Bone Regeneration and Repair - part 4 ppt

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Bone Joint Surg. 82A, 544–554. This is trial version www.adultpdf.com Growth Factor Regulation of Osteogenesis 113 113 From: Bone Regeneration and Repair: Biology and Clinical Applications Edited by: J. R. Lieberman and G. E. Friedlaender © Humana Press Inc., Totowa, NJ 7 Growth Factor Regulation of Osteogenesis Stephen B. Trippel, MD Osteogenesis, the creation of bone, underlies all skeletal development and repair. It encompasses the differentiation of cells along specific developmental pathways and the production by these cells of the matrix required to construct, or to reconstruct, bone. The control of this process is, to a large extent, the responsibility of cell signaling molecules that include hormones, growth factors, and cyto- kines. This chapter reviews some of the factors that participate in regulating the creation of bone at the cellular level. GROWTH HORMONE Growth hormone, or somatotropin, is the prototypical regulator of skeletal growth and develop- ment. Growth hormone deficiency produces severe, generalized failure of osteogenesis at the growth plate and results in clinical dwarfism. The administration of recombinant human growth hormone to children with either growth hormone deficiency or idiopathic short stature can, at least partially, restore the kinetics of osteogenesis at the growth plate and hence the rate of linear bone growth. Excess growth hormone secretion during skeletal development increases longitudinal bone growth and pro- duces clinical gigantism (1). Growth hormone insensitivity due to mutations in the growth hormone receptor are responsible for several forms of dwarfism, ranging from mild to severe (2,3). The ability of growth hormone to influence osteogenesis at the site of bone repair is controversial. Growth hormone has been reported to stimulate the formation of bone in intact bones (4,5) and osseous defects (6), and to enhance healing in fracture models in rats (7–11) and dogs (12). Other investiga- tors, however, have observed that growth hormone has no effect on bone formation (13,14), healing of defects (15), bone graft incorporation (16), or healing of fractures in rat (17,18) or rabbit models (15,19). The differences in the findings of these studies may be explained, in part, by differences in experimental design, growth hormone dosage, site of delivery, species of animal, and outcome mea- sures employed. Whether a deficiency of growth hormone results in failure of fracture healing is similarly controver- sial (20–22). Interestingly, growth hormone deficiency may increase the risk of fracture occurrence (23,24). Early reports of growth hormone treatment of human fractures were encouraging (25,26), but these studies were limited by small sample size and lack of a paralleled control group. Although growth hormone is now widely used to enhance skeletal growth, there currently appears to be little direct support for its clinical application to fracture repair. INSULIN-LIKE GROWTH FACTOR I (IGF-I) IGF-I was discovered in experiments testing the effect of growth hormone on sulfate incorpora- tion into cartilage. These experiments found that a serum factor, later identified as IGF-I, mediated the effect of growth hormone on this tissue (27). Subsequent studies suggested the existence of a growth This is trial version www.adultpdf.com 114 Trippel hormone–IGF axis that includes both endocrine and autocrine/paracrine elements. Growth hormone, secreted by the pituitary, stimulates IGF-I production by the liver (28) and other organs (29). This IGF-I enters the systemic circulation, and from there, acts in an endocrine fashion on multiple tissues includ- ing the skeleton (30,31). Evidence in support of this model, as it applies to skeletal growth, includes the identification of growth hormone receptors (32) and IGF-I receptors (33,34) on growth-plate chon- drocytes, and the ability of anti-IGF-I antibodies to block the growth-enhancing effect of growth hor- mone delivered intraarterially to growing limbs (35). In addition, growth hormone has been shown to stimulate the production of IGF-I mRNA (36), and peptide (37) by growth-plate chondrocytes. The role of IGF-I in the regulation of osteogenesis at the growth plate is further illuminated by studies in transgenic mice. Mice in which the IGF-I gene has been deleted manifest marked intrauter- ine and postnatal skeletal growth deficiency that is not corrected by growth hormone treatment (38, 39). When mice were made transgenic for the IGF-I gene and for ablation of the cells that express growth hormone, the mice carrying both transgenes (IGF-I and absence of growth hormone) grew larger than litter mates that carried only the growth hormone ablation transgene (40). The double-trans- genic animals demonstrated weight and linear growth that were indistinguishable from those of their normal, nontransgenic siblings. IGF-I is capable of at least partly substituting for growth hormone in humans as well as in mice. In recent clinical trials, patients with end-organ insensitivity to growth hormone resulting from an inacti- vating growth hormone receptor mutation were treated with IGF-I (41,42). These children, who mani- fested severe failure of bone growth prior to therapy, experienced a substantial and sustained increase in skeletal growth during IGF-I therapy. Not all of the skeletal effects of growth hormone can be attributed to IGF-I. Growth hormone elicits very rapid anabolic cellular responses that are unlikely to involve such mediators as IGF-I (43). In addition, growth hormone administered systemically to hypophysectomized (and therefore growth hormone–deficient) rats has been found to be a more effective stimulus of skeletal growth than IGF-I, even when growth hormone was administered at 50-fold lower doses (44). The recent use of tissue-specific gene ablation techniques has permitted a partial separation of the effect of IGF-I produced in the liver and of that produced in other tissues. When the hepatic IGF-I gene was rendered nonfunctional, circulating levels of IGF-I fell by 80% while levels of growth hor- mone increased. Interestingly, postnatal (including pubertal) growth remained normal (45). These data raise the possibility that osteogenesis at the growth plate may be less dependent on IGF-I acting by an endocrine route than on IGF-I acting in a paracrine/autocrine fashion. It is also possible that the high level of circulating growth hormone achieved in these animals augmented local production of IGF-I sufficiently to offset the loss of circulating IGF-I. The relative contributions of IGF-I acting via the circulation in an endocrine fashion, that of IGF-I acting in a paracrine/autocrine fashion, and of growth hormone acting independently of IGF-I may differ at different sites and different stages of develop- ment. The specific roles of these various components of the growth hormone–IGF-I axis remain to be elucidated. EPIDERMAL GROWTH FACTOR Unlike growth hormone and IGF-I, epidermal growth factor (EGF) was not initially viewed as being involved in formation of the skeleton. However, as has proved to be the case with many cell signaling molecules, the role of EGF is broader than its name implies. The view that EGF plays a role in the regulation of skeletal development (46) has been supported by the localization of EGF in the growth plate (47), the detection of EGF receptors on growth-plate chondrocytes (48,49), and the observation that EGF is present in the circulation at concentrations that are capable of initiating cellular responses in vitro (50). The potential role of EGF in skeletal growth has been clarified in recent studies that investigated the intereaction of EGF and IGF-I in the regulation of growth-plate chondrocytes. These studies found that This is trial version www.adultpdf.com Growth Factor Regulation of Osteogenesis 115 EGF increased cellular responsiveness to IGF-I with respect to both mitotic activity and proteoglycan synthesis (51). This effect of EGF was associated with an increase in the number of IGF-I receptors per cell, but without a change in IGF-I receptor affinity. The effect of EGF on IGF-I receptors appeared to be a part of a general anabolic effect of EGF rather than a specific effect on the IGF-I receptor. These data suggest that EGF contributes to skeletal growth by increasing growth-plate chondrocyte sensi- tivity to IGF-I. These results may aid in understanding the previously enigmatic observation that the skeletal growth response to IGF-I does not match that achieved with growth hormone (44). The inabil- ity of IGF-I to fully compensate for growth hormone presumably reflects a requirement by the growth plate for growth hormone stimulation of an element in the growth hormone–IGF-I axis other than IGF-I itself. In conjunction with the observation that growth hormone regulates EGF (49), these data suggest that the IGF-I receptor is such an element. FIBROBLAST GROWTH FACTOR The fibroblast growth factors (FGFs) comprise a large family of polypeptides that regulate cell func- tions as diverse as mitogenesis, differentiation, receptor modulation, protease production, and cell main- tenance (1). Several lines of evidence indicate that these factors play an important role in bone formation. FGF-2 (basic FGF) has been immunolocalized to the proliferative and maturation (but not hypertrophic) zones of the growth plate of the fetal rat (52) and to the resting, proliferative, and perichondrial cells of the human fetus (53). Indeed, during fetal development, the highest levels of FGF-2 transcripts were reported to be in the long bones (54). Growth-plate chondrocytes possess high-affinity receptors for FGF-2 (55,56) and, in a variety of models, FGF-2 is a potent mitogen for growth-plate chondrocytes (57–61). In contrast to its repro- ducible effect on chondrocyte mitogenic activity, the role of FGF-2 on matrix synthesis is less clear. FGF-2 has been found to stimulate (62), exert no effect on (61,63), or inhibit (61,63,64) indices of matrix synthetic activity by growth-plate chondrocytes. FGF-2 also influences many of the cellular activities associated with chondrocyte differentiation. For example, FGF-2 effects on growth-plate chondrocytes in culture include a reduction in alkaline phosphatase (61,65), calcium deposition, and calcium content (65). In a fetal rat metatarsal organ culture model of skeletal growth, the effect of FGF was biphasic (66). Matrix production was stimulated by low concentrations (10 ng/mL), but inhibited by high con- centrations (1000 ng/mL), of FGF-2. In this model, as in others, FGF-2 stimulated 3 H-thymidine incor- poration, an index of DNA synthesis. However, the site of incorporation was principally in the peri- chondrium, and labeling was decreased in the proliferative and epiphysial chondrocytes. FGF-2 also caused a marked decrease in the number of hypertrophic chondrocytes. Taken together, these data suggest that the role of FGF-2 in osteogenesis at the growth plate is to promote an immature chondro- cyte phenotype by augmenting chondrocyte proliferation and inhibiting chondrocyte differentiation (55,65). The data also emphasize the complexity imposed on this role by temporal, spatial, and dosage relationships. FGF family members also participate in regulating osteogenesis during fracture repair. FGF-2 has been shown to be widely distributed around the fracture site in a rat fibular fracture model (67). FGF-2 was particularly prominent in the soft callus and periosteum. Application of a single dose of FGF-2 in a fibrin gel in this model augmented callus formation, increased the biomechanical strength of frac- ture repair, and restored the impaired fracture healing associated with diabetes (67). Similarly, FGF-2 in a hyaluronan gel increased callus formation and biomechanical strength when injected into rabbit fibular osteotomies (68). In a subperiosteal osteogenesis model, injection of FGF-2 stimulated exten- sive intramembranous bone formation adjacent to the parietal bone (68). Injection of FGF-1 (acidic FGF) into closed rat femoral fractures resulted in a marked increase in the size of the cartilaginous callus, but also inhibited type II procollagen and proteoglycan core protein gene expression. The net result was a decrease in the mechanical strength at the fracture site (69). This is trial version www.adultpdf.com 116 Trippel The effect of exogenous FGF on osteogenesis in vivo is complex. Local delivery of FGF-2 by direct infusion into the rabbit growth plate increased maximal vascular invasion and accelerated local ossifi- cation (70). Systemic intravenous delivery of 0.1 mg/kg/d of FGF-2 for 7 d to growing rats increased longitudinal growth rate, cartilage cell production rate, bone formation rate, and several histomor- phometric measures of bone quantity (71). Endocortical mineral apposition and bone formation rates were increased, but periosteal mineral apposition and periosteal bone formation rates were depressed. These effects were not matched by the higher dose of 0.3 mg/kg/d. At this dose, FGF-2 decreased longitudinal growth rate, cartilage cell production rate, endocortical bone formation rate, and produced defective calcification of the growth-plate metaphyseal junction. A similar biphasic effect of FGF-2 was observed in a bone chamber model. When injected into the marrow cavity of rat bone implants, a low dose (15 ng) of FGF-2 stimulated bone formation, while a high dose (1900 ng) had a profoundly inhibitory effect (72). In contrast, intraosseous delivery of 400 µg or 1600 µg of FGF-2 in rabbits increased bone mineral density (73). In transgenic mice that overexpress FGF-2, the radii, ulnae, humeri, femora, and tibiae were short- ened by 20–30% (p < 0.001) compared to nontransgenic littermate controls (74). Mean body weights were not significantly different. Growth plates showed significant enlargement of the reserve and pro- liferative zones due to chondrocyte hyperplasia and to enhanced extracellular matrix deposition. In contrast, hypertrophic chondrocytes were substantially diminished (74). Taken together, these data sug- gest that, in vivo, FGF may act to either augment or inhibit osteogenesis, depending on the dose, mode of delivery, and other variables. The contribution of the FGFs to osteogenesis has been further clarified by recent studies of the receptors that mediate FGF actions. There are at least four distinct FGF receptor (FGFR) genes (75), and many variants due to alternative splicing (76). Like the IGF-I receptor, all four FGFRs contain intracellular tyrosine kinase domains that become activated upon FGF binding to the receptor’s extra- cellular ligand-binding domain (Fig. 1). Mutations in these receptors are now known to be respon- sible for a variety of human chondrodysplasias. Studies of these disorders have led to extraordinary advances in our understanding of how growth factor signaling pathways influence osteogenesis dur- ing skeletal growth and development. Achondroplasia, the most common human genetic form of dwarfism, is characterized by rhizomelic (proximal greater than distal) shortening of long bones and by narrow growth plates (77,78). In more than 95% of individuals with achondrodysplasia, the cause is a point mutation in the portion of the gene encoding the transmembrane domain of FGFR3 (79–81) (Fig. 2). Thanatophoric dysplasia, a sporadic perinatal lethal disorder, is also caused by FGFR3 mutations. This severely deforming dysplasia is characterized by micromelic limb shortening, reduced vertebral body height, and disrupted cell distribution in the growth plate (82–84). Death is usually from respira- tory failure associated with marked shortening of the ribs and reduced thoracic cavity volume. Thana- tophoric dysplasia has been divided into two types, based on clinical features. Type I (TD-1) is char- acterized by curved, short femora, and type 2 (TD-2) by relatively longer, straight femora. TD-1 is associated with mutations in the extracellular region of FGFR3 or by a mutation in the stop codon of the gene (85). In contrast, TD-2 is associated with a specific mutation in the intracellular tyrosine kinase domain of FGFR3 (86) (Fig. 3). Hypochondroplasia is a rare autosomal dominant disorder with skeletal deformities similar to those of achondroplasia, but in a milder form (87,88). Slightly over half of individuals with hypochondro- plasia were found in a recent study to have a single mutation in the proximal tyrosine kinase domain of FGFR3 (89). Interestingly, in the remaining individuals with hypochondroplasia, no mutations in FGFR3 were detected, despite screening of more than 90% of the FGFR3 coding sequence and despite the absence of phenotypic differences between the individuals who had or did not have the mutation. Thus, some other gene appears to regulate similar cell functions. Crouzon syndrome, an autosomal dominant condition, is characterized by an abnormally shaped skull, hypertelorism, and proptosis associated with craniosynostosis. The appendicular skeleton is This is trial version www.adultpdf.com Growth Factor Regulation of Osteogenesis 117 spared. Although it is thus quite different in its clinical picture from achondroplasia, it is in some cases similarly associated with a mutation in the transmembrane region of the FGFR3 gene. The Crouzon mutation, however, is at a slightly different location in the gene than the achondroplasia mutation (90). These genetic studies demonstrate a considerable degree of refinement in the regulation of osteogen- esis by FGFR3. Subtle differences in receptor gene sequence may produce subtle, or not-so-subtle, differences in skeletal phenotype. Although the location of the mutation (near an autophosphorylation site, in the transmembrane domain, in the ligand binding region, etc.), may provide clues to the under- lying mechanism of the skeletal disorder, the genotype–phenotype relationships of these receptor abnor- malities are still not understood. Of considerable interest is the demonstration in transgenic mouse models that disruption of the FGFR3 gene promotes, rather than inhibits, bone growth (91,92). Mice lacking FGFR3 [FGFR3 knock- out or FGFR3 (−/−)] mice developed severe, progressive bone dysplasia with expansion of prolifer- ating and hypertrophic chondrocytes in the growth plate. Proliferating cell nuclear antigen, a marker of cell proliferation, was present in greater numbers of cells in FGFR3 (−/−) mice than in wild-type controls (92). Although histological evidence of an increased height of the hypertrophic zone in the growth plate was detectable in the late embryonic period (91), the FGFR3 (−/−) mice showed no obvious skeletal abnormalities during embryonic development (92). By 7 wk of age, all FGFR3 (−/−) femora and 75% of humeri had become bowed. Increased femur length in FGFR3 (−/−) skeletons relative to controls was first observed at 9 wk of age, and by 4 mo or older was 6–20% that of age- matched controls (91). These observations are consistent with the view that FGFR3 activation tends Fig. 1. Schematic illustration of a typical FGF receptor. The extracellular region contains three disulfide (S– S)-linked domains with structural homology to the immunoglobulins (Ig). The receptor traverses the cell mem- brane (red). The cytoplasmic region contains a bipartite kinase domain (orange). (Reproduced with permission from Trippel, S. B. (1994) Biologic regulation of bone growth, in Bone Formation and Repair (Brighton, C. T., Friedlaender, G., and Lane, J. M., eds.), American Academy of Orthopedic Surgeons, Rosemont, IL, pp. 39–60.) (Color illustration appears in e book.) This is trial version www.adultpdf.com 118 Trippel Fig. 2. Schematic illustration of the principal FGFR3 mutation associated with achondroplasia. This point muta- tion in the transmembrane domain of FGFR3 increases FGFR3 function. (Color illustration appears in e book.) to suppress skeletal growth. Indeed, the achondroplasia and TD-2 mutations are associated with ligand- independent activation of FGFR3 (93–95). Thus, both activation and inhibition of FGFR3 produce disordered osteogenesis, the former char- acterized by deficient bone growth and the latter by bone overgrowth. Given that FGFR3 mRNA is expressed in the cartilage rudiments of all bones during endochondral ossification in the developing mouse embryo (96), the observation the FGFR3 (−/−) mice show no obvious abnormalities during embryonic development suggests that alternative pathways are available for regulating the earliest phases of osteogenesis. Other members of the FGF receptor family also participate in osteogenesis. FGFR2 mutations are, as for FGFR3, associated with a variety of craniofacial syndromes. Mutations at several sites in the FGFR-2 extracellular domain (97,98) have recently been linked to Crouzon syndrome (Fig. 4). How- ever, 19 of the 32 Crouzon syndrome patients analyzed did not have mutations in this region and were presumed to have mutations elsewhere in the FGFR-2 gene or in other genes (97). As we have seen, some of these patients have mutations in the FGFR3 gene. Jackson–Weiss syndrome, another form of craniosynostosis, is distinguished by its foot abnormali- ties, including broad great toes with medial deviation and tarsal–metatarsal coalescence (Crouzon syn- drome, by contrast, is characterized by an absence of digital abnormalities [97]). Screening of Jackson– Weiss syndrome families identified a mutation in the FGFR2 extracellular domain only 3 bp away from one of the Crouzon-associated mutations (97). The complexity in the genotype–phenotype relationships of these FGFR-based skeletal disorders is further illustrated by studies of FGFR1. Mutations in the extracellular domain of this gene cause Pfeiffer’s syndrome, one of the classic autosomal dominant craniosynostosis syndromes (99). Pfeiffer’s This is trial version www.adultpdf.com Growth Factor Regulation of Osteogenesis 119 syndrome is associated with multiple digital abnormalities including broad, medially deviated great toes (as in Jackson–Weiss syndrome) and thumbs, with or without variable degrees of syndactyly or brachydactyly of other digits (unlike Jackson–Weiss syndrome) (100). However, Pfeiffer’s syndrome has also been shown to be caused by FGF2R mutations (101), and the identical FGFR2 mutations can cause both Pfeiffer’s and Crouzon’s syndrome phenotypes (102). This confusing lack of correlation between genotype and phenotype is undoubtedly due in part to overlap in the clinical parameters used to identify these syndromes. Such disparities argue for a dif- ferent taxonomy of skeletal anomalies, one based on genotype rather than, or in addition to, phenotype. More interestingly, however, these data demonstrate that the FGFs, acting via their receptors, regu- late osteogenesis through a remarkably refined system of signaling pathways that has only begun to be understood. Knowledge of the specific relationships between FGFR genotype and osteogenesis phenotype has recently been advanced by studies of Apert’s syndrome. Apert’s syndrome is a craniosynostosis asso- ciated with severe syndactyly of the hands and feet. In a recent study of 40 unrelated cases of this syndrome, missense substitutions were identified in adjacent amino acids located between the second and third immunoglobulin domains of FGFR2 (100) (Fig. 5). Both amino acid substitutions resulted from cytidine (C)-to-guanine (G) nucleic acid transversions. The C ♦ G transversion at nucleic acid position 934 (C934G) produced a substitution from serine to tryptophan at amino acid 252. The remain- ing patients showed a C ♦ G transversion at nucleic acid position 937 (C937G), resulting in a proline- to-arginine substitution at amino acid position 253. When syndactyly severity scores were correlated with mutation type, patients with the C937G mutation were found to have a higher syndactyly sever- ity score than patients with the C934G mutation. The difference was not statistically significant for Fig. 3. Schematic illustration of the mutations associated with type I and type II thanatophoric dysplasia. These two mildly different forms of thanatophoric dysplasia are produced by mutations at two widely separated sites in FGFR3, one in the extracellular region of the receptor and the second in an intracellular tyrosine kinase domain. (Color illustration appears in e book.) This is trial version www.adultpdf.com 120 Trippel the hands alone, but was statistically significant for the feet alone (p < 0.005) and for the hands and feet combined (p < 0.025). Of further interest is the fact that the C937G (Pro253Arg) mutation of FGFR-2 in Apert’s syndrome corresponds precisely to the C937G (Pro252Arg) mutation of FGFR1 in some cases with Pfieffer’s syndrome (99,100). These observations raise the possibility that in some circumstances, the particular skeletal developmental event can be dissected down to the level of indi- vidual amino acids and their location in proteins involved in growth-factor signaling. In contrast to the above example of a phenotypic difference associated with mutations that are extre- mely close to each other, some Crouzon patients with FGFR2 mutations on entirely different exons have no obvious phenotypic differences (100). The increasing number of distinct mutations that are being coupled with more carefully defined skeletal phenotypes will provide a potentially valuable resource for better understanding the role of FGF and its receptors in osteogenesis. The existence of at least 13 members of the FGF family and of multiple splice variants of the FGF receptor family yields an astronomical number of potential com- binations of ligands and receptors. This permits a remarkable degree of selectivity and refinement in signaling interactions. It also creates a daunting challenge to define the specific roles of each of them. TRANSFORMING GROWTH FACTOR-BETA (TGF-β) The transforming growth factor-betas are members of a large superfamily of cell signaling mole- cules that include the bone morphogenetic proteins (BMPs), activins, inhibins, and growth and dif- ferention factors (GDFs). Of the five TGF-βs, TGF-β1, TGF-β2, and TGF-β3 are known to be impor- tant in mammalian tissues (103–105). TGF-β family members have a particularly well-established participation in osteogenesis (103,105,106). Fig. 4. Schematic illustration of two of the mutations associated with Crouzon’s syndrome. The two muta- tions in the extracellular region of FGFR2 affect the same amino acid in the receptor and may thus be expected to produce the same clinical picture. However, Crouzon’s syndrome can also be caused by mutations in the transmembrane region of FGFR3. (Color illustration appears in e book.) This is trial version www.adultpdf.com [...]... 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