Richard Feyman first introduced the concept of Nanotechnology in 1959 [2]. Nanotechnology can be defined as the research, design and manufacture of materials with a grain size less than 100nm. These materials follow the laws of quantum physics, not classical mechanics. As a result, they act very differently to their conventional counterparts with similar anatomical structure but grain size greater than 100nm [2]. Manipulation of nanotechnology in bone healing has the potential to be successful as the hierarchical structure of bone is derived from nanoscopic collagen fibres and hydroxyapatite.

Nanotechnology, an emerging field in Orthopaedic Surgery, is a response to the lack of an ideal management option for bone healing in large or non-union fractures. In the USA, trauma is the second most common cause of admission to the Accident and Emergency Department. It is thought that 7.7 million of these admissions will require emergency orthopaedic surgical management [3]. Around 6.5 million of these presentations involve a fracture, with 500,000 requiring a bone graft [4]. A further 700,00 patients will require total joint replacement. Modern joint implants have a 10-15 year life expectancy, with 12.8% of total hip replacements requiring revision [3].

Bone disease such as Osteoporosis and Osteogenesis imperfecta are associated with fracture and disability. Management with bone graft and implants can reduce bone disease associated morbidity [5]. Bone cancers, most notably Osteosarcoma, are rare. It accounts for 1% of all cancer diagnoses in the US and has a bimodal distribution; the first at adolescence and the second in those over sixty years old [6]. The surgical management usually involves large dissection of bone. Bone grafting to optimise pain control, stability and mobilisation and to minimise disability, especially in the adolescent population, is of particular importance in the management of this pathology [5]. Literature suggests that around 10% of fractures result in delayed healing or non-union which severely impedes bone function and may be debilitating for the patient [5].

Bone is biochemically and structurally complex. To a certain extent, implants are able to mimic the structural requirements of bone, and bone grafts the biochemical, but there is not an option currently, which can provide both. This review presents the basic science behind bone structure, formation, function and healing. Furthermore, how these factors are affected by fracture and the current management options when normal bone healing fails. Nanotechnology is discussed, with focus on bone grafts, implants, diagnostics and drug delivery; highlighting promising advances in this novel field.

The structure of bone consists of cells and extracellular matrix (ECM) [7]. Cells include osteoblasts, osteocytes, osteoprogenitor cells and osteoclasts. The ECM is made up of three main constituents; inorganic Hydroxyapatite (60% dry bone weight), an organic matrix (30% dry bone weight) and water [5]. The organic matrix includes collagen molecules (90% Type I with some Type V) [8], peptides, glycoproteins, soluble growth factors and hormones [5].

Manipulation of nanotechnology in bone healing has the potential to be successful as the hierarchical structure of bone is derived from nanoscopic collagen fibres between 100nm -2,000nm in length [7] which provide the architecture for nanoscopic (20-40nm in length) [9] hydroxyapatite Ca₂(PO₄)₃OH to build upon [8].

Type I collagen is derived from a triple helix of two alpha 1 chains and one alpha 2 chain, which first bundles into fibrils, then into collagen fibres [5]. The synthesis of this 300nm molecule is carried out by osteoblast cells [7]. Collagen fibres are bound by Hydroxyapatite (HAp) nanocrystals [9]. Nano composite fibers of these two molecules are formed when the C-axes of hydroxyapatite is parallel to longitudinal fibrils of Type I collagen [10]. These fibres provide stability, allowing bone to maintain form and function under both high tensile and compression stress [9].

The adult skeleton is made up of 80% cortical bone and 20% trabecular (cancellous) bone [5]. Cortical bone is found in the shaft of long bones [9]. It is anisotropic in nature, as the main functional unit, the osteon, is made up of lamellae [9]. These osteons follow a longitudinal direction, parallel to collagen throughout the bone [5], providing strong skeletal support. Central to each osteon are Haversian canals, within which blood vessels, lymphatics and nerves traverse to provide nutrition to the bone [5].

The very porous trabecular bone is located in the epiphysis and metaphysis of long and cuboidal bones [9]. It is also found in the ribs and spine [5]. Compared to cortical bone, the density and weight of Trabecular bone is less due to its larger pores [7]. This increased pore size allows for the greater vascularity of trabecular bone [7].

Bone metabolism occurs more readily in trabecular bone than cortical bone [9]. Trabecular bone contains both woven and lamellae bone in addition to vascular bone marrow. Woven bone is apparent in metaphysial bone growth, fracture associated callus formation and Paget’s Disease. It is isotropic in nature, as it is formed from coarse, disordered collagen [9]. As a result, it provides poor structural support meaning that in times of physiological duress, trabecular bone will undergo compression force [9] (Fig. 1).

Fig. (1). Anatomy of a long bone. (Taken and reproduced from [1]).

Consisting of 206 bones [7], the human skeleton provides structural support, protection of organs, movement of limbs via muscle attachment [9] and a site of calcium and phosphorus storage [8]. Bone, compared to other tissues, is almost unique in its ability to self regenerate when damaged [8]. It is an important buffering mechanism, especially in acidic conditions [9]. Bone marrow, which is found in flat bones and in epiphyseal region of long bones, contains pluripotent mesenchymal stem cells [5]. These cells have the potential to differentiate into bone, cartilage, muscle, skin, soft and adipose tissue [5]. Haemopoiesis is also conducted in the bone marrow [5].

During embryogenesis, pluripotent mesenchymal cells differentiate into osteoprogenitor cells which are either osteoblasts, to produce bone, or chondrocytes, which form the template for endochondral ossification [4]. Bone formation and homeostasis is dependent on a number of different cells. Osteoblasts, Osteocytes and Osteoclasts are found within the bone itself [4]. Whilst Osteoprogenitor and bone lining cells influence bone function [11] (Fig. 2).

Found on the periosteal and endosteal surfaces, osteoblasts produce Type I collagen rich osteoid matrix [7]. This osteoid matrix is bound by non-collagenous proteins which promote calcium and phosphate adhesion [4], resulting in mineralisation and organisation of new bone. Osteoblasts transform into osteocytes if they are within the osteoid matrix when mineralisation takes place [4].

Fig. (2). Bone cells. (Taken and reproduced from [1]).

Osteocytes are located centrally within the osteon [7] and can communicate mechanical stresses with neighbouring osteocytes and bone lining cells through canaliculi [7]. This intercellular communication is thought to facilitate bone formation and resorption in addition to mineral transportation between bone and blood [4] (Fig. 3).

Fig. (3). Periosteum and Endosteum. (Taken and reproduced from [1]).

Osteoclasts are differentiated, cytokine induced, macrophages [8]. Located at the bone surface [4] they resorb mature bone [7] by secreting acids and proteolytic enzymes [5]. Bone lining cells regulate the rate at which minerals from this dissolution are transported from the bone into the blood stream [4].

Non-collagenous proteins, as mentioned briefly, are important in bone formation and repair. Found in the extra cellular matrix, examples include growth factors (TGF beta, PDGF, IGF-1, FGF-a, FGF-b and IL-1), bone sialoprotein, osteopontin, osteonectin, osteocalcin and proteins (fibronectin, vitronectin and laminin) [12]. Osteopontin mediates adhesion of osteoblasts and osteoclasts to the osteoid matrix [13]. Bone sialoprotein, osteonectin and osteocalcin are required for bone mineralisation [14-16]. To note, these extracellular adhesion proteins have enhanced interaction with nanophase implant surfaces, compared to standard implant surfaces, which promotes osseointegration, thus reducing incidence joint loosening [2].

Bone formation follows two main pathways. The first, endochondral bone formation is seen in embryogenesis of long and short bones. Chondrocytes form a collagen rich matrix as they mature. On maturity these cells cease to proliferate and instead produce proteins which calcify the matrix. Resorption of this cartilaginous matrix is performed by phagocytes. At the primary ossification centre located mid-shaft, osteoblasts lay down osteoid to produce a ‘cartilage model’ of the bone. Vascularisation into the matrix facilitates MSC migration from the periosteum. This enables further osteoblastic osteoid deposition and calcification of the collagenous matrix framework forming spongy, trabeculae bone [5]. The medullary cavity of long bones, where the bone marrow is located, is formed by osteoclastic breakdown of this trabecular bone [17].

There is a secondary ossification centre, which is formed at birth. The epiphyseal plate, located between the first and second ossification centres, forms cartilage, which when ossified results in an increase in bone length. This plate is key to growth and development. By the age of twenty, the plate is usually ossified and therefore, ceases to function [17] (Fig. 4).

Fig. (4). Endochondral ossification. (Taken and reproduced from [1]).

The second, intramembranous ossification is associated with formation of flat bones such as the skull. Although similar to endochondral ossification, it differs in that bone is directly formed from mesenchymal tissue without a cartilage framework [18]. During skull formation, neural crest cells group together, some will differentiate to form capillaries, other will become osteoblasts. These osteoblasts secrete the a collagen-proteoglycan (osteoid) matrix which encourages calcium binding and therefore calcification. Calcification results in the formation of bony ‘spicules’, needle like structures of bone, which radiate from the ossification centre and are surrounded by the periosteum, formed from condensed mesenchymal stem cells MSCs. Cells located in the layer between the spicules and the periosteum become osteoblasts. These osteoblasts deposit osteoid matrix in layers, resulting in the structure of flat bones [17].

The structure of bone can be directly related to its function. Weak, woven bone results from disorganised collagen deposition. When collagen fibres become more linear, strong lamellar bone results. Osteoblastic osteoid deposition, and therefore bone density, is greater at cortical bone than primitive cancellous bone.

Bone healing differs from other human systems in that it does not result in scar formation [19]. Primary bone healing is uncommon. Occurrence requires closely opposed fractured bone fragments which are un-displaced with complete stability. Repair occurs via haversian remodelling [20]. Secondary or indirect bone healing occurs by callus formation via both endochondral and intramembranous ossification [21]. Intramembranous ossification recruits osteoprogenitor and MSCs from the periosteum distant from the fracture to form hard callus without a cartilage template [22]. Endochondral ossification follows a stepwise process; haematoma formation, inflammatory phase, repair phase and finally, remodelling [22]. In contrast to intramembranous ossification, healing is aided by periosteum close to the fracture site [22].

Formation of a haematoma is a result of blood vessel rupture in the medullary cavity at the fracture site [5]. The haematoma is replaced by granulation tissue, where cartilage deposition occurs to form a bridging, soft callus crucial for fracture site stabilisation [8]. The inflammatory phase follows when necrotic tissue at the fracture site releases inflammatory mediators promoting chemotaxis of cytokines, neutrophils, platelets, BMPs and MSCs [21]. This promotes osteoprogenitor cell activation, angiogenesis and osteoclast migration to remove necrotic tissue from the fracture site. Within the repair phase, osteoblasts deposit osteoid at the fracture site to form the disorganised weak woven bone [5]. The remodelling phase can, in large fractures, take up to a year to achieve pre-fracture bone strength [18]. Remodelling involves the transformation of woven bone into organised, strong lamellar bone [5].

Nanotechnology in Orthopaedic Surgery, and therefore bone healing, has the following three main avenues of exploration, bone graft and implants, diagnostic and drug delivery [3].

Bone grafts are the second most commonly transplanted tissue in humans [7]. Current bone graft and implant options are limited in their efficacy as bone is complex both in anatomical structure and biological activity. Therefore synthetic replication is not easy. Implants can, to a certain extent, imitate the structural support bone provides, but they cannot mimic the biological functions of bone. Bone grafts have the opposite problem. A material which can provide the structural, mechanical and biological complexities of bone in addition to being asceptic is required [3].

Aseptic joint replacement loosening, usually due to poor osseointegration, is one of the most common causes for implant failure [3]. The surface of nanophase materials imitates that of trabecular bone more than their conventional counterparts. Further more, the smaller material grain size seen in nanophase materials greatly increases their surface area to volume ratio. Both of these assets results in improved osseo-integration, which reduces the risk of joint replacement loosening [2] and fibrous encapsulation [23] compared to conventional implants.

Bone is almost unique in that it can regenerate and remodel without a scar [9]. It also has the ability to interpret mechanical stress such as exercise, which allows it to increase bone mineral content via osteocyte, osteoclast and osteoblast induction, reducing the risk of fracture [7].

There are clinical scenarios where bone can not regenerate and remodel. Large or non-union healing fractures, bone disease and congenital deformity are commonly managed with bone grafts to promote bone healing and therefore improve mechanical function and pain relief [4].

A successful synthetic bone graft, must be sterile, mechanically strong, promote bone growth including osteoid deposition, calcification, remodelling and vascularisation, and biocompatible, with any degradation products being non-toxic to reduce systemic inflammation and morbidity. In addition, replacement joint implants must promote osteointegration to reduce the risk of joint loosening and stress shielding [4, 5].

There are three main forms of bone grafting; autograft, allograft and synthetic [24]. Grafts may be Osteoconductive, osteoinductive or osteogenic. Osteoconductive bone grafts provide a physical scaffold for recruitment and growth of osteoprogenitor cells and neovascularization from host tissue. Examples are cancellous autografts and allografts, demineralized bone matrix, HAp, collagen and calcium phosphate [25]. Osteoinductive grafts are where osteoprogenitor cells are recruited resulting in osteoblastic bone formation [3]. Bone morphogenic proteins and demineralized bone matrix are two examples [25]. Osteogenic grafts contain osteoprogenitor cells or undifferentiated osteoblasts and have the ability to produce bone directly [5]. Only autografts and bone marrow cells are osteogenic [25].

Due to the inadequacy of current bone healing management options, novel strategies which are biochemically and structurally similar to bone are required. As the structure of bone is nanoscopic, Nanotechnology may provide the solution. Critically, nanophase materials show improved bone healing, due to their superior osteoblastic function, compared to their conventional counterparts. This has been noted in various studies of nanophase polymers, ceramics, metals, composites and carbon nanofibres and nanotubes as discussed below [3].

The second area of development for nanotechnology in bone healing is diagnostic. It is has been hypothesized that by altering existing metal implants with microsized pores, the ability for nano-sensors to sit within them would be possible. A multiwall carbon nanotube developed from anodized nanotubular titanium demonstrated promotion of protein redox reactions via direct electron transfer. The potential for development into an implant with inbuilt biological sensor and controlled drug delivery is promising. If successful, implant lifespan could be extended significantly [12].

Research into the third exploratory field of Nanotechnology, drug, protein and gene delivery, has also made advances for bone healing. Nanoparticles are ideal as drug delivery devices as they exhibit high surface area to volume ratio. Their novel size allows their incorporation within, and methodical release from, bone graft scaffolds and other delivery systems [28].

Zhang et al. hypothesise that both genes and proteins could be potentially delivered by a nanoparticle material to promote osteogenesis and therefore bone healing [27]. Examples include, nanosized polymers transporting growth factors, which are released as the polymer degrades. Other reported successful delivery systems include HAp, beta-tricalcium phosphate, carbonate apatite, collagen, hyaluronic acid, chitosan and alignate, micelles [28], PEG-ylation modified particles, liposomes and dendrimers [28, 51].

Implant infection leading to poor osseointegration is a leading cause of implant, and therefore bone healing, failure [3]. Li et al. present a polypeptide nanofilm with the ability to deliver antibiotics. This nanofilm is associated with reduced bacterial load, enhanced osteoblastic activity and promotion of bone healing. The pharmacokinetics of cefazolin release from this nanofilm can be tightly controlled, especially during the critical postimplant period, which is within the first two hours [52]. It is interesting to note that even without drug delivery of antibiotics, the polypeptide nanofilm alone showed markedly reduced Staph Aureus adhesion [52].

Other exciting areas of drug delivery include a silver titanium nanotube coated titanium orthopaedic implant which shows reduced post operative implant associated infection [53]. Scaffolds imbedded with nanophase silver is a future area of development. There introduction into a known or potential site of infection is associated with improved clinical outcomes without the need for long courses of intravenous antibiotics [54].

Yan et al. developed a nanostructured mesoporous bioactive glass (MPG). Compared to its non-mesoporous counterpart, MPG induced greater apatite mineralisation. MPG synthesised as porous 3D scaffolds exhibit enhanced bone regeneration and drug delivery compared to standard MPG forms. In one study, the sustained release of Dexamethasone from an MPG scaffold augmented osteoblastic activity with the potential to induce osteogenesis. The same study looked at a MPG scaffold, loaded with vascular endothelial growth factor (VEGF), which found the mesoporous structure potentiated the bioactivity of VEGF, therefore stimulating angiogenesis, an important aspect of bone healing. The study concluded that both Dexamethasone and VEGF loaded MPG scaffolds are potential nanotechnology bone healing solutions [55].

Harvey et al. studied the behaviour of osteoblasts cultured on 2D surfaces whose topography had been manipulated at the nanoscale level. Examples include alumina, titania, HA, PLGA. These “functionalized allografts” showed improved bone healing compared to surfaces with conventional topography. Surface appearance is thought to be more influential than surface chemistry in promoting bone healing. The authors suggest that rough nano topography induces greater release and adhesion of osteoinductive factors. The paper hypothesises that 3D scaffolds embedded with osteoblasts and that have rough nano-topography, should have similar results dependent that key characteristics, such as pore size, are maintained [28].

Another avenue of exploration within stimulation of bone healing is bone morphogenic proteins (BMPs). They are Food and Drug Association approved [28]. These growth factors are one of the transforming growth factor beta proteins involved in osteogenesis [56]. The disadvantage of using BMPs is their associated cost which is considerable [28].

Nanophase ceramics, metals and polymers are associated with reduced activity of fibroblasts and endothelial cells, which are implicated in the pathophysiology of joint loosening and fibrous encapsulation [3].

During the inflammatory phase of implant introduction, growth and platelet factors are released which attract mesenchymal stem cells. In vitro studies report that MSC migration, proliferation and differentiation to the site of the new implant results in cell adhesion to the extracellular matrix and to the implant. Fibrous tissue may result, which is associated with poor bone healing and implant failure. Nanoscale bone implants have enhanced bone healing properties as they exhibit reduced fibroblast adhesion and proliferation and greater osseointegration. Lavenus et al. hypothesise that the surface topography of nano scale implants may regulate the behaviour of MSCs during the inflammatory phase [23].

Nanoelectromechanical systems (NEMS) are a future direction of study for nanotechnology. Manipulation of this technology may allow for the production of nanosized systems with communication and monitoring functions. Implementation of NEMs at a fracture site could provide important information regarding bone healing for both clinical and research purposes [28].

Core-shell electrospinning is a future direction for nanotechnology. Current electrospun nano-composite fibres are often random in spatial organisation, prohibiting them from mimicking the complex organisation of natural bone. Core-shell electrospinning differs from electrospinning in that the spinneret has two jets, resulting in a dual layered nanofibre. Synthesis of polymers, metals and ceramics using this technology, could potentially further develop bone healing strategies [8].

Documented disadvantages of scaffolds include inflammation, limited cell turnover and growth factor expression [28]. Multiwall carbon nanotubes are associated with mild reversible damage to the liver and tubular damage to the kidneys. Control of these undesirable factors is required for this novel technology is to be clinically viable.

Although nanotechnology is considered to be in its infancy, its manipulation and utilization has developed since its introduction in 1959. Current nanoproducts for bone healing include nano-HA-paste-ostim and nano-beta-tricalcium phosphate-Vitoss [57].

Nanophase synthetic bone grafts, implants and scaffolds are considered superior bone healing solutions to their conventional counterparts. Unfortunately, design and production of these solutions is often expensive and difficult to replicate on mass scale. Further more, the evidence surrounding their biocompatibility, and therefore safety, is conflicting. This is compounded by the fact that many studies are in fact in vitro and not in vivo [9]. It would be prudent for the community to provide greater safety data, as it is estimated that only 3% of the capita spent on nanotechnology research has been devoted to healthy and safety [2].

Nanophase bone healing solutions produced on mass scale with clear safety data is required if Nanotechnology is to fulfill its very promising impact on Orthopaedic Surgery.