Alkaline Phosphatase Activity Of Osteoblasts Research Articles

Osteoblast and osteoclast activity on collagen-based 3D printed scaffolds enriched with strontium-doped bioactive glasses and hydroxyapatite nanorods for bone tissue engineering

Bone tissue engineering (BTE) aims to promote bone regeneration by means of the synergistic effect of biomaterials, cells, and other factors, as potential alternative to conventional treatments for bone fractures. To this aim, a composite material was developed, based on collagen type I, strontium-enriched mesoporous bioactive glasses, and hydroxyapatite nanorods as bioactive and biomimetic components. Nanostructured scaffolds were 3D printed and subsequently chemically crosslinked with genipin to improve mechanical properties and stability. The developed nanostructured system was maintained in culture until 3 weeks with a co-culture of human bone cells to provide anex vivomodel of bone microenvironment and examine the cellular crosstalk and signaling pathways through paracrine cell activities. Human osteoblasts (OBs), derived from trabecular bone, and human osteoclast precursors (OCs), isolated from buffy coat samples were involved, with OBs seeded on the scaffold and OC precursors seeded in a transwell device. When compared to the material without inorganic components, the bioactive and biomimetic scaffold positively influenced cell proliferation and cell metabolic activity, boosting alkaline phosphatase activity of OBs, and reducing OC differentiation. Thus, the bioactive and biomimetic system promoted an enhanced cellular response, highlighting its potential application in BTE.

Development of a composite using calcined bone powder and silane cross-linked alginate as bone substitute material.

Calcined bone is an attractive natural material for use as a bone substitute because of its cost-effectiveness and high biocompatibility, which are comparable to that of synthetic hydroxyapatite. However, the calcination process has significantly weakened the mechanical properties. In this study, a composite of calcined bovine bone powder reinforced with silane cross-linked alginate was prepared to assess its biocompatibility, osteoconductivity, and mechanical compatibility as a bone substitute material. Culture studies with osteoblast-like cells (MC3T3-E1) showed no cytotoxicity toward the composite and exhibited general cell proliferative properties in its presence. In contrast, the composite reduced the alkaline phosphatase activity of osteoblasts but led to significant noncellular apatite deposition on the surface. In addition, quasi-static compression tests of the composite revealed mechanical properties comparable to those of human cancellous bone. The mechanical properties remained stable under wet conditions and did not deteriorate significantly even after 2 weeks of immersion in simulated body fluid at 37°C. The results show that this composite, composed of calcined bone powder and silane cross-linked alginate, is a promising bone substitute material with biocompatibility, osteoconductivity, and mechanical compatibility.

Phosvitin phosphopeptides alleviate the inhibitory effect of oxidative stress on osteoblast function through FoxO/Wnt signaling

Oxidative stress inhibits osteoblast differentiation and function, leading to the development of osteoporosis. The effects of Phosvitin phosphopeptides (PPPs) on the osteogenic function of osteoblast were determined based on oxidative stress MC3T3-E1 cell model induced by hydrogen peroxide. The results showed that 50 μg/mL of PPPs significantly increased cell viability from 56.00% (H2O2) to 106.80% for 72 h. DCFH-DA fluorescence assay demonstrated that PPPs could remarkably reduce reactive oxygen species levels induced by H2O2. The alkaline phosphatase activity of oxidative stress-treated osteoblast increased from 2.36 Kim'unit/100 mL (H2O2 group) to 8.16 Kim'unit/100 mL, along with an increase in the activity of total superoxide dismutase, glutathione peroxidase, catalase, and the number of mineralized nodules in response to PPPs treatment. Overall, the results of this work indicated that PPPs had a stimulating impact on the Wnt/β-catenin signaling pathway while simultaneously inhibiting the forkhead box class O (FoxO) signaling pathway in MC3T3-E1 osteoblast induced by H2O2. Consequently, these findings demonstrate that PPPs have the ability to protect the osteogenic function of MC3T3-E1 osteoblasts against oxidative damage through the activation of the FoxO/Wnt signaling pathway.

Repair of Rat Calvarial Critical-Sized Defects Using Heparin-Conjugated Fibrin Hydrogel Containing BMP-2 and Adipose-Derived Pericytes.

The repair of critical-sized calvarial defects is a challenging problem for orthopedic surgery. One of the promising strategies of bone bioengineering to enhance the efficacy of large bone defect regeneration is the combined delivery of stem cells with osteoinductive factors within polymer carriers. The purpose of the research was to study the regenerative effects of heparin-conjugated fibrin (HCF) hydrogel containing bone morphogenetic protein 2 (BMP-2) and adipose-derived pericytes (ADPs) in a rat critical-sized calvarial defect model. In vitro analysis revealed that the HCF hydrogel was able to control the BMP-2 release and induce alkaline phosphatase (ALP) activity in neonatal rat osteoblasts. In addition, it was found that eluted BMP-2 significantly induced the osteogenic differentiation of ADPs. It was characterized by the increased ALP activity, osteocalcin expression and calcium deposits in ADPs. In vivo studies have shown that both HCF hydrogel with BMP-2 and HCF hydrogel with pericytes are able to significantly increase the regeneration of critical-sized calvarial defects in comparison with the control group. Nevertheless, the greatest regenerative effect was found after the co-delivery of ADPs and BMP-2 into a critical-sized calvarial defect. Thus, our findings suggest that the combined delivery of ADPs and BMP-2 in HCF hydrogel holds promise to be applied as an alternative biopolymer for the critical-sized bone defect restoration.

Tmem119 is involved in bone anabolic effects of PTH through enhanced osteoblastic bone formation in mice

The intermittent administration of parathyroid hormone (PTH) exerts potent bone anabolic effects, which increase bone mineral density (BMD) and reduce fracture risk in osteoporotic patients. However, the underlying mechanisms remain unclear. Tmem119 has been proposed as a factor that is closely linked to the osteoblast phenotype, and we previously reported that PTH enhanced the expression of Tmem119 in mouse osteoblastic cells. However, roles of Tmem119 in the bone anabolic effects of PTH in vivo remain unknown. We herein investigated the roles of Tmem119 in bone anabolic effects of PTH using Tmem119-deficient mice. Tmem119 deficiency significantly reduced PTH-induced increases in trabecular bone volume and cortical BMD of femurs. Effects of Tmem119 deficiency on bone mass seemed predominant in female mice. Histomorphometric analyses with calcein labeling showed that Tmem119 deficiency significantly attenuated PTH-induced increases in the rates of bone formation and mineralization as well as numbers of osteoblasts. Moreover, Tmem119 deficiency significantly blunted PTH-induced decreases in phosphorylation of β-catenin and increases in alkaline phosphatase activity in osteoblasts. In conclusion, the present results indicate that Tmem119 is involved in bone anabolic effects of PTH through osteoblastic bone formation partly related to canonical Wnt-β-catenin signaling in mice.

Janus silk fibroin/polycaprolactone-based scaffold with directionally aligned fibers and porous structure for bone regeneration

To promote bone repair, it is desirable to develop three-dimensional multifunctional fiber scaffolds. The densely stacked and tightly arranged conventional two-dimensional electrospun fibers hinder cell penetration into the scaffold. Most of the existing three-dimensional structural materials are isotropic and monofunctional. In this research, a Janus nanofibrous scaffold based on silk fibroin/polycaprolactone (SF/PCL) was fabricated. SF-encapsulated SeNPs demonstrated stability and resistance to aggregation. The outside layer (SF/PCL/Se) of the Janus nanofiber scaffold displayed a structured arrangement of fibers, facilitating cell growth guidance and impeding cell invasion. The inside layer (SF/PCL/HA) featured a porous structure fostering cell adhesion. The Janus fiber scaffold containing SeNPs notably suppressed S. aureus and E. coli activities, correlating with SeNPs concentration. In vitro, findings indicated considerable enhancement in alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts and upregulation of genes linked to osteogenic differentiation with exposure to the SF/PCL/HA/Se Janus nanofibrous scaffold. Moreover, in vivo, experiments demonstrated successful critical bone defect repair in mouse skulls using the SF/PCL/HA/Se Janus nanofiber scaffold. These findings highlight the potential of the SF/PCL-based Janus nanofibrous scaffold, integrating SeNPs and nHA, as a promising biomaterial in bone tissue engineering.

Lithium-Modified TiO2 Surface by Anodization for Enhanced Protein Adsorption and Cell Adhesion.

Promoting osseointegration is an essential step in improving implant success rates. Lithium has gradually gained popularity for promoting alkaline phosphatase activity and osteogenic gene expression in osteoblasts. The incorporation of lithium into a titanium surface has been reported to change its surface charge, thereby enhancing its biocompatibility. In this study, we applied anodization as a novel approach to immobilizing Li on a titanium surface and evaluated the changes in its surface characteristics. The objective of this study was to determine the effect of Li treatment of titanium on typical proteins, such as albumin, laminin, and fibronectin, in terms of their adsorption level as well as on the attachment of osteoblast cells. Titanium disks were acid-etched by 66 wt % H2SO4 at 120 °C for 90 s and set as the control group. The etched samples were placed in contact with an anode, while a platinum bar served as the counter electrode. Both electrodes were mounted on a custom electrochemical cell filled with 1 M LiCl. The samples were anodized at constant voltages of 1, 3, and 9 V. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) results showed no significant differences in the topography. However, the ζ potentials of the 3 V group were higher than those of the control group at a physiological pH of 7.4. Interestingly, the adsorption level of the extracellular matrix protein was mostly enhanced on the 3 V-anodized surface. The number of attached cells on the Li-anodized surfaces increased. The localization of vinculin at the tips of the stretching cytoplasmic projections was observed more frequently in the osteoblasts on the 3 V-anodized surface. Although the optimal concentration or voltage for Li application should be investigated further, this study suggests that anodization could be an effective method to immobilize lithium ions on a titanium surface and that modifying the surface charge characteristics enables a direct protein-to-material interaction with enhanced biological adhesion.

In vivo corrosion on retrieved hip endoprostheses and in vitro effects of corrosion products on bone mineralization.

In vivo corrosion of modular endoprostheses remains a great concern, as the release of heavy metal ions can impair the implant's service life and the wellbeing of the patient. The detailed corrosion mechanisms that occur in vivo are so far not completely understood. In this context, the effects of implant released cobalt (Co) and chromium (Cr) ions on osteoblast mineralization and gene expression have not been investigated extensively. This comprehensive study aimed at furthering the understanding of in vivo implant corrosion from the clinical signs via prosthesis retrievals and histology of the synovial membranes down to the molecular processes instigated by corrosion products and its effects on bone mineralization. A detailed in vivo failure analysis was performed investigating 22 retrieved hip endoprostheses from different manufacturers and taper material combinations. The aim was to find a correlation of taper damage and especially corrosion to susceptible biomedical alloys and its effect on periprosthetic tissue as well as the clinical implant performance with regard to revision diagnosis and presence of radiolucent lines (RLL). A second part investigated the effects of Co and Cr ions on the in vitro mineralization process of osteoblasts. Cell cultures were exposed to relevant concentrations of CoCl2 and CrCl3 (0μM, 100μM, 200μM) with and without addition of phosphate. Mineralization behavior was analyzed with Alizarin Red assay and Von Kossa staining of calcium depots, alkaline phosphatase activity of osteoblasts and gene expression was analyzed with real time quantitative PCR. The retrieval study provides evidence of in vivo fretting and crevice corrosion on all metallic tapers combined with either ceramic or metal femoral heads. Within the modular taper junctions, selective dissolution of the α phase occurred in wrought TiAl6V4 alloys, and etching of the fine-grained wrought CoCr28Mo6 alloy implants was observed in formed crevices. In addition, significant amounts of wear particles and corrosion products were detected in retrieved synovial membranes. An increased risk for the occurrence of a RLL in the proximal zones was determined for patients with a corroded mixed metal taper. Whereas Co ions have hardly any effects on mineralization, Cr ions cause a significant concentration dependent decrease in mineralization rate of osteoblasts. However, this effect is alleviated by addition of a phosphate source. Our data reveal that Cr ions depleted dissolved phosphates by forming an insoluble complex (CrPO4), which inhibits the phosphate dependent mineralization process. No significant effect of the heavy metal ions on osteoblast activity by means of alkaline phosphate activity as well as on gene expression is determined. This study broadens the understanding of in vivo corrosion of metallic modular implants and its clinically relevant effects on mineralization. Based on these findings, in vivo corrosion of CoCr28Mo6 endoprostheses should be limited to avoid inhibitory effects of Cr3+ on bone mineralization which can contribute to premature implant failure.

Effects of simulated microgravity and vibration on osteoblast and osteoclast activity in cultured zebrafish scales

Zebrafish cultured scales have been used effectively to study cellular and molecular responses of bone cells. In order to expose zebrafish scales to simulated microgravity (SMG) and/or vibration, we first determined via apoptosis staining whether cells of the scale survive in culture for two days and hence, we restricted our analyses to two-day durations. Next, we measured the effects of SMG and vibration on cell death, osteoclast tartrate-resistant acid phosphatase, and osteoblast alkaline phosphatase activity and on the number of Runx2a positive cells. We found that during the SMG treatment, osteoclast tartrate-resistant acid phosphatase activity increased on average, while the number of Runx2a positive cells decreased significantly. In contrast, SMG exposure caused a decrease in osteoblast activity. The vibration treatment showed an increase, on average, in the osteoblast alkaline phosphatase activity. This study demonstrates the effect of SMG and vibration on zebrafish scales and the effects of SMG on bone cells. We also show that zebrafish scales can be used to examine the effects of SMG on bone maintenance.

Comprehensive metabolic profiles of Achyranthes bidentate in rat serum via ultra-high performance liquid chromatography time-of-flight mass spectrometry and their correlation with osteoinductive activity

The osteoinductive effect of crude and salt-processed Achyranthes bidentata is associated with the serum metabolites. Grey relationship analysis between the serum metabolites and osteoinductive effect will help to clarify the bioactive serum metabolites. First, an ultra-high performance liquid chromatography time-of-flight mass spectrometry method was used to develop serum metabolic fingerprint of rats after oral administration of crude and salt-processed Achyranthes bidentata. The MS1 and MS2 data of serum metabolites were scanned in the range of m/z 100–1500 and 50–1200, respectively. The chemical structures of the metabolites were thoroughly elucidated. Two prototypes and twelve metabolites have been identified. Second, osteoblasts were cultured with the drug-containing serum at different time points. The osteoinductive effect of crude and salt-processed Achyranthes bidentata was evaluated by detecting the proliferation rate and alkaline phosphatase activity of osteoblasts. Third, grey correlation analysis was utilized to elucidate the spectral-effect relationship between serum metabolic fingerprints and osteoinductive effect. Finally, the correlation coefficients of ten metabolites, i.e., oleanolic acid, poststerone-M1, chikusetsusaponin V-M1, oleanolic acid-M2, oleanolic acid-M4, spinacoside D-M1, chikusetsusaponin I-M1, betavulgaroside IV-M2, chikusetsusaponin IVa and achyranthoside IV-M1 were above 0.7. Collectively, our work will provide helpful knowledge for the future research on Achyranthes bidentata.

One‐Step Electrodeposition of Multi‐element Doped Hydroxyapatite Nanocoating Leading to Enhanced Cytocompatible and Antibacterial Properties of Titanium Implants

AbstractThe introduction of trace elements into hydroxyapatite (HA) coatings has been widely used for antimicrobial and biocompatible modification of metal implants. In this study, a multifunctional coating of HA co‐doped with Cu2+, Zn2+, Sr2+ and F− (CuZnSrFHA) was synthesized on titanium substrate by one‐step electrodeposition technology. The CuZnSrFHA consisted of nanoscale needle‐like crystals with low roughness and poor hydrophilicity. However, it can continuously release Cu2+, Zn2+ and Sr2+ within 14 days while maintaining good physiological stability. Based on the synergistic effect of Cu and Zn, the CuZnSrFHA coating showed some inhibitory effect on E. coli and S. aureus. Furthermore, the CuZnSrFHA was not significantly cytotoxic and promoted early adhesion and alkaline phosphatase activity of osteoblasts. In conclusion, the multi‐element co‐doped CuZnSrFHA coating was expected to be a novel surface modification material for the prevention of infectious bone defects.

Effect of Hydro-Ethanolic Extract of Cardiospermum halicacabum Linn. on Human Osteoblast Like Cells, MG-63 and Phytochemical Profiling by GC-MS

Background: Ancient Indian Ayurvedic literature prescribes Cardiospermum halicacabum as a remedy for rheumatism, lumbago, stiffness of limbs and allied diseases of musculo-skeletal system. Aim: The present study is to investigate the anti-osteoporotic potential of hydro-ethanolic extract of Cardiospermum halicacabum Linn. on human osteoblast-like cells (MG-63) and their phytochemicals are profiled by GC-MS. Materials and methods: Cell proliferation rate was assessed by standard colorimetric MTT assay on human osteoblast-like cells (MG-63). The osteoblastic and osteoclastic activities in MG-63 cell line were determined by assaying of bone markers like Alkaline phosphatase (ALP) and Lactate dehydrogenase (LDH). Results: The hydro-ethanolic extract of Cardiospermum halicacabum shows a significant increase in both proliferation and ALP activity in osteoblasts, and decreased LDH leakage in the supernatant of cultured human osteoblast-like cells (MG-63). The GC-MS analysis of hydro-ethanolic extract of C. halicacabum revealed the presence of more than 70 phytoconstituents. Conclusion: Hydro-ethanolic extract of Cardiospermum halicacabum showed a potential influence on osteogenesis and hence its use could be explored as a potential anti-osteoporotic agent.

Cell Type-Specific Effects of Implant Provisional Restoration Materials on the Growth and Function of Human Fibroblasts and Osteoblasts.

Implant provisional restorations should ideally be nontoxic to the contacting and adjacent tissues, create anatomical and biophysiological stability, and establish a soft tissue seal through interactions between prosthesis, soft tissue, and alveolar bone. However, there is a lack of robust, systematic, and fundamental data to inform clinical decision making. Here we systematically explored the biocompatibility of fibroblasts and osteoblasts in direct contact with, or close proximity to, provisional restoration materials. Human gingival fibroblasts and osteoblasts were cultured on the "contact" effect and around the "proximity" effect with various provisional materials: bis-acrylic, composite, self-curing acrylic, and milled acrylic, with titanium alloy as a bioinert control. The number of fibroblasts and osteoblasts surviving and attaching to and around the materials varied considerably depending on the material, with milled acrylic the most biocompatible and similar to titanium alloy, followed by self-curing acrylic and little to no attachment on or around bis-acrylic and composite materials. Milled and self-curing acrylics similarly favored subsequent cellular proliferation and physiological functions such as collagen production in fibroblasts and alkaline phosphatase activity in osteoblasts. Neither fibroblasts nor osteoblasts showed a functional phenotype when cultured with bis-acrylic or composite. By calculating a biocompatibility index for each material, we established that fibroblasts were more resistant to the cytotoxicity induced by most materials in direct contact, however, the osteoblasts were more resistant when the materials were in close proximity. In conclusion, there was a wide variation in the cytotoxicity of implant provisional restoration materials ranging from lethal and tolerant to near inert, and this cytotoxicity may be received differently between the different cell types and depending on their physical interrelationships.

The effect of simvastatin-loaded methoxy poly(ethylene glycol)-polylactic acid nanoparticles on osteoblasts

ObjectiveTo investigate the effect of simvastatin-loaded polymer nanoparticles on osteoblast proliferation and differentiation, and to seek a better pharmaceutical dosage form to improve the osteogenic effect of simvastatin and provide a more practical drug carrier for its clinical application. Methodspolymer nanoparticles were prepared via the modified spontaneous emulsifying solvent diffusion (SESD) method, and the effects of simvastatin-loaded methoxy poly(ethylene glycol)-polylactic acid nanoparticles on osteoblasts were investigated. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was adopted to detect the proliferation abilities of the cells. An enzyme-linked immunosorbent assay was used to detect the amount of alkaline phosphatase (ALP) in the cells. A real-time polymerase chain reaction was used to detect the expression of osteocalcin (OC) and bone morphogenetic protein-2 (BMP-2) messenger ribonucleic acid (mRNA). ResultsThe study found that the proliferation of osteocytes was inhibited in the simvastatin-loaded nanoparticle (Sim-NP) group, and this inhibition was stronger in the Sim-NP than in the simvastatin group; the ALP activity of osteoblasts was increased in both groups, and this increase was stronger in the Sim-NP than in the simvastatin group. The expression of ALP, OC and BMP-2 mRNA was increased in both groups, and this increase was stronger in the Sim-NP than in the simvastatin group. ConclusionThe authors thus posited that the modified Sim-NPs SESD preparation method was simple and reproducible, and the prepared nanoparticles, with uniform distribution, a regular morphology and spherical shape could greatly improve the solubility of simvastatin. The polymer nanoparticle carrier system improved the effect of simvastatin on the proliferation and differentiation of osteoblasts so that the Sim-NPs were systematically applied to target bone tissues. These results provide a new direction for research on the drugs and treatment methods for osteoporosis.

Exosomes from artesunate-treated bone marrow-derived mesenchymal stem cells transferring SNHG7 to promote osteogenesis via TAF15-RUNX2 pathway.

Aim: Effect of artesunate (ART)-treated bone marrow-derived mesenchymal stem cells-derived exosomes (BMSC-Exos) on osteogenesis and its underlying mechanisms were investigated. Materials & methods: Proliferation, alkaline phosphatase activity and calcified nodule formation of osteoblasts were determined. A mouse model of osteoporosis was established by ovariectomy. Results: SNHG7was upregulated in BMSC-Exos by twofold, which was further enhanced in ART-BMSC-Exos by about twofold. ART intensified BMSC-Exos-induced proliferation, alkaline phosphatase activity by about fourfold, calcified nodule formation by about threefold and upregulation of osteogenesisrelated molecules RUNX2 (by 50%), BMP2 (by 30%)and ATF4 (by 40%) via delivering SNHG7. Mechanistically, SNHG7 recruited TAF15 to facilitate RUNX2 stability. Conclusion: ART-BMSC-Exos facilitated osteogenesis via delivering SNHG7 by modulating TAF15/RUNX2 axis.

Inhibition of miR‑98‑5p promotes high glucose‑induced suppression of preosteoblast proliferation and differentiation via the activation of the PI3K/AKT/GSK3β signaling pathway by targeting BMP2.

Osteoporosis (OP) is a bone metabolic disease, in which low bone mass and the microarchitectural deterioration of bone tissue contribute to the fragility of bones and increase the risk of fracture. The aim of the present study was to determine the role of microRNA (miR)-98-5p in high glucose (HG)-induced preosteoblasts. HG was used to induce preosteoblasts treated in a differentiation medium to establish an in vitro OP model. Next, miR-98-5p expression was determined using reverse transcription-quantitative PCR. Following the transfection of an miR-98-5p inhibitor into HG-treated osteoblasts, cell viability was assessed using a Cell Counting Kit-8 assay, while alkaline phosphatase (ALP) activity, differentiation ability and the expression of differentiation-regulated genes osteocalcin and osteopontin were measured using the corresponding ALP, Alizarin red staining, reverse transcription-quantitative PCR and western blotting assays. The association between miR-98-5p and the PI3K/AKT/GSK3β signaling pathway was determined using western blotting. Next, the binding relationship between miR-98-5p and bone morphogenetic protein 2 (BMP2) was predicted and verified, and the role of BMP2 in the regulation of the PI3K/AKT/GSK3β signaling pathway was explored using western blotting. The results revealed that miR-98-5p expression was upregulated in HG-induced osteoblasts, and the inhibition of miR-98-5p resulted in enhanced cell viability, alkaline phosphatase activity and differentiation in osteoblasts following HG induction. It was also discovered that miR-98-5p inhibition activated PI3K/AKT/GSK3β signaling, while knockdown of BMP2, which binds to miR-98-5p, enhanced the activation of this signaling pathway and the differentiation ability of osteoblasts. In conclusion, the findings of the present study suggested that the inhibition of miR-98-5p expression may activate PI3K/AKT/GSK3β signaling to promote HG-induced suppression of preosteoblast viability and differentiation by targeting BMP2, which provides a novel insight into future potential molecular markers for OP treatment.

Preparation of a hydroxyapatite–silver gradient bioactive ceramic coating with porous structure by laser cladding: A study of in vitro bioactivity

Porous coatings play a critical role in osseointegration between the artificial joint implant and host bone. Herein, a previously developed hydroxyapatite–silver (HA-Ag) gradient bioactive ceramic coating with a three-layer structure is further improved. Through laser cladding, a bioactive porous ceramic coating was prepared by adding carboxyl multi-walled carbon nanotubes (CMWCNTs) to HA powder. In this study, the effects of the added amount of CMWCNTs on the pore size, porosity, and active substance content were investigated. The results showed that the porous structure of the bioactive surface layer was gradually changed from the round hole shape to the reticular porous shape when the addition amount of CMWCNTs was increased from 5 wt % to 20 wt %, and the most appropriate amount of CMWCNTs was 10 wt %. In addition, the biocompatibility of the porous ceramic coating, as well as its effect on the alkaline phosphatase (ALP) activity of osteoblasts, were evaluated in vitro. Simulated body fluid (SBF) soaking tests were applied to determine the changes in ion concentration and calculate the concentration quotient and transfer free energy of Ca2+ and PO43− during the soaking period in SBF. The effects of the porous ceramic coating on the nucleation and growth of HA were also validated. The results showed that the preparation of the gradient porous ceramic coating was beneficial to the nucleation and growth of apatite, further improving the bioactivity of the coating to achieve osseointegration between the artificial joint implant and host bone.

Identification of a Novel Osteogenetic Oligodeoxynucleotide (osteoDN) That Promotes Osteoblast Differentiation in a TLR9-Independent Manner.

Dysfunction of bone-forming cells, osteoblasts, is one of the causes of osteoporosis. Accumulating evidence has indicated that oligodeoxynucleotides (ODNs) designed from genome sequences have the potential to regulate osteogenic cell fate. Such osteogenetic ODNs (osteoDNs) targeting and activating osteoblasts can be the candidates of nucleic acid drugs for osteoporosis. In this study, the ODN library derived from the Lacticaseibacillus rhamnosus GG genome was screened to determine its osteogenetic effect on murine osteoblast cell line MC3T3-E1. An 18-base ODN, iSN40, was identified to enhance alkaline phosphatase activity of osteoblasts within 48 h. iSN40 also induced the expression of osteogenic genes such as Msx2, osterix, collagen type 1α, osteopontin, and osteocalcin. Eventually, iSN40 facilitated calcium deposition on osteoblasts at the late stage of differentiation. Intriguingly, the CpG motif within iSN40 was not required for its osteogenetic activity, indicating that iSN40 functions in a TLR9-independent manner. These data demonstrate that iSN40 serves as a novel osteogenetic ODN (osteoDN) that promotes osteoblast differentiation. iSN40 provides a potential seed of the nucleic acid drug that activating osteoblasts for osteoporosis therapy.

Dentin Matrix Protein 1 Silencing Inhibits Phosphorus Utilization in Primary Cultured Tibial Osteoblasts of Broiler Chicks.

Three experiments were carried out in the present study to investigate whether dentin matrix protein 1 (DMP1) was involved in regulating phosphorus (P) metabolic utilization in primary cultured tibial osteoblasts of broiler chicks. Experiment 1 was conducted to select the optimal osteogenic inductive culture medium and the optimal induction time in primary cultured tibial osteoblasts of broiler chicks. In experiment 2, the siRNAs against DMP1 were designed, synthesized and transfected into primary cultured tibial osteoblasts of broiler chicks, and then the inhibitory efficiencies of siRNAs against DMP1 were determined, and the most efficacious siRNA was selected to be used for the DMP1 silencing. In experiment 3, with or without siRNA against DMP1, primary cultured tibial osteoblasts of broiler chicks were treated with the medium supplemented with 0.0, 1.0 or 2.0 mmol/L of P as NaH2PO4 for 12 days. The P metabolic utilization-related parameters were measured. The results showed that the osteogenic induced medium 2 and 12 days of the optimal induction time were selected; Among the designed siRNAs, the si340 was the most effective (P < 0.05) in inhibiting the DMP1 expression; DMP1 silencing decreased (P < 0.05) the expressions of DMP1 mRNA and protein, P retention rate, mineralization formation, alkaline phosphatase activity and bone gla-protein content in tibial osteoblasts at all of added P levels. It is concluded that DMP1 silencing inhibited P utilization, and thus DMP1 was involved in regulating P metabolic utilization in primary cultured tibial osteoblasts of broiler chicks, which provides a novel insight into the regulation of the P utilization in the bone of broilers, and will contribute to develop feasible strategies to improve the bone P utilization efficiency of broilers so as to decrease its excretion.

Effect of JUUL e-Liquid Extract on Oral Cell Survival and Function

<h3>Introduction</h3> Electronic vaping devices deliver nicotine in a manner similar to conventional cigarettes. While cigarette smoking has been proven to increase periodontal disease and oropharyngeal cancer risks, extensive stud- ies on the health effects of vaping have yet to be published. A JUUL consists of a device containing a removable cartridge, or JUULpod, that holds an e-liquid of various flavors and nicotine concentrations. Although electronic cigarette use is viewed as a healthy alternative to conventional cigarette smoking, the long-term health effects of vaping are not known at this time. Therefore, this study is to determine the effects of JUUL e-liquid concentrates on the survival and function of oral fibroblasts and osteoblasts. <h3>Materials and Methods</h3> Cells were treated for 1, 6, and 24 hours with various percentages (2%, 4%, 6%, 8%, 10%) of JUULpod eliquid containing 59 mg/ml nicotine in a mix of propylene glycol, glycerine, benzoic acid, and fla- voring agents. The flavors tested included mint, Virginia tobacco, mango, and creme. Cell survival was assessed fluorometrically using Calcein-AM. Alkaline phosphatase activity was measured by enzymatic assay. Osteoblast transcript expression was evaluated using polymerase chain reaction and gel electrophoresis. <h3>Results</h3> Concentrations of JUULpod e-liquid up to 25% extract did not significantly reduce cell survival for all fla- vors. Concentrations of JUULpod e-liquid up to 25% extract drastically reduced alkaline phosphatase activity in osteoblasts. In general, no detectable changes in osteoblast transcript were noted following exposure to JUUL extracts. However, mint flavored extracts selectively reduced expressions of BMP4, TGFbeta1, osteoprotegerin, periostin, as well as collagens III, V, and VIII. <h3>Conclusions</h3> Based on these results, JUULpod e-liquid did not reduce the survival of osteoblasts or oral fibrob- lasts. However, JUULpod e-liquid does significantly reduce alkaline phosphatase activity of osteoblasts. This study supports the likelihood that electronic cigarettes would be less harmful to oral cells than conventional cigarettes.