Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque–associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy 'plane' and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation.     

Targeting Intercellular Communication in the Bone Microenvironment to Prevent Disseminated Tumor Cell Escape from Dormancy and Bone Metastatic Tumor Growth

Metastasis to the bone is a common feature of many cancers including those of the breast, prostate, lung, thyroid and kidney. Once tumors metastasize to the bone, they are essentially incurable. Bone metastasis is a complex process involving not only intravasation of tumor cells from the primary tumor into circulation, but extravasation from circulation into the bone where they meet an environment that is generally suppressive of their growth. The bone microenvironment can inhibit the growth of disseminated tumor cells (DTC) by inducing dormancy of the DTC directly and later on following formation of a micrometastatic tumour mass by inhibiting metastatic processes including angiogenesis, bone remodeling and immunosuppressive cell functions. In this review we will highlight some of the mechanisms mediating DTC dormancy and the complex relationships which occur between tumor cells and bone resident cells in the bone metastatic microenvironment. These inter-cellular interactions may be important targets to consider for development of novel effective therapies for the prevention or treatment of bone metastases.     

骨髓成骨细胞在碱热处理的磷灰石涂层钛表面的分化增殖研究

为了研究骨髓基质细胞转化的成骨细胞在碱热处理的涂层磷灰石钛表面的分化增殖，将钛片经过碱热处理后，浸泡于与类似体液的环境进行表面磷灰石涂层。应用骨髓成骨细胞作为实验工具，观察体外细胞在处理的涂层钛片上的生长，碱性磷酸酶以及降钙素的分泌。在早期，有磷灰石涂层的钛片成骨细胞黏附高于对照组；在晚期，两组之间没有明显差异；成骨细胞分化的标志——碱性磷酸酶活性和降钙素的表达，磷灰石涂层组都明显高于对照组。研究表明：碱热处理的磷灰石涂层钛能够促进成骨细胞黏附，有利于细胞向成骨分化。     

Effects of Lanthanum on Bone Resorbing Activity of Rabbit Mature Osteoclasts Co-Cultured with Osteoblasts

The effects of lanthanum (Ⅲ) on the bone resorbing activity of rabbit mature osteoclasts (OCs) in the presence of osteoblasts (OBs) were studied in vitro by measuring the number and area of absorption pits. La(Ⅲ) at concentrations ranging from 1.00×10-5 to 1.00×10-8 mol·L-1 show no effect on mature OC number (P＞0.05). In the OC-OB co-culture systems without La(Ⅲ), osteoblasts alone did not influence the pit number and area whether the two kinds of cells were in contact or not (P＞0.05). Under the OC-OB not-in-contact condition, the effect of La(Ⅲ) on the bone-resorbing activity of OCs was similar to that of La(Ⅲ) in the absence of OBs (P＞0.05). However, while OCs were in direct contact with OBs, the inhibitory effects of La(Ⅲ) on OCs' bone-resorbing activity decreased at the concentrations of 1.00×10-5, 1.00×10-6 and 1.00×10-7 mol·L-1, and the promotion effects increased at 1.00×10-8 mol·L-1 (P＜0.05). The results suggest that direct cell-cell contact between OC and OB be essential for OBs to play their role in regulating the response of OCs to La(Ⅲ).     

Cell morphology and focal adhesion location alters internal cell stress

Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues.     

Influence of hydroxyvalerate composition of polyhydroxy butyrate valerate (PHBV) copolymer on bone cell viability and in vitro degradation

The objective of this study was to elucidate the role of hydroxyvalerate (HV) composition in polyhydroxy butyrate valerate (PHBV) copolymer film on the degradation of copolymer and osteoblastic cell activity. Degradation was studied by monitoring time‐dependent changes in mass and chemical composition of the macroporous films. The mass loss of PHBV film upon 19 weeks of exposure to pH 7.4 phosphate buffer medium was found to range from 2.8% to 9.2% with a strong dependence on the original composition of the copolyester film and morphology. Tapping mode atomic force microscopy (TMAFM) was used to examine the roughness change of polyester films due to exposure to buffer medium. Chemical analysis of the degraded film was carried out using NMR to aid in the interpretation of the mass loss and TMAFM data. The NMR results showed a significant decrease in the mol % of HV content in the degraded PHBV film. Additionally, we established that UMR‐106 cell proliferation on macroporous PHBV matrix is minimally enhanced by the HV content of PHBV copolymer. Information provided by this study can be used in the selection of appropriate PHBV copolymer for clinical use where the biopolymer needs to remain physically intact and chemically unchanged during the intended period of biomedical application. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

卵黄高磷蛋白磷酸肽及其钙螯合物在双细胞共培养体系中对成骨细胞分化的促进作用

研究了卵黄高磷蛋白磷酸肽（Phosvitin Phosphopeptide,PPP）及其钙螯合物（PPP-Ca）在成骨细胞（MC3T3-E1）和破骨前体细胞（RAW264.7）共培养体系中对成骨细胞分化的调节作用。对细胞毒性、碱性磷酸酶（AKP）、抗酒石酸酸性磷酸酶（TRAP）的活性和TRAP染色进行分析;用RT-PCR技术进一步探究成骨细胞RANKL/OPG通路相关蛋白的mRNA表达情况。结果发现,PPP和PPP-Ca可以使MC3T3-E1体系中的AKP活性分别增加9.5%和12.7%;PPP和PPP-Ca的加入可以使MC3T3-E1体系中OPG基因mRNA的表达量从0.92分别提升至1.25和1.39、使RANKL基因mRNA的表达量从1.00分别提升至1.23和1.45。该研究结果表明,PPP和PPP-Ca可有效促进成骨细胞的分化。实验结果为进一步探索磷酸肽在多细胞模型体系中的作用提供了研究基础,同时为磷酸肽的功能活性开发提供理论依据。