Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity

Surface topography appears to be an important but often neglected factor in implant performance. In this study, fibroblasts were cultured on a range of porous polycarbonate (PC) membranes with well defined surface topography (track-etched micropores, 0.2-8.0 microm in diameter) and wettability gradients. The wettability gradient on the PC membrane surfaces was produced by treating the surfaces with corona from a knife-type electrode whose power increased gradually along the sample length. The PC membrane surfaces were characterized by scanning electron microscopy (SEM) and the water contact angle measurement. Fibroblasts were cultured on the corona-treated PC membrane surfaces with different micropore sizes for 1 and 2 days. The cells attached on the membrane surfaces were examined by SEM and the cell density on the surfaces was estimated by counting the number of attached cells along the wettability gradient. It was observed that the cells were adhered and grew more on the hydrophilic positions of the membrane surfaces than the more hydrophobic ones, regardless of micropore size. It was also observed that cell adhesion and growth decreased gradually with increasing micropore size of the membrane surfaces. It seems that the cell adhesion and growth were progressively inhibited as the membrane surfaces had micropores with increasing size, probably due to surface discontinuities produced by tract-etched pores. On the membrane surfaces with smaller micropore sizes, the cells seemed to override these surface discontinuities.     

In vitro cytocompatibility of MG63 cells on chitosan-organosiloxane hybrid membranes

Chitosan-silicate hybrids were synthesized using gamma-glycidoxypropyltrimethoxysilane (GPSM) as the agent for cross-linking the chitosan chains. CaCl2 was introduced in the hybrids in expectation that it would improve cell adhesion and differentiation of the hybrid surfaces. Fourier-transform infrared (FT-IR) spectroscopy and 29Si CP-MAS NMR spectroscopy were used to analyze the structures of the hybrids. Cytocompatibility of the hybrids was investigated in terms of proliferation of an osteoblastic cell line, MG63. The adhesion and proliferation of the osteoblastic cells cultured on the surface of a chitosan-GPSM hybrid without calcium were similar to those on a control culture plate, and were better than those on a chitosan membrane. The ALP activity of the cells cultured on this hybrid was higher than that on the chitosan membrane. Contrary to expectations, the incorporation of calcium ions into the hybrids did not improve cell attachment and proliferation on their surfaces.     

Activation of phospholipase D1 by surface roughness of titanium in MG63 osteoblast-like cell

Although it is recognized that the surface roughness of titanium (Ti) promotes the osteogenic differentiation, the related mechanisms and factors remain elusive. The purpose of this study was to explore the potential correlation among phospholipase D (PLD) activity, Ti surface roughness and subsequent osteoblast differentiation. The machined Ti disks were sandblasted with aluminum oxide particles to produce surfaces of varying roughness (n = 160). Normal or transfected MG63cells with PLD genes were cultured on roughened Ti specimens and assayed for PLD, alkaline phosphatase (ALP) and osteocalcin. The statistical significance was evaluated by analysis of variance. The activity, mRNA and protein levels of PLD significantly increased in MG63 cells with a roughness-dependent pattern (P < 0.05). The ALP activity and osteocalcin production, promoted by Ti surface roughness, were enhanced by the PLD activator and inhibited by the PLD blocker. It was also found that the PLD1 isoform responds to Ti surface roughness and regulates selectively the ALP activity. These observations strongly suggest that PLD1 mediates the cellular signaling of and modulates osteoblast differentiation induced by Ti surface roughness in MG63 osteoblast-like cell.     

Oxidized NiTi surfaces enhance differentiation of osteoblast-like cells

A new oxidation treatment (OT) on NiTi shape memory alloys was developed in a previous work. This OT treatment significantly decreases Ni ion release into the exterior medium, and therefore is thought to be beneficial for NiTi cytocompatibility. As to confirm this expectation, the in vitro response of MG63 osteoblast-like cells cultured on untreated and oxidized NiTi surfaces was studied. An adhesion test at 1, 4, and 8 h of incubation was performed. Statistical differences were evidenced at 1 h of adhesion depending on the surface treatment and chemical composition of the substrate. However, at larger times of study, there were no statistically significant differences between untreated and oxidized surfaces. The proliferation test (until 9 days) showed that untreated and oxidized NiTi surfaces are not cytotoxic for MG63 cells. The differences of adhesion at short times did not affect the proliferation of MG63 cells. However, after 48 h of stimulation with ascorbic acid and dexamethasone, the MG63 cells cultured on oxidized surfaces showed higher alkaline phosphatase activity and osteocalcin levels. The improvement of osteoblast differentiation due to OT treatment could accelerate bone formation, and, therefore, could allow earlier loading of NiTi devices used in dental and orthopedic applications.     

Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.     

Osteoblast response on Ti- and Zr-based bulk metallic glass surfaces after sand blasting modification

The present study aimed to evaluate the osteoblast response on Ti- and Zr-based BMG surfaces sand blasted with different grit corundums for implant application, with mechanically polished disks before sand blasting as control groups. The surface properties were characterized by scanning electron microscopy (SEM), contact angle, and roughness measurements. Further evaluation of the surface bioactivity was conducted by MG63 cell attachment, proliferation, morphology, and alkaline phosphatase (ALP) activity on the sample surfaces. It was found that corundum sand blasting surfaces significantly increased the surface wettability and MG63 cell attachment, cell proliferation, and ALP activity in comparison with the control group surfaces. Besides, the sample surface treated by large grit corundum is more favorable for cell attachment, proliferation, and differentiation than samples treated by small grit corundum, indicating that it might be effective for improving implant osseointegration in vivo. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

表面粗糙度对种植体钛片表面成骨细胞的影响

目的探讨种植体表面粗糙度对成骨细胞增殖、分化、功能及基因表达的影响。方法种植体钛片分为3组,表面粗糙度(Ra)分别为0.175μm(1组)、1.00μm(2组)及1.689μm(3组)。将人成骨肉瘤细胞系MG63细胞接种于3组材料表面,利用激光共聚焦显微镜观察细胞在各组钛材料表面黏附铺展形态,采用MTY法、碱性磷酸酶(ALP)、骨钙素活性检测评价成骨细胞在钛片表面黏附、增殖和分化水平,通过RT- PCR检测细胞内Cbfa1mRNA表达。结果Mg63在各组材料表面均生长良好,4h时各组细胞均附着,第2组细胞可见突起,24h时各组细胞均伸展,形态不规则。各组细胞在1、5、10d,第2组ALP及骨钙素(OC)活性均逐渐增高,在15d有所下降,在5、10d,ALP及OC活性显著高于其他两组(P<0.05)。第2组的Cbfal mRNA表达高于其他两组(P<0.01)。结论种植体表面适度粗化能够促进成骨细胞的功能,1.00μm是比较适宜的表面粗糙度。     

The effect of cerium valence states at cerium oxide nanoparticle surfaces on cell proliferation

Understanding and controlling cell proliferation on biomaterial surfaces is critical for scaffold/artificial-niche design in tissue engineering. The mechanism by which underlying integrin ligates with functionalized biomaterials to induce cell proliferation is still not completely understood. In this study, poly-l-lactide (PL) scaffold surfaces were functionalized using layers of cerium oxide nanoparticles (CNPs), which have recently attracted attention for use in therapeutic application due to their catalytic ability of Ce⁴⁺ and Ce³⁺ sites. To isolate the influence of Ce valance states of CNPs on cell proliferation, human mesenchymal stem cells (hMSCs) and osteoblast-like cells (MG63) were cultured on the PL/CNP surfaces with dominant Ce⁴⁺ and Ce³⁺ regions. Despite cell type (hMSCs and MG63 cells), different surface features of Ce⁴⁺ and Ce³⁺ regions clearly promoted and inhibited cell spreading, migration and adhesion behavior, resulting in rapid and slow cell proliferation, respectively. Cell proliferation results of various modified CNPs with different surface charge and hydrophobicity/hydrophilicity, indicate that Ce valence states closely correlated with the specific cell morphologies and cell–material interactions that trigger cell proliferation. This finding suggests that the cell–material interactions, which influence cell proliferation, may be controlled by introduction of metal elements with different valence states onto the biomaterial surface.     

Calcium phosphate-chitosan composite scaffolds for bone tissue engineering

Macroporous calcium phosphate-chitosan composite scaffolds were fabricated and evaluated for use in bone tissue engineering. Human osteoblast-like MG63 cells were cultured on the composite scaffolds, and their response to the materials was studied. Cell morphology, total protein content, and expression of classic markers for osteoblast differentiation were characterized. MG63 cells on the hydroxyapatite scaffolds nesting chitosan sponges (HC1) showed significantly higher alkaline phosphatase (ALP) level and osteocalcin (OC) production during the 11-day culture period, compared with the control culture on tissue culture plates. Cells on the chitosan scaffolds incorporated with hydroxyapatite powders (HC2) exhibited lower ALP activity during the 11-day culture period and OC secretion during the first 7 days, in comparison with that on HC1. The addition of calcium phosphate glass as in HC3 scaffolds increased the ALP and OC levels of MG63 cells. Our study indicated that the hydroxyapatite-matrix composite scaffolds might enhance the phenotype expression of MG63 cells, in comparison with chitosan-matrix scaffolds. Soluble calcium phosphate glasses should be added to the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast 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.     

Influences of ionic dissolution products of dicalcium silicate coating on osteoblastic proliferation,differentiation and gene expression

This work aims to explore the influence of the ionic products of dicalcium silicate coating on osteoblastic proliferation and differentiation, as well as on the expression of BMP2 and its signal transducers Smad1, 6 and 7 in MG-63 osteoblast-like cells. Plasma-sprayed dicalcium silicate coatings were soaked in DMEM to obtain culture media containing the ionic dissolution products of dicalcium silicate coating (Ca₂SiO₄–DMEM). MG63 osteoblast-like cells were cultured in Ca₂SiO₄–DMEM (experimental group) for 3–12 days, while those cultured in normal DMEM served as control (control group). MTT assay was used to evaluate cell viability and proliferation. Alkaline phosphatase activity (ALP), osteocalcin (OC) and type I collagen (COLI) were investigated as differentiation markers. Gene expression of BMP2 and Smad1, 6, 7 was also detected. BMP2 protein was examined by ELISA assay. Alizarin Red-S (AR-S) assay was used to detect mineralization. The results demonstrated that Si concentration in Ca₂SiO₄–DMEM is markedly higher than that in normal DMEM. Compared to the control group, MG63 cells of the experimental group exhibited upregulated proliferation on day 3, and markedly upregulated gene expression of the differentiation markers, especially on days 9 and 12 for OC and on days 3, 6 and 9 for ALP. Gene expression of BMP2 and Smad1, as well as BMP2 protein secreted into culture media, was also upregulated in the experimental group, while gene expression of Smad6 and 7 was not influenced. AR-S assay indicated a higher calcium mineral content deposition in cells of the experimental group. In conclusion, the ionic products of plasma-sprayed dicalcium silicate coating are beneficial to the proliferation and differentiation of MG63 osteoblast-like cells.     

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.     

Efficacy of glow discharge gas plasma treatment as a surface modification process for three-dimensional poly (D,L-lactide) scaffolds

Gas plasma surface modification of three-dimensional poly (D,L-lactide) scaffolds fabricated by a novel vibrating particle fabrication technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 10 days in culture using human embryonic palatal mesenchyme cells. Characterization of corresponding two-dimensional treated surfaces revealed decreased contact angle measurements of 54.2 +/- 0.6 degrees for treated surfaces compared to 72.3 +/- 0.7 degrees for control surfaces (p < 0.05). SEM of treated surfaces revealed increased surface roughness combined with marked pitting and erosion. This may contribute to increased cell adhesion. WST-1 cell proliferation assay measurements as an index of cell numbers revealed a statistically significant increase in proliferation activity on treated surfaces on days 1 and 4 compared with controls. There was a fivefold increase in WST-1 activity for both control and treated groups over 10 days. Confocal laser micrographs revealed increased cell numbers on treated specimens throughout all layers of the scaffold, indicating that the glow discharge process enhanced cell proliferation throughout the entire scaffold architecture. Scanning electron micrographs demonstrated increased cell adhesion for treated specimens at the polymer surface most evident after days 1 and 4 of culture. Alkaline phosphatase (ALP)-specific activity peaked by day 7 for control and treated surfaces, indicating cellular differentiation. There was a trend for increased protein production on the treated specimens compared with controls at the initial time points although the differences were not statistically significant. These results demonstrated that gas plasma surface modification enhances osteoblast-like cell function in a three-dimensional scaffold model.     

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.     

Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide)

The impact of the surface topography of polylactone-type polymer on cell adhesion was to be concerned because the micro-scale texture of a surface can provide a significant effect on the adhesion behavior of cells on the surface. Especially for the application of tissue engineering scaffold, the pore size could have an influence on cell in-growth and subsequent proliferation. Micro-fabrication technology was used to generate specific topography to investigate the relationship between the cells and surface. In this study the pits-patterned surfaces of polystyrene (PS) film with diameters 2.2 and 0.45 microm were prepared by phase-separation, and the corresponding scale islands-patterned PLLA surface was prepared by a molding technique using the pits-patterned PS as a template. The adhesion and proliferation behavior of OCT-1 osteoblast-like cells morphology on the pits- and islands-patterned surface were characterized by SEM observation, cell attachment efficiency measurement and MTT assay. The results showed that the cell adhesion could be enhanced on PLLA and PS surface with nano-scale and micro-scale roughness compared to the smooth surfaces of the PLLA and PS. The OCT-1 osteoblast-like cells could grow along the surface with two different size islands of PLLA and grow inside the micro-scale pits of the PS. However, the proliferation of cells on the micro- and nano-scale patterned surface has not been enhanced compared with the controlled smooth surface.     

Evaluation of silicon nanoporous membranes and ECM-based microenvironments on neurosecretory cells

Understanding the interactions between microfabricated synthetic interfaces and cultured cells expressing a neuronal phenotype are critical for advancing research in the field of neural engineering such as neural recording and stimulation and neural microdevice interactions with the human brain. Here we explore the integration of these two components for therapeutic applications of neural prostheses. Microfabricated silicon nanoporous membranes were investigated for their effects on survival, proliferation, and differentiation of the well-known PC12 clonal line. Specifically, cell morphology, examined through fluorescence staining, were comparable in many respects on both silicon membrane and widely-used polystyrene culture surfaces. The attachment and differentiation of PC12 cells cultured on collagen and laminin-modified membranes and standard tissue culture surfaces were similar. Lastly, the differentiation response and tyrosine hydroxylase activity of PC12 cells embedded in a type I collagen matrix on experimental membrane substrates while exposed to NGF were significant and indistinguishable from tissue-culture polystyrene (TC-PS) surfaces. Results from this research suggest that microfabricated silicon nanoporous membranes may be useful, biocompatible permselective structures for neuroprosthetic applications and that collagen may be a useful immobilizing matrix for PC12 cells loaded in implantable macroencapsulation devices designed for the treatment of neurodegenerative disorders.     

Static Magnetic Fields Promote Osteoblast-Like Cells Differentiation Via Increasing the Membrane Rigidity

The aim of this study was to test the differentiative effects of osteoblasts after treatment with a static magnetic field (SMF). MG63 osteoblast-like cells were exposed to a 0.4-T SMF. The differentiation markers were assessed by observing the changes in alkaline phosphatase activity and electron microscopy images. Membrane fluidity was used to evaluate alterations in the biophysical properties of the cellular membranes after the SMF simulation. Our results show that SMF exposure increases alkaline phosphatase activity and extracellular matrix release in MG63 cells. On the other hand, MG63 cells exposed to a 0.4-T SMF exhibited a significant increase in fluorescence anisotropy at 6 h, with a significant reduction in the proliferation effects of growth factors noted at 24 h. Based on these findings, the authors suggest that one of the possible mechanisms that SMF affects osteoblastic maturation is by increasing the membrane rigidity and reducing the proliferation-promoting effects of growth factors at the membrane domain.     

Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.     

Influence of titanium surfaces on attachment of osteoblast-like cells in vitro

Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Studies on preliminary interactions of osteoblast-like cells on implant interface through in vitro systems, can give lucid insights to osseo-integrative efficacies of when in vivo implants. In the present investigation two titanium surfaces of dental implants, a sandblasted and acid-etched surface and an experimental grooved surface were compared through in vitro systems. The titanium implants were seeded with osteoblast-like primary cells and maintained for a period of 1-7 days. Expressions of fibronectin and osteonectin were assessed through immunogold labelling by scanning electron microscopy. The grooved surface, supported better osteoblastic cell adhesion and proliferation than the rough surfaces. Further, osteoblastic cells on the grooved surfaces also displayed a strong labelling for fibronectin at the cytoplasmic extensions coupled with intense osteonectin expression in comparison to the rough surfaced implants. In conclusion, grooved surfaces offered better cell attachment and proliferation than the other rough surfaces studied.