Murine Preosteoblast MC3T3-E1 Cells Research Articles

KCa3.1 regulates cell cycle progression by modulating Ca2+ signaling in murine preosteoblasts

Osteoblasts synthesize and deposit essential components of the extracellular bone matrix and collagen scaffolds, leading to mineralized bone formation. Therefore, the proliferation of preosteoblasts (precursors of mature osteoblasts) helps in regulating skeletal homeostasis. This study demonstrated that the functional expression of KCa3.1, an intermediate-conductance Ca2+-activated K+ channel, is markedly upregulated in murine preosteoblastic MC3T3-E1 cells in the G0/G1 phase. The enhancement of KCa3.1 is involved in the establishment of more negative membrane potentials in MC3T3-E1 cells. This hyperpolarization can promote intracellular Ca2+ signaling because store-operated Ca2+ channels are activated. Treatment with TRAM-34, a specific KCa3.1 inhibitor, attenuated the cell cycle progression from the G0/G1 phase to the S/G2/M phases. In MC3T3-E1 cells, KCa3.1 significantly promoted the transition from the G1 phase to the S phase. KCa3.1 inhibition also caused G0 phase cell accumulation. Furthermore, TRAM-34 decreased the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin, osteoblast differentiation markers in MC3T3-E1 cells, and inhibited the endochondral ossification of murine metatarsals. These results reveal novel ways by which KCa3.1 activity can strongly modulate osteoblast maturation during bone formation.

Piezoelectric PDMS/AlN Film for Osteogenesis in Vitro.

Bone defect and nonunion are complex diseases which are difficult to treat due to insufficient bone regeneration. Electrical stimulation has attracted attention as a promising strategy to induce and enhance bone regeneration. Self-powered and biocompatible materials have been widely explored and used in biomedical devices, owing to their ability to produce electrical stimulation without an external power source. We aimed to prepare a piezoelectric polydimethylsiloxane (PDMS)/aluminum nitride (AlN) film with excellent biocompatibility and osteoconductive ability for the growth of murine calvarial preosteoblast MC3T3-E1 cells. By applying vibration to stimulate body movement, the PDMS/AlN film demonstrated a current density of 2-6 μA cm-2, and the generated continuous alternating current (AC) effectively promoted MC3T3-E1 cell growth, viability, and osteoblastic related gene expression (genes runt-related transcription factor 2 [RUNX2], osteocalcin [OCN], alkaline phosphatase [ALP]) and exhibited higher mineralization. Compared to blank plates and nonvibrated PDMS/AlN films, the vibrated PDMS/AlN film showed rapid and superior osteogenic differentiation. The design of the biocompatible and flexible piezoelectric PDMS/AlN film overcame the poor processability, brittleness, and instability of electrical stimulation of traditional electroactive materials, demonstrating great potential in the application of electrical stimulation for bone tissue engineering.

8-prenylgenistein exerts osteogenic effects via ER α and Wnt-dependent signaling pathway

8-prenylgenistein (8PG) was previously reported to exert stronger osteogenic activity than genistein, a well-known soy phytoestrogen. However, the molecular mechanism underlying the actions of 8PG on osteoblasts was far from clear. In the present study, the osteogenic effects and mechanisms of 8PG and genistein were studied using human BMSC and murine pre-osteoblast MC3T3-E1 cells. Our results indicated that the stimulatory effects of 8PG and genistein on osteoblast differentiation were abolished by co-incubation with MPP (10−6 M, an ERα antagonist), but not PHTPP (10−6 M, an ERβ antagonist). Molecular docking indicated that the binding mode of 8PG toward ERα was similar to that of genistein and therefore could not account for their differential osteogenic actions. In silico target profiling identified the involvement of glycogen synthase kinase-3β (GSK-3β), a key mediator of Wnt/β-catenin pathway, in the actions of 8PG. However, instead of directly inhibiting GSK-3β enzymatic activities, 8PG and genistein were found to induce GSK-3β phosphorylation at Serine-9 in osteoblastic MC3T3-E1 cells. 8PG exerted more potent effects than genistein in stimulating expressions of LRP5, β-catenin, Runx2, osteocalcin, alp, opg, major protein and gene markers involved in Wnt signaling pathway in MC3T3-E1 cells. Moreover, the inhibition of Wnt signaling by DKK1 could be restored by treatment with 8PG and genistein. However, 8PG, but not genistein, stimulated ERα-dependent β-catenin protein expression in MC3T3-E1 cells. Furthermore, the increase in ALP activity, LRP5 and phospho-Akt/Akt expression by 8PG and genistein were abolished by co-treatment with LY294002 (10−5 M, a PI3K pathway inhibitor). Collectively, our results suggested that the osteogenic activities of 8PG was mediated by GSK-3β phosphorylation through the induction of Wnt/β-catenin and ERα-associated PI3K/Akt signaling.

Enhancing Effect of Vitexin on Osteogenic Activity of Murine Pre-Osteoblastic Mc3t3-E1 Cells

Vitexin (5,7,4-trihydroxyflavone-8-glucoside), a natural flavone present in a variety of plants, is well known for its rich pharmacological properties. However, its osteogenic activity remains unclear to date. The purpose of this study was to explore the effects of vitexin on osteogenic activity in murine pre-osteoblastic MC3T3-E1 cells using the MTT assay for cell proliferation, alkaline phosphatase (ALP) activity assay for cell differentiation, and Von Kossa staining for cell mineralization. Quantitative real-time PCR was used for the detection of osteocalcin (OCN) mRNA expression in cells. Furthermore, effects of vitexin on the differentiation and matrix mineralization of dexamethasone (DEX)- suppressed cells was also investigated. The results showed vitexin could significantly enhance cell proliferation in a low concentration range of 10-10-10-6 μg mL-1. ALP activity was significantly increased after the cells were treated with vitexin at 10-8 and 10-6 μg mL-1. The expression levels of the osteogenic OCN gene in cells treated with vitexin at 10-6, 10- 8, and 10-10 μg mL-1 were improved by 3.1-fold, 5.8-fold, and 4.2-fold over the control, respectively. Additionally, vitexin (10-8 μg mL-1) significantly alleviated the inhibitory effect of osteoblast differentiation and mineralization induced by DEX. Collectively, our findings suggest vitexin could enhance cell proliferation and osteogenic differentiation of MC3T3-E1 cells, as well as rescue the inhibitory effect of cell differentiation and matrix mineralization induced by DEX. Therefore, vitexin may be useful as a promising therapeutic agent for bone disease and plays an important role in the prevention of glucocorticoid-induced osteoporosis.

Bioactive Compounds from Abelmoschus manihot L. Alleviate the Progression of Multiple Myeloma in Mouse Model and Improve Bone Marrow Microenvironment.

PurposeAbelmoschus manihot (L.) Medik. (Malvaceae) derived Huangkui capsules (HKC) represent a traditional Chinese medicine that has been widely applied to the clinical therapy of kidney and inflammatory diseases. The present study aimed to determine the potential therapeutic effects and underlying mechanisms of the ingredients on Multiple Myeloma (MM), an incurable disease that exhibits malignant plasma cell clonal expansion in the bone marrow.MethodsA 5TMM3VT syngeneic MM-prone model was established and treated with HKC. Murine pre-osteoblast MC3T3-E1 and pre-osteoclast Raw264.7 cells were treated with nine flavonoid compounds extracted from the flowers of Abelmoschus manihot. MC3T3-E1 and Raw264.7 cells were then examined by alizarin red staining and tartrate-resistant acid phosphatase activity staining, respectively. The proliferation of two human MM cells (ARP1, H929) was examined by performing an MTT assay following treatment with flavonoid compounds. Additionally, the cell cycle was analyzed via staining and flow cytometry. The differential expressions of certain proteins were detected via Western blotting, transcriptomic RNA-sequencing as well as RT-qPCR.ResultsThe results revealed that MM-prone animals appeared to be protected following HKC treatment, as evidenced by a prolonged survival rate. Furthermore, four of the nine flavonoid compounds [Hyperin/Hyperoside, HK-2; Cannabiscitrin, HK-3; 3-O-kaempferol-3-O-acetyl-6-O-(p-coumaroyl)-β-D-glucopyranoside, HK-11; 8-(2’’-pyrrolidione-5’’-yl)-quercetin, HK-B10] induced the differentiation of murine pre-osteoblast MC3T3-E1 cells. In addition, two compounds [Isomyricitrin, HK-8; quercetin-8-(2’’-pyrrolidione-5”-yl)-3ʹ-O-β-D-glucopyranosid, HK-E3] suppressed osteoclastogenesis in murine Raw264.7 cells. HK-11 directly inhibited MM cells (ARP1 and H929) proliferation and induced G0/G1 cell cycle arrest, which may have involved the suppressing β-catenin protein, increasing expressions of IL-6 and TNF-α, as well as activating mature TGF-β1 and some other metabolic pathways.ConclusionThese results of the present study indicated that the bio-active ingredients of HKC exerted protective effects on MM mouse survival through promoting osteoblastogenesis and suppressing osteoclastogenesis, thus improving the bone marrow microenvironment to inhibit MM cell proliferation.

Phlorofucofuroeckol A from Edible Brown Alga Ecklonia Cava Enhances Osteoblastogenesis in Bone Marrow-Derived Human Mesenchymal Stem Cells.

The deterioration of bone formation is a leading cause of age-related bone disorders. Lack of bone formation is induced by decreased osteoblastogenesis. In this study, osteoblastogenesis promoting effects of algal phlorotannin, phlorofucofuroeckol A (PFF-A), were evaluated. PFF-A was isolated from brown alga Ecklonia cava. The ability of PFF-A to enhance osteoblast differentiation was observed in murine pre-osteoblast cell line MC3T3-E1 and human bone marrow-derived mesenchymal stem cells (huBM-MSCs). Proliferation and alkaline phosphatase (ALP) activity of osteoblasts during differentiation was assayed following PFF-A treatment along extracellular mineralization. In addition, effect of PFF-A on osteoblast maturation pathways such as Runx2 and Smads was analyzed. Treatment of PFF-A was able to enhance the proliferation of differentiating osteoblasts. Also, ALP activity was observed to be increased. Osteoblasts showed increased extracellular mineralization, observed by Alizarin Red staining, following PFF-A treatment. In addition, expression levels of critical proteins in osteoblastogenesis such as ALP, bone morphogenetic protein-2 (BMP-2), osteocalcin and β-catenin were stimulated after the introduction of PFF-A. In conclusion, PFF-A was suggested to be a potential natural product with osteoblastogenesis enhancing effects which can be utilized against bone-remodeling imbalances and osteoporosis-related complications.

Sustained release of strontium (Sr2+) from polycaprolactone/poly (D,L-lactide-co-glycolide)-polyvinyl alcohol coaxial nanofibers enhances osteoblastic differentiation.

Electrospun nanofibers mimic the bone’s extra-cellular matrix and represent an alternative implant coating to enhance implant osseointegration. Approximately 99% of the strontium (Sr2+) in our body deposits in the bone and serves as a stimulator of bone formation. The purpose of this study was to develop an Sr2+-doped coaxial polycaprolactone (PCL)/poly(D,L-lactide-co-glycolide) (PLGA)-polyvinyl alcohol (PVA) nanofiber coating to enhance the osseointegration. An optimized PCL/PLGA (1:1 v/v) sheath fiber formula was identified to match the nanofibers degradation to the implant osseointegration physiology. Scanning electron microscopy images showed that the Sr2+-doped nanofibers had a less dense structure and larger porosity than the nanofibers without Sr2+ doping. A sustained Sr2+ release from the PCL/PLGA-PVA coaxial nanofibers was observed for over two months. The indirect and direct cellular contact studies confirmed the biocompatibility of the Sr2+-doped PCL/PLGA-PVA coaxial nanofibers. The Sr2+-doped nanofibers provided further and significant enhancement of the differentiation of murine pre-osteoblast MC3T3-E1 cells. These findings indicate that Sr2+-doped PCL/PLGA-PVA coaxial nanofibers have a great potential to be applied as a new implant coating matrices.

Application of high resolution DLP stereolithography for fabrication of tricalcium phosphate scaffolds for bone regeneration

Bone regeneration requires porous and mechanically stable scaffolds to support tissue integration and angiogenesis, which is essential for bone tissue regeneration. With the advent of additive manufacturing processes, production of complex porous architectures has become feasible. However, a balance has to be sorted between the porous architecture and mechanical stability, which facilitates bone regeneration for load bearing applications. The current study evaluates the use of high resolution digital light processing (DLP) -based additive manufacturing to produce complex but mechanical stable scaffolds based on β-tricalcium phosphate (β-TCP) for bone regeneration. Four different geometries: a rectilinear Grid, a hexagonal Kagome, a Schwarz primitive, and a hollow Schwarz architecture are designed with 400 μm pores and 75 or 50 vol% porosity. However, after initial screening for design stability and mechanical properties, only the rectilinear Grid structure, and the hexagonal Kagome structure are found to be reproducible and showed higher mechanical properties. Micro computed tomography (μ-CT) analysis shows <2 vol% error in porosity and <6% relative deviation of average pore sizes for the Grid structures. At 50 vol% porosity, this architecture also has the highest compressive strength of 44.7 MPa (Weibull modulus is 5.28), while bulk specimens reach 235 ± 37 MPa. To evaluate suitability of 3D scaffolds produced by DLP methods for bone regeneration, scaffolds were cultured with murine preosteoblastic MC3T3-E1 cells. Short term study showed cell growth over 14 d, with more than two-fold increase of alkaline phosphatase (ALP) activity compared to cells on 2D tissue culture plastic. Collagen deposition was increased by a factor of 1.5–2 when compared to the 2D controls. This confirms retention of biocompatible and osteo-inductive properties of β-TCP following the DLP process. This study has implications for designing of the high resolution porous scaffolds for bone regenerative applications and contributes to understanding of DLP based additive manufacturing process for medical applications.

Microstructure, mechanical properties, biocompatibility, and in vitro corrosion and degradation behavior of a new Zn–5Ge alloy for biodegradable implant materials

Zinc (Zn)-based alloys are considered a new class of biodegradable implant materials due to their superior chemical stability and processability compared to biodegradable magnesium (Mg) alloys. In this study, we report a new biodegradable Zn–5Ge alloy with highly desirable mechanical, corrosion, and biological properties. Microstructural characterization revealed the effective grain-refining effect of germanium (Ge) on the Zn alloy. Tensile test results indicated that the hot-rolled Zn–5Ge alloy showed an ultimate tensile strength of 237.0 MPa, a yield strength of 175.1 MPa, and an elongation of 21.6%; while as-cast pure Zn showed an ultimate tensile strength of 33.6 MPa, a yield strength of 29.3 MPa, and an elongation of 1.2%. The corrosion rates measured by potentiodynamic polarization tests in Hank’s solution in ascending order are: as-cast Zn–5Ge (0.1272 mm/y) < as-cast pure Zn (0.1567 mm/y) < hot-rolled Zn–5Ge (0.2255 mm/y) < hot-rolled pure Zn (0.3057 mm/y). Immersion tests revealed that the degradation rate of as-cast Zn–5Ge is 0.042 mm/y, less than half of that of hot-rolled pure Zn and ∼62% of that of as-cast pure Zn. Moreover, the Zn–5Ge alloy showed excellent in vitro hemocompatibility and the addition of 5% Ge effectively enhanced the hemocompatibility of pure Zn. CCK-8 assay using murine preosteoblast MC3T3-E1 cells indicated that the diluted extracts at a concentration <12.5% of both the as-cast Zn–5Ge alloy and pure Zn showed grade 0 cytotoxicity; the diluted extracts at the concentrations of 50% and 25% of Zn-5Ge alloy showed a significantly higher cell viability than those of pure Zn. Statement of SignificanceZinc (Zn)-based alloys are currently considered a new class of biodegradable implant materials due to their excellent processability. Here, we report a novel Zn–5Ge alloy with highly desirable mechanical, corrosion and biological properties. The tensile test results indicated that the hot-rolled Zn–5Ge alloy showed an ultimate tensile strength of 237.0 MPa, a yield strength of 175.1 MPa and an elongation of 21.6%; while as-cast pure Zn showed an ultimate tensile strength of 33.6 MPa, a yield strength of 29.3 MPa and an elongation of 1.2%. The corrosion rate measured by potentiodynamic polarization tests in Hank’s solution in the ascending order is: as-cast Zn–5Ge (0.1272 mm/y) < as-cast pure Zn (0.1567 mm/y) < hot-rolled Zn–5Ge (0.2255 mm/y) < hot-rolled pure Zn (0.3057 mm/y). Immersion tests revealed that the degradation rate of the as-cast Zn–5Ge is 0.042 mm/y, less than half of that of the hot-rolled pure Zn, ∼62% of that of as-cast pure Zn. Moreover, the Zn-5Ge alloy showed excellent in vitro biocompatibility.

Tetrahydroxystilbene Glucoside Regulates Proliferation, Differentiation, and OPG/RANKL/M-CSF Expression in MC3T3-E1 Cells via the PI3K/Akt Pathway.

Tetrahydroxystilbene glucoside (TSG) is a unique component of the bone-reinforcing herb Radix Polygoni Multiflori Preparata (RPMP). It has the ability to promote bone formation and protect osteoblasts. However, the underlying mechanism remains unclear. To better understand its biological function, we determined TSG’s effect on murine pre-osteoblastic MC3T3-E1 cells by the MTT assay, flow cytometry, FQ-PCR, Western blot, and ELISA. The results showed that TSG caused an elevation of the MC3T3-E1 cell number, the number of cells in the S phase, and the mRNA levels of the runt-related transcription factor-2 (Runx2), osterix (Osx), and collagen type I α1 (Col1a1). In addition, the osteoprotegerin (OPG) mRNA level was up-regulated, while the nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) mRNA levels were down-regulated by TSG. Furthermore, TSG activated the phosphoinosmde-3-kinase/protein kinase B (also known as PI3K/Akt) pathway, and blocking this pathway by the inhibitor LY-294002 could impair TSG’s functions in relation to the MC3T3-E1 cells. In conclusion, TSG could activate the PI3K/Akt pathway and thus promote MC3T3-E1 cell proliferation and differentiation, and influence OPG/RANKL/M-CSF expression. TSG merits further investigation as a potential therapeutic agent for osteoporosis treatment.

Estradiol-Loaded Poly(ε-caprolactone)/Silk Fibroin Electrospun Microfibers Decrease Osteoclast Activity and Retain Osteoblast Function

Estrogen, a steroid hormone, plays an important role in modulating osteoclast proliferation and development. Estrogen deficiency boosts osteoclast activity, leading to osteoporosis in elderly women. In this study, 17-ß estradiol (E2)-loaded poly(ε-caprolactone) (PCL)/silk fibroin (SF) electrospun microfibers were developed as a proposed localized E2 delivery system to treat osteoporotic fractures. PCL is a synthetic polymer known for its biocompatibility and excellent mechanical properties. The bioactivity of PCL was enhanced by mixing it with a natural SF polymer that has low immunogenicity and inherent bioactivity. Different ratios of PCL/SF blends were electrospun and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and water contact angle measurement. PCL and SF at a ratio of 50:50 (PCL50/SF50) augmented cell proliferation of murine preosteoblast MC3T3-E1 cells and murine preosteoclast RAW 264.7 cells. Hence, PCL50/SF50 was selected and mixed with three concentrations of E2 to produce electrospun fiber mesh (0.1% E2@PCL/SF, 1% E2@PCL/SF, and 5% E2@PCL/SF). Sustained release of E2 was obtained for about 3 weeks at higher E2 concentration 5% E2@PCL/SF. An E2-loaded PCL50/SF50 elecrospun microfiber (1% E2@PCL/SF and 5% E2@PCL/SF) reduced tartrate-resistant acid phosphate activity, total DNA, and multinucleated cell formation of osteoclasts. On the other hand, the alkaline phosphatase activity and collagen I expression of osteoblasts were retained on all E2-loaded electrospun microfibers. The E2@PCL/SF system shows potential to be used for localized E2 delivery for the treatment of osteoporotic fractures.

Anti-osteoporosis activity of Sanguinarine in preosteoblast MC3T3-E1 cells and an ovariectomized rat model.

Sanguinarine, a benzophenanthridine alkaloid, has been previously demonstrated to exert antimicrobial, anti-inflammatory, and anti-tumor activities. A previous study has identified Sanguinarine as a potential drug candidate for osteoporosis treatment by computational bioinformatics analysis. This study further evaluated the effects of Sanguinarine on the differentiation of murine preosteoblast MC3T3-E1 cells and its anti-osteoporosis activity in an ovarietomized rat model. Sanguinarine treatment (0.25, 0.5, 1, and 2 µm) of MC3T3-E1 cells significantly increased alkaline phosphatase (ALP) activity and the phoshporalyation of AMP-activated protein kinase α subunit (AMPKα), but did not affect cell proliferation. The induction effects of Sanguinarine treatment (2 µm) on ALP activity, AMPKα phosphorylation, Smad1 phosphorylation, and the expression of three osteoblast differentiation-regulators (bone morphogenetic protein 2 [BMP2], osterix [OSX], and osteoprotegerin [OPG]) were partially reversed by Compound C treatment. More importantly, Sanguinarine treatment promoted bone tissue growth in an ovariectomized (OVX) osteoporosis rat model as evaluated by histological examination, micro-CT analysis, and serum parameter detection. In conclusion, these results indicate that Sanguinarine induces the differentiation of MC3T3-E1 cells through the activation of the AMPK/Smad1 signaling pathway. Sanguinarine can stimulate bone growth in vivo and may be an effective drug for osteoporosis treatment.

Silk fibroin-derived peptide directed silver nanoclusters for cell imaging.

Fluorescent silver nanoclusters (Ag NCs) that are capable of emitting green light have been synthesized using a peptide derived from the C terminal of silk fibroin heavy chain (CSH) via a one-pot, green, and facile synthesis method. The emission was also found to be stable at the excitation wavelength and the fluorescence quantum yield of Ag NCs was measured to be 1.1%. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) indicated the presence of a range of Ag species that correspond to Ag1, Ag2, Ag3 and Ag4. Transmission electron microscopic analyses suggested that the formed particles are uniform and well dispersive with an average diameter of 2.5 nm. The Ag NCs were successfully applied to cell imaging in murine preosteoblast MC3T3-E1 cells. Finally, Ag NCs observed by MTT exhibited distinct cytotoxicity at CSH–Ag NCs concentrations of 600 μM. Based on the concept of utilizing a functional peptide from nature, this study demonstrates a novel approach to fabricate aqueous metal nanoclusters for tracking applications in bioimaging.

Alkaline phosphatase levels of murine pre-osteoblastic cells on anodized and annealed titanium surfaces.

PurposeThis study aimed to evaluate the initial adhesion morphology and alkaline phosphatase (ALP) activity of murine pre-osteoblastic MC3T3-E1 cells cultured on anatase/rutile mixed-phase TiO2 thin films with photocatalytical activity with previously confirmed antibacterial properties.Materials and methodsAnatase/rutile mixed-phase TiO2 thin films fabricated by anodization and annealing of cpTi were used to culture MC3T3-E1 cells to evaluate the initial cellular adhesion morphology and ALP activity in vitro.ResultsCompared with MC3T3-E1 cells cultured on cpTi substrates and the control group, cells cultured on anatase/rutile mixed-phase TiO2 thin films exhibited similar ALP levels after cell culture day 9.ConclusionAnodizing and annealing processes fabricate multifunctional surfaces on cpTi with improved osteogenic properties for implants.

Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

Pinostrobin (PI), a natural flavonoid found in a variety of plants, is well known for its rich pharmacological activities. However, its osteogenic function remains unclear. The aim of this study is to evaluate the effect of PI on the proliferation, differentiation, and mineralization of murine pre-osteoblastic MC3T3-E1 cells in vitro using MTT, alkaline phosphatase (ALP) activity, the synthesis of collagen I (Col I) assay, and Von-Kossa staining, respectively. The expression of osteocalcin (OCN) mRNA in cells was detected by real-time PCR. The effect of PI on the differentiation of dexamethasone (DEX)-suppressed cells was also investigated. The results showed that PI greatly promoted the proliferation of MC3T3-E1 cells at 5–80 μg/mL (p < 0.05 or p < 0.01), and caused a significant elevation of ALP activity, Col I content, and mineralization of osteoblasts at 10–40 μg/mL (p < 0.05 or p < 0.01), and the expression levels of OCN gene were greatly upregulated after PI treatment (p < 0.01). Furthermore, PI could rescue the inhibition effect of cell differentiation induced by DEX. Taken together, these results indicated that PI could directly promote proliferation, differentiation, and mineralization of MC3T3-E1 cells and has potential for use as a natural treatment for osteoporosis.

ALP LEVELS OF MURINE PRE-OSTEOBLASTIC CELLS ON ANODIZED AND ANNEALED TITANIUM SURFACES

Purpose: The aim of this study was to evaluate the initial adhesion morphology and alkaline phosphatase activity (ALP) of murine pre-osteoblastic MC3T3-E1 cells on anatase/rutile mixed-phase TiO 2 thin films with photocatalytical activity that previously confirmed antibacterial properties. Materials and Methods: Anatase/rutile mixed-phaseTiO 2 thin films fabricated by anodization and annealing of cpTi were cultured by MC3T3-E1 to evaluate the initial cellular adhesion morphology and ALP activity in vitro. Results: Compared with cpTi substrates and the control group, MC3T3-E1 cells cultured on anodized and annealed titanium substrates exhibited similar ALP levels after cell culture day 9. Conclusion: Anodizing and annealing processes fabricate multifunctional surfaces on cpTi with improved osteogenic properties for implants.

AKT2 is involved in the IL‑17A‑mediated promotion of differentiation and calcification of murine preosteoblastic MC3T3‑E1 cells.

Interleukin (IL)-17A exhibits pleiotropic biological activities and serves a role in the progression of periodontitis. However, data describing the association between IL-17 and osteogenesis are not conclusive. It was previously demonstrated that RAC-β serine/threonine protein kinase (AKT2)-specific knockdown in MC3T3-E1 cells weakened osteogenic effects. The role of AKT2 in the regulation of IL-17A for osteoblast differentiation and calcification remains unclear. The MTT method was adopted in the present study to assess cell proliferation; cell cycle distribution was analyzed by flow cytometry. Following osteogenic induction treatment, the involvement of phosphatidylinositol 3-kinase (PI3K) and phosphorylated-PI3K was evaluated by western blotting. The effects of IL-17A on osteogenesis-associated markers, including Runt-related transcription factor 2 (Runx-2), alkaline phosphatase (ALP) and osteocalcin (OCN) were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. An ALP activity assay and Alizarin Red S staining were used to assess the differentiation and calcification functions. AKT2 knockdown inhibited MC3T3-E1 cell proliferation, inducing significantly increased G0/G1 cell counts, and reduced S and G2/M cell numbers. IL-17A exerted no significant effects. The protein levels of p-PI3K, gene expression levels of IL-17A, Runx-2, ALP and OCN, and relative ALP activity and calcification areas were increased in the induction group, and these effects were markedly promoted by treatment with IL-17A. AKT2 knockdown in MC3T3-E1 cells resulted in reduced IL-17A-induced differentiation and calcification, although it was not completely inhibited. The results of the present study suggested that AKT2 signaling was required for MC3T3-E1 cell proliferation. IL-17A promoted osteoblast differentiation and calcification in a partly AKT2-dependent manner in MC3T3-E1 cells in vitro, possibly reflecting compensation by other signaling pathways. The results of the present study may offer novel perspectives to guide the clinical strategy for the prevention and treatment of periodontitis.

In Vivo Immune Responses of Cross-Linked Electrospun Tilapia Collagen Membrane

Collagen has been used extensively in tissue engineering applications. However, the source of collagen has been primarily bovine and porcine. In view of the potential risk of zoonotic diseases and religious constraints associated with bovine and porcine collagen, fish collagen was examined as an alternative. The aim of this study is to use tilapia fish collagen to develop a cross-linked electrospun membrane to be used as a barrier membrane in guided bone regeneration. As there is limited data available on the cytotoxicity and immunogenicity of cross-linked tilapia collagen, in vitro and in vivo tests were performed to evaluate this in comparison to the commercially available Bio-Gide® membrane. In this study, collagen was extracted and purified from tilapia skin and electrospun into a nanofibrous membrane. The resultant membrane was cross-linked to obtain a cross-linked electrospun tilapia collagen (CETC) membrane, which was characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), degradation studies, cytotoxicity studies, and cell proliferation studies. The membranes were also implanted subcutaneously in rats and the host immune responses were examined. The DSC data showed that cross-linking increased the denaturation temperature of tilapia collagen to 58.3°C ± 1.4°C. The in vitro tests showed that CETC exhibited no cytotoxicity toward murine fibroblast L929 cells, and culture of murine preosteoblast MC3T3-E1 cells demonstrated better proliferation on CETC as compared to Bio-Gide. When implanted in rats, CETC caused a higher production of interleukin IL-6 at early time points as compared to Bio-Gide, but there was no long-term inflammatory responses after the acute inflammation phase. This finding was supported with histology data, which clearly illustrated that CETC has a decreased inflammatory response comparable to the benchmark control group. In all, this study demonstrated the viability for the use of CETC as a tissue engineering scaffold and provides an insight on the in vivo immune responses toward xenogenic collagen scaffolds.

Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells.

Akt activation is required for TGF-β1-induced osteoblast differentiation of MC3T3-E1 pre-osteoblasts.

BackgroundWe have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.Methodology/Principal FindingsMC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.ConclusionsTGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.