Mouse Pre-osteoblast MC3T3-E1 Research Articles

Characterization of natural hydroxyapatite originated from fish bone and its biocompatibility with osteoblasts

Hydroxyapatite (HAP) was very attractive for using as bone implant material for a long period due to the close similarity with natural bone in composition and osteoconductive properties. In this study, three kinds of natural HAP (nHAP) derived from rainbow trout (Onchorynchus mkiss), cod (Gadus) and salmon (Oncorhynchus keta) bones were prepared using thermal calcination method for the first time. Resultant nHAPs were characterized by fourier transform infrared spectroscopy (FT-IR), x-ray diffraction analysis (XRD), field-emission scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. Biocompatibility of calcined nHAP was evaluated through MTT cell viability assay and alkaline phosphatase activity experiment using mouse preosteoblast MC3T3-E1. Results of cell experiment indicated that the nHAP originated from rainbow trout and salmon bones showed better biological compatibility compared with the nHAP originated from cod bone and chemical synthetic HAP (cHAP). This is most likely attributed to the different element composition in nHAP, i.e., the nHAP derived from rainbow trout and salmon bones showed the presence of CO32– and Mg2+. Therefore, the nHAP originated from rainbow trout and salmon bones have a great potential for application as implant material substitute in bone tissue engineering and the natural waste fish bone product can be used for hydroxyapatite synthesis as a part of bio-waste management.

ECM based injectable thermo-sensitive hydrogel on the recovery of injured cartilage induced by osteoarthritis

Intra-articular injection of anti-inflammatory drugs can be a promising strategy for recovery of injured articular cartilage. We prepared a series of injectable thermo-sensitive composite hydrogels, composed of Pluronic F127, glycosaminoglycan (GAG) and bone morphogenetic protein (BMP-2), which was designed to mimic extracellular matrix (ECM). The rheological properties and dissolution rate of composite hydrogels containing chondroitin sulfate or with different hyaluronic acid molecular mass (10k, 90k, 800k) were investigated. Meanwhile, bovine serum albumin (BSA) or FITC-BSA was chosen as model drug loaded into PF/GAG hydrogels to study their sustained release behavior in vitro. The results showed that hydrogels could maintain shapes for more than 16 days and the release rate of BSA in PF/GAG composite gels was much slower than in PF127 gels, due to the affinity between BSA and GAG. Furthermore, increasing the molecular weight of hyaluronic acid correspondingly increased hydrogel dissolution rate and BSA release in the hydrogels. Subsequently, MTT experiments were performed to investigate the toxicity of the hydrogels on mouse pre-osteoblast cell MC3T3-E1. In vivo anti-inflammation results showed that PF/GAG@BMP-2 composite hydrogels had the most efficient efficacy on recovery of injured cartilage, which is induced by osteoarthritis, compared to the control groups (PF127@BMP-2 or BMP-2 saline solution).

ZnO Nanoparticles with Different Sizes and Morphologies for Medical Implant Coatings: Synthesis and Cytotoxicity

Zinc oxide (ZnO) has been used in a large range of technological applications, from light-emitting diodes (LEDs) and solar cells to cancer diagnosis and treatment agents. In the last decades, however, a novel antimicrobial property for ZnO particles has been explored, making this oxide an interesting material to be incorporated or deposited on biomaterials, for example in coatings over metallic implants. In this scenario, ZnO physico-chemical properties, mainly particles’ size and morphology, will play fundamental role on its performance. Thus, in this work, two different procedures of a chemical synthesis route named sonochemistry were used to achieve different morphologies/sizes of ZnO. Then, the obtained particles were used to evaluate possible cytotoxic effects against MC3T3-E1 mouse pre-osteoblasts (bone cells). The obtained results indicated that the sonochemical route is an effective way to produce different ZnO morphologies and the way in which these particles interact with osteoblasts (directly or indirectly) may completely influence their cytotoxicity.

Analysis of osteogenic potential on 3mol% yttria-stabilized tetragonal zirconia polycrystals and two different niobium oxide containing zirconia ceramics.

PURPOSEThis study was performed to evaluate the osteogenic potential of 3mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) and niobium oxide containing Y-TZPs with specific ratios, new (Y,Nb)-TZPs, namely YN4533 and YN4533/Al20 discs.MATERIALS AND METHODS3Y-TZP, YN4533 and YN4533/Al20 discs (15 mm diameter and 1 mm thickness) were prepared and their average surface roughness (Ra) and surface topography were analyzed using 3-D confocal laser microscope (CLSM) and scanning electron microscope (SEM). Mouse pre-osteoblast MC3T3-E1 cells were seeded onto all zirconia discs and evaluated with regard to cell attachment and morphology by (CLSM), cell proliferation by PicoGreen assay, and cell differentiation by Reverse-Transcription PCR and Quantitative Real-Time PCR, and alkaline phosphatase (Alp) staining.RESULTSThe cellular morphology of MC3T3-E1 pre-osteoblasts was more stretched on a smooth surface than on a rough surface, regardless of the material. Cellular proliferation was higher on smooth surfaces, but there were no significant differences between 3Y-TZP, YN4533, and YN4533/Al20. Osteoblast differentiation patterns on YN4533 and YN4533/Al20 were similar to or slightly higher than seen in 3Y-TZP. Although there were no significant differences in bone marker gene expression (alkaline phosphatase and osteocalcin), Alp staining indicated better osteoblast differentiation on YN4533 and YN4533/Al20 compared to 3Y-TZP.CONCLUSIONBased on these results, niobium oxide containing Y-TZPs have comparable osteogenic potential to 3Y-TZP and are expected to be suitable alternative ceramics dental implant materials to titanium for aesthetically important areas.

Phenazine carboxylic acid and its derivative induce osteoblast differentiation in preosteoblastic MC3T3-E1 cells but adipocyte differentiation in pluripotent mesenchymal C3H10T1/2 cells.

Osteoblast and adipocyte are differentiated from mesenchymal stem cells and dysregulation of the differentiation might result in disease, such as osteoporosis and diabetes. To find small compounds that induce osteoblast differentiation, we screened an in-house natural compounds library with mouse preosteoblastic MC3T3-E1 cells using alkaline phosphatase (ALP) expression as an early osteoblast marker. We found that phenazine-1-carboxylic acid (PCA), one of the major phenazine derivatives produced by Pseudomonas, induced osteoblast differentiation in the cells at micromolar concentrations. PCA acted synergistically with an agonist of hedgehog signaling in inducing ALP activity in the cells. We also found that 2-hydroxy-PCA (2H-PCA) induced osteoblast differentiation in the cells but 2-methoxy-PCA and 1-hydroxy-phenazine did not. Unexpectedly, treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with PCA or 2H-PCA induced an obvious morphological change. Oil Red O staining and real-time reverse-transcription PCR analysis revealed that PCA induced not osteoblast differentiation but adipocyte differentiation in C3H10T1/2 cells. These compounds could allow us to investigate the mechanism of osteoblast and adipocyte differentiation in the two model cell systems through a chemical biology approach.

Celecoxib inhibits osteoblast differentiation independent of cyclooxygenase activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects primarily by inhibiting the activity of cyclooxygenase (COX), thus suppressing prostaglandin synthesis. Some NSAIDs are known to perform functions other than pain control, such as suppressing tumour cell growth, independent of their COX-inhibiting activity. To identify NSAIDs with COX-independent activity, we examined various NSAIDs for their ability to inhibit osteoblastic differentiation using the mouse pre-osteoblast cell line MC3T3-E1. Only celecoxib and valdecoxib strongly inhibited osteoblastic differentiation, and this effect was not correlated with COX-inhibiting activity. Moreover, 2,5-dimethyl (DM)-celecoxib, a celecoxib analogue that does not inhibit COX activity, also inhibited osteoblastic differentiation. Celecoxib and DM-celecoxib inhibited osteoblastic differentiation induced by bone morphogenetic protein (BMP)-2 in C2C12 mouse myoblast cell line. Although celecoxib suppresses the growth of some tumour cells, the viability and proliferation of MC3T3-E1 cells were not affected by celecoxib or DM-celecoxib. Instead, celecoxib and DM-celecoxib suppressed BMP-2-induced phosphorylation of Smad1/5, a major downstream target of BMP receptor. Although it is well known that COX plays important roles in osteoblastic differentiation, these results suggest that some NSAIDs, such as celecoxib, have targets other than COX and regulate phospho-dependent intracellular signalling, thereby modifying bone remodelling.

Laser irradiation promotes the proliferation of mouse pre-osteoblast cell line MC3T3-E1 through hedgehog signaling pathway.

Low-level laser could promote osteoblast proliferation, and it has been applied in clinical practice to promote wound healing and tissue regeneration. However, the mechanism related to laser irradiation remains unclear. This study aimed to investigate the effects of low-level laser irradiation on the cell proliferation and the expressions of hedgehog signaling molecules Indian hedgehog (Ihh), Ptch, and Gli in vitro. In our present study, the MTT method was used to evaluate the effect on cell proliferation of laser irradiation on MC3T3-E1 cells. And cell cycle was examined by flow cytometry. Gene and protein expressions of hedgehog signaling molecules, including Ihh, Ptch, Smoothened (Smo), and Gli, were examined by qRT-PCR and western blot analysis. The results showed that laser irradiation at dosage of 3.75J/cm2 enhances the proliferation of MC3T3-E1 cells compared with control groups (p=0.00). Moreover, laser irradiation (3.75J/cm2) increased the cell amount at S phase (p=0.00). In addition, the expressions of Ihh, Ptch, Smo, and Gli were significantly increased compared to the control during laser irradiation (3.75J/cm2)-induced MC3T3-E1 osteoblast proliferation. After adding the hedgehog signaling inhibitor CY (cyclopamine), cell proliferation and Ihh, Ptch, Smo, and Gli expressions were inhibited (p=0.00), and the cell amount at S phase was reduced compared with combination groups (p=0.00). These results indicated that laser irradiation promotes proliferation of MC3T3-E1 cells through hedgehog signaling pathway. Our findings provide insights into the mechanistic link between laser irradiation-induced osteogenesis and hedgehog signaling pathway.

The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration

Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (µCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative µCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.

Sirtuin 3 is required for osteogenic differentiation through maintenance of PGC-1ɑ-SOD2-mediated regulation of mitochondrial function.

Osteogenic differentiation is crucial for the maintenance of bone homeostasis. Sirtuin 3 (SIRT3), a member of sirtuins family, functions as a critical deacetylase that regulates many key proteins. In the current study, we aimed to clarify the role of SIRT3 in osteogenic differentiation and the possible mechanisms, using mouse pre-osteoblastic MC3T3-E1 cells. Expression of SIRT3 was substantially increased in differentiated MC3T3-E1 cells. Knock down of SIRT3 significantly decreased alkaline phosphatase (ALP) staining, and mRNA expression of runt-related transcription factor 2 (Runx2) and collagen type I ɑ 1 (Col1ɑ1), and osteocalcin in differentiated MC3T3-E1 cells. Overexpression of wild type but not mutant SIRT3 could reverse SIRT3 knockdown-resulted decrease of ALP staining. Complex I, II, III, IV, and V activities, oxygen consumption and mitochondrial membrane potential were significantly decreased by SIRT3 knockdown. Moreover, SIRT3 knockdown reduced mitochondrial density, increased mitochondrial size and decreased the expression of NRF1 and TFAM. Knock down of SIRT3 decreased mRNA and protein expression of SOD2 and increased ROS level. Overexpression of SOD2 significantly suppressed SIRT3 knockdown-induced decrease of mitochondrial function and osteogenic differentiation. SIRT3 knockdown resulted in a significant decrease of PGC-1ɑ protein expression but not mRNA expression. Overexpression of wild type but not mutant SIRT3 could reverse SIRT3 knockdown-resulted decrease of PGC-1ɑ protein expression. Moreover, we detected a direct interaction between SIRT3 and PGC-1ɑ and SIRT3 knockdown reduced SIRT3 and PGC-1ɑ interaction, resulting in a reduction of PGC-1ɑ protein stability and PGC-1ɑ-binding in the promoters of SOD2. Overexpression of PGC-1ɑ blocked SIRT3 knockdown-induced decrease of SOD2 expression, increase of ROS level, and decrease of mitochondrial function and biogenesis, leading to improvement of osteogenesis. Overall, the data provide a better understanding of the role of SIRT3 in osteogenic differentiation.

MicroRNA-130b Promotes Wear Particle-Induced Osteolysis via Downregulating Frizzled-Related Protein (FRZB).

Periprosthetic osteolysis induced by wear particles can lead to aseptic loosening, one main reason of arthroplasty failure. However, the role of microRNA-130b (miR-130b) in particle-induced osteolysis (PIO) has not been explored yet. In this study, PIO models were established in C57BL/J6 mice via the implantation of Co-Cr-Mo alloy particles, and evaluated by detecting tartrate-resistant acid phosphatase (TRAP) activity and bone resorption in the calvaria. Mouse preosteoblast MC3T3-E1 cells were cultured to receive particle stimulation in vitro. Real time PCR and western blotting were performed to determine the expression levels of miR-130b and frizzled-related protein (FRZB), one potential target of miR-130b. Results showed upregulated miR-130b and downregulated FRZB in both PIO mice with remarkable osteolysis and particle-treated MC3T3-E1 cells showing inhibited proliferation and differentiation assayed by bromodeoxy urodine (BrdU) incorporation and alkaline phosphatase (ALP) activity respectively. Functional studies were conducted by transfection of miR-130b inhibitor in vitro or the injections of miR-130b inhibitor or small interfering RNA (siRNA) targeting FRZB in vivo. Interestingly, particle-induced inhibition on cell proliferation, differentiation and FRZB expression were all reversed by miR-130b silence. Luciferase report assays demonstrated that miR-130b indeed negatively regulated FRZB expression by targeting, while FRZB could reverse the opposed effect of miR-130b silence on PIO development. Therefore, the upregulated miR-130b in PIO models could act as one key regulator of PIO development, partly due to its negative regulation on FRZB.

Proving the Effectiveness of Homeopathic Remedy Using a Cell-based System

Background: Homeopathy is a medical system based on the principle of “like cures like”. It involves the use of natural substances that can cause a similar illness in a healthy person to stimulate the healing process. Homeopathic medicines use an ultra-diluted concentration of substances, which cannot be measured; therefore, their effectiveness is a matter of debate. Methods: To prove the effectiveness of homeopathic remedies, mRNA and protein expression of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines were evaluated using RT-PCR and immunoblotting in primary cultured mouse chondrocytes and pre-osteoblastic MC3T3-e1 cells stimulated with a homeopathic dilution of Rhus toxicodendron. Results: COX-2mRNA and protein were highly expressed in both chondrocytes and MC3T3-e1 cells stimulated with Rhus toxicodendron. Conclusion: Rhus toxicodendron has been used as a homeopathic medicine to ameliorate arthritic pain and to modulate inflammatory conditions. During the inflammatory process, COX-2 is upregulated, which subsequently increases prostaglandin E2 production. Homeopathic dilution of Rhus toxicodendron increased COX-2 mRNA and protein expression, as confirmed by RT-PCR and immunoblotting. This study provides a unique approach for establishing the effectiveness of homeopathy; however, further studies are needed to examine the exact cellular signaling mechanisms.

The preosteoblast response of electrospinning PLGA/PCL nanofibers: effects of biomimetic architecture and collagen I.

Constructing biomimetic structure and incorporating bioactive molecules is an effective strategy to achieve a more favorable cell response. To explore the effect of electrospinning (ES) nanofibrous architecture and collagen I (COL I)-incorporated modification on tuning osteoblast response, a resorbable membrane composed of poly(lactic-co-glycolic acid)/poly(caprolactone) (PLGA/PCL; 7:3 w/w) was developed via ES. COL I was blended into PLGA/PCL solution to prepare composite ES membrane. Notably, relatively better cell response was delivered by the bioactive ES-based membrane which was fabricated by modification of 3,4-dihydroxyphenylalanine and COL I. After investigation by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, contact angle measurement, and mechanical test, polyporous three-dimensional nanofibrous structure with low tensile force and the successful integration of COL I was obtained by the ES method. Compared with traditional PLGA/PCL membrane, the surface hydrophilicity of collagen-incorporated membranes was largely enhanced. The behavior of mouse preosteoblast MC3T3-E1 cell infiltration and proliferation on membranes was studied at 24 and 48 hours. The negative control was fabricated by solvent casting. Evaluation of cell adhesion and morphology demonstrated that all the ES membranes were more favorable for promoting the cell adhesion and spreading than the casting membrane. Cell Counting Kit-8 assays revealed that biomimetic architecture, surface topography, and bioactive properties of membranes were favorable for cell growth. Analysis of β1 integrin expression level by immunofluorescence indicated that such biomimetic architecture, especially COL I-grafted surface, plays a key role in cell adhesion and proliferation. The real-time polymerase chain reaction suggested that both surface topography and bioactive properties could facilitate the cell adhesion. The combined effect of biomimetic architecture with enhanced surface activity by 3,4-dihydroxyphenylalanine-assisted modification and COL I incorporation of PLGA/PCL electrospun membranes could successfully fill osteogenic defects and allow for better cell proliferation and differentiation.

Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ–induced activity and expression of adipocyte–specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1–PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of osteoporosis.

Effects of RGD immobilization on light-induced cell sheet detachment from TiO2 nanodots films

Light-induced cell detachment is reported to be a safe and effective cell sheet harvest method. In the present study, the effects of arginine–glycine–aspartic acid (RGD) immobilization on cell growth, cell sheet construction and cell harvest through light illumination are investigated. RGD was first immobilized on TiO2 nanodots films through simple physical adsorption, and then mouse pre-osteoblastic MC3T3-E1 cells were seeded on the films. It was found that RGD immobilization promoted cell adhesion and proliferation. It was also observed that cells cultured on RGD immobilized films showed relatively high level of pan-cadherin. Cells harvested with ultraviolet illumination (365nm) showed good viability on both RGD immobilized and unmodified TiO2 nanodot films. Single cell detachment assay showed that cells detached more quickly on RGD immobilized TiO2 nanodot films. That could be ascribed to the RGD release after UV365 illumination. The current study demonstrated that RGD immobilization could effectively improve both the cellular responses and light-induced cell harvest.

Silicon-Doped Titanium Dioxide Nanotubes Promoted Bone Formation on Titanium Implants.

While titanium (Ti) implants have been extensively used in orthopaedic and dental applications, the intrinsic bioinertness of untreated Ti surface usually results in insufficient osseointegration irrespective of the excellent biocompatibility and mechanical properties of it. In this study, we prepared surface modified Ti substrates in which silicon (Si) was doped into the titanium dioxide (TiO2) nanotubes on Ti surface using plasma immersion ion implantation (PIII) technology. Compared to TiO2 nanotubes and Ti alone, Si-doped TiO2 nanotubes significantly enhanced the expression of genes related to osteogenic differentiation, including Col-I, ALP, Runx2, OCN, and OPN, in mouse pre-osteoblastic MC3T3-E1 cells and deposition of mineral matrix. In vivo, the pull-out mechanical tests after two weeks of implantation in rat femur showed that Si-doped TiO2 nanotubes improved implant fixation strength by 18% and 54% compared to TiO2-NT and Ti implants, respectively. Together, findings from this study indicate that Si-doped TiO2 nanotubes promoted the osteogenic differentiation of osteoblastic cells and improved bone-Ti integration. Therefore, they may have considerable potential for the bioactive surface modification of Ti implants.

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells.

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.

A12 マウス前骨芽細胞様細胞MC3T3-E1 における低出力パルス超音波に応答す る遺伝子のネットワーク解析

A12 マウス前骨芽細胞様細胞MC3T3-E1 における低出力パルス超音波に応答す る遺伝子のネットワーク解析

Vinculin expression in MC3T3-E1 cells in response to mechanical stimulus.

Loading frequency is known to influence the expression of the focal adhesions of the adherent cells. A small cyclical tensile force was transmitted to mouse pre-osteoblast MC3T3-E1 cells through PDMS substrates of varying stiffness. Changes in cell behavior with respect to proliferation and characteristics of focal adhesions were quantified through immunofluorescence labeling of vinculin. Amount of inactive vinculin was higher on substrates subjected to cyclic stimulation when compared with the results of the static substrates, whereas the number and area of focal adhesion points underwent a reduction. Inactive vinculin appears as a cloud in the cytoplasm in the vicinity of the nucleus.

Homeopathic Rhus toxicodendron has dual effects on the inflammatory response in the mouse preosteoblastic cell line MC3T3-e1

Homeopathic remedy Rhus toxicodendron (Rhus tox) is used for several symptoms including skin irritations, rheumatic pains, mucous membrane afflictions, and typhoid type fever. Previously, we reported that Rhus tox treatment increased the cyclooxygenase-2 (COX-2) mRNA expression in primary cultured mouse chondrocytes. A preosteoblastic mouse cell line, MC3T3-e1, was treated with different homeopathic dilutions of Rhus tox and the COX-2 mRNA and protein expression was examined using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting. Additionally, nitric oxide (NO) generation was examined in LPS-induced MC3T3-e1 cells using a Griess reaction assay. Stimulation with different concentrations of Rhus tox increased the expression of Cox2 mRNA, with 30X Rhus tox showing the most prominent increase in mRNA expression. In addition, treatment with 30X Rhus tox significantly increased prostaglandin E2 (PGE2) release compared with other homeopathic dilutions. However, the COX-2 protein expression level differed slightly from its mRNA expression, because the 30C Rhus tox treatment increased COX-2 protein to a greater extent compared with other dilutions. NO generation was dramatically decreased in MC3T3-e1 cells after Rhus tox treatment co-stimulated with lipopolysaccharide. Homeopathic dilution of Rhus tox has a dual activity that increases COX-2 expression and decreases NO generation, thus modulating inflammation. Further study is needed to examine the cellular signaling mechanisms that are associated with inflammatory regulation by Rhus tox treatment in greater detail.

An axial distribution of seeding, proliferation, and osteogenic differentiation of MC3T3-E1 cells and rat bone marrow-derived mesenchymal stem cells across a 3D Thai silk fibroin/gelatin/hydroxyapatite scaffold in a perfusion bioreactor

In cell culture, a perfusion bioreactor provides effective transportation of nutrients, oxygen, and waste removal to and from the core of the scaffold. In addition, it provides mechanical stimuli for enhancing osteogenic differentiation. In this study, we used an axial distribution of cell numbers, alkaline phosphatase (ALP) enzyme activity, and calcium content across 4 cross-sections of 10mm thick scaffold, made of Thai silk fibroin (SF)/gelatin (G)/hydroxyapatite (HA), as a tool to evaluate the suitable perfusion flow rate. These evaluations cover all cellular developmental phases starting from seeding, to proliferation, and later osteogenic differentiation. Mouse pre-osteoblastic MC3T3-E1 cell lines were used as a cell model during seeding and proliferation. The bioreactor seeded scaffold provided more uniform cell distribution across the scaffold compared to centrifugal and agitation seeding, while the overall number of adhered cells from bioreactor seeding was slightly lower than agitation seeding. The dynamic culture using 1ml/min perfusion flow rate (initial shear stress of 0.1dyn/cm2) enabled statistically higher MC3T3-E1 proliferation, ALP activity, and calcium deposition than those observed in the static-culturing condition. However, the perfusion flow rate of 1ml/min seemed not to be enough for enhancing ALP expression across all sections of the scaffold. Rat bone marrow derived stromal cells (rMSC) were used in the detachment test and osteogenic differentiation. It was found that perfusion flow rate of 5ml/min caused statistically higher cell detachment than that of 1 and 3ml/min. The perfusion flow rate of 3ml/min gave the highest rMSC osteogenic differentiation on a SF/G/HA scaffold than other flow rates, as observed from the significantly highest number of ALP enzyme activity and the calcium content without any significant cell growth. In addition, all of these parameters were evenly distributed across all scaffold sections.