Current concepts of molecular aspects of bone healing

Injury, Int. J. Care Injured (2005) 36, 1392—1404

www.elsevier.com/locate/injury

REVIEW

Current concepts of molecular aspects of bone healing Rozalia Dimitriou 1, Eleftherios Tsiridis, Peter V. Giannoudis * Academic Department of Trauma and Orthopaedic Surgery, School of Medicine, University of Leeds, St James’s University Hospital, Backett Street, LS9 7TF, UK Accepted 21 July 2005

KEYWORDS Molecular; Bone; Ageing; Fracture healing; Trauma

Summary Fracture healing is a complex physiological process. It involves the coordinated participation of haematopoietic and immune cells within the bone marrow in conjunction with vascular and skeletal cell precursors, including mesenchymal stem cells (MSCs) that are recruited from the surrounding tissues and the circulation. Multiple factors regulate this cascade of molecular events by affecting different sites in the osteoblast and chondroblast lineage through various processes such as migration, proliferation, chemotaxis, differentiation, inhibition, and extracellular protein synthesis. An understanding of the fracture healing cellular and molecular pathways is not only critical for the future advancement of fracture treatment, but it may also be informative to our further understanding of the mechanisms of skeletal growth and repair as well as the mechanisms of aging. # 2005 Elsevier Ltd. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biology of fracture healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct or primary cortical fracture healing . . . . . . . . . . . . . . . . . Indirect or secondary fracture healing . . . . . . . . . . . . . . . . . . . . Molecular aspects of fracture healing. . . . . . . . . . . . . . . . . . . . . . . Signalling molecules and the role of mesenchymal stem cells (MSCs) . Pro-inflammatory cytokines . . . . . . . . . . . . . . . . . . . . . . . . . Growth and differentiation factors . . . . . . . . . . . . . . . . . . . . Metalloproteinases and angiogenic factors. . . . . . . . . . . . . . . . . . . . The role of mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . Sequence of events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Present address: Academic Department of Trauma and Orthopaedics, St. James’s University Hospital, Beckett Street, Leeds LS9 7TF, UK. Tel.: +44 113 20 66460; fax: +44 113 20 65156. E-mail address: [email protected] (P.V. Giannoudis). 1 AO Research Fellow. 0020–1383/$ — see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2005.07.019

Current concepts of molecular aspects of bone healing

Intramembranous bone formation . . . . Endochondral bone formation . . . . . . . Current concepts of systemic enhancement of Parathyroid hormone (PTH) . . . . . . . . . . Growth hormone (GH) . . . . . . . . . . . . . Future directions. . . . . . . . . . . . . . . . . . . Tissue engineering . . . . . . . . . . . . . . . . Muscle stem cells . . . . . . . . . . . . . . . . Clinical applications in trauma . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . .

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........... ........... fracture healing ........... ........... ........... ........... ........... ........... ........... ........... ...........

Introduction Fracture healing remains to a great extent an unknown cascade of complex biological events. It involves intracellular and extracellular molecular signalling for bone induction and conduction. It is a multistage repair process that follows a definable temporal and spatial sequence.20,23,63,68 Molecular mechanisms known to regulate skeletal tissue formation during embryological development are recapitulated during fracture healing.25 Many local and systemic regulatory factors, including growth and differentiation factors, hormones, cytokines, and extracellular matrix, interact with several cell types, including bone and cartilage forming primary cells or even muscle mesenchymal cells, recruited at the fracture-injury site or from the circulation. Cellular and molecular biology provides today the tolls for the investigation and understanding of the fracture healing process. Ongoing research in this field of medicine has improved our understanding of fracture healing at the molecular level. The aim of this review article is to characterise the chain of events contributing to the healing process in order to enhance the clinician’s awareness of the complexity of signalling pathways and molecules involved.

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ongoing to elucidate the unique role of different molecules and their interactions in the healing cascade.

Biology of fracture healing Fracture healing is a complex, however, well orchestrated, regenerative process initiated in response to injury, resulting in optimal skeletal repair and restoration of skeletal function. During the repair process, the pathway of normal embryonic development is recapitulated with the coordinated participation of several cell types.25 All four components involved in the injury site, including the cortex, the periosteum, the bone marrow, and the external soft tissues, contribute in the healing process at different extent, depending on multiple parameters present at the injured tissue such as growth factors, hormones and nutrients, pH, oxygen tension, the electrical environment and the mechanical stability that has been obtained.17,65 In classical histological terms, fracture healing has been divided into direct (primary) and indirect (secondary) fracture healing. Integrated cellular events, and their temporal and spatial characteristics, have been elucidated by using a model of experimental fracture healing in the rat.23

Direct or primary cortical fracture healing

Historical perspectives In 1965, Urist revolutionised the current understanding of fracture healing by hypothesising the existence of bone morphogenetic proteins (BMPs) allocated onto the extracellular collagenous matrix.76,77 The genetic sequences of BMPs were first identified by Wozney et al. in 1988,82 and since then over 40 BMPs have been discovered. Subsequently, with the advances made in molecular medicine and molecular biology extensive research is

Direct fracture healing occurs only when there is anatomic reduction of the fracture fragments by rigid internal fixation and decreased intrafragmentary strain.49 This process involves a direct attempt by the cortex to re-establish new haversian systems by the formation of discrete remodelling units known as ‘cutting cones’, in order to restore mechanical continuity.49 Vascular endothelial cells and perivascular mesenchymal cells provide the osteoprogenitor cells to become osteoblasts. During

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this process, little or no periosteal response is noted (no callus formation).23

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(2) the transforming growth factor-beta (TGF-b) superfamily and other growth factors, and (3) the angiogenic factors (Table 1).33,44

Indirect or secondary fracture healing The majority of fractures heal by indirect fracture healing. It involves a combination of intramembranous and endochondral ossification with the subsequent formation of a callus.23 It is generally enhanced by motion and inhibited by rigid fixation.49 Intramembranous ossification involves the formation of bone directly, without first forming cartilage, from committed osteoprogenitor and undifferentiated mesenchymal cells that resides in the periosteum, farther from the fracture site.23 It results in callus formation, described histologically as ‘hard callus’.23 In this type of healing, the bone marrow’s contribution to the formation of bone is during the early phase of healing, when endothelial cells transform into polymorphic cells, which subsequently express an osteoblastic phenotype.12 Endochondral ossification involves the recruitment, proliferation, and differentiation of undifferentiated mesenchymal cells into cartilage, which becomes calcified and eventually replaced by bone. Its temporal characteristics include six identifiable stages including an initial stage of haematoma formation and inflammation, subsequent angiogenesis and formation of cartilage, cartilage calcification, cartilage removal, bone formation, and ultimately bone remodelling.23 This type of fracture healing, is contributed from the adjacent to the fracture periosteum and the external soft tissues, providing an early bridging callus, histologically characterized as ‘soft callus’, that stabilizes the fracture fragments.23 The classification of fracture healing in direct and indirect healing reflects the histological events that occur during the repair process. However, the ongoing research in bone regeneration provided a further understanding of the cellular and molecular pathways that govern these events, by demonstrating the existence of various signalling molecules and elucidating their contribution in the initiation and control of this physiological process at the molecular level.

Molecular aspects of fracture healing Signalling molecules and the role of mesenchymal stem cells (MSCs) The signalling molecules can be categorized into three groups: (1) the pro-inflammatory cytokines,

Pro-inflammatory cytokines Interleukin-1 (IL-1) and Interleukin-6 (IL-6) as well as tumour necrosis factor-alpha (TNF-a) are shown to play a role in initiating the repair cascade.24,32 They are secreted not only by macrophages and inflammatory cells but also by cells of mesenchymal origin present in the periosteum (Table 1).43 They carry out central functions in the induction of downstream responses to injury by having a chemotactic effect on other inflammatory cells, enhancing extracellular matrix synthesis, stimulating angiogenesis, and recruiting endogenous fibrogenic cells to the injury site.43 They show peak expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and their levels increase again during bone remodelling (Fig. 1).32,43 Cytokines also regulate endochondral bone formation and remodelling.7 TNF-a promotes the recruitment of mesenchymal stem cells, induces apoptosis of hypertrophic chondrocytes during endochondral ossification and stimulates osteoclastic function. Absence of TNF-a result in delayed resorption of mineralized cartilage, prohibiting new bone formation.32 IL-1, IL-6 and TNF-a also show increased levels of expression during fracture callus re-shaping later in the process of fracture healing and remodelling. Growth and differentiation factors The transforming growth factor-beta superfamily. The TGF-b superfamily is a large family of growth and differentiation factors including bone morphogenetic proteins (BMPs), transforming growth factor-beta (TGF-b), growth differentiation factors (GDFs), activins, inhibins and the Mullerian inhibiting substance. At least 34 members have been identified in the human genome.75 They originate from high molecular weight precursors and are activated by proteolytic enzymes.73 They act on serine/threonine kinase membrane receptor on target cells.48 This ligand—receptor interaction activates an intracellular signalling pathway which ultimately affects gene expression in the nucleus. Specific members of this superfamily including bone morphogenetic proteins (BMPs 1—8), growth and differentiation factors (GDF-1, 5, 8, 10) and transforming factor beta (TGF-b1, -b2, -b3), promote the various stages of intramembranous and endochondral bone ossification during fracture healing.20

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Table 1 The essential signalling molecules during fracture healing; their source and targeted cells, and their major functions and expression patterns Cytokines (IL-1, IL-6, TNF-a) Source: macrophages and other inflammatory cells, cells of mesenchymal origin Chemotactic effect on other inflammatory cells, stimulation of extracellular matrix synthesis, angiogenesis, recruitment of endogenous fibrogenic cells to the injury site, and at later stages bone resorption Increased levels from days 1 to 3 and during bone remodelling TGF-b Source: degranulating platelets, inflammatory cells, endothelium, extracellular matrix, chondrocytes, osteoblasts Targeted cells: MSCs, osteoprogenitors cells, osteoblasts, chondrocytes Potent mitogenic and chemotactic for bone forming cells, chemotactic for macrophages Expressed from very early stages throughout fracture healing PDGF Source: degranulating platelets, macrophages, monocytes (during the granulation stage) and endothelial cells, osteoblasts (at later stages) Targeted cells: mesenchymal and inflammatory cells, osteoblasts Mitogenic for mesenchymal cells and osteoblasts, chemotactic for inflammatory and mesenchymal cells Released at very early stages of fracture healing BMPs Source: osteoprogenitors and mesenchymal cells, osteoblasts, bone extracellular matrix and chondrocytes Targeted cells: mesenchymal and osteoprogenitor cells, osteoblasts Differentiation of undifferentiated mesenchymal cells into chondrocytes and osteoblasts and osteoprogenitors into osteoblasts Various temporal expression patterns (Table 2) FGFs Source: monocytes, macrophages, mesenchymal cells, osteoblasts, chondrocytes Targeted cells: mesenchymal and epithelial cells, osteoblasts and chondrocytes Angiogenic and mitogenic for mesenchymal and epithelial cells, osteoblasts, chondrocytes a-FGF mainly effects chondrocyte proliferation, b-FGF (more potent) involved in chondrocytes maturation and bone resorption Expressed from the early stages until osteoblasts formation IGFs Source: bone matrix, endothelial and mesenchymal cells (in granulation stage) and osteoblasts and non-hyperthrophic chondrocytes (in bone and cartilage formation) Targeted cells: MSCs, endothelial cells, osteoblasts, chondrocytes IGF-I: mesenchymal and osteoprogenitor cells recruitment and proliferation, expressed throughout fracture healing IGF-II: cell proliferation and protein synthesis during endochondral ossification Metalloproteinases Source: the extracellular matrix Degradation of the cartilage and bone allowing the invasion of blood vessels during the final stages of endochondral ossification and bone remodelling VEGFs Potent stimulators of endothelial cell proliferation Expressed during endochondral formation and bone formation Angiopoietin (1 and 2) Formation of larger vessel structures, development of co-lateral branches from existing vessels Expressed from the early stages throughout fracture healing

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Figure 1 Schematic summary of the temporal expression patterns of the signalling molecules during fracture healing. (The dashed line represents a difference of opinion amongst scientists in terms of the timing of expression.)

Bone morphogenetic proteins. Members of the BMP family are divided into at least four separate subgroups depending on their primary amino acid sequence. Group one consists of BMP-2 BMP-4, and group two includes BMP-5, -6, and -7. The third group includes GDF-5 (or BMP-14), GDF-6 (or BMP13) and GDF-7 (or BMP-12), and finally group four includes BMP-3 (or osteogenin) and GDF-10 (or BMP3b).67 BMP-1 is not a member of the TGF-b superfamily and it may play a role in modulating BMP actions by the proteolysis of BMP antagonists/binding proteins, such as noggin and chondrin.63 BMPs bind to type II serine/threonine kinase receptors which transphosphorylate type-I receptors.28,57 Subsequently, the Smad intracellular signalling cascade is initiated. The Smad family includes eight members which can be subdivided into three groups: the signal-transducing receptorregulated Smads (R-Smads 1, 2, 3, 5, 8), the common mediator Smad (co-Smad, such as Smad-4), and the inhibitory Smads (I-Smads, such as Smad-6 and Smad-7).37,50 R-Smad 1, 5 and 8 are substrates for BMP receptors and when activated they interact with Smad-4. These heteromeric complexes trans-

locate into the nucleus and regulate the transcription of target genes (Fig. 2).75 BMPs are pleiotropic morphogens and play a critical role in regulating growth, differentiation, and apoptosis of various cell types, including osteoblasts, chondroblasts, neural cells, and epithelial cells.67 Furthermore, it has been shown that BMP heterodimers, such as BMP-4/-7 and BMP-2/-7 have an enhanced osteoinductive activity regulating more efficiently differentiation and proliferation of mesenchymal cells to osteoblasts in vitro and in vivo.38 The extracellular matrix comprises the main source of BMPs being produced by osteoprogenitors, mesenchymal cells, osteoblasts, and chondrocytes (Table 1). BMPs induce a sequential cascade of events for chondro-osteogenesis, including chemotaxis, mesenchymal and osteoprogenitor cells proliferation and differentiation, angiogenesis, and controlled synthesis of extracellular matrix.63,67 Their regulatory effect depends upon the type of the targeted cell, its differentiation stage, the local concentration of the ligand as well as the interaction with other circulating factors.35 Interestingly,

Current concepts of molecular aspects of bone healing

low concentrations of BMPs in vitro favour the differentiation of mesenchymal stem cells into adipocytes.80 BMPs are closely structurally and functionally related, however, each has a unique role as well a distinct temporal expression pattern during the fracture repair process (Fig. 1). Studies of the role

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of BMPs in fracture healing in the mouse and rat have shown a variety of osteogenic effects, temporal expressions, and mitogenic capacities (Tables 2 and 3).9,10,20 In a comprehensive analysis of the osteogenic activity of 14 types of BMPs, Cheng et al. suggested an osteogenic hierarchical model of BMPs. BMP-2,

Table 2 Temporal and functional characteristics of members of the TGF-b superfamily observed during fracture healing in animal models Member of the TGF-b superfamily

Time of expression

Specific responses in vivo and in vitro

GDF-8

Restricted to day 1 20

Potential function as a negative regulator of skeletal muscle growth 20

BMP-2

Days 1—2110,20 (the earliest gene to be induced and second elevation during osteogenesis)

Recruitment of mesenchymal cells Chondrogenesis May initiate the fracture healing cascade and regulate the expression of other BMPs BMP-2, -6, -9 may be the most potent to induce osteoblast lineage-specific differentiation of MSCs 19

BMP-3, -8

Days 14—2120 (restricted expression during osteogenesis)

Temporal data suggest a role in the regulation of osteogenesis

BMP-4

Transient increased expression in the surrounding soft tissues 6 h to day 5 9

Involvement in the formation of callus at a very early stage in the healing process In vitro: BMP-3 and -4 stimulate the migration of human blood monocytes 63

Days 14—21 20 Through out fracture healing 10 BMP-7

Days 14—21 20 From the early stages of fracture healing 9

Regulatory role in both types of ossification In vitro: stimulation of relative mature osteoblasts 19

GDF-10, BMP-5, -6

Days 3—21 20

Regulatory role in both types of ossification BMP-6 may initiate chondrocyte maturation 20

GDF-5, 1

Day 7 (maximal) to day 1420 (restricted expression during chondrogenic phase) GDF-1 at extremely low levels

GDF-5 an exclusive involvement in chondrogenesis is suggested Stimulation of mesenchymal aggregation and induction of angiogenesis through chemotaxis of endothelial cells and degradation of matrix proteins

GDF-3, GDF-6, 9

No detectable levels within the fracture callus 20

GDF-6 may be expressed only in articular cartilage20 and with GDF-5, 7 more efficiently induce cartilage and tendon-like structures in vivo 28

TGF-b1, -b2, -b3

Days 1—21 20

Potent chemotactic for bone forming cells and macrophages Proliferation of undifferentiated mesenchymal and osteoprogenitor cells, osteoblasts, chondrocytes

Days 3—14 20

Days 3—21 20

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Figure 2 BMP-receptor interaction and the intracellular signalling pathway.

-6, and -9 may be the most potent to induce osteoblast differentiation of mesenchymal progenitor cells, whilst most BMPs (except BMP-3 and -13) can promote the terminal differentiation of committed osteoblastic precursors and osteoblasts.19

BMPs may also stimulate the synthesis and secretion of other bone and angiogenic growth factors such as insulin-like growth factor (IGF) and vascularendothelial growth factor (VEGF), respectively.22 They may also stimulate bone formation by directly activating endothelial cells to stimulate angiogenesis.59,79 Recent studies have showed that the expression of the BMP antagonists, most importantly noggin,72 which blocks BMP-2 interaction with its receptor,29 also play an important role in fracture healing regulation.36 It has been suggested that the noggin/ BMP-4 balance could be an important factor in the regulation of callus formation.85 Transforming growth factor beta. Five isoforms of this group have been isolated.42 Platelets release TGF-b during the initial inflammatory phase of bone healing and therefore this factor may be involved in the initiation of callus formation.8,9 TGF-b is also produced by osteoblasts and chondrocytes, and is stored in the bone matrix (Table 1).46 Its effect is exerted via type-I and type-II serine/threonine kinase receptors, activating the Smad pathway (Smad 2 and 3).37 TGF-b is a potent chemotactic stimulator of mesenchymal stem cells and it enhances prolifera-

Table 3 Timing of cellular events and expression of signalling molecules during murine fracture healing Day 1

Haematoma formation, inflammation Recruitment of mesenchymal cells Osteogenic differentiation of MSCs from bone marrow

Day 3

Day 7

Day 14

Day 21

20,33,43

Cytokines: IL-1, IL-6, TNF-a released by inflammatory cells PDGF, TFG-beta released from degranulating platelets BMP-2 expression and restricted to day 1 expression of GDF-8

MSCs proliferation begins Proliferation and differentiation of preosteoblasts and osteoblasts in regions of intramembranous ossification Angiogenesis begins

Decline of cytokines levels Expression of TGF-b2, -b3, GDF-10, BMP-5, -6

Peak of cell proliferation in intramembranous ossification between days 7 and 10 Chondrogenesis and endochondral ossification begins (days 9—14 maturation of chondrocytes)

Peak of TGF-b2 and -b3 expression

Cessation of cell proliferation in intramembranous ossification, but osteoblastic activity continues Mineralization of the soft callus, cartilage resorption, and woven bone formation Neo-angiogenesis which infiltrates along new mesenchymal cells Phase of most active osteogenesis until day 21

Decreased levels of expression for TGF-b2, GDF-5, and probably GDF-1 Expression of BMP-3, -4, -7, and -8

Woven bone remodelled and subsequently replaced by lamellar bone

Angiopoietin-1 is induced

Expression of GDF-5 and probably GDF-1

VEGFs expression Second increase of IL-1 and TNF-a which continues during bone remodelling Decreased expression of TGF-b1 and TGF-b3, GDF-10, and BMPs (2—8)

Current concepts of molecular aspects of bone healing

tion of MSCs, preosteoblasts, chondrocytes and osteoblasts.46 It also induces the production of extracellular proteins such as collagen, proteoglycans, osteopontin, osteonectin, and alkaline phosphatase.68 Its main role is thought to be during chondrogenesis and endochondral bone formation (Table 2).7 TGF-b may also initiate signalling for BMP synthesis by the osteoprogenitor cells,9 while it may inhibit osteoclastic activation and promote osteoclasts apoptosis.51 Recently, it has been suggested that TGF-b2 and possibly TGF-b3 play more important roles in fracture healing than TGF-b1, as their expression peak during chondrogenesis. On the other hand, TGF-b1 has a high basal level of expression in unfractured diaphyseal bone, and the level of expression remains constant throughout the fracture healing process, indicating that TGF-b2 and TGF-b3 may play more important roles in fracture healing than TGF-b1 (Fig. 1).20 Although various studies showed that TGF-b enhances cellular proliferation, its osteoinductive potential seems limited and concern for its unforeseen side effects has been expressed.46 Therefore, its therapeutic potential to enhance bone repair seems to be limited. Platelet-derived growth factor (PDGF). PDGF is a homo- or heterodimeric polypeptide consisting of A and B chains. PDGF effect is exerted via receptors that have tyrosine kinase activity. IL-1, TNF-a, and TGF-b1 affect PDGF’s binding.73 It is synthesized by platelets, monocytes, macrophages, endothelial cells, and osteoblasts and it is a potent mitogen for cells of mesenchymal origin (Table 1).3 PDGF is released by platelets during the early phases of fracture healing and it is a potent chemotactic stimulator for inflammatory cells and a major proliferative and migratory stimulus for MSCs and osteoblasts (Fig. 1).46 Nash et al. showed an increased callus density and volume in tibial osteotomies in rabbits treated with PDGF.56 However, at present, its therapeutic potential remains unclear. Fibroblast growth factor (FGFs). The family of FGFs consists of nine structurally related polypeptides. The acidic and basic FGFs are the most abundant FGFs in normal adult tissue.81 Their action is exerted by binding to tyrosine kinase receptors.83 During bone healing, FGFs are synthesized by monocytes, macrophages, mesenchymal cells, osteoblasts and chondrocytes (Table 1). FGFs promote growth and differentiation of a variety of cells such as fibroblasts, myocytes, osteoblasts, and chondrocytes. FGFs are identified during the early stages of fracture healing and they play a critical role in angiogenesis and mesenchymal cell mitogen-

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esis (Fig. 1). a-FGF mainly effects chondrocyte proliferation and is probably important for chondrocyte maturation, whilst b-FGF is expressed by osteoblasts and is generally more potent than a-FGF.46 In a canine tibial osteotomy model, a single injection of FGF-2 was associated with an early increase in callus size.55 Insulin-like growth factors (IGFs). The sources of IGF-I (or somatomedin-C) and IGF-II (or skeletal growth factor) are the bone matrix, endothelial cells, osteoblasts and chondrocytes. The serum concentration of IGF-I is mainly regulated by the growth hormone (Table 1).46,73 The biological actions of the IGFs are modulated in a cell-specific manner by IGFbinding proteins (IGFBPs).71 IGF-I promotes bone matrix formation (type I collagen and non-collagenous matrix proteins) by fully differentiated osteoblasts15 and is more potent than IGF-II.46 IGF-II acts at a later stage of endochondral bone formation and stimulates type I collagen production, cartilage matrix synthesis, and cellular proliferation (Fig. 1).62 The findings from a number of animal studies assessing the influence of IGF on skeletal repair have varied and therefore further studies are required.7 Metalloproteinases and angiogenic factors Optimal bone regeneration requires adequate blood flow. During the final stages of endochondral ossification as well as during remodelling phase, specific matrix metallopoteinases degrade cartilage and bone, allowing the invasion of blood vessels (Table 1).33 Two separate pathways are believed to regulate angiogenesis: a vascular-endothelial growth factordependent pathway and an angiopoietin-dependent pathway.33 It is speculated that both pathways are functional during fracture repair. VEGFs are essential mediators of neo-angiogenesis and endothelial-cell specific mitogens.26 Whereas, angiopoietin 1 and 2 are regulatory vascular morphogenetic molecules related to the formation of larger vessel and development of co-lateral branches from existing vessels (Fig. 1). However, their contribution in bone repair is not as well understood (Table 1).33 Street et al. showed that fracture repair was enhanced by the exogenous administration of VEGF.74 Recent studies have also shown that BMPs stimulate the expression of VEGF by osteoblasts and osteoblast like cells.22,59,84

The role of mesenchymal stem cells MSCs are undifferentiated cells capable of extensive replication without differentiation.13 MSCs have the

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potential to commit and differentiate along multiple cell lineages. They give rise to those cells that form mesenchymal tissues, including bone, cartilage, tendon, muscle, ligament, and marrow stroma and fat.13,60 During fracture healing, potential sources of stem cells are the bone marrow, the granulation tissue, the deep layer of the periosteum, the endosteum, and the surroundings soft tissues.23,33,53 Also, perivascular mesenchymal stem cells that exist in blood vessels walls contribute to fracture healing.11 The primary tissue source of MSCs is the periosteum53 and studies from Buckwalter et al. showed that the capacity for fracture callus development is diminished if the periosteum is removed.14

Sequence of events Fracture healing, like all other repair responses, is initiated through the induction of an immune response.24 During this initial stage, a haematoma is formed and inflammation occurs. The major players at this initial inflammatory phase include cytokines, platelets, BMPs and MSCs. IL-1, IL-6 and TNF-a secreted by inflammatory cells have a chemotactic effect on other inflammatory cells and on the recruitment of mesenchymal cells.43 Cho et al. reported a peak in expression of IL-1 and -6 one day after fracture followed by a rapid decline until day 3 to near undetectable levels.20 At the same time, platelets, activated by thrombin and subendothelial collagen, release PDGF and TGF-b, which play a role on the initiation of fracture repair.8 These factors induce mesenchymal cell migration, activation and proliferation, angiogenesis, chemotaxis of acute inflammatory cells and further aggregation of platelets. Simultaneously, BMPs not only are released from the bone matrix but also are expressed by recruited primary mesenchymal cells.9 During the subsequent days, MSCs proliferate and differentiate into a chondrogenic or osteogenic lineage.69 During this early phase of events, angiogenesis also takes place and this is a prerequisite for further progression of the regeneration cascade. The vascular ingrowth into the developing callus is regulated by FGF, VEGF and angiopoietin 1 and 2.33,46 Angiopoietic 1 has been suggested to be induced during the initial periods of fracture healing whereas VEGF later on mainly during endochondral and bone formation.33 Intramembranous bone formation The source of the cells that contribute to intramembranous bone formation appears to be the underlying cortical bone, the periosteum within a few

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millimetres from the fracture site and the region of the bone marrow with high cellular density.23 Within the first 24 h of the fracture, the latter cells begin to differentiate into an osteoblastic phenotype. By day 3, osteoblasts from the cortex and committed osteoprogenitors derived from the periosteal cambium divide and differentiate, forming woven bone (hard callus). Their proliferation peaks by days 7 and 10, and it ceases by day 14 while the osteoblastic activity continues.7 In a rat fracture healing model, BMP-2, -4, and -7 showed increased levels of expression during the early stages of intramembranous ossification.9 Endochondral bone formation In regions that are mechanically less stable, endochondral bone formation occurs. This type of ossification mainly occurs at the adjacent to the fracture site periosteum and enhanced by the soft tissues around the fracture site. During this process, MSCs are recruited and begin to proliferate by day 3 after fracture. Their subsequent differentiation into chondroblasts (chondrogenesis) and the proliferation of these new chondrocytes occur from days 7 to 21, resulting to soft callus formation. These cells synthesize and secrete cartilage-specific matrix, including type II collagen and proteoglycans and once firm mechanical stability is established, the cartilage undergoes hypertrophy and mineralization in a spatially organized manner.7 As vasculature begins to invade, the calcifying hypertrophic chondrocytes are being removed by chondroclasts and woven bone formation occurs after the recruitment and osteogenic differentiation of new MSCs.23 Lee et al. hypothesised that this stage of endochondral ossification is the final stage of a genetically programmed process which results to apoptotic chondrocyte death.45 Eventually, this soft callus is replaced by woven marrow-filled bone, which undergoes significant remodelling to become weight-bearing bone following the pathway observed in the growth plate. Recently, Cho et al. demonstrated the different temporal patterns for members of the TGF-b superfamily during murine fracture healing, suggesting a potentially unique role of each BMP (Table 3).20 The stimulation of undifferentiated MSCs and osteoprogenitors cells to differentiate into osteoblasts (osteoinduction)73 is a morphogenetic cascade involving discrete cellular transitions. It is predominantly regulated by the complex interactions that take place between the multiple local (paracrine and autocrine) signals that are presented to mesenchymal stem cells in their natural environment (Table 1).31 Further research is required in order to elucidate the role of each

Current concepts of molecular aspects of bone healing

factor at each discrete stage of fracture healing as well as their combined functioning to promote its various stages. Although the cellular events during fracture healing seem to be predominantly regulated by local factors and cytokines, systemic hormones (parathyroid/thyroid hormone, the growth hormone, the 1,25 dihydroxyvitamin D and the sex steroids) may also modulate these events.51

Current concepts of systemic enhancement of fracture healing Parathyroid hormone (PTH) Contrary to the assumption that PTH has a catabolic effect on the skeleton, intermittent exposure stimulates osteoblasts and results in increased bone formation in rats. It has been shown that low-dose human PTH (1—34) enhances callus formation by stimulating the early proliferation and differentiation of osteoprogenitor cells, increasing the production of bone matrix proteins and enhancing osteoclastogenesis during callus remodelling. PTH effect is likely to be mediated by osteoblastic activity, as PTH receptors are found on osteoblast membranes.1,54

Growth hormone (GH) Growth hormone is a systemic hormone and its effect on the skeleton is mediated by IGF-1 (known as somatomedin-C) which promotes bone matrix formation (type I collagen and non-collagenous matrix proteins) by fully differentiated osteoblasts. Systemically administration of GH in a rat tibial diaphyseal fracture model during the first 3 weeks of healing increased callus formation and enhanced fracture strength.2 Furthermore, in a fracture healing and distraction osteogenesis model in Yucatan micropigs, administration of homologous recombinant porcine GH led to an increase in serum IGF-1, stimulation of fracture healing and acceleration of ossification of bone regenerate in distraction osteogenesis.5

Future directions Tissue engineering Currently, as the molecular and cellular events during the fracture healing cascade are becoming gradually more understood, new strategies are investigated in order to promote or facilitate the

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healing process. Bone tissue engineering, combining the application of the principles of orthopaedic surgery with basic science and engineering, has been heralded as an alternative when bone regeneration is required to replace or restore the function of traumatised, damaged, or lost bone.66 In essence, tissue engineering aims to combine progenitor cells, such as MSCs, or mature cells (for osteogenesis) with biocompatible materials or scaffolds (for osteoconduction), with appropriate growth factors (for osteoinduction), in order to generate and maintain bone.66,78 Although this new strategy is still in its infancy, its clinical application offers great potentials for the treatment of conditions requiring bone repair. A promising technology in the field of bone tissue engineering is the application of gene therapy as a method of growth factor delivery for the clinical management of orthopaedic disorders, including bone healing.18 It involves the transfer of genetic material into targeted cell’s genome, and thus allowing the expression of bioactive factors from the cells themselves and for longer periods of time. The gene transfer can be performed using a viral (transfection) or a non-viral (transduction) vector, by either an in vivo or ex vivo gene-transfer strategy.18,46 With the in vivo technique, a technically easier method, the genetic material is transferred directly into the host. However, there are safety concerns with this strategy. The indirect ex vivo technique involves the collection of cells by tissue harvest and their genetic modification in vitro before transfer back into the host. It is a technically more demanding but safer method, as it allows testing of the cells for any abnormal behaviour before re-implantation as well as selection of those with the greatest gene expression.18 Gene therapy has been used to promote fracture repair through the expression of BMP-26,47 and -470 in animal studies. Although promising, issues of its biosafety and efficacy need to be answered before human trials take place.

Muscle stem cells Since 1965, when Urist first noted the ectopic bone formation after demineralized bone matrix implantation into skeletal muscle,76,77 subsequent studies have reported similar observations. This phenomenon of osteoinduction has been attributed to the mitogenic effects of BMP-2 (in vitro)40 and BMP-3 (in vivo)41 on cells in muscle In skeletal muscle, researchers have identified adult stem cells that can differentiate into cells of different lineages.16,58 The most well-known muscle progenitor cells, termed satellite cells, are the primary source

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of the myoblasts, but they can also exhibit osteogenic and adipogenic differentiation.4 Recently, a population of stem cells that appears to be distinct from the satellite cells has also been discovered: the muscle-derived stem cells (MDSCs). These cells have the ability to differentiate into multiple lineages including osteogenic and haematopoietic lines.21,39 These muscle-based progenitor cells possess a therapeutic potential for tissue repair and regeneration applications in various musculoskeletal as well as cardiac muscle disorders either as a source of inducible progenitor cells or as gene delivery vehicles.21 For bone formation and healing in particular, MDSCs genetically engineered to express BMP-252 and both BMP-4 and VEGF58 have been shown to be capable of stimulating osteogenesis and angiogenesis, respectively.

Clinical applications in trauma Approximately 5—10%, of the 6.2 million fractures occurring annually in the United States, are associated with impaired healing including delayed healing or non-union.61 Various animal studies and clinical trials in humans have been performed and demonstrated the potential use of several of the biological factors described in bone regeneration and skeletal repair, with the BMPs to be the most promising. Riedel and Valentin-Opran were the first to report preliminary results from the use of BMP-2 to augment the treatment of open tibial fractures.64 Govender et al. in a large prospective randomized controlled multi-center trial evaluated the effects of recombinant (rh) BMP-2 on the treatment of open tibial fractures. They reported a higher union rate, an accelerated time to union, improved wound healing, reduced infection rate, and fewer secondary invasive interventions in the group of patients treated with rhBMP-2 and IM nail fixation, compared to the group treated with IM nail fixation alone.34 In 1999, Geesink et al. demonstrated the ability of BMP-7 to cause repair of a critical-sized fibular defect in patients undergoing opening wedge high tibial osteotomy with fibulectomy.30 Friedlaender et al. in a large prospective randomized controlled and partially blinded multicenter study, they assessed the efficacy of rhBMP-7 over iliac crest bone graft in the treatment of 122 patients with 124 tibial non-unions. The authors concluded that whilst no statistical difference was noted between the two groups, BMP-7 was a safe and effective alternative to bone graft in the treatment of tibial non-unions.27

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Currently, recombinant BMP-2 and -7 are commercially available and, although the research is ongoing, they are considered adjuncts in the surgeon’s armamentarium for the treatment of clinically challenging situations.

Conclusion Fracture healing is a complex physiological process which involves a well orchestrated series of biological events. Whilst our knowledge has vastly expanded, with the increasing understanding of the multiple factors and complex pathways involved, a lot of new developments are anticipated in the years to come. It is hoped that many bone disease processes secondary to trauma, aging and metabolic disorders will be successfully treated with novel treatment protocols.

Acknowledgements Rozalia Dimitriou is a research fellow supported via a grant (03-943) by the AO foundation.

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