In vitro cellular response to hydroxyapatite scaffolds with oriented pore architectures

The objective of the present work was to evaluate the in vitro cellular response to hydroxyapatite (HA) scaffolds with oriented pore architectures. Hydroxyapatite scaffolds with approximately the same porosity (65–70%) but two different oriented microstructures, described as ‘columnar’ (pore diameter = 90–110 μm) and ‘lamellar’ (pore width = 20–30 μm), were prepared by unidirectional freezing of suspensions. The response of murine MLO-A5 cells, an osteogenic cell line, to these scaffolds was evaluated using assays of MTT hydrolysis, alkaline phosphatase (ALP) activity, and alizarin red staining. While the cellular response to both groups of scaffolds was better than control wells, the columnar scaffolds with the larger pore width provided the most favorable substrate for cell proliferation and function. These results indicate that HA scaffolds with the columnar microstructure could be used for bone repair applications in vivo.     

The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1)

The aim of this study was to investigate the degree of deacetylation (DD) and molecular weight (MW) of chitosan within chitosan–collagen scaffolds on mouse osteoblasts (MC3T3-E1). The chitosan–collagen scaffolds were fabricated by freeze-drying technique. The studies on cell attachment and proliferation, alkaline phosphatase (ALP) activity, cell morphology, and mineralized nodule formation by osteoblasts on scaffolds were investigated. No statistically significant difference was found on cell attachment, but the chitosan–collagen scaffolds with low-DD chitosan had a statistically significantly (P < 0.05) higher proliferative effect and ALP activity than those scaffolds with high-DD chitosan, regardless of molecular weight. Scanning electron images demonstrated that MC3T3-E1 cells grew well on all test scaffolds; on the contrary, mineralized nodule formation was not found. In conclusion, the DD of chitosan is a crucial factor for MC3T3-E1 cells and it should be considered in further applications for bone tissue engineering.     

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.     

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.     

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.     

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.     

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.     

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。     

Effect of polyvinyl alcohol additive on the pore structure and morphology of the freeze-cast hydroxyapatite ceramics

Porous hydroxyapatite (HAP) ceramics with different morphologies were fabricated by the freeze casting method. The morphologies of HAP ceramics were modified by adjusting the concentration of polyvinyl alcohol (PVA) additive in the HAP slurries. HAP ceramics without PVA additive were composed of non-interconnected macroscopic lamellar pores and porous ceramic walls. With PVA additive, the HAP ceramics were made up of small lamellar pores or three-dimensional reticulate pores and porous ceramic walls. PVA additive had no effect on the phase composition of HAP ceramics. The open porosity and pore connectivity were improved because of the addition of PVA.     

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     

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.     

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.     

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.     

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.     

3D-Cultivation of bone marrow stromal cells on hydroxyapatite scaffolds fabricated by dispense-plotting and negative mould technique

The main principle of a bone tissue engineering (BTE) strategy is to cultivate osteogenic cells in an osteoconductive porous scaffold. Ceramic implants for osteogenesis are based mainly on hydroxyapatite (HA), since this is the inorganic component of bone. Rapid Prototyping (RP) is a new technology in research for producing ceramic scaffolds. This technology is particularly suitable for the fabrication of individually and specially tailored single implants. For tissue engineering these scaffolds are seeded with osteoblast or osteoblast precursor cells. To supply the cultured osteoblastic cells efficiently with nutrition in these 3D-geometries a bioreactor system can be used. The aim of this study was to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. For this, two RP-techniques, dispense-plotting and a negative mould method, were used to produce porous ceramics. The manufactured HA-scaffolds were then cultivated in a dynamic system (bioreactor) with an osteoblastic precursor cell line. In our study, the applied RP-techniques give the opportunity to design and process HA-scaffolds with defined porosity, interconnectivity and 3D pore distribution. A higher differentiation of bone marrow stromal cells could be detected on the negative mould fabricated scaffolds, while cell proliferation was higher on the dispense-plotted scaffolds. Nevertheless, both scaffold types can be used in tissue engineering applications.     

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.     

Structure/processing relationships and mechanical properties of freeze-cast B4C scaffolds with unidirectional channels

Journal of Materials Science - Freeze casting technique has become a promising way to assemble various components into multifunctional nacre-like materials. Surprisingly, although many ceramics...