Alternate Soaking Research Articles

Nonwoven silk fibroin net/nano‐hydroxyapatite scaffold: Preparation and characterization

The purpose of this study was to prepare and characterize a three-dimensional scaffold consisting of porous nonwoven silk fibroin net/nano-apatite composite. The silk fibroin net was mineralized with apatite by alternative soaking in calcium and phosphate solutions. The scaffold exhibited a porous microstructure with open porosity (70-78%), with an average pore size of about 163 +/- 40.0 microm. The swelling ratio and water uptake were 4.55% and 81.93%, respectively, indicating excellent hydrophilicity. The nano-sized hydroxyapatite crystals were needle like, with a length of approximately 100-300 nm and a diameter of approximately 20-60 nm. The results of in vitro cell culture study using rat osteoblast cells demonstrated that the nonwoven silk fibroin net/nano-apatite composite showed excellent cytocompatibility for the growth of osteoblasts and had the capability to improve the viability of osteoblasts.

Hydroxyapatite Deposition by Alternate Soaking Technique on Poly (Vinyl Alcohol)-Coated Poly (ε-Caprolactone) (PCL) Substrate

Biomineralization on surface-modified poly (ε-caprolactone) (PCL) through an alternate soaking process was investigated. A poly(vinyl alcohol) (PVA)-coating on PCL substrate prepared by repetitive adsorption/ drying in an aqueous PVA solution could accelerate hydroxyapatite (HA) deposition while PVA-PCL soaked in aqueous solutions containing Ca2+ and PO3-4 ions alternately. Fourier transform infrared (FTIR) and X-ray diffraction of the apatite verified the formed hydroxyapatite in these processes exhibited a close resemblance with calcium phosphates (HA, Ca10(PO4)6(OH)2). It was also found that the amount of HA formed increased with both soaking with both soaking cycles and deposition amount of PVA on PCL. This study suggested that simple PVA coating on PCL substrate could serve as a novel way to accelerated HAP formation via alternating soaking.

Depositing an Apatite Coating in/on Massive Chitosan Porous Scaffolds by an Alternate Soaking Method

We hope to improve the biological activity and the osteo-conductivity of a porous polymer scaffold by the deposition of a hydroxyapatite (HA) coating. We used an alternate soaking deposition method to prepare HA coating. A massive chitosan (Cs) porous scaffold was used as a deposition template and immersed alternately in calcium chloride and disodium hydrogen phosphate solutions. Finally, a hydroxyapatite coating was deposited in/on the massive chitosan porous scaffold. The compositions, micromorphology, porosity, inorganic deposition amount and compressive strength were characterized by XRD, FT-IR, SEM, porosity measurements, calcination and a compression experiment. Results indicated that the deposition mineral was a low crystallinity carbonate hydroxyapatite with c axis preferential growth and was similar to hydroxyapatite in natural bone. SEM results showed that the sedimentary hydroxyapatite had a gradient distribution in the scaffold. More deposition was observed on the outer pore wall surface than the inner pore wall surface. The exterior pores of the scaffold were partially blocked but the inner pore structure of the scaffold was still interconnected. The porosity of the deposited scaffold was 94.0% and the deposited hydroxyapatite was 13.5% of the total mass after 6 alternate soaking cycles. The compressive strength of the scaffold increased from 0.055 MPa to 0.109 MPa.

Improvement of Biocompatibility of Chitosan Fiber Modified by Ca-Phosphate Deposition through an Alternate Soaking Process

A tube consisted of chitin/chitosan nanofibers is one of the candidates to support nerve regeneration. However, chitin/chitosan would cause inflammation when implanted in human body. This study indicates that chitosan nanofibers (degree of deacetylation; 78% and 93%) could be modified by alternate soaking in calcium and phosphate solutions, resulting in the improvement of their biocompatibility in terms of both in vitro cell culture experiment and in vivo animal test. Deacetylation of the chitosan nanofibers appeared to have little effect on in vitro and in vivo behaviors after 3 days. However, the alternate soaking process clearly improved in vitro and in vivo responses against the chitosan nanofibers. During the process, the surface morphology of the chitosan nanofiber was not changed, i.e. no hydroxyapatite was deposited due to the limited number of soaking. Thus, the surface chemical and physical states of the modified chitosan nanofiber such as surface charge, surface free-energy, and wettability was found to influence both the cell attachment and inflammation.

Scaffold-Free Tissue-Engineered Construct–Hydroxyapatite Composites Generated by an Alternate Soaking Process: Potential for Repair of Bone Defects

Mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC)-hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl(2)/Tris-HCl and Na(2)HPO(4)/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of approximately 1 mum were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. approximately 500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.

Surface modification of P(EMA- co-HEA)/SiO 2 nanohybrids for faster hydroxyapatite deposition in simulated body fluid?

P(EMA- co-HEA)/SiO 2 nanocomposites with 0, 15 and 30 wt% of silica were obtained by copolymerization of ethyl methacrylate, EMA, and hydroxyethyl acrylate, HEA, during the simultaneous acid-catalyzed sol–gel polymerization of tetraethoxysilane, TEOS. A surface modification treatment was applied in order to reduce the induction time for hydroxyapatite (HAp) nucleation, combining a previous NaOH attack to increase the number of surface nucleating sites, and an alternate soaking process in Ca and P solutions to form apatite precursors, prior to the immersion in a simulated body fluid (SBF). The NaOH treatment was not effective by itself in shortening the HAp induction time. It introduced sodium carboxylates in the copolymer but hydrolyzed the silica network excessively, thus reducing the surface nucleating potential of its boundary silanols. Therefore, bioactivity was only due to the surface carboxylate groups of the organic phase. Maybe a controlled dissolution extent of the silica network so as to improve bioactivity could be attained by reducing the duration of the NaOH-treatment. This would be interesting in the hybrid with 30 wt% of silica, because its dense silica network is not able to hydrolyze in SBF without any previous treatment, whereas the silica network in the hybrid with 15 wt% of silica hydrolyzes at the surface promoting the deposition of HAp. The CaP treatment was able to coat the surfaces of the samples with a calcium phosphate layer within minutes. This amorphous calcium phosphate acted as HAp precursor, skipping the induction period in SBF.

Mineralization of chitosan rods with concentric layered structure induced by chitosan hydrogel

Ca ions and P ions absorbed by chitosan hydrogel with a molar ratio of 1.19 were converted into carbonated apatite under ambient condition, by alternate soaking in combination with alkali treatment within a few hours. The alkali treatment helped to convert amorphous calcium phosphate in chitosan hydrogel into carbonated apatite. The bending strength of mineralized chitosan with a concentric layered structure varied from 62.7 MPa to 92.5 MPa, which was 45.8–67.5% as strong as that of rabbit femur. During the process of alternate soaking and alkali treatment, chitosan hydrogel not only provided a medium for the carbonated apatite coating reaction which helped to form the concentric layered structure due to the Liesegang rings phenomenon, but also induced absorption of Ca and P ions in the hydrogel framework via chelation or electrostatic interaction rather than the diffusion model originated from the concentration gradient.

Preparation and characterization of novel β-chitin–hydroxyapatite composite membranes for tissue engineering applications

β-Chitin is a biopolymer principally found in shells of squid pen. It has the properties of biodegradability, biocompatibility, chemical inertness, wound healing, antibacterial and anti-inflammatory activities. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has osteoconductive property. In this work, β-chitin–HAp composite membranes were prepared by alternate soaking of β-chitin membranes in CaCl 2 (pH 7.4) and Na 2HPO 4 solutions for 2 h in each solution. After 1, 3 and 5 cycles of immersion, β-chitin membranes were characterized using the SEM, FT-IR, EDS and XRD analyses. The results showed the presence of apatite layer on surface of β-chitin membranes, and the amounts of size and deposition of apatite layers were increased with increasing number of immersion cycles. Human mesenchymal stem cells (hMSCs) were used for evaluation of the biocompatibility of pristine as well as composite membranes for tissue engineering applications. The presence of apatite layers on the surface of β-chitin membranes increased the cell attachment and spreading suggesting that β-chitin–HAp composite membranes can be used for tissue engineering applications.

Thermal behavior of in vitro mineralized anionic collagen matrices

In the present study porcine skin and bovine pericardium were used as a source of type I collagen. Both were submitted to an alkaline treatment and mineralized by the alternate soaking method. Thermal stability and extent of mineralization have been investigated using DSC and TG. After alkaline hydrolysis there is a decrease in thermal stability but mineralization stabilizes collagen structure. Thermogravimetric data have shown that the amount of hydroxyapatite present in bovine pericardium matrix (45%) was greater than on porcine skin matrix (20%). Presence of hydroxyapatite was confirmed by EDX.

Integration approach for developing a high-performance biointerface: Sequential formation of hydroxyapatite and calcium carbonate by an improved alternate soaking process

Biointerfaces are crucial for regulating biofunctions. An effective method of producing new biomaterials is surface modification, in particular, the hybrid organic–inorganic approach. In this paper, we propose a method for the sequential formation of hydroxyapatite and calcium carbonate on porous polyester membranes by using an improved alternate soaking process. The resulting hybrid membranes were characterized in terms of their calcium and phosphorus ion contents; further, their structure was analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and infrared spectroscopy (IR). As a typical biofunction, protein adsorption by these hybrid membranes was investigated. Sequential hydroxyapatite and calcium carbonate formation on the membranes was successfully achieved, and the total amounts of hydroxyapatite and calcium carbonate formed were precisely regulated by the preparative conditions. The SEM and XRD characterizations were verified by comparing with the IR results. The amount of adsorbed protein correlated well with not only the amount of hydroxyapatite formed but also the combined amounts of hydroxyapatite and calcium carbonate formed. The results indicate that the hybrid membranes can function as high-performance biointerfaces that are capable of loading biomolecules such as proteins.

Anisotropic hydroxyapatite formation inside agarose gels by integration of electrophoretic and alternate soaking approaches

We report here anisotropic hydroxyapatite (HAp) formation inside an agarose gel using an electrophoretic approach and an alternate soaking process. Effective ion migration is shown to be crucial to integrate hydrogel with biominerals, for example, calcium carbonate and HAp. Calcium and phosphate ions easily migrated into the gel interior from ionic solutions when an electric field was applied to both terminals of the gel. The time to reach complete biomineral formation was only 3 min. In this process, roughly 75–300 μg of HAp was formed in 1 mg of dry gel. Moreover, we carried out alternate soaking using the resulting materials and further HAp formation was observed (750 μg HAp/mg dry gel). HAp was characterized by infrared spectroscopy and X-ray diffraction, and was then assigned. Moreover, the HAp was formed in an anisotropic manner. First, (210) and (211) were preferentially formed by the electrophoretic approach. Alternatively, (002) and (211) were formed after the alternate soaking process. This result indicated that preparative conditions were shown to be crucial to regulate anisotropic crystal growth and to keep it the major component. In summary, it is considered that anisotropic ion diffusion inside the hydrogel would be the dominant factor by electrophoretic and alternate soaking approaches.

Mineralization of Chitosan via Alternate Soaking

Hydroxyapatite coating was prepared by alternate soaking strategy in short period time and ambient condition. According to the pH near equality between chitosan acidic acid solution and H2PO4 - aqueous solution, we proposed to choose H2PO4 - as one of hydroxyapatite precursor instead of traditional candidate (HPO4 2-). The properties of chitosan hydrogel containing plenty of water provide enough spaces for hydroxyapatite precursor to diffuse into framework spontaneously. XRD, FTIR and SEM were used to characterize the component and microstructure of mineralized chitosan. The ageing process helps to transfer amorphous calcium phosphate in chitosan framework into hydroxyapatite. The hydroxyapatite coating is gradient structure according to the result of SEM.

Formation of Hydroxyapatite Provides a Tunable Protein Reservoir within Porous Polyester Membranes by an Improved Soaking Process

Biomineralization on porous polyester membranes was examined using an improved alternate soaking process (ASP). The effect of ion migration for the formation of hydroxyapatite (HAp) was shown to be crucial. Ion migration was improved by reducing the surface tension by mixing ethanol into an aqueous solution. The resulting hybrid materials were evaluated in terms of calcium content; structure using scanning electron microscopy (SEM), X-ray diffraction (XRD), and infrared spectroscopy (IR); and protein adsorption. The amount of formed HAp was controlled by the number of ASP cycles and also through the ethanol content of the mixed solvent. As the formation of HAp increased, the formed structure could be verified using SEM, IR, and XRD. Protein adsorption was investigated using albumin, gamma-globulin, and fibrinogen, and the amount of adsorbed protein was well-correlated with that of the formed HAp. This result shows that the total amount of the adsorbed proteins can be regulated by the HAp content. In summary, a tunable protein reservoir was formed on a porous polyester membrane.

Apatite coating on anionic and native collagen films by an alternate soaking process

The present study focuses on apatite coating on collagen films, with various different densities of carboxyl groups, using an alternate soaking process. Anionic collagen (AC), which has different densities of carboxylic groups compared to native collagen (NC), was obtained by hydrolysis of carboxyamides of asparagine and glutamine residues. From X-ray diffraction analysis, apatite was found to be coated on AC and NC films. Peaks ascribed to apatite were observed at 26° and 32° in the diffraction patterns of hydroxyapatite crystals. The amount of apatite coated on both AC and NC collagen films continued to increase up to 100 reaction cycles. However, there is a significant difference in apatite coating between the two films. The amount of apatite formed on the surface of AC film increased 1.24 times faster than on NC film. The scanning electron photomicrograph images of the mineralized NC and the AC film coatings formed after 100 cycles show that regular porous apatite coating had formed within the collagen fibrils. These results suggest that the higher content of carboxyl groups in AC plays an effective role in the heterogeneous nucleation of apatite in the body environment.

Effects on gingival cells of hydroxyapatite immobilized on poly(ethylene‐co‐vinyl alcohol)

Hydroxyapatite was immobilized on poly(ethylene-co-vinyl alcohol) (EVA) by alternate soaking in aqueous CaCl(2) and Na(2)HPO(4) solutions, followed by carboxyl groups introduction through ozone exposure in order to investigate the nature of the gingival cells, to control their proliferation and properties and to develop a highly organized hybrid implant possessing periodontium. Human gingival cells were cultured on the ozone-exposed EVA, collagen-immobilized EVA, hydroxyapatite-immobilized EVA, and a conventional tissue culture dish. Cell proliferation was highest on the tissue culture dish and lowest on the hydroxyapatite-immobilized EVA. The results of RT-PCR of gingival cells on hydroxyapatite-immobilized EVA shows that mRNAs expressed in bone and periodontal ligament were determined. Furthermore, alkaline phosphatase activity and ELISA assay revealed that gingival cells acquired the osteoblastic properties when cultured on hydroxyapatite-immobilized EVA, suggesting that the periodontium might be regenerated around implants using gingival cells.

Calcium Phosphate Hybridized with Human Semitendinosus and Gracilis Tendon Grafts

We hybridized calcium phosphate (CaP) with human semitendinosus and gracilis (ST/G) tendon grafts using an alternate soaking process. To evaluate quantitatively and histologically assess the CaP hybridized human ST/G tendon grafts, we classified them into three groups according to their soaking time – number of soaking cycle: 30 sec – 20 cycles (Group A), 1 min – 15 cycles (Group B), 3 min – 5 cycles (Group C). The tendon grafts were divided into three parts: tibial end (TE), femoral end (FE) and intra-articular (IA) portion. TE was secured using the Krackow technique with No. 2 nonabsorbable sutures, and an Endobutton-CL (Smith &amp; Nephew, USA) was passed through the looped FE, as performed clinically. Then, the IA portion was covered with the sleeve of a rubber glove to prevent CaP hybridization. More soaking cycles induced greater deposition of CaP in the tendon grafts when the total soaking time was the same. Covering the IA portion with a rubber sleeve prevented of CaP deposition. A large amount of CaP in TE was deposited because suture holes increased the total contact area with the solutions.

Bioactive Apatite Coating on Titanium Using an Alternate Soaking Process

We developed a novel apatite coating method that consisted of two-step of chemical treatment: a combined pretreatment of concentrated acid etching and alkaline treatment, followed by alternate soaking. In this study, the effects of the number of reaction cycles, solution temperature, and soaking time on apatite deposition on titanium surface using alternate soaking were investigated. Results revealed that the deposited amount of apatite mainly depended on the number of reaction cycles, and was independent of solution temperature and soaking time. Characterization results revealed that apatite formation using alternate soaking basically depended on ion exchange and adsorption on the pretreated surface. Further, apatite coating using alternate soaking on a 200-grid titanium mesh confirmed that this coating method was applicable for substrates with complicated shapes.

Study of biomineralization of poly(vinyl alcohol)‐based scaffolds using an alternate soaking approach

AbstractThe present work adds to the continuing efforts of designing a natural bone‐like structure by synthesizing a semi‐interpenetrating polymer network (IPN) of poly(vinyl alcohol)–poly[(acrylic acid)‐co‐acrylonitrile] and impregnating hydroxyapatite (HAP) into the polymer matrix by an alternate soaking process. The prepared HAP–polymer scaffolds were characterized using techniques like Fourier transform infrared spectroscopy, X‐ray diffraction, differential scanning calorimetry, thermogravimetric analysis and environmental scanning electron microscopy. The biomineralized semi‐IPN was evaluated for water sorption capacity and the data were utilized for calculating network parameters such as average molecular weight between crosslinks (Mc) and crosslink density (q). The impregnated HAP was quantified as a function of the chemical architecture of the semi‐IPN, number of reaction cycles and temperature of the swelling bath. Copyright © 2006 Society of Chemical Industry

交互浸漬法によるチタンへのアパタイトコーティング（第3報）－チタンメッシュへの応用－

交互浸漬法によるチタンへのアパタイトコーティング（第3報）－チタンメッシュへの応用－

Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation

We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.