On the effect of pepsin incubation on type I collagen from horse tendon: Fine tuning of its physico-chemical and rheological properties.

Type I collagen is commonly recognized as the gold standard biomaterial for the manufacturing of medical devices for health-care related applications. In recent years, with the final aim of developing scaffolds with optimal bioactivity, even more studies focused on the influence of processing parameters on collagen properties, since processing can strongly affect the architecture of collagen at various length scales and, consequently, scaffolds macroscopic performances. The ability to finely tune scaffold properties in order to closely mimic the tissues' hierarchical features, preserving collagen's natural conformation, is actually of great interest. In this work, the effect of the pepsin-based extraction step on the material final properties was investigated. Thus, the physico-chemical properties of fibrillar type I collagens upon being extracted under various conditions were analyzed in depth. Correlations of collagen structure at the supramolecular scale with its microstructural properties were done, confirming the possibility of tuning rheological, viscoelastic and degradation properties of fibrillar type I collagen.

Multiscale Label-Free Imaging of Fibrillar Collagen in the Tumor Microenvironment.

With recent advances in cancer therapeutics, there is a great need for improved imaging methods for characterizing cancer onset and progression in a quantitative and actionable way. Collagen, the most abundant extracellular matrix protein in the tumor microenvironment (and the body in general), plays a multifaceted role, both hindering and promoting cancer invasion and progression. Collagen deposition can defend the tumor with immunosuppressive effects, while aligned collagen fiber structures can enable tumor cell migration, aiding invasion and metastasis. Given the complex role of collagen fiber organization and topology, imaging has been a tool of choice to characterize these changes on multiple spatial scales, from the organ and tumor scale to cellular and subcellular level. Macroscale density already aids in the detection and diagnosis of solid cancers, but progress is being made to integrate finer microscale features into the process. Here we review imaging modalities ranging from optical methods of second harmonic generation (SHG), polarized light microscopy (PLM), and optical coherence tomography (OCT) to the medical imaging approaches of ultrasound and magnetic resonance imaging (MRI). These methods have enabled scientists and clinicians to better understand the impact collagen structure has on the tumor environment, at both the bulk scale (density) and microscale (fibrillar structure) levels. We focus on imaging methods with the potential to both examine the collagen structure in as natural a state as possible and still be clinically amenable, with an emphasis on label-free strategies, exploiting intrinsic optical properties of collagen fibers.

Segment-Long-Spacing (SLS) and the Polymorphic Structures of Fibrillar Collagen.

The diverse and complex functions of collagen during the development of an organism are closely related to the polymorphism of its supramolecular structures in the extracellular matrix. SLS (segment-long-spacing) is one of the best understood alternative structures of collagen. SLS played an instrumental role in the original studies of collagen more than half a century ago that laid the foundation of nearly everything we know about collagen today. Despite being used mostly under in vitro conditions, the natural occurrence of SLS in tissues has also been reported. Here we will provide a brief overview of the major findings of the SLS and other structures of collagen based on a wealth of work published starting from the 1940s. We will discuss the factors that determine the stability and the structural specificity of the different molecular assemblies of collagen in light of the new studies using designed fibril forming collagen peptides. At the end of the chapter, we will summarize some recent discoveries of the alternative structures of collagen in tissues, especially those involved in pathogenic states. A revisit of SLS will likely inspire new understandings concerning the range of critical roles of fibrillar collagen in terms of its organizational diversity in the extracellular matrix.

Molecular Basis of Pathogenic Variants in the Fibrillar Collagens.

The fibrillar collagen family is comprised of the quantitatively major types I, II and III collagens and the quantitatively minor types V and XI. These form heterotypic collagen fibrils (composed of more than a single collagen type) where the minor collagens have a regulatory role in controlling fibril formation and diameter. The structural pre-requisites for normal collagen biosynthesis and fibrillogenesis result in many places where this process can be disrupted, and consequently a wide variety of phenotypes result when pathogenic changes occur in these fibrillar collagen genes. Another contributing factor is alternative splicing, both naturally occurring and as the result of pathogenic DNA alterations. This article will discuss how these factors should be taken into account when assessing DNA sequencing results from a patient.

Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.

Developing exquisite collagen fibrillar assemblies in the presence of keratin nanoparticles for improved cellular affinity.

Recently, the collagen-keratin (CK) composites have received much attention for the purpose of biomedical applications due to the intrinsic biocompatibility and biodegradability of these two proteins. However, few studies have reported the CK composites developed by the self-assembly approach and the influence of the keratin on the collagen self-assembly in vitro was still unknown. In this study, the keratin nanoparticles (KNPs) were successfully prepared by the reduction method, and we focused on investigating the effect of the varying concentrations of KNPs on the mechanism of the fibrillogenesis process of collagen. The intermolecular interaction between the two proteins revealed by the ultraviolet spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy and circular dichromatic (CD) spectroscopy showed that KNPs would interact with the collagen, and keratin significantly influenced the hydrogen bonding interaction existed in collagen molecules. The SEM images exhibited the formation of exquisite fibrillar networks after incorporating the KNPs into collagen, and it was conspicuous that the KNPs could uniformly distribute on the surface of collagen fibrils via electrostatic interaction, for both of the two proteins possessed many charged moieties. In addition, the AFM images confirmed the presence of the characteristic D-periodicity of collagen fibrils, indicating that the introduction of KNPs did not disrupt the self-assembly nature of the native collagen. The cell adhesion, proliferation and migration experiments on the CK fibrils were also performed in this study. The results demonstrated that the CK composites showed a better cellular affinity compared with the collagen, thus it might be a promising candidate for the biomedical applications.

Molecular Basis for Mechanical Properties of ECMs: Proposed Role of Fibrillar Collagen and Proteoglycans in Tissue Biomechanics.

Collagen and proteoglycans work in unison in the ECM to bear loads, store elastic energy and then dissipate excess energy to avoid tissue fatigue and premature mechanical failure. While collagen fibers store elastic energy by stretching the flexible regions in the triple helix, they do so by lowering their free energy through a reduction in the entropy and a decrease in charge-charge repulsion. Entropic increases occur when the load is released that drive the reversibility of the process and transmission of excess energy. Energy is dissipated by sliding of collagen fibrils by each other with the aid of decorin molecules that reside on the d and e bands of the native D repeat pattern. Fluid flow from the hydration layer associated with the decorin and collagen fibrils hydraulically dissipates energy during sliding. The deformation is reversed by osmotic forces that cause fluid to reform a hydration shell around the collagen fibrils when the loads are removed. In this paper a model is presented describing the organization of collagen fibers in the skin and cell-collagen mechanical relationships that exist based on non-invasive measurements made using vibrational optical coherence tomography. It is proposed that under external stress, collagen fibers form a tensional network in the plane of the skin. Collagen fiber tension along with forces generated by fibroblasts exerted on collagen fibers lead to an elastic modulus that is almost uniform throughout the plane of the skin. Tensile forces acting on cells and tissues may provide a baseline for stimulation of normal mechanotransduction. We hypothesize that during aging, changes in cellular metabolism, cell-collagen interactions and light and UV light exposure cause down regulation of mechanotransduction and tissue metabolism leading to tissue atrophy.

Fibrillar biopolymer-based scaffolds to study macrophage-fibroblast crosstalk in wound repair.

Controlled wound healing requires a temporal and spatial coordination of cellular activities within the surrounding extracellular matrix (ECM). Disruption of cell-cell and cell-matrix communication results in defective repair, like chronic or fibrotic wounds. Activities of macrophages and fibroblasts crucially contribute to the fate of closing wounds. To investigate the influence of the ECM as an active part controlling cellular behavior, coculture models based on fibrillar 3D biopolymers such as collagen have already been successfully used. With well-defined biochemical and biophysical properties such 3D scaffolds enable in vitro studies on cellular processes including infiltration and differentiation in an in vivo like microenvironment. Further, paracrine and autocrine signaling as well as modulation of soluble mediator transport inside the ECM can be modeled using fibrillar 3D scaffolds. Herein, we review the usage of these scaffolds in in vitro coculture models allowing in-depth studies on the crosstalk between macrophages and fibroblasts during different stages of cutaneous wound healing. A more accurate mimicry of the various processes of cellular crosstalk at the different stages of wound healing will contribute to a better understanding of the impact of biochemical and biophysical environmental parameters and help to develop further strategies against diseases such as fibrosis.

Tendon-inspired fibers from liquid crystalline collagen as the pre-oriented bioink.

Nature provides rich bionic resources for the construction of advanced materials with excellent mechanical properties. In this work, inspired by animal tendons, a bionic collagen fiber was developed using collagen liquid crystals as the pre-oriented bioink. The texture of liquid crystalline collagen observed from polarized optical microscopy (POM) showed the specific molecular pre-orientation. Meanwhile, the collagen spinning liquids exhibited a minimal rise in viscosity upon increasing concentration from 60 to 120 mg/mL, indicating the feasible processability. The collagen fiber, which was prepared via wet spinning without being denatured, exhibited the favorable orientation of fibrils along its axis as observed with FESEM and AFM. Thanks to the synergistic effects between pre-orientation and shearing orientation, the maximum tensile strength and Young's modulus of collagen fibers reached 9.98 cN/tex (219.29 ± 22.92 MPa) and 43.95 ± 1.11 cN/tex (966.20 ± 24.30 MPa), respectively, which were also analogous to those of tendon. In addition, the collagen fiber possessed a desirable wet strength. Benefiting from the natural tissue affinity of collagen, the as-prepared bionic collagen fiber possessed excellent wound suture performance and biodegradability in vivo, which offers a new perspective for the potential of widespread applications of collagen fibers in biomedical fields.

Suppression of pancreatic ductal adenocarcinoma growth and metastasis by fibrillar collagens produced selectively by tumor cells.

Pancreatic ductal adenocarcinoma (PDAC) has a collagen-rich dense extracellular matrix (ECM) that promotes malignancy of cancer cells and presents a barrier for drug delivery. Data analysis of our published mass spectrometry (MS)-based studies on enriched ECM from samples of progressive PDAC stages reveal that the C-terminal prodomains of fibrillar collagens are partially uncleaved in PDAC ECM, suggesting reduced procollagen C-proteinase activity. We further show that the enzyme responsible for procollagen C-proteinase activity, bone morphogenetic protein1 (BMP1), selectively suppresses tumor growth and metastasis in cells expressing high levels of COL1A1. Although BMP1, as a secreted proteinase, promotes fibrillar collagen deposition from both cancer cells and stromal cells, only cancer-cell-derived procollagen cleavage and deposition suppresses tumor malignancy. These studies reveal a role for cancer-cell-derived fibrillar collagen in selectively restraining tumor growth and suggest stratification of patients based on their tumor epithelial collagen I expression when considering treatments related to perturbation of fibrillar collagens.

Mechanical and structural contributions of elastin and collagen fibers to interlamellar bonding in the arterial wall.

The artery relies on interlamellar structural components, mainly elastin and collagen fibers, for maintaining its integrity and resisting dissection propagation. In this study, the contribution of arterial elastin and collagen fibers to interlamellar bonding was studied through mechanical testing, multiphoton imaging and finite element modeling. Steady-state peeling experiments were performed on porcine aortic media and the purified elastin network in the circumferential (Circ) and longitudinal (Long) directions. The peeling force and energy release rate associated with mode-I failure are much higher for aortic media than for the elastin network. Also, longitudinal peeling exhibits a higher energy release rate and strength than circumferential peeling for both the aortic media and elastin. Multiphoton imaging shows the recruitment of both elastin and collagen fibers within the interlamellar space and points to in-plane anisotropy of fiber distributions as a potential mechanism for the direction-dependent phenomena of peeling tests. Three-dimensional finite element models based on cohesive zone model (CZM) of fracture were created to simulate the peeling tests with the interlamellar energy release rate and separation distance at damage initiation obtained directly from peeling test. Our experimental results show that the separation distance at damage initiation is 80 µm for aortic media and 40 µm for elastin. The damage initiation stress was estimated from the model for aortic media (Circ: 60 kPa; Long: 95 kPa) and elastin (Circ: 9 kPa; Long: 14 kPa). The interlamellar separation distance at complete failure was estimated to be 3 - 4 mm for both media and elastin. Furthermore, elastin and collagen fibers both play an important role in bonding of the arterial wall, while collagen has a higher contribution than elastin to interlamellar stiffness, strength and toughness. These results on microstructural interlamellar failure shed light on the pathological development and progression of aortic dissection.

Quantification of load-dependent changes in the collagen fiber architecture for the strut chordae tendineae-leaflet insertion of porcine atrioventricular heart valves.

Atrioventricular heart valves (AHVs) regulate the unidirectional flow of blood through the heart by opening and closing of the leaflets, which are supported in their functions by the chordae tendineae (CT). The leaflets and CT are primarily composed of collagen fibers that act as the load-bearing component of the tissue microstructures. At the CT-leaflet insertion, the collagen fiber architecture is complex, and has been of increasing focus in the previous literature. However, these previous studies have not been able to quantify the load-dependent changes in the tissue's collagen fiber orientations and alignments. In the present study, we address this gap in knowledge by quantifying the changes in the collagen fiber architecture of the mitral and tricuspid valve's strut CT-leaflet insertions in response to the applied loads by using a unique approach, which combines polarized spatial frequency domain imaging with uniaxial mechanical testing. Additionally, we characterized these microstructural changes across the same specimen without the need for tissue fixatives. We observed increases in the collagen fiber alignments in the CT-leaflet insertion with increased loading, as described through the degree of optical anisotropy. Furthermore, we used a leaflet-CT-papillary muscle entity method during uniaxial testing to quantify the chordae tendineae mechanics, including the derivation of the Ogden-type constitutive modeling parameters. The results from this study provide a valuable insight into the load-dependent behaviors of the strut CT-leaflet insertion, offering a research avenue to better understand the relationship between tissue mechanics and the microstructure, which will contribute to a deeper understanding of AHV biomechanics.

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools.

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named "CT-FIRE" and "CurveAlign" that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Fibrillogenesis of acrylic acid-grafted-collagen without self-assembly property inspired by the hybrid fibrils of xenogeneic collagen.

Along with advancements in both protein and chemistry science, the chemical modification of proteins is attracting more and more attention. More specifically, the attachment of polymers or reactive moieties into collagen offers a method to add novel functions to this protein. However, the fibrillogenesis of the modified collagen with high grafting density cannot always be achieved. Here, inspired by the hybrid fibrils of xenogeneic collagen, fibrillogenesis of acrylic acid-grafted-collagen (AAc-g-Col) without self-assembly property was achieved by the induction of natural collagen (Col). The step-by-step co-assembly process of AAc-g-Col and Col was confirmed by turbidity assay. The formation of Col/AAc-g-Col hybrid fibrils was verified by TEM since the acryloyl groups of the hybrid fibrils were labelled using HS-AuNPs based on the Michael addition. Moreover, rheology, SEM, and MTT assays revealed that the fibrillary structures and biocompatibility of the Col/AAc-g-Col hydrogel were comparable to that of the Col hydrogel, although they presented a lower viscoelasticity.

Biocompatibility of a Marine Collagen-Based Scaffold In Vitro and In Vivo.

Scaffold material is essential in providing mechanical support to tissue, allowing stem cells to improve their function in the healing and repair of trauma sites and tissue regeneration. The scaffold aids cell organization in the damaged tissue. It serves and allows bio mimicking the mechanical and biological properties of the target tissue and facilitates cell proliferation and differentiation at the regeneration site. In this study, the developed and assayed bio-composite made of unique collagen fibers and alginate hydrogel supports the function of cells around the implanted material. We used an in vivo rat model to study the scaffold effects when transplanted subcutaneously and as an augment for tendon repair. Animals' well-being was measured by their weight and daily activity post scaffold transplantation during their recovery. At the end of the experiment, the bio-composite was histologically examined, and the surrounding tissues around the implant were evaluated for inflammation reaction and scarring tissue. In the histology, the formation of granulation tissue and fibroblasts that were part of the inclusion process of the implanted material were noted. At the transplanted sites, inflammatory cells, such as plasma cells, macrophages, and giant cells, were also observed as expected at this time point post transplantation. This study demonstrated not only the collagen-alginate device biocompatibility, with no cytotoxic effects on the analyzed rats, but also that the 3D structure enables cell migration and new blood vessel formation needed for tissue repair. Overall, the results of the current study proved for the first time that the implantable scaffold for long-term confirms the well-being of these rats and is correspondence to biocompatibility ISO standards and can be further developed for medical devices application.

From Food Waste to Innovative Biomaterial: Sea Urchin-Derived Collagen for Applications in Skin Regenerative Medicine.

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Origin of transparency in scattering biomimetic collagen materials.

Living tissues, heterogeneous at the microscale, usually scatter light. Strong scattering is responsible for the whiteness of bones, teeth, and brain and is known to limit severely the performances of biomedical optical imaging. Transparency is also found within collagen-based extracellular tissues such as decalcified ivory, fish scales, or cornea. However, its physical origin is still poorly understood. Here, we unveil the presence of a gap of transparency in scattering fibrillar collagen matrices within a narrow range of concentration in the phase diagram. This precholesteric phase presents a three-dimensional (3D) orientational order biomimetic of that in natural tissues. By quantitatively studying the relation between the 3D fibrillar network and the optical and mechanical properties of the macroscopic matrices, we show that transparency results from structural partial order inhibiting light scattering, while preserving mechanical stability, stiffness, and nonlinearity. The striking similarities between synthetic and natural materials provide insights for better understanding the occurring transparency.

Chitosan-glucan complex hollow fibers reinforced collagen wound dressing embedded with aloe vera. II. Multifunctional properties to promote cutaneous wound healing.

This study presents an innovative multifunctional system in fabricating new functional wound dressing (FWD) products that could be used for skin regeneration, especially in cases of infected chronic wounds and ulcers. The innovation is based on the extraction, characterization, and application of collagen (CO)/chitosan-glucan complex hollow fibers (CSGC)/aloe vera (AV) as a novel FWS. For the first time, specific hollow fibers were extracted with controlled inner (500-900 nm)/outer (2-3 µm) diameters from mycelium of Schizophyllum commune. Further on, research and evaluation of morphology, hydrolytic stability, and swelling characteristics of CO/CSGC@AV were carried out. The obtained FWS showed high hydrolytic stability with enhanced swelling characteristics compared to native collagen. The hemostatic effect of FWS increased significantly in the presence of CSGC, compared to native CO and displayed excellent biocompatibility which was tested by using normal human dermal fibroblast (NHDF). The FWS showed high antibacterial activity against different types of bacteria (positive/negative grams). From in vivo measurements, the novel FWS increased the percentage of wound closure after one week of treatment. All these results imply that the new CO/CSGC@AV-FWD has the potential for clinical skin regeneration and applying for controlled drug release.

Wavy nature of collagen fibrils deduced from the dispersion of their second-order nonlinear optical anisotropy parameters ρ.

From P-SHG experiments, second-order nonlinear optical anisotropy parameters ρ = χZZZ/χZXX of collagen tissues are calculated assuming the same model of supercoiled collagen fibril characterized by a variable angle θ. Dispersion of experimental ρ values is converted into distribution of θ values based on the wavy nature of collagen fibrils deduced from EM studies. For tendon, the results show that the dispersion of experimental ρ values is mainly due to Poisson photonic shot noise assuming a slight fibrillar undulation with θ = 2.2° ± 1.8°. However for skin and vessels, the dispersion of experimental ρ values is mainly due to a stronger fibrillar undulation with θ = 16.2° ± 1.3°. The results highlight that this undulation is reduced during the development of liver fibrosis therefore, contributing to the rigidity of the tissue.

Extracellular matrix-mimetic composite hydrogels of cross-linked hyaluronan and fibrillar collagen with tunable properties and ultrastructure.

A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results showed that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach.