Alkaline Phosphatase Gene Expression Research Articles

Biomimetic hydroxyapatite/gelatin composites for bone tissue regeneration: Fabrication, characterization, and osteogenic differentiation in vitro

Herein, we prepared a series of novel hydroxyapatite (HA) nanowire and gelatin composite films with varying HA to gelatin ratios, attempting to simulate the major features of the natural extracellular matrix of bone. The results showed that the ratio of HA nanowires to gelatin had a great influence on the physicochemical and biological properties of the composites. The morphology of the HA/gelatin 7/3 composite film had a fibrous and porous architecture, while the 5/5 and 3/7 samples led to the formation of non-porous rough surfaces. Additionally, an increase in HA content enhanced the composite mechanical properties, with a maximum tensile strength of 114 ± 8 MPa, respectively. The HA/gelatin composite films exhibited good cell adhesion and proliferation. Osteogenic differentiation of mouse bone marrow mesenchymal stem cells on the 7/3 composite film was superior to that of the remaining films, as evident by alkaline phosphatase activity, calcium deposition, and gene expression. Thus, the HA/gelatin 7/3 composite film prepared herein has potential as a bone substitute for bone repair and regeneration.

Tailoring weight ratio of PCL/PLA in electrospun three-dimensional nanofibrous scaffolds and the effect on osteogenic differentiation of stem cells.

Three-dimensional (3D) scaffolds as artificial ECMs have been extensively studied to mimic the critical features of natural ECMs. To develop more clinically relevant 3D scaffolds, electrospun nanofibrous scaffolds with different weight ratios of PCL/PLA (i.e., 100/0, 60/40, and 20/80) were fabricated via the thermally induced (nanofiber) self-agglomeration (TISA) method. The hypothesis was that, with the weight ratio increase of stiffer and more bioactive PLA in the 3D PCL/PLA blend scaffolds, the osteogenic differentiation of human mesenchymal stem cells (hMSCs) would be enhanced. The results indicated that, all of the 3D scaffolds were elastic/resilient and possessed interconnected and hierarchical pores with sizes from sub-microns to ∼300 μm; therefore, the morphological structures of these scaffolds were similar to those of natural ECMs. The PLA80 scaffolds exhibited the best overall properties in terms of density, porosity, water absorption capacity, mechanical properties, bioactivity, and cell viability. Furthermore, with increasing the PLA weight ratio, the alkaline phosphatase (ALP) activity, calcium content, and gene expression level were also increased, probably due to the improved stiffness/bioactivity of scaffold. Hence, the novel 3D electrospun PLA80 nanofibrous scaffold might be desired/favorable for the osteogenic differentiation of hMSCs.

Iliac and mandible osteoblasts exhibit varied responses to LMHF vibration.

The facial and long bones have distinct developmental origins, structures, and cellular compositions. This study aimed to compare the in vitro responses of human mandible and long bone osteoblasts to low-magnitude, high-frequency (LMHF) mechanical vibration in terms of expression of mediators of bone remodeling. Osteoblast-like cell cultures were prepared from iliac crest and mandibular bone specimens from three individuals and cultured in osteogenic induction media. Induction of mature osteoblastic phenotypes was confirmed by analysis of DNA content, alkaline phosphatase activity and gene expression every 3 days for 27 days. Based on gene expression, mature osteoblasts formed by day 15 of induction culture. After 15 days of culture in induction media, mature osteoblasts were subjected to vibration (0, 30, or 60 Hz) for 30 min every 24 h. After 48 h, RANKL, OPG, IL-1β, IL-6 and TGF-β gene, and protein expression were determined by real-time PCR analysis of total cellular mRNA and ELISAs of the cell supernatants. Both iliac and mandible osteoblasts responded to LMHF vibration: IL-1β and RANKL mRNA were downregulated and IL-6 mRNA was upregulated. However, TGF- β mRNA was unaltered and OPG mRNA was upregulated in iliac osteoblasts, whereas both TGF-β and OPG mRNA were downregulated in mandible osteoblasts. As a result, LMHF reduced the RANKL/OPG mRNA ratio in iliac osteoblasts but did not alter the RANKL/OPG mRNA ratio in mandible osteoblasts. This study suggests mature iliac osteoblasts exhibit a more potent anti-resorptive response to vibration, while this tendency was not obviously apparent in mature mandible osteoblasts.

Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells.

Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids. The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation. Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.

Relation of placental alkaline phosphatase expression in human term placenta with maternal and offspring fat mass.

Alkaline phosphatase is implicated in intestinal lipid transport and in the development of obesity. Placental alkaline phosphatase is localised to the microvillous plasma membrane of the placental syncytiotrophoblast at the maternal-fetal interface, but its role is unclear. We investigated the relations of placental alkaline phosphatase activity and mRNA expression with maternal body composition and offspring fat mass in humans. Term human placentas from the UK Birthright cohort (n = 52) and the Southampton Women's Survey (SWS) (n = 95) were studied. In the Birthright cohort, alkaline phosphatase activity was measured in placental microvillous plasma membrane vesicles. In the SWS, alkaline phosphatase mRNA was measured using Nanostring. Alkaline phosphatase gene expression was compared to other lipid-related genes. In Birthright samples placental microvillous plasma membrane alkaline phosphatase activity was positively associated with maternal triceps skinfold thickness and BMI (β = 0.04 (95% CI: 0.01-0.06) and β = 0.02 (0.00-0.03) µmol/mg protein/min per SD, P = 0.002 and P = 0.05, respectively) after adjusting for potential confounders. In SWS samples placental alkaline phosphatase mRNA expression in term placenta was positively associated with maternal triceps skinfold (β = 0.24 (0.04, 0.44) SD/SD, P = 0.02), had no association with neonatal %fat mass (β = 0.01 (-0.20 to 0.21) SD/SD, P = 0.93) and was negatively correlated with %fat mass at ages 4 (β = -0.28 (-0.52 to -0.04) SD/SD, P = 0.02), 6-7 (β = -0.25 (-0.49 to -0.02) SD/SD, P = 0.03) years. When compared with placental expression of other genes, alkaline phosphatase expression was positively related to genes including the lysophosphatidylcholine transporter MFSD2A (major facilitator superfamily domain containing 2A, P < 0.001) and negatively related to genes including the fatty acid transport proteins 2 and 3 (P = 0.001, P < 0.001). Our findings suggest relationships between placental alkaline phosphatase and both maternal and childhood adiposity. The inverse relationship between placental alkaline phosphatase gene expression and childhood %fat mass suggests that placental alkaline phosphatase may help to protect the foetus from the adverse effects of maternal obesity.

Plasma ion implantation enabled bio-functionalization of PEEK improves osteoblastic activity.

Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and tropoelastin-coated triggered the most favorable osteoblast-like responses. Surface treatment or tropoelastin coating did not alter alkaline phosphatase gene expression and activity of bound cells but did influence the expression of other bone markers including osteocalcin, osteonectin, and collagen I. We conclude that the surface modification of PEEK improves osteoblast interactions, particularly with respect to bone apposition, and enhances the orthopedic utility of PEEK.

In vitro and in vivo biological performance of porous Ti alloys prepared by powder metallurgy.

Titanium (Ti) and Ti-6 Aluminium-4 Vanadium alloys are the most common materials in implants composition but β type alloys are promising biomaterials because they present better mechanical properties. Besides the composition of biomaterial, many factors influence the performance of the biomaterial. For example, porous surface may modify the functional cellular response and accelerate osseointegration. This paper presents in vitro and in vivo evaluations of powder metallurgy-processed porous samples composed by different titanium alloys and pure Ti, aiming to show their potential for biomedical applications. The porous surfaces samples were produced with different designs to in vitro and in vivo tests. Samples were characterized with scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and elastic modulus analyses. Osteogenic cells from newborn rat calvaria were plated on discs of different materials: G1—commercially pure Ti group (CpTi); G2—Ti-6Al-4V alloy; G3—Ti-13 Niobium-13 Zirconium alloy; G4—Ti-35 Niobium alloy; G5—Ti-35 Niobium-7 Zirconium-5 Tantalum alloy. Cell adhesion and viability, total protein content, alkaline phosphatase activity, mineralization nodules and gene expression (alkaline phosphatase, Runx-2, osteocalcin and osteopontin) were assessed. After 2 and 4 weeks of implantation in rabbit tibia, bone ingrowth was analyzed using micro-computed tomography (μCT). EDS analysis confirmed the material production of each group. Metallographic and SEM analysis revealed interconnected pores, with mean pore size of 99,5μm and mean porosity of 42%, without significant difference among the groups (p>0.05). The elastic modulus values did not exhibit difference among the groups (p>0.05). Experimental alloys demonstrated better results than CpTi and Ti-6Al-4V, in gene expression and cytokines analysis, especially in early experimental periods. In conclusion, our data suggests that the experimental alloys can be used for biomedical application since they contributed to excellent cellular behavior and osseointegration besides presenting lower elastic modulus.

Indocyanine green-mediated photobiomodulation on human osteoblast cells.

Photobiomodulation (PBM) and photodynamic therapy (PDT) share similar mechanisms but have opposite aims. Increased levels of reactive oxygen species (ROS) in the target tissue in response to light combined photosensitizer (PS) application may lead to cell proliferation or oxidative damage depending on the ROS amount. The purpose of the present study is to investigate the effect of indocyanine green (ICG)-mediated PBM on osteoblast cells by measuring cell viability, proliferation, alkaline phosphatase (ALP) activity, mineralization, and gene expressions of three phenotypic osteoblast markers. A diode laser irradiating at 809nm (10W output power, 50mW/cm2 power density) was used at 0.5, 1, and 2J/cm2 energy densities (10, 20, and 40s respectively) was applied following ICG incubation. No inhibitory effect was observed in cell viability and proliferation according to the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Alamar Blue assays. ICG-mediated PBM did not alter cell viability but increased ALP activity and enhanced mineralization of existing osteoblasts. These results were also confirmed by real-time polymerase chain reaction (RT-PCR) analysis of osteoblastic markers. PS can be combined to PBM not only to damage the malignant cells as aimed in PDT studies, but also to promote cellular activity. The findings of this in vitro study may contribute to in vivo studies and ICG-mediated PBM can have promising outcomes in bone healing and regeneration therapies in future.

Inhibitory Effects of Human Primary Intervertebral Disc Cells on Human Primary Osteoblasts in a Co-Culture System.

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.

Effect of Brain-Derived Neurotrophic Factor on the Neurogenesis and Osteogenesis in Bone Engineering.

During bone growth, the lack of a neuralized vascular network in the regenerating area can affect subsequent bone quality. This study aimed to investigate if brain-derived neurotrophic factor (BDNF) could promote neurogenesis and osteogenesis in human bone mesenchymal stem cells (hBMSCs) to improve bone formation during tissue engineering. Initially, a safe and effective BDNF concentration that facilitated hBMSC proliferation in vitro was determined. Subsequently, examination of mineralized nodule formation and evaluation of alkaline phosphatase (ALP) activity and ALP gene expression revealed that the most effective concentration of BDNF to elicit a response in hBMSCs was 100 ng/mL. In addition, we found out that by binding with TrkB receptor, the downstream Erk1/2 was phosphorylated, which promoted the expression of transcription factors, such as Runx2 and Osterix that are associated with osteoblast differentiation. We also found that by day 7 post-treatment, the neurogenic biomarkers, p75 and s100, were highly expressed in 100 ng/mL BDNF-treated hBMSCs. Finally, the effects of BDNF on osteogenesis and neurogenesis in newly formed tissues were assessed using animal models with a β-tricalcium phosphate scaffold. This revealed that treatment with 100 ng/mL BDNF promoted the osteogenesis and neurogenesis of hBMSCs in vivo by increasing expression of the osteogenic marker osteocalcin and various neurogenic biomarkers, including microtubule-associated protein 2, glial fibrillary acidic protein, neural/glial antigen 2, and β-tubulin III. This study has demonstrated that BDNF promotes hBMSC osteogenesis and neurogenesis in vitro and in vivo, and that BDNF may indirectly promote osteogenesis through increased neurogenesis. This further suggests that encouraging neutralization during bone engineering will lead to effective repairing of bone defects. The study may also provide insight into related fields, such as osseoperception and stress feedback regulation after dental implantation.

Cytocompatibility and Bone-Formation Potential of Se-Coated 316L Stainless Steel with Nano-Pit Arrays.

316L stainless steel is still widely applied in joint replacement and orthopedic surgery due to its mechanical properties, corrosion resistance and relatively low price. In this study, electrochemical oxidation and nanoscale coating were used to fabricate Se-coated 316L stainless steel with nano-pit arrays to enhance its surface characteristics, biocompatibility and osseointegration ability. The modified 316L stainless steel was tested via field emission scanning microscopy (FESEM), energy-dispersive X-ray spectrometry (EDS), X-ray photoelectron spectroscopy (XPS) and Se release studies. The results of this study showed that the nano-pit arrays were 50 nm in diameter and that the Se coating consisted primarily of elemental Se and exhibited sustained release. The biological response of the samples was evaluated using in vitro rat bone marrow mesenchymal stem cell (rBMSCs) experiments and in vivo animal experiments. The modified 316L stainless steel displays enhanced abilities of cell adhesion, proliferation and osteogenic activity, as shown by FESEM, CCK-8 assay, immunofluorescence microscopy (IF) and alkaline phosphatase (ALP) activity assay in vitro and additional new bone formation in vivo, indicating its outstanding cytocompatibility and osteogenic differentiation ability. More importantly, the Se coating can upregulate gene expression of OPN, RUNX-2 and ALP, indicating that the nano-Se-coated 316L stainless steel with nano-pit arrays is a promising biomedical material for implants in orthopedic or dental clinic applications.

A comparison study on the behavior of human endometrial stem cell-derived osteoblast cells on PLGA/HA nanocomposite scaffolds fabricated by electrospinning and freeze-drying methods

BackgroundAn engineered tissue structure is an artificial scaffold combined with cells and signaling factors. Among various polymers, the polylactide-co-glycolide/hydroxyapatite (PLGA/HA) has attracted much attention due to their optimal properties. The aim of this study was to study the behavior of human endometrial stem cell (hEnSC)-derived osteoblast cells cultured on PLGA/HA nanocomposite scaffolds.MethodshEnSCs were isolated and exposed to osteogenic media for 21 days. Differentiated cells were cultured on PLGA/HA synthetic scaffolds. The PLGA/HA-based nanocomposite scaffolds were fabricated using either electrospinning or freeze-drying methods. Behavior of the cells was evaluated a week after seeding hEnSC-derived osteoblast-like cells on these scaffolds. Osteogenesis was investigated in terms of alkaline phosphatase activity, gene expression, immunocytochemistry (ICC), proliferation, and scanning electron microscopy (SEM). Moreover, scaffold properties, such as pore size and morphology of the cells, onto the scaffolds were evaluated using SEM. Furthermore, biocompatibility of these scaffolds was confirmed by 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.ResultsThe matrix mineralization was proved by alizarin red staining, and the osteogenic media-treated cultures positively expressed osteocalcin and osteopontin markers. Moreover, qRT-PCR results confirmed the positive gene expression of osteopontin and osteonectin in the differentiated osteoblast-like cells. The results of behavior assessment of the cultured cells on electrospinning and freeze-dried scaffolds showed that the behavior of the cultured cells on the freeze-dried PLGA/HA scaffolds was significantly better than the electrospinning PLGA/HA scaffolds.ConclusionIt has been shown that the freeze-dried PLGA/HA nanocomposite scaffolds can appropriately support the attachment and proliferation of the differentiated osteoblast cells and are a suitable candidate for bone tissue engineering.

Synergism of Electrospun Nanofibers and Pulsed Electromagnetic Field on Osteogenic Differentiation of Induced Pluripotent Stem Cells.

According to the current therapies failure for bone fractures and lesions, tissue engineering showed a great potential to help solve these challenges. Because the use of growth factors is very limited in the clinic, it could be very useful that could be introducing an alternative to it. Extremely low frequency pulsed electromagnetic fields (PEMF, 1 mT, 50 Hz) were used for achieving this aim. The PEMF potential in combination with electrospun polycaprolactone (PCL) nanofibers was used to investigate the osteogenic potential of human induced pluripotent stem cells (iPSCs). Several relevant osteogenic markers, such as Alizarin red staining, alkaline phosphatase activity, calcium content, gene expression, and immunocytochemistry, were used to evaluate osteoinductivity of PEMF. Results were shown that PEMF alone can induce osteogenic differentiation, but this capability increased when used in combination with PCL nanofibers significantly. In addition, simultaneous use of osteogenic medium, PEMF and PCL surprisingly increased osteogenic differentiation potential of iPSCs. According to the results, PEMF alone, iPSCs-seeded PCL, and both of them could be considered as a promising candidate for use in bone tissue engineering applications.

Effect of recombinant vascular endothelial growth factor and translationally controlled tumor protein on 2‑hydroxyethyl methacrylate‑treated pulp cells.

Vascular endothelial growth factor (VEGF)-A is a potential signaling protein that may promote angiogenesis. VEGF also helps cells survive in stressfull or hazardous conditions. The present study aimed to compare the effect of VEGF with translationally controlled tumor protein (TCTP), an anti‑apoptotic protein in human dental pulp cells (HDPCs), following exposure to 2‑hydroxyethyl methacrylate (HEMA), which is a major residual monomer from resin restorative dental materials. Cell viability, alkaline phosphatase (ALP) activity, mineralization and gene expressions for odontogenic and osteogenic differentiation markers of HDPCs were investigated, following exposure to HEMA and in combination with TCTP and VEGF. The results revealed that TCTP at 1ng/ml and VEGF at 10ng/ml significantly promoted the proliferation of HDPCs (P<0.05). TCTP (1ng/ml) and VEGF (10ng/ml) maintained the cell viability of 4mM HEMA‑treated cells at the same percentage as the control. However, cells treated with HEMA+TCTP+VEGF had a lower cell viability than the groups treated with HEMA and TCTP or VEGF alone. TCTP and VEGF promoted cell proliferation, ALP activity and mineralization, and upregulated of DSPP, DMP‑1, BMP‑2, and ALP mRNA expression compared with the control. Furthermore, the HEMA+TCTP and HEMA+VEGF groups had significantly higher percentages of calcium deposition than HEMA‑treated cells (P<0.001). HEMA was cytotoxic to HDPCs, reduced ALP activity and caused the significant downregulation of odontogenic and osteogenic gene expressions (P<0.05). It was concluded that VEGF and TCTP promoted pulp cell growth and the survival of HEMA‑treated cells without synergistic effects. TCTP was required in lower concentrations for these effects. VEGF and TCTP enhanced cell differentiation and mineralization.

Downregulation of Heat Shock Protein 70 Impairs Osteogenic and Chondrogenic Differentiation in Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) show promise for bone and cartilage regeneration. Our previous studies demonstrated that hMSCs with periodic mild heating had enhanced osteogenic and chondrogenic differentiation with significantly upregulated heat shock protein 70 (HSP70). However, the role of HSP70 in adult tissue regeneration is not well studied. Here, we revealed an essential regulatory mechanism of HSP70 in osteogenesis and chondrogenesis using adult hMSCs stably transfected with specific shRNAs to knockdown HSP70. Periodic heating at 39 °C was applied to hMSCs for up to 26 days. HSP70 knockdown resulted in significant reductions of alkaline phosphatase activity, calcium deposition, and gene expression of Runx2 and Osterix during osteogenesis. In addition, knockdown of HSP70 led to significant decreases of collagens II and X during chondrogenesis. Thus, downregulation of HSP70 impaired hMSC osteogenic and chondrogenic differentiation as well as the enhancement of these processes by thermal treatment. Taken together, these findings suggest a putative mechanism of thermal-enhanced bone and cartilage formation and underscore the importance of HSP70 in adult bone and cartilage differentiation.

Effect of calcitonin gene-related peptide on proliferation and osteogenic differentiation of bone mesenchymal stem cells in osteoporotic rats

Objective To research the effects of calcitonin gene-related peptide (CGRP) on the proliferation and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) of osteoporotic rats in vitro. Methods Three-month-old SD rats were used, taking bilateral ovariectomy through the abdomen. Micro-computed tomography (Micro-CT) analysis was used to determine the success of modeling at 6 months after surgery. BMSCs were isolated from osteoporotic rats by blood adherence method. The biological characteristics of BMSCs were examined by flow cytometry. Different concentrations of CGRP (10-7-10-10 mol/L) were added to the culture medium of third generation BMSCs for examination of proliferation according CCK-8 method, alkaline phosphatase (ALP) activity and alizarin red staining were used to evaluate the ability of cells differentiation and mineralization. And reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of ALP, Collagen I, BMP-2, Osteonectin and RunX2 gene. Western blotting was used to detect the expression of RunX2 and Osteonectin protein. Results The bone marrow stromal cells of osteoporotic rats were isolated successfully. The optical density (A) values of CCK-8 method increased in a dose-dependent manner, at 7 days, the control group of A value was 0.580 4±0.015 0, the CGRP group 10-7 mol/L was to 0.804 1±0.029 9(P=0.000), 10-8 mol/L to 0.788 6±0.028 0(P=0.000), 10-9 mol/L to 0.743 6±0.028 8 (P=0.001) and 10-10 mol/L to 0.693 3±0.029 2(P=0.004), difference was significant; The ALP activity was positive, at 14 days, the control group of A value was 4.633±0.423, the CGRP group 10-7 mol/L was to 44.047±2.446 (P=0.000), 10-8 mol/L to 42.030±2.626 (P=0.000), 10-9 mol/L to 39.333±0.527 (P=0.000), 10-10 mol/L to 24.647±1.135 (P=0.000) [unit: 10-3 μmol/(min·mg)}, difference was significant (P<0.05); The formation of calcium nodules were proved by alizarin red staining; The target genes expression were all increased, at 7 days, control group of ALP was to 0.999 5±0.205 0, 10-7 mol/L to 2.578 4±0.210 1(P=0.001), 10-10 mol/L to 2.351 2±0.201 2(P=0.001); The control group of BMP-2 was to 0.979 5±0.200 1, 10-7 mol/L to 3.427 9±0.255 2(P=0.000), 10-10 mol/L to 2.914 9±0.202 3(P=0.000); The control group of COLL-I was to 1.029 5±0.202 2, 10-7 mol/L to 2.518 7±0.240 0(P=0.001), 10-10 mol/L to 2.219 8±0.250 3; The control group of RunX2 was to 0.993 2±0.213 2, 10-7 mol/L to 2.498 9±0.262 0(P=0.002), 10-10 mol/L to 2.219 8±0.250 3(P=0.003); The control group of osteonectin was to 1.019 5±0.200 1, 10-7 mol/L to 1.972 8±0.212 3(P=0.005), 10-10 mol/L to 1.864 2±0.250 1 (P=0.010), difference was significant. The proteins expression of RunX2 and osteonectin were all increased. Conclusion Appropriate concentration (10-7 -10-10 mol/L) of CGRP can directly stimulate the proliferation, differentiation and mineralization of the BMSCs of osteoporotic rats in vitro, and CGRP may play an important role in the treatment of osteoporotic bone fracture repair. Key words: Calcitonin gene-related peptide; Osteoporosis; Bone mesenchymal stem cells; Osteogenic differentiation

Effect of &lt;i&gt;Eucommia ulmoides&lt;/i&gt; extract on osteoblast proliferation

Purpose: To evaluate the effect of Eucommia ulmoides extract (EUE) on osteoblast proliferation as well as investigate its probable mechanisms of action. Methods: EUE was pharmacologically evaluated at three doses. Osteoblast cells were divided as follows: Group I: negative control; groups II–IV: received EUE (180, 360 and 540 μg/ml, respectively). We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay to determine osteoblast viability following treatment. Alkaline phosphatase (ALP), osteocalcin, and collagen I levels in osteoblasts were quantified using commercially available kits. Thereafter, mRNA and protein expression of ALP, collagen I, osteocalcin, transforming growth factor-β1 (TGF-β1) were measured using real-time quantitative PCR (qPCR) and western blot, respectively. Results : EUE significantly (p < 0.01) promoted osteoblast proliferation at three treatment doses (180, 360, and 540 μg/mL). Furthermore, ALP, osteocalcin, collagen I and TGF-β1 expression at both mRNA and protein levels increased significantly (all p < 0.05) following EUE treatment. Conclusion : The results suggest that EUE may promote osteoblast cell proliferation and that ALP, osteocalcin, collagen I and TGF-β1 gene expression may be involved in the mechanism of action. Keywords: qPCR, collagen I, Bone, Liver, Eucommia ulmoides extract

Ipriflavone promotes proliferation and osteogenic differentiation of periodontal ligament cells by activating GPR30/PI3K/AKT signaling pathway.

ObjectivesThis study was performed to investigate the effects and mechanism of ipriflavone (IP) on the proliferation and osteoblastic differentiation of periodontal ligament cells in vitro and periodontal tissue remodeling following orthodontic tooth movement (OTM) in vivo.Materials and methodsHuman periodontal ligament cells (hPDLCs) were cultured in vitro and cell counting kit-8, alkaline phosphatase (ALP) activity assay, plate clone formation assay, and alizarin red staining were used to test proliferation and osteogenic differentiation of hPDLCs. What is more, the expression of ALP, Runx2, and GPR30 was examined by real-time polymerase chain reaction and Western blot. To find out if PI3K/AKT signaling pathway was involved in the process, AKT and p-AKT were examined by Western blot. LY294002 (PI3K signaling pathway inhibitor) and small interfering RNA targeting GPR30 mRNA (siGPR30) were used to verify the function of GPR30-mediated PI3K/AKT pathway in this process. Twenty-four male Wistar rats were randomized into 2 groups, the control group with force application and the IP group with force application plus IP. Morphological changes in the periodontal tissue between roots of teeth were investigated using hematoxylin and eosin (HE) staining and bone morphogenetic protein-2 was detected to assess bone remodeling by immunohistochemical staining.ResultsIn vitro, 10−7 M IP was selected significantly promoting proliferation, ALP activity, colony forming efficiency, and mineral deposition (P<0.05) on hPDLCs. Gene expressions of ALP, Runx2, GPR30, and p-AKT were all upregulated than the control group (P<0.05). According to the mechanism, promotion of ALP and Runx2 interdicted by LY294002 and siGPR30 reduced the activation of PI3K/AKT signaling pathway. In addition, HE staining and immunohistochemical staining results showed that the IP group had more new bone formation in the periodontal tissue compared to the control group in vivo.ConclusionIP can promote the expression of ALP and Runx2 which was probably related to the GPR30-mediated PI3K/AKT signaling pathway. Moreover, IP coordination seemed to have the potential to prevent relapsing following OTM.

Anti-osteoporotic activities of the collagen extracted from Carapacis et Plastri Testudinis on a ROS1728 cell line in vitro

Purpose: The decoction of collagen extracted from Carapacis et Plastri Testudinis (CCPT) has been reported to have an effect of anti-osteoporosis. The present study was to investigate the in vitro molecular mechanisms effect of CCPT on ROS1728 differentiation and proliferation. Methods: Rat osteoblast cell line (ROS 1728) was cultured and exposed to CCPT at different concentrations. The effect of CCPT on the proliferation of ROS1728 was evaluated through detecting the activity of Alkaline Phosphatase (ALP) and the type I collagen (Coll-I) content. The gene expression of ALP, Coll-I, Osteocalcin (OC), Runx2, Osterix (Osx), Osteoprotegerin (OPG) and RANKL (receptor activator of NF-MB ligand) was observed using RT-PCR analysis. Results: The results showed that CCPT treatment (2.5, 5.0 and 10.0 mg/ml) affected the proliferation of ROS1728 in a concentration-dependent manner. The highest expression was obtained by treatment with 5 mg/ml of CCPT after a nine-day treatment duration based on ALP activity and Coll-I content (P<0.01) with the control group, whereas, the expression of ALP, Coll-I, OC, Runx2, Osx and OPG mRNA was facilitated (P<0.01). Only the level of RANKL mRNA decreased compared with the control group (P<0.01). Conclusion: Use of CCPT can induce ROS1728 proliferation into osteoblasts and enhance the function of osteogenesis through regulating the gene expression of Runx2 and the osteogenesis-specific transcription factors of Osx, and inhibit bone absorption by OPG/RANKL/RANK signaling pathways.

Human β-defensin 3-combined gold nanoparticles for enhancement of osteogenic differentiation of human periodontal ligament cells in inflammatory microenvironments.

ObjectiveIt is a great challenge to absorb and conduct biophysicochemical interactions at the nano-bio interface. Peptides are emerging as versatile materials whose function can be programmed to perform specific tasks. Peptides combined nanoparticles might be utilized as a new approach of treatment. Human β-defensin 3 (hBD3), possesses both antimicrobial and proregeneration properties. Gold nanoparticles (AuNPs) have shown promising applications in the field of tissue engineering. However, the coordinating effects of AuNPs and hBD3 on human periodontal ligament cells (hPDLCs) remain unknown. In this study, we systematically investigated whether AuNPs and hBD3 would be able to coordinate and enhance the osteogenic differentiation of hPDLCs in inflammatory microenvironments, and the underlying mechanisms was explored.MethodshPDLCs were stimulated with E. coli-LPS, hBD3 and AuNPs. Alkaline phosphatase (ALP) and alizarin red S staining were used to observe the effects of hBD3 and AuNPs on the osteogenic differentiation of hPDLCs. Real-time PCR and western blot were performed to evaluate the osteogenic differentiation and Wnt/β-catenin signaling pathway related gene and protein expression.ResultsIn the inflammatory microenvironments stimulated by E. coli-LPS, we found that AuNPs and hBD3 increased the proliferation of hPDLCs slightly. In addition, hBD3-combined AuNPs could significantly enhance ALP activities and mineral deposition in vitro. Meanwhile, we observed that the osteogenic differentiation-related gene and protein expressions of ALP, collagenase-I (COL-1) and runt-related transcription factor 2 (Runx-2) were remarkably upregulated in the presence of hBD3 and AuNPs. Moreover, hBD3-combined AuNPs strongly activated the Wnt/β-catenin signaling pathway and upregulated the gene and protein expression of β-catenin and cyclin D1. Furthermore, hBD3-combined AuNPs induced osteogenesis, which could be reversed by the Wnt/β-catenin signaling pathway inhibitor (ICG-001).ConclusionThe present study demonstrated that hBD3 combined AuNPs could significantly promote the osteogenic differentiation of hPDLCs in inflammatory microenvironments via activating the Wnt/β-catenin signaling pathway.