MC 3T3 Research Articles

An Eco-Friendly, Simple, and Inexpensive Method for Metal-Coating Strontium onto Halloysite Nanotubes

Osteoporosis increases the risk of bone fracture by reducing bone mass and thereby increasing bone fragility. The addition of strontium (Sr) nanoparticles in bone tissue results in a strengthening of the bone, induction bone formation by osteoblasts, and reduction of bone reabsorption by osteoclasts. The use of Sr for bone tissue regeneration has gained significant research interest in recent years due to its beneficial properties in treating osteoporotic-induced bone loss. We hypothesized that Sr-coated and antibiotic-doped HNTs could be used in antimicrobial coatings and as an antibacterial drug delivery vehicle. Accordingly, we coated HNTs with strontium carbonate (SrHNT) using a simple, novel, and effective electrodeposition method. We tested the antibacterial properties of SrHNT on Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermis using the disc diffusion method. We assessed the potential cytotoxic and proliferative effects of SrHNTs on pre-osteoblasts using a Live/Dead cytotoxicity and cell proliferation assay. We successfully coated HNTs with strontium using a one-step benign coating method that does not produce any toxic waste, unlike most HNT metal-coating methods. Antibacterial tests showed that the SrHNTs had a pronounced growth inhibition effect, and cell culture studies using MC 3T3 cells concluded that SrHNTs are cytocompatible and enhance cell proliferation.

Investigating the structure, solubility, and antibacterial properties of silver- and copper-doped hydroxyapatite.

Hydroxyapatite (HA) powders were synthesized by the wet precipitation method in which two experimental compositions were synthesized (10mol% Ag-HA and Cu-HA) where the CaNO3 content was partially substituted with AgNO3 and Cu(NO3 )2 . X-ray diffraction (XRD) was employed to characterize changes to the HA structure as the dopants (Cu2+ , Ag+ ) were incorporated into the materials structure. Energy-dispersive X-ray spectroscopy (EDS) determined confirmed the compositions and found that the Ca/P ratio was 1.63 for the control (HA) while Ag-HA and Cu-HA exhibited (X + Ca)/P ratios of 1.79 and 1.65, respectively. Antibacterial efficacies were evaluated against E. coli and S. aureus, as a function of surface area and incubation time. The more prominent antibacterial effects were observed with both Ag-HA and Cu-HA and the materials antibacterial influence was maintained with respect to time. Ion release studies of each HA composition (15, 30, and 45 days) showed that Cu-HA released significantly more Cu2+ (36.1 ± 5.1mg/L) than Ag+ (2.9 ± 1.2mg/L) from Ag-HA. Analysis of each composition incubated in simulated body fluid (SBF) exhibited surface depositions that are likely calcium phosphate (CaP). Cytocompatibility testing in MC 3T3 Osteoblasts showed slight reductions in cell viability when tested using MTT assay, however cell adhesion studies were positive for each composition.

Synthesis, Processing and the Effect of Thermal Treatment on the Solubility, Antioxidant Potential and Cytocompatibility of Y2O3 and CeO2 doped SiO2-SrO-Na2O Glass-Ceramics.

Thermal treatment of a 0.52SiO2-0.24SrO-0.24-xNa2O-xMO glass-ceramic series (where x = 0.08 and MO = Y2O3 or CeO2) was conducted in order to synthesize yttrium (Y3+) and cerium (Ce3+) crystalline species that may act as radical oxygen specie (ROS) scavengers. The prominent phase for the Control is a sodium-strontium-silicate while the experimental glass-ceramics (HY, YCe, and HCe) present sodium-Y/Ce-silicate and oxide phases. Disk shrinkage during thermal processing ranges from 1-7% for Control, HY, YCe, and HCe in both diameter and thickness. Solubility studies determined that the release of Si4+ and Na+ are greatest from the Control disks which peaks at 1550µg/mL. Release from the Y3+ and Ce3+ glass-ceramics reached 320µg/mL for Si4+ and 630µg/mL for Na+. The range of antioxidant capacity (ABTS assay) for all samples was 0.31-3.9 mMTE. No significant reduction in MC 3T3 Osteoblast cell viability was observed for any composition tested.

Large fuzzy biodegradable polyester microspheres with dopamine deposition enhance cell adhesion and bone regeneration in vivo

The biodegradable polymer microparticles with different surface morphology and chemical compositions may influence significantly the behaviors of cells, and thereby further the performance of tissue regeneration in vivo. In this study, multi-stage hierarchical textures of poly(D,L-lactic-co-glycolide) (PLGA)/PLGA-b-PEG (poly(ethylene glycol)) microspheres with a diameter as large as 50–100 μm are fabricated based on interfacial instability of an emulsion. The obtained fuzzy structures on the microspheres are sensitive to annealing, which are changed gradually to a smooth one after treatment at 37 °C for 6 d or 80 °C for 1 h. The surface microstructures that are chemically dominated by PEG can be stabilized against annealing by dopamine deposition. By the combination use of annealing and dopamine deposition, a series of microspheres with robust surface topologies are facilely prepared. The fuzzy microstructures and dopamine deposition show a synergetic role to enhance cell-material interaction, leading to a larger number of adherent bone marrow-derived mesenchymal stem cells (BMSCs), A549 and MC 3T3 cells. The fuzzy microspheres with dopamine deposition can significantly promote bone regeneration 12 w post surgery in vivo, as revealed by micro-CT, histological, western blotting and RT-PCR analyses.

Fabrication and In vitro Bioactivity of Robust Hydroxyapatite Coating on Porous Titanium Implant

Compared with the traditional dental implant, TixOs® manufactured by direct laser metal forming(DLMF) technology exhibits improved capability for bone osteointegration due to its porous surface structure, and has achieved remarkable clinical effect. However, like the traditional titanium and other alloyed implants, the porous titanium implant TixOs® also has relatively weak bioactivity. To address this issue, a proper surface modification method may be needed. Hydroxyapatite(HA) has been widely used in implant surface coating for its similar chemical composition to bone tissue and its osteoconductive properties. Thus, combining TixOs® implants with hydroxyapatite can be an efficient way to enhance their bioactivity. We herewith reported a competent pulsed laser deposition(PLD) method of coating nano-sized HA thin film onto the porous TixOs® implant. The HA coatings were characterized by means of scanning electron microscopy(SEM), energy dispersive X-ray spectroscopy(EDS), X-ray photoelectron spectroscopy(XPS) and focused ion beam(FIB) method, and nanocrystal sized thin HA films were identified on the surface of TixOs® implants. The low cytotoxicity and improved cell proliferation ability of HA coated implants were further tested and verified using MC 3T3 E1 cells with the consideration of the controlling group. Our results show that a stable and bioactive HA thin film is able to form on the surface of the porous titanium implant by PLD method. This may benefit the further clinical application of TixOs® implants.

Role of Reactive Oxygen Species during Low-Intensity Pulsed Ultrasound Application in MC-3 T3 E1 Pre-osteoblast Cell Culture

We evaluated the activation of mitogen-activated protein kinase (MAPK) activation through reactive oxygen species (ROS) by application of low-intensity ultrasound (LIPUS) to MC-3 T3 E1 pre-osteoblasts. The cells were subjected to one LIPUS application for either 10 or 20 min, and the control group was exposed to a sham transducer. For ROS inhibition, 10 μM diphenylene iodonium (DPI) was added to the cells an hour before LIPUS application. Samples were collected 1, 3, 6, 12 and 24 h after LIPUS application, and cells were evaluated for ROS generation, cell viability, gene expression and MAPK activation by immunoblot analyses. LIPUS caused a significant increase in ROS and cell viability in the non-DPI-treated group. Expression of RUNX2, OCN and OPN mRNA was higher in the LIPUS-treated groups at 1 h in both the DPI-treated and non-DPI-treated groups; RUNX2 and OCN mRNA levels increased at 6 h. ERK1/2 activation was increased in the LIPUS-treated groups. These results indicate that LIPUS activates MAPK by ROS generation in MC-3 T3 E1 pre-osteoblasts.

Co-cultured tissue-specific scaffolds for tendon/bone interface engineering

The tendon/ligament-to-bone interface has a complex organization to enable transfer of forces through the tendon/ligament to the bone. The purpose of this study is to create a co-culture environment enabling a tissue-specific tendon region and tissue-specific bone region on a degradable scaffold, using NIH 3T3 fibroblast–deposited extracellular matrix and MC 3T3 osteoblast–deposited extracellular matrix, respectively. Before full characterization of the deposited extracellular matrix coating can be analyzed, co-culture parameters including culture medium and seeding technique should be addressed. An appropriate medium formulation was developed to reduce fibroblast to osteoblast mineralization by adjusting beta-glycerophosphate concentrations. Standard growth medium with fetal bovine serum + 3 mM beta-glycerophosphate + 25 µg/mL ascorbic acid was found to be the most suitable formulation evaluated in these study conditions. Seeding and cell migration studies of co-cultured fibroblast- and osteoblast-specific scaffolds were performed to identify whether tissue regions could be created on the scaffold. Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding. Finally, a preliminary analysis of basic extracellular matrix components was measured in the fibroblast, osteoblast, and transition regions. Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions. In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold.

Culturing Primary Human Osteoblasts on Electrospun Poly(lactic-co-glycolic acid) and Poly(lactic-co-glycolic acid)/Nanohydroxyapatite Scaffolds for Bone Tissue Engineering

In this work, we fabricated polymeric fibrous scaffolds for bone tissue engineering using primary human osteoblasts (HOB) as the model cell. By employing one simple approach, electrospinning, we produced poly(lactic-co-glycolic acid) (PLGA) scaffolds with different topographies including microspheres, beaded fibers, and uniform fibers, as well as the PLGA/nanohydroxyapatite (nano-HA) composite scaffold. The bone-bonding ability of electrospun scaffolds was investigated by using simulated body fluid (SBF) solution, and the nano-HA in PLGA/nano-HA composite scaffold can significantly enhance the formation of the bonelike apatites. Furthermore, we carried out in vitro experiments to test the performance of electrospun scaffolds by utilizing both mouse preosteoblast cell line (MC 3T3 E1) and HOB. Results including cell viability, alkaline phosphatase (ALP) activity, and osteocalcin concentration demonstrated that the PLGA/nano-HA fibers can promote the proliferation of HOB efficiently, indicating that it is a promising scaffold for human bone repair.

Interaction of cell culture with composition effects on the mechanical properties of polycaprolactone-hydroxyapatite scaffolds fabricated via selective laser sintering (SLS)

In the current study PCL/HA composites were fabricated using SLS as two- and three-dimensional lattice structures and exposed to a cellular component (MC 3T3 osteoblast-like cells). The main aims were to determine the mechanical differences due to powder composition and to observe the physical and mechanical changes pertaining to cell presence. These structures were characterized by compressive mechanical testing, and the effects of cell culturing and degradation on mechanical properties of the scaffolds with different PCL/HA compositions were determined. Moreover, changes in the scaffold morphology due to the cell culture conditions were determined by μ-CT analysis.Cells steadily grew on the scaffolds for 21days with preferential distribution around the macropores and initially PCL/HA(15%) composites had higher cell numbers. Removal of loosely sintered parts was observable during the culturing period. Cell culture conditions did not change the compressive moduli significantly but had a distinct effect on compressive strength. For PCL/HA(15%) composites, an initial loss in strength caused by cell culture was reversed by longer cell exposure, with compressive strength of the structures restored to the initial properties (p≤0.05). μ-CT measurements showed widespread morphological changes in the scaffolds, such as a decrease in the roughness of the struts. In general, in the initial period composites with lower HA content (15wt.%) showed better metabolic activity compared to the higher HA content, however by day 14 the performance of the two compositions was equal. These results suggest that changes in sintering due to the differences in powder composition can have profound effects on the short and long term mechanical properties of the scaffold particularly under cell culture conditions, and this should be closely considered for SLS processing of scaffolds.

Evaluation of a bio‐based hydrophobic cellulose laurate film as biomaterial—Study on biodegradation and cytocompatibility

The study aims to validate an original bio-based material, obtained by grafting fatty chains, and more especially lauric chains (C12) onto cellulose, for medical applications. The mechanical properties of the synthesized cellulose laurate (C12) are close to those of petrochemical ones such as low density polyethylene. This cellulose-based polymer is transparent, flexible, and hydrophobic. To evaluate the stability of the cellulosic films in biological fluids the samples are soaked in simulated body fluid or blood plasma for a few hours to 6 months, and then submitted to mechanical and chemical analyses. The simultaneously performed cytocompatibility tests were the colony-forming viability, the vitality and cell proliferation tests using NIH 3T3 fibroblasts and MC 3T3 osteoblast-like cells. The results show the stability, the biocompatibility, and the noncytotoxicity of the synthesized cellulose laurate films. This biomaterial may so be considered for surgical applications.

A simple PDMS-based microfluidic channel design that removes bubbles for long-term on-chip culture of mammalian cells

This report shows methods to fabricate polydimethylsiloxane (PDMS) microfluidic systems for long-term (up to 10 day) cell culture. Undesired bubble accumulation in microfluidic channels abruptly changes the microenvironment of adherent cells and leads to the damage and death of cells. Existing bubble trapping approaches have drawbacks such as the need to pause fluid flow, requirement for external vacuum or pressure source, and possible cytotoxicity. This study reports two kinds of integrated bubble trap (IBT) which have excellent properties, including simplicity in structure, ease in fabrication, no interference with the flow, and long-term stability. IBT-A provides the simplest solution to prevent bubbles from entering microfluidic channels. In situ time-lapse imaging experiments indicate that IBT-B is an excellent device both for bubble trapping and debubbling in cell-loaded microfluidics. MC 3T3 E1 cells, cultured in a long and curved microfluidic channel equipped with IBT-B, showed high viability and active proliferation after 10 days of continuous fluid flow. The comprehensive measures taken in our experiments have led to successful long-term, bubble-free, on-chip culture of cells.

Transcriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ.

Core-binding factor 1 (Cbfa1; also called Runx2) is a transcription factor belonging to the Runt family of transcription factors that binds to an osteoblast-specific cis-acting element (OSE2) activating the expression of osteocalcin, an osteoblast-specific gene. Using the yeast two-hybrid system, we identified a transcriptional coactivator, TAZ (transcriptional coactivator with PDZ-binding motif), that binds to Cbfa1. A functional relationship between Cbfa1 and TAZ is demonstrated by the coimmunoprecipitation of TAZ by Cbfa1 and by the fact that TAZ induces a dose-dependent increase in the activity of osteocalcin promoter-luciferase constructs by Cbfa1. A dominant-negative construct of TAZ in which the coactivation domains have been deleted reduces osteocalcin gene expression down to basal levels. NIH 3T3, MC 3T3, and ROS 17/2.8 cells showed the expected nuclear localization of Cbfa1, whereas TAZ was distributed throughout the cytoplasm with some nuclear localization when transfected with either Cbfa1 or TAZ. Upon cotransfection by both Cbfa1 and TAZ, the transfected TAZ shows predominant nuclear localization. The dominant-negative construct of TAZ shows minimal nuclear localization upon cotransfection with Cbfa1. These data indicate that TAZ is a transcription coactivator for Cbfa1 and may be involved in the regulation of osteoblast differentiation.

Role of interleukin-6 in ß2-microglobulin–induced bone mineral dissolution

beta2-microglobulin (beta2m) amyloidosis is commonly seen in patients undergoing long-term dialysis. beta2m has been shown to induce in vitro bone mineral dissolution. The present study was designed to investigate the effect of beta2m on osteoblast function and the role of interleukin-6 (IL-6) on beta2m-induced bone resorption. Using neonatal mouse calvariae as well as primary osteoblasts and MC 3T3 osteoblast-like cells, IL-6 production, release, and gene expression were investigated with enzyme-linked immunosorbent assay (ELISA) and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques, respectively. In calvariae, beta2m induced a time- and dose-dependent calcium release, which was maximum following a 48-hour incubation at a concentration of 10-5 mol/L. beta2m (10-6 mol/L) also induced a significant release of IL-6 from calvarial and primary osteoblastic cultures. Using 10-6 mol/L beta2m, the amount of IL-6 mRNA in MC 3T3 cells increased in a time-dependent fashion, which peaked at 3 hours and declined to baseline by 12 hours. In primary osteoblast cells, beta2m maximally increased IL-6 mRNA levels at 6 hours; however, they remained elevated up to 24 hours. Compared with control, the presence of beta2m significantly increased cell proliferation of both primary osteoblasts and MC 3T3 cells. To investigate osteoblastic function further, osteocalcin mRNA was quantitated. Incubation with beta2m for 3 to 24 hours did not alter the amount of osteocalcin mRNA in the MC 3T3 osteoblast cells. beta2m affects bone metabolism by mechanisms that include increasing IL-6 gene expression and release, and enhancing osteoblast proliferation without affecting osteocalcin gene expression.

Mitogenic action of osteogenic growth peptide (OGP) Role of amino and carboxy-terminal regions and charge

We have recently reported the discovery of a 14-amino-acid osteogenic growth peptide (OGP). In vivo OGP increases bone formation and trabecular bone density. Physiologically it is found in serum complexed to an OGP binding protein (OGPBP). In vitro OGP has a biphasic effect on osteoblastic MC 3T3 E1 and fibroblastic NIH 3T3 cell proliferation; at low concentrations (0.01–1.0 and 1.0–100.0 pM, respectively) it is highly stimulatory with an inhibition at higher doses. To assess possibilities of labeling synthetic OGP to obtain radio- or fluorescent ligands, OGP analogues were extended at the N- or C-termini with Cys or Cys(S-NEtSucc) or the OGP Tyr-10 replaced by 3-I(Tyr). All analogues with N-terminal modifications, as well as the [Cys 15]OGP-NH 2 retained the OGP-like dose-dependent effect on proliferation of the MC 3T3 E1 and NIH 3T3 cells, although the magnitude of stimulation was lower, approx. 2 3 that of the native-like synthetic OGP. The [Cys 15(S-NEtSucc)]OGP-NH 2 and [3-I(Tyr 10)]OGP shared only the inhibitory activity of OGP. This suppression is further shared by a number of other positively and negatively net charged, but not net neutral, peptides. Both N-terminal-modified analogues displayed a decreased binding activity to the OGPBP. All analogues except reverse OGP, [3-I(Tyr 10)]OGP and [Cys 15(S-NEtSucc)]OGP-NH 2 reacted with anti-OGP antibodies. These data are not only important for labeling purposes but suggest a respective role for the OGP N-and C-terminal regions in binding to the OGPBP and putative OGP receptor. It appears that the OGP proliferative activity represents the net effect of stimulation specific to the OGP structure and nonspecific inhibition associated with the peptide's high positive net charge.

Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.