Ex vivo Bone Models and Their Potential in Preclinical Evaluation.

PURPOSE OF REVIEW: Novel therapies for damaged and diseased bone are being developed in a preclinical testing process consisting of in vitro cell experiments followed by in vivo animal studies. The in vitro results are often not representative of the results observed in vivo. This could be caused by the complexity of the natural bone environment that is missing in vitro. Ex vivo bone explant cultures provide a model in which cells are preserved in their native three-dimensional environment. Herein, it is aimed to review the current status of bone explant culture models in relation to their potential in complementing the preclinical evaluation process with specific attention paid to the incorporation of mechanical loading within ex vivo culture systems. RECENT FINDINGS: Bone explant cultures are often performed with physiologically less relevant bone, immature bone, and explants derived from rodents, which complicates translatability into clinical practice. Mature bone explants encounter difficulties with maintaining viability, especially in static culture. The integration of mechanical stimuli was able to extend the lifespan of explants and to induce new bone formation. Bone explant cultures provide unique platforms for bone research and mechanical loading was demonstrated to be an important component in achieving osteogenesis ex vivo. However, more research is needed to establish a representative, reliable, and reproducible bone explant culture system that includes both components of bone remodeling, i.e., formation and resorption, in order to bridge the gap between in vitro and in vivo research in preclinical testing.

Characterizing selection in black-throated blue warblers using a sexual network approach.

Our understanding of trait evolution is built upon studies that examine the correlation between traits and fitness, most of which implicitly assume all individuals experience similar selective environments. However, accounting for differences in selective pressures, such as variation in the social environment, can advance our understanding of how selection shapes individual traits and subsequent fitness. In this study, we test whether variation in the social environment affects selection on individual phenotype. We apply a new sexual network framework to quantify each male's social environment as the mean body size of his primary competitors. We test for direct and social selection on male body size using a 10-year data set on black-throated blue warblers (Setophaga caerulescens), a territorial species for which body size is hypothesized to mediate competition for mates. We found that direct selection on body size was weak and nonsignificant, as was social selection via the body size of the males' competitors. Analysing both types of selection simultaneously allows us to firmly reject a role for body size in competitive interactions between males and subsequent male fitness in this population. We evaluate the application of the sexual network approach to empirical data and suggest that other phenotypic traits such as song characteristics and plumage may be more relevant than body size for male-male competition in this small passerine bird.

Requirement analysis for an AI-based AR assistance system for surgical tools in the operating room: stakeholder requirements and technical perspectives.

PURPOSE: We aim to investigate the integration of augmented reality (AR) within the context of increasingly complex surgical procedures and instrument handling toward the transition to smart operating rooms (OR). In contrast to cumbersome paper-based surgical instrument manuals still used in the OR, we wish to provide surgical staff with an AR head-mounted display that provides in-situ visualization and guidance throughout the assembly process of surgical instruments. Our requirement analysis supports the development and provides guidelines for its transfer into surgical practice. METHODS: A three-phase user-centered design approach was applied with online interviews, an observational study, and a workshop with two focus groups with scrub nurses, circulating nurses, surgeons, manufacturers, clinic IT staff, and members of the sterilization department. The requirement analysis was based on key criteria for usability. The data were analyzed via structured content analysis. RESULTS: We identified twelve main problems with the current use of paper manuals. Major issues included sterile users' inability to directly handle non-sterile manuals, missing details, and excessive text information, potentially delaying procedure performance. Major requirements for AR-driven guidance fall into the categories of design, practicability, control, and integration into the current workflow. Additionally, further recommendations for technical development could be obtained. CONCLUSION: In conclusion, our insights have outlined a comprehensive spectrum of requirements that are essential for the successful implementation of an AI- and AR-driven guidance for assembling surgical instruments. The consistently appreciative evaluation by stakeholders underscores the profound potential of AR and AI technology as valuable assistance and guidance.

Stimulation of human endothelial cell prostacyclin synthesis by select leukotrienes.

Cultured endothelial cells from human umbilical cord labeled with [3H]20:4 release radiolabel when exposed to leukotrienes C or D (LTC or LTD). The major radiolabeled 20:4 metabolite recovered in the culture medium was prostacyclin. Both leukotrienes produced a dose-dependent synthesis of prostacyclin, with a maximal response at 10(-7) M leukotriene. LTC promoted a twofold greater response than did LTD at all concentrations tested (10(-9) to 10(-7) M). In contrast, no release of radiolabel above basal levels was evident with a challenge of LTE or LTB at the same concentrations. Endothelial cells metabolize approximately 40-50% of exogenously supplied LTC to LTD and LTE in 60 min. Levels of alpha-glutamyltranspeptidase (gamma-GTPase), the ectoenzyme reported to convert LTC or LTD, were detected in intact endothelial cells with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide at levels sufficient to account for the observed rate of LTC metabolism. High concentrations of the gamma-GTPase inhibitors, glutathione and AT-125, blocked the metabolism of LTC by endothelium. These results suggest that degradation of leukotrienes by endothelium may be one mechanism for inactivation of these lipid mediators.

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis.

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.

The effect of neutrophil migration on epithelial permeability.

To reach an inflammatory lesion, neutrophils must frequently traverse the epithelium of an infected organ. Whether the actual migration of neutrophils alters the epithelial permeability is unknown. Through the use of an in vitro model system it was possible to directly determine the effect of neutrophil emigration on the transepithelial electrical resistance of the monolayer. Human neutrophils (5 X 10(6) cells/ml) were placed in the upper compartment of a combined chemotaxis/resistance chamber and stimulated for 40 min by a gradient of 10(-7) M n-formyl-methionyl-leucyl-phenylalanine to traverse a confluent monolayer of canine kidney epithelial cells grown on micropore filters. Neither the chemoattractant alone (10(-5)-10(-9) M) nor the accumulation of an average of eight neutrophils per millimeter of epithelium lowered the transepithelial electrical resistance. However, under certain conditions the migration of neutrophils temporarily increased the permeability of the monolayer. The resistance fell approximately 48% within 5 min if the migratory cells were stimulated to reverse their migration across the same monolayer. As re-migration continued, the resistance returned to its initial levels within 60 min. Doubling the initial neutrophil concentration to 10 X 10(6) cells/ml resulted in the accumulation of an average of 66 neutrophils per millimeter of epithelium and an average fall in resistance of 46% (r = 0.98; P less than 0.001) in 40 min. If the resistance had fallen less than 45%, removal of the neutrophils remaining in the upper compartment resulted in a return of the transepithelial electrical resistance to its initial level within 65 min. However, when the fall was greater than 45%, the resistance only recovered to 23.5% of its initial levels within the same time frame. Thus, these results suggest that the integrity of an epithelium can, under certain conditions, be affected by the emigration of neutrophils, but that this effect is either completely or partially reversible within 65 min.

Epithelial permeability and the transepithelial migration of human neutrophils.

Although polymorphonuclear leukocytes (PMN's) can migrate through every epithelium in the body regardless of its permeability, very little is known about the effect of epithelial permeability on PMN migration and the effect of emigrating PMN's on the permeability of the epithelium. In an in vitro model system of transepithelial migration, human PMN's were stimulated by 0.1 micrometer fMet-Leu-Phe to traverse confluent, polarized canine kidney epithelial monolayers of varying permeabilities. Epithelial permeability was determined by both conductance measurement and horseradish peroxidase (HRP) tracer studies. As epithelial permeability increased, the number of PMN invasion sites as well as the number of PMN's that traversed the monolayer increased. The effect of PMN migration on epithelial permeability was examined using the ultrastructural tracers HRP and lanthanum nitrate. PMN's traversing the monolayer made close cell-to-cell contacts with other invading PMNs and with adjacent epithelial cells. These close contacts appeared to prevent leakage of tracer across invasion sites. Following PMN emigration, epithelial junctional membranes reapproximated and were impermeable to the tracers. These results indicated that, in the absence of serum and connective tissue factors, (a) the number of PMN invasion sites and the number of PMN's that traversed an epithelium were a function of the conductance of the epithelium and (b) PMN's in the process of transepithelial migration maintained close cell-cell contacts and prevented the leakage of particles (greater than 5 nm in diameter) across the invasion site.

Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current.

Bovine microvascular endothelial cells (BMECs) proliferated to confluence on the stromal surface of human amniotic membrane that had been denuded of its natural epithelium. The resulting cultures had the following characteristics: (a) The endothelial cells formed a thin, continuous monolayer and, like their in vivo counterparts, contained basal adhesion plaques and large numbers of cytoplasmic vesicles and 10-nm filaments. In addition, the endothelial cells elaborated a basement membrane-like structure. (b) The borders of the BMECs reacted with AgNO3 to produce the "flagstone" pattern typical of endothelium stained with this reagent in vivo. (c) More than 90% of the zones of contact between endothelial cells examined 8 d after plating prevented passage of a macromolecular probe (wheat germ agglutinin conjugated to horseradish peroxidase) across the BMEC monolayer. (d) 8 d-old cultures displayed a transendothelial electrical resistance that averaged 69 +/- 28 omega X cm2. Monolayers of BMECs maintained on amnion thus resemble in vivo endothelium in several respects and should provide a useful and relevant model for the in vitro study of various phenomena that occur at the microvascular wall.

Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.

P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells.

P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P-selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P-selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules.