Visualization of elementary DNA replication units in human nuclei corresponding in size to DNA loop domains

Newly synthesized DNA in mammalian nuclei is concentrated in discrete nuclear granules called replication foci. These foci may be visualized using antibodies against 5-bromodeoxyuridine. In the early S-phase cells 100–250 foci are usually detected. On average, individual foci range between 0.5 and 2 µm in diameter and can be seen as clusters of more than ten average-sized (60–100 kb) synchronously activated replicons. In this study, employing minor modifications of the previous methods, we report the visualization of small replication foci of about 0.3 µm diameter (mini-foci). Some foci are clustered into folded chains consisting of 2–40 subunits. DNA content of one mini-focus is estimated to be 50–120 kb and there are 500–1500 mini-foci per cell in the early S-phase. Experimentally induced decrease in replicon size does not affect the size of mini-foci, suggesting that these represent elementary units of DNA replication in mammalian nuclei and are probably identical to the basic structural DNA loop domains.     

Differential expression of the L- and S-isoforms of myelin associated glycoprotein (MAG) in oligodendrocyte unit phenotypes in the adult rat anterior medullary velum

We have previously demonstrated differences in the expression of carbonic anhydrase II (CAII) in oligodendrocyte units myelinating small and large diameter fibres in the anterior medullary velum (AMV) of the adult rat (each unit comprises the cell body, processes and myelin sheaths). Others have indicated that myelin composition may also vary with respect to myelin basic protein (MBP) and proteolipid protein (PLP), and the small (S)- and large (L)-isoforms of myelin associated glycoprotein (MAG). In this study, we have determined the expression of myelin proteins in oligodendrocyte unit phenotypes I–IV, which myelinate fibres ranging in diameter from 0.3–12 m diameter in the AMV, by using double immunolabelling for Rip, which labels entire units, and MBP, PLP, myelin oligodendrocyte glycoprotein (MOG), L-MAG and S-MAG. We show differences in the expression of L- and S-MAG in units which myelinate different diameter fibres: (1) type I/II units myelinating small diameter fibres had a L-MAG+/S-MAG–/CAII+ phenotype; (2) type II/III units myelinating different diameter fibres had a L-MAG+/S-MAG+/CAII+ phenotype; (3) type III/IV units myelinated large diameter fibres had a L-MAG+/S-MAG+/CAII– phenotype. All units, irrespective of fibre diameter, expressed Rip, MBP, PLP and MOG. The results indicate that type I–IV units may be variants of a single oligodendrocyte population and that phenotypic differences are determined by the diameter of fibres within the unit. The possible significance of metabolic and biochemical differences between oligodendrocytes myelinating small and large diameter axons are discussed with reference to the pathology of demyelination.     

Acute osmotic/stress stimuli induce a transient decrease of transcriptional activity in the neurosecretory neurons of supraoptic nuclei

Axon-oligodendrocyte relations of Rip-immunolabelled and dye-injected oligodendrocyte units are characterised in the adult rat anterior medullary velum (AMV). Each oligodendrocyte unit comprised the oligodendrocyte cell body, processes and the internodal myelin segments they support. Oligodendrocyte units corresponded to classically described type I/II or type III/IV unit phenotypes which respectively myelinated discrete populations of small and large diameter axons, delineated by a myelinated fire diameter of 2–4 m (diameter of the axon plus its myelin sheath). Within units, mean fibre diameter was directly related to mean internodal length and inversely related to the number of myelin sheaths in the unit. The relationship between fibre diameter and internodal length was retained in units which myelinated axons of different diameters, indicating that axon diameter was an important determinant of the longitudinal dimensions of myelin sheaths. We also show that type III/IV units maintained a far greater volume of myelin than type I/II units. It was concluded that type I/II and III/IV oligodendrocytes represent two functionally and morphologically distinct phenotypes whose distribution densities were determined by the diameter and spatial dispersion of axons.     

Janus magnetic cellular spheroids for vascular tissue engineering

Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly.     

Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography

Microcomputed tomography (micro-CT) is increasingly being used to analyze the three-dimensional structure and architecture of microvascular networks. Therefore we have evaluated a micro-CT analysis of VEGF-induced vessel ingrowth into a porous polyurethane scaffold through comparison with analyses by CD31 immunohistochemistry, vascular perfusion by intravital Lycopersicon esculentum lectin perfusion and vascular corrosion casting. Micro-CT scanning found a similar level of vascularisation within the VEGF treated scaffolds to that determined by the other analytical methods. However, although the relative increase in vascularisation (17 fold above PBS controls p < 0.05) induced by VEGF determined by micro-CT was similar to the perfusion based analyses (20.1 and 10.4 fold for lectin perfusion and vascular corrosion respectively p < 0.05), it differed substantially from that determined by CD31 immunohistochemistry (3.2 fold p < 0.05). This difference was due to a large proportion of unperfused vessels in the PBS control that were not present in the VEGF group. The increase in perfusion probably resulted in part from an increase in average vessel diameter. Though this increase was detected by micro-CT, the actual diameters were overestimated by 60–90% most likely as a consequence of a merging effect for juxtaposed vessels. Thus whilst micro-CT gives an accurate three-dimensional quantification of the VEGF-induced increase in perfused vessels, resolution needs to be maximized for accurate sizing of a microvascular network's components.     

Design of custom-shaped vascularized tissues using microtissue spheroids as minimal building units

Tissue engineering strategies are gathering clinical momentum in regenerative medicine and are expected to provide excellent opportunities for therapy for difficult-to-treat human pathologies. Being aware of the requirement to produce larger artificial tissue implants for clinical applications, we used microtissues, produced using gravity-enforced self-assembly of monodispersed primary cells, as minimal tissue units to generate scaffold-free vascularized artificial macrotissues in custom-shaped agarose molds. Mouse myoblast, pig and human articular-derived chondrocytes, and human myofibroblast (HMF)-composed microtissues (microm3 scale) were amalgamated to form coherent macrotissue patches (mm3 scale) of a desired shape. Macrotissues, assembled from the human umbilical vein endothelial cell (HUVEC)-coated HMF microtissues, developed a vascular system, which functionally connected to the chicken embryo's vasculature after implantation. The design of scaffold-free vascularized macrotissues is a first step toward the scale-up and production of artificial tissue implants for future tissue engineering initiatives.     

Influence of nanopatterns on endothelial cell adhesion: Enhanced cell retention under shear stress

In this study, nanopatterned crosslinked films of collagen Type I were seeded with human microvascular endothelial cells and tested for their suitability for vascular tissue engineering. Since the films will be rolled into tubes with concentric layers of collagen, nutrient transfer through the collagen films is quite crucial. Molecular diffusivity through the collagen films, cell viability, cell proliferation and cell retention following shear stress were studied. Cells were seeded onto linearly nanogrooved films (groove widths of 332.5, 500 and 650 nm), with the grooves aligned in the direction of flow. The nanopatterns did not affect cell proliferation or initial cell alignment; however, they significantly affected cell retention under fluid flow. While cell retention on unpatterned films was 35 ± 10%, it was 75 ± 4% on 332.5 nm patterned films and even higher, 91 ± 5%, on 650 nm patterned films. The films were found to have diffusion coefficients of ca. 10⁻⁶ cm² s⁻¹ for O₂ and 4-acetaminophenol, which is comparable to that observed in natural tissues. This constitutes another positive asset of these films for consideration as a scaffold material for vascular tissue engineering.     

Mechanical properties of arteries cryopreserved at −80 °C and −150 °C

A new protocol for cryopreservation of arteries frozen at −80 °C was compared to the reference protocol for cryopreservation at −150 °C and to freshly harvested arteries. The aim of the study is to evaluate both protocols as global procedures to freeze and thaw arteries commonly used in tissue banks. Changes in mechanical properties of rabbit common carotid arteries were studied. Vascular segments were tested in vitro under dynamics loading conditions. Pressure and diameter were recorded simultaneously by a high fidelity transducer and an echotracking device, respectively. The pressure–diameter relationship was fitted by the arctangent Langewouters’ model and the arterial thickness was derived from histological measurements. Histological sections showed that the fresh and −80 °C groups were less damaged by hemodynamic load and histological preparation than the −150 °C group (p < 0.05). No differences between fresh and cryopreserved arteries regarding the structural (diameter, intimal-media thickness) and mechanical parameters (distensibility, circumferential stress, elastic modulus) were found. The isobaric circumferential stress was reduced in frozen arteries. These results demonstrate that the cryopreservation at −80 °C preserves the histological structure and mechanical properties better than the cryopreservation at −150 °C, suggesting that the new cryopreservation protocol at −80 °C is a method of choice for treating vessel replacement in vascular surgery.     

Bioresorbable elastomeric vascular tissue engineering scaffolds via melt spinning and electrospinning

Current surgical therapy for diseased vessels less than 6 mm in diameter involves bypass grafting with autologous arteries or veins. Although this surgical practice is common, it has significant limitations and complications, such as occlusion, intimal hyperplasia and compliance mismatch. As a result, cardiovascular biomaterials research has been motivated to develop tissue-engineered blood vessel substitutes. In this study, vascular tissue engineering scaffolds were fabricated using two different approaches, namely melt spinning and electrospinning. Small diameter tubes were fabricated from an elastomeric bioresorbable 50:50 poly(l-lactide-co-ε-caprolactone) copolymer having dimensions of 5 mm in diameter and porosity of over 75%. Scaffolds electrospun from two different solvents, acetone and 1,1,1,3,3,3-hexafluoro-2-propanol were compared in terms of their morphology, mechanical properties and cell viability. Overall, the mechanical properties of the prototype tubes exceeded the transverse tensile values of natural arteries of similar caliber. In addition to spinning the polymer separately into melt-spun and electrospun constructs, the approach in this study has successfully demonstrated that these two techniques can be combined to produce double-layered tubular scaffolds containing both melt-spun macrofibers (<200 μm in diameter) and electrospun submicron fibers (>400 nm in diameter). Since the vascular wall has a complex multilayered architecture and unique mechanical properties, there remain several significant challenges before a successful tissue-engineered artery is achieved.     

Engineering the binding properties of the T cell receptor:peptide:MHC ternary complex that governs T cell activity

The potency of a T cell is determined in large part by two interactions, binding of a cognate peptide to the MHC, and binding of the T cell receptor (TCR) to this pepMHC. Various studies have attempted to assess the relative importance of these interactions, and to correlate the corresponding binding parameters with the level of T cell activity mediated by the peptide. To further examine the properties that govern optimal T cell activity, here we engineered both the peptide:MHC interaction and the TCR:pepMHC interaction to generate improved T cell activity. Using a system involving the 2C TCR and its allogeneic pepMHC ligand, QL9–L^d, we show that a peptide substitution of QL9 (F5R), increased the affinity and stability of the pep–L^d complex (e.g. cell surface t_1/2-values of 13 min for QL9–L^d versus 87 min for F5R–L^d). However, activity of peptide F5R for 2C T cells was not enhanced because the 2C TCR bound with very low affinity to F5R–L^d compared to QL9–L^d (K_D = 300 μM and K_D = 1.6 μM, respectively). To improve the affinity, yeast display of the 2C TCR was used to engineer two mutant TCRs that exhibited higher affinity for F5R–L^d (K_D = 1.2 and 6.3 μM). T cells that expressed these higher affinity TCRs were stimulated by F5R–L^d in the absence of CD8, and the highest affinity TCR exhibited enhanced activity for F5R compared to QL9. The results provide a guide to designing the explicit binding parameters that govern optimal T cell activities.     

A method of obtaining clone lines of cells

Summary A method of using microcapillaries filled with a small amount of nutrient medium is suggested for trapping and planting single cells in test-tubes. Trapping is done from a drop of cellular suspension on a slide, which is fixed under a microscope. Since the time of cloning is minimized cloning, cellular injury decreases, and the planting efficiency rises. The yield of colonies for A-1 strain was 63%, HeLa strain –50%, No. 630 strain –37% and Cave strain –10%. The number of planted single cells is unlimited. The use of test tubes simplified obtaining of clonal cell lines.(Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 3, pp. 120–121, March, 1964.     

Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients

In this report, we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell–substratum interactions. A single cell adhesion substratum with the constant ridge width (1 μm), and depth (400 nm) and variable groove widths (1 –9.1 μm) allowed us to characterize the dependence of cellular responses, including cell shape, orientation, and migration, on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges, vs. those on the sparser areas, exhibiting a biphasic dependence of the migration speed on the pattern density. In addition, cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term), suggesting that single cells can sense the topography gradient. Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result, in addition to demonstrating a more sophisticated method to study aspects of wound healing processes, strongly suggests that even in the presence of adhesive cell–cell interactions, the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell–matrix interactions, but also contribute to bioengineering of devices with the optimally structured design of cell–material interface.     

An electron microscopic study of autonomic nerve cells in the cloacal region of the lamprey,Lampetra japonica

Summary Two types of autonomic nerve cell in the cloacal region of lamprey,Lampetra japonica have been studied by electron microscopy. Large ganglion cells (LGC) were unipolar and individually invested with a satellite cell sheath. The LGC-satellite cell complex measured 24 m × 38 m on average. Granular endoplasmic reticulum and cored vesicles (80–140 nm in diameter) were scattered in the perikaryon. Two kinds of peculiar cytoplasmic filament were seen in LGC: one type was about 20 nm in diameter with periodic dense material on the surface and the other had a diameter of about 8 nm and showed an undulating profile. Nerve endings containing abundant small clear vesicles (30–50 nm in diameter) and a few larger cored vesicles (50–100 nm in diameter) were found in synaptic contact with LGC. Small ganglion cells (SGC) were also unipolar and covered incompletely by a satellite cell sheath. The SGC-satellite cell complex measured 6 and 12 m on average. The SGC was packed with organelles and the perikaryon appeared more electron dense than that of LGC. SGC perikaryonal cytoplasm contained dispersed granular endoplasmic reticulum and numerous large cored vesicles (55–220 nm in diameter). Nerve endings containing numerous large cored vesicles (70–170 nm in diameter) and variable numbers of small clear vesicles (30–50 nm in diameter) were seen on the surface of SGC.     

Time relationships in the development of cytopathic response to vaccinia virus in tissue culture tubes

Summary The time of appearance of cytopathic changes was studied in vaccinia virus infected tissue culture tubes. From proportions of consecutively recorded positive tubes it was inferred that the cumulative percentages of newly formed plaques plotted on a probit scale formed a straight line against time. In a given experiment this distribution was of the same type irrespective of the virus dose.In repeatedly read virus titrations an approximately linear relationship was found to exist between the logarithm of virus dilution and the reciprocal of incubation time (= the period from the moment of inoculation until the first positive reading). The relation was rectilinear within a dose interval of 4–5 log units but outside this range departure from linearity was found.For practical reasons the incubation time method was less useful as a routine procedure for virus assay.End-point titers in monkey kidney tubes increased by 0.15–0.20 log from day 6 to day 12.     

Long term catheterization of the portal vein in cats

Summary A method of chronic catheterization of the major blood vessels, including the portal vein, may be successfully used for studying the interstitial metabolism and hemodynamics. For catheterization of the portal vein in cats in conditions of chronic experiment we used catheters made of polyethylene, 1–1.5 mm in diameter and 120–180 mm in length with a dilatation at the peripheral end. The catheter was introduced surgically through one of the branches of the mesenteric vein with the aid of a special guide. To avoid thrombosis during the postoperative period the catheters were washed once in 2–3 days with heparin solution (400–500 units/ml).(Presented by Academician V. N. Chernigovskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 55, No. 1, pp. 122–123, January 1963     

Bilayered vascular grafts based on silk proteins

A major block in the development of small diameter vascular grafts is achieving suitable blood vessel regeneration while minimizing the risk of thrombosis, intimal hyperplasia, suture retention, and mechanical failure. Silk-based tubular vessels for tissue engineering have been prepared by molding, dipping, electrospinning, or gel spinning, however, further studies are needed to improve the mechanical and blood compatibility properties. In the present study a bilayered vascular graft based on silk fibroin (SF) was developed. The graft was composed of an inner silk fiber-reinforced SF tube containing heparin and a highly porous SF external layer. Compared with previously fabricated SF tubes the fiber-reinforcement provided a comparable or higher mechanical strength, burst pressure, and suture retention strength, as well as mechanical compliance, to saphenous veins for vascular grafts. Heparin release was sustained for at least 1 month, affording blood compatibility to the grafts. The outer layer of the grafts prepared through lyophilization had a highly porous structure in which the macropore walls were composed of nanofibers similar to extracellular matrix, which offered an excellent environment for cell growth. In vitro studies showed good cytocompatibility and hemocompatibility.     

Cyclo-DfKRG peptide modulates in vitro and in vivo behavior of human osteoprogenitor cells on titanium alloys

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.     

Influence of micro-well biomimetic topography on intestinal epithelial Caco-2 cell phenotype

A microfabrication approach was utilized to create topographic analogs of intestinal crypts on a polymer substrate. It was hypothesized that biomimetic crypt-like micro-architecture may induce changes in small intestinal cell (i.e. Caco-2 cell) phenotype. A test pattern of micro-well features with similar dimensions (50, 100, and 500 μm diameter, 50 μm spacing, 120 μm in depth) to the crypt structures found in native basal lamina was produced in the surface of a poly(dimethylsiloxane) (PDMS) substrate. PDMS surfaces were coated with fibronectin, seeded with intestinal-epithelial-cell-like Caco-2 cells, and cultured up to fourteen days. The cells were able to crawl along the steep side walls and migrated from the bottom to the top of the well structures, completely covering the surface by 4–5 days in culture. The topography of the PDMS substrates influenced cell spreading after seeding; cells spread faster and in a more uniform fashion on flat surfaces than on those with micro-well structures, where cell protrusions extending to micro-well side walls was evident. Substrate topography also affected cell metabolic activity and differentiation; cells had higher mitochondrial activity but lower alkaline phosphatase activity at early time points in culture (2–3 days post-seeding) when seeded on micro-well patterned PDMS substrates compared to flat substrates. These results emphasize the importance of topographical design properties of a scaffolds used for tissue engineered intestine.     

Role of VEGF gene variability in longevity: a lesson from the Italian population

Vascular endothelial growth factor (VEGF) gene polymorphisms have been associated with an increased risk of developing a wide variety of disorders from diabetes to neurodegenerative diseases suggesting functions not confined to its vascular effects originally described. Based on the VEGF protective roles undisclosed in pathological conditions, we evaluate whether VEGF variability might be a determinant also for longevity. Four polymorphisms (−2578C/A, −1190G/A, −1154G/A and −634G/C) within the VEGF gene promoter region in 490 unrelated Italian healthy subjects have been analysed. Significant changes of allele, genotype (−2578/AA versus −2578/CC: OR = 2.08, p = 0.007; −1190/AA versus −1190/GG: OR = 2.01, p = 0.011) and haplotype (AAGG: 10.4% versus 14.9%, p = 0.03) frequency distributions were observed between young/elderly (25–84 years old) and long-lived (85–99 years old) subjects. These results suggest that VEGF gene variability can be inserted among the genetic factors influencing the lifespan.     

Development of a reinforced PMMA-based hip spacer adapted to patients’ needs

Two-stage reimplantation using an interval hip prosthesis (spacer) of antibiotic-impregnated bone cement has become a well-accepted method to eradicate infection and prevent limb shortening. However, custom made as well as prefabricated spacers share a weakness of limited strength and hence several fractures of spacers have been observed, even for partial weight bearing. The purpose of this study was therefore to improve the strength of the custom made spacer, used at the Orthopaedic Department of the Saarland University Hospital (Germany). As the material strength of bone cement is limited, several reinforced spacers with a metal core consisting of titanium grade two have been developed and investigated. Loading procedure was close to the ISO 7206/4, though small adaptation was made. An inserted rod of titanium grade two increased the collapse load up to 1000–1300 N, but considering a maximum expected load of about three times the body weight, still below the required value. A “full-stem” reinforced spacer, i.e. spacer with a titanium endoskeleton and a minimum of 2–3 mm PMMA-coating in order to assure drug release, provides a mobile and functional joint through the treatment course. Those with 8 mm thickness of titanium endoskeleton endured up to one million load cycles in a load range of 300–2300 N. To give further support for individual cases a meaningful S–N curve for this device was determined.