Stress transfer in collagen fibrils reinforcing connective tissues: effects of collagen fibril slenderness and relative stiffness

Unlike engineering fibre composite materials which comprise of fibres that are uniform cylindrical in shape, collagen fibrils reinforcing the proteoglycan-rich (PG) gel in the extra-cellular matrices (ECMs) of connective tissues are taper-ended (paraboloidal in shape). In an earlier paper we have discussed how taper of a fibril leads to an axial stress up-take which differs from that of a uniform cylindrical fibre and implications for fibril fracture. The present paper focuses on the influence of fibre aspect ratio, q (slenderness), and Young's modulus (stiffness), relative to that of the gel phase, E(R), on the magnitude of the axial tensile stresses generated within a fibril and wider implications on failure at tissue level. Fibre composite models were evaluated using finite element (FE) and mathematical analyses. When the applied force is low, there is elastic stress transfer between the PG gel and a fibril. FE modelling shows that the stress in a fibril increases with E(R) and q. At higher applied forces, there is plastic stress transfer. Mathematical modelling predicts that the stress in a fibril increases linearly with q. For small q values, fibrils may be regarded as fillers with little ability to provide tensile reinforcement. Large q values lead to high stress in a fibril. Such high stresses are beneficial provided they do not exceed the fracture stress of collagen. Modulus difference regulates the strain energy release density, u, for interfacial rupture; large E(R) not only leads to high stress in a fibril but also insures against interfacial rupture by raising the value of u.     

Proteoglycan-collagen XV in human tissues is seen linking banded collagen fibers subjacent to the basement membrane.

Type XV is a large collagen-proteoglycan found in all human tissues examined. By light microscopy it was localized to most epithelial and all nerve, muscle, fat and endothelial basement membrane zones except for the glomerular capillaries or hepatic/splenic sinusoids. This widespread distribution suggested that type XV may be a discrete structural component that acts to adhere basement membrane to the underlying connective tissue. To address these issues, immunogold ultrastructural analysis of type XV collagen in human kidney, placenta, and colon was conducted. Surprisingly, type XV was found almost exclusively associated with the fibrillar collagen network in very close proximity to the basement membrane. Type XV exhibited a focal appearance directly on the surface of, or extending from, the fibers in a linear or clustered array. The most common single arrangement was a bridge of type XV gold particles linking thick-banded fibers. The function of type XV in this restricted microenvironment is expected to have an intrinsic dependence upon its modification with glycosaminoglycan chains. Present biochemical characterization showed that the type XV core protein in vivo carries chains of chondroitin/dermatan sulfate alone, or chondroitin/dermatan sulfate together with heparan sulfate in a differential ratio. Thus, type XV collagen may serve as a structural organizer to maintain a porous meshwork subjacent to the basement membrane, and in this domain may play a key role in signal transduction pathways.     

Innervation pattern of different cartilaginous tissues in the rat

BACKGROUND: The innervation of skeletal tissues by sensory nerves is poorly understood - especially of nerve fibres which reach into the bony and cartilaginous tissue. METHODS: Samples of rat cartilaginous tissues from different locations (knee joint, vertebral column, temporomandibular joint) were fixed by perfusion and decalcified. The distribution of protein gene product (PGP) 9.5-, calcitonin gene-related peptide (CGRP)- and tachykinin (TK)-immunoreactive axons was analysed using fluorescence immunohistochemistry. RESULTS: Nerve fibres were detected in the outer regions of the hyaline cartilage of the knee joint, in the hyaline cartilage of the vertebral body, in the fibrocartilage of the intervertebral disc and menisci, and in the articular disc of the temporomandibular joint. Predominantly, they were found to be CGRP-immunoreactive. CONCLUSION: The neuropeptidergic innervation of the hyaline cartilage in different locations and the presence of nerve fibres in the fibrocartilage might indicate that in addition to the classical neuronal afferent and efferent pathway these fibres may also mediate trophic actions like tissue adaptation and repair.     

The stiffness of different sections of the cartilaginous surface of the femur head

As a result of investigations on microstiffness of the cartilagenous covering of the human femoral bone head, normal (20), different values of microstiffness have been defined in various zones, depending on their functional load and thickness of the cartilage. The data obtained can serve for construction of subtotal endoprostheses of the coxal joint.     

A composites theory predicts the dependence of stiffness of cartilage culture tissues on collagen volume fraction.

The tensile stiffness of tissue grown from chondrocyte culture was both measured experimentally and predicted using a composites model theory relating tissue microstructure to macroscopic material stiffness. The tissue was altered by several treatment protocols to provide a wide range of collagen fibril volume fraction (0.015-0.15). The rate of change of tissue modulus with change in collagen volume fraction predicted by the theory was within 14% of the slope of the linear fit through the experimental data, without the use of fitting parameters for the theoretical value of the slope. Use of the model to simulate cytokine mediated tissue digestion suggests that the action of IL-1beta and retinoic acid is mainly removal of proteoglycans and some removal of collagen. The model also indicates that the matrix and collagen remaining in the tissue has the same elastic properties as the untreated tissue, and is not damaged due to the alteration. Young's modulus of the collagen fibrils is predicted to be 120 MPa, a value in the range of previous studies. This value is dependent mainly on the matrix modulus and collagen fibril volume fraction and not on Poisson's ratio of either matrix or fibril. Poisson's ratio of the tissue depends primarily on the Poisson's ratio of the matrix.     

Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage

The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (π_PG), and the collagen network (CN) provides the restraining forces to counterbalance π_PG. Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-π_PG relationship; 2), compute π_PG and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on π_PG in human aging. Extrafibrillar FCD (FCD_EF) and π_PG increased with bovine growth due to an increase in CN concentration, whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD_EF and π_PG decreased. The π_PG-values were close to equilibrium stress (σ_EQ) in all bovine and young human cartilage, but were only approximately half of σ_EQ in old human cartilage. Depth-related variations in the strain, FCD_EF, π_PG, and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-π_PG relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage.     

Contribution of the musculature to rotatory laxity and torsional stiffness at the knee

The relationships between the mean rectified EMG from two muscle groups crossing the knee joint and the rotational stiffness and laxity about the longitudinal axis of the lower leg were investigated. The EMG signals from three of the quadricep muscle group and two of the hamstring muscle group were monitored using surface electrodes. Each subject sustained self-induced muscle activity from specific muscle combinations while the foot was twisted internally and externally by the researcher. Joint rotation was measured using an electrogoniometer. Analyses of the data showed increased joint stiffness with increased numbers of active muscles. The stiffness measurements ranged from 0.16 to 2.54 Nm degree-1 depending upon the combination of active muscles. The stiffness measured in different tests were very repeatable with standard deviations ranging from 0.02 to 0.25 Nm degree-1. Increases in joint stiffness of over 400% by activation of these muscles were measured.     

Contribution of dipole-dipole interactions to the stability of the collagen triple helix

Unveiling sequence-stability and structure-stability relationships is a major goal of protein chemistry and structural biology. Despite the enormous efforts devoted, answers to these issues remain elusive. In principle, collagen represents an ideal system for such investigations due to its simplified sequence and regular structure. However, the definition of the molecular basis of collagen triple helix stability has hitherto proved to be a difficult task. Particularly puzzling is the decoding of the mechanism of triple helix stabilization/destabilization induced by imino acids. Although the propensity-based model, which correlates the propensities of the individual imino acids with the structural requirements of the triple helix, is able to explicate most of the experimental data, it is unable to predict the rather high stability of peptides embedding Gly-Hyp-Hyp triplets. Starting from the available X-ray structures of this polypeptide, we carried out an extensive quantum chemistry analysis of the mutual interactions established by hydroxyproline residues located at the X and Y positions of the Gly-X-Y motif. Our data clearly indicate that the opposing rings of these residues establish significant van der Waals and dipole-dipole interactions that play an important role in triple helix stabilization. These findings suggest that triple helix stabilization can be achieved by distinct structural mechanisms. The interplay of these subtle but recurrent effects dictates the overall stability of this widespread structural motif.     

Contribution of passive tissues to the intersegmental moments at the hip

Many anatomic structures around the hip contribute either actively (i.e., muscle contractile elements) or passively (i.e., capsule, ligaments, non-contractile portions of muscles) to the intersegmental resultant forces and moments. Investigators have often assumed that the passive elements contribute negligibly to those moments, but without substantial supporting data. We explored this assumption by measuring the 'passive moments' in 15 normal subjects from near full extension to 60 degrees of hip flexion at speeds used in slow and normal walking. The data suggest that throughout most of the gait cycle and normal stair climbing, the passive structures contribute a small portion of the total moment, usually well less than 10%. Thus, for this limited range of activities, the assumption of negligible contribution of passive structures is reasonable.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

X-ray diffraction of reconstituted collagen fibers

The X-ray diffraction of fibers reconstituted from purified rat tail tendon collagen has been compared with that of native rat tail tendon. The axial structure is very similar in the two specimens, while the ordered lateral array found in the native state is only poorly reproduced in the reconstituted fiber. Thus, the axial order is determined by the collagen molecules alone, while the native lateral packing may depend, in part at least, on other tissue components.     

Polytonic studies of reconstituted collagen fibers

The tensile force-contraction characteristics of cross-linked reconstituted collagen fibers have been studied for 30 different water-soluble reagents. The data were obtained via a “polytonic” loading technique, which was neither isometric nor isotonic. Certain data were found to fit a master curve, which did not depend strongly on the denaturant. For many denaturants the contractile force appeared to be a maximum at a denaturant concentration corresponding to all the solvent being consumed in denaturant hydration. Also, the maximum contractile force appeared to vary in the same manner as does the hydration number of the individual cations and anions in the solution (i.e., H > Li > Na > K, and I > Br > Cl > F). Several denaturants were found to be as effective as the well-known LiBr and KSCN in contracting the collagen.     

Contribution of intermediate filaments to cell stiffness, stiffening, and growth

It has been shown previously thatintermediate filament (IF) gels in vitro exhibit stiffening athigh-applied stress, and it was suggested that this stiffening propertyof IFs might be important for maintaining cell integrity at largedeformations (Janmey PA, Evtenever V, Traub P, and Schliwa M, JCell Biol 113: 155-160, 1991). In this study, the contribution ofIFs to cell mechanical behavior was investigated by measuring cellstiffness in response to applied stress in adherent wild-type andvimentin-deficient fibroblasts using magnetic twisting cytometry. Itwas found that vimentin-deficient cells were less stiff andexhibited less stiffening than wild-type cells, except at the lowestapplied stress (10 dyn/cm²) where the difference in thestiffness was not significant. Similar results were obtained frommeasurements on wild-type fibroblasts and endothelial cells aftervimentin IFs were disrupted by acrylamide. If, however, cells wereplated over an extended period of time (16 h), they exhibited asignificantly greater stiffness before than after acrylamide, even atthe lowest applied stress. A possible reason could be that theinitially slack IFs became fully extended due to a high degree of cellspreading and thus contributed to the transmission of mechanical stressacross the cell. Taken together, these findings were consistent withthe notion that IFs play important roles in the mechanical propertiesof the cell during large deformation. The experimental data also showedthat depleting or disrupting IFs reduced, but did not entirely abolish,cell stiffening. This residual stiffening might be attributed to theeffect of geometrical realignment of cytoskeletal filaments in thedirection of applied load. It was also found that vimentin-deficientcells exhibited a slower rate of proliferation and DNA synthesis thanwild-type cells. This could be a direct consequence of the absence ofthe intracellular IFs that may be necessary for efficient mediation ofmechanical signals within the cell. Taken together, results of thisstudy suggest that IFs play important roles in the mechanical properties of cells and in cell growth.