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: Safe, effective methods for bone and cartilage regeneration are needed to reverse bone loss caused by trauma, disease, tumor resection and osteoarthritis. Unfortunately, all current or emerging therapies have serious limitations. As will be developed in this chapter, gene therapy offers a promising approach for musculoskeletal regeneration because it can mimic the natural biological processes of bone development and fracture healing. This chapter will provide an overview of normal skeletal development and fracture repair, and describe how gene therapy in combination with tissue engineering can model critical aspects of these natural processes. Current gene therapy approaches for bone and cartilage regeneration will then be summarized, as well as recent work where combinatorial gene therapy is used to express groups of molecules that synergistically interact. Lastly, proposed new directions will be described that incorporate regulated gene expression and cells seeded in precise three-dimensional confi gurations on synthetic scaffolds to control both temporal and spatial distribution of regenerative factors. These and related approaches may eventually allow us to achieve the ultimate goal of bone tissue engineering: to reconstruct entire bones with associated joints, ligaments or sutures.
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ABSTRACT: Fibroblast growth factor-2 (FGF-2) is a well-characterized protein that is used in the treatment of healing-impaired wounds. We previously reported that fragmin/protamine microparticles (F/P MPs) are useful as biodegradable carriers for the controlled release of cytokines. We examined the ability of FGF-2-containing (FGF-2/) F/P MPs to prevent limb loss in an experimentally induced ischemic hindlimb model using adult Balb/c-nu/nu male mice. One day after inducing ischemia, intramuscular injections of 100 μL of FGF-2/F/P MPs turbid suspension (10 μg/mL FGF-2 and 6 mg/mL F/P MPs) were administered into eight sites of the ischemic hindlimb. A 100-μL suspension of each of the following-10 μg/mL FGF-2, 6 mg/mL F/P MPs, and phosphate-buffered saline (PBS; the control)-was similarly injected into the hindlimb. From 5 days onward after the injections, recovery from ischemia was observed in the FGF-2/F/P MP-treated group, but only partial recovery occurred in the FGF-2-treated group. The F/P MP-treated and PBS-treated groups (i.e., control) exhibited no recovery from the ischemia. The histological evaluations of the hindlimbs also confirmed that the capillary (i.e., mature vessels) density was significantly higher in the FGF-2/F/P MP-treated group than in the other groups. The mice injected with FGF-2/F/P MPs also recovered hindlimb blood flow, as reflected by oxygen saturation and surface temperature evaluation. Our present approach using FGF-2/F/P MPs could be considered a valuable option for the therapeutic treatment of peripheral ischemic diseases.Tissue Engineering Part A 06/2012; · 4.64 Impact Factor
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ABSTRACT: Angiogenesis and osteogenesis are critically linked, though the role of angiogenesis is not well understood in osteogenic mechanical loading. In this study, either damaging or non-damaging cyclic axial compression was used to generate woven bone formation (WBF) or lamellar bone formation (LBF), respectively, at the mid-diaphysis of the adult rat forelimb. αvβ3 integrin targeted nanoparticles or vehicle was injected intravenously following mechanical loading. β3 integrin subunit expression on vasculature was maximal 7 days after damaging mechanical loading, but was still robustly expressed 14 days after loading. Accordingly, targeted nanoparticle delivery in WBF loaded limbs was increased compared to non-loaded limbs. Vascularity was dramatically increased after WBF loading (+700% on day 14) and modestly increased after LBF loading (+50% on day 14). This increase in vascularity was inhibited by nanoparticle treatment in both WBF and LBF loaded limbs at days 7 and 14 after loading. Decreased vascularity led to diminished woven, but not lamellar, bone formation. Decreased woven bone formation resulted in impaired structural properties of the skeletal repair, particularly in post-yield behavior. These results demonstrate that αvβ3 integrin mediated angiogenesis is critical for recovering fracture resistance following bone injury, but is not required for bone modeling after modest mechanical strain. © 2014 American Society for Bone and Mineral ResearchJournal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 03/2014; · 6.04 Impact Factor