Hannu T. Aro

Recombinant human bone morphogenetic protein-2 in open tibial fractures. A subgroup analysis of data combined from two prospective randomized studies.

BACKGROUND The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) to improve the healing of open tibial shaft fractures has been the focus of two prospective clinical studies. The objective of the current study was to perform a subgroup analysis of the combined data from these studies. METHODS Two prospective, randomized clinical studies were conducted. A total of 510 patients with open tibial fractures were randomized to receive the control treatment (intramedullary nail fixation and routine soft-tissue management) or the control treatment and an absorbable collagen sponge impregnated with one of two concentrations of rhBMP-2. The rhBMP-2 implant was placed over the fracture at the time of definitive wound closure. For the purpose of this analysis, only the control treatment and the Food and Drug Administration-approved concentration of rhBMP-2 (1.50 mg/mL) were compared. Patients who anticipated receiving planned bone-grafting as part of a staged treatment were excluded from enrollment. RESULTS Fifty-nine trauma centers in twelve countries participated, and patients were followed for twelve months postoperatively. Two subgroups were analyzed: (1) the 131 patients with a Gustilo-Anderson type-IIIA or IIIB open tibial fracture and (2) the 113 patients treated with reamed intramedullary nailing. The first subgroup demonstrated significant improvements in the rhBMP-2 group, with fewer bone-grafting procedures (p = 0.0005), fewer patients requiring invasive secondary interventions (p = 0.0065), and a lower rate of infection (p = 0.0234), compared with the control group. The second subgroup analysis of fractures treated with reamed intramedullary nailing demonstrated no significant difference between the control and the rhBMP-2 groups. CONCLUSIONS The addition of rhBMP-2 to the treatment of type-III open tibial fractures can significantly reduce the frequency of bone-grafting procedures and other secondary interventions. This analysis establishes the clinical efficacy of rhBMP-2 combined with an absorbable collagen sponge implant for the treatment of these severe fractures.

Lisfranc joint injuries: trauma mechanisms and associated injuries

A retrospective review of the roentgenograms of 750 patients treated in our institution for tarsometatarsal joint injuries or metatarsal fractures over a 10-year-period was performed. Injuries of the Lisfranc joint were found in 66 patients (9%). Twelve patients (19%) had a total dislocation, 47 patients (71%) a partial dislocation, and seven patients (11%) a subtle injury of the Lisfranc joint. Four main groups of trauma mechanisms were identified (low-energy injuries, falls from a height, direct crush injuries, and high-energy vehicular crashes). Lisfranc joint injuries caused by low-energy injuries (21 patients) were as frequent as those caused by high-energy vehicular crashes (22 patients). There was no apparent relationship between the mechanism of injury and the type of Lisfranc joint dislocation. All patients except three had associated metatarsal fractures, most commonly in the second metatarsal. The location of associated metatarsal fractures was different from that in patients with metatarsal fractures alone (p < 0.001). Fractures, dislocations, or fracture-dislocations of midtarsal bones (cuneiforms, cuboid, navicular) occurred in 26 patients (39%). Multiple metatarsal fractures and midtarsal bone injuries were more frequent in high-energy vehicular crashes than in low-energy injuries (p = 0.016 and p = 0.033, respectively). In 23 patients (35%), the treatment had been focused on multiple metatarsal fractures or midtarsal bone injuries without full appreciation of the concomitant Lisfranc joint incongruity. Increasing the knowledge of normal foot anatomy and the appreciation of the risk of Lisfranc joint injury even in seemingly trivial stumbling accidents obviously may improve the diagnosis and treatment of these potentially disabling injuries.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression during bone repair.

Detailed understanding of the basic events in fracture healing constitutes a foundation for the development of new approaches to stimulate bone healing. Since the fracture healing process repeats, in an adult organism, several stages of skeletal growth in the same temporal order, it offers an interesting model for developmental regulation of cellular phenotypes and tissue-specific genes. Molecular biology has introduced new methods to study the regulatory phenomena during the process of fracture repair. Gene technology has also produced purified growth factors for research, which will help to understand their roles in fracture healing. This review summarizes data on the regulation of genes coding for extracellular matrix components and growth regulatory molecules during fracture healing. The information available focuses on the sequential expression of genes coding for collagens, proteoglycans, and some other matrix proteins during secondary (callus) healing. The temporal and spatial appearance of the different connective tissue components, mesenchyme, cartilage, and bone, are closely linked to the expression of genes coding for their characteristic constituents. Members of the transforming growth factor-beta superfamily, such as the bone morphogenetic proteins (BMP), are currently the most interesting ones among the factors that regulate chondrogenesis and osteogenesis. In the coming years, the availability of new cloned probes combined with sensitive analytical methods, as reviewed here, will add greatly to our understanding of the various aspects of gene expression during bone repair. This information should provide answers to some of the unresolved questions in fracture callus development.

Accelerated Turnover of Metaphyseal Trabecular Bone in Mice Overexpressing Cathepsin K

This study is based on a hypothesis that overexpression of an osteoclast enzyme, cathepsin K, causes an imbalance in bone remodeling toward bone loss. The hypothesis was tested in transgenic (TG) mice harboring additional copies of the murine cathepsin K gene (Ctsk) identifiable by a silent mutation engineered into the construct. For this study, three TG mouse lines harboring 3‐25 copies of the transgene were selected. Tissue specificity of transgene expression was determined by Northern analysis, which revealed up to 6‐fold increases in the levels of cathepsin K messenger RNA (mRNA) in calvarial and long bone samples of the three TG lines. No changes were seen in the mRNA levels of other osteoclast enzymes, indicating that the increase in cathepsin K mRNA was not a reflection of activation of all osteoclast enzymes. Immunohistochemistry confirmed that cathepsin K expression in the TG mice was confined to osteoclasts and chondroclasts. Histomorphometry revealed a significantly decreased trabecular bone volume (BV), but, surprisingly, also a marked increase in the number of osteoblasts, the rate of bone turnover, and the amount of mineralizing surface (MS). However, monitoring of bone density in the proximal tibias of the TG mice with peripheral quantitative computed tomography (pQCT) failed to reveal statistically significant changes in bone density. Similarly, no statistically significant alterations were observed in biomechanical testing at the age of 7 months. The increases in parameters of bone formation triggered by increased cathepsin K expression is an example of the tight coupling of bone resorption and formation during the bone‐remodeling cycle.

Bone-healing patterns affected by loading, fracture fragment stability, fracture type, and fracture site compression.

The major factors determining the mechanical milieu of a healing fracture are the rigidity of the selected fixation device, the fracture configuration, the accuracy of fracture reduction, and the amount and type of stresses occurring at the bone ends dictated by functional activity and loading at the fracture gap. Of the effects of these factors on fracture healing and remodeling in the canine tibia under unilateral external fixation, the two most significant factors in promoting periosteal callus formation were the amount of physiologic loading as dictated by the body weight and the presence of a significant fracture gap. Uniform axial loading and motion, performed at two or four weeks, did not increase callus formation but did reduce the existing fracture gap. The time-related diminution of periosteal callus (external remodeling) was related to the progress of cortical healing. Cortical reconstruction was fast in stable transverse fractures and delayed in unstable oblique fractures. Motion with loading tended to promote external callus maturation in secondary bone healing. Intracortical new bone formation and porosity were related to the healing pattern and not to the loading magnitude. Endosteal new bone formation showed a strong correlation with the presence of a fracture gap. In terms of torsional strength and energy absorption at failure, the fractures healing through a combination of primary and secondary bone union mechanisms showed the mechanical strength of the healing bone closest to intact bone.

The effect of rigidity on fracture healing in external fixation.

Knowledge of the basic biomechanics of external fixation is necessary to obtain the full benefits of the technique for bone fracture treatment. The rigidity of external fixation, including pin-bone interface stresses, is discussed and bone healing and remodeling under different fixation stiffnesses and fracture gap conditions are described. The rigidity of fixation ultimately depends on the biomechanical characteristics of the fracture, the accuracy of reduction, and the amount of physiologic loading. Comparative experiments using a canine tibial fracture model have suggested that fixation rigidity is important in early bone healing and in the prevention of pin loosening. Bone union can be achieved under external fixation through different pathways, ranging from callus-free gap healing under a rigid neutralization configuration to direct-contact healing with periosteal new bone formation under axially dynamized stable fixation. Cortical reconstruction by secondary osteons seems to be important for the ultimate strength of the bone union.

Mouse cathepsin K: cDNA cloning and predominant expression of the gene in osteoclasts, and in some hypertrophying chondrocytes during mouse development☆

We have constructed cDNA clones covering the entire coding region of mouse, human and rabbit preprocathepsin K mRNA for studies on bone turnover. The clone pMCatK‐1 for mouse cathepsin K shares 87% nucleotide homology with the corresponding human and rabbit sequences. Analysis of a panel of mouse tissues for tissue distribution of cathepsin K mRNA revealed the highest levels in musculoskeletal tissues: bone, cartilage and skeletal muscle. In situ hybridization of developing mouse embryos was performed to identify the cellular source of cathepsin K mRNA. The strongest mRNA signal was detected in osteoclasts of bone, identified in serial sections by positive TRAP staining. Cathepsin K mRNA was also observed in some hypertrophic chondrocytes of growth cartilages. Association of cathepsin K production with degradation of bone and cartilage matrix suggests that this enzyme and its mRNA levels could serve as markers for matrix degradation in diseases affecting these tissues.

Recombinant Human Bone Morphogenetic Protein-2: A Randomized Trial in Open Tibial Fractures Treated with Reamed Nail Fixation

BACKGROUND Recombinant human bone morphogenetic protein-2 (rhBMP-2) improves healing of open tibial fractures treated with unreamed intramedullary nail fixation. We evaluated the use of rhBMP-2 in the treatment of acute open tibial fractures treated with reamed intramedullary nail fixation. METHODS Patients were randomly assigned (1:1) to receive the standard of care consisting of intramedullary nail fixation and routine soft-tissue management (the SOC group) or the standard of care plus an absorbable collagen sponge implant containing 1.5 mg/mL of rhBMP-2 (total, 12.0 mg) (the rhBMP-2/ACS group). Randomization was stratified by fracture severity. The absorbable collagen sponge was placed over the fracture at wound closure. The primary efficacy end point was the proportion of subjects with a healed fracture as demonstrated by radiographic and clinical assessment thirteen and twenty weeks after definitive wound closure. RESULTS Two hundred and seventy-seven patients were randomized and were the subjects of the intent-to-treat analysis. Thirteen percent of the fractures were Gustilo-Anderson Type IIIB. The proportions of patients with fracture-healing were 60% and 48% at week 13 (p = 0.0541) and 68% and 67% at week 20 in the rhBMP-2/ACS and SOC groups, respectively. Twelve percent of the subjects underwent secondary procedures in each group; more invasive procedures (e.g., exchange nailing) accounted for 30% of the procedures in the rhBMP-2/ACS group and 57% in the SOC group (p = 0.1271). Infection was seen in twenty-seven (19%) of the patients in the rhBMP-2/ACS group and fifteen (11%) in the SOC group (p = 0.0645; difference in infection risk = 0.09 [95% confidence interval, 0.0 to 0.17]). The adverse event incidence was otherwise similar between the treatment groups. CONCLUSIONS The healing of open tibial fractures treated with reamed intramedullary nail fixation was not significantly accelerated by the addition of an absorbable collagen sponge containing rhBMP-2.

Silica-based bioactive glasses modulate expression of bone morphogenetic protein-2 mRNA in Saos-2 osteoblasts in vitro

A chemical exchange of the silica gel layer forming on the surface of bioactive glasses is thought to be the principal reaction for bone-bioactive glass bonding. The contribution of biological molecules on cell-bioactive glass interaction is largely unknown. To further analyze the mechanisms involved in efficient bone bonding to bioactive glass, Saos-2 osteoblastic cells with proven osteogenic phenotype were cultured for 4, 7 and 14 days on two bioactive glasses with different Si contents. Culture plates and dishes made of bioactive (BAG, 53 % SiO2), biocompatible (BCG, 58% SiO2) and control (GO) glasses were extensively conditioned with phosphate buffer and DMEM medium before seeding the cells. Northern hybridization was used for analysis of mRNA levels of collagen type I (Col-I), alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). A significant increase was observed in Col-I mRNA levels in cells grown on the two bioactive glasses when compared with those grown on controls at 4 and 7 days (p < 0.04). The mRNA level for ALP in the cultures of bioactive glasses-made plates and dishes was also increased over control at 7 days (p < 0.02) and remained this way between BAG and G0 at 14 days. Striking differences in BMP-2 mRNA levels existed between BAG and G0 plates and dishes at 7 days (p < 0.05). BMP-2 mRNA level in BAG group was higher than in BCG group at 4, 7 and 14 days, but without statistical significance. Saos-2 osteoblastic cells with strong ALP staining were mostly seen on BAG plates under a light microscope. In confocal microscopy, a bright FITC-stained F-actin ring was present in the cytoplasm of cells grown on BAG dish, demonstrating an active functional status. Stimulation of the expression of BMP-2 and other bone mRNAs by bioactive glasses in osteoblastic cells suggests biological involvement of bone related growth factors, peptides and cytokines in bone-bioactive glass bonding.

Effects of axial dynamization on bone healing

The effects of early dynamization (physiologic axial compression) on canine fracture healing at six weeks were studied. Bilateral transverse mid-tibial osteotomies were created and initially stabilized with a 2-mm gap using relatively rigid external fixators. Seven days after osteotomy, the telescoping mechanism of one of the fixators on each dog was released (dynamized), resulting in physiologic loading of the osteotomy, while the contralateral fixator remained locked as a rigid control. The dynamized osteotomy closed, and increased functional weight bearing resulted from 3 weeks on. Radiographically the amount of periosteal callus increased over time, but no difference in callus size was seen between the dynamized fractures and the controls. Torsional mechanical testing found the dynamized osteotomies to be significantly stiffer, and they tended to tolerate more maximum torque than the controls. Microscopic evaluation found no difference in the volume of the periosteal and endosteal calluses or in the tissues constituting them. However, a significantly greater proportion of the dynamized osteotomy gap was filled with new bone. These results suggest that dynamization in this delayed union model improved fracture healing by reducing fracture gap size and increasing weight bearing, not by altering the pathway of fracture healing.