Coatings from micropatterned sulfobetaine polymer brushes as substrates for MC3T3-E1 cells

In the last decades, polymer brush coatings have proven to be excellent anti-fouling materials by preventing protein adhesion. When using this property to restrict cell growth laterally in cell culture, it is crucial to ensure that other cell functions remain unaffected. The present study therefore examines MC3T3-E1 cell growth and morphology on patterned PSBMA brush substrates and probes their proliferation potential at mRNA level. The osteoblastic cells display a more elongated morphology than cells on the control substrates, but show no sign of elevated levels of the apoptosis marker p53 or diminished levels of Ki-67 or H4, which serve as indicators of proliferation. Therefore, patterned polymer brushes do not seem to influence cells in their proliferation state and are suitable cell culture substrates. Nevertheless, the use of polymer brush surfaces in long-term cell culture was found to be limited by their instability in cell culture medium.     

Proliferation and function of MC3T3-E1 cells on freeze-cast hydroxyapatite scaffolds with oriented pore architectures

Previous work by the authors showed that hydroxyapatite (HA) scaffolds with different types of oriented microstructures and a unique ‘elastic–plastic’ mechanical response could be prepared by unidirectional freezing of suspensions. The objective of the present work was to evaluate the in vitro cellular response to these freeze-cast HA scaffolds. Unidirectional scaffolds with approximately the same porosity (65–70%) but different pore architectures, described as ‘lamellar’ (pore width = 25 ± 5 μm) and ‘cellular’ (pore diameter = 100 ± 10 μm), were evaluated. Whereas both groups of scaffolds showed excellent ability to support the proliferation of MC3T3-E1 pre-osteoblastic cells on their surfaces, scaffolds with the cellular-type microstructure showed far better ability to support cell proliferation into the pores and cell function. These results indicate that freeze-cast HA scaffolds with the cellular-type microstructure have better potential for bone repair applications.     

三维多肽支架中前成骨细胞MC3T3-E1成骨分化的初步研究

在多肽EAK16水凝胶支架上接种小鼠前成骨细胞MC3T3-E1,采用倒置显微镜观察细胞形态,CCK-8(细胞计数试剂盒)检测细胞增殖情况。细胞在诱导培养基中培养1周后,观察不同时间段细胞碱性磷酸酶的分泌活性。采用ALP染色和茜素红-S染色作为定性实验研究MC3T3-E1向成骨方向的分化情况。结果表明,MC3T3-E1细胞在水凝胶支架EAK16上有较好的黏附和增殖能力,诱导培养后细胞有较高水平的碱性磷酸酶表达和矿化基质沉积。多肽水凝胶支架对前成骨细胞MC3T3-E1具有较好的生物相容性。     

Regulation of bone-related genes expression by bone-like apatite in MC3T3-E1 cells

Bone-like apatite on HA/TCP ceramics sintered at 1,100 °C (HT1) and 1,200 °C (HT2) could be obtained via immersing substrates into simulated body fluid (SBF) for 3 days. When MC3T3-E1 preosteoblastic cells cultured on the surface of the bone-like apatite for 3 days, SEM observations revealed cell membrane features with secreted crystals very similar to in vivo bone formation during intramembranous ossification with a direct bone apposition on the ceramics. According to semi-quantitative RT-PCR method, mRNA expressions of osteocalcin (marker of late-stage differentiation) and type 1 collagen were increased in cultures with HT1S and HT2S when compared to HT1 and HT2 after cultured for 6 days. The results indicated that bone-like apatite had the ability to support the growth of osteoblast-like cells in vitro and to promote osteoblast differentiation by stimulating the expression of major phenotypic markers. Taken together, our findings will be helpful in understanding the mechanism of osteoinductivity of calcium phosphate ceramics and in constructing more appropriate biomimetic substrate.     

Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts

There is an increased use of nanophase titanium dioxide (TiO₂) in bone implants and scaffolds. However, nano-debris is generated at the bone-biomaterial interface. Therefore, TiO₂ nanoparticles (NPs) of many sizes were investigated for cytotoxic effects on murine MC3T3-E1 preosteoblasts. These TiO₂ NPs induced a time- and dose-dependent decrease in cell viability. There was a significant increase in lactate dehydrogenase (LDH) release, apoptosis and mitochondrial membrane permeability following short-term exposure of the cells to TiO₂ NPs. These NPs also increased granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) gene expression. Compared with the 32 nm TiO₂ NPs, 5 nm TiO₂ NPs were more toxic, induced more apoptosis, increased mitochondrial membrane permeability and stimulated more GM-CSF expression at a high concentration (≥100 μg/ml). The results implied that the differential toxicity was associated with variations in size, so more attention should be given to the toxicity of small NPs for the design of future materials for implantation.     

The inducible expression of the long pentraxin PTX3 in osteoblast-like MC 3T3-E1 cells by E. coli lipopolysaccharide

In this study, we have found that Escherichia coli lipopolysaccharide (LPS) could induce the expression of pentraxin 3 (PTX3) mRNA in osteoblast-like cells (MC 3T3-E1) dose dependently. The relation between this expression and alkaline phosphatase (ALP), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa B (NF-κB) was also analyzed. The results show that the mRNA expression of PTX3 was related to that of ALP. It was also related to the mRNA expression of ERK-1 and p38 but not JNK-1 and NF-κB. These results suggest that ERK-1 and p38 are involved in the regulation of PTX3 expression and PTX3 promotes the differentiation of MC 3T3-E1 cells in response to LPS.     

The effect of adsorbed vitamin D and K to hydroxyapatite on ALP activity of MC3T3-E1 cell

This study describes the adsorptive property of vitamins on HA and the effect of the HA adsorbed vitamin on the alkaline phosphatase (ALP) activity for effective use as a bone graft substitute. The vitamins used were calciferol (D₃), menaquirone (K₂) and 25-hydroxycholecalciferol (25(OH)D₃). These vitamins were adsorbed on HA at 4, 10, 20, 37 and 50 °C. The adsorption amount was constant below 20 °C, and decreased as the incubation temperature increased over 20 °C. The order of the adsorption amount was: 25(OH)D₃ > K₂ > D₃. The HA adsorbed vitamins (HA/D₃, HA/25(OH)D₃ and HA/K₂) were suspended in physiological saline for 48 h for the release test. The release ratio of all vitamins increased with incubation time. The order of the release ratio was: 25(OH)D₃ > K₂ > D₃, which was proportional to that of the adsorption amount. The ALP activity of MC3T3-E1 osteoblast-like cells on HA, HA/D₃, HA/25(OH)D₃ and HA/K₂ was also investigated. The ALP activity was higher on HA/25(OH)D₃ than on any other samples. However, HA/K₂ and HA/D₃ showed similar ALP activity to HA.     

Effect of microstructure of titanium surface on the behaviour of osteogenic cell line MC3T3-E1

Three different microstructures were obtained on a titanium surface via immersion in HCl, H3PO4, or mixed acid of HNO3 and HF (HNO3/HF) solution. The microstructure and Rmax of the acid-treated surfaces were dependent on the acid type and immersion conditions. The growth rate of the osteogenic cell line MC3T3-E1 on each acid-treated sample, which was measured using MTT-formazan assay, was significantly higher than that of the standard which was ground with #400 SiC grit paper. Moreover, both the H3PO4 treated sample and the HNO3/HF-treated surface showed a tendency to enhance the alkaline phosphatase activity of MC3T3-E1 cells, which were grown on each acid-treated surface. These results suggest that the acid treatment of titanium is effective for the improvement of its osteocompatibility. © 1998 Chapman & Hall     

Preparation,surface properties,and MC3T3-E1 cell response of mesoporous hydroxyapatite thin films

Mesoporous hydroxyapatite (meso-HA) thin films were fabricated by a sol–gel method using cetyltrimethyl ammonium bromide as the template. The phase, surface morphology, and mesoporous structure of the meso-HA films were characterized by X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The uniform thin films consisted of meso-HA spherical particles with different pore size (2.0 or 3.1 nm) were prepared on the glass substrate at different pH value (pH 3.0 or 7.0). The introduction of mesopores would enhance the surface area of HA. Water contact angle was also measured on the non-mesoporous and meso-HA thin films, revealing the promotion of surface wettability in the mesoporous ones. In vitro cytocompatibility of HA films were evaluated by cell adhesion and proliferation tests using MC3T3-E1 cells. After 3 days of culture on the samples, the cells spread in an elongated shape and were well adhered to the surface of the meso-HA films. Moreover, the cells proliferation on the meso-HA films was higher than that on the non-mesoporous films. There are significant differences in the cell density between the control group and the meso-HA films with the pores sized in 2.0 nm after being cultured for 2 and 3 days (P < 0.05). The results suggested that the presence of mesopores could influence the surface and biological properties of HA films, and the mesoporous structure would enhance the cell response of HA.     

In Vitro Comparative Effect of Three Novel Borate Bioglasses on the Behaviors of Osteoblastic MC3T3-E1 Cells

Most related investigations focused on the effects of borate glass on cell proliferation/biocompatibility in vitro or bone repair in vivo; however, very few researches were carried out on other cell behaviors. Three novel borate bioglasses were designed as scaffolds for bone regeneration in this wok. Comparative effects of three bioglasses on the behaviors of osteoblastic MC3T3-E1 cells were evaluated. Excellent cytocompatibility of these novel borate bioglasses were approved in this work. Meanwhile, the promotion on cell proliferation, protein secretion and migration with minor cell apoptosis were also discussed in details, which contributed to the potential clinical application as a new biomaterial for orthopedics.     

Investigation of osteoblast-like MC3T3-E1 cells on a novel recombinant collagen-like protein surface with triple helix structure

Fol-8Col is a novel recombinant collagen-like protein incorporated with foldon sequences derived from the native T4 phage fibritin. In this paper, we examined the potential of using Fol-8Col as scaffold for bone tissue engineering. Circular dichroism (CD) spectra indicate that the triple helix structure of Fol-8Col exists at temperatures ranging from 4 to 40 °C. Lactate dehydrogenase assay results and live/death cell staining of osteoblast-like MC3T3-E1 cells, cultivated on Fol-8Col for 24 h, showed evidence of cell cytocompatibility comparable to that of native type I collagen. Attachment and spreading of osteoblast-like MC3T3-E1 cells seeded on Fol-8Col were studied by immunofluorescence staining of cell nuclei, vinculin, and F-actin. Intensive focal adhesion patches and an elongated cortical actin cytoskeleton were observed after 24 hours’ cultivation. Proliferation assays of MC3T3-E1 cells cultivated on Fol-8Col for 2 weeks revealed no consistent differences in rate and pattern compared to growth on type I collagen. Alkaline phosphatase activity assay and osteogenic gene expression, detected by RT-PCR, evaluated the osteogenic differentiation of MC3T3-E1 cells on Fol-8Col. This study shows that Fol-8Col, with a triple helix structure, has good potential for application in bone regeneration as a replacement for native collagen, thereby reducing the risk of contamination.     

Synergistic effects of bioactive ions and micro/nano-topography on the attachment,proliferation and differentiation of murine osteoblasts (MC3T3)

Surface topography and chemical nature of biological materials play an important role in regulating cell behaviors. For the intention of improving the biological performance of Ti₆Al₄V, the hierarchical micro/nano-topographies containing bioactive ions (Ca²⁺ and Mg²⁺) were fabricated in this study. Briefly, the hierarchical micro/nano-topography was constructed on Ti₆Al₄V surface via sandblasting, acid etching and alkali-hydrothermal treatment. Then Na⁺ existing in the nano-topography was replaced by Ca²⁺ and Mg²⁺ through hydrothermal reaction. The surface topographies and chemical nature of native and treated samples were characterized using laser scanning microscope, X-ray Photoelectron Spectroscopy and field emission scanning electron microscopy with the energy-dispersive spectroscopy. Surface wettability was measured with a contact angle goniometer. A series of biological tests were carried out to evaluate the synergistic effects of bioactive ions and micro/nano-topography on the attachment, proliferation and differentiation of murine osteoblastic MC3T3 cells. The results of in vitro tests indicated that Ca²⁺ and Mg²⁺ in the titanium alloy surface had an affirmative effect on cells attachment, proliferation and differentiation. Cells grown onto micro/nano-structured surface with Ca²⁺ implantation exhibited significantly higher differentiation levels of alkaline phosphatase activity and mineralization compared to that on micro/nano-structured surface with Mg²⁺ implantation. This study provided a novel method to construct a favorable biological environment between tissues and implants.     

Fabrication of chitosan microparticles loaded in chitosan and poly(vinyl alcohol) scaffolds for tissue engineering application

In recent decades, the use of microparticle-mediated drug delivery is widely applied in the field of biomedical application. Here, we report the new dressing material with ciprofloxacin-loaded chitosan microparticle (CMP) impregnated in chitosan (CH) and poly(vinyl alcohol) (PVA) scaffold for effective delivery of drug in a sustained manner to the wound site. Moreover, the peculiar physiochemical and structural properties of the CH–CMP scaffold has proved better tensile strength and excellent swelling to achieve 82% of drug release. In vitro biocompatibility was done for both scaffold using NIH 3T3 fibroblasts and human keratinocytes (HaCaT) cell lines. In vitro fluorescent activity showed distinct biocompatibility with good cell adhesion and proliferation. However, the CH–CMP scaffold showed best result to act as promising biomaterial in effective drug delivery in tissue engineering.     

Expression of cell adhesion and differentiation related genes in MC3T3 osteoblasts plated on titanium alloys: role of surface properties

It is important to understand the cellular and molecular events that take place at the cell–material interface of implants used for bone repair. An understanding of the mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material is fundamental in deciding the fate of the cells that come in contact with it. In this study, we compared the relative gene expression of markers that are known to be associated with cell adhesion and differentiation in MC3T3 osteoblast cells, at various time points after plating the cells on surfaces of titanium (Ti) and its two alloys, titanium–zirconium (TiZr) and titanium–niobium (TiNb) by using Quantitative Real Time Polymerase Chain Reaction (RT-PCR). Our analysis indicated that expression of adhesion supporting genes was higher on TiZr surface as compared to Ti and TiNb. The behavior of these genes is possibly driven by a higher surface energy of TiZr. However no significant difference in the expression of differentiation related genes could be seen between the two alloys, although on both substrates it was higher as compared to unalloyed Ti. We propose that substrate composition of the alloys can influence the adhesion and differentiation related gene expression and that Ti alloys are better substrates for inducing osteogenesis as compared to unalloyed Ti.     

Metabolomic profiling of ZrO2 nanoparticles in MC3T3‐E1 cells

The authors'' previous study showed that zirconium oxide nanoparticles (ZrO₂ NPs) induce toxic effects in MC3T3‐E1 cells; however, its toxicological mechanism is still unclear. Liquid chromatography–mass spectrometry/time‐of‐flight mass spectrometry was used to reveal the metabolite profile and toxicological mechanism of MC3T3‐E1 cells in response to ZrO₂ NPs. The results demonstrated that MC3T3‐E1 cells treated with ZrO₂ NPs for 24 and 48 h presented different metabolic characteristics. Following ZrO₂ NP treatment for 24 h, 96 upregulated and 129 downregulated metabolites in the positive ion mode, as well as 91 upregulated and 326 downregulated metabolites in the negative ion mode were identified. Following ZrO₂ NP treatment for 48 h, 33 upregulated and 174 downregulated metabolites were identified in the positive ion mode, whereas 37 upregulated and 302 downregulated metabolites were confirmed in the negative ion mode. Among them, 42 differential metabolites were recognised as potential metabolites contributing to the induced toxic effects of ZrO₂ NPs in MC3T3‐E1 cells. Most of the differential metabolites were lysophosphatidylcholine and lysophosphatidylethanolamide, indicating that exposure to ZrO₂ NPs may have a profound impact on human cellular function by impairing the membrane system. The results also provide new clues for the toxicological mechanism of ZrO₂ NP dental materials.     

Metal ion effects on different types of cell line,metal ion incorporation into L929 and MC3T3-E1 cells,and activation of macrophage-like J774.1 cells

V ions showed high cytotoxicity for mouse fibroblast L929, osteoblastic MC3T3-E1, and macrophage-like J774.1 cells compared with Pb, Cu, Ni, Co, Zn, and Mo ions. The quantities of metal ions incorporated into the L929 and MC3T3-E1 cells increased with increasing metal concentration in the medium, depending on the metal ion type. In particular, the quantities of V incorporated into the cells were markedly higher than those of other metals. It was suggested that the cytotoxicity of a metal ion changes with the quantity of the metal ion incorporated into cells. It was also considered that V ions are incorporated into cells through xanthine derived from fetal bovine serum by high-performance liquid chromatography (HPLC). The strong interaction of Co, Ni and Mo with amino acids was analyzed by HPLC. The rate of increase of nitric oxide (NO) concentration released with the activation of the mouse macrophage-like J774.1 cells increased at a concentration of V ions ten times lower than that of Ni ions. The release of the cytokine tumor necrosis factor-α (TNF-α) from the J774.1 cells started at approximately 0.5 ppm V; interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) showed a marked increase in the rate of increase at more than 1 ppm V. No increase in the concentration of IL-1α, IL-8, IL-15 or granulocyte macrophage-colony stimulating factor (GM-CSF) was observed for V and Ni ions.     

Effect of process parameters on the properties of selective laser sintered Poly(3-hydroxybutyrate) scaffolds for bone tissue engineering

Porous scaffolds are biocompatible and bioactive temporary substrates. They should present appropriated microstructure, mechanical properties and surface properties for guiding bone tissue regeneration. In this work, scaffolds of Poly(3-hydroxybutyrate) (PHB) were printed by Selective Laser Sintering (SLS). The effect of scan spacing (SS) and powder layer thickness (PLT) on the morphology, mechanical properties and dimensional deviations related to the digital model of sintered scaffolds was evaluated. Curling was observed in the first built layers of scaffolds, mainly for scaffolds printed with the lowest PLT. Besides designed pores, the scaffolds also presented micropores derived from the incomplete sinterisation of PHB particles. This morphology can be advantageous for bone regeneration. The variation of PLT caused a higher difference between the values of scaffold mechanical properties than the variation of SS. The scaffolds, except the one printed with the highest PLT or SS, showed mechanical properties within the lower range of human trabecular bone.