Calcium Carbonate Skeletal Material Is Synthesized via Phase Transition of the Calcium Carbonate Cartilaginous Material.

The formation mechanism of calcium carbonate (CC) skeletal tissues in biomineralization has remained poorly understood for a long time. Here, we propose an artificial CC biomineralization system equivalent to the natural one in terms of the primary physicochemical mechanism. Our system is constructed of a polymer gel and a CC solution unsaturated by a dissociated anionic polymer. The gel network consists of proton donor and proton acceptor polymers, which are analogues of polymers in the natural biomineralization system and have affinity for each other through hydrogen bonding interaction. Artificial biomineralization takes place within the polymer gel to produce a monolithic composite of the network and CC, whose powder X-ray diffraction pattern indicates calcite or calcite/vaterite. Scanning electron microscopy and energy-dispersive X-ray spectroscopy observation of the composite during the mineralization process revealed a two-phase structure (network/CC solid solution phase and CC hypercomplex gel phase). As artificial biomineralization proceeds, the solid phase grows in size at the cost of the gel phase as if the latter is substituted with the former, until the solid phase occupies the whole depth of the composite. These results suggest that the hypercomplex gel is the precursor of the resultant network/CC solid solution, and its discontinuous change is a phase transition to the solid solution. Despite minute differences in higher-order structures between our model system and the natural system, the fundamental structure of CC skeletal tissues in the latter can be interpreted as a network/CC solid solution, whereas that of CC cartilaginous tissues as a CC hypercomplex gel. Then, it can be deduced that, in biomineralization, the CC skeletal tissue is in principle formed via a phase transition of the CC cartilaginous tissue.

Formation of Hydroxyapatite Skeletal Materials from Hydrogel Matrices via Artificial Biomineralization.

Several kinds of hydrogels were prepared as mimics for the collagen/acidic protein hydrogel employed as the polymer matrix for mineralization in natural bone formation. The hydrogels prepared as mineralization matrices were employed for synthesizing artificial bones. The artificial bone made from a network of poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) prepared by heating (PVA/PAA-h-network) exhibited mechanical properties comparable with those of fish scales. To elucidate the formation mechanism of the artificial bone, we synthesized four further kinds of matrix. Artificial bones were obtained from both a PVA/PAA network prepared by repeated freezing and thawing (PVA/PAA-ft-network) and a chitosan/PAA network, in which hydrogen bonding exists between the two constituent polymers, similar to that observed in a natural collagen/acidic protein network. The artificial bone made from the chitosan/PAA network was confirmed to be formed by the phase transformation of a cartilaginous precursor by a process similar to the transformation of cartilaginous tissue to natural bone. In addition, skeletal phase material, i.e., a homogeneous solid phase of hydroxyapatite/polymers, was formed in the cartilaginous phase, i.e., the hypercomplex gel. The skeletal phase grew thicker at the expense of the cartilaginous phase until it formed the entirety of the composite. Artificial bones were also obtained from a gelatin/PAA network and a poly[N-(2-hydroxyethyl)acrylamide]-co-(acrylic acid) network. These experimental results suggested that the coexistence of proton donor and proton acceptor functions in the hydrogel is a key factor for bone formation. The hydroxyapatite content of our artificial bones was almost conterminous with those of natural bones.

Construction of a new artificial biomineralization system.

Hydroxyapatite (HAP) was mineralized in poly(vinyl alcohol) (PVA)/poly(acrylic acid) (PAA) complex hydrogel immersed in a salt solution containing PAA. The transparent HAP/polymer composite swelled in water depending on the HAP content; high HAP content gave small swelling and vice versa. The HAP content reached about 80 wt % at most. Observation of the cross section of the composite by energy-dispersive analysis of X-ray (EDAX) revealed that the composite consisted of two phases, i.e., a hard HAP-rich phase and a soft polymer-rich phase. In the HAP-rich phase, the space inside the hydrogel was occupied by HAP, while HAP was not mineralized in the polymer-rich phase. The nucleation seemed to take place, at first, at the middle depth of the hydrogel where the HAP-rich phase was formed. The HAP-rich phase grew its size toward the surface of the hydrogel at the cost of the polymer-rich phase. The presence of phosphorus, P, in the polymer-rich phase indicated the adsorption of HPO(4)(2-) on the polymer chain of the hydrogel via hydrogen bonding, accompanied with Ca(2+) because of electrostatic constraints. This adsorption of ions in addition to Donnan distribution of ions leads to the formation of a hypercomplex that can be regarded as a precursor of the HAP-rich phase. The change of the hypercomplex into the HAP-rich phase is discontinuous and hence concluded as a phase transition. By comparison of our mineralization system with the biomineralization system of HAP and CaCO(3), the physicochemical mechanism of the mineralization process in the present system was found to be similar to that in biological systems. In this sense, we termed the present system an artificial biomineralization system.

Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that alpha-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of alpha-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of alpha-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type alpha-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A alpha-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with alpha-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A alpha-synuclein instead of wild-type alpha-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A alpha-synuclein with synphilin-1 compared with wild-type alpha-synuclein. Expression of S129A alpha-synuclein instead of wild-type alpha-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H(2)O(2) increased alpha-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of alpha-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of alpha-synuclein at S129 may be important for the formation of inclusions in PD and related alpha synucleinopathies.