A number of natural and synthetic bone-graft materials currently in use are produced from coralline HAp. As a result of the conversion process, commercial coralline HAp has retained coral or CaCO₃ and the structure possesses nanopores within the inter-pore trabeculae, resulting in high dissolution rates.

Under certain conditions, these features reduce durability and strength, respectively, and are not utilised where high structural strength is required. To overcome these limitations, a double-stage conversion technique was developed by Ben-Nissan and co-workers [1, 2]. The method involves a two-stage application route whereby, in the first stage, a complete conversion of coral to 100 % HAp is achieved. In the second stage, a sol-gel-derived HAp nanocoating is applied directly to cover the meso- and nanopores within the intra-pore material, while maintaining the large pores for appropriate bone growth (Fig. 16.2).

Mechanical properties such as fracture toughness, Young’s modulus, and compression and biaxial strengths were each improved as a result of this unique double treatment. Application of the treatment method is expected to result in an enhanced bioactivity due to the nanograin size – and hence large surface area – that increases the reactivity of the nanocoating. It is anticipated that this material could be applied to load-bearing bone-graft applications where high-strength requirements are pertinent.

For these studies, the coral was obtained from the Australian Great Barrier Reef and contained micropores of 100–300 μm size. The coral was shaped and treated with boiling water and 5 % NaClO solution. A hydrothermal conversion was carried out in a Parr reactor with a Teflon liner at 220 °C and 3.8 MPa pressure with excess (NH₄)₂HPO₄. A total conversion to HAp was achieved in this way. Nanocrystalline ceramic coatings were produced using the sol-gel process. For this, the precursor solution was formed using the previously reported method [1, 2]. Coatings were formed using these solutions, followed by subsequent heat treatments. Mechanical testing involved a standard, four-point bend test according to ASTM C1161, to measure the flexural strength and flexural modulus of the natural coral. Comparative compression and biaxial strength tests were also carried out.

Characterisation studies of the natural and converted corals using XRD, SEM, DTA/TGA, nuclear magnetic resonance (NMR), and Raman spectroscopy have been previously reported. These results showed a large increase in all mechanical properties, specifically the compression strength, due to hydrothermal conversion and nanocoatings methods. The bioactivity was enhanced through the nanocrystalline formation, due to the HAp nanocoating [66].

In addition to enhancing the bioactivity and the mechanical properties, HAp coatings can also be used in controlling the rate of drug release from coralline apatite delivery system. A bone-stimulator drug, simvastatin, was successfully loaded with the coral-derived β-tricalcium phosphate, and in an attempt to control the release of simvastatin and reduce its release rate, a lipid coating was utilised [67].

Liposomes are one of the most clinically established nanometer-scale systems currently employed to deliver non-toxic and antifungal drugs, genes, and vaccines. Liposomes consist of a single layer, or multiple concentric lipid bilayers, that encapsulate an aqueous compartment. When compared to other delivery systems, the outstanding clinical profile of liposomes is based on their biocompatibility, biodegradability, reduced toxicity, and capacity for size and surface manipulations [68].

The encapsulation of nanoparticles such as calcium phosphate within liposomes may lead to an increase in nanoparticle hydrophilicity, stability in plasma, and an overall improvement in their biocompatibility [68–70]. Moreover, by utilising the ability of liposomes to carry hydrophilic and hydrophobic moieties, combinatory therapy/imaging modalities can be achieved by incorporating therapeutic and diagnostic agents into a single liposome delivery system [68].

Based on their excellent biocompatibility, calcium phosphate-based hybrid nanoparticles have shown great promise as candidates for drug delivery and bone regeneration systems [71–73]. Recently, composite scaffolds composed of collagen and HAp with liposomes were proposed by Wang et al. [72] to provide a sustained drug release platform in bone regeneration and repair. The liposomes were composed of distearoylphosphodioline, cholesterol, distearoylphosphoethanolamine-poly(ethylene glycol), and a bone-binding bisphosphonate attached to the liposome. By taking advantage of the specific interaction between the liposomal bisphosphonate and the HAp incorporated into the scaffold, the bisphosphonate-decorated liposomes were shown to display a strong affinity to collagen-HAp scaffolds. They reported there were no differences in drug release from the liposomes whether the liposomes were bisphosphonate decorated or not. Whereas unencapsulated drugs and drugs encapsulated in PEG-liposomes displayed rapid release from the scaffolds, the drugs entrapped in bisphosphonate liposomes showed a slower release from the collagen-HAp scaffolds.

HAp-coated liposomes were successfully manufactured by Xu et al. [71] and filled with a model hydrophobic (lipophilic) drug, namely, indomethacin (IMC). In this process, the HAp layer was precipitated onto the liposomes, the aim being to provide the HAp-coated liposomes with two functions: (1) that the inner core liposome provides a sustained drug release and (2) that the outer HAp layer provides the osteoconductivity for bone cells. The liposomes were formed from 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The results reported by the authors indicated that precipitating HAp onto the liposome reduced the release rate of IMC compared to uncoated liposomes. In fact, under the conditions used, the 5 h period required to release 70 % of the IMC from the liposomes was extended to 20 h when the liposomes were coated with HAp. Perhaps, more importantly, IMC release from the uncoated liposomes occurred more rapidly at pH 7.4 than at pH 4, whereas the HAp coating reduced the release rate at pH 7.4 compared to that at pH 4. Based on these findings, they suggested that this effect might open up the possibility of creating “smart” (pH-controlled) targeted drug delivery devices.

Osteoporosis, a degenerative bone disorder, is one of the leading causes of morbidity in the elderly. Lewis [73] examined the possibility of using liposomes to encapsulate various micro- and nanosized calcium-based mineral compounds for direct delivery to the bone which may be beneficial to bone health. Calcium phosphate mineral including HAp, dicalcium phosphate dihydrate, as well as multiphase and substituted calcium phosphates was produced using biomimetic process. Preliminary cell culture studies showed no direct effect on osteoblast-like Mg63 or Saos-2 cells or osteoclast resorption, measured by bone collagen release. Macrophage response was explored using U937 cell line. Expression of TNF-α and IL-1, markers of inflammation, increased with liposome treatments compared to the negative control but decreased compared the positive control. The Mg63 cells given U937 supernatants showed liposomes increased OPG production, but this was regardless of mineralisation (Fig. 16.3).

The clinical significance of hydroxyapatite (HAp) as a bone substitute has become apparent in recent years, and bone morphogenetic protein (BMP) has attracted much attention. In a study conducted by Ono et al. [74], 1.2 cm-diameter bone defects created on rabbit cranium were treated with the BMP-2 gene (cDNA plasmid) introduced with porous HAp after completion of haemostasis, and the effect of using cationic liposomes as a vector to the amounts of new bone formation was examined. Four groups of rabbits were compared. In the HAp group, the cranial bone defect was treated with HAp containing 40 μg of liposomes and a dummy gene. The BMP gene HAp group was treated with HAp soaked in liposomes and 10 μg of the BMP-2 gene. Although new bone formation was evident surrounding the scaffold 3 weeks post-operation, the induced bone tissue did not fill all the pores of the scaffold even at 9 weeks post-operation. They hypothesised the clinical usefulness of gene therapy for bone formation, using the BMP-2 gene combined with cationic liposomes as a vector.

When Huang et al. [75] used liposome-coated HAp and tricalcium phosphate as bone implants in the mandibular bony defect of miniature swine; they found the liposome-coated materials to be biocompatible. Moreover, the clinical endpoint was enhanced compared to that in the absence of liposomes. It was hypothesised that HAP coating and tricalcium phosphate with negatively charged liposomes might improve the nucleation process for new bone formation. In experiments conducted in miniature swine, artificial bony defects on one side were implanted with either HAp-coated or tricalcium phosphate-coated liposomes, while defects on the other side served as controls. Histology and radiography performed at 3 and 6 weeks after surgery showed the coated liposome materials to be biocompatible. At 3 weeks, the implant material was surrounded by dense connective tissues, while by 6 weeks new bone formation was visible near the implanted material. Liposomes immobilised in agarose gel and implanted in the defects also showed new bony bridge formation.

Despite being one of the most extensively investigated biomaterial in tissue engineering, the bioactivity of chitosan needs to be enhanced for specific tissues and making them not as ideal when used as a stand-alone material [76]. As a result, new composites are created by combining chitosan with other bioactive materials such as HAp [76–79]. Nano-HAp and its composites with variety of chitosan content were investigated by Tavakol et al. [76], and they observed that the degree of bone regeneration potential was greater in nano-HAp powder than in nano-HAp-chitosan composites. Cell culture studies were attempted by Tomoaia et al. [77] on fibrous biocomposite scaffolds made of nano-HAp particles, chitosan, and type I collagen. They noticed that the incorporation of small amounts of silicon, magnesium, and zinc within the nano-HAP lattice could improve the in vitro biological activity of human osteoblasts on these scaffolds. Im et al. [78] hypothesised that chitosan nanocomposite with both nano-HAp and single-walled carbon nanotubes (SWCNT) could significantly enhance the mechanical properties of the scaffold and synergistically improve scaffold cytocompatibility for osteoblast adhesion and proliferation. Nanocomposites containing magnetically synthesised single-walled CNT (SWCNT) had superior cytocompatibility when compared with nonmagnetically synthesised SWCNT. Their study also revealed that osteoblasts favoured magnetically synthesised SWCNT which has smaller diameters and were twice as long as their nonmagnetically prepared counterparts indicating that the dimensions of SWCNTs can have a substantial effect on osteoblast attachment.

Electrospun composite nanofibrous substrates of chitosan and HAp nanoparticles were fabricated by Venugopal et al. [79