Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.

Combined in vivo and in silico investigations of activation of glycolysis in contracting skeletal muscle.

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.

Unchanged muscle fiber conduction velocity relates to mild acidosis during exhaustive bicycling.

Muscle fiber conduction velocity (MFCV) has often been shown to decrease during standardized fatiguing isometric contractions. However, several studies have indicated that the MFCV may remain constant during fatiguing dynamic exercise. It was investigated if these observations can be related to the absence of a large decrease in pH and if MFCV can be considered as a good indicator of acidosis, also during dynamic bicycle exercise. High-density surface electromyography (HDsEMG) was combined with read-outs of muscle energetics recorded by in vivo (31)P magnetic resonance spectroscopy (MRS). Measurements were performed during serial exhausting bouts of bicycle exercise at three different workloads. The HDsEMG recordings revealed a small and incoherent variation of MFCV during all high-intensity exercise bouts. (31)P MRS spectra revealed a moderate decrease in pH at the end of exercise (~0.3 units down to 6.8) and a rapid ancillary drop to pH 6.5 during recovery 30 s post-exercise. This additional degree of acidification caused a significant decrease in MFCV during cycling immediately after the rest period. From the data a significant correlation between MFCV and [H(+)] ([H(+)] = 10(-pH)) was calculated (p < 0.001, Pearson's R = -0.87). Our results confirmed the previous observations of MFCV remaining constant during fatiguing dynamic exercise. A constant MFCV is in line with a low degree of acidification, considering the presence of a correlation between pH and MFCV after further increasing acidification.

Computational modeling of mitochondrial energy transduction.

Mitochondria are the power plant of the heart, burning fat and sugars to supply the muscle with the adenosine triphosphate (ATP) free energy that drives contraction and relaxation during each heart beat. This function was first captured in a mathematical model in 1967. Today, interest in such a model has been rekindled by ongoing in silico integrative physiology efforts such as the Cardiac Physiome project. Here, the status of the field of computational modeling of mitochondrial ATP synthetic function is reviewed.

Varying Ti-6Al-4V surface roughness induces different early morphologic and molecular responses in MG63 osteoblast-like cells.

Osteoblast response to Ti implants depends not only on the chemistry of the implant but also on the physical properties of the implant surface, such as microtopography and roughness. This study was undertaken to examine early changes in cell morphology and gene expression during the early phase of osteoblast interaction with titanium alloy (Ti-6Al-4V) surfaces of two different roughnesses. MG63 osteoblast-like cells were cultured for 2, 6, 24, and 72 h on smooth (Ra=0.18+/-0.03 microm) and rough (Ra=2.95+/-0.23 microm) Ti-6Al-4V surfaces. Changes in cell proliferation were assessed by measuring cell number after 72 h in culture. Morphological characteristics were observed by scanning electron microscopy after 2, 6, and 24 h of culture. Changes in gene expression for extracellular signal-regulated kinase 2 (Erk2), type I collagen (alpha2[I] collagen), phospholipase C-gamma2 (Plc-gamma2), and beta-actin were measured by RT-PCR after 6 and 24 h in culture. Cell number was significantly higher on the smooth surface. In scanning electron micrographs, cells on smooth Ti-6Al-4V were spherical and raised up from the surface after 2 h in culture. In contrast, cells on the rough surface adopted an irregular, elongated shape that spanned across pits in the surface. At 24 h, cells on the smooth surface had flattened, become elongate, and covered the surface. In contrast, cells on the rough surface appeared more differentiated in shape and the margins of the cells were irregular, with many processes extending out, following the contour of the surface. Of the genes examined, only Erk2 and beta-actin showed a change in expression with surface roughness. Both genes were upregulated (p<0.05) on the rough surface at 6 h. These results indicate that Ti-6Al-4V surface roughness affects osteoblast proliferation, morphology, and gene expression, and that these effects can be measured after periods as short as 2-6 h.

Effect of biofluid environment on the dissolution and flexural strength of calcium phosphate bone cements.

This study investigates the dissolution of calcium (Ca2+), phosphorus (P5+), and the transverse strength of commercially available calcium phosphate (CaP) bone cements after immersion in fetal bovine serum and a tissue fluid substitute. It was observed that although a continual increase in Ca2+ and P5+ dissolution was detected in all three test media throughout the 21-day period, no statistical difference existed in the overall Ca2+ release after incubating the cements in the three different media. However, P5+ release after immersion in Tris solution (0.37 +/- 0.02 microgram/mm2) and fetal bovine serum solution (0.347 +/- 0.06 microgram/mm2) was significantly higher when compared with P5+ released (0.03 +/- 0.002 microgram/mm2) in tissue fluid substitute. In addition, no significant difference in transverse strength was observed for samples immersed in the three solutions during the 21-day period. However, the transverse strength for immersed CaP cement bars at 37 degrees C was statistically greater than non-immersed bars set aside at room temperature for the 21-day period (7.78 +/- 1.82 N and 3.19 +/- 0.93 N, respectively). It was concluded from this study that the transverse strength of the CaP bone cements was not significantly affected by the dissolution process but by the temperature at which the bone cement was exposed.

Cytotoxicity of poly(96L/4D-lactide): the influence of degradation and sterilization.

The cytotoxicity of poly(96L/4D-lactide) (PLA96), and of its accumulated degradation products, was investigated following different sterilization methods and pre-determined heat-accelerated degradation intervals. PLA96 samples sterilized by either steam, ethylene oxide, or gamma irradiation were left untreated (S0 samples), or were degraded for 30 h or 60 h (S30 and S60 samples) at 90 degrees C in water. Extracts of the samples and of the remaining degradation fluids (F30 and F60) were prepared. The toxicity of both unfiltered and filtered extracts was analyzed in a cell growth inhibition (CGI) assay and a lactate dehydrogenase (LDH) leakage assay. Physical analysis of the extracted samples and of the degradation fluids also was performed. The S0 extracts demonstrated no significant CGI. The CGI of the S30 extracts ranged from 37 to 78%, whereas the CGI of the S60 extracts ranged from 6 to 33%. The CGI of the F30 extracts ranged from 19 to 38% and the CGI of the F60 extracts was 98 to 123%. The LDH leakage assay only showed a high response to the unfiltered F60 extracts. Neither sterilization nor filtration appeared to influence the cytotoxicity of the extracts. Particle accumulation, however, might affect cell membrane permeability resulting in LDH leakage. The results of this study suggest that the cytotoxicity of PLA96 is related to the pH and possibly the osmolarity of the tested extracts. The pH and osmolarity, in turn, may depend on variations in the amounts of solubilized lactic acid and oligomers. These variations appear to result from degradation stage-dependent differences in crystallinity, molecular weight and molecular weight distribution of the PLA96 samples.

The future of biodegradable osteosyntheses.

In the last 3 decades, much progress has been made in the development of biodegradable osteosyntheses. Despite this progress, these materials are still only used in small numbers, and the scope of their application has been limited. The limitations of biodegradable osteosyntheses mainly are related to problems with their mechanical properties and, in particular, biocompatibility. These problems need to be solved so that biodegradable osteosyntheses can perform up to their full potential and thus, eventually, make their general clinical application routine. This paper presents a historical perspective on the development of biodegradable osteosyntheses, discusses the successful developmental achievements and the still-existing problems, and gives a perspective on their future development.

Iron-dependent generation of free radicals: plausible mechanisms in the progressive deterioration of the temporomandibular joint.

PURPOSE: The purposes of this study were 1) to determine whether iron concentrations detected in temporomandibular joint (TMJ) lavage fluid samples obtained from symptomatic patients are sufficient to catalyze the degradation of specific extracellular matrix (ECM) molecules in vitro, and 2) to provide evidence of oxidative stress in symptomatic TMJs by the detection of protein carbonyls in lavage fluids. PATIENTS AND METHODS: Iron concentrations in TMJ lavage samples (19 joints in 14 patients) were determined colorimetrically, and the ability of the sample to produce free radicals in the presence of hydrogen peroxide was determined with the chromogen 2,2'-azinobis (3-ethylbenzothizoline-6-sulfonic acid), diammonium salt (ABTS). The presence of oxidized proteins was measured fluorimetrically using Bodipy FL hydrazide (Molecular Probes, Eugene, OR). Degradation of fibronectin was visualized by Western blot. Relative susceptibilities of fibronectin and collagen I to free radical cleavage were measured with the Fenton reaction. RESULTS: Redox-active iron concentration in lavage samples was found to be as high as 3.66 micromol/L. A 70-kd protein band, presumed to be albumin, was found to contain higher levels of carbonyls than peripheral serum albumin, which correlated with a greater degree of oxidative damage. Fibronectin was found to be more susceptible than collagen I to free radical degradation, and fragments of the former were found in the lavage. The TMJ lavage fluid was capable of producing free radicals in the presence of peroxide. CONCLUSION: Circumstantial evidence is provided that the presence of modified and cleaved proteins isolated from lavage of symptomatic TMJs may have been subjected to oxidative stress.

Particulate retrieval of hydrolytically degraded poly(lactide-co-glycolide) polymers.

This article describes a technique for the retrieval of polymeric particulate debris following advanced hydrolytic in vitro degradation of a biodegradable polymer and presents the results of the subsequent particle analysis. Granular 80/20 poly(L-lactide-co-glycolide) (PLG) was degraded in distilled, deionized water in Pyrextrade mark test tubes at 80 degrees C for 6 weeks. Subsequently, a density gradient was created by layering isopropanol over the water, followed by a 48-h incubation. Two opaque layers formed in the PLG tubes, which were removed and filtered through 0.2-micrometer polycarbonate membrane filters. In addition, Fourier transform IR spectroscopy (FTIR) was performed to confirm the presence of polymer in the removed layers. The filters were gold sputter coated, and scanning electron microscopy (SEM) images were made. FTIR analysis confirmed that the removed material was PLG. SEM images of the extracts from the upper (lowest density) opaque layer showed a fine, powderlike substance and globular structures of 500-750 nm. The SEM images of the lower (highest density) opaque layer showed particles with a crystalline-like morphology ranging in size from 4 to 30 micrometer. Particulate PLG debris generated with the described technique can be useful for further studies of its biological role in complications associated with poly(alpha-hydroxy)ester implants. This study shows the presence of very persistent nano- and microparticles in the degradation pathway of PLG.

Reconstruction of bone using calcium phosphate bone cements: a critical review.

The calcium phosphate cements (CPCs) are rapidly emerging as a new technology in craniofacial surgery and will soon impact many areas of orthopedic and maxillofacial reconstructive surgery as well. These materials are, in many ways, substantially different from the previously marketed dense, crystalline, hydroxyapatite (HA) ceramic materials of the 1980s. The CPCs are blends of amorphous and crystalline calcium phosphate compounds and set to produce HA. These materials 1) have x-ray diffraction spectra similar to the mineral phase of bone, 2) set endothermically at body temperature, 3) are capable of being injected into fractures or bone defects, 4) have compressive strengths equal to or greater than bone, 5) form chemical bonds to the host bone, and 6) may exhibit osteoconductive properties. This review provides an overall commentary on the different types of CPCs, emphasizing those materials currently on the market or soon to emerge in the marketplace.

The use of the Naugle orbitometer in maxillofacial trauma.

BACKGROUND AND OBJECTIVES: Objective measuring of globe position is not a universal practice in the management of orbital trauma. Few studies in the literature advocate its routine use. METHODS AND MATERIALS: The Hertel exophthalmometer is the most widely used instrument; however, in trauma involving the lateral orbital rim (e.g., in zygoma fractures), the results are inaccurate because the displacement of the zygomatic bone interferes with its reference point on the lateral orbital rim. A more recent measuring device, the Naugle orbitometer, was introduced in 1992. It uses the superior orbital rim (frontal bar) and inferior orbital rim (malar eminence) as reference points. RESULTS AND/OR CONCLUSIONS: This article reports experience with this instrument in objective measuring the position of the globe in orbital trauma. These measurements are used 1) to monitor fractures that may not require repair but should be followed and observed for dystopia or enophthalmos, 2) to determine the adequacy of fracture repair, and 3) to determine the volume adjustment required for correcting enophthalmos. Future studies will be directed to compare the accuracy of Naugle and Hertel exophthalmometers.

Detection and preliminary characterization of matrix metalloproteinase activity in temporomandibular joint lavage fluid.

In this study, lavage fluid was fractionated from the superior joint space in patients with temporomandibular joint (TMJ) dysfunction. A hide powder azure protease assay was used to assess protease activity in lavage fluid. No correlation between a patient's pain and the level of protease activity was demonstrated. Latent as well as active proteases were detected in the sample lavage fluid. Latent matrix metalloproteinases (MMPs) were activated using trypsin. Stromelysin-1 was detected in an active form in lavage fluid by immunozymography. The presence of high molecular weight species with protease activity was also demonstrated. This study validates the presence of stromelysin-1 as well as other MMPs in TMJ lavage fluid and proposes a mechanism for their physiologic activation.

Oxidative stress and degenerative temporomandibular joint disease: a proposed hypothesis.

The molecular events that underlie degenerative temporomandibular joint diseases are poorly understood. Recent studies have provided evidence that a variety of molecular species, including cytokines, matrix degrading enzymes, neuropeptides, and arachidonic acid catabolites may be involved. This paper advances the theory that mechanical stresses lead to the accumulation of damaging free radicals in affected articular tissues of susceptible individuals. This condition is called oxidative stress. The authors postulate mechanisms that may be involved in the production of free radicals in the temporomandibular joint and in the subsequent induction of molecular events that may amplify damage of articular tissues initiated by free radicals. If the proposed model is correct, then future therapeutic strategies directed at the control of oxidative stress could be effective in the management of degenerative temporomandibular joint diseases.

Presence of denatured hemoglobin deposits in diseased temporomandibular joints.

PURPOSE: The purpose of this study was to test the hypotheses that hemoglobin recovered by arthrocentesis of the superior joint space of symptomatic human temporomandibular joints (TMJs) is "old" hemoglobin that was not iatrogenically introduced by the arthrocentesis procedure and that it exists primarily in a non-native or denatured conformation state that may be sufficient to catalyze a reaction leading to the formation of damaging free radicals. PATIENTS AND METHODS: Twelve patients diagnosed with a unilateral articular disk displacement with TMJ arthralgia were included in this study. A superior joint space arthrocentesis was performed in the affected TMJ, and outflow lavage volumes were collected in serial 2-mL fractions. alpha-Hemoglobin/albumin ratios were determined for each collected fraction by densitometric analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, 3,3',5,5'-tetramethylbenzidine (TMB) assays were used to determine the conformation state of the recovered hemoglobin. RESULTS: High alpha-hemoglobin/albumin ratios relative to that of serum (at least 10 times greater) were observed in several collected fractions of TMJ lavage fluid in all subjects studied. Because the tissue half-life of hemoglobin is significantly longer than that of albumin, these findings indicate that much of the hemoglobin recovered by arthrocentesis of symptomatic TMJs represents "old" hemoglobin that was present in the joint before the procedure. Furthermore, based on reactivity in the TMB assay, we estimate that up to 89% of the alpha-hemoglobin present in TMJ lavage fluid samples exists in a denatured state. CONCLUSIONS: These results indicate that a significant amount of hemoglobin recovered by arthrocentesis of symptomatic TMJs exists in a denatured state and was present in the joint before arthrocentesis. Recent studies suggest that denatured hemoglobin may contribute redox active iron that can catalyze a reaction, leading to the formation of damaging free radicals. Such a process may represent one of the earliest molecular events involved in the pathogenesis of degenerative TMJ disease.

Elution of proteins by continuous temporomandibular joint arthrocentesis.

PURPOSE: The purpose of this study was to determine whether specific proteins recovered from human temporomandibular joints (TMJs) by superior space arthrocentesis are eluted at different outflow volumes. MATERIALS AND METHODS: Twenty subjects with unilateral TMJ pain and restricted mandibular range of motion underwent superior space arthrocentesis of the affected TMJ. Sixteen serial fractions of the arthrocentesis outflow volume were collected for analysis. The protein content of each fraction was determined by a BCA protein assay and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, samples from each collected fraction were assayed for protease activity. RESULTS: The average amount of protein recovered in the total 32 mL of collected arthrocentesis fluid was 1.5 mg (0.72 to 2.1 mg). Significant differences (P = .03) in total protein recovered from arthrocentesis fluid were observed between males (0.824 +/- 0.43 mg/20 mL) and females (1.389 +/- 0.54 mg/20 mL). In general, protein concentration declined serially in collected TMJ lavage fluid fractions. Specific proteins and proteases detected in the lavage fluid were eluted at different outflow volumes. CONCLUSIONS: Although specific proteins are eluted from the TMJ at different outflow volumes during arthrocentesis, the procedure effectively reduces the protein concentration of the lavage fluid in a volume-dependent manner. Based on empirical assumptions, it is estimated that approximately 100 mL of total arthrocentesis volume is sufficient for a therapeutic lavage of the superior joint space of the human TMJ.