Osteoblastic MC3T3-E1 Research Articles

Novel peptides extracted from Muraenesox cinereus bone promote calcium transport, osteoblast differentiation, and calcium absorption

Extracting peptides from fish bones as calcium supplements is of great significance for patients with osteoporosis. Novel peptides were obtained from Muraenesox cinereus bone (MCBP) by enzymatic hydrolysis and membrane separation. Molecular dynamics simulation and electron microscope analysis shown MCBP could chelate with calcium ions. A Caco-2 cell monolayer model was used to investigate the effect of peptide–calcium chelates on promoting calcium transport. MC3T3-E1 osteoblasts were treated with different concentrations of MCBP (0, 0.25, 0.5, or 1 mg/mL) to analyze the effects of MCBP on calcium absorption and cell differentiation factors. The bone formation of zebrafish evaluate the therapeutic effect of MCBP on the zebrafish OP model. The results showed that there were at least 13 kinds of calcium chelation modes in the mixed peptide, and peptide–calcium chelate could significantly improve calcium transport in the Caco-2 cell monolayer. After 14 days of culture with different concentrations of MCBP, the calcium deposition in MC3T3-E1 cells increased significantly. The type I collagen (COL-I), osteocalcin (OCN) and run-related transcription factor 2 (RUNX2), and activities of alkaline phosphatase (ALP) were dose-dependent with MCBP. MCBP can increase bone mineral density in an OP model of zebrafish. After being treated with MCBP, the expression levels of ALP, OCN, osteoprotegerin (OPG) and transforming growth factor-β (TGF-β) in the model group increased, while the expression levels of tartrate-resistant acid phosphatase (TRAP) decreased. The results indicated that MCBP may act as a nutritional food and provide an effective means for dietary supplementation in OP patients.

Inhibitory Effect of Biotransformed-Fucoidan on the Differentiation of Osteoclasts Induced by Receptor for Activation of Nuclear Factor-κB Ligand.

Bone homeostasis is regulated by constant remodeling through osteogenesis by osteoblasts and osteolysis by osteoclasts and osteoporosis can be provoked when this balance is broken. Present pharmaceutical treatments for osteoporosis have harmful side effects and thus, our goal was to develop therapeutics from intrisincally safe natural products. Fucoidan is a polysaccharide extracted from many species of brown seaweed, with valuable pharmaceutical activities. To intensify the effect of fucoidan on bone homeostasis, we hydrolyzed fucoidan using AMG, Pectinex and Viscozyme. Of these, fucoidan biotransformed by Pectinex (Fu/Pec) powerfully inhibited the induction of tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts differentiated from bone marrow macrophages (BMMs) by the receptor for activation of nuclear factor-κB ligand (RANKL). To investigate potential of lower molecular weight fucoidan it was separated into >300 kDa, 50-300 kDa, and <50 kDa Fu/Pec fractions by ultrafiltration system. The effects of these fractions on TRAP and alkaline phosphatase (ALP) activities were then examined in differentiated osteoclasts and MC3T3-E1 osteoblasts, respectively. Interestingly, 50-300 kDa Fu/Pec suppressed RANKL-induced osteoclasts differentiation from BMMs but did not synergistically enhance osteoblasts differentiation induced by osteogenic agents. In addition, this fraction inhibited the expressions of NFATc1, TRAP, OSCAR, and RANK, which are all key transcriptional factors involved in osteoclast differentiation, and those of Src, c-Fos and Mitf, as determined by RT-PCR. In conclusion, enzymatically low-molecularized 50-300 kDa Fu/Pec suppressed TRAP by downregulating RANKL-related signaling, contributing to the inhibition of osteoclasts differentiation, and represented a potential means of inducing bone remodeling in the background of osteoporosis.

EPLINβ Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINβ. In this study, we investigated the role of EPLINβ in osteoblasts using EPLINβ-deficient (EPLINβGT/GT) mice. The skeletal phenotype of EPLINβGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINβGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINβGT/GT mice, suggesting aberrant cell adhesion. In EPLINβGT/GT osteoblasts, α- and β-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINβ knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINβ knockdown cells, suggesting an important role for EPLINβ in osteoblast formation. In conclusion, we propose that EPLINβ is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Osteoblasts in a Perfusion Flow Bioreactor-Tissue Engineered Constructs of TiO2 Scaffolds and Cells for Improved Clinical Performance.

Combining biomaterial scaffolds with cells serves as a promising strategy for engineering critical size defects; however, homogenous cellular growth within large scaffolds is challenging. Mechanical stimuli can enhance bone regeneration by modulating cellular growth and differentiation. Here, we compare dynamic seeding in a perfusion flow bioreactor with static seeding for a synthetic bone scaffold for up to 21 days using the cell line MC3T3-E1 and primary human osteoblast, confocal laser scanning microscopy, and real-time reverse transcriptase-polymerase chain reaction. The secretion of bone-related proteins was quantified using multiplex immunoassays. Dynamic culture improved cellular distribution through the TiO2 scaffold and induced a five-fold increase in cell number after 21 days. The relative mRNA expression of osteopontin of MC3T3-E1 was 40-fold enhanced after 7 and 21 days at a flow rate of 0.08 mL/min, and that of collagen type I alpha I expression was 18-fold after 21 days. A flow rate of 0.16 mL/min was 10-fold less effective. Dynamic culture increased the levels of dickkopf-related protein 1 (60-fold), osteoprotegrin (29-fold), interleukin-6 (23-fold), interleukin-8 (36-fold), monocyte chemoattractant protein 1 (28-fold) and vascular endothelial growth factor (6-fold) in the medium of primary human osteoblasts after 21 days compared to static seeding. The proposed method may have clinical potential for bone tissue engineering.

The Single-Step Fabrication of a Poly (Sodium Vinylsulfonate)-Grafted Polyetheretherketone Surface to Ameliorate Its Osteogenic Activity

Polyetheretherketone (PEEK) is considered a potential material for replacing traditional biomedical metals used in orthopedic implants because of its similar elastic modulus to human bone. However, the poor osteogenic activity of PEEK itself hinders its clinical application. In this study, a PEEK surface was grafted with poly (sodium vinylsulfonate) through a single-step ultraviolet-initiated graft polymerization method to ameliorate its osteogenic activity. X-ray photoelectron spectroscopy and water contact angle measurements confirmed that different amounts of poly (sodium vinylsulfonate) were grafted onto the PEEK surface upon varying the ultraviolet irradiation time. Atomic force microscopy revealed that the surface topography and roughness of PEEK before and after surface grafting did not change significantly. The in vitro results showed that grafting with poly (sodium vinylsulfonate) rendered the PEEK surface with improved MC3T3-E1 osteoblast compatibility and osteogenic activity. Moreover, a PEEK surface with a higher grafting amount of poly (sodium vinylsulfonate) was observed to be more beneficial to the proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts. Collectively, by employing this simple and one-step method, the osteogenic activity of PEEK can be enhanced, paving the way for the clinical application of PEEK in orthopedic implants.

Dietary Collagen Hydrolysates Retard Estrogen Deficiency-Induced Bone Loss through Blocking Osteoclastic Activation and Enhancing Osteoblastic Matrix Mineralization

Osteoporosis manifest in postmenopausal women is an osteolytic disease characterized by bone loss, leading to increased susceptibility to bone fractures and frailty. The use of complementary therapies to alleviate postmenopausal osteoporosis is fairly widespread among women. The current study examined that Pangasius hypophthalmus fish skin collagen hydrolysates (fsCH) inhibited ovariectomy (OVX)-induced bone loss by conducting inter-comparative experiments for anti-osteoporotic activity among 206–618 mg/kg fsCH, 2 mg/kg isoflavone, 15 mg/kg glycine–proline–hydroxyproline (GPH) tripeptide, and calcium lactate. Surgical estrogen loss of mice for 8 weeks reduced serum 17β-estradiol levels with uterus atrophy, which was ameliorated by orally administering fsCH or isoflavone to mice. Similar to isoflavone, fsCH containing GPH-enhanced bone mineral density reduced levels of cathepsin K and proton-handling proteins, and elevated collagen 1 level in OVX bones. The treatment with fsCH and isoflavone enhanced the serum levels of collagen synthesis-related procollagen type 1 carboxy/amino-terminal propeptides reduced by OVX, whereas serum levels of osteocalcin and alkaline phosphatase, as well as collagen breakdown-related carboxy/amino-terminal telopeptides of type 1 collagen were reduced in OVX mice treated with fsCH, isoflavone, and calcium lactate. The trabecular bones were newly formed in OVX bones treated with isoflavone and fsCH, but not with calcium lactate. However, a low-dose combination of fsCH and calcium lactate had a beneficial synergy effect on postmenopausal osteoporosis. Furthermore, similar to isoflavone, 15–70 μg/mL fsCH, with its constituents of GPH and dipeptides of glycine–proline and proline–hydroxyproline, enhanced osteogenesis through stimulating differentiation, matrix mineralization, and calcium deposition of MC3T3-E1 osteoblasts. Accordingly, the presence of fsCH may encumber estrogen deficiency-induced bone loss through enhancing osteoclastogenic differentiation and matrix collagen synthesis. Therefore, fsCH may be a natural compound retarding postmenopausal osteoporosis and pathological osteoresorptive disorders.

An In Vitro Evaluation of Selenium Nanoparticles on Osteoblastic Differentiation and Antimicrobial Properties against Porphyromonas gingivalis.

Despite numerous treatment methods, there is no gold standard for the treatment of peri-implantitis—an infectious peri-implant disease. Here, we examined selenium nanoparticles (SeNPs) at a wide range of concentrations to investigate their cytotoxicity, regulation of osteoblastic differentiation, and assessed the antibacterial effect against Porphyromonas gingivalis. SeNPs (mean size: 70 nm; shape: near-spherical; concentration: 0–2048 ppm) were tested against the MC3T3-E1 osteoblast precursor cell line and P. gingivalis red complex pathogen. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis was used to evaluate the bone morphogenetic protein 2 (BMP-2) signaling pathway. SeNPs at concentrations of 2–16 ppm showed no obvious cytotoxicity and promoted good mineralization and calcification. SeNPs at concentrations 64 ppm and below influenced gene expression promoting osteoblastic differentiation, whereas at high concentrations inhibited the expression of Runt-related transcription factor 2 (Runx2). The growth of P. gingivalis was significantly inhibited at SeNP concentrations of more than 4 ppm. SeNPs at low concentrations promoted osteoblastic differentiation while strongly inhibiting peri-implantitis pathogen growth. This study represents one of the few in vitro assessments of SeNPs against a red complex pathogen and the regulatory effect on osteoblastic differentiation. The findings demonstrate SeNPs could potentially be used for future application on implant coating.

Exploring In Vitro Biological Cellular Responses of Pegylated β-Cyclodextrins.

βCDPEG5 and βCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to βCD. As βCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of βCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO2− overproduction in macrophages, we found that βCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to βCDPEGs and the parent compounds, a similar pattern was observed: the effect of βCDPEG5 on cell viability and cell cycle progression was larger than that of βCDPEG2; PEG2 affected cell viability and cell cycle more than βCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of βCDPEG5 with cell migration was smaller than that of βCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of βCD and PEGs, particularly to yield βCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of βCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from βCD and PEGs.

Osteogenic and angiogenic bioactive collagen entrapped calcium/zinc phosphates coating on biodegradable Zn for orthopedic implant applications.

Zinc is becoming one of the leading candidate materials for biodegradable orthopedic implants owing to its attractive properties in terms of degradation behavior and mechanical properties. However, the insufficient surface bio-activities postpone its clinical application. In this study, an organic-inorganic collagen entrapped calcium/zinc phosphates coating was constructed on Zn surface to lessen Zn2+ releasing rate and to leverage the surface osteogenic and angiogenic properties. Collagen molecules were immobilized onto Zn substrate and subsequently coordinated with calcium and zinc ions to promote the CaZnP inorganic phase growth, ensuing an intertwined collagen-CaZnP hybrid system. Consequently, the hybrid coating was highly coalesced and compact. Such high quality warranted the contained Zn2+ releasing in a tolerable rate favorable for cells viability. The collagen-CaZnP coated Zn showed remarkedly stronger osteogenicity as compared to the untreated Zn, ascertained by the MC3T3-E1 osteoblast cell proliferation and differentiation assays, such as alkaline phosphatase expression and calcium nodule formation results. In addition, this hybrid coating supported human umbilical vein endothelial cells (HUVECs) migration and tube formation. The enhanced osteogenic and angiogenic properties could be ascribed to the nature of collagen and calcium/zinc phosphate components, the hybrid micro/nano-structure as well as the ability of controlling the Zn2+ release of Zn substrate into a suitable concentration range. Our strategy provides a new avenue to surface modification of biodegradable metals for bone regenerative perspective.

Cortistatin attenuates titanium particle-induced osteolysis through regulation of TNFR1-ROS-caspase-3 signaling in osteoblasts.

Aseptic loosening is a major complication of prosthetic joint surgery and is associated with impaired osteoblast homeostasis. Cortistatin (CST) is a neuropeptide that protects against inflammatory conditions. In this study, we found that expression of CST was diminished in patients with prosthetic joint loosening and in titanium (Ti) particle-induced animal models. A Ti particle-induced calvarial osteolysis model was established in wild-type and CST gene knockout mice; CST deficiency enhanced, while exogenously added CST attenuated, the severity of Ti particle-mediated osteolysis. CST protected against inflammation as well as apoptosis and maintained the osteogenic function of MC3T3-E1 osteoblasts upon stimulation with Ti particles. Furthermore, CST antagonized reactive oxygen species production and suppressed caspase-3-associated apoptosis mediated by Ti particles in osteoblasts. Additionally, CST protects against Ti particle-induced osteolysis through tumor necrosis factor receptor 1. Taken together, CST might provide a therapeutic strategy for wear debris-induced inflammatory osteolysis.

Arctiin elevates osteogenic differentiation of MC3T3-E1 cells by modulating cyclin D1

ABSTRACT Osteoporosis is a systemic disorder of bone metabolism. This study aimed to investigate the impacts and possible mechanisms of Arctiin, a lignin isolated from Arctium lappa on MC3T3-E1 osteoblast differentiation. In this study, after treatment with different concentrations of Arctiin, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to estimate the expression of osteogenesis markers. Then, the activity of alkaline phosphatase (ALP) was detected by an ALP assay kit and calcium nodules staining was evaluated by alizarin red staining (ARS). Additionally, the regulatory effects of Arctiin on cyclin D1 (Ccnd1) was assessed by measurement of protein expression. Subsequently, the functions of Ccnd1 silencing on the osteogenic differentiation was examined in Arctiin-treated MC3T3-E1 cells. Results indicated that Arctiin dose-dependently upregulated the expression of runt-related transcription factor 2 (RUNX2), collagen type 1 (COL1A1), osteocalcin (OCN) and osteopontin (OPN). Elevated ALP activity and calcification degree was prominently observed in the Arctiin-treated groups. Moreover, Ccnd1 expression was notably enhanced after Arctiin intervention. Importantly, Ccnd1-knockdown abrogated the impacts of Arctiin on osteogenic differentiation of MC3T3-E1. To conclude, findings in this study suggested that Arctiin could regulate MC3T3-E1 osteoblast differentiation via up-regulating Ccnd1, supporting that Arctiin might be a therapeutic target for osteoporosis.

Effect of Milling Parameters on Mechanical Properties and In Vitro Biocompatibility of Mg-Zn-Co Ternary Alloy

Magnesium (Mg) is a potential candidate for biomedical implants, but its susceptibility to suffer corrosion attack in human body fluid limits its practical use. Thus, alloying Mg with other metal elements is the most effective strategy to improve its mechanical properties and biocompatibility. Herein, we report a Mg-Zn-Co ternary alloy (85-10-5 wt %) synthesized by the mechanical alloying technique. Ball-milling parameters such as ball size and milling time were varied to obtain better alloy properties. After compaction and sintering, the obtained alloy samples were subjected to various characterizations, including grain, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy, X-ray diffraction (XRD), microhardness and nanoindentation analyses. In vitro biocompatibility analysis of different alloys was also performed with MC3T3-E1 osteoblasts. Grain analysis confirmed the even dispersion of particles, while SEM analysis showed the formation of laminates, spherical and fine particles with an increase in time and varied ball size. XRD results further confirmed the formation of intermetallic compounds. The microhardness of samples was increased with the increase in milling time. The Young’s modulus of ternary alloys obtained from nanoindentation analysis was comparable to the modulus of human bone. Moreover, in vitro analysis with osteoblasts showed that the developed alloys were noncytotoxic and biocompatible.

PCL nanofibrous incorporating unique matrix fusion protein adsorbed mesoporous bioactive glass for bone tissue engineering

Mesoporous bioactive glass (MBG) is a potential biomedical material in bone defect repairment because of its bioactivity, biocompatibility, and osteoinduction properties. Here we report that Mg-doped MBG scaffold with 3:1 Ca/Mg ratio (MBG-Ca/Mg-3) is good for MC3T3-E1 osteoblast differentiation and mineralization. Mimicking bone extracellular matrix structure by electrospinning, we used MBG-Ca/Mg-3 adsorbed with Osteocalcin-Osteopontin-Biglycan (OOB), a new unique matrix fusion protein, to form OOB@MBG-Ca/Mg-3 scaffold, which has multifunctional ability in calvarial bone defect repairment in vivo. Intriguingly, we found that OOB@MBG-Ca/Mg-3 scaffold increases the expression of osteoblastic marker genes, including bone morphogenetic protein (Bmp2), osteopontin (Opn), Osterix, Runx2 through activation of ERK1/2. We concluded that OOB@MBG-Ca/Mg-3 scaffold promotes osteoblast differentiation and mineralization through ERK1/2 pathway and it can also enhance bone formation in vivo, which provides a new biomaterial in bone tissue engineering.

Dual-Function Semaphorin 4D Released by Platelets: Suppression of Osteoblastogenesis and Promotion of Osteoclastogenesis.

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.

Fermented Oyster (Crassostrea gigas) Extract Cures and Prevents Prednisolone-Induced Bone Resorption by Activating Osteoblast Differentiation.

Osteoporosis is a bone resorptive disease characterized by the loss of bone density, causing an increase in bone fragility. In our previous study, we demonstrated that gamma aminobutyric acid-enriched fermented oyster (Crassostrea gigas) extract (FO) stimulated osteogenesis in MC3T3-E1 preosteoblast cells and vertebral formation in zebrafish. However, the efficacy of FO in prednisolone (PDS)-induced bone resorption remains unclear. In this study, we evaluated the osteogenic potential of FO in MC3T3-E1 preosteoblast cells and zebrafish larvae under both PDS-pretreated and PDS-post-treated conditions. We found that FO recovered osteogenic activity by upregulating osteoblast markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2, and osterix, in both PDS-pretreated and post-treated MC3T3-E1 osteoblast cells and zebrafish larvae. In both conditions, PDS-induced decrease in calcification and ALP activity was recovered in the presence of FO. Furthermore, vertebral resorption in zebrafish larvae induced by pretreatment and post-treatment with PDS was restored by treatment with FO, along with the recovery of osteogenic markers and downregulation of osteoclastogenic markers. Finally, whether FO disturbs the endocrine system was confirmed according to the Organization for Economic Cooperation and Development guideline 455. We found that FO did not stimulate estrogen response element-luciferase activity or proliferation in MCF7 cells. Additionally, in ovariectomized mice, no change in uterine weight was observed during FO feeding. These results indicate that FO effectively prevents and treats PDS-induced osteoporosis without endocrine disturbances.

Biological properties of direct grafting by ultraviolet irradiation of vinyl benzyl phosphonic acid onto titanium surfaces

Titanium (Ti) and its alloys are the most effective metals for structural implantable device applications. However, Ti-based materials are passively integrated into the bone, resulting in a purely mechanical attachment. Consequently, the loss of osseointegration often leads to implant failure. Therefore, enhancing bone formation surrounding the implant is primordial. In previous investigations conducted in our laboratory, grafting bioactive polymers with sulfonate groups, such as poly(sodium styrene sulfonate) (polyNaSS), was demonstrated to increase the adherence and differentiation of osteoblast cells. In this context, this contribution proposes to functionalize Ti with a phosphonic acid-based polymer, poly(vinyl benzyl phosphonic acid) (poly(VBP)). A two-step UV-initiated grafting polymerization was developed to covalently graft VBP into Ti surfaces. The surfaces were characterized using colorimetry, Fourier-transformed infrared spectra recorded in an attenuated total reflection (FTIR-ATR), X-ray photoelectron spectroscopy (XPS) techniques, and water contact angle (WCA) measurements. The Ti substrates were evaluated for cell viability, spreading, alkaline phosphatase activity, and calcium formation using MC3T3-E1 osteoblast cells. The interaction of Ti grafted samples with osteoblast cells, was significantly improved as well as the cell/surface interaction. Together, these findings demonstrated that poly(VBP) grafted on Ti surfaces improved osteoblasts' early cell adhesion and spreading activities, crucial for osseointegration applications.

Abstract P5-12-04: Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression

Abstract Breast cancer is frequently characterized by calcifications in mammography. Although breast calcifications were previously thought to be produced passively as the final stage of cell regression, recent studies have shown active production. In another context, vascular smooth muscle cells have been found to produce vascular calcifications by acquiring osteoblast-like characteristics. The same mechanism has been suggested to account for breast calcifications. A report showed that breast calcifications are clearly generated by the mouse breast cancer cell line 4T1 through osteoblast-like characteristics. However, the details underlying such a mechanism of mammary calcification is unknown. Understanding this mechanism will improve diagnostic accuracy for breast cancer. We induced calcifications using osteogenic cocktail on the human breast cancer cell line MDA-MB-231. Our hypothesis was that breast cancer calcifications are generated in a process similar to that of osteoblast differentiation. By a comprehensive analysis of transcription compared with osteoblastic cell line MC3T3-E1 in a microarray, we showed that placental alkaline phosphatase (PLAP) is involved in producing calcifications through PI3K-Akt signaling pathways. Herein, we demonstrated that calcifications occur and that alkaline phosphatase enzyme activity increases in MDA-MB-231 cells cultured using an osteogenic cocktail-containing medium. Microarray transcript analysis showed that the PI3K-Akt signaling pathway was significantly involved, with recruitment of placental alkaline phosphatase. Calcifications and alkaline phosphatase enzyme activity were suppressed by silencing placental alkaline phosphatase using a small interfering RNA. Inhibition of the PI3K-Akt signaling pathway suppressed phospho-c-Jun and placental alkaline phosphatase and resulted in absence of calcifications. These findings reveal that breast cancer cells acquire alkaline phosphatase enzyme activity via placental alkaline phosphatase expression and suggest that breast calcification formation is closely associated with the PI3K-Akt signaling pathway. Our results suggest that PLAP produces ALP enzyme activity required for calcification in MDA-MB-231 cells. PLAP is clinically expressed in some breast cancers. A report showed that dexamethasone induces PLAP enzyme activity and mRNA in the human breast cancer BC-M1 cell line. Therefore, dexamethasone in osteogenic cocktail may be associated with PLAP expression in the MDA-MB-231 cells. On the other hand, c-Jun binds to the promoter region of ALPP in MDA-MB-231, and we showed that c-Jun phosphorylation appears in MDA-MB-231 cells cultured using osteogenic cocktail. We suggest that PLAP transcript would be activated when phospho-c-Jun enters the nucleus and binds to the promoter region of PLAP. Our proposed mechanism in breast calcifications is that osteogenic cocktail induces c-Jun phosphorylation via PI3K-Akt signaling pathway and that PLAP transcription would be activated when phospho-c-Jun enters the nucleus and binds to the promoter region of PLAP. Future research might assess whether increased PLAP levels are the main driver of intracellular calcifications in breast cancer, as well as whether the PI3K-Akt signaling pathway has a similar effect in vivo that we demonstrated to cause calcifications in vitro. Citation Format: Atsushi Fushimi, Hiroshi Takeyama, Toshiaki Tachibana, Yoshinobu Manome. Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-04.

Balancing the biocompatibility and bacterial resistance of polypyrrole by optimized silver incorporation

Polypyrrole (PPy) which is a conductive polymer with excellent biocompatibility has enormous potential in implantable electronics. However, pristine PPy does not have sufficient bacterial resistance and hence, bacterial infection poses serious threats in vivo. Silver is an excellent antibacterial agent but the optimal concentration is critical because excessive silver is detrimental to human health. In this study, electrochemical polymerization is carried out to fabricate PPy coatings and silver ions (Ag) are introduced by plasma immersion ion implantation (PIII). The optimal Ag ion fluence is determined by monitoring the antibacterial efficiency and cytotoxicity. Our results show that the optimal balance between the antibacterial ability and cytocompatibility can be attained from sample Ti-PPy@Ag-4 implanted with a silver ion fluence of 4 × 1016 ions cm−2. In addition to retaining good cytocompatibility, 92% of the bacteria Staphylococcus aureus (S. aureus) can be eliminated. The intricate balance between antibacterial effects and biocompatibility arises from the levels of intracellular reactive oxygen species (ROS) in S. aureus and MC3T3-E1 osteoblasts on Ti-PPy@Ag-4. The antibacterial capability and biocompatibility are verified by the subcutaneous infection model in rats in vivo. The results reveal a simple strategy to improve the bacterial resistance of polymers such as PPy while not compromising the inherent biosafety of the materials. To the best of our knowledge, this is the first attempt to functionalize PPy by Ag PIII to create the proper balance between the antibacterial capacity and biosafety of biomedical implants.

PPAR-γ agonist pioglitazone alleviates inflammatory response induced by lipopolysaccharidesin osteoblast cells.

Osteomyelitis is an acute or chronic inflammatory bone disease with a high disability rate. As an anti-inflammatory factor, peroxisome proliferator activated receptor-γ (PPAR-γ) is not only implicated in a variety of inflammatory responsesbut also regulates osteoblast differentiation and bone mass. However, the role of PPAR-γ in osteomyelitis is not fully understood. In the present study, we demonstrated that PPAR-γ showed a lower expression level in infected bone tissue of osteomyelitis patients as compared with uninfected bone tissue from nonosteomyelitis patients with fracture of the hip. We applied lipopolysaccharides (LPSs) in MC3T3-E1 osteoblast precursor cell line as an in vitro model for osteomyelitis. LPS treatment increased osteomyelitis-associated inflammatory cytokines such as interleukin-6(IL-6) and tumor necrosis factor-α (TNF-α), whereas PPAR-γ levels and cell viability in MC3T3-E1 cells were suppressed. PPAR-γ antagonist GW9662 further enhanced IL-6 and TNF-α levels, and decreased cell viability in the presence of LPS treatment. In contrast, PPAR-γ agonist pioglitazone antagonized the effect of LPS treatment in MC3T3-E1 cells. These findings suggest that PPAR-γ downregulation is associated with the inflammation and progression of osteomyelitis, and PPAR-γ agonist could serve as a therapeutic strategy to attenuate inflammatory responses. This study provides novel insights into the physiopathogenesis of osteomyelitisand future study is required to validate the findings in animal model and uncover the molecular mechanism of PPAR-γ-dependent anti-inflammation in osteoblasts.

Synthesis and Characterization of Sol-Gel Derived Strontium Doped S53P4 Bioglass

This study concerns the evaluation of the bioactivity and cells response of strontium (Sr) doped sol-gel derived S53P4 bioglass due to Sr induced osteoblast. Moreover it prevents in-vitro osteoclastic activity and is clinically used as osteoporosis treatment. The different amount of Sr was doped into the S53P4 bioglass formulation (53.82%SiO2-1.72%P2O5-22.64%Na2O-(21.76-x)%CaO-x%SrO) (x=0, 3 and 5 mol %) and synthesized via sol-gel method. These samples were denoted as 0Sr, 3Sr and 5Sr respectively. After soaking in Hank's balanced salt solution (HBSS) for 7 and 14 days, the apatite formation was examined using X-ray powder diffraction (XRD) and scanning electron microscope (SEM) techniques. Proliferation and alkaline phosphatase activity were evaluated using osteoblastic cell line MC3T3-E1. The XRD and SEM findings confirmed the hydroxyapatite (HA) structure on the bioglass surface after soaking. More intense HA peaks were observed in 3Sr specimen on 7 day while in 5Sr specimen on 14 day. Meanwhile, 3Sr specimen showed the highest cells proliferation and ‌ significant difference in alkaline phosphatase (ALP) activity than 0Sr and 5Sr. As a result, this finding indicates that S53P4 bioglass with 3 mol % SrO (3Sr) is a good candidate for bone tissue engineering because it allows for optimum cell proliferation and ALP activity while also having a high bioactivity efficiency.