Optimizing the physical properties of collagen/hyaluronan hydrogels by inhibition of polyionic complexes formation at pH close to the collagen isoelectric point.

Collagen/hyaluronan hydrogels with physical properties well suited for biomedical applications are challenging to synthesize due to the formation of polyionic complexes (PICs). A systematic physicochemical study was thus performed to determine novel conditions to inhibit the formation of collagen/hyaluronan PICs and obtain composite hydrogels with high physical properties. Using a range of pH from 1 to 5.5 and the addition of NaCl, type I collagen and tyramine-substituted hyaluronic acid (THA) solutions were mixed and analyzed by cryo-scanning electron microscopy and ATR-FTIR. PIC formation was inhibited at pH 1 without salt and at pH 2.5 and 5.5 in the presence of 400 mM NaCl. Interestingly, collagen fibrils were observed in solution at pH 5.5 before mixing with THA. After collagen gelling by pH increase, a homogeneous hydrogel consisting of collagen fibrils was only observed when PICs were inhibited. Then, the THA gelling performed by photo-crosslinking increased the rheological properties by four when hydrogels were formed with collagen/THA mixtures at pH 1 or 5.5 with salt. Taken together, these results show that a pH of 5.5, close to the collagen isoelectric point, enables the formation of collagen fibrils in solution, inhibits the PICs formation, and allows the formation of homogenous collagen/THA composite hydrogels compatible with cell survival.

Mechanical loading activates the YAP/TAZ pathway and chemokine expression in the MLO-Y4 osteocyte-like cell line.

Osteocytes are mechanosensitive cells that control bone remodeling in response to mechanical loading. To date, specific signaling pathways modulated by mechanical loading in osteocytes are not well understood. Yes associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the main effectors of the Hippo pathway, are reported to play a role in mechanotransduction and during osteoblastogenesis. Here, we hypothesized that YAP/TAZ signaling mediates osteocyte mechanosensing to target genes of the bone remodeling process. We aimed to investigate the contribution of YAP/TAZ in modulating the gene expression in an osteocyte-like cell line MLO-Y4. We developed a 3D osteocyte compression culture model from an MLO-Y4 osteocyte cell line embedded in concentrated collagen hydrogel. 3D-mechanical loading led to the increased expression of mechanosensitive genes and a subset of chemokines, including M-csf, Cxcl1, Cxcl2, Cxcl3, Cxcl9, and Cxcl10. The transcription regulators YAP and TAZ translocated to the nucleus and upregulated their target genes and proteins. RNAseq analysis revealed that YAP/TAZ knockdown mediated the regulation of several genes including osteocyte dendrite formation. Use of YAP/TAZ knockdown partially blunted the increase in M-csf and Cxcl3 levels in response to MLO-Y4 compression. These findings demonstrate that YAP/TAZ signaling is required for osteocyte-like cell mechano-transduction, regulates the gene expression profiles and controls chemokine expression.

Extracellular versus Intracellular Degradation of Nanostructured Silica Particles.

Silica nanoparticles appear as promising drug carriers for intracellular delivery. However, the mechanisms by which they are degraded within cells remain largely unknown. In this context, we have prepared three types of PEGylated fluorescent silica nanoparticles with various internal structures (core-shell biocomposite, multilayered, and hollow mesoporous) and studied their degradation in a buffer, in a culture medium, and in contact with human dermal fibroblasts. All particles were prone to dissolve in solution, leading to an increase of porosity and/or the precipitation of new colloids and eventually fragmentation, with a faster rate in the medium compared to that in the buffer. All particles were also uptaken by the cells without significant cytotoxic effect. Their intracellular degradation occurred faster than in suspension, but following almost similar dissolution mechanisms. These results strongly suggest that in these conditions, silica nanoparticles must be primarily considered as hydrolytically degraded and not biodegraded, a point of importance for their future applications in drug delivery.

Impact of Polyethylenimine Conjugation Mode on the Cell Transfection Efficiency of Silica Nanovectors.

The conjugation of polyethylenimine (PEI) to silica nanoparticles has emerged as a useful strategy in gene delivery. Here we investigate the influence of the PEI conjugation mode on the transfection ability of plain silica nanoparticles. Surface functionalization with sulfonate- and chloride-bearing silanes modulates the amount and conformation of PEI and therefore the particles' affinity for the plasmid, without impacting on cytotoxicity. However, transfection efficiency in both immortalized and primary cells is more directly correlated to the nature and strength of the particle-PEI interactions. It suggests that PEI detachment from the particle surface at the stage of endosomal escape is a key event in the plasmid delivery process. These data should provide fruitful guidelines for the fine tuning of colloidal surfaces intended for intracellular delivery of bioactive molecules.

Silicon impacts collagen remodelling and mineralization by human dental pulp stem cells in 3D pulp-like matrices.

OBJECTIVES: Silicon-releasing biomaterials are widely used in the field of dentistry. However, unlike bone, very little is known about the role of silicon on dental tissue formation and repair. This study investigates the influence of silicic acid on the survival, differentiation and mineralizing ability of human dental pulp stem cells (hDPSCs) in 3D pulp-like environments METHODS: Dense type I collagen hydrogels seeded with hDPSCs were cultured over 4 weeks in the presence of silicic acid at physiological (10 µM) and supraphysiological (100 µM) concentrations. Cell viability and proliferation were studied by Alamar Blue and live/dead staining. The collagen network was investigated using second harmonic generation imaging. Mineral deposition was monitored by histology and scanning electron microscopy. Gene expression of mineralization- and matrix remodeling-associated proteins was studied by qPCR. RESULTS: Presence of silicic acid did not show any significant influence on cell survival, metabolic activity and gene expression of key mineralization-related proteins (ALP, OCN, BSP). However, it induced enhanced cell clustering and delayed expression of matrix remodeling-associated proteins (MMP13, Col I). OPN expression and mineral deposition were inhibited at 100 µM. It could be inferred that silicic acid has no direct cellular effect but rather interacts with the collagen network, leading to a modification of the cell-matrix interface. SIGNIFICANCE: Our results offer advanced insights on the possible role of silicic acid, as released by pulp capping calcium silicates biomaterials, in reparative dentine formation. More globally, these results interrogate the possible role of Si in pulp pathophysiology.

Role of Physico-Chemical and Cellular Conditions on the Bone Repair Potential of Plastically Compressed Collagen Hydrogels.

Since their first description nearly 20 years ago, dense collagen hydrogels obtained by plastic compression have become popular scaffolds in tissue engineering. In particular, when seeded with dental pulp stem cells, they have demonstrated a great in vivo potential in cranial bone repair. Here, we investigated how physico-chemical and cell-seeding conditions could influence the formation and in vitro mineralization of these cellularized scaffolds. A qualitative assessment demonstrated that the gel stability before and after compression was highly sensitive to the conditions of fibrillogenesis, especially initial acid acetic and buffer concentrations. Gels with similar rheological properties but different fibrillar structures that exhibited different stabilities when used for the 3D culture of Stem cells from Human Exfoliated Deciduous teeth (SHEDs) could be prepared. Finally, in our optimal physico-chemical conditions, mineralization could be achieved only using human dental pulp stem cells (hDPSCs) at a high cell density. These results highlight the key role of fibrillogenic conditions and cell type/density on the bone repair potential of cell-laden plastically compressed collagen hydrogels.

Anisotropic dense collagen hydrogels with two ranges of porosity to mimic the skeletal muscle extracellular matrix.

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle ECM was developed reproducing these four crucial characteristics of the native ECM. An anisotropic hydrogel mimicking the muscle fascia was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic network of pores (100 µm) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. This artificial matrix was thus evaluated for myoblast differentiation. The addition of large channels (600 µm) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. Skeletal myoblasts embedded in Matrigel®, seeded within the channels, organized in 3D, and differentiated into multinucleated myotubes. These results show that porous and anisotropic dense collagen hydrogels are promising biomaterials to model skeletal muscle ECM.

Combining biomimetic collagen/hyaluronan hydrogels with discogenic growth factors promotes mesenchymal stroma cell differentiation into Nucleus Pulposus like cells.

Based on stem cell injection into degenerated Nucleus Pulposus (NP), novel treatments for intervertebral disc (IVD) regeneration were disappointing because of cell leakage or inappropriate cell differentiation. In this study, we hypothesized that mesenchymal stromal cells encapsulated within injectable hydrogels possessing adequate physico-chemical properties would differentiate into NP like cells. Composite hydrogels consisting of type I collagen and tyramine-substituted hyaluronic acid (THA) were prepared to mimic the NP physico-chemical properties. Human bone marrow derived mesenchymal stromal cells (BM-MSCs) were encapsulated within hydrogels and cultivated in proliferation medium (supplemented with 10% fetal bovine serum) or differentiation medium (supplemented with GDF5 and TGFß1) over 28 days. Unlike pure collagen, collagen/THA composite hydrogels were stable over 28 days in culture. In proliferation medium, the cell viability within pure collagen hydrogels was high, whereas that in composite and pure THA hydrogels was lower due to the weaker cell adhesion. Nonetheless, BM-MSCs proliferated in all hydrogels. In composite hydrogels, cells exhibited a rounded morphology similar to NP cells. The differentiation medium did not impact the hydrogel stability and cell morphology but negatively impacted the cell viability in pure collagen hydrogels. A high THA content within hydrogels promoted the gene expression of NP markers such as collagen II, aggrecan, SOX9 and cytokeratin 18 at day 28. The differentiation medium potentialized this effect with an earlier and higher expression of these NP markers. Taken together, these results show that the physico-chemical properties of collagen/THA composite hydrogels and GDF5/TGFß1 act in synergy to promote the differentiation of BM-MSCs into NP like cells.

3D models of dilated cardiomyopathy: Shaping the chemical, physical and topographical properties of biomaterials to mimic the cardiac extracellular matrix.

The pathophysiology of dilated cardiomyopathy (DCM), one major cause of heart failure, is characterized by the dilation of the heart but remains poorly understood because of the lack of adequate in vitro models. Current 2D models do not allow for the 3D organotypic organization of cardiomyocytes and do not reproduce the ECM perturbations. In this review, the different strategies to mimic the chemical, physical and topographical properties of the cardiac tissue affected by DCM are presented. The advantages and drawbacks of techniques generating anisotropy required for the cardiomyocytes alignment are discussed. In addition, the different methods creating macroporosity and favoring organotypic organization are compared. Besides, the advances in the induced pluripotent stem cells technology to generate cardiac cells from healthy or DCM patients will be described. Thanks to the biomaterial design, some features of the DCM extracellular matrix such as stiffness, porosity, topography or chemical changes can impact the cardiomyocytes function in vitro and increase their maturation. By mimicking the affected heart, both at the cellular and at the tissue level, 3D models will enable a better understanding of the pathology and favor the discovery of novel therapies.

Generation of an Adequate Perfusion Network within Dense Collagen Hydrogels Using Thermoplastic Polymers as Sacrificial Matrix to Promote Cell Viability.

Dense collagen hydrogels are promising biomaterials for several tissue-engineering applications. They exhibit high mechanical properties, similar to physiological extracellular matrices, and do not shrink under cellular activity. However, they suffer from several drawbacks, such as weak nutrient and O2 diffusion, impacting cell survival. Here, we report a novel strategy to create a perfusion system within dense and thick collagen hydrogels to promote cell viability. The 3D printing of a thermoplastic filament (high-impact polystyrene, HIPS) with a three-wave shape is used to produce an appropriate sacrificial matrix. The HIPS thermoplastic polymer allows for good shape fidelity of the filament and does not collapse under the mechanical load of the collagen solution. After the collagen gels around the filament and dissolves, a channel is generated, allowing for adequate and rapid hydrogel perfusion. The dissolution process does not alter the collagen hydrogel's physical or chemical properties, and the perfusion is associated with an increased fibroblast survival. Here, we report the novel utilization of thermoplastics to generate a perfusion network within biomimetic collagen hydrogels.

Nanostructured Dense Collagen-Polyester Composite Hydrogels as Amphiphilic Platforms for Drug Delivery.

Associating collagen with biodegradable hydrophobic polyesters constitutes a promising method for the design of medicated biomaterials. Current collagen-polyester composite hydrogels consisting of pre-formed polymeric particles encapsulated within a low concentrated collagen hydrogel suffer from poor physical properties and low drug loading. Herein, an amphiphilic composite platform associating dense collagen hydrogels and up to 50 wt% polyesters with different hydrophobicity and chain length is developed. An original method of fabrication is disclosed based on in situ nanoprecipitation of polyesters impregnated in a pre-formed 3D dense collagen network. Composites made of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) but not polycaprolactone (PCL) exhibit improved mechanical properties compared to those of pure collagen dense hydrogels while keeping a high degree of hydration. Release kinetics of spironolactone, a lipophilic steroid used as a drug model, can be tuned over one month. No cytotoxicity of the composites is observed on fibroblasts and keratinocytes. Unlike the incorporation of pre-formed particles, the new process allows for both improved physical properties of collagen hydrogels and controlled drug delivery. The ease of fabrication, wide range of accessible compositions, and positive preliminary safety evaluations of these collagen-polyesters will favor their translation into clinics in wide areas such as drug delivery and tissue engineering.

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.

Baicalein-modified hydroxyapatite nanoparticles and coatings with antibacterial and antioxidant properties.

Aseptic loosening and bacterial infections are the two main causes of failure for metallic implants used for joint replacement. A coating that is both bioactive and possesses antimicrobial properties may address such shortcomings and improve the performance of the implant. We have sought to study the properties of combining hydroxyapatite-based nanoparticles or coatings with baicalein, a plant-extracted molecule with both antibacterial and antioxidant properties. (B-type) carbonated hydroxyapatite nanoparticles prepared by a chemical wet method could subsequently adsorbed by soaking in a baicalein solution. The amount of adsorbed baicalein was determined to be 63 mg.g-1 by thermogravimetric measurements. In a second approach, baicalein was adsorbed on a biomimetic calcium-deficient hydroxyapatite planar coating (12 µm thick) deposited on Ti6Al4V alloy from an aqueous solution of calcium, phosphate, sodium and magnesium salts. Soaking of the hydroxyapatite coated on titanium alloy in a baicalein solution induced partial dissolution/remodeling of the upper surface of the coating. However, the observed remodeling of the surface was much more pronounced in the presence of a baicalein solution, compared to pure water. The presence of adsorbed baicalein on the HAp layer, although it could not be precisely quantified, was assessed by XPS and fluorescence analysis. Planar coatings exhibited significant antibacterial properties against Staphylococcus epidermidis. Baicalein-modified nanoparticles exhibited significant antioxidant properties. These results illustrate the potential of hydroxyapatite used as a carrier for natural biologically-active molecules and also discuss the challenges associated with their applications as antibacterial agents.

Dense fibrillar collagen matrices for tissue repair.

The preparation of dense fibrillar collagen matrices, through a sol/gel transition at variable concentrations, offers routes to produce a range of simple, non toxic materials. Concentrated hydrogels entrapping cells show enhanced properties in terms of reduced contraction and enhanced cell proliferation . Dense fibrillar matrices attain tissue like mechanical properties and show ultrastructures described in connective tissues, namely liquid crystalline cholesteric geometries. Their colonization by cells and possible association with a mineral phase in a tissue like manner validate their use as biomimetic materials for regenerative medicine.

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.

Native Collagen: Electrospinning of Pure, Cross-Linker-Free, Self-Supported Membrane.

Rebuilding biological environments is crucial when facing the challenges of fundamental and biomedical research. Thus, preserving the native state of biomolecules is essential. We use electrospinning (ES), which is an extremely promising method for the preparation of fibrillar membranes to mimic the ECM of native tissues. Here, we report for the first time (1) the ES of pure and native collagen into a self-supported membrane in absence of cross-linker and polymer support, (2) the preservation of the membrane integrity in hydrated media in absence of cross-linker, and (3) the preservation of the native molecular structure and recovery of the hierarchical assembly of collagen. We use a multiscale approach to characterize collagen native structure at the molecular level using circular dichroism, and to investigate collagen hierarchical organization within the self-supported membrane using a combination of multiphoton and electron microscopies. Finally, we show that the membranes are perfectly suited for cell adhesion and spreading, making them very promising candidates for the development of biomaterials and finding applications in biomedical research.

USPIO-PEG nanoparticles functionalized with a highly specific collagen-binding peptide: a step towards MRI diagnosis of fibrosis.

Fibrosis is characterized by a pathologic deposition of collagen I, leading to impaired function of organs. Tissue biopsy is the gold standard method for the diagnosis of fibrosis but this is an invasive procedure, subject to sampling errors. Several non-invasive techniques such as magnetic resonance imaging (MRI) using non-specific probes have been developed but they are not fully satisfying as they allow diagnosis at a late stage. In this study, collagelin, a collagen-binding peptide has been covalently linked using click chemistry to pegylated Ultra Small Super Paramagnetic Iron Oxide Nanoparticles (USPIO-PO-PEG-collagelin NPs) with the aim of diagnosing fibrosis at an early stage by MRI. USPIO-PO-PEG-collagelin NPs showed a high affinity for collagen I, two times higher than that of free collagelin whereas not peptide labeled USPIO NPs (USPIO-PO-PEG-yne) did not present any affinity. NPs were not toxic for macrophages and fibroblasts. Diffusion through collagen hydrogels concentrated at 3 and 10 mg mL-1 revealed a large accumulation of USPIO-PO-PEG-collagelin NPs within the collagen network after 72 hours, ca. 3 times larger than that of unlabeled USPIO, thereby evidencing the specific targeting of collagen I. Moreover, the quantity of USPIO-PO-PEG-collagelin NPs accumulated within hydrogels was proportional to the collagen concentration. Subsequently, the NPs diffusion through collagen hydrogels was monitored by MRI. The MRI T2 time relaxation decreased much more significantly with depth for USPIO-PO-PEG-collagelin NPs compared to unlabeled ones. Taken together, these results show that USPIO-PEG-collagelin NPs are promising as effective MRI nanotracers for molecular imaging of fibrosis at an early stage.

Differentiation of neural-type cells on multi-scale ordered collagen-silica bionanocomposites.

Cells respond to biophysical and biochemical signals. We developed a composite filament from collagen and silica particles modified to interact with collagen and/or present a laminin epitope (IKVAV) crucial for cell-matrix adhesion and signal transduction. This combines scaffolding and signaling and shows that local tuning of collagen organization enhances cell differentiation.

Exploring the cell-protein-mineral interfaces: Interplay of silica (nano)rods@collagen biocomposites with human dermal fibroblasts.

The benefits of associating biological polymers with nanomaterials within functional bionanocomposite hydrogels have already been evidenced both in vitro and in vivo. However their development as effective biomaterials requires to understand and tune the interactions at the cell-protein-mineral ternary interface. With this purpose, we have studied here the impact of silica (nano)rods on the structural and rheological properties of type I collagen hydrogels â€‹and on the behavior of human dermal fibroblasts. High collagen concentrations were beneficial to the material mechanical properties, whereas silica rods could exert a positive effect on these at both low and high content. Electron microscopy evidenced strong bio-mineral interactions, emphasizing the true composite nature of these materials. In contrast, adhesion and proliferation studies showed that, despite these interactions, fibroblasts can discriminate between the protein and the inorganic phases and penetrate the collagen network to limit direct contact with silica. Such a divergence between physicochemical characteristics and biological responses has major implications for the prediction of the in vivo fate of nanocomposite biomaterials.

Hyperglycemia acts in synergy with hypoxia to maintain the pro-inflammatory phenotype of macrophages.

Diabetic foot ulcers (DFUs) are characterized by a chronic inflammation state which prevents cutaneous wound healing, and DFUs eventually lead to infection and leg amputation. Macrophages located in DFUs are locked in an pro-inflammatory phenotype. In this study, the effect of hyperglycemia and hypoxia on the macrophage phenotype was analyzed. For this purpose, a microarray was performed to study the gene expression profile of macrophages cultivated in a high glucose concentration. Hyperglycemia upregulated the expression of pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, chemokines and downregulated the expression of two receptors involved in phagocytosis (CD 36 and Class B scavenger type I receptors). In addition, eleven anti-apoptotic factors were upregulated whereas three pro-apoptotic genes were downregulated. Subsequently, the contribution of hypoxia and hyperglycemia to chronic inflammation and their potential synergistic effect was evaluated on activated THP-1 derived macrophages. A long term post activation effect (17 hours) was only observed on the upregulation of pro-inflammatory cytokines when hypoxia was combined with a high glucose concentration. In contrast, hyperglycemia and hypoxia did not have any effect on wound healing molecules such as TGF-ß1. Taken together, the results show that hyperglycemia acts in synergy with hypoxia to maintain a chronic inflammation state in macrophages.