A delay in consolidation is observed in a heterozygous conditional BMP2 deficient mouse model of distraction osteogenesis

Distraction osteogenesis (DO) is a surgical technique used to treat limb length discrepancies, limb deformities, long bone non-unions and bone loss due to trauma, infection or malignancies. In this surgical method a transverse osteotomy is performed and the fractured bone is stabilized using the Ilizarov fixation system. After a short latency period, the two ends of the fractured bone are slowly pulled apart, stimulating new bone formation within the distracted gap. After the distraction is completed, the newly formed bone is allowed to fully consolidate. One of the main limitations of DO is the long consolidation period required for the bone to heal. Different methods have been researched to accelerate the consolidation phase of DO, including the exogenous application of bone morphogenetic proteins (BMPs). BMPs are growth factors that are required in the bone developmental pathway. Although numerous studies have tested pharmacological doses of BMP2 and BMP7 using different animal models of DO, the physiological role of BMPs during DO still remains poorly understood. Hence, in this study we investigated the physiological role of BMP2 in a heterozygous conditional BMP2 knockout model of distraction osteogenesis. Distraction osteogenesis was performed on the right tibia of forty wild-type BMP2 fl/+ mice and heterozygous BMP2 fl/+ cre mice using a miniature version of the Ilizarov fixator. Mice underwent a latency period of 5 days, a distraction period of 12 days (distraction rate of 0.2 mm every 12 hours) and a consolidation period of 34 days. Distracted samples were collected from four time points: 11 days (mid-distraction phase), 17 days (end of distraction phase), 34 days (mid-consolidation phase) and 51 days (end of consolidation phase). Samples were studied using µCT, Faxitron x-ray, immunohistochemistry, histology, Real Time-quantitative PCR and biomechanical testing. Results from this study showed that reducing BMP2 expression through gene dL’ostéogenèse par distraction (OD) est une technique chirurgicale utilisée pour traiter des dissymétries des membres et des défauts osseux suite à un traumatisme, une infection ou une maladie. Dans cette méthode, une ostéotomie est faite et l'os fracturé est stabilisé par un fixateur externe de type Ilizarov. Durant la période de distraction, les extrémités de l'os sont tirées lentement l'une de l'autre, ce qui stimule la nouvelle formation d'os dans la zone de distraction. Ensuite, la phase de consolidation permet l’ossification de tissu nouvellement formé.Le problème principal de l’OD est que la période de consolidation est très longue. De recherches ont été faites afin d’accélérer la phase de consolidation de l’OD tel que l'application des protéines de la morphogenèse osseuse (BMP). Les BMP sont nécessaires pour le développement osseux. Bien que beaucoup d’études ont analysé les effets de doses pharmacologiques de BMP2 et BMP7 en utilisant des modèles animaux de l’OD, le rôle physiologique des BMP est encore inconnu. Donc, pour cette étude nous avons analysé le rôle physiologique de BMP2 durant l’OD chez des souris ayant une déficience en BMP2 localisée dans les membres. Nous avons soumis le tibia droit de souris contrôle-BMP2 fl/+ et de souris hétérozygotes-BMP2 fl/+ cre à l’ostéogenèse par distraction en utilisant un fixateur Ilizarov miniaturisé. Les souris ont subi une période de latence de 5 jours, une période de distraction de 12 jours (avec un taux de distraction de 0.2 mm chaque 12 heures), et une période de consolidation de 34 jours. Les échantillons ont été recueillis à 11, 17, 34 et 51 jours, et analysés par µCT, rayons X, immunohistochimie, histologie, par PCR en temps réel, et par un examen biomécanique.Les résultats de cette étude ont montré que la diminution de l’expression de BMP2 dans les membres hétérozygotes avait pour effet de ralentir le processus de co

Biomechanical testing results.

<p>Biomechanical testing parameters to compare HS-injected bones and saline-injected (control) bones at 34 days and 51 days post-osteotomy. For statistical analysis, a two-tailed un-paired t test was performed between the HS-injected group and controls, in which * indicates p<0.05.</p

Summarized results for Immunohistochemistry analysis.

<p>Immunohistochemistry results of dissected tibiae at 34 days and 51 days post-osteotomy.</p

Microcomputed tomography (μCT) results.

<p>μCT histomorphometric analysis of distracted bone injected with 5 µg HS or saline (Control), at 34 and 51 days post-osteotomy. For statistical analysis, a two-tailed un-paired t test was performed between the HS-injected group and controls, in which * indicates p<0.05.</p

Classification of Bone Fill Scores.

<p>Classification of Bone Fill Scores.</p

Frequency of post-operative complications.

<p>The frequency of infection and early euthanasia was increased in the HS-injected group compared to controls. For statistical analysis, a two-tailed un-paired t test was performed between the HS-injected group and controls, in which * indicates p<0.05.</p

Average bone fill scores.

<p>Data is represented as a mean of Bone-fill scores, as blindly graded by radiological assessment.</p

Faxitron, microcomputed tomography (μCT) and histology images.

<p>Analysis and radiological images of Faxitron X-ray (top row) and μCT projection (middle-row) of distracted mouse tibiae collected at 34 and 51days post-osteotomy. The third and bottom rows show histology images of distracted tibiae after Goldner-Trichrome staining and reveal regions of the distracted zone that are occupied with soft, connective tissue (red-stained) vs. calcified tissue and/or bone matrix (green-stained). Images were taken at the center of the callus area at 100× magnification (scale bar represents 150 µM).</p

Histochemistry images of distracted mouse tibiae.

<p>Mouse tibiae immunostained for members of the BMP signaling pathway (BMP-2, BMPR1a, BMP-3) at 34 and 51 days. Representative images taken at 400× magnification, scale bar represents 50 µM. Chondrocytes and fibroblastic cells are indicated by the white arrows and letters “C” and “F”, respectively.</p