Cardiovascular prosthetic bypass grafts do not endothelialize spontaneously in humans, and so they pose a thrombotic risk. Seeding with cells improves their performance, particularly in small-caliber applications. Knitted tubular polyethylene-terephthalate (PET) vascular prostheses (6 mm) with commercial type I collagen (PET/Co) were modified in the lumen by the adsorption of laminin (LM), by coating with a fibrin network (Fb) or a combination of Fb and fibronectin (Fb/FN). Primary human saphenous vein endothelial cells were seeded (1.50 × 10(5)/cm2), cultured for 72 h and exposed to laminar shear stress 15 dyn/cm(2) for 40 and 120 min. The control static grafts were excluded from shearing. The cell adherence after 4 h on PET/Co, PET/Co +LM, PET/Co +Fb and PET/Co +Fb/FN was 22%, 30%, 19% and 27% of seeding, respectively. Compared to the static grafts, the cell density on PET/Co and PET/Co +LM dropped to 61% and 50%, respectively, after 120 min of flow. The cells on PET/Co +Fb and PET/Co +Fb/FN did not show any detachment during 2 h of shear stress. Pre-coating the clinically-used PET/Co vascular prosthesis with LM or Fb/FN adhesive protein assemblies promotes the adherence of endothelium. Cell retention under flow is improved particularly on fibrin-containing (Fb and Fb/FN) surfaces.

Nanocrystalline apatites (NCA) are the inorganic components of mineralized tissues and they have been recently proposed as biomaterials for drug delivery systems. Bisphosphonates (BPs) are currently the reference drugs used to treat diseases involving bone disorders such as osteoporosis. Nevertheless, the interaction phenomena between BP molecules and apatite nanocrystals of bone are not well understood. Therefore, the adsorption characteristics have been examined and cellular activity of tiludronate molecules on NCA as models of bone mineral has been investigated. Adsorption experiments of tiludronate onto NCA were carried out and revealed a Langmuir-type adsorption isotherm. The uptake of tiludronate molecules is associated with a release of phosphate ions, indicating that the main reaction is an ion exchange process involving surface anions. The results evidence the strong affinity of BP molecules for the apatitic surface. The interactions of NCA-tiludronate associations with human osteoprogenitor cells and human bone marrow stromal cells do not reveal any cytotoxicity and evidence the activity of adsorbed tiludronate molecules. Moreover, an evolution of the physico-chemical characteristics of the apatitic substrate during biological study was observed, highlighting the existence of dynamic interactions. This work contributes to clarifying the reaction mechanisms between BPs and biomimetic apatites.

The configuration of a CYANEX 272 solvent-extraction plant can vary significantly depending on the composition of the pregnant leach solution. Parameters such as the organic-to-aqueous flowrate ratio, the extractant concentration, the pH profile, and the number of stages must be optimized for the given feed conditions. MINCHEM is a modeling program that was specifically designed by Cytec Industries Inc. to allow a rapid assessment of these parameters. The software was used in the present study to determine the optimum pH profile and stage requirements for the extraction and scrubbing circuits of a hypothetical cobalt/nickel plant using CYANEX 272 extractant. The simulations were completed to maximize cobalt recovery from the feed and minimize both impurity transfer in the loaded organic phase and the need for cobalt reprocessing from the scrub stage.

The first aim of the present study was to investigate the capacity of a cyclo-DfKRG-coated hydroxyapatite-titanium alloy (Ti-HA-RGD) to activate in vitro human osteoprogenitor cells adhesion and differentiation. The second purpose was to examine in vivo the role of a autologous cell seeding on cyclo-DfKRG-functionalized materials to provide bone repair after implantation in femoral condyle of rabbits. Our in vitro results have demonstrated that both titanium alloy functionalized with hydroxyapatite (Ti-HA-RGD and Ti-HA) contributed to higher cell adhesion than titanium alloy alone respectively 85 and 55% vs 15% compared to tissue culture polystyrene after one hour of cell seeding. As for differentiation, after 3 days of culture, Ti-HA presented the highest increase of ALP mRNA of all surfaces studied. Ti-HA-RGD showed an intermediate value about half as high as Ti-HA. Moreover after 3 days, both Ti-HA and Ti-HA-RGD surfaces showed the highest increase of cbfa1 mRNA expression. Two weeks following implantation, in vivo findings revealed that percentage of lacunae contact observed with pre-cellularized Ti-HA-RGD samples remains significantly lower than with Ti-HA group (10.5+/-9.6 % vs 33.7+/-11.5 %, P<0.03). Meanwhile, RGD peptide coating had no significant additional effect on the bone implant contact and area. Moreover, histomorphometry analysis revealed that implantation of pre-cellularized RGD coated materials with ROP cells increased significantly peri-implant fibrous area (24+/-11.6% vs 3+/-1.7% for Ti-HA-RGD, P<0.02). RGD coatings demonstrated osteoblastic adhesion, differentiation and in vivo bone regeneration at most equivalent to HA coatings.

Osteogenesis occurs in striking interaction with angiogenesis. There is growing evidence that endothelial cells are involved in the modulation of osteoblast differentiation. We hypothesized that primary human umbilical vein endothelial cells (HUVEC) should be able to modulate primary human osteoprogenitors (HOP) function in an in vitro co-culture model. In a previous study we demonstrated that a 3 day to 3 week co-culture stimulates HOP differentiation markers such as Alkaline Phosphatase (ALP) activity and mineralization. In the present study we addressed the effects induced by the co-culture on HOP within the first 48 hours. As a prerequisite, we validated a method based on immuno-magnetic beads to separate HOP from HUVEC after co-culture. Reverse transcription-real time quantitative PCR studies demonstrated up-regulation of the ALP expression in the co-cultured HOP, confirming previous results. Surprisingly, down-regulation of runx2 and osteocalcin was also shown. Western blot analysis revealed co-culture induced down-regulation of Connexin43 expression in both cell types. Connexin43 function may be altered in co-cultured HOP as well. Stimulation of the cAMP pathway was able to counterbalance the effect of the co-culture on the ALP activity, but was not able to rescue runx2 mRNA level. Co-culture effect on HOP transcriptome was analyzed with GEArray cDNA microarray showing endothelial cells may also modulate HOP extracellular matrix production. In accordance with previous work, we propose endothelial cells may support initial osteoblastic proliferation but do not alter the ability of the osteoblasts to produce extracellular mineralizing matrix.

In the field of osseous substitution, the possibilities being offered to the surgeons prove sometimes difficult to apply in particular in the case of great losses of osseous substance. For these reasons, it is necessary to develop innovative techniques to satisfy the request increasing for substitutes and to see appearing on the market solutions combining availability, perenniality and biosecurity of the implants. The implantation of stem cells in a biomaterial opens a way of development of therapeutic substitute. Moreover, in order to optimize the rehabitation of the biomaterials by the cells and the host tissues, the second approach consists in modifying the surface of materials by the coating or the grafting of adhesive factors in order to stimulate their colonization. At least, one cannot consider a tissue mechanism of repair without a better knowledge of the respective role of the various cell populations implied in the rebuilding of this tissue and their cell to cell communication processes.

This study takes place in the field of development of a bioactive surface of titanium alloys. In this paper, titanium was functionalized with cyclo-DfKRG peptide by coating or grafting using different anchors (thiol or phosphonate) as spacers between the surface and the peptide. Cell adhesion, and differentiation of human osteoprogenitor (HOP) cells arising from human bone marrow were investigated. Our results seem to demonstrate that cyclo-DfKRG peptide coating with a phosphonate anchor and grafting procedure contributes to higher cell adhesion and a strong ALP and Cbfa1 mRNA expression, after 10 days of cell seeding. At the contrary, this peptide coated with a thiol anchor stimulates differentiation of HOP within 3 days of culture.

In three different endothelial cell (EC) cultures (primary human umbilical cord vein, so-called HUVEC; and immortalized cell lines HBMEC and EA-hy-926), the effects of different xenobiotics were studied in order to standardize vascular EC models for in vitro pharmacotoxicological studies. Cell characteristics were first investigated by the production and the mRNA levels of known endothelial markers in the three EC culture models. EC secretory products, tissue plasminogen activator (tPA) and von Willebrand factor (vWF), were present in the supernatant of the immortalized cell lines. The mRNA levels of vWF, tPA, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and β -integrin subunit, which are involved in the control of platelet function, coagulation, and fibrinolysis as well as in cell–matrix interactions, were investigated in all EC types. For at least three parameters, cultured cells provided marked characteristics of EC phenotype, in HUVEC and in immortalized cell lines, regardless of their origin from the macro- or microcirculation. Toxicity experiments were assessed after 24 h exposure to cadmium, cyclosporin A and cisplatin by MTT assay. These experiments show nonsignificant difference in susceptibility to cyclosporin A and cadmium on HUVEC, HBMEC, and EA-hy-926. However, HBMEC, seems to be highly susceptible to cisplatin compared to HUVEC, the latter being more sensitive than EA-hy-926. For experiments conducted with cyclosporin and cadmium, cell lines could constitute an alternative material for routine cytotoxicity studies.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly endothelial cells that may be pivotal members of a complex interactive communication network in bone. While cell cooperation was previously established between Human OsteoProgenitor cells (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) the aim of our study was to investigate if this interaction is specific to Human Endothelial cell types (ECs) from different sources. Osteoblastic cell differentiation analysis performed using different co-culture models with direct contact revealed that Alkaline Phosphatase (Al-P) activity was only increased by the direct contact of HOP with human primary vascular endothelial cell types including endothelial precursor cells (EPCs) isolated from blood cord, endothelial cells from Human Saphen Vein (HSV) while a transformed cell line, the Human Bone Marrow Endothelial Cell Line (HBMEC) did not modify osteoblastic differentiation of HOP. Because connexin 43, a specific gap junction protein, seemed to be involved in HUVEC/HOP cell cooperation, expression by RT-PCR and immunocytochemistry of this gap junctional protein was investigated in EPCs, HSV and HBMEC. Both endothelial cells are positive to this protein and the disruption of gap junction communication using 18α-glycyrrhetinic acid treatment decreased the positive effect of these endothelial co-cultures on HOP differentiation as was previously demonstrated for HUVEC and HOP co-cultures. These data seem to indicate that this cross talk between HOP and ECs, through gap junction communication constitutes an additional concept in cell differentiation control.

The metabolic clearance rates (MCR) and interconversions [( rho]BB) values for estrone (E1) and estradiol (E2) in female rhesus (Macaca mulatta) monkeys on Days 9, 14, and 23 of the menstrual cycle were measured using constant infusions of [3H] estradiol and [14C] estrone. The menstrual cycles in these monkeys were reproduced by using Silastic capsules of E2 and progesterone after bilateral ovariectomy. The serum levels of E2 and progesterone were measured by radioimmunoassay and were similar to those for the intact menstrual cycle. The MCR of E2 on Day 14 (52.8 +/- 6.8 l/day/kg) was significantly greater (p less than 0.05) than that measured on Day 9 (31.1 +/- 3.6 l/day/kg) or Day 23 (35.4 +/- 2.1 l/day/kg). The MCR of E1 was also different (p less than 0.05) on Day 14 (77.6 +/- 14.9 l/day/kg) compared to the values on Days 9 and 23 (50.2 +/- 4.9 and 48.2 +/- 3.9 l/day/kg, respectively. There was no change in percentage of free E2, percentage of albumin-bound E2, or sex hormone-binding globulin levels on those 3 days of the cycle. The interconversions between E2 and E1 were not influenced by the day of the cycle. We conclude that the high levels of E2 occurring at the time of the E2 peak result in increases in the MCRs of both E2 and E1 that are not associated with changes in the pattern of protein-binding or in the activity of the 17 beta-hydroxy steroid dehydrogenase.

We studied the dynamics of androgen, estrogen, and cortisol (F) production, metabolism, and protein binding in cynomolgus monkeys (M. fascicularis) to provide baseline data and to compare these parameters with those obtained in other primates. Constant infusions of 3H-labeled androgens, 14C-labeled estrogens, and [3H]F were administered to 11 male cynomolgus monkeys (M. fascicularis) for 3.5 h. Blood samples were obtained from a peripheral vein during the infusion, and all urine was collected for 96 h. In each of 3 monkeys, a catheter was inserted into the hepatic vein, and during the infusions blood samples were obtained from the hepatic and peripheral veins and the femoral artery. All blood and urine samples were analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E2), and estrone (E1). When indicated, blood samples were also analyzed for radioactivity as F. Blood samples taken before the infusions were analyzed for endogenous T, A, DHT, E1, E2, and F concentrations; percent free T, free E2, and free F; and sex hormone-binding globulin and F-binding globulin capacities. The mean +/- SE MCRs for T, A, E2, E1, and F were 44 +/- 4, 407 +/- 40, 175 +/- 17, 315 +/- 28, and 57 +/- 6 liters/day, respectively. The mean blood production rates were 128 +/- 19, 91 +/- 14, 3.3 +/- 0.5, and 9.2 +/- 1.1 micrograms/day and 13.4 +/- 1.9 mg/day for T, A, E2, E1, and F, respectively. The aromatization of androgens was 1.30 +/- 0.10% for A to E1 and 0.28 +/- 0.03% for T to E2. The percent free F (4.34 +/- 0.42%) was greater than the percent free T (1.73 +/- 0.16%) or free E2 (2.75 +/- 0.22%), and the concentration of F-binding globulin was greater than that of sex hormone-binding globulin (227 +/- 35 vs. 60 +/- 7 nM). In the three monkeys who had hepatic venous catheterization, the mean extraction, across the splanchnic bed of T was 32 +/- 3%, that of E was 62 +/- 2%, and that of cortisol was 12 +/- 5%. Across peripheral tissues (leg) the mean extractions were 13 +/- 1%, 18 +/- 1%, and 6 +/- 4%, respectively. In general, the dynamics of androgen, estrogen, and F production and metabolism are similar in male cynomolgus and rhesus monkeys and in man. The similarity is especially close for peripheral aromatization despite differences in adipose tissue content between man and nonhuman primates.

Male cynomologous monkeys (M. fascicularis) were infused with [3H]androgens, [14C]estrogens and [3H]cortisol before and after the administration of l-thyroxine, (l-T4) 150 μg/day for 6 wk, dexamethasone 8 mg every 8 h for 3 doses and dexamethasone 1.0 mg/day for 8 days. Blood samples were obtained before each of the infusions and analyzed for endogenous T, A, E1, E2 and F concentrations, % free T and % free E2, sex hormone-binding globulin (SHBG) and cortisol binding globulin (CBG) capacity. When l-T4 was being administered, T4 and triiodothyronine (T3) concentrations were also measured. Blood samples were obtained during the infusions and analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E1), estrone (E2), and cortisol (F). All urine was collected for 96 h and an aliquot of the pooled urine was analyzed for radioactivity as estrone and estradiol glucuronide. The administration of l-T4 for 6 wk to 3 monkeys resulted in a marked rise in T4 and T3 levels, from 4.8 ± 0.4 μg/dl to 24.8 ± 0.8 μg/dl and from 136 ± 6 to 515 ± 71 ng/dl, respectively. MCRT, MCRE2 and MCRE1 did not change, but MCRA values increased slightly and MCRF increased 2–3-fold. [ϱ]T,E2 did not change but [ϱ]BMA,E1 showed a slight but significant increase. The inter-conversions between the androgens and between the estrogens were not altered. There was a 2–3-fold increase in SHBG and a decrease in %FT but no change in %FE2 or CBG. The concentrations of T, A and DHT rose but there was no trend in the levels of the estrogens. The administration of dexamethasone 8 mg every 8 h for 3 doses or 1 mg/day for 8 days caused no changes in the MCRs for T, A, E1 and E2 but did not cause a significant decrease in MCRF. Measurement of splanchnic and peripheral tissue extractions before and after acute dexamethasone administration in 1 monkey showed that the decrease in MCRF was the result of a marked decrease, 11-2%, in splanchnic extraction of F. The extractions of T and E2 were relatively unaffected. The concentrations of T and F fell but E2 remained the same. % FT and % FE2 rose slightly and the concentrations of SHBG and CBG were unchanged. The androgen interconversions and estrogen interconversions were not affected but [ϱ]BMT,E2 and [ϱ]BMA,E1 showed slight decreases. The changes in steroid dynamics as a result of l-T4 are caused in part by increases in SHBG and tissue blood flow which for T and E2 act as opposing forces to maintain their dynamics relatively normal, but for A, E1 and especially F the effects of increases in tissue blood flow are dominat. Dexamethasone, acutely and chronically, decreases T and F concentration, but only MCRF is decreased. The decrease in MCRF is associated with a marked decrease in the hepatic extractions of F. Peripheral aromatization is decreased by both treatments, but more so by the longer treatment.

Male rabbits were infused at a constant rate with 3H-androstenedione/14C-estrone (n = 5) or 3H-testosterone/14C-estradiol-17 beta (n = 3) for 3 1/2 hr and blood samples were obtained over the last hour and analyzed for radioactivity as androstenedione (A), testosterone (T), estrone (E1), estradiol-17 beta (E2 beta) and estradiol-17 alpha (E2 alpha). The mean value for the metabolic clearance rate of androstenedione (MCRA) was 85 +/- 10 l/day/kg, which was significantly greater than the mean MCRE1 59 +/- 10 l/day/kg. MCRT, 42 +/- 8 l/day/kg, and MCRE2 beta, 45 +/- 9 l/day/kg were not different. The conversion ratio of androstenedione to testosterone (CRA,T) was greater than CRT,A but for the estrogens, CRE2 beta, E1 was greater than CRE1,E2 beta. CRE2 beta, E2 alpha was greater than CRE1,E2 alpha. The overall aromatization of androstenedione to estrone, the fraction of 3H-androstenedione infused into the blood and measured as 3H-estrone in blood [( rho]A,E1BB) was 0.0005 +/- 0.0001 and for [rho]T,E2 beta BB was 0.0012 +/- 0.0006. In the rabbit both sex hormone binding globulin (SHBG) and albumin binding may effect the MCRs, and peripheral aromatization of androgens occurs to a far lesser degree than in humans and primates.