Differential effects of altered patterns of movement and strain on joint cell behaviour and skeletal morphogenesis.

OBJECTIVE: There is increasing evidence that joint shape is a potent predictor of osteoarthritis (OA) risk; yet the cellular events underpinning joint morphogenesis remain unclear. We sought to develop a genetically tractable animal model to study the events controlling joint morphogenesis. DESIGN: Zebrafish larvae were subjected to periods of flaccid paralysis, rigid paralysis or hyperactivity. Immunohistochemistry and transgenic reporters were used to monitor changes to muscle and cartilage. Finite Element Models were generated to investigate the mechanical conditions of rigid paralysis. Principal component analysis was used to test variations in skeletal morphology and metrics for shape, orientation and size were applied to describe cell behaviour. RESULTS: We show that flaccid and rigid paralysis and hypermobility affect cartilage element and joint shape. We describe differences between flaccid and rigid paralysis in regions showing high principal strain upon muscle contraction. We identify that altered shape and high strain occur in regions of cell differentiation and we show statistically significant changes to cell maturity occur in these regions in paralysed and hypermobile zebrafish. CONCLUSION: While flaccid and rigid paralysis and hypermobility affect skeletal morphogenesis they do so in subtly different ways. We show that some cartilage regions are unaffected in conditions such as rigid paralysis where static force is applied, whereas joint morphogenesis is perturbed by both flaccid and rigid paralysis; suggesting that joints require dynamic movement for accurate morphogenesis. A better understanding of how biomechanics impacts skeletal cell behaviour will improve our understanding of how foetal mechanics shape the developing joint.

Within-guild dietary discrimination from 3-D textural analysis of tooth microwear in insectivorous mammals.

Resource exploitation and competition for food are important selective pressures in animal evolution. A number of recent investigations have focused on linkages between diversification, trophic morphology and diet in bats, partly because their roosting habits mean that for many bat species diet can be quantified relatively easily through faecal analysis. Dietary analysis in mammals is otherwise invasive, complicated, time consuming and expensive. Here we present evidence from insectivorous bats that analysis of three-dimensional (3-D) textures of tooth microwear using International Organization for Standardization (ISO) roughness parameters derived from sub-micron surface data provides an additional, powerful tool for investigation of trophic resource exploitation in mammals. Our approach, like scale-sensitive fractal analysis, offers considerable advantages over two-dimensional (2-D) methods of microwear analysis, including improvements in robustness, repeatability and comparability of studies. Our results constitute the first analysis of microwear textures in carnivorous mammals based on ISO roughness parameters. They demonstrate that the method is capable of dietary discrimination, even between cryptic species with subtly different diets within trophic guilds, and even when sample sizes are small. We find significant differences in microwear textures between insectivore species whose diet contains different proportions of 'hard' prey (such as beetles) and 'soft' prey (such as moths), and multivariate analyses are able to distinguish between species with different diets based solely on their tooth microwear textures. Our results show that, compared with previous 2-D analyses of microwear in bats, ISO roughness parameters provide a much more sophisticated characterization of the nature of microwear surfaces and can yield more robust and subtle dietary discrimination. ISO-based textural analysis of tooth microwear thus has a useful role to play, complementing existing approaches, in trophic analysis of mammals, both extant and extinct.

Virtual experiments, physical validation: dental morphology at the intersection of experiment and theory.

Computational models such as finite-element analysis offer biologists a means of exploring the structural mechanics of biological systems that cannot be directly observed. Validated against experimental data, a model can be manipulated to perform virtual experiments, testing variables that are hard to control in physical experiments. The relationship between tooth form and the ability to break down prey is key to understanding the evolution of dentition. Recent experimental work has quantified how tooth shape promotes fracture in biological materials. We present a validated finite-element model derived from physical compression experiments. The model shows close agreement with strain patterns observed in photoelastic test materials and reaction forces measured during these experiments. We use the model to measure strain energy within the test material when different tooth shapes are used. Results show that notched blades deform materials for less strain energy cost than straight blades, giving insights into the energetic relationship between tooth form and prey materials. We identify a hypothetical 'optimal' blade angle that minimizes strain energy costs and test alternative prey materials via virtual experiments. Using experimental data and computational models offers an integrative approach to understand the mechanics of tooth morphology.

What makes an accurate and reliable subject-specific finite element model? A case study of an elephant femur.

Finite element modelling is well entrenched in comparative vertebrate biomechanics as a tool to assess the mechanical design of skeletal structures and to better comprehend the complex interaction of their form-function relationships. But what makes a reliable subject-specific finite element model? To approach this question, we here present a set of convergence and sensitivity analyses and a validation study as an example, for finite element analysis (FEA) in general, of ways to ensure a reliable model. We detail how choices of element size, type and material properties in FEA influence the results of simulations. We also present an empirical model for estimating heterogeneous material properties throughout an elephant femur (but of broad applicability to FEA). We then use an ex vivo experimental validation test of a cadaveric femur to check our FEA results and find that the heterogeneous model matches the experimental results extremely well, and far better than the homogeneous model. We emphasize how considering heterogeneous material properties in FEA may be critical, so this should become standard practice in comparative FEA studies along with convergence analyses, consideration of element size, type and experimental validation. These steps may be required to obtain accurate models and derive reliable conclusions from them.

Finite element modelling of squirrel, guinea pig and rat skulls: using geometric morphometrics to assess sensitivity.

Rodents are defined by a uniquely specialized dentition and a highly complex arrangement of jaw-closing muscles. Finite element analysis (FEA) is an ideal technique to investigate the biomechanical implications of these specializations, but it is essential to understand fully the degree of influence of the different input parameters of the FE model to have confidence in the model's predictions. This study evaluates the sensitivity of FE models of rodent crania to elastic properties of the materials, loading direction, and the location and orientation of the models' constraints. Three FE models were constructed of squirrel, guinea pig and rat skulls. Each was loaded to simulate biting on the incisors, and the first and the third molars, with the angle of the incisal bite varied over a range of 45°. The Young's moduli of the bone and teeth components were varied between limits defined by findings from our own and previously published tests of material properties. Geometric morphometrics (GMM) was used to analyse the resulting skull deformations. Bone stiffness was found to have the strongest influence on the results in all three rodents, followed by bite position, and then bite angle and muscle orientation. Tooth material properties were shown to have little effect on the deformation of the skull. The effect of bite position varied between species, with the mesiodistal position of the biting tooth being most important in squirrels and guinea pigs, whereas bilateral vs. unilateral biting had the greatest influence in rats. A GMM analysis of isolated incisor deformations showed that, for all rodents, bite angle is the most important parameter, followed by elastic properties of the tooth. The results here elucidate which input parameters are most important when defining the FE models, but also provide interesting glimpses of the biomechanical differences between the three skulls, which will be fully explored in future publications.

Shape and mechanics in thalattosuchian (Crocodylomorpha) skulls: implications for feeding behaviour and niche partitioning.

Variation in modern crocodilian and extinct thalattosuchian crocodylomorph skull morphology is only weakly correlated with phylogeny, implying that factors other than evolutionary proximity play important roles in determining crocodile skull shape. To further explore factors potentially influencing morphological differentiation within the Thalattosuchia, we examine teleosaurid and metriorhynchid skull shape variation within a mechanical and dietary context using a combination of finite element modelling and multivariate statistics. Patterns of stress distribution through the skull were found to be very similar in teleosaurid and metriorhynchid species, with stress peaking at the posterior constriction of the snout and around the enlarged supratemporal fenestrae. However, the magnitudes of stresses differ, with metriorhynchids having generally stronger skulls. As with modern crocodilians, a strong linear relationship between skull length and skull strength exists, with short-snouted morphotypes experiencing less stress through the skull than long-snouted morphotypes under equivalent loads. Selection on snout shape related to dietary preference was found to work in orthogonal directions in the two families: diet is associated with snout length in teleosaurids and with snout width in metriorhynchids, suggesting that teleosaurid skulls were adapted for speed of attack and metriorhynchid skulls for force production. Evidence also indicates that morphological and functional differentiation of the skull occurred as a result of dietary preference, allowing closely related sympatric species to exploit a limited environment. Comparisons of the mechanical performance of the thalattosuchian skull with extant crocodilians show that teleosaurids and long-snouted metriorhynchids exhibit stress magnitudes similar to or greater than those of long-snouted modern forms, whereas short-snouted metriorhynchids display stress magnitudes converging on those found in short-snouted modern species. As a result, teleosaurids and long-snouted metriorhynchids were probably restricted to lateral attacks of the head and neck, but short-snouted metriorhynchids may have been able to employ the grasp and shake and/or 'death roll' feeding and foraging behaviours.

Cranial design and function in a large theropod dinosaur.

Finite element analysis (FEA) is used by industrial designers and biomechanicists to estimate the performance of engineered structures or human skeletal and soft tissues subjected to varying regimes of stress and strain. FEA is rarely applied to problems of biomechanical design in animals, despite its potential to inform structure-function analysis. Non-invasive techniques such as computed tomography scans can be used to generate accurate three-dimensional images of structures, such as skulls, which can form the basis of an accurate finite element model. Here we have applied this technique to the long skull of the large carnivorous theropod dinosaur Allosaurus fragilis. We have generated the most geometrically complete and complex FEA model of the skull of any extinct or extant organism and used this to test its mechanical properties and examine, in a quantitative way, long-held hypotheses concerning overall shape and function. The combination of a weak muscle-driven bite force, a very 'light' and 'open' skull architecture and unusually high cranial strength, suggests a very specific feeding behaviour for this animal. These results demonstrate simply the inherent potential of FEA for testing mechanical behaviour in fossils in ways that, until now, have been impossible.

Mechanisms determining course and outcome of diabetic patients who have had acute myocardial infarction.

PURPOSE: To review the pathogenic mechanism that lead to the poor prognosis of diabetic patients after myocardial infarction and to determine the efficacy of current interventions for myocardial infarction in these patients. DATA SOURCES: Search of the MEDLINE database from 1985 to 1995, using the keywords diabetes, myocardial infarction, and cardiomyopathy, and a search of the reference citations of relevant articles. STUDY SELECTION: Experimental and clinical studies on myocardial infarction in diabetic patients and basic research studies relevant to this topic. DATA SYNTHESIS: The excess in-hospital mortality of diabetic patients results primarily from an increased incidence of congestive heart failure. Several combined mechanisms reduce the compensatory ability of the noninfarcted myocardium; such mechanisms include preexisting congestive heart failure caused by diabetic cardiomyopathy, severe coronary artery disease, decreased vasodilatory reserve of epicardial and resistance arteries, and possibly abnormal metabolism of myocardial substrate. Late mortality results from increased reinfarction rates caused by the diffuse nature of the atherosclerotic disease and hypercoagulable state. Platelet hyperactivity, reduced fibrinolytic capacity, increased concentrations of hemostatic proteins, and endothelial dysfunction promote thrombosis at the site of plaque rupture. Autonomic neuropathy predisposes patients to ventricular arrhythmias. Thrombolytic agents, aspirin, beta-blockers, and angiotensin-converting enzyme inhibitors are effective in patients with diabetes. CONCLUSIONS: In the thrombolytic era, mortality rates of diabetic patients who have had acute myocardial infarction remain 1.5 to 2 times higher than those in nondiabetic patients. This increased mortality rate is caused by diverse mechanisms that affect myocardial function and blood supply and by the tendency toward thrombosis in diabetic patients. Current therapies for myocardial infarction are effective in these patients. Improved metabolic control may also decrease mortality rates.

Potential mechanisms promoting restenosis in diabetic patients.

Diabetes is associated with greater restenosis rates after successful balloon angioplasty. The metabolic alterations that occur as a result of hyperglycemia or hyperinsulinemia can accelerate many of the pathophysiologic processes that lead to restenosis. Diabetes results in endothelial dysfunction and accelerated platelet deposition, which increase the propensity to thrombosis. Several growth factors known to promote the restenosis process are overexpressed in the presence of hyperglycemia. Advanced glycosylation promotes inflammatory cell recruitment and smooth muscle cell proliferation. Many of the potential mechanisms promoting restenosis in diabetic patients can be ameliorated by improved metabolic control.

Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure.

In diabetes and ageing, glucose-derived advanced glycosylation endproducts (AGEs) cross-link proteins and cause vascular tissue damage. Elimination of circulating low-molecular weight AGE-modified molecules (LMW-AGEs) by the kidney is impaired in diabetic patients with end-stage renal disease, a group subject to accelerated atherosclerosis. We determined the effectiveness of current renal replacement treatments on elimination of serum LMW-AGEs in diabetic and non-diabetic patients with end-stage renal disease. Although diabetic patients receiving high-flux haemodialysis achieved 33% lower steady-state serum LMW-AGE than did those in conventional haemodialysis (p < 0.005), LMW-AGE concentrations remained 3.5-6 fold above normal, whether high-flux dialysis, conventional haemodialysis, or chronic ambulatory peritoneal dialysis were used. High-flux haemodialysis markedly reduced AGE during each treatment session (47.9% in the diabetic, p < 0.001 and 60.6% in the non-diabetic group, p < 0.001) but concentrations returned to pre-treatment range within 3 hours. In contrast, normal LMW-AGE concentrations were maintained in patients with functioning renal transplants. We found that LMW-AGEs with an apparent molecular weight of 2000-6000 circulate and retain strong inherent chemical reactivity--when exposed to collagen in vitro, up to 77% attached covalently to form AGE-collagen, and the AGE-crosslink inhibitor aminoguanidine completely inhibited this reaction. The results suggest that LMW-AGEs comprise a set of chemically-reactive molecules that are refractory to removal by current dialysis treatments. Through covalent reattachment onto vascular matrix or serum components, LMW-AGEs may exacerbate vascular pathology associated with end-stage renal disease.

Erectile function and penile blood pressure in diabetes mellitus.

Thirty-seven diabetic men selected to exclude the confounding effects of other medical illnesses and nondiabetic medications and 53 healthy controls underwent extensive psychosexual and medical evaluations and penile blood pressure assessments by ultrasonic Doppler measurement and mercury strain-gauge plethysmography. There was a significant negative correlation between age and the penile-brachial index (PBI) in the diabetic but not in the control group. The impotent diabetic group had significantly lower PBI than nondysfunctional diabetic and healthy control subjects. Diabetic type, complications, and adequacy of metabolic control were not statistically related to PBI. Although the PBI may not have diagnostic utility for individual patients, it may provide a valuable noninvasive physiologic measure of penile vascular changes in studies on the aged and the medically ill. The processes that mediate the interaction of diabetes and aging on penile blood pressure and erectile capacity deserve further investigation.

Diabetes mellitus and male sexual function: a controlled study.

There is an extensive clinical literature on the erectile disorders of diabetic men but a paucity of controlled studies that have taken into account the effects of age, concurrent illnesses and medication on sexual function. This investigation was carried out on 40 diabetic men free from other illness or drugs that could affect sexual capacity and 40 age-matched healthy control subjects. Each subject and his female partner underwent semistructured interviews and the men had comprehensive medical evaluations and polygraphic assessment of sleep and nocturnal penile tumescence in the sleep laboratory during three nights. In comparison to control subjects, diabetic patients reported significant decreases in sexual desire, subjective arousal, erectile capacity, coital frequency and sexual satisfaction. The diabetic group also had significant decrements in duration of rapid eye movement sleep and in frequency, duration and degree of nocturnal penile tumescent episodes. There were no differences between Type 1 (insulin-dependent) and Type 2 (non-insulin dependent) diabetic patients in prevalence of sexual problems or in nocturnal tumescent measures. Significant relations were observed between lack of metabolic control, diabetic complications and impaired nocturnal tumescence. Sexually non-dysfunctional diabetic men had significant nocturnal penile tumescence abnormalities. Diabetic men without coital failures may have a subclinical impairment in erectile function which, although of not significant magnitude to interfere with penetration, is reflected in nocturnal penile tumescent measures. This result raises a note of caution in the interpretation of the nocturnal penile tumescence test for the differential diagnosis of diabetic erectile impotence.

Diabetes, sleep disorders, and male sexual function.

The prevalence and role of sleep disorders in the sexual problems of diabetic patients remain unexplored. This study was conducted on 40 diabetic men carefully screened to exclude the confounding effects of other medical illnesses or drugs likely to impair sexual function and 40 age-matched healthy volunteers. They underwent an extensive psychosexual interview, medical and psychiatric evaluations, and three recorded nights in a sleep laboratory. Electroencephalogram, eye movements, muscle tone, and nocturnal penile tumescence were monitored continuously. Respiratory airflow and bilateral anterior tibialis recordings were obtained during the first sleep session. Diabetic men had significantly higher prevalences of respiratory and periodic leg movement disturbances during sleep. There was clinical, although not nocturnal penile tumescence, evidence suggesting that respiratory abnormalities during sleep are associated with erectile difficulties in diabetic men. Future studies should include blood oxygenation and respiratory effort measures to clarify the significance of sleep-related airflow disturbances in diabetic patients.

Hemoglobin-AGE: a circulating marker of advanced glycosylation.

Advanced glycosylation end products (AGEs) form spontaneously from glucose-derived Amadori products and accumulate on long-lived tissue proteins. AGEs have been implicated in the pathogenesis of several of the complications of aging and diabetes, including atherosclerosis and renal disease. With the use of recently developed AGE-specific antibodies, an AGE-modified form of human hemoglobin has been identified. Termed hemoglobin-AGE (Hb-AGE), this modified species accounts for 0.42 percent of circulating hemoglobin in normal individuals but increases to 0.75 percent in patients with diabetes-induced hyperglycemia. In a group of diabetic patients treated with the advanced glycosylation inhibitor aminoguanidine, Hb-AGE levels decreased significantly over a 1-month period. Hemoglobin-AGE measurements may provide an index of long-term tissue modification by AGEs and prove useful in assessing the contribution of advanced glycosylation to a variety of diabetic and age-related complications.

Advanced glycosylation end products in patients with diabetic nephropathy.

BACKGROUND: Glucose reacts nonenzymatically with proteins in vivo, chemically forming covalently attached glucose-addition products and cross-links between proteins. The excessive accumulation of rearranged late-glucose-addition products, or advanced glycosylation end products (AGEs), is believed to contribute to the chronic complications of diabetes mellitus. METHODS: To elucidate the relation of AGEs to diabetic complications, we used a radioreceptor assay to measure serum and tissue AGEs in diabetic (Types I and Type II) and nondiabetic patients with different levels of renal function. Serum AGEs were measured as a low-molecular-weight (less than or equal to 10 kd) peptide fraction and a high-molecular-weight (greater than 10 kd) protein fraction. RESULTS: The mean (+/- SD) AGE content of samples of arterial-wall collagen from 9 diabetic patients was significantly higher than that of samples from 18 nondiabetic patients (14.5 +/- 5.2 vs. 3.6 +/- 1.5 AGE units per milligram, P less than 0.001). Moreover, diabetic patients with end-stage renal disease had almost twice as much AGE in tissue as diabetic patients without renal disease (21.3 +/- 2.8 vs. 11.5 +/- 1.9 AGE units per milligram, P less than 0.001). The AGE levels in both serum fractions were elevated in the patients with diabetes, and the levels of AGE peptides correlated directly with serum creatinine (P less than 0.001) and inversely with creatinine clearance (P less than 0.005), suggesting that levels of AGE peptides increased with the severity of diabetic nephropathy. In six patients with diabetes who required hemodialysis, the levels of AGE peptides were five times higher than in eight normal subjects (82.8 +/- 9.4 vs. 15.6 +/- 3.4 AGE units per milliliter, P less than 0.001). In another group of diabetic patients the mean serum creatinine level, which decreased by 75 percent during a session of hemodialysis, whereas the level of AGE peptides decreased by only 24 percent. Serum levels of AGE peptides were normal in two patients with normal serum creatinine levels after renal transplantation. CONCLUSIONS: AGEs accumulate at a faster-than-normal rate in arteries and the circulation of patients with diabetes; the increase in circulating AGE peptides parallels the severity of renal functional impairment in diabetic nephropathy.

Randomized, controlled trial of diabetic patient education: improved knowledge without improved metabolic status.

We randomized 749 insulin-treated patients on the rolls of the Mount Sinai Medical Center Diabetes Clinic in a controlled trial of diabetic patient education; 345 agreed to participate, of whom 165 were assigned to the education group and 180 to the control group. Cognitive scores increased from 5.3 +/- 1.6 to 5.8 +/- 1.6 in the education group, but there was no change in the control group, whose score was 5.3 +/- 1.7 before and after the intervention (P = .0073). HbA1c fell from 6.8 +/- 2.1 to 6.1 +/- 2.0% in the education group and from 6.6 +/- 2.0 to 6.3 +/- 2.0% in the control group, an insignificant difference (P = .1995). The fasting blood glucose decreased from 223 +/- 94 to 179 +/- 73 mg/dl in the education group and from 199 +/- 81 to 185 +/- 76 mg/dl in the controls (P = .1983). Triglycerides, high- and low-density lipoprotein cholesterol, and insulin dosage also failed to show significant variation among groups. The foot-lesion score showed similar progression in the education and control groups. Neither diastolic nor systolic blood pressure showed significantly greater change in the education or the control group, with falls noted, particularly in diastolic pressures, in both patient groups. Differences between the groups were not significant for sick days, hospitalizations, emergency room visits, or outpatient visits. The sample sizes of the study and control populations were sufficiently large to detect a difference in means between the education and control groups in the HbA1c, the primary outcome variable, of greater than 1.0%, with alpha = .05 and a power of .95. Thus, our study suggests that patient education may not be an efficacious therapeutic intervention in most adults with insulin-treated diabetes mellitus.