Growth Factors

Periodontology 2000, Vol. 41, 2006, 4872 Printed in Singapore.
All rights reserved
Copyright Blackwell Munksgaard 2006
PERIODONTOLOGY 2000
Effects of growth factors and cytokines on osteoblast differentiation

F R A N C I S J. H U G H E S , W E N D Y T U R N E R , G E O R G I O S B E L I B A S A K I S & GIANLUCA MARTUSCELLI
The therapeutic management of new bone formation remains one of the key issues in periodontology and dental implantology. A full understanding of the nature of the bone-forming cells (osteoblasts) and their lineage, the factors that may regulate osteoblast behavior and bone formation locally, and how these different regulatory mechanisms could interact, has been the subject of extensive study for many years, particularly with the aspiration that such an understanding will lead to the development of novel biologically based therapies for the management of bone regeneration, not only in dental applications but also for many other disciplines, such as orthopedic surgery, and maxillofacial and craniofacial surgery. The aim of this review was as follows. to describe the nature of the osteoblast phenotype, its lineage and the genetic regulation of osteoblast differentiation and function. to discuss the role of a range of cytokines and growth factors in regulating osteoblast differentiation and function. to review how some of these cytokine pathways may be regulated and interact with each other in the co-ordinated control of osteoblast function. to review, briey, studies that have investigated the pharmacological application of cytokines for therapeutic management of bone formation locally in vivo. Cytokines are soluble, secreted glycoproteins which act as local signaling molecules to control and co-ordinate cellular behavior and function. They function as ligands which bind to cell-surface receptors, triggering a series of intracellular signaling events, ultimately resulting in the modulation of gene expression in their target cells. Growth and differentiation factors are generally considered to be a subset
of cytokines with anabolic physiological and pathophysiological roles in the regulation of tissue growth and healing. Although this terminology is widely used, the distinction between growth factors and cytokines is probably an articial one, particularly given the different complex roles that these molecules may all have on cell and tissue function. However in keeping with convention, here we have used the term growth factors to describe this distinct subgroup of cytokines, and have separately considered the role of cytokines which are particularly associated with inammation on bone cell regulation.
Osteoblast lineage and function

The osteoblast phenotype
Osteoblasts are specialized mesenchymal-derived cells whose function is the deposition and maintenance of skeletal tissues. As with any cell type, they are dened ultimately by their expression of genes that are a subset of the total genome and which dene their unique function. Specically, osteoblasts express the genes coding for the bone matrix proteins, high levels of the tissue nonspecic form of alkaline phosphatase and are also frequently characterized by their expression of parathyroid hormone/parathyroid hormone receptors, which results in a typical cAMP response on stimulation with the osteotropic parathyroid hormone. The bone matrix proteins include type I collagen and a number of other matrix proteins, which to a greater or lesser extent are characteristic or specically expressed in bone. Collagen type I makes up 90% of
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the organic matrix of bone and provides the strength, structure and elasticity of the mature bone tissue. The noncollagenous proteins of bone include osteocalcin (bone GLA protein) osteopontin, osteonectin and bone sialoprotein. In addition, the proteoglycans decorin and biglycan are signicant components of the bone organic matrix. Although a full description of the function of the different matrix constituents of bone is beyond the scope of this review, these proteins collectively have calcium-binding activity, which is probably responsible for the exquisite regulation of hydroxyapatite crystal deposition, orientation and specic crystal size seen in the mineralized bone matrix. In addition, it is known that bone sialoprotein and osteopontin contain the ArgGlyAsp (RGD) peptide cell-attachment sequences and can mediate attachment of both osteoblasts and osteoclasts to the bone matrix. Of the noncollagenous proteins of bone, osteocalcin is considered to be the most bone-specic. Expression of osteocalcin in the mature organism is restricted to bone, dentin and cementum. Evidence particularly from mouse knockout models suggest that the bone matrix proteins play an important role in regulating the overall control of bone mass. For example, the osteocalcin knockout mouse exhibits an overall increase in bone mass as its main phenotype (54). The expression of bone-specic genes and proteins are valuable markers for demonstration of bone osteoblast phenotype in vitro, and understanding of the transcriptional regulation of these genes is of fundamental importance in the regulation of osteoblast differentiation and function.
The osteoblast lineage

Osteoblasts are derived from multipotent mesenchymal stem cells that give rise to progenitor cells of restricted osteoblast lineage and which may then undergo a series of proliferation/amplication stages before expressing recognizable specic osteoblastic markers. Early studies of the kinetics of normal osteoblast turnover using pulse-chase 3H-thymidine labeling demonstrate the recruitment of osteoprogenitor cells from the surrounding connective tissues toward the periosteal layer on the surface of the bone (188, 189). The periosteum consists of two layers in these studies: a broperiosteum on the outside most distant from the bone surface; and an inner osteogenic periosteum where the rst directly recognizable cell of the differentiating osteoblast lineage, the preosteoblast, is found. Pre-osteoblasts retain a prolif-
erative capcacity but express many of the proteins associated with the mature osteoblast phenotype, including alkaline phosphatase and osteopontin. The mature osteoblast layer lies immediately adjacent to the bone and is a post-mitotic cell expressing all of the features of the mature phenotype (189). These descriptions give rise to the concept of recruitment of proliferating osteoprogenitor cells from adjacent connective tissues, which undergo further differentiation, resulting in the expression of the mature phenotype. These studies also suggest that mature osteoblasts have an average lifespan of 1 month, after which either they undergo apoptosis, to be replaced by newly differentiated osteoblasts or alternatively about one-third of these cells may be incorporated into deposited bone matrix as osteocytes. The osteocyte is thus the most differentiated cell of the osteoblast lineage and may persist in bone matrix for very lengthy periods of time. Although its functions are not fully understood, there is compelling evidence that the osteocyte has a critical role in the maintenance of bone mass and is the principal bone cell responsible for mechanotransduction. It is well established that maintenance of bone mass relies on regular mechanical stimulation. It is thought that the subtle exing seen in bones in response to any loading causes pulsatile uid ow through osteocyte canaliculi, resulting in signal transduction by osteocytes in order to retain bone mass (24, 128, 228). The precise molecular mechanisms whereby this occurs are unknown. However, mechanical strain and pulsatile uid ow are able to induce signaling molecules, such as nitric oxide, by osteocytes in vitro (127, 194). Interestingly, the recently described bone morphogenetic protein (BMP) inhibitor, sclerostin, has also been localized specically to osteocytes and may contribute to the regulation of overall bone mass (264). Around the periodontal tissues, similar pulse chase 3H-thymidine-labeling experiments demonstrate the continual recruitment of proliferating cells from bone marrow stroma through vascular channels that communicate with the periodontal ligament. These cells appear to preferentially migrate to the osteoblast (and cementoblast) surfaces, consistent with the recruitment of osteoprogenitor cells from stem cells located in the bone marrow stroma through to the periodontal ligament and ultimately undergo osteoblastic differentiation, akin to the kinetics described in other bony tissues (159). Thus, the periodontal ligament is constantly repopulated by relatively undifferentiated mesenchymal cells and is a
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rich source of progenitor cells capable of giving rise to new bone formation (see Fig. 1).
Mesenchymal stem cells

Stem cells are dened as undifferentiated selfrenewing cells. In the mature organism they tend to occur with relatively low frequency and although, by denition, they have extremely high proliferative potential, they are typically not continually cycling. In addition, many (but not all) stem cells have multiple potentials, that is they have the capacity to give rise to a range of differentiated phenotypes. The existence of undifferentiated self-renewing cells is clearly essential for the organism to replenish continually differentiated cells which have reached the end of their normal lifespan. Classic studies by Owen & Fredenstein were the rst to describe the existence of multipotent mesenchymal stem cells isolated from bone marrow stromal cell populations (66, 67, 190). In these experiments it was demonstrated that colonies from bone marrow stroma (and therefore of clonal origin) could give rise to a wide range of connective tissues, including bone, cartilage, muscle, brous and adipose tissues, when re-implanted into a suitable host animal. More formal demonstration of the true multipotential nature of mesenchymal stem cells was subsequently demonstrated in clonally derived cell lines in vitro including the murine embryonic broblastic cell line, C3H10T1/2 (132), and the rat calvaria-derived line, RCJ3.1 (84). These and other multipotent mesenchymal cell lines have been valuable tools for using to investigate the role of a range of different factors that may regulate the commitment of multipotent stem cells to cells of restricted lineages, such as osteoprogenitor cells. In recent years there has been considerable debate about the plasticity of stem cells, that is, the ability of committed progenitor cells to dedifferentiate and subsequently redifferentiate along a different lineage. There is considerable evidence for the existence of bipotent osteoblastadipocyte cells from bone marrow in both human and other species (4, 18, 42) and these also exhibit extensive potential for undergoing cell renewal. These data suggest the possibility that restriction to specic differentiation pathways may not occur as a single event but it is possible for multipotent stem cells to exhibit increasing lineagerestricted specication. Further evidence of this is provided by studies of C3H10T1/2 cells, where subclones have also been shown to exhibit tripotent properties (132) and these potentials have also been
Fig. 1. Demonstration of continual recruitment of progenitor cells from bone marrow to the periodontal ligament. Cycling cells were labeled by a bromodeoxyuridine (BrdU) pulse on day 0 and followed-up for periods of up to 10 days in Wistar rats. (A) Day 0. A bone marrow space is seen within the bone, communicating directly with the PDL via a vascular channel. Labeled cycling cells (red) are largely restricted to the bone marrow stroma. (B) Day 6. Labeled cells have now migrated into the periodontal tissue spaces and are seen adjacent to vascular channel openings into the ligament. (C) Day 10. Labeled cells are relatively evenly distributed throughout the ligament, tending toward the bony surface. B, bone; T, tooth; PDL, periodontal ligament. Original magnication 100 [King GN, King N, Hughes FJ, unpublished, based on experiments described by McCulloch et al. (159)].
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reported in primary marrow stromal cells, particularly as osteoblast/chondrocyte/adipocyte stem cells (2, 187, 195). There is much less clear evidence of cells at an advanced stage of differentiation exhibiting true dedifferentiation and redifferentiation along distinct pathways (132). However, studies suggest that this phenomenon may occur in osteoblasts redifferentiating into chondrocytes. In support of the possibility of a differentiated cell subsequently redifferentiating as a distinct phenotype, it has been shown that osteoblastic cells, when transfected with the tissue-specic transcription factor, PPAR-c 2, can be converted to express an adipocytic phenotype (4). In addition to these complexities of the stem cell system, it has proved extremely difcult to identify specic cells of the mesenchymal lineages with labels that specically reect a particular stage in the stem cell/early commitment phases of connective tissue cell lineages. Typically, in stem cell studies using culture methods, in vitro differentiation potentials are elucidated by directly demonstrating differentiation outcomes and ascribing those to cultured cells in retrospect. In addition, at least one antibody (Stro1) has proved extremely useful in identifying the cells in marrow stromal cell cultures with osteogenic potential (225). Stro-1 staining in combination with alkaline phosphatase expression using uorescenceactivated cell sorting has demonstrated the presence of early osteogenic precursor cells in a Stro-1-positive/alkaline phosphatase-negative population of marrow stromal cells (51, 85, 222). During differentiation it has been shown that Stro-1 expression is lost, whilst alkaline phosphatase expression is upregulated (257). Despite these observations and many other attempts, it has still proven difcult and impossible at the present time to dene a marker or markers that would unequivocally identify a single multipotent mesenchymal stem cell in a mixed stromal cell population.
Transcriptional regulation of osteoblastic commitment and differentiation

Lineage-specic gene expression is ultimately under the control of transcription factors that act to regulate specic gene expression. They act as the key switching mechanisms to induce gene transcription. Considerable progress has been made in identifying those transcription factors which act as master switches during commitment of multipotent cells to specic lineages. Basic helix-loop-helix genes of the myoD family are known to specify commitment and differentiation of mesenchymal stem cells to the
myoblast lineage (260). Similarly, the transcription factor, ppar-c 2, can specify adipocyte differentiation (209), and sox-9 expression is required for chondrocyte differentiation (44). A major breakthrough in the understanding of genetic regulation of osteoblast differentiation was made with the identication of the role of the transcription factor core binding factor 1 (Cbfa-1/Runx-2) (55, 130). Cbfa-1/Runx-2 expression is an absolute requirement for osteoblast differentiation. In cbfa-1 knockout mice there is a normal cartilaginous skeleton seen but a complete absence of bone (and tooth) formation. Cbfa-1/Runx2 is known to interact directly with the osteocalcin promoter to induce its expression (56). However an additional transcription factor, osterix, which is a downstream target for Cbfa-1/Runx-2, has also been shown to be an absolute requirement for normal osteoblast differentiation in knockout mice experiments (170). More recent studies have shown the existence of distinct isoforms of Cbfa-1, which may have subtly different roles during normal tissue formation, including regulation of cartilage expression in addition to bone. The expression of Cbfa-1/Runx-2 occurs quite late in the osteoblast lineage scheme and physiologically probably does not specify osteoblastic commitment events in multipotent stem cell populations. Recently, the role of the transcription factor, TAZ, has been reported, which appears to act by specifying osteoblastic cell fate in bipotent osteoblast/adipocyte stem cells, by activating Cbfa-1/Runx-2 whilst simultaneously repressing the adipocyte transcription factor, ppar-c 2 (95), Experimental overexpression of TAZ in primary mesenchymal stem cells appears to preferentially specify osteoblastic differentiation in mesenchymal stem cells. A number of other transcription factors, whilst not being absolute requirements for osteoblast differentiation, are also known to regulate osteoblast differentiation. These include Msx-2, Dlx-5 and the AP-1 family of transcription factors formed by heterodimers of members of fos and jun families. Although a full review of these molecules is beyond the scope of this review, msx-2 appears to act upstream of Cbfa-1/Runx-2 (91) whilst Dlx-5 may be a transcriptional regulator of late stage osteoblast differentiation acting downstream of Cbfa-1/Runx-2.
Summary
In summary, the osteoblast lineage consists of mesenchymal stem cells which exhibit self-renewal and may include both multipotent cells and those of
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Fig. 2. Schematic diagram of stages in the osteoblast lineage and some phenotypic features seen at different stages of the lineage. Cbfa-1, transcription factor expression. AIP, alkaline phosphatase; AP-1, activator-protein-1; BSP, bone sialoprotein; OSX, osterix; PTH, parathyroid hormone; PTHrP R, PTH-related protein receptor.
more restricted potentials. Commitment of these cells to the osteoblast lineage is likely to be mediated by the transcription factor TAZ, resulting in committed monopotent osteoprogenitor cells with extensive proliferative capacity. Differentiation of the mature osteoblast is ultimately regulated by transcription factors, including Cbfa-1/Runx-2 and osterix. A diagramatic schema of the osteoblast lineage and its features is shown in Fig. 2.
The effects of growth factors on osteoblast differentiation

Huge advances have been made in the understanding of cellular and molecular control of bone formation in the past decade. The establishment of in vitro models of osteoblast differentiation and formation (11) have been essential for determining the effects of specic growth factors and growth factor-induced transcription factors on osteogenesis. These models include the use of primary cell cultures and established cell lines from a range of different species and
with distinct properties (98). Interpretation of the large amount of data from these in vitro models can sometimes be difcult owing to the variations seen in different model systems and the fact that cells may show different responses according to the precise stage of target cells through the lineage at different stages of differentiation. For example, as reviewed below, broblast growth factor-2 treatment of osteoprogenitor cells in vitro results in a mitotic response but suppression of the expression of osteoblast differentiation markers, but paradoxically, when applied in vivo, may result in an overall stimulation of bone formation. This can be explained by the suggestion that broblast growth factor-2 results in the expansion of relatively undifferentiated cells, which is seen as a suppression of differentiation in vitro but ultimately results in increased mature osteoblast numbers in vivo. Tissue engineering aims to produce tissues that are both structurally and functionally identical to the original tissues they are replacing, and strategies for growing bone therapeutically are emerging based on the knowledge of the physiological role of different
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signaling molecules in the bone-forming process. The potential therapeutic application of growth factors in bone tissue engineering is considered further below. Bone formation is directed by the co-ordinated expression of many molecules, including growth factors, BMPs and specic transcription factors, which utilize developmentally derived signals to induce cellular and molecular stimuli to guide cellular commitment and differentiation in the proper spatial and temporal sequence. It is envisaged that future periodontal tissue engineering may involve the use of appropriate gene expression systems for controlled delivery of regenerative growth factors within the appropriate biological scaffold. Growth factors are soluble proteins that act as signaling agents for cells and inuence critical functions, such as cell division, matrix synthesis and tissue differentiation, by receptorligand binding. Results of experimental studies have established that growth factors play many important roles in bone formation and bone repair. Within the periodontal environment, growth factors found in bone, cementum and healing tissues include transforming growth factor-b, basic broblast growth factor, insulin-like growth factors, platelet-derived growth factor and BMPs. Recently, with the advent of recombinant growth factor proteins there has been considerable interest in their use as therapeutic agents in the treatment of bone disorders (including periodontal disease), and as growth factors become available as therapeutic agents, it is essential that we understand their biological characteristics and biological potential for periodontal tissue engineering. During bone formation, a number of candidate growth factors and their respective signaling pathways have been delineated, although there is a paucity of understanding of the complexities in the way that these growth factors interact to regulate bone formation and repair. It seems biologically sensible that there is involvement of multiple bioactive factors in osteoblast development and bone formation, with factors acting in a sequential manner. A number of growth factors, and their downstream molecular targets, have been characterized during osteoblast differentiation. These ndings suggest that at least some of the growth factors reviewed in this article, or their genetic effectors, may be potential therapeutic targets for regenerating bone. Growth factors that are known to affect osteogenic cells include transforming growth factor-b, broblast growth factors, platelet-derived growth factor and insulin-like growth factor.
Transforming growth factor-b

Transforming growth factor-b belongs to a large superfamily of related proteins that also includes BMPs, growth and differentiation factors, activins, inhibitins and anti-Mullerian hormone. All members play important roles in regulating cell proliferation and differentiation and the production of extracellular matrix. There are ve isoforms of transforming growth factor-b (transforming growth factor-b1 to transforming growth factor-b5). Most cells synthesize and respond to transforming growth factor-b, but high levels are found in bone, platelets and cartilage. Transforming growth factor-b1 is the most abundant isoform at the protein level (for a recent comprehensive review see Janssens et al. (111)). The activation of transforming growth factor-b is highly regulated, and once activated it interacts with transmembrane serine/threonine kinase receptors. Pivitol genetic studies identied that intracellular transforming growth factor-b signaling is mediated through mothers against dpp (Mad) in Drosophila melanogaster and subsequently the homologous genes in vertebrates (Smads) have been identied [reviewed in Massague and Chen (158)]. Although Smads are the key mediators in the transforming growth factor-b signaling pathway, BMPs have also been shown to signal through the Ras/mitogen-activated protein kinase (MAPK)/activator-protein-1 (AP-1) pathway [for review see Nohe et al. (176)]. During the early stages of bone formation, the action of transforming growth factor-b is to recruit and stimulate osteoprogenitor cells to proliferate, providing a pool of early osteoblasts (36, 206). In contrast, during later phases of osteoblast differentiation, transforming growth factor-b blocks differentiation and mineralization (154). These effects appear to be highly dependent on bone cell source, dose applied and the local environment, which may be a result of the inhibition of DNA synthesis at high transforming growth factor-b concentrations (35). Additionally, transforming growth factor-b inhibits the expression of the Runx2 and osteocalcin genes, whose expression is controlled by Cbfa1/ Runx2 in osteoblast-like cell lines, and this was found to be mediated by Smad3 (5). Transforming growth factor-b interacts with a range of other growth factors in bone with a resulting complex response. Further work is needed to clarify the role of transforming growth factor-b during periodontal regeneration and wound healing and to determine the inter-relationship between transforming growth factor-b and other growth factors that
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have effects during different stages of osteoblast differentiation.
Bone morphogenetic proteins

Bone morphogenetic proteins are secreted signaling molecules which have a variety of functions during development (92) and cell differentiation (266). They were discovered for their remarkable ability to induce cartilage and bone formation from nonskeletal mesenchymal cells (252, 253) by recapitulating the entire sequence of events occurring during endochondral ossication. They have been subsequently cloned, sequenced and recombinant proteins manufactured (219, 268). These recombinant proteins have been the subject of many studies in vivo and in vitro and it is likely that these recombinant proteins will be used therapeutically in the near future for a range of applications in orthopedics, craniofacial surgery and dentistry (267). Like transforming growth factor-b, BMPs are expressed during embryonic development as well as into adulthood. Bone morphogenetic proteins are synthesized as a precursor molecule, and the active homodimeric or heterodimeric forms of BMP follow release of the C terminal by proteolysis (268). More than 20 BMP-related proteins have been identied, and there is diversity of biological activity as each BMP binds to their receptor with different afnities. Bone morphogenetic proteins bind to two distinct type I and II serine/threonine kinase receptors, both of which are required for signal transduction, although the type I receptor determines specicity of intracellular signals. Once activated, signals are transmitted intracellularly via Smaddependent and Smad-independent pathways [for review see Miyazono et al. (161)]. Among the BMP family, BMP-2, BMP-4 and BMP-7 have key roles during osteoblast commitment and differentiation. The primary effect of BMPs is on the pluripotent cells that are capable of differentiating into other mesenchymal cell types (10, 119, 258), and BMP-2 can direct these cells to commit to an osteoblastic pathway, as can BMP-4 and BMP-6 (102). Bone morphogenetic proteins can also increase the differentiation of committed cells to the osteoblast lineage, with the formation of bone nodules and expression of markers of the mature osteoblast phenotype (39, 102). Gene-expression studies, directly delivering an adenovirus with BMP-7 to gingival or dermal broblasts, leads to a robust osteogenic response (61). In recent years there has been an explosion of interest in potential molecular targets of BMPs, both
from the basic molecular pathway and potential therapeutic perspectives. This has given considerable insight into the genetic cascade leading to bone formation. Among these specic targets are the transcription factors Cbfa1/Runx2 (55), osterix (170) and TAZ (95). Bone morphogenetic proteins can upregulate Cbfa1/Runx2 under certain conditions during osteoblast differentiation; therefore, this is a candidate downstream target of BMPs, although Smad complexes can also directly interact and activate target genes independently of Cbfa1/Runx2 (115). Evidence is also emerging to suggest that some aspects of BMP-2-induced differentiation may be mediated through Wnt/b-catenin signaling, although co-operation between b-catenin and Smads are needed to activate late osteoblast gene expression (13). b-catenin is the classical effector of Wnt protein signaling, suggesting a role for this group of proteins in BMP-2 signal transduction.
Fibroblast growth factor

The broblast growth factors are a family of structurally related polypeptides that are known to play a critical role in angiogenesis and mesenchymal cell mitogenesis. To mediate their range of effects, broblast growth factor proteins signal via membrane-spanning tyrosine kinases (FGFR) and there are a wide variety of mechanisms for receptor regulation and availability. Mutations in these receptors are associated with abnormalities in ossication and activating mutations in FGFR2 cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts (185), highlighting a key role for these molecules in the control of bone formation. In normal adult tissues, the most abundant proteins are broblast growth factor-1 and broblast growth factor-2. Fibroblast growth factor-2 is expressed by osteoblasts and is generally more potent than broblast growth factor-1 (29), although the expression of other broblast growth factors are not nearly as ubiquitous. Although broblast growth factor signaling has been implicated in bone development, studies on null mutant mice have not yet fully shown the role of this family in skeletal development (166). Fibroblast growth factor-1 and broblast growth factor-2 in vitro stimulate osteoblast proliferation (in calvarial cells, ROS 17/2.8 and MC3T3-E1) but do not increase collagen production or alkaline phosphatase in differentiated osteoblasts (28, 106, 207), although these effects may be differentiation stage-specic as constitutive broblast growth factor signaling inhibits
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osteoblastic differentiation (49) and dramatically increases apoptosis when cells are exposed to differentiating conditions (157). Fibroblast growth factors are strongly mitogenic to bone marrow stromal cells and are able to maintain the self-renewal of these cells in culture (135). It seems that short-term treatment with broblast growth factor, followed by its removal from culture, has an overall stimulatory effect on osteoblasts, and these growth factors may have potential as an adjunctive agent to increase bone formation. Their general effects in vitro are consistent with them acting mainly to stimulate proliferation in immature cells (whilst concomitantly inhibiting differentiation), resulting in expansion of the osteoblast progenitor pool.
Platelet-derived growth factor

Platelet-derived growth factor is secreted by platelets during the early phases of fracture healing, but its presence has been found in various tissues, including bone. Owing to its expression by a range of tissues, it is thought to have both systemic and local actions. Platelet-derived growth factor is composed of two polypeptide chains and can exist in three different isoforms of two gene products (AA,BB,AB) and these bind to two separate a and b receptors. Platelet-derived growth factor is a powerful mitogen for connective tissue cells, and although it can stimulate, and is synthesized by, mesenchymal cells and osteoblast like cells (83), it does not have powerful bone-induction properties. Platelet-derived growth factor isoforms have a strong chemotactic effect on osteoblasts and other connective tissue cells (100, 101), and may act to recruit mesenchymal cells during bone development and remodeling. Platelet-derived growth factor may also have direct and indirect effects on bone resorption by the upregulation of collagenase transcription (215) and an increase in interleukin-6 (IL-6) expression (64) in osteoblasts. In addition to platelet-derived growth factor autoregulation (214) in osteoblasts, there is paracrine regulation by other growth factors, such as transforming growth factor-b (213). Overall, the main physiological actions for platelet-derived growth factor in bone regulation are as nonspecic mitogens, but their effects on cell chemotaxis and neovasculogenesis may be particularly important during would healing.
mone participates in the regulation of skeletal growth and triggers the release of insulin-like growth factor in target cells. The insulin-like growth factors are bound to binding proteins, adding another crucial tier to modulate the activity of insulin-like growth factor. Two insulin-like growth factors have been identied insulin-like growth factor-1 and insulin-like growth factor-2 both of which are found in high concentration in serum. In bone, whilst insulin-like 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 (208). 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 signicant effects on skeletal insulin-like growth factor, as do many growth factors, such as BMP-2 (27), transforming growth factor- (181) and broblast growth factor (70). 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 insulin-like growth factor 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 Cbfa1/Runx2. In addition, insulin-like growth factor-1, in combination with BMP-2, acts synergistically on osterix expression (34). Although it is widely accepted that insulin-like growth factors have a dening role in bone remodeling, their actual role is still unclear and needs to be understood within the complex inter-relationships of the components of the insulin-like growth factor 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.
Wnt signaling
There is increasing evidence for the role of Wnt signaling in the control of early stages of the osteoblast lineage. Wnts are a group of over 15 related extracellular signaling molecules that show ligand binding with their receptor, frizzled, and co-receptors LRP5/6. Ligand binding sets off a series of intracellular reactions, resulting in stabilization of the protein b-catenin (200). This is then translated to the nucleus, where it acts as a co-factor with other DNA-binding proteins to regulate gene transcription.
Insulin-like growth factors

Growth hormone and insulin-like growth factors play critical roles in skeletal development. Growth hor-
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This Wnt/b-catenin signaling pathway is also known as the canonical Wnt signaling pathway. In addition, Wnt ligand binding can regulate gene expression by noncanonical pathways without the involvement of b-catenin. The canonical Wnt signaling pathway has been shown to have important roles in the maintenance of self-renewal of stem cells in epidermal and hematopoietic cells, and there is increasing evidence for its role in the regulation of bone formation (31, 200, 262). First, it has been shown that canonical Wnt signaling can directly promote osteogenesis through actions on the Cbfa-1/Runx-2 gene (72). Second, genetic defects and polymorphisms of the Wnt co-receptor, LRP5, are associated with an osteoporotic phenotype (14, 129). The relationship between Wnt signaling and other growth factormediated osteoblast effects is, to date, confusing, with evidence for Wnt signaling acting through the induction of BMPs, and, conversely, BMPs acting through the induction of Wnt signaling in mesenchymal cells (13, 199, 265). At present there is little or no information about the role of Wnt signaling in local bone formation during wound healing, although it is likely that this signaling pathway may play an important role in the early events of local bone formation.
BMP expression by direct interaction with bmp gene promoters (121).
Enamel matrix derivatives

Enamel matrix derivatives (EMD) are the major component of commercially available Emdogain and are discussed here in order to compare information on their biological activity with that presented for growth factors above. The main biological effects of EMDs have been attributed to their predominant protein, amelogenin (37), with the remaining fraction comprising less-characterized factors. Amelogenin is not a classic growth factor, but rather a cell-adhesion matrix-bound protein, and specic amelogenin gene products are thought to have activity as epithelialmesenchymal signaling molecules. In addition, there is evidence that the alternate splice variant of the amelogenin gene, leucine-rich amelogenin peptide may also have direct signaling activities on cementoblasts, and, thus at least by implication, osteoblasts (20). Enamel matrix derivative has been used clinically for periodontal regeneration, and its therapeutic effectiveness has been variously attributed to amelogenin, nonamelogenin enamel matrix proteins and growth factors. While EMDs may induce periodontal regeneration, the precise mechanism of this is not known. In vitro studies show that EMDs can increase matrix production (86), proliferation and bone nodule formation of periodontal ligament cell cultures (73) and the differentiation of human and murine osteoblast cell lines (221), with the stimulation of phenotypic bone markers in some osteoblast cell lines (270). In contrast, some studies show no effect of EMDs on osteoblastic differentiation, although other growth factors were stimulated (183). In addition, noncommercial EMDs have been shown to contain both transforming growth factor-b and BMPlike growth factors (233). The osteogenic activity of EMDs may be mediated by the induction of BMP-like molecules, as EMDs induce Cbfa1/Runx2 expression and the phosphorylation of Smad1, both of which can be blocked by the BMP inhibitor, noggin (237). The search for downstream target genes has also revealed EMD response elements in the rat bone sialoprotein gene promoter that may mediate the effects of EMDs on bone sialoprotein gene transcription (223). A cDNA microarray study examining EMD-mediated changes in gene expression in periodontal ligament cells in vitro has reported the downregulation of genes involved in the early inammatory phases of wound healing, while simulta-
Other growth factors

There are many other growth factors that can interact with cells of the osteoblast lineage and may play a role in the local regulation of bone formation. For example, osteoprogenitor cells are responsive to epidermal growth factor stimulation, although epidermal growth factor receptors may be downregulated in mature osteoblastic cells (48). Treatment of cell cultures in vitro with epidermal growth factor results in increased cell proliferation and suppression of bone module formation (7). Epidermal growth factor utilizes many of the signal transduction pathways in common with platelet-derived growth factor although, using a proteomic approach, subtle differences in intracellular signaling have recently been reported in these two signaling mechanisms (137). Sonic hedgehog (Shh) is another growth factor-like protein which binds to a specic cell-surface receptor, patched (Ptc). Shh can induce osteoblastic commitment in cultured multipotent mesenchymal stem cells and promote osteoblast differentiation (126, 230, 271). These actions can be blocked with BMP antagonists, suggesting that Shh is an upstream regulator of BMP production and the Shh-activated intracellular signaling molecule, Gli, can upregulate
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Fig. 3. Simplied schematic diagram showing the main stages of the osteoblast lineage where different growth factors may act. BMP, bone morphogenetic protein; IGF, insulin-like growth factor; FGF, broblast growth factor;
PDGF, platelet-derived growth factor; Shh, Sonic hedgehog; TGF-b, transforming growth factor-b; Wnts, a group of >15 related extracellular signaling molecules.
neously upregulating genes encoding growth and repair-promoting molecules (193). The in vitro treatment of cementoblasts with EMDs was found to decrease osteocalcin expression and to increase osteopontin expression (249). Overall, the data support the positive effect of EMDs on osteoblast differentiation, although further studies are needed to clarify which molecules in EMDs stimulate osteogenesis and to dene their precise modes of action.
outcome of these cellular responses may be both anabolic and catabolic. This section will provide an insight into the capacity of the major pro-inammatory cytokines to regulate osteoblast differentiation and function. Finally, the coupling of the osteoblast to the regulation of bone resorption will be introduced.
Interleukin-1
Interleukin-1 is a multipotent cytokine comprising two individual peptides, namely IL-1a and IL-1b, which exhibit similar biological activities (52). Both hematopoietic and mesenchymal/osteoblastic cells can produce IL-1 (89). There are two IL-1 receptors, namely type-I and type-II. All cellular responses elicited by IL-1 are known to be mediated through the type-I receptor (227), whereas type-II acts as a nonsignaling decoy receptor for the cytokine (41). Interleukin-1 also has a natural inhibitor, termed IL-1 receptor antagonist (IL-1ra). This binds to, but does not activate, IL-1 receptors (88), thus blocking the biological activity of the cytokine (9). The signaling events downsteam of the IL-1 receptor are mediated by IL-1 receptor-associated kinase (IRAK), and include the recruitment of phosphatidylinositol 3-kinase (PI3K), phosphorylation of MAPKs, and ultimately activation of the transcription factor, nuclear factor kappa B (NF-jB) (12). In the context of bone, IL-1 is known to regulate both resorption (149) and formation (25). However, the outcomes of different studies on the effects of IL-1 on osteoblast function are rather divergent. On the one
Summary
Considerable advances have been made in describing the regulation of osteoblasts by growth factors and in identifying some of the molecular mechanisms involved in these processes. Although the data are at times complex and sometimes contradictory, an overall pattern emerges of a carefully regulated, temporal sequence of co-ordinated growth factor expression, which can be modulated at any stage of the differentiation cycle. A schematic representation of this is shown in Fig. 3.
Effects of inammatory cytokines on osteoblast differentiation

Apart from responding to growth and differentiation factors, osteoblasts are also responsive to a number of locally acting cytokines, which are molecules that regulate cellular behavior of bone under situations of inammation, infection and wound healing. The
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hand, both IL-1a and IL-1b have been shown to inhibit osteoblast proliferation and enhance bone formation, as demonstrated by enhanced alkaline phosphatase activity and bone nodule formation (50, 87, 179). On the other hand, depending on the differentiation stage of the cell, prolongation of the culture period and concentration of the cytokines, IL-1a and IL-1b, may stimulate osteoblast proliferation, including DNA and protein synthesis, and inhibit bone formation (25, 57, 202, 241), and inhibit osteocalcin and type I collagen production (231, 232). Interleukin-1 can also stimulate osteoblasts to produce other pro-inammatory cytokines, such as IL-6 (38, 110), IL-7 (261) and tumor necrosis factor-a (TNF-a) (259), or other inammatory mediators, such as prostaglandin E2 and nitric oxide (104, 105, 120, 197, 198).
extracellular signal-regulated protein kinase 1/2 MAPK, as well as the signal transducers and activators of transcription 1 and 3 (STAT-1 and STAT-3) (59, 65). Desensitization of IL-6 signaling is accomplished by endocytosis of the receptorligand complex, a process dependent on gp130. However, attenuation of IL-6-induced STAT-1 and STAT-3 expression, is independent of IL-6/IL-6R, or gp130 endocytosis (247, 248). Apart from the direct effects on osteoblast proliferation and differentiation, it is also possible that the IL-6/sIL-6R complex may indirectly regulate the expression of other factors known to control these cellular events. These include insulin-like growth factors (63, 62), BMPs (269), and a number of other paracrine and endocrine regulators of osteoblasts.
Tumor necrosis factor-a Interleukin-6

The IL-6 family of cytokines includes IL-6, IL-11, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor (216). Interleukin-6 is a pleiotropic cytokine crucial for the regulation of bone metabolism, reportedly more so than other members of the IL-6 family. The major source of this cytokine in bone is osteoblasts and stromal cells (94). Its involvement in bone remodeling is particularly evident in situations of increased bone turnover, rather than in physiological conditions (65, 93, 134, 156). It was initially demonstrated in vitro that IL-6 does not affect, or may slightly downregulate osteoblast differentiation (99, 110, 146, 147). These ndings were further strengthened by the absence of any pathological bone phenotype in IL-6 knockout mice (133). However, it is now established that the cellular expression of IL-6R, the cognate receptor to this cytokine, occurs at low levels. Therefore, the presence of the soluble receptor, sIL6R, an agonist for IL-6, is imperative for the activation of the downstream effects of this cytokine, in a process called trans-signaling (59, 65, 117). In the light of this requirement, studies on the osteoblast demonstrate that the IL-6/sIL-6R complex, IL-11, leukemia inhibitory factor and oncostatin M inhibit proliferation and stimulate osteoblastic cell differentiation via a cascade of intracellular events mediated by gp130, a signal-transducing receptor subunit (17, 58, 235). This molecule is more ubiquitously expressed than IL-6R (216). Binding of IL-6 to either its membrane-bound or its soluble receptor, results in homodimerization of gp130 and initiation of downstream signaling pathways, including the activation of the Janus Kinases and subsequently the Tumor necrosis factor-a is a member of the TNF family of inammatory cytokines that share common signaling pathways. It has a pivotal role in bone pathophysiology, because it has been shown to stimulate bone resorption and inhibit bone formation (19, 171). The major source of TNF-a is the T cell, and the cognate receptors to TNF-a are TNFR-1 (or p55) and TNFR-2 (or p75) (171). The former is required to initiate cell signaling, whereas the latter may contribute to the sensitivity of the cell response to the cytokine (77). Ligandreceptor binding activates TNF receptor-associated factors (TRAFs), mainly TRAF-1 and TRAF-2, in osteoblasts (16, 33), which in turn evoke a series of interconnected events, ultimately leading to the activation of NF-jB. In brief, NF-jB is a set of ve polypeptide transcription factors, called p50, p52, p65 (or Rel A), Rel B and c-Rel, which form heterodimers. The NF-jB heterodimers form an inactive complex with their I-jB inhibitors. Once phosphorylated by the I-jB kinases, I-jB is ubiquitinated and degraded in proteosomes. This results in the liberation and translocation of NF-jB into the nucleus, where it activates the transcription of a number of genes (21). Optimal induction of the respective target genes also requires phosphorylation of the NF-jB proteins (254). It should be noted that NF-jB knockout mice develop osteopetrosis as a result of a defect in osteoclast differentiation (108). Tumor necrosis factor-a is known to inhibit osteoblast function and differentiation. Suppression of the osteoblastic phenotype is characterized by the inhibition of cell proliferation and decreased production of a number of extracellular matrix components, such as type I collagen (82, 172, 210, 229). Indeed, genetic analyses have identied TNF-a-
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responsive regions in the promoter of the respective genes (COL1A1 and COL1A2), indicating that NF-jB mediates the inhibition of collagen transcription (109, 116, 136, 163). Tumor necrosis factor-a also inhibits alkaline phosphatase and osteocalcin expression. However, the nature of osteocalcin inhibition is different from that of type I collagen, because this does not require binding of NF-jB to the promoter of the osteocalcin gene (173, 171). Apart from suppressing extracellular matrix production by osteoblasts, TNF-a also inhibits the differentiation of pre-osteoblasts as a result of the suppression of Cbfa1/Runx2 (1, 76) and osterix (171). In addition, TNF-a stimulates IL-6 production by osteoblasts (47, 110, 203) in an NF-jB-dependent mechanism (139). However, recent evidence suggests that other pathways such as p38 MAPK, and protein kinase C may also be involved in this event (138, 167). Tumor necrosis factor-a also induces prostaglandin E2 production by osteoblasts, an event at least partially mediated by nitric oxide (104).
Regulation of osteoclastogenesis and bone resorption by the osteoblast

In recent years it has become evident that osteoblasts have a global role in orchestrating the bone remodeling process. Their function is not restricted solely to bone formation, but it is now rmly established that they are responsible for initiating bone resorption. In cellular terms, apart from forming the mineral and organic extracellular compartment of bone, the osteoblast provides the essential and sufcient stimuli that control the behavior of the osteoclast, an event that occurs via cellcell interaction. The molecular determinants of this interaction are two factors produced by osteoblasts/stromal cells, namely M-CSF and RANKL, the former secreted and the latter mainly cell-membrane bound (244). M-CSF binds to c-Fms on the surface of osteoclast precursors, and this event enhances their proliferation and survival. RANKL, a member of the TNF ligand superfamily, is the factor that actually triggers the differentiation of osteoclast precursors, into mature osteoclasts and activates bone resorption, and is an absolute requirement for osteoclast formation (131, 140). Therefore, upon binding of RANKL to its cognate RANK receptor on the surface of the M-CSFtriggered osteoclast precursors, a number of downstream signaling pathways are activated, committing the cell to the osteoclast lineage. These are initially mediated by TRAF-6, subsequently leading to the activation of NF-jB, the AP-1 transcription factor
complex, and ultimately nuclear factor of activated T cells (NFAT) c1 (238, 245). The effects of RANKL can be blocked by the soluble nonsignaling decoy receptor, osteoprotegerin (OPG), a member of the TNFR superfamily with high homology to RANK (226). OPG is produced and secreted by osteoblasts and stromal cells, and by binding to RANKL it prevents RANKL/RANK interaction and consequently the downstream events described. RANKL-decient mice lack functional osteoclasts and develop osteopetrosis, whereas OPG-decient mice develop earlyonset osteoporosis, demonstrating the indispensable role of a balanced RANKL/OPG expression in physiological bone remodeling (23, 131). An increased RANKL/OPG expression ratio has been demonstrated in diseases involving inammation-induced bone loss, including periodontitis (148, 243, 246) and rheumatoid arthritis (60). Interleukin-1, IL-6 and TNF-a are all potent inducers of osteoclast differentiation and bone resorption. It is postulated that their catabolic effects on bone are mediated, to a great extent, through osteoblasts/stromal cells, and more specically through the regulation of RANKL and OPG expression in these cells (144, 148, 256). Indeed, it has been demonstrated that IL-6/sIL-6R, IL-11, leukemia inhibitory factor and oncostatin M increase RANKL and OPG expression in osteoblasts, in a manner dependent on gp130 and its downstream effector STAT-3 (177, 178, 191). Similarly, both IL-1 and TNFa induce RANKL expression, the former possibly being dependent on activation of STAT-3, whereas the latter is dependent on phosphorylation of p38 (47, 90, 259). OPG expression is also enhanced by these two cytokines (22, 255). Although both RANKL and OPG are upregulated in these systems, the overall outcome is an increased RANKL/OPG expression ratio, resulting in stimulation of osteoclastogenesis and bone resorption.
Other inammatory cytokines

A number of other cytokines have been reported to regulate bone cell behavior. Interleukin-7 (78) and IL-10 (71) have an inhibitory action on osteoblast differentiation. Interleukin-4 and IL-13 are known to inhibit osteoblast differentiation (251), and mice over-expressing IL-4 appear to have a decreased bone formation, owing to a reduction in the number and activity of bone-lining osteoblasts (145). Interleukin18 is structurally and functionally homologous to IL-1, but it employs differential signal-transducing pathways, preferentially activating p38 MAPK rather
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than NF-jB (53, 143). It is produced by osteoblasts/ stromal cells, to which it acts as a mitogen, and is also known to inhibit osteoclast differentiation (43, 250). This may, in part, account for its capacity to stimulate osteoprotegerin, but not affect RANKL expression in osteoblasts/stromal cells (155). Interferon-c is a lymphokine produced by activated T cells, with a strong potency to inhibit osteoclastogenesis (80, 81, 239), It also inhibits osteoblast proliferation, as well as collagen and noncollagen protein synthesis (82, 172, 229). These effects appear to be dependent on nitric oxide production (103, 152, 153), rather than on prostaglandin E2 synthesis (229).
Summary
In summary, inammatory cytokines may have both anabolic and catabolic effects on osteoblast function, and consequently on bone physiology. However there is relatively little information on how these effects might also interact directly with growth factor pathways, but these interactions are likely to be very important. Studies have shown that both IL-1 and TNF-a may inuence the effects of BMPs on osteoblasts, but the nature of these interactions have not been explored at a mechanistic level (169, 240). Therefore, any tissue engineering approach targeting osteoblasts for bone regeneration must take into consideration the putative cellular responses elicited by cytokines upon them during inammation. This should be a concern, not only in the case of exogenously administered cytokines as a direct treatment strategy. It is highly probable that several other primary molecular agents may induce secondary inammatory responses by the target cells, inuencing, in turn, the cellular dynamics of bone in an autocrine or paracrine manner. This issue is particularly important in the instance of catabolic effects, which may occur either as inhibition of bone formation, or, most reportedly, as enhancement of bone resorption. Tackling efciently the potential sideeffects of these cytokines may help to ensure that the bone-remodeling balance would tend favorably toward bone formation, ultimately resulting clinically in bone regeneration.
in vitro or, on some occasions, from transgenic and null mutant (knockout) mouse models. These methodologies are extremely powerful for dissecting out specic signaling pathways as it is possible to control many of the extrinsic variables, thus facilitating the study of, for example, the role of individual specic molecules and signaling pathways. However, cells in vivo are exposed to a wide range of these signaling pathways simultaneously, and it is the total sum of these interactions which results in the carefully co-ordinated control of osteoblast function that is seen in vivo. Although there are some data on how different pathways may interact during the co-ordinated regulation of bone growth, at the present time it is only possible to provide examples of such interactions, rather than give a complete picture of the complex interactions in cytokine signaling pathways during bone growth. There are a number of mechanisms which can regulate different growth factor pathways. Different signaling pathways can also interact with each other in order to ne tune the nal observed outcome. These mechanisms are listed below. the regulation of growth factor activity by the secretion of soluble antagonists which are often regulated in both autocrine and paracrine fashions. the regulation of receptor expression on specic cells the interaction between intracellular signaling mechanisms in both antagonistic and synergistic ways.
Growth factor antagonists and binding proteins

Growth factors may bind to common proteins, such as a-2 macroglobulin, and be sequestered, for example, in matrices. Bone matrix is a rich source of many growth factors, including transforming growth factor-b and platelet-derived growth factor (45). In addition, many growth factors also appear to have specic binding proteins, which bind with their target factor with high afnity to inactivate them, or in some cases to increase ligand binding. Regulation of expression of growth factor antagonists and the balance of factor to antagonist is a clear mechanism for growth factors to control each others function in a sequential cascade, such as that described above. The activity of BMPs is regulated by a number of inhibitor proteins, including noggin, gremlin, follistatin and sclerostin, which bind to BMPs with high afnity in order to inactivate them (30). In addition, a novel BMP inhibitor, ectodin, has recently been
Regulation and interaction of growth factor pathways

Most of the information regarding the mechanisms of action of growth factors and cytokines on their target cells have been derived from studies carried out
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described, whose expression is reportedly restricted to ectodermal-derived tissues in regulating epithelialmesenchymal interactions that are mediated by BMPs (142). The important actions that these inhibitors may have are suggested by a number of observations. First, mutations in the SOST gene (which codes for sclerostin) result in the rare bone disease, sclerosteosis, which is characterized by increased bone mass (15). Second, the overexpression of noggin in transgenic mice results in blocking the production of BMP-4-induced heterotopic bone formation (79). Third, evidence suggests that BMP-4-induced expression of noggin and gremlin is dysfunctional in patients with brodysplasia ossicans progressiva, a multilating condition where heterotopic bone forms in subdermal tissues (3). Finally, normal expression of ectodin in mice blocks enamel formation on one surface of the mouse incisor, and mice lacking ectodin have a number of defects in tooth formation, including an altered pattern of cusp formation and the formation of extra teeth (118, 142). Distinct BMP inhibitors may have tissue-specic activities and may also interact with different BMPs. For example, sclerostin expression is largely restricted to bone cells, particularly osteocytes, and may be involved in the osteocyte-mediated regulation of bone mass (264). In general, the expression of BMP antagonists is transcriptionally regulated by BMP signaling, giving rise to a negative feedback loop to regulate BMP signaling (30, 180). In addition, other pathways may regulate the expression of BMPs. In the case of ectodin, broblast growth factor and Shh signaling downregulates its expression, providing a mechanism whereby other growth factor signaling pathways may effectively upregulate BMP responses (142). The insulin-like growth factors are regulated by a series of at least six different binding proteins (insulin-like growth factor-binding proteins). As with other antagonists, insulin-like growth factor-binding proteins bind to the insulin-like growth factors with high afnity and typically may inactivate activity by preventing ligand binding. However, it is known that insulin-like growth factor-binding proteins may also act to mediate or regulate the ligand binding of insulin-like growth factors to their receptors on some occasions (26, 160). Insulin-like growth factors have been shown to differentially regulate insulin-like growth factor-binding protein gene expression (69). Insulin-like growth factor-binding proteins may also be regulated by a number of other osteotropic growth factors, including BMPs and transforming growth factor-b (68, 107, 160).
Fibroblast growth factors have complex interactions with a range of proteins and matrix molecules that regulate their function. First, it is known that broblast growth factors are normally matrix bound to heparan sulphate-containing proteoglycans and that heparan sulphate-containing proteoglycan binding is required for normal broblast growth factor ligandreceptor interactions (217). Alternatively spliced broblast growth factor receptor genes can give rise to broblast growth factor soluble receptors, which inactivate broblast growth factor by preventing normal receptorligand binding. Finally, there are additional broblast growth factorbinding proteins that also interact with broblast growth factors to control their function (141). The presence of these complex different levels of regulation together suggest a nely balanced set of mechanisms to control physiological broblast growth factor activity, although the specic functional signicance of broblast growth factor antagonists remains to be determined in bone. Transforming growth factor-b is also regulated closely by its association with binding proteins. Transforming growth factor-b is secreted in an inactive form bound to a specic binding peptide known as latency associated peptide. This latent transforming growth factor-b complex is then normally bound to one of at least four latent transforming growth factor-b-binding proteins (LTBPs) (182, 213). Latent transforming growth factor-b-binding proteins are extracellular matrix proteins found in bone matrix, and may be responsible for the large amounts of latent transforming growth factor-b sequestered in bone matrix. They may act as a delivery and release vehicle for transforming growth factor-b, and active transforming growth factor-b can be released from the complex by the enzymatic action of the plasminogen-activating system (6). In addition, a number of other proteins, including matrix metalloproteinases and thrombospondin, can release active transforming growth factor-b from latency associated peptide. The importance of these proteins is supported by the reports that LTBP-2 null mice die in utero at around stage e3.5; LTBP-3 knockout mice have a range of bone and craniofacial defects associated with a low turnover of bone (46). In contrast to most other growth factors, specic binding proteins have not been described for platelet-derived growth factor. However, platelet-derived growth factor binds to the matrix protein osteonectin (SPARC) and this may promote sequestering of platelet-derived growth factor in bone matrix and other sites (196).
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Overall, it is clear that the production of growth factor-binding proteins is a mechanism whereby the activity of these proteins can be carefully controlled, and represents one level of regulation allowing careful cross-talk between different factors in a carefully co-ordinated way.
Interaction of growth factor intracellular signaling pathways

There are a number of examples described where different growth factor pathways regulate each other during intracellular signaling. Of particular interest, studies have demonstrated the regulation of SMAD signaling following BMP stimulation by additional growth factor signaling pathways (161). Recent results have demonstrated the downregulation of BMP-activated SMAD signaling by both transforming growth factor-b, via stimulating the expression of inhibitory SMAD (154), and by extracellular signalregulated protein kinase and PI3 kinase pathways, mediated by insulin-like growth factor-1 and other growth factor signals (186). These observations suggest many mechanisms whereby different growth factors regulate responsiveness to other signaling pathways. In this way it is possible that specic growth factor expression and signaling activity can be very delicately regulated to balance proliferation and differentiation events within cells of the osteoblast lineage. Overall, it is clear that although different cytokines may have distinct regulatory functions, during the control of bone formation, the interaction of these different pathways can be exquisitely regulated at a number of points. These observations, taken together, are consistent with a model of cytokine signaling consisting of sequential and overlapping expressions of factors to regulate nal bone-forming outcomes.
Therapeutic application of growth factors for bone regeneration

The topic of growth factor-induced bone and periodontal regeneration has been discussed extensively in other recent reviews (74, 234, 267), and it is not the intention in this article to revisit the detail of these experiments. However, here we consider some of the basic issues surrounding the use of exogenously applied growth factors to modulate bone growth, particularly in the periodontal tissues, and
briey describe the general ndings of some of these studies. Given the limitations of currently used clinical techniques, it is unsurprising that there is great interest in the possible utility of recombinant growth factors for tissue regeneration therapies. From a theoretical standpoint, the pharmacological application of growth factors results in a local single dose of the factor, which may be present at a concentration thousands of times higher than that normally seen physiologically. The pharmacological effects of the factor may be distinct from its physiological activity, for example because the normal regulatory feedback mechanisms which control that factor are temporarily overwhelmed by such a large dose. It seems quite likely that therapeutic application of any one of a number of growth factors could perturb the normal bone-forming process in a positive way and, indeed, preclinical studies support this idea. For example, based on the knowledge of physiological function, discussed above, BMPs may increase osteoblastic stem cell recruitment and commitment, broblast growth factor may stimulate early progenitor cell number expansion, platelet-derived growth factor may also increase progenitor cell recruitment and proliferation, and insulin-like growth factors might act to increase nal osteoblast differentiation and function. Any one of these actions may result in the desired nal outcome of increased bone formation. With this in mind, it is likely that one of the main critical determinants of success for these treatments is achieving sufcient substantivity for the factor to provide a continual therapeutic dose over a lengthy period of time (122). Although there is little published data on the pharmacokinetics of growth factor delivery, one study demonstrated that delivery of platelet-derived growth factor with insulin-like growth factor-I in a collagen matrix had a half-life of <4 h, with complete loss of activity by 96 h (151). Thus, in order to address this issue a number of different delivery systems have been used for these applications, including collagen matrix (123, 212), cross-linked gelatin pellets (124), calcium phosphate ceramics (112, 174), and polylactic-acid (PLA)-based resorbable polymers (192). In a different approach to provide persisting growth factor delivery, somatic cell gene transfer of platelet-derived growth factor has also been tested in vitro and in animal models (8, 61, 114, 113, 272). This approach might prove particularly successful in providing a sustained active concentration of growth factor to the site being treated. However, at present, serious concerns about the safety of the
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adenovirus vectors used for gene transfer, and other ethical and practical issues, will need to be overcome before this could be thought of as a viable clinically applicable technique.
Platelet-derived growth factor and periodontal regeneration

A number of studies have tested the potential for tissue regeneration stimulated by platelet-derived growth factor and also with platelet-derived growth factor combined with insulin-like growth factor-1 or dexamethasone (40, 97, 112, 150, 151, 211, 212). In general terms, these studies (carried out in animals) have demonstrated an enhanced rate and an increased total amount of regenerated tissue compared with controls. However, there is little evidence of the efcacy of insulin-like growth factor-1, either on its own or in combination with platelet-derived growth factor (75). Most interestingly, a large multicentre human study (involving 180 patients) of platelet-derived growth factor has recently been reported (175). In that study, recombinant human platelet-derived growth factor-BB, with a b-tricalcium phosphate carrier, demonstrated improved bone ll and enhanced rate of clinical attachment gain compared with carrier-only controls. This is probably the rst large human study to assess the role of recombinant growth factor therapy and these interesting results will need further evaluation over time (201).
growth factors (162, 242). There is also clinical interest in the application of platelet-rich plasma, an autologous preparation obtained from a patients own platelets, which is rich in a number of growth factors, including platelet-derived growth factor and trefoil factor family-b, and which has been applied clinically in both periodontal and implant applications (32, 184, 218, 220). However, disappointingly, to date there is very little proper scientic evidence which evaluates the efcacy of this procedure, and well-controlled clinical studies of platelet-rich plasma would be valuable in ascertaining whether this treatment has a similar potential, for example, as the pharmacological use of recombinant growth factors.
Summary
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 specic parts of the intracellular signaling pathways. There remains an enormous challenge to convert some of the knowledge from basic studies of bone cell physiology to therapeutically useful techniques for the future. We are optimistic that such novel approaches may result in real qualitative improvements in clinical outcomes over currently available techniques.
Other growth factors

Bone morphogenetic proteins have also been widely tested for their ability to be used pharmacologically to enhance new bone formation. Bone morphogenetic proteins can powerfully promote bone formation in critical-size segmental defects in animals and have shown some promise for their ability to regulate bone and periodontal regeneration (123, 125, 205, 204, 224, 263). As these studies are the subject of a further separate review in this volume, they will not be considered further here. The effects of broblast growth factor-2 on animal models of periodontal regeneration have also been tested in animal models. Results of these studies are again promising, with broblast growth factor-2 showing a stimulation of parameters of bone and periodontal regeneration in surgically created defects (96, 164, 165, 168, 236). Other growth factors to have been tested include transforming growth factor-b where initial results of studies were disappointing when compared with the effects of these other
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Growth Factors

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Periodontology 2000, Vol. 41, 2006, 4872 Printed in Singapore.

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Effects of growth factors and cytokines on osteoblast differentiation

Osteoblast lineage and function

Effects of growth factors and cytokines on osteoblast differentiation

The osteoblast lineage

Mesenchymal stem cells

Effects of growth factors and cytokines on osteoblast differentiation

Transcriptional regulation of osteoblastic commitment and differentiation

The effects of growth factors on osteoblast differentiation

Effects of growth factors and cytokines on osteoblast differentiation

Transforming growth factor-b

have effects during different stages of osteoblast differentiation.

Bone morphogenetic proteins

Fibroblast growth factor

Effects of growth factors and cytokines on osteoblast differentiation

Platelet-derived growth factor

Insulin-like growth factors

BMP expression by direct interaction with bmp gene promoters (121).

Enamel matrix derivatives

Other growth factors

Effects of growth factors and cytokines on osteoblast differentiation

Effects of inammatory cytokines on osteoblast differentiation

Tumor necrosis factor-a Interleukin-6

Effects of growth factors and cytokines on osteoblast differentiation

Regulation of osteoclastogenesis and bone resorption by the osteoblast

Other inammatory cytokines

Growth factor antagonists and binding proteins

Regulation and interaction of growth factor pathways

Effects of growth factors and cytokines on osteoblast differentiation

Interaction of growth factor intracellular signaling pathways

Therapeutic application of growth factors for bone regeneration

Effects of growth factors and cytokines on osteoblast differentiation

Platelet-derived growth factor and periodontal regeneration

Other growth factors

Effects of growth factors and cytokines on osteoblast differentiation

59. 60. 61.

Effects of growth factors and cytokines on osteoblast differentiation

Effects of growth factors and cytokines on osteoblast differentiation

Effects of growth factors and cytokines on osteoblast differentiation

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