Collagen Mechanical Properties Research Articles

Novel sulfonated calixarenes derived crosslinker for reinforcing and toughening collagen: Relevance between structure and properties

AbstractCollagen is widely used in the development of new functional materials due to its high structural stability, good biocompatibility, and excellent hydrophilicity, but the collagen protein denatures easily, which makes its thermal stability and mechanical strength difficult to reach some of the parameters required in the application. To solve this, protein crosslinking is an effective way to better use collagen. In this paper, we compared the thermal stability and morphological stability of three kinds of sulfonated calixarenes crosslinked collagen before and after the treatment. The results showed that sulfonated calixarenes could significantly improve the thermal stability and morphological stability of dermal collagen. The reinforcing and toughening mechanism of the sulfonated calixarenes was revealed by scanning electron microscopy (SEM), infrared spectroscopy (FT‐IR), attenuated total reflection‐Fourier transform infrared spectroscopy (ATR‐FTIR), ultraviolet–visible–near‐infrared (UV–vis–NIR) spectrometer, X‐ray diffraction detection (XRD), atomic force microscopy (AFM), and circular dichroism (CD) detection. The results showed that sulfonated calixarenes were evenly distributed in collagen fibers and formed hydrogen bonds or electrovalent bonds with amino groups, peptide groups, or amide groups in collagen to improve the thermal stability of the collagen. Three kinds of sulfonated calixarenes enter collagen fiber to support the collagen, increase the three‐dimensional weave angle of collagen, and improve the physical and mechanical properties of collagen, thereby toughening and enhancing the collagen. Based on the above studies, the reinforcing and toughening mechanism of the sulfonated calixarenes crosslinked collagen was established. Confirmed the existence of hydrogen bonds between the three sulfonated calixarenes and collagen, and the nonchemical bond energy of the system was proportional to the molecular size of sulfonated calixarenes.

Using celluloses in different geometries to reinforce collagen-based composites: Effect of cellulose concentration

Cellulose is frequently used to strengthen biocomposite films, but few literature systematically deliberates the effects of concentration of celluloses in different geometries on the reinforcement of these composites. Here we prepared three types of celluloses, including rod-like cellulose nanocrystalline (CNC), long-chain cellulose nanofiber (CNF) and microscopic cellulosic fines (CF). The effect of concentration of the three celluloses was examined on the barrier properties to water and light, thermostability, microstructure, and mechanical properties of collagen (COL) films. The addition of celluloses increased the watertightness and thermostability of composite films. Besides, FTIR showed a increased hydrogen bonding for COL/CNF and COL/CNC composite films, but decrease for COL/CF composites. As the concentration of CF and CNF increased, the strength of composites improved. The TS for COL/CNF (124 MPa) and COL/CF composites (113 MPa) were largely increased, compared with that of collagen ones (90 MPa). Considering the factors of crystallinity, hydrogen bonding, and interfacial tortuosity, COL/CNF composites possessed better mechanical behaviors than that of COL/CF and COL/CNC composites. Furthermore, Halpin-Kardos and Ouali models well predicted the modulus of COL/CNF composites when CNF was below and above percolation threshold (2.7 wt%), respectively.

Collagen and its derivatives: From structure and properties to their applications in food industry

Collagen is the most abundant extracellular matrix protein in food-producing animals. Gelatin is partially degraded collagen. Collagen peptides refer to the peptides with specific properties identified from collagen hydrolysate who produced by hydrolysis of collagen/gelatin. Due to the specific structural and bio- and physical-chemical properties, collagen and its derivatives are used in the field of food industry. In this review, the structure of the collagen molecule and its biosynthetic process in vivo are introduced, and the production methods and structures of gelatin and collagen peptides described. Then the inherent self-assembly property of collagen, the mechanical properties of collagen and gelatin gels, functional properties of collagen and gelatin, and bioactive properties of collagen peptides are reviewed. Finally, the applications of collagen and its derivatives that are correlated with their properties in food industry are summarized. The mechanisms and advantages of the applications of collagen and its derivatives in food industry are raised, and the limitations and challenges of these applications are also discussed. And possible studies to address the challenges of the applications in different areas are indicated. • Gelatin is partially degraded collagen. • Collagen peptide is the peptide separated from collagen hydrolysate. • Collagen derivatives refer to gelatin and collagen peptides. • The structure and properties of collagen and its derivatives were reviewed. • The application of collagen and its derivatives in food industry were discussed.

Multiscale Modeling of Skin Mechanical Behavior: Effect of Dehydrating Agent on Collagen's Mechanical Properties

Multiscale Modeling of Skin Mechanical Behavior: Effect of Dehydrating Agent on Collagen's Mechanical Properties

Enhancement of engineered cardiac tissues by promotion of hiPSC-cardiomyocyte proliferation

Abstract Background/Introduction Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modelling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Purpose Determining the effects of the induction of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocyte proliferation post-fabrication. Methods hiPSCs were differentiated into cardiomyocytes, embedded with or without CHIR990121 at three concentrations in a collagen pre-gel, and cast. The engineered cardiac tissues were then cultured in the absence or presence of CHIR99021 for up to 35 days. Hydrogels and engineered cardiac tissues were analysed utilizing rheology and assays to determine viability, proliferation, calcium flow, and contractility. Results Here, we show that the integration of CHIR99021 in collagen I hydrogels promotes proliferation of hiPSC-cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered cardiac tissues. Presence of CHIR99021 has no effect on the gelation kinetic or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ∼2-fold within 7 days post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 days in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increases speed of contraction and contraction amplitude, and reduces peak-to-peak time. The CHIR99021-stimulated engineered cardiac tissues exhibited spontaneous rhythmic contractions for at least 35 days. Conclusion Collectively, our data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac tissues by increasing cardiomyocyte density and reducing arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

CHIR99021 Promotes hiPSC-Derived Cardiomyocyte Proliferation in Engineered 3D Microtissues.

Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modeling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Here, it is shown that the integration of the glycogen synthase kinase-3β inhibitor CHIR99021 in collagen I hydrogels promotes proliferation of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered 3D cardiac microtissues. CHIR99021 has no effect on the gelation kinetics or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ≈2-fold within 7 d post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 d in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increased speed of contraction and contraction amplitude, and reduced peak-to-peak time. The CHIR99021-stimulated engineered cardiac microtissues exhibit spontaneous rhythmic contractions for at least 35 d. Collectively, the data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac microtissues by increasing cardiomyocyte density.

Identification of Glucosepane Cross-Link Breaking Enzymes

Glucosepane is a member of the class of advanced glycation end-products (AGEs), which form non-enzymatically in the human body. Glucosepane, is the most abundant AGE found in human collagen and has been implicated in the pathophysiology of various conditions ranging from diabetes to normal human aging. Glucosepane crosslinks impact the structural and mechanical properties of collagen and contribute to stiffening of collagenous tissues and vascular dysfunction. We have previously demonstrated the total synthesis of glucosepane, enabling methods for detecting and targeting glucosepane. The present study seeks to identify enzymes that are capable of catalyzing the decomposition of synthetic glucosepane crosslinks. To identify glucosepane crosslink breaking enzymes, we developed a novel screening technology based on metagenomics. Using this strategy, four enzymes were identified for in vitro validation of glucosepane degradation activity. Thus far, our efforts have focused on identifying metabolites of one class I-like enzyme in particular, since it is heterologously expressed at high levels and does not require any unusual cofactors. In vitro enzymatic assays showed that incubation of glucosepane with the class I-like enzyme led to a product consistent with citrulline. We are currently in the process of evaluating the enzyme’s mechanisms of action and identifying other metabolites generated. This is the first demonstration that glucosepane can be broken down enzymatically. Our findings may provide new insight into the role of glucosepane in aging and disease and aid in the development of novel therapeutic strategies for targeting glucosepane. Disclosure M. Streeter: None. T.N. Goddard: None. J.M. Crawford: None. D.A. Spiegel: None.

The promising indicators of the thermal and mechanical properties of collagen from bass and tilapia: synergistic effects of hydroxyproline and cysteine.

Collagen has been widely documented as one of the most promising and competitive biomaterials for tissue engineering and medical applications. However, the properties of collagen differ from one source to another. In the present study, type I collagen (COL-I) was extracted and purified from the skins of Japanese sea bass (Lateolabrax japonicus) and Nile tilapia (Oreochromis niloticus). Ultraviolet (UV) spectroscopy, Fourier transform infrared spectroscopy (FTIR) and SDS-PAGE were performed to characterize both COL-Is. The denaturing temperature of bass collagen (BC) was observed to be 27.2 °C, and 35.3 °C for tilapia collagen (TC). The content of hydroxyproline was 13.4% in TC, which was similar to that in porcine collagen (PC, 13.6%) and higher than that in BC (10.3%), while the content of cysteine in TC (0.87%) was significantly higher than that in PC (0.04%) and BC (0.35%). After incubation at different temperatures for 9 h, more degraded collagen bands appeared in the BC hydrogel (BCH) group than in the TC hydrogel (TCH) group, indicating that TCH exhibited better thermal stability than BCH. The thermal stabilities of TCH and PC hydrogel (PCH) were similar. The compressive stress of TCH was up to 0.099 MPa, while it was 0.047 MPa for BCH and 0.003 MPa for PCH. These results demonstrated that the content of amino acids (especially hydroxyproline and cysteine) has a synergistic effect on the thermal and mechanical properties of BCH, TCH and PCH, which would be an indicator of the thermal and mechanical properties of collagen hydrogels in future studies.

Parallels between DNA and collagen \u2013 comparing elastic models of the double and triple helix

Multi-stranded helices are widespread in nature. The interplay of polymeric properties with biological function is seldom discussed. This study probes analogies between structural and mechanical properties of collagen and DNA. We modeled collagen with Eulerian rotational and translational parameters of adjacent rungs in the triple-helix ladder and developed statistical potentials by extracting the dispersion of the parameters from a database of atomic-resolution structures. The resulting elastic model provides a common quantitative way to describe collagen deformations upon interacting with integrins or matrix metalloproteinase and DNA deformations upon protein binding. On a larger scale, deformations in Type I collagen vary with a periodicity consistent with the D-periodic banding of higher-order fibers assemblies. This indicates that morphologies of natural higher-order collagen packing might be rooted in the characteristic deformation patterns.

Improved thermal-stability and mechanical properties of type I collagen by crosslinking with casein, keratin and soy protein isolate using transglutaminase

The inferior thermal- stability of collagen hinders its extensive application in food industry, including edible packaging. To improve the thermal- stability and mechanical properties of collagen, we attempted to crosslink collagen with some proteins possessing excellent thermal stability (i. e., casein, keratin and soy protein isolate (SPI)). Observed from the SDS- PAGE and particle size distribution, some complexes with higher molecule weight and relative bigger size particle occurred in the protein mixture, especially after TGase crosslinking. Importantly, the crosslinking greatly improved the thermal- stable property of protein complex, especially that of the collagen- casein complex judged from differential scanning calorimetric (DSC). Moreover, the crosslinking enhanced the mechanical properties of the combined films in terms of tensile strength (TS) and elongation at break (EAB). Also, some obvious differences in morphology of proteins before and after TGase crosslinking were observed by scanning electron microscopy (SEM). These impacts of TGase crosslinking with heat- resistant proteins on collagen features were associated with the conformational changes of the protein complex analyzed by Fourier transform infrared spectroscopy (FTIR). In conclusion, TGase crosslinking with higher thermally stable proteins could be an effective method to contribute to collagen’ application in food packaging field.

Very early osteoarthritis changes sensitively fluid flow properties of articular cartilage

In this study, fibril-reinforced poroelastic (FRPE) modeling was used for rabbit knee after anterior cruciate ligament transection (ACLT) to assess how the mechanical properties of collagen, proteoglycans, and fluid in articular cartilage change in early osteoarthritis, and how site-specific these changes are.Unilateral ACLT was performed in eight skeletally mature, female New Zealand white rabbits. A separate control (CTRL) group consisted of knee joints of five non-operated rabbits. Animals were sacrificed at four weeks after ACLT and cartilage-on-bone samples from femoral groove, medial and lateral femoral condyles, and tibial plateaus were harvested. A stress–relaxation protocol in indentation geometry was applied and the FRPE model was fitted to the experimental force–time curve by minimizing the mean absolute error between experiment and simulation. The optimized parameters were: fibril network modulus (Ef), representing the collagen network; non-fibrillar matrix modulus (Enf), representing the PG matrix; and permeability (k), representing fluid flow.Permeability was increased significantly in the ACLT group compared to the CTRL group knees at all sites except for the medial tibial plateau. ACLT also caused a decrease in the Ef at all sites except for the medial and lateral tibial plateaus. The Enf of the ACLT group knees was altered only for the lateral femoral condyle.The results of this study suggest that early osteoarthritis primarily affects cartilage permeability and impairs the collagen network stiffness in a site-specific manner. These findings from early osteoarthritis indicate that fluid flow velocity in articular cartilage may change prior to quantifiable structural alterations in the tissue.

A three-dimensional computational model of collagen network mechanics.

Extracellular matrix (ECM) strongly influences cellular behaviors, including cell proliferation, adhesion, and particularly migration. In cancer, the rigidity of the stromal collagen environment is thought to control tumor aggressiveness, and collagen alignment has been linked to tumor cell invasion. While the mechanical properties of collagen at both the single fiber scale and the bulk gel scale are quite well studied, how the fiber network responds to local stress or deformation, both structurally and mechanically, is poorly understood. This intermediate scale knowledge is important to understanding cell-ECM interactions and is the focus of this study. We have developed a three-dimensional elastic collagen fiber network model (bead-and-spring model) and studied fiber network behaviors for various biophysical conditions: collagen density, crosslinker strength, crosslinker density, and fiber orientation (random vs. prealigned). We found the best-fit crosslinker parameter values using shear simulation tests in a small strain region. Using this calibrated collagen model, we simulated both shear and tensile tests in a large linear strain region for different network geometry conditions. The results suggest that network geometry is a key determinant of the mechanical properties of the fiber network. We further demonstrated how the fiber network structure and mechanics evolves with a local formation, mimicking the effect of pulling by a pseudopod during cell migration. Our computational fiber network model is a step toward a full biomechanical model of cellular behaviors in various ECM conditions.

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.

Elastic properties of collagen in bone determined by measuring the Debye–Waller factor

Force constant values for thermal vibrational motion of a collagen molecule along the helix axis in tendon, completely demineralized bone (CDB), and partially demineralized bone (PDB) were estimated by determining the Debye–Waller factor (DW factor) for the diffracted X-ray intensity from these specimens. The DW factor for nominal value of 0.286nm meridional diffraction representing a period along the helical axis of a collagen molecule was measured. As the atomic scattering factor of mineral constituents is much larger than that of collagen, it is difficult to detect the diffraction from collagen in bone specimen. Therefore, PDB was used in this study. In order to compare obtained force constant value for CDB with mechanical properties of collagen in the literature, the value was translated into Young's modulus value using the cross-sectional area of a collagen molecule. In the case of collagen in PDB, i.e., collagen with the close presence of HAp mineral particles, as the DW factor of the diffracted intensity by hydroxyapatite (HAp) was considered to be negligible compared with that of collagen, the DW factor determined was interpreted as that of collagen molecule in PDB specimen. The force constant value obtained for collagen in PDB was significantly larger than that of collagen in CDB. This result was thought to be a manifestation of the hardening of collagen matrix in bone by HAp mineral particles and the first straightforward evidence for a difference in collagen properties depending on the presence of HAp mineral particles. The method employed in this study can be utilized for detecting mechanical properties of the individual constituents of composite materials.

Effects of tissue hydration on nanoscale structural morphology and mechanics of individual Type I collagen fibrils in the Brtl mouse model of Osteogenesis Imperfecta

Type I collagen is the most abundant protein in mammals, and is a vital part of the extracellular matrix for numerous tissues. Despite collagen's importance, little is known about its nanoscale morphology in tissues and how morphology relates to mechanical function. This study probes nanoscale structure and mechanical properties of collagen as a function of disease in native hydrated tendons. Wild type tendon and tendon from the Brtl/+ mouse model of Osteogenesis Imperfecta were investigated. An atomic force microscope (AFM) was used to image and indent minimally-processed collagen fibrils in hydrated and dehydrated conditions. AFM was used because of the ability to keep biological tissues as close to their native in situ conditions as possible. The study demonstrated phenotypic difference in Brtl/+ fibril morphology and mechanics in hydrated tendon which became more compelling upon dehydration. Dried tendons had a significant downward shift in fibril D-periodic spacing versus a shift up in wet tendons. Nanoscale changes in morphology in dry samples were accompanied by significant increases in modulus and adhesion force and decreased indentation depth. A minimal mechanical phenotype existed in hydrated samples, possibly due to water masking structural defects within the diseased fibrils. This study demonstrates that collagen nanoscale morphology and mechanics are impacted in Brtl/+ tendons, and that the phenotype can be modulated by the presence or absence of water. Dehydration causes artifacts in biological samples which require water and this factor must be considered for studies at any length scale in collagen-based tissues, especially when characterizing disease-induced differences.

Micromechanical analysis of native and cross-linked collagen type I fibrils supports the existence of microfibrils

The mechanical properties of individual collagen fibrils of approximately 200 nm in diameter were determined using a slightly adapted AFM system. Single collagen fibrils immersed in PBS buffer were attached between an AFM cantilever and a glass surface to perform tensile tests at different strain rates and stress relaxation measurements. The stress–strain behavior of collagen fibrils immersed in PBS buffer comprises a toe region up to a stress of 5 MPa, followed by the heel and linear region at higher stresses. Hysteresis and strain-rate dependent stress–strain behavior of collagen fibrils were observed, which suggest that single collagen fibrils have viscoelastic properties. The stress relaxation process of individual collagen fibrils could be best fitted using a two-term Prony series. Furthermore, the influence of different cross-linking agents on the mechanical properties of single collagen fibrils was investigated. Based on these results, we propose that sliding of microfibrils with respect to each other plays a role in the viscoelastic behavior of collagen fibrils in addition to the sliding of collagen molecules with respect to each other. Our finding provides a better insight into the relationship between the structure and mechanical properties of collagen and the micro-mechanical behavior of tissues.

Structural and mechanical profiles of native collagen fibers in vaginal wall connective tissues

Collagen, an ubiquitous biomaterial, confers robustness and resilience to connective tissues. In this study, we analyzed the structure and elasticity profile of collagen from the vaginal wall connective tissue of healthy pre-menopausal (pre-M) and postmenopausal (post-M) women. The histological staining assisted study with an atomic force microscope renders the examination of native collagen fibers on site of the connective tissue from nanoscopic scale to microscopic scale with high spatial resolution. Our results suggest that during menopause, collagen’s structure and elasticity are subject to changes at all levels of organization- between individual collagen fibers, between collagen and muscle, and between collagen and other matrix elements. The systematic analysis of the native structure and mechanical properties of collagen within a tissue provides a potential way to study non-fatal conditions such as pelvic organ prolapse and other genito-urinary disorders, where the initial symptoms are subtle and multivariate, and where early detection of patient’s condition may allow better non-invasive interventions and reduce the number of women undergoing surgical correction of these common disorders.

Effect of Liquid/Powder Ratio on the Setting, Handling and Mechanical Properties of Collagen–Apatitic Cements

Due to their insufficient mechanical stability and brittle nature, calcium phosphate cements (CPCs) have not been used for the treatment of vertebral fractures. Mechanical stability of human bone is provided by a complex interaction of type I collagen fibres and hydroxyapatite crystals. In the present study, fibre reinforcement of an apatitic calcium phosphate prepared at different liquid/powder ratio (LPR) was investigated. Different lengths of type I collagen fibres sourced from bovine Achilles tendon were used. Compressive strength and fracture behaviour were examined. Fibre addition of up to 5 wt.% had a significant influence on the compressive properties of the CPC. The mechanism of fibre reinforcement appeared to be crack bridging. Setting time and injectability of the CPC with fibre reinforcement was also investigated and decreased with fibre volume fraction. Increasing the LPR, improved the injectability and delayed the setting reaction. However, the compressive properties of the hardened cement were reduced as a consequence.

Preparation and Characterization of PVA-Collagen Composite Fibers

The collagen was blended with polyvinyl alcohol (PVA) with the maximum maintenance of the natural structure as precondition. The apparent viscosity and rheology property of PVA-collagen blended solution were studied. the mechanical properties of the blend membrane formed from PVA-collagen blended solution were also determined. The PVA-collagen blended solution was wet spinned with the sodium sulfate as coagulant to prepare PVA-collagen composite fibers. SEM analysis and X-ray diffraction analysis of the PVA-collagen composite fibers were studied. The results indicated that, blended with PVA, the spinning property and mechanical properties of collagen were improved. The figure of the aim fiber transect structure was similar as the kidney, and it had a uniform size. The crystallization degree of the fiber was 55.7%, and it was increased with the increase of the hot extending temperature and the extending ratio.

Chemical and thermal cross-linking of collagen and elastin hydrolysates

Chemical and thermal cross-linking of collagen soluble in acetic acid and elastin hydrolysates soluble in water have been studied. Solutions of collagen and elastin hydrolysates were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Films made of collagen and elastin hydrolysates were also treated with temperature at 60 °C and 100 °C to get additional cross-links. The effect of cross-linking has been studied using FTIR spectroscopy, thermal analysis, AFM and SEM microscopy. Mechanical and surface properties of materials have been studied after cross-linking. It was found that thermal and mechanical properties of collagen and elastin materials have been altered after thermal treatment and after the reactions with EDC/NHS and/or DEPE. Surface properties of collagen materials after chemical cross-linking have been modified. Thermal and chemical cross-linking of collagen films lead to alteration of polarity of the surface.