Effect of sample geometry on the apparent biaxial mechanical behaviour of planar connective tissues

Mechanical testing methodologies developed for engineering materials may result in artifactual material properties if applied to soft planar connective tissues. The use of uniaxial tissue samples with high aspect ratios or biaxial samples with slender cruciform arms could lead to preferential loading of only the discrete subset of extracellular fibres that fully extend between the grips. To test this hypothesis, cruciform biaxial connective tissue samples that display distinctly different material properties (bovine pericardium, fish skin), as well as model textile laminates with predefined fibrous orientations, were repeatedly tested with decreasing sample arm lengths. With mechanical properties determined at the sample centre, results demonstrated that the materials appeared to become stiffer and less extensible with less slender sample geometries, suggesting that fibre recruitment increases with decreasing sample arm length. Alterations in the observed shear behaviour and rigid body rotation were also noted. The only truly reliable method to determine material properties is through in vivo testing, but this is not always convenient and is typically experimentally demanding. For the in vitro determination of the biaxial material properties, appropriate sample geometry should be employed in which all of the fibres contribute to the mechanical response.     

Molecular mechanisms of ageing in connective tissues

The outward manifestations of tissue ageing occurring in the elderly primarily involve the two major structural proteins of the body, collagen and elastin. The changes in these proteins are associated with intermolecular cross-linking and side-chain modifications. Cross-linking involves two different mechanisms, a precise enzymic process during development and maturation, and a subsequent non-enzymic adventitious reaction with glucose during ageing. The latter glycation reactions are the major cause of tissue dysfunction in the elderly due to cross-linking, which stiffens the tissues, and to side-chain modification, which alters normal cell-matrix interactions. Photoageing by UV involves two competing reactions, chain cleavage and cross-linking, the former predominating on long-term exposure. The molecular mechanisms involved are gradually being unravelled and will lead to the development of inhibitors of these deleterious effects of ageing.     

Structural and mechanical profiles of native collagen fibers in vaginal wall connective tissues

Collagen, an ubiquitous biomaterial, confers robustness and resilience to connective tissues. In this study, we analyzed the structure and elasticity profile of collagen from the vaginal wall connective tissue of healthy pre-menopausal (pre-M) and postmenopausal (post-M) women. The histological staining assisted study with an atomic force microscope renders the examination of native collagen fibers on site of the connective tissue from nanoscopic scale to microscopic scale with high spatial resolution. Our results suggest that during menopause, collagen’s structure and elasticity are subject to changes at all levels of organization- between individual collagen fibers, between collagen and muscle, and between collagen and other matrix elements. The systematic analysis of the native structure and mechanical properties of collagen within a tissue provides a potential way to study non-fatal conditions such as pelvic organ prolapse and other genito-urinary disorders, where the initial symptoms are subtle and multivariate, and where early detection of patient’s condition may allow better non-invasive interventions and reduce the number of women undergoing surgical correction of these common disorders.     

Molecular mechanisms for intrafibrillar collagen mineralization in skeletal tissues

The critical role of the self-assembled structure of collagen in skeletal mineralization is long recognized, yet the angstrom to tens of nanometers length-scale nucleation mechanism of calcium phosphate mineral (Ca–P_i) remains unclear. Here, by constructing three-dimensional structure of collagen fibril, we report direct computational evidence of intrafibrillar Ca–P_i nucleation in the collagen matrix and illustrate the crucial role of charged amino acid sidechains of collagen molecules in nucleation. The all-atom Hamiltonian replica exchange molecular dynamics simulation shows that these charged sidechains are oriented toward the fibril “hole zones” and significantly template nucleation with amorphous Ca–Pi phase, ∼1.3–1.6 nm in size, thus explaining the empirical observations that Ca–P_i nucleates principally in these regions. We also show that the low water density of about 0.70 g cm⁻³ in these zones may further benefit nucleation by lowering the enthalpic penalty for ion desolvation. This work provides insight, at the atomistic level, into the nucleation mechanism of bone crystals within a collagen matrix for understanding mineral deposition, interpreting mineralization experiments and guiding the design of new implantable materials.     

A stereologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover

The purpose of this study was to assess the importance of the phagocytic mechanism of collagen resorption in the normal turnover and remodelling of soft connective tissues. Collagen phagocytosis by fibroblasts in rat skin, attached gingiva, and periodontal ligament was quantitated using the methodology of electron microscopic stereology. Periodontal ligament contained five and 15 times as much phagocytosed collagen as attached gingiva and skin respectively. Also, for each tissue examined, a positive correlation was observed between the amount of collagen phagocytosed and the known rate of mature collagen turnover.     

Collagen fibril diameter and its relation to collagen turnover in three soft connective tissues in the rat

Collagen fibril diameters were measured in electron micrographs of rat skin, gingiva and periodontal ligament. Gingiva was divided into two zones, termed elastin-containing gingiva and attached gingiva, depending on the presence or absence of elastic fibrils. The results revealed that skin had the largest fibrils, followed by elastin-containing gingiva, attached gingiva and periodontal ligament respectively. These differences in fibril diameter were highly significant. The observed trend in fibril diameter was the inverse of that documented for collagen turnover and collagen phagocytosis in the same tissues. A link between fibril diameter and collagen turnover is discussed.     

Hydrothermal isometric tension curves from different connective tissues. Role of collagen genetic types and noncollagenous components

Variations in hydrothermal isometric tension (HIT) were recorded in tendons, ligaments, skin, blood vessels, nerves, palatal mucosa, lungs, muscles, cartilages, demineralized bones and dentine from donors of different ages and species. The curves obtained during a linear rise in temperature from 37 degrees C to 100 degrees C at a rate of 1.15 degrees C/min were classified into three major families, A, B and C, depending on whether these curves displayed an early maximum, two shoulders or a late maximum. The ratio of heat-labile to heat-stable cross-links in the tissue's collagen network was shown to determine the type of curve obtained, but the genetic types of collagen in the tissue and the amount and quality of its noncollagenous components were not important in this respect. These results are discussed in the light of the accepted view that HIT variations are due to the rubber-elastic properties of gelatin.     

Autonomous informational stability in connective tissues.

No coherent theories currently explain connective tissue stability (i.e. 'memory') as well as spatial and temporal adaptability in the face of continual flux of its constituents. Furthermore, explanations of stability based exclusively upon DNA raise certain inherent problems, particularly with the spatial concordance of somatic tissues. As an alternative explanation, it is hypothesized that while connective tissue cells produce extracellular protein precursors through DNA-dependent processes, the assembly, location, orientation and configuration of the extracellular macromolecules as well as their degree of cell attachment depend primarily upon local micro-environmental conditions and/or self-organization rather than strictly cellular processes. The resulting extracellular matrix (ECM) serves as a time- and spatially-variable filter about each cell to afford a relatively consistent micro-environment for all similar cells, regardless of the more variable macro-environment. By insuring a consistent set of signals to the cell, the filter provides a non-genetic memory complementary to genetic memory. The half-lives of constituent molecules define the duration of the filter, allowing the filter to adapt to new environmental demands, yet to maintain a consistent milieu for the cell. The cell/matrix construct permits local, self-optimizing, non-deterministic tissue autonomy obviating the need to postulate certain intricate mechanisms coordinating spatial morphology and temporal behavior.     

Cell transplantation in wounded mixed connective tissues

Direct transplantation of multipotent precursor cells into the periodontium could provide a therapeutic approach for restoring periodontal tissues destroyed by periodontitis or trauma. To improve the understanding of cell migration, proliferation, and differentiation, we used a rodent model combining orthodontic tooth movement and transplantation of Lac-Z-positive murine-cultured periodontal ligament (PL) or femur-derived bone marrow precursor cells into a defined mandibular wound site, thus promoting tissue regeneration in wounded periodontium. Our results show that in orthodontically traumatized tissues, transplanted PL and bone marrow cells migrated systemically, contributing to the repopulation of sites with reduced cell/matrix density. The transplanted PL cells proliferated in adjacent alveolar bone marrow spaces, thus migrating to vascular tissues in the PL. The capillary walls in the PL serve as delivery sites for these cells and other marrow-derived hematopoietic cells, including monocytes. The transplanted marrow cells, extracted from femur of transgenic (TgR) mice exhibited similar behavior to those of transplanted PL cells, showing high proliferative activity in alveolar marrow as well as intensive repopulating capacity in wounded periodontium. On the other hand, the buccal skin fibroblasts failed to migrate and home effectively and thus the transplantation of these cells had no effect on periodontium regeneration. Based on these results, we conclude that the transplanted PL and bone marrow cells migrate systemically and following a cyclical process of growth and development and differentiate into PL fibroblasts, osteoblasts, and cementoblasts, thereby contributing to periodontal regeneration.     

Development and evaluation of microdevices for studying anisotropic biaxial cyclic stretch on cells

Mechanical effects on cells have received more and more attention in the studies of tissue engineering, cellular pathogenesis, and biomedical device design. Anisotropic biaxial cyclic stress, reminiscent of the in vivo cellular mechanical environment, may promise significant implications for biotechnology and human health. We have designed, fabricated and characterized a microdevice that imparts a variety of anisotropic biaxial cyclic strain gradients upon cells. The device is composed of an elastic membrane with microgroove patterns designed to associate cell orientation axes with biaxial strain vectors on the membrane and a Flexcell stretcher with timely controlled vacuum pressure. The stretcher generates strain profile of anisotropic biaxial microgradients on the membrane. Cell axes determined by the microgrooves are associated with the membrane strain profile to impose proper biaxial strains on cells. Using vascular smooth muscle cells as a cell model, we demonstrated that the strain anisotropy index of a cell was likely one of the determinant mechanical factors in cell structural and functional adaptations. The nuclear shape and cytoskeleton structure of smooth muscle cells were influenced by mechanical loading, but were not significantly affected by the strain anisotropy. However, cell proliferation has profound responses to strain anisotropy.     

Physical training and connective tissues in young mice-heart

Summary The effect of physical training on the chemical properties of the heart tissues was studied in male mice of NMRI-strain. The mice to be trained and their controls were about 2 weeks old at the beginning of the training, which took place on a 5° inclined treadmill 5 days a week for 3–22 weeks. The duration of daily exercise was progressively increased over the first 3 weeks. The final daily exercise bouts were 50 and 80 min for moderate programs and 180 min for the intensive program at a speed of 30 cm/s. The whole heart or the ventricles were used for the analyses. We found no significant changes related to training in the concentrations of nitrogen, hexosamines, and hydroxyproline both in the normal-sized and the hypertrophied hearts. The hydroxyproline concentration of the ventricles was lower than that of the whole heart tissue, but no difference was observed in the concentration of nitrogen. The hydroxyproline concentration of the heart tissue increased with age both in the trained and untrained mice (39% over 19 weeks). We conclude that collagen and non-collagen proteins in the heart tissue of young mice are stimulated in equal proportions by physical training.     

Changes in collagen fibril diameters in a lathyritic connective tissue

Collagen fibril diameters were measured in molar and incisor periodontal ligaments of adult rats given aminoacetonitrile (0.15% in their drinking water). Fibrils were also measured in the periodontal ligaments of pair-fed control animals and animals fed ad libitum. The lathyrogen produced a significant change in the distribution of fibril diameters in both molars and incisors and an increase in mean fibril diameter in incisors relative to pair-fed controls. The reduced food intake of the pair-fed control group produced a significant decrease in mean fibril diameter in incisors relative to the ad libitum control group. These results that lathyrogens may affect collagen fibril diameters in an adult connective tissue in a manner similar to that previously shown for a fetal tissue.     

Properties of collagen in OIM mouse tissues

The deletion of the alpha2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the alpha2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6 degrees and 1.9 degrees C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The alpha2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.     

Micro and macro rheology of planar tissues

Tissues are intrinsically non-linear, anisotropic, viscoelastic, and undergo a process of mechanical adaptation (preconditioning). Previous constitutive laws considered one or two of these response aspects, often resulting in inadequate fit to data. Here we developed a general constitutive formulation encompassing the entire set of features. To exemplify this novel approach, constitutive equation for the skin was developed by stochastic incorporation of the fibers' orientation and undulation distributions. Predictions were contrasted with biaxial data of rabbit skin. The significance of each micro-feature was examined by sensitivity analysis. The results show that micro-structure based rheological characterization provides reliable representation under multiple biaxial protocols. Parametric investigation points to the essential roles of the fibers' orientation distributions (elastin and collagen) and waviness (collagen), their respective stress–strain relationship, and their viscoelasticity and preconditioning adaptation. The effect of ground substance is small but significant for model-to-data fit. Although the collagen is two order of magnitude stiffer, the contribution of elastin is predominant at low strains, and still significant (up to 20%) at high strains at which collagen carries the major load. The results are consistent with collagen preconditioning steming from stretch induced increase in the reference length, while in elastin it is the Mullins effect (strain softening). The most important impact of the study is that for the first time the entire scope of multi-axial tissue properties are unified in a single constitutive formulation. The potential implications are on the procedures of tissues characterization and on the design and analysis of artificial tissue scaffolds.