Search Results (15)

An innovative strategy for collagen self-assembly with accelerated intra and extrafibrillar mineralisation is introduced to generate bone scaffolds with biomimetic properties. This method, termed Rapid Fibrillogenic Mineralisation (RFM), leverages coprecipitation with 10× Simulated Body Fluid (10× SBF) during fibril formation to maximise nucleation, particularly within intrafibrillar zones at molecular termini. Densification is achieved within minutes via plastic compression driven by capillary action, producing bone-like scaffold density without compromising the collagen matrix. Transmission electron microscopy confirms intrafibrillar hydroxyapatite crystals within 15 min, while X-ray diffraction demonstrates distinct HA peaks across groups. Scanning electron microscopy verified extrafibrillar mineralisation after 4 h, with saturation by 6 h, yielding ‘nanoflower’ crystal clusters. Infrared spectra showed increased carbonate content over time, indicating lattice substitutions characteristic of natural bone. Enhanced mineralisation translated into significant mechanical gains as Dynamic Mechanical Analysis revealed compressive moduli approaching cancellous bone (up to 283 ± 31 MPa). In addition, a decrease in the piezoelectric coefficient occurs with increased mineralisation process, highlighting the effects of mineral inclusions on collagen fibre composition and anisotropy. Biologically, mineralised scaffolds supported cellular growth compared to collagen controls. RFM thus enables rapid, reproducible fabrication of biomimetic bone scaffolds that closely emulate native mineralisation patterns and mechanical behaviour. Beyond offering a practical route for scaffold production in tissue engineering, the process also provides new insights into bone physiology and in vitro modelling. By reshaping collagen into a synthetic echo of nature’s bone, RFM establishes a rapid approach for designing functional biomaterials with translational potential. Full article

Mineralising avian tendon is a widely used experimental model for studying collagen-guided mineralisation. Yet, the three-dimensional organisation and topology of its internal canal system have never been quantitatively characterised. We combined high-resolution micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to provide the first morphometric and topological analysis of the canalicular network in mineralised turkey gastrocnemius tendon. micro-CT revealed that unmineralised canals occupy approximately 34.6% of the mineralised tissue volume and form a single continuously connected network (99.8% of void volume), with a connectivity density of ~1.3 × 10² mm⁻³, a fractal dimension of 2.58, a degree of anisotropy DA = 0.87 [BoneJ convention, range 0–1], and a closed-loop topology. SEM revealed marked ultrastructural heterogeneity of the mineral phase across fascicle cross-sections, consistent with graded intrafibrillar-to-interfibrillar deposition. These findings establish the first quantitative morphometric framework for physiologically mineralising collagen tissue and support the use of turkey gastrocnemius tendon as a tractable model for studying mineralisation dynamics, enthesis biology, and the design of biomimetic scaffolds with controlled porosity and anisotropy. Full article

Purpose: Collagen membranes with biomimetic mineralization are emerging as promising materials for bone regeneration, owing to their high biocompatibility. In this study, we developed a biogenic collagen membrane by combining citrate (C) pretreatment and carboxymethyl chitosan (CMC)-mediated mineralization and further evaluated its bone healing potential. Methods: C-CMC collagen membranes were prepared by lyophilization. The mineral composition and content were tested through X-ray diffraction (XRD), Fourier transform infrared (FTIR), and thermogravimetric analysis (TGA). The micromorphology was observed using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning probe microscopy (SPM). Physical and mechanical properties, including the swelling rate, porosity, hydrophilicity, tensile strength, Young’s modulus, degradation, and barrier function, were also evaluated. Bone mesenchymal stem cells (BMSCs) were cultured in vitro to observe their behavior. An in vivo critical-size rat calvarial defect model was used to validate the effects of the membrane on bone regeneration. Results: The C-CMC collagen membrane was successfully synthesized as a collagen–hydroxyapatite complex with intrafibrillar mineralization, exhibiting improved mechanical properties and an optimal swelling rate, porosity, hydrophilicity, and degradation rate. Additionally, the C-CMC collagen membrane promoted BMSC proliferation, adhesion, and osteogenesis while preventing epithelial cell infiltration. In vivo experiments indicated that C-CMC collagen membranes significantly stimulated bone regeneration without causing systemic toxicity. Conclusions: Our findings suggest that the C-CMC collagen membrane possesses satisfactory physical and mechanical properties, along with good biocompatibility and efficacy in bone defect regeneration, making it a potential candidate for a bioactive guided bone regeneration membrane in clinical applications. Full article

Collagen fibrils hierarchically assemble from microscale to macroscale, which endows the natural composite bone with good mechanical properties and remodeling functions. Revealing the intrafibrillar growth process of hydroxyapatite nanocrystals of collagen will guide the research of bone repair or collagen-based composites. Herein, we investigated the mineralization of multiscale collagen matrices and strongly proved the intrafibrillar hydroxyapatite nanocrystals in the collagen fibrils. The hydroxyapatite nanocrystals were deposited within collagen fibrils with co-orientation along the (002) crystal plane, which is the longitude of the fibril. The whole growth process was captured by TEM to demonstrated the five stages of the intrafibrillar growth process of hydroxyapatite nanocrystals. The infiltration and transformation of amorphous calcium phosphate in isolated collagen fibrils are both demonstrated. The intrafibrillar growth process of hydroxyapatite nanocrystals in collagen film was also investigated, showing that the growth area of collagen films increased linearly with time and the growth process. By studying the in situ mineralization under different reaction conditions, the kinetic equation of the mineralized area of collagen film under each condition was obtained, and the optimal hydroxyapatite mineralized solution was proved to be a solution with polyacrylic acid of 50 μG/mL and a pH of 7.5. Our work provides more detailed information of the growth process of HAP nanocrystals during the mineralization of collagen at different scales and would contribute to future research on the formation process of more minerals in collagen. Full article

Overcoming the short lifespan of current dental adhesives remains a significant clinical need. Adhesives rely on formation of the hybrid layer to adhere to dentin and penetrate within collagen fibrils. However, the ability of adhesives to achieve complete enclosure of demineralized collagen fibrils is recognized as currently unattainable. We developed a peptide-based approach enabling collagen intrafibrillar mineralization and tested our hypothesis on a type-I collagen-based platform. Peptide design incorporated collagen-binding and remineralization-mediating properties using the domain structure conservation approach. The structural changes from representative members of different peptide clusters were generated for each functional domain. Common signatures associated with secondary structure features and the related changes in the functional domain were investigated by attenuated total reflectance Fourier-transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopy, respectively. Assembly and remineralization properties of the peptides on the collagen platforms were studied using atomic force microscopy (AFM). Mechanical properties of the collagen fibrils remineralized by the peptide assemblies was studied using PeakForce-Quantitative Nanomechanics (PF-QNM)-AFM. The engineered peptide was demonstrated to offer a promising route for collagen intrafibrillar remineralization. This approach offers a collagen platform to develop multifunctional strategies that combine different bioactive peptides, polymerizable peptide monomers, and adhesive formulations as steps towards improving the long-term prospects of composite resins. Full article

Objective: To observe ultrastructural changes during the process of carboxymethyl chitosan (CMC)-mediated intrafibrillar mineralization, we evaluated the biomimetic remineralization potential of CMC in type-I collagen fibrils and membranes, and further explored the bond strength as well as the bond interfacial integrity of the biomimetic remineralized artificial caries-affected dentin (ACAD). Methods: A mineralized solution containing 200 μg/mL CMC was used to induce type-I collagen biomimetic remineralization in ACAD, while traditional mineralization without CMC was used as a control. The process and pattern of mineralization were investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) as well as structured illumination microscopy (SIM). The Vickers hardness test was used to quantify the dentin hardness, while the microtensile bond strength (µTBS) test was used to assess the bond strength and durability. The bond interfacial integrity was evaluated by a confocal laser scanning microscope (CLSM). Results: TEM, SEM, and SIM images showed that CMC had a positive effect on stabilizing amorphous calcium phosphate (ACP) and promoting intrafibrillar mineralization, while extrafibrillar mineralization was formed without CMC. Furthermore, hardness evaluation and µTBS proved that CMC significantly increased dentin hardness and bond strength. CLSM indicated that CMC could create a significantly better bond interfacial integrity with less of a micro-gap in ACAD. Significance: CMC possessed the ability to promote intrafibrillar mineralization and remineralization in demineralized caries dentin lesions, as well as improve bond performance, which implied its potential in carious dentin demineralization or dentin hypersensitivity and possibly even as a possible material for indirect pulp-capping, to deal with deep caries. Highlights: CMC possessed the ability to induce intrafibrillar mineralization effectively; the bond strength and bond durability of demineralized caries dentin were improved via CMC-induced remineralization; the CMC-induced remineralization complex is a potential material for indirect pulp-capping, to deal with deep caries. Full article

Cellular strategies and regulation of their crystallization mechanisms are essential to the formation of biominerals, and harnessing these strategies will be important for the future creation of novel non-native biominerals that recapitulate the impressive properties biominerals possess. Harnessing these biosynthetic strategies requires an understanding of the interplay between insoluble organic matrices, mineral precursors, and soluble organic and inorganic additives. Our long-range goal is to use a sea anemone model system (Nematostella vectensis) to examine the role of intrinsically disordered proteins (IDPs) found in native biomineral systems. Here, we study how ambient temperatures (25–37 °C) and seawater solution compositions (varying NaCl and Mg ratios) will affect the infiltration of organic matrices with calcium carbonate mineral precursors generated through a polymer-induced liquid-precursor (PILP) process. Fibrillar collagen matrices were used to assess whether solution conditions were suitable for intrafibrillar mineralization, and SEM with EDS was used to analyze mineral infiltration. Conditions of temperatures 30 °C and above and with low Mg:Ca ratios were determined to be suitable conditions for calcium carbonate infiltration. The information obtained from these observations may be useful for the manipulation and study of cellular secreted IDPs in our quest to create novel biosynthetic materials. Full article

Mineralized collagen is the basic unit in hierarchically organized natural bone with different structures. Polyacrylic acid (PAA) and periodic fluid shear stress (FSS) are the most common chemical and physical means to induce intrafibrillar mineralization. In the present study, non-mineralized collagen, extrafibrillar mineralized (EM) collagen, intrafibrillar mineralized (IM) collagen, and hierarchical intrafibrillar mineralized (HIM) collagen induced by PAA and FSS were prepared, respectively. The physical and chemical properties of these mineralized collagens with different microstructures were systematically investigated afterwards. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that mineralized collagen with different microstructures was prepared successfully. The pore density of the mineralized collagen scaffold is higher under the action of periodic FSS. Fourier transform infrared spectroscopy (FTIR) analysis showed the formation of the hydroxyapatite (HA) crystal. A significant improvement in the pore density, hydrophilicity, enzymatic stability, and thermal stability of the mineralized collagen indicated that the IM collagen under the action of periodic FSS was beneficial for maintaining collagen activity. HIM collagen fibers, which are prepared under the co-action of periodic FSS and sodium tripolyphosphate (TPP), may pave the way for new bone substitute material applications. Full article

At the molecular scale, bone is mainly constituted of type-I collagen, hydroxyapatite, and water. Different fractions of these constituents compose different composite materials that exhibit different mechanical properties at the nanoscale, where the bone is characterized as a fiber, i.e., a bundle of mineralized collagen fibrils surrounded by water and hydroxyapatite in the extra-fibrillar volume. The literature presents only models that resemble mineralized collagen fibrils, including hydroxyapatite in the intra-fibrillar volume only, and lacks a detailed prescription on how to devise such models. Here, we present all-atom bone molecular models at the nanoscale, which, differently from previous bone models, include hydroxyapatite both in the intra-fibrillar volume and in the extra-fibrillar volume, resembling fibers in bones. Our main goal is to provide a detailed prescription on how to devise such models with different fractions of the constituents, and for that reason, we have made step-by-step scripts and files for reproducing these models available. To validate the models, we assessed their elastic properties by performing molecular dynamics simulations that resemble tensile tests, and compared the computed values against the literature (both experimental and computational results). Our results corroborate previous findings, as Young’s Modulus values increase with higher fractions of hydroxyapatite, revealing all-atom bone models that include hydroxyapatite in both the intra-fibrillar volume and in the extra-fibrillar volume as a path towards realistic bone modeling at the nanoscale. Full article

With the aging population, there is a growing need for mineralized tissue restoration and synthetic bone substitutes. Previous studies have shown that a polymer-induced liquid-precursor (PILP) process can successfully mineralize collagen substrates to achieve compositions found in native bone and dentin. This process also leads to intrafibrillar apatitic crystals with their [001] axes aligned roughly parallel to the long axis of the collagen fibril, emulating the nanostructural organization found in native bone and dentin. When demineralized bovine bone was remineralized via the PILP process using osteopontin (OPN), the samples were able to activate mouse marrow-derived osteoclasts to similar levels to those of native bone, suggesting a means for fabricating bioactive bone substitutes that could trigger remodeling through the native bone multicellular unit (BMU). In order to determine if OPN derived from bovine milk could be a cost-effective process-directing agent, the mineralization of type I collagen scaffolds using this protein was compared to the benchmark polypeptide of polyaspartic acid (sodium salt; pAsp). In this set of experiments, we found that OPN led to much faster and more uniform mineralization when compared with pAsp, making it a cheaper and commercially attractive alternative for mineralized tissue restorations. Full article

Biomineralization is the process by which organisms produce hard inorganic matter from soft tissues with outstanding control of mineral deposition in time and space. For this purpose, organisms deploy a sophisticated “toolkit” that has resulted in significant evolutionary innovations, for which calcium phosphate (CaP) is the biomineral selected for the skeleton of vertebrates. While CaP mineral formation in aqueous media can be investigated by studying thermodynamics and kinetics of phase transitions in supersaturated solutions, biogenic mineralization requires coping with the inherent complexity of biological systems. This mainly includes compartmentalization and homeostatic processes used by organisms to regulate key physiological factors, including temperature, pH and ion concentration. A detailed analysis of the literature shows the emergence of two main views describing the mechanism of CaP biomineralization. The first one, more dedicated to the study of in vivo systems and supported by researchers in physiology, often involves matrix vesicles (MVs). The second one, more investigated by the physicochemistry community, involves collagen intrafibrillar mineralization particularly through in vitro acellular models. Herein, we show that there is an obvious need in the biological systems to control both where and when the mineral forms through an in-depth survey of the mechanism of CaP mineralization. This necessity could gather both communities of physiologists and physicochemists under a common interest for an enzymatic approach to better describe CaP biomineralization. Both homogeneous and heterogeneous enzymatic catalyses are conceivable for these systems, and a few preliminary promising results on CaP mineralization for both types of enzymatic catalysis are reported in this work. Through them, we aim to describe the relevance of our point of view and the likely findings that could be obtained when adding an enzymatic approach to the already rich and creative research field dealing with CaP mineralization. This complementary approach could lead to a better understanding of the biomineralization mechanism and inspire the biomimetic design of new materials. Full article

Hydroxyapatite (HA) reinforced collagen fibrils serve as the basic building blocks of natural bone and dentin. Mineralization of collagen fibrils play an essential role in ensuring the structural and mechanical functionalities of hard tissues such as bone and dentin. Biomineralization of collagen can be divided into intrafibrillar and extrafibrillar mineralization in terms of HA distribution relative to collagen fibrils. Intrafibrillar mineralization is termed when HA minerals are incorporated within the gap zone of collagen fibrils, while extrafibrillar mineralization refers to the minerals that are formed on the surface of collagen fibrils. However, the mechanisms resulting in these two types of mineralization still remain debatable. In this review, the evolution of both classical and non-classical biomineralization theories is summarized. Different intrafibrillar mineralization mechanisms, including polymer induced liquid precursor (PILP), capillary action, electrostatic attraction, size exclusion, Gibbs-Donnan equilibrium, and interfacial energy guided theories, are discussed. Exemplary strategies to induce biomimetic intrafibrillar mineralization using non-collagenous proteins (NCPs), polymer analogs, small molecules, and fluidic shear stress are discussed, and recent applications of mineralized collagen fibers for bone regeneration and dentin repair are included. Finally, conclusions are drawn on these proposed mechanisms, and the future trend of collagen-based materials for bone regeneration and tooth repair is speculated. Full article

The hierarchical structure of bone and intrinsic material properties of its two primary constituents, carbonated apatite and fibrillar collagen, when being synergistically organized into an interpenetrating hard-soft composite, contribute to its excellent mechanical properties. Lamellar bone is the predominant structural motif in mammalian hard tissues; therefore, we believe the fabrication of a collagen/apatite composite with a hierarchical structure that emulates bone, consisting of a dense lamellar microstructure and a mineralized collagen fibril nanostructure, is an important first step toward the goal of regenerative bone tissue engineering. In this work, we exploit the liquid crystalline properties of collagen to fabricate dense matrices that assemble with cholesteric organization. The matrices were crosslinked via carbodiimide chemistry to improve mechanical properties, and are subsequently mineralized via the polymer-induced liquid-precursor (PILP) process to promote intrafibrillar mineralization. Neither the crosslinking procedure nor the mineralization affected the cholesteric collagen microstructures; notably, there was a positive trend toward higher stiffness with increasing crosslink density when measured by cantilever-based atomic force microscopy (AFM) nanoindentation. In the dry state, the average moduli of moderately (X51; 4.8 ± 4.3 GPa) and highly (X76; 7.8 ± 6.7 GPa) crosslinked PILP-mineralized liquid crystalline collagen (LCC) scaffolds were higher than the average modulus of bovine bone (5.5 ± 5.6 GPa). Full article

This in vitro study aimed to accelerate the remineralization of a completely demineralized dentine collagen block in order to regenerate the dentinal microstructure of calcified collagen fibrils by a novel electric field-aided biomimetic mineralization system in the absence of non-collagenous proteins. Completely demineralized human dentine slices were prepared using ethylene diamine tetraacetic acid (EDTA) and treated with guanidine hydrochloride to extract the bound non-collagenous proteins. The completely demineralized dentine collagen blocks were then remineralized in a calcium chloride agarose hydrogel and a sodium hydrogen phosphate and fluoride agarose hydrogel. This process was accelerated by subjecting the hydrogels to electrophoresis at 20 mA for 4 and 12 h. X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) were used to evaluate the resultant calcification of the dentin collagen matrix. SEM indicated that mineral particles were precipitated on the intertubular dentin collagen matrix; these densely packed crystals mimicked the structure of the original mineralized dentin. However, the dentinal tubules were not occluded by the mineral crystals. XRD and EDX both confirmed that the deposited crystals were fluorinated hydroxyapatite. TEM revealed the existence of intrafibrillar and interfibrillar mineralization of the collagen fibrils. A novel electric field-aided biomimetic mineralization system was successfully developed to remineralize a completely demineralized dentine collagen matrix in the absence of non-collagenous proteins. This study developed an accelerated biomimetic mineralization system which can be a potential protocol for the biomineralization of dentinal defects. Full article

The nanostructure of bone has been replicated using a polymer-induced liquid-precursor (PILP) mineralization process. This polymer-mediated crystallization process yields intrafibrillar mineralization of collagen with uniaxially-oriented hydroxyapatite crystals. The process-directing agent, an anionic polymer which we propose mimics the acidic non-collagenous proteins associated with bone formation, sequesters calcium and phosphate ions to form amorphous precursor droplets that can infiltrate the interstices of collagen fibrils. In search of a polymeric agent that produces the highest mineral content in the shortest time, we have studied the influence of various acidic polymers on the in vitro mineralization of collagen scaffolds via the PILP process. Among the polymers investigated were poly-L-aspartic acid (PASP), poly-L-glutamic acid (PGLU), polyvinylphosphonic acid (PVPA), and polyacrylic acid (PAA). Our data indicate that PASP and the combination of PGLU/PASP formed stable mineralization solutions, and yielded nano-structured composites with the highest mineral content. Such studies contribute to our goal of preparing biomimetic bone graft substitutes with composition and structure that mimic bone. Full article