ALP Expression Research Articles

A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats

AimTo compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects. MethodThe endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/β-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting. ResultEndogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, β-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower. ConclusionThe superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/β-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.

PTH 1-34-functionalized bioactive glass improves peri-implant bone repair in orchiectomized rats: Microscale and ultrastructural evaluation

The objective of this work was to investigate the use of Biogran® functionalized with parathyroid hormone (PTH) 1–34 by sonochemistry for the local delivery of this anabolic agent to the implant site. The effects of Biogran® and topical administration of PTH 1–34 on peri-implant bone regeneration were evaluated from the microscale to ultrastructural levels in healthy (SHAM) and orchiectomized (ORQ). While some animals only received a titanium implant in their tibial metaphyses (CLOT group), in others the peri-implant defect was first filled with Biogran® either without or with PTH 1–34 functionalization (BG and BGPTH groups, respectively) prior to implant installation. Osseointegration was characterized from a biomechanical perspective by measuring the removal torque with the counter-torque technique. Micro-CT was used to evaluate the percentage of bone volume, trabecular thickness, number and separation, and bone-implant contact (BIC). Dynamics of new bone formation were assessed by measuring fluorochrome area, daily mineral apposition rate, and neoformed bone area using confocal laser microscopy. RT-PCR was performed to evaluate ALP and osteocalcin expression. The interface between newly formed bone and Biogran® was examined using scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) at the micro-and nanoscale, respectively, while elemental analyses were completed in SEM with energy-dispersive X-ray spectroscopy (EDS). STEM imaging demonstrated the intimate attachment of bone to Biogran® (nanoscale level). Overall, the results suggest that the effectiveness of the topical administration of PTH 1–34 at the implant site seems enhanced in osteoporotic bone, promoting peri-implant bone regeneration to comparable levels in healthy conditions.

MiR-30b-5p inhibits osteoblast differentiation through targeting BCL6

ABSTRACT Human bone marrow mesenchymal stem cells (hBMSCs) are attractive candidates for new therapies to improve bone regeneration and repair. This study was to identify the function of the miR-30b-5p/BCL6 axis in osteogenic differentiation of hBMSCs. Realtime-quantitative PCR (RT-qPCR) and Western blotting were used to measure the relative expression of ALP, OCN, RUNX2, miR-30b-5p, and BCL6 during osteogenic differentiation of hBMSCs. The relationship between miR-30b-5p and BCL6 in hBMSCs was identified using dual-luciferase reporter system and RNA pull-down assay. Alizarin red S staining (ARS) was used to detect the calcium nodules in hBMSCs. We found that the expression of miR-30b-5p was downregulated, whereas that of BCL6 was upregulated during osteogenic differentiation of hBMSCs. Downregulating miR-30b-5p enhanced the expression of OCN, RUNX2, and ALP, and promoted calcium deposition. Conversely, transfection with si-BCL6 had the opposite effect that it inhibited osteogenic differentiation. However, the inhibitory effect of si-BCL6 was abrogated by miR-30b-5p inhibitor. miR-30b-5p inhibits the osteogenic differentiation of hBMSCs by targeting BCL6.

Odontogenic induction of human amniotic membrane scaffold for dental pulp regeneration

ObjectiveThis study aimed to investigate the ability of glycerol or cryopreserved de-epithelialized human amniotic membrane, coded as GAM or CAM respectively, to induce odontogenic differentiation of deciduous teeth stem cells (DTSCs) cultured on their stromal (coded as GAMS or CAMS) or basal sides (coded as GAMB or CAMB). MethodsThe DTSCs attachment, proliferation, dentinogenic differentiation (COL-1, DMP1, ALP, and Nestin) and inflammatory cytokines (TGF-β, IL-β, IL-6, IL-8, GM-CSF and TNF-α) expression were evaluated. Furthermore, dental pulp stem cells (DPSCs)-seeded on GAM was transplanted into a prepared root canal for 3-day, and cells attachment to dentine was investigated. ResultsBoth GAM and CAM supported DTSCs attachment. DTSCs showed significantly higher proliferation after 7 days when seeded on both sides of CAM than on GAM, but both were still significantly lower than the control. There was no significant difference in cell proliferation between the stromal and basal sides of the same membrane. The expression of odontogenic markers of cells seeded on the membranes was comparable to the control cells, except for ALP. The expression of ALP on both sides of GAM was significantly higher than those on CAM, indicating the possible odontogenic differentiation potential of GAM. The differentiation could also explain the higher proliferation of cells grown on CAM compared to GAM. This finding was also consistent with the higher expression of IL-8 on CAM than GAM. When GAM was used to deliver DPSCs to the dentin of the root canal, no cells were observed on the dentin surface, indicating the strong attachment of cells to GAM. The findings warrant further investigation. ConclusionGAM could be used as a scaffold for pulp regeneration while vascularization and toning the degradation of GAM to be consistent with the new tissue formation will be considered for future work.

Mechanism of CircANKRD36 regulating cell heterogeneity and endothelial mesenchymal transition in aortic valve stromal cells by regulating miR-599 and TGF-β signaling pathway

ObjectiveTo explore the mechanism of CircANKRD36 regulating cell heterogeneity and endothelial mesenchymal transition in aortic valve stromal cells by regulating miR-599 and TGF-β signaling pathway. MethodsHuman tissue specimens were divided into Control group (n = 25) and CAVD group (n = 25). The mRNA expressions of CircANKRD36 and miR-599 in tissue samples were analyzed by qRT-PCR. Western blot was used to analyze the protein expression of osteogenic differentiation related factors induced by OM.The expressions of ALP, osteocalcin, osteopontin, Runx2 and Cadherin11 were detected by Western blot. ResultsThe expression of CircANKRD36mRNA in CAVD tissue was lower than that in Control tissue (P < 0.05), and the expression of miR-599mRNA in CAVD tissue was higher than that in Control tissue (P < 0.05). CircANKRD36 was negatively correlated with ALP, osteocalcin, osteopontin, Runx2, Cadherin11 expression level after OM induced osteogenic differentiation. The expression level of miR-599 was positively correlated with ALP, osteocalcin, osteopontin, Runx2 and Cadherin11 after OM induced osteogenic differentiation.The expression of ALP, osteocalcin, osteopontin, Runx2 and Cadherin11 protein in circ+miR-599 group was lower than that in circ+miR-NC group (P < 0.05). Compared with Vector+miR-NC group, the protein expressions of TGF-β1, TGF-β2 and SMAD4 in circ+miR-NC group decreased (P < 0.05), while the protein expressions of TGF-β1, TGF-β2 and SMAD4 in circ+miR-599 group increased (P < 0.05). ConclusionCircANKRD36 can inhibit the expression of miR-599 and the activation of TGF-β signaling pathway, thus inhibiting the expression of differentiation-related factors of VIC osteogenesis and the formation of calcified nodules. Therefore, circANKRD36-miR-599-TGF-β axis can be a new theoretical basis for treating CAVD.

Brown Adipose Tissue Rescues Bone Loss Induced by Cold Exposure.

Cold temperature activates the sympathetic nervous system (SNS) to induce bone loss by altering bone remodeling. Brown adipose tissue (BAT) is influenced by the SNS in cold environments. Many studies have confirmed a positive relationship between BAT volume and bone mass, but the influence and mechanism of BAT on bone in vivo and in vitro is still unknown. Two-month-old C57/BL6j male mice were exposed to cold temperature (4°C) to induce BAT generation. BAT volume, bone remodeling and microstructure were assessed after 1 day, 14 days and 28 days of cold exposure. CTX-1, P1NP and IL-6 levels were detected in the serum by ELISA. To determine the effect of BAT on osteoclasts and osteoblasts in vitro, brown adipocyte conditional medium (BAT CM) was collected and added to the differentiation medium of bone marrow-derived macrophages (BMMs) and bone marrow mesenchymal stem cells (BMSCs). Micro-CT results showed that the bone volume fraction (BV/TV, %) significantly decreased after 14 days of exposure to cold temperature but recovered after 28 days. Double labeling and TRAP staining in vivo showed that bone remodeling was altered during cold exposure. BAT volume enlarged after 14 days of cold stimulation, and IL-6 increased. BAT CM promoted BMSC mineralization by increasing osteocalcin (Ocn), RUNX family transcription factor 2 (Runx2) and alkaline phosphatase (Alp) expression, while bone absorption was inhibited by BAT CM. In conclusion, restoration of bone volume after cold exposure may be attributed to enlarged BAT. BAT has a beneficial effect on bone mass by facilitating osteogenesis and suppressing osteoclastogenesis.

Development and characterization of cannabidiol-loaded alginate copper hydrogel for repairing open bone defects in vitro

The clinical treatment of open bone defects caused by accidental bone trauma, bone tumors, bone diseases and bone infections is challenging. In this study, we designed and fabricated a multifunctional alginate-based hydrogel that contains cannabidiol (CBD), SA@Cu/CBD hydrogel, for repairing open bone defects. The results of physicochemical characterization showed that the SA@Cu/CBD hydrogel was successfully prepared and showed a suitable swelling ratio, high thermal stability, and stable mechanical properties. In vitro evaluation of antibacterial activity indicated that more than 90% of S. aureus and E. coli were inhibited compared to the control group. The ALP activity assay showed that the ALP expression level of MC3T3-E1cells in SA@Cu/CBD hydrogel was approximately 2-fold higher than that in the control group on day 7 and 14. Additionally, compared to the control group, the level of mineralized deposits in SA@Cu/CBD hydrogel was also improved by about 2 times on day 14. The PCR results indicated the mRNA expression levels of osteogenic markers (ALP, Col1α1, OCN, and RUNX2 genes) and angiogenic markers (EGFL6 and VEGF genes) in SA@Cu/CBD hydrogel were significantly upregulated compared to that in the control group, and the mRNA expression levels of critical inflammatory cytokines (TNF-α and IL-1β) in the SA@Cu/CBD hydrogel were significantly down-regulated compared to that in SA@Cu hydrogel. Taken together, these results demonstrated that the SA@Cu/CBD hydrogel showed significantly anti-bacterial, anti-inflammation, angiogenic and osteogenic activities in vitro studies. Thus, SA@Cu/CBD hydrogels may be a promising candidate in repairing open bone defects.

Lycium barbarum polysaccharide protects against osteonecrosis of femoral head via regulating Runx2 expression

BackgroundOsteonecrosis of femoral head (ONFH) is a pathological state caused by lack of blood supply in femoral head. This study aimed to explore the function of Lycium barbarum polysaccharide (LBP), an antioxidant agent extracted from L. barbarum, on ONFH. MethodsOsteonecrosis rat model was generated using lipopolysaccharide (LPS) and methylprednisolone followed by examination of body weight, blood glucose, morphology, and BMSC osteoblast differentiation. The effect and underlying mechanism of LBP on the proliferation, apoptosis, and osteoblast differentiation of BMSC were determined with or without LPS or hypoxia treatment using CCK-8. Alizarin Red S staining, flow cytometry, and western blot, respectively. ResultLBP could protect against glucocorticoid-induced ONFH in rats, resulting in improved sparse trabecular bone, empty lacunae and bone cell coagulation. Moreover, LBP promoted the proliferation and osteoblast differentiation of bone mesenchymal-derived stem cells (BMSCs) in a dose-dependent manner. Furthermore, LBP enhanced osteoblast differentiation of BMSCs under hypoxia condition. Mechanistically, we found that LBP treatment enhanced Runx2 and ALP expression in BMSCs. LBP restored the expression of Runx2 and ALP under hypoxia, suggesting that LBP might be involved in regulating Runx2/ALP expression and contributed to osteoblast differentiation. Knockdown of Runx2 significantly inhibited BMSCs proliferation, while LBP treatment did not rescue the osteoblast differentiation ability of BMSCs with Runx2 knockdown. ConclusionOur findings suggested that LBP protects against ONFH via regulating Runx2 expression, which could be utilized to treat patients suffering ONFH.

Improvement on biosafety and bioactivity of Ti–6Al–4V alloys by construction the three-dimensional grid structure though electrochemical dealloying

Bioactive porous structure coatings have received extensive attention because they can significantly improve the surface bioactivity of medical metal implants. However, traditional porous making technologies were restricted by the “upward growth” mode, with an obvious interface with the substrate, resulting in weak bonding and biological safety problems. This research innovatively proposed to selectively dissolve harmful Al element on the surface of Ti6Al4V alloy through an electrochemical dealloying process, forming a three-dimensional grid porous structure in situ on the surface, with the following three notable advantages: (1) The dealloying process was a “downward growth” mode, there was no obvious interface between the porous layer and the substrate, so the bond was firm; (2) The harmful element Al was effectively removed, and the biological safety was greatly improved; (3) The three-dimensional grid porous structure owned high-efficiency osteogenic activity, which solved the problem of Ti6Al4V biological inertness. The electrode potential and corrosion current density difference of metal elements were studied by electrochemical methods to analyze the corrosion and dissolution mechanism, the successful removal of the element Al, and the thickness of the porous layer were analyzed by XPS sputtering. Furthermore, the bioactivity was verified by co-cultivation with osteoblasts. Interestingly, the results of ALP expression, collagen secretion, and calcification all implied that the dealloyed porous structure could significantly promote the osteogenic differentiation of cells. Employing the electrochemical dealloying, a “subtractive” structure was formed on the surface of Ti6Al4V, which greatly improves the bioactivity and at the same time increased its osteogenic activity, which displayed extremely high research value and bright application value.

A mouse model of disuse osteoporosis based on a movable noninvasive 3D-printed unloading device.

ObjectiveDisuse osteoporosis is a major type of bone loss disease characterized by regional bone loss and microstructure alterations. The condition is induced by a marked decrease in weight bearing over time, which usually occurs due to limb immobilization, therapeutic bed rest or space flight. To date, the most commonly used mouse model of disuse osteoporosis is constructed using the classical tail suspension method, which causes tail injury, movement inconvenience and mental stress. This study aimed to propose a noninvasive and effective method for the establishment of a mouse model of disuse osteoporosis and compared this method with the tail suspension method.Methods3D printing technology was applied to construct a movable unloading device. A movable noninvasive 3D-printed unloading device (3D-ULD) was used to unload the hindlimbs of the mice. The bone microstructure and bone volume of unloaded femurs were analysed through micro-CT and H&E staining, and von Kossa staining was performed for the detection of bone mineralization in the femurs. TRAP staining, IHC-CTSK and Q-PCR were performed for evaluation of the bone resorption ability, and double labelling, IHC-DMP1, ALP staining and Q-PCR assays were conducted to assess the osteogenic ability. The mechanical properties of disused bone were detected using the three-point bending test. The body, thymus and spleen weights of the mice were recorded, and the serum corticosterone level of the mice was assayed by enzyme-linked immunosorbent assay (ELISA).ResultsThe micro-CT results showed significant trabecular bone loss, and 3D-ULD induced cortical bone loss in disused femurs as well as a decrease in the bone mineral density in the unloaded mice. TRAP staining and IHC-CTSK staining results indicated increases in the osteoclast number per bone perimeter (Oc.N/B.Pm) and the osteoclast surface per bone surface (Oc.S/BS) in the unloaded mice. The Ctsk, Trap and Mmp9 expression levels were significantly increased in the unloaded mice. Decreases in the ratio of the mineral surface to bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS) were found in unloaded mice in the 3D-ULD by double labelling. The IHC-DMP1 and ALP staining results showed decreases in the osteoblast number per bone perimeter (Ob.N/B.Pm) and osteoblast surface per bone surface (Ob. S/BS) in the mice unloaded in the 3D-ULD, and these mice also showed decreased Runx2, Alp and Dmp1 expression levels. Three-point bending test results showed that the mechanical properties were attenuated in the disused femurs of the unloaded mice. Less skin rupture and rare alterations in the thymus and spleen weights were found in the unloaded mice in the 3D-ULD. The ELISA results indicated the serum corticosterone level of the mice unloaded in the 3D-ULD was significantly lower than that of mice suspended by their tail.ConclusionThis new disuse osteoporosis mouse model based on 3D-ULD could induce effective disuse bone loss with significantly alleviated side effects.Translational potential of this articleThis study proposes a new disuse osteoporosis mouse model based on 3D-ULD that can be used to better understand disuse bone loss in the future.

Increased Expression of RUNX2 and ALP Resulting from a Combination of Propolis Extract and Bovine Bone Graft in Socket Preservation Material

The aim of this study was to determine the inductive effect of a combination of propolis and BBG extract on RUNX2 and ALP expression in the tooth extraction sockets of Cavia cobaya. Fifty- six Cavia cobaya were divided into four groups: polyethylene glycol (PEG), propolis extract + PEG, BBG + PEG, and propolis extract + BBG + PEG. The lower left incisor was extracted, and the socket subsequently filled with material according to the specific group of which the subject was a member. The subjects were sacrificed on the 14th and 30th days. Immunohistochemical staining was carried out under a light microscope at 400x magnification. Statistical analysis was then carried out by means of One-Way ANOVA and Tukey HSD tests. The mean number of RUNX2 and ALP expressions in each group was significantly different. The highest number of RUNX2 and ALP expressions occurred in the propolis + BBG + PEG group on the 30th day, while the lowest expressions were observed in the control group on the 14th day. A combination of propolis and BBG extract at a concentration of 2% of active substance effectively increases the expression of RUNX2 and ALP in preserving the tooth extraction sockets of Cavia cobaya.

CDK5 inhibition promotes osteoblastic differentiation of MSCs and blocks the migration of osteosarcoma MG-63 cells

CDK5 belongs to the cyclin-dependent kinase family. CDK5 is multifunctional and plays an important role in neural differentiation. However, the role of CDK5 in osteoblastic differentiation remains unclear. The present study investigated functions and molecular mechanism of CDK5 in osteoblastic differentiation. It was found that, CDK5 inhibition promoted the expression of Runx2, ALP, OCN and OPN of MSCs and the mineralization of MC-3T3E1 cells and MSCs. CDK5 inhibition enhanced the development of F-actin, nuclear localization of β-catenin and YAP, as well as the expression of RMRP RNA. When F-actin was suppressed by Blebbistatin, the nuclear localization of YAP and β-catenin, and expression of RMRP RNA as well as Runx2 and ALP were decreased. These indicate Seliciclib promotes osteoblastic differentiation mainly by F-actin. Moreover, Seliciclib also suppressed the migration of MG-63, suggesting a potential application for Seliciclib in bone defect repair and inhibition of the migration of osteosarcoma cells after osteosarcoma surgical resection.

Highly elastic 3D-printed gelatin/HA/placental-extract scaffolds for bone tissue engineering.

Bioengineering scaffolds have been improved to achieve efficient regeneration of various damaged tissues. In this study, we attempted to fabricate mechanically and biologically activated 3D printed scaffold in which porous gelatin/hydroxyapatite (G/H) as a matrix material provided outstanding mechanical properties with recoverable behavior, and human placental extracts (hPE) embedded in the scaffold were used as bioactive components.Methods: Various cell types (human adipose-derived stem cells; hASCs, pre-osteoblast; MC3T3-E1, human endothelial cell line; EA.hy926, and human dermal fibroblast; hDFs) were used to assess the effect of the hPE on cellular responses. High weight fraction (~ 70 wt%) of hydroxyapatite (HA) in a gelatin solution supplemented with glycerol was used for the G/H scaffold fabrication, and the scaffolds were immersed in hPE for the embedding (G/H/hPE scaffold). The osteogenic abilities of the scaffolds were investigated in cultured cells (hASCs) assaying for ALP activity and expression of osteogenic genes. For the in vivo test, the G/H and G/H/hPE scaffolds were implanted in the rat mastoid obliteration model.Results: The G/H/hPE scaffold presented unique elastic recoverable properties, which are important for efficient usage of implantable scaffolds. The effects of G/H and G/H/hPE scaffold on various in vitro cell-activities including non-toxicity, biocompatibility, and cell proliferation were investigated. The in vitro results indicated that proliferation (G/H = 351.1 ± 13.3%, G/H/hPE = 430.9 ± 8.7% at day 14) and expression of osteogenic markers (ALP: 3.4-fold, Runx2: 3.9-fold, BMP2: 1.7-fold, OPN: 2.4-fold, and OCN: 4.8-fold at day 21) of hASCs grown in the G/H/hPE scaffold were significantly enhanced compared with that in cells grown in the G/H scaffold. In addition, bone formation was also observed in an in vivo model using rat mastoid obliteration.Conclusion: In vitro and in vivo results suggested that the G/H/hPE scaffold is a potential candidate for use in bone tissue engineering.

Relevance of Cellular Redox Homeostasis for Vital Functions of Human Dental Pulp Cells.

Odontogenic MSCs are vulnerable to LPS-triggered bacterial infections, and they respond by secreting inflammatory mediators, such as IL-6, and with mineralization. Since both processes might be prone to a disturbance of the redox homeostasis, the oxidative stress influence on vital functions of human dental pulp cells (HPCs) was investigated. With these aims, a model of LPS-stimulated primary HPCs was established, and anti- and pro-oxidant substances were administered up to 21 days to measure inflammation and mineralization parameters. LPS-stimulated HPCs retained mineralization potential, which was decreased with the antioxidants NAC and fisetin and the pro-oxidant BSO. The expression of surface markers related to odontogenic commitment was influenced accordingly but counteracted by the enhanced expression of BMP2 and ALP at the transcriptional level. LPS triggers an early IL-6 production in non-odontogenic conditions, while it can be measured only after 15 days in the presence of the differentiation medium. The present study shows that HPCs functions causally depend on a tightly regulated cellular redox balance. Our data demonstrate a redox control of pulp MSC odontogenic commitment along with a potential association between an IL-6 late secretion and mineralization. These findings lay the groundwork for investigations on the molecular role of IL-6 in dental hard tissue metabolism.

M6A Methylation Regulates Osteoblastic Differentiation and Bone Remodeling.

Osteoporosis is a prevalent bone disease of the aging population, which is characterized by a decrease in bone mass because of the imbalance of bone metabolism. Although the prevention and treatment of osteoporosis have been explored by different researchers, the mechanisms underlying osteoporosis are not clear exactly. N6 methyladenosine (m6A) is a methylated adenosine nucleotide, which functions through its interaction with the proteins called “writers,” “readers” and “erasers.” The epigenetic regulation of m6A has been demonstrated to affect mRNA processing, nuclear export, translation, and splicing. At the cellular level, m6A modification has been known to affect cell proliferation, differentiation, and apoptosis of bone-related cells, such as bone marrow mesenchymal stem cells (BMSC), osteoblasts, and osteoclasts by regulating the expression of ALP, Runx2, Osterix, VEGF, and other related genes. Furthermore, PTH/Pth1r, PI3K‐Akt, Wnt/β‐Catenin, and other signaling pathways, which play important roles in the regulation of bone homeostasis, are also regulated by m6A. Thus, m6A modification may provide a new approach for osteoporosis treatment. The key roles of m6A modification in the regulation of bone health and osteoporosis are reviewed here in this article.

The effect of AMP kinase activation on differentiation and maturation of osteoblast cultured on titanium plate

Background/purpose5′ Adenosine monophosphate-activated protein kinase (AMPK) is known as an enzyme that maintains intracellular homeostasis and has various biological activity. The purpose of this study is evaluation effect of AMPK activation on implant prognosis. Materials & methodsMC3T3-E1 osteoblast-like cells were cultured on titanium using a 24-well plate. The experimental group was divided into the following 3 groups: (1) the normal culture group (control group), (2) the osteogenic induction group, and (3) the osteogenic induction + AMPK activation group. The cell counts were measured; real-time PCR was used to assess the expression of ALP and Osterix as osteogenic related genes at Day 0,7,14 and 21 after experiments. Additionally, ALP activity and calcification were assessed. ResultsThe results of the real-time PCR assessments revealed that the expression of ALP, which is a marker for the initial stages of calcification, was significantly increased by AMPK activation compared to the normal culture or osteogenic induction. A significant increase was also observed in the expression of Osterix, which is a marker for the later stages of calcification. Because significant increases were observed in ALP activity and calcification potential, this suggested that AMPK activation could elicit an increase in osteoblast calcification potential. ConclusionAMPK activation promotes implant peripheral osteoblast differentiation and maturation and enhances calcification. Our results suggest that AMPK activation may help to maintain implant stability.

IL-18-Mediated SLC7A5 Overexpression Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via the c-MYC Pathway.

Objective: To investigate the role of IL-18 in the regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Methods: To assess whether IL-18 affects the osteogenic differentiation of hBMSCs through the c-MYC/SLC7A5 axis, IL-18 dose-response and time-course experiments were performed to evaluate its impact on osteogenic differentiation. To confirm osteogenic differentiation, alizarin red staining calcium measurement were performed. RT-qPCR and western blotting were used to determine the expression levels of bone-specific markers ALP, RUNX2, and BMP2, as well as those of SLC7A5 and c-MYC. Furthermore, SLC7A5 and c-MYC expression was evaluated via immunofluorescence. To elucidate the roles of SLC7A5 and c-MYC in osteoblast differentiation, cells were transfected with SLC7A5 or c-MYC siRNAs, or treated with the SLC7A5-specific inhibitor JPH203 and c-MYC-specific inhibitor 10058-F4, and the expression of SLC7A5, c-MYC, and bone-specific markers ALP, RUNX2, and BMP2 was assessed. Results: Our results demonstrated that IL-18 increased calcium deposition in hBMSCs, and upregulated the expression of SLC7A5, c-MYC, ALP, RUNX2, and BMP2. Silencing of SLC7A5 or c-MYC using siRNA reduced the expression of ALP, RUNX2, and BMP2, while IL-18 treatment partially reversed the inhibitory effect of siRNA. Similar results were obtained by treating hBMSCs with SLC7A5 and c-MYC specific inhibitors, leading to significant reduction of the osteogenesis effect of IL-18 on hBMSCs. Conclusion: In conclusion, our results indicate that IL-18 promotes the osteogenic differentiation of hBMSCs via the SLC7A5/c-MYC pathway and, therefore, may play an important role in fracture healing. These findings will provide new treatment strategies for delayed fracture healing after splenectomy.

In vitro evaluation of periapical lesion-derived stem cells for dental pulp tissue engineering.

Dental pulp tissue engineering is a promising alternative treatment for pulpitis and periapical periodontitis, and dental pulp stem cells (DPSCs) are considered to be the gold standard for dental seed cell research. Periapical lesions harbor mesenchymal stem cells with the capacity for self‐renewal and multilineage differentiation. However, it remains unknown whether these periapical lesion‐derived stem cells (PLDSCs) are suitable for dental pulp tissue engineering. To investigate this possibility, PLDSCs and DPSCs were isolated using the tissue outgrowth method and cultured under identical conditions. We then performed in vitro experiments to investigate their biological characteristics. Our results indicate that PLDSCs proliferate actively in vitro and exhibit similar morphology, immunophenotype and multilineage differentiation ability as DPSCs. Simultaneously, PLDSCs exhibit stronger migrative ability and express more vascular endothelial growth factor and glial cell line‐derived neurotrophic factor than DPSCs, and PLDSC‐derived conditioned medium was more effective in tube formation assay. The mRNA expression levels of immunomodulatory genes HLA‐G, IDO and ICAM‐1 were also higher in PLDSCs. However, regarding osteo/odontogenic differentiation, PLDSCs showed weaker alkaline phosphatase staining and lower calcified nodule formation compared to DPSCs, as well as lower expression of ALP, RUNX2 and DSPP, as confirmed by a quantitative RT‐PCR. The osteo/odontogenic protein expression levels of DSPP, RUNX2, DMP1 and SP7 were also higher in DPSCs. The present study demonstrates that PLDSCs demonstrate potential use as seed cells for dental pulp regeneration, especially for achieving enhanced neurovascularization.

SHED's Response to Various Pulpotomy Materials: Cytotoxicity and Gene Expression Analysis.

To examine the cytotoxicity and genetic expression of SHEDs cultured in eluates of various calcium silicate based pulpotomy materials. MTT assay, flow cytometry, alizarin red staining and scratch assay was used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration respectively. The gene expression of ALP, OCN and BMP -2, were measured with rtPCR. One way ANNOVA and Bonferroni post test was used for statistical analysis. MTT assay analysis reported that all the test specimen had no cytotoxic effects. The highest number of live cells [ % ] was found in RetroMTA. The highest percentage of cell migration was observed in SHEDs cultured in EndoCem Zr. The mean absorbance for calcium matrix deposition was higher or similar in all test specimens, when compared to control groups. The expression of BMP -2 and OCN were significantly higher in cells exposed to RetroMTA and NeoMTA respectively after 24 hrs of incubation. After 72 hrs of incubation the mRNA expression of ALP was significantly higher in MTA. SHEDs cultured in eluates of various calcium silicate based cements exhibited cytocompatibility and maintained odontogenic like phenotype differentiation in SHEDs.

Metformin enhances the osteogenesis and angiogenesis of human umbilical cord mesenchymal stem cells for tissue regeneration engineering.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a potential clinical material in regenerative medicine applications. Metformin has shown safety and effectiveness as a clinical drug. However, the effect of metformin as a treatment on hUC-MSCs is unclear. Our research aimed to explore the effects of metformin on the osteogenesis, adipogenesis and angiogenesis of hUC-MSCs, and attempted to explain the molecular fluctuations of metformin through the mapping of protein profiles. Proliferation assay, osteogenic and adipogenic differentiation induction, cell cycle, flow cytometry, quantitative proteomics techniques and bioinformatics analysis were used to detect the influences of metformin treatment on hUC-MSCs. Our results demonstrated that low concentrations of metformin promoted the proliferation of hUC-MSCs, but high concentrations of metformin inhibited it. Metformin exhibited promotion of osteogenesis but inhibition of adipogenesis. Metformin treated hUC-MSCs up-regulated the expression of osteogenic marker ALP, OCN and RUNX2, but down-regulated the expression of adipogenic markers PPARγ and LPL. Proteomics analysis found that up-regulation of differentially expressed proteins in metformin treatment group involved the biological process of cell migration in Gene Ontology analysis. Metformin enhanced cell migration of HUVEC in a co-culture system, and hUC-MSCs treated with metformin exhibited stronger angiogenesis in vitro and in vivo compared to the hUC-MSCs group. The results of RT-qPCR revealed that the SCF and VEGFR2 were raised in metformin treatment. This study can promote the application of hUC-MSCs treated with metformin to tissue engineering for vascular reconstruction and angiogenesis.