Expression Levels Of Alkaline Phosphatase Research Articles

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.

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.

Effect of Lactoferrin on decreased calcification by DEX of osteoblast-like MC3T3-E1 cells

Lactoferrin (LF) is a protein contained in milk of mammalians including human. We have already reported that LF prevented dexamethasone (DEX)-induced osteopenia in mice (Global Drugs and Therapeutics 2018). We investigated the effect of LF on decreased calcification by DEX with mouse osteoblastic cell line (MC3T3-E1), in this study. In addition, RNA expression levels of Alkaline phosphatase (ALP) and osteocalcin were measured in MC3T3-E1 cells by Real-time PCR. MC3T3-E1 cells were cultured in α-MEM containing 10%FBS. On day 4, medium was replaced by α-MEM containing 2% FBS, ascorbic acid (AA; 50µg/ml), beta-glycerophosphate (b-GP; 5mM) and DEX (3.3µg/mL) with/without LF (100µg/mL). On day11, hydroxyapatite was added into the culture medium. After 3hrs, the cells were stained with Alizarin red to evaluate calcification levels. In RT-PCR, sampling for RT-PCR was collected without exposing hydroxyapatite. In result of Alizarin red staining, decreased calcification by DEX was significantly suppressed by adding of LF. In result of RT-PCR, osteocalcin as a bone formation maker significantly decreased with exposure of DEX. The decreased osteocalcin expression level was significantly suppressed by adding of LF. These results suggested that LF suppressed reduction of calcification by DEX.

The potential application of concentrated growth factor in regenerative endodontics.

To investigate the potential application of concentrated growth factor (CGF) to promote pulp regeneration within immature teeth. Concentrated growth factor clots produced from peripheral blood samples were investigated histologically by haematoxylin-eosin (HE) staining and evaluated morphologically by scanning electron microscope (SEM). The cytokines were extracted from the CGF, and representative cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). The biological effects of the CGF on human stem cells from the apical papilla (SCAPs) were then investigated and quantified, including cell proliferation, cell migration, mineralized nodule formation, and the gene expression of alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP) and dentine matrix protein (DMP)-1. The results were analysed statistically using one-way analysis of variance (anova). Concentrated growth factor had a complex three-dimensional structure with a high density of platelets and nucleated cells. Representative growth factors including PDGF-BB, IGF-1, TGF-β1, bFGF and VEGF were detected. The growth rate and migratory cell numbers of the CGF groups were significantly greater than those in the control groups (P<0.05). The mineralization areas in the CGF groups were significantly larger than those in the control groups (P<0.05). The expression levels of ALP, DSPP and DMP-1 were significantly up-regulated after induction by CGF (P<0.05). Concentrated growth factor promoted the proliferation, migration and differentiation of SCAPs and could be a promising biomaterial applied in regenerative endodontics.

Strategy for In Situ Imaging of Cellular Alkaline Phosphatase Activity Using Gold Nanoflower Probe and Localized Surface Plasmon Resonance Technique.

In this work, a simple and ultrasensitive localized surface plasmon resonance (LSPR) method that use Au nanoflowers (AuNFs) as a probe was designed for in situ monitoring of alkaline phosphatase (ALP) activity. The AuNFs were fabricated by hydrogen tetrechloroaurate-induced oxidative disruption of polydopamine-coated Au nanoparticles (AuNPs), and subsequently, growth of Au nanopetals on AuNPs occurred. The as-prepared AuNFs showed a much higher LSPR capability and stronger scattering color change than AuNPs. The strategy for in situ cellular ALP activity detection relied on the deposition of Ag on the AuNFs surface, which changed the morphology of AuNFs and led to a tremendous LSPR response and scattering color change. The deposition of Ag shell on AuNFs was related to ALP activity, where ALP catalyzed the hydrolysis of l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to form l-ascorbic acid (AA), and then AA reduced Ag+ to Ag and deposited onto AuNFs. With this concept, the ALP activity could be monitored with a detection limit of 0.03 μU L-1. Meanwhile, the ALP activity of single HepG2 cells and HEK 293 cells was tracked with a proposed approach, which indicated the trace expression level of ALP in HEK 293T cell and overexpressed level of ALP in HepG2 cells. After treatment with drugs, the cellular ALP activity of HepG2 cells was decreased with the treating time and dose increasing. Therefore, the proposed strategy could be used for tracking the cellular ALP activity, which paved a new avenue for cell studies and held great potential for discovering novel ALP-based drugs applications.

Expression and influence of BMP-4 in human dental pulp cells cultured in vitro.

Effects of bone morphogenetic protein (BMP)-4 on proliferation and differentiation capacities of dental pulp cells through BMP-4 acting on human dental pulp cells cultured in vitro were investigated. Dental pulp tissues of lesion-free teeth extracted from patients due to orthodontics were taken, and human dental pulp cells were cultured in vitro using the tissue explant method. Immunocytochemical staining was used for the identification of vimentin and keratin. The dental pulp cells were divided into groups A and B. A total of 100 ng/ml BMP-4 was added into group A, while no inducer was added into group B as the control group. The cell growth curves at day 1, 2, 3, 5 and 7 after culture were drawn. At day 7, the cell count, alkaline phosphatase (ALP) activity, number of calcified nodules, and expression levels of dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1) and each gene related to dentinogenesis in each group were detected, respectively. Human dental pulp cells were conformed to the biological characteristics of dental pulp cells according to the identification of vimentin and keratin via immunocytochemical staining. With the prolongation of culture time, the number of cells in both groups was gradually increased, reaching the peak at day 5 and began to decline at day 7. The number of cells in group A was significantly greater than that in group B (p<0.05). According to the results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the relative messenger ribonucleic acid (mRNA) expression levels of ALP, DSPP and DMP-1 in group A were significantly higher than those in group B (p<0.05). BMP-4 can promote the growth of dental pulp cells and remarkably enhance the differentiation of dental pulp cells into odontoblasts.

Increased serum levels of miR-214 in patients with PCa with bone metastasis may serve as a potential biomarker by targeting PTEN.

MicroRNAs (miRNAs/miRs) are identified to serve key functions in the progression of various tumors. miR-214 is aberrantly expressed in various types of cancer. In the present study, the function of miR-214 and its feasibility as a potential non-invasive biomarker for patients with prostate cancer (PCa) in a hyperplasia group and a control group were investigated. First, RNA was isolated from the serum of 75 patients with PCa with bone metastasis, 65 patients with PCa with no bone metastasis and 70 healthy controls. The level of miR-214 expression was significantly upregulated in the serum of the bone metastasis group compared with the healthy control and non-bone metastasis groups. Expression levels of alkaline phosphatase (ALP), bone sialoprotein (BSP), collagen type I pyridine crosslinking peptide (ICTP) were also evaluated. The results indicated that serum levels of BSP, ALP and ICTP were increased in the bone metastasis group compared with that in the non-bone metastasis group, hyperplasia group and the control group (P<0.05). The expression level of miR-214 is positively associated with poorly differentiated tumors in patients with PCa with a Gleason score >7 (P<0.05). Western blot analysis demonstrated that phosphatase and tensin homolog (PTEN) was a target gene of miR-214. Additionally, silencing of PTEN significantly increased the invasive ability of PC3 cells even when miR-214 expression was inhibited. In summary, serum miR-214 expression may serve as a potential novel non-invasive biomarker for PCa screening through targeting PTEN.

Expression level of Dickkopf-1 protein in bone mesenchymal stem cells in Type 2 diabetic rats and its relationship with osteogenic activity

To examine the growth activity and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in rats with Type 2 diabetes mellitus (T2DM) as well as the expression level of Dickkopf-1 (DKK-1) in bone marrow, and to explore the relationship between the osteogenic activity of BMSCs and the expression of DKK-1. Methods: The BMSCs were isolated from T2DM rats and were cultured in vitro. The BMSCs were divided into a T2DM group and a control group. The proliferation of BMSCs was detected by cell counting kit-8 (CCK8). Apoptosis rate was detected by annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. In the osteogenic induction phase, the expression level of alkaline phosphatase (ALP) in BMSCs was detected by ALP staining and ALP activity assay kit. The osteogenic differentiation of BMSCs was analyzed by alizarin red staining and mineralized nodule quantification. In addition, the expression of Runx2 and DKK-1 in BMSCs was detected by qRT-PCR. Results: Compared with the control group, the proliferation of BMSCs was decreased and the apoptosis was increased in the T2DM group (both P<0.01). In the osteogenic induction process of BMSCs, the expression of ALP significantly decreased, the formation of calcium nodules reduced, and the expression of osteoblast transcription factor Runx2 was down-regulated in the T2DM group compared with those in the control group (all P<0.01). The levels of DKK-1 protein and mRNA were up-regulated in the T2DM group, which were higher than those in the control group (both P<0.01). The levels of DKK-1 protein and mRNA were related to the increase of Runx2 (both P<0.01). Conclusion: The growth activity of BMSCs and the potential of osteogenic differentiation are attenuated in the T2DM rats, which may be related to the increase of DKK-1 expression in BMSCs.

Enhanced in vitro biocompatibility and osteogenesis of titanium substrates immobilized with dopamine-assisted superparamagnetic Fe3O4 nanoparticles for hBMSCs.

Titanium (Ti) is an ideal bone substitute due to its superior bio-compatibility and remarkable corrosion resistance. However, in order to improve the osteoconduction and osteoinduction capacities in clinical applications, different kinds of surface modifications are typically applied to Ti alloys. In this study, we fabricated a tightly attached polydopamine-assisted Fe3O4 nanoparticle coating on Ti with magnetic properties, aiming to improve the osteogenesis of the Ti substrates. The PDA-assisted Fe3O4 nanoparticle coatings were characterized by scanning electron microscopy, energy dispersive spectroscopy, atomic force microscopy and water contact angle measurements. The cell attachment and proliferation rate of the human bone mesenchymal stem cells (hBMSCs) on the Ti surface significantly improved with the Fe3O4/PDA coating when compared with the pure Ti without a coating. Furthermore, the results of in vitro alkaline phosphatase (ALP) activity at 7 and 14 days and alizarin red S staining at 14 days showed that the Fe3O4/PDA coating on Ti promoted the osteogenic differentiation of hBMSCs. Moreover, hBMSCs co-cultured with the Fe3O4/PDA-coated Ti for approximately 14 days also exhibited a significantly higher mRNA expression level of ALP, osteocalcin and runt-related transcription factor-2 (RUNX2). Our in vitro results revealed that the present PDA-assisted Fe3O4 nanoparticle surface coating is an innovative method for Ti surface modification and shows great potential for clinical applications.

A Comparative Evaluation of Concentrated Growth Factor and Platelet-rich Fibrin on the Proliferation, Migration, and Differentiation of Human Stem Cells of the Apical Papilla

IntroductionConcentrated growth factor (CGF) is considered to be a natural biomaterial that is better than platelet-rich fibrin (PRF) in bone regeneration, but there is little information acquired in regenerative endodontics. Therefore, the purpose of this study was to evaluate their effects on the proliferation, migration, and differentiation of human stem cells of the apical papilla (SCAPs). MethodsCGF- and PRF-conditioned medium were prepared using the freeze-dried method. SCAPs were isolated and identified. The proliferative potential of SCAPs was investigated using the Cell Counting Kit-8 (KeyGen Biotech, Nanjing, China). The migration capacity was analyzed using transwell assays, and the mineralization ability was determined by alizarin red S staining. The expression levels of alkaline phosphatase, bone sialoprotein, dentin matrix protein 1, and dentin sialophosphoprotein were determined by quantitative polymerase chain reaction. ResultsThe cultured cells exhibited mesenchymal stem cell characteristics. The growth rate and migratory cell numbers of the CGF and PRF groups were significantly greater than those of the control group. The mineralized areas in the CGF and PRF groups were significantly larger than those in the control group after incubation for 7 days and 14 days. The expression levels of osteogenic/odontoblast-related genes were reduced on day 7, but they were dramatically enhanced on day 14, and the related gene expression levels in the PRF group were higher than those in the CGF group. ConclusionsBoth CGF and PRF can promote the proliferation, migration, and differentiation of SCAPs. CGF may be a promising alternative in regenerative endodontics.

Effects of inhibitory concentration minocycline on the proliferation, differentiation, and mineralization of osteoblasts

To study the effect of the inhibitory concentration minocycline on the proliferation, differentiation, and expression of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteopontin (OPN) mRNA of osteoblasts. Primary osteoblasts were cultured in osteogenic induction medium containing 0, 0.1, 0.5, 1, 10 μg·mL⁻¹ minocycline. Cell counting kit-8 was used to observe cell proliferation. ALP activity assay, alizarin red S staining, and real-time quantitative polymerase chain reaction (PCR) were used to determine cell differentiation and mineralization. The groups with 0.1, 0.5, 1 μg·mL⁻¹ minocycline promoted cell proliferation. The mRNA expression levels of ALP and Runx2 were up-regulated. Osteoblast-mediated mineralization was increased. The group with 1 μg·mL⁻¹ showed maximal promotion effect (P<0.05). When the concentration increased to 10 μg·mL⁻¹, the promoting effect began to decline, and the ALP activity and OPN expression were significantly inhibited (P<0.01). Appropriate concentration of minocycline can promote osteoblasts proliferation, up-regulate the expression levels of Runx2, ALP and OPN, and increase the differentiation and mineralization of osteoblasts.

Effects of platelet-rich plasma on angiogenesis and osteogenesis-associated factors in rabbits with avascular necrosis of the femoral head.

Platelet-rich plasma (PRP) contains various growth factors and cytokines that can enhance the recovery of the damaged tissues. The present study aimed to examine the effects of PRP on the recovery of avascular necrosis of the femoral head (ANFH), and to provide novel insights into the clinical treatment of this disease. A total of 24 New Zealand white rabbits were randomly divided into the normal control group, ANFH model and PRP-treated groups (n =1 2 each). Blood samples were extracted from the auricular vein at 4, 8 and 12 weeks after establishing the model to determine the hemorheological indexes, as well as the content of serum osteocalcin bone Gla-protein (BGP) and vascular endothelial growth factor (VEGF). In addition, femoral head tissue was collected, with part of it used for hematoxylin and eosin (HE) staining to observe the histological changes. The remaining was used to detect the mRNA expression levels of alkaline phosphatase (AKP), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), bone morphogenetic protein 2 (BMP-2) and platelet-derived growth factor B (PDGF-B) by reverse transcription-polymerase chain reaction. Compared with the model group, PRP treatment significantly improved the hemorheological indexes, as well as significantly increased the contents of BGP and VEGF. In the PRP group, the expression levels of TGF-β1, bFGF, BMP-2 and PDGF-B were significantly upregulated, while AKP expression was downregulated compared with the model group. Furthermore, PRP evidently improved the histological structure of the ANFH tissue. PRP was able to improve the hemorheological indexes following femoral neck fracture, repair the local blood vessels, and promote the expression of osteoblast-associated and angiogenesis-associated factors, which suggested a high efficiency in repairing ANFH.

AMP-activated protein kinase stimulates osteoblast differentiation and mineralization through autophagy induction.

Previous studies have reported that adenosine mono-phosphate-activated protein kinase (AMPK) activation can enhance osteoblast differentiation and mineralization; however, the underlying mechanism is not fully understood. Autophagy also serves an important role in osteoblast mineralization and bone homeostasis. The present study aimed to explore whether activation of AMPK could enhance osteoblast differentiation and mineralization via the induction of autophagy. The fracture healing and nonunion animal models were established and verified by X-ray imaging. Bone maturation was measured by Masson staining and the expression of AMPK, p-AMPK, microtubule-associated proteins 1A/1B light chain 3B II, and p62 in the fracture ends were detected by immunohistochemical staining. The mRNA expression levels of alkaline phosphatase (ALP), osteocalcin, runt-related transcription factor 2 and BCN1 were determined by reverse transcription-quantitative polymerase chain reaction. 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to determine ALP activity and alizarin red staining was adopted to examine mineralization. Western blot analysis was performed to detect protein expression. Autophagosome was observed by Transmission electron microscopy. Small interfering (si)RNA was used to knock down the expression of target gene. In vivo experiments demonstrated that new bone mineralization and maturation was markedly restrained in the nonunion group, alongside decreased AMPK activation and autophagic activity, compared with in the fracture healing group. The results of an in vitro study indicated that AMPK activation stimulated the osteogenic differentiation of MC3T3-E1 cells, with increases in ALP activity, mineralization, and the mRNA expression levels of ALP, osteocalcin and runt-related transcription factor 2. Furthermore, AMPK activation induced autophagy, as determined by upregulation of microtubule-associated proteins 1A/1B light chain 3B, increased autophagosome density and downregulation of p62. In addition, inhibition of autophagy reversed the effects of AMPK activation on osteoblast differentiation. These results suggested that AMPK activation may stimulate osteoblast differentiation and mineralization via the induction of autophagy, and provides evidence to suggest that enhancing AMPK activation and autophagic activity may be a potential novel approach to promote fracture healing.

Silk Biomaterials-Mediated miRNA Functionalized Orthopedic Devices.

Silk-based bioresorbable medical devices, such as screws, plates, and rods, have been under investigation due to their promising properties for orthopedic repairs. Options to functionalize these new devices for enhanced control of bone regeneration would also exploit the compatible processing methods used to generate the devices. MicroRNAs are important regulators of bone maintenance and formation, and miRNA-based therapeutics have the potential to aid bone repair, utilizing a transient therapeutic approach with local bioactivity. We hypothesized that silk-based orthopedic devices could be used for the local delivery of miRNAs, using anti-sense miR-214 (AS-miR-214), to inhibit endogenous expression of osteoinductive antagonist and thereby supporting the upregulation of osteoinductive target molecules activating transcription factor 4 (ATF4) and Osterix (Osx). AS-miR-214 silk devices, prepared using surface coating, demonstrated continuous release of miRNA inhibitors up to 7 days in vitro. Additionally, human mesenchymal stem cells seeded on AS-miR-214 silk films expressed higher levels of osteogenic genes ATF4, Osx, Runx2, and Osteocalcin. Interestingly, these cells exhibited lower cell viability and DNA content over 21 days. Conversely, the cells demonstrated significantly higher levels of alkaline phosphatase expression and calcium deposition compared with cells seeded on silk films with nontargeting miRNA controls. The study demonstrated that the silk-based orthopedic devices, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel system for localized bone tissue engineering, enhancing osteogenesis at the implant interface while avoiding detrimental systematic side effects.

Leptin Induces Odontogenic Differentiation and Angiogenesis in Human Dental Pulp Cells via Activation of the Mitogen-activated Protein Kinase Signaling Pathway

IntroductionUp-regulation of odontogenic differentiation, dentin formation, and angiogenesis in dental pulp are key factors in vital pulp therapy. The aim of this study was to investigate whether leptin could promote odontogenic differentiation and angiogenesis in human dental pulp cells (hDPCs). In addition, the involvement of the intracellular signaling pathway in these effects was determined. MethodsThe viability of hDPCs treated with leptin was examined using the water soluble tetrazolium salt-1 assay. Real-time polymerase chain reaction was performed to determine messenger RNA (mRNA) expression levels of odontogenic and angiogenic markers. Western blot analysis was used to measure odontogenic and angiogenic protein expression levels and assess mitogen-activated protein kinase (MAPK) pathway involvement. Alkaline phosphatase (ALP) and alizarin red staining were used to evaluate expression levels of ALP and calcified nodule formation after treatment with leptin and/or the presence of MAPK inhibitors. ResultsAll concentrations of leptin used in this study did not significantly affect the viability of hDPCs. However, mRNA and protein levels of odontogenic and angiogenic markers, ALP activity, and calcified nodule formation were significantly increased in the leptin-treated group compared with those in the control group. Leptin enhanced phosphorylation of extracellular signal-related kinases, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases within 5 minutes after treatment. However, leptin-induced dentin sialophosphoprotein and vascular endothelial growth factor protein expression and mineralization were appreciably blocked by the presence of MAPK inhibitors. ConclusionsLeptin can induce angiogenesis, odontogenic differentiation, and mineralization in hDPCs via activating the MAPK signaling pathway.

Effect of low-level laser therapy on the healing of sites grafted with coagulum, deproteinized bovine bone, and biphasic ceramic made of hydroxyapatite and β-tricalcium phosphate. In vivo study in rats.

The aim of this study was to evaluate the effect of low-level laser therapy (LLLT) on the healing of biomaterial graft areas (i.e., coagulum, deproteinized bovine bone, and biphasic ceramics comprising hydroxyapatite and β-tricalcium phosphate). Ninety rats were divided into two groups according to laser irradiation use (λ 808 nm, 100 mW, φ ∼600 μm, seven sessions with 28 J of irradiation dose in total): a laser group and a control group. Each of these groups was divided into three subgroups of 15 animals each according to the type of biomaterial used: Coagulum (COA), deproteinized bovine bone (DBB), and hydroxyapatite/β-tricalcium phosphate (HA/βTCP). Biomaterials were inserted into Teflon domes, and these domes were grafted to the lateral aspect of the mandibular branch of the rats. The animals were sacrificed after 30, 60, and 90 days. Scarring patterns were evaluated by microtomography and histometry. The expression levels of BMP2, osteocalcin (OCN), and alkaline phosphatase (ALP) were evaluated by immunohistochemistry. The mRNA expression levels of ALP, BMP2, Jagged1, Osterix, Runx2, and TGFβ1 were determined by RT-qPCR. The animals treated with LLLT exhibited increased mineralized tissues and bone, particularly after 90 days. These increases were associated with increased BMP2, OCN, and ALP protein expression and ALP, BMP2, and Jagged1 mRNA expression. LLLT improved the osteoconductive potential of DBB and HA/βTCP grafts and bone formation in ungrafted areas. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Icariin Restores Bone Structure and Strength in a Rat Model of Chronic High-Dose Alcohol-Induced Osteopenia

Background/Aims: Chronic alcohol abuse is an important risk factor for osteopenia. However, few studies have focused on the efficacy and mechanism of action of icariin on alcohol-induced osteopenia. The aim of this study was to investigate the efficacy and underlying mechanism of action of icariin in the treatment of chronic high-dose alcohol-induced osteopenia in a rat model. Methods: Thirty-six adult male Sprague-Dawley rats were randomly divided into four groups: sham, alcohol, and low-dose and high-dose icariin groups. Bone volume fraction (BV/TV), bone mineral density (BMD), bone biomechanical properties, and bone morphology were assessed after 16 weeks. Reverse-transcription PCR was used to detect mRNA expression levels of alkaline phosphatase (ALP), collagen type I (Col I), osteocalcin (OC), runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP-2), and osteoprotegerin (OPG). Results: Bone metabolic markers and biomechanical properties in the alcohol group were decreased significantly compared with the sham group. BV/TV, BMD, mineral apposition rate (MAR), percent trabecular area (%Tb.Ar), and bone biomechanical properties were elevated in the low-dose and high-dose icariin groups relative to the alcohol group. ALP, Col I, OC, Runx2, BMP-2, and OPG mRNA levels in the icariin group were significantly elevated in comparison with the alcohol group. Conclusion: Icariin can prevent overall progression of chronic high-dose alcohol-induced osteopenia in a rat model, in a dose-dependent manner. Icariin promotes bone formation and inhibits bone loss, and effectively restores bone structure and strength in chronic high-dose alcohol-induced osteopenic rats. Bone metabolism reversal is evidenced by increased BV/TV, BMD, MAR, %Tb.Ar, and biomechanical properties and elevated ALP, Col I, OC, Runx2, BMP-2, and OPG mRNA levels.

Effects of 1,25-dihydroxyvitamin D3 on the differentiation of MC3T3-E1 osteoblast-like cells.

PurposeThe purpose of this study was to evaluate the effects of 1,25-dihydroxyvitamin D3 on the proliferation, differentiation, and matrix mineralization of MC3T3-E1 osteoblast-like cells in vitro. MethodsMC3T3-E1 osteoblastic cells and 1,25-dihydroxyvitamin D3 were prepared. Cytotoxic effects and osteogenic differentiation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) activity assay, ALP staining, alizarin red S staining, and reverse transcription-polymerase chain reaction (RT-PCR) for osteogenic differentiation markers such as ALP, collagen type I (Col-I), osteocalcin (OCN), vitamin D receptor (VDR), and glyceraldehyde 3-phosphate dehydrogenase.ResultsThe MTT assay showed that 1,25-dihydroxyvitamin D3 did not inhibit cell growth and that the rate of cell proliferation was higher than in the positive control group at all concentrations. ALP activity was also higher than in the positive control group at low concentrations of 1,25-dihydroxyvitamin D3 (10−10, 10−12, and 10−14 M). RT-PCR showed that the gene expression levels of ALP, Col-I, OCN, and vitamin D receptor (VDR) were higher at a low concentration of 1,25-dihydroxyvitamin D3 (10−12 M). Alizarin red S staining after treatment with 1,25-dihydroxyvitamin D3 (10−12 M) showed no significant differences in the overall degree of calcification. In contrast to the positive control group, formation of bone nodules was induced in the early stages of cell differentiation.ConclusionsWe suggest that 1,25-dihydroxyvitamin D3 positively affects cell differentiation and matrix mineralization. Therefore, it may function as a stimulating factor in osteoblastic bone formation and can be used as an additive in bone regeneration treatment.

Conductive nanofibrous composite scaffolds based on in-situ formed polyaniline nanoparticle and polylactide for bone regeneration

Conducting polymers and biodegradable polylactide (PLA) scaffolds are both promising biomaterials applied in bone tissue engineering. It is necessary to develop a composite scaffold combining their properties of osteogenic differentiation promotion and three-dimension matrix. To conquer the problem of poor processability of conductive polymers, we use a novel in-situ polymerization/thermal induced phase separation (TIPS) method to fabricate conductive nanofibrous PLA scaffolds with well-distributed polyaniline (PANI) nano-structures. The simple preparation technique provides the possibility to scale-up production of these conductive nanofibrous composite scaffolds. The scaffold structure and content of in-situ formed polyaniline nanoparticles was thoroughly characterized with 1H NMR, FT-IR, XPS, TGA, SEM and UV–vis, and the conductivity/electrochemical properties of the composite scaffolds were controlled with varied feed ratios of aniline to PLA. Meanwhile, the good cytocompatibility of these composite scaffolds was evaluated by culturing bone marrow derived mesenchymal stem cells (BMSCs) on them. The effect of conductive nanofibrous scaffolds on osteogenic differentiation was studied with expression levels of alkaline phosphatase (Alp), osteocalcin (Ocn) and runt-related transcription factor 2 (Runx2) during the culture of BMSCs for three weeks. The calcium mineralization of BMSCs is determined by alizarin red staining. These results indicated that a moderate content of PANI in the conductive nanofibrous scaffolds significantly promoted osteogenic differentiation of BMSCs for engineering bone tissues.

The differential effect of basic fibroblast growth factor and stromal cell‑derived factor‑1 pretreatment on bone morrow mesenchymal stem cells osteogenic differentiation potency.

In situ tissue engineering has become a novel strategy to repair periodontal/bone tissue defects. The choice of cytokines that promote the recruitment and proliferation, and potentiate and maintain the osteogenic differentiation ability of mesenchymal stem cells (MSCs) is the key point in this technique. Stromal cell-derived factor-1 (SDF-1) and basic fibroblast growth factor (bFGF) have the ability to promote the recruitment, and proliferation of MSCs; however, the differential effect of SDF-1 and bFGF pretreatment on MSC osteogenic differentiation potency remains to be explored. The present study comparatively observed osteogenic differentiation of bone morrow MSCs (BMMSCs) pretreated by bFGF or SDF-1 in vitro. The gene and protein expression levels of alkaline phosphatase (ALP), runt related transcription factor 2 (Runx-2) and bone sialoprotein (BSP) were detected using reverse transcription-quantitative polymerase chain reaction and western blotting. The results showed that the expression of ALP mRNA on day 3, and BSP and Runx-2 mRNA on day 7 in the bFGF pretreatment group was significantly higher than those in SDF-1 pretreatment group. Expression levels of Runx-2 mRNA, and ALP and Runx-2 protein on day 3 in the SDF-1 pretreatment group were higher than those in the bFGF pretreatment group. However, there was no significant difference in osteogenic differentiation ability on day 14 and 28 between the bFGF- or SDF-1-pretreatment groups and the control. In conclusion, bFGF and SDF-1 pretreatment inhibits osteogenic differentiation of BMMSCs at the early stage, promotes it in the medium phase, and maintains it in the later stage during osteogenic induction, particularly at the mRNA level. Out of the two cytokines, bFGF appeared to have a greater effect on osteogenic differentiation.