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- Int Wound J
- v.12(3); 2015 Jun
- PMC7950494
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Int Wound J. 2015 Jun; 12(3): 238–247.
Published online 2014 Feb 21. doi:10.1111/iwj.12231
PMCID: PMC7950494
PMID: 24618334
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Abstract
Fracture healing is a complex physiological process, which involves a well‐orchestrated series of biological events. Repair of large bone defects resulting from trauma, tumours, osteitis, delayed unions, non‐unions, osteotomies, arthrodesis and multifragmentary fractures is a current challenge of surgeons and investigators. Different therapeutic modalities have been developed to enhance the healing response and fill the bone defects. Different types of growth factors, stem cells, natural grafts (autografts, allografts or xenografts) and biologic‐ and synthetic‐based tissue‐engineered scaffolds are some of the examples. Nevertheless, these organic and synthetic materials and therapeutic agents have some significant limitations, and there are still no well‐approved treatment modalities to meet all the expected requirements. Bone tissue engineering is a newer option than the traditional grafts and may overcome many limitations of the bone graft. To select an appropriate treatment strategy in achieving a successful and secure healing, more information concerning injuries of bones, their healing process and knowledge of the factors involved are required.
The main goals of this work are to present different treatment modalities of the fractured bones and to explain how fractures normally heal and what factors interfere with fracture healing. This study provides an overview of the processes of fracture healing and discusses the current therapeutic strategies that have been claimed to be effective in accelerating fracture healing.
Keywords: Bone healing, BMPs, Grafts, Tissue engineering
Introduction
A bone fracture or an osteotomy is a complete or incomplete break in the anatomic continuity of bone, which leads to mechanical instability of the bone. A fracture is accompanied by various degrees of injury to the surrounding soft tissues, including blood supply, and in most instances results in compromised function of the locomotor system. Fractures commonly happen because of falls, car accidents or sports injuries. Fractures are often associated with penetration injuries on the battlefield. Other factors such as lower bone density and osteoporosis increase the incidence of fracture. Bone fractures are common and costly to the public because of high health care expenditures. In the USA alone, an estimated 15 million fractures occurs annually, including 1·6 million hospital admissions for traumatic fractures and 2 million osteoporotic fractures, costing over 60 billion dollars and calling for 1·6 million bone grafts each year 1.
Repair of large bone defects resulting from trauma, tumours, osteitis, delayed unions, non‐unions, osteotomies, arthrodesis and multifragmentary fractures is a current challenge of surgeons and investigators 2, 3, 4. In order to find proper ways of fracture healing, it is essential to understand how fractures normally heal and what factors interfere with fracture healing. This study provides an overview of the process of fracture healing at the cellular and molecular levels and discusses several key situations complicating fracture healing. The current therapeutic strategies that are aimed to accelerate fracture healing are also discussed.
Bone structure
Bone tissue has very interesting structural properties. This is essentially because of the composite structure of bone, composed of hydroxyapatite, collagen, small amount of proteoglycans, non‐collagenous proteins and water 5. Inorganic components are mainly responsible for the compression strength and stiffness, whereas organic components provide the corresponding tensional properties. This composition varies with species, age, sex, type and anatomical position of bone and whether or not the bone has been affected by a disease 6. Another important aspect characterising this peculiar mechanical behaviour of bone is its hierarchical organisation. Weiner and Wagner 7 described this criterion, starting from the nanometric level and ending at the macroscopic levels, relating the latter to the mechanical properties. From a macroscopic point of view, bone tissue is non‐hom*ogeneous, porous and anisotropic. Although porosity can vary continuously from 5% to 95%, most bone tissues have either very low or very high porosity. Accordingly, we usually distinguish two types of bone tissue (Figure (Figure1).1). The first type is trabecular or cancellous bone with 50–95% porosity, usually found in cuboidal bones, flat bones and at the ends of long bones. The pores are interconnected and are filled with marrow. This marrow is a tissue composed of blood vessels, nerves and various types of cells and its main function is to produce the basic blood cells. While the bone matrix has a thickness of about 200 µm and is arranged to form plates and struts, called trabeculae having variable arrangements 8. The second type is cortical or compact bone with 5–10% porosity and different types of pores 8. Vascular porosity is the largest (50 µm diameter) that is formed by the Haversian canals (aligned along the long axis of bone) and Volkmann's canals (transverse canals). Other porosities are associated with lacunae (cavities connected through small canals known as canaliculi) and with the space between collagen and hydroxyapatite (very small, around 10 nm). Cortical bone consists of cylindrical structures known as osteons or Haversian systems (Figure (Figure1),1), having a diameter of about 200 µm formed by cylindrical lamellae surrounding the Haversian canal. The boundary between the osteon and the surrounding bone is known as the cement line.
Cortical bone is usually found in the shafts of long bones and surrounding the trabecular bone forming the external shell of flat bones. This combination of trabecular and cortical bones forms a ‘sandwich‐type” structure, well known in engineering for its optimal structural properties 9. Throughout their functional life, both types of bones are formed by two different tissues: woven and lamellar bones. The bone in an embryo consists of woven bone, which is later replaced by lamellar bone. Normally there is no woven bone in the skeleton after 4 or 5 years but it reappears during the healing process after fracture. The two types of bones have many differences in composition, organisation, growth and mechanical properties. Woven bone is quickly formed and poorly organised with a more or less random arrangement of collagen fibres and mineral crystals. While the lamellar bone is slowly formed, is highly organised and have parallel layers or lamellae that make it stronger than the woven bone. Bones can grow, modify their shape (external remodelling or modelling), self‐repair when fractured (fracture healing) and continuously renew themselves by internal remodelling. All these processes are governed by mechanical, hormonal and physiological patterns. Growth and modelling mostly occur during childhood, fracture healing only occurs during fracture repair and internal remodelling occurs throughout our life span, playing a fundamental role in the evolution of the bone microstructure and, consequently, in the adaptation of structural properties and repair of the microdamages.
Bone remodelling only occurs on the internal surfaces of the bone matrix (trabecular surfaces of the cancellous bone and the Haversian systems of cortical bone). Bone can only be added or removed by bone cells on these surfaces. There are four types of bone cells, which can be classified according to their functions.
Osteoblasts are the differentiated mesenchymal cells that produce bone. They are created at the periosteum layer or stromal tissue of bone marrow.
Osteoclasts remove bone, demineralising it with acid and dissolving collagen with enzymes. These cells originate from the bone marrow.
Bone lining cells are inactive osteoblasts that are not buried in new bone. They remain on the surface when bone formation stops and can be reactivated in response to chemical and/or mechanical stimuli 10. Like bone lining cells, osteocytes are former osteoblasts that are buried in the bone matrix. They are located in lacunae and communicate with other cells via canaliculi. Many authors 11, 12, 13 suggested that osteocytes are the mechanosensor cells that control bone remodelling, but this has not been proven yet. Furthermore, it is quite reasonable to assume that osteocytes, the only cells embedded in the bone matrix, are affected by processes that damage the bone matrix. Matrix disruption may be expected to directly injure osteocytes, disrupting their attachments to bone matrix, interrupting their communication through canalicular or altering their metabolic exchange. Fatigue microdamage may therefore create a situation resembling disuse at the level of the osteocyte cell body and lead to bone remodelling, starting with osteoclast recruitment.
The remodelling process is not performed individually by each cell, but by groups of cells functioning as organised units, which Frost 14 named ‘basic multicellular units’ (BMUs). They operate on bone periosteum, endosteum, trabecular surfaces and cortical bone, replacing old bone by new bone in discrete packets. The BMUs always follow a well‐defined sequence of processes, normally known as the activation–resorption–formation (A–R–F) sequence (Figure (Figure22).
Bone is also anisotropic. Cortical bone has a very low porosity and its anisotropy is mainly controlled by lamellar and osteonal orientation. On the other hand, trabecular bone has a higher porosity and its anisotropy is determined by trabecular orientation 15. In fact, structural anisotropy has a direct influence on stiffness properties as well as strength.
Types of bone fracture
Fractures may be classified on many bases, and all are useful in describing the fracture. These include causal factors, presence of a communicating external wound, location, morphology, severity of the fracture and stability of the fracture after axial reduction of the fragments 16. All fractures can be broadly described as closed (no skin break) or open (skin break). Open fractures are always associated with more damage to the surrounding soft tissues, including the periosteum, have a higher risk of infection and often have a higher incidence of non‐union than the closed fractures. Fractures of long bones, such as the femur, humerus, tibia and other long bones can be classified according to the characteristics of the forces resulting in such fractures 17. Simple and comminuted fractures in which the bones are broken into two or several pieces, respectively, are caused by a single injury. Stress fracture is an overuse injury, which has been resulted from repetitive loading. Simple fracture occurs when a bending force or twisting force is applied to a bone, resulting in two fragments with transverse, oblique or long curved (spiral) edges of the broken bones. This type of fracture heals through the spontaneous repair processes we will discuss later 16 (Figure (Figure33).
Comminuted fracture is characterised by breaking a bone into several small pieces and is the result of high‐velocity injuries, such as car accidents or falls from a height. Repair of comminuted fractures follows a healing pattern similar to that of simple fractures, but on a larger scale. Such fractures generally are very difficult to treat and may result in a deformity of the injured part even after treatment. Stress fractures result when low‐magnitude cyclically repeated force is applied over a long period of time, causing progressive accumulation of microdamages. Unlike simple and comminuted fractures, stress fractures and their associated fatigue damage heal via normal bone remodelling. This process involves the sequential and coordinated activity of osteoclasts and osteoblasts that remove and replace the damaged bone, respectively. If the repetitive loading is prolonged and/or microdamage cannot be repaired, the bone may eventually fail through propagation of the microdamage 17, 18.
Primary and secondary repair mechanisms
Repair of fractured long bones is a unique process that results in restoration of the anatomy and function of a normal bone after injury. This repair can be divided into primary and secondary healing based on differences in the local motion between the fracture fragments. Primary healing involves a direct attempt by the cortex to reestablish continuity between the fracture fragments. This process appears to occur only when the alignment stability and decrease in interfragmentary motion of the fracture fragments are established by rigid internal fixation. Osteoblasts derived from mesenchymal stem cells (MSCs) lay down osteoid on the exposed bone surfaces. New Haversian systems will be reestablished across the original fracture line through intracortical remodelling. Secondary (spontaneous) healing involves a response of the periosteum and the surrounding soft tissues at the fracture site (Figure (Figure4).4). The response from the periosteum is a fundamental reaction to bone injury; it is enhanced by limited fragment motion and is inhibited by rigid fixation. Mesenchymal cells and osteoprogenitor cells contribute to the process of repair by recapitulation of embryonic intramembranous ossification and endochondral bone formation.
Secondary (spontaneously) healing has occurred in an oblique fracture in tibia without any fixation. Excessive callus formation is notable.
The new bone formed by intramembranous ossification is found peripheral to the site of the fracture. Osteoblast progenitor cells in the inner layer of the periosteum differentiate into osteoblasts in response to molecular signals produced during fracture and directly synthesise new bone matrix on the bone surface without first forming cartilage. This process does not contribute to directly bridging the fracture. The callus that forms by endochondral ossification is formed within the fracture site and involves the development of cartilage in response to hypoxia caused by the lack of blood supply (Figure (Figure5).5). The chondrocytes are derived from MSCs in the periosteum and endosteum. They proceed through a state of hypertrophy and the cartilage matrix becomes calcified. The hypertrophic chondrocytes undergo apoptosis and the calcified matrix is removed by invasion of osteoclasts and blood vessels, followed by osteoblast‐induced bone formation. Fracture repair is clearly related to external factors, including the mechanical environment at the fracture site. Motion at the fracture site results in healing primarily through cartilage formation (endochondral ossification) and stability favours the direct formation of bone (intramembranous ossification). Most long bone fractures heal by a combination of intramembranous and endochondral ossification. Both endochondral and intramembranous ossification produce woven bone with poorly organised hydroxyapatite matrix. This is extremely important during fracture healing, as rapid new bone formation is required in order to quickly consolidate fracture fragments to restore the mechanical stability of bone. The mineral appositional rate of woven bone formation can be 2–4 times greater than the lamellar bone formation. The woven bone will be later remodelled by osteoclasts to achieve lamellar bone.
Stages of fracture repair
Secondary fracture healing of long bones can be considered as a series of four discrete stages occurring in sequence and overlapping to a certain extent (Figure (Figure66).
Stage 1: Immediately following trauma, the first stage of fracture healing is blood clotting (haematoma formation) and inflammation, which begin within the fracture site. Stage 2: Repair phase, the granulation tissue is replaced by soft (cartilaginous) and then hard callus of woven bone at the fracture site. Stage 3: The osteoblasts form woven bone on the calcified matrix. Stage 4: The remodelling process proceeds with osteoclasts and osteoblasts facilitating the conversion of woven bone into lamellar bone and eventually recreating the appropriate anatomical shape.
Inflammatory response
The inflammatory phase begins immediately after the initial disruption of bone and the surrounding soft tissues and persists until the formation of cartilage or bone is initiated. This phase therefore lasts 3–4 days and potentially longer, depending on the extent of the force causing the fracture. Clinically, the end of the inflammatory stage coincides with a decrease in pain and swelling. Fractures inevitably cause a disruption of the medullary vessels and subsequent extravasation of blood. Although contraction and thrombosis of the disrupted vessels minimise blood loss, the traumatic interruption of the blood flow leads to ischaemic necrosis of bone, characterised histologically by the presence of empty lacunae. A fibrin‐rich clot forms at the fracture site, initiating spontaneous fracture healing. The lack of mechanical support provided by this blood coagulum is well‐recognised. However, its biological contribution to fracture healing remains somewhat controversial. In 1969, Ham observed that much of the fracture repair process took place around, rather than within, the interfragmentary haematoma and questioned its significance 19. In fact, large clots persisting unchanged at the fracture site for an extended period of time have been described as potentially hindering bone repair 20. Others suggest that the haematoma acts as a scaffold for cells and a spacer guiding the size and shape of the callus 21. However, there is growing evidence to support the concept that the haematoma sets the stage for the repair phase by releasing growth factors, thereby stimulating angiogenesis and bone formation. Transplantation of fracture haematoma has been found to induce endochondral bone formation in ectopic sites, which would be consistent with the presence of osteoinductive growth factors within the haematoma 22. Platelets are likely to be the first source of mitogenic factors at a traumatised site 23. In addition to coagulation factors, they release platelet‐derived growth factor (PDGF) and transforming growth factor‐β1 (TGF‐β1), both of which stimulate bone production 24.
The angiogenic properties of fracture haematoma appear to be mediated via vascular endothelial growth factor (VEGF) 22. Local acidity and cytokines, contained in the exudate accumulating in the injured area, complement this effect. Indeed, inflammatory mediators such as prostaglandins E1 and E2 may stimulate angiogenesis, and may also be responsible for signalling early bone resorption by osteoclasts and proliferation of osteoprogenitor cells 25. Finally, mast cells containing vasoactive substances are abundant during this stage and contribute to the formation of new vessels 26. Within hours, a transient extraosseous blood supply emerges from the surrounding soft tissues, revascularising the hypoxic fracture site 27. Mononuclear phagocytes delivered by these new vessels assist in the removal of necrotic bone and aid in construction of the callus. Resorption of fragment ends is particularly obvious in spontaneous fracture healing when the fracture gap widens, thereby lowering interfragmentary strain and minimising the deformation of local tissues 21. Macrophages are also believed to orchestrate the orderly sequence of cutaneous wound healing and would play a similar role in fracture repair. They contain several growth factors, such as fibroblast growth factor (FGF) and initiate fibroplasia both in soft tissue as well as in bone repair 24. The proliferative vascular response and the degree of bone resorption may be affected by soft‐tissue compromise, either traumatic or iatrogenic. On the other hand, angiogenesis has been enhanced by a muscle flap and has improved the healing of experimental tibial osteotomies in dogs 28. This illustrates the importance of optimising the role of the soft tissues surrounding the fracture. As medullary blood flow resumes, this extraosseous blood supply subsides. The haematoma is resorbed by the end of the first week unless infection, excessive motion or extensive necrosis of the surrounding soft tissues persists at the fracture site 29.
Soft callus (cartilage) formation
Within a few days, capillary ingrowth, together with mononuclear cells and fibroblasts, begins the transformation of haematoma into granulation tissue. This initial stage of the repair phase coincides with a slight gain in mechanical strength, as granulation tissue can withstand a tensile force up to 0·1 nm/mm221. The ability of granulation tissue to elongate to twice its original length explains its formation at this stage as interfragmentary deformation remains high. As granulation tissue matures into connective tissue collagen fibres become more abundant. They have an ultimate tensile strength of 1–60 nm/mm2 and resist elongation up to a maximum of 17%. Types I, II and III collagen are initially deposited, but as the maturation process continues, type I collagen predominates 30. This interfragmentary fibrous tissue is organised in a diagonal pattern, optimising its ability to elongate 21.
Low oxygen tension, poor vascularity, growth factors and interfragmentary strain influence the elaboration of a cartilaginous callus 30, 31. Mesenchymal cells within the cambium layer of the periosteum, the endosteum, bone marrow and adjacent soft tissues start proliferating during the inflammatory phase and differentiate into chondrocytes during the repair phase. Chemotaxis, proliferation, coordination and differentiation of these stem cells into chondrocytes or osteoblasts are orchestrated by numerous growth factors, among which TGF‐β and bone morphogenic proteins (BMPs) play a major role. Although the exact timing of this induction phase remains unclear, it may be initiated during the inflammatory phase and is crucial to the orderly formation and maturation of tissues within the fracture gap. The periosteum surrounding the fracture site thickens prior to undergoing chondrogenic transformation, thereby producing an external callus entirely vascularised by extraosseous vessels 30. An internal or medullary callus develops from the endosteal cell layer and is confined to the medullary canal and receives its blood supply derived from the medullary arterioles. Presence of a fibrocartilage layer within the medullary canal temporarily interrupts the medullary blood flow across the fracture gap. Both the external and internal callus constitute the ‘bridging callus’ 32. This early ‘soft callus’ formed during the first three weeks after injury resists compression, but its ultimate tensile strength (4–19 nm/mm2) and elongation at rupture (10–12·8%) are similar to those of fibrous connective tissue 33.
Hard callus formation (endochondral ossification)
Production of a prominent external callus is a common finding in well‐vascularised unstable fractures. The resulting enlargement in the cross‐sectional diameter of the injured area greatly increases its resistance to bending, as its strength efficiency increases by the third power of the distance to the neutral axis of the bone and its rigidity increases by the fourth power 21. Increasing proteoglycan concentrations within the fibrocartilage also contribute to the stiffening of the interfragmentary gap 31. Although the mechanical properties of this calcified fibrocartilaginous tissue have not been reported, these structures contribute greatly to the restoration of strength and stiffness within the fracture gap, thus allowing formation of compact bone. Mineralisation of the soft callus proceeds from the fragment ends toward the centre of the fracture site and forms a ‘hard callus’ 21. Rather than being a uniform process, chondrocytes initiate and control the formation of mineralised clusters 31. Although the exact mechanism of this calcification remains unclear, it is thought that mitochondria in the fracture gap behave as they do in growth plates 34. They appear to accumulate calcium‐containing granules that are released in the hypoxic environment created by anaerobic metabolism. Intramitochondrial deposits of calcium phosphate are released in the extracellular matrix and become the seeds for growth of apatite microcrystallites. Other steps of bony substitution at the fracture site closely resemble endochondral ossification. Vascular invasion of fibrocartilage is coupled with the degradation of non‐mineralised matrix compartments by macrophages. Following this resorbing front, blood vessels and osteoprogenitor cells form new trabeculae. Provided there is sufficient vascularity and mechanical support from the mineralised callus, the fibrous tissue within the fracture gap can undergo intramembranous bone formation 21.
The ultimate tensile strength of compact bone is around 130 nm/mm2, but its modulus of elasticity (resistance to deformation) is high (10 000 nm/mm2) and its ability to elongate is limited to 2% 21. At the end of the repair phase, bone union is achieved, but the structure of the fracture site differs from that of the original bone. The time required to achieve union varies greatly according to fracture configuration and location, status of the adjacent soft tissues, as well as patient characteristics (species, age, food ingredient, health status and concurrent injuries/diseases). At the end of the repair phase, the injured bone has regained enough strength and rigidity to allow low‐impact exercise 35.
Bone remodelling
This final phase of fracture repair is characterised by a morphological adaptation of bone to regain optimal function and strength. This slow process may last for 6–9 years in humans, representing 70% of the total healing time of a fracture 30. The balanced action of osteoclastic resorption and osteoblastic deposition is governed by Wolff's law and modulated by piezoelectricity, a phenomenon in which electrical polarity is created by pressure exerted in a crystalline environment 30. In spontaneous healing of a fracture, progression from soft to hard callus depends upon an adequate blood supply and a gradual increase in stability at the fracture site. Compromised vascularisation and excessive instability will merely permit the formation of fibrous tissue and the development of an atrophic non‐union. If the fracture gap is well‐vascularised, but there is uncontrolled interfragmentary motion, the fracture will progress to a cartilaginous callus, but this may be unable to stabilise the fragments. Thus, a hypertrophic non‐union or pseudoarthrosis may develop. On the other hand, a stable fracture with an adequate blood supply will allow the formation of mineralised callus. Nevertheless, initial displacement of bone fragments because of trauma and muscle contraction frequently results in malunion 16.
Assessment of fracture healing
Methods of evaluation
Fracture healing involves a dynamic interplay of biological processes to restore the original anatomic structure and mechanical function of bone. Therefore, both structural and biomechanical evaluations are used to assess fracture repair. The extent and quality of structural repair can be evaluated, using radiographic and histological methods. The mechanical properties of a healing bone are assessed by mechanical tests.
Clinical assessment of healing requires non‐invasive methods, including clinical symptoms (pain or tenderness when bearing weight) and radiographic indicators. Plain radiography is a ubiquitous method used to evaluate fracture healing in both laboratory and clinical settings, because of its non‐invasive nature. The most common radiographic definitions of fracture healing in clinical setting involve the bridging of fracture site by callus, obliteration of the fracture line and continuity. At radiological level, the defect area is observed at different consequential times, and the quality and morphology of the healing tissue regenerated in the defect area is scored. These scores include presence of bone indicating a complete union (+3 score), presence of cartilage (+2 score), existence of soft tissue within the defect indicating a possible unstable union (+1 score) or complete instability at the defect area indicating non‐union (0 score) 36. To investigate different stages of healing and to measure the rate of bone formation, union and remodelling, radiographs of the fracture site are taken. Radiographic examination is also considered as a diagnostic tool for evaluating the type of fracture and for the surgical scheming 37.
To test the mechanical function of a healing bone, torsion and four‐point bending tests are logical choices when studying fracture healing in long bones. The choice of the type of test is mainly determined by technical considerations. In general, torsion is a better choice than four‐point bending because torsion tests subject every cross section of the callus to the same torque, whereas four‐point bending might create a non‐uniform bending moment throughout the callus. As a result, failure of the callus during a four‐point bending test does not necessarily occur at the weakest cross section of the callus. It should be highlighted that the three‐point bending should not be recommended to estimate the mechanical properties of a healing bone, especially during early stages of the healing process, because the site to which the force is applied is located at the original fracture line, which is composed primarily of cartilage, calcified cartilage or less mature bone tissue depending on the healing stages. The outcome measures that can be obtained from mechanical tests, such as ultimate strength, stiffness, energy to failure and torque in the torsion test are structural rather than material properties 38, 39.
Light microscopy is the most commonly used morphological technique and allows histomorphometric measurement of different parameters in the healing bones (e.g. callus size, cartilaginous and mineralised volume of callus, transverse cross‐sectional area of bone cortex, transverse cross‐sectional area of bone marrow cavity, the ratio of the latter/former, cell constituents and counts in the healing area and many other parameters such as differential cell count, collagen typing and glycosaminoglycan composition) 40. The histopathological examination of a healing bone by light microscopy is routinely carried out using haematoxylin and eosin staining, and is usually scored for example according to the Emery's scoring system as follows: when the gap is empty (score = 0), filled with fibrous connective tissue only (score = 1), more fibrous tissue than cartilage (score = 2), more cartilage than fibrous tissue (score = 3), cartilage only (score = 4), more cartilage than bone (score = 5), more bone than cartilage (score = 6) and ultimately if it is filled only with bone (score = 7) 41, 42, 43.
Immunohistochemistry, molecular and biochemical analyses are other methods whose popularity is recently increasing in the field of experimental medicine 22, 23, 44, 45. Other techniques that could be used include magnetic resonance imaging, microcomputed tomography scan (to measure bone volume of the callus and bone densitometry) and microcomputed tomography angioscan are used to assess quality of bone healing both in experimental in vivo and in clinical practice 20, 46.
Bone fracture healing accelerators
Recently, various animal studies and clinical trials in humans have demonstrated the potential use of several biological factors in bone regeneration and skeletal repair, such as natural and/or synthetic materials and implants, grafts and scaffolds with or without cell seeding. Some of these studies are summarised in Table Table11.
Table 1
Recent studies on bone‐defect healing with several natural or synthetic materials
| Method of treatment | Species of animal | Methods of evaluation | Results of study |
|---|---|---|---|
| Autogenous bone marrow with static magnetic 47 | Rabbit radial bone model | Radiological, histological | Positive effects on bone healing |
| Fresh cortical autograft versus fresh cortical allograft 48 | Rabbit radial bone model | Radiological, histological, biomechanical | Autograft was superior to allograft |
| Bovine foetal growth plate 49 | Rabbit radial bone model | Radiological, histological, biomechanical | Growth plate lead to enhanced bone healing |
| Demineralised calf foetal growth plate 50 | Rabbit Radial bone model | Radiological, histological | Demineralised growth plate lead to enhanced bone healing |
| Combined hydroxyapatite and human platelet‐rich plasma (hPRP) 38 | Rabbit radial bone model | Radiological, histological, biomechanical | Positive effects recorded with hydroxyapatite and hPRP |
| Human platelet‐rich plasma plus Persian Gulf coral 39 | Rabbit radial bone model | Radiological, histological, biomechanical | Positive effects recorded with coral and hPRP |
| Bone morphogenetic protein‐2 (BMP‐2) gene therapy 51 | Sheep, iliac crest defects | CT scan, histological analysis | Slow down of bone formation because of antibodies produced against the adenovirus (viral vector) |
| Adipose tissue stem cell (ADSc) concurrent with greater omentum 52 | Dog, radius defect | Radiological, histological, | ADSc did not lead to enhanced bone healing and act as control group |
| Autogenous free graft of greater omentum 19 | Dog, radius defect | Radiological, histological | Enhances bone healing |
| The effect of heparan sulphate application on bone formation 21 | Mouse model, tibia defect | MicroCT, radiology, biomechanical testing, immunohistochemistry and histology | Heparan sulphate injection had negative effects on bone formation |
| Bone morphogenic protein‐2‐coated tricalcium phosphate/hydroxyapatite 22 | Rat model, femoral bone defect | MicroCT, histology and real‐time polymerase chain reaction (PCR) | Lead to enhanced osteogenesis |
| Platelet‐rich plasma (PRP) on bone healing in combination with autogenous bone 23 | Forehead region of a miniature pig | Microradiographically and immunohistologically | PRP with autogenous bone enhanced bone formation |
| Effect of aspirin on bone healing 24 | Rabbit, ulnar osteotomy | Radiological, histological, biomechanical | Aspirin delayed bone healing |
| The effects of ibuprofen and rofecoxib on rabbit fibula osteotomy healing 25 | Rabbit, fibula osteotomy | Histomorphometry, biomechanical | Despite the ibuprofen‐induced delay, rofecoxib treatment produced worse fracture (osteotomy) healing than ibuprofen treatment. |
| Cortical autograft, commercial DBM, calf foetal demineralized bone matrix (DBM), omentum and omentum‐calf foetal DBM 31 | Dog, tibia defects | Radiological, histological | The omentum and omentum‐DBM groups were superior to the control group, but inferior to the autograft, commercial DBM, calf foetal DBM and calf foetal cartilage groups. |
The number of patients suffering from bone tumour resections, fracture defects or chronic infection is increasing rapidly owing to disease and trauma, and more than 1·5 million bone graft operations are performed in the USA every year 26. For the treatment of non‐union and bone defects, autograft is the gold standard for bone repair. However there are some disadvantages associated with the autografts, such as the limited abundance in supply, new nerve damage, persistent pain and new fractures. Allograft has been used successfully in the orthopaedic operations owing to its excellent osteoconductivity and abundance in supply. However, allografts have the potential risk of infecting, disease transmission and immune response. On the other hand, allografts are inferior in promoting bone regeneration compared with the autografts, because they require processing, sterilisation steps and preservation before they are used 27, 28, 48. Thus, tissue engineering might provide a promising approach for bone regeneration 29. An ideal bone grafting material should not only provide mechanical strength, a void space for vascularisation and tissue infiltration but also be both osteoinductive and osteoconductive, and serve as a carrier for relevant therapeutical factors 30, 35. At present, we have various biomaterials for bone regeneration such as bone‐derived collagen, decalcified bone matrix, fibrin, nano‐hydroxyapatite/collagen (nHAC), synthetic poly (glycolic‐co‐lactic acid) polymer, true bone ceramics or sintered bovine bone and titanium 31, 32, 33, 34, 53. Each has its own advantages, at the same time a variety disadvantages still remain. For example, ceramic and polymer‐based bone graft substitutes are mostly osteoconductive but are deficient in osteoinductivity. Other problems may include unsuitable degradation rates and inferior mechanical properties. In addition, protein‐ or growth factor‐based bone graft substitutes usually requires addition of an osteoconductive scaffold for structural support 54, 55. Previous studies have showed that natural bone is a three‐dimensional composite, which has an intricate hierarchical structure of mineralised collagen fibre by nano‐hydroxyapatite crystals 7. Inspired from this, many researchers have fabricated nano‐hydroxyapatite/collagen by biomimetic strategy and it shows great promise in clinical applications because its composition and structure are similar to natural bone 56, 57.
Discussion
Different types of glycosaminoglycans, growth factors, stem cells, natural grafts (autografts, allografts or xenografts) and biologic‐ and synthetic‐based tissue‐engineered scaffolds are some of the examples. Nevertheless, these organic and synthetic materials and therapeutic agents have some significant limitations, and there are still no well‐approved treatment modalities to meet all the expected requirements. Bone tissue engineering is a newer option than traditional grafts, which may overcome many limitations of the bone graft usage. To select an appropriate treatment strategy in achieving a successful and secure healing, more information concerning injuries of bones, their healing process and knowledge of the factors involved are required. Hence, this study reviews how the bone fractures heal. The aim of this review is to provide useful information to orthopaedic surgeons and investigators working in the field of bone healing 46.
The limitations in autograft and allograft restricted their clinical application. Alternatively, tissue engineering approach may offer a new solution to produce bone substitutes for clinical use. Over the last 20 years, tissue engineering of the bone has made remarkable progress, although the problems of translating into clinical application still remain in its infancy. Various types of stem cells have been used to form mineralised bone in vitro. In contrast, much fewer studies have focused on the healing efficacy and its potential side effects. One main barrier is the complicated in vivo environment, which has profound interactions with the implanted cell types. This is especially so for allogeneic cells, where the host immune reaction is likely to play a very important role, with the macrophage system currently being under intense investigations 58, 59.
BMPs are members of the transforming growth factors β superfamily. The major biological effect of BMP lies in its ability to stimulate the aggregation, proliferation and differentiation of mesenchymal cells, which accelerates bone and cartilage formation. Among all the BMP family, BMP‐2 has the strongest biological activity 55. However, an important issue to consider in the use of BMPs is their side effects 60. BMPs are reported to be safe if they are used appropriately and the side effects include heterotopic ossification, local erythema and swelling, and immune response. Critical issues include the potential risk that BMPs will induce heterotopic bone formation, especially when implanted near to neural tissues. An unforeseen issue is their role in osteoclast activation and formation. Osteoclasts are formed before osteoblasts, which may lead to a weave of resorption that precedes the appearance and effect of osteoblasts 61. rhBMP‐2 or other growth factors alone does not achieve the expected efficacy of bone or cartilage formation because of the short retention of protein in vivo, thus an ideal scaffold material as a delivery carrier is necessary 62. As a delivery carrier for growth factors, it should have two primary functions: first, it should maintain growth factors' bioactivity and optimal release amount at the implantation site to maximise the osteogenic effect of growth factor; second, it should be an osteoconducitve scaffold with suitable pore structure for vascularisation and new bone formation 63. Nowadays, a large number of natural and synthetic biomaterials have been fabricated for the economical delivery of rhBMP‐2 55, 64. However, critical views on the use of BMPs have been emerging lately because of their short half‐lives, expensiveness and ineffectiveness 65, 66, 67.
The use of biomaterials and development of scaffolds are especially important for engineering bone grafts, because they need to provide mechanical support during bone repair and bioactive aspect for bone formation. In order to obtain optimal mechanical properties, and high biocompatibility, numerous composite materials have been designed to acquire integrated properties from the individual components. Recently, more stringent requirements brought forth in the scaffold design for complete bone healing efficacy, such as inducing neovascularisation during the formation of bone. The achievements in engineering bone tissue so far are encouraging, while new challenges and opportunities are bringing the perspective of bone tissue engineering to a new height in clinical application 68.
Conclusion
Fracture healing is a complex physiological process, which involves a well‐orchestrated series of biological events. This study provides more recent basic information about bone fracture and healing cascades. These information are necessary for researchers who are going to design a new study about enhancing the bone healing and regeneration. Knowledge of bone biology has vastly expanded with the increased understanding at the molecular level, resulting in development of many new treatment methods, with many others (or improvements to current ones) anticipated in the years to come. Research is ongoing within all relevant fields, and it is hoped that many bone disease processes secondary to trauma, bone resection because of ablative surgery, ageing and metabolic or genetic skeletal disorders will be successfully treated with novel bone regeneration protocols that may address both local and systemic enhancement to optimise outcome.
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