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.

Interactions of Organosilanes with Fibrinogen and Their Influence on Muscle Cell Proliferation in 3D Fibrin Hydrogels.

Silanization of biomacromolecules has emerged as a fruitful approach to prepare hybrid biohydrogels. However, very little is known about interactions between organosilanes and biopolymers in solution. Here we focused on fibrin, a protein of interest in the biomedical field, whose self-assembly process and resulting gel structure are highly sensitive to experimental conditions. Three main silanes were selected to decipher the relative influence of the silanol groups and organic functions. Whereas no protein denaturation was observed, silanes bearing hydrophobic groups had a surfactant-like behavior and could improve the dispersion of fibrinogen molecules, impacting gel formation kinetics and rheological properties. 3D cultures of myoblasts evidenced that organosilanes could promote or impede cell proliferation, suggesting interactions of silanols with fibrin. These results demonstrate that the two sides of the coin of organosilane reactivity are relevant at different stages of fibrin gel formation and must be considered for future development of hybrid biomaterials.

Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.

Cell migration plays a major role in many fundamental biological processes, such as morphogenesis, tumor metastasis, and wound healing. As they anchor and pull on their surroundings, adhering cells actively probe the stiffness of their environment. Current understanding is that traction forces exerted by cells arise mainly at mechanotransduction sites, called focal adhesions, whose size seems to be correlated to the force exerted by cells on their underlying substrate, at least during their initial stages. In fact, our data show by direct measurements that the buildup of traction forces is faster for larger substrate stiffness, and that the stress measured at adhesion sites depends on substrate rigidity. Our results, backed by a phenomenological model based on active gel theory, suggest that rigidity-sensing is mediated by a large-scale mechanism originating in the cytoskeleton instead of a local one. We show that large-scale mechanosensing leads to an adaptative response of cell migration to stiffness gradients. In response to a step boundary in rigidity, we observe not only that cells migrate preferentially toward stiffer substrates, but also that this response is optimal in a narrow range of rigidities. Taken together, these findings lead to unique insights into the regulation of cell response to external mechanical cues and provide evidence for a cytoskeleton-based rigidity-sensing mechanism.

Tunable biomimetic materials elaborated by ice templating and self-assembly of collagen for tubular tissue engineering.

Synthetic tubular grafts currently used in clinical context fail frequently, and the expectations that biomimetic materials could tackle these limitations are high. However, developing tubular materials presenting structural, compositional and functional properties close to those of native tissues remains an unmet challenge. Here we describe a combination of ice templating and topotactic fibrillogenesis of type I collagen, the main component of tissues' extracellular matrix, yielding highly concentrated yet porous tubular collagen materials with controlled hierarchical architecture at multiple length scales, the hallmark of native tissues' organization. By modulating the thermal conductivity of the cylindrical molds, we tune the macroscopic porosity defined by ice. Coupling the aforementioned porosity patterns with two different fibrillogenesis routes results in a new family of tubular materials whose textural features and the supramolecular arrangement of type I collagen are achieved. The resulting materials present hierarchical elastic properties and are successfully colonized by human endothelial cells and alveolar epithelial cells on the luminal side, and by human mesenchymal stem cells on the external side. The proposed straightforward protocol is likely to be adapted for larger graft sizes that address ever-growing clinical needs, such as peripheral arterial disease or tracheal and bronchial reconstructions.

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization.

Circulating tumor cells (CTCs) represent an interesting source of biomarkers for diagnosis, prognosis, and the prediction of cancer recurrence, yet while they are extensively studied in oncobiology research, their diagnostic utility has not yet been demonstrated and validated. Their scarcity in human biological fluids impedes the identification of dangerous CTC subpopulations that may promote metastatic dissemination. In this Perspective, we discuss promising techniques that could be used for the identification of these metastatic cells. We first describe methods for isolating patient-derived CTCs and then the use of 3D biomimetic matrixes in their amplification and analysis, followed by methods for further CTC analyses at the single-cell and single-molecule levels. Finally, we discuss how the elucidation of mechanical and morphological properties using techniques such as atomic force microscopy and molecular biomarker identification using nanopore-based detection could be combined in the future to provide patients and their healthcare providers with a more accurate diagnosis.

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies.

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with successful development of diseased 3D myotubes displaying reduced contraction. The miniaturized myotube technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should substantially improve drug screening capability.

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.

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.

Type I Collagen-Fibrin Mixed Hydrogels: Preparation, Properties and Biomedical Applications.

Type I collagen and fibrin are two essential proteins in tissue regeneration and have been widely used for the design of biomaterials. While they both form hydrogels via fibrillogenesis, they have distinct biochemical features, structural properties and biological functions which make their combination of high interest. A number of protocols to obtain such mixed gels have been described in the literature that differ in the sequence of mixing/addition of the various reagents. Experimental and modelling studies have suggested that such co-gels consist of an interpenetrated structure where the two proteins networks have local interactions only. Evidences have been accumulated that immobilized cells respond not only to the overall structure of the co-gels but can also exhibit responses specific to each of the proteins. Among the many biomedical applications of such type I collagen-fibrin mixed gels, those requiring the co-culture of two cell types with distinct affinity for these proteins, such as vascularization of tissue engineering constructs, appear particularly promising.

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.

Substrate topography induces a crossover from 2D to 3D behavior in fibroblast migration.

In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are plated on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin II activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally, the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars.

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.

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.

Microfabricated substrates as a tool to study cell mechanotransduction.

Mechanical cell-substrate interactions affect many cellular functions such as spreading, migration, and even differentiation. These interactions can be studied by incorporating micro- and nanotechnology-related tools. The design of substrates based on these technologies offers new possibilities to probe the cellular responses to changes in their physical environment. The investigations of the mechanical interactions of cells and their surrounding matrix can be carried out in well-defined and near physiological conditions. In particular, this includes the transmission of forces as well as rigidity and topography sensing mechanisms. Here, we review techniques and tools based on nano- and micro-fabrication that have been developed to analyze the influence of the mechanical properties of the substrate on cell functions. We also discuss how microfabrication methods have improved our knowledge on cell adhesion and migration and how they could solve remaining problems in the field of mechanobiology.

Relaxing the actin cytoskeleton for adhesion and movement with Ena/VASP.

At cell-cell contacts, as well as at the leading edge of motile cells, the plasticity of actin structures is maintained, in part, through labile connections to the plasma membrane. Here we explain how and why Drosophila enabled/vasodilator stimulated phosphoprotein (Ena/VASP) proteins are candidates for driving this cytoskeleton modulation under the membrane.

VASP governs actin dynamics by modulating filament anchoring.

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.