Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor
ABSTRACT Chondrogenesis is an essential component of endochondral fracture healing, though the molecular and cellular events by which it is regulated have not been fully elucidated. In this study, we used a rat model of closed fracture healing to determine the spatial and temporal expression of genes for cartilage-specific collagens. Furthermore, to determine the effects of basic fibroblast growth factor (bFGF) on chondrogenesis in fracture healing, we injected 100 μg recombinant human bFGF into the fracture site immediately after fracture.In normal calluses, pro-(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-(X) collagen mRNA (COL10A1) in hypertrophic chondrocytes beginning on day 7. In FGF-injected calluses, the cartilage enlarged in size significantly. On day 14, both COL2A1-and COL10A1-expressing cells were more widely distributed, and the amounts of COL2A1 and COL10A1 mRNAs were both approximately 2-fold increased when compared with uninjected fractures. Temporal patterns of expression for these genes were, however, identical to those found in normal calluses. The number of proliferating cell nuclear antigen-positive cells was increased in the non-cartilaginous area in the bFGF-injected calluses by day 4.The present molecular analyses demonstrate that a single injection of bFGF enhances the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. However, maturation of chondrocytes and replacement of the cartilage by osseous tissue are not enhanced by exogenous bFGF, and this results in the prolonged cartilaginous callus phase. We conclude that, in the healing of closed fractures of long bones, exogenous bFGF has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.
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ABSTRACT: To test whether mechanical loading produces faster healing in aged mice, fractured femurs of aged 1-year-old mice were subjected to low-intensity pulsed ultrasound (LIPUS), a treatment that is routinely used to help heal fractures in humans. Cyclooxygenase-2 knockout mice (COX-2(-/-)), which lack an immediate early mediator of mechanical stimulation, were also studied by histochemistry, microcomputed tomography and quantitative polymerase chain reaction to determine the role of COX-2. The healing in the aged COX-2(-/-) mice is slow during the endochondral bone remodeling (>30 d), a period generally prolonged in senescence. For aged wild-type mice, LIPUS halved the endochondral phase to about 10 d, whereas that was not the case for aged COX-2(-/-) mice, which showed no apparent shortening of the prolonged endochondral-phase healing time. Injecting prostaglandin E(2) receptor agonists, however, rescued the COX-2(-/-) callus from insensitivity to LIPUS. In conclusion, COX-2 is a limiting factor in the delayed endochondral bone healing and is induced by LIPUS, which normalizes healing rate to the wild-type level.Ultrasound in medicine & biology 07/2010; 36(7):1098-108. · 2.46 Impact Factor
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ABSTRACT: GFP reporter mice previously developed to assess levels of osteoblast differentiation were employed in a tibial long bone fracture model using a histological method that preserves fluorescent signals in non-decalcified sections of bone. Two reporters, based on Col1A1 (Col3.6GFPcyan) and osteocalcin (OcGFPtpz) promoter fragments, were bred into the same mice to reflect an early and late stage of osteoblast differentiation. Three observations were apparent from this examination. First, the osteoprogenitor cells that arise from the flanking periosteum proliferate and progress to fill the fracture zone. These cells differentiate to osteoblasts, chondrocytes, to from the outer cortical shell. Second, the hypertrophic chondrocytes are dispersed and the cartilage matrix mineralized by the advancing Col3.6+ osteoblasts. The endochondral matrix is removed by the following osteoclasts. Third, a new cortical shell develops over the cartilage core and undergoes a remodeling process of bone formation on the inner surface and resorption on the outer surface. The original fractured cortex undergoes resorption as the outer cortical shell remodels inward to become the new diaphyseal bone. The fluorescent microscopy and GFP reporter mice used in this study provide a powerful tool for appreciating the molecular and cellular processes that control these fundamental steps in fracture repair, and may provide a basis for understanding fracture nonunion.Journal of Orthopaedic Research 10/2010; 28(10):1338-47. · 2.88 Impact Factor
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ABSTRACT: We developed a fatigue loading protocol in mice to produce a non-displaced ulnar fracture in vivo, and characterized the early healing response. Using adult (5 month) C57Bl/6 mice, we first determined that cyclic compression of the forelimb under load-control leads to increasing applied displacement and, eventually, complete fracture. We then subjected the right forelimbs of 80 mice to cyclic loading (2 Hz; peak force approximately 4N) and limited the displacement increase to 0.75 mm (60% of the average displacement increase at complete fracture). This fatigue protocol created a partial, non-displaced fracture through the medial cortex near the ulnar mid-shaft, and reduced ulnar strength and stiffness by >50%. Within 1 day, there was significant upregulation of genes related to hypoxia (Hif1a) and osteogenesis (Bmp2, Bsp) in loaded ulnae compared to non-loaded, contralateral controls. The gene expression response peaked in magnitude near day 7 (e.g., Osx upregulated 8-fold), and included upregulation of FGF-family genes (e.g., Fgfr3 up 6-fold). Histologically, a localized periosteal response was seen at the site of the fracture; by day 7 there was abundant periosteal woven bone surrounding a region of cartilage. From days 7 to 14, the woven bone became denser but did not increase in area. By day 14, the woven-bone response resulted in complete recovery of ulnar strength and stiffness, restoring mechanical properties to normal levels. In the future, the fatigue loading approach can be used create non-displaced bone fractures in transgenic and knockout mice to study the mechanisms by which the skeleton rapidly repairs damage.Bone 03/2010; 46(6):1604-12. · 3.82 Impact Factor