Alkaline Phosphatase Activity In Cells Research Articles

Dried root of Rehmannia glutinosa extracts prevents steroid-induced avascular necrosis of femoral head by activating the wingless-type (Wnt)/β-catenin signal pathway

Steroid-induced avascular necrosis of femoral head (SANFH) is one of the most common complications caused by long-term or excessive clinical use of glucocorticoids. This study aimed to investigate the effects of dried root of Rehmannia glutinosa extracts (DRGE) in SANFH. First, SANFH rat model was established by dexamethasone (Dex). Tissue change and proportion of empty lacunae were detected by hematoxylin and eosin staining. Protein levels were detected by western bloting analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess apoptosis of femoral head tissue. Cell viability and apoptosis of MC3T3-E1 cells were assessed by Cell Counting Kit-8 assay and flow cytometry. ALP activity and cell mineralization were detected by ALP staining assay and Alizarin red staining. The findings showed that DRGE improved tissue damage, inhibited apoptosis, and promoted osteogenesis in SANFH rats. In vitro, DRGE increased cell viability, inhibited cell apoptosis, promoted osteoblast differentiation, reduced the levels of p-GSK-3β/GSK-3β, but increased the levels of β-catenin in cells treated with Dex. Furthermore, DKK-1, an inhibitor of the wingless-type (Wnt)/β-catenin signaling pathway, reversed the effect of DRGE on cell apoptosis and ALP activity in cells treated with Dex. In conclusion, DRGE prevents SANFH by activating the Wnt/β-catenin signaling pathway, indicating that DRGE may be a hopeful choice drug to prevent and treat patients with SANFH.

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

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

Diatom silica frustules-doped fibers for controlled release of melatonin for bone regeneration

Sustained release of a bioactive agent from a tissue engineering scaffold is one of the most common strategies to improve regenerative potential of the construct. However, depending on the chemistry of the agent, achieving high enough loading and controlled release can be challenging depending on the scaffold materials. These shortcomings can be solved by novel scaffold design fabricated by appropriate techniques and materials for the target tissue. In this study, an electrospun scaffold was designed to improve osteogenic activity of cells and diatom silica frustules were used to sustain loading and controlled release of a hydrophilic molecule, melatonin. Fibrous scaffolds were produced via wet electrospinning of the polymer blend solution poly(hydroxybutyrate-co-hydroxyvalerate (PHBV)/poly(ε-caprolactone) (PCL) which contains melatonin loaded diatom frustules. In the 3D fiber matrix diatom frustules were covered with a polymer coat which successfully lowered melatonin release more than half through 7 days achieving a controlled release. Melatonin had a concentration dependent effect on ALP activity of cells, while scaffolds bearing melatonin loaded frustules have significantly improved ALP activity of Saos-2 cells. Developed scaffold system has successfully induced osteogenic activity by controlled melatonin delivery and silica nature of diatom frustules which hold potential use for bone tissue engineering.

Gelatin-hydroxyapatite Fibrous Nanocomposite for Regenerative Dentistry and bone Tissue Engineering

Aims: This study aimed to prepare and physicochemically evaluate as well as assess the cytotoxicity and stimulation of early osteogenic differentiation of dental pulp stem cells of gelatin-hydroxyapatite (Gel-HA) fibrous nanocomposite scaffold. Background: Recently, the electrospinning approach in nanotechnology has been considered due to its application in the preparation of biomimetic nanofibers for tissue engineering. Objective: The main objective of this study was to evaluate Gel-HA fibrous nanocomposite for regenerative dentistry and bone tissue engineering material. Methods: The nano-scaffold was prepared via the electrospinning method. Then, the physicochemical properties (particle size, surface charge, morphology, hydrophilicity, specific surface area, crystalline state and the characterization of functional groups) and the proliferative effects of nano-scaffolds on dental pulp stem cells were assessed. The alkaline phosphatase activity was assessed for evaluation of early osteogenic differentiation of dental pulp stem cells. Results: The prepared nano-scaffolds had a negative surface charge (-30 mv±1.3), mono-dispersed nano-scale diameter (98 nm±1.2), crystalline state and fibrous uniform morphology without any bead (structural defects). The nanofibrous scaffold showed increased hydrophobicity compared to gelatin nanofibers. Based on Brunauer-Emmett-Teller analysis, the specific surface area, pore volume and pore diameter of Gel-HA nanofibers decreased compared to gelatin nanofibers. The Gel-HA nano-fibers showed the proliferative effect and increased the alkaline phosphatase activity of cells significantly (P<0.05). Conclusion: The prepared Gel-HA nanofibers can be considered potential candidates for application in bone tissue engineering and regenerative dentistry. Other: Gel-HA nanofibers could be a potential material for bone regeneration and regenerative dentistry in the near future.

In Vitro and In Vivo Analysis of the Effects of 3D-Printed Porous Titanium Alloy Scaffold Structure on Osteogenic Activity.

The effect of titanium scaffold geometry on the bone regeneration ability of the scaffold remains unclear. Here, selective laser melting as a 3D printing technology was used to create porous titanium alloy scaffolds with two unit structures: a hollow hexagonal prism (group A) and a hollow triangular prism (group B). The structures and morphologies of the scaffolds were characterized before mechanical properties were simulated. Cell adhesion behaviors, osteoblast activity and proliferation, and alkaline phosphatase (ALP) activity were evaluated, in addition to in vivo testing in an animal model. The results showed that the two scaffolds made of Ti6Al4V had compression moduli similar to that of human cortical bone (116.91 ± 0.01 and 174.29 ± 2.21 MPa vs. 89–164 MPa). The two scaffolds were nontoxic to cells and had good biocompatibility, while group A scaffolds facilitated cell adhesion. The number of cells increased gradually in culture. The ALP activity of cells on group A scaffolds demonstrated higher osteogenic ability than that of group B scaffolds. The in vivo tests showed that all scaffolds retained their shape well after implantation, and no obvious inflammatory reaction or infection in surrounding tissues was found. Based on fluorescence staining, mature new bone formation was found at week 12. Group A scaffolds showed better bone integration ability compared with group B scaffolds. The percentage of new bone area in group A (7.5%) was higher than that in group B (6.5%). This research suggests that the hollow hexagonal prism structure of porous scaffolds can promote osteogenic differentiation and osseointegration better than the triangular prism structure.

Vancomycin Containing PDLLA and PLGA/β-TCP Inhibit Biofilm Formation but Do Not Stimulate Osteogenic Transformation of Human Mesenchymal Stem Cells.

AimsChronic osteomyelitis, including implant-related prosthetic joint infection, is extremely difficult to cure. We develop vancomycin containing release systems from poly(d,l-lactide) (PDLLA) and poly(d,l-lactide-co-glycolide) (PLGA) composites with beta-tricalcium phosphate (β-TCP) to treat methicillin-resistant Staphylococcus aureus osteomyelitis. We ask whether vancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites will prevent early biofilm formation, allow cell proliferation and osteogenic differentiation, and stimulate osteogenic signaling molecules in the absence of an osteogenic medium.MethodsComposites were produced and characterized with scanning electron microscopy. In vitro vancomycin release was assessed for 6 weeks. Biofilm prevention was calculated by crystal violet staining. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and osteosarcoma cell (SaOS-2) proliferation and differentiation were assessed with water soluble tetrazolium salt and alkaline phosphatase (ALP) staining. Real-time quantitative polymerase chain reaction defined osteogenic signaling molecules for hBM-MSCs.ResultsTotally, 3.1 ± 0.2 mg and 3.4 ± 0.4 mg vancomycin released from PDLLA/β-TCP and the PLGA/β-TCP composites, respectively, and inhibited early biofilm formation. hBM-MSCs and SaOS-2 cells proliferated on the composites and stimulated ALP activity of cells. Runt-related transcription factor 2 (RUNX2) and SRY-Box transcription Factor 9 (SOX9) expressions were, however, lower with composites when compared with control.ConclusionVancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites inhibited early biofilm formation and proliferated and differentiated hBM-MSCs and SaOS-2 cells, but osteogenesis-related RUNX2 and SOX9 transcription factors were not strongly expressed in the absence of an osteogenic medium for 14 days.

Fast and sensitive near-infrared ratiometric fluorescent probe with a self-immolative spacer for imaging of endogenous alkaline phosphatase activity in cells and in vivo

Alkaline phosphatase (ALP), a vital hydrolase widely distributed in organisms, is regarded as a critical biomarker strongly associated with many physiological and pathological processes. Therefore, fast and efficient detection of ALP activity in vivo is of great value for clinical diagnosis. Herein, a novel near-infrared (NIR) ratiometric fluorescent probe (HP) was designed based on ESIPT for trapping ALP activity in cells and in vivo. Notably, incorporating a self-immolative spacer dramatically reduces the response time (8.5 min) of HP. Moreover, the probe exhibits excellent water solubility, large Stokes shift (147 nm), the ratiometric determination of ALP at 570 nm and 689 nm, low detection limit (3.98 U L−1). More importantly, the probe was also successfully applied to detect and monitor variations in endogenous ALP activity in zebrafish due to the drug (APAP) induced organ damages.

Fast and Sensitive Near-Infrared Ratiometric Fluorescent Probe with a Self-Immolative Spacer for Imaging of Endogenous Alkaline Phosphatase Activity in Cells and <i>In Vivo</i>

Fast and Sensitive Near-Infrared Ratiometric Fluorescent Probe with a Self-Immolative Spacer for Imaging of Endogenous Alkaline Phosphatase Activity in Cells and <i>In Vivo</i>

Effect and mechanism of propranolol on promoting osteogenic differentiation and early implant osseointegration.

The present study aimed to investigate the effect of β-receptor blocker propranolol on early osseointegration of pure titanium implants and the underlying molecular regulatory mechanisms. An implant osseointegration model using the tibial metaphysis of New Zealand rabbits was established. The rabbits were divided into control and low-, medium- and high-dose propranolol groups. The formation of implant osseointegration was detected by X-ray scanning. Mesenchymal stem cells (MSCs) and osteoblasts (OBs) were isolated and cultured in vitro, isoproterenol was supplemented to simulate sympathetic action and propranolol was subsequently administrated. The effect of propranolol on cell proliferation and osteogenic differentiation were assessed by EdU, flow cytometry, alizarin red staining and alkaline phosphatase (ALP) detection. The expression levels of bone morphogenetic protein (BMP)2, RUNX family transcription factor (RunX)2, collagen (COL)-1, osteocalcin (OCN) and β2-adrenergic receptor (AR) were detected by immunofluorescence, reverse transcription-quantitative PCR and western blot assay. Propranolol effectively promoted implant osseointegration in vivo, facilitated proliferation of OBs, inhibited proliferation of MSCs and enhanced osteogenic differentiation of OBs and MSCs. The calcium content and ALP activity of cells treated with propranolol were markedly higher than in the control group. Propranolol also elevated mRNA and protein expression levels of BMP2, RunX2, COL-1 and OCN in tissue and cells, and decreased the expression of β2-AR. The present study demonstrated that the β-receptor blocker propranolol promoted osteogenic differentiation of OBs and MSCs and enhanced implant osseointegration. The present study provided a novel insight into the application and regulatory mechanisms of propranolol.

Self-powered pulsed direct current stimulation system for enhancing osteogenesis in MC3T3-E1

Abstract Promoting the differentiation of osteoblasts is critical to maintain bone homeostasis for treatment osteoporosis and fracture healing. For these orthopedic diseases, a portable, highly patient compliance therapy device remains a great challenge. Here, we proposed a biomechanical-energy-driven shape memory piezoelectric nanogenerator (sm-PENG) that integrated with fixation splint to promote osteogenic differentiation. The pulsed direct current (DC) generated from the sm-PENG effectively promote MC3T3-E1 preosteoblast cell proliferation, orientation and increase intracellular calcium ion. At the same time, the ALP activity of cells is also improved by pulsed-DC under long-term culture conditions. Ultimately, increasing calcium deposition, extracellular matrix mineralization and osteogenesis. Our work demonstrates the potential of sm-PENG as a power source for pulsed-DC stimulation of bone repair, and shows great prospect self-powered and portable electronic medical device.

MC3T3 E1 cell response to mineralized nanofiber shish kebab structures.

Block copolymers (BCPs) are of growing interest because of their extensive utility in tissue engineering, particularly in biomimetic approaches where multifunctionality is critical. We synthesized polycaprolactone-polyacrylic acid (PCL-b-PAA) BCP and crystallized it onto PCL nanofibers, making BCP nanofiber shish kebab (BCP NFSK) structures. When mineralized in 2× simulated body fluid, BCP NFSK mimic the structure of mineralized collagen fibrils. We hypothesized that the addition of a calcium phosphate layer of graded roughness on the nano-structure of the nanofiber shish kebabs would enhance preosteoblast alkaline phosphatase (ALP) activity, which has been shown to be a critical component in bone matrix formation. The objectives in the study were to investigate the effect of mineralization on cell proliferation and ALP activity, and to also investigate the effect of BCP NFSK periodicity, a structural feature describing the distance between PCL-b-PAA crystals on the nanofiber core, on cell proliferation, and ALP activity. ALP activity of cells cultured on the mineralized BCP NFSK template was significantly higher than the nonmineralized BCP NFSK templates. Interestingly, no statistical difference was observed in ALP activity when the periodic varied, indicating that surface chemistry seemed to play a larger role than the surface roughness.

Cabbage-derived three-dimensional cellulose scaffold-induced osteogenic differentiation of stem cells.

Herbal-derived three-dimensional scaffolds have a unique structure that represents the natural cellular microenvironment and can be potentially used for tissue engineering applications. In the present study, cabbage (Cb) leaves were decellularized and then their characteristics, such as surface roughness, wettability, porosity, mechanical properties, and specific surface area, were investigated. After that, scaffold osteoinductivity was studied by bone-marrow-derived mesenchymal stem cells (BM-MSCs) osteogenic differentiation while growing on the decellularized Cb leaves. Cells mineralization, calcium secretion, alkaline phosphatase (ALP) activity, and expression levels of bone-related genes were determined during the differentiation process. Our results from the structural characterization of the scaffolds demonstrated that decellularized Cb leaves are good candidates for bone differentiation in terms of surface roughness, mechanical properties,and interconnected pores. Osteogenic differentiation evaluation of the BM-MSCs determined that the cell's ALP activity and mineralization were increased significantly while cultured on the decellularized Cb leaves compared to the cells cultured on the culture plate as a control. Besides, Runx2, ALP, collagen-1 (Col-I), and osteocalcin genes were expressed in cells cultured on decellularized Cb leaves significantly higher than cells cultured on the culture plate. Based on these results, it can be concluded that the decellularized Cb scaffold has great potential for promoting BM-MSCs proliferation and osteogenic differentiation.

Simultaneous enhancement of anti-corrosion, biocompatibility, and antimicrobial activities by hierarchically-structured brushite/Ag3PO4-coated Mg-based scaffolds.

Development of bone graft substitutes with appropriate integration of mechanical, biodegradable, and biofunctional properties, which promote bone formation while simultaneously preventing implant-associated infections, remains a great challenge. Herein we designed and synthesized a brushite/Ag3PO4-coated Mg-Nd-Zn-Zr scaffolds through chemical solution deposition of a composite coating onto the fluorinated Mg-based scaffolds generated with template replication method. The coated Mg-based open-porous scaffolds exhibit hierarchically-structured surface with cube-shaped Ag3PO4 nanoparticles uniformly distributed on top of microsized brushite grains. Immersion test reveals that the initial degradation rate of the coated scaffolds could be reduced by ~81% compared to the original scaffolds. The mean corrosion rate in 4weeks falls into 0.10-0.15mm/year to meet clinical requirements. The compatibility and ALP activity of cells grown in the extracts from the coated Mg-based scaffolds were increased compared with Ti control and original scaffolds, mainly due to the favorable microenvironment generated by Mg biodegradation. Besides, the coated Mg-based scaffold demonstrated potent antimicrobial activity via the synergistic actions of alkaline degradation products of Mg and the Ag species in the coating, achieving >99.5% antibacterial rate against both gram-positive and gram-negative bacteria with relatively low silver content. Taken together, this study presents a new candidate of brushite/Ag3PO4-coated Mg-based scaffold with appropriate degradation characteristics, cytocompatibility, and antimicrobial activities for bone tissue engineering applications.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.

Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS).Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively.ResultsFAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces.Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Bacterial cellulose-reinforced boron-doped hydroxyapatite/gelatin scaffolds for bone tissue engineering

Today, many people are suffering from bone defects due to trauma, tumor or bone related diseases. Mimicking bone in terms of composition and structure has been a challenge for tissue engineers. In this study, a novel 3D porous tissue-engineered construct composed of natural and easily-accessible biomaterials, namely (1) an exopolysaccharide; bacterial cellulose (BC), (2) mineral crystals; boron-doped nano-hydroxyapatite (BHA), and (3) a natural protein; gelatin (GEL) as the main constituting biomaterial was prepared by a simple and cost-effective technique; lyophilization, for bone tissue engineering applications. BC was produced by Gluconacetobacter xylinum bacteria species and hydroxyapatite (HA) and BHA were synthesized via sol–gel technique. Characterizations of GEL-BC, GEL-BC/HA, and GEL-BC/BHA scaffolds showed that they all possessed porous structure and pores became more irregular with the addition of HA or BHA. Scaffolds exhibited high water absorption, suitable degradation rate while having in vitro bioactivity with a Ca/P ratio similar to that of bone (Ca/P < 1.67). Thermo-gravimetric analysis showed that structural stability of the scaffolds was improved with the addition of HA and BHA. The porosity of scaffolds was similar (68.49–80.94%). HA and BHA-incorporation into scaffolds further improved the mechanical properties. Cell culture studies conducted with Saos-2 cell line showed that cells attached, proliferated on the GEL-BC/BHA scaffolds at a higher level demonstrating that the scaffolds were cytocompatible. ALP activity of cells seeded on GEL-BC/BHA scaffolds was statistically highest at day 14 which was also correlated with the results of intracellular calcium deposition. Thus, GEL-BC/BHA scaffolds hold potential for use in bone tissue engineering applications.

Porous clinoptilolite—nano biphasic calcium phosphate scaffolds loaded with human dental pulp stem cells for load bearing orthopedic applications

Clinoptilolite (Cpt)-nanohydroxyapatite (HA) (Cpt-HA) scaffolds were fabricated as a potential material for loadbearing orthopaedic applications. Cpt-HA materials were successfully synthesized by using microwave assisted reflux method followed by the fabrication of three-dimensional (3D) porous scaffold via thermal decomposition process using polyethylene glycol (PEG)/ polyvinyl alcohol (PVA) as porogens. The scaffold materials were characterized using x-ray diffraction, Fourier transform Infra-red, Scanning electron microscopy and Energy dispersive spectroscopy techniques. Incorporation of Cpt in HA scaffold significantly increased the compressive strength and surface hardness while scaffolds retained an interconnected porous structure with 64% porosity. Human dental pulp stem cells (DPSCs) were isolated from the third molar and used as pluripotent-like cell model to evaluate the biological properties of Cpt-HA scaffolds. Highest cellular attachment and proliferation were observed for DPSCs seeded on 2.0 g Cpt-HA scaffolds compare to pure HA. Similarly, significantly higher ALP activity of cells was observed on Cpt-HA scaffolds compared to pure HA. The enhanced proliferation and osteogenic response of the DPSCs cultured on Cpt-HA scaffolds suggest that the fabricated scaffolds can be used in bone tissue engineering. In this work, we have successfully shown that the interconnected porous Cpt-HA scaffolds have superior mechanical biological properties compared to pure HA scaffold.

Baghdadite nanoparticle-coated poly l-lactic acid (PLLA) ceramics scaffold improved osteogenic differentiation of adipose tissue-derived mesenchymal stem cells.

Bone repair has been a new approach in regenerative medicine especially by application of stem cells. Discovering a suitable combination of scaffolds to stimulate osteogenesis is one of the major concerns in this issue. Porous polymeric scaffolds such as poly l-lactic acid (PLLA) have been attracted a lot of attention because of their biodegradability. In the present study, we have been coated Baghdadite on the plasma-treated surface of PLLA and evaluated osteogenic potential of mesenchymal stem cells (MSCs). Adipose tissue-derived mesenchymal stem cells (AD-MSCs) were cultured on PLLA and PLLA-Baghdadite scaffolds, and cell properties were characterized by MTT assay, scanning electron microscope, and FTIR analysis. Then, osteogenic differentiation potential of AD-MSCs has been investigated, such as alkaline phosphatase (ALP) activity, calcium mineral deposition, and the expression of bone-related genes (RUNX2, ALP, and OCN). The results have been indicated that calcium content and ALP activity of cells cultured on PLLA-Baghdadite nanofibers were higher than that of tissue culture polystyrenes (TCPs). Gene expression analysis showed that PLLA-Baghdadite had effectively induced osteogenesis-related genes. Taken together, these results suggest that porous nanofiber scaffolds which coated with Baghdadite can enhance osteogenic differentiation of AD-MSC, and PLLA-Baghdadite can be used as a new biodegradable scaffold for bone regeneration. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1284-1293, 2019.

Down-regulation of miR-339 promotes differentiation of BMSCs and alleviates osteoporosis by targeting DLX5.

It was the aim of this study to investigate whether miR-339 may affect osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) by targeting DLX5, thereby alleviating osteoporosis. BMSCs were isolated from the bone marrow of mice. The expression levels of miR-339 and DLX5 during the process of osteogenesis was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Meanwhile, the expression of downstream osteogenesis-associated proteins, such as runt-related transcription factor 2 (RUNX2) and osteopontin (OPN), were also detected after overexpression or inhibition of miR-339. The alkaline phosphatase (ALP) activity was measured in cells by ALP activity assay kit. Alizarin red staining was performed to reveal the cell mineralization ability. The luciferase reporter gene assay was used to identify the targeted pairings of miR-339 and DLX5 genes. In addition, the expression of DLX5 was detected by Western blot analysis after overexpression or knockdown of miR-339. Rescue test was applied to evaluate whether miR-339 could affect the differentiation of BMSCs by inhibiting the expression of DLX5. QRT-PCR showed that miR-339 expression gradually decreased while the expression of DLX5 increased during the induction culture of BMSCs. After overexpression of miR-339 in BMSCs, the expression levels of ALP, RUNX2, and OPN were reduced. Besides, ALP activity assay showed a decreased cell ALP activity. RUNX2 protein expression was also decreased. In addition, Alizarin red staining detected a significant increase in cell mineralization, whereas silencing miR-339 resulted in an opposite result. These results indicated that miR-339 could regulate the osteogenic differentiation of BMSCs. Subsequently, we predicted using bioinformatics software that miR-339 might target DLX5, and validated this hypothesis by luciferase reporter assay. Finally, Western blot and ALP activity assay revealed that DLX5 could reverse the inhibitory effect of overexpression of miR-339 on osteogenic differentiation of BMSCs. Down-regulation of miR-339 can promote osteogenic differentiation of BMSCs by targeting DLX5, thereby relieving osteoporosis.

The in vitro effects of CCN2 on odontoblast-like cells

ObjectiveTo investigate the in vitro effects of CCN2 on odontoblast-like cells proliferation and differentiation. DesignMDPC-23 cells were cultured in DMEM supplemented with 5% FBS. CCN2 was either added to culture media or coated onto culture polystyrene, addition or coating of dH2O was served as control. In the addition group, CCN2 (100 ng/mL) was added into culture media. In the coating group, CCN2 at the concentration of 1000 ng/mL was employed. Cell proliferation was performed using CCK-8 assay. Cell differentiation and mineralization were analyzed by ALPase activity assay, real time RT-PCR and alizarin red staining. One-way ANOVA with post-hoc tukey HSD test was used for statistical analysis. ResultsMDPC-23 cells exhibited robust proliferative activity upon exposure to either soluble or immobilized CCN2. ALP activity of cells cultured on CCN2-modified surface was continuously strengthened from day six (0.831 ± 0.024 units/μg protein versus 0.563 ± 0.006 units/μg protein of control) till day eight (1.035 ± 0.139 units/μg protein versus 0.704 ± 0.061 units/μg protein of control). Gene expression of BSP, OCN and OPN were promoted by soluble CCN2 after 48 h exposure. Moreover, gene expression of BSP, OCN, OPN, ALP, COL1 A1, Runx-2, DSPP and DMP-1 was significantly enhanced by immobilized CCN2. Finally, mineralization of MDPC-23 cells was accelerated by both soluble and immobilized CCN2 to different extent. ConclusionsThe findings indicate that CCN2 promoted proliferation, odontogenic gene expression and mineralization of MDPC-23 cells. It is proposed that CCN2 may be a promising adjunctive formula for dentin regeneration.

MiR-374b promotes osteogenic differentiation of MSCs by degrading PTEN and promoting fracture healing.

To investigate whether miR-374b can promote the differentiation of MSCs into osteoblasts by mediating PTEN, thus promoting fracture healing. Primary cultured mouse mesenchymal stem cells were obtained for following experiments. Flow cytometry was used to determine the expression of MSCs surface antigens to identify the purity. Alizarin red staining was used to detect whether MSCs could differentiate into osteoblasts. QRT-PCR was used to detect the expression of osteogenic marker genes as well as miR-374b and PTEN in bone marrow-derived MSCs from fractured mice model. ALP activity detection kit was used to detect ALP activity in cells. Changes of osteogenic proteins in cells were evaluated by Western blot. Bioinformatics methods were used to predict the binding sites between miR-374 and target genes. Luciferase reporter assay was used to confirm whether miR-374 could bind to the target gene. Under normal culture, MSCs grew into a long fusiform shape on the 4th day. After induced in the osteogenic induction medium for seven days, calcified nodules appeared. The results of the detection of MSCs surface antigen markers showed that CD90 was 99.12%, and CD45 was 0.23%. Alizarin red staining showed that MSCs possess the ability to differentiate into osteogenic. The expression level of MiR-374b and PTEN increased significantly in the early stage of fracture in mice, but no significant difference was observed at a later stage. After overexpression of miR-374b, the cell ALP activity, the expression of osteogenesis-related genes and osteogenesis-related proteins was significantly increased, while after knocking out miR-374b, the opposite result was observed. The result of luciferase reporting assay showed that miR-374b can bind to PTEN. As mentioned above, overexpression of miR-374b resulted in upregulation of osteogenic-related genes and proteins, while over-expression of both PTEN and miR-374b could partly reverse the outcomes. miR-374b can promote osteogenic differentiation of MSCs by degrading PTEN, thereby promoting fracture healing.