Yasutaka Sekijima

Surface Electric Fields Increase Osteoblast Adhesion through Improved Wettability on Hydroxyapatite Electret

Osteoblasts are susceptible to the surface characteristics of bioceramics and stimulation from outside the cells. The purpose of this study was to evaluate the effects of electrical polarization on surface characteristics and osteoblastic adhesion. The surface characteristics revealed that electrical polarization had no effect on the surface roughness, crystallinity, and constituent elements. According to contact-angle measurements, electrically polarized hydroxyapatite (HA), which provides two kinds of surfaces, negatively charged HA (N-HA) and positively charged HA (P-HA), was even more hydrophilic than that of normal HA (O-HA). Morphological observations and quantitative analyses revealed that the typical adhered cells had a round shape on O-HA but had a spindle or fanlike spreading configuration on N-HA and P-HA 1 h after seeding. After 3 h of cultivation, the rate of the number of spread cells and the size of the focal adhesions on O-HA increased and approached that of N-HA and P-HA. However, the cell areas positively stained for actin, which indicates the degree of cell spreading, were distinctly larger on N-HA and P-HA than that on O-HA. The number of focal adhesions per cell was also less than that on N-HA and P-HA.

Polarized hydroxyapatite promotes spread and motility of osteoblastic cells

Osteoblast adhesion to surfaces of implant substrates is recognized as playing a fundamental role in the process of osteoconduction. The purpose of this study was to evaluate the in vitro adhesion of osteoblasts cultured on polarized hydroxyapatite (HA), which provides two kinds of surfaces; negatively charged HA (N-HA) and positively charged HA (P-HA). Those surfaces have been proved to enhance the osteobonding capabilities. Osteoblastic cells were seeded onto normal and polarized HA; adhesion and motility of each was observed. Polarization did not affect the percentage of the spread cells against all the adhered cells, but had a significant effect on the spreading of each cell as shown by the measured elongation of the adhered cells by fluorescence observation. The elongation of each cell was especially enhanced on the N-HA and P-HA, when compared with normal HA (O-HA). In addition, the polarization affected cell motility shown by wound healing. Motility analysis showed that the same number of cells started to migrate toward the wound areas on each type of surface. However, the migration of each cell type towards the wound area was accelerated on the N-HA and P-HA. The charges induced on the HA surface accelerated the cytoskeleton reorganization of the adhered cells. The acceleration was appeared as cell shape, actin filament pattern such as stress fiber formation, and prolongation of cell motility distance.

Regulation of Osteoblast-Like Cell Behaviors on Hydroxyapatite by Electrical Polarization

The osteoblast adhesion to the substrates are recognized to play a fundamental role in osteoconduction process. The purpose of this study was to evaluate the in vitro behavior of osteoblasts cultured on polarized hydroxyapatite (HA), having the enhanced osteobonding abilities. Osteoblast-like cells were seeded onto the polarized HA and investigated the adhesion and motility. The polarization had no effects on the percentage of the number of the spreaded cells against all the adhered cells, but had significant effects on the elongation of adhered cells from fluorescent observation and on the cell motility showed by the wound healing assay. The charges induced on the HA surface accelerated the cytoskeleton reorganization of the adhered cells cultured on HA specimens. The acceleration was emerged as the cells shape, actin filament pattern such as stress fiber formation, and the prolongation of the cell movement distances.

Immediate Mechanism of the Osteoconductivity of the Polarized Hydroxyapatite

We investigated the immediate response in the vicinity of the implanted polarized hydroxyapatite (HA). One of the important immediate events was found to be the fibrin adsorption on the implanted HA. The activation of the fibrin adsorption on the polarized HA was confirmed by the observation by SEM observation and immunohistochemical detection. After the implantation, the coagulation cascade induced the wound healing. The adsorbed fibrin was formed network structure and worked for later cell responses as the scaffolds. The acceleration of fibrin scaffolds formation in the vicinity of the implanted polarized HA contributed to the stimulation and activation of plate ets and osseous cells. The platelets were activated from the results of the signal transduction and cytoskeleton changes and released growth factors. The growth factors released from platelets led to bone regeneration by stimulating migration and proliferation of the osseous cells. In the vicinity of the implanted HA samples, the coagulation cascade reaction to repair wound healing led to the new bone formation by mediation of the platelets and osseous cells. The mechanism of osteoconduction at earlier stage had close relationships to the electrostatic properties of the implant.

Comparison of Hydroxyapatite with Carbonate Apatite in Osteoclastic Cell Resorptive Activity

Various bioactive calcium phosphates such as hydroxyapatite (HA) and carbonate apatite (CA) bone substitutes have been studied because of the biocompatibility and osteoconductivity when implanted into bone defects. In this study, the interaction between bioceramics and osteoclast-like cell using the cell-line such a RAW264 was examined for the investigation of the important factors of the osteoclastic responses. From the results, the possibility of effectiveness by surface geometry and chemical property means solubility was suggested. Moreover, it was considerable that the CA induced much stronger responses to osteoclast-like cells than the HA.

Osseous Cell Response to Electrostatic Stimulations of Poled Hydroxyapatite Ceramics in Canine Diaphyses

Cell responses of electrically charged surfaces of poled hydrox yapatite (HA) ceramics were investigated by implantation in wide defects of canine femora , c mpared with the uncharged surfaces. The HA ceramic specimens were poled in a dc field i n air at 300-400°C. Base on the thermo stimulated current measurements, the stored charge and the half–value period of HA ceramics polarized at 300°C and 1.0 kVcm —1 were 4.2 μCcm and 7.3×10 s (at 37.0°C), respectively. Although the non-polarized HA ceramic surfaces were still isolat ed from osteoid tissues by dominant fibrin multi-layers 7 days after the implantation, newly formed bone lay rs of 0.01-0.02 mm in thickness contacted the negatively charged surfaces without any incl usion. On the contrary, The osteoid tissues surrounded by osteoblastic cells occupied the gap betwee n the positively charged surface and the cortical bone. We have demonstrated that the polarize d HA ceramic had a large and long durable charge suitable for biomedical applications and that the HA surf ace charges induced by electrical polarization stimulated osseous cells and promoted bone reconstruction. Introduction Surface charges of biomaterials are recognized as one of the im portant factors to determine cell responses [1, 2]. The cells receiving the stimulation exhibited vari ous modulated reactions, such as migration, alteration of differentiation, cell phase, and extracellular matr ix secretion. We have more recently disclosed that exclusively large surface charges we re inducible on hydroxyapatite (HA) ceramics by proton transport polarization [3, 4] and demonstrated that the negatively charged surface of the HA ceramics enhanced their osteobonding ability in canine femor a [5, 6]. In the present study, the cell responses of the negatively and the positively charged surf aces of the electrically poled HA ceramics were investigated by implantation in wide defects of c anine femora, compared with the uncharged surfaces. Materials and Methods HA powder was precipitated from calcium hydroxide aqueous suspension and phos phoric acid solution. The HA powder, calcined at 850°C, was pressed in a mold at 200 MPa . Dense HAp ceramics prepared by sintering at 1250°C for 2 h in saturated water vapo were employed as the specimens. The HA ceramic specimens with a size of 5.0 ×8.0×1.0 mm were electrically poled in a dc field of 1.0 kVcm with a pair of Pt electrodes in air at 300-400°C for 1 h (Fig. 1a). The confirmation of the poling charges of the samples chosen at random was examined by the thermally stimulated current (TSC) method using a handmade measurement cell [4]. Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 849-852 doi:10.4028/www.scientific.net/KEM.254-256.849

Molecular Biologic Detections of Cell Activation Related Proteins on Polarized Hydroxyapatite

Effect of polarized hydroxyapatite implantation on its osteoconductivit y was estimated using biochemical and immunohistochemical detections. In this study, alka line phosphatase (ALP) activity, type I collagen concentration, osteocalcin concentration wer e d tected in serum. The localization of osteocalcin (bone Gla protein) known as the later expr ession proteins during new bone formation was immunohistochemistrically detected in the vicinity of the N-surface of the polarized HA ceramics. In the polarized HA surface, the new bone formation w as promoted, compared with the non-polarized HA surface. The results suggested that biological mineralizati on was in close relation to the electrostatic properties of the implant surface.