Kinetic study of biogas production from energy crops and animal waste slurry: effect of organic loading rate and reactor size.

Biogas production in agriculture is processed mostly continuously at mesophilic temperatures in completely stirred tank reactors. Therefore, reactor performance data were studied in long-term semi-continuous laboratory-scale experiments with maize silage, whole-crop rye silage and fodder beet silage as mono-substrate and cattle slurry at mesophilic temperatures. For calculation of biogas yield as function of the organic loading rate, a hyperbolic equation was developed on the base of a first-order reaction rate for substrate degradation. The biogas yield depends also on the maximum biogas yield, the concentration of volatile solids of the input, the density of the effluent, the density of the biogas and the reaction rate constant, which are all substrate- or process-specific. Values of the theoretical maximum biogas yield and the reaction rate constant were observed in the range 0.61-0.93 m3 per kg volatile solids and 0.032-0.316 d(-1), respectively. By means of the hyperbolic equation, the proportion of the biogas yield from the maximum can be calculated for the first and a second reactor which also depends on the volume of each reactor.

Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis.

AIMS: The Archaea diversity was evaluated in an agricultural biogas plant supplied with cattle liquid manure and maize silage under mesophilic conditions. METHODS AND RESULTS: Two different genes (16S rRNA; methyl-coenzyme-M-reductase, MCR) targeted by three different PCR primer sets were selected and used for the construction of three clone libraries comprising between 104 and 118 clones. The clone libraries were analysed by restriction fragment polymorphism (RFLP). Between 11 and 31 operational taxonomic units (OTUs) were detected and assigned to orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. Over 70% of all Archaea OTUs belong to the order Methanomicrobiales which mostly include hydrogenotrophic methanogens. Acetotrophic methanogens were detected in minor rates. Similar relative values were obtained by a quantitative real-time PCR analysis. CONCLUSIONS: The results implied that in this biogas plant the most of the methane formation resulted from the conversion of H(2) and CO(2). SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports, for the first time, a molecular analysis of the archaeal community in this type of agricultural biogas plants. Therein the hydrogenotrophic methanogenesis seems to be the major pathway of methane formation. These results are in contrast with the common thesis that in biogas fermentations the primary substrate for methanogenesis is acetate.

Mechanical properties of 18 different AO bone plates and the clamp-rod internal fixation system tested on a gap model construct.

OBJECTIVE: To compare the stiffness and strength of AO bone plates (DCP, LC-DCP, VCP, RCP, and LP) and the Clamp-Rod Internal Fixation System (CRIF). STUDY DESIGN: In vitro. SAMPLE SIZE: 12 individual implants of 18 plate dimensions and four sizes of CRIF, each corresponding to 2.0, 2.4/2.7, 3.5, or 4.5 mm screw sizes. METHODS: Implant-constructs of each plate and CRIF were created using Canevasit rods as a bone substitute in an unstable gap fracture model. Six implant-constructs of each type were tested under single cycle four-point bending loading, and six were tested under single cycle torsional loading until permanent plastic deformation occurred. RESULTS: Torsional stiffness and yield load of the DCP were always significantly greater than the CRIF within the same group. Bending properties of the 2.0 DCP were not significantly different to the 2.0 CRIF. The 2.7 DCP had significantly higher bending values than the 2.7 CRIF. The bending stiffness of the 3.5 DCP and 4.5 DCP was significantly less than their CRIF counterparts. While the bending yield load of the 3.5 DCP was significantly greater than the 3.5 CRIF, the bending yield load of the 4.5 DCP was significantly less than the 4.5 CRIF. CONCLUSION: A weakness was found in the torsional resistance of the CRIF constructs compared to the DCP constructs. CLINICAL SIGNIFICANCE: Bone holding power and applied screw torque should be considered when using the CRIF system in clinical application.

Factors affecting the stability of screws in human cortical osteoporotic bone: a cadaver study.

We investigated several factors which affect the stability of cortical screws in osteoporotic bone using 18 femora from cadavers of women aged between 45 and 96 years (mean 76). We performed bone densitometry to measure the bone mineral density of the cortical and cancellous bone of the shaft and head of the femur, respectively. The thickness and overall bone mass of the cortical layer of the shaft of the femur were measured using a microCT scanner. The force required to pull-out a 3.5 mm titanium cortical bone screw was determined after standardised insertion into specimens of the cortex of the femoral shaft. A significant correlation was found between the pull-out strength and the overall bone mass of the cortical layer (r(2) = 0.867, p < 0.01) and also between its thickness (r(2) = 0.826, p < 0.01) and bone mineral density (r(2) = 0.861, p < 0.01). There was no statistically significant correlation between the age of the donor and the pull-out force (p = 0.246), the cortical thickness (p = 0.199), the bone mineral density (p = 0.697) or the level of osteoporosis (p = 0.378). We conclude that the overall bone mass, the thickness and the bone mineral density of the cortical layer, are the main factors which affect the stability of a screw in human female osteoporotic cortical bone.

BRIGEP--the BRIDGE-based genome-transcriptome-proteome browser.

The growing amount of information resulting from the increasing number of publicly available genomes and experimental results thereof necessitates the development of comprehensive systems for data processing and analysis. In this paper, we describe the current state and latest developments of our BRIGEP bioinformatics software system consisting of three web-based applications: GenDB, EMMA and ProDB. These applications facilitate the processing and analysis of bacterial genome, transcriptome and proteome data and are actively used by numerous international groups. We are currently in the process of extensively interconnecting these applications. BRIGEP was developed in the Bioinformatics Resource Facility of the Center for Biotechnology at Bielefeld University and is freely available. A demo project with sample data and access to all three tools is available at https://www.cebitec.uni-bielefeld.de/groups/brf/software/brigep/. Code bundles for these and other tools developed in our group are accessible on our FTP server at ftp.cebitec.uni-bielefeld.de/pub/software/.

Osteosynthesis of supracondylar humerus fractures in children: a biomechanical comparison of four techniques.

BACKGROUND: Crossed k-wire osteosynthesis is a widely used procedure for displaced supracondylar humerus fractures in children, but the rate of secondary displacements is up to 31%. Alternative techniques including casts, elastic stable intramedullary nailing, and the fixateur extern, have been used, but there are no biomechanical data comparing these methods. We developed a biomechanical model to compare four osteosynthesis techniques for stabilizing supracondylar humerus fractures in children. METHODS: An osteotomy to simulate a fracture was made in a total of 32 adult cadaver humeri. The pseudofractures were then stabilized by crossed k-wires, elastic nailing, a fixateur extern with either k-wires, or Schanz screws. We measured the stiffness values in flexion and extension and torsion with static loading. The movements in cyclic loading were chosen to resemble the mechanism described in the development of a clinical cubitus varus. FINDINGS: No significant differences were found with static loading. With cyclic loading all methods showed an irreversible torsional deformation less than 20 degrees . Crossed k-wires and elastic nailing showed significantly lower reversible torsional deformation than the external fixateurs. INTERPRETATION: Our biomechanical data reveal that the crossed k-wires have the highest stiffness and lowest loss of reduction under cyclic loading. The external fixators proved to be good alternatives.

Systematic review: isocaloric ketogenic dietary regimes for cancer patients.

The efficacy and benefits of ketogenic diets (KD) have recently been gaining worldwide and remain a controversial topic in oncology. This systematic review therefore presents and evaluates the clinical evidence on isocaloric KD dietary regimes and reveals that evidence supporting the effects of isocaloric ketogenic dietary regimes on tumor development and progression as well as reduction in side effects of cancer therapy is missing. Furthermore, an array of potential side effects should be carefully considered before applying KD to cancer patients. In regard to counseling cancer patients considering a KD, more robust and consistent clinical evidence is necessary before the KD can be recommended for any single cancer diagnosis or as an adjunct therapy.

Improved anchorage in osteoporotic vertebrae with new implant designs.

The goal of our study was to evaluate two newly developed implant designs and their behavior in terms of subsidence in lumbar vertebral bodies under cyclic loading. The new implants were evaluated in two different configurations (two small prototypes vs. one large prototype with similar load-bearing area) in comparison to a conventional screw-based implant (MACS TL). A pool of 13 spines with a total of 65 vertebrae was used to establish five testing groups of similar bone mineral density (BMD) distribution with eight lumbar vertebrae each. In additional to BMD assessment via dual-energy X-ray absorptiometry, cancellous BMD and structural parameters were determined using a new generation in vivo 3D-pQCT. The specimens were loaded sinusoidally in force control at 1 Hz for 1000 cycles at three load levels (100, 200, and 400 N). A survival analysis using the number of cycles until failure (Cox regression with covariates) was applied to reveal differences between implant groups. All new prototype configurations except the large cylinder survived significantly longer than the control group. The number of cycles until failure was significantly correlated with the structural parameter Tb.Sp. and similarly with the cancellous BMD for three of five implants. In both large prototypes the cycle number until failure significantly correlated with the preoperative distance to the upper endplates. Although the direct relationship between bone structure or density and mechanical breakage behavior cannot be conclusively proven, all the prototypes adapted for poor bone structure performed better than the comparable conventional implant.

Use of UITma DNA polymerase improves the PCR detection of rearranged immunoglobulin heavy chain CDR3 junctions.

The development of rapid PCR protocols for amplification of rearranged IgH gene sequences has greatly facilitated the identification of clonal IGH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias. However, the 15-35% incidence of false negative results with this approach has been a constant and unresolved problem. To assess the reliability of a previously published framework region 3 (FR3A) IgH-CDR3-PCR for detection of monoclonal IgH gene rearrangements we compared the PCR and Southern results in a series of 44 NHL and leukemias of B cell lineage showing a JH-rearrangement in Southern analysis with genomic DNA and hybridization with a IgH joining region (JH) probe. IgH-CDR3 regions were amplified using DNA extracted from clinical specimens by PCR using fluorescent dye-labeled consensus primers homologous to conserved regions within the variable (VH) and the joining (JH) gene segments. The PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. With commonly used DNA polymerases monoclonal IgH-CDR3 junctions were identified in 36/44 samples (82%). However, in the remaining eight cases (18%) with pathohistologically clearly demonstrated B cell malignancies which were also monoclonal on JH-Southern analysis, monoclonality could be demonstrated by FR3A-IgH-CDR3-PCR only with the proofreading UITma DNA polymerase. In four of these monoclonal VH--N--DH--N--JH junctions sequence analysis was performed which showed a point mutation in one and a single nucleotide deletion at the 3' terminus of the primer target site in the other case. In the remaining two cases no primer mismatches could be identified. Thus we conclude that the marked improvement of the PCR-detection rate of monoclonal IgH-CDR3 junctions was achieved at least in part due to the ability of UITma DNA polymerase to remove mismatched bases at the 3' terminus of the primers with respect to the target during the first amplification cycles. Our results suggest, that UITma is the DNA polymerase of choice for amplification of IgH-CDR3 junctions with consensus FR3A-VH- and JH-primers.

Identification and structural analysis of rearranged immunoglobulin heavy chain genes in lymphomas and leukemias.

The third complementarity determining region (CDR3) of the hypervariable domain of immunoglobulin heavy chain (IgH) genes represents a highly variable and clone-specific IgH-CDR3 sequences in 10 non-Hodgkin's lymphomas (NHL), five chronic lymphocytic leukemias (CLL) and five acute lymphoblastic leukemias (ALL) of B cell lineage. The IgH-CDR3 sequences were amplified using DNA extracted from clinical specimens (bone marrow, peripheral blood and fresh-frozen or paraffin-embedded lymph nodes) by a semi-nested PCR with consensus primers directed to conserved regions within the variable (VH) and the joining (JH) gene segments. In 17/20 samples (85%), a distinct IgH-CDR3 PCR product was obtained. Individual PCR products were sequenced after cloning. The nucleotide sequences of 134 randomly chosen recombinant vectors were determined demonstrating in 17/20 cases (85%) monoclonal VH-N-DH-N-JH junctions. Analysis of PCR products by temperature-gradient gel electrophoresis (TGGE) confirmed the specificity of the IgH-CDR3 PCR/sequencing results. Moreover, the combination of PCR/TGGE technology allowed the rapid and specific characterization of clonal IgH-CDR3 junctions in B cell proliferations by direct sequencing even in the presence of admixed polyclonal B cells.

Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.

The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene sequences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the present report we have explored the recently described improved strategy for assessment of clonality of rearranged immunoglobulin heavy chain (IgH) genes in more detail in a series of 101 B cell malignancies and 50 polyclonal controls. The assay is based on an IgH-PCR with an automated fluorescence-based strategy for PCR detection of IgH gene rearrangements. Third complementarity determining region (IgH-CDR3) sequences were amplified using fluorescent dye labeled consensus primers homologous to the corresponding variable (V[H]) and joining (J[H]) gene segments in combination with a thermostable proofreading DNA polymerase. PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. PCR findings obtained with the optimized IgH-CDR3-PCR assay showed an overall monoclonality detection rate of 97% (97 of 101 cases with B cell neoplasms). The specificity was 100% as determined by analysis of 50 controls, all of which gave polyclonal PCR results. We found a high rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular lymphoma, where a high rate of false negative results is frequently reported in the literature. In summary, we identified monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, immunocytoma and multiple myeloma. The results demonstrate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination with allele-specific primers the procedure may improve current experimental approaches to detect occult malginant B cells during initial staging and follow-up of NHL and ALL patients.

Localized gastric non-Hodgkin's lymphoma of high-grade malignancy in patients with pre-existing chronic lymphocytic leukemia or immunocytoma.

Analyses for clonality in cases of Richter's syndrome have provided evidence for a clonal evolution of high-grade lymphoma in most patients, while in others an independent cellular clone seems to exist in the secondary neoplasm. Richter's syndrome with an isolated high-grade lymphoma of the stomach has been rarely reported in patients with pre-existing B cell chronic lymphocytic leukemia (CLL). We investigated four cases of CLL or lymphoplasmacytoid immunocytoma (LPIC) with development of a localized high-grade B cell lymphoma in the stomach. Southern blotting showed different rearrangements of the immunoglobulin light and heavy chain genes in the tumor cells of the low-grade lymphoma and the gastric tumor in two cases. Comparison of the DNA sequences of the CDR3 region of the immunoglobulin genes revealed different clones in another case. By means of chromosomal in situ hybridization, trisomy 3 was detected in two cases of high-grade lymphoma of the stomach, but not in the cells of the associated low-grade tumor. Our findings indicate that high-grade non-Hodgkin's lymphomas arising localized in the stomach of patients with CLL or immunocytoma are not clonally related to the pre-existing low-grade lymphoma and, therefore indeed, present true secondary neoplasms.

Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas.

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.

Aspects of internal fixation of fractures in porotic bone. Principles, technologies and procedures using locked plate screws.

Fractures of the bones of elderly people occur more often and have a more important effect because of a generally diminished ability to coordinate stance and walking. These fractures occur at a lower level of load because of lack of strength of the porotic bone. Prompt recovery of skeletal support function is essential to avoid respiratory and circulatory complications in the elderly. To prevent elderly people from the risks of being bedridden, demanding internal fixation of fractures is required. The weak porotic bone and the high level of uncontrolled loading after internal fixation pose complex problems. A combination of several technical elements of design, application and aftercare in internal fixation are proposed. Internal fixators with locked screws improve the biology and the mechanics of internal fixation. When such fixators are used as elevated splints they may stimulate early callus formation because of their flexibility, the limit of flexibility being set by the demands of resistance and function of the limb. Our own studies of triangulation of locked screws have demonstrated their beneficial effects and unexpected limitations.

Platelets from flowing blood attach to the inflammatory chemokine CXCL16 expressed in the endothelium of the human vessel wall.

Endothelial chemokine CXC motif ligand 16 (CXCL16) expression is associated with atherosclerosis, while platelets, particularly those attaching to atherosclerotic plaque, contribute to all stages of atherosclerotic disease. This investigation was designed to examine the role of CXCL16 in capturing platelets from flowing blood. CXCL16 was expressed in human atherosclerotic plaques, and lesion severity in human carotid endarterectomy specimens was positively correlated with CXCL16 levels. CXCL16 expression in plaques was co-localised with platelets deposited to the endothelium. Immobilised CXCL16 promoted CXCR6-dependent platelet adhesion to the human vessel wall, endothelial cells and von Willebrand factor during physiologic flow. At low shear, immobilised CXCL16 captured platelets from flowing blood. It also induced irreversible platelet aggregation and a rise in intra-platelet calcium levels. These results demonstrate that endothelial CXCL16's action on platelets is not only limited to platelet activation, but that immobilised CXCL16 also acts as a potent novel platelet adhesion ligand, inducing platelet adhesion to the human vessel wall.

Amperometric biosensor for in vivo glucose sensing based on glucose oxidase immobilized in a redox hydrogel.

A potentially implantable glucose sensor, based on glucose oxidase immobilized in a redox hydrogel, is considered. The redox hydrogel consisted of glucose oxidase immobilized in a cross-linkable poly(vinylpyridine) complex of [Os(bis-bipyridine)2Cl]+1/+2 that communicates electrically with the flavin adenine dinucleotide (FADH2) redox centres of the glucose oxidase. The implantable electrode consisted of a Teflon insulated platinum wire (0.25 mm diameter) which was coated at the tip with a cross-linked redox polymer/glucose oxidase film and covered with a thin layer of polycarbonate. In a three-electrode system at +400 mV (Ag/AgCl) the response to increasing glucose concentrations in isotonic phosphate buffer and human plasma was approximately 0.2-0.3 nA/mM, linear in the range between 0 and 15 mM glucose. No oxygen dependence was observed. To determine the in vivo performance, the electrode was implanted into the subcutaneous tissue of a dog. The sensor currents after an oral glucose load paralleled the plasma glucose measurements, with a time lag of 10 min. Three-day implantations in cultured cells showed that the electrode did not affect the growth and differentiation of cell monolayers.

The function of a hydrogen peroxide-detecting electroenzymatic glucose electrode is markedly impaired in human sub-cutaneous tissue and plasma.

Electroenzymatic glucose sensors implanted into sub-cutaneous (s.c.) tissue of human subjects and experimental animals exhibit lower sensitivities to glucose than in buffer solutions before implantation. The mechanism of the decrease of sensitivity is not known. Sensors used in this study were fabricated from platinum wires (diameter 0.125 mm) with covalently bound glucose oxidase at the tip of the wire. After coating the tip with polyurethane, wires were placed into 27 gauge steel needles. Sensors were operated potentiostatically at 700 mV against Ag/AgCl pseudo-reference electrodes. These sensors were implanted s.c. in 6 diabetic patients for 7 h. In 4 patients, sensors were responsive to successive increases of plasma glucose levels. Mean sensitivity to glucose in s.c. tissue was 29% of in vitro sensitivity. In 2 patients there was a sudden decrease of sensor currents, unrelated to glucose, shortly after implantation. Sensors were inhibited in human plasma to a similar extent. When sensors were exposed to native plasma and to plasma ultrafiltrate (mol. wt. < 10 kDa) for 10 h, identical decreases of signals were found. Exposure to dialysed plasma (mol. wt. > 12 kDa) caused much less decrease of sensor signals. Losses of sensor sensitivities to glucose in s.c. tissue and in plasma were totally reversible upon re-exposure of sensors to buffer solutions. We conclude that sensor inactivation in plasma and possibly in s.c. tissue is caused by low molecular weight substances not retained by the polyurethane membrane.

Development of a mechanical testing system for a mandibular distraction wound.

The purpose of this study was to develop a mechanical testing system to estimate stiffness of an experimental porcine mandibular distraction osteogenesis (DO) wound. The system was designed to function without changing the morphology of the healing mandible. A customized jig was designed to allow cantilever-bending tests of the Yucatan mini-pig hemi-mandible. Experimental and control hemi-mandibles were placed in the jig and the proximal segment was secured. A material testing unit applied progressively increasing downward force on the pre-molar occlusal surface. The maximum force applied was 0.030 kN. The stiffness value for each hemi-mandible was represented by the slope of the plot of force (kN) vs displacement (mm). Radiographs were taken before and after mechanical testing to demonstrate any gross morphologic changes or identifiable fractures across the distraction wound. A total of 24 mini-pigs underwent DO of the right mandible with 0-day latency and distraction rates of 1, 2, and 4 mm per day resulting in a 12 mm gap. At the completion of 0, 8, 16, and 24 days of neutral fixation, two animals for each of three different distraction rates were sacrificed for mechanical testing. Stiffness of control hemi-mandibles ranged between 0.018 and 0.317 kN/mm (median 0.063; mean 0.099 +/- 0.080). Stiffness of experimental hemi-mandibles ranged between 0 and 0.025 kN/mm (median 0.004; mean 0.005). The subset that was tested at the end of neutral fixation had stiffness between 0.005 and 0.025 (median 0.011; mean 0.012 +/- 0.011). No morphologic changes were evident on the X-rays after testing. The results indicate that the cantilever-bending model is useful for testing stiffness of an experimental mandibular DO wound without destroying its morphology.

Correlation of biomechanical stiffness with plain radiographic and ultrasound data in an experimental mandibular distraction wound.

The aim of this study was to determine the stiffness of a porcine mandibular distraction wound at the end of neutral fixation.Twenty-four Yucatan minipigs underwent unilateral mandibular distraction: zero-day latency, rates of 1, 2, and 4 mm per day, distraction gap of 12 mm. Radiographic and ultrasound bone fill scores at 0, 8, 16 and 24 days of neutral fixation were used to evaluate interval healing. At sacrifice, biomechanical stiffness was determined using an Instron machine to produce a downward force of 2 mm/min at the premolar occlusal surface. Force (kilo-Newton, kN) and displacement (mm) were recorded at a rate of 10,000 points/second. Stiffness across the distraction wound, plain radiographic and ultrasound bone fill scores all increased with duration of neutral fixation and were highest for the 1 mm per day group at all time points. At the end of fixation, even in the most clinically stable group, stiffness across the distraction wound was only 25.5% of control.Despite clinical stability and high bone fill scores by X-ray and ultrasound, the distraction wound did not achieve normal stiffness at the end of neutral fixation in this model. This may have implications for the management of patients who undergo large movements by distraction.

Immunological detection of osteocalcin in meat and bone meal: a novel heat stable marker for the investigation of illegal feed adulteration.

A sandwich ELISA was developed for the detection of bovine meat and bone meal (BMBM) in feed, based on polyclonal rabbit antibodies raised against the synthetic N-terminal amino acid sequence 1-9 (YLDHWLGAP) of bovine osteocalcin. To set up a sandwich ELISA pair, a commercial mouse monoclonal capture antibody binding to a highly conserved epitope in the mid-fragment of the peptide was employed. It is shown that the bone marker osteocalcin is immunologically well detectable in BMBM extracts obtained by a simple EDTA-based procedure even in a sample heated up to 145°C. Furthermore, a genus-specific restriction of the major specificity to cattle and horse was possible. The observed bi-specificity is consistent with theoretical predictions. The assay sensitivity with bovine osteocalcin of 1 ng was sufficient to enable the detection of 0.1% BMBM in compound plant feed or fish meal, for which no cross reaction was observed. In general the quantification of osteocalcin in extracts is possible using a standard curve procedure with pure bovine osteocalcin.