Synthesis of Hybrid Polyphenol/Hydroxyapatite Nanomaterials with Anti-Radical Properties.

Plant-derived natural bioactive molecules are of great therapeutic potential but, so far, their application in nanomedicine has scarcely been studied. This work aimed at comparing two methodologies, i.e., adsorption and in situ incorporation, to prepare hybrid polyphenol/hydroxyapatite nanoparticles. Two flavonoids, baicalin and its aglycone derivative baicalein, and two phenolic acids derived from caffeic acid, rosmarinic and chlorogenic acids, were studied. Adsorption of these polyphenols on pre-formed hydroxyapatite nanoparticles did not modify particle size or shape and loading was less than 10% (w/w). In contrast, presence of polyphenols during the synthesis of nanoparticles significantly impacted and sometimes fully inhibited hydroxyapatite formation but recovered particles could exhibit higher loadings. For most hybrid particles, release profiles consisted of a 24 h burst effect followed by a slow release over 2 weeks. Antioxidant properties of the polyphenols were preserved after adsorption but not when incorporated in situ. These results provide fruitful clues for the valorization of natural bioactive molecules in nanomedicine.

Porous yet dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance.

Standard in vitro cell cultures are one of the pillars of biomedical sciences. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement with in vivo observations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrient exchange, as well as dense collagen walls, which mimic locally the extracellular matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for faster and more extensive colonisation when compared with equivalent non-porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towards in vitro systems applicable to other cell types and with higher physiological relevance.

Synthesis of Fibrin-Type I Collagen Biomaterials via an Acidic Gel.

Fibrin-Type I collagen composite gels have been widely studied as biomaterials, in which both networks are usually formed simultaneously at a neutral pH. Here, we describe a new protocol in which mixed concentrated solutions of collagen and fibrinogen were first incubated at acidic pH to induce fibrinogen gel formation, followed by a pH change to neutral inducing collagen fiber formation. Thrombin was then added to form fibrin-collagen networks. Using this protocol, mixed gels containing 20 mg.mL-1 fibrin and up to 10 mg.mL-1 collagen could be prepared. Macroscopic observations evidenced that increasing the content of collagen increases the turbidity of the gels and decreases their shrinkage during the fibrinogen-to-fibrin conversion. The presence of collagen had a minor influence on the rheological properties of the gels. Electron microscopy allowed for observation of collagen fibers within the fibrin network. 2D cultures of C2C12 myoblasts on mixed gels revealed that the presence of collagen favors proliferation and local alignment of the cells. However, it interferes with cell differentiation and myotube formation, suggesting that further control of in-gel collagen self-assembly is required to elaborate fully functional biomaterials.

Cellulose Nanocrystal-Fibrin Nanocomposite Hydrogels Promoting Myotube Formation.

Cellulose nanocrystals (CNCs) have been widely studied as fillers to form reinforced nanocomposites with a wide range of applications, including the biomedical field. Here, we evaluated the possibility to combine them with fibrinogen and obtain fibrin hydrogels with improved mechanical stability as potential cellular scaffolds. In diluted conditions at a neutral pH, it was evidenced that fibrinogen could adsorb on CNCs in a two-step process, favoring their alignment under flow. Composite hydrogels could be prepared from concentrated fibrinogen solutions and nanocrystals in amounts up to 0.3 wt %. CNCs induced a significant modification of the initial fibrin fibrillogenesis and final fibrin network structure, and storage moduli of all nanocomposites were larger than those of pure fibrin hydrogels. Moreover, optimal conditions were found that promoted muscle cell differentiation and formation of long myotubes. These results provide original insights into the interactions of CNCs with proteins with key physiological functions and offer new perspectives for the design of injectable fibrin-based formulations.

Core-Shell Pure Collagen Threads Extruded from Highly Concentrated Solutions Promote Colonization and Differentiation of C3H10T1/2 Cells.

The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration.

Self-assembly/condensation interplay in nano-to-microfibrillar silicified fibrin hydrogels.

Fibrin-based gels are used in clinics as biological glues but their application as 3D cellularized scaffolds is hindered by processing and stability issues. Silicification of fibrin networks appears as a promising strategy not only to address these limitations but also to take advantage of the bioactivity of Si. However, it raises the question of the influence of silica sources on fibrin self-assembly. Here tetraethoxysilane, aminopropyltriethoxysilane and silica nanoparticles were used to design hybrid and nanocomposite fibrin-based hydrogels. By varying the concentration in silica source, we could evidence two regimes of interactions that depend on the extent of inorganic condensation. These interactions modulated the fibrillar structure of the fibrin network from more than 500 nm to less than 100 nm. These nanofibrillar hydrogels could exhibit higher mechanical properties than pure fibrin while preserving their capacity to support proliferation of myoblasts, opening promising perspectives for the use of fibrin-silica constructs in tissue engineering.

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.

Topotactic Fibrillogenesis of Freeze-Cast Microridged Collagen Scaffolds for 3D Cell Culture.

Type I collagen is the main component of the extracellular matrix (ECM). In vitro, under a narrow window of physicochemical conditions, type I collagen self-assembles to form complex supramolecular architectures reminiscent of those found in native ECM. Presently, a major challenge in collagen-based biomaterials is to couple the delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions. In this work, an ice-templating approach promoting the structuration of collagen into macroporous monoliths is used. Instead of common solvent removal procedures, a new topotactic conversion approach yielding self-assembled ordered fibrous materials is implemented. These collagen-only, non-cross-linked scaffolds exhibit uncommon mechanical properties in the wet state, with a Young's modulus of 33 ± 12 kPa, an ultimate tensile stress of 33 ± 6 kPa, and a strain at failure of 105 ± 28%. With the help of the ice-patterned microridge features, normal human dermal fibroblasts and C2C12 murine myoblasts successfully migrate and form highly aligned populations within the resulting three-dimensional (3D) collagen scaffolds. These results open a new pathway to the development of new tissue engineering scaffolds ordered across various organization levels from the molecule to the macropore and are of particular interest for biomedical applications where large-scale 3D cell alignment is needed such as for muscular or nerve reconstruction.

Highly concentrated collagen solutions leading to transparent scaffolds of controlled three-dimensional organizations for corneal epithelial cell colonization.

This study aimed at controlling both the organization and the transparency of dense collagen scaffolds making use of the lyotropic mesogen properties of collagen. Cholesteric or plywood-like liquid crystal phases were achieved using mixtures of acetic and hydrochloric acids as solvents. The critical pH at which the switch between the two phases occurred was around pH = 3. The use of the two acids led to fibrillated collagen I scaffolds, whose visual aspect ranged from opaque to transparent. Rheological investigations showed that viscoelastic properties of the plywood-like solutions were optimized for molding due to faster recovery. They also confirmed the correlation between the elastic modulus and the diameter of collagen fibrils obtained after fibrillogenesis under ammonia vapor. Human corneal epithelial cells, grown from donor limbal explants, were cultured both on transparent plywood-like matrices and on human amniotic membranes for 14 days. The development of corneal epithelium and the preservation of epithelial stem cells were checked by optical microscopy, colony formation assay, immuno-fluorescence and quantitative polymerase chain reaction. A higher level of amplification of limbal stem cells was obtained with collagen matrices compared with amniotic membranes, showing the high biocompatibility of our scaffolds. We therefore suggest that collagen solutions presenting both plywood-like organization and transparency might be of interest for biomedical applications in ophthalmology.

Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases.

Collagen and its denatured form, gelatin, are biopolymers of fundamental interest in numerous fields ranging from living tissues to biomaterials, food, and cosmetics. This study aims at characterizing mixtures of those biopolymers at high concentrations (up to 100 mg·mL-1) at which collagen has mesogenic properties. We use a structural approach combining polarization-resolved multiphoton microscopy, polarized light microscopy, magnetic resonance imaging, and transmission electron microscopy to analyze gelatin and collagen/gelatin dense phases in their sol and gel states from the macroscopic to the microscopic scale. We first report the formation of a lyotropic crystal phase of gelatin A and show that gelatin must structure itself in particles to become mesogenic. We demonstrate that mixtures of collagen and gelatin phase segregate, preserving the setting of the pure collagen mesophase at a gelatin ratio of up to 20% and generating a biphasic fractal sample at all tested ratios. Moreover, differential scanning calorimetric analysis shows that each protein separates into two populations. Both populations of gelatins are stabilized by the presence of collagen, whereas only one population of collagen molecules is stabilized by the presence of gelatin, most probably those at the interface of the fibrillated microdomains and of the gelatin phase. Although further studies are needed to fully understand the involved mechanism, these new data should have a direct impact on the bioengineering of those two biopolymers.

From Tendon Injury to Collagen-based Tendon Regeneration: Overview and Recent Advances.

Tendon injury is a clinical, societal and economical issue. Moreover, tendon repair represents an important clinical challenge, partly due to the mechanical constraints that occur at the junctions with muscle and bone. Several strategies have been developed for tendon repair. In this review, we first assess the importance of tendon injuries from different sites and their causes. After a short overview of tendon three-dimensional organization, the complexity of the perfect repair quest is presented ranging from current clinical procedures to new engineering scaffolds. We then sum up tendon engineering requirements and focus on new collagen-based scaffolds, which raise promising prospects to mimic and repair tendon. In particular, we survey quantitatively a large panel of techniques to produce these scaffolds, detailing their principle and recent improvements.

Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy.

This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules.

Evaluation of dense collagen matrices as medicated wound dressing for the treatment of cutaneous chronic wounds.

Cutaneous chronic wounds are characterized by an impaired wound healing which may lead to infection and amputation. When current treatments are not effective enough, the application of wound dressings is required. To date, no ideal biomaterial is available. In this study, highly dense collagen matrices have been evaluated as novel medicated wound dressings for the treatment of chronic wounds. For this purpose, the structure, mechanical properties, swelling ability and in vivo stability of matrices concentrated from 5 to 40 mg mL(-1) were tested. The matrix stiffness increased with the collagen concentration and was associated with the fibril density and thickness. Increased collagen concentration also enhanced the material resistance against accelerated digestion by collagenase. After subcutaneous implantation in rats, dense collagen matrices exhibited high stability without any degradation after 15 days. The absence of macrophages and neutrophils evidenced their biocompatibility. Subsequently, dense matrices at 40 mg mL(-1) were evaluated as drug delivery system for ampicillin release. More concentrated matrices exhibited the best swelling abilities and could absorb 20 times their dry weight in water, allowing for an efficient antibiotic loading from their dried form. They released efficient doses of antibiotics that inhibited the bacterial growth of Staphylococcus Aureus over 3 days. In parallel, they show no cytotoxicity towards human fibroblasts. These results show that dense collagen matrices are promising materials to develop medicated wound dressings for the treatment of chronic wounds.

Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration.

Several diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of ΔNp63α and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%).

Solid-state NMR study reveals collagen I structural modifications of amino acid side chains upon fibrillogenesis.

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {(1)H}-(13)C solid-state NMR spectroscopy. We interpret (13)C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a (13)C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.

Controlling the nano-bio interface to build collagen-silica self-assembled networks.

Bio-hybrid networks are designed based on the self-assembly of surface-engineered collagen-silica nanoparticles. Collagen triple helices can be confined on the surface of sulfonate-modified silica particles in a controlled manner. This gives rise to hybrid building blocks with well-defined diameters and surface potentials. Taking advantage of the self-assembling properties of collagen, collagen-silica networks are further built-up in solution. The structural and specific recognition properties of the collagen fibrils are well-preserved within the hybrid assembly. A combination of calorimetry, dynamic light scattering, zetametry and microscopy studies indicates that network formation occurs via a surface-mediated mechanism where pre-organization of the protein chains on the particle surface favors the fibrillogenesis process. These results enlighten the importance of the nano-bio interface on the formation and properties of self-assembled bionanocomposites.

Long-term fate of silica nanoparticles interacting with human dermal fibroblasts.

The long-term fate of fluorescent non-porous FITC-SiO(2) nanoparticles of various sizes (10-200 nm) and charge is studied in the presence of human dermal fibroblasts. Particle aggregates are formed in the culture medium and uptaken, at least partially, by macropinocytosis. The smallest particles have a strong impact on cell viability and genotoxic effects can be observed for negatively-charged colloids 10 nm in size. Largest particles do not impact on cellular activity and can be monitored in cellulo via fluorescence and transmission electron microscopy studies over two weeks. These observations reveal a significant decrease in the size of silica particles located in endocytic vesicles. The dissolution process is confirmed by monitoring the cell culture medium that contains both colloidal and soluble silica species. Such dissolution can be explained on the sole basis of silica solubility and has great implication for the use of non-porous silica particles as intra-cellular drug release systems.

Nanorods of Well-Defined Length and Monodisperse Cross-Section Obtained from Electrostatic Complexation of Nanoparticles with a Semiflexible Biopolymer.

We show by combining small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (cryo-TEM) that anionic silica nanoparticles (SiNPs) assemble into well-defined 1D cluster when mixed with a dilute solution of semiflexible chitosan polycation. The nanorods are stable in excess of SiNPs and composed of 10 SiNPs well-ordered into straight single strands with length Lrod ≈ 184.0 nm and radius Rrod = 9.2 nm = RSiNPs. We point out that the ratio between the chitosan persistence length and the SiNP radius, which is here equal to 1, can be the determining condition to obtain such original objects.

In vitro studies and preliminary in vivo evaluation of silicified concentrated collagen hydrogels.

Hybrid and nanocomposite silica-collagen materials derived from concentrated collagen hydrogels were evaluated in vitro and in vivo to establish their potentialities for biological dressings. Silicification significantly improved the mechanical and thermal stability of the collagen network within the hybrid systems. Nanocomposites were found to favor the metabolic activity of immobilized human dermal fibroblasts while decreasing the hydrogel contraction. Cell adhesion experiments suggested that in vitro cell behavior was dictated by mechanical properties and surface structure of the scaffold. First-to-date in vivo implantation of bulk hydrogels in subcutaneous sites of rats was performed over the vascular inflammatory period. These materials were colonized and vascularized without inducing strong inflammatory response. These data raise reasonable hope for the future application of silica-collagen biomaterials as biological dressings.

Synthesis and in vivo integration of improved concentrated collagen hydrogels.

Normal collagen hydrogels, currently used as the dermal layer of skin substitute Apligraf®, are obtained by encapsulating dermal fibroblasts in a collagen hydrogel at low concentration (0.66 mg/ml). However they suffer from extensive contraction by cells and weak resistance against degradation, which limits their use as permanent graft. We have previously shown that concentrated collagen hydrogels at 3 mg/ml exhibit an improved performance in this respect but nevertheless degrade in vivo to ca. 50% of their initial area after 1 month. We have now investigated a new procedure to synthesize more concentrated collagen hydrogels at 5 mg/ml in order to improve hydrogel resistance and integration capability. The constructs were implanted in subcutaneous pockets in a rat model and analysed after 15 and 30 days. They were still visible after 1 month without any reduction of their area. Histological analysis revealed rapid colonization of the implants by host cells. Neovascularization was observed and reached the core of the implant at day 15. Moreover, cell colonization was not associated with a severe host response. The absence of apoptotic cells evidenced cell viability and the neosynthesis of collagen III a remodelling process. These novel non-crosslinked and cost-effective materials show superior stability and in vivo integration compared to less concentrated collagen hydrogels and appear promising for the treatment of skin lesions.