Quantitative kinetic analysis of gene expression during human osteoblastic adhesion on orthopaedic materials

Little information was found in the literature about the expression on hydroxyapatite (HA) materials of genes specific of cellular adhesion molecules although more were found on titanium-based substrates. Hence, the goal of this work was to study by a kinetic approach from 30 min to 4 days the adhesion of Saos-2 cells on microporous (mHA) and non-microporous hydroxyapatite (pHA) in comparison to polished titanium. Our strategy associated the visualization of adhesion proteins inside the cells by immunohistochemistry and the quantitative expression of genes at mRNA level by real-time PCR. The cell morphology was assessed using scanning electron microscopy and the number of cells thanks to biochemical techniques. The cellular attachment was the highest on mHA from 30 min to 24 h although the cell growth on mHA was the lowest after 4 days. Generally, the Saos-2 osteoblastic cells morphology on mHA was radically different than on other surfaces with the particularity of the cytoplasmic edge, which appeared un-distinguishable from the surface. The revelation by specific antibodies of proteins of the cytoskeleton (actin) and the focal adhesions (FAK, phosphotyrosine) confirmed that adhesion and spreading were different on the 3 materials. The actin stress fibres were less numerous and shorter on mHA ceramics. Cells had more focal contacts after 4 h on mHA compared to other substrates but less after 24 h. The highest values of total proteins were extracted from mHA at 0.5 and 24 h and from pHA at 1, 4, and 96 h. The alphav and beta1 integrin, actin, FAK, and ERK gene expression were found to be different with adhesion time and with materials. C-jun expression was comparable on mHA, titanium and plastic but was largely higher than on pHA at 0.5 and 1 h. On the contrary, c-fos expression was the highest on pHA after 0.5 h and the lowest after 1h. This difference between c-fos and c-jun expression on pHA after 0.5 h could be related to the fact that these two genes may differ in their signalling pathways. The expression of the alkaline phosphatase gene after 4 days was lower on mHA compared to other materials demonstrating that the microstructure of the mHA ceramic was not favourable to Saos-2 cells differentiation. Finally, it was demonstrated in this study that HA and titanium surfaces influence as well gene expression at early times of adhesion as the synthesis of adhesion proteins but also proliferation and differentiation phases. Indeed, the signal transduction pathways involved in adhesion of Saos-2 cells on HA and titanium were confirmed by the sequential expression of alphav and beta1 integrins, FAK, and ERK genes followed by the expression of c-jun and c-fos genes for proliferation and alkaline phosphatase gene for differentiation.     

Morphology of rod stock and compression-moulded sheets of ultra-high-molecular-weight polyethylene used in orthopaedic implants

Compression-moulded sheets and extruded rods of ultra-high-molecular-weight polyethylene (UHMWPE) are currently used in the production of joint replacement prostheses. Crystallographic texture present in rods and sheets of UHMWPE was measured using a combination of small-angle X-ray scattering and wide-angle X-ray diffraction. Crystallographic texture can induce anisotropy in macroscopic properties of polymers, such as modulus and yield stress. Both rods and sheets of UHMWPE revealed a low but discernible degree of preferred orientation of polyethylene chains within crystallites. There was a spatial variation in Crystallographic orientation in extruded rods. The direction of chain alignment within crystallites located near the outer surface of rods was orthogonal to the radial direction, whereas the chain direction was orthogonal to the axial or extrusion direction in crystallites located near the centreline of extruded rods. Crystallographic texture was spatially uniform in compression-moulded sheets with the chain direction within crystallites aligned orthogonal to the moulding direction. In both cases the induced crystallographic texture can be explained in terms of crystallization from an oriented melt.     

Costimulatory molecule expression following exposure to orthopaedic implants wear debris

Patients with long-term orthopedic implants may develop inflammatory reactions due to the accumulation of biomaterial particles both around the implant and in distant organs. The exact impact of these particles on the normal immune cell function still remain relatively unclear. Activation of T-cells following exposure to biomaterial particles is driven by macrophages and requires synergistic signals primed by both antigen presentation and costimulation. The pattern of costimulatory molecule expression (CD80,CD86) was primarily examined using immunohistochemistry on tissue specimens of bone/implant interface membranes taken from sites of bone erosion. Additionally, costimulatory molecule expression was also assessed in the monocytic leukemia cell line U937 following exposure to clinically relevant titanium aluminum vanadium (TiAlV) and stainless steel particles (FeCrNi) cultured in vitro. This study demonstrates the induction and prominent expression of CD86 on almost all macrophage subsets at the bone/implant interface, including fused forms and large multinucleated giant cells (MNGC). In vitro analysis also indicated phagocytosis of metal particles by differentiated U937 caused significant induction of both CD80 and CD86 (p < 0.01), although the expression of CD86 dominated following prolonged exposure. The data presented highlights that CD86 is the predominant costimulatory molecule ligating to the complementary CD28 molecule at the inflammatory lesion of the interface. We propose that the intracellular presence of indigestible implant material, in addition to elevated costimulatory molecule expression, may promote T-cell inflammatory reactions at sites close to and distant from the orthopedic implant.     

Numerical model to predict the longterm mechanical stability of cementless orthopaedic implants

The objective of this research was to develop a purely biomechanical model, intended to predict the long-term secondary stability of the implant starting from the biomechanical stability immediately after the operation. A continuous rulebased adaptation scheme was formulated as a dynamic system, and the work verified if such a model produced unique and clinically meaningful solutions. It also investigated whether this continuous model provided results comparable with those of a simpler, discrete-states model used in a previous study. The proposed model showed stable convergence behaviour with all investigated initial conditions, with oscillatory behaviour limited to the first steps of the simulation. The results obtained with the wide range of initial conditions support the hypothesis of the existence and uniqueness of the solution for all initial conditions. The differences between the continuous model and the simpler and more efficient finite-states model were found to be extremely modest (less than 4% over the predicted bonded area). Because of these minimal differences, the use of the much faster finite-states model is recommended to investigate asymptotic conditions, and the continuous model described should be used to investigate the evolution over time of the adaptive process.     

Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography

The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 microm in diameter, whereas they did not recognize the 10 microm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 microm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 microm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.     

A jet impingement investigation of osteoblastic cell adhesion

When designing dental and orthopedic implants, it is important to consider phenomena occurring at the microscopic level, particularly at the bone-implant interface. The presence of hard tissue at this interface is essential to implant viability. The integrity of this tissue-biomaterial interface is dependent on appropriate osteoblast functions (adhesion, matrix deposition, etc.) in the immediate area. Researchers have modified various materials with cell-adhesive peptides with the ultimate goal of controlling osteoblast functions. This study used microjet impingement to compare the strength of adhesion of osteoblastic cells (at varying populations) and fibroblasts to peptide-modified substrates in the presence and absence of fetal bovine serum. In the presence of the serum, there was no significant difference in cellular adhesion strength between substrates. In the absence of serum, all cells tested adhered more strongly to underlying substrates, and the strength of cellular adhesion was greater on modified surfaces than on plain glass surfaces. In the absence of serum, second-passage osteoblastic cells generally adhered to substrates more strongly than first-passage osteoblastic cells; fibroblasts adhered similarly to second-passage osteoblastic cells. Fundamental studies such as the present increase the understanding of cell adhesion to various substrates--knowledge that may be ultimately useful in creating an optimal bone-implant interface.     

骨科内植物多功能涂层研究进展

无菌性松动和植入物周围感染是骨科内固定术失败的两个主要原因,随着手术数量的日益增长,如何降低此类手术失败风险变得格外重要。在这个领域中,近期大量的研究工作致力于研制各种各样的内植物涂层,而这些涂层大多只具有抗感染作用而无骨整合作用;或者只具有骨整合作用而不具备抗感染作用。但是要使内植物长期有效,理想的涂层应兼顾骨整合和抗感染这两个功能。本文将对同时具有骨整合性和抗感染性的多功能涂层近期的研究进展和未来研究方向进行综述。     

Mechanisms of platelet adhesion to the basal lamina.

The human glomerular basal lamina (HGBL) is composed of collagenous and noncollagenous glycoproteins. We assessed the role played by each costituent in platelet-basal-lamina interaction by selective cleavage and removal of each component by clostridial collagenase or by pepsin. When noncollagenous proteins are removed from HGBL, human platelets exhibit littel reactivity toward the residual collagen framework of the isolated basal lamina. With the noncollagen matrix of basal lamina, after removal of the bulk of the collagen, platelet adhesion and spreading proceed normally in the presence of divalent cations, similar to what occurs on intact basal lamina. No platelet degranulation or aggregation is observed. The results indicate that the basal lamina collagen, even in its native packing arrangement, lacks affinity for platelet adhesion and is incapable of triggering platelet release reactions. Platelet adhesion and spreading on the basal lamina appears to depend primarily on the presence of the noncollagen components and to require divalent cations. The data suggest the presence on platelets of receptors for basal lamina distinct from those for interstitial collagens. These receptors activate a unique modulation of platelet behavior, ie, adhesion and spreading without degranulation. A difference in biologic function of the basal lamina and interstitial collagens is apparent in these experiments.     

Mechanisms and clinical relevance of bacterial adhesion to polymers

The mechanisms of bacterial adhesion to polymers with regard to their significance in the development of foreign-body infections are discussed. The morphological, physico-chemical and biological aspects are treated with special emphasis on the adhesion of coagulase-negative staphylococci to medical polymers. Strategies for the prevention of bacterial adhesion to biomaterials by developing antiadhesive polymers are given.     

Effects of commonly used immunosuppressants on graft-derived fibroblasts

In acute rejection of transplanted organs intragraft fibroblasts increase their production of hyaluronan. Hyaluronan has strong water binding capacity and an increased tissue content of hyaluronan thus contributes to the development of interstitial oedema. The present study examined the effects of commonly used immunosuppressants (prednisolone, cyclosporin, tacrolimus, mycophenolic acid and sirolimus) on fibroblast proliferation, hyaluronan production and cell surface receptor expression. Fibroblasts isolated from rejecting tissue and from normal, non-transplanted tissue were studied in parallel. All substances investigated, except tacrolimus, were found to affect fibroblasts in one way or another. The most striking effect was the almost total inhibition of fibroblast proliferation in the presence of mycophenolic acid. Cyclosporin reduced the proliferation by about 50% and prednisolone had an inhibiting effect on hyaluronan production (50% reduction). These effects were observed on fibroblasts isolated from rat cardiac allografts undergoing rejection as well as on fibroblasts obtained from normal heart tissue. In contrast, sirolimus was found to stimulate the proliferation of fibroblasts from rejecting tissue (100% increase), but not that of normal fibroblasts. The majority of the fibroblasts expressed the hyaluronan receptor CD44, with a more intense expression in cultures of fibroblasts derived at rejection. None of the immunosuppressants affected the staining pattern (number of positive cells or intensity). The inhibitory effects of prednisolone, cyclosporin and mycophenolic acid on fibroblasts may contribute to the overall beneficial effects of these drugs when used for prevention or treatment of rejection.     

Physiological strains remodel extracellular matrix and cell-cell adhesion in osteoblastic cells cultured on alumina-coated titanium alloy

The effects of mechanical strains on cellular activities were assessed in an in vitro model using human osteoblastic MG-63 cells grown on titanium alloy discs coated with porous alumina and exposed to chronic intermittent loading. Strain was applied with a Dynacell device for three 15-min sequences per day for several days with a magnitude of 600 microepsilon strain and a frequency of 0.25 Hz. We have previously demonstrated that this regimen increased alkaline phosphatase activity in confluent cultures on ceramic coated titanium (alumina and hydroxyapatite) (Biomaterials 24 (2003) 3139). In this study, we analysed the production of bone matrix proteins. Osteocalcin secretion quantified by ELISA between day 5 and 11 was not affected by mechanical strain. Strain had even no quantifiable effect on collagen production from day 1 to 5 as measured by carboxy terminal collagen type I propeptide release. On the other hand, stress stimulation resulted in increased expression of fibronectin (FN) measured by Western blot after 1 day stretching. This upregulation of FN production was followed by reorganisation of the FN network after 5 days stretching observed by immunostaining. The receptors for collagen and FN, alpha2beta1, alpha5beta1 and beta1 integrins were not quantitatively affected by the strains as measured by flow cytometry. A modification of cell morphology was seen after 5 days of loading that appeared to increase cell spreading, implying consequences on intercellular contacts. For this reason, N, C11 and E-adherins were examined. We noted a selective effect characterised by increased expression of N-cadherin using both RT-PCR and Western blot analyses. We concluded that reinforcement of cell-cell adhesion and remodelling of the FN network are important adaptive responses to physiological strains for human osteoblasts grown on alumina-coated biomaterials.     

Mechanisms of lymphocyte adhesion to endothelial cells: studies using a LFA-1-deficient cell line

In order to investigate the role of lymphocyte function-associated antigen 1 (LFA-1) in lymphocyte adhesion to endothelial cells (EC), we have studied the adhesion of a LFA-1-deficient lymphoblastoid cell line, ICH-KM, which has < 10% of the cell surface LFA-1 expressed on a normal lymphoblastoid cell line, ICH-BJ. The adhesion of ICH-KM cells to unstimulated EC was 49.9 +/- 8.6% (mean +/- SD) that of ICH-BJ cells. Moreover, phorbol ester-stimulated ICH-KM cells showed a considerably weaker increase in adhesion to unstimulated EC compared with ICH-BJ cells (mean +/- SD increase in percentage adhesion, 3.8 +/- 2.3 compared with 18.5 +/- 8.0; P<0.025). In contrast, there was no significant difference between the enhanced adhesion of ICH-KM cells and ICH-BJ cells to interleukin-1 (IL-1)-stimulated EC. Thus ICH-KM cells showed a 22.7 +/- 11.0 (mean +/- SD) increase in percentage adhesion to IL-1-stimulated EC compared with the 24.8 +/- 8.5 increase in percentage adhesion of ICH-BJ cells. Anti-LFA-1 monoclonal antibodies had no effect on the enhanced adhesion of ICH-KM and ICH-BJ cells to IL-1-stimulated EC but abolished the differences in adhesion between the two cell lines. The study therefore indicates that although a major part of unstimulated and phorbol ester-stimulated lymphocyte-EC adhesion is dependent upon LFA-1, the enhanced adhesion due to stimulation of EC with IL-1 is not dependent upon this molecule. The data therefore supports the existence of cytokine-inducible LFA-1-independent adhesion molecules for lymphocytes on EC.     

聚醚醚酮内植物骨整合改性的研究进展

聚醚醚酮（PEEK）材料因有着诸多优点而在骨缺损修复等医学领域得到越来越多的关注和应用,同时对于克服其生物惰性,提高其骨整合性能的改性研究成为热点。本文通过查阅国内外关于PEEK材料骨整合改性的相关文献,综述了近年来对骨科植入物PEEK材料进行改性以提高其骨整合性能的相关研究现状,并重点从改性原理的角度进行总结。     

MG63 osteoblastic cell adhesion to the hydrophobic surface precoated with recombinant osteopontin fragments

The hydrophobicity of biomaterials has been recognized as a limitation to the adequate function of anchorage-dependent cells when hydrophobic biomaterials are used for tissue engineering. This is due to flawed solid-state signals from cell adhesion. In this study, a recombinant osteopontin (rOPN17-169) fragment containing the cell adhesion motifs was expressed in E. coli and was precoated on the hydrophobic surface prior to osteoblastic MG63 cell culture. Precoating the hydrophobic surface with rOPN17-169 improved osteoblastic cell adhesion, which was blocked by soluble RGDS. The adhesion of MG63 cells to rOPN17-169 pre-coated surface-activated mitogen-activated protein kinases (MAPK) such as extracellular signal-receptor kinase 1/2, p38, and c-Jun N-terminal kinase (JNK). In addition, p38 MAPK was activated in response to a soluble factor of transforming growth factor-beta in the cells adhered to the hydrophobic surface via rOPN17-169. This suggests that rOPN17-169 precoated on the hydrophobic surface can allow osteoblastic cells to generate adhesion signals sufficient for cell adhesion, MAPK activation, and the cytokine activation of osteoblastic cells.     

Significance of nano- and microtopography for cell-surface interactions in orthopaedic implants

Cell-surface interactions play a crucial role for biomaterial application in orthopaedics. It is evident that not only the chemical composition of solid substances influence cellular adherence, migration, proliferation and differentiation but also the surface topography of a biomaterial. The progressive application of nanostructured surfaces in medicine has gained increasing interest to improve the cytocompatibility and osteointegration of orthopaedic implants. Therefore, the understanding of cell-surface interactions is of major interest for these substances. In this review, we elucidate the principle mechanisms of nano- and microscale cell-surface interactions in vitro for different cell types onto typical orthopaedic biomaterials such as titanium (Ti), cobalt-chrome-molybdenum (CoCrMo) alloys, stainless steel (SS), as well as synthetic polymers (UHMWPE, XLPE, PEEK, PLLA). In addition, effects of nano- and microscaled particles and their significance in orthopaedics were reviewed. The significance for the cytocompatibility of nanobiomaterials is discussed critically.     

Case of Mycobacterium marinum infection with unusual patterns of susceptibility to commonly used antibiotics

Mycobacterium marinum, found commonly in salt water and freshwater, is the causative agent of disease in many species of fish and occasionally in humans. MICs to most antimicrobial agents are relatively low. Susceptibility testing is not routinely performed, and single-drug therapy is used for the treatment of most infections. Here, we report an infection caused by a drug-resistant M. marinum strain in an otherwise healthy patient.     

Effect of ion modification of commonly used orthopedic materials on the attachment of human bone-derived cells.

Biomaterials which combine optimum properties of strength and biocompatibility are desirable in improving the long-term performance of implantable medical devices. Our study is aimed at developing technology designed to alter the outer atomic layers of a material to give the desired compatibility with the tissue while retaining the properties of the bulk substratum. Materials used in this study were titanium vanadium alloy (Ti-6Al-4V) and cobalt chromium molybdenum alloy (Co-Cr). Soda lime glass discs and polyethylene terephthalate (PET) acted as controls. A cathode of either Ti-6Al-4V or Co-Cr was used to simultaneously deposit and implant identified substrata. The attachment of human bone-derived cells (HBDC) to various materials was determined using radiolabeling or colorimetric assays. Results show that HBDC adhere preferentially to the unmodified surfaces of Ti-6Al-4V and Ti-6Al-4V on glass compared to the unmodified Co-Cr surfaces and to that of the Co-Cr on glass. Depositing Ti-6Al-4V on Co-Cr gives significantly better attachment of HBDC than when depositing Co-Cr onto Ti-6Al-4V. While cellular attachment to the created surfaces reflects that of the cathodic materials, it is not identical to these materials. Ion deposition/implantation is capable of creating permanent surfaces which reflect the adhesion of source materials not bulk substrata.