Nanomechanical Mapping of Hydrated Rat Tail Tendon Collagen I Fibrils

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 μm in length, were acquired at indentation speeds around 10⁵ nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10⁵ nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.     

Nanomechanical Mapping of Hydrated Rat Tail Tendon Collagen I Fibrils

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 μm in length, were acquired at indentation speeds around 10⁵ nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10⁵ nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Fibrous long spacing type collagen fibrils have a hierarchical internal structure

Nanodissection of single fibrous long spacing (FLS) type collagen fibrils by atomic force microscopy (AFM) reveals hierarchical internal structure: Fibrillar subcomponents with diameters of approximately 10 to 20 nm were observed to be running parallel to the long axis of the fibril in which they are found. The fibrillar subcomponent displayed protrusions with characteristic approximately 270 nm periodicity, such that protrusions on neighboring subfibrils were aligned in register. Hence, the banding pattern of mature FLS-type collagen fibrils arises from the in-register alignment of these fibrillar subcomponents. This hierarchical organization observed in FLS-type collagen fibrils is different from that previously reported for native-type collagen fibrils, displaying no supercoiling at the level of organization observed.     

Sequential assembly of collagen revealed by atomic force microscopy.

Most polymers which comprise biological filaments assemble by two mechanisms: nucleation and elongation or a sequential, stepwise process involving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filament. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correlated with turbidimetric alterations in the assembly mixture. The assembly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of assembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments approximately 2-6 nm in diameter (> 10 microns in length) many with a conspicuous approximately 67-nm axial period were observed. Occasional mature collagen fibrils with a approximately 67-nm axial repeat were found late in the course of assembly. Our results are consistent with initial end-to-end axial association of monomers to form oligomers followed by lateral association into higher-order filaments. On this basis, there appears to be at least two distinctive types of structural interactions (axial and lateral) which are operative at different levels in the assembly hierarchy of collagen.     

Analysis of collagen fibril diameter distribution in connective tissues using small-angle X-ray scattering

Analysis of the diameters of collagen fibrils provides insight into the structure and physical processes occurring in the tissue. This paper describes a method for analyzing the frequency distribution of the diameters of collagen fibrils from small-angle X-ray scattering (SAXS) patterns. Frequency values of fibril diameters were input into a mathematical model of the form factor to calculate the equatorial intensity which best fits the experimentally derived data from SAXS patterns. A minimization algorithm utilizing simulated annealing (SA) was used in the fitting procedure. The SA algorithm allowed for random sampling of the frequency values, and was run iteratively to build up an optimized frequency distribution of fibril diameters. Results were obtained for collagen samples from sheep spine ligaments. The mean fibril diameter value obtained from this data-fitting method was 73 nm+/-20 nm (S.D.). From scanning transmission electron microscopy, the mean diameter was found to be 69 nm+/-14 nm (S.D.). The good agreement between the two methods demonstrates the reliability of the SAXS method for the tissue examined. The non-destructive nature of this technique, as well as its statistical robusticity and capacity for large sampling, means that this method is both quick and effective.     

Mechanical properties of mineralized collagen fibrils as influenced by demineralization

Dentin and bone derive their mechanical properties from a complex arrangement of collagen type-I fibrils reinforced with nanocrystalline apatite mineral in extra- and intrafibrillar compartments. While mechanical properties have been determined for the bulk of the mineralized tissue, information on the mechanics of the individual fibril is limited. Here, atomic force microscopy was used on individual collagen fibrils to study structural and mechanical changes during acid etching. The characteristic 67 nm periodicity of gap zones was not observed on the mineralized fibril, but became apparent and increasingly pronounced with continuous demineralization. AFM-nanoindentation showed a decrease in modulus from 1.5 GPa to 50 MPa during acid etching of individual collagen fibrils and revealed that the modulus profile followed the axial periodicity. The nanomechanical data, Raman spectroscopy and SAXS support the hypothesis that intrafibrillar mineral etches at a substantially slower rate than the extrafibrillar mineral. These findings are relevant for understanding the biomechanics and design principles of calcified tissues derived from collagen matrices.     

Growth and development of collagen fibrils in immature tissues from rat and sheep

The collagen fibril diameter distribution of four immature tissues from both rat and sheep have been determined from the transverse sections observed in the transmission electron microscope. In many instances before birth, the form of the distribution for the tissues is both unimodal and sharp and the mean diameters of the distributions lie close to a multiple of 80 A. For some tissues, the collagen fibril diameter distributions may be resolved into a number of components, each of which represents a population of fibrils with a diameter close to a multiple of 80 A (8 nm). These data confirm and extend previous observations by the authors that small collagen fibrils growth occurs by the accretion of 80 A units. The form of the collagen fibrils diameter distribution at birth is broad for the sheep tissues but narrow for the rat tissues, thus confirming that the range of fibril diameters at this stage of life reflects the differing degree of development of precocious and altricious animals.     

Collagen: the organic matrix of bone

Collagen is the principal organic matrix in bone. The triple helical region of the molecule is 1014 amino acids long. In fibrils these molecules are staggered axially by integers of 234 residues or 68 nm (D). This axial shift occurs by self-assembly and can be understood in terms of a periodicity in the occurrence of apolar and polar residues in the amino acid sequence. Because the molecular length L = 4.47 D, there are gaps 1.5 X 36.5 nm regularly arrayed throughout the fibrils. The three-dimensional molecular arrangement is a quasi-hexagonal lattice with three distinct values for the principal interplanar spacings. Analysis of the intensity distribution in the medium-angle X-ray diffraction patterns from tendons has produced the following picture of the molecular arrangement in fibrils (Fraser et al. 1983). The molecular helices have a coherent length of 32 nm and are tilted parallel to a specific place within the lattice. A regular azimuthal interaction exists between these helices. This crystalline region could be the overlap region with a non-crystalline gap region. However, the gap is still regular axially and the molecular helices retain their structure; their lateral packing is perturbed although they retain a 'gap'. Neutron and X-ray scattering experiments have shown that calcium hydroxyapatite crystals occur in the gap and are nucleated at a specific though unknown location within the gap. The c-axis of the apatite crystals is parallel to the fibril axis and its length c = 0.688 nm is close to the axial periodicity in a protein with an extended beta-conformation. If the telopeptides at the end of a collagen molecule do have this conformation they would either have a highly heterogeneous conformation or exist in a folded manner because the overall length of the telopeptides is shorter than a regular collagen repeat of 0.029 nm would allow.