Collagen self-assembly and the development of tendon mechanical properties

The development of the musculoskeleton and the ability to locomote requires controlled cell division as well as spatial control over deposition of extracellular matrix. Self-assembly of procollagen and its final processing into collagen fibrils occurs extracellularly. The formation of crosslinked collagen fibers results in the conversion of weak liquid-like embryonic tissues to tough elastic solids that can store energy and do work. Collagen fibers in the form of fascicles are the major structural units found in tendon. The purpose of this paper is to review the literature on collagen self-assembly and tendon development and to relate this information to the development of elastic energy storage in non-mineralizing and mineralizing tendons. Of particular interest is the mechanism by which energy is stored in tendons during locomotion. In vivo, collagen self-assembly occurs by the deposition of thin fibrils in recesses within the cell membrane. These thin fibrils later grow in length and width by lateral fusion of intermediates. In vitro, collagen self-assembly occurs by both linear and lateral growth steps with parallel events seen in vivo; however, in the absence of cellular control and enzymatic cleavage of the propeptides, the growth mechanism is altered, and the fibrils are irregular in cross section. Results of mechanical studies suggest that prior to locomotion the mechanical response of tendon to loading is dominated by the viscous sliding of collagen fibrils. In contrast, after birth when locomotion begins, the mechanical response is dominated by elastic stretching of crosslinked collagen molecules.     

An experimental model for studying radiation-induced suppression of antitumor resistance

In experiments on mice (CBA × C57Bl)F₁ an experimental model is developed for studying the effect of radiation on antitumor resistance. Adequate ways are offered for statistical processing of the data on enhancement of tumor growth in the irradiated organism. The validated model is a solid tumor developing after inoculation of a mouse with 1000 cells of Ehrlich ascites tumor into a hind shin, combined with suppression of antitumor resistance upon a 6-Gy (¹³⁷Cs) whole-body exposure. The recommended parameters for assessing tumor growth are: share of animals with tumors by day 30 after inoculation, length of the latent period, probability of tumor development, and tumor volume by day 30. For data processing, besides the common Fisher, χ² and Mann-Whitney tests, we recommend the Kaplan-Meier method and Log-rank. The proposed model can be a reliable tool in solving problems at the junction of radiobiology, oncology, immunology, pharmacology, etc.     

Shedding light on the system: studying embryonic development with light sheet microscopy

Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications. There is an outstanding potential in applying this technology to the quantitative study of embryonic development. Here, we provide an overview of the different basic implementations of LSFM, review recent technical advances in the field and highlight applications in the context of embryonic development. We conclude with a discussion of promising future directions.     

Determining the contribution of glycosaminoglycans to tendon mechanical properties with a modified shear-lag model

Tendon has a complex hierarchical structure composed of both a collagenous and a non-collagenous matrix. Despite several studies that have aimed to elucidate the mechanism of load transfer between matrix components, the roles of glycosaminoglycans (GAGs) remain controversial. Thus, this study investigated the elastic properties of tendon using a modified shear-lag model that accounts for the structure and non-linear mechanical response of the GAGs. Unlike prior shear-lag models that are solved either in two dimensions or in axially symmetric geometries, we present a closed-form analytical model for three-dimensional periodic lattices of fibrils linked by GAGs. Using this approach, we show that the non-linear mechanical response of the GAGs leads to a distinct toe region in the stress–strain response of the tendon. The critical strain of the toe region is shown to decrease inversely with fibril length. Furthermore, we identify a characteristic length scale, related to microstructural parameters (e.g. GAG spacing, stiffness, and geometry) over which load is transferred from the GAGs to the fibrils. We show that when the fibril lengths are significantly larger than this length scale, the mechanical properties of the tendon are relatively insensitive to deletion of GAGs. Our results provide a physical explanation for the insensitivity for the mechanical response of tendon to the deletion of GAGs in mature tendons, underscore the importance of fibril length in determining the elastic properties of the tendon, and are in excellent agreement with computationally intensive simulations.     

Experimental model for studying the mechanical effects of +Gz accelerations on the brain

Bovine brains were excised and placed into a transparent mold equipped with a pump and perfusion system. This unit was then centrifuged and changes in brain contour were video recorded. Analysis of the resulting images showed that changes occurred in brain structures as a result of crushing. The effects of perfusion on the amount of deformation and other results are discussed.     

Region-specific mechanical properties of the human patella tendon.

The present study investigated the mechanical properties of tendon fascicles from the anterior and posterior human patellar tendon. Collagen fascicles from the anterior and posterior human patellar tendon in healthy young men (mean +/- SD, 29.0 +/- 4.6 yr, n = 6) were tested in a mechanical rig. A stereoscopic microscope equipped with a digital camera recorded elongation. The fascicles were preconditioned five cycles before the failure test based on pilot data on rat tendon fascicle. Human fascicle length increased with repeated cycles (P < 0.05); cycle 5 differed from cycle 1 (P < 0.05), but not cycles 2-4. Peak stress and yield stress were greater for anterior (76.0 +/- 9.5 and 56.6 +/- 10.4 MPa, respectively) than posterior fascicles (38.5 +/- 3.9 and 31.6 +/- 2.9 MPa, respectively), P < 0.05, while yield strain was similar (anterior 6.8 +/- 1.0%, posterior 8.7 +/- 1.4%). Tangent modulus was greater for the anterior (1,231 +/- 188 MPa) than the posterior (583 +/- 122 MPa) fascicles, P < 0.05. In conclusion, tendon fascicles from the anterior portion of the human patellar tendon in young men displayed considerably greater peak and yield stress and tangent modulus compared with the posterior portion of the tendon, indicating region-specific material properties.     

Evidence that sclerotomal cells do not migrate medially during normal embryonic development of the rat.

During embryonic development the medial part of the somite disorganizes or breaks up into sclerotomal cells which, according to many published reports, migrate medially to surround the notochord. The purpose of the study was to determine whether these cells actually migrate medially toward the notochord. Distances were measured between the notochord and the adjacent neural tube and the somite or its remnant during the period of somite disorganization. Serially sectioned, normal 10.5- to 13.5-day (d) rat embryos were used. Only transverse sections through the middle of the fourth cervical (C-4) body segment were measured, corresponding to the level of somite No. 8 (10.5 d) or its dermatomyotome remnant (10.5-11.5d) or spinal nerve C-4 (12.5-13.5d). Measurements were taken at six stages from photographic montages, all of which were made at precisely the same magnification. The notochord was the central axial structure from which the measurements were determined. The changes in distance show that during the period of somite breakup the neural tube grows dorsally, away from the notochord which lies adjacent to its ventral surface. Simultaneously the somite remnant moves laterally and dorsally, all the while maintaining its position relative to the overlying ectoderm and leaving behind a trail of sclerotomal cells. Also at each stage cell counts were made on the medial sclerotomal region of the C-4 segment. The average counts reveal that not only does the total number of cells increase substantially over the three-day period (42-7,546), but also the total number of mitoses (3.5-200), while the mitotic index decreases (9.0-2.7). High proliferative activity is apparent in the medial sclerotomal cells throughout the 3-day period. The evidence supports the conclusion that local proliferation of the trailing cells, which were left by the somite remnant as it moved dorsolaterally, causes the subsequent increase in density of the perichordal tissue, rather than an influx of migrating cells. Instead of sclerotomal cells migrating medially toward the notochord, the present study suggests that these cells retain their position relative to the notochord or central axis and that the medial sclerotomal region forms as a result of the growth movements of the surrounding structures.     

气温上升对模拟森林生态系统影响实验的介绍

气温上升对森林生态系统结构和功能有重要的影响。该文简要介绍了鼎湖山森林生态系统定位研究站开展的大型实验——气温上升对模拟森林生态系统的影响。介绍了实验设计及其创新性, 实验研究内容等, 为相关实验的设计提供指导与依据。     

Xenopus: An emerging model for studying congenital heart disease

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes.     

The human erythrocyte ghost: A new experimental model for studying adenosine transport

Previous work on adenosine transport has always had problems with the interference of adenosine metabolism, due to its high metabolic rate and because the enzymes involved are consistently present in most tissues. A new experimental model for studying adenosine transport in human erythrocyte ghosts is presented in this work: Human erythrocyte ghosts were sealed in the presence of erythro-3(2-hydroxynonyl)adenine and P1-P5-di(adenosine)5′-pentaphosphate, inhibitors of adenosine deaminase and adenosine kinase, respectively. These ghosts proved to lack adenosine metabolism when incubated in [U-¹⁴C]adenosine at 10 μm concentration at 37 °C for 60 min. Ghosts were 99.4% sealed in the correct orientation and had constant intracellular water volume. With these characteristics, the erythrocyte ghost preparation has many advantages for studying adenosine transport without adenosine metabolism interference. Adenosine transport was studied following the technique of 24., 25. Experiments to study Zero-trans influx and efflux, equilibrium exchange, and infinite-trans influx and efflux are presented. Adenosine transport did not behave linearly in any of these experimental procedures. Adenosine basic kinetic constants, calculated according to the procedure of Lieb and Stein, were R_1→-2 = 4.1 × 10⁻⁴, R_2→-1 = 3.97 × 10⁻⁴, R_ee = 1.94 × 10⁻⁴, R_oo = 6.08 × 10⁻⁴, K_1→-2 = 125.67 μm, andK_2→-1 = 84.36 μm. Lieb and Stein rejection criteria were used to distinguish a simple pore from a simple carrier. The data accumulated indicate that adenosine transport is carried out by a system that satisfies the criteria used for the simple carrier model. Asymmetric behavior was observed indicating lower affinity of the carrier for adenosine influx, although V_max values for influx and efflux were similar.     

Effects of early incubation constancy on embryonic development: An experimental study in the herring gull Larus argentatus

1.

In many birds, parental nest attendance in early incubation is variable, with eggs incubated only intermittently. The effect of this on chick hatching success is unknown.     

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BackgroundSubcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.MethodsFLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.Results5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca^2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB₄. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.ConclusionThe cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.General significanceWe present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.     

The Mouse Limb Anatomy Atlas: An interactive 3D tool for studying embryonic limb patterning

Background  

The developing mouse limb is widely used as a model system for studying tissue patterning. Despite this, few references are available that can be used for the correct identification of developing limb structures, such as muscles and tendons. Existing textual references consist of two-dimensional (2D) illustrations of the adult rat or mouse limb that can be difficult to apply when attempting to describe the complex three-dimensional (3D) relationship between tissues.     

A fine structural study of the development of the chick flexor digital tendon: a model for synovial sheathed tendon healing.

The development of the synovial sheathed flexor digital tendon in the chick was studied by light and electron microscopy in 12-day embryos to 22-day post-hatched chickens. Areas of specialized connective tissue differentiation were identified in this complex structure consisting of a lubricated synovial sheath, elastic vincula and fibrocartilaginous adaptations on the surface of the tendon. The presence of some of these specialized adaptations may be related to the specific types of mechanical forces and stresses applied to the developing connective tissue system. This model system appears to be appropriate for the experimental study of tendon injuries related to the human hand.     

An experimental study on in vivo dynamic mechanical properties of the knees of cats in valgus loading

In these experiments an attempt was made to investigate in vivo dynamic mechanical properties of the knee in valgus loading. Fifteen adult cats were used. Sinusoidal bending oscillation of 0.3, 1.0, 1.6, 3.3, 5.0, 8.6 and 10.0 Hz was transmitted to the knee-leg complex under each pre-bending strain in the valgus direction, and the bending moment was measured. Dynamic mechanical properties of the knee-leg complex were given as a complex bending elastic coefficient. Bending elastic and bending damping coefficient of the knee could be calculated by the least square method. As a first approximation, the knee was represented as a non-linear Kelvin model. Both bending elastic and bending damping coefficient of the knee increased as pre-bending moment was increased, and decreased as amplitude of bending oscillation was increased. Mechanical loss tangent of the knee-leg complex decreased within the range of pre-bending moment larger than 1.0 X 10(6) dyn cm as bending moment was increased, and decreased as amplitude of bending oscillation was increased.     

Discrete probability of a model for information provision on early embryonic development

A percolation model of the diffuse redistribution of morphogenetic information in early regulative development is analyzed. It is demonstrated that the statistical average values of cell connectedness remaining below the percolation threshold of the spatial redistribution of developmental determinants do not provide for the formation of cell structures of the necessary size. The average number of cell interactions should exceed the percolation threshold, and, therefore, the carriers of morphogenetic information in early development can move over distances comparable with the size of the entire embryo. The assumption concerning the percolation mechanism of cell death is used as a basis for estimating the statistical average value of cell connectedness at which the predicted number of cells theoretically isolated from the flow of signal molecules corresponds to the observed frequencies of dying embryonic cells. The estimated average number of cell interactions significantly exceeds the threshold of information resource percolation in the embryonic space and agrees with estimations of other authors, based on direct observations. The probable role of the diffusion front, or percolation cluster shell, in the regionalization of embryonic structures differing in their prospective values is discussed. It is shown that the duration of the communicative period, along with the statistical average number of channels providing for the intercellular transfer of signal molecules by diffusion, is a parameter controlling the processes of determination of embryonic structures.