Extrafibrillar Mineral Research Articles

Relationship between the heterogeneity in mechanical properties, bone density and composition parameters of cortical bone to design and develop bone scaffolds and implants: Analysis of bone microstructure

Bone is heterogeneous and anisotropic because its mechanical characteristics vary by anatomic location and loading direction. Previous research established a correlation between bone density, mineral, organic content, and porosity and mechanical qualities. Medical and bioengineering researchers can learn about bone heterogeneity and anisotropy by analysing bone density and later composition parameters across the bone diaphysis in both longitudinal and transverse orientations. This research examines bone density and composition parameters in bovine femoral cortical bone from higher, middle, and lower diaphysis regions and longitudinal and transverse orientations. At three homogeneous diaphysis sites, these properties did not alter in longitudinal or transverse orientations. At the upper and lower diaphysis locations, mineral and organic content were statistically different in both longitudinal and transverse orientations, contributing to anisotropy. At the middle location, all measured parameters were not statistically different. Transverse bone density and water heterogeneity were greater, while longitudinal values were higher for the other metrics. Upper and lower sites had increased bone variability due to %water, although mineral content was more homogenous. The longitudinal and transverse organic content and apparent density heterogeneity was greater at the middle position. At the later site, bone density and mineral content were more homogenous longitudinally and transversely. Lamellar bone microstructure with canalicular/vascular network, cavities, and holes contributed to bone materials' %water content. Collagen fibres contributed to bone material's organic composition, whereas intrafibrillar and extra fibrillar minerals contributed to its mineral content.

The influence of neutral MDP-Na salt on dentin bond performance and remineralization potential of etch-&-rinse adhesive

ObjectivesTo investigate the effect of neutral 10-methacryloyloxydecyl dihydrogen phosphate salt (MDP-Na) on the dentin bond strength and remineralization potential of etch-&-rinse adhesive.MethodsTwo experimental etch-&-rinse adhesives were formulated by incorporating 0 wt% (E0) or 20 wt% (E20) neutral MDP-Na into a basic primer. A commercial adhesive, Adper Single Bond 2 (SB, 3 M ESPE), served as the control. Sixty prepared teeth were randomly allocated into three groups (n = 20) and bonded using either one of the experimental adhesives or SB. Following 24 h of water storage, the bonded specimens were sectioned into resin-dentin sticks, with four resin-dentin sticks obtained from each tooth for microtensile bond strength (MTBS) test. Half of the sticks from each group were immediately subjected to tensile loading using a microtensile tester at a crosshead speed of 1 mm/min, while the other half underwent tensile loading after 6-month incubation in artificial saliva (AS). The degree of conversion (DC) of both the control and experimental adhesives (n = 6 in each group) and the adsorption properties of MDP-Na on the dentin organic matrix (n = 5 in each group) were determined using Fourier-transform infrared spectrometry. Furthermore, the effectiveness of neutral MDP-Na in promoting the mineralization of two-dimensional collagen fibrils and the adhesive-dentin interface was explored using transmission electron microscopy and selected-area electron diffraction. Two- and one-way ANOVA was employed to assess the impact of adhesive type and water storage on dentin bond strength and the DC (α = 0.05).ResultsThe addition of MDP-Na into the primer increased both the short- and long-term MTBS of the experimental adhesives (p = 0.00). No difference was noted in the DC between the control, E0 and E20 groups (p = 0.366). The MDP-Na remained absorbed on the demineralized dentin even after thorough rinsing. The intra- and extra-fibrillar mineralization of the two-dimensional collagen fibril and dentin bond hybrid layer was confirmed by transmission electron microscopy and selected-area electron diffraction when the primer was added with MDP-Na.ConclusionsThe use of neutral MDP-Na results in high-quality hybrid layer that increase the dentin bond strength of etch-&-rinse adhesive and provides the adhesive with remineralizing capability. This approach may represent a suitable bonding strategy for improving the dentin bond strength and durability of etch-&-rinse adhesive.

Bone collagen tensile properties of the aging human proximal femur

Despite the dominant role of bone mass in osteoporotic fractures, aging bone tissue properties must be thoroughly understood to improve osteoporosis management. In this context, collagen content and integrity are considered important factors, although limited research has been conducted on the tensile behavior of demineralized compact bone in relation to its porosity and elastic properties in the native mineralized state. Therefore, this study aims (i) at examining the age-dependency of mineralized bone and collagen micromechanical properties; (ii) to test whether, and if so to which extent, collagen properties contribute to mineralized bone mechanical properties.Two cylindrical cortical bone samples from fresh frozen human anatomic donor material were extracted from 80 proximal diaphyseal sections from a cohort of 24 female and 19 male donors (57 to 96 years at death). One sample per section was tested in uniaxial tension under hydrated conditions. First, the native sample was tested elastically (0.25 % strain), and after demineralization, up to failure. Morphology and composition of the second specimen was assessed using micro-computed tomography, Raman spectroscopy, and gravimetric methods. Simple and multiple linear regression were employed to relate morphological, compositional, and mechanical variables with age and sex.Macro-tensile properties revealed that only elastic modulus of native samples was age dependent whereas apparent elastic modulus was sex dependent (p < 0.01). Compositional and morphological analysis detected a weak but significant age and sex dependency of relative mineral weight (r = −0.24, p < 0.05) and collagen disorder ratio (I∼1670/I∼1640, r = 0.25, p < 0.05) and a strong sex dependency of bone volume fraction while generally showing consistent results in mineral content assessment. Young's modulus of demineralized bone was significantly related to tissue mineral density and Young's modulus of native bone.The results indicate that mechanical properties of the organic phase, that include collagen and non-collagenous proteins, are independent of donor age. The observed reduction in relative mineral weight and corresponding overall stiffer response of the collagen network may be caused by a reduced number of mineral-collagen connections and a lack of extrafibrillar and intrafibrillar mineralization that induces a loss of waviness and a collagen fiber pre-stretch.

Exploring the hierarchical structure of lamellar bone and its impact on fracture behaviour: A computational study using a phase field damage model

Bone is a naturally occurring composite material composed of a stiff mineral phase and a compliant organic matrix of collagen and non-collagenous proteins (NCP). While diverse mineral morphologies such as platelets and grains have been documented, the precise role of individual constituents, and their morphology, remains poorly understood. To understand the role of constituent morphology on the fracture behaviour of lamellar bone, a damage based representative volume element (RVE) was developed, which considered various mineral morphologies and mineralised collagen fibril (MCF) configurations. This model framework incorporated a novel phase-field damage model to predict the onset and evolution of damage at mineral-mineral and mineral-MCF interfaces. It was found that platelet-based mineral morphologies had superior mechanical performance over their granular counterparts, owing to their higher load-bearing capacity, resulting from a higher aspect ratio. It was also found that MCFs had a remarkable capacity for energy dissipation under axial loading, with these fibrillar structures acting as barriers to crack propagation, thereby enhancing overall elongation and toughness. Interestingly, the presence of extrafibrillar platelet-based minerals also provided an additional toughening through a similar mechanism, whereby these structures also inhibited crack propagation. These findings demonstrate that the two primary constituent materials of lamellar bone play a key role in its toughening behaviour, with combined effect by both mineral and MCFs to inhibit crack propagation at this scale. These results have provided novel insight into the fracture behaviour of lamellar bone, enhancing our understanding of microstructure-property relationships at the sub-tissue level.

Micromechanical modelling of transverse fracture behaviour of lamellar bone using a phase-field damage model: The role of non-collagenous proteins and mineralised collagen fibrils

At the tissue-scale and above, there are now well-established structure-property relationships that provide good approximations of the biomechanical performance of bone through, for example, power-law relationships that relate tissue mineral density to elastic properties. However, below the tissue-level, the individual role of the constituents becomes prominent and these simple relationships tend to break down, with more detailed theoretical and computational models are required to describe the mechanical response. In this study, a two-dimensional micromechanics damage-based representative volume element (RVE) of lamellar bone was developed, which included a novel implementation of a phase-field damage model to describe the behaviour of non-collagenous proteins at mineral-mineral and mineral-fibril interface regions. It was found that, while the stiffness of the tissue was governed by the relative proportion of extra-fibrillar mineral and mineralised collagen fibrils, the strength and toughness of the tissue in transverse direction relied on the interactions occurring at mineral-mineral and mineral-fibril interfaces, highlighting the prominence of non-collagenous proteins in determine fracture-based processes at this scale. While fractures tended to initiate in mineral rich areas of the extra-fibrillar mineral matrix, it was found that the presence of mineralised collagen fibrils at low density did not provide a substantial contribution to crack propagation behaviour under transverse loading. However, at physiological volume fraction (VfMCF=50%), different scenarios could arise depending on the relative strength value of the interphase around the MCFs (σfInterphaseMCF) to the interphase between individual minerals (σfInterphaseHA): (i) When γ=σfInterphaseMCFσfInterphaseHA<1, MCFs appear to facilitate crack propagation with MCF-mineral debonding being the dominant failure mode; (ii) once γ>1, the MCFs hinder the microcracks, leading to inhibition of crack propagation, which can be regarded as an energy dissipation mechanism. The effective fracture properties of the tissue also experience a sudden increase in fracture work density (J-integral) once the crack is arrested by MCFs or severely deflected. Collectively, the predicted behaviour of the model compared well to those reported through experimental and computational methods, highlighting its potential to provide further understanding into the mechanistic response of bone ultrastructure alterations related to the structural and compositional changes resulting from disease and aging.

Elucidating the role of diverse mineralisation paradigms on bone biomechanics - a coarse-grained molecular dynamics investigation.

Bone as a hierarchical composite structure plays a myriad of roles in vertebrate skeletons including providing the structural stability of the body. Despite this critical role, the mechanical behaviour at the sub-micron levels of bone's hierarchy remains poorly understood. At this scale, bone is composed of Mineralised Collagen Fibrils (MCF) embedded within an extra-fibrillar matrix that consists of hydroxyapatite minerals and non-collagenous proteins. Recent experimental studies hint at the significance of the extra-fibrillar matrix in providing the bone with the stiffness and ductility needed to serve its structural roles. However, due to limited resolution of experimental tools, it is not clear how the arrangement of minerals, and in particular their relative distribution between the intra- and extra-fibrillar space contribute to bone's remarkable mechanical properties. In this study, a Coarse Grained Molecular Dynamics (CGMD) framework was developed to study the mechanical properties of MCFs embedded within an extra-fibrillar mineral matrix and the precise roles extra- and intra-fibrillar mineralisation on the load-deformation response was investigated. It was found that the presence of extra-fibrillar mineral resulted in the development of substantial residual stress in the system, by limiting MCF shortening that took place during intra-fibrillar mineralisation, resulting in substantial compressive residual stresses in the extra-fibrillar mineral phase. The simulation results also revealed the crucial role of extra-fibrillar mineralisation in determining the elastic response of the Extrafibrillar mineralised MCF (EFM-MCF) system up to the yield point, while the fibrillar collagen affected the post-yield behaviour. When physiological levels of mineralisation were considered, the mechanical response of the EFM-MCF systems was characterised by high ductility and toughness, with micro-cracks being distributed across the extra-fibrillar matrix, and MCFs effectively bridging these cracks leading to an excellent combination of strength and toughness. Together, these results provide novel insight into the deformation mechanisms of an EFM-MCF system and highlight that this universal building block, which forms the basis for lamellar bone, can provide an excellent balance of stiffness, strength and toughness, achieving mechanical properties that are far beyond the capabilities of the individual constituents acting alone.

A glycol chitosan derivative with extrafibrillar demineralization potential for self-etch dentin bonding

ObjectivesExtrafibrillar demineralization is an etching technique that removes only minerals from around the collagen fibrils for resin infiltration. The intrafibrillar minerals are left intact to avoid their replacement by water that is hard for adhesive resin monomers to displace. The present work reported the synthesis of a water-soluble methacryloyloxy glycol chitosan-EDTA conjugate (GCE-MA) and evaluated its potential as an extrafibrillar demineralization agent for self-etch dentin bonding. MethodsGlycol chitosan-EDTA was functionalized with a methacryloyloxy functionality. Conjugation was confirmed using Fourier transform-infrared spectroscopy. The GCE-MA was used to prepare experimental self-etch primers. Extrafibrillar demineralization of the primers was evaluated with scaning electron microscopy and transmission electron microscopy. The feasibility of this new self-etch bonding approach was evaluated using microtensile bond strength testing and inhibition of dentin gelatinolytic activity. The antibacterial activity and cytotoxicity of GCE-MA were also analyzed. ResultsConjugation of EDTA and the methacryloyloxy functionality to glycol chitosan was successful. The functionalized conjugate was capable of extrafibrillar demineralization of mineralized collagen fibrils. Tensile bond strength of the experimental self-etch primer to dentin was comparable to that of phosphoric acid-etched dentin and the commercial self-etch primer Clearfil SE Bond 2. The GCE-MA also inhibited soluble rhMMP-9. In-situ zymography detected minimal fluorescence in hybrid layers conditioned with the experimental primer. The GCE-MA was noncytotoxic and possessed antibacterial activities against planktonic bacteria. SignificanceSynthesis of GCE-MA brought into fruition a self-etch conditioner that selectively demineralizes the extrafibrillar mineral component of dentin. A self-etch primer prepared with GCE-MA achieved bond strengths comparable to commercial reference adhesive systems.

Ultrastructure and Nanoporosity of Human Bone Shown with Correlative On-Axis Electron and Spectroscopic Tomographies.

Mineralized collagen fibrils are the building block units of bone at the nanoscale. While it is known that collagen fibrils are mineralized both inside their gap zones (intra-fibrillar mineralization) and on their outer surfaces (extra-fibrillar mineralization), a clear visualization of this architecture in three dimensions (3D), combining structural and compositional information over large volumes, but without compromising the resolution, remains challenging. In this study, we demonstrate the use of on-axis Z-contrast electron tomography (ET) with correlative energy-dispersive X-ray spectroscopy (EDX) tomography to examine rod-shaped samples with diameters up to 700 nm prepared from individual osteonal lamellae in the human femur. Our work mainly focuses on two aspects: (i) low-contrast nanosized circular spaces ("holes") observed in sections of bone oriented perpendicular to the long axis of a long bone, and (ii) extra-fibrillar mineral, especially in terms of morphology and spatial relationship with respect to intra-fibrillar mineral and collagen fibrils. From our analyses, it emerges quite clearly that most "holes" are cross-sectional views of collagen fibrils. While this had been postulated before, our 3D reconstructions and reslicing along meaningful two-dimensional (2D) cross-sections provide a direct visual confirmation. Extra-fibrillar mineral appears to be composed of thin plates that are interconnected and span over several collagen fibrils, confirming that mineralization is cross-fibrillar, at least for the extra-fibrillar phase. EDX tomography shows mineral signatures (Ca and P) within the gap zones, but the signal appears weaker than that associated with the extra-fibrillar mineral, pointing toward the existence of dissimilarities between the two types of mineralization.

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

AbstractOsteons, 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.

Extrafibrillarly Demineralized Dentin Matrix for Bone Regeneration.

Dentin is a natural extracellular matrix, but its availability in bone grafting and tissue engineering applications is underestimated due to a lack of proper treatment. In this study, the concept of extrafibrillar demineralization is introduced into the construction of dentin-derived biomaterials for bone regeneration for the first time. Calcium chelating agents with large molecular weights are used to selectively remove the extrafibrillar apatite minerals without disturbing the intrafibrillar minerals within dentin collagen, resulting in the formation of an extrafibrillarly demineralized dentin matrix (EDM). EDM with distinctive nanotopography and bone-like mechanical properties is found to significantly promote cell adhesion, migration, and osteogenic differentiation in vitro while enhancing in vivo bone healing of rat calvarial defects. The outstanding osteogenic performance of EDM is further confirmed to be related to the activation of the focal adhesion-cytoskeleton-nucleus mechanotransduction axis. Overall, this study shows that extrafibrillar demineralization of dentin has great potential to produce hierarchical collagen-based scaffolds for bone regeneration, and this facile top-down fabrication method brings about new ideas for the biomedical application of naturally derived bioactive materials.

Applications of collagen extrafibrillar demineralization in dentin bonding

The existing dentin bonding systems based on acid-etching technique lead to the loss of both extrafibrillar and intrafibrillar minerals from dentin collagen, causing excessive demineralization. Because resin monomers can not infiltrate the intrafibrillar spaces of demineralized collagen matrix, degradation of exposed collagen and resin hydrolysis subsequently occur within the hybrid layer, which seriously jeopardizing the longevity of resin-dentin bonding. Collagen extrafibrillar demineralization can effectively avoid the structural defects within the resin-dentin interface caused by acid-etching technique and improve the durability of resin-dentin bonding, by preserving intrafibrillar minerals and selectively demineralizing extrafibrillar dentin. The mechanism and research progress of collagen extrafibrillar demineralization in dentin bonding are reviewed in the paper.

Sodium carboxymethyl cellulose-based extrafibrillar demineralization to optimize dentin bonding durability.

To investigate the effects of a sodium carboxymethyl cellulose (SCMC)-based extrafibrillar demineralization conditioner on dentin bonding durability and explore the possible mechanisms. The SCMC-based extrafibrillar demineralization conditioner was facilely developed by dissolving SCMC into deionized water at an appropriate concentration. A single layer collagen mineralization/demineralization model was designed to visualize extrafibrillar demineralization in detail. Dentin surfaces of human third molars were conditioned with 3 % SCMC or 37 % phosphoric acid (PA). The morphology, composition, and mechanical properties of conditioned dentin from each group were characterized. To evaluate dentin bonding performance, SCMC- and PA- conditioned dentin were applied with adhesive restoration using the dry-bonding technique. The microtensile bond strength (MTBS), interface nanoleakage, and in situ zymography were measured after 24h of water storage, 10,000 thermocycles, or one month of collagenase aging. The inhibitory effect of SCMC on recombinant human matrix metalloproteinase-2 (rhMMP-2) and cell toxicity were also investigated. After SCMC conditioning, both demineralization of extrafibrillar minerals and retention of intrafibrillar minerals were observed in the single layer collagen model and the dentin ultrastructure. The mechanical properties of SCMC-conditioned dentin were largely preserved. Compared with PA, SCMC conditioning produced greater MTBS values and less nanoleakage expression after aging. Endogenous gelatinolytic activity was suppressed in SCMC-conditioned dentin. In addition to being nontoxic, the inhibition of rhMMP-2 by SCMC was confirmed to be dose-dependent. From the perspective of minimal intervention, the SCMC-based extrafibrillar demineralization conditioner could improve dentin bonding durability, suggesting a promising strategy to extend the service life of adhesive restorations.

Biomineralized dipeptide self-assembled hydrogel with ultrahigh mechanical strength and osteoinductivity for bone regeneration

Peptide self-assembled hydrogels are ideal scaffolds for three-dimentional cell culture and tissue engineering due to their native extracellular matrix enabling the transport of nutrients as well as the cells growth and expansion. However, the use of peptide hydrogels in the hard tissues regeneration such as alveolar and skull bone has been largely unexplored, hindered by the insufficient mechanical strength and osteogenesis activity. Herein, peptide self-assembled nanostructures are used as templates to emulate natural biomineralization process. The vaterite nanoparticles loaded with thermolysin act as a role of mature osteoblasts secreting collagen fibrils to initiate dipeptide self-assembly as biomimetic extracellular matrix. Meanwhile, the vaterite nanoparticles with a high specific surface area form strong intermolecular and interfacial interaction with hydrogel networks, acting as a cross-linker to achieve extrafibrillar mineralization. Moreover, vaterite nanoparticles function as a mineral reservoir to release Ca2+, inducing intrafibrillar mineralization, further enhancing the mechanical property. The resulting hydrogel exhibits an unprecedently high value of storage modulus (473 kPa), excellent biocompatibility and osteoinductivity. In rat calvarial defect model, the hydrogel significantly accelerates regeneration of osteogenesis tissue, thus indicating the potential of such peptide hydrogels for application in bone regeneration.

Insight on the Role of Poly(acrylic acid) for Directing Calcium Phosphate Mineralization of Synthetic Polymer Bone Scaffolds.

Bone is a complex tissue with robust mechanical and biological properties originating from its nanoscale composite structure. Although much research has been conducted on designing bioinspired artificial bone, the role of biological macromolecules such as noncollagenous proteins (NCPs) in influencing the formation of biominerals is not fully understood. In this work, we have designed nanofiber shish-kebab (NFSK) structures that can template mineral location by recruiting calcium cations from an ion-rich mineralization solution. Poly(acrylic acid) (PAA) is used as the NCP analogue to understand the role of polyelectrolytes in scaffold mineralization. We demonstrate that the addition of PAA in the mineralization solution suppresses the development of extrafibrillar minerals as well as slows down the accumulation and development of mineral phases within NFSKs. We probe the mechanism behind this effect by monitoring the free calcium ion concentration, investigating the PAA molecular weight effect, and conducting mineralization in membrane-partitioned solutions. Our results suggest the 2-fold effect of PAA as a solution stabilizer and physical barrier on the NFSK surface. This work could shed light on the understanding of the NCP effect in biomineralization.

Promotion Effect of Carboxymethyl Chitosan on Dental Caries via Intrafibrillar Mineralization of Collagen and Dentin Remineralization.

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.

Multifunctional Nanomachinery for Enhancement of Bone Healing (Adv. Mater. 9/2022)

Bone Tissue Engineering In article number 2107924, Kai Jiao, Franklin R. Tay, Li-na Niu, and co-workers present RNA-ACP nanomachines that induce extrafibrillar and intrafibrillar mineralization of collagen fibrils and regenerate new bone in a dynamic and programmable manner. These multifunctional nanomachines are comparable to the function of osteoblasts and bring new opportunities and challenges to bone tissue engineering.

A multiscale finite element investigation on the role of intra- and extra-fibrillar mineralisation on the elastic properties of bone tissue

Lamellar bone is one of the fundamental structural units of bone tissue and it consists of mineralised collagen fibrils (MCFs) embedded within an extra-fibrillar matrix comprised of hydroxyapatite minerals distributed throughout a matrix of non-collagenous proteins (NCPs). While both intra- and extra-fibrillar phases provide a critical contribution to tissue-level behaviour, the mechanical implications of their structural arrangement, and in particular the relative distribution of HA minerals between both phases, remains poorly understood. This study presents a multiscale finite element framework to investigate the role of intra- and extra-fibrillar mineralisation on the elastic properties of bone tissue by considering two levels of structural hierarchy. At the nanoscale, representative volume elements (RVEs) of both MCFs and the extra-fibrillar matrix were developed, and a homogenisation strategy was used to determine the effective elastic properties of each phase. At the sub-micron level, an RVE of lamellar bone that accounted for newly reported patterns of mineral platelets encircling collagen fibrils was used to predict the effective response of lamellar bone tissue, with material properties established from the previous length scale. The results demonstrated that the overall mineral content in the tissue is the biggest contributor to the effective elastic properties of lamellar bone. While this is perhaps unsurprising, importantly, it was demonstrated that the extra-fibrillar matrix (and mineral therein) is the phase that makes the primary contribution to the elastic response of the tissue. The two main reasons that the extra-fibrillar matrix dominated the load-bearing response are (i) the greater proportion of mineral content compared to the intra-fibrillar regions and (ii) the highly ordered arrangement of mineral platelets that are aligned to the longitudinal axis of MCFs. Both of these features resulted in extra-fibrillar mineral strain ratios that were consistently higher than intra-fibrillar mineral strain ratios under axial loading. As a result, the predicted elastic properties of MCFs were much lower than the extra-fibrillar matrix, indicating that intra-fibrillar mineralisation only provided a modest contribution to the stiffness of bone tissue. Collectively, the predicted results of the multiscale approach compared well to the range properties measured through various experimental testing methods, highlighting its potential to provide further insight into the role of sub-tissue features of tissue biomechanics.

Multifunctional Nanomachinery for Enhancement of Bone Healing.

The visionary idea that RNA adopts nonbiological roles in today's nanomaterial world has been nothing short of phenomenal. These RNA molecules have ample chemical functionality and self-assemble to form distinct nanostructures in response to external stimuli. They may be combined with inorganic materials to produce nanomachines that carry cargo to a target site in a controlled manner and respond dynamically to environmental changes. Comparable to biological cells, programmed RNA nanomachines have the potential to replicate bone healing in vitro. Here, an RNA-biomineral nanomachine is developed, which accomplishes intrafibrillar and extrafibrillar mineralization of collagen scaffolds to mimic bone formation in vitro. Molecular dynamics simulation indicates that noncovalent hydrogen bonding provides the energy source that initiates self-assembly of these nanomachines. Incorporation of the RNA-biomineral nanomachines into collagen scaffolds in vivo creates an osteoinductive microenvironment within a bone defect that is conducive to rapid biomineralization and osteogenesis. Addition of RNA-degrading enzymes into RNA-biomineral nanomachines further creates a stop signal that inhibits unwarranted bone formation in tissues. The potential of RNA in building functional nanostructures has been underestimated in the past. The concept of RNA-biomineral nanomachines participating in physiological processes may transform the nanoscopic world of life science.

Progress on Biomimetic Mineralization and Materials for Hard Tissue Regeneration.

Biomineralization is a process in which natural organisms regulate the crystal growth of inorganic minerals, resulting in hierarchical structured biominerals with excellent properties. Typical biominerals in the human body are the bones and teeth, and damage to these hard tissues directly affect our daily lives. The repair of bones and teeth in a biomimetic way, either by using a biomimetic mineralization strategy or biomimetic materials, is the key for hard tissue regeneration. In this review, we briefly introduce the structure of bone and tooth, and highlight the fundamental role of collagen mineralization in tissue repair. The recent progress on intra-/extrafibrillar collagen mineralization by a biomimetic strategy or materials is presented, and their potential for tissue regeneration is discussed. Then, recent achievements on bone and tooth repair are summarized, and these works are discussed in the view of materials science and biological science, providing a broader vision for the future research of hard tissue repair techniques. Lastly, recent progress on hard tissue regeneration is concluded, and existing problems and future directions are prospected.

Molecular weight and concentration of poly (acrylic acid) dual‐responsive homogeneous and intrafibrillar collagen mineralization using an in situ co‐organization strategy

AbstractIntrafibrillar mineralization is crucial in the synthesis of collagen‐hydroxyapatite (HAP) composites with hierarchical structure resembling natural bone. The polymer‐induced liquid precursor method, which involves immersing the matrix into polymer‐stabilized amorphous calcium phosphate (ACP) solutions, has been widely used to regulate collagen mineralization; however, heterogeneous infiltration and crystallization of ACP precursors from the surface to the inner matrix remain a concern. In this study, the polymer‐mediated co‐organization strategy in which poly (acrylic acid) (PAA)‐stabilized ACP solutions were initially introduced to collagen solutions before sol–gel transformation was employed to achieve homogeneous mineralization. Moreover, the effects of PAA with various molecular weights (2, 10, and 450 kDa) and concentrations (40, 80, and 120 mg/L) on collagen mineralization were evaluated. Increasing concentration and decreasing molecular weight both improved the stability of ACP precursors, which affected collagen mineralization. A lower concentration (40 mg/L) of 2 kDa PAA and higher concentration (80 and 120 mg/L) of 100 and 450 kDa PAA effectively retarded ACP crystallization, thereby inducing intrafibrillar mineralization. Extremely unstable ACP precursors failed to infiltrate the interior of fibrils using 100 and 450 kDa of PAA at 40 mg/L, resulting in extrafibrillar mineralization. Inversely, collagen fibrils were unmineralized until 7 days owing to excessive stability of ACP precursors using 2 kDa of PAA at higher concentrations. The present results further explain the mechanisms of intrafibrillar mineralization, and may provide a facile method for producing biomimetic materials.