Fractures are the most common large-organ, traumatic injuries to humans. The repair of bone fractures is a postnatal regenerative process that recapitulates many of the ontological events of embryonic skeletal development. Although fracture repair usually restores the damaged skeletal organ to its pre-injury cellular composition, structure and biomechanical function, about 10% of fractures will not heal normally. This article reviews the developmental progression of fracture healing at the tissue, cellular and molecular levels. Innate and adaptive immune processes are discussed as a component of the injury response, as are environmental factors, such as the extent of injury to the bone and surrounding tissue, fixation and the contribution of vascular tissues. We also present strategies for fracture treatment that have been tested in animal models and in clinical trials or case series. The biophysical and biological basis of the molecular actions of various therapeutic approaches, including recombinant human bone morphogenetic proteins and parathyroid hormone therapy, are also discussed.

Fracture healing is a specialized post-natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing, these processes take place in a post-natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the embryonic differentiation and growth of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (1) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular differentiation during repair; (4) the role of three key groups of soluble factors, pro-inflammatory cytokines, the TGF-beta superfamily, and angiogenic factors, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture.

Skeletal development and turnover occur in close spatial and temporal association with angiogenesis. Osteoblasts are ideally situated in bone to sense oxygen tension and respond to hypoxia by activating the hypoxiainducible factor α (HIFα) pathway. Here we provide evidence that HIFα promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIFα in osteoblasts through selective deletion of the von Hippel-Lindau gene (Vhl) expressed high levels of Vegf and developed extremely dense, heavily vascularized long bones. By contrast, mice lacking Hif1a in osteoblasts had the reverse skeletal phenotype of that of the Vhl mutants: long bones were significantly thinner and less vascularized than those of controls. Loss of Vhl in osteoblasts increased endothelial sprouting from the embryonic metatarsals in vitro but had little effect on osteoblast function in the absence of blood vessels. Mice lacking both Vhl and Hif1a had a bone phenotype intermediate between those of the single mutants, suggesting overlapping functions of HIFs in bone. These studies suggest that activation of the HIFα pathway in developing bone increases bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone. IntroductionThe development of the mammalian skeleton takes place in distinct phases involving the initial migration of cells to the site of future bone, condensation of mesenchymal cells, and finally the differentiation of progenitors into chondrocytes and osteoblasts. During intramembranous bone formation, which gives rise to the flat bones of the skull, mesenchymal cells differentiate directly into bone-forming osteoblasts. By contrast, in endochondral bone formation, bones are formed through a 2-stage mechanism that begins with the formation of a chondrocyte anlage, onto which osteoblasts then differentiate and deposit bone. Endochondral bone formation occurs in close spatial and temporal association and proximity to capillary invasion, suggesting that angiogenesis and osteogenesis are coupled.The initial signals for blood vessel invasion into bone are unknown, but tissue hypoxia is believed to be critical for commencement of the angiogenic cascade (1). Hypoxia triggers the changes in oxygen-regulated gene expression via the activation of the Per/Arnt/Sim (PAS)

Adult bones have a notable regenerative capacity. Over 40 years ago, an intrinsic activity capable of initiating this reparative response was found to reside within bone itself, and the term bone morphogenetic protein (BMP) was coined to describe the molecules responsible for it. A family of BMP proteins was subsequently identified, but no individual BMP has been shown to be the initiator of the endogenous bone repair response. Here we demonstrate that BMP2 is a necessary component of the signaling cascade that governs fracture repair. Mice lacking the ability to produce BMP2 in their limb bones have spontaneous fractures that do not resolve with time. In fact, in bones lacking BMP2, the earliest steps of fracture healing seem to be blocked. Although other osteogenic stimuli are still present in the limb skeleton of BMP2-deficient mice, they cannot compensate for the absence of BMP2. Collectively, our results identify BMP2 as an endogenous mediator necessary for fracture repair.

Fracture healing is a unique postnatal repair process in which the events of endochondral and intramembranous bone formation follow a definable temporal sequence. The temporal patterns of messenger RNA (mRNA) expression for members of the transforming growth factor ␤ (TGF-␤) superfamily were examined over a 28-day period of fracture healing in mouse tibias. Bone morphogenetic protein 2 (BMP-2) and growth and differentiation factor 8 (GDF8) showed maximal expression on day 1 after fracture, suggesting their roles as early response genes in the cascade of healing events. Restricted expression of GDF8 to day 1, in light of its known actions as a negative regulator of skeletal muscle growth, suggests that it may similarly regulate cell differentiation early in the fracture healing process. GDF5, TGF-␤2, and TGF-␤3 showed maximal expression on day 7, when type II collagen expression peaked during cartilage formation. In contrast, BMP-3, BMP-4, BMP-7, and BMP-8 showed a restricted period of expression from day 14 through day 21, when the resorption of calcified cartilage and osteoblastic recruitment were most active. TGF-␤1, BMP-5 and BMP-6, and GDF10 were constitutively expressed from day 3 to day 21. However, during the same time period, GDF3, GDF6, and GDF9 could not be detected, and GDF1 was expressed at extremely low levels. These findings suggest that several members of the TGF-␤ superfamily are actively involved in fracture healing and although they are closely related both structurally and functionally, each has a distinct temporal expression pattern and potentially unique role in fracture healing. (J Bone Miner Res 2002;17:513-520)

The hypoxia-inducible factor-1alpha (HIF-1alpha) pathway is the central regulator of adaptive responses to low oxygen availability and is required for normal skeletal development. Here, we demonstrate that the HIF-1alpha pathway is activated during bone repair and can be manipulated genetically and pharmacologically to improve skeletal healing. Mice lacking pVHL in osteoblasts with constitutive HIF-1alpha activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1alpha in osteoblasts had impaired angiogenesis and bone healing. The increased vascularity and bone regeneration in the pVHL mutants were VEGF dependent and eliminated by concomitant administration of VEGF receptor antibodies. Small-molecule inhibitors of HIF prolyl hydroxylation stabilized HIF/VEGF production and increased angiogenesis in vitro. One of these molecules (DFO) administered in vivo into the distraction gap increased angiogenesis and markedly improved bone regeneration. These results identify the HIF-1alpha pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.

Fracture healing and distraction osteogenesis have important applications in orthopedic, maxillofacial, and periodontal treatment. In this review, the cellular and molecular mechanisms that regulate fracture repair are contrasted with bone regeneration that occurs during distraction osteogenesis. While both processes have many common features, unique differences are observed in the temporal appearance and expression of specific molecular factors that regulate each. 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. A complete summary of biological activities and functions of various bioactive factors may be found at COPE (Cytokines & Cells Online Pathfinder Encyclopedia), http://www.copewithcytokines.de/cope.cgi.

ABSTRACTwere analyzed over a 28-day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M-CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF-␣, lymphotoxin-␤ (LT-␤), IL-1␣, and IL-1␤ showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF-␣ receptors (p55 and p75) and the IL-1RII receptor showed identical patterns of expression to their ligands, while the IL-1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF-␣ and IL-1␣ expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF-␣ expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M-CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF-␣ and IL-1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.