The influence of temperature during water-quench rapid heat treatment on the microstructure, mechanical properties and biocompatibility of Ti6Al4V ELI alloy.

This study investigates the influence of a rapid heat treatment followed by water-quenching on the mechanical properties of Ti6Al4V ELI alloy to improve its strength for use in implants. Prior to the experiment, a dilatometry test was performed to understand the progressive α-to ß-phase transformation taking place during heating. The results were then used to carry out heat treatments. Microstructure was analysed using SEM, EBSD, EDX and XRD techniques. Vickers micro-hardness, tensile and high cycle rotating bending tests were used to analyse the influence of the $\alpha'$-phase fraction on the strength of the studied alloy. Results show that this process can provide a Ti6Al4V ELI alloy with a better Yield Strength (YS)/uniform deformation (εu) ratio and improved high cycle fatigue strength than those observed in the current microstructure used in medical implants. Lastly, cytotoxicity tests were performed on two types of human cells, namely MG63 osteoblast-like cells and fibroblasts. The results reveal the non-toxicity of the heat-treated Ti6Al4V ELI alloy.

Degradation of paraoxon (VX chemical agent simulant) and bacteria by magnesium oxide depends on the crystalline structure of magnesium oxide.

In this work, our goal was to study the capability of a single metallic oxide to neutralize a chemical agent and to exhibit an antibacterial effect. We tested two types of magnesium oxides, MgO. The first MgO sample tested, which commercial data size characteristic was -325 mesh (MgO-1) destroyed in 3 h, 89.7% of paraoxon and 93.2% of 4-nitrophenol, the first degradation product. The second MgO sample, which commercial data size was <50 nm (MgO-2) neutralized in the same time, 19.5% of paraoxon and 10.9% of 4-nitrophenol. For MgO-1 no degradation products could be detected by GC-MS. MgO-1 had a bactericidal activity on Escherichia coli (6 log in 1 h), and showed a decrease of almost 3 log on a Staphylococcus aureus population in 3 h. MgO-2 caused a decrease of 2 log of a E.coli culture but had no activity against S. aureus. Neither of these two products had an activity on Bacillus subtilis spores. Analytical investigations showed that the real sizes of MgO nanoparticles were 11 nm for MgO-1 and 25 nm for MgO-2. Moreover, their crystalline structures were different. These results highlighted the importance of the size of the nanoparticles and their microscopic arrangements to detoxify chemical products and to inhibit or kill microbial strains.

Wear study of Total Ankle Replacement explants by microstructural analysis.

The implantation of Total Ankle Replacement (TAR) prostheses generally gives satisfactory results. However, a high revision rate is associated with the Ankle Evolutive System (AES) implant, due to periprosthetic osteolysis that generates significant cortical lesions and bone cysts in the periprosthetic region. Radioclinical and histological analyses of peri-implant tissues show the presence of numerous foreign particles that may come from the implant. It is known that a precocious wear of materials may lead to an important rate of foreign body in tissues and may generate osteolysis lesions and inflammatory reactions. Thus the objectives of this retrospective study of 10 AES TAR implants (recovered after revision surgeries) are to understand how the prostheses wear out, which part is the most stressed and to determine the nature and size of foreign body particles. A better understanding of friction mechanisms between the three parts of the implant and of the nature and morphology of foreign particles generated was needed to explain the in vivo behavior of the implant. This was achieved using microstuctural and tomographic analysis of both implants parts and periprosthetic tissues.

Endovascular Treatment of Intracranial Aneurysms with the WEB Device: A Systematic Review of Clinical Outcomes.

BACKGROUND AND PURPOSE: Despite the improvement in technology, endovascular treatment of bifurcation intracranial wide-neck aneurysms remains challenging, mainly due to the difficulty of maintaining coils within the aneurysm sac without compromising the patency of bifurcation arteries. The Woven EndoBridge (WEB) device is a recent intrasaccular braided device specifically dedicated to treating such aneurysms with a wide neck by disrupting the flow in the aneurysmal neck and promoting progressive aneurysmal thrombosis. MATERIALS AND METHODS: Using several health data bases, we conducted a systematic review of all published studies of WEB endovascular treatment in intracranial aneurysms from 2010 onward to evaluate its efficacy and safety profile. RESULTS: The literature search identified 6 relevant studies (7 articles) including wide-neck bifurcation aneurysms in ≥80% of cases. Clinical data supporting the efficacy and safety of the WEB are limited to noncomparative cohort studies with large heterogeneity from a methodologic standpoint. The WEB deployment was feasible with a success rate of 93%-100%. Permanent morbidity (mRS of >1 at last follow-up) and mortality were measured at 2.2%-6.7% and 0%-17%, respectively. The adequate occlusion rate (total occlusion or neck remnant) varied between 65% and 85.4% at midterm follow-up (range, 3.3-27.4 months). CONCLUSIONS: Endovascular treatment of bifurcation wide-neck aneurysms with the WEB device is feasible and allows an acceptably adequate aneurysm occlusion rate; however, the rate of neck remnants is not negligible. The WEB device needs further clinical and anatomic evaluation with long-term prospective studies, especially of the risk of WEB compression. Prospective controlled studies should be encouraged.

Significance of the serum CTX-II level in an osteoarthritis animal model: a 5-month longitudinal study.

OBJECTIVE: The aim of this study was to investigate the clinical value of serum measurement of C-telopeptide of type II collagen (CTX-II). In correlation with late stages of osteoarthritis (OA) evaluated with histological assessment, the evolution of serum CTX-II concentration was followed during a 20-week longitudinal study in rabbit anterior cruciate ligament transection (ACLT) OA model in adult and growing animals. METHODS: OA was induced in five adult and nine growing rabbits. Four adult and four young rabbits were unoperated. Serum sampling was made at week 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16 and 20 after the surgery in all rabbits. Animals were euthanized 20 weeks after the surgery. Serum CTX-II levels were analyzed with a recently available enzyme-linked immunosorbent assay (ELISA) kit, the protocol of which has been modified to increase the sensitivity of the test. RESULTS: Significant differences for the CTX-II levels at W3, W6, W8, W10, W12, W14, W16 and W20 were observed between the adult ACLT and the control groups. A negative correlation between CTX-II levels and cartilage thickness of the medial compartment of the knee at W8, W10, W12 and a positive correlation between the CTX-II levels and the histomorphological score of the medial compartment of the knee at W3, W6, W8, W10, W12 were noted in adult animals. In young animals, operated or not, we observed high CTX-II levels at the beginning of the study, which decreased until the end. CONCLUSION: Our results suggest the interest of the serum CTX-II monitoring for the OA progression and the relevance of the multiple time point analysis of this biomarker. Moreover, they address the question of the importance of correctly choosing the age of the animals used in the pre-clinical studies of OA.

Knee joint ultrasonography of the ACLT rabbit experimental model of osteoarthritis: relevance and effectiveness in detecting meniscal lesions.

OBJECTIVES: To develop a protocol for rabbit knee joint ultrasonography (US); to grade ultrasonographically the meniscal injuries of the anterior cruciate ligament transection (ACLT) rabbit model of osteoarthritis (OA); to assess with US the effectiveness of the ACLT; to compare final US with macroscopy for the evaluation of medial and lateral meniscal injuries depending on the age and weight when ACLT is performed. METHODS: Twenty-two skeletally mature and adolescent New Zealand white rabbits were housed during the same period at the Institut Claude-Bourgelat, Lyon, France. Surgical ACLT was performed in the left knee of nine adolescent and five adult rabbits. Final US and macroscopic semi-quantitative grading of the meniscal injuries were compared 5 months after ACLT. RESULTS: A standardised protocol was developed to evaluate the rabbit knee joint. US was performed in both control and ACLT knees. Normal and abnormal meniscal US appearances were described. A semi-quantitative scale to grade US meniscal injuries was created. Macroscopic and US total meniscal scores were significantly positively correlated (P<0.001, r=0.70). US detection of meniscal injuries was 92% sensitive and 87.5% specific compared to macroscopy. Positive and negative predictive values of US were, respectively, 92% and 87.5%. US detection of the ACLT effectiveness was 100% specific and 78.5% sensitive. CONCLUSION: A significant relationship was found between ultrasonographic and macroscopic grading of meniscal injuries. US was both specific and sensitive in detecting meniscal lesions. We propose US as a non-invasive, non-expensive, in vivo imaging technique for preclinical studies in the ACLT rabbit OA model.

Electrophoretic and immunochemical study of collagens from Sepia officinalis cartilage.

Electrophoretic and Western blot studies were conducted on collagen fractions extracted from Sepia officinalis (cuttlefish) cartilage using a modified salt precipitation method developed for the isolation of vertebrate collagens. The antibodies used had been raised in rabbit against the following types of collagen: Sepia I-like; fish I; human I; chicken I, II, and IX; rat V; and calf IX and XI. The main finding was that various types of collagen are present in Sepia cartilage, as they are in vertebrate hyaline cartilage. However, the main component of Sepia cartilage is a heterochain collagen similar to vertebrate type I, and this is associated with minor forms similar to type V/XI and type IX. The cephalopod type I-like heterochain collagen can be considered a first step toward the evolutionary development of a collagen analogous to the typical collagen of vertebrate cartilage (type II homochain). The type V/XI collagen present in molluscs, and indeed all phyla from the Porifera upwards, may represent an ancestral collagen molecule conserved relatively unchanged throughout evolution. Type IX-like collagen seems to be essential for the formation of cartilaginous tissue.

Age-dependent changes in the expression of matrix components in the mouse eye.

Although the presence of 'cartilage-specific' collagens in the eye has been documented earlier, very little is known about their synthesis rates during ocular development, growth and aging. The purpose of the present study was to follow changes in the mRNA levels and distribution of key components of the extracellular matrix in the eyes of normal and transgenic Del1 mice, harboring a short deletion mutation in the type II collagen gene, during ocular growth and aging. Total RNAs extracted from mouse eyes were studied by Northern analysis for mRNA levels of type I, II, III, VI, IX and XI collagens, biglycan, fibromodulin and decorin. A predominant finding of the present study was the marked reduction in the mRNA levels of type I and II collagens in the eye upon aging. The changes in the mRNA levels of type III and VI collagen and proteoglycans were smaller. Localization of type II and IX collagen in the eye was performed by immunohistochemistry. Despite the reduction in the type II collagen mRNA levels, immunohistochemistry confirmed widespread distribution of the protein also in aging mouse eyes, suggesting its slow turnover. Although the Del1 mutation caused gradual degenerative lesions in the eyes, the distribution of the protein remained essentially unchanged. The widespread distribution and marked downregulation of type II collagen production in the mouse eye upon aging probably explain the gradual development of degenerative lesions, particularly in the eyes of transgenic Del1 mice, where production of mutant type II collagen chains also contributes to the process.

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.

Mycophenolate mofetil reduces myofibroblast infiltration and collagen III deposition in rat remnant kidney.

BACKGROUND: Myofibroblasts have been shown to play a pivotal role in the synthesis of extracellular matrix components in several animal models of renal fibrosis. The purpose of the present study was to investigate whether mycophenolate mofetil (MMF) reduces interstitial myofibroblast infiltration and collagen III deposition in 5/6 nephrectomized rats. METHODS: Forty-five Wistar rats underwent 5/6 renal ablation and received by daily oral gavage either vehicle (N = 20) or MMF (N = 25) during the 60 days following surgery. Groups of five treated and five untreated rats were killed at two, four, and eight weeks after subtotal nephrectomy. Four untreated and three treated rats were killed at week 12, one month after treatment withdrawal. At the time of sacrifice, proteinuria, plasma, and urine creatinine were determined. Immunohistochemistry was performed on renal tissue for alpha-smooth muscle actin (alpha-SMA), a cytoskeletal marker of myofibroblasts, for type III collagen, and for proliferating cell nuclear antigen (PCNA). Moreover, in order to study the in vitro effects of MMF on fibroblast proliferation, rat fibroblasts were cultured in the presence or absence of mycophenolic acid (MPA). RESULTS: At all periods studied, MMF treatment improved renal functional parameters and progressively decreased remnant kidney hypertrophy and glomerular volume increment. Proliferating cells in renal tubules, interstitium, and glomeruli, as well as interstitial myofibroblast infiltration and interstitial type III collagen deposition, were also significantly reduced by MMF treatment. In addition, MPA exhibited a dose-dependent inhibitory effect on in vitro proliferation of rat fibroblasts. CONCLUSION: Reduction of interstitial myofibroblast infiltration may be an important event by which MMF significantly prevents renal injury following subtotal renal ablation. Thus, our results suggest that MMF could be useful to limit the progression of chronic renal disease toward end-stage renal failure.

Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in patients with cirrhosis.

BACKGROUND/AIMS: Severe alcoholic hepatitis occurs mainly in patients with cirrhosis, and has a high death rate. Corticosteroid therapy has been particularly advocated as reducing mortality in patients with severe histologic lesions. However, identification of these patients is difficult, requiring transvenous liver biopsy. Extracellular matrix serum markers have been proposed as non-invasive diagnostic tools in alcoholic liver disease. The aim of this study was to determine the accuracy of 5 extracellular matrix serum markers, i.e. laminin (Lam), N-terminal peptide of type III procollagen (PIIINP), type I (CI), type III (CIII) and type IV (CIV) collagens in identifying patients with severe histologic alcoholic hepatitis from among those with cirrhosis and suspected alcoholic hepatitis. METHODS: We studied 80 consecutive patients with alcoholic cirrhosis and clinical suspicion of alcoholic hepatitis referred for transvenous liver biopsy. Clinical severity of alcoholic hepatitis was assessed according to Maddrey's score. Histological severity was scored using the sum of the 3 following items: polynuclear infiltration (0-3); hepatocytes alterations (0-3); Mallory bodies (0-2). According to this score, patients were divided into 3 groups: mild (1-3), moderate (4-6), and severe (7-8) alcoholic hepatitis. Serum levels of the 5 extracellular matrix serum markers were measured at the time of biopsy using radioimmunoassays. Diagnostic value for histologically severe alcoholic hepatitis of the 5 extracellular matrix serum markers was assessed using receiver operating characteristic curves. RESULTS: Histological alcoholic hepatitis was present in 67 patients (mean alcoholic hepatitis score: 3.4+/-2.3). Maddrey's score was 66% sensitive and 69% specific for the diagnosis of severe histologic alcoholic hepatitis. The serum Lam and CIV concentrations were the most accurate in identifying correctly patients with severe histologic alcoholic hepatitis. At a cut-off of 4.1 UI/ml, Lam was 90% sensitive and 77% specific, whereas at a cut-off of 150 ng/ml, CIV was 89% sensitive and 77% specific. Combination of markers did not result in improved diagnostic value. CONCLUSION: In patients with cirrhosis, determination of serum Lam or CIV could represent a simple and accurate non-invasive method for identification of patients with histologically severe alcoholic hepatitis eligible for corticosteroid treatment.

Modifications of bone and connective tissue after orthostatic bedrest.

Eight male volunteers were submitted to a 6-week anti-orthostatic bedrest trial followed by a 1-month reambulation period. We prospectively monitored whole-body composition by dual-energy X-ray absorptiometry, bone and connective tissue metabolism by biochemical markers and calcium regulating hormones by 1-84 parathyroid hormone and 1,25-dihydroxyvitamin D(3). Bone mineral density (BMD) did not vary significantly; however, a trend toward an increase in head BMD and a decrease in trunk, lumbar vertebrae and lower limb BMD was observed. A decrease in the lower limb lean content occurred by day 27 and was maximum by day 42 after the beginning of bedrest; it normalized by day 30 after bedrest. The serum levels of both osteocalcin and C-terminal crosslinked telopeptide of type I collagen increased as a consequence of bedrest. A slight increase in the serum levels of the N-terminal propeptide of type III collagen, a marker of connective tissue metabolism, was observed during the bedrest period. Except for the C-terminal extension propeptide of type I collagen, all markers decreased to baseline pre-immobilization levels during the 1-month recovery phase. Serum PTH and 1,25-dihydroxyvitamin D(3) levels were low during the bedrest period and rose during the reambulation phase. These results seem to reflect early changes in bone and connective tissue metabolism as a result of bedrest unloading, but their order of magnitude remains moderate, thus emphasizing the necessity to perform longer-duration trials.

Immunohistochemical study of collagens of the extracellular matrix in cartilage of Sepia officinalis.

We used various anti-collagen antibodies to perform indirect immunofluorescent staining of cartilage sections from cuttlefish (S. officinalis). On ultrathin sections and collagen fibril preparations from the same tissue, we performed immunostaining with colloidal gold. The extracellular matrix (ECM) of S. officinalis cartilage reacted intensely and homogeneously with an antibody directed against type I-like collagen isolated from the cartilage of cuttlefish and with anti-rat type V collagen antibody. A weak reaction was observed with anti-fish and anti-chicken type I collagen antibodies, while no reaction was observed with anti-rat type I and anti calf type II collagen antibodies. Anti-chicken type II, anti calf type IX and type XI collagen antibodies reacted weakly with ECM, while stained cell bodies and cell processes reacted more intensely. A similar pattern of reaction was observed on cartilage section and isolated collagen fibrils prepared for electron microscopy. These findings suggest that ECM of cuttlefish cartilage may be composed of molecules similar to the type I, type V, type IX and type XI collagen molecules of vertebrates. Cephalopods have evolved a cartilage of structure and macromolecular organisation similar to that of vertebrate cartilage. However, the main molecular components of S. officinalis cartilage--type I-like and type V collagens--differ from those of vertebrate cartilage. We suggest that this type I-like collagen can be considered an initial step toward the evolution of type II collagen typical of vertebrates.

Effect of calcitonin on bone and connective tissue metabolism in hemiplegic patients: a two-year prospective study.

OBJECTIVES: To test the effects of 200 IU/day of a nasal spray of salmon calcitonin in modulating the increased bone tissue metabolism observed in hemiplegic patients. DESIGN: A two-year, prospective, randomized, double-blind, placebo-controlled study. PATIENTS: Thirty-four patients with hemiplegia due to a cerebrovascular accident were included in the study during the first month after onset of hemiplegia after giving their informed consent. All patients followed a suitably planned course of active rehabilitation according to Bobath. They were randomly allocated to either the calcitonin or the placebo group but all of them received a daily oral calcium supplementation of 1000 mg. RESULTS: Biochemical markers of bone formation, serum total alkaline phosphatase, osteocalcin and type I procollagen did not vary during the two years of follow-up. Cross-linked carboxy-terminal telopeptide of type I collagen, a biochemical marker of bone resorption, was elevated initially after stroke and then decreased significantly (p <0.001), stabilizing as of month 12. Urinary calcium/creatinine and total hydroxyproline/creatinine ratios presented with similar profiles. The serum levels of the type III procollagen, a marker of connective tissue metabolism, were also elevated upon entry and decreased thereafter. No significant difference could be found between the calcitonin-treated and the placebo group for any of the biochemical markers at any time point. CONCLUSION: This prospective study demonstrated that biochemical markers of bone and connective tissue metabolism were elevated shortly after the occurrence of stroke and then gradually decreased during the first two years after injury. The daily administration of 200 IU of intranasal calcitonin did not influence the levels of these markers.

Relationships between several markers of extracellular matrix turn-over and ultrasonography in human Schistosomiasis mansoni.

We measured the concentrations of several serum and urinary fibrosis markers, which are metabolites of extracellular matrix, in schistosomiasis patients to investigate their relationship with the ultrasonographic scoring system and with parasitologic data. This study was conducted in patients with various stages of the disease evaluated by ultrasonography (intestinal disease with no organ involvement, with minor hepatosplenic involvement and with severe disease) and in endemic controls. The level of hyaluronan, which were increased in infected patients compared with controls (P < 0.01), was the only fibrosis marker that correlated with the ultrasonographic score (P = 0.003) and is thus a potential serum marker of schistosomiasis-associated morbidity. Urinary free pyridinoline levels were lower (P < 0.001) in infected patients with fibrosis (score > or = 1) than in nonfibrotic patients. A two-year follow-up of the patients treated with praziquantel showed that type I collagen and hyaluronan decreased during the first year post-treatment, whereas free pyridinolines peaked after 12 months and decreased thereafter.

Replication and extension analyzing the factor structure of Locus of Control scales for substance-abusing behaviors.

Two research questions were addressed in this study. First, what are the dimensions of the concept of control orientation with respect to substance-abusing behaviors as measured by Keyson and Janda's 1972 Drinking-related Locus of Control Scale and by an adaptation of that scale developed for users of other drugs. Second, utilizing a large sample of both male and female clients who abused both alcohol and other drugs, how does that dimensional structure differ from the one presented by Donovan and O'Leary in 1978 for male alcoholics at a VA hospital. Since the present sample was a large (N = 542) representative sample of publicly subsidized clients in Missouri, it provided an opportunity to extend earlier work across type of drug abused and sex. Like Donovan and O'Leary we used a principal components analysis with a varimax rotation to examine the dimensions of the 25-item locus of control scales. Like those authors, the analysis for male alcohol users produced a three-factor solution with interpretations resembling their "intrapersonal" and "interpersonal" factors but with a more fully developed third factor interpretable as a fate or luck dimension. These dimensions were apparent for the other groups as well, although the first two factors were reversed for male users of other drugs and the analysis for female alcohol abusers showed a more consistent sociability dimension than for males. While the derived structures are therefore similar to Donovan and O'Leary's results and consistent with the theoretical uses of control orientation in discussion of alcohol and other drug abuse, they also show some differences for type of drug abused and by sex. These are necessary but not sufficient conditions for including such measures in more specific models relating clients' motivation and propensity for change to outcomes.

Monitoring of extracellular matrix metabolism and cross-linking in tissue, serum and urine of patients with chromoblastomycosis, a chronic skin fibrosis.

BACKGROUND: Chromoblastomycosis is a fungal disease leading to a granulomatous reaction associated with dermal fibrosis. METHODS: In an attempt to elucidate the mechanisms leading to improvement in the cutaneous lesions after treatment with terbinafine, a new antifungal drug, we analysed collagen content and cross-linking before and at the end of the treatment. The turnover of extracellular matrix was monitored for 1 year by following up serum and urinary metabolites. RESULTS: The serum levels of type III collagen and its N-terminal propeptide were correlated with the lesion size (P < 0.035) after 4 and 12 months of treatment respectively. After 4 months of treatment, urinary pyridinoline was higher (P = 0.04) in patients whose lesion size was reduced by more than 50% and serum hyaluronan was lower in patients who had lesions active for less than 5 years (P < 0.05). The treatment increased pyridinoline and pentosidine cross-links in the lesions but significantly reduced the collagen content (P = 0.05). CONCLUSION: This is the first demonstration that, in addition to its fungicidal activity, terbinafine acts in vivo as an antifibrotic drug.

Development of an ELISA for pantothenic acid (vitamin B5) for application in the nutrition and biological fields.

Immunological assays appear to be the only alternative to the microbiological method for analysis of pantothenic acid in foods and blood. In order to evaluate the influence of the linker on the immunogenicity of the hapten, we have tried to raise antisera against pantothenic acid in rabbits using different conjugates. The hapten was coupled to a carrier protein (BSA or thyroglobulin) using adipoyl dichloride (adipoyl conjugate) or bromoacetyl bromide (acetyl conjugate). Only the acetyl conjugate has induced the production of a specific antibody. With this antibody, an assay on microplate using the ELISA inhibition technique was developed to measure pantothenic acid. The use of pantothenic acid coupled to thyroglobulin with adipoyl dichloride as the capture antigen has improved the sensitivity of the ELISA. This assay was applied to food products and blood.

Spatiotemporal patterns of expression of extracellular matrix molecules in the developing and adult rat olfactory system.

Using immunocytochemical methods, we have examined extensively the spatial and temporal patterns of expression of three extracellular matrix molecules-laminin, fibronectin, and type IV collagen-in the embryonic, postnatal (days 2 and 11) and adult rat olfactory system. The study started at embryonic day 14 when olfactory fibres and their associated migrating cells course through the nasal mesenchyme. From embryonic day 14 to the adult, a sheet-like pattern of labelling for laminin, fibronectin and type IV collagen was observed along the basal surface of the olfactory epithelium and around the telencephalon. This type of labelling was continuous around the telencephalic vesicle, whereas it appeared disrupted in the basal lamina of the olfactory epithelium to permit exit of the olfactory axons and their associated migrating cells into the mesenchyme. From embryonic day 14 to day 20, punctate labelling for the three molecules studied was observed along the mesenchymal olfactory pathway, the ventral part of the olfactory bulb, the olfactory nerve layer and the presumptive glomerular layer, respectively. By embryonic day 17, the punctate labelling initially detected in the mesenchymal olfactory pathway was replaced by a sheet-like pattern related to the mature basal lamina surrounding the olfactory axon fascicles. Punctate labelling for laminin and type IV collagen persisted in the olfactory nerve layer and around the glomeruli through adult life whereas that of fibronectin declined and disappeared by postnatal day 2. The spatiotemporal distribution of the punctate pattern for laminin, fibronectin and type IV collagen observed in the embryonic olfactory system suggests a role in delineating the pathway for olfactory axon elongation. The continuous expression of laminin and type IV collagen in the adult olfactory bulb may be related to the regenerative activity and high plasticity of the olfactory system.

Biochemical markers of bone metabolism in draught and warmblood horses.

Concentrations of the cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin (OC) have been determined in the serum of one hundred clinically healthy adult Draught or Warmblood horses. The correlation between these two markers has been evaluated and the influence of gender, age and type of horse described. No significant variations were observed between animals of different sex, but a significant inverse correlation (P < 0.0001) with age was observed for both measured parameters. After correction for age, serum levels of OC were found to be lower in Draught [adjusted least square mean (LSM) = 6.612 micrograms.L-1] than in Warmblood horses (adjusted LSM = 8.596 micrograms.L-1), whereas levels of ICTP were higher in Draughts (adjusted LSM = 8.035 micrograms.L-1) than in Warmbloods (adjusted LSM = 6.643 micrograms.L-1). A significant correlation (P < 0.0001) was observed between OC and ICTP. This correlation was stronger if the type of horse was taken into account in the statistical model. The ratio OC:ICTP was independent of gender and age. A higher OC:ICTP ratio in Warmbloods compared to the Draught horses might reflect a higher bone remodelling level of horses submitted to regular daily work. It was concluded that ICTP and OC are influenced by the type of horse, and probably reflect a physiological difference in bone remodelling between these animals.