. prepared novel porous nanocomposite scaffolds containing PCL and forsterite nanopowder by a SC/PL method and investigated the effects of forsterite nanopowder on the mechanical properties, bioactivity, biodegradability and cytotoxicity of the scaffolds.[44] The elastic modulus of the scaffolds increased from 3.1 to 6.9 MPa and the compressive strength increased from 0.0024 to 0.3 MPa when the forsterite nanopowder was increased to 30 wt% in the composites, probably because the forsterite nanopowder acts as a stiff filler within the PCL matrix and, thus, decreases the porosity and pore sizes. The in vitro tests of cytotoxicity and osteoblast proliferation showed that the nanocomposite scaffolds were noncytotoxic, therefore, allowing cells to adhere, grow and proliferate on the surface of these scaffolds. Increasing the content of forsterite nanopowder in the composites promoted cell growth and differentiation in the nanocomposite scaffolds (Figure 2). This is due to the formation of more silanol groups on the surface of scaffolds, which in turn leads to increased cell adhesion and generation of higher cellular activity.

Figure 2.

Scanning electron microscopy morphology images.
(A) Pure poly(ε-caprolactone), and nanocomposite scaffolds containing (B) 10, (C) 20, (D) 30, (E) 40 and (F) 50 wt% forsterite cultured for 2 days with SaOS-2 cells.
Reproduced with permission from [44].

Recently, several research groups have developed different types of nanofibrous composite scaffolds composed of a fibrous matrix and HA nanocrystals.[45] The fibrous matrices can be made of different materials, such as PCL–PEG–PCL copolymer,[46,47] PLLA[48] and PCL.[49]

Fu et al. successfully prepared an electrospun n-HA/PCL–PEG–PCL fibrous scaffold.[46,47] The results of hydrolytic analysis revealed that the composite scaffold was degradable and its weight was reduced by approximately 32.6 ± 3.5% after 10 weeks. The in vivo biocompatibility and degradability were examined by implanting the scaffold into the muscle pouches. When implantation time was increased from 4 to 20 weeks, the newly formed bone increased from 28.1 to 82.6% in the treated defect. By contrast, the newly formed bone for the control sample increased from 22.8 to 56.9%. The results confirmed that the electrospun n-HA/PCL–PEG–PCL fibrous scaffold possessed excellent osteogenesis and a high efficiency of bone formation. However, the tensile strength and elongation-at-break of this scaffold is relatively low, therefore, the authors suggested that the scaffold could be used for guided bone regeneration in non-load-bearing bone defects.

From the above discussion, it can be concluded that ceramic–polymer nanocomposites have been shown to be more effective for enhancement of both mechanical properties and bioactivity compared with ceramics and polymers and, thus, they are suitable for application in bone tissue regeneration.

Bioceramic/Nonbiodegradable Polymer Nanocomposites Nonbiodegradable polymers have been used in bone tissue engineering due to their better mechanical properties and chemical stability than biodegradable polymers. Currently, synthetic nonbiodegradable polymers used for bone tissue engineering are polyethylene, polypropylene, polytetrafluoroethylene, poly(vinyl chloride), polyamide (PA), poly(methyl methacrylate), polycarbonate, poly(ethylene terephtalate), poly(ether ether ketone), acrylics and silicones, among others.[9,17,50] HA/nonbiodegradable nanocomposites are usually used when tissue cannot be regenerated due to large losses or for elderly patients with a less effective self-healing ability of the tissue.[17]

UHMWPE has attracted considerable attention as a matrix for HA/polymer nanocomposites for biomedical applications. Fang et al. developed HA-reinforced UHMWPE nanocomposites by combined swelling/twin-screw extrusion, compression molding and then hot drawing.[51] The HA nanoparticles were homogeneously dispersed in UHMWPE. The tensile strength of the composite (100 ± 22 MPa) after hot drawing was comparable with that of cortical bone, and much higher than that of other HA/UHMWPE nanocomposites.[52] This biocomposite showed an excellent ability to induce calcium phosphate formation in simulated body fluid, which was promising for applications in load-bearing bone substitutes.

PA is considered to be a useful polymer for biomedical application because PA contains CONH groups in the molecular chains that are similar to the chemical structure of collagen. It also has good biocompatibility with human tissues. PA6,6/n-HA composites showed mechanical properties similar to those of natural bone, and the in vivo results proved that these nanocomposites have good biocompatibility to bone and can bond to hard tissue directly.[53] Wang et al. synthesized n-HA/PA composite scaffolds by a thermally induced phase inversion processing technique.[54] These n-HA/PA composite scaffolds with 70% porosity presented a compressive strength comparative with that of natural cancellular bone. In the in vitro study the n-HA/PA scaffolds exhibited no negative effects on the growth, proliferation and osteoblastic differentiation of marrow-derived mesenchymal stem cells (MSCs). The in vivo study proved that n-HA/PA composite scaffolds have a good biocompatibility and extensive osteoconductivity with host bone. Therefore, this nanocomposite can be applied in orthopedic, reconstructive and maxillofacial surgery.

Li and Yang prepared n-HA/PA6 nanocomposites via in situ hydrolytic ring-opening polymerization of ε-caprolactam in the presence of n-HA aqueous slurry.[55] The tensile strength, bending strength and bending modulus of these composites were 78.2 MPa, 120.5 MPa and 5.79 GPa, respectively, which are close to natural bone (60–120 MPa, 80–210 MPa and 3–25 GPa, respectively).

Recently, Cheng et al. synthesized the n-HA/PA6 composite scaffold by a combined method of phase separation and particle leaching.The scaffold was cultured with and without MSCs.[56] They found that the scaffolds acted as a template for the adhesion, growth, differentiation and proliferation of cells, and had no negative effects on MSCs in vitro. In animal experiments, both pure scaffolds and MSC-hybridized scaffolds exhibited a favorable biocompatibility and osteogenesis, but the hybrid scaffolds accelerated bone reconstruction compared with the pure scaffolds. These studies indicate that n-HA/PA composite materials are suitable for bone tissue engineering and that they can be used in orthopedics for clinical application. Besides these polymer nanocomposites, other HA/nondegradable polymer nanocomposites such as HA/poly(ether ether ketone),[57,58] HA/polyoxymethylene[17] and HA/poly(methyl methacrylate) have been used for bone tissue engineering.

Bioceramic/Natural Polymer Nanocomposites

Natural biopolymers are currently of interest in tissue engineering because their biological recognition may positively support cell adhesion and function. However, they have poor mechanical properties. As the mechanical properties of a scaffold are important for bone, there is a need to improve the mechanical strength and biological property of natural polymer scaffolds in order to make them suitable for bone tissue engineering. Currently different bioceramics such as HA, β-TCP and calcium phosphate are being incorporated into natural polymers to improve the mechanical as well as biological properties.[59–61] Composites containing collagen and calcium phosphate have received much more attention because they mimic the basic composition of bone. Cunniffe et al. developed a novel collagen–n-HA nanocomposite scaffold via suspension and immersion methods.[62] The composite scaffolds produced by the suspension method were up to 18-times stiffer than the collagen (5.50 ± 1.70 vs 0.30 ± 0.09 kPa). The in vitro analysis suggested that there was no significant difference in cell number observed in the case of nanocomposites prepared by the suspension method as compared with the collagen control. The collagen–n-HA nanocomposite scaffold exhibited higher mechanical properties and the same high biological activity as the collagen control scaffold, demonstrating its potential as a bone graft substitute in orthopedic regenerative medicine. Fu et al. fabricated a biomimetic hydrogel composite based on the triblock PEG–PCL–PEG copolymer, collagen and n-HA, and investigated in vivo biocompatibility and biodegradability by implanting the hydrogel composite in muscle pouches of rats for 3, 7 and 14 days.[63] The results showed that a slight inflammatory response appeared at 7 days postsurgery owing to the degradation of the implant, which indicated that the composite had a good biocompatibility and biodegradability. In vivo bone regeneration was evaluated by implanting the composite material in cranial defects of New Zealand white rabbits for 4, 12 and 20 weeks. Radiological examination and histological analysis showed that new bone tissue formed initially from the edge of the defects and the surface of the native bone and grew towards the center (Figure 3).

Figure 3.

Hematoxylin and eosin staining of cranial bone defect sections.
(A–C) Control and (D–F) treatment groups at 4, 12 and 20 weeks postsurgery. The treatment group did not exhibit serious inflammatory responses or foreign body reactions. Both groups allowed bone ingrowth, but the treatment group showed faster and more effective osteogenesis at the defect area than that of the control. The arrows indicate the new O originally formed at the margin of the HB. The dotted line indicates the interface between the HB and the defect region.
Magnification: 20×.
BM: Bone marrow; HB: Host bone; IM: Implanted material; NB: New bone; O: Osteoid; VT: Vascular tissue.
Reproduced with permission from [63].

Barbani et al. synthesized gelatin/HA nanocomposite-based porous scaffolds using freeze drying.[64] The elastic modulus of the nanocomposites was very close to natural bone. Biological tests showed good adhesion and proliferation of human MSCs. Luo et al. prepared a HA–gelatin nanocomposite using a coprecipitation method in the presence of aminosilane as a chemical linker to facilitate the binding and solidification of the HA–gelatin nanocomposite.[65] This nanocomposite exhibited a compressive strength of 133 MPa and showed a good biocompatibility based on cell adhesion, proliferation, alkaline phosphate synthesis and mineralization studies.

Chitosan (CS) and its derivatives are very attractive candidates for scaffold composites in orthopedic and other biomedical applications because of their biocompatibility, biodegradability, pore formation behavior, suitability for cell ingrowth and intrinsic antibacterial nature.[66] Other advantages of CS scaffolds are the formation of highly porous scaffolds with interconnected pores, osteoconductivity and the ability to enhance bone formation both in vitro and in vivo.[67] Reves et al. fabricated nanocomposite scaffolds based on CS and n-HA using coprecipitation and investigated the effects of the degree of deacetylation (DDA), drying method, HA content and acid washing on scaffold properties.[68] They found that the freeze-dried 61% DDA scaffolds exhibited a lower compressive modulus of 0.12 ± 0.01 MPa, but a higher degradation extent, with a weight loss 3.5 ± 0.5%, compared with the air-dried 61% DDA scaffolds and 80% DDA scaffolds. On the other hand, the air-dried 80% DDA scaffolds presented the highest compressive modulus (3.79 ± 0.51 MPa) and the degradation of these scaffolds increased from 1.3 ± 0.1 to 4.4 ± 0.4% after acid washing at pH 6.1. However, plain 80% DDA microspheres without HA exhibited higher proliferation of SAOS-2 cells compared with composite microspheres with HA. This result was not supported by previous results observed by the same authors who reported greater cell proliferation on the composite scaffolds compared with plain scaffolds.[69,70] This may be due to the different cell types used for these different studies. Other groups have also reported that the preosteoblast proliferation on CS–n-HA scaffolds was greater than pure CS scaffolds.[71]

The mechanical strength, physicochemical and biological properties of scaffolding matrices depend on the molecular weight (MW) of CS, the temperature and water content of the scaffold.[72] Biomimetic nanocomposites consisting of CS and n-HA with different MWs of CS were obtained by Thein-Han et al. using freezing and lyophilization processes.[73] The compression modulus of the high-MW CS scaffold was higher than the medium-MW CS, where the compression modulus of medium-MW CS with 2 wt% n-HA was nearly the same as the high-MW CS with 1 wt% n-HA. The compression modulus of the high-MW CS scaffolds was increased from 6.0 to 9.2 kPa by the addition of 1 wt% n-HA, which was attributed to the strong interaction between CS and HA. The biological response of preosteoblasts (MC 3T3-E1) was superior on nanocomposite scaffolds; for example, improved cell attachment, higher proliferation and a well-spread morphology compared with CS scaffold (Figure 4). Cell culture after 7 days and beyond showed that the cell proliferation on the composite scaffolds was approximately 1.5-times greater than that on pure CS. This study demonstrated that the CS–n-HA composite is a potential scaffold material for bone regeneration.

Figure 4.

Scanning electron micrographs illustrating morphology of preosteoblasts seeded on high-molecular-weight chitosan and chitosan–nanohydroxyapatite scaffolds (saggital section).
Preosteoblasts on the chitosan surface after (A) days 1, (B) 3, (C) 7 and (D) 21 of cell culture; and on the chitosan–nanohydroxyapatite surface after (E) days 1, (F) 3, (G) 7 and (H) 21 of cell culture.
n-HA: Nanohydroxyapatite.
Reproduced with permission from [73].

Silk fibroin (SF) polymers are very useful for biomedical applications, due to their excellent biocompatibility and biodegradability, superior mechanical properties and versatility in processing. Liu et al. fabricated a porous composite scaffold based on HA/SF by a two-step method. Initially, they fabricated the nanosized HA/SF composite powders by coprecipitation, and then blended them with SF solution to prepare the 3D porous HA/SF composite scaffolds.[74] These porous scaffolds prepared by the two-step method showed the higher compressive modulus and strength of 3.06 MPa and 350 kPa, respectively, compared with those of the conventional HA/SF composite scaffolds prepared by one-step blending (2.13 MPa and 261 kPa, respectively). This is due to the uniform dispersion of nanosized HA/SF composite powders in the SF matrix. Cell culture assay and MTT assay confirmed that the HA/SF composites scaffolds prepared by the two-step method improved the cell proliferation and osteogenic differentiation compared with the conventional composites because of the better cell attachment on the HA/SF composite prepared by the two-step method.

Niu et al. synthesized the HA/SF composite by biomimetic synthesis.[75] MG-63 cell viability was studied by MTT assay for the HA/SF composites and HA discs. The HA/SF exhibited excellent biocompatibility in vitro, accelerated osteoblast attachment and adhesion, and enhanced proliferation and differentiation. This is due to the fact that SF enhanced the attachment, adhesion and growth of cells. A rabbit model was used to determine the biocompatibility of the HA/SF composite. In the HA/SF group, the HA/SF composite particles were adsorbed and new bone was filled into the cavity faster than the pure HA group. This is because of the good biocompatibility and bioactivity of the HA/SF composites.

Tanaka et al. prepared the nanoscaled, sintered HA/SF sheets.[76] They studied the cell adhesion, proliferation and differentiation of the HA/SF sheets, SF sheets and tissue culture polystyrene dishes as controls using rat marrow mesenchymal cells. They observed a large number of viable cells on the HA/SF sheets compared with the controls after 1 h of culture. After 14 days of culture, the alkaline phosphatase activity and bone-specific osteocalcin secretion of the cells on HA/SF sheets were higher compared with the controls under osteogenic conditions. This is due to the good adhesion of the cells to the HA/SF sheets compared with the controls. Therefore, the HA/SF composites are promising materials for bone replacement and regeneration.

This article previously discussed that BMP-2 and HA can help to produce high-performance bone-regenerative materials. Recently, Notodihardjo et al. studied the effect of bone formation on critical size defects of 4 mm in rat calvarial bone by applying BMP-2 with or without HA to the defects.[77] The authors used four groups of five male Wistar rats: control, HA, BMP-2 and mixed BMP-2/HA. Osteochondrogenesis was highest for the mixed BMP-2/HA group (85.29 ± 8.21%), followed by the BMP-2 (77.34 ± 7.39%) and HA groups (59.82 ± 11.23%), and lowest for the control group (40.27 ± 7.44%) within 4 weeks. Therefore, the results suggested that the BMP-2/HA group exhibited the highest level of bone induction.

Ono et al. also proved that BMP-2 has strong bone induction properties with HA by studying the cranial bone of rabbits.[78,79] They treated HA rods with 1 or 5 µg of recombinant human BMP-2 and implanted them into the cranium of rabbits. These HA rods were removed after implantation for 3, 6 or 9 weeks, and the authors observed that the group with recombinant human BMP-2-treated HA rods had significantly improved bending strength at 3 weeks, and new bone formation was also seen in the rod pores, which was more prominent in those receiving the higher dose of 5 µg.

In addition to HA, other ceramics also provide cell adhesion and cell proliferation when combined with CS. Composite scaffolds of CS–gelatin with bioactive glass ceramic nanoparticles showed good bioactivity, and better cell attachment and spreading over CS–gelatin scaffolds.[80]

Cu and Zn ions are utilized in the synthesis of biomaterials for bone tissue engineering because of their high antibacterial activity, low toxicity and chemical stability. The effect of Cu–Zn alloys in n-HA/CS composites scaffolds was studied by Tripathi et al..[81] The scaffolds based on both n-HA/CS and nano-Cu–Zn showed decreased degradation and increased swelling, protein adsorption and antibacterial activity as compared with the n-HA/CS-based scaffolds. The n-HA/nano-Cu–Zn/CS scaffolds did not exhibit any toxicity towards the rat osteoprogenitor cells. In the presence of Cu–Zn nanoparticles, the scaffolds showed better antibacterial and osteoproliferative properties, which help to minimize the implant-associated bacterial infection and to promote bone regeneration.

Recently, bacterial cellulose (BC) materials have been developed for bone tissue regeneration purposes because of their osteoconduction properties.[82] BC possesses high mechanical strength and modulus, as well as good biodegradability compared with other natural biodegradable polymers, which are important when developing scaffolds. Fang et al. developed HA/BC nanocomposite scaffolds that were biocompatible and exhibited improved cell proliferation and differentiation in vitro with stromal cells derived from human bone marrow.[83] Saska et al. developed BC/HA nanocomposites and evaluated them in noncritical bone defects in rat tibiae at 1, 4 and 16 weeks.[84] The in vivo results demonstrated that there was no inflammatory reaction after 1 week. They found that the defects were completely filled in by new bone tissue after 4 weeks. Therefore, from these results it can be concluded that the BC–HA nanocomposites were effective for bone regeneration.

Liu et al. used a thermally induced phase separation technique to prepare nanofibrous gelatin (NF-gelatin) and then fabricated apatite/NF-gelatin composite scaffolds for bone tissue engineering.[85] The NF-gelatin scaffolds showed high porosity, interconnectivity and good mechanical strength. The compressive modulus of the NF-gelatin scaffold was 801 ± 108 kPa, which was more than ten-times higher than that of Gelfoam® (Pfizer, NY, USA) with a similar porosity. With addition of apatite, the apatite/NF-gelatin composite scaffolds demonstrated high mechanical strength as well as favorable osteoblastic cell differentiation, indicating that it was a good candidate for bone tissue engineering.

Carbon Nanotube/Polymer Nanocomposites

The excellent mechanical properties, highly specific surface area and low density of carbon nanotubes (CNTs) make them ideal for the fabrication of tissue engineering scaffolds with high strength and low weight. Although the biocompatibility and cytotoxicity of CNTs are not clear, many researchers have found that biofunctionalized CNTs are water soluble and can be cleared from systemic blood circulation through the renal excretion route, indicating that biofunctionalized CNTs are safe for biomedical applications. Recently, the application of CNT/biopolymer nanocomposites to tissue engineering has attracted increasing attention,[86,87] CNTs have been incorporated into different polymer matrices such as PCL,[88,89] CS[90,91] and PLGA[92] to produce scaffolds for bone tissue engineering.

Pan et al. fabricated multiwalled CNT (MWCNT)/PCL nanocomposites by the solution mixing and evaporation technique.[88] Both of the tensile and compressive moduli were significantly enhanced with the addition of MWCNTs. The compressive modulus of nanocomposites filled with 0.5, 1 and 2 wt% MWCNTs increased by 37.2, 38.8 and 54.5%, respectively, as compared with pure PCL. Moreover, the addition of MWCNTs to PCL facilitated cell growth and promoted cell attachment, proliferation and differentiation. In the cell adhesion assays, the rat bone marrow-derived stroma cells covered the whole surface of the nanocomposite scaffold but only 60% of the surface of the pure PCL scaffold, suggesting the better cellular compatibility of the MWCNT/PCL scaffold, which may result from the rough nanoscaled surface topography of the nanocomposite scaffold. It was found that the nanocomposite scaffold containing 0.5 wt% MWCNT exhibited the best enhancement of the proliferation and differentiation of bone marrow-derived stroma cells while a content of MWCNTs above 2 wt% could lead to a reduced effect on cell growth. Mattioli-Belmonte et al. also fabricated a bone-like structure scaffold based on MWCNT/PCL nanocomposites by solution mixing.[89] The elastic modulus of nanocomposites increased to 75 MPa with 11 wt% MWCNT, which is much higher than that of pure PCL (10 MPa). MTT assay showed that the nanocomposites can sustain osteoblast proliferation and the osteoblast viability depending on the intrinsic rigidity of the substrate, as well as the architecture and morphology of the substrate.

In addition to the fabrication of CNT-filled biopolymers via directly physical mixing, CNTs can be chemically modified and integrated into biomaterials for bone tissue engineering. Kim's group has developed a series of scaffolds based on functionalized MWCNTs (f-MWCNT)/CS nanocomposites for bone tissue engineering.[90] A scaffold for bone tissue engineering necessitates an interconnected highly porous structure as mentioned above. In comparison with the 87.7% porosity of a pure CS scaffold, the addition of only 0.01 wt% f-MWCNT increased the porosity to 88.5%, probably due to the highly specific surface area of f-MWCNT. The mechanical properties of the f-MWCNT/CS nanocomposites were not reported in their study, although it is generally regarded that the incorporation of CNTs could enhance the mechanical properties. The cytotoxicity and cell proliferation for various scaffolds of f-MWCNT/CS were examined using a MTT assay. The cell proliferation of MG-63 cells on the f-MWCNT/CS nanocomposites scaffolds was observed to be twice that of the pure CS scaffold, indicating the better biocompatibility and biofunction of the nanocomposite scaffolds. They attributed this advantage to the ability of f-MWCNTs to easily interact with cells and increase the cell metabolic function, which may be confirmed by their ongoing study. Kim's group also fabricated a tricomponent system composed of CS-grafted MWCNT, HA and CS for bone tissue engineering.[91] The ternary nanocomposite was found to have no cytotoxicity in the MG-63 cell line and the cell viability on the nanocomposite scaffold was even higher than that of the pure CS scaffold. In addition, the cell proliferation of MG-63 cells on the nanocomposite scaffolds was higher than that on the pure CS scaffold.

The cytotoxicity of CNTs is still obscure, but has been proven that its toxicity could be drastically decreased when incorporated into a polymeric matrix,[93] thus making it possible to fabricate CNT/polymer nanocomposites for bone tissue engineering. However, the long-term toxicity of CNTs in human tissues and the influence of CNTs on bone remodeling still need further investigation.

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