The need for the development of bone grafts is continuously stimulated by the unsatisfactory performance of available grafts and, of course, lack of an appropriate technique to fabricate bone-resembling grafts. A novel way of fabricating nanocomposite bone grafts using strategies found in nature have recently received much attention and have been found to be more beneficial than other conventional methods. This section describes more on nanostructured bonelike composite grafts processed by biomimetic self-assembly suitable for bone-grafting applications.
Of particular interest, in this section, attention is given to HA/collagen systems because they are currently widely accepted osteoconductive bone grafts and also they are the basic ingredients of natural bone tissue. Biomimetic HA/collagen composite offers enormous promise for the repair of defective bone tissue owing to its structure, composition, and other factors similar to natural bone. In fact, bone is a family of nanocomposites in which the basic building blocks are the mineralized collagen nanofibers; nature thereby provides critical ideas on how to engineer such biomimetic bone grafts with a well-organized structure and composition. However, it is clear that the current approaches have not yet led to the design of optimal bone grafts owing to the lack of understanding of the complete mechanism involved in the natural bone-building strategy. With the advances of nanotechnology and structural biology, it is now possible to develop a new class of bone-resembling nanocomposite grafts that could help in bone regeneration similar to some extent to naturally occurring bone matrices.
A key step in the biomimetic strategy is to achieve a controlled nucleation and crystal growth of apatite phase onto collagen matrix (see Figure 13.11). This process partly mimics the biological phenomenon and demonstrates a good system for bone regeneration with enhanced osteoconductivity from a pure HA and a pure collagen [133,134]. A biomimetic nanocomposite was developed by self-assembling of HA nanocrystals onto collagen fibers . The direct nucleation of HA nanocrystals onto collagen fibers has been performed by starting from an aqueous suspension of Ca(OH)2 and H3PO4 together with a collagen solution of pH 9 to 10 at 25°C. The obtained composite product was freeze-dried at -40°C and then gradually warmed up to 35°C for 36 h. The characteristic results indicate that the nanocrystals of HA are elegantly aligned with their c-axis preferentially oriented along the collagen fibers, suggesting a close interaction between HA and collagen phases.
As this process mimics biological mineralization to some extent, it was suggested that the HA/collagen nanocomposite can be used for bone repair in orthopedic and maxillofacial surgeries. A similar bonelike nanocomposite consisting of HA and collagen, by a self-organization mechanism using Ca(OH)2, H3PO4> and porcine atelocollagen as precursors, was developed by Kikuchi et al. [133,135]. The composite has elegantly self-organized into a nanostructure quite similar to that of natural bone, which might have occurred through a chemical interaction between HA and collagen. The length of self-assembled fiber bundles was found
to be 20 |im in which each bundle consisted of many collagen fibrils of 300 nm length embedded with bladelike HA nanocrystals of 50 nm in size. They found that increasing the degree of self-assembling eventually increases the bending strength of the composite, suggesting that high mechanical strength can be attained by optimizing the self-assembling mechanism between HA and collagen. The in vivo performance of this nanocomposite was evaluated in beagle dogs by creating an artificial bone defect. The results of the animal study proved excellent biocom-patibility, osteocompatibility, and bioactivity of the composite with surrounding tissues and stimulated the formation of new bone growth with an Haversian system (see Figure 13.12).
Furthermore, it enhanced the conventional biomaterials' rate of bone-healing within a short period, which might have resulted from its nanostructural self-assembly and compositional similarity to natural bone tissue, therefore a biomimet-ically processed HA/collagen system can be considered as a good bone graft. This kind of composite can readily be incorporated into bone metabolism rather than
being a permanent implant. Accordingly, the biomimetic processing of composites is perceived to be highly preferable to conventional processing methods.
It is also good to know that, nowadays, there are many advanced techniques and sophisticated instrumentations available for the study of biomineralization that tremendously helps the development of this interesting multidisciplinary field. Some of them are listed in Table 13.8 with their few critical comments. Recently, Zhai et al.  demonstrated the features of biomineralized recombinant humanlike collagen. They analyzed the mineral growth using various analytical tools. The morphology of the mineralized collagen was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The respective micrographs are shown in Figure 13.13. It can be seen that a flowerlike morphological structure of the mineralized collagen fibrils on the powder surface and the leaves of the flower are entangled and perpendicular to that surface (Figure 13.13a). The length of the fibrils is found to be about 20 nm in width and 500 nm in length. Furthermore, a TEM micrograph (Figure 13.13b) confirmed that biomineralization has taken place on the surface of collagen, that is, deposition of nano HA. The nano HA is about 6 to 25 nm in size and its c-axis is parallel to the collagen fibrils' orientation.
Another interesting method was developed for the self-assembly of HA coating onto collagen membrane . The membrane was soaked in a stimulated body fluid (SBF) solution, very close to human blood plasma, with and without citric acid, the role of which was exclusively investigated during the nucleation of HA. It should be noted that the researchers have chosen collagen membrane that had no HA-forming ability in the SBF solution. They found that there was no nucleation of apatite crystals on the surface of the membrane when it was soaked in SBF without citric acid. Interestingly, the membrane soaked in SBF with citric acid gradually stimulated the nucleation of HA crystals. Therefore, it can be suggested that citric acid has a nucleating ability and can accelerate the coating of apatite onto membrane.
Later, many research groups demonstrated the ability of nucleation of HA crystals onto collagen through a chemical interaction of carboxylate groups of collagen macromolecules [138-140]. All the reports suggested that the nucleation process
Table 13.8 Commonly used techniques in biomineralization study.
X-Ray Diffraction Techniques
Wide angle x-ray diffraction
Small angle x-ray scattering Synchrotron diffraction
Neutron Diffraction Technique
Electron Diffraction Techniques
Scanning electron microscopy
Transmission electron microscopy
Atomic force microscopy
Magnetic resonance force microscopy Infrared spectroscopy
Provides a specific proof for the mineral identification with their crystal structure and unit cell dimensions.
Determines mineral's crystal size, shape, and orientation. It is also widely used in structural analysis of proteins.
A relatively new technique to study mineral composition with high resolution.
Provides qualitative information on mineral texture and crystal orientation.
Provides morphological structure of the minerals with their size, shape, and related structural information.
Provides ultrastructure of minerals and their size and shape. Crystal orientation and texture can also be studied.
Provides details on molecular interaction between minerals and protein components through which mineral binding and deposition can be determined. Surface topology of the crystals can also be identified.
Provides complete details on chemical specificity, composition, three-dimensional subsurface structure.
Facilitates differentiation of symmetric and asymmetric vibration and stretching bonds of the mineral components and allows estimating mineral contents.
Provides all information on symmetric and asymmetric vibration and stretching bonds of the mineral components and allows estimating mineral contents, such as infrared.
is critically dependent on the carboxylate groups of the collagen. On the other hand, mechanical reliability of the nanocomposite does not match exactly with that of the host bone in many cases. It is an essential factor to be considered for all bone grafts as it directly measures their strength. In order to enhance the mechanical reliability of the mineralized collagen, a glutaraldehyde cross-linked porous HA/collagen nanocomposite was developed . Here, Ca(OH)2, H3PO4> and collagen were used as precursors to formulate the composite. An homogeneous suspension of 0.1994 mol of Ca(OH)2 dispersed in 2000 ml of H2O and 59.7 mM of H3PO4 was gradually added to the aqueous solution of 5 g collagen. The pH of the reaction mixture was adjusted to 8.4 and the reaction was maintained at 38°C and finally aged for 12 h by adjusting the pH to 7. In order to mimic the cross-linking process in the toughening of natural bone, 0.2% aqueous glutaraldehyde solution was slowly added into the slurry solution at the same reaction temperature. The composite was thoroughly analyzed and its 3-D porous nanostructure that could be able to support cellular growth was confirmed. They noticed that if the cross-linking agent was added, the size of the reaction precipitate was found
to grow and seemed to be triggered by the assembly of HA nanocrystals (50 nm) around the collagen fibers (300 nm), leading to the formation of a thick composite bundle. The thickness of the mineralized collagen depends on the amount of glutaraldehyde added. Because this system has relatively good toughness, it was suggested for use as a bone graft.
Although many investigations have been carried out to mimic bone artificially, using HA and collagen, with compositional, structural, biological, or mechanical functions comparable to its natural counterpart, it should be noted, however, that bone is not a simple mixture of HA crystals and collagen fibers in many ways. In addition to collagen, bone contains many traces of bioorganics such as glycopro-teins or glycosaminoglycans, which play a pivotal role in controlling the functions of bone cells. In this regard, attempts were made to mimic both the bioorganic compositions of bone and peculiar configurational arrays of HA nanocrystals on thosebioorganics [142,143].
A design strategy for making such a nanocomposite using HA, collagen, and chondroitin sulfate (proteoglycans) was developed with an intention of using it as a biomimetic bone graft for cartilage repair (a premature bone) as well as for bone repair. The hypothesis was that the cartilage is a key tissue of all growing bones because bone formation is initiated from the calcification of cartilage and then it would be replaced by the bone through endochondral ossification. Therefore, cartilage is regarded as a precursor of bone. The chondroitin sulfate is best known for its ability to promote the binding of chondronectin, which is the chon-drocyte attachment factor, to collagen and thus gradually stimulates chondrocyte adhesion. Accordingly, this nanocomposite system is likely to provide specific binding sites for chondrocytes. The crystallographic results confirmed the orientation of the c-axis of the HA nanocrystals along the longitudinal direction of the collagen and chondroitin sulfate complex. The distribution of HA was found to be almost uniform within the matrix and has substantial mechanical strength with fracture toughness 35-50 MPa and hardness 119-219 MPa. In addition, it has a potential capacity of bone remodeling through the process of endochondral ossification. Based on these results, the proteoglycans-immobilized HA/collagen nanocomposite may be used as a promising bone substitute.
Nanocomposite grafts can also be used as a carrier vehicle for the delivery of growth factors. Recently, an HA/collagen nanocomposite system was biomimet-ically developed and used as a carrier for the delivery of rhBMP-2 [144,145]. The performance study of this kind of system is based on the hypothesis that the controlled release of rhBMP-2 biomolecules facilitates early callus and new bone growth upon implantation, as compared to other conventional biomaterials. As discussed earlier, the rhBMP-2 is a well-known osteoinductive protein. It should be noted that both naturally occurring BMPs and recombinant BMPs have long been used as osteogenic initiators in various bone-defective models in order to enhance the bone-healing process quite similar to autogenic bone. The studies found that the mechanical reliability of this system corroborated well with the strength of autogenic cancellous bone. The overall experimental results indicated that the in vivo performance of the nanocomposite with rhBMP-2 is better than the nanocomposite without rhBMP-2.
Early bone formation occurred with the use of rhBMP-2 treated composites, which implies their efficacy as a good bone graft. Based on these findings, this biomimetically processed system is considered as a perfect osteoconductive as well as osteoinductive bone graft. It can be used for anterior fusion of the cervical spine as well as inlay grafting bone defects in weight-bearing orthopedic sites. Although biomimetic design of bone grafts is still in the infant stage, there is a bright future for creating a new generation of bone grafts with improved toughness and structural analogy to natural bone, evidence of which can be found in recent and rapid publications.
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