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С. Сагаловски
КЛЕТОЧНЫЕ И МОЛЕКУЛЯРНЫЕ АСПЕКТЫ ЗАЖИВЛЕНИЯ ПЕРЕЛОМОВ КОСТИ: РОЛЬ ФАКТОРОВ РОСТА И КОСТНОГО МОРФОГЕНЕТИЧЕСКОГО БЕЛКА В АКТИВАЦИИ РЕПАРАТИВНОГО ОСТЕОГЕНЕЗА (ОБЗОР ЛИТЕРАТУРЫ)
Отдел ортопедии клиники Медиан.
Германия, 04651 БадЛаузик, Паркштрассе 4. E-mail: s.sagalovsky@gmail.com
В обзоре литературы отражены современные представления о клеточных и молекулярных механизмах развития заживления перелома кости. Показана значительная роль факторов роста и костного морфогене-тического белка в активации репаративного процесса перелома кости. Отражена ключевая роль в активации репаративного остеогенеза ряда молекул клеточных сигнальных систем и их агонистов/антагонистов, которые представляют интерес как молекулы-мишени для поиска новых лекарственных средств стимуляции репаративного остеогенеза.
Ключевые слова: заживление перелома кости, факторы роста, костный морфогенетический белок.
S. Sagalovsky
CELLULAR AND MOLECULAR ASPECTS OF BONE FRACTURE HEALING: THE ROLE OF GROWTH FACTORS AND BONE MORPHOGENETIC
PROTEINS IN ACTIVATION REPARATIVE OSTEOGENESIS (REVIEW)
Department of Orthopedics Clinic Median, 04651, Bad Lausick, Parkstraße 4, Germany.
E-mail: s.sagalovsky@gmail.com
In a review of the literature reflect the modern understanding of the cellular-molecular mechanisms of bone fracture healing. The importance of the role growth factors and bone morphogenetic proteins in activation bone fracture repair is shown. Noting the key role in the process of bone fracture repair a number of molecules of cell signaling pathway and their agonists/antagonists are of interest as a target molecule to search for new drug for activation reparative osteogenesis.
Keywords: bone fracture healing, growth factors, bone morphogenetic protein, osteogenesis.
Fractures are one of the most frequent injuries of the musculoskeletal system. Fracture repair can be considered as a biologically optimal process resulting in the restoration of injured skeletal tissue to a state of normal structure and function. Although the process leads to healing in the vast majority of cases, a small but significant proportion of fractures result in delayed union or persistent nonunion. Surgical interventions have been directed toward enhancing the fracture repair process, normalizing the rate of healing, and decreasing the likelihood of nonunion. During fracture repair, a number of growth factors, cytokines, and their cognate receptors are present at elevated levels in and around the fracture site.
Many of these proteins are normally expressed in skeletal tissue, and others are released from associated inflammatory cells at the site of injury. The induction of these proteins is regulated both spatially and temporally, suggesting that they play an active role in promoting fracture repair. The following review will summarize the current literature on the roles of the major cytokines and growth factors involved in fracture repair. In addition, the signaling cascades induced by these molecules will be discussed. While many cytokine and growth factor signaling events have not been specifically examined in the context of fracture repair, a large body of literature on signal transduction has emanated from studies on these
molecules in embryonic bone development. Given the conserved nature of these molecules and their signaling cascades from Drosophila to humans, and the similarities between the fracture repair process and embryonic bone development, it seems highly probable that these downstream signaling events are conserved in fracture repair.
General fracture healing
Fracture repair can be envisioned as involving five distinguishable processes, including the immediate response to injury, intramembranous bone formation, chondrogenesis, endochondral bone formation leading to the reestablishment of local bearing function, and bone remodeling. While these processes may be discussed individually, it should be recognized that the first four occur simultaneously during fracture repair and are likely to influence one another. Extensive remodeling of the newly formed bone follows these four concurrent processes and facilitates the reestablishment of the full biomechanical integrity of the bone. A number of investigators [1, 2, 3] have commented on the similarities between the repair process and embryonic bone formation and indeed a number of specific events characteristic of embryonic bone formation are reiterated in fracture repair. This is especially evident as one begins to examine the specifics of local factors regulating fracture repair, specifically those involved in the events of endochondral bone formation.
The first stage after injury: the acute inflammatory response
The immediate response to injury from the fracture trauma is the initiating event of the fracture repair process. The stage after the injury is inflammation (Figure 1, A). Fracture trauma involves not only an interruption of skeletal integrity but also a disruption of the normal vascular structures and nutrient flow at the fracture site. This leads to reduce oxygen tension, disruption of the marrow architecture, and results in the infiltration of inflammatory cells, macrophages, and degranulating platelets during formation of a hematoma [4,5,6]. While it is likely that the mechanical stresses, changes in oxygen tension, and loss of vascular nutrients at the fracture site may signal some aspects of the healing process, the dominant initiators of fracture repair are most likely the numerous cytokines and growth factors released into the fracture site [7]. To date the majority of research on fracture repair has focused on the actions of a relatively limited number of cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-a), and macrophage colony-stimulating factor (M-CSF) as well as the local growth factors: transforming growth factor — beta (TGF-.H), and bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF). The initial proinflammatory response involves of tumor necrosis factor-a, interleukin-1 and interleulin-6. These factors recruit
inflammatory cells and promote angiogenesis [8] . The TNF-a concentration has been shown to peak at 24h and to return to baseline within 72h post trauma
[I]. During this time-frame TNF-a is expressed by macrophages and other inflammatory cells, and it is believed to mediate an effect by inducing secondary inflammatory signals, and act as a chemotactic agent to recruit necessary cells. TNF-a has also been shown in vitro to induce osteogenic differentiation of mesenchymal stem cells (MSC). These effects are mediated by activation of the two receptors TNFRI and TNFRII which are expressed on both osteoblasts and osteoclasts. However, TNFRI is always expressed in bone whereas TNFRII is only expressed following injury, suggesting a more specific role in bone regeneration [9]. Among the different interleukins, IL-1 and IL-6 are believed to be most important for fracture repair. IL-1 expression overlaps with that of TNF-a with a biphasic mode. It is produced by macrophages in the acute phase of inflammation and induces production of IL-6 in osteoblasts, promotes the production of the primary cartilaginous callus, and also promotes angiogenesis at the injured site by activating either of its two receptors, ILRI or ILRII. IL-6 on the other hand, is only produced during the acute phase and stimulates angiogenesis, vascular endothelial growth factor (VEGF) production, and the differentiation of osteoblasts and osteoclasts [10].
The second stage of the bone fracture repair:
recruitment of mesenchymal stem cells (MSC)
In order for bone to regenerate, specific mesenchymal stem cells (MSCs) have to be recruited, proliferate and differentiate into osteogenic cells (Figure 1, B). Exactly where these cells come from is not fully understood. Although most data indicate that these MSCs are derived from surrounding soft tissues and bone marrow, recent data demonstrate that a systemic recruitment of circulating MSCs to the injured site might be of great importance for an optimal healing response
[II]. Which molecular events mediate this recruitment is still under debate. It has long been suggested that bone morphogenetic protein-2 has an important role in this recruitment, but data from [12] indicates that this is not the case. Indeed, BMP-2 is essential for bone repair [7] but other BMPs such as BMP-7 may play a more important in the recruitment of progenitor cells. Current data suggested that mesenchymal cells proliferate and differentiate into fibroblasts, chondrocytes or osteoblasts depending on the biological and mechanical conditions. At each site of the fracture gap near the bone tissue, the mesenchymal cells differentiate into osteoblasts producing intramembranous woven bone. Further away from the bone tissue towards the centre of the callus, MSCs differentiate into either fibroblasts or chondrocytes. Because of this, the callus is gradually stabilized.
The third stage generation of a cartilaginous and periosteal bony callus
In the third stage of the healing process, although indirect fracture healing consist of both intramembranous and endochondral ossification,
the formation of a cartilaginous callus which later undergoes mineralization, resorption and is then replaced with bone is its key feature of this process (Figure 1, C). Following the formation of the primary hematoma, a fibrin-rich granulation tissue forms [13]. Within this tissue, endochondral formation occurs in between the fracture ends, and external to periosteal sites. These regions are also mechanically less stable and the cartilaginous tissue forms a soft callus which gives the fracture a stable structure [14]. In animal models (rat, rabbit, mouse) the peak of soft callus formation occurs 7-9 days post trauma with a peak in both type II procollagen and proteoglycan core protein extracellular markers. At the same time, an intramembranous ossification response occurssubperiostally directly adjacent to the distal and proximal ends of the fracture, generating a hard callus. It is the final bridging of this central hard callus that ultimately provides the fracture with a semi-rigid structure which allows weigth bearing [15]. The generation of these callus tissues is dependent on the recruitment of MSCs from the surrounding soft tissues, cortex, periosteum, and bone marrow as well the systemic mobilization of stem cells into the peripheral blood from remote hematopoietic sites. Once recruited, a molecular cascade involves collagen-I and collagen-II matrix production and the participation of several peptide signaling molecules. In this process the transforming growth factor-beta (TGF-.H) superfamily members have been shown to be of great importance. The TGF-.H superfamily involved in chondrogenesis and endochondral ossification, whereas BMP-5 and -6 have been suggested to induce cell proliferation in intramembranous ossification at periosteal sites [5]. In addition, as noted above, BMP-2 has been shown to be crucial for initiation of the healing cascade, as mice with inactivating mutationsin BMP-2 are not able to
form callus in order to heal their fractures successfully. Whether this is due to effects on mesenchymal stem cell proliferation and differentiation or effects on cell migration is still under debate.
Revascularization and neoangiogenesis at the fracture site
Fracture healing requires a blood supply and revascularization is essential for successful bone repair [16]. In endochondral fracture healing, this not only involves angiogenic pathways, but also chondrocyte apoptosis and cartilaginous degradation as the removal of cells and extracellular matrices are necessary to allow blood vessels in/growth at the repair site. Once this structural pattern is achieved, the vascularization process is mainly regulated by two molecular pathways, an angiopoietin-dependent pathway, and vascular endothelial growth factor (VEGF)-dependent pathway [7]. The angiopoietins, primary angiopoetin-1 and 2, are vascular morphogenetic proteins. Their expressions are induced early in the healing cascade, suggesting that they promote an initial vascular in-growth from existing vessels in the periosteum. However, the VEGF pathway is considered to be the key regulator of vascular regeneration [16]. It has been shown that both osteoblasts and hypertrophic chondrocytes express high levels of VEGF, thereby promoting the invasion of blood vessels and transforming the a vascular cartilaginous matrix into a vascularized osseous tissue. VEGF promotes both vasculogenesis, i.e. aggregation and proliferation of endothelial mesenchymal stem cells into a vascular plexus, and angiogenesis, i.e. growth of new vessels from already existing ones. Hence, VEGF plays a crucial role in the neoangiogenesis and revascularization at the fracture site.
Table 1. Summary of the four stages of fracture healing and the accompanying expression of signaling
molecules (based on published results from [17, 18, 19].
Stage of fracture repair Biological processes Expression of signaling molecules and their proposed functions
Inflammation Hematoma Inflammation Recruitment of mesenchymal stem cells IL-1, IL-6, and TNF-a play a role in initiating the repair cascade. TGF-A, PDGF, and BMP-2 expression increases to initiate callus formation. TGF-.H is restricted to day 1, suggesting its role in controlling cellular proliferation.
Cartilage formation periosteal response Chondrogenesis and endochondral ossification begins Cell proliferation in intramemranous ossification. Vascular in-growth Neo-angiogenesis TGF-.H and BMP peak due to their involveme and in chondrogenesis and endochondral bone formation. BMP-5 and -6 rise. Angiopoietins and VEGFs are induced to stimulate vascular in growth from vessels in the periosteum.
Cartilage resorption and primary bone formation Phase of most active osteogenesis. Bone cell recruitment and woven bone formation. Chondrocyte apoptosis and matrix proteolysis. Osteoclast recruitment and cartilage resorption. Neo-angiogenesis TNF-a rises in association with mineralized cartilage resorption. This promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes. RANKL and MCSF rise in association with mineralized cartilage resorption. BMP-3,-4,-7, and -8 rise in association with the resorption of calcified cartilage. They promote recruitment of cells in the osteoblastic lineage. BMP-5 and -6 remain high during this stage suggesting a regulatory effect on both intramembranous and endochondral ossification. VEGFs are up-regulated to stimulate neo-angiogenesis.
Secondary bone formation and remodeling Bone remodeling coupled with osteoblast activity Establishment of marrow. IL-1 and IL-6 rise again in association with bone remodeling, whereas RANKL and MCSF display diminished levels. Diminished expression of members of the TGF-.H superfamily.
The four stage of fracture healing: mineralization and resorption of the cartilaginous callus and bone remodeling
In order for bone regeneration to progress, the primary soft cartilaginous callus needs to be resorbed and replaced by a hard bony callus. This step of fracture healing, to some extent, recapitulates embryological bone development with a combination of cellular proliferation and differentiation, increasing cellular volume and increasing matrix deposition. As fracture callus chondrocytes proliferate, they become hypertrophic and the extracellular matrix becomes calcified. A cascade orchestrated primarily by macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG) and tumor necrosis factor alpha (TNF-a) initiates the resorbtion of this mineralized cartilage [20]. During this process
M-CSF, RANKL and OPG are also thought to help recruit bone cells and osteoclasts to form woven bone. The calcification mechanism involves the role of mitochondria, which accumulate calcium-containing granules created in the hypoxic fracture environment. After elaboration into the cytoplasm of fracture callus chondrocytes, calcium granules are transported into the extracellular matrix where they precipitate with phosphate and form initial mineral deposits. These deposits of calcium and phosphate become the nidus for homogeneous nucleation and the formation of apatite crystals. The peak of the hard callus formation is usually reached by day 14 in animal models as defined by histomorphometry of mineralized tissue, but also by the measurement of extracellular matrix markers such as type I procollagen, osteocalcin, alkaline phosphatase and osteonectin. As the hard callus formation progresses and the calcified cartilage is replaced with woven bone, the callus becomes
more solid and mechanically rigid (Figure 1, D). Although the hard callus is a rigid structure providing biomechanical stability, it does not fully restore the biomechanical properties of normal bone. In order to achieve this, the fracture healing cascade initiates a second resorptive phase, this time to remodel the hard callus into a lamellar bone structure with a central medullary cavity. This phase is biochemically orchestrated by IL-1 and TNF-D, which show high expression levels during this stage, as opposed to most members of the TGF-.H family which have diminished in expression by this time. The remodeling process is carried out by a balance of hard callus resorption by osteoclasts, and lamellar bone deposition by osteoblasts. Although the process is initiated as early as 3-4 week in animal and human models, the remodeling may take years to be completed to achieve a fully regenerated bone structure. The multiple stages of fracture healing and the accompanying expression of signaling molecules are summarized in Table. Thus, it is clear that fracture repair involves the coordinate regulation of cellular chemotaxis, proliferation, and differentiation and that these events are likely to be regulated through signaling via growth factors
(Table). The major growth factors characterized as being present in the fracture site are TGF-.H1, TGF-H2, BMP-2, BMP-3, BMP-4, and BMP-7 (OP-
1), platelet-derived growth factor (PDGF), and acidic and basic fibroblast growth factor (FGF-1 and FGF-
2), vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) [5, 7, 18, 20 ]. Given the well described roles of these factors in embryonic bone development and in vitro effects on bone cells, these molecules are likely to be important regulators of fracture repair [3, 6]. It is interesting to bone that the signaling pathways utilized by these molecules fall into distinct groups: these signaling through receptor tyrosine kinases (FGFs, PDGF, VEGFs and IGF) and those through serine-threonine kinase receptors (TGF-^s and BMPs) (Figure 2). The common theme is signal transduction through a receptor in which kinase activity is induced by receptor occupancy by ligand. The subtle beauty of such a system is the cross talk that this facilitates between distinct signaling pathways and the ability to further modulate the signal cascade inside the cell after the initial receptor binding (Figure 2).
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GROWTH FACTORS (FGFs, PDGF, VEGFs, and IGF) AND FRACTURE REPAIR: REGULATION VIA THE RECEPTOR
TYROSINE KINASE PATHWAYS
Fibroblast growth factors (FGF). Fibroblast growth factors and corresponding receptors (FGFRs) are known to play important roles during bone development. FGF signaling is essential for maintaining bone homeostasis and during fracture healing. The FGF family currently comprises over 20 structurally related members that bind to tyrosine kinase transmembrane receptors. Upon binding FGFR on its extracellular ligand-binding domain, FGF causes the dimerization of receptor monomers, leading to autophosphorylation of tyrosine residues on the intracellular signal transduction domain [21]. Alternative downstream signal transduction pathways have been described that basically imply the activation of the mitogen-activated protein kinase (MAPK) signaling [22]. The FGFR family consist of four distinct but highly homologous transmembrane proteins (FGFR1 — FGFR4), which act as high affinity receptors for the FGF ligands. Each full-length FGFR contains a signal peptide, three extracellular immunoglobulin-like domains, an acid box domain ( a contiguous box of acidic residues withing the linker domain between Ig1 and Ig2), a transmembrane domain, an intracellular juxtramembrane domain and an intracellular split tyrosine kinase domain. The third immunoglobulin domain provides for FGF ligand specifically, while the acid box confers the ability for glycosaminoglycan modulation of the receptor at a serine residue immediately N terminal to this domain. Presence of the first immunoglobulin domain (Ig1) can prevent receptor glycosaminoglycan modification through steric hindrance and inhibit signaling. Signaling involving exons encoding the first immunoglobulin domain and the acid box determines if these domains will be present in the mature FGFR protein. Fibroblast growth factors are secreted glycoproteins that are commonly sequestered in the extracellular matrix by heparin sulfate proteoglycans. Heparinase or protease liberated FGFs stimulate a diverse array of biologic responses by binding and activating cell surface FGFRs. The majority of FGFs bind with high affinity to FGFRs to stimulate downstream signaling only in the presence of heparin or heparin-like moieties, such as cell surface-bound heparin sulfate glycoproteins or addition of glycosaminoglycan moieties to the receptor. Crystallographic and biochemical studies support a structural model that incorporates two FGFs with two heparin moieties and two FGFRs in symmetric complex [23]. This structure may explain why distinct heparin sulfate motifs are required to elicit the activation of different FGF/FGFR pairs. Upon activation, FGFRs elicit downstream signaling via receptor dimerization, autophosphorylation and recruitment of docking and signaling proteins at the plasma membrane (Figure 2, A). FGFR1 and FGFR2 can directly bind to activate PLC, and can indirectly activate the Ras/Raf/MEK/MAPK signaling pathway [24]. Receptor autophosphorylation of a tyrosine at position 766 in FGFR1 and 769 in FGFR2 creates a specific binding site for the SH2 domain of PLC. Activated PLC hydrolyzes phosphatidyl inositol to form diacylglycerol (DAG) and inositoltriphosphate (IP3), which, in turn, stimulate intracellular calcium release and the activation of protein kinase C (PKC).
Ras activation is achieved through recruitment and tyrosine phosphorylation of the docking protein Grb2, followed by binding and activation of adaptor protein SOS. MAPK activation can also be stimulated via Grb2-bound, atypical PKCs. In either scenario, MAPK activation is dependent upon the binding and tyrosine phosphorylation of Grb2 by the FGF receptor. This major downstream signaling cascades include signals generated through the Ras/Raf (retrovirus associated DNA sequences/factors), MEK (MAP kinase kinase) and MAPK pathway (Figure 2, A). Upon phosphorylation, MAPK translocates to the nucleus where it functions to regulate gene expression by phosphorylation transcription factors (RUNX2/ Cbfa-1). Both FGF1 and FGF2 can be detected in the early stages of fracture repair in the granulation tissue at the fracture site [22, 25]. Macrophages and other inflammatory cells express FGFs and this is the likely source of FGF in the granulation tissue. Subsequently, FGFs are expressed by mesenchymal cells, maturing chondrocytes and osteoblasts and have been demonstrated to enhance TGF-.H expression in osteoblastic cells. The FGFs primarily function as mitogens on a variety of mesenchymal cells including fibroblasts, chondrocytes and osteoblasts. In the case of FGF1, the mitogenic effects of this molecule appear to be primarily on chondrocytes. This is suggested by the expression profile of FGF1 that peaks during chondrogenesis. In addition, FGF2 was tested in rats using a standard closed bilateral femoral fracture model. Injection of FGF2 (percutaneus) during the first 9 days post fracture was associated with an increased callus size, much of which was attributed to increased proliferation of chondrocytes [26]. FGF2 (basic FGF) is generally more potent than FGF1 and has been accorded much more attention in fracture repair studies [22, 27]. FGF2 is expressed by osteoblasts and has also been detected in the upper hypertrophic zones of the growth plate during development, an observation that suggests a role in chondrocyte maturation and endochondral bone formation. In addition to its mitogenic and angiogenic properties, FGF2 also stimulates bone resorption. These properties suggest that FGF2 has the potential to influence many phases of fracture repair, from early post-traumatic events to late remodeling of the callus [28]. This hypothesis is supported by the in vivo data. In a canine tibial osteotomy model, a single injection of FGF2 was associated with an early increase in callus size [29]. This stimulation was attributed to the stimulation of periosteal progenitor cell proliferation and was associated with increased mechanical strength at 16 weeks post fracture. FGF2 treatment was also associated with a more rapid resolution of the soft callus, such that at 32 weeks post fracture there was no difference in mechanical strength between fractures treated with FGF2 or with vehicle. In a fibular fracture model, FGF2 was injected at the time of fibular fracture to the fracture site of normal rats and rats were rendered experimentally diabetic via streptozotocin treatment. In these studies, FGF2 dose-dependently increased the volume and the mineral content of the callus and improved the mechanical properties of the healing fractures [30]. Similar results
have been obtained by using FGF2 suspended in a viscous gel formation of hyaluronan, an extracellular matrix component, and given as a single administration to osteotomies generated in the fibulae of baboons [31]. Unlike most other growth factors, there appears to be a positive correlation between FGF2-induced fracture callus size and mechanical strength. It is tempting to speculate that the stimulation of fibroblast and osteoblast proliferation by FGF2 would result in enhanced collagen synthesis within the callus and concomitantly increase its mechanically stability. This possibility requires further direct study.
Vascular endothelial growth factor (VEGF). Vascular endothelial growth factor is a homodimeric glycoprotein that shares almost 20% amino acid homology with platelet-derived growth factor. VEGF exists in 5 isoforms resulting from alternative splicing of its mRNA, with chain lengths of 121, 145, 165, 189 and 206 amino acids [32]. These 5 forms are commonly referred to as VEGF-A, VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PlGF). The various VEGF forms bind to two tyrosine kinase receptors, VEGFR-1 and VEGFR-2, which are expressed almost exclusively in endothelial cells. These receptors are characterized by the presence of seven immunoglobulin-like domains in their extracellular parts and can therefore be regarded as a new subfamily of tyrosine kinase receptors [33]. Vascular endothelial growth factor is a potent angiogenic factor that plays an important role during skeletal development and fracture healing. In developing mice, blocking VEGF activity results in an enlarged area of hypertrophic cartilage, loss of metaphyseal blood vessels, and impaired trabecular bone formation [34]. Thus, during development, VEGF is essential for normal growth plate morphogenesis, including blood vessel invasion and cartilage remodeling. VEGF has also been implicated in bone repair. During bone repair, VEGF is expressed in a similar pattern as that which occurs in development. The angiogenic activity of the hematoma (which does not exist during development) and the plasma from injured individuals are due primarily to VEGF. Inhibition of VEGF activity delays bone repair in mice and decreases blood flow and leads to non-unions in rabbits [35]. Thus, during bone repair, VEGF is required not only for blood vessel formation, but also for normal callus volume and mineralization [36]. These results indicate that normal angiogenesis is central to tissue repair, and that VEGF may be the major signal to couple angiogenesis and osteogenesis during bone repair [20]. VEGF may be at least one of the mediators of growth factor stimulation of bone repair in animal models and clinical trials. Most osteo-inductive growth factors, as well as ultrasound, induce VEGF expression. In fact, inhibition of VEGF blocks angiogenesis induced by either FGF2 or BMP2 and induction of primary osteoblast differentiation by BMP7 (OP-1) or bone formation by BMP4. VEGF affects chemotaxis, proliferation, survival and activity of several cell types, including endothelial cells, osteoblasts and osteoclasts [35]. Consistent with the fact that endogenous VEGF is important for normal bone repair, exogenous VEGF can promote angiogenesis and bone formation in mouse femur fractures and
rabbit radial critical-sized defects, and can synergize with BMP4 [37]. VEGF treatment has been shown to increase bone blood flow in radial fractures and during tibial distraction osteogenesis. Finally, VEGF can increase bone formation and decrease bone resorption in intact rabbit femurs [35].VEGF can stimulate endothelial cells to synthesize osteogenic factors and thus indirectly promote bone formation. In addition, osteoblasts make and respond toVEGF [38, 39]. Many pro-osteogenic factors stimulate VEGF production by osteoblasts [35, 37], and VEGF receptor expression is regulated during osteoblast differentiation [34, 35]. VEGF can induce osteoblast chemotaxis, proliferation, differentiation, and cAMP production. VEGF stimulates bone formation in organ cultures and in vivoduc, at least in part, to direct effects on osteoblasts [20, 40]. Inhibition of VEGF impairs in vitro osteoblast differentiation and bone growth ex vivo [40]. VEGF can also have direct effects on bone-resorbing osteoclasts. In both wildtype and osteopetrotic (op/op) mice, VEGF regulates normal osteoclastic resorption during endochondral ossification [41], by affecting osteoclast recruitment, survival, and activity and differentiation [40]. While VEGF is likely involved in lamellar bone remodeling in mouse fractures, VEGF may have a different role in resorption in intact rabbit femurs [37]. Inhibiting endogenous VEGF or adding exogenous VEGF can also alter cartilage tissue through effects on chondrocyte apoptosis and differentiation and cartilage resorption. These in vivo effects of VEGF on cartilage may be indirect through its stimulation of vascularity and cartilage resorbing cells. However, VEGF treatment induces phosphorylation of VEGF receptors in hypertrophic chick chondrocytes, suggesting that chondrocytes may respond directly to VEGF.
Platelet-derived growth factor (PDGF). PDGF is a dimeric molecule consisting of disulfide-bonded A- and B-polypeptide chains. PDGF can exist either as a homodimeric (PDGF-AA, PDGF-BB) or heterodimeric form (PDGF-AB) according to the relative levels of each subunit generating a level of ligand complexity in cells in which both polypeptides are expressed [41]. The different PDGF isoforms (AA, AB, BB) exert their effect on target cells by binding with different specificity to two structurally related protein tyrosine kinase receptors (Figure 2, A), denoted as the a-receptors and H-receptors [41]. The PDGF receptors are activated by ligand binding and subsequent receptor dimerization. Because each subunit of the dimeric PDGF molecule contains a receptor-binding site, one complete PDGF molecule binds two receptor molecules simultaneously. The receptors also display polypeptide preference in that the a-receptor will bind either an A-chain or B-chain but the H-receptor only binds the B-chain. Thus, PDGF-AA induces aa receptor dimmers, PDGF-AB induces aa or a. receptor dimmers, and PDGF-BB induces all three possible combinations. Receptor dimerization leads to autophosphorylation, which regulates the intrinsic tyrosine kinase activity. Downstream signal transduction molecules associate with activated PDGF receptor complexes through their Grb homology
2 domains (SH2) (Figure 2, A). Molecules demonstrated as able to bind with PDGF a-receptors and ^-receptors include phosphatidylinositol 3 kinase, phospholipase C, the Grb/Sos family of tyrosine kinases, Ras/Raf, and signal transducer and activation of transcription MAPK/ERK pathway. Platelet-derived growth factor enhances DNA synthesis, increases collagen deposition, and stimulates synthesis of extracellular matrix [17]. In vitro, PDGF has been shown to stimulate type I collagen production and messenger RNA expression in osteoblasts and chondrocytes [17, 20]. Platelet-derived growth factor has enhanced chemotactic and proliferative effects and the ability to initiate differentiation of osteoprogenitor cells toward an osteoblastic lineage [5]. Platelet-growth factor functions in a macrophage autocrine feedback loop stimulating production and release of growth factors or cytokines [7]. PDGF is initially released by degranulating platelets in the fracture hematoma and may be important in promoting chemotaxis [13]. In this early stage PDGF-A is more abundant than PDGF-B and most cells appear to be making the homodimeric PDGF-AA. PDGF is also expressed by macrophages that migrate into the fracture site in response to the fracture trauma and initial release of PDGF by platelets. Later in fracture repair, PDGF protein is detectable in both early and mature hypertrophic chondrocytes, although this is primarily PDGF-A. Osteoblasts express only PDGF-B, suggesting the PDGF-BB form is the primary isotype in these cells [5, 7]. Studies using of exogenous PDGF/BB in vivo include a unilateral tibial osteotomy model in rabbits. PDGF or vehicle was suspended in a collagen carrier and injection into osteotomies. PDGF treatment was associated with a subjective increase in callus density [42, 43]. After 28 days post surgery, PDGF-treated osteotomies were as strong as non-operated control tibiae, whereas vehicle-treated osteotomies were still weaker than controls. These PDGF effects were also associated with an earlier return to normal weight bearing. This pilot study used a small number of animals, and these promising results need further support through larger studies. The mechanisms by which exogenous PDGF might influence fracture repair have yet to be defined. In studies Arvidson and colleagues [17] analyzed the effect of daily injection of PDGF into uninjured newborn rat femurs. Their findings suggest that PDGF initiated osteogenesis and chondrogenesis processes. Daily injections of PDGF resulted in a dose-dependent increase in mesenchymal cell proliferation with a mass of new bone formation. Analysis the expression of PDGF along with a- and ^-receptor messenger RNA to further elucidate its role in the inflammatory phase (days 2-4) after fracture [44]. These investigators theorized that the function mediated by the ^-receptor, including cell migration, might be a prerequisite to the recruitment of mesenchymal cells in the initial step and to the interaction between osteoclasts and osteoblasts in the bone remodeling phase. Platelet-derived growth factor has been identified at fracture sites in humans throughout the stages of healing. Many investigators demonstrated PDGF expression from many cell types throughout a normal human healing fracture
process including endothelial and mesenchymal cells in granulation tissue and osteoblasts, chondrocytes, and osteoclasts during later stages of fracture healing [2, 7, 17, 20]. One study demonstrated that PDGF actually inhibits the bone regeneration induced by osteogenin (BMP3), in a rat cranial defect model [41]. The mechanism for this inhibition was attributed to a significant stimulation of soft tissue repair with PDGF, which may have mitigated the effects of BMP3 on osteogenesis. These studies shed light on the potential for PDGF to influence bone formation and on its limitations in effecting the full cascade of events required for fracture repair.
Insulin-like growth factor (IGF).Growth hormone and insulin-like growth factor play critical roles in skeletal development and bone fracture repair. Growth hormone participates in the regulation of skeletal growth and triggers the release of insulinlike growth factor in target cells. The insulinlike growth factors are bound to binding proteins, adding another crucial tier to modulate the activity of IGF. Two insulin-like growth factors have been identified — insulin-like growth factor-1 and insulinlike growth factor-2 both of which are found in high concentration in serum. In bone, whilst insulinlike growth factor-2 is more abundant, insulin-like growth factor-1 may be more potent, although this might be different both between and within species. Insulin-like growth factor (IGF-1) is a potent anabolic growth factor. IGF-1 activity is mediated by the IGF type 1 receptor (IGF1R) a ligand-dependent tyrosine kinase receptor. The binding of IGF-1 to the IGF1R activates the autophosphorylation of its cytoplasmic kinase domain (Figure 2, A) which through interaction with various docking proteins, including the insulin-receptor-substrate-1 and Grb2, activates downstream signaling pathways. The two main signaling activated by IGF1R are the Ras/Raf/MAPK pathway. Insulin-like growth factor 1 stimulates proliferation of osteoblast precursors and early- stage osteoblasts and promotes bone matrix formation by fully differentiated osteoblasts [19]. In vivo, endogenous and exogenous IGF has been associated with induces of active bone matrix production. Insulin-like growth factor expression was also greatest in osteoblasts that were involved in active bone remodeling. The data suggest that IGF is involved in cell proliferation or differentiation of mesenchymal cells, periosteal cells, osteoblasts, and chondrocytes by way of an autocrine or paracrine fashion. The regulation of insulin-like growth factor is complex, and the growth hormone mode of action in skeletal cells is largely unknown. Of the major hormones that regulate the skeleton, all have significant effects on skeletal IGF, as do many growth factors, such as BMP-2, TGF-fl and FGF [45]. Insulin-like growth factors increase proliferation and play a major role in stimulating mature osteoblast function. As with other growth factors detailed in this section, the way that osteoblasts respond to IGF signals may well depend on both the differentiation status of the cell and cell type. At the molecular level, insulin-like growth factor-1 upregulates the osteoblast-associated transcription factor, Osterix, but not RUNX2/Cbfa1. In addition, insulin-like growth
factor-1, in combination with bone morphogenetic protein -2 (BMP-2), acts synergistically on Osterix expression [45]. Although it is widely accepted that insulin-like growth factors have a defining role in bone remodeling, their actual role is still unclear and needs to be understood within the complex interrelationships of the components of the IGF system that evidently occur in vivo. Overall, the evidence suggests that the major effects of insulin-like growth factors are to promote the late-stage differentiation and activity of osteoblasts.
GROWTH FACTORS (TGF-.H AND BMPS) AND FRACTURE REPAIR: REGULATION VIA RECEPTOR SERINE-THREONINE KINASE PATHWAYS
Transforming growth factor-H (TGF-H). The transforming growth factor-^ molecules are members of a large family of secreted factors collectively referred to as the TGF-.H superfamily. This superfamily contains not only the TGF-.H isoforms but also the bone morphogenetic proteins (BMPs) and activins. All members of the TGF-H superfamily are synthesized as large precursors which are proteolytically cleaved to yield carboxy-terminal mature protein dimmers. These evolutionarily conserved molecules play important roles in cell differentiation and proliferation during development and appear to play a variety of regulatory roles in the adult organism. Five isoforms of TGF-H have been identified to date [18]. Two isoforms, TGF-H1 and TGF-H2, have received the most attention regarding fracture repair and for discussion purposes may be referred to collectively as TGF-H [46]. TGF-H signaling involves two receptor types, TGF-H receptor type I and type II (Figure 2, B). Most cells within the fracture site as well as elsewhere in the body express TGF-.H receptors on their surface. The specificity of downstream signals is generated according to which of the various type I receptors is associated with the receptor ligand complex. TGF-.H ligand initially binds an oligomeric form of type II receptor followed by the recruitment of a type I receptor into the complex, possibly also in an oligomeric form, resulting in a heterotetrameric receptor complex associated with the ligand. The type II receptor has a constituitively active serine-threonine kinase activity, which phosphorylates the type I receptor in the glycine and serine —rich domain, thus activating the type I receptor serine-threonine kinase activity. The activated type I receptor kinase is responsible for the downstream transmission of the signal through the superfamily signal effector (SMAD) family of molecules. Signaling through the downstream SMAD family of molecules is characteristic of the TGF-.H superfamily member receptors (Figure 2, B). SMADs, a family of proteins, are important mediators in the TGF-H signaling cascade. SMAD2 and SMAD3 are bound to SARA (SMAD anchor for receptor activation) in the cytoplasm which presents SMAD2 and SMAD3 to the activated TGF-.H receptor complex. TGF-.H type I receptor then directly phosphorylates the carboxy terminal of SMAD2 and SMAD3, resulting
in decreased affinity to SARA and heterotrimerization of SMAD2 and SMAD3 with SMAD4. This entire complex then translocates into the nucleus via the nucleoporins within the nuclear pore complex, and transcriptionally regulates multiple effector genes. The SMAD2/3/4 complex's stay within the nucleus is transient, as it becomes dephosphorylated, and shuttled back out to the cytoplasm, where it becomes rephosphorylated to repeat its trip once again. In addition, several other lines of evidence point to the involvement of MAPK signaling pathways in transmitting TGF-.H signals from receptor to nucleus. In vitro kinase assays have demonstrated that TGF-.H can activate all three MAPK pathways, leading to ERK, c-Jun N-terminal kinase (c-JNK) and p38 MAPK and phosphorylation of members of the c- Jun,c- Fos, c-Myc and transcription factor families, which homo — and heterodimerize to form the activator protein (AP-1) (see reviewed in Ref.18). Crosstalk between SMAD and MAPK pathways adds to the complexity of TGF-.H signaling. Signaling by TGF-.H family proteins regulates the differentiation and function of the bone-matrix-depositing osteoblasts and of the bone-matrix resorbing osteoclasts, as well as the cross-talk between both cell types, which controls bone remodeling and homeostasis [47].
Roles of TGF-fl family in bone remodeling. Bone remodeling is a complex process involved a number of cellular functions directed toward the co-ordinated resorption and formation of new bone. Bone remodeling is regulated by systemic hormones and by local factors [48]. Hormones regulate the synthesis, activation, and effects of the local factors that have a direct action on cellular metabolism, and they modify the replication and differentiated function of cells of the osteoblast or osteoclast lineage. Throughout life, bone tissue is continuously remodeled by the balanced processes of bone resorption and consecutive bone formation. Formation, deposition, and mineralization of bone tissue are executed by the osteoblasts that differentiate from mesenchymal precursor cells. The key transcription factor that drives the mesenchymal precursor cell toward the osteoblast lineage and controls bone formation is RUNX2 (Cbfa 1), which regulates the expression of all known marker genes expressed by the osteoblast [49]. Bone resorption by the osteoclasts involves demineralization of the inorganic matrix by acidification followed by enzymatic degradation of the organic matrix by cathepsin K and matrix metalloproteinases [50].Osteoclasts are large, multinucleated cells of hematopoietic origin that differentiate from monocyte/macrophage precursor cells within the bone environment. The recognition that osteoclast differentiation requires the presence of marrow stromal cells or osteoblasts led to the discovery of the two osteoblast-derived factors essential and sufficient to promote osteoclastogenesis: macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL). Upon binding to their respective receptors on the osteoclast precursor cell surface (c-fms and RANK), two prominent transcription factor complexes, the NF-kB and NFATc-1 proteins, are activated, and signaling cascades essential for proper
osteoclast differentiation, fusion, function, motility, and survival are initiated (Figure 3). Osteoblasts also secrete a soluble inhibitor of osteoclast differentiation, osteoprotegerin (OPG), which acts as a "decoy" receptor for RANKL. OPG inhibits activation of the RANK receptor [49]. A balance of these osteoclast promoting and inhibitory signals allows calibration and coordination of bone deposition and bone resorption [51]. A pivotal role in the bone-remodeling process has been assigned to TGF-.H because it was proven to affect both bone resorption and formation. Bone formation by TGF-.H is promoted through chemotactic attraction of osteoblasts, enhancement of osteoblast proliferation and the early stages of differentiation with production of extracellular matrix proteins that compose the bone matrix, e.g. type I and II collagen, osteopontin, and osteonectin, as well as by the expression of the osteoblast differentiation markers, alkaline phosphatase (ALP) and, in a later stage osteocalcin. To better understand the complex roles of TGF-H in bone metabolism, Karst M. et al. [52] examined the impact of a range of TGF-H concentrations on osteoclast differentiation. In co-
cultures of support cells and spleen or marrow osteoclast precursors, low TGF-H concentrations stimulated while high concentrations inhibited differentiation. Authors investigated the influences of TGF-.H on macrofage colony stimulating factor (M-CSF), receptor activator of NF-kB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M-CSF and RANKL expression with a dramatic increase in OPG (see Figure 3). From their findings, they conclude that osteoclast differentiation is stimulated at low TGF-.H concentrations because both the RANKL to OPG ratio and M-CSF levels are high. In contrast, at high TGF-H concentrations, the RANKL to OPG ratio is repressed as TGF-.H suppresses RANKL expression and increases OPG expression by the osteoblast [52]. In combination with the dose-dependent inhibition by TGF-H of M-CSF expression, this results in inhibition of osteoclast differentiation. Regarding the diversity of processes in which TGF-H is involved, it is not surprising that this cytokine is of major importance both during embryogenesis and in maintaining bone homeostasis during life.
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Role transforming growth factor-^ in bone fracture healing. TGF-.H is pleiotropic growth factor initially released by the degranulating platelets in the hematoma and the bone extracellular matrix at the fracture site [53]. In the initial stages of fracture repair, TGF-.H can be immunolocalized to the region of the hard callus where it defines the region of periosteal proliferation and intramembranous bone formation. Evidence suggests that TGF-.H is likely to be primarily involved in the stimulation of proliferation by the preosteoblasts in this region. In addition, the expression of TGF-.H is elevated during chondrogenesis and endochondral bone formation with an initial peak in mRNA levels detected around day 6 post fracture followed by a nadir at day 10. TGF-.H expression peaks again by day 14 and remains elevated until week 4. The nadir of TGF-.H expression correlates with the peak in type II collagen expression, and the subsequent peak temporally coincides with chondrocyte hypertrophy [54]. TGF-.H is primarily thought to be a stimulator of undifferentiated mesenchymal cell and chondrocyte proliferation and extracellular matrix production during chondrogenesis and endochondral bone formation (see Table). TGF-.H may also be involved in the normal coupling of bone formation with resorbtion [53]. The role of endogenous TGF-.H in normal fracture repair is inherently difficult to resolve. However, the importance of TGF-.H to this process is implied by the ability of exogenous TGF-.H to stimulate fracture repair in several models. The ability of TGF-.H to stimulate long bone healing was first demonstrated in midtibial osteotomies in rabbits treated with a compression plate. Continuous infusion of the osteotomy size with high doses of TGF-.H (110 |g/day) for 6 weeks resulted in a dose-dependent increase in callus volume and increased mechanical strength compared with untreated osteotomies. In a rat tibial fracture study, TGF-.H (4- 40 ng) was injected around the fracture site every 2 days during a 40-day healing period. TGF-.H dose dependently increased the cross-sectional area of the callus, and mechanical testing demonstrated a higher ultimate load in fractures treated with the high dose of TGF-.H [55]. The results of these studies suggest that the ability of TGF/- to stimulate fracture repair may require persistent dosing or very high concentrations.
Bone morphogenetic proteins (BMPs).The BMPs are a subfamily of the TGF-.H superfamily of polypeptides. The BMPs play critical roles in regulating cell growth, differentiation, and apoptosis in a variety of cells during development, including osteoblasts and chondrocytes. Compared with TGF-.H, BMPs have more selective effects on bone and also have shown more promising results in animal models of fracture healing. BMP signal transduction occurs by a mechanism similar to the other members of the TGF-.H superfamily (Figure 2). BMP ligand can associate with several serine-threonine kinase receptors, including BMP receptor type II, receptor type IA, and receptor type IB as well as the related activin receptors (ActR-II, ActR-I) [56]. As with TGF-.H, the BMP ligand binds to the type II receptor, and this receptor occupancy leads to association of the complex with an appropriate type I receptor forming
an active receptor-ligand complex. This interaction can be blocked by the antagonists of BMPs, noggin and chordin, which can bind and block BMP activity by preventing receptor binding [57, 58]. This antagonist function of noggin and chordin has been specificallz demonstrated in osteoblastic cells. The expression of the BMP receptors is dramatically increased in osteogenic cells of the periosteum near the ends of the fracture in the early post fracture period. Therefore, BMP signaling involves a complex receptor pattern in addition to the multitude of BMPs expressed during fracture repair. BMP receptor signaling, as with the TGF-^s, is transmitted through the SMAD family of signal effectors, again providing for a high degree of cross-talk between signals generated by multiple members of the TGF-.H superfamily of polypeptides [55]. During fracture repair, the BMPs reported to be expressed include BMP-2, BMP-3 (osteogenin), BMP-4 and BMP-7 (osteogenetic protein, OP-1). Several reports have demonstrated that BMPs are expressed in the early stages of fracture repair where it is likely that small amounts are released from the extracellular matrix of the fractured bone (Table) [59].During intramembranous bone formation, osteoprogenitor cells in the cambium layer of the periosteum may respond to this initial low level of release from the extracellular matrix and begin differentiating. BMP-4 mRNA levels do transiently increase in osteoprogenitor cells in this region, and immunolocalization demonstrates an increase in detectable BMP-2 and BMP-4 near the fracture ends in the cambium region of the periosteum. By days 7-14 post fracture, the expression of BMP-2 and -4 is maximal in chondroid precursors, while hypertrophic chondrocytes and osteoblasts only show moderate levels of expression. The current view of the role of BMPs in fracture repair is that these molecules are primarily activators of differentiation in osteoprogenitor and mesenchymal cells destined to become osteoblasts and chondrocytes (Table). This activation by BMPs, specifically BMP-2, is inhibited by the molecules noggin and chordin which have been demonstrated to block BMP-2 interaction with its receptor [60]. As these primitive cells mature, BMP expression is dramatically reduced. BMP expression emerges transiently in chondrocytes and osteoblasts during their respective periods of matrix formation, and returns to low levels during callus remodeling. It is interesting to note that while mature osteoblasts and chondrocytes do not express significant levels of BMPs in normal bone, they both have greatly increased BMP expression later in fracture repair.
Сonclusion
The explosion of knowledge and the understanding of the role of growth factors, their mechanisms of action and molecular signaling pathways, which have been reviewed in this article, suggest the potential for many novel therapeutic targets, not only for applying growth factors but also for the potential use of growth factor inhibitors or agents that target specific parts of the intracellular signaling pathways. There remains an enormous challenge to convert some of the
knowledge from basic studies of bone cell physiology in real qualitative improvement in clinical outcomes to therapeutically useful techniques for the future. We over currently available techniques. are optimistic that such novel approaches may result
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