Mouse Pre-osteoblast MC3T3-E1 Research Articles

Versatile cell cultivation on injectable poly(butylene adipate-co-terephthalate) microcarriers: Impact of surface properties across different cell types.

Injectable cell therapies offer several advantages compared with traditional open surgery, including less trauma to the patient, shorter recovery time, and lower risk of infection. However, a significant problem is the difficulty in developing effective cell delivery carriers that are cyto-compatible and maintain cell viability both during and after injection. In the presented study, it was aimed to develop poly(butylene adipate-co-terephthalate) (PBAT) microcarriers using the emulsion preparation-solvent evaporation technique. The optimized diameter of the PBAT microcarriers was determined as 104 ± 15 μm at 700 rpm and there would be no blockage after injection due to the nonswelling feature of microcarriers. Furthermore, the cellular activities of PBAT microcarriers were evaluated in static culture for 7 days using L929 mouse fibroblasts, MC3T3-E1 mouse pre-osteoblasts, and rat adipose-derived mesenchymal cells (AdMSCs). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide results and Sscanning electron microscope images showed that PBAT microcarriers increased the adhesion and proliferation properties of pre-osteoblasts and stem cells, while L929 fibroblasts formed aggregates by adhering to certain regions of the microcarrier surface and did not spread on the surface. These results emphasize that PBAT microcarriers can be used as injectable carriers, especially in stem cell therapies, but their surface properties need to be modified for some cells.

Emulsion electrospun epigallocatechin gallate-loaded silk fibroin/polycaprolactone nanofibrous membranes for enhancing guided bone regeneration

Guided bone regeneration (GBR) membranes play an important role in oral bone regeneration. However, enhancing their bone regeneration potential and antibacterial properties is crucial. Herein, silk fibroin (SF)/polycaprolactone (PCL) core-shell nanofibers loaded with epigallocatechin gallate (EGCG) were prepared using emulsion electrospinning. The nanofibrous membranes were characterized via scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, water contact angle (CA) measurement, mechanical properties testing, drug release kinetics, and 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) free radical scavenging assay. Mouse pre-osteoblast MC3T3-E1 cells were used to assess the biological characteristics, cytocompatibility, and osteogenic differentiation potential of the nanofibrous membrane. Additionally, the antibacterial properties againstStaphylococcus aureus (S. aureus)andEscherichia coli (E. coli)were evaluated. The nanofibers prepared by emulsion electrospinning exhibited a stable core-shell structure with a smooth and continuous surface. The tensile strength of the SF/PCL membrane loaded with EGCG was 3.88 ± 0.15 Mpa, the water CA was 50°, and the DPPH clearance rate at 24 h was 81.73% ± 0.07%. The EGCG release rate of membranes prepared by emulsion electrospinning was reduced by 12% within 72 h compared to that of membranes prepared via traditional electrospinning.In vitroexperiments indicate that the core-shell membranes loaded with EGCG demonstrated good cell compatibility and promoted adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells. Furthermore, the EGCG-loaded membranes exhibited inhibitory effects onE. coliandS. aureus. These findings indicate that core-shell nanofibrous membranes encapsulated with EGCG prepared using emulsion electrospinning possess good antioxidant, osteogenic, and antibacterial properties, making them potential candidates for research in GBR materials.

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.

Blackcurrant extract promotes differentiation of MC3T3‑E1 pre‑osteoblasts.

Osteoporosis risk increases in menopausal individuals owing to the decrease in estrogen secretion. Blackcurrant extract (BCE) ameliorates osteoporosis; however, the underlying mechanisms are unclear. Furthermore, although BCE has phytoestrogenic activity, its effects on osteoblasts are unknown. In the present study, we investigated BCE-mediated attenuation of osteoporosis using mouse MC3T3-E1 pre-osteoblasts, with a focus on osteogenesis. After treating MC3T3-E1 cells with BCE for 48 h, cell proliferation was assessed using Cell Counting Kit-8. Levels of osteoblast differentiation markers, namely alkaline phosphatase activity and total collagen content in the cells, were evaluated after 3 and 14 days of BCE treatment, respectively. The expression of genes encoding osteoblast differentiation markers, including collagen type I (Col-I), alkaline phosphatase (Alp), bone γ-carboxyglutamate protein (Bglap), and runt-related transcription factor 2 (Runx2), was evaluated using reverse transcription-quantitative polymerase chain reaction. Mineralization of the cells was evaluated using Alizarin Red staining. Femoral tissues of ovariectomized (OVX) rats with or without 3% BCE were stained using ALP to evaluate osteogenic differentiation in femoral tissue. After treating MC3T3-E1 cells with BCE, cell proliferation had increased. BCE treatment increased Alp activity and total collagen content. Moreover, the expression of Col-I, Alp, Bglap, and Runx2 increased in BCE-treated cells. Furthermore, when MC3T3-E1 cells were treated with BCE for 21 days, the levels of calcified nodules increased. Alp staining intensity was stronger in the epiphyses on femoral tissue of OVX rats treated with 3% BCE than in those of untreated OVX rats. The results suggest that BCE may promote osteogenesis by inducing osteoblast differentiation.

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.

Peroxiredoxin 1 promotes proliferation and inhibits differentiation of MC3T3-E1 cells via AKT1 / NF-κB signaling pathway

ObjectivesOsteoporosis is the most common metabolic bone disease worldwide. The decrease in bone mass is primarily accompanied by a decrease in the number and activity of osteoblasts. Peroxiredoxins (PRDXs) are proteins that detect extremely low peroxide levels and act as sensors that regulate oxidation signals, thereby regulating various cellular functions. This study aimed to evaluate the effects of PRDX1 and estrogen on the biological behavior of osteoblasts, including their proliferation and differentiation. MethodsOvariectomized (OVX) mice were used to establish a model of osteoporosis and perform morphological and immunohistochemical analyses. Prdx1 gene knockout and overexpression were performed in mouse MC3T3-E1 pre-osteoblasts to assess proliferation and osteogenic differentiation using the cell counting kit-8, quantitative reverse transcription polymerase chain reaction, western blotting (WB), Alizarin Red S staining, etc. ResultsThe OVX mice exhibited osteoporosis and PRDX1 expression increased. In vitro experiments showed that during the osteogenic differentiation of osteoblasts, PRDX1 expression decreased, while the expression of COL1 and RUNX2 increased. After Prdx1 knockout, the proliferation of osteoblasts decreased; expression of Runx2, ALP, and COL1 increased; and mineralization increased. However, after Prdx1 overexpression, osteoblast proliferation was enhanced, whereas osteogenic differentiation and mineralization were inhibited. Estrogen inhibits the H2O2-induced decrease in osteoblastic differentiation and increase in PRDX1 expression. WB revealed that when LY294002 inhibited the AKT signaling pathway, the levels of p-AKT1, p-P65, and PRDX1 protein in MC3T3-E1 cells decreased. However, when pyrrolidine dithiocarbamate (PDTC) inhibited the NF-κB signaling pathway, the expression of p-AKT1 and PRDX1 did not change except for a significant reduction of p-P65 expression. Furthermore, PDTC reversed the decreased expression of RUNX2, ALP, and COL1 caused by PRDX1 overexpression. ConclusionsPRDX1 promotes the proliferation of osteoblasts and inhibits osteogenic differentiation. Estrogen regulated osteoblastic differentiation by affecting the expression of PRDX1 in osteoblasts, and the effect is related to the AKT1/NF-κB signaling pathway.

Human umbilical cord mesenchymal stem cells protect MC3T3-E1 osteoblasts from dexamethasone-induced apoptosis via induction of the Nrf2-ARE signaling pathway

ObjectiveTo investigate the protective effect human umbilical cord mesenchymal stem cells (hUC-MSCs) have on Dexamethasone (Dex)-induced apoptosis in osteogenesis via the Nrf2-ARE signaling pathway. MethodsGlucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) was developed in rats through the administration of lipopolysaccharide and methylprednisolone. The incidence of femoral head necrosis, cavity notch, apoptosis of osteoblasts, and bone density were observed by HE staining, TUNEL staining, and Micro-CT. HUC-MSCs were co-cultured with mouse pre-osteoblast MC3T3-E1. The survival rate of osteoblasts was determined by CCK8, and apoptosis and ROS levels of osteoblasts were determined by flow cytometer. The viability of antioxidant enzymes SOD, GSH-Px, and CAT was analyzed by biochemistry. Nrf2 expression levels and those of its downstream proteins and apoptosis-related proteins were analyzed by Western blotting. ResultsIn rats, hUC-MSCs can reduce the rates of empty bone lacuna and osteoblast apoptosis that are induced by glucocorticoids (GCs), while reducing the incidence of GC-ONFH. hUC-MSCs can significantly improve the survival rate and antioxidant SOD, GSH-Px, and CAT activity of MC3T3-E1 cells caused by Dex, and inhibit apoptosis and oxidative stress levels. In addition, hUC-MSCs can up-regulate the expression of osteoblast antioxidant protein Nrf2 and its downstream protein HO-1, NQO-1, GCLC, GCLM, and apoptosis-related protein bcl-2, while also down-regulating the expression of apoptosis-related protein bax, cleaved caspase-3, cleaved caspase-9, and cytochrome C in MC3T3-E1 cells. hUC-MSCs improve the ability of MC3T3-E1 cells to mineralize to osteogenesis. However, the promoting effects of hUC-MSCs were abolished following the blocking of the Nrf2-ARE signaling pathway for osteoblasts. ConclusionThe results reveal that hUC-MSCs can reduce Dex-induced apoptosis in osteoblasts via the Nrf2-ARE signaling pathway.

Exploring the mechanical strength, antimicrobial performance, and bioactivity of 3D-printed silicon nitride-PEEK composites in cervical spinal cages

 In this study, our goal was to assess the suitability of a polyether-ether-ketone (PEEK) and silicon nitride (Si3N4) polymer composite for antimicrobial three-dimensional (3D)-printed cervical cages. Generic cage designs (PEEK and 15 vol.% Si3N4-PEEK) were 3D-printed, including solid and porous cage designs. Cages were tested in static compression, compression shear, and torsion per ASTM F2077. For antibacterial testing, virgin and composite filament samples were inoculated with Staphylococcus epidermidis and Escherichia coli. In vitro cell testing was conducted using MC3T3-E1 mouse preosteoblasts, where cell proliferation, cumulative mineralization, and osteogenic activity were measured. The 3D-printed PEEK and Si3N4-PEEK cages exhibited adequate mechanical strength for all designs, exceeding 14.7 kN in compression and 6.9 kN in compression shear. Si3N4-PEEK exhibited significantly lower bacterial adhesion levels, with a 93.9% reduction (1.21 log), and enhanced cell proliferation when compared to PEEK. Si3N4-PEEK would allow for custom fabrication of 3D-printed spinal implants that reduce the risk of infection compared to unfilled PEEK or metallic alloys.  

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.

Production and Characterization of Bioactive and Antimicrobial Titanium Oxide Surfaces with Silver Nanoparticles and a Poly(lactic Acid) Microfiber Coating

Silver nanoparticles (AgNp) were deposited on highly porous TiO2 surfaces by the dripping of a colloidal AgNp solution to provide antimicrobial activity. Micro-porous TiO2 surfaces were obtained on commercially pure titanium by micro-arc oxidation in an electrolyte containing Ca and P precursors. In addition, as silver can be toxic to cells, these surfaces were uniformly covered with the biocompatible and bioresorbable poly(lactic acid) (PLA) polymer by electrospinning, aiming at promoting a controlled release of silver ions to the medium. The resulting AgNp-containing surfaces were characterized by scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and X-ray diffraction (XRD), and in vitro assays were performed to evaluate their antimicrobial activity and bioactivity. Tests revealed that the surfaces showed antimicrobial activity against Staphylococcus aureus, with better results for the surfaces without PLA. However, all the surfaces presented good biocompatibility in assays with mouse MC3T3-E1 pre-osteoblasts, and greater cell differentiation for the polymer-coated surfaces. Finally, the PLA ultrafine fibers electrospun on the TiO2/AgNp surfaces allowed a controlled release of silver ions in the phosphate-buffered saline (PBS) medium.

Isofraxidin inhibits the proliferation and differentiation of the osteoblast MC3T3-E1 subclone 14 cell line.

Ankylosing spondylitis (AS) is an autoimmune inflammatory disease associated with joint inflammation and destruction. Current treatment modalities alleviate symptoms; however, they cannot cure the disease and are associated with significant side effects. Thus, we aimed to confirm the inhibitory effect of isofraxidin, a herbal extract, on pathological osteogenesis in ankylosing spondylitis to better treat patients affected by the disease. Mouse preosteoblast MC3T3-E1 subclone 14 cells were used in vitro to establish control and isofraxidin intervention groups. Cell viability was then determined using the MTT assay; the expression of osteogenic factors, including Runx2, OSX, collagen I, and ALP was measured using qRT-PCR and western blotting. Final osteogenic mineralization was performed by alizarin red staining. The results showed that isofraxidin could inhibit osteoblast viability; however, this effect was nullified at concentrations of 0-20 µM after adding 1% serum. Gene and protein expression of the osteogenic factors RUNX2, OSX, Collagen I, and ALP was inhibited, and a similar trend was exhibited at 7, 14, and 21 days after isofraxidin treatment. This trend was further verified by alizarin red staining of the final osteogenic mineralized nodules on days 7, 14, 21, and 35. Isofraxidin inhibits MC3T3-E1 subclone 14 proliferation and differentiation and may be considered a potential drug therapy for treating pathological osteogenesis in ankylosing spondylitis.

Obtaining of Antibacterial Nanoporous Layer on Ti7.5Mo Alloy Surface Combining Alkaline Treatment and Silver: In Vitro Studies

In the present study, a combination of alkaline treatment and silver was used to produce an antibacterial nanolayer on the Ti7.5Mo alloy surface. The antibacterial response and osteogenesis were evaluated by assessing the adhesion and proliferation of S. aureus and S. epidermidis, as well as the adhesion, viability, and expression levels of genes involved in osteogenic differentiation in the mouse pre-osteoblast cell line MC3T3-E1. The potential stimulus of extracellular remodeling was evaluated using zymography. Our results showed that there is no difference in cytotoxicity after silver immobilization. Protein activity (MMP9) progressively increased for theTi7.5Mo alloy, both untreated and after alkaline treatment. However, the highest increase in protein activity was observed when the alloy was in direct contact with immobilized silver nanoparticles. The surfaces containing silver showed a better response in terms of colony formation, meaning that less bacterial adhesion was detected. The results showed that the layer formed was effective in reducing bacterial activity without altering cell viability.

FBXO11 regulates bone development.

FBXO11 is the substrate-recognition component of a ubiquitin ligase complex called SKP1-cullin-F-boxes. The role of FBXO11 in bone development is unexplored. In this study, we reported a novel mechanism of how bone development is regulated by FBXO11. FBXO11 gene knockdown by lentiviral transduction in mouse pre-osteoblast MC3T3-E1 cells leads to reduced osteogenic differentiation, while overexpressing FBXO11 accelerates their osteogenic differentiation in vitro. Furthermore, we generated two osteoblastic-specific FBXO11 conditional knockout mouse models, Col1a1-ERT2-FBXO11KO and Bglap2-FBXO11KO mice. In both conditional FBXO11KO mouse models, we found FBXO11 deficiency inhibits normal bone growth, in which the osteogenic activity in FBXO11cKO mice is reduced, while osteoclastic activity is not significantly changed. Mechanistically, we found FBXO11 deficiency leads to Snail1 protein accumulation in osteoblasts, leading to suppression of osteogenic activity and inhibition of bone matrix mineralization. FBXO11 knockdown in MC3T3-E1 cells reduced Snail1 protein ubiquitination and increased Snail1 protein accumulation in the cells, which eventually inhibited osteogenic differentiation. In conclusion, FBXO11 deficiency in osteoblasts inhibits bone formation through Snail1 accumulation, inhibiting osteogenic activity and bone mineralization.

Resveratrol induces proliferation and differentiation of mouse pre-osteoblast MC3T3-E1 by promoting autophagy

BackgroundIn mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact.Methods(1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention.Results(1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group’s alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05).ConclusionThe present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.

Fish scale containing alginate dialdehyde-gelatin bioink for bone tissue engineering

The development of biomaterial inks suitable for biofabrication and mimicking the physicochemical properties of the extracellular matrix is essential for the application of bioprinting technology in tissue engineering (TE). The use of animal-derived proteinous materials, such as jellyfish collagen, or fish scale (FS) gelatin (GEL), has become an important pillar in biomaterial ink design to increase the bioactivity of hydrogels. However, besides the extraction of proteinous structures, the use of structurally intact FS as an additive could increase biocompatibility and bioactivity of hydrogels due to its organic (collagen) and inorganic (hydroxyapatite) contents, while simultaneously enhancing mechanical strength in three-dimensional (3D) printing applications. To test this hypothesis, we present here a composite biomaterial ink composed of FS and alginate dialdehyde (ADA)-GEL for 3D bioprinting applications. We fabricate 3D cell-laden hydrogels using mouse pre-osteoblast MC3T3-E1 cells. We evaluate the physicochemical and mechanical properties of FS incorporated ADA-GEL biomaterial inks as well as the bioactivity and cytocompatibility of cell-laden hydrogels. Due to the distinctive collagen orientation of the FS, the compressive strength of the hydrogels significantly increased with increasing FS particle content. Addition of FS also provided a tool to tune hydrogel stiffness. FS particles were homogeneously incorporated into the hydrogels. Particle-matrix integration was confirmed via scanning electron microscopy. FS incorporation in the ADA-GEL matrix increased the osteogenic differentiation of MC3T3-E1 cells in comparison to pristine ADA-GEL, as FS incorporation led to increased ALP activity and osteocalcin secretion of MC3T3-E1 cells. Due to the significantly increased stiffness and supported osteoinductivity of the hydrogels, FS structure as a natural collagen and hydroxyapatite source contributed to the biomaterial ink properties for bone engineering applications. Our findings indicate that ADA-GEL/FS represents a new biomaterial ink formulation with great potential for 3D bioprinting, and FS is confirmed as a promising additive for bone TE applications.

Gene Expression in MC3T3-E1 Cells Treated with Diclofenac and Methylprednisolone.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids (GCs) are often used to treat articular-skeletal disorders. The extended use of NSAIDs and GCs have adverse effects on bone metabolism, reducing bone quality and impairing fracture healing. In the present study, we used mouse pre-osteoblast cells MC3T3-E1 to demonstrate the effects of diclofenac (DF) and methylprednisolone (MP) on cell proliferation and gene expression. Cells were incubated with three doses of DF or MP: 0.5 µM, 5 µM, and 50 µM. MP decreased cell viability even after 24 h, but DF inhibited cell viability after only seven days of treatment. The cells were lysed after one, two, three, and seven days of treatment, and gene expression was analyzed by reverse transcription and quantitative PCR (RT-qPCR) assays. DF did not significantly affect the expression of the osteogenic marker genes. MP modified the expression of Osx, Runx, and Col1a1. We concluded that MP is a more potent inhibitor of mouse pre-osteoblast differentiation and viability than is DF. Our results suggest that prolonged DF treatment could be less harmful to osteoblasts than MP treatment.

Drug/bioactive eluting chitosan composite foams for osteochondral tissue engineering

Joint defects associated with a variety of etiologies often extend deep into the subchondral bone leading to functional impairment and joint immobility, and it is a very challenging task to regenerate the bone-cartilage interface offering significant opportunities for biomaterial-based interventions to improve the quality of life of patients. Herein drug-/bioactive-loaded porous tissue scaffolds incorporating nano-hydroxyapatite (nHAp), chitosan (CS) and either hydroxypropyl methylcellulose (HPMC) or Bombyx mori silk fibroin (SF) are fabricated through freeze drying method as subchondral bone substitute. A combination of spectroscopy and microscopy (Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX), and X-ray fluorescence (XRF) were used to analyze the structure of the porous biomaterials. The compressive mechanical properties of these scaffolds are biomimetic of cancellous bone tissues and capable of releasing drugs/bioactives (exemplified with triamcinolone acetonide, TA, or transforming growth factor-β1, TGF-β1, respectively) over a period of days. Mouse preosteoblast MC3T3-E1 cells were observed to adhere and proliferate on the tissue scaffolds as confirmed by the cell attachment, live-dead assay and alamarBlue™ assay. Interestingly, RT-qPCR analysis showed that the TA downregulated inflammatory biomarkers and upregulated the bone-specific biomarkers, suggesting such tissue scaffolds have long-term potential for clinical application.

Protective Effect of Photobiomodulation against Hydrogen Peroxide-Induced Oxidative Damage by Promoting Autophagy through Inhibition of PI3K/AKT/mTOR Pathway in MC3T3-E1 Cells.

Photobiomodulation (PBM) has been repeatedly reported to play a major role in the regulation of osteoblast proliferation and mineralization. Autophagy is closely associated with various pathophysiological processes in osteoblasts, while its role in oxidative stress is even more critical. However, there is still no clear understanding of the mechanism of the role of autophagy in the regulation of osteoblast mineralization and apoptosis under oxidative stress by PBM. It was designed to investigate the impact of 808 nm PBM on autophagy and apoptosis in mouse preosteoblast MC3T3-E1 treated with hydrogen peroxide (H2O2) through PI3K/AKT/mTOR pathway. PBM could inhibit MC3T3-E1 cell apoptosis under oxidative stress and promote the expression of osteogenic proteins, while enhancing the level of autophagy. In contrast, 3-methyladenine (3-MA) inhibited the expression of osteoblast autophagy under oxidative stress conditions, increased apoptosis, and plus counteracted the effect of PBM on osteoblasts. We also found that PBM suppressed the activated PI3K/AKT/mTOR pathway during oxidative stress and induced autophagy in osteoblasts. PBM promoted autophagy of MC3T3 cells and was further blocked by 740 Y-P, which reversed the effect of PBM on MC3T3 cells with H2O2. In conclusion, PBM promotes autophagy and improves the level of osteogenesis under oxidative stress by inhibiting the PI3K/AKT/mTOR pathway. Our results can lay the foundation for the clinical usage of PBM in the treatment of osteoporosis.

Involvement of PI3K/Akt signaling pathway in promoting osteogenesis on titanium implant surfaces modified with novel non-thermal atmospheric plasma.

Non-thermal atmospheric plasma (NTAP) modification to induce a hydrophilic titanium (Ti) surface with less carbon contamination, has been demonstrated to boost the osteogenic responses. In this study, we investigated the underlying bone formation mechanism of NTAP-Ti, and the involvement of PI3K/Akt signaling pathway in regulating osteogenic activities on NTAP-Ti surfaces. NTAP was employed for Ti activation, and PI3K inhibitor, LY294002, was applied to the suppression of PI3K/Akt pathway. We systematically and quantitatively detected the cell morphology, attachment, proliferation, osteogenic differentiation and mineralization of MC3T3-E1 mouse preosteoblasts, and molecular expressions involved in osteogenesis and PI3K/Akt signaling pathway in vivo and in vitro. A descent in osteoblast proliferation on Ti surfaces in relation to LY294002. Alkaline phosphatase (ALP) activity, as well as matrix mineralization, was mitigated by PI3K inhibitor in NTAP-Ti. Likewise, the expression levels of osteogenesis-related genes [ALP, osteocalcin (Ocn), osteopontin (Opn) and runt-related transcription factor 2 (Runx2)] on NTAP-Ti were notably attenuated by LY294002, as confirmed by the results of osteogenesis-related proteins (ALP, and Runx2) expression analysis. In addition, the expression of PI3K/Akt signal pathway proteins further verified the inhibition of LY294002 on Ti surfaces modified by NTAP. Collectively, the PI3K/Akt signal pathway was involved in the amelioration of osteogenesis induced by NTAP modification. NTAP treatment for Ti activation is promising in augmented osteogenic potential through the activation of PI3K/Akt signal pathway.

The promoting effect of the TiO2 nano-porous arrays after polydopamine-based modification and Arg-Gly-Asp peptide immobilization on biomineralization and osteogenesis

TiO2 nano-porous arrays with appropriate structural parameters, roughness and wettability have application potential in implantable titanium-based (Ti-based) alloys. In this research, the polydopamine (PDA) and Arg-Gly-Asp (RGD) peptide are adopted to construct a favorable osteogenic microenvironment on the anodized TiO2 nano-porous arrays before and after heat treatment. The characterization of surface morphologies and chemical compositions has been conducted to confirm the feasibility of the PDA-based modification and RGD peptide immobilization. Via immersion in the modified-simulated body fluid (m-SBF) for 7 days, the PDA-based modified samples have represented enhanced biomineralization performance. Moreover, the cell adhesion and proliferation assay, alkaline phosphatase (ALP) activity assay and quantitative real-time polymerase chain reaction (qRT-PCR) assay in vitro have indicated that the heat-treated TiO2 nano-porous arrays modified with PDA and RGD peptide have endowed the seeded MC3T3-E1 mouse pre-osteoblasts with excellent osteogenic performance. Such modified TiO2 nano-porous arrays have great latent capacity in implantable materials for biomineralization and osteogenesis.