Novel anti-decay self-setting paste of hydroxyapatite/collagen nanocomposite utilizing GPTMS

Bone is a typical inorganic/ortganic nanocomposite mainly composed of hydroxyapatite (HAp) nanocrystals and collagen molecules. The composition and nanostructure is closely related to bone’s biomechanical and biochemical properties. One of the most important things for bone is a bone remodeling process that maintains mechanical strength of bone to allow walking and running as well as homeostasis of calcium in our body. In fact, sintered HAp composed of HAp crystals approximately 1 µm in particle size is considered as non-bioresorbable; however HAp nanocrystals easily resorbed by osteoclasts. A hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) was successfully synthesized by the authors via self-organizing process. [1] The HAp/Col is incorporated into bone remodeling process completely as the same as the autologous bone transplanted and substituted with new bone in 3 months. Porous type HAp/Col was also developed by the authors and shows spoge-like viscoelastity in wet condition. [2] The porous HAp/Col had been tested clinically and has been being sold in Japan from April, 2013 as ReFit®. According to the clinical test [3], the HAp/Col shows higher rate of remarkable efficiency in comparison to Osferion®, porous β-tricalcium phosphate, for substitution with newly formed bone. In addition, sponge-like deformability allows to fit irregular shape of bone defects as well as to press to pack the porous HAp/Col in cavity created by removal of benign bone tumor. On the other hand, recent need from surgeon for bone filler is a self-setting bone paste with bioresorbability. Previously, the HAp/Col anti-decay self-setting paste was prepared successfully with the use of sodium alginate and calcium compounds. It showed tolerant paste properties; however, the paste contained 30 % in mass of calcium compounds that could interfered excellent biological activity of the HAp/Col. In this study, the HAp/Col paste with injectability, self-setting and anti-decay abilities was prepared using the HAp/Col powder and 1 % in volume of 3-glycidoxypropyl)methyldiethoxysilane (GPTMS) aqueous solution. The paste obtained was evaluated by viscosity, hardening behavior and anti-decay property tests. The HAp/Col was synthesized according to ref 1. The HAp/Col was compacted with specially designed mold by uniaxially press squeezing of water from the HAp/Col. The HAp/Col compact was then crushed and ball-milled to obtain the HAp/Col particles of 100 µm or less in size. The GPTMS was dissolved in pure water at 1 % in volume and steadily placed in 25 °C for 1 h to allow hydrolysis of the GPTMS to form silanol groups. The HAp/Col powder (powder phase, P) and the GPTMS aqueous solution (liquid phase, L) were mixed at 0.20-2.00 of P/L ratio in g/cm3 to obtain a HAp/Col paste. A viscosity of the HAp/Col paste obtained was measured according to ref 1. Briefly, the paste obtained was shaped to cylinder at 5 mm in diameter and 5.1 mm in hight and start pressing at 10 min after mixing by 2 kg weight for 10 min. A spread area of the paste was measured from digital photo with Image-J. Hardening behavior was measured using the viscosity test as a function of time, because softness of the paste did not allow to apply conventional the needle method. Anti-decay property was tested according to JIS T 0330-4:2012 [5]; briefly, the paste shaped in cylinder 4.8 mm in diameter and 16.5 mm in height was placed on wire mesh and, at 5 min after mixing, soaked in phosphate buffered saline for 72 h. Debris were then collected and measured their mass, and decay ratio was calculated as debris/original masses. The paste with P/L ratio of 0.2 could not shaped by its high fluidity and that of 2.0 could not shaped because of its aggregation. The paste with P/L ratio of 0.33 could injected through 18G needle and others could injected through syringe with 1.8 mm in inner diameter. The viscosity of the paste increased with increasing in P/L ratio and did not depend on the amounts of GPTMS. The initial hardening was observed first 30-40 min from mixing and gradually hardened. The paste with P/L ratio of 1.5 showed mechanical strength more than 1 MPa with viscoelastic property. No significant decay was observed for all pastes. The HAp/Col-GPTMS paste can be good candidate for high performance injectable bone filler as well as a raw material for 3D printing

A Case of Hypocalcaemia Due to Vitamin D Deficiency in ‘Hikikomori’ Syndrome

Objective: To describe hypocalcaemia due to vitamin D deficiency in ‘hikikomori’ syndrome. Materials and methods: A 37-year-old man with ‘hikikomori’ syndrome for a year was admitted with hypocalcaemia (serum ionic calcium 1.17 mmol/l). Serum 1,25(OH)2-vitamin D3 determined by liquid chromatography–tandem mass spectrometry was depressed at 12.1 pg/ml (29.0 pmol/l) and plasma intact PTH elevated at 324 ng/l. Administration of 1 μg/day 1α(OH)-vitamin D3 and 1 g/day calcium lactate for 1 week normalized calcium and PTH, and raised 1,25(OH)2-vitamin D3 to low normal levels. Conclusion: This is the first report of hypocalcaemia due to vitamin D deficiency in a patient with ‘hikikomori’ syndrome

Solid-state nuclear magnetic resonance study of setting mechanism of beta-tricalcium phosphate-inositol phosphate composite cements

Solid-state nuclear magnetic resonance (NMR) spectroscopy is a technique, which can be used to provide insight into the chemical structure of non-crystalline and crystalline materials. Hence, the present study aimed to elucidate the setting mechanism of CPC, which was fabricated using beta -tricalcium phosphate (beta -TCP)-inositol phosphate (IP6) composite powder using NMR In addition, the effect of IP6 on the local chemical structure of the beta -TCP-IP6 composite powder and its hardened cement would also be investigated. The H-1 -> P-31 heteronuclear correlation NMR spectrum revealed that an amorphous hydrated layer, along with small amount of hydroxyapatite (HA) was formed on the surface of beta -TCP during the ball-milling process. Results demonstrated that the IP6 in the hydrated layer on the surface of beta -TCP inhibited the formation of HA. Moreover, the setting reaction of the cement was mainly triggered by the dissolution of the amorphous hydrated layer on beta -TCP surface, and subsequent precipitation, followed by the inter-entanglement between the HA crystals on the beta -TCP

Potential Application of Protamine for Antimicrobial Biomaterials in Bone Tissue Engineering

Bacterial infection of biomaterials is a serious problem in the field of medical devices. It is urgently necessary to develop new biomaterials with bactericidal activity. Antimicrobial peptides and proteins (AMPs), alternative antibacterial agents, are expected to overcome the bacterial resistance. The aim of this study was to develop a new intelligent material in bone tissue engineering based on protamine-loaded hydroxyapatite (protamine/HAp) that uses AMPs rather than antibiotics. It was found that the adsorption of protamine to HAp followed the Langmuir adsorption model and was due to electrostatic and/or hydrophobic interactions. In vitro bacterial adhesion and growth on protamine/HAp was inhibited in a protamine dose-dependent manner. Adherent bacteria exhibited an aberrant morphology for high dosages of protamine/HAp, resulting in the formation of large aggregates and disintegration of the membrane. The released protamine from protamine/HAp also prevented the growth of planktonic bacteria in vitro. However, a high dosage of protamine from powders at loading concentrations over 1000 μg·mL−1 induced a cytotoxic effect in vitro, although those exhibited no apparent cytotoxicity in vivo. These data revealed that protamine/HAp (less than 1000 μg·mL−1) had both antimicrobial activity and biocompatibility and can be applied for bone substitutes in orthopedic fields