Pulsed electromagnetic field stimulation may improve fusion rates in cervical arthrodesis in high-risk populations.

OBJECTIVES: Pulsed electromagnetic field (PEMF) stimulation was evaluated after anterior cervical discectomy and fusion (ACDF) procedures in a randomized, controlled clinical study performed for United States Food and Drug Administration (FDA) approval. PEMF significantly increased fusion rates at six months, but 12-month fusion outcomes for subjects at elevated risk for pseudoarthrosis were not thoroughly reported. The objective of the current study was to evaluate the effect of PEMF treatment on subjects at increased risk for pseudoarthrosis after ACDF procedures. METHODS: Two evaluations were performed that compared fusion rates between PEMF stimulation and a historical control (160 subjects) from the FDA investigational device exemption (IDE) study: a post hoc (PH) analysis of high-risk subjects from the FDA study (PH PEMF); and a multicentre, open-label (OL) study consisting of 274 subjects treated with PEMF (OL PEMF). Fisher's exact test and multivariate logistic regression was used to compare fusion rates between PEMF-treated subjects and historical controls. RESULTS: In separate comparisons of PH PEMF and OL PEMF groups to the historical control group, PEMF treatment significantly (p < 0.05, Fisher's exact test) increased the fusion rate at six and 12 months for certain high-risk subjects who had at least one clinical risk factor of being elderly, a nicotine user, osteoporotic, or diabetic; and for those with at least one clinical risk factor and who received at least a two- or three-level arthrodesis. CONCLUSION: Adjunctive PEMF treatment can be recommended for patients who are at high risk for pseudoarthrosis.Cite this article: D. Coric, D. E. Bullard, V. V. Patel, J. T. Ryaby, B. L. Atkinson, D. He, R. D. Guyer. Pulsed electromagnetic field stimulation may improve fusion rates in cervical arthrodesis in high-risk populations. Bone Joint Res 2018;7:124-130. DOI: 10.1302/2046-3758.72.BJR-2017-0221.R1.

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway.

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50microM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7microg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7microg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation.

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.

Three-dimensional morphometry of the L6 vertebra in the ovariectomized rat model of osteoporosis: biomechanical implications.

This article summarizes the results of a three-dimensional study of changes in the morphology of the L6 rat vertebra at 120 days after ovariectomy (OVX), with estrogen replacement therapy used as a positive control. Synchrotron radiation microtomography was used to quantify the structural parameters defining trabecular bone architecture, while finite-element methods were used to explore the relationships between these parameters and the compressive elastic behavior of the vertebrae. There was a 22% decrease in trabecular bone volume (TBV) and a 19% decline in mean trabecular thickness (Tb.Th) with OVX. This was accompanied by a 150% increase in trabecular connectivity, a result of the perforation of trabecular plates. Finite-element analysis of the trabecular bone removed from the cortical shell showed a 37% decline in the Young's modulus in compression after OVX with no appreciable change in the estrogen-treated group. The intact vertebrae (containing its trabecular bone) exhibited a 15% decrease in modulus with OVX, but this decline lacked statistical significance. OVX-induced changes in the trabecular architecture were different from those that have been observed in the proximal tibia. This difference was a consequence of the much more platelike structure of the trabecular bone in the vertebra.

Computational methods for ultrasonic bone assessment.

Ultrasound has been proposed as a means to noninvasively assess bone and, particularly, bone strength and fracture risk. Although there has been some success in this application, there is still much that is unknown regarding the propagation of ultrasound through bone. Because strength and fracture risk are a function of both bone mineral density and architectural structure, this study was carried out to examine how architecture and density interact in ultrasound propagation. Due to the difficulties inherent in obtaining fresh bone specimens and associated architectural and density features, simulation methods were used to explore the interactions of ultrasound with bone. A sample of calcaneal trabecular bone was scanned with micro-CT and subjected to morphological image processing (erosions and dilations) operations to obtain a total of 15 three-dimensional (3-D) data sets. Fifteen two-dimensional (2-D) slices obtained from the 3-D data sets were then analyzed to evaluate their respective architectures and densities. The architecture was characterized through the fabric feature, and the density was represented in terms of the bone volume fraction. Computer simulations of ultrasonic propagation through each of the 15 2-D bone slices were carried out, and the ultrasonic velocity and mean frequency of the received waveforms were evaluated. Results demonstrate that ultrasound propagation is affected by both density and architecture, although there was not a simple linear correlation between the relative degree of structural anisotropy with the ultrasound measurements. This study elucidates further aspects of propagation of ultrasound through bone, and demonstrates as well as the power of computational methods for ultrasound research in general and tissue and bone characterization in particular.

Tissue engineered bone repair of calvarial defects using cultured periosteal cells.

Periosteum has been demonstrated to have cell populations, including chondroprogenitor and osteoprogenitor cells, that can form both cartilage and bone under appropriate conditions. In the present study, periosteum was harvested, expanded in cell culture, and used to repair critical size calvarial defects in a rabbit model. Periosteum was isolated from New Zealand White rabbits, grown in cell culture, labeled with the thymidine analog bromodeoxyuridine for later localization, and seeded into resorbable polyglycolic acid scaffold matrices. Thirty adult New Zealand White rabbits were divided into groups, and a single 15-mm diameter full-thickness calvarial defect was made in each animal. In group I, defects were repaired using resorbable polyglycolic acid implants seeded with periosteal cells. In group II, defects were repaired using untreated polyglycolic acid implants. In group III, the defects were left unrepaired. Rabbits were killed at 4 and 12 weeks postoperatively. Defect sites were then studied histologically, biochemically, and radiographically. In vitro analysis of the cultured periosteal cells indicated an osteoblastic phenotype, with production of osteocalcin upon 1,25(OH)2 vitamin D3 induction. In vivo results at 4 weeks showed islands of bone in the defects repaired with polyglycolic acid implants with periosteal cells (group I), whereas the defects repaired with untreated polyglycolic acid implants (group II) were filled with fibrous tissue. Collagen content was significantly increased in group I compared with group II (2.90 +/- 0.80 microg/mg dry weight versus 0.08 +/- 0.11 microg/mg dry weight, p < 0.006), as was the ash weight (0.58 +/- 0.11 mg/mg dry weight versus 0.35 +/- 0.06 mg/mg dry weight, p < 0.015). At 12 weeks there were large amounts of bone in group I, whereas there were scattered islands of bone in groups II and III. Radiodensitometry demonstrated significantly increased radiodensity of the defect sites in group I, compared with groups II and III (0.740 +/- 0.250 OD/mm2 versus 0.404 +/- 0.100 OD/mm2 and 0.266 +/- 0.150 OD/mm2, respectively, p < 0.05). Bromodeoxyuridine label, as detected by immunofluorescence, was identified in the newly formed bone in group I at both 4 and 12 weeks, confirming the contribution of the cultured periosteal cells to this bone formation. This study thus demonstrates a tissue-engineering approach to the repair of bone defects, which may have clinical applications in craniofacial and orthopedic surgery.

Three-dimensional in vivo morphometry of trabecular bone in the OVX rat model of osteoporosis.

This paper describes the application of synchrotron radiation microtomography to osteoporosis research. By taking advantage of the high intensity, collimation, and monochromaticity of synchrotron radiation, we have been able to image the three-dimensional trabecular bone structure in living rats, thus providing serial data on the earliest architectural changes that occur with estrogen loss. Results from these in vivo animal experiments demonstrate that one of the earliest manifestations of estrogen loss, in addition to a decrease in the amount of trabecular bone, is decreased connectivity. We demonstrate that estrogen replacement therapy, when initiated soon after significant changes have occurred, restores bone mass to baseline levels but does not recover the trabecular connectivity. Even without an associated recovery in trabecular connectivity, finite element calculations on the three-dimensional images suggest that estrogen recovers the original structural modulus of elasticity. We believe the recovery of the elastic properties is due to an increase in trabecular thickness above baseline values.

Clinical effects of electromagnetic and electric fields on fracture healing.

The clinical use of electric and electromagnetic fields for fracture healing applications began in the early 1970s. Since then, several technologies have been developed and shown to promote healing in difficult to heal fractures. The development of these devices has been aided in recent years by basic research and several well controlled clinical trials. This review provides a brief description of the different techniques and their respective clinical utility. Finally, future directions in basic and clinical research are outlined to exploit fully the usefulness of these noninvasive bone growth stimulation technologies.

Influence of marrow on ultrasonic velocity and attenuation in bovine trabecular bone.

Measurements of ultrasonic velocity and specific differential attenuation (SDA) were obtained on 24 bovine trabecular bone specimens from the femoral condyles. The measurements were obtained using two pairs of ultrasonic transducers, one with a low nominal center frequency (500 kHz) and the other pair with a high nominal center frequency (1 MHz). The ultrasonic velocity and specific differential attenuation associated with the bone samples were determined both with and without marrow, i.e., replacing the marrow with water in the pores of the trabecular bone. Significant increases (2.1% and 2.9%) in the velocity of ultrasound were observed after removal of the marrow, for the low and high frequency transducer pairs, respectively. In contrast, significant decreases (-6.5% and -8.8%) in SDA were observed after removal of the marrow, for the low and high frequency transducer pairs, respectively. The bone densities (BD) of the samples were also determined using single photon absorptiometry (SPA). Correlations between ultrasonic parameters and bone densities for samples both with and without marrow were found to be similar. For example, for the 1 MHz transducer pair, the correlation between BD and velocity was r = 0. 86 with marrow, and r = 0.89 without marrow. This study also compared the results obtained using a contact (no water bath) technique and an insertion (with a water bath) technique of ultrasonic measurements. For the high frequency transducer pair, the correlation coefficients between the two methods were r = 0.99 and r = 0.93, for the velocity and specific differential attenuation, respectively. Similar results were found for the low frequency transducer pair as well. In addition, approximately equal correlations between BD and ultrasonic velocity and SDA were also found, indicating that contact and insertion measurements provide essentially equivalent information.

Ultrasonic assessment of human and bovine trabecular bone: a comparison study.

A comparison study is reported on the ultrasonic assessment of human trabecular and bovine trabecular bone samples. Both ultrasonic velocity and ultrasonic attenuation were evaluated through a transmission insertion technique and correlated with bone mineral density as determined with single photon absorptiometry. For a 1-MHz ultrasonic transducer pair and the human cancellous bone samples the correlations were 0.91 and 0.89 between density and velocity and attenuation, respectively. For a 500-kHz ultrasonic transducer pair the correlations were 0.89 and 0.81 between density and velocity and attenuation, respectively. For the bovine bone samples, the correlations were 0.90 and -0.31 for the velocity and attenuation, respectively, for the 1 MHz transducer pair. For the 500-kHz transducers, the correlations were 0.85 and -0.17 for the velocity and attenuation, respectively. By combining both velocity and attenuation in a multivariate regression, an improvement was achieved in the estimation of bone density in the human samples for both the 500-kHz and 1-MHz transducer pairs. No significant improvement was achieved in the multivariate regressions for the bovine bone samples. In conclusion, the results indicate that ultrasonic measurements are in general highly correlated with bone mineral density in trabecular bone samples. This correlation is more consistent and strong in relatively low density human samples compared with the higher density bovine samples.

Uma nova e simplificada técnica ultra-sônica clínica para a caracterizaçäo de tecido ósseo / A new and simplified clinical ultrasound technical for bone tissue caracterization

A atenuação (BUA), medida pela técnica ultra-sônica de caracterização de tecido ósseo, pode ser substituída pela frequência instantânea média (MIF), que é um parâmetro mais simples de ser medido e sem perda de informação, utilizando-se a técnica de inserção por contato no calcanhar do paciente. Uma nova forma de classificação da osteoporose poderá ser criada baseada neste novo parâmetro.

An easy to measure ultrasonic feature namely mean instantanaeous frequency (MJF) has been demonstrated to provide about the sarne information as attenuation (BUA) in a new contact clinical ultrasonic system. A new classification of osteoporosis may be achieved using this new parameter.

Combined magnetic fields increase insulin-like growth factor-II in TE-85 human osteosarcoma bone cell cultures.

In vitro exposure to low-energy, combined magnetic fields (CMF) increased the release of insulin-like growth factor (IGF)-II from human TE-85 osteosarcoma cells. Short-term CMF exposure of only 10 min increased IGF-II levels in conditioned medium 1 h post CMF exposure. IGF-II levels were measured with a radioreceptor assay using H-35 cells that contain abundant IGF-II but not IGF-I receptors. This assay also uses a recently validated BioGel P-10 acid gel filtration method to remove IGF binding protein before quantitation of either IGF-I or IGF-II. In addition to an increase in IGF-II levels, DNA synthesis, as an index of cell proliferation, was increased during the 24-h period post CMF exposure. A monoclonal antibody against IGF-II blocked the increase in cell proliferation following CMF exposure, whereas a control monoclonal antibody against osteocalcin did not attenuate the mitogenic action of CMF exposure. The effect of CMF exposure to increase both cell proliferation and IGF-II was cell-density dependent with greater stimulation by CMF observed at lower densities. Together, these data are consistent with the hypothesis that CMF exposure stimulates release/production of IGF-II from bone cells and that increased IGF-II then promotes an increase in cell proliferation.

IGF-II receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields.

Human osteosarcoma-derived osteoblast-like cells, TE-85, were used to assess the effect of a low frequency alternating magnetic field in combination with a controlled static magnetic field (combined magnetic fields, CMF) on insulin-like growth factor receptor regulation. In our culture system, application of a 15.3 Hz CMF induces a calculated maximum electrical potential in the culture media of 10(-5) V/m. Initial characterization of TE-85 cells demonstrated that (a) TE-85 cells contain both type I insulin-like growth factor (IGF-I) and IGF-II receptors and (b) dose dependence for IGF-stimulated cell proliferation were comparable to the affinities of the IGF's binding to membrane binding sites (i.e., receptors had dissociation constants in the low nanomolar concentration range). The studies with CMF exposure revealed that CMF treatment for 30 minutes increased the number of IGF-II receptors in a frequency-dependent manner without affecting the number of IGF-I receptors. The CMF-dependent increase in IGF-II receptor number was associated with a significant increase in the IGF-II dissociation constant. These results indicate that a membrane receptor levels can be altered by short-term exposure to low-energy, low-frequency electromagnetic fields and suggest a potential biochemical mechanism for electromagnetic effects on bone formation and remodeling.

Combined magnetic fields increased net calcium flux in bone cells.

Low energy electromagnetic fields (EMF) exhibit a large number of biological effects. A major issue to be determined is "What is the lowest threshold of detection in which cells can respond to an EMF?" In these studies we demonstrate that a low-amplitude combined magnetic field (CMF) which induces a maximum potential gradient of 10(-5) V/m is capable of increasing net calcium flux in human osteoblast-like cells. The increase in net calcium flux was frequency dependent, with a peak in the 15.3-16.3 Hz range with an apparent bandwidth of approximately 1 Hz. A model that characterizes the thermal noise limit indicates that non-spherical cell shape, resonant type dynamics, and signal averaging may all play a role in the transduction of low-amplitude EMF effects in biological systems.

Direct modulation of osteoblastic activity with estrogen.

Estrogens play an important but poorly understood role in the maintenance of skeletal mass. Whereas the mechanisms of estrogen action on bone may be complex, the finding that osteoblasts express estrogen receptors suggests that this class of hormones exerts direct effects on bone cells. To understand how estrogens regulate osteoblastic function, the physiologically active estrogen metabolite 17 beta-estradiol was tested to determine its effects on the well characterized murine osteoblastic cell-line MC3T3-E1. Experiments were designed to identify the effects of estrogen on osteoblastic activities associated with both the formation and the resorption of bone. Estrogen treatment coordinately increased DNA content and alkaline phosphatase activity in MC3T3-E1 cells as much as twofold. The stimulatory effect on alkaline phosphatase was stereospecific, dose-dependent between 0.1 and ten nanomolar, and dependent on the time in culture when the hormone was administered. The effect was also persistent, since alkaline phosphatase activity remained elevated for several days after withdrawal of the hormone. Estrogen increased the levels of messenger RNA for alkaline phosphatase and type-I collagen as well, and these effects also persisted after removal of the hormone. The levels of messenger RNA for osteopontin, another bone-matrix protein, were only slightly affected by estrogen. Finally, estrogen inhibited the activation of adenylate cyclase by three osteotropic agents known to stimulate the resorption of bone: parathyroid hormone, prostaglandin E2, and the beta-adrenergic agonist isoproterenol. Thus, estrogen promoted the expression of traits associated with the formation of bone while reducing cellular responsiveness to hormones that may trigger the resorption of bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteotropic agents induce the differential secretion of granulocyte-macrophage colony-stimulating factor by the osteoblast cell line MC3T3-E1.

Osteoblasts play a central role in the regulation of bone remodeling. Not only are they responsible for the formation of new bone, but they also regulate bone resorption. These cells also exert regulatory influences outside the bone in that they are able to regulate hematopoiesis. However, obtaining pure populations of osteoblasts devoid of contaminating cell types remains problematic. One approach to this problem is the use of cloned osteoblastic cell lines. To this end we have used MC3T3-E1, a cloned murine osteoblast cell line of C57BL/6 origin. We report that MC3T3-E1 cells respond to lipopolysaccharide (LPS) and, to a lesser extent, parathyroid hormone (PTH) by the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). However, 1,25-(OH)2D3, a potent activator of osteoblasts, fails to induce these cells to secrete GM-CSF. These results suggest that MC3T3-E1 cells respond to osteotropic agents in a hierarchical fashion. Secretion of GM-CSF is not constitutive but rather requires active induction of the cells. MC3T3 cells fail to secrete detectable levels of interleukin-2 (IL-2), IL-3, or IL-4, regardless of whether or not the cells are activated. The data indicate that MC3T3-E1 cells secrete cytokines in response to osteotropic agents in a way similar to that of normal primary osteoblasts. Therefore, MC3T3-E1 cells may serve as a good in vitro model for primary osteoblasts.