Autologous Versatile Vesicles‐Incorporated Biomimetic Extracellular Matrix Induces Biomineralization

Restoring extracellular matrix (ECM) with supportive and osteoinductive abilities is of great significance for bone tissue regeneration. Current approaches involving cell‐based scaffolds or nanoparticle‐modified biomimic‐ECM have been met with additional biosafety concerns. Herein, the natural biomineralization process is first analyzed and is found that mesenchymal stem cells‐derived extracellular vesicles (EVs) from early and late stages of osteoinduction play different roles during the mineralization process. The functional EVs hierarchically with blood‐derived autohydrogel (AH) are then incorporated to form an osteoinductive biomimetic extracellular matrix (BECM). The alkaline phosphatase‐rich EVs are released from the outer layers to induce osteoblast differentiation during early stages. Thereafter, as the degradation of AH occurred, calcium/phosphorus (Ca/P)‐rich EVs are liberated to promote the nucleation of extracellular mineral crystals. Additionally, BECM contains considerable collagen fibrils that provide additional nucleation sites for crystallites deposition, thus reaching self‐mineralization in situ. In conclusion, this research provides a promising, versatile mineralization‐instructive platform to tackle the challenges faced in bone‐tissue engineering.     

Thermodynamic Aspects of Molluscan Shell Ultrastructural Morphogenesis

Over the years, molluscan shells have become an exemplar model system to study the process of mineral formation by living organisms, the process of biomineralization. Typically, the shells consist of a number of mineralized ultrastructural motifs, each exhibiting a specific mineral‐organic composite architecture. These are made of calcium carbonate building blocks having a well‐defined three‐dimensional morphology that is significantly different from the shape of inorganically formed counterparts. Shell ultrastructures are known to form via a biologically controlled extracellular mineralization pathway in which the organism has no direct control over mineral formation. The cellular tissue, responsible for shell biomineralization, forms an organic framework and sets‐up the physical conditions necessary for the deposition of a specific morphology, whereas the growth of the mineral part of the shell proceeds spontaneously via the process of self‐assembly. In this feature article, the ability to employ thermodynamic models from classical materials science to describe the process of self‐assembly and structural evolution of a variety of shell architectures is reviewed. Having the potential to offer an analytical framework to express ultrastructure formation in time and in space, these models not only provide a deeper insight into shell biomineralization, but also suggest tools for novel composite materials design.     

Appearance of electron-dense segments: indication of possible conformational changes of pre-mineralizing collagen fibrils in the osteoid of rat bones

To elucidate precise mechanisms of appositional mineralization of bone, structural features of mineralizing collagen fibrils of the osteoid in normal and hypocalcaemic rats were examined in detail by transmission electron microscopy. Ultrathin sections of the osteoid of various types of bones of the rats fed with regular or normal calcium diet often displayed electrondense segments in the specific regions of the collagen fibrils located immediately adjacent to the mineralization front or to the mineralization nodules. Such dense segments appeared only after Ur-Pb staining and were more distinct in undecalcified specimens. Dense segments were undetectable in ultrathin sections picked up on ethylene glycol instead of water in the trough, even after Ur-Pb staining. Collagen fibrils in the widened osteoid of hypocalcaemic rats fed with calcium-free diet failed to show electron-dense segments. A careful comparison between the hydrously or anhydrously processed adjacent sections of a normal rat bone indicated a drastic dissolution of electron-dense material from the bone matrix near the mineralization front in hydrously processed sections and, thus, implicated the presence of labile mineral-matrix complexes in the recently mineralized bone matrix. Such labile sediments were readily dissociated within the ultrathin sections while the sections were floating on water and immediately adsorbed onto the pre-mineralizing collagen fibrils, where some conformational changes might have occurred. These data indicate that highly electron-dense segments appearing in the osteoidal collagen fibrils are a type of process-induced product, which indirectly represent possible structural alterations in the segmental portions of pre-mineralizing collagen fibrils in the osteoid of rat bones.     

Hierarchical Intrafibrillar Nanocarbonated Apatite Assembly Improves the Nanomechanics and Cytocompatibility of Mineralized Collagen

Nanoscale replication of the hierarchical organization of minerals in biogenic mineralized tissues is believed to contribute to the better mechanical properties of biomimetic collagen scaffolds. Here, an intrafibrillar nanocarbonated apatite assembly is reported, which has a bone‐like hierarchy, and which improves the mechanical and biological properties of the collagen matrix derived from fibril‐apatite aggregates. A modified biomimetic approach is used, which based on the combination of poly(acrylic acid) as sequestration and sodium tripolyphosphate as templating matrix‐protein analogs. With this modified dual‐analog‐based biomimetic approach, the hierarchical association between collagen and the mineral phase is discerned at the molecular and nanoscale levels during the process of intrafibrillar collagen mineralization. It is demonstrated by nanomechanical testing, that intrafibrillarly mineralized collagen features a significantly increased Young's modulus of 13.7 ± 2.6 GPa, compared with pure collagen (2.2 ± 1.7 GPa) and extrafibrillarly‐mineralized collagen (7.1 ± 1.9 GPa). Furthermore, the hierarchy of the nanocarbonated apatite assembly within the collagen fibril is critical to the collagen matrix's ability to confer key biological properties, specifically cell proliferation, differentiation, focal adhesion, and cytoskeletal arrangement. The availability of the mineralized collagen matrix with improved nanomechanics and cytocompatibility may eventually result in novel biomaterials for bone grafting and tissue‐engineering applications.     

Stabilization of Amorphous Calcium Carbonate with Nanofibrillar Biopolymers

Calcium carbonate is the most abundant biomineral that is biogenically formed with a vast array of nano and microscale features. Among the less stable polymorphs present in mineralized organisms, the most soluble, amorphous calcium carbonate (ACC), formed in chitin exoskeletons of some crustacea, is of particular interest since aqueous stability of isolated ACC is limited to a few hours in the absence of polyanions or magnesium. Here the influence of a selection of biopolymer gels on the mineralization of calcium carbonate is investigated. Mineralization is achieved in all biopolymers tested, but is particularly abundant in collagen hydrogels, in which a significant proportion of the calcium carbonate (≈18%) is found to be amorphous. In dense collagen gels, this amorphous fraction does not crystallize for up to six weeks in deionized water at room temperature. The reason why collagen in particular should stabilize this phase remains obscure, although the results suggest that the fiber diameter, fiber spacing, and the amphoteric nature of collagen fibers are important. Upon immersion in phosphate containing solutions, the calcium carbonate present within the collagen hydrogels is readily converted to carbonated hydroxyapatite, enabling the formation of a stiff bone‐like composite containing 78 wt% mineral, essentially equivalent to cortical bone.     

Ultrastructural and histochemical evaluation of appositional mineralization of circumpulpal dentin at the crown- and root-analog portions of rat incisors

Mineralization of circumpulpal dentin has been interpreted in such a way that predentin matrix is abruptly converted to almost fully mineralized dentin at the mineralization front. A group of investigators pointed out the existence of intermediary layer along the mineralization front of rat incisor dentin and claimed that dentin mineralization is a rather transient process. Owing to a paucity of information, however, the entity of transient mineralization of dentin has remained elusive. Here we confirmed the existence of a lightly mineralized layer (LL) along the mineralization front of rat incisor dentin, recognizable by both light and electron microscopy, in routinely processed specimens. LL less than 3?μm thick was shown to be located along the mineralization front of crown-analog dentin and tapered out toward the root analog of the incisor. Electron microscopy revealed that mineral deposition first occurred in the non-collagenous matrix of LL and that mineralization of collagen fibers took place sometime later at the conventional mineralization front. Microscopic appearance of the mineral phase of LL varied considerably depending on the histological processing of ultrathin sections, thus explaining the inconsistent interpretation of dentin mineralization in previous studies. These data suggest that mineralization of circumpulpal dentin in rat incisors proceeds in a stepwise or a transient manner, initiated by crystal deposition in the non-collagenous matrix followed by massive mineral deposition in collagen fibers at the mineralization front. The thickness of LL where only the non-collagenous matrix is mineralized may vary in relation to differences in the local non-collagenous matrix and also the rate of collagen mineralization in the respective portions of circumpulpal dentin.     

A 3D Cell-Free Bone Model Shows Collagen Mineralization is Driven and Controlled by the Matrix

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization, and different degrees of mineralization where the intrafibrillar and extrafibrillar minerals are intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement are demonstrated. Then, this cell-free in vitro system is explored as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicate that glycosaminoglycans (GAGs) play a more prominent role than generally assumed in the matrix–mineral interactions. The experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.     

Complementarity and Uncertainty in Intrafibrillar Mineralization of Collagen

Biomineralization in vertebrates is a ubiquitous and tightly regulated process which creates hierarchical structures for the skeleton. Because of the lack of understanding and applicability of cell‐based or biological systems to achieve intrafibrillar mineralization, scientists adopted various in vitro methods to elucidate the mechanism of intrafibrillar mineralization. In this article, biomimetic intrafibrillar mineralization of collagen in its wide ramifications is reviewed. It is intriguing how prevailing intrafibrillar mineralization mechanisms derived from two potentially discordant crystallization philosophies were equally adept, depending on the experimental context, at theorizing the formation of calcium phosphate within a fibrillar template. This complementarity is not unique to biomineralization and has precedence in other fundamental physical interpretations. A new intrafibrillar mineralization process based on the use of polycationic process‐directing agent added uncertainty to the use of existing mechanisms in accounting for the observations.     

Oriented Strontium Carbonate Nanocrystals within Collagen Films for Flexible Piezoelectric Sensors

Bone, assembled by mineralized collagen fibrils, displays piezoelectric properties under external stimulation to affect tissue growth. The mineralized collagen fibrils consist of collagen and oriented inorganic nanocrystals. Inspired from the unique structures and piezoelectric effect of mineralized collagen fibrils, the intrafibrillar mineralization of oriented strontium carbonate nanocrystals is achieved in vitro, which also exhibits good piezoelectric properties. The amorphous strontium carbonate precursors penetrate from the gap zones and fill gradually into the whole space within the collagen fibrils, and transform into a co-oriented crystalline phase. Isolated mineralized collagen fibrils with organized SrCO₃ nanocrystals acquire good flexible properties and inverse piezoelectric responses with an effective piezoelectric coefficient of 3.45 pm V⁻¹, much higher than individual collagen (1.12 pm V⁻¹) and SrCO₃ crystals (0.092 pm V⁻¹). These results may indicate that the organic and inorganic components synergistically contribute to the piezoelectric effect of bone. Furthermore, devices of flexible piezoelectric thin films assembled by SrCO₃ mineralized collagen fibrils exhibit a regular open-circuit voltage of 1.2 V under compressive stress and a stable cycling short-circuit current of 80 nA under a bending mode. It can also facilitate the development of promising piezoelectric sensors.     

Osteoconductive Performance of Carbon Nanotube Scaffolds Homogeneously Mineralized by Flow‐Through Electrodeposition

The treatment of bone lesions, including fractures, tumor resection and osteoporosis, is a common clinical practice where bone healing and repair are pursued. It is widely accepted that calcium phosphate‐based materials improve integration of biomaterials with surrounding bone tissue and further serve as a template for proper function of bone‐forming cells. Within this context, mineralization on preformed substrates appears as an interesting and successful alternative for mineral surface functionalization. However, mineralization of “true” 3D scaffolds –in which the magnitude of the third dimension is within the same scale as the other two– is by no means a trivial issue because of the difficulty to obtain a homogeneous mineral layer deposited on the entire internal surface of the scaffold. Herein, a “flow‐through” electrodeposition process is applied for mineralization of 3D scaffolds composed of multiwall carbon nanotubes and chitosan. It is demonstrated that, irrespective of the experimental conditions used for electrodeposition (e.g., time, temperature and voltages), the continuous feed of salts provided by the use of a flow‐through configuration is the main issue if one desires to coat the entire internal structure of 3D scaffolds with a homogeneous mineral layer. Finally, mineralized scaffolds not only showed a remarkable biocompatibility when tested with human osteoblast cells, but also enhanced osteoblast terminal differentiation (as early as 7 days in calcifying media).     

Synthetic Bone-Like Structures Through Omnidirectional Ceramic Bioprinting in Cell Suspensions

The integration of hierarchical structure, chemistry, and functional activity within tissue-engineered scaffolds is of great importance in mimicking native bone tissue. Bone is a highly mineralized tissue which forms at ambient conditions by continuous crystallization of the mineral phase within an organic matrix in the presence of bone residing cells. Despite recent advances in the biofabrication of complex engineered tissues, replication of the heterogeneity of bone microenvironments has been a major challenge in constructing biomimetic bone scaffolds. Herein, inspired by the bone biomineralization process, the first example of bone mimicking constructs by 3D writing of a novel apatite-transforming ink in a supportive microgel matrix with living cells is demonstrated. Using this technique, complex bone-mimicked constructs are made at room temperature without requiring invasive chemicals, radiation, or postprocessing steps. This study demonstrates that mineralized constructs can be deposited within a high density of stem cells, directing the cellular organization, and promoting osteogenesis in vitro. These findings offer a new strategy for fabrication of bone mimicking constructs for bone tissue regeneration with scope to generate custom bone microenvironments for disease modeling, multicellular delivery, and in vivo bone repair.     

Statistical analysis of collagen alignment in ligaments byscale-space analysis

Injuries to ligaments in the knee are common in sports and other physical activities. Some clinical methods are available for qualitatively evaluating the degree of ligament injury and healing. It is, however, desirable to objectively assess the healing of ligaments and to predicate optimal treatment on quantitative measurements of their structure. Information such as areas of coverage and spatial orientations of collagen fibrils, for example, may provide important information about the internal structure of ligament tissues. Since normal ligament tissues are made up of collagen fibrils which are highly organized, they can be considered as oriented piecewise linear patterns. In this paper, we propose a computational technique for statistical analysis of collagen alignment in ligament images using the scale-space approach. In this method, a ligament image is preprocessed by a sequence of filters which are second derivatives of two-dimensional Gaussian functions with different scales. This gives a set of zero-crossing maps (the scale space) from which a stability map is generated. Significant linear patterns are captured by analyzing the stability map. The directional information in terms of orientation distributions of the collagen fibrils in the image and the area covered by the fibrils in specific directions are extracted for statistical analysis. Examples illustrating the performance of this method with scanning electron microscope images of the collagen fibrils in healing rabbit medial collateral ligaments are presented in this paper.     

Imidazole‐Grafted Nanogels for the Fabrication of Organic–Inorganic Protein Hybrids

Here, a platform for the development of highly responsive organic–inorganic enzyme hybrids is provided. The approach entails a first step of protein engineering, in which individual enzymes are armored with a porous nanogel decorated with imidazole motifs. In a second step, by mimicking the biomineralization mechanism, the assembly of the imidazole nanogels with CuSO₄ and phosphate salts is triggered. A full characterization of the new composites reveals the first reported example in which the assembly mechanism is triggered by the sum of Cu(II)–imidazole interaction and Cu₃(PO₄)₂ inorganic salt formation. It is demonstrated that the organic component of the hybrids, namely the imidazole‐modified polyacrylamide hydrogel, provides a favorable spatial distribution for the enzyme. This results in enhanced conversion rates, robustness of the composite at low pH values, and a remarkable thermal stability at 65 °C, exhibiting 400% of the activity of the mineralized enzyme lacking the organic constituent. Importantly, unlike in previous works, the protocol applies to the use of a broad range of transition metal cations (including mono‐, di‐, and trivalent cations) to trigger the mineralization mechanism, which eventually broadens the chemical and structural diversity of organic–inorganic protein hybrids.     

Phase‐Transited Lysozyme as a Universal Route to Bioactive Hydroxyapatite Crystalline Film

A key factor for successful design of bioactive complex, organic–inorganic hybrid biomaterials is the facilitation and control of adhesion at interfaces, as many current synthetic biomaterials are inert, lacking interfacial bioactivity. In this regard, the development of a simple, unified way to biofunctionalize diverse organic and inorganic materials toward biomineralization remains a critical challenge. In this report, a universal biomimetic mineralization route that can be applied to virtually any type and morphology of scaffold materials is provided to induce nucleation and growth of hydroxyapatite (HAp) crystals based on phase‐transited lysozyme (PTL) coating. Surface‐anchored abundant functional groups in the PTL enrich the interface with strongly bonded calcium ions, facilitating the formation of HAp crystals in simulated body fluid with the morphology and alignment being similar to that observed in natural HAp in mineralized tissues. By the adhesion of amyloid contained in the PTL, such protein assembly could readily integrate HAp on ceramics, metals, semiconductors, and synthetic polymers irrespective of their size and morphology, with robust bonding stability and corresponding ultralow wear extent under normal bone pressure. This strategy successfully improves the in vivo osteoconductivity of Ti‐based implant, underpinning the expectation for such biomaterial in future biointerface and tissue engineering.     

Silk Fibroin‐Regulated Crystallization of Calcium Carbonate

Mollusk shell is one of the best studied of all calcium carbonate biominerals. Its silk‐like binder‐matrix protein plays a pivotal role during the formation of aragonite crystals in the nacre sheets. Here, we provide novel experimental insights into the interaction of mineral and protein compounds using a model system of reconstituted Bombyx mori silk fibroin solutions serving as templates for the crystallization of calcium carbonate (CaCO₃). We observed that the inherent (self‐assembling) aggregation process of silk fibroin molecules affected both the morphology and crystallographic polymorph of CaCO₃ aggregates. This combination fostered the growth of a novel, rice‐grain‐shaped protein/mineral hybrid with a hollow structure with an aragonite polymorph formed after ripening. Our observations suggest new hypotheses about the role of silk‐like protein in the natural biomineralization process, but it may also serve to shed light on the formation process of those ‘ersatz’ hybrids regulated by artificially selected structural proteins.     

A Fourier domain directional filtering method for analysis of collagen alignment in ligaments

Collagen fibers and their component fibrils make up the protenaceous " backbone" of most tissues and provide the majority of their resistance to tensile loading. Spatial orientation of collagen fibrils is an important factor in determining tissue properties. This is particularly true in ligament tissue, since ligaments must be loose enough to allow joints to move but tight enough to prevent joint surfaces from separating. A method is presented here to reproducibly quantify this collagen arrangement, which should be useful in studies on ligament healing and growth.     

A Drug-Mineralized Hydrogel Orchestrated by Spontaneous Dynamic Mineralization

Fabrication of mineralized materials through the nature-mimicking dynamic biomineralization process has attracted great interest but remains a huge challenge till now. Here, a drug-mineralized hydrogel (DMH) with spontaneous dynamic mineralization behaviors is reported, which highly mimics the dynamic mineralization process of natural tissues. The hydrogel is facilely fabricated via directly mixing sodium hyaluronate (HA) with alendronate sodium (ADA) and CaCl₂ in deionized water. The resultant hydrogel possesses a white-to-semi-transparent transition and a soft-to-hard transition during incubation at 37 °C. Mechanism studies reveal that the spontaneous dynamic change of the hydrogel is ascribed to the HA-regulated dynamic mineralization of ADA/Ca²⁺ complexes, where amorphous ADA/Ca²⁺ complexes will gradually transform into mineral crystals with the prolongation of incubation time. Owing to the dynamic transformation process, the DMH shows excellent injectability and moldability at the initial stage. More importantly, the DMH exhibits a sustained release behavior of ADA in vitro, superior adhesiveness with bone, biodegradability, and good biocompatibility. As a result, the developed DMH demonstrates promising uses for accelerating bone repair in a rat cranial defect model. Overall, the research provides a general strategy to prepare drug-mineralized hydrogels with spontaneous dynamic mineralization behaviors, which may find potential uses in various biomedical fields.     

Mussel‐Inspired Polydopamine Coating as a Universal Route to Hydroxyapatite Crystallization

Bone tissue is a complex biocomposite material with a variety of organic (e.g., proteins, cells) and inorganic (e.g., hydroxyapatite crystals) components hierarchically organized with nano/microscale precision. Based on the understanding of such hierarchical organization of bone tissue and its unique mechanical properties, efforts are being made to mimic these organic–inorganic hybrid biocomposites. A key factor for the successful designing of complex, hybrid biomaterials is the facilitation and control of adhesion at the interfaces, as many current synthetic biomaterials are inert, lacking interfacial bioactivity. In this regard, researchers have focused on controlling the interface by surface modifications, but the development of a simple, unified way to biofunctionalize diverse organic and inorganic materials remains a critical challenge. Here, a universal biomineralization route, called polydopamine‐assisted hydroxyapatite formation (pHAF), that can be applied to virtually any type and morphology of scaffold materials is demonstrated. Inspired by the adhesion mechanism of mussels, the pHAF method can readily integrate hydroxyapatites on ceramics, noble metals, semiconductors, and synthetic polymers, irrespective of their size and morphology (e.g., porosity and shape). Surface‐anchored catecholamine moieties in polydopamine enriches the interface with calcium ions, facilitating the formation of hydroxyapatite crystals that are aligned to the c‐axes, parallel to the polydopamine layer as observed in natural hydroxyapatites in mineralized tissues. This universal surface biomineralization can be an innovative foundation for future tissue engineering.     

Biomimetic Mineralized Organic–Inorganic Hybrid Macrofiber with Spider Silk‐Like Supertoughness

Spider silk fibers (SSF) have a hierarchical structure composed of proteins with highly repetitive sequences and biomineralization is sophisticated in hierarchical organic–inorganic constructions. By using inorganic hydroxyapatite (HAP) and organic polyvinyl alcohol (PVA) to simulate the rigid crystalline and flexible amorphous protein blocks of SSF, respectively, biomimetic mineralization is herein attempted for the large‐scale preparation of SSF‐like macrofibers with a hierarchical ordered structure, a superhigh tensile strength of 949 ± 38 MPa, a specific toughness of 296 ± 12 J g^?1, and a stretch ability of 80.6%. The hybrid macrofibers consist of microfibers, and their outstanding performance (e.g., extreme tolerance to temperatures ranging from ?196 to 80 °C and superior ability to inhibit the transverse growth of cracks) is attributed to the hierarchical arrangement as well as the organic–inorganic integrated structure within the oriented mineralized polymer chains. The biomineralization‐inspired technique provides a promising tactic that can be used to synthesize functional organic–inorganic fibers that are structurally complex and, furthermore, industrially manufacture SSF‐like artificial fibers with a supertoughness.     

Multiphase Biomineralization: Enigmatic Invasive Siliceous Diatoms Produce Crystalline Calcite

Diatoms are considered unicellular eukaryotic organisms exclusively depositing biogenic silica. Heretofore there has been no report of calcification by these algae. Here it is shown that calcium carbonate within the stalks of Didymosphenia geminata, a nuisance species that has prolifically colonized streams and rivers globally, is biogenic in origin and occurs as a network of calcite nanofibers. The nanofibrous framework in the mineralized polysaccharide matrix imparts mechanical support to the stalks, providing stability in variable flow conditions. The results demonstrate that D. geminata possesses cellular and periplasmic carbonic‐anhydrases that contribute to carbon fixation and biomineralization, respectively. The activity of external carbonic‐anhydrase was more than 50% of the total activity, which points to its role in anchoring this bioeroding diatom on hard surfaces. The first evidence of multiphase biomineralization by diatoms that deposit both biogenic silica and crystalline biogenic calcite which are imparting distinct functional advantage to the organism is provided.