Regional regenerative potential of meniscal cartilage exposed to recombinant insulin-like growth factor-I in vitro

It is well recognised that meniscal tears situated within the inner, avascular region do not heal. We investigated the potential effect of insulin-like growth factor-I (IGF-I) in promoting regeneration of meniscal tissue in the inner, middle and outer zones of the meniscus. Sheep menisci were harvested and monolayer cell cultures prepared. Various concentrations of IGF-I were used in the presence or absence of 10% fetal calf serum (FCS). We measured the uptake of radioactive thymidine, sulphur, and proline to assess cell proliferation and formation of extracellular matrix (ECM). IGF-I, in the presence or absence of FCS, increased the formation of DNA and ECM in all meniscal zones. However, the response of the cells from the avascular zone was greater than that from the vascular zone. Our findings indicate that fibrochondrocytes cultured from avascular meniscal tissue have the ability to regenerate when exposed to anabolic cytokines such as IGF-I.     

A comparison of the torsional performance of stainless steel and titanium alloy tibial intramedullary nails: a clinically relevant approach

In recent years there has been a tendency to design and manufacture intramedullary nails from titanium alloy rather than from stainless steel. The aim of this project was to compare the torsional performance of one manufacturers standard stainless steel and titanium alloy tibial intramedullary nails, using their distal locking screw holes and dedicated cross screws to secure each nail distally. A custom built test rig and materials testing machine were used to determine the torsional rigidity of the nails. Theory was used to calculate the torsional rigidity of the central parts of each nail. From the mechanical testing, the mean torsional rigidity of the titanium alloy nail system was 40.9 N m2 while that of the stainless steel nail system was 34.6 N m2, for all distal interlocking screw positions tested. Based on theoretical calculations the torsional rigidity of the central part of the nail was 83 N m2 for the stainless steel nail and 66 N m2 for the titanium alloy nail. This study shows the importance of using the distal locking screw holes and dedicated cross screws to secure intramedullary nails during mechanical testing so that clinically relevant results are obtained about the whole nail system and not just the nail.     

Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids

BACKGROUND: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. METHODS: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. RESULTS: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. CONCLUSIONS: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.     

A decrease in physical activity affects appetite, energy, and nutrient balance in lean men feeding ad libitum

BACKGROUND: It is not clear how decreased activity quantitatively affects energy balance (EB) in subjects feeding ad libitum. OBJECTIVE: We assessed the effect of an imposed sedentary routine on appetite, energy intake (EI), EB, and nutrient balance in lean men for 7 d. DESIGN: Six men with a mean (+/-SD) age of 23.0 +/- 2.3 y, weight of 69.2 +/- 11.4 kg, and height of 1.76 +/- 0.07 m were each studied twice during a sedentary [1.4 x resting metabolic rate (RMR)] and a moderately active (1.8 x RMR) regimen. During each treatment, they resided in the whole-body indirect calorimeter for the 7 d and had ad libitum access to a medium-fat diet of constant, measurable composition. Meal size, frequency, and composition were continually monitored. Motivation to eat was recorded during waking hours. Subjects were weighed in light clothing each morning, and their weight was corrected to nude. RESULTS: Energy expenditure was 9.7 and 12.8 MJ/d [P < 0.01; SE of the difference between means (SED) = 0.41] during the sedentary and active regimens, respectively. EI was 13.5 and 14.4 MJ/d (P = 0.463, SED = 1.06), respectively. There was no regimen effect on hunger, appetite, or body weight. By day 7, cumulative EB was 26.3 and 11.1 MJ, respectively. CONCLUSIONS: Reducing a level of physical activity from 1.8 to 1.4 x RMR can markedly affect EB. A sedentary routine does not induce a compensatory reduction of EI and leads to a significantly positive EB, most of which is stored as fat.     

Molecular cloning and pharmacological characterization of the guinea pig 5-HT1E receptor

The human 5-HT(1E) receptor gene was cloned more than a decade ago. Little is known about its function, and there have been no reports of its existence in the genome of small laboratory animals. In this study, attempts to clone the 5-HT(1E) gene from the rat and mouse were unsuccessful. In fact, a search of the mouse genome database revealed that the 5-HT(1E) receptor gene is missing from the mouse genome. However, the 5-HT(1E) gene was cloned from guinea pig genomic DNA and was characterized. The guinea pig 5-HT(1E) receptor gene encodes a protein of 365 amino acids. It shares 88% (nucleic acid) and 95% (amino acid) homology with the human receptor. The guinea pig 5-HT(1E) receptor showed similar pharmacology to the human 5-HT(1E) receptor in radioligand binding assays. Serotonin (5-hydroxytryptamine, 5-HT) dose-dependently stimulated [35S]GTPgammaS binding to the guinea pig 5-HT(1E) receptor with an EC(50) of 13.6+/-1.92 nM, similar to that of the human 5-HT(1E) receptor (13.7+/-1.78 nM). Activation of the guinea pig 5-HT(1E) receptor was also achieved by ergonovine, alpha-methyl-5-HT, 1-naphthylpiperazine, methysergide, tryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Methiothepin exhibited antagonist activity. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 5-HT(1E) mRNA was present in the guinea pig brain with the greatest abundance in the hippocampus, followed by the olfactory bulb. Lower levels were detected in the cortex, thalamus, pons, hypothalamus, midbrain, striatum, and cerebellum. Our current study marks the first identification of the 5-HT(1E) receptor gene in a commonly used laboratory animal species. This finding should allow the elucidation of the receptor's role(s) in the complex coordination of central serotonergic effects.     

Influence of short-term dietary weight loss on cortisol secretion and metabolism in obese men

OBJECTIVES: Obesity is associated with increased inactivation of cortisol by hepatic A-ring 5alpha- and 5beta-reductases, impaired hepatic regeneration of cortisol from cortisone by 11beta-hydroxysteroid dehydrogenase type 1 (11HSD1), but increased subcutaneous adipose 11HSD1 activity enhancing local cortisol levels in fat. Cause and effect between obesity and abnormal cortisol metabolism is untested. DESIGN: Acute weight loss was induced by very low calorie diet (VLCD) or starvation in obese men. METHODS: Otherwise healthy males (aged 20-55 years; body mass index (BMI) 30-40 kg/m2) were studied after 6 days on a weight maintenance diet; then after either 6 days of starvation (n=6) or 3 weeks of VLCD (2.55 MJ; n=6); then after 1 week of weight maintenance; and finally after 2 weeks of being allowed to feed ad libitum. Plasma samples were obtained from indwelling cannulae at 0930 h and 1815 h and a 24 h urine collection was completed for analysis of cortisol metabolites by gas chromatography/mass spectrometry. RESULTS: Data are mean+/-S.E.M. BMI fell (kg/m3) from 34.8+/-0.8 at baseline to 31.8+/-1.4 on VLCD and 32.7+/-1.1 on starvation. Starvation caused a rise in plasma cortisol (at 0930 h from 143+/-17 to 216+/-11 nM, P<0.001) but no change in total urinary cortisol metabolites. VLCD did not alter plasma cortisol and markedly reduced cortisol metabolite excretion (from 15.8+/-1.1 mg/day at baseline to 7.0+/-1.1 mg/day, P<0.001). Relative excretion of 5alpha-reduced cortisol metabolites fell on both diets, but there were no changes in cortisol/cortisone metabolite ratios reflecting 11HSD activities. CONCLUSIONS: Weight loss with VLCD in obesity reverses up-regulation of hepatic A-ring reductases and normalises cortisol production rate; in contrast, starvation produces acute stress and further activation of cortisol secretion. We suggest that activation of cortisol secretion is not an irreversible intrinsic abnormality in obese patients, and speculate that dietary content has an important influence on the neuroendocrine response to weight loss.     

Endothelization and adherence of leucocytes to nanostructured surfaces

We analyse the leucocyte and endothelial cell response to polybromostyrene-polystyrene (PS/PBrS) and the poly-n-butylmethacrylate-polystyrene (PnBMA/PS) systems, both in flat form or nanostructured surfaces consisting of nanohills with increasing hill height (13-95nm). Experiments were carried out first with blood leucocytes alone, endothelial cells (of three different types) alone, and finally, using blood cells and endothelized nanosurfaces. Blocking monoclonal antibodies specific for CD11, CD29, CD31, CD54, CD166 were used to analyse whether and to what extent adhesion molecules could be involved in the adherence of both blood leucocytes and endothelial cells to different nanosurfaces. Expression of CD29 (beta-1 integrin), CD54 (ICAM-1) and CD166 (ALCAM) on blood leucocytes was dependent on the hill height, being most prominent with 13nm (PS/PBrS) and 45nm hill (PnBMA/PS) nanosurfaces. Adherence of a human microvascular endothelial cell line and umbilical primary endothelial cells was also related to hill height, being most prominent with 13nm hill height. An indirect correlation was observed between the extent of endothelization and the degree of leucocyte adherence. In cases of low to medium extent of endothelization, the adherence of monocytes and granulocytes was mediated by the expression of CD166, CD29 and CD11a (alpha-L integrin), CD29, CD31 (PECAM-1), respectively. Scanning electron microscopy studies showed the predominant emission of pseudopodia at the holes of the surfaces and the focal contacts with the nanosurfaces. Our studies emphasize the relevance of testing functional properties in co-culture experiments in the development and optimization of nanosurfaces for biomedical application.     

Nonadhesive nanotopography: fibroblast response to poly(n-butyl methacrylate)-poly(styrene) demixed surface features

It is becoming clear that cells do not only respond to micrometric scale topography, but may also respond to topography at the nanometric scale. Nano-fabrication methods such as electron beam lithography are, however, expensive and time consuming. Polymer demixing of poly(styrene) and poly(4-bromostyrene) has been found to produce nano-scale islands of reproducible height, and the islands have been previously shown to effect cell events such as adhesion, spreading, proliferation, and differentiation. This study uses demixed poly(styrene) and poly(n-butyl methacrylate) to produce nano-islands with closer packing and narrower widths compared with those previously studied. Observations have been made of morphological and cytoskeletal changes in human fibroblasts interacting with 10- and 50-nm-high islands. The methods used included scanning electron microscopy, fluorescent microscopy, and optical microscopy. The results indicated that the cells do not respond differently to the 10-nm islands compared with planar samples but, in contrast, the 50-nm islands are nonadhesive.