Single molecule mechanical properties of type II collagen and hyaluronan measured by optical tweezers

Type II collagen and hyaluronan are the two major components of extracellular molecules in cartilage and play an important role in mechanical functions of extracellular matrix. Currently, their mechanical properties have been investigated only at the gross-level. In this study, the mechanical properties of single type II collagen and hyaluronan molecules were directly measured using optical tweezers technique. The persistence length was found to be 11.2+/-8.4 nm in type II collagen and 4.5+/-1.2 nm in hyaluronan. This result suggested that type II collagen is stiffer than hyaluronan at the individual molecule level, which supports the general concept that collagen is responsible for resisting tensile force. The experimental system developed here also provides a powerful tool for quantifying mechanical properties of extracellular matrix at the single molecule level.     

Effect of thermal damage and biaxial loading on the optical properties of a collagenous tissue

Thermal denaturation can induce marked changes in the optical and mechanical properties of collagenous tissues. The optical properties are important in both therapeutic and diagnostic applications of lasers in medicine. Although mechanical stress can be caused by collagen shrinkage in laser-based therapies, how the mechanical loading state affects the optical properties is not well understood. We used a new computer-controlled biaxial testing system to subject bovine epicardium to various loading conditions both before and after multiple levels of thermal damage. An integrating sphere technique was used to measure transmittance and diffuse reflectance, from which absorption and scattering coefficients were calculated using a Monte Carlo method. Results showed that the scattering coefficient increased with increasing mechanical load but decreased as the degree of thermal damage increased. There was no significant change in the absorption coefficient due to thermal damage over the ranges studied.     

A Quantitative Study of the Relationship between the Distribution of Different Types of Collagen and the Mechanical Behavior of Rabbit Medial Collateral Ligaments

The mechanical properties of ligaments are key contributors to the stability and function of musculoskeletal joints. Ligaments are generally composed of ground substance, collagen (mainly type I and III collagen), and minimal elastin fibers. However, no consensus has been reached about whether the distribution of different types of collagen correlates with the mechanical behaviors of ligaments. The main objective of this study was to determine whether the collagen type distribution is correlated with the mechanical properties of ligaments. Using axial tensile tests and picrosirius red staining-polarization observations, the mechanical behaviors and the ratios of the various types of collagen were investigated for twenty-four rabbit medial collateral ligaments from twenty-four rabbits of different ages, respectively. One-way analysis of variance was used in the comparison of the Young''s modulus in the linear region of the stress-strain curves and the ratios of type I and III collagen for the specimens (the mid-substance specimens of the ligaments) with different ages. A multiple linear regression was performed using the collagen contents (the ratios of type I and III collagen) and the Young''s modulus of the specimens. During the maturation of the ligaments, the type I collagen content increased, and the type III collagen content decreased. A significant and strong correlation () was identified by multiple linear regression between the collagen contents (i.e., the ratios of type I and type III collagen) and the mechanical properties of the specimens. The collagen content of ligaments might provide a new perspective for evaluating the linear modulus of global stress-strain curves for ligaments and open a new door for studying the mechanical behaviors and functions of connective tissues.     

Designed to fail: a novel mode of collagen fibril disruption and its relevance to tissue toughness

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules-meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils-were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils.     

Mechanical and biological performances of new scaffolds made of collagen hydrogels and fibroin microfibers for vascular tissue engineering

A microstructured composite material made of collagen hydrogel (matrix) and silk fibroin microfibers (randomly oriented reinforcing fibers) is investigated in order to conjugate the mechanical resistance of fibroin with the suitable biological performance of collagen to design new scaffolds for vascular tissue engineering. Results show that fibroin microfibers and collagen fibrils have suitable interfacial adhesion, and the scaffold exhibits improved mechanical properties if compared with a pure collagen hydrogel. Furthermore, the overall biological performance is improved.     

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.     

Tensile properties of human collagen fibrils and fascicles are insensitive to environmental salts

To carry out realistic in vitro mechanical testing on anatomical tissue, a choice has to be made regarding the buffering environment. Therefore, it is important to understand how the environment may influence the measurement to ensure the highest level of accuracy. The most physiologically relevant loading direction of tendon is along its longitudinal axis. Thus, in this study, we focus on the tensile mechanical properties of two hierarchical levels from human patellar tendon, namely: individual collagen fibrils and fascicles. Investigations on collagen fibrils and fascicles were made at pH 7.4 in solutions of phosphate-buffered saline at three different concentrations as well as two HEPES buffered solutions containing NaCl or NaCl + CaCl₂. An atomic force microscope technique was used for tensile testing of individual collagen fibrils. Only a slight increase in relative energy dissipation was observed at the highest phosphate-buffered saline concentration for both the fibrils and fascicles, indicating a stabilizing effect of ionic screening, but changes were much less than reported for radial compression. Due to the small magnitude of the effects, the tensile mechanical properties of collagen fibrils and fascicles from the patellar tendon of mature humans are essentially insensitive to environmental salt concentration and composition at physiological pH.     

Structural and micromechanical characterization of type I collagen gels

In this paper we report a study where we use a novel optical tweezers technique to measure the local viscoelastic properties of type I collagen solutions spanning the sol-to-gel transition. We use phase contrast optical microscopy to reveal dense and sparse regions of the rigid fibril networks, and find that the spatial variations in the mechanical properties of the collagen gels closely follow the structural properties. Within the dense phase of the connected network in the gel samples, there are regions that exhibit drastically different viscoelastic properties. Within the sparse regions of the gel samples, no evidence of elasticity is found. In type I collagen gels, we find a high degree of structural inhomogeneity. The inhomogeneity in the structural properties of collagen gels and the corresponding viscoelastic properties provide benchmark measurements for the behavior of desirable biological materials, or tissue equivalents.     

Controlling the mechanical properties of three-dimensional matrices via non-enzymatic collagen glycation

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.     

Biomimetic mineralization of woven bone-like nanocomposites: role of collagen cross-links

Ideal biomaterials for bone grafts must be biocompatible, osteoconductive, osteoinductive and have appropriate mechanical properties. For this, the development of synthetic bone substitutes mimicking natural bone is desirable, but this requires controllable mineralization of the collagen matrix. In this study, densified collagen films (up to 100 μm thick) were fabricated by a plastic compression technique and cross-linked using carbodiimide. Then, collagen-hydroxyapatite composites were prepared by using a polymer-induced liquid-precursor (PILP) mineralization process. Compared to traditional methods that produce only extrafibrillar hydroxyapatite (HA) clusters on the surface of collagen scaffolds, by using the PILP mineralization process, homogeneous intra- and extrafibrillar minerals were achieved on densified collagen films, leading to a similar nanostructure as bone, and a woven microstructure analogous to woven bone. The role of collagen cross-links on mineralization was examined and it was found that the cross-linked collagen films stimulated the mineralization reaction, which in turn enhanced the mechanical properties (hardness and modulus). The highest value of hardness and elastic modulus was 0.7 ± 0.1 and 9.1 ± 1.4 GPa in the dry state, respectively, which is comparable to that of woven bone. In the wet state, the values were much lower (177 ± 31 and 8 ± 3 MPa) due to inherent microporosity in the films, but still comparable to those of woven bone in the same conditions. Mineralization of collagen films with controllable mineral content and good mechanical properties provide a biomimetic route toward the development of bone substitutes for the next generation of biomaterials. This work also provides insight into understanding the role of collagen fibrils on mineralization.     

A novel model-gel-tissue assay analysis for comparing tumor elastic properties to collagen content

In previous work, a new assay was realized for determining soft-tissue mechanical properties. The method, named the model-gel-tissue (MGT) assay, couples material testing with a finite element model built from a micro-CT image acquisition of a gel-embedded tissue specimen to determine its mechanical properties. Given recent reports demonstrating that increased stromal collagen promotes mammary tumor initiation and proliferation, in this paper, the MGT assay is used to evaluate the modulus of murine mammary tumors and is subsequently correlated quantitatively to type I collagen content. In addition, preliminary testing of the assay sensitivity with respect to gel-volume to tissue-mass ratio is reported here. The results demonstrate a strong linear correlation between tumor mechanical properties and collagen content (R ² = 0.9462). This result is important because mechanical stiffness as provided by the MGT assay is very similar to parameters under clinical investigation using elastographic imaging techniques. The sensitivity tests indicated that an approximate gel-volume to tissue-mass ratio threshold of 16.5 ml g⁻¹ is needed for successful analysis. This is an important result in that it presents guideline constraints for conducting this analysis.     

The dynamic mechanical, dielectric, and melting behavior of reconstituted collagen

The dielectric, dynamic mechanical, and melting (denaturation) behavior of reconstituted collagen has been investigated. Dynamic mechanical properties are reported for the range of temperature from ?100 to 220°C. Dielectric properties are reported for the range of temperature from ?120 to 90°C. Possible origins of dynamic mechanical and dielectric relaxations are discussed. Effect of moisture content on mechanical and dielectric behavior is also presented. The melting process of collagen immersed in diluents as well as that of dry collagen were studied by the techniques of hot-stage polarizing microscopy, differential thermal analysis, thermal mechanical analysis, and small-angle light scattering. It was concluded that the melting point of dry collagen is about 217°C.     

Collagen fiber alignment does not explain mechanical anisotropy in fibroblast populated collagen gels

Many load-bearing soft tissues exhibit mechanical anisotropy. In order to understand the behavior of natural tissues and to create tissue engineered replacements, quantitative relationships must be developed between the tissue structures and their mechanical behavior. We used a novel collagen gel system to test the hypothesis that collagen fiber alignment is the primary mechanism for the mechanical anisotropy we have reported in structurally anisotropic gels. Loading constraints applied during culture were used to control the structural organization of the collagen fibers of fibroblast populated collagen gels. Gels constrained uniaxially during culture developed fiber alignment and a high degree of mechanical anisotropy, while gels constrained biaxially remained isotropic with randomly distributed collagen fibers. We hypothesized that the mechanical anisotropy that developed in these gels was due primarily to collagen fiber orientation. We tested this hypothesis using two mathematical models that incorporated measured collagen fiber orientations: a structural continuum model that assumes affine fiber kinematics and a network model that allows for nonaffine fiber kinematics. Collagen fiber mechanical properties were determined by fitting biaxial mechanical test data from isotropic collagen gels. The fiber properties of each isotropic gel were then used to predict the biaxial mechanical behavior of paired anisotropic gels. Both models accurately described the isotropic collagen gel behavior. However, the structural continuum model dramatically underestimated the level of mechanical anisotropy in aligned collagen gels despite incorporation of measured fiber orientations; when estimated remodeling-induced changes in collagen fiber length were included, the continuum model slightly overestimated mechanical anisotropy. The network model provided the closest match to experimental data from aligned collagen gels, but still did not fully explain the observed mechanics. Two different modeling approaches showed that the level of collagen fiber alignment in our uniaxially constrained gels cannot explain the high degree of mechanical anisotropy observed in these gels. Our modeling results suggest that remodeling-induced redistribution of collagen fiber lengths, nonaffine fiber kinematics, or some combination of these effects must also be considered in order to explain the dramatic mechanical anisotropy observed in this collagen gel model system.     

Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy

The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.     

Effect of preconditioning and stress relaxation on local collagen fiber re-alignment: inhomogeneous properties of rat supraspinatus tendon

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.     

Reinforced bioartificial dermis constructed with collagen threads

In this work, a novel type of composite scaffold was designed, which has the suitability of both high biocompatibility and strong mechanical properties, for use in bioartificial dermis applications. The reinforced scaffold consisted of a lyophilized collagen sponge formed around a cross-linked collagen meshwork with an average thread diameter of approximately 55 μm. Fibroblasts were cultured in the reinforced collagen sponge for 7 days, during which time the pores in the sponge became filled with cells that secreted extracellular matrix (ECM) to form a bioartificial dermis. Results of ultimate tensile strength (UTS) measurements and compression tests indicated that the bioartificial dermis formed around the reinforced collagen sponge showed about ten times the strength of the bioartificial dermis formed around a typical collagen sponge (1.5 ± 0.05 vs. 0.15 ± 0.05 and 2.5 ± 0.1 vs. 0.2 ± 0.08 MPa, respectively). As a result, reinforced collagen mesh improved mechanical properties and this technique will be possible to make stronger scaffolds, not only for artificial skin applications but also various artificial tissues, such as synthetic cartilage, bone, and blood vessels.     

Biomimetically mineralized salmon collagen scaffolds for application in bone tissue engineering

Biomimetic mineralization of collagen is an advantageous method to obtain resorbable collagen/hydroxy-apatite composites for application in bone regeneration. In this report, established procedures for mineralization of bovine collagen were adapted to a new promising source of collagen from salmon skin challenged by the low denaturation temperature. Therefore, in the first instance, variation of temperature, collagen concentration, and ionic strength was performed to reveal optimized parameters for fibrillation and simultaneous mineralization of salmon collagen. Porous scaffolds from mineralized salmon collagen were prepared by controlled freeze-drying and chemical cross-linking. FT-IR analysis demonstrated the mineral phase formed during the preparation process to be hydroxyapatite. The scaffolds exhibited interconnecting porosity, were sufficiently stable under cyclic compression, and showed elastic mechanical properties. Human mesenchymal stem cells were able to adhere to the scaffolds, cell number increased during cultivation, and osteogenic differentiation was demonstrated in terms of alkaline phosphatase activity.     

Mechanical properties of rat tail tendon in relation to proximal-distal sampling position and age

The mechanical properties of 3, 15 and 25 month-old rat tail tendons were investigated in relation to proximal-distal sampling location along the fibre length. For the 15 and 25 month-old tendons maximum load as well as collagen content per mm fibre length (unit collagen) increased markedly from the proximal to the distal location. A linear regression analysis of the collagen content and mechanical parameters (maximum load, maximum slope of the load-strain curve and energy absorption) showed that these parameters were linearly correlated to the collagen content. However, normalization of the mechanical parameters with regard to the collagen content did not cancel the dependency of the parameters on proximal-distal sampling location. Normalized load and energy values for the 3 month-old tendons and normalized slope values for the 15 and 25 month-old tendons were found to decrease from proximal to distal location. These findings showed that tail tendons are heterogeneous along their length in respect to mechanical strength. The regression analysis also indicated the existence of an inverse relationship between unit collagen and mechanical quality of the collagen. Alternatively, the mechanical properties of tendon fibres might be influenced by other components than collagen.     

Characterizing local collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse supraspinatus tendon model

BackgroundCollagen fiber re-alignment and uncrimping are two postulated mechanisms of tendon structural response to load. Recent studies have examined structural changes in response to mechanical testing in a postnatal development mouse supraspinatus tendon model (SST), however, those changes in the mature mouse have not been characterized. The objective of this study was to characterize collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse SST.Method of approachA tensile mechanical testing set-up integrated with a polarized light system was utilized for alignment and mechanical analysis. Local collagen fiber crimp frequency was quantified immediately following the designated loading protocol using a traditional tensile set up and a flash-freezing method. The effect of number of preconditioning cycles on collagen fiber re-alignment, crimp frequency and mechanical properties in midsubstance and insertion site locations were examined.ResultsDecreases in collagen fiber crimp frequency were identified at the toe-region of the mechanical test at both locations. The insertion site re-aligned throughout the entire test, while the midsubstance re-aligned during preconditioning and the test's linear-region. The insertion site demonstrated a more disorganized collagen fiber distribution, lower mechanical properties and a higher cross-sectional area compared to the midsubstance location.ConclusionsLocal collagen fiber re-alignment, crimp behavior and mechanical properties were characterized in a mature mouse SST model. The insertion site and midsubstance respond differently to mechanical load and have different mechanisms of structural response. Additionally, results support that collagen fiber crimp is a physiologic phenomenon that may explain the mechanical test toe-region.     

Biomechanics of the lung parenchyma: critical roles of collagen and mechanical forces.

The biomechanical properties of connective tissues play fundamental roles in how mechanical interactions of the body with its environment produce physical forces at the cellular level. It is now recognized that mechanical interactions between cells and the extracellular matrix (ECM) have major regulatory effects on cellular physiology and cell-cycle kinetics that can lead to the reorganization and remodeling of the ECM. The connective tissues are composed of cells and the ECM, which includes water and a variety of biological macromolecules. The macromolecules that are most important in determining the mechanical properties of these tissues are collagen, elastin, and proteoglycans. Among these macromolecules, the most abundant and perhaps most critical for structural integrity is collagen. In this review, we examine how mechanical forces affect the physiological functioning of the lung parenchyma, with special emphasis on the role of collagen. First, we overview the composition of the connective tissue of the lung and their complex structural organization. We then describe how mechanical properties of the parenchyma arise from its composition as well as from the architectural organization of the connective tissue. We argue that, because collagen is the most important load-bearing component of the parenchymal connective tissue, it is also critical in determining the homeostasis and cellular responses to injury. Finally, we overview the interactions between the parenchymal collagen network and cellular remodeling and speculate how mechanotransduction might contribute to disease propagation and the development of small- and large-scale heterogeneities with implications to impaired lung function in emphysema.