Heat generation during ulnar osteotomy with microsagittal saw blades.
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Ulnar shortening osteotomy is a surgical treatment option for patients with symptomatic ulnar positive variance for a variety of reasons. Delayed healing and nonunion of the osteotomized sites have been reported and present problematic complications of this procedure. Studies have shown nonunion rate with transverse cuts ranging from 8-15%. The goal is to achieve parallel cuts, thus maximizing the contacting bony surface area for a better union rate. The senior surgeon attempted using a custom thick blade to insure parallel cuts. The concern is whether the heat generated during such a cut would contribute to non-union. It is our hypothesis that complications with ulnar shortening osteotomy using a thick blade are secondary to excess heat generation. When generated heat surpasses the threshold temperature of bone tissue, the organic matrix is irreversibly damaged and necrosis of the bony ends may occur. The present study measured the heat generation during ulnar osteotomy using different blade thicknesses. Thirty-five fresh turkey femurs, having similar size and cortical thickness of the human ulna, were used. Loading was done at three different speeds of 0.66, 1.0, and 1.5 mm/second corresponding respectively to 30, 20, and 10 seconds for the complete cut. A general linear statistical model was fitted relating temperature rise to three predictive factors: blade thickness, sensor distance, and initial bone temperature. There was a statistically significant relationship between temperature rise and all three predictor variables at the 99% confidence level. There was no statistically significant relationship between temperature rise and the number of cuts with the same blade up to 10 times. Compared with the single microsagital saw blade, the temperature rise for the double thickness blade was 14% higher and for the triple thickness blade was 23% higher. The temperature rise was inversely related to the speed of the cut. The temperature rise for the bone cut in 30 seconds was 1.5 times higher than the temperature rise when the bone was cut in 10 seconds. Complications with ulnar shortening osteotomy may be secondary to excess heat generation. A new thick saw blade design and the use of proper internal/external irrigation may overcome the problem. (+info)
Dose-dependent effects of combined IGF-I and TGF-beta1 application in a sheep cervical spine fusion model.
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Combined IGF-I and TGF-beta1 application by a poly-(D,L-lactide) (PDLLA) coated interbody cage has proven to promote spine fusion. The purpose of this study was to determine whether there is a dose-dependent effect of combined IGF-I and TGF-beta1 application on intervertebral bone matrix formation in a sheep cervical spine fusion model. Thirty-two sheep underwent C3/4 discectomy and fusion. Stabilisation was performed using a titanium cage coated with a PDLLA carrier including no growth factors in group 1 ( n=8), 75 micro g IGF-I plus 15 micro g TGF-beta1 in group 2 ( n=8), 150 micro g IGF-I plus 30 micro g TGF-beta1 in group 3 ( n=8) and 300 micro g IGF-I plus 60 micro g TGF-beta1 in group 4 ( n=8). Blood samples, body weight and temperature were analysed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8, and 12 weeks. At the same time points, disc space height and intervertebral angle were measured. After 12 weeks, the animals were killed and fusion sites were evaluated using quantitative computed tomographic (CT) scans to assess bone mineral density, bone mineral content and bony callus volume. Biomechanical testing was performed and range of motion, and neutral and elastic zones were determined. Histomorphological and histomorphometrical analysis were carried out and polychrome sequential labelling was used to determine the time frame of new bone formation. In comparison to the group without growth factors (group 1), the medium- and high-dose growth factor groups (groups 3 and 4) demonstrated a significantly higher bony callus volume on CT scans, a higher biomechanical stability, an advanced interbody bone matrix formation in histomorphometrical analysis, and an earlier bone matrix formation on fluorochrome sequence labelling. Additionally, the medium- and high-dose growth factor groups (groups 3 and 4) demonstrated a significantly higher bony callus volume, a higher biomechanical stability in rotation, and an advanced interbody bone matrix formation in comparison to the low-dose growth factor group (group 2). No significant difference could be determined between the medium- and the high-dose growth factor groups (groups 3 and 4, respectively). The local application of IGF-I and TGF-beta1 by a PDLLA-coated cage significantly improved results of interbody bone matrix formation in a dose-dependent manner. The best dose-response relationship was achieved with the medium growth factor dose (150 micro g IGF-I and 30 micro g TGF-beta1). With an increasing dose of these growth factors, no further stimulation of bone matrix formation was observed. Although these results are encouraging, safety issues of combined IGF-I and TGF-beta1 application for spinal fusion still have to be addressed. (+info)
Metabolic activation stimulates acid secretion and expression of matrix degrading proteases in human osteoblasts.
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BACKGROUND: Both cellular and matrix components of healthy bone are permanently renewed in a balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases. Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix proteins. OBJECTIVES: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to proton secretion expressed on osteoblasts. METHODS: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer. RESULTS: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is associated partially with proton pump ATPases. CONCLUSION: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone degradation. (+info)
Immunohistochemistry of matrix markers in Technovit 9100 New-embedded undecalcified bone sections.
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Trabecular bone is routinely analysed by histomorphological-histometrical and immunohistochemical techniques as means of assessing the differentiation status of bone deposition and growth. Currently few embedding resins exist for which both morphological and immunohistochemical analyses can be performed on mineralised tissue. Paraffin, the standard embedding medium for bone enzyme and immunohistochemistry, can only be used on demineralised tissue, but then trabecular structure may be badly preserved. Methyl methacrylate (MMA), the resin of choice for undecalcified bone histology can only be used for bone immunohistochemistry if the usual, highly exothermic polymerisation procedure is avoided which destroys tissue antigenicity. Consequently, most current practices involve cutting samples in half to be processed in separate resins when more than one type of analysis is required. Technovit 9100 New is a low temperature MMA embedding system that is purported to significantly improve tissue antigenicity preservation allowing polymerisation at -20 degrees C. In this study, Technovit 9100 New-embedded undecalcified trabecular bone samples (adult human, young bovine and ovine) yielded immunolabelling with several bone matrix markers and preserved morphological features in 7 microm sections when stained with Masson-Goldner, von Kossa, or toluidine blue. Bone samples from all resins used were immunolabelled with antibodies against osteocalcin, alkaline phosphatase, osteopontin, osteonectin, bone sialoprotein and procollagen type I amino-terminal propeptide. Technovit-embedded bone yielded more reliable immunolabelling of the matrix proteins when compared with heat or cold-cured LR White or standard embedded MMA samples. Technovit 9100 New provided better routine histology than LR White, and was comparable to MMA. Results demonstrated that Technovit 9100 New can be used as a low-temperature acrylic resin embedding method for routine undecalcified bone histology, as well as for immunohistochemistry. (+info)
A central role for the notochord in vertebral patterning.
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The vertebrates are defined by their segmented vertebral column, and vertebral periodicity is thought to originate from embryonic segments, the somites. According to the widely accepted 'resegmentation' model, a single vertebra forms from the recombination of the anterior and posterior halves of two adjacent sclerotomes on both sides of the embryo. Although there is supporting evidence for this model in amniotes, it remains uncertain whether it applies to all vertebrates. To explore this, we have investigated vertebral patterning in the zebrafish. Surprisingly, we find that vertebral bodies (centra) arise by secretion of bone matrix from the notochord rather than somites; centra do not form via a cartilage intermediate stage, nor do they contain osteoblasts. Moreover, isolated, cultured notochords secrete bone matrix in vitro, and ablation of notochord cells at segmentally reiterated positions in vivo prevents the formation of centra. Analysis of fss mutant embryos, in which sclerotome segmentation is disrupted, shows that whereas neural arch segmentation is also disrupted, centrum development proceeds normally. These findings suggest that the notochord plays a key, perhaps ancient, role in the segmental patterning of vertebrae. (+info)
The roles of annexins and alkaline phosphatase in mineralization process.
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In this review the roles of specific proteins during the first step of mineralization and nucleation are discussed. Mineralization is initiated inside the extracellular organelles-matrix vesicles (MVs). MVs, containing relatively high concentrations of Ca2+ and inorganic phosphate (Pi), create an optimal environment to induce the formation of hydroxyapatite (HA). Special attention is given to two families of proteins present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases (TNAPs). Both families participate in the formation of HA crystals. AnxAs are Ca2+ - and lipid-binding proteins, which are involved in Ca2+ homeostasis in bone cells and in extracellular MVs. AnxAs form calcium ion channels within the membrane of MVs. Although the mechanisms of ion channel formation by AnxAs are not well understood, evidence is provided that acidic pH or GTP contribute to this process. Furthermore, low molecular mass ligands, as vitamin A derivatives, can modulate the activity of MVs by interacting with AnxAs and affecting their expression. AnxAs and other anionic proteins are also involved in the crystal nucleation. The second family of proteins, TNAPs, is associated with Pi homeostasis, and can hydrolyse a variety of phosphate compounds. ATP is released in the extracellular matrix, where it can be hydrolyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophosphohydrolases. However, TNAP is probably not responsible for ATP-dependent Ca2+/phosphate complex formation. It can hydrolyse pyrophosphate (PPi), a known inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this respect, antagonistic activities of TNAPs and NTP pyrophosphohydrolases can regulate the mineralization process. (+info)
Endogenous TGF-beta signaling suppresses maturation of osteoblastic mesenchymal cells.
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Transforming growth factor-beta (TGF-beta), one of the most abundant cytokines in bone matrix, has positive and negative effects on bone formation, although the molecular mechanisms of these effects are not fully understood. Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, induce bone formation in vitro and in vivo. Here, we show that osteoblastic differentiation of mouse C2C12 cells was greatly enhanced by the TGF-beta type I receptor kinase inhibitor SB431542. Endogenous TGF-beta was found to be highly active, and induced expression of inhibitory Smads during the maturation phase of osteoblastic differentiation induced by BMP-4. SB431542 suppressed endogenous TGF-beta signaling and repressed the expression of inhibitory Smads during this period, possibly leading to acceleration of BMP signaling. SB431542 also induced the production of alkaline phosphatase and bone sialoprotein, and matrix mineralization of human mesenchymal stem cells. Thus, signaling cross-talk between BMP and TGF-beta pathways plays a crucial role in the regulation of osteoblastic differentiation, and TGF-beta inhibitors may be invaluable for the treatment of various bone diseases by accelerating BMP-induced osteogenesis. (+info)
Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro.
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Osteocalcin detected from serum samples is considered a specific marker of osteoblast activity and bone formation rate. However, osteocalcin embedded in bone matrix must also be released during bone resorption. To understand the contribution of each type of bone cell in circulating osteocalcin levels, we used immunoassays detecting different molecular forms of osteocalcin to monitor bone resorption in vitro. Osteoclasts were obtained from rat long bones and cultured on bovine bone slices using osteocalcin-depleted fetal bovine serum. In addition, human osteoclasts differentiated from peripheral blood mononuclear cells were used. Both rat and human osteoclasts released osteocalcin from bovine bone into medium. The amount of osteocalcin increased in the presence of parathyroid hormone, a stimulator of resorption, and decreased in the presence of bafilomycin A1, an inhibitor of resorption. The amount of osteocalcin in the medium correlated with a well characterized marker of bone resorption, the C-terminal telopeptide of type I collagen (r > 0.9, p < 0.0001). The heterogeneity of released osteocalcin was determined using reverse phase high performance liquid chromatography, and several molecular forms of osteocalcin, including intact molecule, were identified in the culture medium. In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation. (+info)