- Email: [email protected]
Overview of the fracture healing cascade
Injury, Int. J. Care Injured (2005) 36S, S5—S7
www.elsevier.com/locate/injury
Overview of the fracture healing cascade A.M. Phillips Consultant Trauma & Orthopaedics, Kings Hospital London, Denmark Hill, London SE5 9RS, UK
KEYWORDS Bone healing; Callus formation; Growth factors; Mesenchymal stem cells
Summary Fracture healing is a dynamic process being governed by a variety of cellular elements and stimulating agents. Our knowledge in understanding the molecular and cellular processes occurring in healing fractures has vastly expanded as a result of the advances made in all aspects of medicine. The ability to manipulate mesenchymal stem cells and to deliver locally growth factors in order to create new bone has led to a proliferation of research papers. The purpose of this article is to provide a brief overview of the current level of understanding of the physiological processes regulating the healing of fractures. # 2005 Published by Elsevier Ltd.
Introduction A detailed understanding of the molecular and cellular processes occurring in healing fractures is essential for manipulation of the process for patient benefit. Significant advances are currently being made in this field in the clinical and basic science arena. The ability to manipulate pluripotential mesenchymal stem cells (MSCs) to create bone by stimulating them with exogenous signalling molecules, or encouraging cells to express proteins favourable for other cells to create bone by genetic modification, has led to a proliferation of research papers on this subject.11 The purpose of this manuscript is to provide a brief overview of the current level of understanding of this process as is relevant to an orthopaedic surgeon with an interest in promoting fracture healing as quickly as possible, reducing the need for intervention, and reducing the incidence of non-union.
E-mail address: [email protected]. 0020–1383/$ — see front matter # 2005 Published by Elsevier Ltd. doi:10.1016/j.injury.2005.07.027
Biological considerations of fracture healing Primary and secondary fracture healing Much has been made of the concept of ‘primary fracture healing’. The concept is that if the fracture is anatomically reduced, at the micrometric level, ‘osteonal’ healing occurs. Osteoclasts create ‘cutting cones’ and primarily cross the fracture site. This requires very high stability, and in practice is the rarest type. More commonly, ‘secondary’ fracture healing occurs and a larger a mass of callus is created. This type of healing benefits from micromotion.10 The description of fracture healing in this presentation refers to this latter, ‘secondary’ fracture healing.
The contribution of the different spatial areas in a healing fracture Fracture healing is not homogeneous throughout the callus. The four main zones are the medullary canal,
S6
the area between the cortices, the subperiosteal layer and the surrounding soft tissues. Essentially, the medullary canal and inter-cortical areas create ‘soft callus’ and go on to create bone by enchondral ossification. The subperiosteal area and the soft tissues immediately surrounding the fracture form ‘hard callus’ and create bone by intramembranous ossification. These are discussed in more detail in the section on healing phases in rodent models.
A.M. Phillips
osteoblasts, and osteoprogenitor cells into osteoblasts. FGF, released from macrophages, MSCs, chondrocytes and osteoblasts, is mitogenic for MSCs, chondrocytes and osteoblasts. IGF, released from bone matrix, osteoblasts and chondrocytes, promotes proliferation and differentiation of osteoprogenitor cells. PDGF, released from platelets and osteoblasts, is mitogenic for MSCs and osteoblasts and responsible for macrophage chemotaxis.
Five temporal stages of fracture healing Healing phases in the rat model In addition to the four ‘spatial’ divisions of fracture healing, there are five ‘temporal’ phases. In chronological order, these are: haematoma formation, inflammation, angiogenesis, cartilage formation (with subsequent calcification, cartilage removal and then bone formation) and bone remodelling.
Three simultaneous processes There are three discrete simultaneous process involved, which are all interdependent including cellular processes leading directly to production of calcified bone, the process of angiogenesis and formation of the matrix. Any intervention that affects any or all of these processes will affect the quality of bone formation.
The role of the inflammatory cascade Without an inflammatory process, bone will not heal. Inhibition of the cyclo-oxygenase pathway appears to inhibit bone healing. Newer, more selective non-steroidal ani-inflammatory drugs promised fewer side effects. However, even blocking the cyclo-oxygenase 2 pathway (COX-2) appears to significantly halt bone formation.12 The major signalling molecules involved in, and controlling the cascade are: interleukin 1 (IL-1) and interleukin 6 (IL-6)4, transforming growth factor b (TGF b)8, insulin like growth factor (IGF)13, fibroblast growth factor (FGF)9, platelet-derived growth factor (PDGF)2 and the bone morphogenetic proteins (BMPs).12 Essentially, the actions of the growth factors can be summarised as follows7: TGFb, released from platelets, bone and cartilage extracellular matrix, is a pleiotropic growth factor responsible for stimulation of undifferentiated MSCs. BMPs, released from osteoprogenitor cells, osteoblasts and bone extracellular matrix, promote the differentiation of MSCs into chondrocytes and
Although there are limitations in extrapolation of animal data to human practice, it is useful to understand the process in an animal model. The phases proceed at about twice the speed of a human fracture. In the first hours after fracture, haematoma forms and platelets degranulate, releasing mostly TGFb and PDGF.1 Inflammatory cells release IL-1 and IL-6.4 The cellular and molecular events occurring during fracture healing in rodents have been described in detail by Einhorn.5 Within 24 h, there is the first evidence of mesenchymal stem cells expressing BMPs. There are probably more than 40 BMPs, and some of them have been shown to induce angiogenesis, chemotaxis, mitogenesis, cell differentiation and proliferation. Endothelial cells in the marrow transform to polymorphic cells and begin to express an osteoblastic phenotype and form bone. Osteoprogenitor cells (OPCs) are already present in area under periosteum, ready to begin intramembranous ossification. Primitive MSCs express BMPs on the first day. BMPs begin the differentiation of OPCs into osteocytes. It seems that BMPs 2, 6 and 9 are important in the differentiation of pluripotent MSCs into OPCs, and then BMPs 2, 4, 7 and 9 further differentiate them to become osteoblasts. Most of the BMPs are then able to differentiate osteoblasts into osteocytes, excluding BMPs 3 and 12. From day 2 to 5, in the subperiosteal area, hard callus begins to form. Intramembranous ossification begins, promoted by motion and inhibited by rigid internal fixation. At this time, in this area, there is an increased proliferation of OPCs and undifferentiated MSCs. The adhesion molecule osteonectin is evident between days 4 and 7. In the area between the bone ends, in the soft callus, early cartilage formation begins. Undifferentiated MSCs begin to proliferate by day 3. At day 5 there is evidence of increased expression of mRNA for type II collagen, from cells that later acquire a chondrocytic phenotype.
Overview of the fracture healing cascade
From day 6 to 10, osteocalcin is expressed in the hard callus. In both the soft and the hard callus, there is high cellular proliferation. Osteopontin is present in both the osteocytes and the OPCs at the junction between the hard and soft callus. In the soft callus chondrocytes begin to proliferate. Type II collagen mRNA expression peaks at around day 9. Other minor collagens appear. Adhesion and migration molecules, especially fibronectin, are found in fibroblasts, chondrocytes and ostoeblasts. In days 11—20, in the areas of hard callus formation, cellular proliferation ceases. The predominant collage type is type II, but expression of mRNA for collagen type II is absent. Fibronectin levels decline at day 14. In the soft callus, the cartilage begins to calcify. There is ‘budding’ of hypertrophic chondrocytes. The matrix vesicles release calcium and enzymes. The soft callus takes on the structure of a growth plate, with primary and secondary spongiosa. From days 21 to 25, there is no more cellular proliferation. In the hard callus area, the structure is now woven bone. In the soft callus, chondrocytes begin to undergo apoptosis and there is some cellular necrosis. Solid union with woven bone is present by around 35 days. Remodelling and formation of lamellar bone follows.
Orchestration of fracture healing It is now clear that BMPs play a highly significant role in the control of fracture healing. The expression of BMPs is variable throughout the phases of fracture healing.3 BMPs 2 and 4 are expressed early by primitive MSCs, and seem to be expressed throughout the fracture healing process. BMP 7 is expressed later, by osteogenic cells, from day 7, and peaking at 2—4 weeks. BMPs 2, 4, 6, 7 and 9 increase osteocalcin expression and alkaline phosphatase expression in pre-osteoblastic cells, leading to mineralisation. BMPs bind to cells via receptors, of which there seems to be two main types. Once bound, they cause phosphorylation of intracellular messengers called Smads.7 There are several different Smads. Once phosphorylated, an intracellular cascade leads to expression of BMP dependent genes within the nucleus of any cell that is BMP sensitive. The cell, depending on the phenotype and degree of differentiation, may then differentiate, express proteins or cause other growth factors to be upregulated. Angiogenesis may be induced and mitogenesis may be stimulated. Extracellular BMPs can cause chemotaxis of cells via the concentration gradient. It is not known
S7
precisely what local concentrations, at the cellular level, of BMPs are required to produce these effects. There are several inhibitory proteins that serve to provide negative feedback loops to control BMP activity, e.g. noggin.6 It may be that antibodies to these could increase the effects of BMPs and could be used therapeutically. When inhibitory proteins are combined with other inhibitory effects on the action of BMPs, such as receptor down-regulation, and antibodies to BMPs, it is clear that simply adding more BMPs to the fracture environment may not have enhanced beneficial effects in terms of bone healing. We now know from many animal and some clinical trials that exogenous BMPs can work to our advantage. The challenge now is to find out how they work, how to manipulate them for best effect and to make sure that they are not harmful in the clinical setting.
References 1. Bolander ME. Regulation of fracture repair by growth factors. Proc Soc Exp Biol Med 1992;200:165—70. 2. Canalis E, McCarthy TL, Centrella M. Effects of plateletderived growth factor on bone formation in vitro. J Cell Physiol 1989;140:530—7. 3. Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res 2002;17(3):513—20. 4. Einhorn TA, Majeska RJ, Rush EB, et al. The expression of cytokine activity by fracture callus. J Bone Miner Res 1995;10:1272—81. 5. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orth Relat Res Suppl 1998;355S:S7—S21. 6. Hannallah D, Peng H, Young B, et al. Retroviral delivery of noggin inhibits the formation of heterotopic ossification induced by BMP4, demineralised bone matrix, and trauma in an animal model. J Bone Joint Surg 2004;86A:80—91. 7. Lieberman JR, Daluiski A, Einhorn TA. The role of growth factors in the repair of bone. J Bone Joint Surg 2002;84A: 1032—44. 8. Lind M, Schumacher B, Soballe K, et al. Transforming growth factor beta enhances fracture healing in rabbit tibiae. Acta Orthop Scand 1993;64:553—6. 9. Lind M. Growth factor stimulation of bone healing. Effects on osteoblasts, osteotomies, and implant fixation. Acta Orthop Scand Suppl 1998;283:2—37. 10. McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg 1978;60B:150—62. 11. Schmitt JM, Hwang K, Winn SR, Hollinger JO. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999;17:269—78. 12. Simon AM, Manigrasso MB, O’Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing. J Bone Miner Res 2002;17(6):963—76. 13. Trippel SB. Potential role of insulin like growth factors in fracture healing. Clin Orthop Relat Res Suppl 1998;355S: S301—13.
www.elsevier.com/locate/injury
Overview of the fracture healing cascade A.M. Phillips Consultant Trauma & Orthopaedics, Kings Hospital London, Denmark Hill, London SE5 9RS, UK
KEYWORDS Bone healing; Callus formation; Growth factors; Mesenchymal stem cells
Summary Fracture healing is a dynamic process being governed by a variety of cellular elements and stimulating agents. Our knowledge in understanding the molecular and cellular processes occurring in healing fractures has vastly expanded as a result of the advances made in all aspects of medicine. The ability to manipulate mesenchymal stem cells and to deliver locally growth factors in order to create new bone has led to a proliferation of research papers. The purpose of this article is to provide a brief overview of the current level of understanding of the physiological processes regulating the healing of fractures. # 2005 Published by Elsevier Ltd.
Introduction A detailed understanding of the molecular and cellular processes occurring in healing fractures is essential for manipulation of the process for patient benefit. Significant advances are currently being made in this field in the clinical and basic science arena. The ability to manipulate pluripotential mesenchymal stem cells (MSCs) to create bone by stimulating them with exogenous signalling molecules, or encouraging cells to express proteins favourable for other cells to create bone by genetic modification, has led to a proliferation of research papers on this subject.11 The purpose of this manuscript is to provide a brief overview of the current level of understanding of this process as is relevant to an orthopaedic surgeon with an interest in promoting fracture healing as quickly as possible, reducing the need for intervention, and reducing the incidence of non-union.
E-mail address: [email protected]. 0020–1383/$ — see front matter # 2005 Published by Elsevier Ltd. doi:10.1016/j.injury.2005.07.027
Biological considerations of fracture healing Primary and secondary fracture healing Much has been made of the concept of ‘primary fracture healing’. The concept is that if the fracture is anatomically reduced, at the micrometric level, ‘osteonal’ healing occurs. Osteoclasts create ‘cutting cones’ and primarily cross the fracture site. This requires very high stability, and in practice is the rarest type. More commonly, ‘secondary’ fracture healing occurs and a larger a mass of callus is created. This type of healing benefits from micromotion.10 The description of fracture healing in this presentation refers to this latter, ‘secondary’ fracture healing.
The contribution of the different spatial areas in a healing fracture Fracture healing is not homogeneous throughout the callus. The four main zones are the medullary canal,
S6
the area between the cortices, the subperiosteal layer and the surrounding soft tissues. Essentially, the medullary canal and inter-cortical areas create ‘soft callus’ and go on to create bone by enchondral ossification. The subperiosteal area and the soft tissues immediately surrounding the fracture form ‘hard callus’ and create bone by intramembranous ossification. These are discussed in more detail in the section on healing phases in rodent models.
A.M. Phillips
osteoblasts, and osteoprogenitor cells into osteoblasts. FGF, released from macrophages, MSCs, chondrocytes and osteoblasts, is mitogenic for MSCs, chondrocytes and osteoblasts. IGF, released from bone matrix, osteoblasts and chondrocytes, promotes proliferation and differentiation of osteoprogenitor cells. PDGF, released from platelets and osteoblasts, is mitogenic for MSCs and osteoblasts and responsible for macrophage chemotaxis.
Five temporal stages of fracture healing Healing phases in the rat model In addition to the four ‘spatial’ divisions of fracture healing, there are five ‘temporal’ phases. In chronological order, these are: haematoma formation, inflammation, angiogenesis, cartilage formation (with subsequent calcification, cartilage removal and then bone formation) and bone remodelling.
Three simultaneous processes There are three discrete simultaneous process involved, which are all interdependent including cellular processes leading directly to production of calcified bone, the process of angiogenesis and formation of the matrix. Any intervention that affects any or all of these processes will affect the quality of bone formation.
The role of the inflammatory cascade Without an inflammatory process, bone will not heal. Inhibition of the cyclo-oxygenase pathway appears to inhibit bone healing. Newer, more selective non-steroidal ani-inflammatory drugs promised fewer side effects. However, even blocking the cyclo-oxygenase 2 pathway (COX-2) appears to significantly halt bone formation.12 The major signalling molecules involved in, and controlling the cascade are: interleukin 1 (IL-1) and interleukin 6 (IL-6)4, transforming growth factor b (TGF b)8, insulin like growth factor (IGF)13, fibroblast growth factor (FGF)9, platelet-derived growth factor (PDGF)2 and the bone morphogenetic proteins (BMPs).12 Essentially, the actions of the growth factors can be summarised as follows7: TGFb, released from platelets, bone and cartilage extracellular matrix, is a pleiotropic growth factor responsible for stimulation of undifferentiated MSCs. BMPs, released from osteoprogenitor cells, osteoblasts and bone extracellular matrix, promote the differentiation of MSCs into chondrocytes and
Although there are limitations in extrapolation of animal data to human practice, it is useful to understand the process in an animal model. The phases proceed at about twice the speed of a human fracture. In the first hours after fracture, haematoma forms and platelets degranulate, releasing mostly TGFb and PDGF.1 Inflammatory cells release IL-1 and IL-6.4 The cellular and molecular events occurring during fracture healing in rodents have been described in detail by Einhorn.5 Within 24 h, there is the first evidence of mesenchymal stem cells expressing BMPs. There are probably more than 40 BMPs, and some of them have been shown to induce angiogenesis, chemotaxis, mitogenesis, cell differentiation and proliferation. Endothelial cells in the marrow transform to polymorphic cells and begin to express an osteoblastic phenotype and form bone. Osteoprogenitor cells (OPCs) are already present in area under periosteum, ready to begin intramembranous ossification. Primitive MSCs express BMPs on the first day. BMPs begin the differentiation of OPCs into osteocytes. It seems that BMPs 2, 6 and 9 are important in the differentiation of pluripotent MSCs into OPCs, and then BMPs 2, 4, 7 and 9 further differentiate them to become osteoblasts. Most of the BMPs are then able to differentiate osteoblasts into osteocytes, excluding BMPs 3 and 12. From day 2 to 5, in the subperiosteal area, hard callus begins to form. Intramembranous ossification begins, promoted by motion and inhibited by rigid internal fixation. At this time, in this area, there is an increased proliferation of OPCs and undifferentiated MSCs. The adhesion molecule osteonectin is evident between days 4 and 7. In the area between the bone ends, in the soft callus, early cartilage formation begins. Undifferentiated MSCs begin to proliferate by day 3. At day 5 there is evidence of increased expression of mRNA for type II collagen, from cells that later acquire a chondrocytic phenotype.
Overview of the fracture healing cascade
From day 6 to 10, osteocalcin is expressed in the hard callus. In both the soft and the hard callus, there is high cellular proliferation. Osteopontin is present in both the osteocytes and the OPCs at the junction between the hard and soft callus. In the soft callus chondrocytes begin to proliferate. Type II collagen mRNA expression peaks at around day 9. Other minor collagens appear. Adhesion and migration molecules, especially fibronectin, are found in fibroblasts, chondrocytes and ostoeblasts. In days 11—20, in the areas of hard callus formation, cellular proliferation ceases. The predominant collage type is type II, but expression of mRNA for collagen type II is absent. Fibronectin levels decline at day 14. In the soft callus, the cartilage begins to calcify. There is ‘budding’ of hypertrophic chondrocytes. The matrix vesicles release calcium and enzymes. The soft callus takes on the structure of a growth plate, with primary and secondary spongiosa. From days 21 to 25, there is no more cellular proliferation. In the hard callus area, the structure is now woven bone. In the soft callus, chondrocytes begin to undergo apoptosis and there is some cellular necrosis. Solid union with woven bone is present by around 35 days. Remodelling and formation of lamellar bone follows.
Orchestration of fracture healing It is now clear that BMPs play a highly significant role in the control of fracture healing. The expression of BMPs is variable throughout the phases of fracture healing.3 BMPs 2 and 4 are expressed early by primitive MSCs, and seem to be expressed throughout the fracture healing process. BMP 7 is expressed later, by osteogenic cells, from day 7, and peaking at 2—4 weeks. BMPs 2, 4, 6, 7 and 9 increase osteocalcin expression and alkaline phosphatase expression in pre-osteoblastic cells, leading to mineralisation. BMPs bind to cells via receptors, of which there seems to be two main types. Once bound, they cause phosphorylation of intracellular messengers called Smads.7 There are several different Smads. Once phosphorylated, an intracellular cascade leads to expression of BMP dependent genes within the nucleus of any cell that is BMP sensitive. The cell, depending on the phenotype and degree of differentiation, may then differentiate, express proteins or cause other growth factors to be upregulated. Angiogenesis may be induced and mitogenesis may be stimulated. Extracellular BMPs can cause chemotaxis of cells via the concentration gradient. It is not known
S7
precisely what local concentrations, at the cellular level, of BMPs are required to produce these effects. There are several inhibitory proteins that serve to provide negative feedback loops to control BMP activity, e.g. noggin.6 It may be that antibodies to these could increase the effects of BMPs and could be used therapeutically. When inhibitory proteins are combined with other inhibitory effects on the action of BMPs, such as receptor down-regulation, and antibodies to BMPs, it is clear that simply adding more BMPs to the fracture environment may not have enhanced beneficial effects in terms of bone healing. We now know from many animal and some clinical trials that exogenous BMPs can work to our advantage. The challenge now is to find out how they work, how to manipulate them for best effect and to make sure that they are not harmful in the clinical setting.
References 1. Bolander ME. Regulation of fracture repair by growth factors. Proc Soc Exp Biol Med 1992;200:165—70. 2. Canalis E, McCarthy TL, Centrella M. Effects of plateletderived growth factor on bone formation in vitro. J Cell Physiol 1989;140:530—7. 3. Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res 2002;17(3):513—20. 4. Einhorn TA, Majeska RJ, Rush EB, et al. The expression of cytokine activity by fracture callus. J Bone Miner Res 1995;10:1272—81. 5. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orth Relat Res Suppl 1998;355S:S7—S21. 6. Hannallah D, Peng H, Young B, et al. Retroviral delivery of noggin inhibits the formation of heterotopic ossification induced by BMP4, demineralised bone matrix, and trauma in an animal model. J Bone Joint Surg 2004;86A:80—91. 7. Lieberman JR, Daluiski A, Einhorn TA. The role of growth factors in the repair of bone. J Bone Joint Surg 2002;84A: 1032—44. 8. Lind M, Schumacher B, Soballe K, et al. Transforming growth factor beta enhances fracture healing in rabbit tibiae. Acta Orthop Scand 1993;64:553—6. 9. Lind M. Growth factor stimulation of bone healing. Effects on osteoblasts, osteotomies, and implant fixation. Acta Orthop Scand Suppl 1998;283:2—37. 10. McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg 1978;60B:150—62. 11. Schmitt JM, Hwang K, Winn SR, Hollinger JO. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999;17:269—78. 12. Simon AM, Manigrasso MB, O’Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing. J Bone Miner Res 2002;17(6):963—76. 13. Trippel SB. Potential role of insulin like growth factors in fracture healing. Clin Orthop Relat Res Suppl 1998;355S: S301—13.