• Bone is the second most common transplanted tissue with approximately 3 million grafting procedures performed each year worldwide.
• Bone substitutes are being increasingly used especially in oncological surgery, trauma, revision prosthetic surgery and spine surgery.
• Autografts still represent the gold standard for bone substitution, although the morbidity and the inherent limited availability are the main limitations.
• Grafts are by definition taken without a blood supply so need to be transplanted into a vascular bed.
• When a tissue is taken with its blood supply such as a free fibula transplant it is an osseous flap.

• To provide structural/mechanical stability (cortical bone best).
• To provide linkage, i.e. replace missing bone. Cortical or segmental defect.
• To stimulate osteogenesis and bone healing in fractures.

• The term bone graft substitute describes a spectrum of products that have various effects on bone-healing.
• Bone graft substitutes can be broadly categorised into bone grafts (autograft, allograft, xenograft), ceramics (hydroxyapatite, TCP, calcium sulphate) and growth factors (DBM, PRP, bone morphogenetic protein (BMP)).
• The ideal bone graft substitute should be osteoconductive, osteoinductive, biocompatible, bioresorbable, structurally similar to bone, easy to use, and cost effective.

• Autograft is bone transplanted from one area to another in the same individual.
• Allograft is bone transplanted from one individual to another.
• Xenograft is bone transplanted between animals of different species.
• Isograft is bone transferred between monozygotic twins.
• Grafts maybe fresh or preserved.

• Osteogenesis, osteoinduction and osteoconduction are the three essential elements of bone regeneration, along with the final bonding between host bone and grafting material which is called osteointegration.

• This is the formation of new bone by cells derived from precursors such as mesenchymal stem cells that are in the grafting material.
• In suitable host conditions, these precursors proliferate and differentiate and then generate new bone.

• Defined as “a process that supports the mitogenesis of undifferentiated mesenchymal cells leading to the formation of osteoprogenitor cells with the capacity to form new bone.”¹
• Graft derived factors (BMP, etc.) stimulate the proliferation and differentiation of bone-producing cells from precursors such as mesenchymal stem cells in the surrounding host tissues.

• The graft provides a scaffold for bone deposition. This promotes the development of new bone and also integration with the host skeleton. Success depends on several factors including surface roughness and porosity.

• The surface bonding between the host bone and the grafting material.

• Low cost.
• Abundant quantity.
• Low immunogenicity and not evoke any adverse inflammatory response.
• Biocompatible and biodegradable with mechanical properties similar to the surrounding bone.
• It should be easily moulded into the bone defect within a short setting time.
• It should provide signalling molecules, osteoprogenitor cells and a supporting scaffold to support osteoinduction, osteogenesis and osteoconduction.

Cortical

• Less biologically active than cancellous bone and incorporated through slow remodelling which initially weakens the graft.
• Approximately 40–60% weaker than normal bone from 6 weeks to 6 months after transplantation.
• Returns to normal 1–2 years after transplant.
• Provides more structural support.

Cancellous

• Revascularised more quickly.
• Osteoblasts lay new bone down on old trabeculae which is later remodelled.
• Three dimensional scaffold.
• Osteocytes and stem cells (osteogenic).
• Little initial support.

Osteochondral

• For tumour surgery.
• Need tissue typing if allograft used.
• Osteochondral graft survival enhanced by immersion in glycerol.

Initial phase

• Analogous to fracture healing.
• Little difference between cortical and cancellous grafts.

Secondary phase

• Important differences between cortical and cancellous bone.

Table 1. Difference between cortical and cancellous graft incorporation

• A bone graft produces a response leading to the accumulation of inflammatory cells, followed by the chemotaxis of host mesenchymal cells to the graft site.
• The primitive host cells differentiate into chondroblasts and osteoblasts, a process under the influence of various osteoinductive factors.
• The additional processes of bone graft revascularisation and necrotic graft resorption occur concurrently.
• Finally, bone production from the osteoblasts onto the graft’s three-dimensional framework occurs, followed by bone remodelling in response to mechanical stress.

Autografts

• From the same person.
• Gold standard in bone substitution.
• Iliac crest is the most frequent donor site as it provides easy access to good quality cancellous autograft.
• No problems with immunogenicity or infection-related risks.
• Highest osteogenic and osteoinductive capacity.
• Revascularised more quickly than allograft.
• Problems with donor site morbidity (20%) that include haematoma, residue chronic pain at donor site, fracture, wound infection, neurovascular injury and cosmetic deformity.
• Limited supply especially in paediatric and elderly patients.
• Increased anaesthetic times to harvest.
• Vascularised flaps, e.g. fibula but can have considerable donor site morbidity.
• Best reserved for area of large bone loss or irradiated tissues.
• No resorption and either end of the transplanted segment heals as a fracture.

Allografts

• Donor bone from another person either living (THA patients) or non-living donors and must be processed within a bone tissue bank.
• Regarded as the surgeons’ second choice.
• No donor site morbidity.
• Reasonable amounts are available.
• Can be used off the shelf.
• Require sterilisation with gamma radiation ® alters collagen, denatures proteins ® depresses graft incorporation ® immunological response and less reliable incorporation.
• The osteoprogenitor cells are destroyed by the sterilisation process, thus allografts are not osteogenic.
• Osteoconductive and weakly osteoinductive (growth factors may still be present depending on the processing).
• The sequence of events of incorporation is qualitatively similar to that for autografts, but it is delayed and less extensive as allografts are biologically inferior to autografts.
• Allograft infection rate 10–12% and more than 80% of infected allografts are associated with clinical failure.
• Risk of transmission of infection, e.g. HIV, Hep B, Hep C.
• Processing methods may vary between companies.

Types

• Fresh – requires no preservation. There is little time to test for disease or sterility, therefore used less frequently than processed allografts. There is an intense immune response. The application of fresh allograft is limited to joint resurfacing using shape-matched osteochondral allografts. Highest risk of disease transmission.
• Frozen – frozen allograft involves freezing to below –60°C. This diminishes enzyme degradation affording decreased immunogenicity without changes in the biomechanical properties. BMP preserved and therefore osteoinductive. Time to test for disease.
• Freeze-dried – this involves removing the water from the frozen tissue and then vacuum packing the graft with storage for up to 5 years at room temperature. This process decreases the antigenicity. It is osteoconductive only as the process destroys all osteogenic cells and leaves very limited osteoinductive capacity. The main disadvantage is biomechanical alteration on rehydration. The host immune response is less robust than the response to fresh or fresh-frozen allogaft. lowest likelihood of viral transmission, BMP depleted and least structural integrity.

Graft incorporation

• Incorporation of allograft bone differs according to the type of graft used.
• Cortical strut grafts are incorporated by creeping substitution through the process of intramembranous bone formation at the cortical junctions.
• Cortical graft ends with an exposed medullary canal are incorporated by enchondral ossification. This process involves weakening of the initial structural strength of the cortical graft as it is resorbed. Strength is recovered as new bone formation occurs.
• Cancellous allograft chips or powders are incorporated solely by enchondral bone formation along the osteoconductive framework of the graft, which strengthens the construct over time.

Summary of allografts

• Allografts are excellent for structural integrity and reasonable for osteoconduction. However, osteoinductive potential is limited and osteogenic potential completely absent. Allografting results are poor when grafting on to an unfavourable bed, as in the infective case. Allografts require augmentation for the formation of new bone.

Bone marrow

• Contains osteoprogenitor cells.
• Grows well into ceramics.
• Bone marrow contains osteoprogenitor cells in the order of one per 50,000 nucleated cells. Techniques have been developed to increase the number five-fold. These may be used with an inorganic matrix (to provide osteoconduction).
• Bone marrow grows well into ceramics. Studies have demonstrated that bone marrow will successfully treat non-unions when adequate amounts are utilised.
• Bone marrow is harvested by aspirating 2–3 ml from either the proximal humerus or the ilium, diluting with blood and using immediately.
• The best clinical indication for bone marrow use is when augmentation (osteogenesis) is required.

Xenograft

• From a different species, i.e. porcine or bovine bone that can be freeze dried or demineralised and deproteinised.
• Coral based xenografts are usually calcium carbonate while natural human bone is made of hydroxyapatite along with calcium phosphate and carbonate. Coral material is therefore either transformed industrially into hydroxyapatite through a hydrothermal process, yielding to a non-resorbable xenograft, or simply the process is omitted and the coralline material remains in its calcium carbonate state for better resorption of the graft by the natural bone.² The coral xenograft is then saturated with growth enhancing gels and solutions.
• Advantages include easy of availability, osteoconductivity, low cost, avoidance of donor site morbidity, utilisation of potentially superior mechanical properties, elimination of the risk of human blood-borne diseases (e.g. HIV, hepatitis C) and a reduction of surgical/anaesthetic time.³
• Disadvantages include uncertain contradictory clinical results, antigenicity, potential transmission of zoonoses (e.g. BSE), the potential to compromise the tissue’s biomechanical properties secondary to processing methods used to mitigate the risks of infection and reduce immunogenicity.

• Several categories of bone graft and bone graft substitutes exist and include a wide variety of materials, material sources, and origins (natural vs. synthetic). Many are formed from composites of one or more types of material. These composites are usually built on a base material.
• Laurencin et al.⁴ classified bone grafts and graft substitutes into five categories:

1. Allograft-based bone graft substitutes involve allograft bone, used alone or in combination with other materials.
2. Factor-based bone graft substitutes are natural and recombinant growth factors, used alone or in combination with other materials such as transforming growth factor-beta (TGF-b), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and BMP.
3. Cell-based bone graft substitutes use cells to generate new tissue alone or are seeded onto a support matrix.
4. Ceramic-based bone graft substitutes include calcium phosphate, calcium sulfate, and bioglass used alone or in combination.
5. Polymer-based bone graft substitutes, degradable and non-degradable polymers, are used alone or in combination with other materials.

Synthetic bone grafts

• As bone grafting technology advances, synthetic alternatives are being used more frequently to augment or replace autograft or allograft.
• Synthetic bone materials offer surgeons greater freedom in grafting solutions because they are unlimited in quantity, lack possible viral contamination, decrease surgical time, and avert the need for additional surgical procedures.

Table 2. Bone graft substitutes. From Laurencin et al. (2014)⁴

Ceramic-based bone graft substitutes

• As there is an obvious need for a bone graft substitute to serve as an “off the shelf” alternative to autograft attention has turned to alternatives such as ceramics. Ceramics are osteoconductive only. They are excellent in compression.
• Ceramics are non-inflammatory and inert when in a structural arrangement. Small granules, however, may elicit a foreign body-giant cell reaction and subsequent partial resorption.
• Ceramics are brittle with little tensile strength and hence their applications are limited. Ceramics must be shielded from loading forces until ingrowth has occurred.
• Calcium phosphate based ceramics are synthetic scaffolds that have been the subject of investigation for decades due to their compositional correlation to the mineral component of natural bone.
• Much research has centered on the use of hydroxyapatite and tricalcium phosphate, as coatings, porous constructs, or solid blocks.
• The intellectual task has been to put the appropriate calcium salts into the correct three-dimensional form, so that bone can be guided into a prescribed location. Porosity allows soft tissue and bone to regenerate within the pore space. The optimal pore size is 150–500 mm.
• Ceramics are produced as porous implants, non-porous dense implants and granular particles with pores. The shape of ceramics is important. For example, dense blocks with small surface areas degrade much slower.
• Types of ceramics include:
• Calcium sulfate
• Tricalcium phosphate
• Hydroxyapatite
• Coralline hydroxyapatite

Ceramics

Calcium sulfate

• Also known as plaster of Paris.
• Osteoconductive void filler that completely resorbs as newly formed bone remodels and restores anatomical features and structural properties.
• Low compressive strength – no structural support therefore questionable choice for load bearing applications.
• It is biocompatible, bioactive and rapidly resorbs after 30–60 days.
• May be used as, filling for bone cysts, bone cavities, benign bone cysts, segmental bone defects and as a autogenous graft extender.

Tricalcium phosphate (TCP)

• TCP composition is similar to calcium and phosphate (mineral) phase of human bone.
• The small particle size and interconnected sponge-like microporosity are believed to improve osteoconductive properties and promote timely resorption.
• Bioabsorbable and biocompatible.
• Wet compressive strength slightly less than cancellous bone.
• Available as blocks, wedges and granules.
• Numerous trade names.
• Tricalcium phosphate is more porous and biological degradation is ten to 20 times faster than hydroxyapatite with complete resorption at 6 months.
• Degradability occurs by combined dissolution and osteoclastic resorption.
• Hydroxyapatite is resorbed by foreign body giant cells, which stop ingesting once 2–10 µm of hydroxyapatite has been consumed. Consequently, hydroxyapatite may remain in the body for up to 10 years. Tricalcium phosphate, on the other hand, is better remodelled but weaker. For this reason, tricalcium phosphate is not ideal for compression.

Hydroxyapatite (HA)

• Hydroxyapatite is the major constituent of the inorganic component of bone that is biocompatible, osteoconductive, bioactive and nonimmunogenic.
• Its unique property is the chemical similarity with the mineralised phase of bone.
• The intellectual task has been to put the appropriate calcium salts into the correct three-dimensional form, so that bone can be guided into a prescribed location. Porosity allows soft tissue and bone to regenerate within the pore space. The optimal pore size is 150–500 µm.
• Ceramics are produced as porous implants, non-porous dense implants and granular particles with pores. The shape of ceramics is important. For example, dense blocks with small surface areas degrade much slower.
• HA is usually used as a coating for orthopaedic implants.
• The optimal pore size is around 500 µm and porosity 60%.
• Osteoblasts can produce osteoid directly on the surface of HA and form an intimate bond of new bone.
• Porosity and interconnected pores are critical features for bone graft substitutes because they allow for a greater degree and faster rate of bone ingrowth.
• Porosity is defined as the percentage of void space in a solid and it is a morphological property independent of the material.
• Pores in bone graft substitutes can be categorised as macropores (pores larger than 100 μm), and micropores (pores smaller than 50 μm). It is generally agreed that pores must be at least 100 μm for bone ingrowth.
• Pores are necessary for bone tissue formation because they allow migration, penetration and proliferation of osteoblasts and mesenchymal cells. They also allow for nutrient and waste transport and vascularisation.
• A porous surface also improves mechanical interlocking between the implant biomaterial and the surrounding natural bone, providing greater mechanical stability at this critical interface.
• The mechanical performance of synthetic HA can be very poor compared to bone. Studies have shown, however, that porous HA is more resorbable and more osteoconductive than dense HA. This has stimulated much research into the development of synthetic porous HA bone replacement materials for the filling of both load-bearing and non-load-bearing osseous defects.

Bioactive glass

• In 1969, Hench et al.⁵ discovered that rat bone can bond chemically to certain silicate-based glass compositions . This group of glasses was later termed “bioactive,” being “a material that elicits a specific biological response at the material surface which results in the formation of a bond between the tissues and the materials.”
• Bioactive glass ceramics are osteoconductive and bond to bone without an intervening fibrous connective tissue interface.
• The bone-bonding reaction results from a sequence of reactions in the glass and its surface.
• A major concern with bioactive glasses is their brittleness and low fracture toughness. Their limited strength and low fracture toughness (i.e. ability to resist fracture when a crack is present) has so far prevented their use for load-bearing implants.

Coralline ceramics

• These may be natural coral or replamineform ceramics. Replamineform ceramics are porous hydroxyapatites derived from the calcium carbonate skeletal structure of sea coral. The replamineform process converts calcium carbonate to hydroxyapatite. These are produced from marine specimens by hydrothermal exchange replacement of coral carbonate with calcium phosphate replicas. The pore structure is highly organised and is similar to human cancellous bone.
• Replanineform ceramics include those derived from goniopora (large pores 500–600 µm) and porites (pores of 200–250 µm). The trade name is ProOsteon. The number following the trade name designates the nominal pore diameter, either 500 or 200 p.m. To be considered for commercialisation and amenable to conventional manufacturing methods, the corals must be naturally in abundance and grow in large hemispherical forms. On the whole, autograft is superior to ceramics in studies but some do show favourable results (coralline hydroxyapatite used as a defect filler in small tibial defects in dogs). Once incorporated, coralline ceramics obey Wolff’s law and do not stress-shield the regenerated bone. These grafts have mainly been used in dentistry and maxillofacial surgery. There is one human study of tibial plateau fractures, which shows a favourable result.

Demineralised bone matrix (DBM)

• Demineralised bone matrix is formed by the acid extraction of bone leaving non-collagenase proteins, bone growth factors and collagen in continuity in a composite. The material produced, the DBM, retains the trabecular collagenous structure of the original tissue and can serve as a biologic osteoconductive scaffold despite the loss of structural strength once contributed by the pre-existing bone mineral.
• DBM is quickly revascularised (osteoconductive), moderately osteoinductive (due to bone morphogenic proteins), but offers no structural support.
• The process of incorporation involves inflammation, cellular differentiation of mesenchymal cells into chondrocytes (5 days), a cartilaginous phase, mineralisation and eventual complete absorption of the DBM.
• The source of DBM and its processing are important. If DBM is kept at room temperature for more than 24 hours it becomes biologically inactive. Ethylene oxide and radiation (more than 2.5 MRad) reduces the osteoconductive potential.
• There are numerous demineralised bone matrix formulations based on refinements of the manufacturing process. DBM is available as powder, crushed granules, chips, putties, strips and pastes. They have also been developed as combination products with other materials such as allogeneic bone.
• DBM is most effective in conjunction with internal fixation and as an adjunct to other grafting materials. Its applications include augmentation of autogenous and traditional allogeneic bone grafts in the repair of cysts, fractures, non-unions and stable fusions.
• Clinical results are variable and while level IV evidence exists demonstrating good therapeutic benefit there is a lack of evidence from level I trials.

Collagen-based matrices

• Highly purified type I bovine dermal fibrillar collagen.
• Bone marrow is added to provide bone forming cells to provide osteoprogenitor cells and other growth factors.

BMPs

• Recombinant gene technology has been used to develop individual BMPs.
• Their association with adverse events and their application in settings where appropriate stability has not yet been achieved has led to results that have been unfavourable at the time.
• In posterolateral spine fusion procedures prospective randomised trials have shown better outcomes (fusion rate, blood loss, operative time, reoperation rate) using BMP-2 compared to autogenous iliac crest graft. Safety is an issue with complications reported of ectopic bone formation in and around the spinal canal, postoperative radiculitis, vertebral osteolysis and allergic/hyperinflammatory response.⁶

Yasko AW, Lane JM, Fellinger EJ, Rosen V, Wozney JM, Wang A. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2): a radiographic, histological, and biomechanical study in rats. J Bone Joint Surg Am 1992; 74: 659–670.

• The osteoinductive properties of rhBMP-2 in promoting bone healing were firmly established in this animal model. The mechanism of action of rhBMP-2 involves osteoinductive signalling and regulation of a number of gene expression pathways involving the differentiation of mesenchymal progenitor cells into osteoblasts.
• This was an animal study that provided significant evidence that rhBMP-2 was capable of inducing union of a bony defect. By showing a 100% union rate the authors concluded that rhBMP-2 could act as alternative or enhancer of autologous bone graft in bone regeneration.
• The authors used a well defined femoral-defect model that had been extensively used to study osteoinductive, osteoconductive and osteogenic factors. The model results in a predictable non-union, observations and quantification of bone formation by radiographs can be easily determined and the rigid fixation obtained with the plate maintains a coaxial relationship of the ends of the bone for mechanical testing.
• Bone morphogenetic proteins constitute at least 15 growth factors originally identified to stimulate de novo bone formation. They play a critical role in regulating cell growth, differentiation and apoptosis in a variety of cells during development including osteoblasts and chondrocytes. It is thought BMPs are expressed in the early stages of facture healing. It is thought BMPs are primary activators of differentiation in osteoprogenitor and mesenchymal cells destined to become osteoblasts and chondrocytes.
• rhBMP-2 is one of the most studied osteoinductive molecules. Purified rhBMP-2 has been established to induce de novo bone formation and bone repair in adult animals and chondrogenic and osteogenic differentiation in various cellular systems in vitro.
• Studies have shown BMP-2 (rhBMP-2) can induce osteogenic differentiation in multipotent cells, progenitor cells and osteoblasts.

Younger EM, Chapman MW. Morbidity at Bone Graft Donor Sites. J Orthop Trauma. 1989; 3(3): 192–195.

• Younger and Chapman report a major complication rate of 8.6% and a minor complication rate of 20.6% in their series of 243 autogenous grafts, including 215 grafts from the iliac crest.
• The authors reported that, overall, patients with pre-existing medical illness were at higher risk of complication than their healthier cohorts.
• Complication rates at autologous bone graft donor sites were acceptable and comparable to clean orthopedic surgeries.
• An increased complication rate occurred when the same incision was used for surgery and donor site and inner iliac table donor site.
• This article puts into perspective the complications at donor site for autogenous bone graft. There were anecdotal mentions of iliac crest fracture, pelvic instabilities, lateral femoral cutaneous nerve or thigh injuries in iliac crest bone graft harvest surgery but none in which an overall picture of the risks involved in bone graft harvest until this paper was published.
• Younger and Chapman divided complications at the donor site into minor and major. Minor complications were defined as self-limited events that did not require an additional surgical procedure and responded to non-operative management. Major complications were those that led to prolonged hospitalisation and required additional surgery. Early complications were those that occurred in the periopertive period, usually while the patient was still in hospital. This definition has been used in subsequent studies of iliac crest complication rates.

Creeping substitution

• This refers to the process whereby necrotic bone is resorbed and replaced by new tissue moving along the channels created by invasive host blood vessels. This is analogous to fracture healing. The process is well under way by 6 months and complete by 1 year.

Table 3. Stages of graft incorporation

Urist MR. Bone: formation by autoinduction Science 1965; 150(3698): 893–899.

• Over 50 years ago Urist made the key discovery that demineralised bone fragments implanted either subcutaneously or intramuscularly in animals induced bone formation.
• The extracellular matrix of bone contains substances that can stimulate new bone formation when implanted into extraskeletal sites in a host. These substances were later identified as bone morphogenetic proteins (BMPs).
• Urist provided conclusive evidence on the induction of cartilage and bone by demineralised segments of bone. The osteoinductive activity was found to be induced by a family of proteins present in bone, which were named BMPs.
• BMPs have become the subject of intense research and development for their therapeutic use in the restoration and treatment of skeletal injuries Further research has led to the isolation of individual BMPs.
• The initial understanding was that bone matrix – demineralised bone matrix in particular – contains some property that can induce new bone formation when implanted into an extraskeletal site.
• Urist and his colleagues soon identified a protein that they named bone morphogenetic protein (BMP). This led to a programme of investigation to identify and characterise an entire family of osteoinductive molecules. By the mid-1990s, it had become clear that this family included at least 15 BMPs and was part of the larger transforming growth factor-beta (TGF-b) superfamily of molecules.

• Vascularity
• Infection
• Foreign material
• Malnutrition
• Drugs, e.g. NSAIDs, diphosphonates

Femoral heads

• Written consent from the donor to use the bone and perform blood tests (HIV, Hep B, VDRL, rhesus).
• Swabs taken of acetabulum and femoral head ® MC&S.
• Head placed in two sterile bags, sterile container and unsterile bag.
• Stored in –70°C ultra-cold freezer.
• All bone for re-implantation receives 2.5 MRad of gamma irradiation.