CELLULAR AND MOLECULAR MECHANISMS OF BONE HEALING
Floreasca Emmergency Hospital
Osul are o capacitate substantiala pentru repararea si regenerarea in raspuns la leziune sau la tratamentul chirurgical. Ambele procese implica integrarea celulara, a factorilor de crestere si a matricei extracelulare. Repararea in mod simplu restaureaza continuitatea tesutului lezat fara a fi necesara cresterea in volum. Regenerarea, in contrast, implica diferentierea a noi celule si formarea osului nou care rezulta in cresterea totala in volum a noilor tesuturi osoase. Importanta relativa a citokinelor inflamatorii in vindecarea normala si in diabet, superfamilia factorilor de crestere beta a mediatorilor morfogenici ai osului si procesul angiogenezei sunt discutate intrucat acestia sunt legati de repararea osoasa.
Cuvinte cheie: fractura osoasa, citokine inflamatorii, TGF, angiogeneza
Bone has a substantial capacity for repair and regeneration in response to injury or surgical treatment. Both processes involve a complex integration of cells, growth factors, and the extracellular matrix. Repair simply restores the continuity of the injured tissues, without necessarily increasing bone volume. Regeneration, in contrast, involves the differentiation of new cells and the formation of new bone tissue that results in an overall increase in volume of new skeletal tissues. The relative importance of inflammatory cytokines in normal and diabetic healing, the transforming growth factor beta superfamily of bone morphogenetic mediators, and the process of angiogenesis are discussed as they relate to bone repair.
Key words: bone fracture, inflamatory cytokines, transforming growth factor, angiogenesis
Fracture healing is a multistage repair process that involves complex yet well-orchestrated steps that are initiated in response to injury, resulting eventually in the repair and restoration of function.Inflammatory cytokines involved in fracture repair are believed to play a role in initiating the repair cascade following injury. These cytokines are produced and function immediately after injury for a limited time period. At a mid-stage in healing, some of the inflammatory cytokines are up-regulated, so osteoclastogenesis is stimulated to remove mineralized cartilage, and others are induced at a later stage during bone remodeling.Interleukins-1 and -6 (IL-1 and IL-6) and TNF-? have been shown to play a role in initiating the repair cascade. They induce a downstream response to injury by recruiting other inflammatory cells, enhancing extracellular matrix synthesis, and stimulating angiogenesis  are secreted at the injury site by macrophages, inflammatory cells, and cells of mesenchymal origin. Their expression peaks within the first 24 hrs, then declines rapidly to nearly undetectable levels by day 3 [1;2]. The expression of IL-1 and IL-6 rises again in association with bone remodeling during secondary bone formation, whereas the expression of TNF-? rises in association with mineralized cartilage resorption by the end of the endochondral phase of fracture repair . In addition to stimulating osteoclast function, TNF-? promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes during endochondral bone formation. Its absence delays the resorption of mineralized cartilage and, consequently, prevents the formation of new bone. In situations where TNF-? is over-expressed, such as diabetic healing, there is premature cartilage removal that is associated with deficient bone formation and healing.The expression of RANKL and OPG (two members of the TNF-? superfamily), as well as macrophage colony-stimulating factor (MCSF), which are key regulatory factors in osteoclastogenesis, increases after initial injury as well as during the period of mineralized cartilage resorption. During the phase of secondary bone formation and bone remodeling, RANKL, OPG, and MCSF showed expression levels diminished from those seen during cartilage resorption. In contrast, IL-1 and IL-6 expression rose during late remodeling .
Pro-inflammatory Cytokines and Impaired Diabetic Fracture Healing
Diabetes causes diminished bone formation and increases the risk of fracture[6; 7]. Moreover, fracture healing is impaired in diabetic humans and in animal models[8; 9]. There are likely to be multiple mechanisms through which diabetes may affect bone, including the expression of genes that regulate osteoblast differentiation and the expression of growth factors that promote bone formation [10;11] To gain insight into how diabetes affects fracture healing, investigators have carried out experiments in a type 1 diabetic model, focusing on the impact of diabetes on the transition from cartilage to bone . There was relatively little difference in the initial amount of callus formed. However, diabetes caused an increase in mRNA levels of TNF-?, MCSF, and RANKL. The increase in these cytokines was accompanied by a similar increase in osteoclast numbers and a more rapid degradation of cartilage. The greater loss of cartilage also coincided with increased mRNA levels of ADAMTS 4 and 5 and aggrecanases that degrade cartilage. The accelerated loss of cartilage may be physiologically significant, since it may leave a reduced template for endochondral bone formation This may explain a decrease in callus size and a decrease in the strength of healing bone that is typically found in diabetic fracture healing [12;13]. It is striking that the more rapid removal of cartilage, greater osteoclast formation, and enhanced expression of pro-inflammatory cytokines are the opposite of that observed in TNF receptor-deficient mice . Thus, in diabetes, high levels of TNF-? and other pro-inflammatory cytokines may increase osteoclastogenesis, which leads to excessive removal of cartilage, which in turn may interfere with the transition from cartilage to bone and impair fracture healing. In contrast, TNF receptor-deficient mice exhibit delayed osteoclast formation, failure to remove cartilage in timely fashion, and a longer time required for healing of fractured bone.
The Role of the Transforming Growth Factor Beta Superfamily in Fracture Repair
The transforming growth factor-beta (TGF-?) superfamily consists of a large number of growth and differentiation factors that include bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-?), growth differentiation factors (GDFs), activins, inhibins, and Müllerian inhibiting substance. Specific members of this family—such as BMPs (2-8), GDF (1, 5, 8, and 10), and TGF-?1-3—promote various stages of intramembranous and endochondral bone ossification during fracture healing All three isoforms of this group of morphogens are involved in fracture repair (TGF-?1-3). They are produced by degranulated platelets after initial injury, which suggests their involvement in the initiation of callus formation . They are also produced by osteoblasts and chondrocytes at later stages, which enhances the proliferation of these cells as well as that of mesenchymal cells and pre-osteoblasts . TGF-? is thought to play an important role in chondrogenesis and endochondral bone formation . It induces the expression of extracellular matrix proteins. The expression of TGF-?2 and TGF-?3 peaks on day 7 post-fracture in the mouse, when type II collagen expression rises, and appears to be associated with cartilage formation. The expression of TGF-?1 remains constant throughout the fracture-healing process. This suggests that TGF-?2 and TGF-?3 may play a more important role during fracture healing, since their expression peaks during the critical phase of chondrogenesis
Bone Morphogenetic Proteins
During fracture repair, BMPs are produced by mesenchymal cells, osteoblasts, and chondrocytes. Different BMPs function independently of or in collaboration with each other, as well as with other members of the TGF-? superfamily, to trigger a cascade of events that promote the formation of cartilage and bone. Cellular processes stimulated include chemotaxis, mesenchymal cell proliferation and differentiation, angiogenesis, and synthesis of extracellular matrix [14; 15]. Although different BMPs are closely related structurally and functionally, they exhibit different temporal patterns of expression at different stages of fracture healing, based on several animal experiments. In studies of murine fracture healing, BMP-2 mRNA expression showed maximal levels within 24 hrs of injury, suggesting that this BMP plays a role in initiating the repair cascade. Consistent with this finding are recent studies showing that BMP-2 is necessary for post-natal bone repair and is genetically associated with the maintenance of normal bone mass ;. In contrast, BMP-2 is apparently not needed for embryological bone formation. Other in vitro studies examining marrow stromal stem cell differentiation have shown that BMP-2 controls the expression of several other BMPs, and when its activity is blocked, marrow stromal stem cells fail to differentiate into osteoblasts .BMP- 3, -4, -7, and -8 show a restricted period of expression during fracture healing (days 14 through 21), when the resorption of calcified cartilage and osteoblastic recruitment are most active, and coupled bone formation takes place. BMP-5 and -6 and other members of the TGF-? superfamily are constitutively expressed from days 3-21 during fracture in mice, suggesting that they have a regulatory effect on both intramembranous and endochondral ossificationIt has been proposed that BMP-2, -6, and -9 may be the most potent inducers of mesenchymal cell differentiation to osteoblasts, while the remaining BMPs promote the maturation of committed osteoblasts . BMP antagonists also play an important role in fracture repair. It has been reported that the expression of noggin, which blocks BMP-2, -4, and-7, is modulated during fracture healing . The pattern of noggin expression is similar to that of BMP-4, suggesting that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing. This is supported by findings that, in the absence of noggin, there is excess bone and cartilage formation during development, indicating that noggin plays an important role in limiting the formation of these tissues .All three isoforms of this group of morphogens are involved in fracture repair (TGF-?1-3). They are produced by degranulated platelets after initial injury, which suggests their involvement in the initiation of callus formation . They are also produced by osteoblasts and chondrocytes at later stages, which enhances the proliferation of these cells as well as that of mesenchymal cells and pre-osteoblasts . TGF-? is thought to play an important role in chondrogenesis and endochondral bone formation . It induces the expression of extracellular matrix proteins. The expression of TGF-?2 and TGF-?3 peaks on day 7 post-fracture in the mouse, when type II collagen expression rises, and appears to be associated with cartilage formation. The expression of TGF-?1 remains constant throughout the fracture-healing process. This suggests that TGF-?2 and TGF-?3 may play a more important role during fracture healing, since their expression peaks during the critical phase of chondrogenesis (.
The Role of Angiogenic Factors in Fracture Repair
Optimal bone healing is dependent on adequate vascularization and therefore requires the development of new blood vessels. During endochondral fracture healing, the transition from a cartilaginous callus to new bone formation represents a crucial stage in the repair process.This stage includes four coordinated biological events: chondrocyte apoptosis; cartilaginous matrix degradation and removal; vascularization of the repair site; and osteogenic cell recruitment, differentiation, and bone matrix production. Disruption of any one of these can lead to delayed or impaired healing . In other biological processes, such as growth plate development, disruption of any of these events has been shown to interfere with the formation of skeletal bone. For example, disruption of cartilaginous matrix degradation through the loss of MMP-9 expression leads to a massive expansion of the hypertrophic zone , a consequence of abnormal regulation of the apoptotic process. Moreover, in mmp-9-/- mice, a delay is observed in the progression of fracture repair, and this effect can be rescued by the addition of exogenous VEGF, suggesting that angiogenesis is linked to the apoptosis of chondrocytes.The above observations have led to the suggestion that the molecular regulation of angiogenesis is linked with the removal of cartilage during endochondral bone formation. The interdependence of the various biologic processes in fracture healing was clearly demonstrated in data from our laboratory, which showed that lack of TNF-? signaling delays chondrocyte apoptosis, which then leads to delays in the resorption of mineralized cartilage and, ultimately, a delay in fracture healing [3,22].Angiogenesis is regulated by 2 pathways, a vascular endothelial growth factor (VEGF)-dependent pathway and an angiopoietin-dependent pathway . Both pathways are speculated to be functional during fracture repair. The VEGF-related family of proteins includes endothelial cell mitogens and essential mediators of neo-angiogenesis. It has been demonstrated that VEGF signaling plays a central role in neo-angiogenesis and in endochondral bone formation [(27]. Furthermore, fracture repair is enhanced by exogenous VEGF . Osteoblasts are known to express elevated amounts of VEGF, and therefore have been implicated as primary regulators of angiogenesis in fracture healing. Moreover, several studies have shown that BMPs stimulate the expression of VEGF and their receptors, suggesting an intimate relationship between these two families that promotes the formation of new bone .A second pathway that regulates vascular growth includes angiopoietin-1 and -2 and their receptors. Angiopoietins are vascular morphogenetic proteins that are associated with the formation of larger vessels and the development of collateral branches from existing vessels. The role of angiopoietin in fracture repair is not as well-understood as the VEGF pathway. The expression of angiopoietin-1 is induced during the initial stages of fracture repair, suggesting that initial vascular in-growth from vessels in the periosteum plays an important role in the repair process .In literature data related to fracture healing, comparison of the expression profiles of angiogenic regulators demonstrated that the most prevalent factors expressed over the time-course of repair are angiopoietin-2, pigment epithelial-derived factor, pleiotrophin, Tie1, and vascular endothelial growth inhibitor . The VEGF gene family members detectable during fracture healing are VEGF-D, VEGF-A, and VEGF-C. They are expressed throughout the chondrogenic phase of healing, reaching maximal levels of expression during the late phases of calcification of the cartilage tissues, at the time when resorption is initiated. A relationship between the expression of some angiogenic factors and pro-inflammatory cytokines has been shown in mice lacking TNF receptors. The absence of TNF receptor signaling diminishes the expression of angiopoietins, metalloproteinases, and vascular endothelial growth inhibitor during fracture healing. However, the expression of VEGF family members that directly promote new vessel formation is not inhibited.
Taken together, the results from this study suggest that, after injury, existent vessels are first dissociated into a pool of non-dividing endothelial cells through the actions of angiopoietin-2 and vascular endothelial growth inhibitor, the latter limiting proliferation. At the time when cartilage resorption and primary bone remodeling are initiated, VEGF levels rise, stimulating cell division of this pool of progenitors and promoting participation of these endothelial cells in neo-angiogenesis.These results suggest that TNF-? signaling in chondrocytes controls vascularization of cartilage through the regulation of angiopoietin and vascular endothelial growth inhibitor factor, which play counterbalancing roles in the induction of growth arrest and apoptosis of endothelial cells.
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