Dominique Lauzier

Temporal and spatial expression of bone morphogenetic protein-2, -4, and -7 during distraction osteogenesis in rabbits

The Ilizarov method of limb lengthening makes use of the fact that osteogenesis is induced at an osteotomy site when distraction is applied. It is unknown at present how the mechanical forces created by distraction are translated into biological signals. Because bone morphogenetic proteins (BMPs) are potent inducers of osteogenesis in many experimental systems, they are obvious candidates for playing a role in this process. In this study, we investigated the temporal and spatial expression of BMP-2, -4, and -7 proteins during distraction osteogenesis using immunohistochemistry. An osteotomy was performed on the right tibiae of white New Zealand rabbits. After a delay of 7 days, distraction was started at a rate of 0.25 mm/12 h for 3 weeks, followed by a 3 week consolidation phase. Each week after osteotomy one rabbit was killed for immunohistochemical studies. Staining for BMP-2, -4, and -7 was evident before distraction was applied and was mainly localized to mesenchymal cells and osteoblastic cells in the periosteal region. After distraction was started, the typical fibrous interzone developed between the osteotomy fragments, where both intramembranous and endochondral ossification were noted. In this area, cells resembling fibroblasts and chondrocytes, but not mature osteoblasts, showed intense staining for all three BMPs. This high level of expression was maintained during the entire distraction phase and then gradually disappeared during the consolidation phase. These results are compatible with the hypothesis that BMPs play an important role in the signaling pathways that link the mechanical forces created by distraction to biological responses.

Effects of osteogenic protein-1 on distraction osteogenesis in rabbits

In this study we tested the effect of locally applied osteogenic protein 1 (OP-1) on distraction osteogenesis in rabbits. Seven days after tibial osteotomy, distraction was started at a rate of 0.25 mm per 12 h for 3 weeks. At the end of the distraction period, OP-1 was injected at the site of osteotomy. Four different dosages were tested (0, 80, 800, or 2000 microg; eight rabbits per dose group). Rabbits were sacrificed 3 weeks later, and histologic, densitometric, and biomechanical parameters were assessed. No significant differences were found between groups for any parameter. To explain why this approach was only modestly successful, the expression of BMP receptor protein in the newly formed tissue was analyzed by immunohistochemistry. Strong expression of BMP receptor IA, IB, and II was found during the early distraction phase, but not during later stages of the process. Thus, it appears that the lack of receptor protein in the target tissue impairs the effect of OP-1 given at the end of the distraction period. Possibly, OP-1 could be more useful when applied early in the distraction phase.

Effects of locally applied transforming growth factor-β1 on distraction osteogenesis in a rabbit limb-lengthening model

In this study we tested the effect of locally applied transforming growth factor-beta1 (TGF-beta1) on distraction osteogenesis in rabbits. A total of 61 rabbits were studied. Seven days after tibial osteotomy, distraction was started at a rate of 0.25 mm/12 h for 3 weeks. Starting with distraction, TGF-beta1 was applied in four different dosages (0, 10, 20, and 40 ng/day) at the site of osteotomy through a catheter connected to a subcutaneously implanted miniosmotic pump. Rabbits were killed at the end of the distraction period or 3 weeks later, and histological, densitometric, and biomechanical parameters were assessed. TGF-beta1 treatment had no detectable effect on bone mineral density or histologically determined bone volume in the distraction gap but it increased the amount of fibrous tissue in the callus region. Load to failure in uniaxial tension tended to be lower in TGF-beta1-treated animals. In conclusion, TGF-beta1 treatment during distraction osteogenesis did not have a beneficial effect in this model.

Immunohistochemical localization of bone morphogenetic protein-signaling Smads during long-bone distraction osteogenesis.

In this study we investigated the expression of bone morphogenetic protein (BMP)-signaling Smads in distraction osteogenesis (DO). Osteotomy of the right tibia was performed in 14 skeletally mature white New Zealand male rabbits. Lengthening was started 1 week later at a rate of 0.5 mm/12 hr and was maintained for 3 weeks. Expression of Smad proteins 1, 4, 5, 6, 7, and 8 and Smad ubiquitin regulatory factors (Smurfs) 1 and 2 was evaluated in the distracted zone using immunohistochemistry. Expression of receptor-regulated Smads (R-Smads) 1, 5, and 8 showed a significant increase during the distraction phase, followed by a gradual decrease during the consolidation phase. Smad 4 showed significant expression during both distraction and the beginning of the consolidation phase. Smad 6 and Smad 7 were highly expressed during the consolidation phase. Staining for both Smurfs 1 and 2 was maximal at the end of the distraction period. Staining for all proteins was most intense in chondrocyte and fibroblast-like cells. Expression pattern of R-Smads correlated with our previously reported expression pattern of BMPs 2, 4, and 7 and their receptors. These results therefore suggest a role for the whole BMP signaling pathway including the Smad proteins in DO.

Prospective heterotopic ossification progenitors in adult human skeletal muscle

Skeletal muscle has strong regenerative capabilities. However, failed regeneration can lead to complications where aberrant tissue forms as is the case with heterotopic ossification (HO), in which chondrocytes, osteoblasts and white and brown adipocytes can arise following severe trauma. In humans, the various HO cell types likely originate from multipotent mesenchymal stromal cells (MSCs) in skeletal muscle, which have not been identified in humans until now. In the present study, adherent cells from freshly digested skeletal muscle tissue were expanded in defined culture medium and were FACS-enriched for the CD73(+)CD105(+)CD90(-) population, which displayed robust multilineage potential. Clonal differentiation assays confirmed that all three lineages originated from a single multipotent progenitor. In addition to differentiating into typical HO lineages, human muscle resident MSCs (hmrMSCs) also differentiated into brown adipocytes expressing uncoupling protein 1 (UCP1). Characterizing this novel multipotent hmrMSC population with a brown adipocyte differentiation capacity has enhanced our understanding of the contribution of non-myogenic progenitor cells to regeneration and aberrant tissue formation in human skeletal muscle.

Overview of the radiology, histology, and bone morphogenetic protein expression during distraction osteogenesis of the mandible.

INTRODUCTION Distraction osteogenesis (DO) is a form of in vivo tissue engineering during which an osteotomy and controlled distraction are used to lengthen bone. The molecular signals that govern distraction-induced bone formation have not been fully elucidated. Specifically, the role of bone morphogenetic proteins (BMPs) in DO of the mandible remains unclear. OBJECTIVE To characterize the radiologic and histologic evolution of newly formed bone during DO of the mandible and to relate these changes to the expression of BMPs. METHODS Fourteen skeletally mature male rabbits were used. A distractor device was surgically applied to one side of the mandible following osteotomy. After 1 week (latency period), distraction was started at a rate of 0.25 mm every 12 hours for 3 weeks (distraction period) and was followed by a 3-week consolidation period. Two animals were sacrificed each week after surgery (weeks 1 to 7). The mandible was resected and the new bone assessed by radiography and histology. The expression of BMPs was also analyzed using immunohistochemistry. RESULTS There was radiographic and histologic evidence of bone formation during the distraction period. By week 6, there was mature woven bone within the distraction zone. Bone morphogenetic proteins 2 and 4 were strongly expressed in osteoblasts during distraction and in chondrocytes during consolidation. The expression of BMP-7 was relatively minor. CONCLUSION The temporal and spatial pattern of BMP expression suggests that these proteins are important mediators of mandibular DO. Understanding the expression of BMPs may facilitate the use of recombinant proteins to enhance the rate and quality of bone generation during craniofacial DO.

BMP-9 expression in human traumatic heterotopic ossification: a case report

BackgroundHeterotopic ossification (HO) is defined as the abnormal formation of mature bone in soft tissue, notably skeletal muscle. The morbidity of HO in polytraumatized patients impacts the functional outcome, impairs rehabilitation, and increases costs due to subsequent surgical interventions.Case presentationWe present the case of a 34-year-old African male who developed severe HO around his right hip 11 days after a major trauma. Immunohistochemical analyses of resected tissue revealed that several BMPs were expressed in the HO, including highly osteogenic BMP-9.ConclusionsTo the best of our knowledge, this is the first report of local BMP expression, notably BMP-9, in traumatic HO, and suggests that BMP-9, possibly through mrSCs, can contribute to HO formation in soft tissues when a suitable microenvironment is present.

The effect of heparan sulfate application on bone formation during distraction osteogenesis.

Bone morphogenetic proteins (BMPs) are recognized for their ability to induce bone formation in vivo and in vitro. Their osteogenic and osteoinductive properties are tightly regulated by the secretion of specific BMP antagonists, which have been shown to physically bind and sometimes be blocked by the extracellular proteoglycan heparan sulphate side chains (from hereon referred to as HS). The purpose of this study was to investigate if local application of 5 µg of HS proteoglycan to a bone regenerate site in a mouse model of distraction osteogenesis (DO) can accelerate bone healing and affect the expression of key members of the BMP signaling pathway. DO was performed on the right tibia of 115 adult male wild-type mice. At mid-distraction (day 11), half the group was injected locally with 5 µg of HS, while the other half was injected with saline. The mice were sacrificed at 2 time-points: mid-consolidation (34 days) and full consolidation (51 days). The distracted tibial zone was then collected for analysis by μCT, radiology, biomechanical testing, immunohistochemistry, and histology. While μCT data showed no statistically significant difference in bone formation, the results of biomechanical testing in stiffness and ultimate force were significantly lower in the HS-injected bones at 51 days, compared to controls. Immunohistochemistry results also suggested a decrease in expression of several key members of the BMP signaling pathway at 34 days. Furthermore, wound dehiscence and infection rates were significantly elevated in the HS group compared to the controls, which resulted in a higher rate of euthanasia in the treatment group. Our findings demonstrate that exogenous application of 5 µg of HS in the distracted gap of a murine model had a negative impact on bone and wound healing.

Serial bone densitometry during mandibular distraction osteogenesis.

The purpose of this study was to obtain serial measurements of bone mineral density (BMD) of the mandible during distraction osteogenesis. Fourteen skeletally mature male rabbits were subjected to unilateral mandibular osteodistraction at a defined rate. Two animals were sacrificed each week after surgery for 7 wk. The mandible and overlying soft tissue were resected and the BMD measured using dual-energy X-ray absorptiometry (DXA). Measurements obtained from the generate bone were compared to the contralateral (control) hemi-mandible. In the control hemi-mandible, BMD remained stable throughout the distraction protocol at 0.5 gm/cm2. In the distracted hemi-mandible, BMD sharply decreased to 0.35 gm/cm2 by the second week of distraction but steadily increased starting the third week of distraction. BMD surpassed control levels by wk 7. BMD measurements may provide a noninvasive assessment of bone mineralization and strength during distraction osteogenesis.