Differentiation Of MC3T3 Cells Research Articles

VEGF mitigates bisphosphonate-induced apoptosis and differentiation inhibition of MC3T3-E1 cells.

The present study aimed to investigate whether VEGF was involved in bisphosphonate (BP)-induced apoptosis and differentiation of osteoblasts. Murine MC3T3-E1 osteoblasts were stimulated with zoledronic acid (ZA) for 7 days. VEGF mRNA and protein expression levels were determined via reverse transcription-quantitative PCR and western blot analysis, respectively. Cell viability was evaluated using Cell Counting Kit-8 assay. In addition, the cell apoptotic rate and the expression levels of apoptosis-related proteins were measured using a TUNEL staining kit and western blot analysis, respectively. To evaluate mineralization, cells were stained with alizarin red, while the secretion levels of alkaline phosphatase (ALP) were measured using the corresponding assay kit. Finally, the expression levels of differentiation-related proteins and proteins of the Nod-like receptor family pyrin domain-containing 3 (NLRP3)/caspase 1/gasdermin D (GSDMD) pyroptosis pathway were measured by western blot analysis. VEGF expression level was notably decreased in ZA-stimulated MC3T3-E1 cells. However, the viability of these cells was enhanced following VEGF addition. Furthermore, VEGF attenuated apoptosis, promoted mineralization and increased ALP activity in ZA-stimulated MC3T3-E1 cells. The ZA-mediated decrease in the protein expression of the osteogenic genes osteopontin, osteocalcin and runt-related transcription factor 2 was restored after MC3T3-E1 cell treatment with 10 ng/ml VEGF. The present study demonstrated that VEGF could attenuate BP-induced apoptosis and differentiation of MC3T3 cells by regulating the NLRP3/caspase 1/GSDMD pathway.

Biocompability evaluation of micro textures coated with zinc oxide on Ti-6Al-4V treated by nanosecond laser

Surface morphology of implants plays an important role in enhancing cell response at the interface. Zinc ions (Zn 2+ ) have a promising potential for osseointegration by stimulating osteoblast cell multiplication and differentiation. In this study, regular micro grooves coated with zinc oxide (ZnO) were successfully manufactured on Ti-6Al-4V surface by two-step nanosecond laser treatment to investigate the synergetic effects of micro/nano structures and Zn 2+ on the cell adhesion, proliferation and differentiation. Surface characteristics including surface morphology, roughness, wettability and phase composition were characterized. Results showed that the roughness and hydrophilicity of laser-processing surface were significantly improved compared with those of the acid etching surface. Moreover, XRD results indicated that the ZnO diffraction peak appeared on the surface. The biocompatibility of different samples was investigated by cell experiment in vitro. The spreading morphology of cells along grooves, cell multiplication rate, and activity of alkaline phosphatase and osteocalcin indicated that the micro grooves coated with ZnO on Ti-6Al-4V surface had great potential to promote the adhesion, multiplication and differentiation of MC3T3 cells. This study provides a new and effective method for surface modification of Ti-6Al-4V implants. • A micro textured surface coated with zinc oxide was fabricated on surface of Ti-6Al-4V by two-step nanosecond laser treatment, the precise control of surface morphology and close combination of bioactive coating are realized. • Micro textured surface coated with zinc oxide displayed increased roughness and enhanced hydrophilicity comparing with acid etching surface. • The cell adhesion, proliferation and differentiation of MC3T3s was improved obviously by the synergistic effects of micro grooves and zinc ions released.

Osteogenic differentiation of an osteoblast precursor cell line using composite PCL-gelatin-nHAp electrospun nanofiber mesh

Designing a temporary substrate to adhere, multiply and form new bone tissue for the bone-forming cells is a challenge in bone tissue engineering. In this article, a new biodegradable and composite scaffolding system was fabricated using an electrospinning technique to augment bone formation using poly-caprolactone (PCL), natural polymer gelatin (GL) and nano-hydroxyapatite (nHAp). The effect of fabrication parameters on nanofiber scaffolds (PCL, PCL + GL, PCL + GL + nHAp) production, as well as MC3T3-E1 proliferation and osteogenic differentiation was investigated. Physical properties of scaffolds, such as morphology, surface area, wettability, were examined using SEM, BET method, and contact angle measurements, respectively. A pre-osteoblastic cell line, MC3T3-E1 was seeded on these scaffolds to study the proliferation by DNA assay and cell viability using live-dead assay. After 21 days of in vitro culture, more than 89% of the MC3T3 cells remained viable within PCL + GL + nHAp scaffold, in contrast to 61.9% viability observed in the plain PCL scaffold. The differentiation of MC3T3 cells into the bone phenotype was determined quantitatively using alkaline phosphatase (ALP) activity and calcium deposition as well as qualitatively using alizarin red staining. At day 21, the DNA content of PCL + GL + nHAp scaffold was 4.5 times higher than its day 1 DNA contents. At day 21, the normalized intracellular ALP activity in the PCL + GL + nHAp group was 1.2 times higher than that in the PCL + GL group, which in turn was 1.7 times larger than that in the PCL group. Moreover, at day 21, PCL + GL + nHAp scaffold showed a 12.43-fold increase in ALP activity and deposited nearly 35-fold higher calcium relative to their respective values at day 1, thus indicating that the inclusion of gelatin and nHAp significantly enhanced cell binding, long-term cell survivability, proliferation, and stimulated osteogenesis in vitro. The findings from current work show that incorporation of nHAp into the nanofibrous scaffold of PCL + GL and the use of our optimized electrospinning process provides a favorable substrate to promote bone healing.

Mitigative effect of erythromycin on PMMA challenged preosteoblastic MC3T3-E1 cells.

Background. Aseptic loosening (AL) is a major complication of total joint replacement. Recent approaches to limiting AL have focused on inhibiting periprosthetic inflammation and osteoclastogenesis. Questions/Purposes. The purpose of this study was to determine the effects of erythromycin (EM) on polymethylmethacrylate (PMMA) particle-challenged MC3T3 osteoblast precursor cells. Methods. MC3T3 cells were pretreated with EM (0–10 μg/mL) and then stimulated with PMMA (1 mg/mL). Cell viability was evaluated by both a lactate dehydrogenase (LDH) release assay and cell counts. Cell differentiation was determined by activity of alkaline phosphatase (ALP). Gene expression was measured via real-time quantitative RT-PCR. Results. We found that exposure to PMMA particles reduced cellular viability and osteogenetic potential in MC3T3 cell line. EM treatment mitigated the effects of PMMA particles on the proliferation, viability and differentiation of MC3T3 cells. PMMA decreased the gene expression of Runx2, osterix and osteocalcin, which can be partially restored by EM treatment. Furthermore, EM suppressed PMMA- induced increase of NF-κB gene expression. Conclusions. These data demonstrate that EM mitigates the effects of PMMA on MC3T3 cell viability and differentiation, in part through downregulation of NF-κB pathway. EM appeared to represent an anabolic agent on MC3T3 cells challenged with PMMA particles.

PTH Stimulates Osteoblastic VEGF-A Production and Remodels Bone Marrow Micro-Endothelial Structures

Abstract 722In their niches within the bone marrow (BM) microenvironment, hematopoietic stem cells (HSCs) interact with a number of other cellular and molecular factors that can affect their regulation. We previously demonstrated that activation of osteoblasts (OBs) by intermittent Parathyroid Hormone (PTH) stimulation or by expression of a constitutively active PTH receptor (Col1caPTH1R mice, hereafter referred to as TG) expands HSCs with long term repopulating ability. PTH treatment has also been found to improve survival after BM injuries that severely disrupt the hematopoietic system as well as the BM vasculature; however the mechanism for this effect is unknown. Osteoblastic cells respond directly to PTH administration, whereas HSCs lack expression of the PTH1R, indicating that PTH-mediated HSC expansion occurs through the BM microenvironment. Both endosteal osteoblastic cells and the vasculature constitute HSC niches within the BM. Intermittent PTH exerts an anabolic effect on bone, expanding trabecular bone and bone-lining stromal cells. New vessel formation is required for adult bone remodeling. To test if PTH also increases the BM vasculature, we treated mice with an intensive regimen of systemic PTH that expanded hematopoietic stem and progenitor cells (HSPCs) and analyzed hindlimb histology as well as BM cell immunophenotype. In the tibial and femoral metaphyseal region, PTH increased microvessels (167 ± 18.1 vs 348 ± 39.3 microvessels/hindlimb, n=5 mice per group, p=0.0030) and vascular area ((mm2): 0.135 ± 0.0194 vs 0.281 ± 0.00951, n=5 mice per group, p=0.0001) measured by histomorphometry. PTH also increased the frequency of PECAM+ BM endothelial cells (0.0935 ± 0.0173 vs 0.196 ± 0.0172, n=4–5 mice per group, p=0.0043) measured flow cytometrically. To determine if PTH increases osteoblast-derived angiogenic signals, mouse calvarial MC3T3 cells were treated with PTH(1-34) at various stages of osteoblastic maturation. Differentiation of MC3T3 cells did not affect baseline Vegfa expression. PTH strongly induced total Vegfa expression in day 7 cells at two hours, an effect that peaked at six hours (3.83 ± 1.76 vs 50.7 ± 9.72 fold change above baseline, n=3, p=0.0090) and was sustained 24 hours after treatment. The magnitude of the effect increased throughout osteoblastic differentiation. In vitro PTH also increased levels of secreted VEGF-A protein at corresponding time points (VEGF-A(pg/mL): 43.4 ± 2.27 vs 464 ± 24.2, n=3, p<0.0001). Similarly, PTH induced Vegfa expression two hours post-stimulation in a second osteoblastic cell line, derived from rat osteosarcoma (1.133 ± 0.2963 vs 8.500 ± 1.320 fold change above baseline, n=3, p=0.0055). Vegfa pre-mRNA comprises eight exons that undergo alternative splicing to form variants encoding several VEGF-A isoforms. These isoforms differ functionally primarily due to their varying heparan-binding affinity. Specifically, VEGF189 remains mostly cell and matrix-associated due to its interactions with heparan sulfate proteoglycans in extracellular matrix and on cell surfaces. Because PTH stimulation dramatically increases extracellular matrix and bone volume, expression of the splice variant encoding matrix-associated VEGF189 was assayed in PTH-stimulated MC3T3 cells. PTH strongly increased expression of the VEGF189-encoding variant six hours after treatment of maturing MC3T3 cells (1.70 ± 1.100 vs 113 ± 25.1, n=3, p=0.0416), suggesting that PTH may favor expression of a more highly localized VEGF-A isoform. Analysis of BM plasma revealed that soluble VEGF-A levels were significantly decreased both in TG mice (VEGF-A(pg/mL): 74.2 ± 7.05 vs 44.0 ± 3.67, n=3-5 mice per group, p=0.0054) and in mice treated systemically with PTH (VEGF-A(pg/mL): 105 ± 6.62 vs 78.6 ± 4.95, n=9 mice per group, two independent experiments, p=0.0054), while serum VEGF-A levels were unchanged. Since PTH strongly stimulates osteoblastic expression of matrix-bound VEGF-A but less soluble VEGF-A is detected in the BM during PTH stimulation in vivo, we speculated that PTH-dependent proangiogenic signals in the BM microenvironment may be highly localized via modulation of Vegfa pre-mRNA splicing in osteoblastic cells. Because the blood perfusion status of BM niches profoundly affects HSPC behavior, these findings may suggest a mechanism by which PTH establishes highly localized niches to instruct the fate of resident HSCs. Disclosures:No relevant conflicts of interest to declare.

Investigating the role of the extracellular matrix on differentiation of human mesenchymal stem cells and MC3T3 cells

Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but due to their limited number and donor variation, other cell types are used to answer relevant questions in bone tissue engineering. Since the extracellular matrix (ECM) is a complex entity with instructive properties, it is of key importance to analyze its role in osteogenic differentiation in different cell types used for bone tissue engineering. To investigate the role of the ECM on osteogenic differentiation, we interfered with collagen assembly using an irreversible inhibitor of lysyl oxidase, the enzyme responsible for collagen crosslinking, and analyzed its effect on differentiation of MC3T3 cells and hMSCs. MC3T3 cells and hMSCs were differentiated into the osteogenic lineage using BMP-2 and dexamethasone, respectively. β-aminopropionitrile (BAPN) was added to the medium at different concentrations and alkaline phosphatase expression and calcium deposition were quantified. To analyze whether collagen crosslinking is essential for osteogenic differentiation, we exposed MC3T3 cells and hMSCs to BAPN. In the case of MC3T3 cells, we observed a decrease in ALP expression with increasing concentrations of BAPN. In contrast, different concentrations of BAPN did not affect ALP expression of hMSCs when compared to the control. Next, we analyzed whether differences in the level of collagen crosslinking, due to the presence of BAPN, would have an effect on matrix mineralization. To analyze this, cells were grown in the presence or absence of dex and with or without BAPN. Surprisingly, when BAPN was added to the medium in combination with dex, hMSCs showed a 5-fold increase in calcium deposition. Nevertheless, BAPN alone is not able to induce mineralization. These results demonstrated that BAPN does not affect ALP expression in hMScs, in contrast to MC3T3 cells. Interestingly, BAPN treatment greatly enhances mineralization of hMSCs. These data are in line with our previous results that show that collagen does not affect ALP expression by hMSCs besides the one provided by dex (data not shown).

Cloning of a Novel cDNA Expressed during the Early Stages of Fracture Healing

Using differential mRNA display (DD-PCR), a novel cDNA, FxC1 (Fracture Callus 1) was isolated from the early stages of a healing fractured femur. Utilizing 5′ RACE PCR, a 598-bp full-length cDNA was obtained for FxC1 that contains an open reading frame (ORF) of 243 bp, encoding for an 80 amino acid protein. Within this ORF, a leucine zipper motif was present.In vitrotranscription/translation of the full-length cDNA generated the expected 9-kDa protein. Northern analysis reveals that this gene is expressed in calluses harvested from post-fracture day 5, 7 and 10, as well as in several other tissues and bone-derived cell lines. During the differentiation of MC3T3 cells along the osteoblast lineage, FxC1 expression increases 3- to 4-fold during the production and deposition of matrix proteins, suggesting a possible role for this protein in cell differentiation.