Rotational Dynamics of Water near Osmolytes by Molecular Dynamics Simulations.

The behavior of water molecules around organic molecules has attracted considerable attention as a crucial factor influencing the properties and functions of soft matter and biomolecules. Recently, it has been suggested that the change in protein stability upon the addition of small organic molecules (osmolytes) is dominated by the change in the water dynamics caused by the osmolyte, where the dynamics of not only the directly interacting water molecules but also the long-range hydration layer affect the protein stability. However, the relation between the long-range structure of hydration water in various solutions and the water dynamics remains unclear at the molecular level. We performed density-functional tight-binding molecular dynamics simulations to elucidate the varying rotational dynamics of water molecules in 15 osmolyte solutions. A positive correlation was observed between the rotational relaxation time and our proposed normalized parameter obtained by dividing the number of hydrogen bonds between water molecules by the number of nearest-neighbor water molecules. For the 15 osmolyte solutions, an increase or a decrease in the value of the normalized parameter for the second hydration shell tended to result in water molecules with slow and fast rotational dynamics, respectively, thus illustrating the importance of the second hydration shell for the rotational dynamics of water molecules. Our simulation results are anticipated to advance the current understanding of water dynamics around organic molecules and the long-range structure of water molecules.

Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers.

Isolating cancer cells from tissues and providing an appropriate culture environment are important for a better understanding of cancer behavior. Although various three-dimensional (3D) cell culture systems have been developed, techniques for collecting high-purity spheroids without strong stimulation are required. Herein, we report a 3D cell culture system for the isolation of cancer spheroids using enzymatically synthesized cellulose oligomers (COs) and demonstrate that this system isolates only cancer spheroids under coculture conditions with normal cells. CO suspensions in a serum-containing cell culture medium were prepared to suspend cells without settling. High-purity cancer spheroids could be separated by filtration without strong stimulation because the COs exhibited antibiofouling properties and a viscosity comparable to that of the culture medium. When human hepatocellular carcinoma (HepG2) cells, a model for cancer cells, were cultured in the CO suspensions, they proliferated clonally and efficiently with time. In addition, only developed cancer spheroids from HepG2 cells were collected in the presence of normal cells by using a mesh filter with an appropriate pore size. These results indicate that this approach has potential applications in basic cancer research and cancer drug screening.

Association Between Reduced Posterior Occlusal Contact and Alzheimer's Disease Onset in Older Japanese Adults: Results from the LIFE Study.

BACKGROUND: An association between poor oral health and cognitive decline has been reported. Most of these studies have considered the number of teeth as a criterion, only a few studies have analyzed the relationship between occlusal status and Alzheimer's disease (AD). OBJECTIVE: To elucidate whether posterior occlusal contact is associated with AD, focusing on the Eichner classification, among an older population aged 65 years or older in Japan. METHODS: This study used monthly claims data of National Health Insurance in Japan from April 2017 to March 2020. The outcome was newly diagnosed AD defined according to ICD-10 code G30. The number of teeth was estimated by dental code data, and occlusal contact was divided into three categories, namely A, B, and C, according to the Eichner classification. Multivariate Cox proportional hazards models were used to analyze the association between a new diagnosis of AD and the Eichner classification. RESULTS: A total of 22,687 participants were included, 560 of whom had newly diagnosed AD during a mean follow-up period of 12.2 months. The AD participants had a lower proportion of Eichner A and a higher proportion of Eichner C. After adjusting for covariates, hazard ratios (95% confidence intervals) with Eichner B and C were 1.34 (1.01-1.77) and 1.54 (1.03-2.30), respectively. CONCLUSION: In older people aged≥65 years old, reduced posterior occlusal contact as well as tooth loss have an impact on AD. This study emphasizes the importance of paying attention to occlusal contacts to reduce the risk of AD.

Simple Detection and Culture of Circulating Tumor Cells from Colorectal Cancer Patients Using Poly(2-Methoxyethyl Acrylate)-Coated Plates.

Here we aimed to establish a simple detection method for detecting circulating tumor cells (CTCs) in the blood sample of colorectal cancer (CRC) patients using poly(2-methoxyethyl acrylate) (PMEA)-coated plates. Adhesion test and spike test using CRC cell lines assured efficacy of PMEA coating. A total of 41 patients with pathological stage II-IV CRC were enrolled between January 2018 and September 2022. Blood samples were concentrated by centrifugation by the OncoQuick tube, and then incubated overnight on PMEA-coated chamber slides. The next day, cell culture and immunocytochemistry with anti-EpCAM antibody were performed. Adhesion tests revealed good attachment of CRCs to PMEA-coated plates. Spike tests indicated that ~75% of CRCs from a 10-mL blood sample were recovered on the slides. By cytological examination, CTCs were identified in 18/41 CRC cases (43.9%). In cell cultures, spheroid-like structures or tumor-cell clusters were found in 18/33 tested cases (54.5%). Overall, CTCs and/or growing circulating tumor cells were found in 23/41 CRC cases (56.0%). History of chemotherapy or radiation was significantly negatively correlated with CTC detection (p = 0.02). In summary, we successfully captured CTCs from CRC patients using the unique biomaterial PMEA. Cultured tumor cells will provide important and timely information regarding the molecular basis of CTCs.

Roles of interfacial water states on advanced biomedical material design.

When biomedical materials come into contact with body fluids, the first reaction that occurs on the material surface is hydration; proteins are then adsorbed and denatured on the hydrated material surface. The amount and degree of denaturation of adsorbed proteins affect subsequent cell behavior, including cell adhesion, migration, proliferation, and differentiation. Biomolecules are important for understanding the interactions and biological reactions of biomedical materials to elucidate the role of hydration in biomedical materials and their interaction partners. Analysis of the water states of hydrated materials is complicated and remains controversial; however, knowledge about interfacial water is useful for the design and development of advanced biomaterials. Herein, we summarize recent findings on the hydration of synthetic polymers, supramolecular materials, inorganic materials, proteins, and lipid membranes. Furthermore, we present recent advances in our understanding of the classification of interfacial water and advanced polymer biomaterials, based on the intermediate water concept.

Effect of Osmolytes on Water Mobility Correlates with Their Stabilizing Effect on Proteins.

There is a long, ongoing debate on how small molecules (osmolytes) affect the stability of proteins. The present study found that change in collective rotational dynamics of water in osmolyte solutions likely has a dominant effect on protein denaturation. According to THz spectroscopy analysis, osmolytes that stabilize proteins are accompanied by bound hydration water with slow dynamics, while the collective rotational dynamics of water is accelerated in the case of denaturant osmolytes. Among 15 osmolytes studied here, there is a good correlation between the change in mobility in terms of water rotational dynamics and the denaturation temperature of ribonuclease A. The changes in water dynamics due to osmolytes can be regarded as a pseudo-temperature-change, which agrees well with the change in protein denaturation temperature. These results indicate that the molecular dynamics of water around the protein is a key factor for protein denaturation.

Modulation of Biological Responses of Tumor Cells Adhered to Poly(2-methoxyethyl acrylate) with Increasing Cell Viability under Serum-Free Conditions.

Circulating tumor cells in body fluids are important biomarkers in cancer diagnosis. The culture of tumor cells isolated from body fluids can provide intrinsic information about tumors and can be used to screen for the best anticancer drugs. However, the culture of primary tumor cells has been hindered by their low viability and difficulties in recapitulating the phenotype of primary tumors in in vitro culture. The culture of tumor cells under serum-free conditions is one of the methodologies to maintain the phenotype and genotype of primary tumors. Poly(2-methoxyethyl acrylate) (PMEA)-coated substrates have been investigated to prolong the proliferation of tumor cells under serum-free conditions. In this study, we investigated the detailed behavior and the mechanism of the increase in tumor cell viability after adherence to PMEA substrates. The blebbing formation of tumor cells on PMEA was attributed not to apoptosis but to the low adhesion strength of cells on PMEA. Moreover, blebbing tumor cells showed amoeboid movement and formed clusters with other cells via N-cadherin, leading to an increase in tumor cell viability. Furthermore, the behaviors of tumor cells adhered to PMEA under serum-free conditions were involved in the activation of the PI3K and Rho-associated protein kinase pathways. Thus, we propose that PMEA would be suitable for the development of devices to cultivate primary tumor cells under serum-free conditions for the label-free diagnosis of cancer.

Effect of bound water content on cell adhesion strength to water-insoluble polymers.

Adhesion of cells on biomaterials plays an essential role in modulating cellular functions. Although hydration of biomaterials occurs under biological conditions, it is challenging to systematically evaluate the correlation of hydrated water content in biomaterials with the cell adhesion strength. In this report, we investigated the effect of bound water content on the adhesion strength of cells on poly(2-methoxyethyl acrylate) (PMEA) analogue substrates. Water-insoluble PMEA analogues were synthesized to fabricate substrates with a systemically controlled bound water content. To assess the surface properties of their substrates, contact angle measurement, atomic force microscopy (AFM), and fluorescence measurement were conducted. To reflect the effect of bound water of PMEA analogues, the relationship between the bound water content and cell adhesion behavior was evaluated under serum-free condition. From the single cell force spectrometry (SCFS) and microscopic analysis, it revealed that the increment of bound water content on the substrates decreased cell adhesion strength and cell spreading on the substrates. The bound water content exhibited a good correlation with adhesion strength, spreading area, circularity, and aspect ratio of cells. Our findings indicate that the bound water content could contribute to the development of a novel biomaterial and evaluation of cell behaviors on biomaterials. STATEMENT OF SIGNIFICANCE: For coordinating cell functions, such as growth, mobility, and differentiation, modulating the adhesion strength between cells and their environments is important. Although the hydration to biomaterials has been reported to be closely related to a antifouling property, the effect of hydration water on the cell adhesion behavior is not well understood. We present the first demonstration of essential relationship between cell adhesion strength and hydrated water on a biomaterials surface using the water-insoluble polymers with different hydrated water content. The results reveal that the hydrated water content of polymer substrates strong correlation with adhesion strength of cells. Collectively, the hydrated water content of the biomaterials will be a predominant factor affecting the cell adhesion strength and behavior.

Impact of simultaneous hydrolysis of OCP and PLGA on bone induction of a PLGA-OCP composite scaffold in a rat femoral defect.

Effect of the simultaneous hydrolysis of octacalcium phosphate (OCP) and poly (lactic-co-glycolic acid) (PLGA) was investigated on its osteoconductivity. PLGA soaked in phosphate buffered saline with 0%, 20%, and 40% OCP at 37°C for eight weeks indicated that when the OCP dose was increased, 1) the weight loss of PLGA increased, 2) the glass transition temperature of the PLGAs decreased, 3) the saturation degree in the saline moved to nearly saturated condition with respect to hydroxyapatite (HA) but was undersaturated with respect to OCP, and 4) OCP tended to convert to HA by X-ray diffraction and Fourier transform infrared spectroscopy. OCP/PLGA composites of 20% and 40% with more than 92% porosity were produced by combining OCP granules with 1,4-dioxane-solubilizing PLGA followed by lyophilization and then subjected to four- and eight-week in vivo implantation tests in 3 mm diameter rat femora defects. Microfocus X-ray computed tomography, histochemical and histomorphometric analyses showed that while bone formation was very limited with PLGA implantation, the extent of repair tended to increase with increasing OCP content in the PLGA, coupled with PLGA degradation, and bridge the defects with trabecular bone. Tartrate-resistant acid phosphatase-positive osteoclast-like cells were accumulated four weeks after implantation, while osteocalcin-positive osteoblastic cells appeared later at eight weeks, especially in 40% OCP/PLGA. These results suggest that OCP hydrolysis, with phosphate ion release, enhances PLGA hydrolysis, probably through the acid catalysis function of the protons supplied during the hydrolysis of OCP, thereby inducing PLGA biodegradation and new bone formation in the femoral defects. STATEMENT OF SIGNIFICANCE: Octacalcium phosphate (OCP) enhances osteoblasts and osteocytes differentiations during its hydrolysis accompanying inorganic ions exchange in this material. The present study found that the advancement of OCP hydrolysis under physiological conditions had an effect on poly (lactic-co-glycolic acid) (PLGA) degradation through its chemical environmental change around OCP, which was ascertained by the decreases in weight loss and glass transition temperature of PLGA with increasing the dose of OCP co-present. Rat femur-penetrated standardized severe defects were found to repair through bridging the cortical region defect margin. PLGA degradation could be enhanced through an acid catalyst function by protons derived from inorganic phosphate (Pi) ions through OCP hydrolysis under bone forming condition, resulting in showing a prominent bone regenerative capacity in OCP/PLGA composite materials.

Effect of surface modification of Ti-6Al-4V alloy by electron cyclotron resonance plasma oxidation.

Ti-6Al-4V alloy is used as biomaterials for dental and orthopedic implants because of their excellent biocompatibilities and mechanical properties. However, it is unclear that electron cyclotron resonance (ECR) plasma oxidation can create the oxide films on Ti-6Al-4V alloy surface, and this technique improves the ability of its osseointegration. The purpose of this study was to investigate the characteristics and calcification ability of the oxide films. X-ray diffraction (XRD) peaks of rutile phase were intensified with increasing the temperature. Scanning electron microscopy (SEM) images showed a crater-like structure, and bonding strengths between the substrate and oxide film reached a maximum at 400°C. Calcium phosphate (CaP) compounds after calcification process were identified as octacalcium phosphate (OCP) and precipitation amount was maximized at 400°C. The results suggested that the altered surface of Ti-6Al-4V alloy by ECR plasma oxidation might have the potential of accelerating the ability of its osseointegration through enhancement of OCP.

Chemical Stability-Sensitive Osteoconductive Performance of Octacalcium Phosphate Bone Substitute in an Ovariectomized Rat Tibia Defect.

The purpose of this study was to investigate whether the chemical condition of osteoporotic serum affects the chemical stability of octacalcium phosphate (OCP) and its osteoconductive property. The in vitro chemical dissolution in osteoporotic ovariectomized (OVX)-simulated conditions was analyzed. OCP and its composite form with gelatin (OCP/Gel), containing specific amounts of OCP (either 17% or 44% by weight), were used as experimental materials. The degrees of supersaturation (DS) of the OVX-simulated buffer solutions, containing distinct inorganic phosphate (Pi) ion concentrations, after immersing OCP or OCP/Gel, were determined. The rod-shaped OCP/Gel was then implanted into the OVX and Sham rat tibia defects, exhibiting a similar shape and size, and assessed at 4, 8, and 12 weeks. Increasing Pi concentration in OVX-simulated buffer solution increased the DS, with respect to OCP, upon the introduction of OCP and 44% OCP/Gel, but decreased the DS to a slightly saturated condition with 17% OCP/Gel, indicating that increasing the OCP in the Gel matrix tends to inhibit the hydrolysis of OCP into hydroxyapatite (HA). Histomorphometric analyses of bone formation and the appearance of osteoblasts and osteoclast-like cells, together with OCP resorption, confirmed that while 44% OCP/Gel showed higher bone formation than 17% OCP/Gel at intramedullary bone defect sites in Sham rat tibia, both OCP/Gels tended to enhance cortical bone formation in the OVX group, concomitant with the higher resorption of OCP within 17% OCP/Gel. The appearance of osteoclast-like cells in the OVX group increased as the OCP dose decreased from 44% to 17% in the Gel matrix, with an approximately 4 times higher bone formation rate, 8-12 weeks after the implantation. Additional in vitro assays showed that bone marrow mesenchymal stem cells isolated from OVX and wild-type (WT) rats treated with OCP had similar proliferation and differentiation rates, up to 21 days. These results show that OCP can enhance cortical bone repair even in osteoporotic bone if suitable thermodynamic metastable dissolution conditions are provided in relation to the mass of OCP.

Histomorphometric assessments of peri-implant bone around Ti-Nb-Sn alloy implants with low Young's modulus.

Many ß-Ti alloys have been developed for, and used in, medical devices because of the corrosion resistance, biocompatibility, and exceptionally low Young's modulus. The aim of the present study was to investigate the histomorphometric aspects of peri-implant bone around Ti-Nb-Sn alloy implants and compare them with those in the case of commercially pure Ti (Ti). Fluorescent morphological observations of ST-2 cells on the substrate were performed and bone morphogenesis around implants in rat femur was evaluated. There was no difference between the cell morphology on Ti and those on the Ti-Nb-Sn alloy during observation for 24 h. A comparison of the Ti-Nb-Sn alloy implant and the Ti implant showed no significant differences between the bone-to-implant contact ratios or the bone fractions. These results suggest that the biological adaptations with Ti-Nb-Sn implants during a healing period are similar to those with Ti. Ti-Nb-Sn is therefore suitable for use in dental implants.

Vascularized Bone-Mimetic Hydrogel Constructs by 3D Bioprinting to Promote Osteogenesis and Angiogenesis.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.

Culture of hybrid spheroids composed of calcium phosphate materials and mesenchymal stem cells on an oxygen-permeable culture device to predict in vivo bone forming capability.

Three-dimensional (3-D) cell culture can better mimic physiological conditions in which cells interact with adjacent cells and the extracellular matrix than monolayer culture. We have developed a 3-D cell culture device, the Oxy chip, which can be used to generate and supply oxygen to cell spheroids to prevent hypoxia. Here, we used the Oxy chip to generate hybrid spheroids comprising calcium phosphate (CaP) particles (hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP) or octacalcium phosphate (OCP)) and mesenchymal stem cells (MSCs, C3H10T1/2 cells or D1 cells) that can be used to analyze cell differentiation mechanisms. We showed that the 3-D cell-cell and cell-material interactions and oxygenation offered by the Oxy chip promoted osteoblastic differentiation of MSCs. We also used histomorphometric analysis of hematoxylin and eosin staining, quality analyses by µCT and collagen orientation observation with picrosirius red staining in bone regeneration following implantation of three CaPs in a critical-sized defect in mouse calvaria. The in vivo bone formation capacity of the three tested CaP materials was OCP ≥ ß-TCP > HA: the newly formed bone by OCP had a structure relatively close to that of the calvaria intact bone. When MSCs were 3-D cultured with the CaP materials using the Oxy chip, the in vitro osteogenic capacity of these materials was highly similar to trends observed in vivo. The in vitro alkaline phosphatase activity of D1 cells had the highest correlation with in vivo bone volume (R = 0.900). Chemical and FTIR spectroscopic analyses confirmed that differentiation of D1 cells could be associated with amorphous calcium phosphate (ACP) precipitation concomitant with OCP hydrolysis. Taken together, hybrid spheroid cultures using the Oxy chip can be used to screen and predict bone forming potential of bone substitute materials. STATEMENT OF SIGNIFICANCE: An oxygen permeable-culture chip (Oxy chip) can be used to induce formation of cell spheroids by mesenchymal stem cells (MSCs). Use of the Oxy chip avoids hypoxia in the spheroid core and enhances MSC osteoblastic differentiation relative to conventional spheroid culture methods. The present study showed that the Oxy chip mimics the in vivo environment associated with bone formation and can be used to generate hybrid spheroids consisting of calcium phosphates and MSCs that are useful for analyzing cell differentiation mechanisms. Bone formation analysis following implantation of calcium phosphate materials in mouse calvaria defects showed positive correlation with the in vitro results. We propose that hybrid spheroids cultured on the Oxy chip can be used to screen and predict the bone forming potential of bone substitute materials.

Angiogenesis involvement by octacalcium phosphate-gelatin composite-driven bone regeneration in rat calvaria critical-sized defect.

Effect of octacalcium phosphate/gelatin composite (OCP/Gel) on angiogenesis was studied by its implantation in rat calvaria critical-sized defect in relation to bone regeneration for 2 and 4 weeks. The implantation of OCP/Gel disks was analyzed by radiomorphometry using a radiopaque material perfusion (Microfil®) method and histomorphometry by hematoxylin and eosin-staining before and after the decalcification. Effect of the OCP dose in the range up to 4 mg per well on the capillary-like tube formation by human umbilical vein endothelial cells (HUVECs) was also examined in a transwell cell culture. The results showed that the blood vessels formation by OCP/Gel group was significantly higher at 2 weeks than other groups but decreased at 4 weeks during the advancement of new bone formation. The capillary-like tube formation was highest in an OCP dose of 1 mg per well while other OCP doses above or below 1 mg did not show such a stimulatory effect. The results established both in vivo and in vitro confirmed that OCP has a positive effect on angiogenesis during bone regeneration in a suitable dose ranges, suggesting that the angiogenesis stimulated by OCP could be involved in the OCP/Gel-enhanced bone regeneration. STATEMENT OF SIGNIFICANCE: We have reported that octacalcium phosphate (OCP) materials display stimulatory capacities on the bone tissue-related cells. However, the effect of OCP on the angiogenesis and its relation to the OCP-enhanced bone regeneration is unknown. This study confirmed the capacity of OCP on angiogenesis before increasing the new bone mass after the implantation of a composite of OCP and gelatin (OCP/Gel). The blood vessels formation took place associated with the area beginning of the new bone formation, which finally decreased together with development of bone formation. Because OCP was ascertained stimulating the capillary-like tube formation in HUVEC culture with a certain OCP dose, the present study is the first report showing the capacity of OCP on angiogenesis during the OCP/Gel-enhanced bone regeneration.

Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling.

Timely controlled oxygen (O2) delivery is crucial for the developing liver. However, the influence of O2 on intercellular communication during hepatogenesis is unclear. Using a human induced pluripotent stem cell-derived liver bud (hiPSC-LB) model, we found hypoxia induced with an O2-permeable plate promoted hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. Conversely, extensive hypoxia generated with an O2-non-permeable plate elevated TGFBs and cholangiocyte marker expression. Single-cell RNA sequencing revealed that TGFB1 and TGFB3 are primarily expressed in the human liver mesenchyme and endothelium similar to in the hiPSC-LBs. Stromal cell-specific RNA interferences indicated the importance of TGFB signaling for hepatocytic differentiation in hiPSC-LB. Consistently, during mouse liver development, the Hif1a-mediated developmental hypoxic response is positively correlated with TGFB1 expression. These data provide insights into the mechanism that hypoxia-stimulated signals in mesenchyme and endothelium, likely through TGFB1, promote hepatoblast differentiation prior to fetal circulation establishment.

Osteogenic cellular activity around onlaid octacalcium phosphate-gelatin composite onto rat calvaria.

The activity and the distribution of osteogenic cells around octacalcium phosphate (OCP) granules combined with gelatin matrix (OCP/Gel) onlaid on rat calvaria were analyzed histomorphometrically and immunohistochemically during vertical bone augmentation under mechanical or nonmechanical environment until 8 weeks. OCP/Gel disk was placed in subperiosteal pocket on the calvaria with or without polytetrafluoroethylene (PTFE) support. The latter is a nonmechanical stress model to alleviate the mechanical stress from the subcutaneous tissues. Onlay grafts of gelatin (Gel) sponge disk and OCP granules were also carried out for the comparison purpose. When bone augmentation was evaluated in first area from bone surface (area until 150 µm high from bone surface) and second area above the newly formed bone (area until 150 µm high from the first area), bone formation was enhanced most in first area followed by second area of OCP/Gel with PTFE. The appearance of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells was suppressed more in the newly formed tissue with PTFE than those without PTFE with an emphasis of the presence of gelatin. Although Runx2 positive-cells were accumulated more in both OCP/Gel with and without PTFE, osteocalcin-positive cells were abundant in OCP/Gel with PTFE than that without PTFE, suggesting that nonmechanical stress condition is more suitable for osteoblast differentiation. The appearance of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-positive cells was restrained in OCP/Gel and Gel with PTFE while osteoprotegerin-positive cells were most accumulated in OCP/Gel without PTFE. The results suggest that OCP/Gel composite under the mechanical stress-alleviated condition can enhance bone augmentation through balanced osteoblastic and osteoclastic cellular activities. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1322-1333, 2018.

Capacity of octacalcium phosphate to promote osteoblastic differentiation toward osteocytes in vitro.

Octacalcium phosphate (OCP) has been shown to act as a nucleus for initial bone deposition and enhancing the early stages of osteoblastic differentiation. However, the effect on differentiation at the late stage into osteocytes has not been elucidated. The present study was designed to investigate whether OCP can promote the differentiation lineage from osteoblasts to late osteocytes using a clonal cell line IDG-SW3 compared to commercially available sintered ß-tricalcium phosphate (ß-TCP) and hydroxyapatite (HA) in a transwell cell culture. Special attention was paid to detect the progress of OCP hydrolysis associated with ionic dissolution products from this material. OCP induced the appearance of an alkaline phosphatase (ALP) peak in the IDG-SW3 cells compared to ß-TCP and HA and increased SOST/sclerostin and FGF23 gene expression after 35 days of incubation. Analyses by X-ray diffraction, curve fitting of Fourier transform infrared spectra, and acid phosphate inclusion of the materials showed that OCP tended to hydrolyze to an apatitic structure during the incubation. Since the hydrolysis enhanced inorganic phosphate ion (Pi) release from OCP in the media, IDG-SW3 cells were further incubated in the conditioned media with an increased concentration of Pi in the presence or absence of phosphonoformic acid (PFA), which is an inhibitor of Pi transport within the cells. An increase in Pi concentration up to 1.5 mM raised ALP activity, while its positive effect was eliminated in the presence of 0.1 to 0.5 mM PFA. Calcium ions did not show such an effect. These results indicate the stimulatory capacity of OCP on osteoblastic differentiation toward osteocytes. STATEMENT OF SIGNIFICANCE: Octacalcium phosphate (OCP) has been shown to have a superior osteoconductivity due to its capacity to enhance initial stage of osteoblast differentiation. However, the effect of OCP on the late osteoblastic differentiation into osteocyte is unknown. This study showed the capacity associated with the structural change of OCP. The data show that OCP released inorganic phosphate (Pi) ions while the hydrolysis advanced if soaked in the media, determined by chemical and physical analyses, and enhanced osteocytes differentiation of IDG-SW3 cells more than hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP). Conditioned elevated Pi-containing media in the absence of OCP enhanced the osteocyte differentiation in the range of the concentration induced by OCP, the effect of which was cancelled by the inhibitor of Pi-transporters.

Surface reactivity of octacalcium phosphate-derived fluoride-containing apatite in the presence of polyols and fluoride.

The present study was designed to characterize co-precipitated fluoridated apatitic materials from octacalcium phosphate (OCP) precursor and to investigate their surface reactions with polyols including glycerol in the presence of fluoride ions. Laboratory-synthesized fluoridated apatite crystals (LS-FA) were obtained in a solution containing fluoride (F) from 25 to 500 ppm. LS-FAs and commercially available fluoroapatite (FA) and hydroxyapatite (HA) were characterized by physical techniques, such as X-ray diffraction. LS-FA obtained in the presence of 100 ppmF (100 ppm-LS-FA) had an apatitic structure, but its solubility was close to HA in a culture medium (α-MEM) despite the fact it contains over 3 wt % of F. 100 ppm-LS-FA, FA, and HA were then subjected to the human serum albumin (HSA) adsorption test at pH 7.4 (in a 150 mM Tris-HCl buffer) and the dissolution and re-mineralization experiments in the presence of xylitol, D-sorbitol, or glycerol, and F under acidic and neutral conditions. Adsorption affinity of HSA was estimated as highest for FA and lowest for LS-FA. LS-FA, FA, and HA were immersed in a lactic acid solution with the polyols and/or F ion-containing solution up to 200 ppm to analyze the dissolution behavior. LS-FA had the highest dissolution tendency in the conditions examined. Glycerol enhanced the dissolution of phosphate from apatite crystals in particular from LS-FA. The results suggest that the apatite crystals, obtained through the hydrolysis of OCP in the presence of F, provide a more reactive surface than FA or HA under physiological environments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2235-2244, 2018.