Human Osteoblastic Cells Research Articles

Orthodontic force regulates metalloproteinase-3 promoter in osteoblasts and transgenic mouse models

Background/purposePreviously we demonstrated up-regulation of matrix metalloproteinase-3 (MMP-3) in human osteoblasts under compression and in bony specimens of experimental orthodontic tooth movement (OTM). Here, we studied the temporal characteristics of compression stimulation in human and mouse osteoblast cell lines, and generated a transgenic mouse model for assessing the MMP-3 expression during OTM. Materials and methodsWe investigated MMP-3 expressions in human and murine osteoblasts through RT-PCR and luciferase assay, after compressive force loading. Inhibitors were added to identify the possible mechanisms for signal transduction. A human MMP-3 promoter was isolated, cloned and transfected to generate a transgenic mouse with a green fluorescent protein reporter. OTM was then initiated to observe the location and time course of transcriptional regulation of MMP-3 signals. ResultsWe found changes in the transcription of MMP-3 in response to mechanical force applied to both human and mouse osteoblast cell lines, suggesting that the response is positive across species. Cloned human MMP-3 promoter may cause the response of luciferase to 1% compression. Moreover, p38 inhibitor exerted a down-regulatory effect on MMP-3 promoter expression, although the inhibitory effect didn't reach a significant level. In the transgenic mouse OTM model, we again found increased expression of MMP-3 in response to mechanical force loading around the periodontal ligament. ConclusionMechanical force can stimulate MMP-3 expression, possibly through the p38 MAPK pathway, with its strongest signal occurring at 24 h. The mechanical responsiveness in MMP-3 promoter regions can be observed in both humans and rodents in vitro and in vivo.

Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (μCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

MiR-382-5p’s Function as a Suppressor in Osteosarcoma (OS) by Targeting PDPK1

ABSTRACT miR-382-5p engages in development of osteosarcoma (OS). However, the regulatory system of miR-382-5p in osteosarcoma remains to be revealed. This research studied the interplay between PDPK1 and miR- 382-5p in OS. RT-PCR was used to evaluate miR-382-5p and PDPK1 expression in OS cells and normal human osteoblast cells. Dual-luciferase reporter assay validated PDPK1 as a miR-382-5p target. CCK-8 evaluated the cell viability. Flow cytometric method determined cell apoptosis rate. Transwell and Scratch assays estimated the cell metastasis. miR-382-5p was inhibited in OS cells. Further functional results showed miR-382-5p upregulation reduced cell viability, and mobility by mediating PDPK1 in OS cells

Why SNP rs3755955 is associated with human bone mineral density? A molecular and cellular study in bone cells.

SNP rs3755955 (major/minor allele: G/A) located in Iduronidase-Alpha-L- (IDUA) gene was reported to be significant for human bone mineral density (BMD). This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone. We tested the effects of single nucleotide polymorphisms (SNP) rs3755955 (defined allele G as wild-type and allele A as variant-type) on osteoblastic and osteoclastic functions, as well as protein phosphorylation in stably transfected human fetal osteoblast (hFOB) cell and mononuclear-macrophage (RAW264.7) cell. In hFOB cells, transfection with variant-type IDUA significantly decreased osteoblastic gene expression (OPN, COL1A1 and RANKL) (p < 0.01), impeded cell proliferation (p < 0.05), stimulated cell apoptosis (p < 0.001) and decreased ALP enzyme activity, as compared with that of wild-type IDUA transfection. In RAW264.7 cells, transfection with variant-type IDUA significantly inhibited cell apoptosis (p < 0.01), promoted osteoclastic precursor cell migration (p < 0.0001), growth (p < 0.01), osteoclastic gene expression (TRAP, RANK, Inte-αv and Cath-K) (p < 0.05) and TRAP enzyme activity (p < 0.001), as compared with that of wild-type IDUA transfection. In both hFOB and RAW264.7 cells, the total protein and IDUA protein-specific phosphorylation levels were significantly reduced by variant IDUA transfection, as compared with that of wild-type IDUA transfection (p < 0.05). Variant allele A of phosSNP rs3755955 in IDUA gene regulates protein phosphorylation, inhibits osteoblast function and promotes osteoclastic activity. The SNP rs3755955 could alter IDUA protein phosphorylation, significantly regulates human osteoblastic and osteoclastic gene expression, and influences the growth, differentiation and activity of osteoblast and osteoclast, hence to affect BMD.

Mineralized nodule formation in primary osteoblasts culture in titanium doped phosphate glass and in-house prepared freeze dried demineralized bone extracts

ObjectivesMineral trioxide aggregate (MTA) is widely used in dentistry as root perforation repair material, however due to its high alkalinity, tissue necrosis might occur. This study aimed to investigate the mineralization potential of titanium dioxide doped phosphate glass (TDPG) and in-house prepared freeze dried demineralized bone (FDDB) as substitutes for MTA. MethodsTDPG and FDDB were prepared and characterized using scanning electron microscopy coupled with energy dispersive X-ray and Raman spectroscopy. The extract of FDDB, TDPG and FDDB + TDPG was used for culture of primary human osteoblast cells; cells grown in the presence of tissue culture medium was used as control. Mineral trioxide aggregate (ProRoot® MTA White) was used as a control material. Cell viability was evaluated up to 72 h using XTT assay. The mineralization potential was assessed in the presence of osteogenic and non-osteogenic media using Alizarin Red staining after 7 and 14 days. Cell morphology was captured using phase contrast microscopy. ResultsAll tested materials showed irregular-shaped morphology with variation in particle sizes. Both TDPG and FDDB contain calcium, phosphorus, and oxygen as main elements. Phosphorus however is absent from ProRoot® MTA. The cells grown in FDDB, TDPG or FDDB + TDPG extract showed no significant difference from the control. ProRoot® MTA showed the lowest cell viability at 72 h. All tested extracts showed nodule formation at 7 and 14 days even in absence of osteogenic supplements. The mineralization significantly improved in the presence of osteogenic supplements and increased with time for all extracts except for ProRoot® MTA that showed a reduction in mineralization at day 14. FDDB + TDPG extract showed significantly higher mineralization than TDPG alone. ConclusionsFDDB, TDBG or combination could be potentially used as alternative to ProRoot® MTA.

Calcium phosphate composites to synergistically promote osteoconduction and corrosion resistance on bone materials via plasma polymerized hexamethyldisilazane coatings

A facile and effective plasma polymerized hexamethyldisilazane (ppHMDSZ) combining with calcium phosphate (Ca-P) coating to promote osteoconduction meanwhile provide resistance to corrosion is developed in this study. The composites of Ca-P and ppHMDSZ thin films were deposited on bone substitutes to investigate the osteoblast compatibility. The amount of the Ca-P coatings was modulated with a linear deposition rate of 7.37 μg/(cm2∙cycle) by alternative immersion cycles (1, 2, 5, and 10) in solutions containing calcium and phosphate, measured by quartz crystal microbalance (QCM). Importantly, cell behaviors including cell adhesion, proliferation, and functional activity responded positively toward the surface wettability and roughness of the Ca-P layers. Moreover, the human osteoblast cells (hFOB1.19) revealed excellent compatibility on the stainless steel coated with Ca-P/ppHMDSZ composite films. The combination of bioceramics and corrosion resisting plasma polymerized HMDSZ films showed a synergistic effect on the biocompatibility and osteoconductive for bone tissue engineering applications.

Exosomal miR-21-5p derived from bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion by targeting PIK3R1.

Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can release a large number of exosomes which act as paracrine mediators in tumour‐associated microenvironment. However, the role of MSC‐derived exosomes in pathogenesis and progression of cancer cells especially osteosarcoma has not been thoroughly clarified until now. In this study, we established a co‐culture model for human bone marrow‐derived MSCs with osteosarcoma cells, then extraction of exosomes from induced MSCs and study the role of MSC‐derived exosomes in the progression of osteosarcoma cell. The aim of this study was to address potential cell biological effects between MSCs and osteosarcoma cells. The results showed that MSC‐derived exosomes can significantly promote osteosarcoma cells’ proliferation and invasion. We also found that miR‐21‐5p was significantly over‐expressed in MSCs and MSC‐derived exosomes by quantitative real‐time polymerase chain reaction (qRT‐PCR), compared with human foetal osteoblastic cells hFOB1.19. MSC‐derived exosomes transfected with miR‐21‐5p could significantly enhance the proliferation and invasion of osteosarcoma cells in vitro and in vivo. Bioinformatics analysis and dual‐luciferase reporter gene assays validated the targeted relationship between exosomal miR‐21‐5p and PIK3R1; we further demonstrated that miR‐21‐5p‐abundant exosomes derived human bone marrow MSCs could activate PI3K/Akt/mTOR pathway by suppressing PIK3R1 expression in osteosarcoma cells. In summary, our study provides new insights into the interaction between human bone marrow MSCs and osteosarcoma cells in tumour‐associated microenvironment.

The Mechanosensing and Global DNA Methylation of Human Osteoblasts on MEW Fibers.

Cells interact with 3D fibrous platform topography via a nano-scaled focal adhesion complex, and more research is required on how osteoblasts sense and respond to random and aligned fibers through nano-sized focal adhesions and their downstream events. The present study assessed human primary osteoblast cells’ sensing and response to random and aligned medical-grade polycaprolactone (PCL) fibrous 3D scaffolds fabricated via the melt electrowriting (MEW) technique. Cells cultured on a tissue culture plate (TCP) were used as 2D controls. Compared to 2D TCP, 3D MEW fibrous substrates led to immature vinculin focal adhesion formation and significantly reduced nuclear localization of the mechanosensor-yes-associated protein (YAP). Notably, aligned MEW fibers induced elongated cell and nucleus shape and highly activated global DNA methylation of 5-methylcytosine, 5-hydroxymethylcytosine, and N-6 methylated deoxyadenosine compared to the random fibers. Furthermore, although osteogenic markers (osterix-OSX and bone sialoprotein-BSP) were significantly enhanced in PCL-R and PCL-A groups at seven days post-osteogenic differentiation, calcium deposits on all seeded samples did not show a difference after normalizing for DNA content after three weeks of osteogenic induction. Overall, our study linked 3D extracellular fiber alignment to nano-focal adhesion complex, nuclear mechanosensing, DNA epigenetics at an early point (24 h), and longer-term changes in osteoblast osteogenic differentiation.

Sex Determining Region Y-Box 12 Promotes Proliferation, Migration and Invasion of Osteosarcoma Cells by Activating β-Catenin/T Cell Factor Axis

Objectives: This study aims to clarify the role of sex determining region Y-box 12 (SOX12) in accelerating the proliferative, migratory and invasive abilities of osteosarcoma (OS) via β-catenin/TCF axis. Materials and Methods: SOX12 levels in human osteosarcoma cell lines and human fetal osteoblastic cell line were determined by RT-qPCR. The proliferation rates of osteosarcoma cells were both determined by CCK-8 assay and EdU staining. In addition, osteosarcoma cell migration and migration were determined by wound healing assay and trans-well assay, respectively. TOPFlash/FOPFlash reporter activity assay and western blot assay were simultaneously performed for the detection of β-catenin/TCF axis. Results: SOX12 was elevated in osteosarcoma cell lines, developing the critical role in proliferation, migration and invasion of osteosarcoma cells. The β-catenin/TCF pathway was activated in osteosarcoma. SOX12 overexpression exerted promotive effects on activation of β-catenin/TCF pathway and SOX12 knockdown showed the opposite effects. Conclusions: SOX12 accelerates proliferation, migration and invasion of osteosarcoma cells by activating β-catenin/TCF axis, thus stimulating the progression of OS.

Physicochemical and Biological Characterization of Ti6Al4V Particles Obtained by Implantoplasty: An In Vitro Study. Part I.

Implantoplasty is a mechanical decontamination technique that consists of polishing the supra-osseous component of the dental implant with peri-implantitis. This technique releases metal particles in the form of metal swarf and dust into the peri-implant environment. In the present in vitro study, the following physicochemical characterization tests were carried out: specific surface area, granulometry, contact angle, crystalline structure, morphology, and ion release. Besides, cytotoxicity was in turn evaluated by determining the fibroblastic and osteoblastic cell viability. As a result, the metal debris obtained by implantoplasty presented an equivalent diameter value of 159 µm (range 6–1850 µm) and a specific surface area of 0.3 m2/g on average. The particle had a plate-like shape of different sizes. The release of vanadium ions in Hank’s solution at 37 °C showed no signs of stabilization and was greater than that of titanium and aluminum ions, which means that the alloy suffers from a degradation. The particles exhibited cytotoxic effects upon human osteoblastic and fibroblastic cells in the whole extract. In conclusion, metal debris released by implantoplasty showed different sizes, surface structures and shapes. Vanadium ion levels were higher than that those of the other metal ions, and cell viability assays showed that these particles produce a significant loss of cytocompatibility on osteoblasts and fibroblasts, which means that the main cells of the peri-implant tissues might be injured.

Mechanically Stable β-TCP Structural Hybrid Scaffolds for Potential Bone Replacement

The authors report on the manufacturing of mechanically stable β-tricalcium phosphate (β-TCP) structural hybrid scaffolds via the combination of additive manufacturing (CerAM VPP) and Freeze Foaming for engineering a potential bone replacement. In the first step, load bearing support structures were designed via FE simulation and 3D printed by CerAM VPP. In the second step, structures were foamed-in with a porous and degradable calcium phosphate (CaP) ceramic that mimics porous spongiosa. For this purpose, Fraunhofer IKTS used a process known as Freeze Foaming, which allows the foaming of any powdery material and the foaming-in into near-net-shape structures. Using a joint heat treatment, both structural components fused to form a structural hybrid. This bone construct had a 25-fold increased compressive strength compared to the pure CaP Freeze Foam and excellent biocompatibility with human osteoblastic MG-63 cells when compared to a bone grafting Curasan material for benchmark.

Naturally prefabricated 3D chitinous skeletal scaffold of marine demosponge origin, biomineralized ex vivo as a functional biomaterial

Solutions developed by nature for structural and functional optimization of three-dimensional (3D) skeletal structures provide unique windows not only into the evolutionary pathways of organisms, but also into bioinspired materials science and biomimetics. Great examples are naturally formed 3D chitinous scaffolds of marine sponge remain a focus of modern biomedicine and tissue engineering. Due to its properties like renewability, bioactivity, and biodegradability such constructs became very interesting players as components of organic-inorganic biocomposites. Herein, we developed chitin-based biocomposites by biomimetic ex vivo deposition of calcium carbonate particles using hemolymph from the cultivated mollusk Cornu aspersum and chitinous matrix from the marine demosponge Aplysina fistularis. The biological potential of the developed biofunctionalized scaffolds for bone tissue engineering was evaluated by investigating the spreading and viability of a human fetal osteoblast cell line has been determined for the first time. Performed analyses like dynamic mechanical analysis and atomic force microscopy shown that biofunctionalized scaffold possess about 4 times higher mechanical resistance. Moreover, several topographical changes have been observed, as e.g., surface roughness (Rq) increased from 31.75 ± 2.7 nm to 120.7 ± 0.3 nm. The results are indicating its potential for use in the modification of cell delivery systems in future biomedical applications.

Tannic acid enhances cisplatin effect on cell proliferation and apoptosis of human osteosarcoma cell line (U2OS).

The increase in cases of chemoresistance of cisplatin for osteosarcoma treatment has called for the need to establish a new treatment regime. Tannic acid (TA) possesses a potent antiproliferative effect against various cancers. Therefore, this study investigated the effect of TA combined with cisplatin on human osteosarcoma cell lines (U2OS). MTT assay was used to determine the half-maximal inhibitory concentration (IC50), while the combination index (CI) value was utilized to analyze the interaction within each combination. The antiproliferative effect of the treatment was evaluated by trypan blue exclusion assay. The morphological changes of cells were observed under a phase-contrast inverted microscope. The nuclear morphology and percentage of apoptosis cells were evaluated by using the Hoechst 33258 staining and annexin V/PI assay, respectively. The U2OS cells showed cytotoxic effect when treated with TA and cisplatin, with IC50 at 4.47µg/mL and 16.25µg/mL, respectively. The TA demonstrated no significant inhibition effect on the normal human fetal osteoblast cells (hFOB 1.19); yet, interestingly, a potent proliferative effect was indicated. Synergistic interaction was triggered when TA was combined with cisplatin at percentage ratios of 90:10 and 85:15. Meanwhile, antagonistic interaction was induced in the combination at percentage ratios of 75:25 and 50:50. On the other hand, a significant antiproliferative effect with prominent morphological alteration was detected in the cells treated with a combination of TA and cisplatin at the percentage ratio of 90:10. Additionally, combination-treated cells demonstrated the highest percentage of apoptosis cells, with distinct chromosomal condensation, nuclear fragmentation, reduction of nuclear volume, and notable apoptotic body. Therefore, there is a high potential for the inclusion of TA in the cisplatin-based chemotherapeutic regimen of osteosarcoma.

Thymosin‐β4, and Human Vitronectin peptides Grafted to Collagen Tune Adhesion or VEGF Gene Expression in Human Cell Lines**

AbstractIn order to improve collagen bioactivity for regenerative medicine approaches, thymosin‐β4 (Tβ4P) and Human Vitronectin (HVP) derived peptides are grafted to collagen by thiol‐ene Michael addition. Tβ4P and HVP are known to exert a pro‐angiogenic and a pro‐adhesive activity respectively and HVP is involved in osteogenesis promotion. The ability of these peptides to increase collagen cell adhesion and angiogenesis properties is assessed on human cell lines. In particular, HVP‐grafted collagen increased human osteoblast adhesion and cell proliferation: after 24 h, both adhesion and proliferation roughly showed a 4‐fold increase, if compared to pristine collagen. Tβ4P‐grafted collagen promotes Vascular Endothelial Growth Factor (VEGF) gene expression in human vascular cell lines by more than 7 times. These results suggest that HVP‐grafted collagen may be an interesting biomaterial for bone tissue regeneration, while Tβ4P‐grafted collagen is useful for angiogenesis promotion.

Evaluation of the Growth and Differentiation of Human Fetal Osteoblasts (hFOB) Cells on Demineralized Bone Matrix (DBM)

ABSTRACT Cells with osteogenic potential are believed to be an ideal source for bone tissue bioengineering. Large bone defects require temporary substitution of the damaged parts. In this respect, the transplantation of bone cells cultured on osteogenic substrates has been investigated. To use the natural bone matrix, one approach is the so-called demineralized bone matrix (DBM). In this study, we evaluated the interaction of human fetal osteoblasts (hFOB 1.19 cells, a human fetal osteoblastic cell line) with DBM fragments. No additional bone differentiation inducer was used other than the DBM itself. The samples were processed, had adhesion pattern evaluated and analyzed by light microscopy (cytochemical and immunocytochemical analysis) and electron microscopy (scanning and transmission). The adhesion pattern of hFOB cells on DBM was similar to what was observed on the cell culture plate. Morphological analysis showed that the hFOB cells had emitted filopodia and cellular projections on both controls and DBM. On DBM, the adhered cells emitted prolongations and migrated into the matrix. The monolayer growth pattern was observed as well as the accumulation of filamentous and reticulate extracellular materials when hFOB cells were cultured on the DBM surface. EDS analysis revealed the deposition of calcium on DBM. Immunocytochemical data showed that the hFOB cells were able to secrete extracellular matrix molecules such as fibronectin and laminin on DBM. Our data indicate that DBM successfully stimulates the osteoblastic phenotype of osteoblast-like cells and corroborate with the fact that DBM is a considerable natural matrix that promotes fractured-bone healing.

Mir-Oz Kegulates Osteoblast Apoptosis, Proliferation and Differentiation through Targeted Inhibition of Runx2 in Osteoporosis Running Title: Role and Potential Mechanism of Mir-152 in Osteoporosis

Objective: To explore the role and potential mechanism of miR-152 in osteoporosis. Methods: Fifty-four osteoporotic patients and 54 healthy subjects were recruited from August 2017 to January 2019. Serum samples of the two groups were obtained, and the miR-152 expression in serum was detected and compared. The human osteoblast cell line hFOB1.19 was obtained and miR-152 in cells was increased. The biological behavior changes such as cell proliferation, apoptosis and differentiation were observed by MTT, flow cytometry and detection of osteoblast differentiation markers (ALP, OCN). Results: miR-152 was elevated in osteoporosis patients, and AUC value of serum miR-152 in diagnosing osteoporosis was 0.939. After miR-152 in osteoblasts was elevated, cell proliferation was inhibited, cell apoptosis rate increased, and ALP and OCN content in cells reduced, while increasing cell RUNX2 simultaneously was totally different. Dual luciferase report showed that RUNX2 could be targeted and regulated by miR-152. Conclusion: miR-152 is elevated in serum of osteoporosis patients and can be used as a biological indicator for diagnosing osteoporosis. In addition, miR-152 can inhibit osteoblast proliferation, differentiation and induce apoptosis through negative regulation of RUNX2.

Cellulose nanocrystal reinforced silk fibroin coating for enhanced corrosion protection and biocompatibility of Mg-based alloys for orthopedic implant applications

In this work, a novel coating material based on silk fibroin (SF) and cellulose nanocrystals (CNCs) was developed on biodegradable AZ31 Mg alloy and assessed for corrosion resistance and biocompatibility. Before the application of the coating, AZ31 substrate was modified with a layer of polydopamine to enhance the adhesion of the protective coating to the metal surface as confirmed by the adhesion tape test. SEM images revealed the formation of a defect-free and uniform SF-CNC coating with a thickness of 11.2 ± 2.5 μm on AZ31 alloy. The results of the electrochemical corrosion and in vitro immersion tests clearly demonstrated an enhanced corrosion resistance of the SF coating after the incorporation of CNCs. Compared to the unmodified Mg alloy, the SF-CNC coated AZ31 exhibited a remarkably improved cytocompatibility with a viability of 114% and excellent adhesion and spreading of human fetal osteoblast cells onto the coating surface. The findings of this work highlight the great potential of SF and CNC as bio-based nature-derived anticorrosive nanofillers for fabrication of protective and biocompatible coatings on Mg-based biodegradable orthopedic implants.

In vitro studies of B2O3–SiO2–Na2O–CaO–ZnO bioactive glass system

The main purpose of the present investigation is to examine in detail in vitro studies like bioactivity and cytocompatibility of a novel bioactive glass system i.e., B2O3–SiO2–Na2O–CaO–ZnO glass composition. Glasses are prepared by the traditional melt quenching method. In vitro studies are performed with ISO standards. Bioactivity has been studied using standard Kokubo's method. Highly sophisticated analytical techniques were utilized for the assessment of apatite forming ability. Scanning electron microscopy micrographs have shown visible evidence of apatite forming ability through cauliflower type of precipitation on the surface of the glass. Cytocompatibility was performed using human osteoblast cells (MG-63). Cytocompatibility of zinc-containing soda lime borosilicate glasses has shown significant results for bone tissue engineering applications.

Anti-Osteoporotic Activity of Pueraria lobata Fermented with Lactobacillus paracasei JS1 by Regulation of Osteoblast Differentiation and Protection against Bone Loss in Ovariectomized Mice

Osteoporosis is the most common bone disease associated with low bone mineral density. It is the process of bone loss and is most commonly caused by decreased estrogen production in women, particularly after menopause. Pueraria lobata, which contains various metabolites, especially isoflavone, is widely known as regulator for bone mineral contents. In this study, the effects of the P. lobata extract (PE) with or without fermentation with Lactobacillus paracasei JS1 (FPE) on osteoporosis were investigated in vitro and in vivo. The effects of PE and FPE on human osteoblastic MG63 cells, RAW 264.7 cells, and ovariectomized (OVX)-induced model mice were analyzed at various ratios. We found that FPE increased calcium deposition and inhibited bone resorption by in vitro assay. Furthermore, treatment with PE and FPE has significantly restored destroyed trabecular bone in the OVX-induced bone loss mouse model. Overall, FPE demonstrated bioactivity to prevent bone loss by decreasing bone turnover.

Sequential alendronate delivery by hydroxyapatite-coated maghemite for enhanced bone fracture healing

Accelerating the healing of bone fractures in the presence of infection is a serious challenge that has increased hopes for reducing this challenge by using nanotechnology. This study tried to effectively reduce bacterial infection by producing alendronate (An)-maghemite nanosphere-hydroxyapatite (An-MNS-HA) through co-precipitation, in addition to increasing the rate of proliferation and differentiation of human primary osteoblasts cells. After production of An-MNS-HA, their physicochemical properties were investigated by TEM, DLS and FTIR methods as well as the level of An release. The toxicity of An-MNS-HA was determined by MTT assay and the antibacterial activities of An-MNS-HA were also performed on both Gram-negative and Gram-positive bacteria. In addition, cell differentiation markers including ALP, OC, ODF, OPG and RANKL genes were analyzed by real time PCR technique. The results revealed that An-MNS-HA is spherical with a rough surface with a size of around 105.5 ± 2.08 nm. Also, DLS and FTIR results confirmed the potential loading of HA and An on MNS with a drug loading efficiency of 55.39 ± 4.68% and a drug release rate of 80.93 ± 5.28% from An-MNS-HA. The antibacterial activity of An-MNS-HA on both Gram-negative and Gram-positive bacteria exhibited a significant reduction in infection, despite a greater effect on Gram-negative bacteria. Also, cell proliferation and differentiation in the presence of An-MNS-HA were observed which were associated with an increase in the expression of the ALP, OC, ODF and OPG mRNA as well as decreased expression of RANKL gene. Finally, An-MNS-HA with high biological compatibility, antibacterial activity and induction of ossification, has a good ability to accelerate and repair bone defects.