Differentiation Of MC3T3-E1 Pre-osteoblasts Research Articles

Microstructural evolution of strontium‑zinc-phosphate coating on titanium via changing Zn2+ concentration in phosphate solution for enhanced osteogenic activity

The microstructure of the phosphate coating on implant surface is critical for successful osseointegration. Based on the morphological variability of phosphate crystals, the microstructure of the coating can be regulated by designing the composition of the phosphate solution. In this study, a strontium‑zinc-phosphate (SrZn2(PO4)2, SZP) coating was prepared on titanium (Ti) surface by phosphate chemical conversion (PCC) method. The effect of Zn2+ concentration in phosphate solution, which was regulated by increasing the addition amount of Zn(H2PO4)2·2H2O, on the microstructure and properties of the SZP coating was investigated. The characterization of the coating was carried out by scanning electron microscope (SEM), X-ray diffractometer (XRD), Fourier transform infrared spectrometer (FTIR), X-ray photoelectron spectrometer (XPS), laser scanning confocal microscope (LSCM), contact angle goniometer, and electrochemical workstation system. The results revealed that the increase of Zn2+ concentration in phosphate solution could induce the crystal morphology of the SZP coating to evolve from irregular granules (Zn(H2PO4)2·2H2O: 1 g/L) to regular cubic blocks (Zn(H2PO4)2·2H2O: 8 g/L). This morphological evolution process was accompanied by an increase in thickness (from 4.8 ± 1.2 to 20.6 ± 1.9 μm), an improvement in wettability and corrosion resistance, as well as a decrease in the release amount of Sr2+ (from 3.97 ± 0.26 to 0.86 ± 0.12 ppm) and Zn2+ (from 0.31 ± 0.01 to 0.18 ± 0.01 ppm) after 21 days of immersion. In vitro evaluations proved that the adhesion, proliferation, and differentiation of MC3T3-E1 pre-osteoblasts on SZP coatings, especially for the coating with tiny cubic-block crystals, were significantly enhanced compared with pure Ti. This study clarified the rule of morphological evolution of the SZP crystal that is caused by changing the Zn2+ concentration in the phosphate solution, which contributes to the structural optimization of the SZP coating to obtain high osteogenic activity.

Chitosan-based double-faced barrier membrane coated with functional nanostructures and loaded with BMP-6.

In the present study, a chitosan-based, multifunctional and double-faced barrier membrane was developed for the periodontitis therapy. The porous surface of the membrane was coated with bone-like hydroxyapatite (HA) produced by microwave-assisted biomimetic method and enriched with bone morphogenetic factor 6 (BMP-6) to enhance the bioactivity of chitosan. This surface of the membrane was designed to be in contact with the hard tissue that was damaged due to periodontitis. Otherwise the nonporous surface of membrane, which is in contact with the inflammatory soft tissue, was coated with electrospun polycaprolactone (PCL) fibers to prevent the migration of epithelial cells to the defect area. PrestoBlue, Scanning Electron Microscope (SEM) and real-time PCR results demonstrated that while porous surface of the membrane was enhancing the proliferation and differentiation of MC3T3-E1 preosteoblasts, nonporous surface of membrane did not allow migration of epithelial Madine Darby Bovine Kidney (MDBK) cells. The barrier membrane developed here is biodegradable and can be easily manipulated, has osteogenic activity and inactivity for epithelial cells. Thus, by implanting this membrane to the damaged periodontal tissue, bone regeneration will take place and integrity of periodontal tissues will be preserved.

Simvastatin delivery on PEEK for bioactivity and osteogenesis enhancements

A strategy developed for obtaining positive cellular responses remains to be focused in the filed of functional biomimetics. In this study, a hydrogel covered simvastatin-loaded polyetheretherketone (PEEK) bio-composites was constructed with the purpose of bone tissue regeneration therapy. Briefly, a three-dimensional (3D) porous structure was fabricated on PEEK surface; then the substrate was functionalized with the poly(L-lactic acid)/simvastatin porous film and hyaluronic acid hydrogel subsequently. In vitro cell attachment, proliferation, and cytoskeletal observation experiments reveal that our scaffolds show better bio-affinity due to the layer of hyaluronic acid hydrogel compared with control. Furthermore, the alkaline phosphatase activity, calcium mineral deposition evaluation, and gene expression for osteogenic potential all exhibit that the superior osteogenic differentiation of MC3T3-E1 pre-osteoblasts on our scaffolds. Therefore, our PEEK samples loaded with simvastatin and covered with hyaluronic acid hydrogel hold great potential in clinical applications for bone repair.

MC3T3-E1 pre-osteoblast response and differentiation after graphene oxide nanosheet uptake

Nano-graphene oxide (GO) and its functionalized derivatives have aroused a great interest for drug delivery, tissue engineering and photothermal cancer therapy, but their biocompatibility has not yet been fully assessed. The aim of the present study was to evaluate the proliferation and differentiation of MC3T3-E1 pre-osteoblasts after the uptake of GO nanosheets (c.a. 400nm), functionalized with poly(ethylene glycol-amine) (PEG) and labelled with fluorescein isothiocyanate (FITC). Significant proliferation decrease and apoptosis increase were observed 3days after incorporation of FITC-PEG-GO by MC3T3-E1 cells. However, alterations on healthy pre-osteoblast differentiation into cells exhibiting osteoblast phenotype were not observed, as they showed normal alkaline phosphatase levels and matrix mineralization 12days after nanosheet uptake. The results suggest that 40μg/mL concentrations of these nanosheets would not affect the differentiation of healthy pre-osteoblasts, thus these PEG-GO nanosheets have potential to be used for biomedical applications after their internalization, as the induction of local hyperthermia on bone cancer.

Bovine collagen peptides compounds promote the proliferation and differentiation of MC3T3-E1 pre-osteoblasts.

ObjectiveCollagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells.MethodsMouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0.ResultsCell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days.ConclusionsBovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis.

Identification of a Growth Factor Mimicking the Synergistic Effect of Fetal Bovine Serum on BMP-9 Cell Response

The bone morphogenetic proteins (BMPs) are potent osteogenic molecules that are used for bone repair in delivery systems and in regenerative medicine. We studied the responses of murine MC3T3-E1 preosteoblasts to doses of recombinant human (rh)BMP-9 with and without fetal bovine serum (FBS). rhBMP-2 was used as a control since it is currently approved by the Food and Drug Administration for bone application. We analyzed the major cell signaling pathways and the expression of osteogenic markers. Without FBS, BMP-9 had a similar effect on MC3T3-E1 preosteoblast differentiation in comparison to BMP-2. In contrast, FBS reduced the EC50 of BMP-9 fourfold to sixfold, as determined by osterix gene expression and alkaline phosphatase (ALP) activity, while it had no influence on EC50 of BMP-2. As suggested by MAPK inhibitor assays, FBS could induce an intracellular signaling environment that favors cell response to BMP-9 by inhibiting ERK1/2 activation and increasing p38 phosphorylation. Finally, IGF-2 (100 ng/mL) could mimic the effect of FBS on BMP-9 cell response in terms of MAPK signaling and ALP activity. Thus, the action of BMP-9 on preosteoblast differentiation can be greatly improved by IGF-2. This finding may well be critical for developing optimal growth factor delivery systems and bone tissue engineering strategies.

Osteogenic activities of MC3T3-E1 cells on heparin-immobilized poly(caprolactone) membranes

The aim of this study was to investigate the effects of heparin on the activity of osteoblast-like cells seeded on poly(caprolactone) (PCL) membranes. The membranes were prepared by solvent-casting technique in ~150 µm thickness. Then they were treated with 1,6-hexanediamine solution and functionalized with covalently bound heparin. The morphology, proliferation, and differentiation of MC3T3-E1 preosteoblasts on these membranes were investigated in vitro. The heparin functionalized PCL membranes, compared to non-functionalized membranes, significantly stimulated osteoblast proliferation. The Scanning electron microscope images confirmed the stimulative effect of covalently bound heparin on the osteoblast-like cell proliferation. The alkaline phosphatase and osteocalcin levels for cells proliferated on heparin containing PCL membranes were higher than that of nonfunctionalized membranes.

Heparin-functionalized chitosan scaffolds for bone tissue engineering

The aim of this study is to investigate the effects of heparin-functionalized chitosan scaffolds on the activity of preosteoblasts. The chitosan scaffolds having the pore size of ∼100 μm were prepared by a freeze-drying method. Two different methods for immobilization of heparin to chitosan scaffolds were successfully performed. In the first method, functionalization of the scaffolds was achieved by means of electrostatic interactions between negatively charged heparin and positively charged chitosan. The covalent immobilization of heparin to chitosan scaffolds by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC) and N-hydroxysuccinimide (NHS) was used as a second immobilization method. Morphology, proliferation, and differentiation of MC3T3-E1 preosteoblasts on heparin-functionalized chitosan scaffolds were investigated in vitro. The results indicate that covalently bound heparin containing chitosan scaffolds (CHC) stimulate osteoblast proliferation compared to other scaffolds, that is, unmodified chitosan scaffolds (CH), electrostatically bound heparin containing chitosan scaffolds (EHC), and CH+free heparin (CHF). SEM images also proved the stimulative effect of covalently bound heparin on the proliferation of preosteoblasts. Alkaline phosphatase (ALP) and osteocalcin (OCN) levels of cells proliferated on CHC and EHC were also higher than those for CH and CHF. In vitro studies have demonstrated that chitosan scaffolds increase viability and differentiation of MC3T3-E1 cells especially in the presence of immobilized heparin.

Pbx1 Represses Osteoblastogenesis by Blocking Hoxa10-Mediated Recruitment of Chromatin Remodeling Factors

Abdominal-class homeodomain-containing (Hox) factors form multimeric complexes with TALE-class homeodomain proteins (Pbx, Meis) to regulate tissue morphogenesis and skeletal development. Here we have established that Pbx1 negatively regulates Hoxa10-mediated gene transcription in mesenchymal cells and identified components of a Pbx1 complex associated with genes in osteoblasts. Expression of Pbx1 impaired osteogenic commitment of C3H10T1/2 multipotent cells and differentiation of MC3T3-E1 preosteoblasts. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) increased expression of osteoblast-related genes. Studies using wild-type and mutated osteocalcin and Bsp promoters revealed that Pbx1 acts through a Pbx-binding site that is required to attenuate gene activation by Hoxa10. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. Our results show that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. We propose that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis.

Ucma, a Novel Secreted Cartilage-specific Protein with Implications in Osteogenesis

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.

Modulation of Src Activity by Low Molecular Weight Protein Tyrosine Phosphatase During Osteoblast Differentiation

Background: Src kinase plays a critical role in bone metabolism, particularly in osteoclasts. However, the ability of Src kinase to modulate the activity of other bone cells is less well understood. In this work, we examined the expression and activity of Src and low molecular weight protein tyrosine phosphatase (LMWPTP) during osteoblast differentiation and assessed the modulation of Src kinase by LMWPTP. Methods: Differentiation of MC3T3-E1 pre-osteoblasts was induced by incubation with ascorbic acid and β-glycerophosphate for up to 28 days. Src phosphorylation and LMWPTP expression were analyzed by immunoblotting. Src dephosphorylation in vitro was assessed by incubating immunoprecipitated Src with LMWPTP followed by assay of the residual Src activity using Sam68 as substrate. The importance of LMWPTP in Src dephosphorylation was confirmed by silencing pre-osteoblasts with siRNA-LMWPTP and then assessing Src phosphorylation. Results: Pre-osteoblast differentiation was accompanied by a decrease in phosphorylation of the activator site of Src and an increase in phosphorylation of the inhibitory site. The expression of total Src was unaltered, indicating that post-translational modifications play a pivotal role in Src function. LMWPTP expression was higher in periods when the activator site of Src was dephosphorylated. LMWPTP dephosphorylated pY₅₂₇-Src and pY₄₁₆-Src in vitro, with greater specificity for pY₅₂₇Src. Activation of LMWPTP produced strong activation of Src mediated by fast dephosphorylation of pY₅₂₇-Src, followed by slower deactivation of this kinase via dephosphorylation of pY₄₁₆Src. Conclusion: These results provide new insight into the mechanisms governing the dynamics of Src activity during osteoblast differentiation. A fuller understanding of these mechanisms will improve our knowledge of bone metabolism and of the regulation of Src in other types of cells.

Quantifying the relation between adhesion ligand-receptor bond formation and cell phenotype.

One of the fundamental interactions in cell biology is the binding of cell receptors to adhesion ligands, and many aspects of cell behavior are believed to be regulated by the number of these bonds that form. Unfortunately, a lack of methods to quantify bond formation, especially for cells in 3D cultures or tissues, has precluded direct probing of this assumption. We now demonstrate that a FRET technique can be used to quantify the number of bonds formed between cellular receptors and synthetic adhesion oligopeptides coupled to an artificial extracellular matrix. Similar quantitative relations were found between bond number and the proliferation and differentiation of MC3T3-E1 preosteoblasts and C2C12 myoblasts, although the relation was distinct for each cell type. This approach to understanding 3D cell-extracellular matrix interactions will allow one to both predict cell behavior and to use bond number as a fundamental design criteria for synthetic extracellular matrices.