RESUMO
Distraction osteogenesis (DO) is one of the most dramatic in vivo applications of mechanical stimulation as a means of inducing bone regeneration. A simple and reproducible murine model of tibia distraction osteogenesis was developed using a monolateral fixator. Bone formation was assessed histologically over a 35-day time course. The steady state expression of a broad family of angiogenesis-associated genes was assessed by microarray hybridization analyses over the same time course, while the immediate gene response that was induced during each cycle of distraction was assessed at 30 min and 8 h after the first and last rounds of activation of the fixator. Distraction osteogenesis promoted new bone formation primarily through an intramembranous process with maximal osteogenesis during the active distraction period. Histological analysis also showed that dense cortical bone continued to be formed, during the consolidation phase, for 2 weeks after distraction ended. The analysis of steady state mRNA expression levels over the time course of DO showed that VEGF-A and neuropilin, an alternate receptor for VEGF-A, both angiopoietin (Ang) 1 and 2 factors, and the Ang receptor Tie2 were the critical angiogenic factors during DO. A key transcriptional regulator of many of the angiogenic factors, hypoxia-induced factor1alpha (Hif-1a), the FGF binding protein pleiotropin/OSF1, and multiple MMP(s), were also induced during the active distraction period. Examination of the expression of angiogenic factors that were induced after each cycle of activation, demonstrated that Hif-1a, Nrp1, and VEGF-A were all cyclically induced after each increment of distraction. These results suggest that these factors are early mediators that are produced by distraction and contribute toward the processes that promote bone formation. These experiments represent the first step in defining the molecular mechanisms that regulate skeletal regeneration and the functional relationship between angiogenesis and osteogenesis during distraction osteogenesis.
Assuntos
Neovascularização Fisiológica , Osteogênese por Distração , Animais , Masculino , Metaloproteases/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Distraction osteogenesis is a unique and effective way to treat limb length inequality resulting from congenital and posttraumatic skeletal defects. However, despite its widespread clinical use, the cellular and molecular mechanisms by which this surgical treatment promotes new bone formation are not well understood. Previous studies in distraction osteogenesis have noted increased blood flow and vessel formation within the zone of distraction. These observations suggest that distraction osteogenesis may be driven in part by an angiogenic process. Using immunohistological analysis, the expression of two different angiogenic factors (VEGF and bFGF) was shown to localize at the leading edge of the distraction gap, where nascent osteogenesis was occurring. These cells were spatially adjacent to new vessels that were identified by staining for factor VIII. Microarray analysis detected maximal mRNA expression for a wide variety of angiogenic factors including angiopoietin 1 and 2, both Tie receptors, VEGF-A and -D, VEGFR2, and neuropilin 1. Expression of these factors was found to be maximal during the phase of active distraction. Expression of mRNA for extracellular matrix proteins and BMPs was also maximal during this period. A comparison between the patterns of gene expression in fracture healing and distraction osteogenesis revealed similarities; however, the expression of a number of genes showed selective expression in these two types of bone healing. These data suggest that bone formation during distraction osteogenesis is accompanied by the robust induction of factors associated with angiogenesis and support further investigations to elucidate the mechanisms by which angiogenic events promote bone repair and regeneration.
Assuntos
Indutores da Angiogênese/metabolismo , Perfilação da Expressão Gênica , Osteogênese por Distração , Angiopoietinas/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte/genética , Colágeno/genética , Citocinas/genética , Fêmur/metabolismo , Fêmur/patologia , Fêmur/cirurgia , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/genética , Consolidação da Fratura/genética , Consolidação da Fratura/fisiologia , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Masculino , Neovascularização Fisiológica/genética , Neuropilinas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Osteocalcina/genética , Osteogênese/genética , Osteopontina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de TIE/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Sialoglicoproteínas/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta2 , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética , Fatores de Crescimento do Endotélio Vascular/genética , Fator de von Willebrand/análiseRESUMO
Distraction osteogenesis has proven to be of great value for the treatment of a variety of musculoskeletal problems. Little is still known, however, about the phenotypic changes in the cells participating in the bone formation process, induced by the procedure. Recent findings of the expression of a contractile muscle actin isoform, alpha-smooth muscle actin (SMA), in musculoskeletal connective tissue cells prompted this immunohistochemical study of the expression of SMA in cells participating in distraction osteogenesis in a rat model. The tissues within and adjacent to the distraction site could be distinguished histologically on the basis of cell morphology, density, and extracellular matrix make-up. The percentage of SMA-containing cells within each tissue zone was graded from 0 to 4. The majority of the cells in each of the zones stained positive for SMA within five days of the distraction period. The SMA-containing cells included those with elongated morphology in the center of the distraction site and the active osteoblasts on the surfaces of the newly forming bone. These finding warrant further investigation of the role of this contractile actin isoform in distraction osteogenesis and investigation of the effects of modulation of this actin isoform on the procedure.
Assuntos
Actinas/análise , Fêmur/cirurgia , Fibras Musculares Esqueléticas/química , Osteoblastos/química , Osteogênese por Distração , Animais , Imuno-Histoquímica , Masculino , Modelos Animais , Fibras Musculares Esqueléticas/fisiologia , Osteoblastos/fisiologia , Osteotomia , Ratos , Ratos Sprague-DawleyRESUMO
The control of postoperative pain in the pediatric orthopaedic patient is a challenging endeavor. Several studies have shown the efficacy of ketorolac tromethamine in the pediatric general surgical population, but its efficacy in the pediatric orthopaedic population remains unproven. Twenty-seven consecutive patients (age 6 months to 18 years) who underwent long-bone osteotomies or foot procedures by a group of three pediatric orthopaedic surgeons were given a ketorolac protocol (1 mg/kg loading, 0.5 mg/kg every 6 h for 24 h). Breakthrough pain was managed with morphine until the patient was able to take oral pain medication, as was any pain after the 24-h period for ketorolac expired. Thirty-seven age- and case-matched patients were used as retrospective controls. The patients in the study who received ketorolac required significantly fewer doses of morphine than did the control group (2.29 +/- 3.98 vs. 10.02 +/- 3.39; p < 0.05). In addition the patients on the ketorolac protocol experienced fewer gastrointestinal side effects (4% vs. 32%; p < 0.05). Finally, the patients in the ketorolac group had a significantly shorter length of stay (3.63 +/- 1.64 days vs. 4.74 +/- 1.76 days; p < 0.05). There were no bleeding complications in either group. Ketorolac is thus a safe and effective means of controlling postoperative pain in the pediatric orthopaedic population while avoiding the troubling maleffects seen with the exclusive use of morphine.
