Growth Factor Regulation of Fracture Repair (1999)

Fracture repair can be considered as a biologically optimal process resulting in the restoration of injured skeletal tissue to a state of normal structure and function. Although the process leads to healing in the vast majority of cases, a small but significant proportion of fractures result in delayed union or persistent nonunion. Surgical interventions have been directed toward enhancing the fracture repair process, normalizing the rate of healing, and decreasing the likelihood of nonunion. During fracture repair, a number of growth factors, cytokines, and their cognate receptors are present at elevated levels in and around the fracture site. Many of these proteins are normally expressed in skeletal tissue, and others are released from associated inflammatory cells at the site of injury. The induction of these proteins is regulated both spatially and temporally, suggesting that they play an active role in promoting fracture repair. The following review will summarize the current literature on the roles of the major cytokines and growth factors involved in fracture repair. In addition, the signaling cascades induced by these molecules will be discussed. While many cytokine and growth factor signaling events have not been specifically examined in the context of fracture repair, a large body of literature on signal transduction has emanated from studies on these molecules in embryonic bone development. Given the conserved nature of these molecules and their signaling cascades from Drosophila to humans, and the similarities between the fracture repair process and embryonic bone development, it seems highly probable that these downstream signaling events are conserved in fracture repair. Fracture repair can be envisioned as involving five distinguishable processes, including the immediate response to injury, intramembranous bone formation, chondrogenesis, endochondral bone formation leading to the reestablishment of load bearing function, and bone remodeling. While these processes may be discussed individually, it should be recognized that the first four occur simultaneously during fracture repair and are likely to influence one another. Extensive remodeling of the newly formed bone follows these four concurrent processes and facilitates the reestablishment of the full biomechanical integrity of the bone. A number of investigators have commented on the similarities between the repair process and embryonic bone formation and indeed a number of specific events characteristic of embryonic bone formation are reiterated in fracture repair. This is especially evident as one begins to examine the specifics of local factors regulating fracture repair, specifically those involved in the events of endochondral bone formation. The immediate response to injury from the fracture trauma is the initiating event of the fracture repair process. Fracture trauma involves not only an interruption of skeletal integrity but also a disruption of the normal vascular structures and nutrient flow at the fracture site. This leads to reduce oxygen tension, disruption of the marrow architecture, and results in the infiltration of inflammatory cells, macrophages, and degranulating platelets during formation of a hematoma.1, 2 While it is likely that the mechanical stresses, changes in oxygen tension, and loss of vascular nutrients at the fracture site may signal some aspects of the healing process, the dominant initiators of fracture repair are most likely the numerous cytokines and growth factors released into the fracture site.3 To date the majority of research on fracture repair has focused on the actions of a relatively limited number of cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha, and macrophage colony-stimulating factor as well as the local growth factors fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), and the bone morphogenetic proteins (BMPs)-2, -3, -4, and -7. It is interesting to note that the majority of the factors mentioned have well described roles in the formation and patterning of the embryonic skeleton or in the homeostatic maintenance of the mature skeleton. During the immediate injury response, the first detectable factors include PDGF and TGF-β released by the degranulating platelets.3 This is closely followed by infiltrating macrophages and other inflammatory cells that secrete FGF and additional PDGF and TGF-β. Presumably, these inflammatory cells also release a variety of cytokines including IL-1 and IL-6; however, this has only been demonstrated indirectly and no comprehensive data are available on the spatiotemporal profile of the proinflammatory and immune cytokine expression during the fracture repair process.4 Thus within the first hours post fracture, a complex milieu of factors involved in bone repair can be detected in the fracture site. Some or all of these factors are presumably the initiators of the active repair process acting on the cells of the bone marrow, periosteum, and external soft tissues adjacent to the fracture site. One of the first tissues to visibly respond to the milieu of factors released by the immediate response to injury is the bone marrow, and in fact preliminary evidence suggests the bone marrow may be a major contributor of some of these factors.2 It has been demonstrated that within hours following the fracture event, the bone marrow undergoes a loss of normal architecture, disappearance of the blood vessels adjacent to the fracture callus clot, and a reorganization of the cellular complement into regions of high and low cellular density (Fig. 1A). Within 24 h post fracture, cells in the high cellular density regions undergo differentiation and take on an osteoblastic phenotype.2 In addition to changes in the bone marrow, osteoblasts lining the cortical bone surface are activated, and adjacent periosteal cambium derived preosteoblasts divide and begin differentiating.1 These resident and differentiating osteoblasts lay down new bone via an intramembranous pathway, thus forming the woven bone or so-called “hard” callus adjacent to the fracture site. These events can be detected as early as 3 days post fracture and continue with proliferation peaking between 7–10 days and disappearing in this region by 14 days post fracture (Figs. 1B, and 2A, 2B).5 Osteocalcin expression in this region is maximal around day 15, indicating continued osteoblastic activity post cessation of proliferation. Under specific conditions in which the fracture is rigidly fixed by a surgical plate and screws, this newly formed bone can bridge the fracture gap and produce fracture repair without the production of a cartilaginous callus.6 This, however, is a relatively rare situation. In the majority of fractures, healing involves an additional chemotactic response by periosteally derived undifferentiated mesenchymal cells and fibroblasts from the external soft tissues that infiltrate and divide replacing the hematoma. Photomicrographs of early fracture callus formation in the mouse. (A) Fracture site 24 h after fracture showing no detectable fracture callus. (B) Fracture site at day 7 post fracture showing early callus formation and organization. Intramembranous bone formation is detectable under the periosteum (open arrow) and the early chondroid callus representing the initial stages of the endochondral repair process is visible adjacent to the fracture site (closed arrow) (hematoxylin and eosin, ×40). Low power photomicrographs of fracture callus in the rat. (A) Fracture callus at day 9 post fracture showing increased intramembranous bone formation under the periosteum and increased chondroid tissue overlying the fracture site. (B) Fracture callus at 14 days post fracture showing the continued intramembranous bone formation under the periosteum referred to as the hard callus and increased volume of chondroid tissue referred to as the soft callus. (C) Fracture callus 21 days after fracture showing newly formed woven bone adjacent to the fracture site formed as a result of endochondral bone formation (closed arrow) and the residual chondroid tissue still to be replaced with new bone. (hematoxylin and eosin, ×4). Mesenchymal cell proliferation can be detected as early as day 3 post fracture and remains high for several days.5 The subsequent chondrogenesis by these mesenchymal cells and proliferation of these new chondrocytes continues from day 7 to day 21, leading to the formation of a cartilaginous callus that bridges the fracture site. This cartilaginous callus is referred to as the “soft” callus and provides the initial stabilization of the fracture site (Figs. 1B and 2A–2C). This cascade of events is the initiation of an endochondral bone formation process and is remarkably reminiscent of the proliferative zone of the growth plate. By 9 days post fracture, the chondrocytes of the soft callus adjacent to the woven bone of the hard callus begin to elongate, form elaborate vesicular structures, and turn the and expression characteristic of These chondrocytes undergo and the characteristic expression of of chondrocytes of the growth In addition, these cells produce and release that begin down the cartilaginous including in for 9 expression by day 14 and is followed days by a in indicating an in The of the soft callus in a spatially with of chondrocytes and at the between the and newly formed woven bone (Figs. and closely after of the is and the formation of new vascular structures which infiltrate with The resident chondrocytes begin to undergo cell or and the is replaced by woven bone down by the osteoblasts (Fig. the fracture site is with new bone which will be by bone which is from While the process by which fracture repair is well relatively is the of the events leading to repair. is the process can leading to of delayed and repair. It is that fracture repair involves the of cellular and differentiation and that these events are likely to be regulated signaling via growth factors The major growth factors as present in the fracture site are and and and FGF and Given the well described roles of these factors in embryonic bone and in on bone cells, these molecules are likely to be of fracture repair. It is interesting to note that the signaling by these molecules into those signaling and and those receptors and The is signal transduction a in which activity is induced by by The of a is the that this facilitates between signaling and the to the signal cascade the cell after the initial (Fig. the between the signaling of the major growth factors and cytokines involved in fracture repair. involved in and are not The are a of by a high to The FGF expressed in The most in normal tissues are FGF and also and and are expressed in a variety of tissues and are in the bone presumably to their for The FGF of molecules to the via a of receptors with have been five FGF receptors to The FGF receptors for of the of the FGF and are expressed in a variety of tissues including no have the FGF profile during the fracture repair process. with many of the of the activity in response to additional may be to the the of FGF by or in These molecules may by the to the thus the complex and the downstream The major downstream signaling cascades include the associated and (Fig. and can be detected in the early stages of fracture repair in the tissue at the fracture and other inflammatory cells and this is the likely of FGF in the are expressed by mesenchymal cells, chondrocytes and osteoblasts and have been demonstrated to TGF-β expression in osteoblastic are also expressed by The as on a variety of mesenchymal cells including chondrocytes and osteoblasts In the of the of this to be on This is by the expression profile of that during In addition, in a fracture of into fracture during the first 9 days post fracture associated with an increased callus of which to increased proliferation of biomechanical of fractures but preliminary data a of fracture with This result the that fracture callus is a of fracture repair and the major role of in proliferation. is and has been in fracture repair is expressed by osteoblasts and has also been detected in the of the growth plate during development, an that suggests a role in and endochondral bone formation In addition to and also bone These that has the to influence many of fracture repair, from early events to remodeling of the callus. 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The TGF-β molecules are of a large of factors referred to as the TGF-β This not only the TGF-β but also the the growth and differentiation factors or and several other proteins are in all the and the of the TGF-β are as large which are to mature These conserved molecules play roles in cell differentiation and proliferation during and to play a variety of roles in the of TGF-β have been to and have the most fracture repair, and for may be referred to as TGF-β. TGF-β signaling involves TGF-β and cells within the fracture site as well as in the body TGF-β receptors on their The of downstream is to which of the receptors is associated with the TGF-β an form of followed by the of a into the also in an resulting in a complex associated with the The has a active which the in the thus the The is for the downstream of the signal the signal of the downstream of molecules is characteristic of the TGF-β receptors (Fig. Given the of the signaling and the for between of the TGF-β of several of which will be discussed in the context of the fracture repair process, a on signaling molecules is TGF-β is a growth factor released by the degranulating platelets in the and by the bone at the fracture In the initial stages of fracture repair, TGF-β can be to the region of the hard callus it the region of periosteal proliferation and intramembranous bone suggests that TGF-β is likely to be involved in the of proliferation by the preosteoblasts in this In addition, the expression of TGF-β is elevated during chondrogenesis and bone formation with an initial in levels detected around day post fracture followed by a at day TGF-β expression by day 14 and remains elevated The of TGF-β expression with the in and the subsequent with TGF-β is to be a of undifferentiated mesenchymal cell and proliferation and production during chondrogenesis and endochondral bone formation TGF-β may also be involved in the normal of bone formation with The role of or of growth in normal fracture repair is to the of TGF-β to this process is by the of TGF-β to fracture repair in several evidence for the of TGF-β from studies on repair in the A of TGF-β induced of that with While healing in this via intramembranous bone formation, this the first of a growth factor bone repair in the of bone morphogenetic The of TGF-β to bone healing first demonstrated in in with a of the site with high of TGF-β for in a in callus volume and increased mechanical with In a fracture TGF-β around the fracture site 2 days during a healing TGF-β increased the of the and mechanical demonstrated a load in fractures with the high of TGF-β. The results of these studies be to the or into the fracture site have limited and studies have the healing of fractures after a of into the callus days after by or and healing 14 days post This cartilaginous callus to form but is not to cortical not in this of associated with significant around the site. In fractures, the high of an in callus volume but not the bone of the callus. In fractures, not callus volume or bone but the of the callus. 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