- Email: [email protected]
Growth Factor Targets for Orthodontic Treatments
Growth Factor Targets for Orthodontic Treatments Elisabeth R. Barton and Charles Crowder The use of growth factors to modulate the craniofacial skeleton is poised to become a new avenue of therapy for the orthodontic community. Growth factor actions occur not only directly on cells within bone but may also stimulate other tissues, such as skeletal muscle that can indirectly modify skeletal growth. The interaction of direct and indirect targets that lead to skeletal changes must be recognized to design effective treatment regimens. As an example of this concept, this review will focus on insulin-like growth factor-1; its actions in muscle, bone, and cartilage; and how the interplay among its effects on multiple targets could ultimately modulate craniofacial growth. (Semin Orthod 2010;16:128-134.) © 2010 Published by Elsevier Inc.
ecause much of orthodontic tribulations are created by the disproportionate growth of the jaws and craniofacial complex, a clearer understanding of the etiological process of growth into the malocclusion and dentofacial deformity must be understood. How is facial growth regulated? Can these processes be modulated through exogenous factors to treat pathologic growth?
B
Growth in the Craniofacial Complex The bones of the skull can be subdivided into 3 parts: the neurocranium (calvaria, cranial vault), the splanchocranium/viscerocranium (facial skeleton), and the basicranium/chondrocranium (cranial base). Two distinct ossification processes, endochondral and intramembranous, govern the formation of the skull. Endochondral ossification is the process by which the general bone shape is
From the Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA. Department of Orthodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA. Address correspondence to Elisabeth R. Barton, Department of Anatomy and Cell Biology, 441A Levy Building, 240 S. 40th Street, University of Pennsylvania, Philadelphia, PA 19104. Phone: 215-5730887; Fax: 215-573-2324; E-mail: [email protected] © 2010 Published by Elsevier Inc. 1073-8746/10/1602-0$30.00/0 doi:10.1053/j.sodo.2010.02.004
128
laid down first by a cartilage scaffold followed by progressive replacement of bone. Alternatively, intramembranous ossification represents bone that develops directly from the dense fibrous membrane covering the brain without a cartilaginous precursor.1 Although the cranial-base bones are primarily formed by endochondral ossification, the cranial vault and facial bones are primarily formed by intramembranous ossification. Within the viscerocranium (facial skeleton), the mandible is formed by a combination of these ossification processes with endochondral growth at the condylar head and intramembranous ossification at the ramus and mandibular body.
Modulation of Craniofacial Growth The functional matrix theory posits that neither bone nor cartilage is the primary determinate of bone growth in the craniofacial complex but that the soft tissue surrounding these hard tissues dictates growth. Described by Moss in the late 1960s, this theory suggests that growth of the jaw occurs not by the mandible cartilage or nasal septum but instead the face grows as a response to functional needs and neurotrophic influences to which the jaws are embedded.2-4 It hypothesized that the enlargement of nasal and oral cavities, resulting from increased functional needs, determined growth of the maxilla and
Seminars in Orthodontics, Vol 16, No 2 (June), 2010: pp 128-134
Growth Factor Targets for Orthodontic Treatments
mandible. It further hypothesized that cranial vault size is determined by brain size and pressure on the sutures. Therefore, although the site of growth is within the craniofacial skeleton, modulators of growth exist external to the mandible and craniofacial complex. Although some researchers still may believe that cartilage is the growth “captain,” the functional matrix theory has many proponents. The impact of aberrant function on the mandible and craniofacial complex has been well documented. Ankylosis of the mandibular condyle can greatly impair mandibular growth.5 A lateral functional shift can cause asymmetric mandibular growth.6 A posterior crossbite of the dental occlusal plane can promote aberrant growth of the maxilla and mandible.6 Clinicians, in turn, use physical forces to alter maxillary growth via the introduction of headgear and facemask therapy, along with distraction osteogenesis through maxillary palatal expansion, which can also induce bone growth.7 There are many who believe that the mandible and nasomaxillary complexes respond differently to soft-tissue changes. It is well established that maxillary growth can be modified with orthopedic intervention; however, the literature on mandibular plasticity is somewhat conflicting. Many sources suggest that modifications of mandibular morphology are caused by changes in function/soft tissue, whereas nearly equal numbers of investigators report that the mandible cannot be influenced by soft-tissue/functional alterations and that any morphologic modifications observed are not attributable to altered growth of the condyle but rather compensatory changes in the dental arches. However, data from several animal models have demonstrated the ability of the mandibular condyle to adapt to an altered functional position,8-10 suggesting it may be possible to influence mandibular condyle changes. All of these examples suggest that physical forces associated with function are critical to the resultant growth of the mandible and craniofacial complex.
Insulin-Like Growth Factors Actions in General Insulin-like growth factor-1 (IGF-1) is essential for normal growth and development. IGF-1 is a major mediator of postnatal growth within the
129
growth hormone (GH)/IGF-1 axis. The main source of IGF-1 is the liver,11 which is delivered in an endocrine fashion to target tissues. However, many cell types, including muscle, brain, bone, and kidney, produce and respond to IGF-1 via autocrine or paracrine actions. In both the circulation and the extracellular matrix, IGF-1 is stabilized by associating with IGF binding proteins (IGFBPs), of which there are 6.12 Release from IGFBPs allows IGF-1 to bind to its transmembrane tyrosine kinase receptor, IGF-1R (Fig 1). Upon IGF-1 binding, IGF-1R autophosphorylates on several sites in the cytoplasmic domain, which initiates multiple signaling cascades. Through the adapter proteins, grb2 and mSos, the Ras-raf-1-MEK-Erk pathway is stimulated and has been shown to mediate proliferation.13 A second signaling arm is stimulated by the recruitment of phospho inositol 3-kinase, which coordinates the activation of Akt to promote translation and survival. Proper embryonic development relies on IGF-1 signaling; IGF-IR knockout mice die at birth as the result of cardiac failure, and IGF-1 knockout mice are dwarfs that rarely survive past birth.14 The IGF-1⫺null mice that survive continue to grow slowly,15 and mice overexpressing IGF-1 systemically are 1.3 times as large as control mice,16 indicating that IGF-1 signaling is also essential for postnatal growth.
Direct Actions of IGF-1 in Bone Many growth factors are involved in the formation of bone in addition to IGF-1, including the bone morphogenetic proteins, transforming growth factor beta, fibroblast growth factor, platelet-derived growth factor, and vascular endothelial growth factor.17,18 Furthermore, there is coordination in timing and actions of these growth factors during bone development, growth, and healing. Thus, from the vast literature on growth factor actions, it is clear that the use of a single growth factor in the absence of the battery of additional proteins is unlikely to be sufficient to promote growth of the craniofacial skeleton. However, for the purposes of this review, we will discuss the direct actions of IGF-1 on craniofacial growth. The primary site of action for IGF-1 is at the growth plate, where GH stimulates production of IGF-1 from the chondrocytes.19-21 GH and
130
E.R. Barton and C. Crowder
Figure 1. Signaling pathways for IGF-1. Most IGF-1 in the extracellular space is bound to IGF binding proteins (IGFBPs). Release from IGFBPs allows IGF-1 to bind to its transmembrane tyrosin kinase receptor, IGF-(R), which leads to autophosphorylation of the receptor cytoplasmic domain and activation of insulin receptor substrates (IRS1-4). The adapter proteins, grb2 and mSos, associate with phosphorylated IRS, after which association with the G protein, ras, occurs and leads to stimulation of the ras-raf-MEK-ERK pathway. A second signaling arm is stimulated by the recruitment of phospho inositol 3-kinase (PI-3K) to IRS, which coordinates the activation of Akt pathways. The ERK pathway promotes cell proliferation, and Akt promotes translation and survival. (Color version of figure is available online.)
IGF-1 then help to regulate the differentiation and maturation of chondrocytes, directly stimulate osteoblasts and ultimately lead to endochondral ossification. In the IGF-1⫺null mouse described previously, there was a generalized decrease in craniofacial size.22 However, there was also a change in the craniofacial proportions, suggesting that IGF-1 differentially regulated growth of the skull. Specifically, the cranium was more rounded, and the nasomaxillary complex was smaller in all dimensions, leading to the shorter snout. In mice subjected to daily IGF-1 injections, the nasomaxillary complex exhibited increased osteoblasts compared with age-matched control mice, indicating the anabolic actions of IGF-1 in bone formation.23 The duration of the study did not enable examination of the effects of IGF-1 on the craniofacial proportions. However, the results support the hypothesis that postnatal IGF-1 delivery primarily affects growth plate bone formation.
Direct Actions of IGF-1 in Skeletal Muscle IGF-1 has long been recognized as one of the critical factors for coordinating muscle growth, enhancing muscle repair, and increasing muscle
mass and strength. IGF-1 is a potent skeletal muscle growth factor that mediates GH’s effects on skeletal muscle.24 IGF-1 can help in 2 main ways. First, IGF-1 acts directly on the muscle fibers to increase protein synthesis and muscle mass. Second, it also drives activated satellite cells (a stem cell-like population residing close to muscle fibers and a source for replenishing nuclear content of the muscle) to fuse to existing muscle fibers, to help repair damaged regions of the fibers, and to promote muscle growth25 (Fig 2). This process is very similar to that which occurs during myogenesis, which is also regulated by IGF-1. When IGF-IR is inactivated specifically in skeletal muscle, mice have 10% to 30% smaller muscles than wild-type control mice,26 exemplifying the IGF-1 requirement in postnatal skeletal muscle growth. Increasing IGF-1 in muscle by infusion of recombinant IGF-1,27 tissue-specific transgenic overexpression of IGF-128,29 or via the injection of muscle with recombinant viral vectors containing igf-1 cDNA,30 causes hypertrophy, can ameliorate dystrophic muscle pathology and improve function.31,32 Overexpression of IGF-1 also supplements the natural hypertrophy caused by resistance training.33 Taken together, IGF-1 administration to muscle results in increased
Growth Factor Targets for Orthodontic Treatments
131
Figure 2. Satellite cells in muscle differentiation. Satellite cells are found associated with muscle fibers outside of the sarcolemma and within the basal lamina (in yellow). Activation by hepatocyte growth factor (HGF) can occur with growth or damage, and leads to the expression of IGF-IR on the satellite cells. IGF-1 binding to its receptor is important for satellite cell proliferation and differentiation into myoblasts. Single myoblasts can then fuse into existing fibers to repair damage and increase fiber size, or they can fuse to each other to create new muscle fibers. (Color version of figure is available online.)
mass and strength, and this functional hypertrophy could create sufficient physical force to modulate bone growth.
Indirect Actions of Muscle to Bone It seems clear that the mandible is translated in space by the growth of muscles and other adjacent soft tissues and that addition of new bone at the condyle is in response to the soft tissue changes.34 For quite some time, it has been accepted that the degree of muscle and bone mass are associated.35 Other studies since have found more supporting evidence that the levels of muscle and bone mass are correlated and that the resulting cause-and-effect relationship has been termed the “muscle-bone unit.”36-39 Experimentally, attempts to increase muscle mass and strength required vigorous exercise, which introduced confounding variables (eg, increased bone blood flow and osteogenic factor changes like GH).40,41 However, stimulating hypertrophy through growth factors eliminates these variables and can test the effects of muscle mass changes on the craniofacial complex. Increased masticatory muscle mass found in the mdx mouse (a mouse model for Duchenne muscular dystrophy) significantly changes mandibular condyle maturation.9 The profound increase in muscle size in mice lacking myostatin, a negative regulator of muscle growth,42 causes increased sagittal suture complexity43 and craniofacial skeleton shape.44 Thus, regardless of how muscle hypertrophy occurs, there are apparent changes in craniofacial skeletal morphology.
Because IGF-1 can directly modulate both muscle and bone, one would predict that there would be synergy between the muscle and bone effects, potentially leading to more noticeable changes in the craniofacial complex (Fig 3). A systematic study of the effects of IGF-1⫺mediated hypertrophy has not been performed to date but requires tissue specific targeting of IGF-1 to eliminate the direct effects of this growth factor on bone.
Delivery of IGF-1 Several clinical trials have assessed the efficacy of systemic delivery of recombinant IGF-1 in patients who could benefit from muscle mass and strength gains. These include elderly patients,
Figure 3. Direct and indirect actions of IGF-1 on bone growth. GH stimulates production of circulating IGF-1 from the liver, as well as IGF-1 expression at the growth plates. Both GH and IGF-1 appear to regulate the formation of bone. IGF-1 also directly modulates the growth of skeletal muscle, which in turn, can modify the growth of bone.
132
E.R. Barton and C. Crowder
patients with GH deficiency, and those who suffer from amyotrophic lateral sclerosis and myotonic dystrophy.45-51 Because IGF-1 is a potent growth factor in many tissues of the body and poses a potential carcinogenic risk, investigators have introduced IGF-1 in limiting amounts. Thus, these trials have produced mixed results because the ability for IGF-1 to provide any benefit to skeletal muscle is constrained by both the low level of protein administered as well as the limited distribution of IGF-1 to the muscle by the circulation.45,48,49 Two strategies have been considered that could reduce the risks of systemic effects. First, one can “mask” the IGF-1 by chelating it to an agent that reduces its bioavailability until it is needed. A new compound, which is a complex of recombinant IGF-1 and IGFBP3 called IPlexTM,52 has been developed and received approval from the Food and Drug Administration in 2007. IGFBP3 is the major circulating binding protein and regulates the levels of free IGF-1 in the bloodstream.12 Greater levels of IGF-1 can be delivered via IPlexTM to patients with less systemic risk because cleavage of the complex and IGF-1 release occurs only at target tissues. This extends the bioavailability for these targets. Subsequent legal action has prevented the use of Iplex for growth therapy. However, clinical trials are underway to assess the efficacy of IPlexTM in myotonic dystrophy patients (http://www.mdausa.org/news/060104 insmed_iplex_mmd.html). A second strategy relies on the production of IGF-1 in the target tissue. Liver is the predominant source of circulating IGF-1,11 but this growth factor can also be produced by skeletal muscle. In mice lacking liver IGF-1 through tissue-specific gene targeting, endogenous expression of IGF-1 by the muscle is sufficient to maintain normal muscle mass.53 Thus, increasing the local muscle production of IGF-1 could provide an effective method for increasing muscle mass. Previous demonstration of this concept used transgenic animals expressing IGF-1 specifically in muscle.28,29 In these mice, muscles exhibit a 40% increase in muscle mass, including the masticatory muscles, with no increase in circulating IGF-1 (Fig 4). This mouse model would be ideal to separate the direct and indirect effects of IGF-1 on the craniofacial skeleton. Obviously, germline transmission of any gene is not a rational approach for people. However, viral admin-
Figure 4. Muscle-specific expression of IGF-1 in transgenic mice leads to a 40% increase in mass of limb (represented by tibialis anterior) and masticatory (represented by masseter) muscles (upper panel). Tissue content of IGF-1 is more than 4-fold greater in the transgenic muscles (lower left panel), whereas there is no change in total circulating IGF-1 with restricted transgenic expression (lower right panel). WT, wild type; TG, transgenic. (Color version of figure is available online.)
istration of the cDNA encoding for IGF-1 into target tissues is a viable option for experimental studies30,33,54 and possibly for gene therapy in patients. Indeed, clinical trials for genetic muscle diseases could help to pave the way for this tool to be used routinely to deliver therapeutic genes to skeletal muscle. For orthodontic procedures, this could drastically streamline the current clinical approaches. Instead of highly invasive methods for modulating the craniofacial complex, a single set of injections could provide sufficient and durable expression of growth factors and allow the masticatory muscles to do the moving.
References 1. de Crombrugghe B, Lefebvre V, Nakashima K: Regulatory mechanisms in the pathways of cartilage and bone formation. Curr Opin Cell Biol 13:721-727, 2001 2. Moss ML: A theoretical analysis of the functional matrix. Acta Biotheor 18:195-202, 1968 3. Moss ML, Rankow RM: The role of the functional matrix in mandibular growth. Angle Orthod 38:95-103, 1968
Growth Factor Targets for Orthodontic Treatments
4. Moss ML: The functional matrix hypothesis revisited. 1. The role of mechanotransduction. Am J Orthod Dentofac Orthop 112:8-11, 1997 5. Ko EW, Huang CS, Chen YR, et al: Cephalometric craniofacial characteristics in patients with temporomandibular joint ankylosis. Chang Gung Med J 28:456-466, 2005 6. Kecik D, Kocadereli I, Saatci I: Evaluation of the treatment changes of functional posterior crossbite in the mixed dentition. Am J Orthod Dentofac Orthop 131: 202-215, 2007 7. Mew J: Re: Handelman CS, Wang L, BeGole EA, Haas AJ. Nonsurgical rapid maxillary expansion in adults: Report on 47 cases. Angle Orthod 70:129-144, 1999. Angle Orthod 71:3, 2001 8. Ghafari J, Heeley JD: Condylar adaptation to muscle alteration in the rat. Angle Orthod 52:26-37, 1982 9. Ghafari J, Cowin DH: Condylar cartilage in the muscular dystrophic mouse. Am J Orthod Dentofac Orthop 95: 107-114, 1989 10. Ghafari J, Degroote C: Condylar cartilage response to continuous mandibular displacement in the rat. Angle Orthod 56:49-57, 1986 11. Schwander JC, Hauri C, Zapf J, et al: Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: Dependence on growth hormone status. Endocrinologist 113:297-305, 1983 12. Firth SM, Baxter RC: Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 23:824-854, 2002 13. Coolican SA, Samuel DS, Ewton DZ, et al: The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem 272:66536662, 1997 14. Liu JP, Baker J, Perkins AS, et al: Mice carrying null mutations of the genes encoding insulin-like growth factor I (IGF-1) and type 1 IGF receptor (Igf1r). Cell 75:59-72, 1993 15. Baker J, Liu JP, Robertson EJ, et al: Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73-82, 1993 16. Mathews LS, Hammer RE, Behringer RR, et al: Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology 123:2827-2833, 1988 17. Solheim E: Growth factors in bone. Int Orthop 22:410416, 1998 18. Spears R, Svoboda KKH: Growth factors and signaling proteins in craniofacial development. Semin Orthod 11: 184-198, 2005 19. Lindahl A, Isgaard J, Isaksson OG: Growth hormone in vivo potentiates the stimulatory effect of insulin-like growth factor-1 in vitro on colony formation of epiphyseal chondrocytes isolated from hypophysectomized rats. Endocrinologist 121:1070-1075, 1987 20. Isaksson OG, Ohlsson C, Nilsson A, et al: Regulation of cartilage growth by growth hormone and insulin-like growth factor I. Pediatr Nephrol 5:451-453, 1991 21. Ohlsson C, Isaksson O, Lindahl A: Clonal analysis of rat tibia growth plate chondrocytes in suspension culture— Differential effects of growth hormone and insulin-like growth factor I. Growth Regul 4:1-7, 1994
133
22. McAlarney ME, Rizos M, Rocca EG, et al: The quantitative and qualitative analysis of the craniofacial skeleton of mice lacking the IGF-1 gene. Clin Orthod Res 4:206219, 2001 23. Tokimasa C, Kawata T, Fujita T, et al: Effects of insulinlike growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice. Arch Oral Biol 48:31-38, 2003 24. Kim H, Barton E, Muja N, et al: Intact insulin and insulin-like growth factor-I receptor signaling is required for growth hormone effects on skeletal muscle growth and function in vivo. Endocrinologist 146:1772-1779, 2005 25. Florini JR, Ewton DZ, Coolican SA: Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17:481-517, 1996 26. Fernandez AM, Dupont J, Farrar RP, et al: Muscle-specific inactivation of the IGF-1 receptor induces compensatory hyperplasia in skeletal muscle. J Clin Invest 109: 347-355, 2002 27. Adams GR, McCue SA: Localized infusion of IGF-1 results in skeletal muscle hypertrophy in rats. J Appl Physiol 84:1716-1722, 1998 28. Coleman ME, DeMayo F, Yin KC, et al: Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem 270:12109-12116, 1995 29. Musaro A, McCullagh K, Paul A, et al: Localized IGF-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 27:195200, 2001 30. Barton-Davis ER, Shoturma DI, Musaro A, et al: Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci U S A 95:15603-15607, 1998 31. Barton ER, Morris L, Musaro A, et al: Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice. J Cell Biol 157:137-148, 2002 32. Lynch GS, Cuffe SA, Plant DR, et al: IGF-1 treatment improves the functional properties of fast- and slowtwitch skeletal muscles from dystrophic mice. Neuromuscul Disord 11:260-268, 2001 33. Lee S, Barton ER, Sweeney HL, et al: Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J Appl Physiol 96:10971104, 2004 34. Proffit WR, Fields HW Jr.: Contemporary Orthodontics. Philadelphia, Mosby, 2000 35. Doyle F, Brown J, Lachance C: Relation between bone mass and muscle weight. Lancet 1:391-393, 1970 36. Andreoli A, Monteleone M, Van Loan M, et al: Effects of different sports on bone density and muscle mass in highly trained athletes. Med Sci Sports Exerc 33:507-511, 2001 37. Kaye M, Kusy RP: Genetic lineage, bone mass, and physical activity in mice. Bone 17:131-135, 1995 38. Schoenau E, Frost HM: The “muscle-bone unit” in children and adolescents. Calcif Tissue Int 70:405-407, 2002 39. Rauch F, Bailey DA, Baxter-Jones A, et al: The “musclebone unit” during the pubertal growth spurt. Bone 34: 771-775, 2004
134
E.R. Barton and C. Crowder
40. Godfrey RJ, Madgwick Z, Whyte GP: The exercise-induced growth hormone response in athletes. Sports Med 33:599-613, 2003 41. Yao Z, Lafage-Proust MH, Plouet J, et al: Increase of both angiogenesis and bone mass in response to exercise depends on VEGF. J Bone Miner Res 19:1471-1480, 2004 42. McPherron AC, Lawler AM, Lee SJ: Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83-90, 1997 43. Byron CD, Hamrick MW, Wingard CJ: Alterations of temporalis muscle contractile force and histological content from the myostatin and Mdx deficient mouse. Arch Oral Biol 51:396-405, 2006 44. Vecchione L, Byron C, Cooper GM, et al: Craniofacial morphology in myostatin-deficient mice. J Dent Res 86: 1068-1072, 2007 45. Borasio GD, Robberecht W, Leigh PN, et al: A placebocontrolled trial of insulin-like growth factor-I in amyotrophic lateral sclerosis. European ALS/IGF-I Study Group. Neurologist 51:583-586, 1998 46. Butterfield GE, Thompson J, Rennie MJ, et al: Effect of rhGH and rhIGF-I treatment on protein utilization in elderly women. Am J Physiol 272:E94-E99, 1997 47. Cusi K, DeFronzo R: Recombinant human insulin-like growth factor I treatment for 1 week improves metabolic control in type 2 diabetes by ameliorating hepatic and muscle insulin resistance. J Clin Endocrinol Metab 85: 3077-3084, 2000
48. Friedlander AL, Butterfield GE, Moynihan S, et al: One year of insulin-like growth factor I treatment does not affect bone density, body composition, or psychological measures in postmenopausal women. J Clin Endocrinol Metab 86:1496-1503, 2001 49. Lai EC, Felice KJ, Festoff BW, et al: Effect of recombinant human insulin-like growth factor-I on progression of ALS. A placebo-controlled study. The North America ALS/IGF-I Study Group. Neurologist 49:1621-1630, 1997 50. Mauras N, O’Brien KO, Welch S, et al: Insulin-like growth factor I and growth hormone (GH) treatment in GH-deficient humans: Differential effects on protein, glucose, lipid, and calcium metabolism. J Clin Endocrinol Metab 85:1686-1694, 2000 51. Waters D, Danska J, Hardy K, et al: Recombinant human growth hormone, insulin-like growth factor 1, and combination therapy in AIDS-associated wasting. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 125:865-872, 1996 52. Kemp SF, Fowlkes JL, Thrailkill KM: Efficacy and safety of mecasermin rinfabate. Exp Opin Biol Ther 6:533-538, 2006 53. Sjogren K, Liu JL, Blad K, et al: Liver-derived insulin-like growth factor I (IGF-1) is the principal source of IGF-1 in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci U S A 96:7088-7092, 1999 54. Barton ER: Viral expression of insulin-like growth factor-I isoforms promotes different responses in skeletal muscle. J Appl Physiol 100:1778-1784, 2006
ecause much of orthodontic tribulations are created by the disproportionate growth of the jaws and craniofacial complex, a clearer understanding of the etiological process of growth into the malocclusion and dentofacial deformity must be understood. How is facial growth regulated? Can these processes be modulated through exogenous factors to treat pathologic growth?
B
Growth in the Craniofacial Complex The bones of the skull can be subdivided into 3 parts: the neurocranium (calvaria, cranial vault), the splanchocranium/viscerocranium (facial skeleton), and the basicranium/chondrocranium (cranial base). Two distinct ossification processes, endochondral and intramembranous, govern the formation of the skull. Endochondral ossification is the process by which the general bone shape is
From the Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA. Department of Orthodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA. Address correspondence to Elisabeth R. Barton, Department of Anatomy and Cell Biology, 441A Levy Building, 240 S. 40th Street, University of Pennsylvania, Philadelphia, PA 19104. Phone: 215-5730887; Fax: 215-573-2324; E-mail: [email protected] © 2010 Published by Elsevier Inc. 1073-8746/10/1602-0$30.00/0 doi:10.1053/j.sodo.2010.02.004
128
laid down first by a cartilage scaffold followed by progressive replacement of bone. Alternatively, intramembranous ossification represents bone that develops directly from the dense fibrous membrane covering the brain without a cartilaginous precursor.1 Although the cranial-base bones are primarily formed by endochondral ossification, the cranial vault and facial bones are primarily formed by intramembranous ossification. Within the viscerocranium (facial skeleton), the mandible is formed by a combination of these ossification processes with endochondral growth at the condylar head and intramembranous ossification at the ramus and mandibular body.
Modulation of Craniofacial Growth The functional matrix theory posits that neither bone nor cartilage is the primary determinate of bone growth in the craniofacial complex but that the soft tissue surrounding these hard tissues dictates growth. Described by Moss in the late 1960s, this theory suggests that growth of the jaw occurs not by the mandible cartilage or nasal septum but instead the face grows as a response to functional needs and neurotrophic influences to which the jaws are embedded.2-4 It hypothesized that the enlargement of nasal and oral cavities, resulting from increased functional needs, determined growth of the maxilla and
Seminars in Orthodontics, Vol 16, No 2 (June), 2010: pp 128-134
Growth Factor Targets for Orthodontic Treatments
mandible. It further hypothesized that cranial vault size is determined by brain size and pressure on the sutures. Therefore, although the site of growth is within the craniofacial skeleton, modulators of growth exist external to the mandible and craniofacial complex. Although some researchers still may believe that cartilage is the growth “captain,” the functional matrix theory has many proponents. The impact of aberrant function on the mandible and craniofacial complex has been well documented. Ankylosis of the mandibular condyle can greatly impair mandibular growth.5 A lateral functional shift can cause asymmetric mandibular growth.6 A posterior crossbite of the dental occlusal plane can promote aberrant growth of the maxilla and mandible.6 Clinicians, in turn, use physical forces to alter maxillary growth via the introduction of headgear and facemask therapy, along with distraction osteogenesis through maxillary palatal expansion, which can also induce bone growth.7 There are many who believe that the mandible and nasomaxillary complexes respond differently to soft-tissue changes. It is well established that maxillary growth can be modified with orthopedic intervention; however, the literature on mandibular plasticity is somewhat conflicting. Many sources suggest that modifications of mandibular morphology are caused by changes in function/soft tissue, whereas nearly equal numbers of investigators report that the mandible cannot be influenced by soft-tissue/functional alterations and that any morphologic modifications observed are not attributable to altered growth of the condyle but rather compensatory changes in the dental arches. However, data from several animal models have demonstrated the ability of the mandibular condyle to adapt to an altered functional position,8-10 suggesting it may be possible to influence mandibular condyle changes. All of these examples suggest that physical forces associated with function are critical to the resultant growth of the mandible and craniofacial complex.
Insulin-Like Growth Factors Actions in General Insulin-like growth factor-1 (IGF-1) is essential for normal growth and development. IGF-1 is a major mediator of postnatal growth within the
129
growth hormone (GH)/IGF-1 axis. The main source of IGF-1 is the liver,11 which is delivered in an endocrine fashion to target tissues. However, many cell types, including muscle, brain, bone, and kidney, produce and respond to IGF-1 via autocrine or paracrine actions. In both the circulation and the extracellular matrix, IGF-1 is stabilized by associating with IGF binding proteins (IGFBPs), of which there are 6.12 Release from IGFBPs allows IGF-1 to bind to its transmembrane tyrosine kinase receptor, IGF-1R (Fig 1). Upon IGF-1 binding, IGF-1R autophosphorylates on several sites in the cytoplasmic domain, which initiates multiple signaling cascades. Through the adapter proteins, grb2 and mSos, the Ras-raf-1-MEK-Erk pathway is stimulated and has been shown to mediate proliferation.13 A second signaling arm is stimulated by the recruitment of phospho inositol 3-kinase, which coordinates the activation of Akt to promote translation and survival. Proper embryonic development relies on IGF-1 signaling; IGF-IR knockout mice die at birth as the result of cardiac failure, and IGF-1 knockout mice are dwarfs that rarely survive past birth.14 The IGF-1⫺null mice that survive continue to grow slowly,15 and mice overexpressing IGF-1 systemically are 1.3 times as large as control mice,16 indicating that IGF-1 signaling is also essential for postnatal growth.
Direct Actions of IGF-1 in Bone Many growth factors are involved in the formation of bone in addition to IGF-1, including the bone morphogenetic proteins, transforming growth factor beta, fibroblast growth factor, platelet-derived growth factor, and vascular endothelial growth factor.17,18 Furthermore, there is coordination in timing and actions of these growth factors during bone development, growth, and healing. Thus, from the vast literature on growth factor actions, it is clear that the use of a single growth factor in the absence of the battery of additional proteins is unlikely to be sufficient to promote growth of the craniofacial skeleton. However, for the purposes of this review, we will discuss the direct actions of IGF-1 on craniofacial growth. The primary site of action for IGF-1 is at the growth plate, where GH stimulates production of IGF-1 from the chondrocytes.19-21 GH and
130
E.R. Barton and C. Crowder
Figure 1. Signaling pathways for IGF-1. Most IGF-1 in the extracellular space is bound to IGF binding proteins (IGFBPs). Release from IGFBPs allows IGF-1 to bind to its transmembrane tyrosin kinase receptor, IGF-(R), which leads to autophosphorylation of the receptor cytoplasmic domain and activation of insulin receptor substrates (IRS1-4). The adapter proteins, grb2 and mSos, associate with phosphorylated IRS, after which association with the G protein, ras, occurs and leads to stimulation of the ras-raf-MEK-ERK pathway. A second signaling arm is stimulated by the recruitment of phospho inositol 3-kinase (PI-3K) to IRS, which coordinates the activation of Akt pathways. The ERK pathway promotes cell proliferation, and Akt promotes translation and survival. (Color version of figure is available online.)
IGF-1 then help to regulate the differentiation and maturation of chondrocytes, directly stimulate osteoblasts and ultimately lead to endochondral ossification. In the IGF-1⫺null mouse described previously, there was a generalized decrease in craniofacial size.22 However, there was also a change in the craniofacial proportions, suggesting that IGF-1 differentially regulated growth of the skull. Specifically, the cranium was more rounded, and the nasomaxillary complex was smaller in all dimensions, leading to the shorter snout. In mice subjected to daily IGF-1 injections, the nasomaxillary complex exhibited increased osteoblasts compared with age-matched control mice, indicating the anabolic actions of IGF-1 in bone formation.23 The duration of the study did not enable examination of the effects of IGF-1 on the craniofacial proportions. However, the results support the hypothesis that postnatal IGF-1 delivery primarily affects growth plate bone formation.
Direct Actions of IGF-1 in Skeletal Muscle IGF-1 has long been recognized as one of the critical factors for coordinating muscle growth, enhancing muscle repair, and increasing muscle
mass and strength. IGF-1 is a potent skeletal muscle growth factor that mediates GH’s effects on skeletal muscle.24 IGF-1 can help in 2 main ways. First, IGF-1 acts directly on the muscle fibers to increase protein synthesis and muscle mass. Second, it also drives activated satellite cells (a stem cell-like population residing close to muscle fibers and a source for replenishing nuclear content of the muscle) to fuse to existing muscle fibers, to help repair damaged regions of the fibers, and to promote muscle growth25 (Fig 2). This process is very similar to that which occurs during myogenesis, which is also regulated by IGF-1. When IGF-IR is inactivated specifically in skeletal muscle, mice have 10% to 30% smaller muscles than wild-type control mice,26 exemplifying the IGF-1 requirement in postnatal skeletal muscle growth. Increasing IGF-1 in muscle by infusion of recombinant IGF-1,27 tissue-specific transgenic overexpression of IGF-128,29 or via the injection of muscle with recombinant viral vectors containing igf-1 cDNA,30 causes hypertrophy, can ameliorate dystrophic muscle pathology and improve function.31,32 Overexpression of IGF-1 also supplements the natural hypertrophy caused by resistance training.33 Taken together, IGF-1 administration to muscle results in increased
Growth Factor Targets for Orthodontic Treatments
131
Figure 2. Satellite cells in muscle differentiation. Satellite cells are found associated with muscle fibers outside of the sarcolemma and within the basal lamina (in yellow). Activation by hepatocyte growth factor (HGF) can occur with growth or damage, and leads to the expression of IGF-IR on the satellite cells. IGF-1 binding to its receptor is important for satellite cell proliferation and differentiation into myoblasts. Single myoblasts can then fuse into existing fibers to repair damage and increase fiber size, or they can fuse to each other to create new muscle fibers. (Color version of figure is available online.)
mass and strength, and this functional hypertrophy could create sufficient physical force to modulate bone growth.
Indirect Actions of Muscle to Bone It seems clear that the mandible is translated in space by the growth of muscles and other adjacent soft tissues and that addition of new bone at the condyle is in response to the soft tissue changes.34 For quite some time, it has been accepted that the degree of muscle and bone mass are associated.35 Other studies since have found more supporting evidence that the levels of muscle and bone mass are correlated and that the resulting cause-and-effect relationship has been termed the “muscle-bone unit.”36-39 Experimentally, attempts to increase muscle mass and strength required vigorous exercise, which introduced confounding variables (eg, increased bone blood flow and osteogenic factor changes like GH).40,41 However, stimulating hypertrophy through growth factors eliminates these variables and can test the effects of muscle mass changes on the craniofacial complex. Increased masticatory muscle mass found in the mdx mouse (a mouse model for Duchenne muscular dystrophy) significantly changes mandibular condyle maturation.9 The profound increase in muscle size in mice lacking myostatin, a negative regulator of muscle growth,42 causes increased sagittal suture complexity43 and craniofacial skeleton shape.44 Thus, regardless of how muscle hypertrophy occurs, there are apparent changes in craniofacial skeletal morphology.
Because IGF-1 can directly modulate both muscle and bone, one would predict that there would be synergy between the muscle and bone effects, potentially leading to more noticeable changes in the craniofacial complex (Fig 3). A systematic study of the effects of IGF-1⫺mediated hypertrophy has not been performed to date but requires tissue specific targeting of IGF-1 to eliminate the direct effects of this growth factor on bone.
Delivery of IGF-1 Several clinical trials have assessed the efficacy of systemic delivery of recombinant IGF-1 in patients who could benefit from muscle mass and strength gains. These include elderly patients,
Figure 3. Direct and indirect actions of IGF-1 on bone growth. GH stimulates production of circulating IGF-1 from the liver, as well as IGF-1 expression at the growth plates. Both GH and IGF-1 appear to regulate the formation of bone. IGF-1 also directly modulates the growth of skeletal muscle, which in turn, can modify the growth of bone.
132
E.R. Barton and C. Crowder
patients with GH deficiency, and those who suffer from amyotrophic lateral sclerosis and myotonic dystrophy.45-51 Because IGF-1 is a potent growth factor in many tissues of the body and poses a potential carcinogenic risk, investigators have introduced IGF-1 in limiting amounts. Thus, these trials have produced mixed results because the ability for IGF-1 to provide any benefit to skeletal muscle is constrained by both the low level of protein administered as well as the limited distribution of IGF-1 to the muscle by the circulation.45,48,49 Two strategies have been considered that could reduce the risks of systemic effects. First, one can “mask” the IGF-1 by chelating it to an agent that reduces its bioavailability until it is needed. A new compound, which is a complex of recombinant IGF-1 and IGFBP3 called IPlexTM,52 has been developed and received approval from the Food and Drug Administration in 2007. IGFBP3 is the major circulating binding protein and regulates the levels of free IGF-1 in the bloodstream.12 Greater levels of IGF-1 can be delivered via IPlexTM to patients with less systemic risk because cleavage of the complex and IGF-1 release occurs only at target tissues. This extends the bioavailability for these targets. Subsequent legal action has prevented the use of Iplex for growth therapy. However, clinical trials are underway to assess the efficacy of IPlexTM in myotonic dystrophy patients (http://www.mdausa.org/news/060104 insmed_iplex_mmd.html). A second strategy relies on the production of IGF-1 in the target tissue. Liver is the predominant source of circulating IGF-1,11 but this growth factor can also be produced by skeletal muscle. In mice lacking liver IGF-1 through tissue-specific gene targeting, endogenous expression of IGF-1 by the muscle is sufficient to maintain normal muscle mass.53 Thus, increasing the local muscle production of IGF-1 could provide an effective method for increasing muscle mass. Previous demonstration of this concept used transgenic animals expressing IGF-1 specifically in muscle.28,29 In these mice, muscles exhibit a 40% increase in muscle mass, including the masticatory muscles, with no increase in circulating IGF-1 (Fig 4). This mouse model would be ideal to separate the direct and indirect effects of IGF-1 on the craniofacial skeleton. Obviously, germline transmission of any gene is not a rational approach for people. However, viral admin-
Figure 4. Muscle-specific expression of IGF-1 in transgenic mice leads to a 40% increase in mass of limb (represented by tibialis anterior) and masticatory (represented by masseter) muscles (upper panel). Tissue content of IGF-1 is more than 4-fold greater in the transgenic muscles (lower left panel), whereas there is no change in total circulating IGF-1 with restricted transgenic expression (lower right panel). WT, wild type; TG, transgenic. (Color version of figure is available online.)
istration of the cDNA encoding for IGF-1 into target tissues is a viable option for experimental studies30,33,54 and possibly for gene therapy in patients. Indeed, clinical trials for genetic muscle diseases could help to pave the way for this tool to be used routinely to deliver therapeutic genes to skeletal muscle. For orthodontic procedures, this could drastically streamline the current clinical approaches. Instead of highly invasive methods for modulating the craniofacial complex, a single set of injections could provide sufficient and durable expression of growth factors and allow the masticatory muscles to do the moving.
References 1. de Crombrugghe B, Lefebvre V, Nakashima K: Regulatory mechanisms in the pathways of cartilage and bone formation. Curr Opin Cell Biol 13:721-727, 2001 2. Moss ML: A theoretical analysis of the functional matrix. Acta Biotheor 18:195-202, 1968 3. Moss ML, Rankow RM: The role of the functional matrix in mandibular growth. Angle Orthod 38:95-103, 1968
Growth Factor Targets for Orthodontic Treatments
4. Moss ML: The functional matrix hypothesis revisited. 1. The role of mechanotransduction. Am J Orthod Dentofac Orthop 112:8-11, 1997 5. Ko EW, Huang CS, Chen YR, et al: Cephalometric craniofacial characteristics in patients with temporomandibular joint ankylosis. Chang Gung Med J 28:456-466, 2005 6. Kecik D, Kocadereli I, Saatci I: Evaluation of the treatment changes of functional posterior crossbite in the mixed dentition. Am J Orthod Dentofac Orthop 131: 202-215, 2007 7. Mew J: Re: Handelman CS, Wang L, BeGole EA, Haas AJ. Nonsurgical rapid maxillary expansion in adults: Report on 47 cases. Angle Orthod 70:129-144, 1999. Angle Orthod 71:3, 2001 8. Ghafari J, Heeley JD: Condylar adaptation to muscle alteration in the rat. Angle Orthod 52:26-37, 1982 9. Ghafari J, Cowin DH: Condylar cartilage in the muscular dystrophic mouse. Am J Orthod Dentofac Orthop 95: 107-114, 1989 10. Ghafari J, Degroote C: Condylar cartilage response to continuous mandibular displacement in the rat. Angle Orthod 56:49-57, 1986 11. Schwander JC, Hauri C, Zapf J, et al: Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: Dependence on growth hormone status. Endocrinologist 113:297-305, 1983 12. Firth SM, Baxter RC: Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 23:824-854, 2002 13. Coolican SA, Samuel DS, Ewton DZ, et al: The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem 272:66536662, 1997 14. Liu JP, Baker J, Perkins AS, et al: Mice carrying null mutations of the genes encoding insulin-like growth factor I (IGF-1) and type 1 IGF receptor (Igf1r). Cell 75:59-72, 1993 15. Baker J, Liu JP, Robertson EJ, et al: Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73-82, 1993 16. Mathews LS, Hammer RE, Behringer RR, et al: Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology 123:2827-2833, 1988 17. Solheim E: Growth factors in bone. Int Orthop 22:410416, 1998 18. Spears R, Svoboda KKH: Growth factors and signaling proteins in craniofacial development. Semin Orthod 11: 184-198, 2005 19. Lindahl A, Isgaard J, Isaksson OG: Growth hormone in vivo potentiates the stimulatory effect of insulin-like growth factor-1 in vitro on colony formation of epiphyseal chondrocytes isolated from hypophysectomized rats. Endocrinologist 121:1070-1075, 1987 20. Isaksson OG, Ohlsson C, Nilsson A, et al: Regulation of cartilage growth by growth hormone and insulin-like growth factor I. Pediatr Nephrol 5:451-453, 1991 21. Ohlsson C, Isaksson O, Lindahl A: Clonal analysis of rat tibia growth plate chondrocytes in suspension culture— Differential effects of growth hormone and insulin-like growth factor I. Growth Regul 4:1-7, 1994
133
22. McAlarney ME, Rizos M, Rocca EG, et al: The quantitative and qualitative analysis of the craniofacial skeleton of mice lacking the IGF-1 gene. Clin Orthod Res 4:206219, 2001 23. Tokimasa C, Kawata T, Fujita T, et al: Effects of insulinlike growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice. Arch Oral Biol 48:31-38, 2003 24. Kim H, Barton E, Muja N, et al: Intact insulin and insulin-like growth factor-I receptor signaling is required for growth hormone effects on skeletal muscle growth and function in vivo. Endocrinologist 146:1772-1779, 2005 25. Florini JR, Ewton DZ, Coolican SA: Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17:481-517, 1996 26. Fernandez AM, Dupont J, Farrar RP, et al: Muscle-specific inactivation of the IGF-1 receptor induces compensatory hyperplasia in skeletal muscle. J Clin Invest 109: 347-355, 2002 27. Adams GR, McCue SA: Localized infusion of IGF-1 results in skeletal muscle hypertrophy in rats. J Appl Physiol 84:1716-1722, 1998 28. Coleman ME, DeMayo F, Yin KC, et al: Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem 270:12109-12116, 1995 29. Musaro A, McCullagh K, Paul A, et al: Localized IGF-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 27:195200, 2001 30. Barton-Davis ER, Shoturma DI, Musaro A, et al: Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci U S A 95:15603-15607, 1998 31. Barton ER, Morris L, Musaro A, et al: Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice. J Cell Biol 157:137-148, 2002 32. Lynch GS, Cuffe SA, Plant DR, et al: IGF-1 treatment improves the functional properties of fast- and slowtwitch skeletal muscles from dystrophic mice. Neuromuscul Disord 11:260-268, 2001 33. Lee S, Barton ER, Sweeney HL, et al: Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J Appl Physiol 96:10971104, 2004 34. Proffit WR, Fields HW Jr.: Contemporary Orthodontics. Philadelphia, Mosby, 2000 35. Doyle F, Brown J, Lachance C: Relation between bone mass and muscle weight. Lancet 1:391-393, 1970 36. Andreoli A, Monteleone M, Van Loan M, et al: Effects of different sports on bone density and muscle mass in highly trained athletes. Med Sci Sports Exerc 33:507-511, 2001 37. Kaye M, Kusy RP: Genetic lineage, bone mass, and physical activity in mice. Bone 17:131-135, 1995 38. Schoenau E, Frost HM: The “muscle-bone unit” in children and adolescents. Calcif Tissue Int 70:405-407, 2002 39. Rauch F, Bailey DA, Baxter-Jones A, et al: The “musclebone unit” during the pubertal growth spurt. Bone 34: 771-775, 2004
134
E.R. Barton and C. Crowder
40. Godfrey RJ, Madgwick Z, Whyte GP: The exercise-induced growth hormone response in athletes. Sports Med 33:599-613, 2003 41. Yao Z, Lafage-Proust MH, Plouet J, et al: Increase of both angiogenesis and bone mass in response to exercise depends on VEGF. J Bone Miner Res 19:1471-1480, 2004 42. McPherron AC, Lawler AM, Lee SJ: Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83-90, 1997 43. Byron CD, Hamrick MW, Wingard CJ: Alterations of temporalis muscle contractile force and histological content from the myostatin and Mdx deficient mouse. Arch Oral Biol 51:396-405, 2006 44. Vecchione L, Byron C, Cooper GM, et al: Craniofacial morphology in myostatin-deficient mice. J Dent Res 86: 1068-1072, 2007 45. Borasio GD, Robberecht W, Leigh PN, et al: A placebocontrolled trial of insulin-like growth factor-I in amyotrophic lateral sclerosis. European ALS/IGF-I Study Group. Neurologist 51:583-586, 1998 46. Butterfield GE, Thompson J, Rennie MJ, et al: Effect of rhGH and rhIGF-I treatment on protein utilization in elderly women. Am J Physiol 272:E94-E99, 1997 47. Cusi K, DeFronzo R: Recombinant human insulin-like growth factor I treatment for 1 week improves metabolic control in type 2 diabetes by ameliorating hepatic and muscle insulin resistance. J Clin Endocrinol Metab 85: 3077-3084, 2000
48. Friedlander AL, Butterfield GE, Moynihan S, et al: One year of insulin-like growth factor I treatment does not affect bone density, body composition, or psychological measures in postmenopausal women. J Clin Endocrinol Metab 86:1496-1503, 2001 49. Lai EC, Felice KJ, Festoff BW, et al: Effect of recombinant human insulin-like growth factor-I on progression of ALS. A placebo-controlled study. The North America ALS/IGF-I Study Group. Neurologist 49:1621-1630, 1997 50. Mauras N, O’Brien KO, Welch S, et al: Insulin-like growth factor I and growth hormone (GH) treatment in GH-deficient humans: Differential effects on protein, glucose, lipid, and calcium metabolism. J Clin Endocrinol Metab 85:1686-1694, 2000 51. Waters D, Danska J, Hardy K, et al: Recombinant human growth hormone, insulin-like growth factor 1, and combination therapy in AIDS-associated wasting. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 125:865-872, 1996 52. Kemp SF, Fowlkes JL, Thrailkill KM: Efficacy and safety of mecasermin rinfabate. Exp Opin Biol Ther 6:533-538, 2006 53. Sjogren K, Liu JL, Blad K, et al: Liver-derived insulin-like growth factor I (IGF-1) is the principal source of IGF-1 in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci U S A 96:7088-7092, 1999 54. Barton ER: Viral expression of insulin-like growth factor-I isoforms promotes different responses in skeletal muscle. J Appl Physiol 100:1778-1784, 2006