Amorphous Calcium Phosphate Precursor Research Articles

STMP and PVPA as Templating Analogs of Noncollagenous Proteins Induce Intrafibrillar Mineralization of Type I Collagen via PCCP Process.

The phosphorylated noncollagenous proteins (NCPs) play a vital role in manipulating biomineralization, while the mechanism of phosphorylation of NCPs in intrafibrillar mineralization of collagen fibril has not been completely deciphered. Poly(vinylphosphonic acid) (PVPA) and sodium trimetaphosphate (STMP) as templating analogs of NCPs induce hierarchical mineralization in cooperation with indispensable sequestration analogs such as polyacrylic acid (PAA) via polymer-induced liquid-like precursor (PILP) process. Herein, STMP-Ca and PVPA-Ca complexes are proposed to achieve rapid intrafibrillar mineralization through polyelectrolyte-Ca complexes pre-precursor (PCCP) process. This strategy is further verified effectively for remineralization of demineralized dentin matrix both in vitro and in vivo. Although STMP micromolecule fails to stabilize amorphous calcium phosphate (ACP) precursor, STMP-Ca complexes facilely permeate into intrafibrillar interstices and trigger phase transition of ACP to hydroxyapatite within collagen. In contrast, PVPA-stabilized ACP precursors lack liquid-like characteristic and crystallize outside collagen due to rigid conformation of PVPA macromolecule, while PVPA-Ca complexes infiltrate into partial intrafibrillar intervals under electrostatic attraction and osmotic pressure as evidenced by intuitionistic 3D stochastic optical reconstruction microscopy (3D-STORM). The study not only extends the variety and size range of polyelectrolyte for PCCP process but also sheds light on the role of phosphorylation for NCPs in biomineralization.

Biomimetic Intrafibrillar Mineralization of Native Tendon for Soft-Hard Interface Integration by Infiltration of Amorphous Calcium Phosphate Precursors.

Soft and hard tissues possess distinct biological properties. Integrating the soft-hard interface is difficult due to the inherent non-osteogenesis of soft tissue, especially of anterior cruciate ligament and rotator cuff reconstruction. This property makes it difficult for tendons to be mineralized and integrated with bone in vivo. To overcome this challenge, a biomimetic mineralization strategy is employed to engineer mineralized tendons. The strategy involved infiltrating amorphous calcium phosphate precursors into collagen fibrils, resulting in hydroxyapatite deposition along the c-axis. The mineralized tendon presented characteristics similar to bone tissue and induced osteogenic differentiation of mesenchymal stem cells. Additionally, the interface between the newly formed bone and tendon is serrated, suggesting a superb integration between the two tissues. This strategy allows for biomineralization of tendon collagen and replicating the hallmarks of the bone matrix and extracellular niche, including nanostructure and inherent osteoinductive properties, ultimately facilitating the integration of soft and hard tissues.

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.

Stereolithography printing of bone scaffolds using biofunctional calcium phosphate nanoparticles

Calcium phosphate (CaP) has been widely used for bone defect repair due to good biocompatibility and osteoconductivity. Additive manufacture of calcium phosphate bioceramics with tailored architectures and improved mechanical properties has recently attracted great attention. Herein, calcium phosphate nanoparticles with the size of ∼89−164 nm were synthesized by the hydrothermal treatment of amorphous calcium phosphate (ACP) precursors at 180 °C for 24 h. Biofunctional elements including Mg, Sr and Zn have been doped into these calcium phosphate nanoparticles. Our results revealed that Mg2+ ions played critical roles in formation of whitlockite-type calcium phosphate (not hydroxyapatite) from ACP precursors. Moreover, gyroid scaffolds with bionic triply periodic minimal surface structures were fabricated using stereolithography printing of these calcium phosphate nanoparticles, which are likely used as biofunctional scaffolds for bone repair.

Protein nanoribbons template enamel mineralization

As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue-based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.

Biomineralization Precursor Carrier System Based on Carboxyl-Functionalized Large Pore Mesoporous Silica Nanoparticles.

Bone and teeth are derived from intrafibrillarly mineralized collagen fibrils as the second level of hierarchy. According to polymer-induced liquid-precursor process, using amorphous calcium phosphate precursor (ACP) is able to achieve intrafibrillar mineralization in the case of bone biomineral in vitro. Therefore, ACP precursors might be blended with any osteoconductive scaffold as a promising bone formation supplement for in-situ remineralization of collagens in bone. In this study, mesoporous silica nanoparticles with carboxyl-functionalized groups and ultra large-pores have been synthesized and used for the delivery of liquid like biomimetic precursors (ACP). The precursor delivery capacity of the nanoparticles was verified by the precursor release profile and successful mineralization of 2D and 3D collagen models. The nanoparticles could be completely degraded in 60 days and exhibited good biocompatibility as well. The successful translational strategy for biomineralization precursors showed that biomineralization precursor laden ultra large pore mesoporous silica possessed the potential as a versatile supplement in demineralized bone formation through the induction of intrafibrillar collagen mineralization.

Low temperature preparation of α-tricalcium phosphate up-converted luminescence nano phosphor

In this work, fluorescent α-tricalcium phosphate (α-TCP: Yb3+,Er3+) was successfully prepared by the thermal transformation of amorphous calcium phosphate (ACP) precursor. β-cyclodextrin (β-CD) was used for preparation of ACP precursor and played an important role in designing its special structure. Due to the new low temperature preparation of α-tricalcium phosphate, the obtained microsphere has a uniform distribution with rational strength (15.0 Mpa). Futhermore, the two ions (Yb3+,Er3+) were doped into α-tricalcium phosphate (α-TCP), the obtained phosphor can emit exhibit bright green luminescence under the excitation of a 980 nm laser diode. The α-TCP: Yb3+,Er3+ may have great potential in the fields of in vivo bio-imaging and a drug delivery system.

Contribution of Mitophagy to Cell-Mediated Mineralization: Revisiting a 50-Year-Old Conundrum.

Biomineralization in vertebrates is initiated via amorphous calcium phosphate (ACP) precursors. These precursors infiltrate the extracellular collagen matrix where they undergo phase transformation into intrafibrillar carbonated apatite. Although it is well established that ACP precursors are released from intracellular vesicles through exocytosis, an unsolved enigma in this cell‐mediated mineralization process is how ACP precursors, initially produced in the mitochondria, are translocated to the intracellular vesicles. The present study proposes that mitophagy provides the mechanism for transfer of ACP precursors from the dysfunctioned mitochondria to autophagosomes, which, upon fusion with lysosomes, become autolysosomes where the mitochondrial ACP precursors coalesce to form larger intravesicular granules, prior to their release into the extracellular matrix. Apart from endowing the mitochondria with the function of ACP delivery through mitophagy, the present results indicate that mitophagy, triggered upon intramitochondrial ACP accumulation in osteogenic lineage‐committed mesenchymal stem cells, participates in the biomineralization process through the BMP/Smad signaling pathway.

Citrate Improves Collagen Mineralization via Interface Wetting: A Physicochemical Understanding of Biomineralization Control.

Biological hard tissues such as bones always contain extremely high levels of citrate, which is believed to play an important role in bone formation as well as in osteoporosis treatments. However, its mechanism on biomineralization is not elucidated. Here, it is found that the adsorbed citrate molecules on collagen fibrils can significantly reduce the interfacial energy between the biological matrix and the amorphous calcium phosphate precursor to enhance their wetting effect at the early biomineralization stage, sequentially facilitating the intrafibrillar formation of hydroxyapatite to produce an inorganic-organic composite. It is demonstrated experimentally that only collagen fibrils containing ≈8.2 wt% of bound citrate (close to the level in biological bone) can reach the full mineralization as those in natural bones. The effect of citrate on the promotion of the collagen mineralization degree is also confirmed by in vitro dentin repair. This finding demonstrates the importance of interfacial controls in biomineralization and more generally, provides a physicochemical view about the regulation effect of small biomolecules on the biomineralization front.

Disordered Conformation with Low Pii Helix in Phosphoproteins Orchestrates Biomimetic Apatite Formation.

The interplay between noncollagenous proteins and biomineralization is widely accepted, yet the contribution of their secondary structure in mineral formation remains to be clarified. This study demonstrates a role for phosvitin, an intrinsically disordered phosphoprotein, in chick embryo skeletal development, and using circular dichroism and matrix least-squares Henderson-Hasselbalch global fitting, unravels three distinct pH-dependent secondary structures in phosvitin. By sequestering phosvitin on a biomimetic 3D insoluble cationic framework at defined pHs, access is gained to phosvitin in various conformational states. Induction of biomimetic mineralization at near physiological conditions reveals that a disordered secondary structure with a low content of PII helix is remarkably efficient at promoting calcium adsorption, and results in the formation of biomimetic hydroxyapatite through an amorphous calcium phosphate precursor. By extending this finding to phosphorylated full-length human recombinant dentin matrix protein-1 (17-513 AA), this bioinspired approach provides compelling evidence for the role of a disordered secondary structure in phosphoproteins in bone-like apatite formation.

Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide.

Previously, we have shown that serine-16 phosphorylation in native full-length porcine amelogenin (P173) and the Leucine-Rich Amelogenin Peptide (LRAP(+P)), an alternative amelogenin splice product, affects protein assembly and mineralization in vitro. Notably, P173 and LRAP(+P) stabilize amorphous calcium phosphate (ACP) and inhibit hydroxyapatite (HA) formation, while non-phosphorylated counterparts (rP172, LRAP(−P)) guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases. To test this hypothesis, we have utilized Fourier transform infrared spectroscopy (FTIR) to determine the secondary structure of LRAP(−P) and LRAP(+P) in the absence/presence of calcium and selected mineral phases relevant to amelogenesis; i.e., hydroxyapatite (HA: an enamel crystal prototype) and (ACP: an enamel crystal precursor phase). Aqueous solutions of LRAP(−P) or LRAP(+P) were prepared with or without 7.5 mM of CaCl2 at pH 7.4. FTIR spectra of each solution were obtained using attenuated total reflectance, and amide-I peaks were analyzed to provide secondary structure information. Secondary structures of LRAP(+P) and LRAP(−P) were similarly assessed following incubation with suspensions of HA and pyrophosphate-stabilized ACP. Amide I spectra of LRAP(−P) and LRAP(+P) were found to be distinct from each other in all cases. Spectra analyses showed that LRAP(−P) is comprised mostly of random coil and β-sheet, while LRAP(+P) exhibits more β-sheet and α-helix with little random coil. With added Ca, the random coil content increased in LRAP(−P), while LRAP(+P) exhibited a decrease in α-helix components. Incubation of LRAP(−P) with HA or ACP resulted in comparable increases in β-sheet structure. Notably, however, LRAP(+P) secondary structure was more affected by ACP, primarily showing an increase in β-sheet structure, compared to that observed with added HA. These collective findings indicate that phosphorylation induces unique secondary structural changes that may enhance the functional capacity of native phosphorylated amelogenins like LRAP to stabilize an ACP precursor phase during early stages of enamel mineral formation.

Proton Environments in Biomimetic Calcium Phosphates Formed from Mesoporous Bioactive CaO-SiO2-P2O5 Glasses in Vitro: Insights from Solid-State NMR.

When exposed to body fluids, mesoporous bioactive glasses (MBGs) of the CaO–SiO2–P2O5 system develop a bone-bonding surface layer that initially consists of amorphous calcium phosphate (ACP), which transforms into hydroxy-carbonate apatite (HCA) with a very similar composition as bone/dentin mineral. Information from various 1H-based solid-state nuclear magnetic resonance (NMR) experiments was combined to elucidate the evolution of the proton speciations both at the MBG surface and within each ACP/HCA constituent of the biomimetic phosphate layer formed when each of three MBGs with distinct Ca, Si, and P contents was immersed in a simulated body fluid (SBF) for variable periods between 15 min and 30 days. Directly excited magic-angle-spinning (MAS) 1H NMR spectra mainly reflect the MBG component, whose surface is rich in water and silanol (SiOH) moieties. Double-quantum–single-quantum correlation 1H NMR experimentation at fast MAS revealed their interatomic proximities. The comparatively minor H species of each ACP and HCA component were probed selectively by heteronuclear 1H–31P NMR experimentation. The initially prevailing ACP phase comprises H2O and “nonapatitic” HPO42–/PO43– groups, whereas for prolonged MBG soaking over days, a well-progressed ACP → HCA transformation was evidenced by a dominating O1H resonance from HCA. We show that 1H-detected 1H → 31P cross-polarization NMR is markedly more sensitive than utilizing powder X-ray diffraction or 31P NMR for detecting the onset of HCA formation, notably so for P-bearing (M)BGs. In relation to the long-standing controversy as to whether bone mineral comprises ACP and/or forms via an ACP precursor, we discuss a recently accepted structural core–shell picture of both synthetic and biological HCA, highlighting the close relationship between the disordered surface layer and ACP.

Low temperature preparation of α-tricalcium phosphate and its mechanical properties

In this work, ?-tricalcium phosphate (?-TCP) was successfully prepared by the thermal transformation of amorphous calcium phosphate (ACP) precursor. ?-cyclodextrin (?-CD) was used for preparation of ACP precursor and played an important role in designing its special structure. The phase composition and microstructures of the obtained ?-TCP at different annealing temperature were analysed by X-ray diffraction and scanning electron microscope, and confirmed that ?-TCP can be prepared at 650?C for 3 h using ACP as precursor, which is much lower than the phase transition temperature of ?-TCP. Mechanical properties were tested 24 h after mixing the obtained ?-TCP with 30 wt.% of deionised water. The compressive strength and the flexural strength were 26.4MPa and 12.0MPa, respectively. The flexural strength was higher than that of ?-TCP prepared by other methods.

The synergic role of collagen and citrate in stabilizing amorphous calcium phosphate precursors with platy morphology.

Citrate is an important component of the bone organic matrix but its specific role in bone mineralization is presently unclear. In this work, bioinspired in vitro collagen mineralization experiments in the presence of citrate have been carried out and the combined role of collagen and citrate in controlling mineral formation has been addressed for the first time. Through X-ray scattering and Atomic Force Microscopy characterizations on mineralized and non-mineralized collagen fibrils, we have found that citrate in synergy with collagen stabilizes an amorphous calcium phosphate (ACP) phase with platy morphology over one week and controls its deposition on collagen fibrils.

Preparation, characterization and in vitro dissolution behavior of porous biphasic α/β-tricalcium phosphate bioceramics

The ideal bone tissue engineering scaffolds are long-cherished with the properties of interconnected macroporous structures, adjustable degradation and excellent biocompatibility. Here, a series of porous α/β-tricalcium phosphate (α/β-TCP) biphasic bioceramics with different phase ratios of α-TCP and β-TCP were successfully synthesized by heating an amorphous calcium phosphate precursor. The chemical and morphological characterization showed that α- and β-TCP phases co-existed in the α/β-TCP bioceramics and they had interconnected pore structures with size between 200 and 500μm. The in vitro dissolution behavior and bioactivity of the dual α/β-TCP were also probed in static and dynamic SBF for the first time. The results revealed that α/β-TCP scaffolds had good in vitro bioactivity, as the formation of bone-like apatite layers was induced on the scaffolds after mineralization in SBF. Moreover, dissolution rate of α/β-TCP bioceramics in dynamic environment was higher than that under static condition. Compared with monophasic TCP ceramics, these porous α/β-TCP bioceramics displayed a tailored dissolution rate proportionate to the TCP content (α and β) in the materials. Further, the degradation profile of porous α/β-TCP was well-described by Avrami equation. The porous dual α/β-TCP bioceramics with controllable degradation behavior hold great potential to be applied in bone tissue engineering as bone substitutes.

Shear-mediated crystallization from amorphous calcium phosphate to bone apatite

The contribution of fluid shear stress (FSS) on the conversion of amorphous calcium phosphate (ACP) to bone apatite is investigated. The ACP precursors are prepared by using a wet-chemistry method and further exposed to the constant FSS environment with values of 0.5, 1.0, 1.5, and 2.0Pa. At the designated time points, the apatites are characterized by transmission electron microscopy, X-ray diffraction, and inductively coupled plasma-mass spectroscopy. The results show that, the low FSS (≤1.0Pa) has positive effects on the transition of ACP, characterized by the accelerated crystallization velocity and the well-organized calcium-deficient hydroxyapatite (CDHA) structure, whereas the high FSS (>1.0Pa) has negative effects on this conversion process, characterized by the poor CDHA crystal morphologies and the destroyed structures. The bioactivity evaluations further reveal that, compared with the FSS-free group, the CDHA prepared under 1.0Pa FSS for 9h presents the more biocompatible features with pre-osteoblast cells. These results are helpful for understanding the mechanism of apatite deposition in natural bone tissue.

Biomimetic Intrafibrillar Mineralization of Type I Collagen with Intermediate Precursors-loaded Mesoporous Carriers.

Limited continuous replenishment of the mineralization medium is a restriction for in-situ solution-based remineralization of hypomineralized body tissues. Here, we report a process that generated amine-functionalized mesoporous silica nanoparticles for sustained release of biomimetic analog-stabilized amorphous calcium phosphate precursors. Both two-dimensional and three-dimensional collagen models can be intrafibrillarly mineralized with these released fluidic intermediate precursors. This represents an important advance in the translation of biomineralization concepts into regimes for in-situ remineralization of bone and teeth.

Role of mineralization inhibitors in the regulation of hard tissue biomineralization: relevance to initial enamel formation and maturation.

Vertebrate mineralized tissues, i.e., enamel, dentin, cementum, and bone, have unique hierarchical structures and chemical compositions. Although these tissues are similarly comprised of a crystalline calcium apatite mineral phase and a protein component, they differ with respect to crystal size and shape, level and distribution of trace mineral ions, the nature of the proteins present, and their relative proportions of mineral and protein components. Despite apparent differences, mineralized tissues are similarly derived by highly concerted extracellular processes involving matrix proteins, proteases, and mineral ion fluxes that collectively regulate the nucleation, growth and organization of forming mineral crystals. Nature, however, provides multiple ways to control the onset, rate, location, and organization of mineral deposits in developing mineralized tissues. Although our knowledge is quite limited in some of these areas, recent evidence suggests that hard tissue formation is, in part, controlled through the regulation of specific molecules that inhibit the mineralization process. This paper addresses the role of mineralization inhibitors in the regulation of biological mineralization with emphasis on the relevance of current findings to the process of amelogenesis. Mineralization inhibitors can also serve to maintain driving forces for calcium phosphate precipitation and prevent unwanted mineralization. Recent evidence shows that native phosphorylated amelogenins have the capacity to prevent mineralization through the stabilization of an amorphous calcium phosphate precursor phase, as observed in vitro and in developing teeth. Based on present findings, the authors propose that the transformation of initially formed amorphous mineral deposits to enamel crystals is an active process associated with the enzymatic processing of amelogenins. Such processing may serve to control both initial enamel crystal formation and subsequent maturation.

Biomimetic mineralization of a single-layer reconstituted type I collagen model induced by sodium tripolyphosphate and polyacrylic acid

To investigate the functions of sodium tripolyphosphate (STTP) and polyacrylic acid (PAA) in the process of collagen biomimetic mineralization. This would allow future applications to other collagen matrices such as bone collagen or 3-D collagen scaffolds. Glass cover slips and transmission electron microscopy (TEM) grids were coated with reconstituted typeIcollagen fibrils. Mineralization of the reconstituted collagens was demonstrated with scanning electron microscopy (SEM) and TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of PAA and STTP. The rest were immersed in a biomimetic remineralization medium without PAA and/or STTP (control). In the presence of PAA and STTP in the mineralization medium, intrafibrillar mineralization based on the non-classical crystallisation pathway could be identified. Mineral phases were evident within the collagen fibrils as early as 12 h after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Collagens at 72 h were heavily mineralized with periodically arranged intrafibrillar apatite platelets. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium (control). Intrafibrillar apatite deposition can be achieved via biomimetic mineralization system containing PAA and STTP when amorphous calcium phosphate precursor is stabilized.

Nanostructured Calcium Phosphates for Biomedical Applications

This work is focused on the phase transformation from amorphous calcium phosphate (ACP) to nanostructured hydroxyapatite (HA) or tricalcium phosphate (TCP). Amorphous calcium phosphates with Ca/P molar ratio near 1.67 and 1.5 were synthesized by wet-chemical precipitation method and treated with ethanol. Upon thermal treatment, ACP clusters about 50 nm create a nanostructured HA or TCP. The highlights of this research: The precipitate treatment with ethanol provided a pure α-TCP that was found to be stable up to 1000 °C. HA is obtained from the ACP precursor synthesized using also ammonium dihydrogen phosphate.