Structure-mechanics relationships of collagen fibrils in the Osteogenesis Imperfecta Mouse model

The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry.United States. Dept. of Defense. Presidential Early Career Award for Scientists and EngineersNational Science Foundation (U.S.) (CAREER Award

Biochemical changes in bone in a model of weightlessness

The amounts of nonmineralized and mineralized collagen in bone from control, immobilized, and immobilized reambulated monkeys were examined. In order to understand structure function relationships of bone collagen and the reponse of a variety of conditions on control of the three dimensional structure of the collagen fibril, the stereochemistry of the cross-linking reactions as well as the stereospecific packing of the collagen molecules were studied

Tissue engineering a fetal membrane

The aim of this study was to construct an artificial fetal membrane (FM) by combination of human amniotic epithelial stem cells (hAESCs) and a mechanically enhanced collagen scaffold containing encapsulated human amniotic stromal fibroblasts (hASFs). Such a tissue-engineered FM may have the potential to plug structural defects in the amniotic sac after antenatal interventions, or to prevent preterm premature rupture of the FM. The hAESCs and hASFs were isolated from human fetal amniotic membrane (AM). Magnetic cell sorting was used to enrich the hAESCs by positive ATP-binding cassette G2 selection. We investigated the use of a laminin/fibronectin (1:1)-coated compressed collagen gel as a novel scaffold to support the growth of hAESCs. A type I collagen gel was dehydrated to form a material mimicking the mechanical properties and ultra-structure of human AM. hAESCs successfully adhered to and formed a monolayer upon the biomimetic collagen scaffold. The resulting artificial membrane shared a high degree of similarity in cell morphology, protein expression profiles, and structure to normal fetal AM. This study provides the first line of evidence that a compacted collagen gel containing hASFs could adequately support hAESCs adhesion and differentiation to a degree that is comparable to the normal human fetal AM in terms of structure and maintenance of cell phenotype

Hierarchical nanomechanics of collagen microfibrils

Collagen constitutes one third of the human proteome, providing mechanical stability, elasticity and strength to connective tissues. Collagen is also the dominating material in the extracellular matrix (ECM) and is thus crucial for cell differentiation, growth and pathology. However, fundamental questions remain with respect to the origin of the unique mechanical properties of collagenous tissues, and in particular its stiffness, extensibility and nonlinear mechanical response. By using x-ray diffraction data of a collagen fibril reported by Orgel et al. (Proceedings of the National Academy of Sciences USA, 2006) in combination with protein structure identification methods, here we present an experimentally validated model of the nanomechanics of a collagen microfibril that incorporates the full biochemical details of the amino acid sequence of the constituting molecules. We report the analysis of its mechanical properties under different levels of stress and solvent conditions, using a full-atomistic force field including explicit water solvent. Mechanical testing of hydrated collagen microfibrils yields a Young’s modulus of ≈300 MPa at small and ≈1.2 GPa at larger deformation in excess of 10% strain, in excellent agreement with experimental data. Dehydrated, dry collagen microfibrils show a significantly increased Young’s modulus of ≈1.8 to 2.25 GPa (or ≈6.75 times the modulus in the wet state) owing to a much tighter molecular packing, in good agreement with experimental measurements (where an increase of the modulus by ≈9 times was found). Our model demonstrates that the unique mechanical properties of collagen microfibrils can be explained based on their hierarchical structure, where deformation is mediated through mechanisms that operate at different hierarchical levels. Key mechanisms involve straightening of initially disordered and helically twisted molecules at small strains, followed by axial stretching of molecules, and eventual molecular uncoiling at extreme deformation. These mechanisms explain the striking difference of the modulus of collagen fibrils compared with single molecules, which is found in the range of 4.8±2 GPa or ≈10-20 times greater. These findings corroborate the notion that collagen tissue properties are highly scale dependent and nonlinear elastic, an issue that must be considered in the development of models that describe the interaction of cells with collagen in the extracellular matrix. A key impact the atomistic model of collagen microfibril mechanics reported here is that it enables the bottom-up elucidation of structure-property relationships in the broader class of collagen materials such as tendon or bone, including studies in the context of genetic disease where the incorporation of biochemical, genetic details in material models of connective tissue is essential

The effect of collagenase, water and calcium chloride on the removal of <i>Salmo salar</i> (salmon) and <i>Oncorhynchus mykiss</i> (trout) pin bones

The aim of this study was to determine the influence of the fillet structure on the deboning force required to remove salmon and trout pin bones. Salmon and trout fillets with differing fillet structure were used, in order to study the importance of the fillet structure on the deboning process. In the first test naturally gaping and non-gaping fillets were compared. To confirm the role that the collagen plays within the fillet structure, the fillets underwent series of treatments. Fillets were put into (i) a collagenase solution to remove the collagen in the fillet (ii) a calcium chloride solution to determine if collagen was the main influential factor. Both treated salmon and trout fillets were again compared to untreated fillets from the same batch. The results indicate that collagenase and calcium chloride have a large interaction on deboning force compared to water or no treatments

The structure and performance of collagen biomaterials : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering, Massey University, Palmerston North, New Zealand

Type I collagen materials are used in a wide range of industrial applications. Some examples include leather for shoes and upholstery, acellular dermal matrix (ADM) materials for surgical applications, and bovine pericardium for the fabrication of heart valve replacements. The structure of these materials is based on a matrix of collagen fibrils, largely responsible for the physical properties and strength of the materials. How the collagen fibrils themselves contribute to the overall bulk properties of these materials is not fully understood. The first part of this work investigates a collagen structure defect in leather, known as looseness. Looseness occurs in around 5-10% of bovine leather, and is a result of the collagen fibril layers separating during processing from raw skin to leather. A greater understanding of why looseness develops in leather and a method of detecting looseness early in processing is needed to save tanners a significant amount on wasted processing time and costs. In addition, an environmentally safe method of disposing of defect and waste leather is sort after since the current method of disposing to landfill is causing environmental concern due to the possibility of chromium leaching from leather into the soil as it biodegrades. Synchrotron based small angle X-ray scattering (SAXS) revealed that loose leather has a more aligned and layered collagen fibril arrangement, meaning there is less fibril overlap, particularly in the grain-corium boundary region. This results in larger gaps in the internal structure of loose leather compared with tight. These gaps could be detected using ultrasonic imaging in partially processed pickle and wet-blue hides as well as leather. Incorporating an ultrasound system into the leather processing line could be a viable method for identifying hides deemed to develop looseness earlier in processing, and these could be diverted down a separate processing line or removed. Disposing of waste leather by first forming biochar prior to land fill proved to be an effective way of reducing chromium from leaching into the environment. XAS revealed that heating leather to temperatures above 600°C in the absence of oxygen formed a char where chromium was bound in the stable form of chromium carbide. The stability of this structure makes chromium less available to form the toxic hexavalent form in the environment and presents a possible alternative option for environmentally safe disposal of leather. The second part to this work looks at the correlation between collagen fibril structure in a range of biomaterials in relation to material strength. Leather, ADM and pericardium are three type I collagen based materials which rely on sufficient strength to carry out their industrial and medical applications. These three materials were studied to try and identify collagen fibril characteristics that relate to high material strength. SAXS on a range of leather samples from various species revealed that collagen fibril diameter had only a small influence over material strength in bovine leather, and no correlation to strength in leather from other species. Therefore it can be said that the influence of fibril orientation on leather strength takes precedence over that of fibril diameter. Fibril diameter, d-spacing and orientation were studied in pericardium using SAXS while simultaneously applying strain. It was revealed collagen materials undergo two distinct stages of deformation when strain is applied and incrementally increased. The first stage, at low strain, involves a re-orientation of fibrils to become more aligned. When strain is increased further, the fibrils themselves take up the strain, causing fibrils to stretch and decrease in diameter. The Poisson ratio of the collagen fibrils was calculated to be 2.1 ± 0.7. This high Poisson's ratio indicates the fibrils decrease in diameter at a faster rate than they elongate with strain, and as a result the volume of the fibrils decreases. This feature of collagen could help explain some of the unique behaviours and strength of collagen based materials and could be useful for optimizing industrial applications of collagen materials. ADM materials, derived from human, porcine and bovine skin was the third collagen material studied. SAXS revealed that each species of ADM material had a slightly different collagen fibril arrangement when viewing the samples perpendicular to the surface. Human ADM was highly isotropic in arrangement, porcine was largely anisotropic, and bovine was somewhere in between the two. Bovine has a more layered fibril arrangement edge on and was the strongest material, followed by human ADM, and porcine was significantly weaker. Bovine was also the most porous material of the three. The discovery of the variations in strength, porosity and fibril arrangement between the three types of ADM materials may help medical professionals select the most suitable material for specific surgical procedures and could lead to a greater number of successful surgeries taking place

Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.This was supported by the Biotechnology and Biological Sciences Research Council

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure

Second-harmonic generation microscopy analysis reveals proteoglycan decorin is necessary for proper collagen organization in prostate.

Collagen remodeling occurs in many prostate pathologies; however, the underlying structural architecture in both normal and diseased prostatic tissues is largely unexplored. Here, we use second-harmonic generation (SHG) microscopy to specifically probe the role of the proteoglycan decorin (Dcn) on collagen assembly in a wild type (wt) and Dcn null mouse (Dcn  -    /    -  ). Dcn is required for proper organization of collagen fibrils as it regulates size by forming an arch-like structure at the end of the fibril. We have utilized SHG metrics based on emission directionality (forward-backward ratio) and relative conversion efficiency, which are both related to the SHG coherence length, and found more disordered fibril organization in the Dcn  -    /    -  . We have also used image analysis readouts based on entropy, multifractal dimension, and wavelet transforms to compare the collagen fibril/fiber architecture in the two models, where all these showed that the Dcn  -    /    -   prostate comprised smaller and more disorganized collagen structures. All these SHG metrics are consistent with decreased SHG phase matching in the Dcn  -    /    -   and are further consistent with ultrastructural analysis of collagen in this model in other tissues, which show a more random distribution of fibril sizes and their packing into fibers. As Dcn is a known tumor suppressor, this work forms the basis for future studies of collagen remodeling in both malignant and benign prostate disease