The in vitro behavior of as-prepared and pre-immersed RF-sputtered calcium phosphate thin films in a rat bone marrow cell model.

In this paper we focus on the behavior of radio frequency (RF)-sputtered calcium phosphate (CaP) thin films in a rat bone marrow (RBM) cell model. Two issues are addressed. Firstly, we benchmarked the in vitro cell behavior of these CaP coatings by comparing their proliferation, differentiation and mineralization behavior and the structure of the formed interface to similar coatings of alumina and titania. We found that the CaP coatings showed reduced proliferation, enhanced early differentiation and enhanced activity of mature osteoblasts compared to the alumina coatings. Enhanced production of mineralized extracellular matrix (ECM) was seen for both CaP and titania. Two types of CaP precipitates could be observed, one directly bonded CaP layer at the coating interface and one of globular accretions associated with the ECM. The directly bonded layer was not observed on the alumina coatings. Further, no thin film effects were found. Secondly, the effect of pre-immersion of the CaP coatings in SBF2 was explored. We found that the early formation of a directly bonded CaP layer is obstructed by the absence of CaP nuclei. After approximately 8 days, cell activity induces the nucleation of CaP crystals on both the surface and the ECM, and growth is enhanced. By initially providing these coatings with CaP crystals, growth of the directly bonded CaP layer is immediate. Hence, the formation of the interfacial CaP layer and the matrix-associated CaP accretions can effectively be decoupled.

Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review.

The adhesion of osteoblasts to substrates is mediated through proteins that have adsorbed to the substrate, providing integrins on the cell membrane with ligands to connect to. The integrins regulate cell behavior through bi-directional signaling pathways. This critical review has the purpose to consider the research that has been performed with osteoblasts, integrins, and bone replacing materials. Until now, most research has been done to investigate the integrin expression of osteoblasts in culture during cellular adhesion. However, it remains difficult to draw general conclusions from this research. Nevertheless, it can be concluded that the used substrates and protein or peptide coatings can influence the integrin expression and cellular behavior. Additional research has to be done to fully understand all the parameters involved in integrin expression, the adhesion of cells to substrates, and the subsequent cellular behavior. For this purpose, model substrates are under development. The signaling pathway is receiving more and more attention, but for biomaterial purposes, too little consideration is paid to the translation of the in vitro results to the in vivo situation, and to practical applications.

Modulation of integrin expression on rat bone marrow cells by substrates with different surface characteristics.

Biomaterials have been shown to be able to influence the growth and differentiation of osteogenic cells cultured on the surface. Although the precise mechanisms by which the materials influence osteogenic cells are unclear, it is possible that the materials manipulate the expression of integrins by the cells. We therefore studied the expression of a number of integrins by rat bone marrow (RBM) cells, after culture on culture polystyrene, on machined and grit-blasted titanium, and on calcium phosphate-coated titanium. Integrin expression was studied by FACS analysis. We found a large variation in the expression of integrins by cells in replicate experiments. After culture on polystyrene for 7 days, cells expressed alpha1, alpha2, alpha3, alpha5, alpha6, beta1, and beta3, although some of the subunits were expressed only occasionally. The cells did not express the alpha4 subunit. After culture of RBM cells for 8 days on coated and noncoated titanium substrates, cells always expressed alpha3, alpha5, alpha6, and beta1. The alpha1 and beta3 subunits were only expressed in some of the experiments. Frequently, the expression of alpha5, alpha6, and beta1 was higher on the coated than on the noncoated titanium substrates. Based on our results, we conclude that the studied materials are capable of influencing the expression of integrins by RBM cells cultured on relevant implant materials.

Initial interaction of U2OS cells with noncoated and calcium phosphate coated titanium substrates.

From previous studies, we know that calcium phosphate (CaP) coated implants stimulate bone formation compared to uncoated implants. Nevertheless, the mechanism by which substrate surface characteristics affect cell function is unclear. In this study, we examined the initial interaction (30 min to 24 h) of U2OS cells with titanium substrates with or without a CaP coating. The effect of substrate roughness was also studied. When cell attachment was studied, we found that cells attached more readily to rough than to smooth surfaces. Also, more cells attached to the uncoated than to the CaP coated surface. After 24 h, cell numbers were similar for all substrate surfaces. Further, cells spread to a larger area on noncoated titanium than on the CaP coated substrates. At 24 h, the sequence of cell size was smooth titanium > rough titanium > CaP coated titanium. Shape measurements showed differences in cell shape between the cells on the different materials only at 7 h, not at different culture times. Cells expressed alpha2, alpha3, alpha5, alpha6, alphav, and beta1 subunits. Expression of alpha1, alpha4, alphavbeta3, beta3, beta4, and beta7 was extremely low or was not found. The beta1 integrin expression was higher on the coated than on the noncoated titanium at 3 h, but not on the other studied times. Expression of alpha2, alpha5, alpha6, and alphav expression was found to be upregulated at 24 h compared to earlier culture times on coated titanium, but not on uncoated titanium substrates. From this we conclude that the surface characteristics of a material (roughness and composition) can affect the initial interaction of cells with the material.

Initial interaction of rat bone marrow cells with non-coated and calcium phosphate coated titanium substrates.

Initial interactions of rat bone marrow (RBM) cells with smooth titanium, rough titanium or calcium phosphate coated substrates were tested. Cells were seeded onto the substrates, and attachment, integrin expression and spreading and morphology were studied. We found no difference in attachment of RBM cells to the different materials. We did find differences in the percentage of attached cells within a certain time between replicate runs of the experiments. RBM cells on all materials express alpha1, alpha3, alpha5, alpha6 and beta1 subunits. Again there was a large difference in expression patterns on RBM cells in different runs. No difference was found in expression on the various materials. For alpha1, alpha5, alpha6 and beta1, no difference was found in expression between attached and unattached cells. Expression of alpha3 was similar on attached and unattached cells during early culture. At the end of culture, alpha3 expression was downregulated for attached cells and not for unattached cells. This resulted in a higher expression of alpha3 for unattached cells compared to attached cells. Cells did spread on all materials, and reached a larger cell size on smooth titanium than on the rough materials. Morphology of the cells on the materials differed. On smooth titanium, cells usually showed a compact cell body with short cellular extensions. On the rough materials, cells often showed elongated shapes, with many thin cellular extensions. From this we conclude that the substrate surface characteristics of the materials we used do not influence attachment or integrin expression during the initial cell-material interactions. On the other hand, spreading behavior and cell morphology do depend on substrate surface characteristics.

In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone.

The aim of our study was to investigate the osteogenic potential of subcultured rat bone marrow cells. Rat bone marrow (RBM) cells were cultured with or without dexamethasone. Subsequently, osteogenic differentiation and expression was studied. When cells were cultured continuously in the presence of dexamethasone, cultures initially showed high alkaline phosphatase expression and abundant mineralization. Expression of differentiation markers decreased with passaging. After cells were passaged three times, no alkaline phosphatase activity and calcification were found. Primary cells cultured without dexamethasone showed low alkaline phosphatase and no calcification, and remained fibroblast-like. When these cells were subcultured in the presence of dexamethasone, the cells did show osteogenic differentiation. Nevertheless, this occurred at a significant lower level than with cells continuously cultured with dexamethasone. In addition, no differentiation was found after second passage. Our results indicate that subcultured undifferentiated RBM cells show osteogenic differentiation after addition of dexamethasone. Expression of alkaline phosphatase and mineralization is higher in cells continuously supplemented with dexamethasone. Still, even when dexamethasone is added continuously, RBM cells loose their osteogenic potential after several passages. Therefore, we conclude that subculture of undifferentiated rat bone marrow cells results in the loss of osteogenic potential of these cells.

Effect of calcium phosphate coating crystallinity and implant surface roughness on differentiation of rat bone marrow cells.

In this study, we examined the effect of calcium phosphate (Ca-P) coating crystallinity and of surface roughness on growth and differentiation of osteogenic cells. Grit-blasted titanium substrates were provided with Ca-P coatings of different crystallinities. Rat bone marrow (RBM) cells were cultured on these substrates and on noncoated rough and smooth titanium substrates. After specific culture times, expression of osteogenic markers by the cells was studied. Cells cultured on crystalline coatings and on titanium substrates proliferate, express alkaline phosphatase, osteocalcin (OC), and show mineralization of the extracellular matrix. Rough titanium substrates only express low OC levels. Significantly higher OC levels were expressed on smooth titanium, and even higher levels on the crystalline Ca-P coating. No difference was found in calcification between smooth and rough titanium. The crystalline coating showed more calcification than the titanium substrates. When substrates without cells were incubated in medium, precipitation of calcium was found. On the titanium substrates, this precipitate disappeared after prolonged incubation. The precipitate on the crystalline coating was stable and increased with longer incubation times. On the amorphous coatings, no proliferation and differentiation of RBM cells were found. After longer culture periods, substrates showed extensive dissolution. Cells on the amorphous coatings did express high levels of prostaglandin E2. In contrast, prostaglandin E2 expression was low for the other substrates. We conclude that crystalline Ca-P coatings stimulate differentiation of RBM cells, to a higher extent than titanium substrates. Surface roughness only has a limited effect on phenotype expression of the cells. In contrast, thin amorphous coatings show negative effects on the growth and differentiation of cultured RBM cells.

Analysis of integrin expression in U2OS cells cultured on various calcium phosphate ceramic substrates.

Earlier we observed that calcium phosphate (Ca-P)-coated implant substrates stimulated the differentiation of osteoblast-like cells compared to uncoated substrates. This suggests that this difference in osteogenic induction is due to the chemical composition of the substratum. We hypothesized that Ca-P coatings modulate integrin expression patterns, because those receptors are the sensors of the cell. Therefore, in the present study we quantitatively analyzed integrin expression of osteosarcoma cells and their proliferation behavior on various well-defined Ca-P substrates. For this study we used the osteosarcoma cell line U2OS. Five groups of substrates were used: thermanox (Th), uncoated titanium (Ti), dense sintered hydroxyapatite (HA), and two Ca-P-coated titanium discs (TiHA-O% and TiHA-5%). At day 5, cell numbers were significantly lower (p < 0.05) for both types of Ca-P-coated titanium substrates compared to the other substrates. There were no significant differences between HA and uncoated titanium. From day 5 to 8, accumulated cell number was ranking highest to lowest HA > Th = Ti > TiHA-0% > TiHA-5%. Integrin expression at day 5 and day 8 of incubation was analyzed by flow cytometry for integrin subunits beta 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v. Fluorescence-activated cell sorting (FACS) analysis showed that the cells express high levels of beta 1, low levels of alpha 4, alpha 5, and alpha 6, and moderate levels of alpha 3 and alpha v integrin subunits on the various biomaterial substrates. Minor differences in integrin expression between the various substrates were seen. Therefore, the observed differences in proliferation between the coatings may reside in modulating the functional properties of integrins.

Acid stress induces a D1-like dopamine receptor in pituitary MSH cells of Oreochromis mossambicus.

A 7-day exposure of tilapia (Oreochromis mossambicus) to water with a pH of 4.5 activates their pituitary melanophore-stimulating hormone (MSH) cells to preferentially release diacetyl alpha-MSH as an important corticotrope (13). We here focus on the control of alpha-MSH release by dopamine in tilapia exposed to water with low pH ("low-pH tilapia"). The MSH cells of low-pH tilapia showed a decreased sensitivity to inhibitory concentrations (10(-7)-10(-5) M) of dopamine compared with controls. Low concentrations (10(-14)-10(-8) M) of dopamine stimulated the release of alpha-MSH in low-pH tilapia but not in controls. Strong pharmacological evidence for a stimulatory dopamine receptor (D1-like) was obtained: the D1-agonists SKF-38393 and 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazep ine hydrobromide (6-chloro APB) had a stimulatory effect on the release of alpha-MSH in low-pH tilapia MSH cells but not in controls. The selective D2-agonists quinpirole and 2-hydroxy apomorphin inhibited the release of alpha-MSH in controls as well as in low-pH tilapia, and there was no difference in the sensitivity of the cells to these agonists. We conclude that only MSH cells of low-pH exposed tilapia exhibit a D1-like receptor activity. A comparable D2-like receptor activity, as demonstrated by specific D2-receptor agonists, is present in both controls and low-pH-adapted fish. The apparent loss of sensitivity of the MSH cells to inhibitory concentrations of dopamine, therefore, must be caused by the activation of the D1-like receptors and not by changes in the activity of the D2-like receptor proper. Stimulatory concentrations of dopamine not only quantitatively but also qualitatively enhanced the corticotropic activity of the released alpha-MSH, as indicated by the elevated ratio of diacetyl and monoacetyl alpha-MSH. This effect was mimicked by the D1-like agonists SKF-38393 and 6-chloro APB, indicating that the D1-like receptor activity is responsible for the enhancement of the di/mono ratio.