Collagen Gradients Research Articles

Novel collagen gradient membranes with multiphasic structures: Preparation, characterization, and biocompatibility

Scaffolds with multiphasic structures are considered to be superior for guided tissue regeneration. Two types of tilapia skin collagen gradient membranes (stepped gradient and linear gradient) with multiphasic structures were prepared by controlling the collagen concentrations and the freezing rates. The results revealed that collagen gradient membranes were more capable of guiding tissue regeneration compared to homogeneous membranes. These two gradient membranes featured a dense outer layer and a loose inner layer, with good mechanical properties as indicated by tensile strengths of more than 250 Kpa and porosities exceeding 85 %. Additionally, these membranes also showed good hydrophilicity and water absorption, with an inner layer contact angle of less than 91° and a water absorption ratio greater than 40 times. Furthermore, the multiphasic scaffolds were proved to be biocompatible by the acute toxicity assay, the intradermal irritation test and so on. Gradient membranes could effectively promote the adhesion and proliferation of fibroblasts and osteoblasts, through elevating the TGF-β/Smad signaling pathway by TGF-β and Smads, and activating the Wnt/β-catenin signaling pathway by LRP5 and β-catenin, similar to homogenous membranes. Therefore, collagen gradient membranes from tilapia skin show important application value in guiding tissue regeneration.

Mechanical Response of Neural Cells to Physiologically Relevant Stiffness Gradients.

Understanding the influence of the mechanical environment on neurite behavior is crucial in the development of peripheral nerve repair solutions, and could help tissue engineers to direct and guide regeneration. In this study, a new protocol to fabricate physiologically relevant hydrogel substrates with controlled mechanical cues is proposed. These hydrogels allow the analysis of the relative effects of both the absolute stiffness value and the local stiffness gradient on neural cell behavior, particularly for low stiffness values (1–2 kPa). NG108‐15 neural cell behavior is studied using well‐characterized collagen gradient substrates with stiffness values ranging from 1 to 10 kPa and gradient slopes of either 0.84 or 7.9 kPa mm−1. It is found that cell orientation is influenced by specific combinations of stiffness value and stiffness gradient. The results highlight the importance of considering the type of hydrogel as well as both the absolute value of the stiffness and the steepness of its gradient, thus introducing a new framework for the development of tissue engineered scaffolds and the study of substrate stiffness.

Quantitative second harmonic generation imaging of leporine, canine, and porcine vocal fold collagen.

Vocal fold collagen composition is an important determinant of material properties and mucosal wave propagation. Collagen alignment and straightness are quantitatively characterized by second harmonic generation (SHG) imaging. We examined leporine, canined and porcine vocal folds showing collagen composition variation that is species, location, and strain specific. Animal model. Leporine (n = 5), canine (n = 5), and porcine (n = 5) larynges were harvested and fixed in situ. Samples were transversely sectioned, and SHG images were collected for two inferior-superior sections along five anterior-posterior locations. Additional porcine samples were fixed and imaged under tensile strain (0%, 5%, 10%, 15%, 20%, n = 5 per group). Two-way repeated measures (RM) analysis of variance (ANOVA) tested for section and location differences in each species. Multiway RM-ANOVA tested for section, location, and strain differences in porcine samples. Alignment and straightness were higher inferiorly in the porcine (P = .0047, P = .002) and canine (P = .0011, P < .001) vocal folds, but not in leporine samples (P = .67652, P = .4831). There were significant interactions between elongation and superior-inferior section for both alignment (P = .0047) and straightness (P = .0371). Our results correspond well to findings in the literature that the inferior vocal fold lip is stiffer in porcine and canine larynges. The absence of a collagen gradient in the leporine vocal fold is notable because rabbits are less vocal animals, indicating the collagen gradient may be a result of voice use and an important consideration in model selection when extracellular matrix is of interest. Strain results were also consistent with the role of collagen in strain stiffening behavior of vocal fold tissue. NA Laryngoscope, 129:2549-2556, 2019.

Collagen density gradient on poly(epsilon-caprolactone) surface for the design of 3D printed osteochondral

Event Abstract Back to Event Collagen density gradient on poly(epsilon-caprolactone) surface for the design of 3D printed osteochondral Ugo D'Amora1, Matteo D'Este2, David Eglin2, Antonio Gloria1, Roberto De Santis1, Mauro Alini2 and Luigi Ambrosio1, 3 1 National Research Council of Italy, Institute of Polymers, Composites and Biomaterials, Italy 2 AO Research Institute Davos, Switzerland 3 National Research Council of Italy, Chemical Science & Materials Technology Department, Italy Introduction: The ability to engineer scaffolds which resemble the transition between tissues, like cartilage and bone, has yet to be achieved. Indeed, such constructs should present several continuous gradients in terms of geometry, stiffness and biochemical composition to mimic tissue organization. Although the introduction of fused deposition modeling (FDM) techniques allows tailoring of mechanical, mass transport and morphological properties, the possibility to additionally functionalize such structures by introducing biochemical signal gradients has not been fully explored. Thus, a two-step functionalization method in which collagen was covalently grafted to a polyester surface is presented. A continuous collagen gradient was created along the length of a polymer surface which was characterized for collagen content, topography and cell adhesion. Materials and Methods: Poly(ε-caprolactone) (PCL) surfaces were produced by a melting/molding technique. First, the PCL surfaces were modified by immersing the substrates in a 1,6-hexanediamine/isopropanol solution. An amino-group (NH2) density gradient was achieved along the length of the substrate via the controlled surface exposure into the reactive solution[1]. In the second step, lyophilized collagen type I was grafted using a carbodiimide reaction. Ninhydrin and hydroxyproline assays, contact angle measurements, confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FTIR), fast green and picrosirius red staining were performed to characterize the surfaces and gradients. Results and Discussion: The NH2 concentration varied between 5*10-7 mol/cm2 (15 min processing) and 15*10-7 mol/cm2 (45 min processing). (Fig. 1). Figure 1. NH2 concentration as function of the reaction times and of the position along the PCL surface. The PCL-NH2 substrates were treated with Rhodamine B Isothiocyanate to visualize the gradient by using CLSM. The fluorescence intensity decreased from 80 (a.u.) for the region treated for 45 minutes to 10 (a.u.) for the PCL zone. The region treated for 45 minutes showed a greater roughness, probably due to surface erosion of the material. (Fig. 2). Figure 2. Representative images of PCL-NH2-Rhod surfaces functionalized after right) 45; middle) 30; and left) 15 minutes of treatment. Static water contact angle decreased by about 15° whilst collagen density increased with increased NH2 concentration. The introduction of collagen improved the hydrophilicity of the surfaces. Microscopy imaging/analysis visualized the collagen distribution onto the substrates whilst FTIR confirmed collagen grafting and preservation of its helical structure. Conclusion: A two-step functionalization procedure protein was developed. A continuous collagen gradient along the length of a PCL substrate was confirmed by morphological and physico-chemical analysis. Future studies will apply the methodology to FDM PCL-based scaffolds for the repair/regeneration of focal osteochondral defects. The authors would like to thank FIRB MERIT project (GAE: P0000570) for providing financial support

Opposing Rigidity-Protein Gradients Reverse Fibroblast Durotaxis.

The migration of cells is a complex and dynamic process that is governed by several stimuli acting simultaneously. In vivo, cells receive and process a wide range of cues that guide their motion and migratory characteristics such as speed and directionality. The design of biomaterials that can recapitulate the combinatorial signaling environment can aid in understanding how migrating cells respond to more than one stimulus and when one cue dominates over the other. We have designed hydrogel substrates that exhibit opposing rigidity-and collagen gradients. Within the boundaries of the interfacial region, the values for substrate modulus decreased in one direction with a concomitant increase in the concentration of surface-bound collagen. The well-known durotactic migration of fibroblasts was first validated on substrates that only exhibit a gradient in modulus while keeping the concentration of surface-bound collagen constant. Upon increasing the collagen concentration on the low-modulus regions by 4- or 7-fold compared to the high-modulus side of the interface, cells exhibited directed migration toward the soft regions of the substrate. This effect was more pronounced when the surface-bound collagen concentration was 7-fold greater. Cell displacements, areas, cytoskeleton and focal adhesions were investigated on the opposing rigidity-immobilized collagen gradients. These features were affected by the elastic modulus of the substrate as well as the change in protein concentration. In the future, incorporating multiple gradients within a single substrate will lead to a deeper and more comprehensive understanding of cells navigate through the complex in vivo microenvironment.

Haptotaxis is cell type specific and limited by substrate adhesiveness.

Motile cells navigate through tissue by relying on tactile cues from gradients provided by extracellular matrix (ECM) such as ligand density or stiffness. Mesenchymal stem cells (MSCs) and fibroblasts encounter adhesive or 'haptotactic' gradients at the interface between healthy and fibrotic tissue as they migrate towards an injury site. Mimicking this phenomenon, we developed tunable RGD and collagen gradients in polyacrylamide hydrogels of physiologically relevant stiffness using density gradient multilayer polymerization (DGMP) to better understand how such ligand gradients regulate migratory behaviors. Independent of ligand composition and fiber deformation, haptotaxis was observed in mouse 3T3 fibroblasts. Human MSCs however, haptotaxed only when cell-substrate adhesion was indirectly reduced via addition of free soluble matrix ligand mimetic peptides. Under basal conditions, MSCs were more contractile than fibroblasts. However, the presence of soluble adhesive peptides reduced MSC-induced substrate deformations; increased contractility may contribute to limited migration, but modulating cytoskeletal assembly was ineffective at promoting MSC haptotaxis. When introduced to gradients of increased absolute ligand concentrations, 3T3s displayed increased contractility and no longer haptotaxed. These data suggest that haptotactic behaviors are limited by adhesion and that although both cell types may home to tissue to aid in repair, fibroblasts may be more responsive to ligand gradients than MSCs.

Regulation of endothelial cells migration on poly( d, l-lactic acid) films immobilized with collagen gradients

To investigate the effect of protein surface-density gradient on the motility of endothelial cells, we developed a novel approach for the fabrication of a collagen density gradient onto poly( d, l-lactic acid) (PDLLA) films in this study. The approach involves a sequential alkali hydrolysis of PDLLA films to produce a density gradient of –COOH moieties onto the films, which were activated and then covalently linked with collagen. A collagen surface-density gradient onto PDLLA films was thus generated by this approach. Contact angle measurement and confocal laser scanning microscopy (CLSM) were employed to confirm the formation of –COOH gradient and collagen gradient, respectively. All results proved the feasibility of the fabrication of a collagen density gradient onto PDLLA films via the approach. Endothelial cells cultured on the gradient areas with low and moderate collagen surface-densities displayed a strong motility tendency, with the values such as net displacement, total distance, chemotactic index, migration rate and cell trajectories in parallel to the gradient. However, endothelial cells grew on the gradient area with high collagen density demonstrated a reverse response to the collagen gradient clue. These results suggest that cell motility is regulated by the collagen gradient with a surface-density dependent manner. This study provides an alternative for the fabrication of protein surface-density gradient onto biodegradable substrates to investigate chemical stimuli induced cell directional motility. It is potentially important for understanding the controlled angiogenesis for implantation of tissue-engineered constructs.

Convection-driven generation of long-range material gradients

Natural materials exhibit anisotropy with variations in soluble factors, cell distribution, and matrix properties. The ability to recreate the heterogeneity of the natural materials is a major challenge for investigating cell–material interactions and for developing biomimetic materials. Here we present a generic fluidic approach using convection and alternating flow to rapidly generate multi-centimeter gradients of biomolecules, polymers, beads and cells and cross-gradients of two species in a microchannel. Accompanying theoretical estimates and simulations of gradient growth provide design criteria over a range of material properties. A poly(ethylene-glycol) hydrogel gradient, a porous collagen gradient and a composite material with a hyaluronic acid/gelatin cross-gradient were generated with continuous variations in material properties and in their ability to regulate cellular response. This simple yet generic fluidic platform should prove useful for creating anisotropic biomimetic materials and high-throughput platforms for investigating cell–microenvironment interactions.

The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen–receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.

Microscale control over collagen gradient on poly( l-lactide) membrane surface for manipulating chondrocyte distribution

A collagen gradient was constructed to interrogate cell adhesion on a poly( l-lactide) (PLLA) membrane surface. Utilizing a microinfusion pump, gradients of amino groups were generated on the PLLA surface by an aminolysis method. Immobilization of collagen onto the gradient surfaces was performed by glutaraldehyde (GA) coupling to form the collagen gradients. The –NH 2 and immobilized collagen density profiles on the PLLA membranes were quantitatively determined by ninhydrin and hydroproline (Hyp) analysis, respectively. By using fluorescein isothiocyanate (FITC) labeled collagen (FITC-Col), the profile of the as-prepared collagen gradient was directly monitored by a fluorescence microscope. The scanning force microscopy and water contact angle studies revealed that the morphology and wettability of the modified membranes changed progressively as a function of position along the gradient surface. Rabbit auricular chondrocytes were cultured on the collagen gradient membranes to test their cellular response. The attachment and spreading behaviors of the chondrocytes were dependent on the surface collagen density. These results indicate a surface on which the variation of collagen gradient strongly modifies the biological response of chondrocytes.

Pak1 kinase activity affects the character of cell morphology and movement

As described in the review by Bagrodia and Cerione1xBagrodia, S. and Cerione, R. Trends Cell Biol. 1999; 9: 350–355Abstract | Full Text | Full Text PDF | PubMed | Scopus (323)See all References1, inducible expression of different forms of Pak1 in NIH3T3 cells leads to changes in cell morphology and pattern of movement2xSells, M.A. et al. J. Cell Biol. 1999; 145: 837–849Crossref | PubMed | Scopus (291)See all References2. This figure shows wild-type cells (a), and cells overexpressing either an active form of Pak1 (b) or a kinase-dead form (c). The cells expressing constitutively active Pak1 have large, polarized lamellipodia at their leading edge and are more motile than the control cells (Fig. 1Fig. 1). Sells and colleagues also observed that these cells moved more rapidly in response to a collagen gradient when plated on a fibronectin surface. By contrast, the cells expressing the kinase-dead form of Pak1 had several lamellipodia projecting in different directions simultaneously. They were highly motile, but in a more random manner, and their ability to respond to the collagen gradient by directional movement was impaired. These movement differences correlated with changes in the organization of the actin cytoskeleton and are consistent with previous findings. Sells et al. also found that cells expressing the constitutively active form of Pak1 showed increased myosin light chain (MLC) phosphorylation and proposed that this is involved in the regulation of cell movement by Pak1. However, as described by Bagrodia and Cerione1xBagrodia, S. and Cerione, R. Trends Cell Biol. 1999; 9: 350–355Abstract | Full Text | Full Text PDF | PubMed | Scopus (323)See all References1, this does not agree with previous results in other cell types, where activated Pak reduced MLC phosphorylation. The influence of Pak, both direct and indirect, on MLC phosphorylation and the role of this modification in influencing cell movement both require further investigation.FIGURE 1Pak1 expression was induced in Pak1-overexpressing NIH 3T3 cells by removing tetracycline from the growth medium for 16 hours. The cells were then trypsinized and, after a 30-minute incubation on plates coated with bovine serum albumin, the cells were transferred to fibronectin-coated plates. Phase-contrast micrographs were taken 95 min later. The cells shown express: (a) empty vector (control cells), (b) Pak1L83,L86 (constitutively active) and (c) Pak1R299 (kinase dead). Bar, 50 μm.View Large Image | Download PowerPoint Slide

P21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts.

The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak's effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of Pak1 in regulating cell movement. We established clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which were not inhibited by coexpression of a dominant-negative form of Rac1. Cells inducibly expressing wild-type or constitutively-active Pak1 had large, polarized lamellipodia at the leading edge, were more motile than their normal counterparts when plated on a fibronectin-coated surface, and displayed enhanced directional movement in response to an immobilized collagen gradient. In contrast, cells expressing a kinase-dead form of Pak1 projected multiple lamellipodia emerging from different parts of the cell simultaneously. These cells, though highly motile, displayed reduced persistence of movement when plated on a fibronectin-coated surface and had defects in directed motility toward immobilized collagen. Expression of constitutively activated Pak1 was accompanied by increased myosin light chain (MLC) phosphorylation, whereas expression of kinase-dead Pak1 had no effect on MLC. These results suggest that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly affects cell movement.