Changes in the morphology and synthetic activity of cultured rat tail tendon

Summary Isolated single fascicles from tail tendons of young rats were freed of epitenon cells and cultured in vitro for up to 7 days. The tissue remained viable, as judged by the structural integrity of cell organelles and the ability to synthesize DNA and glycosaminoglycans (GAG). The rate of DNA synthesis peaked after 2 days in culture and decreased slowly thereafter. Concomitantly, an increase in cell number was noted at the periphery of the fascicle. GAG production also increased during culture, sulphated GAG being increased proportionately more than hyaluronic acid. Dermatan sulphate was the predominant sulphated GAG in freshly isolated fascicles, but in cultured tissue, the newly synthesized sulphated GAG was more sensitive to degradation by chondroitinase AC and had an increased electrophoretic mobility. Fine structural changes were observed in cultured tissues such as the retraction of cell processes. rounding up of cell bodies and the appearance of gaps between collagen fibrils. Cultured tenocytes also frequently contained apparently phagocytized collagen fibrils which were not seen in freshly isolated fascicles, and this appearance was suggestive of collagen degradation occurring in vitro, although no change in the total hydroxyproline content was noted. The data show that when individual fascicles are cultured in vitro they undergo a process of matrix remodelling which has features in common with events occurring in vivo when tendons have been surgically manipulated.     

The influence of cryopreservation and quick-freezing on the mechanical properties of tendons

In order to maintain their native properties, cryopreserved tendons are usually used in biomechanical research and in transplantation of allogenic tendon grafts. The use of different study protocols leads to controversy in literature and thus complicates the evaluation of the current literature. The aim of this study consisted in examining the influence of different freezing and thawing temperatures on the mechanical properties of tendons. 60 porcine tendons were frozen at either −80 °C or −20 °C for 7 days and thawed at room or body temperature for 240 or 30 min, respectively. A subgroup of ten tendons was quick-frozen with liquid nitrogen (−196 °C) for 2 s before cryopreservation. Biomechanical testing was performed with a material testing machine and included creep, cyclic and load-to-failure tests. The results showed that freezing leads to a reduced creep strain after constant loading and to an increased secant modulus. Freezing temperature of −80 °C increased the secant modulus and decreased the strain at maximum stress, whereas thawing at room temperature reduced the maximum stress, the strain at initial tendon failure and the Young’s Modulus. Quick-freezing led to increased creep strain after constant loading, increased strain at initial failure in the load-to-failure test, and decreased strain at maximum stress. When cryopreserving, tendons for scientific or medical reasons, freezing temperature of −20 °C and thawing temperature of 37.5 °C are recommended to maintain the native properties of tendons. A treatment with liquid nitrogen in the sterilization process of tendon allografts is inadvisable because it alters the tendon properties negatively.     

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon

Summary The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-m long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 m transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 m and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.     

Thermal stability, flame retardancy and mechanical properties of cotton fabrics treated with inorganic coatings synthesized through sol-gel processes

Cotton was sol-gel treated employing several metal alkoxide precursors (namely, tetraethylortho-silicate, -titanate, -zirconate and aluminium isopropylate) in order to get inorganic phases able to improve the thermal stability and flame retardancy of the fabric, without changing its mechanical features. Indeed, after the sol-gel treatment the fibre/fabric surface was morphologically modified: a homogeneous and compact film located in the fibre interstices (warp and weft) and partially covering their walls was observed for all the systems investigated. These coatings turned out to be responsible of an overall enhancement of the thermal and fire stability of the fabrics preserving, at the same time, the original mechanical properties of the neat cotton in terms of tensile strength and deformation. In addition, such inorganic coatings increased the abrasion resistance of the cotton in a remarkable way.     

PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro

Resident cells in the surface epitenon and internal compartment of flexor tendons are subjected to cyclic mechanical load as muscle contracts to move limbs or digits. Tendons are largely tensile load bearing tissues and are highly matrix intensive with nondividing cells providing maintenance functions. However, when an injury occurs, tendon cells are stimulated to divide by activated endogenous growth factors and those from platelets and plasma. We hypothesize that tendon cells detect mechanical load signals but do not interpret such signals as mitogenic unless an active growth factor is present. We have used an in vitro mechanical load model, application of cyclic strain to cells cultured on flexible bottomed culture plates, to test the hypothesis that tendon cells require platelet-derived growth factor (PDGF-BB) and insulin-like growth factor-I (IGF-I) in addition to mechanical load to stimulate DNA synthesis. In addition, we demonstrate that in avian tendon cells, load and growth factors stimulate phosphorylation of tyrosine residues in multiple proteins, including pp₆₀src, a protein kinase that phosphorylates receptor protein tyrosine kinases. A lack of mitogenic responsiveness to mechanical load alone by tendon cells may be a characteristic of a regulatory pathway that modulates cell division.     

Effects of age and streptozotocin-induced diabetes on biogenic amines in rat tail artery

The changes in amine concentrations in different segments of the rat tail artery have been investigated at different ages and after different durations of streptozotocin-induced diabetes. There was a significant positive slope to the relationship between amine concentrations and age in proximal portion of the normal tail artery, and a significant additional increase in amine concentrations following induction of diabetes. The peak of the latter response occurred between 10 and 20 weeks following the induction of diabetes. There was also a significant dependence on the length of the post-ganglionic neurones, which may be related to the failure of axonal transport of some of the essential enzymes or transporters for these biogenic amines. This model of altered catecholamine metabolism and handling requires further investigation so that alterations in the mechanisms involved in processing of these amines in diabetic autonomic neuropathy may be elucidated. (Mol Cell Biochem 261: 77–82, 2004)     

高脂喂养合并小剂量链脲佐菌素建立2型糖尿病大鼠模型

目的 观察不同配方的高脂饲料,以及不同周龄的大鼠对于该模型的造模成功率和模型病变特点的影响.方法 将26只3周龄SD大鼠分为正常一组(N1组)、模型一组(M1组)和模型二组(M2组);26只5周龄SD大鼠分为正常二组(N2组)、模型三组(M3组)和模型四组(M4组).M1组和M3组给予高脂饲料配方一喂养,M2组和M4组给予高脂饲料配方二喂养.4周后,各模型组大鼠腹腔注射STZ溶液35 mg/kg.连续观察大鼠的空腹血糖(FBG)、空腹胰岛素(FIN)、总胆固醇(TG)、甘油三酯(TC)水平.结果 5周龄SD大鼠的FBG水平在注射STZ后两周即可达到稳定状态,并维持在较高的水平;高脂饲料配方二使大鼠的进食量和体重增加明显,并且成功诱导出胰岛素抵抗( insulin resistance,IR).结论 选取5周龄SD大鼠作为模型动物,并给予配方二高脂饲料喂养,所建立的大鼠模型具备2型糖尿病的主要特征,是值得推广的2型糖尿病动物模型.     

Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

Compressive mechanical properties of rat and pig optic nerve head

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), the primary risk factor for glaucoma, is thought to induce abnormally high strains in optic nerve head (ONH) tissues, which ultimately result in retinal ganglion cell damage and vision loss. The mechanisms by which excessive deformations result in vision loss remain incompletely understood. The ability of computational and in vitro models of the ONH to provide insight into these mechanisms, in many cases, depends on our ability to replicate the physiological environment, which in turn requires knowledge of tissue biomechanical properties. The majority of mechanical data published to date regarding the ONH has been obtained from tensile testing, yet compression has been shown to be the main mode of deformation in the ONH under elevated IOP. We have thus tested pig and rat ONH tissue using unconfined cyclic compression. The material constants C₁, obtained from fitting the stress vs. strain data with a neo-Hookean material model, were 428 [367, 488] Pa and 64 [53, 76] Pa (mean [95% Confidence Interval]) for pig and rat optic nerve head, respectively. Additionally, we investigated the effects of strain rate and tissue storage on C1 values. These data will inform future efforts to understand and replicate the in vivo biomechanical environment of the ONH.     

Aldose reductase,NADPH and NADP+ in normal,galactose-fed and diabetic rat lens

NADPH and NADP⁺ levels were measured in rat lens from normal controls, from galactose-fed and diabetic rats during the first week of cataract formation.The level of NADPH in normal rat lens was determined to be 12.3 ± 0.4 nmol/g wet weight, and that of NADP⁺ 4.6 ± 0.2 nmol/g wet weight. In early cataract formation NADPH levels decreased rapidly during the first 2 days and then remained stable at 76% of control for galactose-fed and 84% for diabetic rats. NADP⁺ levels increased by 38% of control for galactose-fed and 54% for diabetic rats. Calculated NADPH/NADP⁺ ratios dropped from 3.36 ± 0.21 to 1.86 ± 0.16 in galactose fed rats, and from 2.81 ± 0.15 to 1.61 ± 0.16 in diabetic rats (P < 0.001 for both experimental groups). These data are consistent with rapid NADPH oxidation during onset of lens cataracts. No significant changes in aldose reductase enzymatic activity levels were observed in either the galactosemic or the diabetic rats during the times measured.     

Organization of collagen bundles during tendon healing in rats treated with L-NAME

The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely, with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast, in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen. T.C.T. and W.R.N were supported by studentships from CAPES, and S.H. was supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).     

First principles investigation of pressure dependent stability,phonon, Debye temperature,physical, mechanical and thermodynamic properties of Rh3Al intermetallic compound

ABSTRACT

Pressure dependence of stability, phonon, Debye temperature, physical, mechanical and thermodynamic properties of Rh₃Al intermetallic compound were investigated by first-principles The calculated cohesive energy (E_c), formation enthalpy (ΔH) show that Rh₃Al is a thermodynamically stable compound. Properties related to the phonons of Rh₃Al were also obtained. In addition, the transverse sound velocity (ν_s), longitudinal sound velocity (ν_l), average sound velocity (ν_m) and Debye temperature (Θ_D) of Rh₃Al were calculated by using the VRH method along with pressure range from 0 to 60?GPa. The values of lattice parameters, bulk modulus and its first-order pressure derivative are consistent well with other works. The band structure indicates that Rh₃Al compound exhibits a metallic character. Moreover, the total density of states, partial density of states, Mulliken charges and electron density difference have been analysed to explain the physical properties. Based on the stress–strain approach and the Born stability criteria, the mechanical properties were evaluated by elastic constants (C_ij), other modulus (B, E, G), (B/G) ratio, Poisson’s ratio (ν), the anisotropic index (A), hardness (H) and compressibility (K) for this intermetallic compound. Finally, the thermodynamic properties, including enthalpy, free energy, entropy and heat capacity are discussed range from 0 to 1000?K.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

The effects of phosphoproteins on collagen self-assembly in tail tendon and incision dentin from rats

Phosphoproteins retard the rate at which collagen molecules undergo self-assembly into fibrils. The inhibition appears to be dependent on the amount of phosphoprotein present, with increasing phosphoprotein concentrations resulting in greater inhibition. Prior treatment of the phosphoprotein with calcium markedly increases the resultant inhibitory effect. Dentin phosphoproteins are considerably more effective than phosvitin in retarding collagen self-assembly, with retardation times for these hard tissue extracellular matrix proteins being 25–30 times greater than control values.     

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions.The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10^?6 M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.     

Role of the RRM domain in the activity, structure and stability of poly(A)-specific ribonuclease

Poly(A) specific ribonuclease (PARN), which contains a catalytic domain and two RNA-binding domains (R3H and RRM), acts as a key enzyme in eukaryotic organisms to regulate the stability of mRNA by degrading the 3' poly-(A) tail. In this research, the activity, structure and stability were compared between the full-length 74kDa PARN, the proteolytic 54kDa fragment with half of the RRM, and a truncated 46kDa form completely missing the RRM. The results indicated that the 46kDa one had the lowest activity and substrate binding affinity, the most hydrophobic exposure in the native state and the least stability upon denaturation. The dissimilarity in the activity, structure and stability of the three PARNs revealed that the entire RRM domain not only contributed to the substrate binding and efficient catalysis of PARN, but also stabilized the overall structures of the protein. Spectroscopic experiments suggested that the RRM domain might be structurally adjacent to the R3H domain, and thus provide a basis for the cooperative binding of poly(A) by the two RNA-binding domains as well as the catalytic domain.     

Microstructure,mineral and mechanical properties of teleost intermuscular bones

There is an increasing interest in understanding teleost bone biomechanics in several scientific communities, for instance as interesting biomaterials with specific structure-function relationships. Intermuscular bones of teleost fish have previously been described to play a role in the mechanical force transmission between muscle and bone, but their biomechanical properties are not yet fully described. Here, we have investigated intermuscular bones (IBs) of the North Atlantic Herring with regard to their structure and micro-architecture, mineral-related properties, and micro-mechanical tensile properties. A total of 115 IBs from 18 fish were investigated. One cohort of IBs, containing 20 bones from 2 smaller fish and 23 bones of 3 larger fish, was used for mechanical testing, wide-angle X-ray scattering, and scanning electron microscopy. Another cohort, containing 36 bones from 7 smaller fish and 36 bones from 6 larger fish, was used for microCT. Results show some astonishing properties of the IBs: (i) IBs present higher ductility, lower Young's modulus but similar strength and TMD (Tissue Mineral Density) compared to mammalian bone, and (ii) IBs from small fish were 49% higher in Young’s modulus than fish bones from larger fish while their TMD was not statistically different and crystal length was 8% higher in large fish bones. Our results revealed that teleost IB presents a hybrid nature of soft and hard tissue that differs from other bone types, which might be associated with their evolution from mineralized tendons. This study provides new data regarding teleost fish bone biomechanical and micro-structural properties.     

The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice

The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity.A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out (−/−), heterozygous (+/−) and wild-type (+/+). A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties.There was no significant difference in the Young’s modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice.Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated.     

Different regions of bovine deep digital flexor tendon exhibit distinct elastic,but not viscous,mechanical properties under both compression and shear loading

Tendons in different locations function in unique, and at times complex, invivo loading environments. Specifically, some tendons are subjected to compression, shear and/or torsion in addition to tensile loading, which play an important role in regulating tendon properties. To date, there have been few studies evaluating tendon mechanics when loaded in compression and shear, which are particularly relevant for understanding tendon regions that experience such non-tensile loading during normal physiologic function. The objective of this study was to evaluate mechanical responses of different regions of bovine deep digital flexor tendons (DDFT) under compressive and shear loading, and correlate structural characteristics to functional mechanical properties. Distal and proximal regions of DDFT were evaluated in a custom-made loading system via three-step incremental stress-relaxation tests. A two-relaxation-time solid linear model was used to describe the viscoelastic response. Results showed large differences in the elastic behavior between regions: distal region stresses were 4–5 times larger than proximal region stresses during compression and 2–3 times larger during shear. Surprisingly, the viscous (i.e., relaxation) behavior was not different between regions for either compression or shear. Histological analysis showed that collagen and proteoglycan in the distal region distributed differently from the proximal region. Results demonstrate mechanical differences between two regions of DDFT under compression and shear loading, which are attributed to variations of composition and microstructural organization. These findings deepen our understanding of structure–function relationships of tendon, particularly for tissues adapted to supporting combinations of tension, compression, and shear in physiological loading environments.     

Nano- and microscale mechanical properties of erythrocytes in hereditary spherocytosis

Hereditary spherocytosis (HS), an erythrocyte membranopathy, is a heterogeneous disease, even at the level of the erythrocyte population. The paper aims at studying the mechanical properties (the Young’s modulus, median and RMS roughness of friction force maps; fractal dimension, lacunarity and spatial distribution parameters of lateral force maps) of the cell surface layer of the erythrocytes of two different morphologies (discocytes and spherocytes) in HS using atomic force microscopy. The results of spatial-spectral and fractal analysis showed that the mechanical property maps of the HS spherocyte surface were more structurally homogeneous compared to the maps of HS discocytes. HS spherocytes also had a reduced RMS roughness and lacunarity of the mechanical property maps. The Young’s modulus and averaged friction forces over the microscale HS spherocyte surface regions were approximately 20% higher than that of HS discocytes. The revealed significant difference at the nano- and microscales in the structural and mechanical properties of main (discoidal and spheroidal) morphological types of HS erythrocytes can potentially cause blood flow disturbance in the vascular system in HS.