Acidic Osteoid Templates the Plywood Structure of Bone Tissue.

Bone is created by osteoblasts that secrete osteoid after which an ordered texture emerges, followed by mineralization. Plywood geometries are a hallmark of many trabecular and cortical bones, yet the origin of this texturing in vivo has never been shown. Nevertheless, extensive in vitro work revealed how plywood textures of fibrils can emerge from acidic molecular cholesteric collagen mesophases. This study demonstrates in sheep, which is the preferred model for skeletal orthopaedic research, that the deeper non-fibrillar osteoid is organized in a liquid-crystal cholesteric geometry. This basophilic domain, rich in acidic glycosaminoglycans, exhibits low pH which presumably fosters mesoscale collagen molecule ordering in vivo. The results suggest that the collagen fibril motif of twisted plywood matures slowly through self-assembly thermodynamically driven processes as proposed by the Bouligand theory of biological analogues of liquid crystals. Understanding the steps of collagen patterning in osteoid-maturation processes may shed new light on bone pathologies that emerge from collagen physico-chemical maturation imbalances.

Collagen Suprafibrillar Confinement Drives the Activity of Acidic Calcium-Binding Polymers on Apatite Mineralization.

Bone collagenous extracellular matrix provides a confined environment into which apatite crystals form. This biomineralization process is related to a cascade of events partly controlled by noncollagenous proteins. Although overlooked in bone models, concentration and physical environment influence their activities. Here, we show that collagen suprafibrillar confinement in bone comprising intra- and interfibrillar spaces drives the activity of biomimetic acidic calcium-binding polymers on apatite mineralization. The difference in mineralization between an entrapping dentin matrix protein-1 (DMP1) recombinant peptide (rpDMP1) and the synthetic polyaspartate validates the specificity of the 57-KD fragment of DMP1 in the regulation of mineralization, but strikingly without phosphorylation. We show that all the identified functions of rpDMP1 are dedicated to preclude pathological mineralization. Interestingly, transient apatite phases are only found using a high nonphysiological concentration of additives. The possibility to combine biomimetic concentration of both collagen and additives ensures specific chemical interactions and offers perspectives for understanding the role of bone components in mineralization.

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.

Self-Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials.

Extracellular matrices (ECM) rich in type I collagen exhibit characteristic anisotropic ultrastructures. Nevertheless, working in vitro with this biomacromolecule remains challenging. When processed, denaturation of the collagen molecule is easily induced in vitro avoiding proper fibril self-assembly and further hierarchical order. Here, an innovative approach enables the production of highly concentrated injectable collagen microparticles, based on collagen molecules self-assembly, thanks to the use of spray-drying process. The versatility of the process is shown by performing encapsulation of secretion products of gingival mesenchymal stem cells (gMSCs), which are chosen as a bioactive therapeutic product for their potential efficiency in stimulating the regeneration of a damaged ECM. The injection of collagen microparticles in a cell culture medium results in a locally organized fibrillar matrix. The efficiency of this approach for making easily handleable collagen microparticles for encapsulation and injection opens perspectives in active tissue regeneration and 3D bioprinted scaffolds.

Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis.

The transition from osteoblast to osteocyte is described to occur through passive entrapment mechanism (self-buried, or embedded by neighboring cells). Here, we provide evidence of a new pathway where osteoblasts are "more" active than generally assumed. We demonstrate that osteoblasts possess the ability to migrate and differentiate into early osteocytes inside dense collagen matrices. This step involves MMP-13 simultaneously with IBSP and DMP1 expression. We also show that osteoblast migration is enhanced by the presence of apatite bone mineral. To reach this conclusion, we used an in vitro hybrid model based on both the structural characteristics of the osteoid tissue (including its density, texture and three-dimensional order), and the use of bone-like apatite. This finding highlights the mutual dynamic influence of osteoblast cell and bone extra cellular matrix. Such interactivity extends the role of physicochemical effects in bone morphogenesis complementing the widely studied molecular signals. This result represents a conceptual advancement in the fundamental understanding of bone formation.

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.

Water-mediated structuring of bone apatite.

It is well known that organic molecules from the vertebrate extracellular matrix of calcifying tissues are essential in structuring the apatite mineral. Here, we show that water also plays a structuring role. By using solid-state nuclear magnetic resonance, wide-angle X-ray scattering and cryogenic transmission electron microscopy to characterize the structure and organization of crystalline and biomimetic apatite nanoparticles as well as intact bone samples, we demonstrate that water orients apatite crystals through an amorphous calcium phosphate-like layer that coats the crystalline core of bone apatite. This disordered layer is reminiscent of those found around the crystalline core of calcified biominerals in various natural composite materials in vivo. This work provides an extended local model of bone biomineralization.

The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite.

The involvement of collagen in bone biomineralization is commonly admitted, yet its role remains unclear. Here we show that type I collagen in vitro can initiate and orientate the growth of carbonated apatite mineral in the absence of any other vertebrate extracellular matrix molecules of calcifying tissues. We also show that the collagen matrix influences the structural characteristics on the atomic scale, and controls the size and the three-dimensional distribution of apatite at larger length scales. These results call into question recent consensus in the literature on the need for Ca-rich non-collagenous proteins for collagen mineralization to occur in vivo. Our model is based on a collagen/apatite self-assembly process that combines the ability to mimic the in vivo extracellular fluid with three major features inherent to living bone tissue, that is, high fibrillar density, monodispersed fibrils and long-range hierarchical organization.

Fibroblasts within concentrated collagen hydrogels favour chronic skin wound healing.

Apligraf(®), a skin substitute currently used in skin chronic wound treatment, acts as a source of macromolecules and cytokines to promote wound healing. Normal collagen hydrogel (NCH), obtained from collagen at low concentration (0.66 mg/ml), is the base of the dermal layer. Apligraf has several drawbacks, such as poor persistence of fibroblasts within the normal collagen hydrogel. In the present study we have evaluated concentrated collagen hydrogels at 5 mg/ml (CCH5s) as dermal substitutes for the treatment of skin chronic wounds. The effect of raised collagen concentration on hydrogel stability, cell growth, apoptosis and fibroblast phenotype was evaluated over 21 days in culture. In contrast to NCHs, CCH5s were more stable because no contraction was observed during the first week. CCH5 favoured cell proliferation and protected fibroblasts against apoptosis. At day 21, cell number assessed in CCH5 was around one million, i.e about 10 times higher than in NCH. Matrix metalloproteinases detection appeared lower in CCH5 than in NCH. In CCH5, fibroblasts exhibited a sustained collagen I gene expression for 14 days, while it was inhibited from day 4 in NCH. Moreover, gene expression of KGF was constant in CCH5 and that of VEGFA increased from day 7. Taken together, our results demonstrate that concentrated collagen hydrogels at 5 mg/ml can be considered as new candidates for cell therapy in chronic skin wounds. They are stable, enhance cell viability and allow gene expression of matrix macromolecules and cytokines involved in re-epithelialization or neovascularization.

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.

Dense fibrillar collagen matrices sustain osteoblast phenotype in vitro and promote bone formation in rat calvaria defect.

Two pure collagen materials were prepared from acidic collagen solutions at 5 and 40 mg/mL. Benefits of collagen concentration on bone repair were evaluated in vitro with human calvaria cells and in vivo in a rat cranial defect. Both materials exhibited specific structures, 5 mg/mL was soft with an open porous network of fibrils; 40 mg/mL was stiffer with a plugged surface and bundles of collagen fibrils. Osteoblasts seeded on 5 mg/mL formed an epithelioid layer with ultrastructural characteristics of mature osteoblasts and induced mineralization. Numerous osteoblasts migrated inside 5 mg/mL, triggering reorganization of their actin cytoskeleton, whereas on 40 mg/mL osteoblasts remained in a resting state. In rat calvaria defects, both materials induced active bone formation. Dual-energy X-ray absorption bone area measures after 4 weeks averaged 84.0% with 5 mg/mL, 88.4% with 40 mg/mL, and 36.7% in the controls (p < 0.05). Tartrate-resistant acid phosphatase-positive giant cells releasing amounts of metalloproteinase-2 progressively degraded the implants at 76.5% with 5 mg/mL and 38.2% with 40 mg/mL (p < 0.05), whereas alkaline phosphatase-positive osteoprogenitors invaded collagen remnant. Hence, the dense structure of collagen materials allowed cell invasion and raise their mechanical behavior without addition of chemical cross-linkers. Collagen concentration can be tuned to form 3D matrices for in vitro investigations or to fit degradation rate to different bone repair purposes.

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.

Silica-collagen bionanocomposites as three-dimensional scaffolds for fibroblast immobilization.

Silica-collagen bionanocomposite hydrogels were obtained by addition of silica nanoparticles to a protein suspension followed by neutralization. Electron microscopy studies indicated that larger silica nanoparticles (80 nm) do not interact strongly with collagen, whereas smaller ones (12 nm) form rosaries along the protein fibers. However, the composite network structurally evolved with time due to the contraction of the cells and the dissolution of the silica nanoparticles. When compared to classical collagen hydrogels, these bionanocomposite materials showed lower surface contraction in the short term (1 week) and higher viability of entrapped cells in the long term (3 weeks). A low level of gelatinase MMP2 enzyme expression was also found after this period. Several proteins involved in the catabolic and anabolic activity of the cells could also be observed by immunodetection techniques. All these data suggest that the bionanocomposite matrices constitute a suitable environment for fibroblast adhesion, proliferation and biological activity and therefore constitute an original three-dimensional environment for in vitro cell culture and in vivo applications, in particular as biological dressings.

Collagen supramolecular and suprafibrillar organizations on osteoblasts long-term behavior: benefits for bone healing materials.

This study compares the behavior of osteoblastic cells seeded on three structurally distinct collagen-based materials. Adhesion and long-term behavior were evaluated in vitro in regard to collagen scaffolds forming loose or dense fibrillar networks or exempt of fibrils. In this purpose collagen solutions at concentrations of 5 and 40 mg/mL were processed by freeze-drying or by sol/gel fibrillogenesis to form either sponges or hydrogels. Macroscopic and microscopic images of sponges showed a light material exhibiting large pores surrounded by dense collagen walls made of thin unstriated microfibrils of 20 nm in diameter. In comparison collagen hydrogels are more homogeneous materials, at 5 mg/mL the material consists of a regular network of cross-striated collagen fibrils of 100 nm in diameter. At 40 mg/mL the material appears stiffer, the ultrastructure exhibits cross-striated collagen fibrils packed in large bundles of 300-800 nm of width. Human osteoblastic cells seeded on top of the 5 mg/mL matrices exhibit a squared shaped osteoblast-like morphology over 28 days of culture and express both alkaline phosphatase and osteocalcin. Osteoblastic cells seeded on top of sponges or of 40 mg/mL matrices exhibit both flat and elongated resting-osteoblast morphology. Osteoblastic cells have mineralized the three collagen-based materials after 28 days of culture but collagen sponges spontaneously mineralized in absence of cells. These results highlight, in an in vitro cell culture approach, the benefit of fibrils and of dense fibrillar networks close to in vivo-like tissues, as positive criteria for new bone tissue repair materials.

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.

Concentrated collagen hydrogels as dermal substitutes.

Collagen hydrogels first appeared promising for skin repair. Unfortunately, their extensive contraction and their poor mechanical properties constituted major disadvantages toward their utilization as permanent graft. The present study has investigated a way to correct these drawbacks by increasing the collagen concentration in controlled conditions. Concentrated collagen hydrogels (CCH) at 1.5, 3 and 5mg/ml were obtained. The effect of raised collagen concentration on contraction, cell growth and remodeling activities was evaluated for 21 days in culture. Subsequently, in vivo integration of CCH and normal collagen hydrogels (NCH) was assessed. Compared to NCH, CCH contraction was delayed and smaller. At day 21, surface area of CCH at 3mg/ml was 18 times more important than that of NCH. Whatever the initial fibroblast density, CCH favored cell growth that reached about 10 times the initial cell number at day 21; cell proliferation was inhibited in NCH. Gelatinase A activities appeared lower in CCH than within NCH. In vivo studies in rats revealed a complete hydrolysis of NCH 15 days after implantation. In contrast, CCH at 3mg/ml was still present after 30 days. Moreover, CCH showed cell colonization, neovascularization and no severe inflammatory response. Our results demonstrate that concentrated collagen hydrogels can be considered as new candidates for dermal substitution because they are is easy to handle, do not contract drastically, favor cell growth, and can be quickly integrated in vivo.

Dense fibrillar collagen matrices: a model to study myofibroblast behaviour during wound healing.

Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype.

Dense tissue-like collagen matrices formed in cell-free conditions.

A new protocol was developed to produce dense organized collagen matrices hierarchically ordered on a large scale. It consists of a two stage process: (1) the organization of a collagen solution and (2) the stabilization of the organizations by a sol-gel transition that leads to the formation of collagen fibrils. This new protocol relies on the continuous injection of an acid-soluble collagen solution into glass microchambers. It leads to extended concentration gradients of collagen, ranging from 5 to 1000 mg/ml. The self-organization of collagen solutions into a wide array of spatial organizations was investigated. The final matrices obtained by this procedure varied in concentration, structure and density. Changes in the liquid state of the samples were followed by polarized light microscopy, and the final stabilized gel states obtained after fibrillogenesis were analyzed by both light and electron microscopy. Typical organizations extended homogeneously by up to three centimetres in one direction and several hundreds of micrometers in other directions. Fibrillogenesis of collagen solutions of high and low concentrations led to fibrils spatially arranged as has been described in bone and derm, respectively. Moreover, a relationship was revealed between the collagen concentration and the aggregation of and rotational angles between lateral fibrils. These results constitute a strong base from which to further develop highly enriched collagen matrices that could lead to substitutes that mimic connective tissues. The matrices thus obtained may also be good candidates for the study of the three-dimensional migration of cells.

Type I collagen, a versatile liquid crystal biological template for silica structuration from nano- to microscopic scales.

Type I collagen is a suitable and versatile template for the structuration of silica at different length scales.

Liquid crystalline assemblies of collagen in bone and in vitro systems.

Precise descriptions of the three-dimensional arrangements of collagen in bone are essential to understand the mechanical properties of this complex tissue. Transmission electron microscopy (TEM) analysis of decalcified human compact bone in section reveals characteristic patterns forming regular series of nested arcs. Such patterns are a direct consequence of an organization described as a twisted plywood and relate the distribution of collagen fibrils in osteons with that of molecules in cholesteric liquid crystals. The hypothesis that liquid crystalline properties are involved in the morphogenesis of dense collagen matrices was supported by data obtained in vitro. At a molecular level, acid-soluble collagen molecules spontaneously assemble, at concentrations of 50mg/ml or more, in precholesteric-banded patterns and cholesteric phases, identified by polarized light microscopy. In a more physiological context, these results were conforted, with the precursor molecule of collagen, procollagen, soluble at neutral pH. This protein spontaneously forms liquid crystalline precholesteric phases corresponding to banded patterns and birefringent cords. Stabilization of the liquid crystalline collagen, induced by pH modification and fibril formation, shows characteristic morphologies in TEM, which directly mimic arrays described in vivo. Undulating fibrils are indeed similar to crimp morphologies described in tendons and continuously twisting fibrils, and give rise to arced patterns similar to supra-molecular architectures identified in compact bone.