2. Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair Louis C. Gerstenfeld, Cory M. Edgar, Sanjeev Kakar, Kimberly A. Jacobsen, and Thomas A. Einhorn

transforming growth factor β (TGF-β) super- 2.1 Introduction family, angiogenic factors, and parathyroid hormone/parathyroid hormone-related peptide Ontogenetic development is initiated at the (PTH/PTHrP). Major emphasis has been time of fertilization and terminates with the directed to these molecules because their activ- differentiation, growth, and maturation of spe- ities constitute current targets of pharmaco- cialized tissues and organs. These developmen- logical studies to promote or alter bone healing. tal processes are characterized by molecular Short reviews of the fi broblast growth factor specialization that accompanies cellular differ- (FGF) and Wnt families of factors are also pre- entiation and tissue morphogenesis. Most sented in the context of their known functions developmental processes terminate after birth in skeletal development and intended use as or when animals reach sexual maturity, but therapeutic agents. The second half of the some morphogenetic processes are reinitiated review (sections 2.3–5) is focused on the in response to injury in specifi c tissues. One anatomy and cell biology of bone healing, on such regenerative process is the repair of skel- what is known about the temporal and spatial etal fractures and bone tissue after surgery, expression of the various cytokines during a process that recapitulates specifi c aspects bone healing, and how cytokines and morpho- of the initial developmental processes in the gens may therapeutically modify the repair course of healing [58, 209]. Several aspects of process. the postnatal tissue environment of fracture healing, however, are unique and differ from what occurs in embryological and postnatal 2.2 Cytokines, Morphogens, development. Understanding how cytokines and morphogens affect fracture or postsurgical and Growth Factors: healing is essential to the development of The TNF-a Family pharmacological and molecular approaches intended to enhance bone healing after surgery or traumatic injury, as well as to promote skel- 2.2.1 The TNF Family of Cytokines etal tissue engineering. and Their Intracellular Functions The fi rst half of this review (Section 2.2) will focus on several groups of soluble protein TNF was fi rst identifi ed in the early 1980s, and factors that regulate postnatal bone repair: the a large superfamily of related molecules has tumor necrosis factor α (TNF-α) family, the since been identifi ed. So far, 18 members with

17 18 Engineering of Functional Skeletal Tissues

15% to 25% amino acid sequence homology growth, primarily through the activation of the and at least six cell-surface receptors have been nuclear factor κB (NFκB) and c-Jun N-terminal described. The two members of this cytokine kinase (JNK) transcription factors. The dichot- family that have been the most extensively omy of cellular responses to these cytokines characterized are TNF and Fas ligand (FasL). resides in the receptors that are activated and The ligands of this family are all predominantly the downstream signal transduction molecules type II transmembrane proteins. The receptors that interact with these receptors. Signal trans- are all type I transmembrane proteins and are duction is mediated through a two-part system believed to aggregate upon interaction with of docking proteins including MORT/FADD, their ligands. Although the extracellular side TRADD, RIP, and CRADD, which bind to the of the receptors is conserved and composed death domain (DD) of the receptors, and the of cysteine repeats, the cytoplasmic domains of adaptor proteins that have been named TRAFs. the receptors are different and mediate unique Downstream from the coupled responses to activities that lead to a multitude of biological TNFR1 and TNFR2 that mediate cell survival responses through variations in their coupled are the various mitogen-activated protein signal transduction processes. These cytokines (MAP)-related kinases. Downstream from the have been implicated in a wide variety of apoptotic activation of TNFR1 and FAS is the diseases, including tumorigenesis, septic shock, activation of specifi c proteases (caspases) [19, viral replication, bone resorption, rheumatoid 121, 153, 187]. There is a further bifurcation arthritis, diabetes, and other infl ammatory of the apoptotic cascade, with two separate diseases [19, 121, 153, 187]. Recently, several pathways that can mediate apoptosis: an intrin- therapeutic regimens have been approved that sic (mitochondria-dependent low caspase 8) antagonize TNF-α activity to treat a variety of pathway and an extrinsic (mitochondria- autoimmune diseases, including rheumatoid independent high caspase 8) pathway [185]. To arthritis and Crohn’s disease [163, 184]. Pre- understand the complex regulatory functions liminary studies have also examined whether within a tissue that are mediated through the these approaches can be used to impede the actions of the TNF cytokine family, it is neces- loosening of orthopedic prostheses [37]. sary to defi ne the ligands and to specify the The TNF family members with the most actions of specifi c receptors and the specifi c homogeneity are TNF-α, TNF-β (LT-α), and mechanisms of intracellular transduction LT-β. Both TNF-α ligands and TNF-β (LT-α) within that tissue. are homotrimers, whereas LT-β is a heterotri- mer of (LT-α)1(LT-β)2. There are three receptors in this family: TNFR1/p55/death receptor 1/ 2.2.1.1 TNF Cytokines as Arbitrators of the DR1, TNFR2 (p75), and LT-β receptor. Both Tissue Microenvironment by Selective TNF ligands bind both TNF receptors, but LT- Promotion of Cell Death or Survival β/TNF-α trimers only bind to the LT receptor. FasL is a unique family member and is solely The TNF family of cytokines plays a central recognized by its receptor, FAS/Apo1/DR2 [211]. role in the timing of the immune response, Most cells express TNF-α and its receptors, but namely, when to terminate activation of the the expression of TNF-β and its receptor innate infl ammatory response and initiate the appears to be restricted to T cells and natural acquired immune response, and when to termi- killer cells. TNFR1 (p55) is constitutively nate an innate or acquired response and initi- expressed by almost all cells, but TNFR2 (p75) ate local tissue repair and regeneration. Thus is strongly induced in immune and infl amma- both TNFR1 and Fas mediate activation- tory responses. FasL and Fas are also expressed induced cell death in macrophages, T cells, and by many cells but show unique expression B cells [99, 111, 187]. The pathological manifes- during many developmental processes, includ- tations of inappropriate control of the apop- ing the hypertrophy of chondrocytes [72, 174] totic processes in immune function are seen in and the regulation of immune cell differentia- mice that are defi cient in TNFR1, Fas, and FAS/ tion [17, 23, 55, 192]. TNF-α and related cyto- TNFR1. These animals exhibit more severe kines either mediate programmed cell death autoimmune disease and accelerated lym- (apoptosis) or facilitate cell survival and phoproliferation. These responses indicate that Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 19 whereas Fas and TNFR1 receptors both activate 126]. Treatment of human articular chondro- the apoptotic cascade and carry out compensa- cytes with FasL in vitro causes apoptosis. tory or redundant functions, each receptor Because the Fas system is present in growth- mediates a unique set of biological responses plate chondrocytes in vivo, it may play a role in [229]. Thus failure to initiate the programmed chondrocyte apoptosis during endochondral cell death of one or another population of development [6, 83]. In previous studies, carti- immune cells that mediate the transition of the lage cells within the fracture callus [224] have specifi c stages of an immune response leads to been shown to express Fas, and articular chon- a variety of systemic autoimmune pathologies drocytes will undergo programmed cell death [204]. In essence, these cytokines act as the in response to TNF-α [69]. The relationship central arbitrators of a tissue’s microenviron- between the apoptotic process and the normal ment during immune activation. They do so progression of endochondral development can by promoting the survival of one population be observed in pathological conditions such as of cells while causing another to undergo rickets, as well as in the numerous genetically apoptosis. engineered defects that affect growth-cartilage The TNF-α family of cytokines has been the development. The hallmark of almost all of primary focus of many immune function these defects is either a foreshortening or an studies, but the death receptor family also plays expansion of the growth plates. Two examples a pivotal regulatory role in many developmen- of factors causing an expansion of the growth tal processes [43]. It is interesting that during plate are vitamin D defi ciency in growing postnatal tissue repair and regeneration these animals and the genetically engineered abla- cytokines directly and indirectly regulate many tion of matrix metalloproteinase 9 (MMP-9) nonimmune cell types downstream from an [210]. Ablation of the PTHrP gene, on the other initial immune response [82]. The signaling hand, causes an osteochondrodysplasia, functions by immune cell cytokines during primarily manifested in an accelerated hyper- postnatal tissue repair derive from functions trophy and removal of the chondrocytes. A carried out during embryogenesis. Alterna- phenomenon common to these very different tively, these cells may initiate postnatal repair pathologies of the endochondral process is that or regenerative processes that replace mecha- in all three the timing or rate of chondrocyte nisms that functioned during embryological apoptosis has been altered. The consequence of development. TNF-α thus functions within an abnormally timed apoptosis is that the skeletal tissues either during the course of microenvironment of the endochondral tissue normal skeletal homeostasis or in response to is altered by retention or loss of the chondro- tissue injury [158]. It does so by acting on both cytes. This is important because osteogenesis, apoptotic and nonapoptotic events within mes- vascular invasion, and marrow formation enchymal cell types found in skeletal tissues. follow in sequence as the chondrogenic cells This includes specifi c types of mesenchymal hypertrophy and undergo apoptosis [120]. precursors [78], osteogenic cells [1], and syno- Thus, in analogy with their role during the vial fi broblasts [64, 137]. immune response, the death receptors and Recent studies have shown that activation of ligands during endochondral development TNF-α and/or NFκB can affect tissue repair, promote the removal of one cell population response to injury, and arthritic pathology by (chondrocytes) and are permissive for osteo- specifi cally inducing the expression of mor- genic and marrow cell populations to move phogenetic factors of the TGF-α family [64]. It into the space previously occupied by the car- may also alter second signal activity of SMADs tilage tissue. that mediate bone morphogenetic protein (BMP) signaling [21, 36, 57]. It is now well established that cartilage cells 2.2.1.2 Role of the TNF-α Family of undergo apoptosis during normal endochon- Cytokines in Bone Remodeling dral development and during arthritic disease [3, 4, 5, 56, 72]. Currently, three members of the As just discussed, embryologic development TNF family of cytokines have been implicated: and postnatal growth are regulated by ontoge- Fas ligand (FasL), TNF-α, and TRAIL [6, 39, 83, netic and systemic hormonal mechanisms. 20 Engineering of Functional Skeletal Tissues

Fracture and skeletal tissue healing after extreme differences in the avascular microen- surgery, on the other hand, are initiated in vironment of cartilage and bone. Indeed, the response to regulatory mechanisms associated interactions of hematopoietic/lymphopoietic with infl ammation and the innate immune and osteogenic microenvironments in regulat- response [16, 54]. Two discrete types of resorp- ing bone remodeling are emerging as a major tion take place during fracture repair. The fi rst area of research, and changes in cytokines occurs at the end of the endochondral period, that alter lymphopoiesis affect both bone in the course of which mineralized cartilage is homeostasis and immune function [30, 105, removed and primary bone is formed. TNF-α 175, 206]. and its receptors remain largely unexpressed during the initial periods of endochondral dif- ferentiation, but are expressed as the cartilage 2.2.2 The Bone Morphogenetic cells hypertrophy and tissue resorption begins. During this same period, there is an increase Proteins (BMPs) in the concentration of RANKL and osteopro- 2.2.2.1 BMPs and Signaling tegrin (OPG) (two members of the TNF-α superfamily) as well as macrophage colony- On the basis of their distinct structural charac- stimulating factor (M-CSF), all key regulatory teristics, BMPs (with the exception of what has factors in osteoclastogenesis [118]. However, been named BMP-1) are members of the trans- other cytokines that are associated with bone forming growth factor β (TGF-β) superfamily. remodeling, including interleukin 1α (IL-1α), This family also includes activins, inhibins, IL-1β, and IL-6 [115], are not expressed. The and growth and differentiation factors (GDFs). other type of resorption occurs during second- BMP-1 belongs to the astacin family of ary bone formation, which follows the endo- metalloendopeptidases and exhibits BMP-like chondral phase. These events are comparable activity by proteolytically activating mixtures to the process of coupled remodeling seen in that contain the proforms of BMP. normal bone homeostasis. During this period, The TGF-β superfamily of pre-proproteins expression of IL-1 and IL-6 increases, whereas displays extensive amino acid sequence homol- the levels of OPG, M-CSF, and RANKL ogy across species and can carry out a wide decline. diversity of biological functions. The proteins These data suggest that the processes medi- share a characteristic pattern of seven con- ating endochondral resorption and the more served cysteine residues within the carboxy- prolonged phase of secondary bone remodeling terminal mature region that are essential for differ and that the resorption of the mineral- the formation of cysteine knot domains. This ized cartilage is more dependent on the activi- tertiary protein structure is thought to be criti- ties of M-CSF, OPG, and RANKL. In contrast, cal for receptor interaction [220]. The mature- bone remodeling appears to depend on the region cysteines are also important in the levels of RANKL and to be coregulated by the formation of intermonomeric disulfi de bonds activities of the cytokines IL-1, IL-6, and TNF- necessary for the formation of physiologically α found in bone marrow. Differences between functional dimers [119]. As in most secreted bone and cartilage remodeling are apparent proteins, there are numerous potential N- from studies of RANKL (TRANCE)-defi cient linkage glycosylation sites located throughout mice and of mice whose RANKL expression the amino acid sequence. Most BMPs induce was rescued by engineering RANKL expression some level of glycosylation, which varies among in their lymphocytes. When RANKL was species, with mouse BMP inducing the lowest expressed by lymphocytes in the knockout and bovine BMP inducing the highest degree of mice, their osteopetrosis was overcome and glycosylation [183, 212]. osteoclast development was promoted. However, As an example of a typical BMP structure, it was not possible to correct the chondrodys- BMP-2 is translated as a 396-amino-acid pre- plasia of the epiphyseal and metaphyseal proprotein that contains a 19-amino-acid signal regions. The authors therefore concluded that sequence for targeted secretion, a 263-amino- cartilage and bone possess different mecha- acid proregion, and a 114-amino-acid mature nisms that induce RANKL expression [114]. segment. Within the mature region of BMP-2, In this context it is interesting to note the seven cysteines and one N-linked glycosylation Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 21 recognition site are identifi able. The mature type I receptor phosphorylation in the GS protein has a predicted mass of 14 kDa with an domain by the type II receptor [134]. observed mass of 18 kDa, presumably due to Currently, seven type I receptors, termed glycosylation. The functional protein exists as activin receptor-like kinases (ALKs) 1–7, have a homodimer that is linked by two disulfi de been identifi ed in mammals. ALK-3 (BMP type bridges. There is some speculation on the exis- IA) and ALK-6 (BMP type IB) receptors share tence of heterodimeric complexes in some situ- an 85% amino acid sequence identity in the ations, although in normal physiological kinase domains, and both bind BMP-4, BMP-2, settings homodimeric complexes among the GDF-5, and BMP-7 [149]. Truncated forms of BMPs are most common [183, 217]. Consider- the ALK receptors are currently being used to able amino acid sequence similarity exists examine the role of BMP signaling during the between species for the various family members. development of numerous types of tissues. On Approximately 16 BMPs have been character- the other hand, there are only three BMP type ized, with the majority demonstrating a high II receptors that can interact with BMPs. The percentage of amino acid sequence homology BMPR-II receptor seems to bind exclusively to among the different isotypes, in addition to a BMPs, but the activin types IIA and IIB have high level of amino acid conservation between affi nities for specifi c BMPs (BMP-7, BMP-2 and species [119, 217]. GDF-5), in addition to their activin binding BMPs initiate their signaling at the cell [220]. BMPR-II binds all BMPs weakly by itself, surface through interaction with two distinct with a dramatic increase in the binding serine/threonine kinase receptors: a type I affi nity following recruitment of the type I receptor (50–55 kDa) and a type II receptor receptors. (more than 75 kDa) [220]. It appears that they BMP-2 ligand and receptor interactions have weakly interact with certain members of the been carefully studied (160). During BMP-2 type II receptors independently of type I recep- receptor activation, the BMP-2 protein contains tors, but in the presence of both receptors their two distinct domains that facilitate receptor binding affi nity is increased dramatically [133]. interaction. The fi rst is a large, high-affi nity Following receptor dimerization induced by binding site (termed the “wrist epitope”), which BMP ligand binding, the type II receptor trans- interacts with the BMPR-IA. The second is a phosphorylates the type I receptor, which sub- low-affi nity binding site (termed the “knuckle sequently transmits the BMP signal by epitope”), which interacts with BMPR-II [59]. activation of intracellular Smad (Sma and Mad) The wrist epitopes from monomers (BMPs are proteins. This activation is accomplished by dimeric structures) contribute to the binding of the directed phosphorylation of specifi c serine the BMPR-IA receptor, whereas the knuckle or threonine residues within the Smad pro- epitope from only one monomer binds to teins. The structures of the two receptors are BMPR-IA. The juxtapositioning of these regions similar in that they contain N-glycosylated facilitates a close proximity of the receptors extracellular domains, a single membrane- and initiation of intracellular signaling from spanning domain, and an intracellular serine/ inter-receptor type II phosphorylation to type threonine kinase domain. The extracellular I. Transphosphorylation eventually leads to the domains have several conserved cysteine resi- activation of Smad proteins and signal trans- dues believed to facilitate the formation of mission to target downstream responsive genes essential three-dimensional structures involved [149]. in BMP binding [59]. One distinction between Within the cell BMP signals are transduced the two receptor types is the presence of a by the Smad molecules. To date, eight Smad glycine- and serine-rich domain (GS domain) mammalian proteins have been isolated and found on the type I receptor within the intra- characterized. Smad proteins are the direct cellular N-terminal to the serine/threonine downstream signaling molecules of BMPs and kinase domain. This region is important for the other TGF-β superfamily members and are transmission of the BMP signal to intracellular activated directly by their serine/threonine second-messenger proteins by facilitating the kinase receptors. These proteins can be classi- receptors’ ability to interact with Smad pro- fi ed into three distinct groups based on their teins. This was highlighted in an amino acid intracellular function. The receptor-regulated mutagenesis study linking Smad 7 activation to Smads (R-Smads) are the direct signal 22 Engineering of Functional Skeletal Tissues transducers from the BMP receptor complex or lethality. The complete ablation of BMP-2 following receptor transphosphorylation by homologous recombination resulted in events. Smads 1, 5, and 8 interact with types I embryonic lethality when bred to homozygos- and II BMP receptors and are subsequently ity [228]. These animals had distinct cardiac phosphorylated by the type I receptor within defects consistent with the expression pattern- their COOH-terminus at the conserved SSXS ing of BMP-2 in the extraembryonic mesoderm motif [207]. They are then rapidly released and promyocardium [228]. BMP-2 is expressed from the receptor and subsequently interact in a variety of embryonic nonskeletal epithe- with a common mediator Smad (co-Smad). lial and mesenchymal tissues known to play Smad 4 is the only known co-Smad that signals important roles in morphogenesis [139]. For in both the BMP and the TGF-β transduction example, during limb development high levels pathways [207]. The R-Smad and the co-Smad of transcripts were found in the ventral ecto- proteins form active hetero-oligomeric com- derm and apical ectodermal ridges of the plexes, which can then translocate to the developing limb buds. In addition, BMP-2 nucleus and regulate the transcription of spe- expression was detectable in the developing cifi c downstream genes. The nuclear localiza- heart, whisker follicles (ectodermal placodes), tion of the Smad complexes is dependent on tooth buds (epithelial buds, dental papillae, nuclear localization signals present on Smad 4. and odontoblasts), and craniofacial mesen- Consequently, this protein displays constant chyme [139]. Although other studies have vali- nuclear–cytoplasmic shuttling and is capable dated the importance of BMP-2 during a wide of autonomous nuclear import and export array of mesodermal developmental processes, [218]. The third class of Smad proteins consists the protein also plays important roles in regu- of the inhibitory Smads (I-Smads), Smad 6 and lating the postnatal development of mesenchy- Smad 7, which exert their inhibitory effect by mal skeletal tissues [176]. In animals with a binding to the type I receptor and competing homozygous deletion of the mature coding with the R-Smads for binding to the phosphor- region of the BMP-4 gene, development fails at ylated type I receptor. an extremely early stage. The mice fail to All Smads share two conserved regions develop the necessary primordial germ cells termed Mad homology domains 1 (MH1) and (PGCs) to form a functional embryo [123]. 2 (MH2). MH1 is found in the N-terminal Lawson et al. have shown that BMP-4 pro- portion of the protein, whereas MH2 is in the moter-driven LacZ expression in embryos C-terminal portion, with a linker region of prior to gastrulation results in BMP-4 expres- variable length and amino acid sequence sepa- sion in the extraembryonic ectoderm, followed rating the two domains [150]. The MH2 domain by expression in the extraembryonic meso- contains protein–protein interaction sequences derm [123]. These authors concluded that the and is important in R-Smad/co-Smad oligo- initiation of the germ line in the mouse was merization. The MH1 domain seems to carry dependent on secreted BMP-4 signals from the specifi c DNA-binding sequences necessary to previously segregated, extraembryonic, troph- act at the DNA level in the discrimination of ectoderm lineage. This places BMP-4 function gene regulation. However, a putative “Smad at one of the earliest stages of development consensus sequence” has yet to be determined [123]. However, BMPs do not appear to act [107]. individually but in a coordinated network. For example, the above-mentioned PGC cell gen- eration is directed by more than just BMP-4. 2.2.2.2 BMPs and Developmental In fact, one study has demonstrated that BMP- 2 Regulation is primarily expressed in the endoderm of mouse pregastrula and gastrula embryos and BMPs are considered one of the major groups that the PGC generation in the mouse embryo of morphogenetic factors that mediate pat- is regulated not only by extraembryonic ecto- terning and growth of many tissue types derm-derived BMP-4 and BMP-8B, but also by during embryogenesis and organogenesis. In endoderm-derived BMP-2 [223]. the absence of specifi c BMPs, certain systems BMP-7 has been extensively studied. It is fail to develop, resulting in embryonic defects expressed later during mammalian develop- Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 23 ment, but its function is redundant with that of extraskeletal sites induces de novo formation other BMPs, since knockout animals survive of cartilage and bone [203]. This seminal obser- through gestation. However, BMP-7-defi cient vation led to investigations culminating in the mice die shortly after birth because their extensive purifi cation of the osteoinductive kidneys do not develop normally [86]. In situ activity of demineralized bone matrix (DBM) hybridization analysis has shown that the and the sequencing and cloning of the individ- absence of BMP-7 affects the expression of ual BMPs [35, 166, 217]. The subsequent expres- molecular markers of nephrogenesis, such as sion of BMPs in recombinant systems permitted Pax-2 and Wnt-4, between 12.5 and 14.5 days their use in a variety of animal models, in par- postcoitum [138]. In addition, BMP-7-defi cient ticular to demonstrate their stimulating effects mice have defects in the eye that appear to on the repair of fracture and skeletal defects originate during lens development. Skeletal [53, 67, 221]. Even though exogenous BMPs may patterning defects affect the rib cage, the skull, enhance fracture healing, our understanding and the hind limbs; this shows the wide infl u- of their role in skeletal repair and regeneration ence BMPs have in mammalian development remains incomplete. [138]. The importance of BMPs, however, is Using reverse-transcriptase polymerase restricted neither to skeletal development nor chain reaction (PCR) amplifi cation, Nakase to prenatal development. BMP-2 expression has et al. were the fi rst to demonstrate the temporal been reported to be critical for both extraem- and spatial distribution of BMP-4 in fracture bryonic and embryonic development [101], with healing [157]. In an investigation using a mono- BMP-2 shown to be essential for cranial neural clonal antibody against BMP-2 and BMP-4, crest production. Without it, the skeletal and Bostrom et al. delineated the expression of neural derivatives failed to develop. The im- these BMPs over a 4-week period of fracture portance of BMPs during development has healing [26]. Recently, Cho et al. [38] have been most extensively studied in Xenopus. If shown that specifi c members of the TGF-β BMP-4 signaling is disrupted transgenically by superfamily, including the BMPs, may act in expression of a dominant negative form of its combination to promote the various stages of receptor, the ventral mesoderm is converted to intramembranous and endochondral bone for- a dorsal mesoderm [197]. In situ hybridization mation observed during fracture healing. in Xenopus showed that BMP-4 is expressed in Using ribonuclease protection analysis, this a spatially and temporally restricted manner. study demonstrated that BMP-2 has an early Disruption of the pattern of BMP-4 expression peak in expression on day 1 of fracture healing. by localized microinjections of rhBMP-4 This suggests that BMP-2 may be the most severely disturbed embryonic development upstream mediator in the cascade of BMP [49]. These experiments make it clear that expression. BMP-3 appeared to be preferen- BMP-4 regulates dorsal-ventral patterning in tially associated with intramembranous bone terms of both location and temporal expres- formation, whereas BMP-4, -7, and -8 may sion. As a morphogen, BMP-4 modulates meso- function in osteoblast recruitment during both dermal patterning by establishing concentration intramembranous and endochondral ossifi ca- gradients that cells detect during migration. tion. Taken together, these studies suggest that Further evidence of the signifi cant role active the coordinated expression of multiple BMPs BMPs play in the control of differentiation and their receptors during fracture healing is comes from experiments that have examined important in both skeletal development and the regulation and responsive expression of skeletal repair. However, the roles of specifi c specifi c BMP antagonists, such as Noggin [44, BMPs during fracture healing need to be 49]. The coordinated expression of BMP antag- investigated. onists interferes with BMP function in somite and limb development [172, 190]. 2.2.3 Angiogenic Factors 2.2.2.3 BMP Function in Skeletal Repair Angiogenesis is the process by which new blood In 1965, Marshall R. Urist demonstrated that vessels are formed from pre-existent vessels. the implantation of demineralized bone at It is important for almost all embryological 24 Engineering of Functional Skeletal Tissues development and in wound healing, because result, the hematopoietic cells are protected the higher metabolic activities of cells within from myelosuppressive stresses [9]. developing and healing tissues increase their nutrient and oxygen requirements [32]. Two classes of angiogenic factors and their 2.2.3.1 The VEGF Family and Receptors receptors are associated with new vessel forma- The VEGF family of genes is currently known tion [60, 130]. These are the vascular endothe- to comprise fi ve related genes: VEGF A, B, C, lial growth factor (VEGF) [61] and the and D and placental growth factor (PlGF). All angiopoietin (Ang) [98] families. of these have some sequence similarity to plate- VEGFs promote vascular permeability and let-derived growth factor (PDGF). The VEGF stimulate mitogenesis in vascular endothelial proteins are roughly 45 kDa in size and exist as cells. In conjunction with the angiopoietins homodimers. Some of the VEGF isotypes bind (see below), VEGF stimulates endothelial-cell to heparin. This enhances retention in the ECM survival by inhibiting endothelial-cell apopto- and presentation to cellular receptors. Of the sis. The VEGFs are produced primarily in genomic subtypes, VEGF A is the most preva- response to hypoxia-induced transcription lent, based on tissue distribution and expres- factors (Hif 1α and Hif 2α), which are expressed sion levels. Selective exon splicing leads to by many stromal and extracellular matrix variants of VEGF A, of which six have been (ECM)-producing cells in tissues with a high identifi ed. They are denoted as VEGF 121, 145, degree of vascularization. Vascular endothelial 165, 183, 189, and 206, based on their amino cells express most receptors for the various acid lengths. Of these, VEGF 121 and 165 appear VEGF isoforms and are the primary responders to be the most commonly expressed, whereas to VEGF. the 165, 189, and 206 variants maintain exons The angiopoietins, like the VEGFs, are that encode the heparin-binding domains expressed by stromal, mesenchymal, and [201]. smooth-muscle cells of larger vessels. Their VEGFs have multiple receptors, including receptors are expressed primarily on endothe- VEGFR1, also known as Flt-1, VEGFR2 (KDR lial cells. Angiopoietins appear to be intimately or Flk1), and VEGFR3 (Flt-4). Each of these involved in vessel remodeling and may play a receptors is characterized by multiple IgG-like particular role in wound-healing and tissue- extracellular domains, and each is coupled to repair situations where there are pre-existent intracellular signaling networks through an vessels [171, 202]. The expression of Ang 2 is intracellular domain that has tyrosine kinase up-regulated by hypoxia and the associated Hif activity. Two other more distantly related mem- 1α factor, VEGF, angiotensin II, leptin, and brane receptors, neuropilin 1 and 2, also inter- estrogen. Ang 2 expression is down-regulated act selectively with various VEGF molecules. by basic fi broblast growth factor (bFGF). TNF- VEGFR1 also exists in a soluble form that lacks α also regulates Ang 2 expression, with up- or the ability for intracellular signaling and down-regulation dependent on the tissue type antagonizes the less soluble form of VEGFR1. [75]. Ang 1 expression, although not extensively Each of the VEGF receptors differs in its inter- characterized as yet, appears to be up- action with the VEGF isotypes. VEGF A inter- regulated in response to hypoxia [167]. acts with VEGFR1 and 2 and both neuropilins, Unlike VEGF, angiopoietins are not mito- VEGF B interacts with VEGFR1 and neuropilin genic but promote cell survival by blocking 1, and VEGF C and D interact with VEGFR2 apoptotic signals. Ang 1 also has strong che- and 3, whereas PlGF only interacts with moattractant properties for endothelial cells VEGFR1. These receptors also have the ability and promotes the adhesion of hematopoietic to signal utilizing a variety of intracellular stem cells. Angiopoietins appear to stimulate pathways and can activate PLC, Ras, Shc, Nck both dissolution and migration of endothelial PKC, and PI3 kinase. cells from pre-existent vessels and, in conjunc- tion with VEGF promote cell survival and sta- 98 171 bilize newly formed vessels in [ , ]. Recent 2.2.3.2 Angiopoietins and Tie Receptors studies have shown Tie 2/angiopoietin signal- ing to regulate the hematopoietic stem-cell qui- Three angiopoietins (Ang 1, 2, and 3/4) have escence niche in the bone marrow niche. As a been identifi ed. They are made up of 498 amino Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 25 acids and have a coil domain that is separated ablation strategies to assess the contribution of by a hinged region from a fi brinogen-like vessel formation to new bone formation. domain. Angiopoietins exist as multiple splic- Administration of a soluble VEGF receptor 1- ing variants and only interact with the Tie 2 immunoadhesin, mFlt(1–3)-IgG, completely receptor. blocked new vessel formation in the growth Angiopoietins bind to Tie 1 and 2 receptors, plates of mice and impaired chondrocyte apop- a tyrosine kinase with immunoglobulin and tosis and trabecular bone formation [66]. epidermal growth factor homology domains. Studies by Gerber et al. (66) have identifi ed Ligand binding induces receptor dimerization, VEGF as the key factor that regulates capillary which causes autophosphorylation of the recep- invasion, growth-plate morphogenesis, and tor, thereby activating its kinase signaling [143]. cartilage remodeling. In mice, systemic inhibi- Other studies have shown that the Tie 1 recep- tion of VEGF during periods of rapid growth tor is proteolytically modifi ed when endothe- has led to inhibition of angiogenesis and to a lial cells interact with VEGF. This suggests decrease in the number of chondroclasts/osteo- some coordination between the signaling clasts/osteoblasts at the growth plates. Chon- events that are mediated by angiopoietins and droclasts/osteoclasts belong to the monocyte VEGF. Interestingly, although the Tie receptors cell lineage, express VEGFR, and migrate in are tyrosine kinases, they do not signal through response to VEGFR1-selective ligands. This the MAP kinase system used by VEGF, but indicates that VEGFR1 has a role in monocyte appear to recruit various phosphatases selec- migration [15]. Because osteoblasts express tively, including SHP2, a factor that promotes both VEGF receptors and neuropilin 1 [80], the cell migration by altering activities of focal decrease in osteoblasts at the growth plates in adhesion kinases. anti-VEGF-treated mice refl ects an impairment of VEGFR or neuropilin signaling. This in turn has impaired recruitment and/or differentia- 2.2.3.3 The Role of Angiogenic Factors in tion of these cell types. Thus, VEGF contrib- Bone Development utes importantly not only to angiogenesis, but also to osteogenesis. In mice lacking the VEGF Angiogenesis is important during intramem- gene, the long bones demonstrate a disturbed branous and endochondral bone formation. vascular pattern at birth, consistent with Vascularization of the growth plate contributes reduced bone growth [140]. Osteoblast and to the coupling of chondrogenesis and osteo- hypertrophic chondrocyte development are genesis. Chondrocyte apoptosis and osteoclast also impaired [140]. recruitment and activation are essential termi- VEGFs play an important role in regulating nal stages of cartilage hypertrophy. The osteo- bone remodeling. They do so by attracting clasts resorb the mineralized cartilage and endothelial cells, osteoblasts, and osteoclasts thereby permit bone formation by osteoblasts. [46, 147] and by autocrine regulation of chon- Morphological evidence suggests that chon- drocyte function [33]. Local administration of drocyte apoptosis occurs readily following the VEGF also enhances osteoclast number [100]. invasion of endothelial cells [56, 90] and that Further linkage between VEGF and bone for- chondrocyte death is induced by diffusible mation was recently described by studies in factors that arise either from the vasculature or which hypoxia was shown to drive BMP expres- from hematopoietic elements brought in during sion through VEGF [27]. A number of recent angiogenesis [71, 72]. The newly developing studies have also shown that BMPs stimulate blood vessels in addition establish the conduit the expression of VEGF by osteoblasts and for the cells that form primary bone following osteoblast-like cells [47, 222]. Finally, the tissue- resorption of the mineralized trabeculae of specifi c regulation of VEGF expression during cartilage [59]. bone development seems to be dependent on The interrelationship between blood-vessel the expression of Cbfa1/Runx2, known as the formation and osteogenesis has been studied key transcriptional factor that regulates the by various approaches aimed at inhibiting commitment of mesenchymal cells to the skel- VEGF signaling. Because mice whose VEGF etal-cell lineage [226]. Taken together, these has been ablated die as embryos, studies have fi ndings provide a considerable body of evi- utilized inhibitors of VEGF signaling or select dence in support of the concept that VEGF 26 Engineering of Functional Skeletal Tissues mediates bone formation by direct stimulation healing studies, showed that during fracture of osteogenesis and indirectly by its effects on healing Ang 2 was the factor with the highest vascularization. expression. Unlike the VEGF family, which promotes new vessel formation by stimulating 2.2.3.4 The Role of Angiogenic Factors in endothelial cell division, Ang 2 promotes desta- Tissue Healing bilization and regression of blood vessels in the absence of VEGF A or bFGF [87, 135, 141]. Fracture healing and bone or tissue repair Recent fi ndings have suggested that Ang 2, result in an up-regulation of blood fl ow, so that along with VEGF, promotes new vessel forma- bone regeneration can occur within the callus tion by inducing remodeling of the capillary or repair tissues [10, 52, 179]. The importance basal lamina and by stimulating endothelial- of vascularization during fracture repair was cell sprouting and migration [141]. This sug- confi rmed by studies showing that broad- gests that Ang 2 expression plays a role similar spectrum angiogenic inhibitors completely to that of VEGF in bone repair. By itself, Ang 2 prevented fracture healing, callus formation, inhibits blood-vessel formation, but in combi- and the formation of periosteal woven bone nation with VEGF it stimulates new vessel for- [84, 195]. In contrast, treatment of healing frac- mation and plasticity in existing vessels. tures with VEGF improved bone healing and These studies also pointed to collaborative led to more rapid mineralization of the callus interactions between VEGI (vascular endothe- and regaining of mechanical strength [195]. lial growth inhibitor)-induced angiogenesis The role angiogenesis plays in osteogenesis and the TNF-α family of regulators. Interac- following distraction rupture has been exten- tion of VEGI with death receptor 4 and the sively studied with the aid of an artifi cially pro- primary regulator of the progression of vascu- duced gap following osteotomy. When this larization refl ects a dual role: maintaining technique is used, new bone forms primarily growth arrest of endothelial cells in G0/G1 via an intramembranous mechanism with interfaces, while at the same time inducing extensive revascularization of the regenerated apoptosis in cells that enter the S phase [79, 225, bone. Within the marrow space, venous sinu- 227]. Taken together, these results suggest that, soids are formed that parallel the newly grown after injury, vessels are dissociated into a pool trabeculae. Analysis of experimental models of of nondividing endothelial cells through the osteogenesis following distraction has revealed actions of Ang 2. They are then are held in this an early intense vascular response, with the state through the actions of VEGI, which stim- newly formed vessels maturing into sturdier ulates apoptosis of all cells that enter the S vessels capable of withstanding the tensile phase. When endochondral remodeling is forces that are generated in the distraction gap initiated, VEGF levels rise, stimulating cell [129, 179]. division and allowing endothelial cells to con- As discussed above, angiogenesis appears to tribute to neoangiogenic processes. The concept involve two separate pathways: a VEGF-depen- of controlled cell regression and growth is also dent pathway and an angiopoietin-dependent consistent with the role that angiopoietin is pathway. Interestingly, both Ang 1 and Ang 2 thought to play in blood-vessel formation [87]. have been identifi ed in bone cells during devel- opment [89] and in bone cells that arise in osteogenesis following distraction and fracture 2.2.4 Parathyroid Hormone (PTH)/ healing [34, 127, 128]. Indeed, in studies of mice that had undergone distraction fracture, Ang 1, Parathyroid Hormone-Related Ang 2, and their Tie receptors were expressed Peptide (PTHrP) and throughout healing at the same time that VEGF A and VEGF C were expressed. These regula- PTHrP Signaling tors of angiogenesis were expressed throughout 2.2.4.1 PTH Versus PTHrP: Endocrine Versus the chondrogenic phase of healing, reaching Paracrine Effects maximum levels during the late phases of endochondral remodeling and during bone PTH, a peptide, is an hormone that is synthe- formation. These studies, as well as fracture- sized by the parathyroid gland. The mature Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 27 form of the peptide is 84 amino acids in length. 2.2.4.2 PTH Receptor Signal Transduction PTHrP, on the other hand, is an autocrine/ and Nuclear Effects paracrine factor that was fi rst discovered as the primary cause of malignant hypocalcemia in Receptor activities are modulated through many cancers [28]. It is normally expressed interaction with heterodimeric (α, β, γ) G pro- during development and in many postnatal teins that activate or inhibit cyclase production tissues, including cardiac, vascular, mammary, of cAMP. The levels of cAMP then control the cartilage, and renal tissues, as well as a number activity of protein kinase A (PKA), which serves of other epithelial surfaces. The mature form of as the cAMP intracellular second signal trans- PTHrP is 141 amino acids in length. Even ducer [77]. The activation of the receptor by though PTH and PTHrP bind to the same ligand binding also activates phospholipases receptor, the two molecules share only a limited Cβ through Gαq11. Activated phospholipase sequence homology along the fi rst 34 amino generates diacylglycerol and 1,4,5-inositol tri- acids of their amino terminal sequences phosphate (IP3). These two molecules activate and diverge considerably in their carboxyl both protein kinase C (PKC) and Ca2+ release. domains. Study of the “cross-talk” between the PKA- The effects of the major calcitropic hormone arm, PKC kinases [125], and Ca2+ will likely sort PTH on skeletal cells are very important clini- out the many parallel and sometimes antago- cally, owing to the role played by the skeleton nistic functions of the PTH and PTHrP ligands in mineral homeostasis. Both molecules have in different target-cell populations [76]. similar systemic effects on mineral metabo- At the nuclear level, both the PKA and the lism, yet they differ in amino acid composition PKC families of kinases mediate their actions and physiological function. PTH and PTHrP through the phosphorylation of members of share a common receptor (PTHR1) and, when the leucine zipper family of transcription in the circulation, are primarily targeted to the factors [125]. These transcription factors, when kidney and skeleton [62]. In the kidney, PTH phosphorylated, may activate or inhibit the and PTHrP bring about their calcitropic effects transcription of specifi c genes [113, 132] and by stimulating calcium reabsorption and phos- may be classifi ed into two broad groups: the phate excretion in the distal end of the collect- cAMP response element-binding protein family ing tubules. They also regulate formation of (CREBs) and members of the AP-1 family. The the active vitamin D3 metabolite, 1,25- CREBs include the CREB, CREM, and ATF dihydroxyvitamin D3 by activating the enzyme classes of factors; the primary members of the that carries out the 1α-hydroxylation of 25- AP-1 family include fos, jun, and fra [8, 77, 125, hydroxyvitamin D3 in the proximal tubules. 131, 173, 205]. In general, the actions of PKA are This leads to a rise in serum calcium and a mediated through the phosphorylation of lowering of phosphate level. The effects on the members of the CREB family, while PKC skeletal system are less well understood. PTH appears to act on members of the AP-1 family. binds to the receptors of osteoblasts [177], However, phosphorylation may not be restricted which produce paracrine factors that induce to one type of kinase or individual factors. The increased activation and recruitment of factors are active when dimeric. Members of osteoclasts. both families can undergo specifi c heterodi- PTH and PTHrP, like other peptide hor- merization with one another [125]. Heterodi- mones, mediate their effects through interac- merization gives rise to a diversity of specifi c tion with a receptor. Two forms of this receptor transcription factors. As a result, genes may are known, but the two peptides interact pri- be expressed or silenced in a tissue-specifi c marily with PTH1R. This receptor has seven fashion in response to common second signals transmembrane domains and is closely related [77]. Similarly targeted changes or ablation of to a subset of similar receptors that include the these transcriptional regulators give rise to calcitonin and secretin receptors [65]. PTH and specifi c skeletal tissue phenotypes. PTHrP bind almost identically to the receptor, Extensive data have been accumulated to which has both endocrine and autocrine/para- suggest that the leucine zipper family of tran- crine functions in the tissues in which it is scription factors plays a major role in the regu- expressed [2]. lation of gene expression and development in 28 Engineering of Functional Skeletal Tissues the skeleton. Studies in which both the c-fos PTHrP in their growth plates, which are made and the v-fos genes were virally introduced up mainly of hypertrophic chondrocytes. Con- have shown that fos expression generated osteo- versely, animals lacking PTHrP have very small sarcomas [74, 180, 181]. C-fos knockout mice zones of proliferating chondrocytes and exhibit develop osteochondrodysplasia, overproduce a premature transition to cellular hypertrophy hypertrophic cartilage, and cannot replace and mineralization [136, 194]. Interestingly, if bone. This condition in some ways looks like the Ihh-ablated mice are engineered to have a osteopetrosis [213]. Other studies have also constitutively active PTH1R receptor, prema- shown increases in c-fos proto-oncogene in ture chondrocyte hypertrophy is prevented, bone from patients with fi brous dysplasia in but proliferation of the chondrocytes in the whom bone formation is overexpressed or bone growth zones is still diminished. These fi nd- forms ectopically [31]. Studies examining dif- ings suggest molecular mechanisms that are ferent members of the basic leucine zipper regulated by Ihh and not activated by PTHrP protein family have demonstrated that both control cell proliferation [103]. ATF-2 and hXBP are expressed in skeletal A further understanding of the developmen- tissues [40, 173]. Ablation of ATF-2 leads to a tal role of PTHrP has been gained from studies defect in endochondral ossifi cation with a his- in two different human chondrodysplasias. topathology similar to human hypochondro- One type of chondrodysplasia is a nonlethal, plasia [173]. autosomal dominant disorder that was fi rst identifi ed by Jansen in 1934 [95]. It has subse- 2.2.4.3 The Role of PTHrP in quently been characterized at a molecular level Endochondral Development as a constitutively activating mutation in the PTH1R receptor [186]. The patients are charac- The discovery of PTHrP as the primary factor terized by short limbs caused by severe abnor- in malignant hypocalcemia constituted a malities in their growth plates and associated major advance in understanding the systemic hypocalcemia. The second type of chondrodys- effects of many malignancies. However, the plasia was identifi ed by Blomstrand et al. [22]. subsequent characterization of PTHrP as an It is a prenatal lethal chondrodysplasia charac- essential autocrine/paracrine factor in skeletal- terized by abnormal bone ossifi cation and tissue development was equally important. shortened limbs. In these fetuses, the limbs Initial animal studies demonstrated that PTHrP show very advanced endochondral develop- mRNA was fully expressed in perichondral ment, and the disease is characterized by an cells and in the chondrocytes found in the autosomal recessive pattern of inheritance. proliferating zones of endochondral growth Molecular analysis of these patients suggested plates. The PTHrP receptor was expressed the disease is due to an inactivating mutation progressively more fully as endochondral in the PTH1R receptor [97]. chondrocytes matured toward their terminal hypertrophic state. Missense expression studies of PTHrP in developing avian embryo growth 2.2.4.4 PTH as a Therapeutic for plates and studies in transgenic animals with Osteoporosis and Augmentation the targeted ablation of PTHrP have demon- of Fracture Healing strated a complex negative feedback loop that involves Indian hedgehog (Ihh) regulation of Numerous recent studies have focused on the the progression of chondrocyte development systemic effects and potential therapeutic appli- during endochondral bone formation [102, 122, cations of PTH [48, 159, 169]. The continuous 208]. These studies demonstrate that Ihh posi- infusion of PTH into mammals induces cata- tively regulates PTHrP expression, as a result bolic events, increases bone remodeling, and of which cells are maintained in an undifferen- leads to a loss of skeletal bone mass. On the tiated state, with Ihh promoting proliferation other hand, intermittent dosing seems to have and thereby expanding the population. The anabolic effects and results in increased bone increased output of PTHrP causes Ihh expres- mass [116, 151, 199]. Clinical trials utilizing the sion to be down-regulated by expanding the 1–34 PTH peptide have increased bone mineral pool of proliferating immature chondrocytes. density and reduced the risk of vertebral and Ihh-ablated transgenic mice have no detectable nonvertebral fractures in postmenopausal Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 29 women. Intermittent treatment also has im- current family of closely structurally related proved the bone mass in osteoporotic men [85]. proteins is encoded by at least 22 genes. Four The recent approval of PTH(1–34) as an ana- distinct FGF receptors each a unique gene bolic treatment for osteoporosis has been a product, mediate activity through tyrosine major impetus to the use of PTH in bone kinase activity. Each receptor appears to be healing. PTH administration has enhanced activated by all members of the FGF ligand early fracture healing in parathyroidectomized family. The ligands in general have heparin- rats [63], with PTH doses ranging from 10 to binding activity and, when complexed with 200µg/kg having signifi cantly improved the heparin, have improved activity. FGF ligands mechanical and histological aspects of normal regulate a wide variety of cellular functions fracture repair in the rat [7, 88, 155]. PTH and can act as mitogens, chemoattractants, analogs have also been shown to reverse the and mediators of cellular differentiation. FGF inhibition of bone healing in ovariectomized receptor activity appears to directly regulate rats [112] and in corticosteroid-treated rabbits the expression of a number of different pro- [25]. PTH(1–34) is reported to increase bone teins, including metalloproteinases and ingrowth and pullout strength in porous metal- morphogens [142, 164, 165]. lic implants [193]. The roles played by FGF in skeletal develop- One drawback of the rat studies is that the ment have been elucidated by identifying auto- hormone doses were much higher than would somal dominant mutations that constitutively be tolerated in humans. To evaluate the clinical activate the FGF receptors [191, 219]. Mutations potential of PTH for fracture healing, patient- in the receptors lead two types of disorders. appropriate doses of recombinant human para- One, in FGFR3, affects axial long-bone thyroid hormone [PTH(1–34); teriparatide; development and leads to the dwarfi ng chon- ForteoTM] were used in a well-established rat drodysplasia syndromes. These include hypo- model. As early as day 21 of this study, calluses chondroplasia [18], achondroplasia [191], and from the group treated with 30µg/kg of PTH thanatophoric dysplasia [178]. The second showed signifi cant increases over controls in group of mutations, in FGFR2, causes a variety terms of torsional strength, stiffness, bone of craniosynostosis syndromes, including the mineral content (BMC), bone mineral density Apert syndrome [215] and the Crouzon syn- (BMD) and cartilage volume. By day 35, both drome [93]. To date, changes in growth due the 5-µg and the 30-µg/kg PTH-treated groups to inactivating mutations in individual FGF showed signifi cant increases in BMC, BMD, and ligands have not been identifi ed. This suggests total osseous tissue volume; the experimental that the developmental functions of the FGF groups also showed signifi cant decreases in ligands involve collaboration among various void space and cartilage volume. At day 35, tor- molecules. sional strength was also signifi cantly increased FGF family signaling pathways play multiple in the group treated with 30µg of PTH. Even and essential roles in the early stages of skeletal after 84 days, the group that had received 30µg patterning and in the recruitment and ultimate of PTH for 21 days, with treatment discontin- apoptosis of mesenchymal cells. They also ued thereafter, exhibited increases in torsional seem to participate in the control of endochon- strength and BMD over comparable control dral growth in the axial skeleton and of cranial values. Thus, daily systemic administration of bone growth at suture lines. During early limb- a low dose of PTH(1–34) enhanced fracture bud development, FGF signaling plays a role in healing and induced an anabolic effect through- mesenchymal epithelia [144]. As a result, FGF- out the entire remodeling phase. 10 is produced and acts on the FGF receptor 2b in the apical ectodermal ridge. Cells in the latter then express FGF-8, which signals back 2.2.5 Other Growth Factors Within to FGFR1c in the limb mesoderm. The role of FGF signaling in endochondral Skeletal Tissues growth has been made apparent by activating 3 2.2.5.1 Fibroblast Growth Factor (FGF) mutations in FGFR . However, the exact effect of the signaling pathways involved in endo- FGFs were originally isolated as oncogenes and chondral development and the downstream shown to stimulate cell proliferation [196]. The FGF signaling on chondrocytes and osteoblasts 30 Engineering of Functional Skeletal Tissues is less well understood. As with BMPs, multiple dogs, a single dose of bFGF injected into the forms of the FGFs are expressed in the peri- fracture sites resulted in increased callus area chondrium. FGFR1 is expressed in prehyper- and BMC and signifi cant recovery in strength trophic and hypertrophic zones and FGFR3 by by week 16. Thus, FGF has therapeutic poten- proliferating chondrocytes. More recently, the tial to enhance bone healing after surgery or actions of FGF signaling have been shown to injury. depend on the stage of chondrocyte differentia- tion and the nature of the individual ligands. 2.2.5.2 Wnts (Wingless) Thus, specifi c receptors are expressed and interact with specifi c ligands in chondrocytes Wnts are 39- to 46-kDa cysteine-rich, secreted only at specifi c stages of differentiation. Studies glycoproteins that are closely associated with of limb cell cultures have indicated that FGF both the cell surface and the ECM [161, 214]. signaling interacts with both the Ihh/PTHrP Wnts are considered one of the major morpho- and the BMP signaling systems in a complex genetic gene families responsible for appropri- network. FGF signaling seems to accelerate ate embryonic development [146]. Genetic both the onset and the pace of hypertrophic studies fi rst performed in Drosophila have differentiation, in actions that are antagonistic defi ned the function of this gene family. In Dro- to those of BMPs, and to regulate chondrocyte sophila, the wingless gene is required for Ihh expression and hypertrophic differentia- normal patterning in the adult and larval body tion. BMP, on the other hand, seems to rescue segments [13, 14, 162]. The lack of this wingless the remaining proliferating and hypertrophic gene results in the deletion of the posterior chondrocytes in achondroplastic mice. This region of each body segment [14, 162]. Ectopic has led to the conclusion that the interaction of gene expression in Xenopus and gene knockout BMP and FGF in the growth cartilage regulates models in mice have since led to further under- the rate of chondrocyte differentiation and standing of the crucial role that Wnts play in proliferation [148]. organ development, segmentation, CNS pat- The intracellular effects of FGFs are medi- terning, cell fate and growth, limb develop- ated by two signaling pathways: the mitogen- ment, and organization of asymmetric cell activated protein kinase/ERK kinase 1 (MEK1) divisions [11, 45, 168, 216]. To date, approxi- pathway and the Janus kinase-signal trans- mately 100 Wnt genes have been identifi ed in ducer and activator of transcription (JAK- species ranging from Caenorhabditis elegans to STAT) pathway [152, 165, 182]. The JAK-STAT humans [216]. signaling pathway mediates the ability of FGF Once the Wnt proteins are secreted, they signaling to inhibit chondrocyte proliferation bind to two families of cell-surface receptors, and enhances hypertrophic chondrocyte apop- the Frizzled (Fzd) receptors and the low-density tosis, whereas the MEK1 pathway mediates FGF lipoprotein (LDL) receptor-related proteins inhibition of hypertrophic differentiation. (LRPs). The Fzd receptor generally consists of A number of studies have examined whether an extracellular cysteine-rich domain (CRD) FGF has utility in promoting bone formation. that binds the specifi c Wnt protein. This recep- Systemic low doses of basic FGF (FGF-2) stim- tor also consists of a seven membrane spanning ulate endosteal and endochondral bone forma- domain on the cytoplasmic tail towards the tion, but depress periosteal bone formation in carboxy-terminus of the protein. In contrast, growing rats [145, 156]. Local administration the LRP-5 and -6 receptors have a single trans- of acidic FGF (FGF-1) increases new bone for- membrane domain [200]. A variety of secreted mation and bone density, whereas systemic proteins, such as Frizzled-related proteins FGF-1 appears to restore bone microarchitec- (sFRPs), Wnt inhibitory factor 1 (WIF1), and ture and prevent bone loss associated with Cereberus, have been shown to be moderators estrogen withdrawal [50]. Both FGF-1 and of extracellular Wnt signaling. The Dickkopf FGF-2 appear immediately at injury sites after (DKK) protein also exerts regulatory action by fracture. FGF-2 was shown to improve bone directly binding to the LRPs, thereby blocking healing in a study that induced a large seg- signal transduction [11]. mental defect and in another with a metaphy- When the ligand becomes bound to the Fzd seal fracture. In a 32-week study of beagle receptor, three signaling pathways are acti- Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 31 vated: the Wnt/β-catenin (canonical) pathway, 110]. When Wnt 3a expression was analyzed in the Wnt/Ca2+ pathway, and the Wnt/polarity a murine knockout model, severe skeletal pathway. The latter two are defi ned as nonca- defects were observed [91, 198]. Studies of the nonical [11]. It is of interest that a given Wnt direct effects of Wnt 3 on mesenchymal stem protein can activate more than one signaling cells (MSCs) demonstrated that exogenous cascade. The canonical pathway involves stabi- addition of Wnt 3 to murine MSCs inhibited lization of β-catenin, followed by translocation osteogenic differentiation and decreased matrix to the nucleus where transcription genes are mineralization; however, the suppression of activated via the TCF/LEF1 family of transcrip- osteogenesis can be fully reversed when Wnt 3a tion factors [24]. is removed. The noncanonical signaling is not as well The noncanonical effects of Wnt signaling understood, though studies in Drosophila and have been examined through studies of Wnt 5. C. elegans are being continued. The Wnt/Ca2+ In contrast to the inhibitory effects of Wnt 3a, pathway is thought to induce an increase in Wnt 5 appeared to promote osteogenic differ- intracellular Ca2+ and activation of PKC, but entiation of the MSCs. These fi ndings suggest further signaling steps have not yet been iden- that canonical Wnt signaling functions to tifi ed. Genetic studies in Drosophila indicate maintain an undifferentiated, proliferative that the c-Jun N-terminal kinase (JNK) pathway MSC population, whereas the noncanonical is involved in the Wnt/polarity pathway, which Wnts stimulate osteogenic differentiation [24]. in turn regulates cell polarity by controlling Interestingly, ectopic expression of Wnt 5a cytoskeletal organization, utilizing at some delayed chondrocyte maturation and collagen stage the disheveled (Dsh) scaffold protein [11]. type X expression, processes involved in carti- The exact mechanism by which the LRP-5 and lage formation [81, 109]. -6 coreceptors function is not understood, but Many questions remain on the functional they are essential for appropriate signaling. role of the Wnts, their receptors, their intracel- Loss of function of Arrow, the Drosophila lular signaling, and their possible interaction analog to the vertebrate LRP receptor, mimics with other morphogenic factors, such as the the wingless mutation that was fi rst observed TGF-β family. in the early 1980s and therefore provides evidence for the synergism between these receptors [200]. The roles Wnt signaling plays during skeletal development and postnatal bone repair were 2.3 Origins of Postnatal recognized as a result of mutations in humans. One is the autosomal recessive disorder osteo- Skeletal Stem Cells, porosis pseudoglioma, characterized by low Cytokines, and bone mass, frequent deformations and frac- tures, and defects in eye vascularization, all of Morphogenetic Signals which are linked to mutations in LRP-5 [73]. Children with osteoporosis pseudoglioma During Bone Repair have normal endochondral growth and bone turnover, but their trabecular bone volume Bone is unique in that after fracture or surgery, is signifi cantly decreased [106]. Furthermore, it can regenerate the original structure and bio- gain-of-function experiments in humans and mechanical competency of the damaged tissue. in mouse models have shown that organisms Bone repair involves four stages that overlap with an activated LRP-5 mutation exhibit a and cause the various tissue types to interact, high bone mass [12]. as shown in Fig. 2.1. The fracture line in the Because the canonical signal transduction bone determines the spatial relationships of the pathway is fairly well known, Wnt 3a was morphogenetic fi elds during tissue regenera- studied in transgenic mice. Previous studies tion. This is evidenced by the development of had shown that Wnt 3a acts in the apical ecto- two circular centers of cartilage (ECB) that dermal ridge of the limb bud to keep cells in an form symmetrically with respect to the fracture undifferentiated and proliferative state [108, line and taper proximally and distally along the 32 Engineering of Functional Skeletal Tissues

ORIGIN OF CELLS STAGES OF FRACTURE REPAIR & SIGNALS Biological Processes

Muscle D. Initial Injury

Inflammation Marrow response

Marrow Hematoma Periosteum MSC recruitment

Cortical Bone

E. A. Endochondral Formation Cartilage formation

Periosteal Response ECB Vascular ingrowth Intramembranous bone Formation

F. IMB Primary Bone Formation Bone cell recruitment Chondrocyte apoptosis B. Matrix proteolysis Osteoclast* recruitment Endochondral neovascularization

G. Secondary Bone Secondary Bone Formation Establishment of marrow Osteoclast remodeling Coupled osteoblast recruitment

C. Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 33 bone cortices (see the middle microphotograph MSCs may also originate in the surrounding in Fig. 2.1A, B, and E). At the same time, a muscle or marrow space. Data to support a crescent-shaped region of intramembranous muscle origin come from studies showing that bone formation appears at the proximal and demineralized bone powder or purifi ed BMPs, distal ends of the area of periosteal response when implanted or injected into muscle tissue, and tapers inward toward the fracture line deep induce bone formation [92, 96, 203]. Other in the cartilage ring. Thus, endochondral and studies have shown that a variety of premyo- intramembranous bone formation both con- genic cell lines can differentiate into chondro- tribute to bone healing. genic or osteogenic cells when treated with During bone repair, cell interactions are ini- BMPs [41, 70, 104]. Marrow stroma also can tiated between the external soft tissues that differentiate into osteoblasts and chondrocytes surround the injured bone, the underlying [20, 94, 188, 189]. Once recruited, their numbers cortical bone and marrow, and the developing increase as a result of other morphogenetic endochondral and intramembranous bone factors. It is important to identify the source of tissues (Fig. 2.1A). The origin of the MSCs that the stem cells, because they make up much of contribute to bone repair and the identity of the the callus tissue and may make up as much as cells that initiate morphogenetic signals are 30% of the original volume of the uninjured still unresolved. Figure 2.1 shows potential long bone sources of cells and signals that lead to the con- Vascular tissues grow into the developing struction of these developmental fi elds. callus as new periosteal bone develops and pro- MSCs involved in fracture repair may origi- gresses toward the fracture line from the proxi- nate in the periosteum, the surrounding tissues, mal and distal edges. The interaction of the or both (Fig. 2.1A). The periosteum appears to vascular elements and the initiation and propa- be the primary source of MSCs that then give gation of the periosteal response thus appear rise to the intramembranous bone that forms to be the primary driving mechanisms that in the callus [154]. If the periosteum is removed, facilitate intramembranous bone formation. callus development is diminished [29], because Perivascular mesenchymal cells in blood-vessel periosteal cells robustly produce BMPs during walls may also contribute to this process [27]. the initial phases of fracture healing [26]. These Figure 2.2 summarizes the mesenchymal observations suggest that morphogens recruit lineage and types of morphogens that are stem cells locally and induce them to involved in lineage selection, expansion, sur- differentiate. vival, and programmed cell removal.

Figure 2.1. Anatomic characterization of fracture repair. Left panels (A-C) show an overview of the morphogenetic fields of tissue development and the proximate tissue interactions. (A) Histological section of the fracture site immediately postfracture. Potential tissue origins of mesenchymal stem cells (MSCs) and morphogenetic signals are denoted by the arrows and denoted in the figure. (B) Histological section of the fracture site at 7 days postfracture. The two types of bone-formation processes are denoted as endochondral bone (ECB) and intramembraneous bone (IMB) formation. The two proceed in a symmetrical manner around the fracture site. (C) Histological section of the fracture site at 28 days postfracture. Secondary bone formation and coupled remodeling predominate in the late stage of bone repair. Right panels (D-G) show a summary of the multiple stages of fracture healing. Histological sections are presented for each stage, and the various processes associated with each stage are summarized. All histological specimens are from sagittal sections of mouse tibia transverse fractures and were stained with safranin O and fast green; micrographic images are at 200× magnification. (D) Section for the initial injury was taken from the fracture site 24 hours postinjury. (E) Section depicting the initial periosteal response and endochondral formation is from 7 days postinjury. Arrows denote vascular ingrowth from the peripheral areas of the periosteum. (F) Section depicting the period of primary bone formation is from 14 days postinjury. Arrows denote neovascular growth areas in the underlying new bone. Inset depicts images of an osteoclast (*chondroclast) resorbing an area of calcified cartilage. (G) Sections depicting the period of secondary bone formation are from 21 days postinjury. Callus sites. Inset depicts 400× images of an osteoclast resorbing an area of primary bone. Reproduced with permission from Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. 2003 Apr 1;88(5):873–84. Copyright © 2003 Wiley-Liss, Inc., A Wiley Company. 34 Engineering of Functional Skeletal Tissues

Stages during Which Morphogens and Cytokines Regulate Mesencyhymal Stem Cell Differentiation

MSC Recruitment (PTHrP, BMP, TNF-α Family) MSC Commitment (BMP, PTHrP, VEGF) Proliferative Expansioon Cell Survival Apoptosis (BMP, PTHrP, VEGF, Wnts, IGF) (VEGF, FGF, BMPs, IGF) (TNF-α Family) Enhancement of Differentiated Function Matrix Matrix Production and Mineralization Maturation

CollagenII Aggrecan (PG) APase Collagen X Sox 9 PTHrP (R) Collagen IX BSP Fas Mineral Deposition Chondroblast

Mesenchymal Stem Cell Differentiation (MSC) Committed Progenitor Cell Collagen I AlkPhos Osteocalcin Runx2 TCF-b1 BSP Mineral deposition Ostrix Osteopontin Collagen Myoblast Adipoblast Osteoblast Tendon Fibroblast Chondroblast Osteoblast Preosteoblast Mature Osteocyte Osteoblast

Figure 2.2. Schematic summary of the lineage progression of mesenchymal stem-cell (MSC) differentiation. Upper panel: Mul- tiple stages of the life cycle of an MSC. The morphogenetic regulators of each stage are in parentheses. Lower panel: The separate stages of each of the major anabolic skeletal cell lineages are indicated with known markers that define each stage of their lineage progression. PTHrP, parathyroid hormone-related peptide; BMP, bone morphogenetic protein; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; IGF, insulin-like growth factor; FGF, fibroblast growth factor; PG, large proteoglycan; BSP, bone sialoprotein; TGF, transforming growth factor.

Restoration of the original anatomic geome- able relevance, because the infl ammatory try of the tissue is an important aspect of bone signals spread out from the point of origin of repair. For this to occur there must be some the injury [16, 51, 54]. Data that support the role relationship between the original structure of infl ammatory cytokines play in the initiation the tissue and the gradients of the morphogens of skeletal tissue repair come from studies that promote the developmental process and showing that in the absence of TNF-α signaling the characteristics of the injury. One obvious in receptor-null animals, the callus does not functional role must be attributed to the signals develop symmetrically around the fracture that initiate and establish the symmetry of line. The absence of TNF-α signaling also leads bone repair around the fracture line. These to a delay in intramembranous and endochon- signals may be thought of as arising from the dral bone formation. Thus, TNF-α signaling marrow or from the injured cortical bone facilitates the repair process, perhaps by stimu- matrix. In this connection, how the injury lating MSC recruitment or differentiation infl uences tissue responses may have consider- [68]. Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 35

The structural geometry of the callus may skeletal-cell lineages. Of particular interest is also depend on the muscular anatomy or vas- the fact that some factors act at several stages cularization of the tissue and on the biome- during skeletal-cell lineage progression. For chanical environment at the site of injury. The example, BMPs not only are associated with latter seems to be particularly important. When MSC lineage commitment but also are involved bending and shear loading were introduced at in cellular expansion. In contrast, the scope an osteotomy site, osteogenesis was favored of morphogens such as VEGF and the Wnts over chondrogenesis [42]. Other studies have appears to be more restricted, with their pre- similarly shown that mechanical instability dominant effect on proliferative lineage expan- leads to persistence of cartilage tissue at the sion or survival. Members of the TNF-α family, fracture site. This involves up-regulation of which are part of the immune response to molecular signals such as Ihh that regulate injury, regulate the initial stages of MSC recruit- chondrogenesis [124]. How morphogenetic ment and cell survival during the infl ammatory fi elds are established and how biomechanical stage and re-emerge at the end of the MSC cycle factors direct tissue differentiation and the to control apoptosis during tissue remodeling. geometry of the regenerative process are ques- Finally, factors such as the FGFs control the rate tions of considerable importance, because the or timing of entry and exit of committed cells answers may identify the signal molecules and during their period of proliferative expansion. relate them to the origins of MSCs. Defi ning The functional contribution of specifi c cyto- how the morphogenetic fi elds are established kines and morphogens during fracture healing also has clinical importance, since the thera- is presented in Fig. 2.3. These factors are peutic responses to bioactive factors may expressed during different phases of fracture depend on whether they are correctly directed healing and therefore may vary in the roles to the morphogenetic fi eld. they play during healing. For example, TGF-β2, TGF-β3, and GDF-5 show peak mRNA expres- sion during chondrogenic differentiation and as the endochondral phases develop. This sug- 2.4 Bone Repair Is gests that the two factors are functionally Dependent upon restricted to the periods in which chondrogen- esis takes place. Multiple Cellular and Understanding the temporal pattern and Molecular Signals molecular nature of the factors as they are expressed during bone healing can allow targeting and modifi cation of their actions to The cellular and molecular processes that lead to better fracture healing. Knowing the govern bone repair after injury have many fea- spatial nature of the morphogenetic fi elds tures that are similar to what occurs in a growth during the temporal processes of fracture plate during embryonic and postnatal skeletal healing has clinical importance because the development. As reviewed earlier, fracture therapeutic responses to bioactive factors may healing involves several stages and is mediated be infl uenced by the moment in time when they by very different biological processes. Figure contact the correct morphogenetic fi eld. Such 2.2 presents the stages and progression of MSC knowledge will help to develop therapeutic differentiation into cartilage and bone as the agents to treat osteoporosis and can equally skeleton is formed. well be applied to the development of therap- Figure 2.2 also shows the stages at which eutic agents that promote bone formation. various morphogens and cytokines become Table 2.1 lists the biological processes and active and regulate MSC and skeletal-cell dif- approaches that can be modifi ed in coupled ferentiation. In addition the fi gure lists the spe- bone remodeling, either to impede bone loss or cifi c transcription factors (Runx 2, Osterix, and to promote bone regeneration. The table also Sox 9 [117]) involved in lineage commitment lists approaches that could enhance the rate or and identifi es stage-specifi c markers for the two quality of bone healing. 36 Engineering of Functional Skeletal Tissues

STAGES OF FRACTURE REPAIR Initial Injury Endochondral Formation Primary Bone Formation Secondary Bone Formation Inflammation Periosteal Response Cartilage Resorption Coupled Remodeling RELATIVE TIMES

Cytokines M-CSF IL-a IL-1b IL-6 IL-11 RANKL OPG INF-γ TNF-α TRAIL VEG1

Morphogens TGF-b1 TGF-b2 TGF-b3 BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 BMP-8a GDF-1 GDF-5 GDF-8 GDF-10 Ihh PTHrP Wtn 4 Wtn 5b Wtn 10b Wtn 11

Proteases MMP-2 MMP-8 MMP-9 MMP-13 MMMP-14 Angiogenic VEGF A VEGF B VEGF C VEGF D Ang 1 Ang 2 Osteogenic Growth Factors and Cytokines and Their Role in Bone Repair 37

Figure 2.3. Schematic summary of the stages of fracture repair and their associated molecular processes. The relative temporal aspects of each of the stages of the fracture healing process are denoted by basic geometric shapes that also connote the relative intensity of the molecular processes that define each of the stages. The relative levels of expression of various mRNAs that have been examined in our laboratories are denoted by three line widths. The levels of expression are in percent over baseline for each and are not comparable for the various mRNAs. Data for expression levels for the proinflammatory cytokines and the extracellullar matrix (ECM) mRNAs are from Kon et al., 2001 [118]; data for TGF-α family members are from Cho et al., 2002 [38]; data for prote- ases and angiogenic factors from are from Lehmann et al., 2002 [127]; and data for Cox2 are from Gerstenfeld et al., 2002 [70]. Data pertaining to Ihh and iNOs expression are unpublished. M-CSF, macrophage colony-stimulating factor; IL, interleukin; RANKL, RANK ligand; OPG, osteoprotegrin; INF, interferon; TNF, tumor necrosis factor; VEG1, xxx; TGF, transforming growth factor; BMP, bone morphogenetic protein; GDF, growth and differentiation factor; Ihh, Indian hedgehog; PTHrP, parathyroid hormone-related peptide; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; Ang, angiopoietin. Reproduced with permission from Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. 2003 Apr 1;88(5):873–84. Copyright © 2003 Wiley-Liss, Inc., A Wiley Company.

Table 2.1. Comparison of strategies in the development of therapeutic agents to treat osteoporosis versus fracture and bone repair A. Stages of fracture repair and strategies to enhance fracture repair Initial injury Endochondral formation Primary bone formation Secondary bone formation Inflammation Periosteal response Cartilage resorption Coupled remodeling Factors that promote Increase ratio of bone/ Factors that change rates of Factors that enhance coupled stem-cell recruitment cartilage differentiation endochondral remodeling bone formation (TNF family) (PTH, BMPs) (FGFs, Wnts, PTH) (TNF family) B. Stages of coupled remodeling and strategies to enhance bone mass Activation Diminish numbers of osteoclasts (TNF family) Resorption Diminish osteoclast activity/increase rate of osteoclast turnover (TNF family) Formation Increase osteoblast numbers/osteoblast activity (BMPs, PTH, Wnts)

2.5 Future Perspectives vidual factors has had mixed success in pro- moting bone healing. Regaining biomechanical on Therapeutic Uses of competency more quickly is even more compli- cated than promoting stem-cell differentiation. Morphogenetic Factors Biomechanical competency involves many factors, including the restoration of the mate- Reduction of the morbidity associated with rial properties of the tissue and of appropriate some 5% to 10% of fractures and improvement skeletal-tissue geometry. At the same time, it of healing after osteotomies, arthrodeses, will be necessary to defi ne appropriate modali- spinal fusions, and other reconstructive ortho- ties for using repair-promoting factors and to pedic procedures depend on better understand- identify when, where, and how long the factors ing of the biology of fracture and bone healing should be applied. Because many factors, once (170). As discussed above, multiple morphoge- they activate receptors, utilize overlapping netic factors regulate normal skeletal develop- signal-transduction pathways to mediate intra- ment, but it is not clear how they function in cellular effects, signal pathways need to be postnatal healing. Many factors act coopera- identifi ed in the hope of making optimal use of tively or even antagonistically at different the small-molecule pharmaceuticals that are stages of bone development. Single use of indi- being developed. 38 Engineering of Functional Skeletal Tissues

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