Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

A precise analysis of the mechanical response of collagen fibrils in tendon tissue is critical to understanding the ultrastructural mechanisms that underlie collagen fibril interactions (load transfer), and ultimately tendon structure–function. This study reports a novel experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations. An atomic force microscope was used to characterize diameters and periodic banding (D-period) of individual type-I collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10% macroscopic nominal strain, respectively. D-period banding of the collagen fibrils increased with increasing tendon strain (2.1% increase at 10% applied tendon strain, p < 0.05), while fibril diameter decreased (8% reduction, p < 0.05). No statistically significant differences between 0% and 5% applied strain were observed, indicating that the onset of fibril (D-period) straining lagged macroscopically applied tendon strains by at least 5%. This confirms previous reports of delayed onset of collagen fibril stretching and the role of collagen fibril kinematics in supporting physiological tendon loads. Fibril strains within the tissue were relatively tightly distributed in unloaded and highly strained tendons, but were more broadly distributed at 5% applied strain, indicating progressive recruitment of collagen fibrils. Using these techniques we also confirmed that collagen fibrils thin appreciably at higher levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon structure–function concepts data revealed that loading of the collagen network is fairly homogenous, with no apparent predisposition for loading of collagen fibrils according to their diameter.     

Structural changes in human type I collagen fibrils investigated by force spectroscopy

In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin, and tendon. Here, we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy (AFM)-based force spectroscopy (FS). The elongation profiles show that in vitro-assembled human type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurring within the fibrils in the 1.5- to 4.5-nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in the extracellular matrix (ECM) remodeling associated with tissue growth and morphogenesis.     

Investigations into the polymorphism of rat tail tendon fibrils using atomic force microscopy

Collagen type I displays a typical banding periodicity of 67 nm when visualized by atomic force or transmission electron microscopy imaging. We have investigated collagen fibers extracted from rat tail tendons using atomic force microscopy, under different ionic and pH conditions. The majority of the fibers reproduce the typical wavy structure with 67 nm spacing and a height difference between the peak and the grooves of at least 5 nm. However, we were also able to individuate two other banding patterns with 23+/-2 nm and 210+/-15 nm periodicities. The small pattern showed height differences of about 2 nm, whereas the large pattern seems to be a superposition of the 67 nm periodicity showing height differences of about 20 nm. Furthermore, we could show that at pH values of 3 and below the fibril structure gets dissolved whereas high concentrations of NaCl and CaCl(2) could prevent this effect.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

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.     

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.     

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment

Fibrillar collagen–integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril–integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.     

Structural investigations on native collagen type I fibrils using AFM

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.     

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

Electron-microscopic study of the collagen fibrils of the rat tail tendon as revealed by freeze-fracture and freeze-etching techniques

Summary The ultrastructure of the collagen of rat tail tendon was investigated by the freeze-fracture technique. Collagen fibers were pretreated with the digestive enzymes, -amylase, elastase and collagenase to remove matrix substances. Some of the samples were etched for 20 min. Fibrils had an average diameter of 318±12 nm and a banded structure with a mean periodicity of 64.2±0.9 mm; the banding was most marked in -amylase/elastase-treated specimens, although the periodicity was independent of pretreatment. Microfibrils were well-displayed following -amylase/elastase and collagenase pretreatments. A difference in the diameters of microfibrils was, however, observed between etched specimens (8.3±0.3 nm) and those prepared by other experimental methods (11.4±0.5 nm). In replicas of collagenase-treated and etched specimens, the interconnecting filaments in the interfibrillar region formed a network that was continuous with the microfibrils of collagen fibrils. The diameter of the interconnecting filaments was the same as that of microfibrils. Microfibrillar bundles were observed in the interfibrillar region.     

Molecular mobility in the gap regions of type 1 collagen fibrils

Recent studies of the structure of Type I collagen fibrils (Piez and Trus,Biosci. Rep. 1:801–810, 1981; Fraser, MacRae, Miller and Suzuki,J. Mol. Biol. 167:497–521, 1983) suggest that the segments of the collagen molecule which comprise the gap region are more mobile than those which comprise the overlap region. We have analyzed the distribution of amino acid residues and triplet types between the two regions, and find significantly non-uniform distributions for Ala, Gln, His, Hyp, Leu, Phe, and Tyr, and for triplets containing two imino acid residues. Taken together with the lower packing density in the gap region these observations provide a basis for understanding the greater mobility of the molecular segments in the gap region. In addition, we have examined the linear distribution of residue types in the two regions and also the hydropathy profile (Kyte and Doolittle,J. Mol. Biol. 157: 105–113, 1982). These reveal a segment of the gap region comprising helical residues 165–173, 399–407, 633–641 and 867–975 which has the highest hydropathy index, is devoid of charged residues, and contains very high proportions of Ala, Hyp and Phe.     

Interaction of lubricin with type II collagen surfaces: Adsorption,friction, and normal forces

One of the major constituents of the synovial fluid that is thought to be responsible for chondroprotection and boundary lubrication is the glycoprotein lubricin (PRG4); however, the molecular mechanisms by which lubricin carries out its critical functions still remain largely unknown. We hypothesized that the interaction of lubricin with type II collagen, the main component of the cartilage extracellular matrix, results in enhanced tribological and wear properties. In this study, we examined: (i) the molecular details by which lubricin interacts with type II collagen and how binding is related to boundary lubrication and adhesive interactions; and (ii) whether collagen structure can affect lubricin adsorption and its chondroprotective properties. We found that lubricin adsorbs strongly onto denatured, amorphous, and fibrillar collagen surfaces. Furthermore, we found large repulsive interactions between the collagen surfaces in presence of lubricin, which increased with increasing lubricin concentration. Lubricin attenuated the large friction and also the long-range adhesion between fibrillar collagen surfaces. Interestingly, lubricin adsorbed onto and mediated the frictional response between the denatured and native amorphous collagen surfaces equally and showed no preference on the supramolecular architecture of collagen. However, the coefficient of friction was lowest on fibrillar collagen in the presence of lubricin. We speculate that an important role of lubricin in mediating interactions at the cartilage surface is to attach to the cartilage surface and provide a protective coating that maintains the contacting surfaces in a sterically repulsive state.     

Nanoscale structures and mechanics of barnacle cement

Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining. Nanoscale structures exhibiting rod-shaped, globular and irregularly-shaped morphologies were observed in the bulk cement of the barnacle Amphibalanus amphitrite (=Balanus amphitrite) by AFM. SEM coupled with energy dispersive X-ray (EDX) provided chemical composition information, making evident the organic nature of the rod-shaped nanoscale structures. FTIR spectroscopy gave signatures of β-sheet and random coil conformations. The mechanical properties of these nanoscale structures were also probed using force spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the rod-shaped structures when compared with the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both the extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo red and thioflavin-T) revealed that about 5% of the bulk cement were amyloids. A dominant 100 kDa cement protein was found to be mechanically agile, using repeating hydrophobic structures that apparently associate within the same protein or with neighbors, creating toughness on the 1–100 nm length scale.     

Involvement of phospholipase D1 in collagen type I production of human dermal fibroblasts

In the current study, the involvement of phospholipase D (PLD) in the regulation of collagen type I production was examined using human dermal fibroblasts. Procollagen I production in the cells overexpressing PLD1, but not PLD2, was found to be increased compared with those in the vector control cells. To investigate the role of PLD1, we examined the effect of knockdown of endogenous PLD1 by small interference RNA (siRNA) on collagen production. The reduction of expression levels of PLD1 by siRNA transfection was accompanied by diminution of procollagen biosynthesis and also ribosomal S6 kinase 1 (S6K1) phosphorylation. The activity of mammalian target of rapamycin (mTOR) is essential for phosphorylation of S6K1 and the treatment of dermal fibroblasts with rapamycin, a potent inhibitor of mTOR abolished procollagen I production. These results suggest that PLD1 plays a crucial role in collagen type I production through mTOR signaling in human dermal fibroblast.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.     

Solvent specific persistence length of molecular type I collagen

Type I collagen is a fibril‐forming protein largely responsible for the mechanical stability of body tissues. The tissue level properties of collagen have been studied for decades, and an increasing number of studies have been performed at the fibril scale. However, the mechanical properties of collagen at the molecular scale are not well established. In the study presented herein, the persistence length of pepsin digested bovine type I collagen is extracted from the conformations assumed when deposited from solution onto two‐dimensional surfaces. This persistence length is a measure of the flexibility of the molecule. Comparison of the results for molecules deposited from different solvents allows for the study of the effect of the solutions on the flexibility of the molecule and provides insight into the molecule's behavior in situ. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 329–335, 2014.     

The precision of lateral size control in the assembly of corneal collagen fibrils

Collagen fibrils in the corneal stroma have been recognised to have a high degree of uniformity of diameter and spatial arrangement compared with those in other mature connective tissues. The precision of this lateral size control has been determined in this study by mass per unit length measurements on fibrils isolated from adult bovine corneal stroma. At the molecular level, however, there are substantial variations in lateral size, both between fibrils and along individual fibrils. The mean mass per unit length was measured to be 304 kDa nm(-1), equivalent to 347 collagen molecules in transverse section and had a standard deviation of 8.3%. The variation of lateral size along individual fibrils was measured as a mass slope over approximately 7 microm lengths (100 D-periods) and had a mean mass slope equivalent to 0.56 molecules per D-period. Smoothly tapered tips of length approximately 7 microm were also observed with a mass slope of about approximately three molecules per D-period. The frequency of these tips was used to estimate a mean fibril length of approximately 940 microm in the sample tissue. Observations of molecular polarity within the fibril shafts and tips were used to consider possible models of fibril assembly.     

Layer-specific arterial micromechanics and microstructure: Influences of age,anatomical location,and processing technique

The importance of matrix micromechanics is increasingly recognized in cardiovascular research due to the intimate role they play in local vascular cell physiology. However, variations in micromechanics among arterial layers (i.e. intima, media, adventitia), as well as dependency on local matrix composition and/or structure, anatomical location or developmental stage remain largely unknown. This study determined layer-specific stiffness in elastic arteries, including the main pulmonary artery, ascending aorta, and carotid artery using atomic force indentation. To compare stiffness with age and frozen processing techniques, neonatal and adult pulmonary arteries were tested, while fresh (vibratomed) and frozen (cryotomed) tissues were tested from the adult aorta. Results revealed that the mean compressive modulus varied among the intima, sub-luminal media, inner-middle media, and adventitia layers in the range of 1–10 kPa for adult arteries. Adult samples, when compared to neonatal pulmonary arteries, exhibited increased stiffness in all layers except adventitia. Compared to freshly isolated samples, frozen preparation yielded small stiffness increases in each layer to varied degrees, thus inaccurately representing physiological stiffness. To interpret micromechanics measurements, composition and structure analyses of structural matrix proteins were conducted with histology and multiphoton imaging modalities including second harmonic generation and two-photon fluorescence. Composition analysis of matrix protein area density demonstrated that decrease in the elastin-to-collagen and/or glycosaminoglycan-to-collagen ratios corresponded to stiffness increases in identical layers among different types of arteries. However, composition analysis was insufficient to interpret stiffness variations between layers which had dissimilar microstructure. Detailed microstructure analyses may contribute to more complete understanding of arterial micromechanics.     

Failure of mineralized collagen fibrils: modeling the role of collagen cross-linking

Experimental evidence demonstrates that collagen cross-linking in bone tissue significantly influences its deformation and failure behavior yet difficulties exist in determining the independent biomechanical effects of collagen cross-linking using in vitro and in vivo experiments. The aim of this study is to use a nano-scale composite material model of mineral and collagen to determine the independent roles of enzymatic and non-enzymatic cross-linking on the mechanical behavior of a mineralized collagen fibril. Stress–strain curves were obtained under tensile loading conditions without any collagen cross-links, with only enzymatic cross-links (modeled by cross-linking the end terminal position of each collagen domain), or with only non-enzymatic cross-links (modeled by random placement of cross-links within the collagen–collagen interfaces). Our results show enzymatic collagen cross-links have minimal effect on the predicted stress–strain curve and produce a ductile material that fails through debonding of the mineral–collagen interface. Conversely, non-enzymatic cross-links significantly alter the predicted stress–strain response by inhibiting collagen sliding. This inhibition leads to greater load transfer to the mineral, which minimally affects the predicted stress, increases modulus and decreases post-yield strain and toughness. As a consequence the toughness of bone that has more non-enzymatically mediated collagen cross-links will be drastically reduced.