Osteoblastic MC3T3-E1 Cells Research Articles

Novel selenium and/or copper substituted hydroxyapatite-gelatin-chitosan-eggshell membrane nanocomposite scaffolds for bone tissue engineering applications.

Limitations with the majority of bone therapeutic treatments include low availability, ethical constraints and low biological compatibility. Although a number of choice materials have been exploited successfully, there has always been scope for improvement as well as development of the next-generation of materials. Herein, scaffolds - developed from gelatin, chitosan and eggshell membranes - were crosslinked using tannic acid, and further infused with selenium and/or copper substituted hydroxyapatite nanoparticles to generate a novel nanocomposite substrate. FESEM images of the nanocomposite scaffolds revealed the presence of interconnected pores, mostly spread over the whole surface of the scaffold, alongside XRD and FTIR profiling that detailed the formation of hydroxyapatite as a sole phase. Moreover, physical characterisation of the nanocomposite confirmed that the hydroxyapatite particulates and the eggshell membrane fibres were uniformly distributed and contributed to the surface roughness of the scaffold. Biocompatibility and cytotoxicity of the novel constructs were assessed using the mouse-derived osteoblastic cell line, MC3T3-E1, and standard cell culture assays. Metabolic activity assessment (i.e. MTS assay), LDH-release profiles and Live/Dead staining demonstrated good cell adhesion, viability, and proliferation rates. Accordingly, this work summarises the successful development of a novel construct which may be exploited as a clinical/therapeutic treatment for bone repair as well as a possible translational application as a novel biomaterial for the drug development pipeline.

Probing the interaction between metastatic breast cancer cells and osteoblasts in a thread-based breast-bone co-culture device.

Breast cancer metastasis to bone is a leading killer in breast cancer patients. A type I collagen-modified thread (Col-I@thread) was prepared for 3-dimensional cell culture and breast cancer bone metastasis co-culture device assembly. First, the coating of Col-I on nylon threads for promoting cell adhesion and growth was studied. Through SEM, XPS, and protein concentration measurements, it was found that the lyophilization method remarkably preserved the Col-I activity and the internal structure of the thread, thereby promoting cell attachment and proliferation. RNA-sequencing (RNA-Seq) and quantitative PCR analysis showed that osteoblast cells (MC3T3-E1) grown on Col-I@thread had elevated RUNX2, ALP, OPN, and Col-I gene expression to promote osteoblast differentiation. Single-cell analysis found that osteoblast MC3T3-E1 cells growing on Col-I@thread had higher Ca2+ secretion activity and mineralized nodules, suggesting robust cell activity and bone matrix formation than cells growing on 2D culture plates. Col-I@threads were knotted in an interdigital cross-finger frame to assemble the breast cancer-bone co-culture model. Confocal microscopy and flow cytometry tests quantified the invasive breast cancer cells. Moreover, the thread-based co-culture devices allowed us to isolate the invasive and non-invasive breast cancer cells to compare their molecular characteristics. qPCR results showed that expression of CX43, CXCR5, and CSPG4 genes was significantly increased in breast cancer cells with bone metastasis. Meanwhile, the expression of RUNX2 and OPG genes in osteoblasts was inhibited. The co-culture model based on the Col-I@thread mimics the bone tissue microenvironment to reveal the cross-talk between cancer cells and bone tissue. Moreover, the thread-based co-culture device is easy to fabricate and operate, providing a platform for exploring the cellular and molecular mechanisms of breast cancer bone metastasis, and holds potential for high-throughput screening of anti-breast cancer bone metastasis drugs.

CypD-mediated mitochondrial dysfunction contributes to titanium ion-induced MC3T3-E1 cell injury

Titanium (Ti) ion can stimulate osteoblast apoptosis and therefore have a high potential to play a negative role in the aseptic loosening of implants. Mitochondrial abnormalities are closely related to osteoblast dysfunction. However, the mitochondrial molecular mechanism of Ti ion induced osteoblastic cell apoptosis is still unclear. This study investigated in vitro mitochondrial oxidative stress (mtROS) mediated mitochondrial dysfunction involved in Ti ion-induced apoptosis of murine MC3T3-E1 osteoblastic cells. In addition to reducing mitochondrial membrane potential (MMP) and decreasing adenosine triglyceride production, exposure to Ti ions increased mitochondrial oxidative stress. Moreover, mitochondrial abnormalities significantly contributed to Ti ion induction of osteoblastic cellular apoptosis. A mitochondria-specific antioxidant, mitoquinone (MitoQ), alleviated Ti ion-induced mitochondrial dysfunction and apoptosis in osteoblastic cells, indicating that Ti ion mainly induces mitochondrial oxidative stress to produce a cytotoxic effect on osteoblasts. Here we show that the primary regulator of mitochondrial permeability transition pore (mPTP), cyclophilin D (CypD), is involved in mitochondrial dysfunction and osteoblast cell apoptosis induced by Ti ion. Overexpression of CypD exacerbates osteoblast apoptosis and impairs osteogenic function. Moreover, detrimental effects of CypD were rescued by cyclosporin A (CsA), an inhibitor of CypD, which shows its protective effect on mitochondrial and osteogenic osteoblast functions. Based on new insights into the mitochondrial mechanisms underlying Ti ion-induced apoptosis of osteoblastic cells, the findings of this study lay the foundation for the clinical use of CypD inhibitors to prevent or treat implant failure.

The Effect of Apple-Derived Nanovesicles on the Osteoblastogenesis of Osteoblastic MC3T3-E1 Cells.

Osteoporosis is characterized by low bone mass and elevated structural deterioration of the bone tissue, resulting in bone weakness with an increased risk of fracture. Considering biological activities of various phytochemicals extracted from apples, we herein demonstrated the potential antiosteoporotic effects of apple-derived nanovesicles (apple NVs) using osteoblastic MC3T3-E1 cells. Apple NVs significantly stimulated the growth of MC3T3-E1 cells. The cellular alkaline phosphatase (ALP) activity was significantly upregulated in the 5 μg/mL apple NVs-treated group. In addition, the concentrarion of mineralized nodules was significantly increased in the apple NVs-treated groups. Furthermore, apple NVs increased the expression of the genes and proteins associated with osteoblast growth and differentiation, such as Runx2, ALP, OPN, and BMP2/4, which further activated ERK- and JNK-related mitogen-activated protein kinase signaling. These results demonstrate that apple NVs have a potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells through regulating the BMP2/Smad1 pathways.

Preparation, Characterization, and Drug Delivery of Hexagonal Boron Nitride-Borate Bioactive Glass Biomimetic Scaffolds for Bone Tissue Engineering

In this study, biomimetic borate-based bioactive glass scaffolds containing hexagonal boron nitride hBN nanoparticles (0.1, 0.2, 0.5, 1, and 2% by weight) were manufactured with the polymer foam replication technique to be used in hard tissue engineering and drug delivery applications. To create three-dimensional cylindrical-shaped scaffolds, polyurethane foams were used as templates and covered using a suspension of glass and hBN powder mixture. Then, a heat treatment was applied at 570 °C in an air atmosphere to remove the polymer foam from the structure and to sinter the glass structures. The structural, morphological, and mechanical properties of the fabricated composites were examined in detail. The in vitro bioactivity of the prepared composites was tested in simulated body fluid, and the release behavior of gentamicin sulfate and 5-fluorouracil from glass scaffolds were analyzed separately as a function of time. The cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. The findings indicated that the hBN nanoparticles, up to a certain concentration in the glass matrix, improved the mechanical strength of the glass scaffolds, which mimic the cancellous bone. Additionally, the inclusion of hBN nanoparticles enhanced the in vitro hydroxyapatite-forming ability of bioactive glass composites. The presence of hBN nanoparticles accelerated the drug release rates of the system. It was concluded that bioactive glass/hBN composite scaffolds mimicking native bone tissue could be used for bone tissue repair and regeneration applications.

Depletion of Zinc Causes Osteoblast Apoptosis with Elevation of Leptin Secretion and Phosphorylation of JAK2/STAT3.

Zinc (Zn) has been reported to mediate leptin secretion, and thus leptin can be an important candidate molecule linking Zn with bone formation. The present study investigated whether zinc deficiency induces leptin secretion by activating a JAK2/STAT3 signaling pathway and leads to osteoblastic apoptosis. MC3T3-E1 cells were incubated for 24 h in normal osteogenic differentiation medium (OSM) or OSM treated with either 1 μM (Low Zn) or 15 μM (High Zn) of ZnCl2 containing 5 μM TPEN (Zn chelator). Our results demonstrated that low Zn stimulated extracellular leptin secretion and increased mRNA and protein expression of leptin in osteoblastic MC3T3-E1 cells. The OB-Rb (long isoform of leptin receptor) expressions were also elevated in osteoblasts under depletion of Zn. Leptin-signaling proteins, JAK2 and p-JAK2 in the cytosol of low Zn osteoblast conveyed leptin signaling, which ultimately induced higher p-STAT3 expression in the nucleus. Apoptotic effects of JAK2/STAT3 pathway were shown by increased caspase-3 in low Zn osteoblasts as well as apoptotic morphological features observed by TEM. Together, these data suggest that low Zn modulates leptin secretion by activating JAK2/STAT3 signaling pathway and induces apoptosis of osteoblastic MC3T3-E1 cells.

Nobiletin, a NF-κB signaling antagonist, promotes BMP-induced bone formation.

The NF-κB family of transcription factors plays an important role in skeletal development and bone homeostasis. In osteoblast cells, NF-κB signaling has been shown to suppress survival, proliferation, and differentiation. Furthermore, pharmacological suppression of NF-κB enhances osteoblast differentiation and bone formation. Thus, NF-κB antagonists are promising candidates as anabolic agents for enhancing bone mass. In this study, we describe the mechanism by which nobiletin, an inhibitor of NF-κB activity, regulates osteoblast differentiation and mineralization. We found that in MC3T3-E1 osteoblast cells, nobiletin inhibited a TNF-α responsive NF-κB luciferase reporter and also decreased the induction of classical NF-κB target genes by TNF-α. Consistent with this, nobiletin prevented TNF-α -mediated suppression of osteogenesis and potently enhanced the differentiation and mineralization of MC3T3-E1 cells. Likewise, in an in vivo BMP2-induced ectopic bone formation assay, nobiletin markedly enhanced ossicle bone volume. Western blotting and SMAD-responsive luciferase assays also demonstrated that NF-κB suppression of BMP signaling could be inhibited by nobiletin. Thus, our data suggest that mechanistically, nobiletin prevents the endogenous repression of BMP signaling by TNF-α, thereby enhancing osteoblast activity. In conclusion, nobiletin is a novel NF-κB antagonist that may be a useful anabolic agent for bone formation.

Sambucus williamsii Hance maintains bone homeostasis in hyperglycemia-induced osteopenia by reversing oxidative stress via cGMP/PKG signal transduction

BackgroundSambucus williamsii Hance (SWH) has effectively been adopted to treat joint and bone disorders. Diabetes-induced osteopenia (DOP) is caused primarily by impaired bone formation as a result of hyperglycemia. We had previously demonstrated that SWH extract accelerated fracture healing and promoted osteoblastic MC3T3-E1 cell proliferation and osteogenic differentiation. This study assessed the impacts of SWH extract on diabetes-induced bone loss and explored the mechanisms underlying its osteoprotective effects. MethodsThis work employed MC3T3-E1 cell line for evaluating how SWH extract affected osteogenesis, oxidative stress (OS), and the underlying mechanism in vitro. Streptozotocin-induced osteopenia mouse model was applied with the purpose of assessing SWH extract's osteoprotection on bone homeostasis in vivo. ResultsThe increased OS of MC3T3-E1 cells exposed to high glucose (HG) was largely because of the upregulation of pro-oxidant genes and the downregulation of antioxidant genes, whereas SWH extract reduced the OS by modulating NADPH oxidase-4 and thioredoxin-related genes by activating cyclic guanosine monophosphate (cGMP) production and increasing the level of cGMP-mediated protein kinase G type-2 (PKG2). The oral administration of SWH extract maintained bone homeostasis in type 1 diabetes mellitus (T1DM) mice by enhancing osteogenesis while decreasing OS. In bones from hyperglycemia-induced osteopenia mice and HG-treated MC3T3-E1 cells, the SWH extract achieved the osteoprotective effects through activating the cGMP/PKG2 signaling pathway, upregulating the level of antioxidant genes, as well as downregulating the level of pro-oxidant genes. ConclusionSWH extract exerts osteoprotective effects on hyperglycemia-induced osteopenia by reversing OS via cGMP/PKG signal transduction and is a potential therapy for DOP.

Acteoside Derived from Cistanche Improves Glucocorticoid-Induced Osteoporosis by Activating PI3K/AKT/mTOR Pathway

Objective Glucocorticoids are widely used in clinical practice; however, they can cause side effects, such as osteoporosis. Acteoside (ACT) from Cistanche has been used to combat a variety of diseases. The study was conducted to evaluate the efficacy of ACT in glucocorticoid-induced osteoporosis (GIOP) and its potential mechanism. Methods Dexamethasone (Dex) was injected intramuscularly to induce osteoporosis in a rat model, and ACT was given orally. ACT was supplemented in vivo in Dex-stimulated osteoblastic MC3T3-E1 cells. RT-qPCR was performed to assess the mRNA levels of bone formation (Runx2, CoL1A1), and bone resorption (OPG and RANKL). A commercial ELISA kit was applied to assess serum OC and CTX levels. Western blot was performed to assess protein levels in the PI3K/AKT/mTOR signaling pathway. CCK-8 assay and flow cytometry were performed to assess osteoblast viability and apoptosis. Results ACT reduced Dex-induced bone microstructure deterioration, increased serum levels of OC, and decreased the levels of CTX (P < 0.05). In the MC3T3-E1 cells, Dex inhibited cell viability and promoted apoptosis; however, this effect was greatly attenuated by ACT (P < 0.05). Concurrently, ACT reversed the reduction in Runx2, osterix, CoL1A1, and OPG mRNA levels, ALP activity, and the promotion of RANKL by Dex. Additionally, ACT attenuated Dex-induced inhibition of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K protein levels by Dex (P < 0.05), while the PI3K/AKT/mTOR pathway inhibitor LY294002 diminished the potential effect of ACT (P < 0.05). Conclusion ACT from Cistanche may exert osteoprotective effects by activating the PI3K/AKT/mTOR signaling pathway to alleviate Dex-induced osteoporosis.

Effects of Cow Milk-Derived Extracellular Vesicles on the Differentiation and Mineralization of Osteoblastic MC3T3-E1 Cells

Effects of Cow Milk-Derived Extracellular Vesicles on the Differentiation and Mineralization of Osteoblastic MC3T3-E1 Cells

Corrosion stability of GO/silane in simulated inflammatory conditions as a biocompatible sealant

The GO/silane sealant was formed on the oxide coating, prepared by plasma electrolytic oxidation (PEO) on titanium, using the sol–gel process. The interaction with osteoblastic MC3T3-E1 cells revealed that GO/silane had a beneficial effect on promoting cell response and proliferation such that the entire surface was covered after 1 d of culture. The results of electrochemical impedance spectroscopy in the simulated inflammatory medium at 37 °C clarified that the sealant developed a superior impedance modulus of over five times up to 7 d of immersion. The impedance modulus decreased by passing immersion time, but it maintained its superiority over the PEO coating during 14 d. The incorporation of GO delayed the degradation rate of the silane sealant. Moreover, calcium phosphate compounds did not precipitate due to inflammatory conditions.

Fusion of parathyroid hormone (1–34) to an albumin-binding domain improves osteogenesis

Osteoporosis is a common severe orthopedic disease that can lead to bone fractures and significantly reduce a patient's quality of life. Teriparatide (parathyroid hormone, PTH (1–34)) has been approved for the treatment of osteoporosis. However, PTH (1–34) needs to be injected once daily due to its short half-life of 1 h. To improve the pharmacokinetic and pharmacodynamic profiles of PTH (1–34), a G148-ABD3 (GA3) domain, which can bind to albumin non-covalently, was employed to modify PTH (1–34). The resultant PTH34-GA3-GS1 and PTH34-GA3-GS3 fusion proteins were constructed by fusing PTH (1–34) to the N-terminus of GA3 via (G4S)1 and (G4S)3 linkers, respectively. The fusion proteins were highly expressed in Escherichia coli. In vitro results showed that both PTH34-GA3-GS1 and PTH34-GA3-GS3 could bind to human serum albumin (HSA) with high affinity. The fusion proteins significantly increased the proliferation and differentiation of osteoblastic MC3T3-E1 cells, thereby promoting osteogenesis. PTH34-GA3-GS3 increased intracellular and serum calcium levels more than PTH34-GA3-GS1 did. The half-lives of PTH34-GA3-GS1 and PTH34-GA3-GS3 were 8.4 h and 9.1 h, respectively, which are significantly longer than that of PTH (1–34). Injection of PTH34-GA3-GS3 every three days increased bone mineral density and improved trabecular structure in ovariectomized mice, indicating that PTH34-GA3-GS3 has the potential to alleviate osteoporosis in humans. In conclusion, the fusion proteins maintained the biological activity of native PTH (1–34) and were not affected by the GA3 moiety, which extended the half-life of PTH (1–34). Long-acting GA3-fusion PTH (1–34) proteins have therapeutic effects on osteoporosis by improving osteogenesis and by enhancing the proliferation and differentiation of osteoblasts.

3D-Printed scaffolds based on poly(Trimethylene carbonate), poly(ε-Caprolactone), and β-Tricalcium phosphate.

Three-dimensional (3D)-printed scaffolds of biodegradable polymers have been increasingly applied in bone repair and regeneration, which helps avoid the second surgery. PTMC/PCL/TCP composites were made using poly(trimethylene carbonate), poly(ε-caprolactone), and β-tricalcium phosphate. PTMC/PCL/TCP scaffolds were manufactured using a biological 3D printing technique. Furthermore, the properties of PTMC/PCL/TCP scaffolds, such as biodegradation, mechanic properties, drug release, cell cytotoxicity, cell proliferation, and bone repairing capacity, were evaluated. We showed that PTMC/PCL/TCP scaffolds had low cytotoxicity and good biocompatibility, and they also enhanced the proliferation of osteoblast MC3T3-E1 and rBMSC cell lines, which demonstrated improved adhesion, penetration, and proliferation. Moreover, PTMC/PCL/TCP scaffolds can enhance bone induction and regeneration, indicating that they can be used to repair bone defects in vivo.

Role of Solvent Used in Development of Graphene Oxide Coating on AZ31B Magnesium Alloy: Corrosion Behavior and Biocompatibility Analysis.

Clinical applications of bio-absorbable magnesium (Mg) and its alloys can be enhanced by increasing their corrosion resistance, using surface modification and functionality. In this study, we synthesized graphene oxide (GO) through improved Hummers' method and deposited it on biodegradable AZ31B Mg alloy for further characterization. Different suspensions of GO were prepared in various solvents, like deionized water, ethanol, and acetone by ultra-sonication. Electrophoretic deposition (EPD) was used to develop GO coatings on AZ31B Mg using different GO suspensions. Effect of various solvents on corrosion behavior, as well as in vitro biocompatibility, was studied. The optimized EPD parameters were 3 volts and 90 s for coating. Different characterization techniques were used to study GO and prepared coatings. Atomic force microscopy found that the average thickness of GO was ~1 nm. Electrochemical behavior of coatings was studied through electrochemical impedance spectroscopy (EIS) and Tafel analysis in Ringer's lactate solution. Tafel analysis revealed that GO coatings deposited by GO water suspension increased corrosion protection efficiency of AZ31B Mg alloy by ~94%. After 72 h incubation in MC3T3-E1 osteoblast cells extract, in vitro analysis was performed to determine the cell viability and biocompatibility of the GO- coated and bare Mg samples. GO coatings deposited by GO water suspension demonstrated ~2× cell viability, as well as nontoxicity and better biocompatibility compared to the bare and other GO-coated Mg samples.

Aroclor 1254 induced inhibitory effects on osteoblast differentiation in murine MC3T3-E1 cells through oxidative stress.

This study aimed to evaluate the osteotoxicity of polychlorinated biphenyls in murine osteoblastic MC3T3-E1 cells, and to explore the underlying mechanism focused on oxidative stress. The cells were exposed to Aroclor 1254 at concentrations of 2.5-20 µmol/L, and then cell viability, oxidative stress, intracellular calcium concentration, osteocalcin content, and calcium nodules formation were measured. Aroclor 1254 reduced cell viability and induced overproduction of intracellular reactive oxygen species in a dose-dependent manner. Activity of superoxide dismutase was decreased, and malondialdehyde content was promoted after exposure. Moreover, inhibitory effects of Aroclor 1254 on calcium metabolism and mineralization of osteoblasts were observed, as indicated by reduction of the intracellular calcium concentration, osteocalcin content, and modules formation rate. The decreased expression of osteocalcin, alkaline phosphatase, bone sialoprotein, and transient receptor potential vanilloid 6 further confirmed the impairment of Aroclor 1254 on calcium homeostasis and osteoblast differentiation. Addition of the antioxidant N-acetyl-L-cysteine partially restored the inhibitory effects on calcium metabolism and mineralization. In general, Aroclor 1254 exposure reduces calcium homeostasis, osteoblast differentiation and bone formation, and oxidative stress plays a vital role in the underlying molecular mechanism of osteotoxicity.

Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging

The secretions of osteocalcin and bone morphogenetic protein 2 (BMP2) from living osteoblastic cells were visualized for the first time using a method of video-rate bioluminescence imaging. The fusion proteins with Gaussia luciferase (GLase) for mouse osteocalcin and BMP2 (OC-GLase and BMP2-GLase, respectively) expressed in osteoblastic MC3T3-E1 cells were correctly processed and secreted. In the video images of exocytotic secretion, the luminescence spots of OC-GLase and BMP2-GLase disappeared rapidly and gradually, respectively, indicating different manners of these proteins in diffusion. Notably, a deletion mutant of BMP2 (Δ3BMP2-GLase) lacking three basic amino acid residues in the N-terminal region for binding to heparan sulfate showed rapidly disappearing luminescence spots. In our imaging conditions, the half-life of luminescence for the spots of Δ3BMP2-GLase (1.61 ± 0.20 s) was similar to that of OC-GLase (1.22 ± 0.14 s) but not to that of BMP2-GLase (4.31 ± 0.41 s). These results suggest that, in contrast to osteocalcin, the diffusion of BMP2 from cells occurred slowly after exocytosis. Thus, our bioluminescence imaging method is useful to study the diffusion properties of secreted proteins in exocytosis.

Recombinant Unique Cartilage Matrix-associated Protein Potentiates Osteogenic Differentiation and Mineralization of MC3T3-E1 Cells.

The relative balance of osteoblasts in bone formation and osteoclasts in bone resorption is crucial for maintaining bone health. With age, this balance between osteoblasts and osteoclasts is broken, resulting in bone loss. Anabolic drugs are continuously being developed to counteract this low bone mass. Recombinant proteins are used as biotherapeutics due to being relatively easy to produce on a large scale and are cost-effective through various expression systems. This study aimed to develop a recombinant protein that would positively impact osteoblast differentiation and mineralized nodule formation using unique cartilage matrix-associated protein (UCMA). A recombinant glutathione-S-transferase (GST)-UCMA fusion protein was generated in an E.coli system, and purified by affinity chromatography. MC3T3-E1 osteoblast cells and Osterix (Osx)-knockdown stable cells were cultured for 14 days to investigate osteoblast differentiation and nodule formation in the presence of the recombinant GST-UCMA protein. The differentiated cells were assessed by alizarin red S staining and quantitative PCR of the osteoblast differentiation marker osteocalcin. In addition, cell viability in the presence of the recombinant GST-UCMA protein was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell adhesion assay. The isolation of both purified recombinant GST-only and GST-UCMA proteins were confirmed at 26 kDa and 34 kDa, respectively, by Coomassie staining and western blot analysis. Neither dose-dependent nor time-dependent presence of recombinant GST-UCMA affected MC3T3-E1 cell viability. However, MC3T3-E1 cell adhesion to the recombinant GST-UCMA protein increased dose-dependently. Osteoblast differentiation and nodule formation were promoted in both MC3T3-E1 osteoblast cells and Osxknockdown stable cells when cultured in the presence of recombinant GST-UCMA protein. A recombinant GST-UCMA protein induces osteogenic differentiation and mineralization, suggesting its potential use as an anabolic drug to increase low bone mass in osteoporotic patients.

In vitro release of silver ions and expression of osteogenic genes by MC3T3-E1 cell line cultured on nano-hydroxyapatite and silver/strontium-coated titanium plates.

Attempts are ongoing to improve the surface properties of dental implants by application of different coatings, aiming to enhance osseointegration, and decrease the adverse effects of titanium and its alloys used in dental implants. Coating of implant surface with hydroxyapatite (HA) is one suggested strategy for this purpose due to its high biocompatibility and similar structure to the adjacent bone. This study aimed to quantify the release of silver ions and expression of osteogenic genes by MC3T3-E1 cells cultured on nano-HA and silver/strontium (Ag/Sr)-coated titanium plates via the electrochemical deposition method. Plates measuring 10 × 10 × 0.9mm were fabricated from Ti-6Al-4V alloy, and polished with silicon carbide abrasive papers before electrochemical deposition to create a smooth, mirror-like surface. After applying homogenous nano-HA coatings with/without silver/strontium on the surface of the plates, the composition of coatings was confirmed by energy-dispersive X-ray spectroscopy (EDS), and their morphological properties were analyzed by scanning electron microscopy (SEM). The coated specimens were then immersed in simulated body fluid (SBF), and the concentration of released sliver ions was quantified by spectroscopy at 7-14days. The MC3T3-E1 osteoblastic cell line was cultured in osteogenic medium for 7-14days, and after RNA extraction and cDNA synthesis, the expression of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN); osteogenic genes was quantified by polymerase chain reaction (PCR) using SYBR Green Master Mix kit. The expression of genes and the released amount of silver ions were compared between the two groups using the Mann-Whitney U test. The two groups were not significantly different regarding silver ion release at 14days (P > 0.05). However, silver ion release was significantly higher from nano-HA coatings with silver/strontium at 7days (P = 0.03). The difference in expression of RUNX2 (P = 0.04), OPN (P = 0.04), and OCN (P = 0.03) genes was also significant between nano-HA coating groups with and without silver/strontium at 7days, and the expressions were higher in nano-HA with silver/strontium group, but this difference was not significant at 14days. Addition of silver and strontium to specimens coated with nano-HA increased the release of silver ions within the non-toxic range, and enhanced the expression of osteogenic genes particularly after 7days.

Effects of rmBMP-7 on Osteoblastic Cells Grown on a Nanostructured Titanium Surface

This study evaluates the effects of the availability of exogenous BMP-7 on osteoblastic cells’ differentiation on a nanotextured Ti surface obtained by chemical etching (Nano-Ti). The MC3T3-E1 and UMR-106 osteoblastic cell lines were cultured for 5 and 7 days, respectively, on a Nano-Ti surface and on a control surface (Control-Ti) in an osteogenic medium supplemented with either 40 or 200 ng/mL recombinant mouse (rm) BMP-7. The results showed that MC3T3-E1 cells exhibited distinct responsiveness when exposed to each of the two rmBMP-7 concentrations, irrespective of the surface. Even with 40 ng/mL rmBMP-7, important osteogenic effects were noticed for Control-Ti in terms of cell proliferation potential; Runx2, Osx, Alp, Bsp, Opn, and Smad1 mRNA expression; and in situ ALP activity. For Nano-Ti, the effects were limited to higher Alp, Bsp, and Opn mRNA expression and in situ ALP activity. On both surfaces, the osteogenic potential of UMR-106 cultures remained unaltered with 40 ng/mL rmBMP-7, but it was significantly reduced when the cultures were exposed to the 200 ng/mL concentration. The availability of rmBMP-7 to pre-osteoblastic cells at the concentrations used alters the expression profile of osteoblast markers, indicative of the acquisition of a more advanced stage of osteoblastic differentiation. This occurs less pronouncedly on the nanotextured Ti and without reflecting in higher mineralized matrix production by differentiated osteoblasts on both surfaces.

New PCL/PEC Blends: In Vitro Cell Response of Preosteoblasts and Human Mesenchymal Stem Cells.

Simple SummaryA topic of great relevance is the design of new biocompatible materials for their application in tissue engineering. The preparation of new polycaprolactone (PCL)/poly (ethylene carbonate) (PEC) blends has resulted in a series of new biomaterials obtained in a simple manner, which favor cell proliferation and subsequent differentiation using two different cell lines, the osteoblastic cell line (MC3T3-E1) and human mesenchymal cells (hMSCs), whose results indicate that these materials could be used in future to carry out 3D forming with the purpose of testing their potential application in tissue engineering for bone regeneration through “in vivo” assays.In this study, new blends of PCL/PEC have been prepared in an easy manner by casting with the objective of obtaining new biomaterials to apply to tissue engineering and bone regeneration. The PCL/PEC blends obtained, together with neat polymer blends, were characterized by infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). This full characterization is the key to disentangle the miscibility, which means good compatibility, of the polymer blends used in this work. The addition of increasing amounts of PEC, has shown in the new biomaterials obtained, a remarkable improvement in relation with the mechanical properties (manageable materials) and above all, in terms of an increase in their hydrophilic character with respect to the PCL neat polymer. The improvement of all these properties is reflected in their biological properties. With these thoughts in mind, the blends obtained were tested through the assessment of several biological parameters such as cell viability, proliferation, and differentiation of both the MC3T3-E1 osteoblastic cell line and hMSCs to evaluate their cell response to different polymer membranes aimed at bone tissue regeneration. “In vitro” biocompatibility methods have been chosen rather than in vivo studies due to their lower cost, faster procedure time, and minimum ethical concerns, and because it was the first time that the biological effects of these blends were studied. The results show that the PCL/PEC blends obtained, with tunable properties in terms of hydrophilic character and hydrolytic degradation, may be regarded as good candidates to perform “in vivo” tests and check their real-life applicability for bone regeneration. The polymer acronym (the weight percentage in the sub index) is PCLx/PECy as noted in table one with the summary of compositions.