Mechanical Properties Of Collagen Gels Research Articles

Effect of Phytic Acid Addition on the Structure of Collagen-Hyaluronic Acid Composite Gel.

Composite collagen gels with hyaluronic acid are developed tissue-engineered structures for filling and regeneration of defects in various organs and tissues. For the first time, phytic acid was used to increase the stability and improve the mechanical properties of collagen gels with hyaluronic acid. Phytic acid is a promising cross-linker for collagen hydrogels and is a plant-derived antioxidant found in rich sources of beans, grains, and oilseeds. Phytic acid has several benefits due to its antioxidant, anticancer, and antitumor properties. In this work, studies were carried out on the kinetics of the self-assembly of collagen molecules in the presence of phytic and hyaluronic acids. It was shown that both of these acids do not lead to collagen self-assembly. Scanning electron microscopy showed that in the presence of phytic and hyaluronic acids, the collagen fibrils had a native structure, and the FTIR method confirmed the chemical cross-links between the collagen fibrils. DSC and rheological studies demonstrated that adding the phytic acid improved the stability and modulus of elasticity of the collagen gel. The presence of hyaluronic acid in the collagen gel slightly reduced the effect of phytic acid. The presence of phytic acid in the collagen gel improved the stability of the scaffold, but, after 1 week of cultivation, slightly reduced the viability of mesenchymal stromal cells cultured in the gel. The collagen type I gel with hyaluronic and phytic acids can be used to replace tissue defects, especially after the removal of cancerous tumors.

Controlling collagen gelation pH to enhance biochemical, structural, and biomechanical properties of tissue-engineered menisci.

Collagen-based hydrogels have been widely used in biomedical applications due to their biocompatibility. Enhancing mechanical properties of collagen gels remains challenging while maintaining biocompatibility. Here, we demonstrate that gelation pH has profound effects on cellular activity, collagen fibril structure, and mechanical properties of the fibrochondrocyte-seeded collagen gels in both short- and long-terms. Acidic and basic gelation pH, below pH7.0 and above 8.5, resulted in dramatic cell death. Gelation pH ranging from 7.0 to 8.5 showed a relatively high cell viability. Furthermore, physiologic gelation (pH7.5) showed the greatest collagen deposition while glycosaminoglycan deposition appeared independent of gelation pH. Scanning electron microscopy showed that neutral and physiologic gelation pH, 7.0 and 7.5, exhibited well-aligned collagen fibril structure on day 0 and enhanced collagen fibril structure with laterally joined fibrils on day 30. However, basic pH, 8.0 and 8.5, displayed a densely packed collagen fibril structure on day 0, which was also persistent on day 30. Initial equilibrium modulus increased with increasing gelation pH. Notably, after 30 days of culture, gelation pH of 7.5 and 8.0 showed the highest equilibrium modulus, reaching 150 -160 kPa. While controlling gelation pH is simply achieved compared with other strategies to improve mechanical properties, its influences on biochemical and biomechanical properties of the collagen gel are long-lasting. As such, gelation pH is a useful means to modulate both biochemical and biomechanical properties of the collagen-based hydrogels and can be utilized for diverse types of tissue engineering due to its simple application.

Effect of hyaluronic acid on microscale deformations of collagen gels

As fibrous collagen is the most abundant protein in mammalian tissues, gels of collagen fibers have been extensively used as an extracellular matrix scaffold to study how cells sense and respond to cues from their microenvironment. Other components of native tissues, such as glycosaminoglycans like hyaluronic acid, can affect cell behavior in part by changing the mechanical properties of the collagen gel. Prior studies have quantified the effects of hyaluronic acid on the mechanical properties of collagen gels in experiments of uniform shear or compression at the macroscale. However, there remains a lack of experimental studies of how hyaluronic acid changes the mechanical properties of collagen gels at the scale of a cell. Here, we studied how addition of hyaluronic acid to gels of collagen fibers affects the local field of displacements in response to contractile loads applied on length scales similar to those of a contracting cell. Using spherical poly(N-isopropylacrylamide) particles, which contract when heated, we induced displacement in gels of collagen and collagen with hyaluronic acid. Displacement fields were quantified using a combination of confocal microscopy and digital image correlation. Results showed that hyaluronic acid suppressed the distance over which displacements propagated, suggesting that it caused the network to become more linear. Additionally, hyaluronic acid had no statistical effect on heterogeneity of the displacement fields, but it did make the gels more elastic by substantially reducing the magnitude of permanent deformations. Lastly, we examined the effect of hyaluronic acid on fiber remodeling due to localized forces and found that hyaluronic acid partially – but not fully – inhibited remodeling. This result is consistent with prior studies suggesting that fiber remodeling is associated with a phase transition resulting from an instability caused by nonlinearity of the collagen gel.

Fiber stiffness, pore size and adhesion control migratory phenotype of MDA-MB-231 cells in collagen gels.

Cancer cell migration is influenced by cellular phenotype and behavior as well as by the mechanical and chemical properties of the environment. Furthermore, many cancer cells show plasticity of their phenotype and adapt it to the properties of the environment. Here, we study the influence of fiber stiffness, confinement, and adhesion properties on cancer cell migration in porous collagen gels. Collagen gels with soft fibers abrogate migration and promote a round, non-invasive phenotype. Stiffer collagen fibers are inherently more adhesive and lead to the existence of an adhesive phenotype and in general confined migration due to adhesion. Addition of TGF-β lowers adhesion, eliminates the adhesive phenotype and increases the amount of highly motile amoeboid phenotypes. Highest migration speeds and longest displacements are achieved in stiff collagen fibers in pores of about cell size by amoeboid phenotypes. This elucidates the influence of the mechanical properties of collagen gels on phenotype and subsequently migration and shows that stiff fibers, cell sized pores, and low adhesion, are optimal conditions for an amoeboid phenotype and efficient migration.

Development and mechanical characterisation of self-compressed collagen gels

Collagen gels are considered a promising biomaterial for the manufacturing of tissue engineering scaffolds, however, their mechanical properties often need to be improved to enable them to provide enough mechanical support during the course of tissue regeneration process. In this paper, we present a simple self-compression technique for the improvement of the mechanical properties of collagen gels, identified by the fitting of bespoke biphasic finite element models. Radially-confined highly hydrated gels were allowed to self-compress for 18h, expelling fluid, and which were subsequently subjected to unconfined ramp-hold compression. Gels, initially of 0.2%, 0.3% and 0.4% (w/v) collagen and 13mm thickness, transformed to 2.9±0.2%, 3.2±0.3% and 3.6±0.1% (w/w) collagen and 0.45±0.06mm, 0.69±0.04mm and 0.99±0.07mm thickness. Young's moduli of the compressed gels did not increase with increasing collagen fibril density, whilst zero-strain hydraulic permeability significantly decreased from 51 to 21mm4/Ns. The work demonstrates that biphasic theory, applied to unconfined compression, is a highly appropriate paradigm to mechanically characterise concentrated collagen gels and that confined compression of highly hydrated gels should be further investigated to enhance gel mechanical performance.

Multiscale Measurements of the Mechanical Properties of Collagen Matrix

The underlying mechanisms by which extracellular matrix (ECM) mechanics influences cell and tissue function remain to be elucidated because the events associated with this process span size scales from tissue to molecular level. Furthermore, ECM has an extremely complex hierarchical 3D structure and the load distribution is highly dependent on the architecture and mechanical properties of ECM. In the present study, the macro- and microscale mechanical properties of collagen gel were studied. Dynamic rheological testing was performed to study the macroscale mechanical properties of collagen gel. The microscale mechanical properties of collagen gel were measured using optical magnetic twisting cytometry (OMTC). Ferromagnetic beads embedded in the matrix were used as mechanical probes. Our study on the multiscale mechanical properties of collage matrix suggests several interesting differences between macro and microscale mechanical properties originated from the scales of measurements. At the macroscopic scale, storage and loss modulus increase with collagen concentrations. Nonaffine collagen fibril structural network deformation plays an important role in determining the macroscopic mechanical properties of the collagen matrix. At the microscopic scale, however, the local mechanical properties are less sensitive to changes in collagen concentration because of the more immediate/direct deformation of collagen fibrils in the OMTC measurements through forces exerted by locally attached ferromagnetic beads. The loss modulus is more affected by the local interstitial fluid environment, leading to a rather dramatic increase in viscosity with frequency, especially at higher frequencies (>10 Hz). A finite element model was developed to study the geometric factors in the OMTC measurements when the collagen matrix was considered to be hyperelastic. Our results show that the geometric factors are dependent on collagen concentration, or the stiffness of matrix, when nonlinear material properties of the matrix are considered, and thus interpretation of the apparent modulus from OMTC measurements should be conducted carefully.

In situ gelation properties of a collagen–genipin sol with a potential for the treatment of gastrointestinal ulcers

We investigated the potential of collagen–genipin sols as biomaterials for treating artificial ulcers following endoscopic submucosal dissection. Collagen sol viscosity increased with condensation, allowing retention on tilted ulcers before gelation and resulting in collagen gel deposition on whole ulcers. The 1.44% collagen sols containing genipin as a crosslinker retained sol fluidity at 23°C for >20 min, facilitating endoscopic use. Collagen sols formed gel depositions on artificial ulcers in response to body temperature, and high temperature responsiveness of gelation because of increased neutral phosphate buffer concentration allowed for thick gel deposition on tilted ulcers. Finally, histological observations showed infiltration of gels into submucosal layers. Taken together, the present data show that genipin-induced crosslinking significantly improves the mechanical properties of collagen gels even at low genipin concentrations of 0.2–1 mM, warranting the use of in situ gelling collagen–genipin sols for endoscopic treatments of gastrointestinal ulcers.

Mathematical modeling of uniaxial mechanical properties of collagen gel scaffolds for vascular tissue engineering.

Small diameter tissue-engineered arteries improve their mechanical and functional properties when they are mechanically stimulated. Applying a suitable stress and/or strain with or without a cycle to the scaffolds and cells during the culturing process resides in our ability to generate a suitable mechanical model. Collagen gel is one of the most used scaffolds in vascular tissue engineering, mainly because it is the principal constituent of the extracellular matrix for vascular cells in human. The mechanical modeling of such a material is not a trivial task, mainly for its viscoelastic nature. Computational and experimental methods for developing a suitable model for collagen gels are of primary importance for the field. In this research, we focused on mechanical properties of collagen gels under unconfined compression. First, mechanical viscoelastic models are discussed and framed in the control system theory. Second, models are fitted using system identification. Several models are evaluated and two nonlinear models are proposed: Mooney-Rivlin inspired and Hammerstein models. The results suggest that Mooney-Rivlin and Hammerstein models succeed in describing the mechanical behavior of collagen gels for cyclic tests on scaffolds (with best fitting parameters 58.3% and 75.8%, resp.). When Akaike criterion is used, the best is the Mooney-Rivlin inspired model.

Migration, Force Generation and Mechanosensing of Cells in Collagen Gels

Collagen gels are frequently used to study cell migration in a three-dimensional environment. Mechanical properties of collagen gels are governed by non-affine deformation of the collagen fibrils, such as buckling and tautening, resulting at the macroscopic scale in strain stiffening under shear and a strong lateral contraction under stretch. It is currently unknown how these macroscopic properties play out at the scale of a migrating cell, and how this depends on the cell geometry. To explore this question, we develop a non-linear elastic material model for collagen gels based on observations from confocal microscopy that fibrils can evade mechanical stress using their internal degrees of freedom. This non-affine behavior results in a non-linear force length relationship of fibril segments and leads to a macroscopic strain stiffening and lateral contraction. In particular, we show that tautening of fibrils results in a strong material stiffening against expanding forces, e.g. from a migrating cell with a diameter larger than the network pore diameter. By this mechanism, even a soft collagen gel can sterically constrain a migrating cell. Using our material model, we compute cell traction forces, induced stresses as well as material stiffening, from collagen fiber displacements during the migration of MDA-MB 231 breast carcinoma cells through dilute (0.6 mg/ml, Young's modulus 85 Pa) and dense (2.4 mg/ml, Young's modulus 1100 Pa) collagen gels. We find that cells exert highly localized forces onto the matrix, leading to a localized ∼2-fold material stiffening. However, the average traction force magnitude increases with collagen concentration by ∼3-fold, from 40nN in dilute gels to 120nN in dense gels. This observation may help explain why cells can migrate more efficiently in stiffer gels, despite their narrower pore diameter.

Ultrasonic Setup for Testing Hydrogels: Preliminary Experiments on Collagen Gels

The assessment of mechanical properties of highly hydrated natural materials remains a challenge because, in general, their mechanical evaluation implies invasive and finally destructive methods. Acoustic-based tests may represent the appropriate tools to investigate the mechanical properties of such materials, particularly collagen gels, whose acoustic properties are poorly understood. The objective of this work is to develop two experimental setups for the assessment of acoustic properties of such a hydrogels. In the first one, a typical pulse echo reflectometer was implemented. The acoustic parameters were measured at controlled temperature in an especially designed chamber. In the second one, the previous configuration was combined with a setup for compressive tests, allowing to interrogate simultaneously both the acoustic and mechanical properties of the sample under test. The frequency of the acoustic transducer was 10MHz. The acoustic and mechanical properties of collagen gels prepared according to different experimental conditions (pH and collagen concentration) were evaluated. The first set of experiment was useful to accomplish estimation of the speed of sound, attenuation and acoustic impedance. The second one allowed us to monitor the speed of sound during the evolution of the compression test. This approach could be a potential tool to study the changes in hydrogels mass density and bulk compressibility.

Why Mechanical Properties of Collagen Scaffolds Should Be Tested in a Pseudo-Physiological Environment?

Collagen gels constitute an adequate scaffold for supporting the adhesion, proliferation and tissue regeneration of vascular cells inside a bioreactor. However, their mechanical properties should be enhanced not only for their manipulation but also to resist the mechanical constraints applied in the bioreactor. Actually, assessing the mechanical properties of a hydrogel requires many precautions since they are very sensitive to the environmental conditions (temperature, ionic strength, aqueous environment, etc). Whereas mechanical properties are usually measured directly in the air, the aim of this work was to evaluate the effects of a pseudo-physiological environment (PPE) on the mechanical properties of collagen gels. Furthermore, reinforcement was also tested using UV treatments (λ = 254 nm, 20 J/cm2), known to induce crosslinking. Irradiated samples were more resistant to enzymatic degradation and swelling tests showed that the crosslink density was increased by a factor of 30. This increase was thereafter correlated to the mechanical properties. Results showed that the UV-treated samples were stiffer and more brittle than the non-treated ones when tested in air. However, a 20% decrease and 40% increase were respectively measured on the linear modulus and strain at rupture when the gels were tested in the PPE. In the perspective of vascular tissue regeneration, these results show that the mechanical properties of a hydrogel should be performed in PPE in order to take into account the plasticization phenomenon that will occur in a bioreactor.

Fibroblast contractility and growth in plastic compressed collagen gel scaffolds with microstructures correlated with hydraulic permeability

Scaffold microstructure is hypothesized to influence physical and mechanical properties of collagen gels, as well as cell function within the matrix. Plastic compression under increasing load was conducted to produce scaffolds with increasing collagen fibrillar densities ranging from 0.3 to above 4.1 wt % with corresponding hydraulic permeability (k) values that ranged from 1.05 to 0.03 μm², as determined using the Happel model. Scanning electron microscopy revealed that increasing the level of collagen gel compression yielded a concomitant reduction in pore size distribution and a slight increase in average fibril bundle diameter. Decreasing k delayed the onset of contraction and significantly reduced both the total extent and the maximum rate of contraction induced by NIH3T3 fibroblasts seeded at a density of either 6.0 x 10⁴ or 1.5 x 10⁵ cells mL⁻¹. At the higher cell density, however, the effect of k reduction on collagen gel contraction was overcome by an accelerated onset of contraction which led to an increase in both the total extent and the maximum rate of contraction. AlamarBlue™ measurements indicated that the metabolic activity of fibroblasts within collagen gels increased as k decreased. Moreover, increasing seeded cell density from 2.0 x 10⁴ to 1.5 x 10⁵ cells mL⁻¹ significantly increased NIH3T3 proliferation. In conclusion, fibroblast-matrix interactions can be optimized by defining the microstructural properties of collagen scaffolds through k adjustment which in turn, is dependent on the cell seeding density.

Tailoring Mechanical Properties of Collagen-Based Scaffolds for Vascular Tissue Engineering: The Effects of pH, Temperature and Ionic Strength on Gelation

Collagen gels have been widely studied for applications in tissue engineering because of their biological implications. Considering their use as scaffolds for vascular tissue engineering, the main limitation has always been related to their low mechanical properties. During the process of in vitro self-assembly, which leads to collagen gelation, the size of the fibrils, their chemical interactions, as well as the resulting microstructure are regulated by three main experimental conditions: pH, ionic strength and temperature. In this work, these three parameters were modulated in order to increase the mechanical properties of collagen gels. The effects on the gelation process were assessed by turbidimetric and scanning electron microscopy analyses. Turbidity measurements showed that gelation was affected by all three factors and scanning electron images confirmed that major changes occurred at the microstructural level. Mechanical tests showed that the compressive and tensile moduli increased by four- and three-fold, respectively, compared to the control. Finally, viability tests confirmed that these gels are suitable as scaffolds for cellular adhesion and proliferation.

Characterization of type I collagen gels modified by glycation

Chronic exposure to reducing sugars due to diabetes, aging, and diet can permanently modify extracellular matrix (ECM) proteins. This non-enzymatic glycosylation, or glycation, can lead to the formation of advanced glycation end products (AGE) and crosslinking of the ECM. This study investigates the effects of glycation on the properties of type I collagen gels. Incubation with glucose-6-phopshate (G6P), a reducing sugar that exhibits similar but more rapid glycation than glucose, modified the biological and mechanical properties of collagen gels. Measures of AGE formation that correlate with increased complications in people with diabetes, including collagen autofluorescence, crosslinking, and resistance to proteolytic degradation, increased with G6P concentration. Rheology studies showed that AGE crosslinking increased the shear storage and loss moduli of type I collagen gels. Fibroblasts cultured on glycated collagen gels proliferated more rapidly than on unmodified gels, but glycated collagen decreased fibroblast invasion. These results show that incubation of type I collagen gels with G6P increases clinically relevant measures of AGE formation and that these changes altered cellular interactions. These gels could be used as in vitro models to study ECM changes that occur in diabetes and aging.

Effects of the Mechanical Properties of Collagen Gel on the In Vitro Formation of Microvessel Networks by Endothelial Cells

Vascularization by endothelial cells (ECs) is an essential element in tissue-engineering of organoids. Morphogenesis of these cells is regulated not only by the biochemical properties of the extracellular matrix (ECM) but also by its mechanical properties. Here, we investigated the effect of substrate mechanical properties on the formation of capillary-like networks by ECs; in particular, we examined the three-dimensional (3D) configurations of the resulting networks. Bovine pulmonary microvascular ECs (BPMECs) were cultured on a series of collagen gels of different stiffness but the same collagen concentration. Imaging techniques revealed that cells cultured in rigid and flexible gels formed 3D networks via different processes; cells formed dense, thin networks in the flexible gel, whereas thicker and deeper networks were formed in the rigid gel. Cross-sections of the networks revealed that those formed within the rigid gel had large lumens composed of multiple cells, whereas those formed within the flexible gel had small, intracellular vacuoles. The expression of vinculin, a focal adhesion protein, appeared to change with the mechanical properties of collagen gel. Our results indicate that the mechanical properties of adhesion substrates play an important role in regulating 3D network formation.

Mechanical properties of collagen gels derived from rats of different ages

In a previous study, we found that different collagen gels produced using collagen fibrils extracted from 1-, 4- and 8-month-old rat tails essentially influenced the morphogenesis of epithelial cells. More importantly, the youngest collagen gel induces the highest level of cell apoptosis. The objective of this study was to investigate mechanical properties of various collagen gels correlated to the rat ages. A rheometer and dynamic mechanical analyzer were used to measure shear and compressive properties of hydrated collagen gels. Experimental results obtained from both testing modes showed that older age-related collagen gels possessed a larger elastic modulus, possibly due to the enhanced cross-linking degree. The moduli obtained in shear mode were 1.4–2.7-times greater than those in compression. The results of shear test and compressive test consistently indicated the age of rats did have a statistically significant effect on mechanical properties of hydrated collagen gels.

Photo-cross-linking of type I collagen gels in the presence of smooth muscle cells: mechanical properties, cell viability, and function.

The effectiveness of photomediated cross-linking of type I collagen gels in the presence of rat aortic smooth muscle cells (RASMC) as a method to enhance gel mechanical properties while retaining native collagen triple helical structure and maintaining high cell viability was investigated. Collagen was chemically modified to incorporate an acrylate moiety. Collagen methacrylamide was cast into gels in the presence of a photoinitiator along with RASMC. The gels were cross-linked using visible light irradiation. Neither acrylate modification nor the cross-linking reaction altered collagen triple helical content. The cross-linking reaction, however, moved the denaturation temperature beyond the physiologic range. A twelve-fold increase in shear modulus was observed after cross-linking. Cell viability in the range of 70% (n = 4, p > 0.05) was observed in the photo-cross-linked gels. Moreover the cells were able to contract the cross-linked gel in a manner commensurate with that observed for natural type I collagen. Methacrylate-mediated photo-cross-linking is a facile route to improve mechanical properties of collagen gels in the presence of cells while maintaining high cell viability. This enhances the potential for type I collagen gels to be used as scaffolds for tissue engineering.

Influence of synthetic polymers on neutrophil migration in three-dimensional collagen gels.

In vitro studies of cell migration within three-dimensional polymeric materials are essential for understanding cell behavior and for developing new biomedical materials. Human neutrophil motility was examined in hydrated collagen gels containing various synthetic polymers. Physical mixtures of collagen and certain water-soluble polymers formed stable gels that were good substrates for cell migration. Addition of either polyethylene glycol (PEG) or the pluronictrade mark copolymer F68 did not change the morphological or mechanical properties of collagen gels, as determined by SEM and oscillatory rheometry; however, addition of either polymer significantly inhibited cell motility in both a modified 96-well chemotaxis chamber assay and a direct visual assay. Although the mechanism for this observed polymer inhibition of neutrophil migration is not yet clear, these results suggest that PEG and F68, two widely used biomedical polymers that are considered to be relatively "inert," may cause significant inhibition of cell motility.