Activity Of MG63 Cells Research Articles

LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) regulates a variety of malignant properties of cancer cells. It is known that endothelial cells promote tumor progression and chemoresistance. The present study aimed to investigate the roles of LPA5 in cellular functions modulated by endothelial cells and anticancer drug in osteosarcoma cells. Human osteosarcoma MG-63 cells were maintained in endothelial F2 cell supernatants. After culturing for 3 months, MG63-F2 cells were established. LPAR5 expression level in MG63-F2 cells was significantly elevated, compared with MG-63 cells. The cell motile activity of MG63-F2 cells was markedly higher than that of MG-63 cells. To validate the effects of LPA5 on cell motile activity, LPA5 knockdown cells were generated from MG-63 cells. The cell motile activity of MG-63 cells was inhibited by LPA5 knockdown. The cell survival to cisplatin (CDDP) was reduced in MG-63 cells treated with LPA. In the presence of LPA, the cell survival rate was significantly lower in MG63-F2 cells than MG-63 cells, correlating with LPAR5 expression. LPA5 knockdown cells indicated the high cell survival rate to CDDP. Moreover, LPAR5 expression level was increased in the long-term CDDP treated MG63-C cells. The cell survival to CDDP of MG63-C cells was enhanced by LPA5 knockdown. These results suggest that cellular functions are regulated through LPA5-mediatd signaling induced by endothelial cells and CDDP in MG-63 cells.

In Vitro Osteocompatibility and Enhanced Biocorrosion Resistance of Diammonium Hydrogen Phosphate-Pretreated/Poly(ether imide) Coatings on Magnesium for Orthopedic Application.

Magnesium, as a biodegradable metal, is a promising candidate for biomedical applications. To modify the degradation behavior of magnesium and improve its osteocompatibility, chemical conversion and spin coating methods were combined to develop a diammonium hydrogen phosphate-pretreated/poly(ether imide) (DAHP/PEI) co-coating system. The diammonium hydrogen phosphate pretreatment was employed to enhance the attachment between PEI coatings and the magnesium substrate; meanwhile, it could serve as another bioactive and anticorrosion layer when PEI coatings break down. Surface characterization, electrochemical tests, and short-term immersion tests in DMEM were performed to evaluate DAHP/PEI coatings. Electrochemical measurements showed that DAHP/PEI coatings significantly improved the corrosion resistance of pure magnesium. No obvious changes of the chemical compositions of DAHP/PEI coatings occurred after 72 h of immersion in DMEM. An in vitro cytocompatibility study confirmed that viability and LDH activity of human osteoblast-like cells on DAHP/PEI coatings showed higher values than those on the DAHP-pretreated layer and pure magnesium. The DAHP-pretreated layer could still enhance the ALP activity of MG-63 cells after the degradation of PEI in DAHP/PEI coatings. Besides that, the in vitro cellular response to the treated magnesium was investigated to gain knowledge on the differentiation and proliferation of human adipose-derived stem cells (hADSCs). Cell distribution and morphology were observed by fluorescence and SEM images, which demonstrated that DAHP/PEI coatings facilitated cell differentiation and proliferation. The high level of C-terminals of collagen type I production of hADSCs on DAHP/PEI coatings indicated the potential of the coating for promoting osteogenic differentiation. Positive results from long-term cytocompatibility and proliferation tests indicate that DAHP/PEI coatings can offer an excellent surface for hADSCs.

Impact of Biofield Energy Treatment Based Test Formulation on Vital Organ Health Specific Biomarkers Using Cell Line Study

Multiple organ dysfunction syndrome or failure is one of the major concerns against healthcare services in order to maintain the normal function. The present study aimed to explore the impact of the Biofield Energy Treated test formulation on the function of vital organs such as bones, heart, liver, lungs, and brain using standard activity parameters in specific cell-based assays. The test formulation and cells medium was divided into two parts, one untreated (UT) and other part received the Biofield Energy Treatment remotely by a renowned Biofield Energy Healer, Ariadne Esmene Afaganis, Canada and was labeled as the Biofield Treated (BT) test formulation/media. The test formulation was tested for cell viability, and the data suggested that the test formulation was found safe and non-toxic against all the cell lines. Cytoprotective activity among the experimental groups showed a significant improved activity by 94.4% at 1 µg/mL in untreated medium (UT-Med) + Biofield Treated Test Item (BT-TI) group in human cardiac fibroblasts cells (HCF) cells, while 84.4% at 10 µg/mL in BT-Med + BT-TI groups in human hepatoma cells (HepG2), and 124% increased cytoprotective action at 1 µg/mL in UT-Med + BT-TI group in adenocarcinomic human alveolar basal epithelial cells (A549) cells as compared with the untreated test group. ALP activity in MG-63 cells was significantly increased by 85.9% at 10 µg/mL in the UT-Med + BT-TI group, while in Ishikawa cells showed maximum increased ALP activity by 59.2% at 0.1 µg/mL in BT-Med + BT-TI groups as compared to the untreated group. The percent protection of HCF (heart) cells (decreased of LDH activity) was significantly increased by 53% and 40.5% at 1 and 10 µg/mL concentrations respectively, in UT-Med + BT-TI group, while BT-Med + UT-TI group showed increased protection by 68.5%, 70.7%, and 16.8% at 0.1, 1, and 10 µg/mL respectively, and 86.5%, 62.5%, and 34.2% improved cellular protection at 0.1, 1, and 10 µg/mL respectively, in BT-Med + BT-TI group as compared to the untreated test group. The percent protection of HepG2 (liver) cells (decreased of ALT activity) was reported by 33.5%, 63.2%, and 99.2% at 10 µg/mL in the UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI groups, respectively compared to the untreated group. Cellular protection of A549 (lungs) cells (increased of SOD activity) in terms of percentage was increased by increased by 39.8% (at 10 µg/mL), 44% (at 25.5 µg/mL), and 59.7% (at 25.5 µg/mL) in the UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI groups, respectively compared to untreated group. Serotonin level was significantly increased by 59.2% (at 0.1 µg/mL), 190.3% (at 0.1 µg/mL), and 201% (at 1 µg/mL) in the UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI groups, respectively compared to untreated in human neuroblastoma cells (SH-SY5Y). However, the relative quantification (RQ) of vitamin D receptor (VDR) was significantly increased by 159.1% (at 50 µg/mL), 212.7% (at 1 µg/mL), and 278.3% (at 10 µg/mL) in the UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI groups, respectively as compared to the untreated in MG-63 cells. Thus, the present data concluded that the overall multiple organ health using various standard biomarkers in specific cell lines were significantly improved with respect to health of bones, heart, liver, lungs, and brain after treatment with the Biofield Energy treated test formulation (The Trivedi Effect®). Thus, it can be used as a complementary and alternative therapy approach against many multiple organ disorders such as coronary artery disease, arrhythmias, congenital heart disease, cardiomyopathy, cirrhosis, liver cancer, hemochromatosis, asthma, chronic bronchitis, cystic fibrosis, osteoporosis, etc.

EP1 receptor is involved in prostaglandin E2-induced osteosarcoma growth.

Recent studies showed that the activation of prostaglandin (PG) receptor EP1 promotes cell migration and invasion in different cancers. The aim of this study was to investigate the role of EP1 in the proliferation of osteosarcoma (OS) cells in vitro and in vivo. EP1 mRNA and protein levels were analyzed by real-time RT-PCR and Western blot, respectively in human OS cell lines MG63, OS732, U-2OS, and 143B compared to human fetal osteoblastic hFOB 1.19 cells. MG63 cells were treated with PGE2, EP1 specific agonist 17-PT-PGE2, 17-PT-PGE2 + EP1 specific antagonist SC51089, or DMSO (control). EP1R-siRNA or a non-silencing irrelevant RNA duplex (negative control) were used for the transfection of MG63 cells, followed by PGE2 treatment. Nude mice carrying MG63 xenografts were treated with SC51089 (2 mg/kg/day). MG63 cells/xenografts were analyzed by MTT assay, TUNEL assay, PKC enzyme activity assay, and Western blot (EP1 and apoptotic proteins), and tumor growth/volume was evaluated in mice. EP1 levels were significantly higher in OS cells compared to osteoblasts. PGE2 or 17-PT-PGE2 treatment increased the proliferation and decreased the apoptosis of MG63 cells. Inhibition of EP1 by SC51089 or siRNA markedly decreased the viability of MG63 cells. Similarly, SC51089 treatment significantly inhibited MG63 cell proliferation and promoted apoptosis in vivo. The silencing of EP1 receptor by siRNA or blockade of EP1 signaling by SC51089 activated extrinsic and intrinsic apoptotic pathways both in vivo and in vitro, as evidenced by increased levels of Bax, cyt c, cleaved caspase-3, caspase-8 and caspase-9. EP1 appears to be involved in PGE2-induced proliferative activity of MG63 cells. Antagonizing EP1 may provide a novel therapeutic approach to the treatment of OS.

Biofield Energy Treatment Based Test Formulation as a Novel and Efficient Approach on Various Biomarkers in human Bones, Heart, Liver, Lungs, and Brain Cells

Multiple organ dysfunction syndromes (MODS) are one of the common reasons for increased mortality rate against healthcare services. The present experiment aimed to determine the impact of the Biofield Energy Treated test formulation on the function of vital organs such as bones, heart, liver, lungs, and brain using standard activity parameters in specific cell-based assays. The test formulation and cells medium was divided into two parts; one untreated (UT) and other part received the Biofield Energy Treatment remotely by a renowned Biofield Energy Healer, Laura Nelson Streicher, and USA and were labeled as the Biofield Energy Treated (BT) test formulation/media. The test formulation was tested against various activities using cell line assay. The test formulation was tested for cell viability, and the results showed that the test formulation was found non-toxic against all the cell lines at the tested concentrations. Cytoprotective activity among the experimental groups showed a significant improved activity by 156.9% at 1 µg/mL in UT- medium (Med) + BT - test item/formulation (TI) group in human cardiac fibroblasts cells (HCF) cells, while 94.5% at 25.5 µg/mL in the UT-Med + BT-TI group in human hepatoma cells (HepG2), and 92.4% at 63.75 µg/mL in the BT-Med + BT-TI group as compared with the untreated test group in adenocarcinomic human alveolar basal epithelial cells (A549) cells as compared with the untreated test group. Alkaline phosphatase (ALP) activity in MG-63 cells was significantly increased by 80.2% and 93.5% at 10 and 50 µg/mL respectively in the UT-Med + BT-TI group, while in Ishikawa cells showed maximum increased ALP activity by 51.5% at 1 µg/mL in UT-Med + BT-TI group as compared to the untreated group. The percent protection of HCF (heart) cells (decreased of lactate dehydrogenase-LDH activity) was significantly increased by 156.9% and 18.3% at 1 and 10 µg/mL concentrations respectively, in UT-Med + BT-TI group, while BT-Med + UT-TI group showed increased protection by 24.5% and 17.7% at10 and 25.5 µg/mL respectively, and 67.1%, 32.2%, 47.7%, and 73.1% improved cellular protection 1, 10, 25.5, and 63.75 µg/mL respectively, in BT-Med + BT-TI group as compared to the untreated test group. Alanine amino transferase (ALT) in terms of percent protection of HepG2 (liver) cells (decreased of ALT activity) was reported by 93.1% and 64.1% at 1 µg/mL in the UT-Med + BT-TI and BT-Med + UT-TI groups, respectively as compared to the untreated group. Cellular protection of A549 (lungs) cells (increased of superoxide dismutase-SOD activity) in terms of percentage was increased by 20.6% (at 0.1 µg/mL) and 6.9% (at 10 µg/mL) in the UT-Med + BT-TI and BT-Med + BT-TI groups, respectively as compared to untreated group. Serotonin level was significantly increased by 59.3% (at 10 µg/mL), 55.6% (at 1 µg/mL), and 100.1% (at 0.1 µg/mL) in the UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI groups, respectively compared to untreated in human neuroblastoma cells (SH-SY5Y). However, the relative quantification (RQ) of vitamin D receptor (VDR) was significantly increased by 150.6% (at 50 µg/mL), 380.1% (at 10 µg/mL), and 148.1% (at 50 µg/mL) in the UT-Med + BT-TI, BT-Med + UT-TI, and BT-Med + BT-TI groups, respectively as compared to the untreated in MG-63 cells. In conclusion, test formulation would be significantly useful for multiple organ health and improve overall health of bones, heart, liver, lungs, and brain after treatment with the Biofield Energy treated test formulation (The Trivedi Effect®). As a complementary and alternative therapy, Biofield Energy approach can be used against coronary artery disease, arrhythmias, congenital heart disease, cardiomyopathy, cirrhosis, liver cancer, hemochromatosis, asthma, chronic bronchitis, cystic fibrosis, osteoporosis, etc.

The role of the ERK1/2 pathway in simvastatin-loaded nanomicelles and simvastatin in regulating the osteogenic effect in MG63 cells.

ObjectivesThe present study aimed to clarify the role of the ERK1/2 pathway in simvastatin (SV)-loaded nanomicelles (SVNs)- and SV-mediated promotion of cell osteogenic differentiation and explore the molecular mechanisms by which SVNs exhibited a greater efficacy in promoting osteogenic differentiation than SV.Materials and methodsSVNs were synthesized using a dialysis method. MG63 cells were treated with 2.5, 0.25, and 0.025 μmol/L of the drug. The optimal drug dosage was determined by examining the proliferative activity and ALP activity of the MG63 cells. Subsequently, Western blot analysis was performed to analyze the levels of the phosphorylated ERK1/2 proteins in each experimental group at various time points. Finally, the inhibitor PD98059 was used to effectively inhibit the ERK1/2 pathway. The resulting changes in the proliferative activity of MG63 cells and the osteogenesis-related markers were analyzed.ResultsThe SVNs synthesized in the present study had a mean diameter of 27 nm. The encapsulation and drug-loading efficiencies were 52.03% ± 4.05% and 9.42% ± 0.66%, respectively. SVNs and SV exhibited optimum osteogenesis-promoting effects when the drugs were administered at a concentration of 0.25 μmol/L. The drug-induced activation of the ERK1/2 pathway reached a peak at 15 minutes after administration and then declined rapidly. From 24 hours to 7 days, SVNs and SV exerted an inhibitory effect on the ERK1/2 pathway rather than an activating effect. Throughout the whole experimental process, the regulatory effect of SVNs on the ERK1/2 pathway was significantly greater than that of SV. Inhibition of the ERK1/2 pathway by PD98059 markedly reduced the proliferative activity of the cells in all experimental groups. In addition, the ALP activity and the expression levels of the osterix (OSX) and osteocalcin (OC) proteins were drastically increased.ConclusionSVNs significantly increased the effect of SV-induced osteogenic differentiation by strongly inhibiting the ERK1/2 pathway.

Bioactive Sr(II)/Chitosan/Poly(ε-caprolactone) Scaffolds for Craniofacial Tissue Regeneration. In Vitro and In Vivo Behavior.

In craniofacial tissue regeneration, the current gold standard treatment is autologous bone grafting, however, it presents some disadvantages. Although new alternatives have emerged there is still an urgent demand of biodegradable scaffolds to act as extracellular matrix in the regeneration process. A potentially useful element in bone regeneration is strontium. It is known to promote stimulation of osteoblasts while inhibiting osteoclasts resorption, leading to neoformed bone. The present paper reports the preparation and characterization of strontium (Sr) containing hybrid scaffolds formed by a matrix of ionically cross-linked chitosan and microparticles of poly(ε-caprolactone) (PCL). These scaffolds of relatively facile fabrication were seeded with osteoblast-like cells (MG-63) and human bone marrow mesenchymal stem cells (hBMSCs) for application in craniofacial tissue regeneration. Membrane scaffolds were prepared using chitosan:PCL ratios of 1:2 and 1:1 and 5 wt % Sr salts. Characterization was performed addressing physico-chemical properties, swelling behavior, in vitro biological performance and in vivo biocompatibility. Overall, the composition, microstructure and swelling degree (≈245%) of scaffolds combine with the adequate dimensional stability, lack of toxicity, osteogenic activity in MG-63 cells and hBMSCs, along with the in vivo biocompatibility in rats allow considering this system as a promising biomaterial for the treatment of craniofacial tissue regeneration.

MicroRNA-499a-5p inhibits osteosarcoma cell proliferation and differentiation by targeting protein phosphatase 1D through protein kinase B/glycogen synthase kinase 3β signaling.

A number of studies have attempted to elucidate the association between mircoRNAs (miRNAs/miRs) and cancer-associated processes. The aim of the present study was to determine how miR-499a-5p intervenes in human osteosarcoma cell proliferation and differentiation. The cancerous tissues and adjacent non-cancerous tissues of 62 patients with osteosarcoma (OS) were collected. miRNA microarray analysis revealed that 29 miRNAs were upregulated while 26 were downregulated, among which miR-499a-5p expression was the most decreased. Western blot analysis and reverse transcription-quantitative polymerase chain reaction demonstrated that the mRNA and protein expression of miR-499a-5p was lower, while that of protein phosphatase 1D (PPM1D) was higher in OS tissues compared with expression levels in normal tissues. Furthermore, miR-499a-5p expression was markedly decreased in the metastatic tumors and in those at stage III+IV compared with the non-metastatic tumors and those at stage I, respectively. In addition, following transfection of the human OS MG-63 cell line with an miR-499a-5p mimic, the expression of miR-499a-5p was elevated while the protein and mRNA expression of PPM1D was decreased. When combining these findings with the information obtained from the Targetscan predictive software, it was confirmed that PPM1D was targeted by miR-499a-5p. In MG-63 cells transfected with an miR-499a-5p mimic, PPM1D-associated downstream proteins phosphorylated protein kinase B (p-Akt) and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) were significantly downregulated compared with the negative control (NC) group, while the expression of p-Akt and p-GSK-3β were significantly elevated in the tumor tissues compared with the adjacent non-tumor tissues. Simultaneously, the growth and proliferation activity of MG-63 cells were notably reduced when transfected with the miR-499a-5p mimic, compared with the NC group. Therefore, it may be concluded that miR-499a-5p suppresses OS cell proliferation and differentiation by targeting PPM1D through modulation of Akt/GSK-3β signaling.

Synergistic effects of retinoic acid and graphene oxide on the physicochemical and in-vitro properties of electrospun polyurethane scaffolds for bone tissue engineering

AbstractTissue engineering scaffolds simulate extracellular matrixes (ECMs) to promote healing processes of damaged tissues. In this investigation, ECM were simulated by retinoic acid-loaded polyurethane-graphene oxide nanofibers to regenerate bone defects. Scanning electron microscopy (SEM) micrographs, Fourier transform infrared (FTIR) spectrum and X-ray diffraction (XRD) patterns proved the synthesis of graphene oxide (GO) nanosheets. SEM micrographs of nanofibers demonstrated through the formation of homogeneous and bead free fibrous scaffolds that the diameter of fibers were reduced by decreasing the applied voltage in an electrospinning process and the addition of GO. According to the results, the addition of GO to the polyurethane (PU) solution led to an increase in mechanical strength which is the most important parameter in the hard tissue repair. The GO-containing scaffolds showed an increased wettability, swelling, biodegradation and drug release level. Release behavior in nanocomposite scaffolds followed the swelling and biodegradation mechanisms, so osteogenic expression was possible by incorporating retinoic acid (RA) in PU-GO nanofibrous scaffolds. Biological evaluations demonstrated that composite scaffolds are biocompatible and support cellular attachment in which RA-loaded samples represented better cellular spreading. In brief, nanocomposite fibers showed desired that the physicochemical, mechanical and biological properties and synergic effects of GO and RA in osteogenic activity of MG-63 cells produced favorable constructs for hard tissue engineering applications.

Correlation between osteosarcoma and the expression of WWOX and p53.

The objective of this study was to analyze the effect of the expression of WWOX and p53 on the growth of MG-63 osteosarcoma cells and to explore the correlation between osteosarcoma and the expression of WWOX and p53. WWOX and p53-overexpressing MG-63 osteosarcoma cell lines were established by transfection and named the MW and MP cell lines, respectively. Untransfected MG-63 cells (blank control) were used as control. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of WWOX and wild-type p53 mRNA and protein, respectively. The effects of WWOX and p53 (wild-type) on the activity of MG-63 cells were determined by MTT assay and flow cytometry. The expression of mutant p53 protein in 65 cases of osteosarcoma was detected by immunohistochemistry to analyze the correlation between p53 and the development of osteosarcoma. qPCR showed that WWOX and p53 mRNA was overexpressed in MW and MP cells, respectively. Western blot analysis showed that the levels of WWOX and p53 protein in MW and MP cells were higher than in the blank control group. MTT assay showed that the cell proliferation ability of MW and MP cells was significantly lower than in the blank control group. Flow cytometry showed that 78.49% of MW and 66.76% of MP cells were arrested in the G0/G1 phase. Immunohistochemistry showed that mutant p53 was highly expressed in osteosarcoma, with a positive expression rate of 47.7%. The expression rate was positively correlated with the pathological grade of cancer. In conclusion, WWOX can affect the cell cycle of MG-63 osteosarcoma cells to inhibit cell proliferation, which provides new insights into gene therapy for osteosarcoma. The two types of the p53 gene have different functions in the development of osteosarcoma. Wild-type p53 acts as a tumor suppressor, while mutant p53, which is overexpressed in malignant osteosarcoma, has a carcinogenic effect associated with the degree of osteosarcoma.

Effect of polygonimitin C on bone formation and resorption in human osteoblast-like MG63 cells

Purpose: To investigate the effect of polygonimitin C (PC) on bone formation and resorption in human osteoblast-like MG63 cells.Methods: MG63 cells were treated with PC at doses of 0, 20, 40 or 80 μg/mL for 48 h, with an untreated group as control. The effect of PC on alkaline phosphatase (ALP) activity in MG63 cells was investigated by p-nitrophenyl phosphate disodium hexahydrate assay. Western blot assay was used to evaluate the effect of PC on the expressions of osterix (OSX), bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), fibronectin (FN), type I collagen (COL I), osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) proteins in MG63 cells.Results: ALP relative activity in MG63 cells treated with PC at 20, 40 or 80 μg/mL (123.58, 137.74 or 159.62 %, respectively) was significantly (p < 0.05 or 0.01) higher than that in control group (99.37 %). Expressions of OSX, BMP-2, RUNX-2, OC, FN, COL I and OPG proteins in MG63 cells treated with PC at 20, 40 or 80 μg/mL were significantly (p < 0.01) higher than those in control group. However, there were no statistically significant differences in RANKL protein expression between PC-treated MG63 cells and control group.Conclusion: These results show that PC exerts protective effects against osteoporosis by promoting bone formation and inhibiting bone resorption. Thus, PC may be useful in the development of new antiosteoporosis drugs.Keywords: Polygonimitin C, MG63 cells, Bone formation, Bone resorption, Osteoporosis

Hybrid hydrogels with high strength and biocompatibility for bone regeneration

The development of hydrogels for bone regeneration has highlighted the challenge that load-bearing hydrogels need to be biocompatible while achieving high strength. Several approaches have been reported to improve the toughness of hydrogels, but achieving high toughness and biocompatibility simultaneously remains a challenge. Here we report a polyacrylic acid/alginate/demineralized bone matrix (PAA/Alg/DBM) hybrid double network hydrogel, which was synthesized by a two-step sequential polymerization with embedded DBM, possessing both high strength and biocompatibility. With the persistence of DBM, it can promote the synthesis of VEGF and bFGF and the ALP activity of MG63 cells on hydrogel. All the results suggest that this hybrid double network hydrogel have potential for future application in bone regeneration.

Pattern-coated titanium bone fixation plate for dual delivery of vancomycin and alendronate

Metallic bone-fixation devices have been widely used in the treatment of fractured bones. However, there are still some unmet clinical needs associated with post-surgical infection and hampered bone repair. Therefore, to both prevent infection and enhance bone formation, we propose a titanium (Ti)-based bone plate for dual delivery of an antibiotic, vancomycin (VAN), and a bone-forming drug, alendronate (AL). In this work, we prepared the coating with a blend of a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), and a drug, VAN or AL. Instead of coating the whole surface, we patterned the coatings each in the shape of a dot, on a flat surface of the Ti bone plate currently in clinical use (BP0001-PT, U&I, Korea). This way, we could prevent detachment of the polymeric coating from the metallic plate surface while being immersed in an aqueous medium for 23 days. Both VAN and AL were released in a sustained manner for more than 28 days, maintaining their own activity without being influenced by their co-existence in the medium. Thus, the coated plate showed an antibacterial activity against Staphylococcus aureus, while also promoting osteoblast proliferation and alkaline phosphate activity in MG-63 cells. Therefore, we conclude that a Ti-based bone plate coated with patterned dots, each for delivery of VAN or AL, can be a promising strategy to allow for both antibacterial and bone-forming activities.

The response of human osteoblasts, epithelial cells, fibroblasts, macrophages and oral bacteria to nanostructured titanium surfaces: a systematic study.

Nanotopography modification is a major focus of interest in current titanium surface design; however, the influence of the nanostructured surface on human cell/bacterium behavior has rarely been systematically evaluated. In this study, a homogeneous nanofiber structure was prepared on a titanium surface (Nano) by alkali-hydrothermal treatment, and the effects of this Nano surface on the behaviors of human MG-63 osteoblasts, human gingival epithelial cells (HGECs) and human gingival fibroblasts (HGFs) were evaluated in comparison with a smooth titanium surface (Smooth) by polishing and a micro-rough titanium surface (Micro) by sandblasting and acid etching. In addition, the impacts of these different surface morphologies on human THP-1 macrophage polarization and Streptococcus mutans attachment were also assessed. Our findings showed that the nanostructured surface enhanced the osteogenic activity of MG-63 cells (Nano=Micro>Smooth) at the same time that it improved the attachment of HGECs (Nano>Smooth>Micro) and HGFs (Nano=Micro>Smooth). Furthermore, the surface with nanotexture did not affect macrophage polarization (Nano=Micro=Smooth), but did reduce initial bacterial adhesion (Nano<Smooth<Micro). These results suggest that the nanostructured titanium surface may promote bone and soft tissue healing, and thereby increase the success and survival of dental implants.

The Incorporation of Strontium in a Sodium Alginate Coating on Titanium Surfaces for Improved Biological Properties

Orthopedic implant failure is mainly attributed to the poor bonding of the implant to bone tissue. An effective approach to minimize the implant failure would be modifying the surface of the implant. Strontium (Sr) can stimulate the proliferation and differentiation of osteoblasts and reduce the activity of osteoclasts. In this study, a titanium (Ti) surface was successively functionalized by covalently grafting dopamine, sodium alginate (SA), and Sr2+ via the electrostatic immobilization method. The as-prepared coatings on the Ti surface were characterized by using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), and contact angle. The results indicated that the Sr-incorporated coatings were successfully prepared and that Sr distributed uniformly on the surface. A long-lasting and sustained Sr release had been observed in Sr2+ release studies. The Ti/DOPA/SA/Sr exhibited little cytotoxicity and a robust effect of Sr incorporation on the adhesion and spreading of MG63 cells. The proliferation and alkaline phosphatase (ALP) activity of MG63 cells were enhanced by immobilizing Sr2+ on the SA-grafted Ti. The Sr-containing coatings, which displayed excellent biocompatibility and osteogenic activity, may provide a promising solution for promoting the tissue integration of implants.

Dual-functional bone implants with antibacterial ability and osteogenic activity.

A growing number of biomaterial-associated infections cause bone implant failures in early and long-term applications. In this regard, a calcium silicate-gelatine composite bone implant with high strength and superior osteogenic activity was coated with a layer of Ag, chitosan polysaccharide (CS) or water-soluble chitosan oligosaccharide (COS) as a bactericidal agent. The influences of surface modifications to the bone implants on phase composition, microstructure, antibacterial effectiveness, and osteogenic activity in vitro were evaluated. Experimental results revealed the presence of the coating on the implant surface using a simple deposition technique. The in vitro antibacterial evaluation indicated that the antimicrobial effectiveness of the Ag coating against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) was inferior to the 0.4% CS coating, but comparable to those of 0.2% CS and 0.4% COS coatings after 48 h of culture. CS presented a greater bactericidal effect than COS, which was bacteria-independent. CS and COS coatings had no significant cytotoxicity towards L929 cells at coating concentrations of 0.1%, 0.2%, and 0.4%, except for the cells exposed to the 0.4% CS coating, while the 0.004% Ag coating remarkably produced cytotoxicity. The assays of cell functions consistently showed significantly higher osteogenic activity of MG63 cells grown on CS and COS-coated surfaces by increased attachment, proliferation, alkaline phosphatase, osteocalcin, and calcium deposits production, except for the 0.4% CS coating, in comparison with those on the Ag coated surface. It was concluded that, taking antibacterial ability and osteogenic activity into account, 0.2% CS-coated and 0.4% COS-coated calcium silicate-gelatine composite bone implants had a large potential to be used in bone grafts and fracture fixation devices.

Rapid fabrication of dense 45S5 Bioglass® compacts through spark plasma sintering and evaluation of their in vitro biological properties

It is challenging to obtain dense 45S5 Bioglass® (45S5) with controlled crystallinity and satisfactory mechanical properties by conventional sintering processes due to its fast crystallization above the first glass transition temperature. Spark plasma sintering (SPS) has stood out in this respect by virtue of its capability to provide fast heating and densification rates. However, there have been insufficient investigations into the in vitro biological properties of 45S5 compacts obtained by SPS. In this study, we report the fabrication of fully densified 45S5 pellets in the temperature range of 500 °C–600 °C through a rapid SPS process (sintering for 3 min) as well as the assessment of the influence of sintering temperature and aqueous aging on the biological properties of sintered pellets with L929 and MG63 cells. The cell culture results showed that both extended ageing and a lower SPS temperature in the 500–600 °C range could generally lead to faster cell proliferation and higher cell viability. The former was possibly caused by the slower alkalization of the media during cell culture, and the latter may have resulted from the release of more Ca and Si ions. The pellet sintered at 550 °C without aqueous aging led to the highest ALP activity in MG63 cells, which may be attributed to the high interfacial pH at the pellet surface and the leaching of more Si ions. Therefore, dense 45S5 compacts with mild crystallinity consolidated by SPS at 550 °C is a promising candidate for orthopedic implants in loading bearing applications.

Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.

Degradability, cytocompatibility, and osteogenesis of porous scaffolds of nanobredigite and PCL-PEG-PCL composite.

Biocomposite scaffolds were fabricated by incorporation of nanobredigite (n-BD) into the polymer of poly(ε-caprolactone)–poly(ethyleneglycol)–poly(ε-caprolactone) (PCL–PEG–PCL). The results revealed that the addition of n-BD into PCL–PEG–PCL significantly improved water absorption, compressive strength, and degradability of the scaffolds of n-BD/PCL–PEG–PCL composite (n-BPC) compared with PCL–PEG–PCL scaffolds alone. In addition, the proliferation and alkaline phosphatase activity of MG63 cells cultured on n-BPC scaffolds were obviously higher than that cultured on PCL–PEG–PCL scaffolds. Moreover, the results of the histological evaluation from the animal model revealed that the n-BPC scaffolds significantly improved new bone formation compared with the PCL–PEG–PCL scaffolds, indicating good osteogenesis. The n-BPC scaffolds with good biocompatibility could stimulate cell proliferation, differentiation, and bone tissue regeneration and would be an excellent candidate for bone defect repair.

In vitro biological properties of 45S5 bioglass fabricated by spark plasma sintering (SPS)

Event Abstract Back to Event In vitro biological properties of 45S5 bioglass fabricated by spark plasma sintering (SPS) Zhong Li1, Brianna C. Thompson1, Huanlong Hu1 and Khiam Aik Khor1 1 Nanyang Technological University, School of Mechanical & Aerospace Engineering, Singapore Introduction: In our previous study, we reported the first use of Spark Plasma Sintering (SPS) to densify 45S5 Bioglass (45S5) and found that SPS could lead to better early-stage cytocompatibility compared with conventional sintering processes[1]. Although SPS has been utilized to fabricate bulk 45S5 and various 45S5-based composites, there is a lack of comprehensive investigation of the in vitro biological properties of such materials [2]-[4]. In this research, cell culture was carried out for 45S5 sintered by SPS at three different temperatures using two established cell lines: L929 and MG63 Materials and Methods: 45S5 powder (XL Sci-Tech Inc., US) was sintered for 3 min by an SPS system (Dr. Sinter 1050, SCM, Japan) at three different temperatures: 500, 550 and 600 °C. X-ray Diffraction (XRD) was utilized to identify the phase composition of the samples. Growth of L929 and MG63 was assessed with a CyQUANT Cell Proliferation Assay Kit (C7026) after culturing them for 3 days in the extractant media of sintered pellets. Alkaline phosphatase (ALP) activity was quantified using the Abcam ALP Assay Kit (ab83369) for MG63 cells cultured on the pellets’ surface for 4 days. In both experiments, the “new” samples were freshly polished and had not been soaked in any medium, while “old” samples had undergone prolonged soaking (~21 days) for other tests before the current study. Results and Discussion: It can be seen in Fig.1 that 45S5 sintered at 500 °C is amorphous, while pellets sintered at higher temperatures are crystalline with Na2CaSi3O8 as the major phase. Fig. 2 compares the cell number for different pellets. Statistical difference is represented by * (p≤0.05), ** (p≤0.01) and *** (p≤0.001) in Fig. 2 and Fig. 3, with asterisks in parentheses indicating differences between the old samples and their new counterparts. It is explicitly observed that 45S5 pellets all resulted in slower cell growth than the control (tissue culture plastic, TCP). Furthermore, both a smaller sintering temperature (and hence lower crystallinity) and long-term soaking prior to this research (and thus a smaller amount of ions released into the extractant media) could generally promote cell growth. Both old and new pellets compacted at 600 °C led to the slowest cell growth. It is interesting to find in Fig. 3 that 45S5 pellets all brought about remarkably higher ALP activity in MG63 than TCP, which is an important feature of the osteoblast phenotype. In addition, new samples could all result in significantly higher ALP activity than their old counterparts, indicating the presence of more bioactive substances which could facilitate cell differentiation and bone formation. Interestingly, 45S5 sintered at 550 °C led to much higher ALP activity in MG63 than those fabricated at higher or lower temperatures. This phenomenon suggests that neither fully amorphous nor highly crystalline 45S5 favors the differentiation of MG63 and bone formation [1]. Conclusions: This study investigated the in vitro biological properties of 45S5 pellets fabricated by SPS and reported for the first time the difference in ALP activity of MG63 cells cultured on 45S5 caused by different SPS temperatures. It was found that relatively low SPS temperatures (<600 °C) could bring about faster cell proliferation, and a moderate temperature of 550 °C could result in highest ALP activity. Therefore, it can be concluded that 550 °C should be used for sintering 45S5 used as orthopaedic implants. Z. LI thanks NTU for the University Research Scholarship