Trends in bone graft development

There is increasing need for bone grafts that has inherent capability of supporting new bone formation and the complete remodelling of the grafts. The biological fixation of an implant by virtue of native bone is supposed to ensure the longer survival and prolong the need for the revision of orthopaedic and maxillofacial implants compared to inanimate bone grafts. In order to reach this goal various strategies have been developed to enhance the osteoinductive and osteogenic potential of bone grafts by taking the advantage of the recent achievements of nanotechnology and coating techniques that are exploited by tissue engineers.

2.3.4.1 Nanotechnology

When bone substitutes are placed into the human body, interactions between the surface of the bone substitute and the surrounding bone and soft tissues are critical to MSC differentiation and osseointegration. MSCs appear to be one of the first cell types

involved when a nanophase biomaterial is introduced into the host¹¹⁷. Thus, mimicking the nanoscale, three-dimensional extracellular matrix and cell topography may improve the osseointegration by promoting the adhesion, proliferation and differentiation of MSCs¹¹⁸. Supplied with state-of-the-art nanofabrication techniques (nanolithography), scientists have become able to produce well-controlled nano-scale topography on 2-dimensional planar substrates to investigate their effect on stem cell fate under standardized experimental conditions. Nanolithography is a branch of methods that are suitable to control the size, shape, spacing and organizational symmetry of nano-scale features on the surface of various planar materials at least in one lateral dimension between 1-100 nm, like nanopits, nanopillars and nanochannels (Figure 12)119,120,121,122. For instance, the diameter and spacing of nano-fibres or nano-channels produced by electrospinning can be varied in ranges that approach the dimensions of natural basement-membrane fibre sizes of 5–200 nm and pore sizes of 3–80 nm123,124,125. Channels and pillars formed by lithographic techniques can also be varied in ranges that mimic the porosity of natural ECM¹²⁶. Various nano-patterned materials have been shown to enhance osteogenic differentiation of MSCs compared to micro-rough surfaces, including nanophase ceramics, aluminium-oxide, titanium-oxide and titanium alloy, carbon nanotubes and cobalt–chrome alloys¹²⁷. Nanocrystalline hydroxyapatite paste has been used as a filler of bone defects with good clinical outcome¹²⁸. Nano-composite scaffolds that consists of type I collagen and nano-crystalline hydroxyapatite are currently being used in the treatment of osteochondral defects of the knee with encouraging short-term clinical and radiological results¹²⁹. Nanophase delivery systems have recently been studied for the local and precision delivery of drugs and growth factors. For instance, type-2 bone morphogenetic protein loaded nanofibre poly-L-lactic acid enhanced the closure of large calvarial bony defects¹³⁰.

Figure 12. Cultured cells on the 400 nm polymeric nanopillar arrays. Panel A-B show the SEM image of an adhered cell on the array. Panel C shows the combined confocal image of a cell cultured on a 200-nm nanopillar array immunofluorescently stained with tensin (green) and FAK (red) after 24 hours of incubation. Images are adaptations from reference 125.

2.3.4.2 Coatings

The coating of bone grafts with peptides, proteins and other molecules in order to enhance their ability to incorporate into the host tissue is one of the first biomimetic approaches^131,132. More recently novel coatings address the local and controlled delivery of therapeutic molecules at the fracture site. The advance of polymer and composite techniques may enable bone grafts to carry and sustain the release of molecules in appropriate dosage for days or weeks¹³³. For the purpose of delivery of therapeutic molecules synthetic (poly-lactic-acid, poly lactic-co-glycolic acid, polycapronolactone, etc.) and natural (chitosan, silk, alginate, etc.) polymers or their composite with inorganic substances can be used either for the fabrication of scaffolds or for the coating of bone grafts134,135,136. In general, such composite scaffolds or coatings are utilized to carry either antimicrobial agents to prevent local infections, or growth factors and adjuvants that enhance bone regeneration^137,138. The latter approach is also known as in situ tissue engineering, which utilizes the own regenerating capacity of the body by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the site of injury. This approach relies on the development of target-specific biomaterial scaffolding and coating systems that have the capability of controlling the host microenvironment by mechanical and soluble cues that directs the fate of recruited host cells¹³⁹. The release of the therapeutic molecules can be optimized by various methods, including their encapsulation into nano-, or micro-carriers and by the degradation rate of the polymer bed through its chemical composition140,141,142,143. From regulatory point of view, coated bone grafts that are loaded with therapeutic molecules are regarded as

combination products that fall under the scope both of the directives of medical devices and the medicinal products. Growth factors are relatively new as therapeutic molecules, while most of the drugs, such as antimicrobial agents are administered through different routes (oral or parenteral); therefore, the clinical safety and efficacy of such novel combination products are supposed to be proven in controlled, randomized human studies before clinical use.

2.3.4.3 Tissue engineering

The approach of tissue engineering is to generate new, functional tissues with living cells instead of placing non-living scaffolds into bone defects in order to enhance bone regeneration, especially in the case of large bone defects where bone grafting is essential¹⁴⁴. The objective of bone tissue engineering is to populate three-dimensional scaffolds with progenitor and/or mature cells, such as mesenchymal stem cells and epithelial cells so that enhance bone remodelling¹⁴⁵. The recently constructed engineered bone tissues are the artworks of multidisciplinary science that utilize the achievements of biology, material sciences, physics and engineering¹⁹⁴. By the development of nanotechnology and the discovery of biophysical cues the biomimetic approach has spread in the field of tissue engineering¹⁵³. These achievements allowed the developed three-dimensional porous scaffolds to have specific surface topography ranging from the micro-, to nano-size with optimized surface chemistry to improve cell adherence, proliferation and differentiation. The development of additive manufacturing, e.g. 3D printing, stereolithography, fused deposition modelling, and selective laser sintering may also have a significant impact on the engineering of scaffolds that mimic more the mechanical properties of the host bone¹⁴⁶. Dynamic cell culture instruments have been designed to support the continuous oxygen and nutrient supply of the cells in the 3-dimensional scaffolds. Albeit, in clinical setting, the large artificial bone grafts still have a significant weakness because their blood supply is insufficient due to the lack of indwelling blood vessels. The adequate blood supply is

the question of the clinical relevance of bone tissue engineering; or it should be used as an in vitro testing method for the evaluation of the novel biomimetic scaffolds.

2.3.4.4 Bioreactors

In vitro bioreactors have been designed to ensure the homogeneous cell distribution on the surface of 3-dimensional biomaterials and their appropriate oxygen and nutrient supply. Under static cell culture condition the insufficient nutrient and oxygen transport, and waste removal may cause decreased proliferation and differentiation and non-uniform cell distribution ¹⁴⁷. Therefore, more complex instruments needed to improve culture media circulation and convective transport of nutrients to the dwelling cells allowing the development of a more uniform tissue.

Bioreactors have been designed to: a) allow controlled and fast cell expansion, b) support the efficient exchange of nutrients, oxygen and metabolites in all parts of the scaffold, and c) enhanced cell seeding and provision of physical or biochemical stimuli.

The most common bioreactor types are spinner flasks, rotating wall vessels and perfusion systems. Chronologically, spinner flasks and rotating-wall vessels have been the first alternatives to static culture that tried to minimize gradients in nutrient and metabolite concentrations. These types of bioreactors utilize convection to improve the nutrient transport into the porous structure of the scaffold. Perfusion systems constitute a newer generation of bioreactors that are more complex, they can perfuse fluid directly through the pores and interconnecting channels ensuring good mass transport inside the scaffold¹⁴⁷.