Pre-osteoblastic MC3T3-E1 Cells Research Articles

Chondroitin Sulfate and Hyaluronic Acid-Based PolyHIPE Scaffolds for Improved Osteogenesis and Chondrogenesis In Vitro.

Osteochondral damage, affecting the articular cartilage and the underlying subchondral bone, presents significant challenges in clinical treatment. Such defects, commonly seen in knee and ankle joints, vary from small localized lesions to larger defects. Current medical therapies encounter several challenges, such as donor shortages, drug side effects, high costs, and rejection problems, often resulting in only temporary relief. Highly porous emulsion-templated polymers (polyHIPEs) offer numerous potential benefits in the fabrication of scaffolds for tissue engineering and regenerative medicine. Polymeric scaffolds synthesized using a high internal phase emulsion (HIPE) technique, called PolyHIPEs, involve polymerizing a continuous phase surrounding a dispersed internal phase to form a solid, foam-like structure. A dense, porous design encourages cell ingrowth, nutrient delivery, and waste disposal from the scaffold, mimicking the cells' natural microenvironment. This study used hydroxyethyl methacrylate (HEMA) and acrylamide (AAM) polyHIPE scaffolds combined with extracellular matrix (ECM) components of the tissue, such as methacrylated hyaluronic acid (MHA) and methacrylated chondroitin sulfate (MCS), to prepare polyHIPE scaffolds. The mouse preosteoblast MC3T3-E1 cells and primary rat chondrocytes (harvested from male Wistar rats) were seeded on the scaffolds and cultured for 21 days to assess the osteogenesis and chondrogenesis in vitro. When compared to the AAM-MHA and AAM-MCS groups at day 21, scaffold groups HEMA-MHA and HEMA-MCS showed a significant rise in alkaline phosphatase (ALP) and calcium content. Chondrogenic markers such as glycosaminoglycan (GAG) and hydroxyproline were also assessed over a 21-day time point. On day 21, it was found that GAG and hydroxyproline production were considerably higher in the HEMA-MHA and HEMA-MCS scaffolds than in the AAM-MHA and AAM-MCS scaffolds. The overall studies showed that polyHIPE monolith scaffolds could favor cell adherence, survival ability, proliferation, differentiation, and ECM formation over 21 days. Thus, incorporating ECM components enhanced osteogenesis and chondrogenesis in vitro and can be further used as tissue repair models.

Fabrication of alginate composite hydrogel encapsulated retinoic acid and nano Se doped biphasic CaP to augment in situ mineralization and osteoimmunomodulation for bone regeneration

BackgroundBone tissue engineering endows alternates to support bone defects/injuries that are circumscribed to undergo orchestrated process of remodeling on its own. In this regard, hydrogels have emerged as a promising platform that can confront irregular defects and encourage in situ bone repair. MethodsIn this study, we aimed to develop a new approach for bone tissue regeneration by developing an alginate based composite hydrogel incorporating selenium doped biphasic calcium phosphate nanoparticles, and retinoic acid. The fabricated hydrogel was physiochemically evaluated for morphological, bonding, and mechanical behavior. Additionally, the biological response of the fabricated hydrogel was evaluated on MC3T3-E1 pre-osteoblast cells. ResultsThe developed composite hydrogel confers excellent biocompatibility, and osteoconductivity owing to the presence of alginate, and biphasic calcium phosphate, while selenium presents pro osteogenic, antioxidative, and immunomodulatory properties. The hydrogels exhibited highly porous microstructure, superior mechanical attributes, with enhanced calcification, and biomineralization abilities in vitro. SignificanceBy combining the osteoconductive properties of biphasic calcium phosphate with multifaceted benefits of selenium and retinoic acid, the fabricated composite hydrogel offers a potential transformation in the landscape of bone defect treatment. This strategy could direct a versatile and effective approach to tackle complex bone injuries/defects and present potential for clinical translation.

Cytocompatibility, cell-material interaction, and osteogenic differentiation of MC3T3-E1 pre-osteoblasts in contact with engineered Mg/PLA composites.

Bioabsorbable Mg wire-reinforced poly-lactic acid (PLA) matrix composites are potential candidate for load-bearing orthopedic implants offering tailorable mechanical and degradation properties by stacking sequence, volume fraction and surface modification of Mg wires. In this study, we investigated the cytocompatibility, cell-material interaction, and bone differentiation behavior of MC3T3-E1 pre-osteoblast cells for medical-grade PLA, Mg/PLA, and PEO-Mg/PLA (having PEO surface modification on Mg wires) composites. MTT and live/dead assay showed excellent biocompatibility of both composites while cell-material interaction analysis revealed that cells were able to adhere and proliferate on the surface of composites. Cells on the longitudinal surface of composites showed a high and uniform cell density while those on transversal surfaces initially avoided Mg regions but later migrated back after the formation of the passivation layer. Bone differentiation tests showed that cells in extracts of PLA and composites were able to initiate the differentiation process as osteogenesis-related gene expressions, alkaline phosphatase protein quantity, and calcium mineralization increased after 7 and 14 days of culture. Interestingly, the bone differentiation response of PEO-Mg/PLA composite was found to be similar to medical-grade PLA, proving its superiority over Mg/PLA composite.

TXNIP mediated by EZH2 regulated osteogenic differentiation in hBmscs and MC3T3-E1 cells through the modulation of oxidative stress and PI3K/AKT/Nrf2 pathway

ABSTRACT Background Previous research has identified a significant role of Thioredoxin-interacting protein (TXNIP) in bone loss. The purpose of this investigation was to assess the role and the underlying molecular mechanisms of TXNIP in the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and pre-osteoblast MC3T3-E1 cells. Methods Human bone marrow stem cells (hBMSCs) and MC3T3-E1 cells were used to induce osteogenic differentiation. The expression of genes and proteins was assessed using RT-qPCR and western blot, respectively. ChIP assay was used to validate the interaction between genes. The osteogenic differentiation ability of cells was reflected using ALP staining and detection of ALP activity. The mineralization ability of cells was assessed using ARS staining. DCFCA staining was employed to evaluate the intracellular ROS level. Results Initially, downregulation of TXNIP and upregulation of EZH2 were observed during osteogenesis in hBMSCs and MC3T3-E1 cells. Additionally, it was discovered that EZH2 negatively regulates TXNIP expression in these cells. Furthermore, experiments indicated that the knockdown of TXNIP stimulated the activation of the PI3K/AKT/Nrf2 signaling pathway in hBMSCs and MC3T3- E1 cells, thus inhibiting the production of reactive oxygen species (ROS). Further functional experiments revealed that overexpression of TXNIP inhibited the osteogenic differentiation in hBMSCs and MC3T3-E1 cells by enhancing ROS produc-tion. On the other hand, knockdown of TXNIP promoted the osteogenic differentiation capacity of hBMSCs and MC3T3-E1 cells through the activation of the PI3K/AKT/Nrf2 pathway. Conclusion In conclusion, this study demonstrated that TXNIP expression, under the regulation of EZH2, plays a crucial role in the osteogenic differentiation of hBMSCs and MC3T3-E1 cells by regulating ROS production and the PI3K/AKT/Nrf2 pathway.

3D printed hetero-layered composite scaffold with engineered superficial zone promotes osteogenic differentiation of pre-osteoblast MC3T3-E1 cells

In bone tissue engineering, a new design of composite scaffold with various compositions by incorporating both natural and synthetic cues via dual nozzle 3D printing technique could efficiently accelerate the cell differentiation to osteogenic lineage. Based on the hypothesis that a biomimetic scaffold per se can supply the vital cues to provoke bone formation, 3D printed poly(ε-caprolactone) (PCL) scaffold loaded with synthetic hydroxyapatite (HAs) decorated on graphene oxide (GO) sheets (GO@HAs), was developed. To further improve its osteogenic potential, the upper and lower layer/s of scaffolds were 3D printed using another ink containing PCL and naturally allograft HA (HAa). The FE-SEM images of the 3D printed scaffolds revealed a highly regular architecture, demonstrating the successful printability of the developed materials. The compressive mechanical test confirmed the drastic role of GO@HAs on the enhancement of Young's modulus and mechanical strength of the PCL scaffold. Moreover, existence of HAa on the upper and lower sections of scaffold interestingly improved the cellular behavior of the 3D printed scaffold without having an adverse effect on mechanical features. The experimental results indicated that such engineered scaffold significantly facilitated the cell attachment and evoked higher levels of pre-osteoblast MC3T3-E1 differentiation than PCL/GO@HAs scaffold as demonstrated by alizarin red staining, ALP activity and osteo-related gene expression. Taken together, these results indicate that such fascinating scaffold comprising both mechanical and biological cues has a high potency for inducing bone repair and also open new avenues for future advancements in the realm of bone tissue engineering exploration.

Tribological behavior and biocompatibility of novel Nickel-Free stainless steel manufactured via laser powder bed fusion for biomedical applications

Due to the risk of releasing carcinogenic nickel ions from conventional 316L stainless steel under a corrosive human body environment, a new variant of steel termed nickel-free stainless steel (NiFSS) has been investigated. The present study investigates the tribological properties and biocompatibility of NiFSS manufactured via laser powder bed fusion (PBF-LB/M). The ferritic NiFSS exhibited significantly lower coefficient of friction (0.08 to 0.28) and wear rate (1.60 × 10-6 mm3/Nm to 6.60 × 10-6 mm3/Nm) compared to reported values for austenitic 316L SS, under both dry and simulated body fluid (SBF) conditions and various sliding geometries. This improvement is attributed to the superior hardness (3.394 ± 0.1340 GPa) and elastic modulus (238 ± 9.0797 GPa) of NiFSS. To assess the biocompatibility, the viability of mouse pre-osteoblastic MC3T3-E1 cells was evaluated with an Alamar Blue assay when the cells were cultured on top of PBF-LB/M built NiFSS and 316L SS samples. The results indicated that even though cell growth was most optimal on regular cell culture plastic, cell viability was better maintained on PBF-LB/M built NiFSS compared to 316L SS. Therefore, the results of the present study propose that PBF-LB/M fabricated NiFSS holds promise for application in biomedical devices for joint arthroplasty.

Low-temperature aqueous synthesis and biocompatibility of manganese whitlockite

In this work, manganese whitlockite (Mn-WH, Ca18Mn2(HPO4)2(PO4)12) was successfully synthesized and comprehensively characterized. This material is a manganese-containing analog of the biomineral magnesium whitlockite (Mg-WH, Ca18Mg2(HPO4)2(PO4)12). The synthesis was performed via a low-temperature dissolution-precipitation process in aqueous medium under ambient pressure at 75 °C using dicalcium phosphate dehydrate and manganese acetate tetrahydrate as starting materials. The crystal structure of the synthesized powder was determined by powder X-ray diffraction, FTIR and Raman spectroscopies, demonstrating the presence of characteristic structural units. Morphological features and elemental distribution within the synthesized powder were studied by SEM/EDX analysis. XPS, EPR and magnetic studies confirmed the presence of Mn ions in a divalent state. Temperature-dependent magnetic measurements revealed a paramagnetic behavior of Mn-WH down to 5 K. In vitro cytotoxicity experiments were performed with MC3T3-E1 preosteoblastic cell line.

Fabrication and characterization of β-TCP/Zn-1Mg composite scaffolds for orthopedic applications

Bone tissue defects have emerged as a significant health concern, impacting individuals' overall well-being and daily functionality. The implantation of scaffold materials for bone repair is crucial to structurally replace the damaged bone area, providing essential mechanical support and fulfilling other functional requirements. In this study, in order to develop bone repair scaffold materials with enhanced mechanical properties, moderate corrosion behavior, favorable cytocompatibility, and notable antibacterial activity, xβ-TCP/Zn-1Mg composite scaffolds with varying β-TCP concentrations (x = 0, 0.5, 1, 3) were fabricated using an atmospheric pressure infiltration molding method. The resulting scaffolds featured interconnected and uniformly distributed pores, with an average pore size of approximately 250 μm and a porosity of around 60 %. The xβ-TCP/Zn-1Mg composite scaffolds exhibited a plateau stress ranging from 13.22 to 16.89 MPa and an elastic modulus between 0.62 and 0.82 GPa, closely resembling the mechanical properties of human cancellous bone. The incorporation of β-TCP accelerated the degradation process and promoted calcium phosphate deposition on the specimen's surface. Additionally, the β-TCP/Zn-1Mg composite scaffolds demonstrated significant antibacterial activity against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Furthermore, 2-fold diluted extracts derived from xβ-TCP/Zn-1Mg composite scaffolds were found to enhance the proliferation of MC3T3-E1 pre-osteoblast cells, indicating good cytocompatibility. Therefore, the xβ-TCP/Zn-1Mg scaffold materials hold promise as a compelling candidate for orthopedic applications.

In Vitro Evaluation of Optimized PEEK Surfaces for Enhanced Osseointegration

The materials traditionally used for implant applications, such as titanium alloys, cobalt chromium, and zirconium, often require surface modifications to achieve the desired osseointegration. These materials still have the problematic stress-shielding effect. To limit stress shielding, PEEK is the superior alternative to fulfill implant needs. However, the traditional methods of modifying and functionalizing the surface of PEEK are often expensive, time consuming, and are not easily translated into commercialization. Sulfonation is a process, which is dependent on controllable factors. Thus far, no research has been performed to optimize the sulfonation process. Our data suggest that the process factors can be controlled and optimized. Cellular activity was examined on the optimized PEEK surfaces through testing with pre-osteoblast MC3T3-E1 cells through cell viability (MTT assay), cell proliferation (DNA assay), cell differentiation (ALP assay), and cell mineralization (Alizarin red assay). Overall, sulfonated and heat-treated PEEK exhibited a statistically significant increase in DNA content over the course of 21 days, indicating more cell proliferation and viability for that surface. In vitro testing results showed that the optimized sulfonated and heat-treated PEEK exhibited superior cell proliferation and mineralization performance over smooth PEEK and sulfonated-only PEEK.

Fracture healing on non-union fracture model promoted by non-thermal atmospheric-pressure plasma.

Non-thermal atmospheric-pressure plasma (NTAPP) is attracting widespread interest for use in medical applications. The tissue repair capacity of NTAPP has been reported in various fields; however, little is known about its effect on fracture healing. Non-union or delayed union after a fracture is a clinical challenge. In this study, we aimed to investigate how NTAPP irradiation promotes fracture healing in a non-union fracture model and its underlying mechanism, in vitro and in vivo. For the in vivo study, we created normal and non-union fracture models in LEW/SsNSlc rats to investigate the effects of NTAPP. To create a fracture, a transverse osteotomy was performed in the middle of the femoral shaft. To induce the non-union fracture model, the periosteum surrounding the fracture site was cauterized after a normal fracture model was created. The normal fracture model showed no significant difference in bone healing between the control and NTAPP-treated groups. The non-union fracture model demonstrated that the NTAPP-treated group showed consistent improvement in fracture healing. Histological and biomechanical assessments confirmed the fracture healing. The in vitro study using pre-osteoblastic MC3T3-E1 cells demonstrated that NTAPP irradiation under specific conditions did not reduce cell proliferation but did enhance osteoblastic differentiation. Overall, these results suggest that NTAPP is a novel approach to the treatment of bone fractures.

Defining the Most Potent Osteoinductive Culture Conditions for MC3T3-E1 Cells Reveals No Implication of Oxidative Stress or Energy Metabolism.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and β-glycerophosphate (βGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 μg.mL-1/2 mM Asc/βGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone‑induced MC3T3‑E1 cells by activating the Keap1/Nrf2 pathway.

Steroid-induced osteonecrosis of the femoral head (ONFH) is a serious complication caused by long-term or excessive use of glucocorticoids. The present study aimed to ascertain the effects of tripartite motif-containing protein 21 (TRIM21) on the process of steroid-induced ONFH and its hidden action mechanism. TRIM21 expression in dexamethasone (Dex)-treated mouse MC3T3-E1 preosteoblast cells was examined using reverse transcription-quantitative PCR and western blotting. The Cell Counting Kit-8 (CCK-8) method and lactate dehydrogenase release assay were used to respectively measure cell viability and injury. Flow cytometry analysis was used to assay cell apoptosis. Caspase 3 activity was evaluated using a specific assay, while alkaline phosphatase and Alizarin red S staining were used to evaluate osteogenesis. 2,7-dichloro-dihydrofluorescein diacetate fluorescence probe was used to estimate reactive oxygen species generation. Specific assay kits were used to appraise oxidative stress levels. In addition, the expression of apoptosis-, osteogenic differentiation- and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling-associated proteins was assessed using western blotting. In Nrf2 inhibitor (ML385)-pretreated MC3T3-E1 cells exposed to Dex, cell apoptosis, osteogenesis and oxidative stress were detected again as aforementioned. Results revealed that TRIM21 expression was raised in Dex-induced MC3T3-E1 cells and TRIM21 deletion improved the viability and osteogenic differentiation, whereas it hampered the oxidative stress and apoptosis in MC3T3-E1 cells with Dex induction. In addition, silencing of TRIM21 activated Keap1/Nrf2 signaling. Moreover, ML385 partially abrogated the effects of TRIM21 depletion on the oxidative stress, apoptosis and osteogenic differentiation in MC3T3-E1 cells exposed to Dex. In conclusion, TRIM21 silencing might activate Keap1/Nrf2 signaling to protect against steroid-induced ONFH.

Comparison of the osteogenic potential of fibroblasts from different sources

ObjectiveWith the growing interest in the role of fibroblasts in osteogenesis, this study presents a comparative evaluation of the osteogenic potential of fibroblasts derived from three distinct sources: human gingival fibroblasts (HGFs), mouse embryonic fibroblasts (NIH3T3 cells), and mouse subcutaneous fibroblasts (L929 cells). MC3T3-E1 pre-osteoblast cells were employed as a positive control for osteogenic behavior. DesignOur assessment involved multiple approaches, including vimentin staining for cell origin verification, as well as ALP and ARS staining in conjunction with RT-PCR for osteogenic characterization. ResultsOur findings revealed the superior osteogenic differentiation capacity of HGFs compared to MC3T3-E1 and NIH3T3 cells. Analysis of ALP staining confirmed that early osteogenic differentiation was most prominent in MC3T3-E1 cells at 7 days, followed by NIH3T3 and HGFs. However, ARS staining at 21 days demonstrated that HGFs produced the highest number of calcified nodules, indicating their robust potential for late-stage mineralization. This late-stage osteogenic potential of HGFs was further validated through RT-PCR analysis. In contrast, L929 cells displayed no significant osteogenic differentiation potential. ConclusionsIn light of these findings, HGFs emerge as the preferred choice for seed cells in bone tissue engineering applications. This study provides valuable insights into the potential utility of HGFs in the fields of bone tissue engineering and regenerative medicine.

Biodegradable Honeycomb-Mimic Scaffolds Consisting of Nanofibrous Walls.

The scaffold is a porous three-dimensional (3D) material that supports cell growth and tissue regeneration. Such 3D structures should be generated with simple techniques and nontoxic ingredients to mimic bio-environment and facilitate tissue regeneration. In this work, simple but powerful techniques are demonstrated for the fabrication of lamellar and honeycomb-mimic scaffolds with poly(L-lactic acid). The honeycomb-mimic scaffolds with tunable pore size ranging from 70 to 160µm are fabricated by crystal needle-guided thermally induced phase separation in a directional freezing apparatus. The compressive modulus of the honeycomb-mimic scaffold is ≈4 times higher than that of scaffold with randomly oriented pore structure. The fabricated honeycomb-mimic scaffold exhibits a hierarchical structure from nanofibers to micro-/macro-tubular structures. Pre-osteoblast MC3T3-E1 cells cultured on the honeycomb-mimic nanofibrous scaffolds exhibit an enhanced osteoblastic phenotype, with elevated expression levels of osteogenic marker genes, than those on either porous lamellar scaffolds or porous scaffolds with randomly oriented pores. The advanced techniques for the fabrication of the honeycomb-mimic structure may potentially be used for a wide variety of advanced functional materials.

Enhanced osteogenic response by collagen type I coating on surface-modified titanium bone implants

Surface modifications of titanium (Ti) bone implants can activate an osteogenic response and accelerate osseointegration after implantation. In this study, in vitro tests were performed to evaluate different titanium dioxide TiO2 modified surfaces, sandblasted, large grit, acid-etched implant surfaces and surfaces featuring nanotubular structures (nanopore diameters of 20 and 100 nm) produced through potentiostatic anodization before and after subjecting them to covalent treatment for functionalization and subsequent coating with collagen type I (Col I). The physicochemical and morphological properties were evaluated using spectroscopy and microscopy, which confirmed the structural and biochemical modifications of the surfaces after coating with Col I. Studies with pre-osteoblastic MC3T3-E1 cells indicate improved cell adhesion and proliferation on surfaces coated with Col I, whereas cell viability remained similar for all evaluated surfaces. The results of alkaline phosphatase expression and activity, as well as a mineralization assay, demonstrated that surfaces coated with Col I acted positively on the osteogenic differentiation of the cells in comparison with the non-coated surfaces. Our findings suggest that, regardless of the previous modification of the TiO2 surface, the addition of a Col I coating presents a promising modification for bone implant applications.

Molecular mechanism of resveratrol promoting differentiation of preosteoblastic MC3T3-E1 cells based on network pharmacology and experimental validation

The purpose of this study was to investigate the mechanism by which resveratrol (Res) inhibits apoptosis and promotes proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells, laying the groundwork for the treatment of osteoporosis (OP). The TCMSP database was used to find the gene targets for Res. The GeneCards database acquire the gene targets for OP. After discovering the potential target genes, GO, KEGG, and Reactome enrichment analysis were conducted. Verifying the major proteins involved in apoptosis can bind to Res using molecular docking. CCK8 measured the proliferative activity of mouse pre-osteoblasts in every group following Res intervention. Alkaline phosphatase staining (ALP) and alizarin red staining to measure the ability of osteogenic differentiation. RT-qPCR to determine the expression levels of Runx2 and OPG genes for osteogenic differentiation ability of cells. Western blot to measure the degree of apoptosis-related protein activity in each group following Res intervention. The biological processes investigated for GO of Res therapeutic OP involved in cytokine-mediated signaling pathway, negative regulation of apoptotic process, Aging, extrinsic apoptotic signaling pathway in absence of ligand, according to potential therapeutic target enrichment study. Apoptosis, FoxO signaling pathway, and TNF signaling pathway are the primary KEGG signaling pathways. Recactome pathways are primarily engaged in Programmed Cell Death, Apoptosis, Intrinsic Apoptotic Pathway, and Caspase activation via extrinsic apoptotic signaling pathways. This research established a new approach for Res treatment of OP by demonstrating how Res controls the apoptosis-related proteins TNF, IL6, and CASP3 to suppress osteoblast death and increase osteoclastogenesis.

Exploring the Mechanisms of Sanguinarine in the Treatment of Osteoporosis by Integrating Network Pharmacology Analysis and Deep Learning Technology.

Sanguinarine (SAN) has been reported to have antioxidant, antiinflammatory, and antimicrobial activities with potential for the treatment of osteoporosis (OP). This work purposed to unravel the molecular mechanisms of SAN in the treatment of OP. OP-related genes and SAN-related targets were predicted from public databases. Differential expression analysis and VennDiagram were adopted to detect SAN-related targets against OP. Protein-protein interaction (PPI) network was served for core target identification. Molecular docking and DeepPurpose algorithm were further adopted to investigate the binding ability between core targets and SAN. Gene pathway scoring of these targets was calculated utilizing gene set variation analysis (GSVA). Finally, we explored the effect of SAN on the expressions of core targets in preosteoblastic MC3T3-E1 cells. A total of 21 candidate targets of SAN against OP were acquired. Furthermore, six core targets were identified, among which CASP3, CTNNB1, and ERBB2 were remarkably differentially expressed in OP and healthy individuals. The binding energies of SAN with CASP3, CTNNB1, and ERBB2 were -6, -6.731, and -7.162 kcal/mol, respectively. Moreover, the GSVA scores of the Wnt/calcium signaling pathway were significantly lower in OP cases than in healthy individuals. In addition, the expression of CASP3 was positively associated with Wnt/calcium signaling pathway. CASP3 and ERBB2 were significantly lower expressed in SAN group than in DMSO group, whereas the expression of CTNNB1 was in contrast. CASP3, CTNNB1, and ERBB2 emerge as potential targets of SAN in OP prevention and treatment.

Improvement of Osteogenic Differentiation of Mouse Pre-Osteoblastic MC3T3-E1 Cells on Core-Shell Polylactic Acid/Chitosan Electrospun Scaffolds for Bone Defect Repair.

Electrospun hybrid scaffolds composed of synthetic and natural polymers have gained increasing interest in tissue engineering applications over the last decade. In this work, scaffolds composed of polylactic acid electrospun fibers, either treated (P-PLA) or non-treated (PLA) with air-plasma, were coated with high molecular weight chitosan to create a core-shell microfibrous structure. The effective thickness control of the chitosan layer was confirmed by gravimetric, spectroscopic (FTIR-ATR) and morphological (SEM) investigations. The chitosan coating increased the fiber diameter of the microfibrous scaffolds while the tensile mechanical tests, conducted in dry and wet environments, showed a reinforcing action of the coating layer on the scaffolds, in particular when deposited on P-PLA samples. The stability of the Chi coating on both PLA and P-PLA substrates was confirmed by gravimetric analysis, while their mineralization capacity was evaluated though scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) after immersing the scaffolds in simulated body fluids (SBF) at 37 °C for 1 week. Sample biocompatibility was investigated through cell viability assay and SEM analysis on mouse pre-osteoblastic MC3T3-E1 cells grown on scaffolds at different times (1, 7, 14 and 21 days). Finally, Alizarin Red assay and qPCR analysis suggested that the combination of plasma treatment and chitosan coating on PLA electrospun scaffolds influences the osteoblastic differentiation of MC3T3-E1 cells, thus demonstrating the great potential of P-PLA/chitosan hybrid scaffolds for bone tissue engineering applications.

Physicochemical and biological properties of encapsulated Boesenbergia rotunda extract with different wall materials in enhancing antioxidant, mineralogenic and osteogenic activities of MC3T3-E1 cells

Boesenbergia rotunda(L.) comprises bioactive compounds with biological and pharmacological properties, especially flavonoid compounds with osteoblastogenesis-stimulating potential. However, the application ofB. rotundain the food and pharmaceutical industry is restricted by its low solubility and stability. Encapsulation becomes an alternative to overcome these restrictions. The purpose study was to encapsulateB. rotundaextract by freeze-drying and to investigate the effects of different wall materials (maltodextrin (MD), gum arabic (GA), and their combination (MDGA)) and extract contents on the physicochemical, bioactive properties and in vitro enhancement of osteogenesis of MC3T3-E1 cells of the obtained encapsulates. The results revealed that encapsulatedB. rotundacan reduce cytotoxicity, enhance biological activity, and maintain the stability of bioactive compounds. The MD was a good wall material for yield percentage. However, the values of moisture content Aw, and solubility among all the encapsulated powders were no significant differences, with all encapsulated powders having similar structures based on scanning electron microscopy. Fourier transform infrared spectroscopy confirmed the extract was encapsulated by the selected wall materials. Combining the MD and GA encapsulation agents afforded the best protection of the bioactive compounds, increasing EE (MDGA-7 > MDGA-5), pinostrobin content, TPC, and antioxidant activities (MDGA-5 > MDGA-7). The MDGA-5 and MDGA-7 at 10–50 μg/mL were not toxic to cells and promoted MC3T3-E1 cell viability, while also enhancing the Alkaline phosphatase activity, and promoting matrix mineralization of pre-osteoblast MC3T3-E1 cells after 21 and 28 days. This result showed that MDGA was a suitable wall material for B. rotunda encapsulations and a potential source of bioactive ingredients that could applied in food or pharmaceutical products for osteoporosis prevention.

Effect of recombinant human bone morphogenetic protein-2 and osteoprotegerin-Fc in MC3T3-E1 cells.

We compared the osteoblastogenesis by serially administrating recombinant human bone morphogenetic protein-2 (rhBMP-2) and osteoprotegerin-immunoglobulin Fc segment complex (OPG-Fc). The MC3T3-E1 preosteoblast cell line was differentiated for 1, 3, and 7 days with a treatment of OPG-Fc in 10~200 ng/mL concentration and the cell viability was evaluated by Cell Counting Kit-8 analysis. The level of differentiation from MC3T3-E1 cells to osteoblasts was determined by alkaline phosphatase activity. The level of runt domain-containing transcription factor 2 (Runx2) and osteopontin (OPN) manifestation, involved in osteoblast differentiation, was examined by real-time polymerase chain reaction and western blotting. During MC3T3-E1 cell differentiation, the differentiation level was high with 1-day treatment using 100 ng/mL OPG-Fc. The treatment with 50 ng/mL rhBMP-2 for 7 days, followed by 1-day treatment with 100 ng/mL OPG-Fc produced the highest differentiation level, which was approximately 5.3 times that of the control group (p<0.05). The expression of Runx2 mRNA significantly increased, reaching 2.5 times the level of the control group under the condition of 7-day treatment with rhBMP-2 and 1-day treatment with OPG-Fc (p<0.001). The expression of Runx2 protein significantly increased to approximately 5.7 times that of the control group under the condition of 7-day treatment with rhBMP-2, followed by 1-day treatment with OPG-Fc (p<0.01). The expression of OPN protein showed no change from that of the control group under various conditions of rhBMP-2 and OPG-Fc combinations. These results imply that the treating preosteoblasts with rhBMP-2 first and then with OPG-Fc increased osteoblast differentiation efficacy.