Ability of recombinant human bone morphogenetic protein 2 to enhance bone healing in the presence of tobramycin: evaluation in a rat segmental defect model.

OBJECTIVE: To determine whether locally applied tobramycin influences the ability of recombinant human bone morphogenetic protein 2 (rhBMP-2) to heal a segmental defect in the rat femur. METHODS: The influence of tobramycin on the osteogenic differentiation of mesenchymal stem cells was first evaluated in vitro. For the subsequent, in vivo experiments, a 5-mm segmental defect was created in the right femur of each of 25 Sprague-Dawley rats and stabilized with an external fixator and four Kirschner wires. Rats were divided in four groups: empty control, tobramycin (11 mg)/absorbable collagen sponge, rhBMP-2 (11 microg)/absorbable collagen sponge, and rhBMP-2/absorbable collagen sponge with tobramycin. Bone healing was monitored by radiography at 3 and 8 weeks. Animals were euthanized at 8 weeks and the properties of the defect were compared with the intact contralateral femur. Bone formation in the defect region was assessed by dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing. RESULTS: Tobramycin exerted a dose-dependent inhibition of alkaline phosphatase induction and calcium deposition by mesenchymal stem cells cultured under osteogenic conditions. The inhibition was reversed in the presence of 500 ng/mL of rhBMP-2. Segmental defects in the rat femora failed to heal in the absence of rhBMP-2. Tobramycin exerted no inhibitory effects on the ability of rhBMP-2 to heal these defects and increased the bone area of the defects treated with rhBMP-2. Data obtained from all other parameters of healing, including dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing, were unaffected by tobramycin. CONCLUSIONS: Although our in vitro results suggested that tobramycin inhibits the osteogenic differentiation of mesenchymal stem cells, this could be overcome by rhBMP-2. Tobramycin did not impair the ability of rhBMP-2 to heal critical-sized femoral defects in rats. Indeed, bone area was increased by nearly 20% in the rhBMP-2 group treated with tobramycin. This study shows that locally applied tobramycin can be used in conjunction with rhBMP-2 to enhance bone formation at fracture sites.

Regional and cellular localisation of BMPs and their inhibitors' expression in human fractures.

The objective of this study was to determine whether BMP-2 and -14, noggin, and chordin could be detected in human fractures and to assess their regional and cellular distribution. The expression of these proteins was detected by immunohistochemistry in an archive of human fractures. BMP-2 and BMP-14 expression was strongest in areas of cartilage formation and, to a lesser extent, in areas of bone formation. Within areas of cartilage formation, both BMP-2 and BMP-14 were expressed more strongly by the non-hypertrophic chondrocytes. The BMP inhibitors noggin and chordin were also expressed most intensely in areas of cartilage formation and there was no difference in their expression between the non-hypertrophic and hypertrophic chondrocytes. Our study demonstrates the expression of BMP-14 and the BMP inhibitors in human fractures for the first time, and our findings will contribute to an improved understanding of the physiological processes in bone repair.

Altered relative expression of BMPs and BMP inhibitors in cartilaginous areas of human fractures progressing towards nonunion.

The present study was conducted to evaluate the hypothesis that an imbalance in the local production of bone morphogenetic proteins (BMPs) and BMP inhibitors exists within the cartilaginous intermediate of nonhealing fractures. Biopsies were recovered intraoperatively from human fractures that, upon follow-up, were found to heal normally or become nonunions. The samples were examined by immunohistochemistry to determine the expression of BMP-2, BMP-14, and the BMP inhibitors noggin and chordin. Expression was determined semiquantitatively based on the area of positive staining per area of cartilage and by determining the number of positively staining cells and the intensity of staining. There was a significant reduction in BMP-2 and BMP-14 expression in cartilaginous areas of nonhealing fractures compared to healing fractures. However, there was no difference in the expression of the BMP inhibitors between the two groups of fractures. This imbalance in the expression of BMPs and BMP inhibitors within cartilaginous areas of developing nonunions may account for their reduced bone forming ability. These data suggest strategies for preventing the development of nonunions by altering levels of BMPs and their inhibitors within fracture sites.

Recent developments in the biology of fracture repair.

Fracture repair is dependent on local and systemic molecular and cellular processes. During fracture repair, mesenchymal stem cells are systemically recruited to the fracture site, and cytokines are released from the fracture site into the vascular system. In a significant minority of fractures, healing delays result from adverse clinical factors that interfere with these processes. Extrinsic factors, such as aging and smoking, adversely affect the molecular and cellular processes occurring locally in the fracture site. Fracture fixation affects healing through local changes in the biologic signaling within the fracture callus. Current biologic treatment of fractures includes the local application of osteoinductive bone morphogenetic proteins (ie, BMP-2, BMP-7) and cell-based therapies. Although clinical results with bone morphogenetic proteins have been satisfactory, they have not been as impressive as those reported in animal studies. Further understanding of the biology of fracture repair may lead to improved treatment modalities.

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells.

INTRODUCTION: Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro. METHODS: Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test. RESULTS: We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation. CONCLUSION: We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.

Facilitated endogenous repair: making tissue engineering simple, practical, and economical.

Facilitated endogenous repair is a novel approach to tissue engineering that avoids the ex vivo culture of autologous cells and the need for manufactured scaffolds, while minimizing the number and invasiveness of associated clinical procedures. The strategy relies on harnessing the intrinsic regenerative potential of endogenous tissues using molecular stimuli, such as gene transfer, to initiate reparative processes in situ. In the simplest example, direct percutaneous injection of an osteogenic vector is used to stimulate bone healing. If necessary, additional progenitor cells and space-filling scaffolds can be provided by autologous bone marrow, muscle, fat, and perhaps other tissues. These can be harvested, processed, and reimplanted by simple, expedited, intraoperative procedures. Examples of repair of experimental osseous and osteochondral lesions in laboratory animals are described. If successful, these strategies will provide methods for tissue regeneration that are not only effective but also inexpensive, safe, and clinically expeditious. Although orthopaedic examples are given here, the technology should be more generally applicable.

Publication rate of Trauma abstracts presented at an International Orthopaedic conference.

INTRODUCTION: Oral presentations at major conferences are often used to present new material and generate discussion. However, conference abstracts that ultimately fail to be published are of little use to the wider medical community. The aim of this study was to evaluate the publication rate of trauma papers presented at an international orthopaedic conference, and to assess the factors which predict publication. METHODS: All abstracts presented orally at the trauma sessions of the European Federation of National Associations of Orthopaedics and Traumatology (EFORT) Congresses in 1999 (Belgium) and 2001 (Greece) were assessed. A MEDLINE and EMBASE search was performed to identify articles written by the first, second and last authors of each abstract to identify a matching journal article. Subspecialty, country of origin of abstract, study type, journal of publication and publication year were tabulated. RESULTS: Two hundred and seventy eight trauma abstracts were presented orally, and 112 (40.3%) achieved subsequent publication. Abstracts on fractures of the proximal femur were the most common (18.7%) and had one of the highest rates of publication (44.2%). Greece and the UK provided the largest number of abstracts, and randomised trials were the study type with the highest rate of publication (80.0%). Thirty two percent of journal articles appeared within 1 year of the conference and 63% within 2 years. Injury was the journal most likely to publish the articles. CONCLUSION: About 60% of Trauma abstracts presented did not result in a subsequent full-text publication. The citation of conference proceedings should be discouraged, and clinicians should be wary of implementing information gleaned from conference presentations into their clinical practice.