Lateral packing of mineral crystals in bone collagen fibrils

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization. The mineral platelets were found to be very thin (1.5 nm ∼ 2.0 nm), having a narrow thickness distribution. The thickness of the organic layers between adjacent mineral platelets within a stack is more broadly distributed with the average value varying from 6 nm to 10 nm, depending on the extent of mineralization. The two-dimensional analytical scheme also leads to quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c axis.     

Amino and carboxyl propeptides in bone collagen fibrils during embryogenesis

Summary Collagen fibrillogenesis was studied in tibiae of chick embryos, 9, 11, and 14 days old. Specimens were incubated with antibodies against the amino and the carboxyl propeptides of type-I collagen and subjected to ferritin-la-belling immuno-electron microscopy. The amino propeptide was found in thin fibrils, 20–40 nm in diameter, distributed at 60-nm periodicity. The carboxyl propeptide antibody labelled a wide spectrum of fibrils, although the majority were in the range of 40–100 nm, distinctly larger than those labelled with the amino propeptide antibody. The presence of pN (amino propeptide plus collagen) and pC (carboxyl propeptide plus collagen) collagen was also demonstrated by Western blotting in all specimens. This study suggests that the sequence of propeptide removal may regulate collagen fibril diameter.     

Stress relaxation function of bone and bone collagen

Relaxation Young's and shear moduli of bovine bone and bone collagen were investigated. It was found that each relaxation process observed had two stages, which were referred to as process I and process II in order of time. Process II was described by a simple exponential decay while process I was not. The Kohlrausch-Williams-Watts (KWW) function, ψ(t) = exp[t/τ₁)^B] (0 < B < 1), was found to be suitable to describe process I. The normalized relaxation modulus, M_r(t), was expressed by the combination of the simple exponential type relaxation function and the KWW function

M_r=A₁exp[−(t/τ₁)^B]+A₂exp[(t/τ₁)](0<B1)

On the basis of this equation, the relaxation mechanism in bone and bone collagen was identified. According to the model proposed for the KWW relaxation function, the stress relaxation process in bone was considered to be governed by viscoelastic properties of matrix collagen fiber. The model for the KWW relaxation function requires the disordered glassy structure of collagen fiber, which is consistent with the results of the structural investigations.     

Mechanical properties of collagen fibrils

The formation of collagen fibers from staggered subfibrils still lacks a universally accepted model. Determining the mechanical properties of single collagen fibrils (diameter 50-200 nm) provides new insights into collagen structure. In this work, the reduced modulus of collagen was measured by nanoindentation using atomic force microscopy. For individual type 1 collagen fibrils from rat tail, the modulus was found to be in the range from 5 GPa to 11.5 GPa (in air and at room temperature). The hypothesis that collagen anisotropy is due to the subfibrils being aligned along the fibril axis is supported by nonuniform surface imprints performed by high load nanoindentation.     

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions’ orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young’s modulus \(E = 1.90\) GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young’s modulus in the preferential direction of 9–16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.     

The importance of superficial collagen fibrils for the function of articular cartilage

It has been proposed that the superficial tangential zone (STZ) of articular cartilage is essential to the tissue’s load-distributing function. However, the exact mechanism by which the STZ fulfills this function has not yet been revealed. Using a channel-indentation experiment, it was recently shown that compared to intact tissue, cartilage without STZ behaves slightly stiffer and deforms significantly different in regions adjacent to mechanically compressed areas (Bevill et al. in Osteoarthr Cartil 18:1310–1318, 2010). We aim to further explore the role of STZ in the load-transfer mechanism of AC by thorough biomechanical analysis of these experiments. Using our previously validated fibril-reinforced swelling model of articular cartilage, which accounts for the depth-dependent collagen structure and biochemical composition of articular cartilage, we simulated the above-mentioned channel-indenter compression experiments for both intact and STZ-removed cartilage. First, we show that the composition of the deep zone in cartilage is most effective in carrying cartilage compression, which explains the apparent tissue stiffening after STZ removal. Second, we show that tangential fibrils in the STZ are responsible for transferring compressive loads from directly loaded regions to adjacent tissue. Cartilage with an intact STZ has superior load-bearing properties compared to cartilage in which the STZ is compromised, because the STZ is able to recruit a larger area of deep zone cartilage to carry compressive loads.     

Crystalline regions in collagen fibrils

A new image processing technique, content-dependent anisotropic spatial frequency filtering, has been developed to visualize the location and orientation of crystalline regions in collagen fibril cross-sections. The results show that most crystalline regions are oriented with their approximately 4 nm periodicity directed radially from the fibril centre. This periodicity corresponds to the separation between rows of molecular ends in the quasi-hexagonal molecular packing scheme. The extent of crystallinity increases with radius and frequently the lattice is either continuously distorted or interrupted by sharp discontinuities.     

Low strain nanomechanics of collagen fibrils

The high stiffness of collagenous tissues such as tendon and ligament is derived in large part from the mechanics and geometries of the constituent collagen's hierarchical forms. The primary structural unit in connective tissues is the collagen fibril for which there exists little direct mechanical or deformational study. Therefore, the current understanding of the mechanisms involved is extrapolated from whole tissue data. To address this, the elastic response due to bending of readily extractable adult collagen fibrils was studied, and the results were compared to previously reported radial indentation experiments. A demonstration of a material anisotropy arising without loss of the assumptions of homogeneity is presented.     

Viscoelastic properties of isolated collagen fibrils

Understanding the viscoelastic behavior of collagenous tissues with complex hierarchical structures requires knowledge of the properties at each structural level. Whole tissues have been studied extensively, but less is known about the mechanical behavior at the submicron, fibrillar level. Using a microelectromechanical systems platform, in vitro coupled creep and stress relaxation tests were performed on collagen fibrils isolated from the sea cucumber dermis. Stress-strain-time data indicate that isolated fibrils exhibit viscoelastic behavior that could be fitted using the Maxwell-Weichert model. The fibrils showed an elastic modulus of 123 ± 46 MPa. The time-dependent behavior was well fit using the two-time-constant Maxwell-Weichert model with a fast time response of 7 ± 2 s and a slow time response of 102 ± 5 s. The fibrillar relaxation time was smaller than literature values for tissue-level relaxation time, suggesting that tissue relaxation is dominated by noncollagenous components (e.g., proteoglycans). Each specimen was tested three times, and the only statistically significant difference found was that the elastic modulus is larger in the first test than in the subsequent two tests, indicating that viscous properties of collagen fibrils are not sensitive to the history of previous tests.     

The precipitation of toroidal collagen fibrils

The morphology of aggregates of calf-skin tropocollagen, precipitated by continuous injection into neutral phosphate buffers at 35 degrees , has been studied by electron microscopy. Although most of the collagen is precipitated as normal native fibrils, a small proportion forms closed toroidal structures having the usual native band-interband pattern. Theoretical considerations, based on elastic energies in a general microfibril model, predict that the toroids should have a simple super-helical structure, and this is not inconsistent with the observations. From the theoretical energies it was possible to estimate a crude lower limit of 3kcal./mole for the free energy of association of the tropocollagen macromolecules.     

Stabilization of collagen fibrils by hydroxyproline

The substitution of hydroxyproline for proline in position Y of the repeating Gly-X-Y tripeptide sequence of collagen-like poly(tripeptide)s (i.e., in the position in which Hyp occurs naturally) is predicted to enhance the stability of aggregates of triple helices, while the substitution of Hyp in position X (where no Hyp occurs naturally) is predicted to decrease the stability of aggregates. Earlier conformational energy computations have indicated that two triple helices composed of poly(Gly-Pro-Pro) polypeptide chains pack preferentially with a nearly parallel orientation of the helix axes [Nemethy, G., & Scheraga, H.A. (1984) Biopolymers 23, 2781-2799]. Conformational energy computations reported here indicate that the same packing arrangement is preferred for the packing of two poly(Gly-Pro-Hyp) triple helices. The OH groups of the Hyp residues can be accommodated in the space between the two packed triple helices without any steric hindrance. They actually contribute about 1.9 kcal/mol per Gly-Pro-Hyp tripeptide to the packing energy, as a result of the formation of weak hydrogen bonds and other favorable noncovalent interatomic interactions. On the other hand, the substitution of Hyp in position X weakens the packing by about 1.7 kcal/mol per Gly-Hyp-Pro tripeptide. Numerous published experimental studies have established that Hyp in position Y stabilizes an isolated triple helix relative to dissociated random coils, while Hyp in position X has the opposite effect. We propose that Hyp in position Y also enhances the stability of the assembly of collagen into microfibrils while, in position X, it decreases this stability.     

Liquid crystallinity in collagen solutions and magnetic orientation of collagen fibrils

Studies of the optical birefringence of solutions of acid-soluble collagen from rat-tail tendon at 22°C in the pH range 1.0–6.0 show that collagen exhibits an isotropic to mesophase transition only between pH 2.4 and 3.0 at 10% weight concentration. Such liquid crystalline order is probably essential for the orientation of collagen in a magnetic field. When solutions of neutral salt-soluble collagen were precipitated at pH 7.0 by warming to 37°C (“heat gelling”) in a magnetic field of ca. 20 kG, the resulting fibrils wee oriented perpendicular to the direction of the field. Heat gelling is shown to be a useful technique for maintaining the orientation induced in precursor solutions even after the sample is removed from the magnetic field.     

Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde.

Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.     

Elastic model for crimped collagen fibrils

A physiologic constitutive expression is presented in algorithmic format for the nonlinear elastic response of wavy collagen fibrils found in soft connective tissues. The model is based on the observation that crimped fibrils in a fascicle have a three-dimensional structure at the micron scale that we approximate as a helical spring. The symmetry of this wave form allows the force/displacement relationship derived from Castigliano's theorem to be solved in closed form: all integrals become analytic. Model predictions are in good agreement with experimental observations for mitral-valve chordae tendinece.     

Effect of cyclic loading on the nanoscale deformation of hydroxyapatite and collagen fibrils in bovine bone

Cyclic compressive loading tests were carried out on bovine femoral bones at body temperature $(37\,^{\circ }\hbox {C})$ , with varying mean stresses ( $-55$ to $-80$  MPa) and loading frequencies (0.5–5 Hz). At various times, the cyclic loading was interrupted to carry out high-energy X-ray scattering measurements of the internal strains developing in the hydroxyapatite (HAP) platelets and the collagen fibrils. The residual strains upon unloading were always tensile in the HAP and compressive in the fibrils, and each increases in magnitude with loading cycles, which can be explained from damage at the HAP–collagen interface and accumulation of plastic deformation within the collagen phase. The samples tested at a higher mean stress and stress amplitude, and at lower loading frequencies exhibit greater plastic deformation and damage accumulation, which is attributed to greater contribution of creep. Synchrotron microcomputed tomography of some of the specimens showed that cracks are produced during cyclic loading and that they mostly occur concentric with Haversian canals.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.