Differentiation Of MC3T3-E1 Cells Research Articles

Zoledronic acid epigenetically alleviates high-glucose-suppressed osteogenic differentiation of MC3T3-E1 cells.

Due to the impact of excessive glucose on osteogenic differentiation, diabetic osteopathy frequently results in skeletal fragility, osteoporosis, and bone pain. Zoledronic acid, a bisphosphonate (BP) that effectively inhibits osteoclastic bone resorption is given yearly to improve bone mineral density (BMD) in patients with osteoporosis. However, the detailed molecular mechanisms remained unclear. This study investigates the possible pathways by which zoledronic acid regulates osteogenesis when blood glucose levels are high. MC3T3-E1 cells were treated with one mM zoledronic acid or not in a standard or high glucose culture medium. A quantitative polymerase chain reaction (qPCR) assay was utilized to assess the expression of the target candidate genes, including RUNX2, MALAT1, miR-133, miR-20a, and miR-204. In a high-glucose condition, zoledronic acid treatment significantly lowered MALAT1 (p < 0.0001) and miR-20a (p < 0.0001) expression. Conversely, in a high-glucose condition, RUNX2, miR-133, and miR-204 expressions were found to be significantly increased in the zoledronic acid treatment group as compared to no treatment (all p < 0.0001). In conclusion, under a high-glucose environment, zoledronic acid can modulate the expression of the RUNX2 transcription factor through epigenetic regulation.

Curculigo orchioides polysaccharide COP70-1 stimulates osteogenic differentiation of MC3T3-E1 cells by activating the BMP and Wnt signaling pathways

The crude polysaccharide CO70 isolated from Curculigo orchioides could alleviate ovariectomy-induced osteoporosis in rats. To clarify the bioactive components, a new heteropolysaccharide (COP70-1) was purified from CO70 in this study, which was consisted of β-D-Manp-(1→, →4)-α-D-Glcp-(1→, →4)-β-D-Manp-(1→, →3,4)-β-D-Manp-(1→, →4,6)-β-D-Manp-(1→, and →4,6)-α-D-Galp-(1→. COP70-1 significantly promoted the osteoblastic differentiation of MC3T3-E1 cells through improving alkaline phosphatase activity, the deposition of calcium as well as up-regulating the expression of osteogenic markers (RUNX2, OSX, BSP, OCN, and OPN). Furthermore, COP70-1 stimulated the expression of critical transcription factors of the BMP and Wnt pathways, including BMP2, p-SMAD1, active-β-catenin, p-GSK-3β, and LEF-1. In addition, LDN (BMP pathway inhibitor) and DKK-1 (Wnt pathway inhibitor) suppressed the COP70-1-induced osteogenic differentiation of MC3T3-E1 cells. Therefore, COP70-1 was one of the bioactive constituents of C. orchioides for targeting osteoblasts to treat osteoporosis by triggering BMP/Smad and Wnt/β-catenin pathways.

Policosanol Stimulates Osteoblast Differentiation via Adenosine Monophosphate-Activated Protein Kinase-Mediated Expression of Insulin-Induced Genes 1 and 2.

Policosanol is known as a hypocholesterolemic compound and is derived from plants such as sugar cane and corn. Policosanol can lower blood pressure or inhibit adipogenesis, but its effect on osteogenic differentiation and the molecular mechanism is unclear. This study aims to investigate the effect of policosanol on osteogenic differentiation in MC3T3-E1 cells and zebrafish models. Administration of policosanol into MC3T3-E1 induced the expression of the osteogenic genes such as distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). Alkaline phosphatase activity and extracellular mineralization also increased. Policosanol promoted activation of adenosine monophosphate-activated protein kinase (AMPK) and insulin-induced genes (INSIGs) expression and regulation of INSIGs modulated osteoblast differentiation. AMPK activation through transfection of the constitutively active form of AMPK (CA-AMPK) increased INSIGs expression, whereas policosanol-induced INSIGs expression was suppressed by inhibitor of AMPK (Com. C). Furthermore, the osteogenic effects of policosanol were verified in zebrafish. Amputated caudal fin rays were regenerated by policosanol treatment. Taken together, these results show that policosanol increases osteogenic differentiation and contributes to fin regeneration in zebrafish via AMPK-mediated INSIGs expression, suggesting that policosanol has potential as an osteogenic agent.

Extracellular ATP-induced calcium oscillations regulating the differentiation of osteoblasts through aerobic oxidation metabolism pathways.

The increase of ATP concentration in the extracellular space represents one of the effective signals that stimulate the physiological activities of cells when the bone is exposed to external mechanical stimulation such as stretching and shear stress force throughout life. However, the effects of ATP on osteoblast differentiation and related mechanisms are not well understood. In this study, the roles of extracellular ATP on osteoblast differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. Our results showed that 100μM extracellular ATP initiated intracellular calcium ([Ca2+]i) oscillations via the calcium-sensing receptor (P2R) and promoted the differentiation of MC3T3-E1 cells. Metabolomics analysis showed that the differentiation of MC3T3-E1 cells depended on aerobic oxidation, but little glycolysis. Moreover, the differentiation of MC3T3-E1 cells and aerobic oxidation were suppressed with the inhibition of AMP-activated protein kinase (AMPK). These results indicate that calcium oscillations triggered by extracellular ATP can activate aerobic oxidation through AMPK-related signaling pathways and thus promote osteoblast differentiation.

Osteogenic and antibacterial PLLA membrane for bone tissue engineering

Insufficient bone regeneration and bacterial infection are two major concerns of bone repair materials. Poly-L-lactic acid (PLLA) have been widely used in bone tissue engineering (BTE), however, lack of osteogenic and antibacterial properties have greatly limit its clinical application. Herein, PLLA membrane was firstly treated with polydopamine (PDA), and then modified with ε-polylysine (ε-PL) and alginate (ALG) via layer-by-layer method. The (ε-PL/ALG)n composite layer coated PLLA (PLLA@(ε-PL/ALG)n) could facilitates the adhesion and osteoblast differentiation of MC3T3-E1 cells. Furthermore, PLLA@(ε-PL/ALG)n presents an effective antibacterial efficacy against S. aureus and E. coli, and the bacterial survival rates of S. aureus and E. coli on PLLA@(ε-PL/ALG)10 were 21.5 ± 3.5 % and 13 ± 2.1 %, respectively. This work provides a promising method to design PLLA materials with osteogenic and antibacterial activity simultaneously. Furthermore, the method is also an optional choice to construct multifunctional coatings on the other substrate.

Enhanced bone regeneration via local low-dose delivery of PTH1-34 in a composite hydrogel.

Introducing bone regeneration-promoting factors into scaffold materials to improve the bone induction property is crucial in the fields of bone tissue engineering and regenerative medicine. This study aimed to develop a Sr-HA/PTH1-34-loaded composite hydrogel system with high biocompatibility. Teriparatide (PTH1-34) capable of promoting bone regeneration was selected as the bioactive factor. Strontium-substituted hydroxyapatite (Sr-HA) was introduced into the system to absorb PTH1-34 to promote the bioactivity and delay the release cycle. PTH1-34-loaded Sr-HA was then mixed with the precursor solution of the hydrogel to prepare the composite hydrogel as bone-repairing material with good biocompatibility and high mechanical strength. The experiments showed that Sr-HA absorbed PTH1-34 and achieved the slow and effective release of PTH1-34. In vitro biological experiments showed that the Sr-HA/PTH1-34-loaded hydrogel system had high biocompatibility, allowing the good growth of cells on the surface. The measurement of alkaline phosphatase activity and osteogenesis gene expression demonstrated that this composite system could promote the differentiation of MC3T3-E1 cells into osteoblasts. In addition, the in vivo cranial bone defect repair experiment confirmed that this composite hydrogel could promote the regeneration of new bones. In summary, Sr-HA/PTH1-34 composite hydrogel is a highly promising bone repair material.

Osteocytes Exposed to Titanium Particles Inhibit Osteoblastic Cell Differentiation via Connexin 43

Periprosthetic osteolysis (PPO) induced by wear particles is the most severe complication of total joint replacement; however, the mechanism behind PPO remains elusive. Previous studies have shown that osteocytes play important roles in wear-particle-induced osteolysis. In this study, we investigated the effects of connexin 43 (Cx43) on the regulation of osteocyte-to-osteoblast differentiation. We established an in vivo murine model of calvarial osteolysis induced by titanium (Ti) particles. The osteolysis characteristic and osteogenesis markers in the osteocyte-selective Cx43 (CKO)-deficient and wild-type (WT) mice were observed. The calvarial osteolysis induced by Ti particles was partially attenuated in CKO mice. The expression of β-catenin and osteogenesis markers increased significantly in CKO mice. In vitro, the osteocytic cell line MLO-Y4 was treated with Ti particles. The co-culturing of MLO-Y4 cells with MC3T3-E1 osteoblastic cells was used to observe the effects of Ti-treated osteocytes on osteoblast differentiation. When Cx43 of MLO-Y4 cells was silenced or overexpressed, β-catenin was detected. Additionally, co-immunoprecipitation detection of Cx43 and β-catenin binding in MLO-Y4 cells and MC3T3-E1 cells was performed. Finally, β-catenin expression in MC3T3-E1 cells and osteoblast differentiation were evaluated after 18α-glycyrrhetinic acid (18α-GA) was used to block the intercellular communication of Cx43 between MLO-Y4 and MC3T3-E1 cells. Ti particles increased Cx43 expression and decreased β-catenin expression in MLO-Y4 cells. The silencing of Cx43 increased the β-catenin expression, and the over-expression of Cx43 decreased the β-catenin expression. In the co-culture model, Ti treatment of MLO-Y4 cells inhibited the osteoblastic differentiation of MC3T3-E1 cells and Cx43 silencing in MLO-Y4 cells attenuated the inhibitory effects on osteoblastic differentiation. With Cx43 silencing in the MLO-Y4 cells, the MC3T3-E1 cells, co-cultured alongside MLO-Y4, displayed decreased Cx43 expression, increased β-catenin expression, activation of Runx2, and promotion of osteoblastic differentiation in vitro co-culture. Finally, Cx43 expression was found to be negatively correlated to the activity of the Wnt signaling pathway, mostly through the Cx43 binding of β-catenin from its translocation to the nucleus. The results of our study suggest that Ti particles increased Cx43 expression in osteocytes and that osteocytes may participate in the regulation of osteoblast function via the Cx43 during PPO.

High biocompatible polyacrylamide hydrogels fabricated by surface mineralization for subchondral bone tissue engineering

The subchondral bone is an important part of cartilage which contains a large amount of hydroxyapatite. The mineral components of subchondral bone is the key factor which determines the biomechanical strength, and then affects the biological function of articular cartilage. Here, a mineralized polyacrylamide (PAM-Mineralized) hydrogel with good ALP activity, cell adhesion and biocompatibility was fabricated for subchondral bone tissue engineering. The micromorphology, composition and mechanical properties of PAM and PAM-Mineralized hydrogels were studied. The PAM hydrogels showed a porous structure, while the PAM-Mineralized hydrogels had well-distributed layers of hydroxyapatite mineralization on the surface. The XRD results show that the characteristic peak of hydroxyapatite (HA) was measured in PAM-Mineralized, indicating that the main component of the mineralized structure formed on the surface of the hydrogel after mineralization is HA. The formation of HA ectively decreased the rate of equilibrium swelling of the PAM hydrogel, with PAM-M reaching swelling equilibrium at 6 h. Meanwhile, compressive strength of PAM-Mineralized hydrogel (moisture state) reached 290 ± 30 kPa, compressive modulus reached 130 ± 4 kPa. PAM-Mineralized hydrogels did not affect the growth and proliferation of MC3T3-E1 cells. Surface mineralization of PAM hydrogel could significantly improve osteogenic differentiation of MC3T3-E1 cells. These results showed that PAM-Mineralized hydrogel could possess potential application in the field of subchondral bone tissue engineering.

Fabrication and evaluation of 3D printed poly(l-lactide) copolymer scaffolds for bone tissue engineering

The application of poly(L-lactic acid) (PLLA) in tissue engineering is limited due to its brittleness and uncontrollable degradation rate. In this study, the flexible p-dioxanone (PDO) and highly reactive glycolide (GA) units were introduced into PLLA segments by chemical modification to prepare poly(l-lactide-ran-p-dioxanone-ran-glycolide) (PLPG) copolymers. The copolymers were then processed into the PLPG scaffold by a 3D printing technology. The physicochemical properties of the PLPG copolymers were studied by NMR, DSC, XRD, GPC, and SEM. Furthermore, the mechanical properties, degradation properties, and biocompatibility of the PLPG scaffolds were also studied. The results showed that introducing PDO and GA units disrupted the regularity of PLLA, decreasing the crystallinity of the PLPG copolymers. However, introducing PDO and GA units could effectively improve the mechanical and degradation properties of the PLLA scaffolds. In vitro cell culture experiments indicated that the PLPG scaffolds supported proliferation, growth, and differentiation of MC3T3-E1 cells. The PLPG scaffolds reported herein, with controllable degradation rates and mechanical performance, may find applications in bone tissue engineering.

Nano-SiO2 reinforced alginate-chitosan-gelatin nanocomposite hydrogels with improved physicochemical properties and biological activity

Alginate (Alg) hydrogels possess desirable advantages for application in tissue engineering; however, they are limited by their weak mechanical properties, poor chronical stability in phosphate buffered saline, and absence of mammalian cell recognition sites, severely restricting their biomedical applications. To overcome these limitations, we integrated Alg hydrogels with nano-silica (SiO2) to produce nano-SiO2 reinforced Alg–chitosan–gelatin nanocomposite hydrogels (Alg/SiO2–CHI–GA NCH) for biomedical purposes, utilizing Chitosan (CHI) and gelatin (GA) in an alternate electrostatic adsorption. Specifically, we investigated the regulatory and promotional effects of the nano-SiO2 on the morphological structure, mechanical properties, thermal stability, rheological properties, swelling, biodegradability, biomineralization and cytocompatibility of the resultant Alg/SiO2-CHI-GA NCH. The experimental findings demonstrate that the constructed Alg/SiO2-CHI-GA NCH exhibited uniform morphology and a regular structure. Upon freeze–drying, the internal cross-sections of the NCH exhibited a honeycomb porous structure. Furthermore, the physicochemical properties and biological activities of the prepared Alg/SiO2–CHI–GA NCH were regulated to some extent by nano-SiO2 content. Notably, nano-SiO2 inclusion enhanced the attachment and viability of MG63 and MC3T3-E1 cells and induced three-dimensional cell growth in ALG/SiO2–CHI–GA NCH. Among the fabricated NCH, Alg/SiO2–CHI–GA NCH with 0.5% and 1.0% (w/v) nano-SiO2 exhibited significant proliferative activity, which is attributable to their high porosity and uniform cell adhesion. Furthermore, the alkaline phosphatase activity in the cells gradually increased with increasing of nano-SiO2 amount, indicating the favorable effect of nano-SiO2 on the osteogenic differentiation of MG63 and MC3T3-E1 cells. Our study findings provide a comprehensive foundation for the structural- and property-related limitations of Alg hydrogels in biomedicine, thereby expanding their potential applications in tissue engineering.

A cytocompatible microporous Sr-doped titanium dioxide coating fabricated by plasma electrolytic oxidation

Titanium has broad application prospects in the field of hard bone tissue repair and replacement due to its low density, an elastic modulus similar to that of human bone, and better biocompatibility than stainless steel and cobalt-based alloys. However, titanium is a biologically inert material with low biological activity. After implantation, it only physically bonds with bone instead of chemically bonding, which causes complications such as implant loosening. In this study, a strontium-doped titanium dioxide coating was prepared on a titanium surface with micro-arc oxidation. Based on a systematic study of the surface properties of the strontium-doped titanium dioxide coating, the biocompatibility and bioactivity of the coating were evaluated with in vitro cell experiments. The results showed that the strontium-doped titanium dioxide coating was successfully prepared on the titanium surface and exhibited strong bonding with the substrate. The coating surface had a porous structure, and the strontium was dispersed on the surface and inside the coating. The strontium ions were released slowly, which improved the corrosion and wear resistance of the titanium. In vitro cell experiments showed that the coating had good cell compatibility; it promoted the adhesion, proliferation and differentiation of MC3T3-E1 cells and exhibited good biological activity. These results showed that preparation of a strontium-doped titanium dioxide coating by micro-arc oxidation is feasible. The coating had a good surface morphology, improved the biological activity of the titanium and has good potential for clinical application.

Low-intensity pulsed ultrasound enhances the positive effects of high-intensity treadmill exercise on bone in rats.

Moderate exercise benefits bone health, but excessive loading leads to bone fatigue and a decline in mechanical properties. Low-intensity pulsed ultrasound (LIPUS) can stimulate bone formation. The purpose of this study was to explore whether LIPUS could augment the skeletal benefits of high-intensity exercise. MC3T3-E1 osteoblasts were treated with LIPUS at 80mW/cm2 or 30mW/cm2 for 20min/day. Forty rats were divided into sham treatment normal control (Sham-NC), sham treatment high-intensity exercise (Sham-HIE), 80mW/cm2 LIPUS (LIPUS80), and high-intensity exercise combined with 80mW/cm2 LIPUS (LIPUS80-HIE). The rats in HIE group were subjected to 30m/min slope treadmill exercise for 90min/day, 6days/week for 12weeks. The LIPUS80-HIE rats were irradiated with LIPUS (1MHz, 80mW/cm2) for 20min/day at bilateral hind limb after exercise. LIPUS significantly accelerated the proliferation, differentiation, mineralization, and migration of MC3T3-E1 cells. Compared to 30mW/cm2 LIPUS, 80mW/cm2 LIPUS got better promotion effect. 12weeks of high-intensity exercise significantly reduced the muscle force, which was significantly reversed by LIPUS. Compared with the Sham-NC group, Sham-HIE group significantly optimized bone microstructure and enhanced mechanical properties of femur, and LIPUS80-HIE further enhanced the improvement effect on bone. The mechanisms may be related to activate Wnt/β-catenin signal pathway and then up-regulate the protein expression of Runx2 and VEGF, the key factors of osteogenesis and angiogenesis. LIPUS could augment the skeletal benefits of high-intensity exercise through Wnt/β-catenin signal pathway.

Dipeptidyl peptidase 3 is essential for maintaining osteoblastic differentiation under a high-glucose environment by inhibiting apoptosis, oxidative stress and inflammation through the modulation of the Keap1-Nrf2 pathway

Dipeptidyl peptidase 3 (Dpp3) has emerged as a pivotal mediator of bone homeostasis and bone loss pathology. However, whether Dpp3 plays a role in diabetic osteoporosis has not been addressed. Therefore, this work explored the possible role of Dpp3 in osteoblast dysfunction evoked by high glucose (HG), a cellular model for studying diabetic osteoporosis in vitro. Dpp3 expression was decreased in the pre-osteoblast MC3T3-E1 during osteoblastic differentiation under the HG environment. The osteoblastic differentiation impaired by HG was reversed in Dpp3-overexpressing MC3T3-E1 cells. The migration and invasion of MC3T3-E1 cells impeded by HG were reversed by Dpp3 overexpression. Moreover, HG-evoked apoptosis, oxidative stress and inflammation were ameliorated in Dpp3-overexpressing MC3T3-E1 cells. A mechanistic study showed that Dpp3 up-regulated the activation of nuclear factor E2-related factor 2 (Nrf2) depending on Kelch-like ECH-associated protein 1 (Keap1). The blockade of Nrf2 reversed Dpp3-mediated effects on osteoblastic differentiation, apoptosis, oxidative stress and inflammation of HG-stimulated MC3T3-E1 cells. Therefore, Dpp3 plays an essential role in maintaining osteoblastic differentiation under a HG environment associated with the regulation of the Keap1-Nrf2 pathway. This work indicates a possible relationship between Dpp3 and diabetic osteoporosis.

Robo2 promotes osteoblast differentiation and mineralization through autophagy and is activated by parathyroid hormone induction.

As a systemic skeletal disorder, osteoporosis can increase fracture risk. This study wants to discuss the mechanism of osteoporosis and find possible molecular therapy. Bone morphogenetic protein 2 (BMP2) was utilized to stimulate MC3T3-E1 to establish a cellular osteoporosis model in vitro. Initially, the viability of BMP2-induced MC3T3-E1 was assessed with a Cell counting kit-8 (CCK-8) assay. By real-time quantitative PCR (RT-qPCR) and western blot, Robo2 expression after roundabout (Robo) silencing or overexpression was estimated. Besides, alkaline phosphatase (ALP) expression, mineralization level and LC3II green fluorescent protein (GFP) expression were evaluated using ALP assay, Alizarin red staining and immunofluorescence staining, separately. Additionally, the expression of proteins related to osteoblast differentiation and autophagy was analyzed by RT-qPCR and western blot. Then, following autophagy inhibitor 3-methyladenine (3-MA) treatment, osteoblast differentiation and mineralization were measured again. MC3T3-E1 cells were differentiated into osteoblasts under BMP2 induction and Robo2 expression was greatly ascended. After Robo2 silencing, Robo2 expression was markedly diminished. ALP activity and mineralization level in BMP2-induced MC3T3-E1 cells were declined after depleting Robo2. Robo2 expression was conspicuously enhanced after overexpressing Robo2. Robo2 overexpression promoted the differentiation and mineralization of BMP2-induced MC3T3-E1 cells. Rescue experiments revealed that Robo2 silence and its overexpression could regulate the autophagy of BMP2-stimulated MC3T3-E1 cells. After 3-MA treatment, the increased ALP activity and mineralization level of BMP2-induced MC3T3-E1 cells with Robo2 upregulation were reduced. Furthermore, parathyroid hormone 1-34 (PTH1-34) treatment enhanced the expression of ALP, Robo2, LC3II and Beclin-1 and reduced the levels of LC3I and p62 of MC3T3-E1 cells concentration-dependently. Collectively, Robo2, which was activated by PTH1-34, promoted osteoblast differentiation and mineralization through autophagy.

Osteocytes autophagy mediated by mTORC2 activation controls osteoblasts differentiation and osteoclasts activities under mechanical loading

Autophagy is an important mechanosensitive response for cellular homeostasis and survival in osteocytes. However, the mechanism and its effect on bone metabolism have not yet clarified. The objective of this study was to evaluate how compressive cyclic force (CCF) induced autophagic response in osteocytes and to determine the effect of mechanically induced-autophagy on bone cells including osteocytes, osteoblasts, and osteoclasts. Autophagic puncta observed in MLO-Y4 cells increased after exposure to CCF. The upregulated levels of the LC3-II isoform and the degradation of p62 further confirmed the increased autophagic flux. Additionally, ATP synthesis and release, osteocalcin (OCN) expression, and cell survival increased in osteocytes as well. The Murine osteoblasts MC3T3-E1 cells and RAW 264.7 macrophage cells were cultured in conditioned medium collected from MLO-Y4 cells subjected to CCF. The concentration of FGF23 increased and the concentrations of SOST and M-CSF and RANKL/OPG ratio decreased significantly in the conditioned medium. Moreover, the promotion of osteogenic differentiation in MC3T3-E1 cells and inhibition of osteoclastogenesis and function in RAW 264.7 cells were significantly attenuated when osteocytes autophagy was inhibited by siAtg7. Our findings suggested that CCF induced protective autophagy in osteocytes and subsequently enhanced osteocytes survival and osteoblasts differentiation and downregulated osteoclasts activities. Further study revealed that CCF induced autophagic response in osteocytes through mechanistic target of rapamycin complex 2 (mTORC2) activation. In conclusion, CCF-induced osteocytes autophagy upon mTORC2 activation promoted osteocytes survival and osteogenic response and decreased osteoclastic function. Thus, osteocytes autophagy will provide a promising target for better understanding of bone physiology and treatment of bone diseases.

Fabrication of carboxymethyl chitosan-strontium chondroitin sulfate composites for potential bone regeneration

Large bone defects have been a major healthcare concern. These defects are difficult to heal because of the chronic inflammatory environment surrounding the injury area. Osteoconductive composites with anti-inflammatory effects could provide a substrate to support cell growth and guide bone defect repair. Here, we synthesized a biofunctional composite that combined carboxymethyl-chitosan (CMCS) and strontium chondroitin sulfate (SrCS), for potential use in bone regeneration. We hypothesized that the as-prepared CMCS-SrCS composites could stimulate osteogenic activity and suppress inflammatory responses. The resultant composites displayed uniformly porous structures and good swelling capacity. The effect of the composites on MC3T3-E1 cells was investigated in vitro and the live cells showed favorable viability. Moreover, the RT-PCR results indicated that the CMCS-SrCS composites could suppress inflammatory responses and promote the osteogenic differentiation of MC3T3-E1 cells. These results suggested the CMCS-SrCS composites should have osteoconductive characteristics. Thus, we believe that the developed CMCS-SrCS composites should be a promising skeletal candidate for regenerative engineering applications.

Effect of Erxian Decoction-containing serum on H_2O_2-induced proliferation and osteogenic differentiation of MC3T3-E1 cells via BK channels

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.

Plum-Derived Exosome-like Nanovesicles Induce Differentiation of Osteoblasts and Reduction of Osteoclast Activation

Osteoblasts and osteoclasts play crucial roles in bone formation and bone resorption. We found that plum-derived exosome-like nanovesicles (PENVs) suppressed osteoclast activation and modulated osteoblast differentiation. PENVs increased the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells and osteoblasts from mouse bone marrow cultures. Notably, PENVs elevated the expression of osteoblastic transcription factors and osteoblast differentiation marker proteins in MC3T3-E1 cells. Higher levels of phosphorylated BMP-2, p38, JNK, and smad1 proteins were detected in PENV-treated MC3T3-E1 cells. Additionally, the number of TRAP-positive cells was significantly decreased in PENV-treated osteoclasts isolated from osteoblasts from mouse bone marrow cultures. Importantly, osteoclastogenesis of marker proteins such as PPAR-gamma, NFATc1, and c-Fos were suppressed by treatment with PENVs (50 μg/mL). Taken together, these results demonstrate that PENVs can be used as therapeutic targets for treating bone-related diseases by improving osteoblast differentiation and inhibiting osteoclast activation for the first time.

Particulate beta-tricalcium phosphate and hydroxyapatite doped with silver promote invitro osteoblast differentiation in MC3T3-E1 cells.

Calcium phosphates including β-tricalcium phosphate (β-TCP) and hydroxyapatite (HAp) have been widely used for bone regeneration application because of their high osteoconductive activities. In addition, various kinds of inorganic ions enhance differentiation, proliferation, and mineralization of osteoblasts. However, information about the effects of silver-doped β-TCP [β-TCP (Ag)] and HAp [HAp (Ag)] particles on osteogenic differentiation is not available yet. We focused on the impact of β-TCP (Ag) and HAp (Ag) particles on the osteogenic differentiation of MC3T3-E1 osteoblast precursor cells. MC3T3-E1 osteoblast precursor cells were pre-treated by β-TCP (Ag) or HAp (Ag). And then the medium was changed to differentiation medium. Subsequently, osteoblast differentiation-related markers were determined. We found that treatment with β-TCP (Ag) or HAp (Ag) particles increased alkaline phosphatase activity in MC3T3-E1 cells. Expression of osteoblast differentiation-related genes also increased after treatment with β-TCP (Ag) or HAp (Ag) particles, a response thought to be regulated by zinc finger-containing transcription factor osterix. The ratio of the receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) was decreased by β-TCP (Ag) and HAp (Ag) particles. Silver doping of β-TCP and HAp particles is effective for bone regeneration.

Novel antiosteoporotic peptides purified from protein hydrolysates of taihe black-boned silky fowl: By larval zebrafish model and molecular docking

The black-boned silky fowl (BSF) muscle protein hydrolysate was gained by alcalase. The hydrolysate could stimulate MC3T3-E1 cell proliferation, as well as enhance alkaline phosphatas (ALP) activity and deposits of minerals. After isolation and purification, 55 peptide sequences with Mascot score over 40 were identified. Combined with molecular docking simulation and molecular dynamics analysis, two novel peptides (PASTGAAK and PGPPGTPF) were identified with the lowest binding energy of −4.99 kcal/mol and −3.07 kcal/mol with receptor BMPR1A of BMP-2/Smad pathway, showing the ability to increase BMPR1A stability. Moreover, both PASTGAAK and PGPPGTPF revealed strong anti-osteoporosis activities in the zebrafish model induced by dexamethasone. Additionally, the identified peptides could be beneficial for the differentiation of MC3T3-E1 cell for upregulating the expression of some osteoblast-related genes and proteins by stimulating BMP-2/Smad pathway. Overall, the two newly identified peptides could be the potential candidate to prevent osteoporosis.