Osteoconductive Bone Grafts

Osteoconduction is a physical effect wherein the matrix of the graft acts as a scaffold on which cells are able to form new bone tissue. In other words, the bone-healing response is conducted through the graft. Accordingly, osteoconduc-tive bone grafts are to serve as a structural framework through which host bone infiltrates and regenerates new bone tissue. The best examples of osteoconductive grafts are autogenic- and allogenic-cancellous bones, HA, and purified collagen (see Table 13.3). Among them, HA and collagen are widely used not only in bone grafting but also in other medical applications owing to their abundant availability, easy processing, amazing functional properties, and, in fact, ingredients of natural bone tissue; therefore we describe them in this section in a detailed fashion.

13.4.1.1. Hydroxyapatite and Its Use in Bone Grafting

Hydroxyapatite is a class of calcium phosphate-based bioceramic material, frequently used as a bone graft substitute owing to its chemical and structural

Table 13.2 Basic characteristics of an ideal bone graft.

Characteristics

Comments

Biocompatible Bioactive

Biodegradable in a controlled fashion Degradation time Mode of degradation Osteoconductive

Osteoinductive

Vascular supportive

Nontoxic Nonimmunogenic High porosity with interconnected pores

Pore size <50 |m >100 |im >200 |im 3-D structure

High surface area-to-volume ratio

Surface modifiable

Adequate mechanical strength

Ultrapure

Sterilizable

Cost-effective

Biologically compatible to host tissue (i.e., should not provoke any rejection, inflammation, and immune responses).

To facilitate a direct biochemical bonding to host tissue.

The rate of biodegradation has to be adjusted to match the rate of bone tissue formation.

Preferably less than six months, but depends on case by case.

Bulk or surface erosion.

Capable of supporting in-growth of sprouting capillaries, perivascular mesenchymal tissues, and osteoprogenitor cells from the recipient host into the 3-D structure of a graft that acts as a scaffold.

A process in which biomolecules enriched within the graft convert the patient's cells into cells capable of forming a new bone.

Should provide channels for blood supply for fast and healthy bone regeneration.

Should not evoke toxicity to host tissue.

Should not evoke immunogenic response to host tissue.

To maximize the space for cellular adhesion, growth, ECM secretion, revascularization, adequate nutrition, and oxygen supply. However, it should not compromise mechanical strength of the graft.

To allow cell penetration.

Sufficient to in-growth of fibrous tissue.

Needed for new bone generation.

Required for mature osteons to form.

For the assistance of cellular in-growth and transport of nutrition and oxygen.

Needed for high density of cellular attachment.

To functionalize additional chemical or biomolecular groups in order to enhance cellular adhesion and bone tissue growth.

To withstand in vivo stimuli.

Needed for better performance of graft.

To avoid toxic contamination.

Affordable to all; thereby making the patient happier.

similarity with natural bone mineral [47,48]. The HA derived either from natural sources or from synthetic sources is regarded as a bioactive substance because it forms a strong chemical bond with host bone tissue, and hence it is recognized as a good bone graft material. HA is not only bioactive but also osteoconductive, nontoxic, nonimmunogenic, and its structure is crystallographically similar to that of bone mineral. All the critical properties of the HA such as physiochemical, biomechanical, and biological are compiled and listed in Table 13.4 [22,49-53], which makes the HA a possible and perhaps the most appropriate bone graft material. The stoichiometric HA has a chemical composition of Ca1o(PO4)6(OH)2 with a Ca/P ratio of 1.67. Crystallographic studies are one of the important parameters for the selection of stoichiometric HA.

Table 13.3 Classification of bone grafts upon tissue response.

Types of bone grafts

Description

Examples

References

Osteoconductive graft

Bone grafts that serve as a structural framework through which bone-forming cells can attach, migrate, and grow, leading to new bone tissue

AutoB, AlloB, HA, Collagen

[41,42]

Osteoinductive graft

Bone grafts that induce a biochemical process capable of promoting or accelerating new bone tissue

AutoB, BMP, DBM

[43,44]

Osteogenic graft

Bone grafts that convert the transplanted living tissue into new bone tissue

AutoB, BMA

[45,46]

AutoB: Autogeous bone; AlloB: Allogenous bone

A complete understanding of the crystal structure of HA can be acquired from the in-depth knowledge of the spatial orientation or organization of a small number of constituent ions. The unit cell, a basic structural pattern of the constituent ions, of HA is a right rhombic prism that forms a simple hexagonic crystal lattice. The

Table 13.4 Critical properties of HA.a

Properties

Experimental data

Chemical composition

Ca/P molar

Color

Crystal system

Space group

Cell dimensions (A)

Young's modulus (GPa)

Elastic modulus (GPa)

Compressive strength (MPa)

Bending strength (MPa)

Density (g/cm3)

Relative density (%)

Fracture toughness (MPa.m1/2)

Hardness (HV)

Decomposition temp.(°C)

Dielectric constant

Thermal conductivity (W/cm.K)

Biocompatibility

Bioactivity

Biodegradation

Cellular compatibility

Osteoinduction

Osteoconduction

Ca1o(PO4)6(OH)2 1.67

Mostly white

Hexagonal

400-900 115-200 3.16 95-99.5 0.7-1.2 600 >1000 1614

7.40-10.47

0.013

High

High

High

High aCompiled from Refs. [22, 49-53].

Figure 13.6. A diagrammatic view of crystal structure of HA. Courtesy: Prof. H. Aoki, Tokyo Denki University, Japan.

length along an edge of the basal plane of the unit cell is a = b = 9.42 A and the height of the unit cell is c = 6.88 A [54]. The spatial symmetry (i.e., space group) symbolized as P63/m, cannot be completely specified with fewer than this number of atoms. The arrangement of these constituent atoms is projected along the c-axis onto the basal plane. A schema of the crystal structure of HA viewed diagonally is shown in Figure 13.6 [54].

For simplicity, the chemical composition of HA can be written as Ca4(I)Ca6(II)(PO4)6(OH)2, where the Ca atoms occupy two series of nonequiv-alent sites. The Ca(I) atoms are on the fourfold symmetry 4(f) positions and the Ca(II) atoms are in the sixfold symmetry 6(f) position. The P atom is surrounded by four atoms and forms a tetrahedron. The PO4 tetrahedron is almost regular with only slight distortion. The hydroxyl ions lie, in projection, at the corners of the rhombic base of the unit cell. Furthermore, hydroxyl ions are always perpendicular to the nearest plane of calcium with the hydrogen ion facing away from this plane in such a way that the O-H band never overlaps the plane. Of course, the crystal structure and crystallization behavior of HA are strongly dependent on the substitution nature of the ionic species and the pattern ordering. Even the crystal structure can be changed by the manipulation of the calcium to phosphorous (Ca/P) ratio, which leads to possible changes in their solubility.

As per a literature survey, HA has a long history of being used as a good bone graft. Its possible clinical uses range from augmenting atrophic alveolar ridges to repairing long bone defects, ununited bone fractures, middle ear prostheses, spinal fusions, cranioplasty, craniofacial repair, and vertebral fusions. On the other hand, it has also been used in various dental surgeries and biomolecular delivery. Basically, HA is a family of apatite phase. The name apatite was derived from the Greek word, meaning "to deceive," which was coined by Werner in 1786 [55]. Apatite belongs to a family of components having similar structure but not necessarily having identical phase composition. The first synthesis of apatite was carried out by Daubree in 1851 [56], in which apatite was produced by just passing phosphorous trichloride vapor over red hot lime. In 1951, a synthetic HA was prepared by Ray and Ward [57], suitable for bone defects. They implanted their processed HA at surgically created bone defects in the long bones and iliac wings of dogs and the skulls of cats and monkeys and obtained affirmative results. In 1970s and 1980s, Aoki and Kato [58], De Groot [59], and Jarcho [60] pioneered multishapeable HA suitable for clinical orthopedics.

Since then, a variety of preparation methodologies of HA has been reported. It is worthwhile to note that HA can also be processed from animal bone [61,62] and coral exoskeleton [63,64]. In all these, however, different processing methods are involved in the production of microsize HA in various forms such as powder or dense or porous blocks, corresponding to clinical applications. In the past few years, significant research efforts have been devoted to the production of nanosize HA in order to obtain high surface reactivity with adequate physical, chemical, and biological properties similar to natural bone mineral.

The nano HA has unique advantages over conventional micro HA in terms of high surface area-to-volume ratio and ultrafine structure, which are the most imperative properties essential for cell-substrate interactions in vivo. For example, compared to conventionally crystallized HA, nanocrystallized HA promotes osteoblast cell adhesion, differentiation, proliferation, osteointegration, and deposition of calcium-containing minerals on their surface, thereby enhancing new bone growth within a short period [65]. Keeping these points in view, nano HA can be considered as a unique class of bone grafting material. The nano HA can be synthesized by many different methods, which include solid state [66], wet chemical [67,68], hydrothermal [69,70], mechanochemical [71], pH shock wave [72], and more recently microwave processing [73]. Critical comments on these processing methods are specified in Table 13.5 and detailed descriptions about them are given in the following sections, respectively.

(a) Solid-State Reaction

Solid-state reaction has generally been used for the processing of bioceramic powders at high temperature. The HA powders prepared by this method usually have irregular shapes with larger grain size and they quite often exhibit heterogeneity in the phase composition due to chemical reactions resulting from small diffusion coefficients of ions within the solid. An example for the formation of HA using the solid-state method is given below:

Table 13.5 Methods involved in the synthesis of nano HA.

Methods

Grain size (nm)

General remarks

References

Solid state

500

Inhomogeneous, large grain size

(micro to nano), irregular shapes, reaction condition 900 to 1300°C

[66]

Wet chemical

20-200

Nanograin size, low crystallinity, homogeneous, reaction condition: room temp. to 100°C

[67,68]

Precipitation/

10-25

Homogeneous, ultrafine particles, low

[69]

hydrothermal

crystallinity, reaction condition: room temp. to 200°C (1 to 2 MPa)

Hydrothermal

10-80

Homogeneous, fine crystals, high temperature, and high-pressure atmosphere

[70]

Mechanochemical

<20

Easy production, semi-crystallinity, ultrafine crystals, room temp. process

[71]

pH shock wave

20-100

High-energy dispersing, nonporous, monocrystalline particles with Ca/P molar ratio 1.43 to 1.66

[72]

Microwave

100-300

Uniformity, nanosize particles, time and energy saving

[73]

The key ingredients of this reaction are basically in solid phase in which both act as Ca and P precursors in the formation of HA. Typically, solid-state reaction takes place at very high temperature, that is, above 1000°C.

A method for the preparation of HA fibers was established using a solid phase reaction [66]. With this method, the HA fibers were fabricated by heating a compact consisting of calcium metaphosphate fibers with calcium hydroxide particles at 1000°C in air atmosphere, and subsequently treated with dilute aqueous HCl solution to remove unwanted secondary phase substitution such as CaO. The obtained HA fibers were characterized and found that the nanostructural characteristic features of HA are quite similar to the natural bone mineral apatite phase. Therefore, the solid-state reaction can be considered as one of the capable methods for the production of nanostructure HA.

(b) Wet-Chemical Method

The wet-chemical route is one of the promising and most frequently used methods to synthesize HA as it is relatively easy to process even at low temperature, in contrast to solid-state reaction. The materials synthesized by using this method are almost in homogeneous phase composition, but are poorly crystallized owing to the low-temperature process. One common wet-chemical method of synthesizing HA is based on using calcium hydroxide with orthophosphoric acid according to following equation:

A free-flowing form of nanocrystalline HA was synthesized by using this method [74]. The obtained powder agglomerates were consolidated into bulk form to test their mechanical reliability. The experimental data clearly showed that the compressive strength of the bulk nano HA could be increased up to fourfold in comparison to natural bone, whereas bending strength was found to be almost equivalent to that of bone.

Recently, Ahn et al. [75] processed a bioactive nanocrystalline HA through nanostructure processing to achieve superior chemical homogeneity and structural uniformity in order to manufacture high-quality apatites. They have attained nanostructural HA by using the precipitation route with Ca(NO3)2.4H2O and (NH4)2HPO4 and studied the optimal condition by changing the reaction parameters such as precursor pH, aging time, and temperature to achieve nano HA particles with tailored composition and morphology. This investigation clearly indicated that the nanostructure processing offers an alternative solution to challenges encountered in conventional HA processing and concluded that nanostructured HA results in a 70% improvement over the best conventional HA.

Our group has also processed HA nanoparticles by using the wet-chemical method [67,68] and studied their physicochemical and physiological properties at body pH. The investigations clearly showed the resorbable characteristic of nano HA over conventional micro HA owing to their larger surface area-to-volume ratio that ultimately makes the nano HA highly surface reactive. A typical SEM micrograph of HA obtained by using the wet-chemical method is shown in Figure 13.7 [76], indicating a spherical form of HA nanoparticles approximately 100 nm in diameter. With reference to the overall experimental results, the wet-chemical method is one of the most promising methods for the production of nano HA and, perhaps, the easiest method too.

Figure 13.7. SEM micrograph of HA nanoparticles.

(c) Hydrothermal Reaction

The hydrothermal method enables the synthesis of a crystallized phase of HA with homogeneous phase composition at high temperature and at high-pressure atmosphere. The HA processed by using this method are easily sinterable owing to the effects of high temperature and high-pressure aqueous solutions. Zhang and Consalves [69] synthesized nanosize HA by precipitating the calcium and phosphate precursors under hydrothermal conditions, and they studied its thermal stability. By using this technique, they obtained HA with a rod shape and an average crystal size of 10 to 24 nm with respect to compositions of precursors. They produced a variety of calcium phosphate powders with respect to starting materials with a Ca/P ratio ranging from 1.66 to 1.73, meaning that they have different solubility features.

Another interesting method was recently reported by Zhang et al. [69], in which they synthesized HA in the form of nanorods in the presence of anionic starburst dendrimer polyamidoamine with surface carboxylate groups through a hydrothermal method. The reaction conditions are as follows. First, Ca2+ (0.01 mol/l) and PO4- (0.01 mol/l) were prepared separately by dissolving the desired amount of Ca(NO3)2 and (NH4)2HPO4 in distilled water. Then, 0.1 g polyamidoamine was added to the Ca2+ containing solution. The two solutions were kept 3 h to ensure the cooperative interaction and self-assembly process were completed. Then, the (NH4)2HPO4 solution was added dropwise (about 1 s/drop) to the Ca2+ containing solution under stirring condition. After blending, the suspension was kept for about 3 h before separating the precipitate. The resulting white precipitate was filtered, washed thoroughly with distilled water, and oven-dried at about 60°C. The same process was carried out to form a suspension; then it was transferred to an autoclave, sealed tightly, and hydrothermally treated at 150°C for 10 h. The obtained products were analyzed by several techniques, and the results indicated that HA nanorods can be prepared by using this hydrothermal method with narrow particle distribution within nanosize range (80 nm by 10 nm, with aspect ratio about 8). The investigation suggested that the dendrimer-mediated hydrothermal method is a relatively new processing method for the manufacture of HA.

(d) Mechanochemical Route

Production of nano HA by using the mechanochemical route is a versatile process owing to its simplicity, which differs slightly from the conventional wet-chemical route. Although HA have been synthesized by the conventional wet-chemical method, the purity of the HA products depends on their reaction pH, whereas reaction through the mechanochemical route need not be under precise pH control. Nakamura et al. [71] recently reported the synthesis of nano HA by using the mechanochemical method. The HA was obtained directly by milling a mixture comprising Ca(OH)2, an aqueous solution of H3PO4, and a dispersant, an ammonium salt of polyacrylic acid. The reaction was carried out at room temperature. The obtained HA had relatively crystallized particles in the nanometer regime. The average crystallite size of HA was found to be below 20 nm, but the obtained product was calcium-deficient HA (1.51, Ca/P molar ratio) as compared to that of stoichiometric HA (1.67, Ca/P molar ratio). Furthermore, they suggested some of the merits of this mechanochemical route, which include (1) assisting depro-tonation from brushite to form HA, and (2) keeping high dispersability and low viscosity of reaction sol. Overall, the production of nanocrystalline HA through the mechanochemical route is straightforward.

(e) pH Shock Wave Method

Another interesting method for the production of HA is the pH shock wave method. Recently, Koumoulidis et al. [72] reported the processing of HA using this pH shock wave method. They synthesized HA in the form of lathlike monocrys-talline particles by using high-energy dispersing equipment with the precursors of Ca(H2PO4)2.H2O and CaCl2 with a Ca/P molar ratio of 1.66, which is almost similar to the stoichiometric Ca/P ratio of HA. The obtained products were characterized well using various analytical methods to determine their phase composition, structure, and texture. The products have a very different Ca/P molar ratio ranging from 1.43 to 1.66 with respect to the preparation methodology. This method produces nonporous monocrystalline HA particles with average grain size of ~ 140 to 1300 nm in length, 20 to 100 nm in width, and 10 to 40 nm in thickness. The Ca/P atomic ratio of nano HA was found to be 1.66, which is very close to the reported theoretical value of stoichiometric HA (1.67). As per their investigation, this seems to be a unique method, because it creates appropriate hydrodynamic conditions for lathlike particle growth in the [001] direction that could not be obtained by using conventional mechanical stirring equipment.

(f) Microwave Treatment

Microwave processing is relatively a new class of method, which has recently gained much interest and has been perceived to be beneficial in the synthesis of HA. Many investigations reported the processing of HA using microwaves. This method offers high-purity materials with minimized reaction time and energy saving over conventional methods [77-79]. The preparation and characterization of nanosize HA under microwave irradiation was investigated recently [78]. The nano HA was synthesized by the precipitation method using NH4H2(PO4) and Ca(NO3)2.4H2O. The precipitate was subjected to microwave sintering after being washed with distilled water using a high-speed centrifuge. The microwave generated the power input of 980 W and power output of 490 W with a frequency of 2.45 GHz for a period of 5 to 15 minutes.

This method offers several advantages over normal furnace heating corresponding to time and energy savings. The experimental results suggested that there was a slight increase in the microhardness value of the sintered HA with increasing microwave exposure time. The major problem associated with microwave densifi-cation is in getting large samples without microcracks. The formation of cracks is believed to result from the nonuniform packing of crystallite in the green compact as well as due to the nonuniform heating profile occurring inside the microwave cavity. Another interesting investigation is that in which Yang et al. [79] reported the sintering effect of HA by using the microwave-processing technique. They synthesized HA by using the chemical precipitation method at 95°C with a pH from 9 to 11.5. The prepared HA was characterized systematically by various analytical methods. They found that the prepared HA had a Ca/P ratio of 1.67, which is similar to that of stoichiometric HA. The HA green samples were sintered by microwave heating at 1050 to 1100°C for 5 to 10 min, and the results were compared with other HA green samples sintered by using the conventional heating method. The results clearly indicated that the HA prepared by microwave processing were denser and had a finer grain size as compared to those prepared by the conventional heating method. Accordingly, the HA prepared under microwave irradiation seem to be a unique class of material owing to their characteristic functional properties.

Although nano HA is an excellent bone graft material, its inherent low fracture toughness has limited its use in certain orthopedic applications, in particular heavy load-bearing implantation. The fracture toughness of HA is about 1.0 MPa.m1/2, which is very low as compared to natural bone (2-12 MPa.m1/2) and the Weibull modulus is also sufficiently low, such that it depromotes the reliability of HA for heavy-loaded implants. The Weibull modulus of the HA is in the range between 5 and 18, which means that HA behaves as a typically brittle biomaterial; therefore it is used only in low-weight-bearing orthopedic applications such as bone defect filler, coating agent on metallic bioimplant, and biomolecular delivery. In order to improve reliability, it is necessary to introduce some biocompatible reinforcement agents or matrix materials. However, introduction of foreign materials may lead to a decrease in reliability of HA; therefore choosing the reinforcement agents or matrix materials is of great importance. Recently, a composite of nano HA with natural polymer, in particular collagen, has been preferred to improve the reliability of nano HA. Collagen is a natural choice of ECM, abundantly found in human bone tissue; thus choosing collagen as a matrix material could enhance the reliability of bone grafts. Moreover, in fact, they are the basic structural building blocks of natural bone tissue.

13.4.1.2. Collagen and Its Use in Bone Grafting

Collagen is a major protein of the native ECM that primarily serves as a structural protein in biological mineralization. Collagen comprises as much as one third of the total proteins in human body. In general, it has many desirable functional properties favorable for bone metabolism. The purified collagen has excellent biocompatibility, biodegradability, nontoxicity, and nonantigenecity that make the collagen a prime and safe source in a variety of biomedical applications, including bone grafting [80-82]. The way in which it interacts with the host tissue further makes it a more unique class of biomaterial than the other natural or synthetic polymers. Some of the merits and demerits of collagen that make it an excellent substrate for bone tissue engineering are listed in Table 13.6.

There are many types of collagen available based on their molecular sequences. So far, 27 distinct types with at least 42 distinct polypeptide chains of collagen have been identified. The different types of existing collagen are formed according to the twists and turns between the hydrogen bonds holding the structure together. Although many types of collagen exist in a living organism, the most abundant form of

Table 13.6 Merits and demerits of collagen as a bone graft. Collagen

Merits Demerits

Biocompatible High cost

Bioresorbable More hydrophilic

Hemostatic Antigenic if it has telopeptides

Osteoconductive Superior cellular adhesion Regulatable biodegradability Surface modifiable Nontoxic Weak-antigenic Tunable mechanical strength Shapeable Sterilizable Abundant availability Best ion exchanger and binder of electrolyte metabolites collagen in the connective tissue is type I. Type I collagen is composed of two a1 (I) chains and one a2 (I) chain in the form of a triple helix pattern with a fiber diameter of about 50 nm [81,82]. All collagens are composed of three polypeptide chains, designated as a-chains, that are each coiled into a left-handed helix. These three chains are then wrapped around each other into a right-handed triple helix; therefore the final structure is a triple helical ropelike fashion. The triple-helical structure of collagen is shown in Figure 13.8 [83]. The triple-helical domain has a characteristic

Figure 13.8. Triple-helical structure of collagen. Adapted from Ref. [83].

primary structure, where glycine in every third amino acid generates repeating (Gly X-Y)n units; X is alanine or praline, and Y is hydroxyproline [81,82].

In general, collagen extracted from the natural tissues is capable of eliciting immunogenic response to a certain extent upon implantation; thus direct use of this type of collagen is limited. Nowadays, a purified form of collagen known as reconstituted collagen is processed by various biochemical methods and commercially sold. The reconstituted collagen is relatively less immunogenic than native collagen. Collagen can also be chemically modified by various methods (succiny-lation, e.g.) to increase its surface reactivity by inducing more negative charges, which in turn aids the collagen in dissolving even in the neutral pH; therefore its usage can be widened significantly in biomedical applications. However, it does not have significant strength or stiffness and so it cannot be used as such for a variety of bone grafting applications, which leads to the concept of composite bone grafts.

There is a possibility of enhancing its functionality by incorporating other bone graft substances (e.g., HA, bone morphogenetic protein (BMP) and osteoprogen-itor precursors). Such a composite bone graft is also commercially available for clinical use. Collagraft, BioOss, and Healos are a few notable examples of such a kind of bone graft [84-86]. Collagraft (Zimmer Inc., U.S.A.), which is made of calcium phosphates and collagen, is used for low-weight-bearing orthopedic applications,. Bio-Oss (Geistlich Biomaterials Inc., U.S.A.) is a craniofacial graft, which is made of bovine collagen and deorganified bovine bone. Healos (CE Mark, CA) is a spongelike mineralized collagen fiber, mainly used as an osteoconductive matrix. Accordingly, collagen can be considered as an accomplished biomaterial for bone grafting.

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