The Main Challenges Of Bone Tissue Engineering
Bone tissue can become damaged by trauma when a large impact force damages the tissue. Bone tissue can also become damaged pathologically when bones are weakened by cancer, osteoporosis, osteomalacia or osteomyelitis. These diseases all cause the weakening of bone tissue which increases the likelihood of bone defects occurring. When bone tissue acquires a defect, the tissue can often heal itself. Often this occurs without the formation of scar tissue. However, in the case of pathological bone defects and very large defects, as well as insufficient blood supply, the repair mechanisms can fail and result in non-unions, which can lead to pain or loss of function in the affected tissue. To encourage fracture union of a broken bone, large and pathological bone defects are treated with bone grafts. Bone grafts are implanted materials which promote the healing of bone tissue through osteogenesis, osteoinduction and osteoconduction. In order for new bone tissue to grow, there needs to be a source of osteogenic cells, which lay down new bone material, osteoinductive growth factors, which initiate osteogenesis, and a suitably osteoconductive scaffold which provides a surface for new bone tissue to grow on.
Autografts are still considered the gold standard for the promotion of bone tissue regeneration, as they provide a source of all of the components necessary for bone tissue regeneration. However, the use of autografts is limited by the availability of suitable donor tissue, and the increased risk associated with harvesting it. This is particularly relevant in relation to bone defects associated with disease, as there is even less suitable donor tissue.
In the past few decades, tissue engineering has attempted to overcome the limitations of traditional bone grafts. In this review I will outline the main challenges faced when designing tissue engineered bone grafts, and the main strategies which have been developed to overcome these challenges in commercially available products. I will outline the most common biomaterials used for commercially available scaffolds, discuss the current research into the use of synthetic growth factors to promote osteoinduction, and I will outline some cell based therapies currently in development.
Osteoconduction: Biomaterial Scaffolds
An osteoconductive material is one which encourages bone tissue growth on its surface. There are many factors which contribute to scaffold osteoconductivity including porosity, surface topography, hydrophilicity and resorbability. The aim of a bone scaffold is to mimic the extracellular matrix found in natural human bone in terms of mechanical strength and porosity. The mechanical properties of bone range widely depending on the anatomical location of the tissue. Therefore, an ideal material for a bone tissue scaffold would be able to mimic the range of mechanical properties of bone tissue. Porosity affects a scaffold’s osteoconductive properties as it mediates vascularisation, and interconnected pores allow cells to populate the centre of a scaffold. A pore size of 200-350µm is ideal for bone growth and facilitates osteoconduction.
Biomaterials used for tissue engineering scaffolds can be broadly categorised into two categories: natural and synthetic. In general, natural materials have excellent biocompatibility and are bioactive, they contain biomarkers that encourage tissue regeneration. However, they can also have unpredictable effects on the host immune response, and are also not very structurally and chemically predictable. Synthetic materials can be more highly optimised to the case specific needs, but they lack the innate bioactivity of natural materials.
Natural Biomaterials
Collagen
Collagen forms 90% of bone ECM proteins, which are mainly type 1 collagens. Collagen is a major component of the ECM, so is quite widely used across the field of tissue engineering, including for bone tissue. Due to its’ abundance in nature, collagen is easily isolated from natural sources. However, obtaining pure type 1 collagen can be costly
However, due to its very poor mechanical properties the focus is on including collagen with other bone scaffold materials such as synthetic polymers and bioglasses, to increase their bioactivity, osteoconductivity and degradation behaviours.
Synthetic Biomaterials
Synthetic Polymers
Synthetic polymers are very useful because they can be positively tailored, and individual implanted devices can be designed with different structural and mechanical properties. However, synthetic polymers are not bioactive, which limits their abilities. One approach to reducing this limitation is through surface modification. Glass nano particles and fibres have been used to increase bioactivity and osteoconductivity of polymer scaffolds, with the added benefit of structural reinforcement.
Calcium Phosphate derived materials
Bone mineral is made of hydroxyapatite crystals. This biomimicry means that hydroxyapatite has been used extensively in bone tissue scaffolds as it highly encourages osteogenesis. Hydroxyapatite is also used to coat metal orthopedic implants to increase biomaterial bonding with the surrounding tissue. Hydroxyapatite and collagen are very often paired to make electrospun scaffolds.
Bioactive Glasses
Bioglasses can also be used to increase osteoconductivity of scaffolds. Bioglasses have a controllable degradation rate and can be very useful to incorporate a long term drug delivery aspect into a scaffold.
Osteoinduction: Growth Factors
Normal bone tissue healing is mediated by several different classes of growth factors. Bone morphogenic proteins (BMPs), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), transforming growth factor-bets (TGF-β) and vascular endothelial growth factor (VEGF) are all stored in bone ECM and are released after bone tissue injury to initiate osteoinduction. The addition of growth factors to successful biomaterial scaffolds can further enhance their regenerative action. To successfully incorporate growth factors into bone scaffolds for regeneration, the normal growth factor cascade during bone fracture repair must first be understood. In the first few days after a bone fracture or defect occurs, the surrounding area becomes inflamed, a process which is mediated by tumour necrosis factor alpha (TNFα), FGF-2 interleukin-1 (IL-1), IL-6 and macrophage colony stimulating factor (MCSF). These factors increase osteoclast activity and inflammatory cell migration to the site.
Following inflammation, it is important to establish sufficient vascularization which prevents hypoxia and allows signalling and transport of nutrients to newly forming tissue. This occurs via angiogenic factors, which initiate the formation of new vessels from the surrounding tissue. Low oxygen levels in an area of damaged tissue are detected by hypoxia inducible factors (HIFs), which results in upregulation of VEGF. VEGF leads to the formation of very permeable blood vessels which are unstable. For this reason it is thought that VEGF release is only helpful to bone formation when it is released in conjunction with other factors which promote stable tissue formation, and it has been shown that early release of VEGF followed by sustained release of BMP-2 can significantly enhance tissue regeneration compared to BMP-2 alone (7). FGFs (specifically FGF-2) enhance osteoblast proliferation and angiogenesis and have been shown to enhance bone regeneration in vivo.
However, FGFs have a short half-life in vivo, so there are difficulties associated with their therapeutic delivery. Efforts are currently underway to develop methods for prolonging the release of growth factors, for example by incorporating coaxial electrospun nanofibers containing the factors into a scaffold, which can lead to a more long term release of molecules. Once supportive vasculature has been formed, osteogenic factors begin recruiting osteoblast progenitor cells, which then differentiate into osteoblasts, which are capable of laying down new tissue. PDGF, TGFβ, FGF and IGF all have osteogenic effects, but the most well understood osteogenic factors in relation to tissue engineering are the BMPs. In fact, products which contain BMP-2 have had FDA approval since 2002 for use in spinal surgery. However, when used outside of very specific surgical areas, BMP-2 has been associated with uncontrolled bone tissue growth, which has lead to some undesirable neurological effects. These side effects underline the importance of the fact that all growth factors in the body exist in a delicate balance within a dynamic system. For optimum biomimicry of a system, temporal and spatial release needs to be considered, as well as the release of several factors from an individual device.
The rate of release of a growth factor from a scaffold can be controlled to an extent through the choice of scaffold material and method of entrapment of the molecules. Covalent attachment of a factor to a scaffold allows for prolonged, sustained release from the scaffold, but requires that the factor contains suitable functional group and may affect the bioactivity of the factor due to the chemical interaction with the scaffold. Physical entrapment of a factor into a scaffold is the simplest method of delivery, and preserves the molecules bioactivity, but the release rate is harder to control and usually involves a rapid burst release of the factor. Incorporating a growth factor into nanospheres allows for better control of the release rate, and can potentially be used for sequential release of cascade of growth factors. However, the use of nanospheres adds several fabrication steps, which increases production costs. I believe that the use of nanospheres within scaffolds is the best option when designing a system for bone tissue regeneration, as sequential release of several different growth factors has been shown to enhance the formation of stable bone tissue through biomimicry.
Osteogenesis: Osteogenic Cells
There are many different sources of stem cells that can be preferentially differentiated into osteoblasts. Things like pluripotency, risk, abundance and in the case of bone tissue regeneration, osteogenic potential, must be taken into consideration when deciding where to source cells for a specific application. Some stem cell sources have a higher cost associated with their harvesting and expansion, while others are associated with ethical concerns or are less well characterized.
An important consideration when using stem cells seeded onto a scaffold is the vascularisation of the graft. Cells in natural bone tissue are all within 200µm of a blood vessel, as this is the distance that oxygen and nutrients can diffuse across. A major clinical challenge has been creating regenerated bone tissue that is adequately vasculaturised, this is particularly important in the treatment of large bone defects. This has resulted in efforts to develop “pre-vascularized” tissue engineered bone grafts. This can be done using several different methods which employ stem cells.
In vitro pre-vascularisation involves using growth factors to induce the formation of blood vessels throughout a graft pre-implantation. Ectopic pre-vascularisation involves implanting a cell seeded scaffold onto a highly vascularised tissue to allow vascular ingrowth, so that the graft can then be removed and transplanted into the bone defect. Alternatively, orthotopic vascularisation employs the controlled release of growth factors to encourage vascularisation in situ (cells can be aggregated to improve cell survival).
The immune system can have an effect on bone tissue repair. Macrophages can polarize into two (general) groups. M1 macrophages are pro-inflammatory, and inhibit tissue remodelling, whereas M2 macrophages are anti-inflammatory, and can promote successful tissue remodelling. This shows the importance of considering a graft response on the surrounding tissue, as if a graft elicits a strong M1 response, this could inhibit the remodelling of the bone tissue. One way of controlling this macrophage polarisation is with the choice of scaffold material. There has been evidence to show that the size of hydroxyapatite particles can affect the polarization. Generally, hydroxyapatite crystals promote M1 macrophage polarization, but using nano hydroxyapatite crystals has been shown to result in M2 macrophage polarisation, implying a more pro tissue remodelling response.
There are currently many commercially available FDA approved bone graft substitutes composed from calcium phosphates, synthetic polymers, collagen, hydrogels, hydroxyapatite, in the form of pastes, putties, gels, granules, and powders. Most commercially available bone graft substitutes are natural based materials. There is a variation in the claimed mechanism of action of each product. There is a variation in the osteoconductivity and osteoconductivity of the products, as well as levels or types of growth factors included in the scaffold. The choice of product is usually down to the experience and preference of the physician in charge.
While there is a wide range of products available, and there has been much success with bone tissue engineering, there is always room for improvement, and there is further potential for manipulation of stem cells and host tissue cells for the benefit of bone tissue regeneration.
