Microrough titanium surface affects biologic response in MG63 osteoblast-like cells

The purpose of this study was to define the surface properties of prepared titanium (Ti) disks, which served as a model system, and to contrast the biologic response of MG63 cells exposed to Ti disks with different levels of surface roughness. The surface properties interact with each other, resulting in a change of other surface qualities in addition to roughness due to the surface roughening procedure. The machined Ti disks were roughened by sandblasting and electric glow discharging. The surface properties of the Ti specimens were inspected through a comprehensive surface analysis. MG63 cell behaviors were compared along with cell number, alkaline phosphatase (ALP) activity, Runx2 gene expression, and type I collagen production. Statistics were evaluated, using analysis of variance (ANOVA). The sandblasted Ti disks demonstrated well-controlled surface roughness features and meaningful average roughness ranges, including the surface roughness of the "modern" microrough implant, used clinically. With increasing Ti surface roughness, the cell number decreased, while the ALP activity, type I collagen production, and Runx2 gene expression increased significantly. The rougher the Ti surface was, the sooner the Runx2 gene was expressed. Based on these results, we suggest that the microrough Ti surfaces of the 1-3 mum range may contribute effectively to osteogenic differentiation and proliferation in MG63 cells.     

Effect of different surface nanoroughness of titanium dioxide films on the growth of human osteoblast-like MG63 cells

Cell behavior depends strongly on the physical and chemical properties of the material surface, for example, its chemistry and topography. The authors have therefore assessed the influence of materials of different chemical composition (i.e., glass substrates with and without TiO(2) films in anatase form) and different surface roughness (R(a) = 0, 40, 100, or 170 nm) on the adhesion, proliferation, and osteogenic differentiation of human osteoblast-like MG63 cells. On day 1 after seeding, the largest cell spreading area was found on flat TiO(2) films (R(a) = 0 nm). On TiO(2) films with R(a) = 170 nm, the cell spreading area was larger and the number of initially adhering cells was higher than the values on the corresponding uncoated glass. On day 3 after seeding, the cell number was higher on the TiO(2) films (R(a) = 0 and 40 nm) than on the corresponding glass substrates and the standard polystyrene dishes. On day 7, all TiO(2) films contained higher cell numbers than the corresponding glass substrates, and the cells on the TiO(2) films with R(a) = 40 and 100 nm also contained a higher concentration of β-actin. These results indicate that TiO(2) coating had a positive influence on the adhesion and subsequent proliferation of MG63 cells. In addition, on all investigated materials, the cell population density achieved on day 7 decreased with increasing surface roughness. The concentration of osteocalcin, measured per mg of protein, was significantly lower in the cells on rougher TiO(2) films (R(a) = 100 and 170 nm) than in the cells on the polystyrene dishes. Thus, it can be concluded that the adhesion, growth, and phenotypic maturation of MG63 cells were controlled by the interplay between the material chemistry and surface topography, and were usually better on smoother and TiO(2)-coated surfaces than on rougher and uncoated glass substrates.     

Varying Ti-6Al-4V surface roughness induces different early morphologic and molecular responses in MG63 osteoblast-like cells

Osteoblast response to Ti implants depends not only on the chemistry of the implant but also on the physical properties of the implant surface, such as microtopography and roughness. This study was undertaken to examine early changes in cell morphology and gene expression during the early phase of osteoblast interaction with titanium alloy (Ti-6Al-4V) surfaces of two different roughnesses. MG63 osteoblast-like cells were cultured for 2, 6, 24, and 72 h on smooth (Ra=0.18+/-0.03 microm) and rough (Ra=2.95+/-0.23 microm) Ti-6Al-4V surfaces. Changes in cell proliferation were assessed by measuring cell number after 72 h in culture. Morphological characteristics were observed by scanning electron microscopy after 2, 6, and 24 h of culture. Changes in gene expression for extracellular signal-regulated kinase 2 (Erk2), type I collagen (alpha2[I] collagen), phospholipase C-gamma2 (Plc-gamma2), and beta-actin were measured by RT-PCR after 6 and 24 h in culture. Cell number was significantly higher on the smooth surface. In scanning electron micrographs, cells on smooth Ti-6Al-4V were spherical and raised up from the surface after 2 h in culture. In contrast, cells on the rough surface adopted an irregular, elongated shape that spanned across pits in the surface. At 24 h, cells on the smooth surface had flattened, become elongate, and covered the surface. In contrast, cells on the rough surface appeared more differentiated in shape and the margins of the cells were irregular, with many processes extending out, following the contour of the surface. Of the genes examined, only Erk2 and beta-actin showed a change in expression with surface roughness. Both genes were upregulated (p<0.05) on the rough surface at 6 h. These results indicate that Ti-6Al-4V surface roughness affects osteoblast proliferation, morphology, and gene expression, and that these effects can be measured after periods as short as 2-6 h.     

Zirconium oxide: analysis of MG63 osteoblast-like cell response by means of a microarray technology

Zirconium oxide ceramics have outstanding mechanical properties, a high biocompatibility and a high resistance to scratching. Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured on zirconium oxide discs (Cercon, Degussa Dental, Hanau, Germany) several genes whose expression was significantly up or down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport and (c) cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of a zirconium oxide surface. They can be relevant to better understand the molecular mechanism of biocompatibility and as a model for comparing other materials.     

Calcium sulfate: analysis of MG63 osteoblast-like cell response by means of a microarray technology

Calcium sulfate (CaS) is an highly biocompatible material that has the characteristic of being one of the simplest as well as one of the synthetic bone-like graft with the longest clinical history, spanning more than 100 years. Solidified or crystallized CaS is very osteogenic in vivo. As the surface CaS dissolves in body fluid, the calcium ions form calcium phosphate that reprecipitates on the surface forming an osteoblast "friendly" environment. How this "friendly" environment alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identified genes that are differently regulated in osteoblasts exposed to CaS. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured with CaS (Surgiplaster, Classimplant, Roma, Italy) several genes that expression was significantly upregulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) lysosomal enzymes production, (c) cell cycle regulation, (d) and signaling transduction. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of CaS effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.     

In vitro effects on MG63 osteoblast-like cells following contact with two roughness-differing fluorohydroxyapatite-coated titanium alloys

The chances of integration between an implant and the surrounding bone tissue depend on the surface characteristics of the implant itself. Particularly, chemical composition and surface roughness of the material have emerged as crucial factors in affecting the behaviour of cells in contact with the material. Among various surfaces, calcium phosphate coatings seem to favour a rapid initial integration, but their dissolution by extracellular fluids raises some concern about the long-term stability at the bone-implant interface. Fluorinated apatites are known to be more stable than other ceramic coatings, but, at present, little is known on their effects on human cells. In this study, MG63 osteoblast-like cells were seeded onto two fluorohydroxyapatite (FHA)-coated titanium alloy (Ti6Al4V) materials differing in roughness, respectively, LR-FHA (Ra = 5.6 microm) and HR-FHA (Ra = 21.2 microm). Quantification of the cells in contact with the FHA-coated materials by conventional methods involved some technical difficulties, on which we report. Only the indirect esteem by the measure of total content of proteins and a procedure based on cell count, following a double enzymatic treatment to detach the cells, offered plain results, indicating no significant differences between cellular growth in contact with test materials and with plastic control. Differentiation and functionality of the cells were comparatively evaluated by analysis of alkaline phosphatase activity and osteocalcin production. As far as osteocalcin release is concerned, only slight variations were detected on FHA-coated materials in comparison with the control. Both types of coatings showed a significant increase in alkaline phosphatase activity with respect to the control, the roughest surface exhibiting a more prolonged effect on the time.     

Local factor production by MG63 osteoblast-like cells in response to surface roughness and 1,25-(OH)2D3 is mediated via protein kinase C- and protein kinase A-dependent pathways

Titanium (Ti) surface roughness affects bone formation in vivo and osteoblast attachment, proliferation and differentiation in vitro. MG63 cells exhibit decreased proliferation and increased differentiation when cultured on rough Ti surfaces (Ra > 2 microm) and response to 1,25-(OH)2D3 is enhanced, resulting in synergistic increases in TGF-beta1 and PGE2. To examine the hypothesis that surface roughness and 1,25-(OH)2D3 exert their effects on local factor production through independent, but convergent, signaling pathways, MG63 cells were cultured on tissue culture plastic or on smooth (PT, Ra = 0.60 microm) and rough (SLA, Ra = 3.97 microm; TPS, Ra = 5.21 microm) Ti disks. At confluence (5 days), cultures were treated for 24h with 10(-8) M 1alpha,25-(OH)2D3 and active and latent TGF-beta1 in the conditioned media measured by ELISA. Cell layers were digested with plasmin and released TGF-beta1 was also measured. 1,25-(OH)2D3 regulated the distribution of TGF-beta1 between the media and the matrix in a surface-dependent manner; the effect was greatest in the matrix of cells cultured on SLA and TPS. Inhibition of PKA with H8 for the last 24 h of culture increased PGE2 on SLA and TPS, but when present throughout the entire culture period H8 caused an increase in PGE2 on all surfaces. 1,25-(OH)2D3 reduced the effect of H8 on PGE2 production in cultures treated for 24 h. H8 had no effect on TGF-beta1 in the media by itself but caused a complete inhibition of the 1,25-(OH)2D3 dependent increase. Inhibition of PKC with chelerythrine increased PGE2 in a surface-dependent manner and 1,25-(OH)2D3 reduced the effect of the PKC inhibitor. Chelerythrine also increased TGF-beta1 but the effect was not surface dependent; however, 1,25-(OH)2D3 reduced the effects of chelerythrine with the greatest effects on the smooth surface. Thus, the distribution of TGF-beta1 between the media and the matrix is regulated by 1,25-(OH)2D3 in a surface-dependent manner. Surface roughness exerts its effects on TGF-beta1 production via PKC but not PKA. The effect of 1,25-(OH)2D3 on TGF-beta1 production is not via PKC. PKA is involved in the surface-dependent regulation of PGE2 but not in the regulation of PGE2 by 1,25-(OH)2D3 on rough surfaces. Regulation of PKC affects PGE2 production but it is not involved in the surface roughness-dependent response to 1,25-(OH)2D3. These results suggest two independent but interconnected pathways are involved.     

Effect of four acrylic bone cements on transforming growth factor-beta1 expression by osteoblast-like cells MG63.

Based on the hypothesis that bone cements cause changes in the production of transforming growth factor-beta 1 (TGF-beta1) by bone cells, the effects of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2 and CMW 3) were examined using the osteoblast-like cell line MG63. The extracts in MEM of the cements were tested, following 1 h- and 7 day-curing. MG63 cells seldom expressed mRNA specific for TGF-beta1 in basal conditions. The cultures expressed mRNA constantly after incubation with the extract of CMW 1 cured for 1 h. TGF-beta1 specific mRNA was seldom expressed after incubation with the other cement extracts. The release of TGF-beta1 into the conditioned medium was increased significantly by CMW 1 extract at 1 h-curing, but was not changed significantly by CMW 1 extract at 7 day-curing and by the extracts of the other cements, at both curing times. The stimulating effect of CMW 1 on the secretion of TGF-beta1, even with all the restrictions of an in vitro study of continuous cell lines, if confirmed in vivo, might favor the development of the synovial-like membrane around the implant, and therefore impair the chance of success of the prosthesis.     

Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2, and CMW 3) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1beta (0.2 microg ml(-1)) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1 and CMW 2 significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60 and CMW 3 produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1 and CMW 2 increase the production of IL-6 in MG63 cells. The response to Sulfix-60 and CMW 3 is not significantly greater than the negative control.     

Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60^®, CMW 1^®, CMW 2^®, and CMW 3^®) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1 β (0.2 μg ml-1) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1^® and CMW 2^® significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60® and CMW 3® produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1^® and CMW 2^® increase the production of IL-6 in MG63 cells. The response to Sulfix-60^® and CMW 3^® is not significantly greater than the negative control.     

转化生长因子β1对多孔钽/MG63成骨样细胞复合物细胞增殖及分泌功能的影响

BACKGROUND: Previous studies have demonstrated that the Chinese porous tantalum made in China has non-toxicity and good biocompatibility, which can promote osteogenesis.             

Bone cement extracts modulate the osteoprotegerin/osteoprotegerin-ligand expression in MG63 osteoblast-like cells

The osteoprotegerin-ligand (OPG-L) has been identified as the essential factor required for osteoclastogenesis, and its effects are prevented by the osteoprotegerin (OPG). The OPG-L/OPG balance plays a crucial role in coordinating the sequence of osteoclast and osteoblast differentiation during the bone remodeling. The aim of the study was to investigate if polymethylmethacrylate-based cements are able to modulate the expression of OPG-L/OPG in MG63 cells, which are known to have high levels of OPG and inducible expression of OPG-L. Four radio-opaque cements. namely Sulfix-60, CMW1, CMW2 and CMW3, were polymerized for either 1 h or 7 d. MG63 were incubated for 24 h with culture medium only, cement extracts and 2 microg/ml of human recombinant IL-1beta as positive control. An RT-PCR was performed to detect the OPG and OPG-L expression, and the house-keeping gene, GAPDH, was used as a reference for the semi-quantitative analysis. An increase in the OPG-L band density was observed for all cements, and consequently, the OPG-L/OPG ratio also increased. The ability of bone cements to induce the expression of OPG-L could be a co-factor in the development of osteolysis at the bone-cement interface.     

Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)(2)D(3) through regulation of phospholipase A(2) activity and activation of protein kinase A.

Implant surface roughness influences osteoblast proliferation, differentiation, and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones such as 1, 25-(OH)(2)D(3) is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1, 25-(OH)(2)D(3) are synergistic. Prostaglandin E(2) (PGE(2)) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)(2)D(3). However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1, 25-(OH)(2)D(3). The present study examined the potential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), and protein kinase C (PKC) in this process. MG63 osteoblast-like human osteosarcoma cells were cultured on cpTi disks with R(a) values of 0. 54 microm (PT), 4.14 microm (SLA), or 4.92 microm (TPS). PKA was inhibited by adding H8 to the cultures; similarly, PLA(2) was inhibited with quinacrine or activated with melittin, and PKC was inhibited with chelerythrine. Inhibitors or activators were included in the culture media through the entire culture period or for the last 24 h of culture. In addition, cultures were treated for 24 h with inhibitors or activators in the presence of 1,25-(OH)(2)D(3). The effects on cell number and alkaline phosphatase specific activity were determined after 24 h; PKC activity was determined after 9 min and at 24 h. Cell number was reduced on rough surfaces, and alkaline phosphatase activity was increased. 1,25-(OH)(2)D(3) had a synergistic effect with surface roughness on alkaline phosphatase. However, neither surface roughness nor 1,25-(OH)(2)D(3) had an effect on PKC. H8 treatment for 24 h inhibited cell number and alkaline phosphatase on all surfaces; however, when it was present throughout the culture period, the PKA inhibitor had no effect on cell number, but decreased alkaline phosphatase-specific activity. H8 reduced the 1,25-(OH)(2)D(3)-mediated effect on cell number and alkaline phosphatase. Quinacrine inhibited cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25-(OH)(2)D(3)-dependent decreases in both parameters. Melittin had no effect when applied for 24 h and did not modify the 1,25-(OH)(2)D(3) effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity. Chelerythrine, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period. However, it decreased alkaline phosphatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period. The results indicate that surface roughness and 1,25-(OH)(2)D(3) both mediate their effects through PLA(2) which catalyzes the rate-limiting step in PGE(2) production. Further downstream, PGE(2) activates PKA. Surface roughness-dependent effects are also mediated through PKC, but only after the cells have reached confluence and are undergoing phenotypic maturation. The effect of surface roughness on responsiveness to 1,25-(OH)(2)D(3) is mediated through PLA(2)/PKA and not through PKC.     

Beads of collagen-nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells

The aim of this work was to develop a novel method for preparing a three-dimensional bone-like matrix comprising nanohydroxyapatite crystals and fibrous collagen and to apply it for bone tissue engineering. Hydroxyapatite and collagen are the major components of natural hard bone. Therefore, they have been used extensively in orthopedic surgery as bone-filling materials. According to the principle of complex coacervation, three-dimensional collagen beads can be formed by extruding collagen solution into chondroitin sulfate A (CSA) solution. Subsequently, the collagen beads thus formed are soaked in simulated body-fluid solution to biomimic the formation process of natural bone matrix via the fabrication of collagen-nanohydroxyapatite beads. We also investigate the effect of the collagen-nanohydroxyapatite matrix on the proliferation and differentiation of MG63 cells. The presence of crystalline hydroxyapatite structure on the surface of fibrous collagen was confirmed by X-ray diffraction. MG63 cells cultured on the collagen-nanohydroxyapatite beads proliferate at the normal rate. Moreover, alkaline phosphatase (ALP) activity and the expression levels of three osteogenic genes, namely, type I collagen osteopontin and osteocalcin, in MG63 cells were significantly higher when the cells were cultured on collagen-nanohydroxyapatite beads than when they were cultured on collagen alone. The results of this study reveal that, in the presence of nanohydroxyapatite, the three-dimensional cell beads not only provide a substrate for cell growth but could also enhance the osteoblast-like cell differentiation of MG63 cells.     

Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes

Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (P<0.05) between different micropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.     

Enhanced proliferation and osteocalcin production by human osteoblast-like MG63 cells on silicon nitride ceramic discs.

The biocompatibility of silicon nitride (Si3N4) was assessed in an in vitro model using the human osteoblast-like MG-63 cell line. Cells were propagated on the surface of: reaction-bonded silicon nitride discs, sintered after reaction-bonded silicon nitride discs or control polystyrene surface for 48 h. Compared to cells propagated on polystyrene surface, cells grown on the surface of unpolished silicon nitride discs had significantly lower cell yield and decreased osteocalcin production. In contrast, cells on the surface of polished silicon nitride discs showed similar proliferative capacity to control cells propagated on polystyrene surface. Cells propagated on polished discs also produced higher levels of osteocalcin than cells on unpolished discs. SEM analysis showed cells with well-delineated morphology and cytoplasmic extensions when propagated on polished sintered after reaction-bonded discs. Cells appeared more spherical, when grown on polished reaction-bonded discs. The results of this study suggest that silicon nitride is a non-toxic, biocompatible ceramic surface for the propagation of functional human bone cells in vitro. Its high wear resistance and ability to support bone cell growth and metabolism make silicone nitride an attractive candidate for clinical application. Further studies are needed to explore the feasibility of using silicon nitride clinically as an orthopedic biomaterial.     

强磁重力环境对MG63成骨样细胞钙离子/钙调蛋白信号的影响

目的:研究强磁重力环境对MG63成骨样细胞钙离子浓度和钙离子下游信号分子表达的影响。方法:利用大梯度强磁场提供μg(12T),1g(16T)和2g(12T)三组不同强磁重力复合环境处理MG63成骨样细胞后,经Fluo-3/AM标记的细胞用激光共聚焦显微镜检测处理0.5 h对细胞胞内游离钙离子浓度([Ca2+]i)的影响;用Western blot检测处理3 h对钙调蛋白(CaM)和肌球蛋白轻链激酶(MLCK)表达以及钙离子/钙调蛋白依赖蛋白激酶Ⅱ(CaMKⅡ)活性的变化。结果:钙离子浓度检测结果表明,与对照组细胞(1 g,地磁)相比,1 g(16 T)组细胞Fluo-3荧光强度增加,结果显示强磁场导致[Ca2+]i增加;与2 g(12 T)组相比,μg(12 T)组细胞Fluo-3荧光强度下降,结果显示模拟失重导致[Ca2+]i降低,抑制钙离子信号。蛋白质表达的检测结果表明,与对照组相比,1 g组细胞CaM和MLCK表达以及CaMKⅡ活性没有明显变化;与2 g(12 T)组相比,μg(12 T)组细胞CaM表达以及CaMK活性下降,结果显示模拟失重抑制CaM/CaMKⅡ信号。结论:强磁场导致MG63成骨样细胞胞内游离钙离子浓度增加,模拟失重抑制成骨样细胞钙离子/钙调蛋白信号。     

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.