Expression Of Osteogenesis Markers Research Articles

Low-intensity pulsed ultrasound reduces alveolar bone resorption during orthodontic treatment via Lamin A/C-Yes-associated protein axis in stem cells.

The bone remodeling during orthodontic treatment for malocclusion often requires a long duration of around two to three years, which also may lead to some complications such as alveolar bone resorption or tooth root resorption. Low-intensity pulsed ultrasound (LIPUS), a noninvasive physical therapy, has been shown to promote bone fracture healing. It is also reported that LIPUS could reduce the duration of orthodontic treatment; however, how LIPUS regulates the bone metabolism during the orthodontic treatment process is still unclear. To investigate the effects of LIPUS on bone remodeling in an orthodontic tooth movement (OTM) model and explore the underlying mechanisms. A rat model of OTM was established, and alveolar bone remodeling and tooth movement rate were evaluated via micro-computed tomography and staining of tissue sections. In vitro, human bone marrow mesenchymal stem cells (hBMSCs) were isolated to detect their osteogenic differentiation potential under compression and LIPUS stimulation by quantitative reverse transcription-polymerase chain reaction, Western blot, alkaline phosphatase (ALP) staining, and Alizarin red staining. The expression of Yes-associated protein (YAP1), the actin cytoskeleton, and the Lamin A/C nucleoskeleton were detected with or without YAP1 small interfering RNA (siRNA) application via immunofluorescence. The force treatment inhibited the osteogenic differentiation potential of hBMSCs; moreover, the expression of osteogenesis markers, such as type 1 collagen (COL1), runt-related transcription factor 2, ALP, and osteocalcin (OCN), decreased. LIPUS could rescue the osteogenic differentiation of hBMSCs with increased expression of osteogenic marker inhibited by force. Mechanically, the expression of LaminA/C, F-actin, and YAP1 was downregulated after force treatment, which could be rescued by LIPUS. Moreover, the osteogenic differentiation of hBMSCs increased by LIPUS could be attenuated by YAP siRNA treatment. Consistently, LIPUS increased alveolar bone density and decreased vertical bone absorption in vivo. The decreased expression of COL1, OCN, and YAP1 on the compression side of the alveolar bone was partially rescued by LIPUS. LIPUS can accelerate tooth movement and reduce alveolar bone resorption by modulating the cytoskeleton-Lamin A/C-YAP axis, which may be a promising strategy to reduce the orthodontic treatment process.

Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway.

Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms. Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs. Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway. Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway.

The Construction of Foot and Ankle Joint by Autologous Oxygen-Releasing Nano-Bionic Scaffold Combined with Bone Marrow Mesenchymal Stem Cells

This study assesses the role of a scaffold constructed by co-cultivating autologous oxygen-releasing nano-bionic materials and bone marrow stromal cells (BMSCs) in joint repairing. A scaffold constructed of autologous oxygen-releasing nano-bionic materials and BMSCs was transplanted into SD rats. The immunofluorescence detected the expression of nuclear antigen (PCNA) and analyzed the proliferation of BMSCs. Reverse transcription polymerase chain reaction (RT-PCR) examined the expression of osteogenic markers and TUNEL staining analyzed BMSCs apoptosis. There was a significant difference in the apoptosis and proliferation cell number of BMSCs in ankle joint between solely autologous oxygen-releasing nano-bionic material scaffold group and composite BMSCs nano-bionic scaffold (P <0.05). There was a statistical difference in PCNA and TUNEL expression between two groups at 4 weeks and 8 weeks (P <0.05). The expression of osteogenesis markers in ankle joint at 1 week, 4 weeks, and 8 weeks were continuously reduced, and the composite of autologous oxygen-releasing nanomaterials and BMSCs increased the expression of osteogenic markers (P <0.05). The composite scaffold constructed by autologous oxygen-releasing nano-bionic materials and BMSCs has a good two-way immune regulation function and is able to carry lipids, proteins, nutritional factors, and growth factors, which can effectively promote tissue engineering repairing and delay the scaffold degradation. Combined with the nano-release system to repair bone tissue, composite material can effectively promote the proliferation of joint osteoblasts and osteogenic differentiation, thus help repairing the ankle joint.

Cellular Response to Bone Morphogenetic Proteins-2 and -7 Covalently Bound to Photocrosslinked Heparin-Diazoresin Multilayer.

Despite the plethora of research that exists on recombinant human bone morphogenetic protein-2 and -7 (rhBMP-2 and rhBMP-7) and has been clinically approved, there is still a need to gain information that would allow for their more rational use in bone implantology. The clinical application of supra-physiological dosages of these superactive molecules causes many serious adverse effects. At the cellular level, they play a role in osteogenesis and cellular adhesion, migration, and proliferation around the implant. Therefore, in this work, we investigated the role of the covalent binding of rhBMP-2 and rhBMP-7 separately and in combination with ultrathin multilayers composed of heparin and diazoresin in stem cells. In the first step, we optimized the protein deposition conditions via quartz crystal microbalance (QCM). Then, atomic force microscopy (AFM) and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein-substrate interactions. The effect of the protein binding on the initial cell adhesion, migration, and short-term expression of osteogenesis markers was tested. In the presence of both proteins, cell flattening and adhesion became more prominent, resulting in limited motility. However, the early osteogenic marker expression significantly increased compared to the single protein systems. The presence of single proteins resulted in the elongation of cells, which promoted their migration activity.

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein–substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.

Long Noncoding RNA IGFBP7-AS1 Promotes Odontogenesis of Stem Cells from Human Exfoliated Deciduous Teeth via the p38 MAPK Pathway.

Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. Epigenetics refers to heritable changes in gene expression patterns that do not alter DNA sequences. Long noncoding RNAs (lncRNAs) are one of the main methods of epigenetic regulation and participate in cell differentiation; however, little is known regarding the role of lncRNAs during SHED odontogenic differentiation. In this study, RNA sequencing (RNA-seq) was used to obtain the expression profile of lncRNAs and mRNAs during the odontogenic differentiation of SHED. The effect of IGFBP7-AS1 on odontogenic differentiation of SHED was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, quantitative reverse transcription PCR (qRT-PCR), Western blot, and in vivo. The level of p38 and p-p38 protein expression was examined by Western blot, and the result was verified by adding the p38 inhibitor, SB203580. The expression profiles of lncRNAs and mRNAs were identified by RNA-seq analysis, which help us to further understand the mechanism in odontogenesis epigenetically. IGFBP7-AS1 expression was increased during odontogenic differentiation on days 7 and 14. The ALP staining, ARS staining, and expression of odontogenic markers were upregulated by overexpressing IGFBP7-AS1 in vitro, whereas the expression of osteogenesis markers was not significantly changed on mRNA level. The effect of IGFBP7-AS1 was also verified in vivo. IGFBP7-AS1 could further positively regulate odontogenic differentiation through the p38 MAPK pathway. This may provide novel targets for dental tissue engineering.

Arctiin elevates osteogenic differentiation of MC3T3-E1 cells by modulating cyclin D1

ABSTRACT Osteoporosis is a systemic disorder of bone metabolism. This study aimed to investigate the impacts and possible mechanisms of Arctiin, a lignin isolated from Arctium lappa on MC3T3-E1 osteoblast differentiation. In this study, after treatment with different concentrations of Arctiin, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to estimate the expression of osteogenesis markers. Then, the activity of alkaline phosphatase (ALP) was detected by an ALP assay kit and calcium nodules staining was evaluated by alizarin red staining (ARS). Additionally, the regulatory effects of Arctiin on cyclin D1 (Ccnd1) was assessed by measurement of protein expression. Subsequently, the functions of Ccnd1 silencing on the osteogenic differentiation was examined in Arctiin-treated MC3T3-E1 cells. Results indicated that Arctiin dose-dependently upregulated the expression of runt-related transcription factor 2 (RUNX2), collagen type 1 (COL1A1), osteocalcin (OCN) and osteopontin (OPN). Elevated ALP activity and calcification degree was prominently observed in the Arctiin-treated groups. Moreover, Ccnd1 expression was notably enhanced after Arctiin intervention. Importantly, Ccnd1-knockdown abrogated the impacts of Arctiin on osteogenic differentiation of MC3T3-E1. To conclude, findings in this study suggested that Arctiin could regulate MC3T3-E1 osteoblast differentiation via up-regulating Ccnd1, supporting that Arctiin might be a therapeutic target for osteoporosis.

MicroRNA-34a and microRNA-146a target CELF3 and suppress the osteogenic differentiation of periodontal ligament stem cells under cyclic mechanical stretch

Background/purposeDuring orthodontic tooth movement, mechanical forces induce the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), which contributes to alveolar bone remodeling. MicroRNAs (miRNAs) are involved in regulating PDLSC osteogenic differentiation. Therefore, we intended to explore the role of miR-34a and miR-146a in osteogenic differentiation of PDLSCs under cyclic stretch. Materials and methodsPhenotypic identification of PDLSCs was determined by flow cytometry analysis. PDLSCs were incubated with osteogenic differentiation medium for 3 weeks and the osteogenic differentiation capability was detected by Alizarin Red staining. To mimic the orthodontic forces, cyclic mechanical stretch was applied to PDLSCs. Alkaline phosphatase (ALP) activity assay and ALP staining were used for evaluating the ALP activity. The expression of osteogenesis markers in PDLSCs was assessed by western blotting and qRT-PCR. The binding between miR-34a (or miR-146a) and CUGBP Elav-like family member 3 (CELF3) was validated by luciferase reporter assay. ResultsCyclic stretch elevated ALP activity and the expression of osteogenesis markers, osteopontin (OPN), runt-related transcription factor 2 (RUNX2), type I collagen (COL1), ALP, osteocalcin (OCN) and osterix (OSX), in PDLSCs. MiR-146a and miR-34a were downregulated in PDLSCs under cyclic stretch. Either overexpressing miR-146a and miR-34a reduced ALP activity and the expression of osteogenesis markers. CELF3 was a target of both miR-146a and miR-34a. CELF3 silencing attenuated while CELF3 overexpression enhanced ALP activity and the expression of osteogenesis markers. ConclusionMiR-34a and miR-146a repress cyclic stretch-induced osteogenic differentiation of PDLSCs via regulating the expression of CELF3.

Paracrine Effects of Recombinant Human Adiponectin Promote Bone Regeneration.

Bone regeneration is a delicate physiological process. Non-union and delayed fracture healing remains a great challenge in clinical practice nowadays. Bone and fat hold a close relationship to remain balanced through hormones and cytokines. Adiponectin is a well-known protein to maintain the hemostasis, which may be an interesting target for fracture healing. Herein, we provided a facile and efficient method to obtain high-purity and high-yield recombinant human adiponectin (ADPN). The biocompatibility and the pharmaceutical behaviors were evaluated in Sprague–Dawley rats. The paracrine effects of adiponectin on bone fracture healing were investigated with a rat tibia fracture model via intrabone injection. Significantly accelerated bone healing was observed in the medulla injection group, indicating the paracrine effects of adiponectin could be potentially utilized for clinical treatments. The underlying mechanism was primarily assessed, and the expression of osteogenic markers, including bone morphogenic protein 2, alkaline phosphatase, and osteocalcin, along with adiponectin receptor 1 (AdipoR1), was markedly increased at the fracture site. The increased bone healing of ADPN treatment may result from both enhanced osteogenic proliferation as well as differentiation. Cell experiments confirmed that the expression of osteogenesis markers increased significantly in ADPN treatment groups, while it decreased when the expression of AdipoR1 was knocked down by siRNA. Our study provided a feasible and efficacious way for bone fracture treatment with local administration of ADPN, which could be rapidly translated into the clinics.

Calcium silicate-based cements cause environmental stiffness and show diverse potential to induce osteogenesis in human osteoblastic cells

Calcium silicate-based cements differ markedly in their radiopacifiers and the presence of calcium sulfate, aluminates, carbonates and other components that can affect their biological properties. This study aimed to compare the biological properties of six calcium silicate cements in human osteoblastic cell culture (Saos-2 cells): Bio-C Repair (Bio-C), PBS HP (PBS-HP), Biodentine (Biodentine), MTA Repair HP (MTA-HP), NeoMTA Plus (NeoMTA-P), and ProRoot MTA (ProRoot). After exposure to these materials, the cells were analyzed by MTT, wound healing, cell migration, and alkaline phosphatase activity (ALP) assays, real-time PCR (qPCR) analysis of the osteogenesis markers (osteocalcin or bone gamma-carboxyglutamate protein, BGLAP; alkaline phosphatase, ALPL; bone sialoprotein or secreted phosphoprotein 1, BNSP), and alizarin red staining (ARS). Curiously, the migration rates were low 24–48 h after exposure to the materials, despite the cells showing ideal rates of viability. The advanced and intermediate cell differentiation markers BGLAP and BNSP were overexpressed in the Bio-C, MTA-HP, and ProRoot groups. Only the Biodentine group showed ALPL overexpression, a marker of initial differentiation. However, the enzymatic activity was high in all groups except Biodentine. The mineralization area was significantly large in the NeoMTA-P, ProRoot, PBS-HP, MTA-HP, and Bio-C groups. The results showed that cellular environmental stiffness, which impairs cell mobility and diverse patterns of osteogenesis marker expression, is a consequence of cement exposure. Environmental stiffness indicates chemical and physical stimuli in the microenvironment; for instance, the release of cement compounds contributes to calcium phosphate matrix formation with diverse stiffnesses, which could be essential or detrimental for the migration and differentiation of osteoblastic cells. Cells exposed to Bio-C, PBS-HP, ProRoot, NeoMTA-P, and MTA-HP seemed to enter the advanced or intermediate differentiation phases early, which is indicative of the diverse potential of cements to induce osteogenesis. Cements that quickly stimulate osteoblast differentiation may be ideal for reparative and regenerative purposes since they promptly lead to dentin or bone deposition.

Vasoactive Intestinal Peptide Promotes Fracture Healing in Sympathectomized Mice.

Vasoactive intestinal peptide (VIP) as a neuromodulator and neurotransmitter played a significant role in modulating bone homeostasis. Our previous study reported an essential role of VIP in in vitro BMSCs osteogenesis and in vivo bone defect repair. VIP was also revealed to have a promoting effect on embryonic skeletal element development. However, the role of VIP in fracture healing is not known yet. We hypothesized that the disorder of sympathetic nervous system impairs bone structure and fracture healing, whereas VIP may rescue the sympathetic inhibition effects and promote fracture healing. We employed a 6-hydroxydopamine(6-OHDA) induced sympathectomy mice model (sympathectomized mice), in which successful sympathetic inhibition was confirmed by a decreased level of norephedrine (NE) in the spleen. In the sympathectomized mice, the femoral micro-architecture, bone density and mechanical properties were all impaired compared to the vehicle control mice. The femoral fracture was created in the vehicle or sympathectomized mice. Vehicle mice were locally injected with PBS as a negative control, and the sympathectomized mice were treated with injection of PBS or VIP. VIP expression at the fracture site was significantly decreased in sympathectomized mice. The fracture healing was repressed upon 6-OHDA treatment and rescued by VIP treatment. Micro-CT examination showed that the femoral bone micro-architecture at the fracture sites and mechanical properties were all impaired. Simultaneously, the expression level of osteogenic markers OCN and OPN were reduced in sympathectomized mice compared with vehicle group. While the VIP treatment rescued the repression effects of 6-OHDA on bone remodeling and significantly promoted bone quality and mechanical properties as well as increased osteogenesis marker expression in the sympathectomized mice. VIP administration promoted bone fracture healing by inhibiting bone resorption, making it a putative new alternative treatment strategy for fracture healing.

Circular RNA circ_0000020 promotes osteogenic differentiation to reduce osteoporosis via sponging microRNA miR-142-5p to up-regulate Bone Morphogenetic Protein BMP2

ABSTRACT This study was designed to study functions of Circ_0000020 during osteogenic differentiation. First, we used RT-qPCR to detect the expression of Circ_0000020, miR-142-5p and osteogenesis-related genes, whereas western blot analysis detected the expression of osteogenesis markers after the osteogenic differentiation of primary BMSCs isolated from rats. Alkaline phosphatase (ALP) activity and alizarin red Sstaining validated osteoblast phenotypes. Flow cytometry was used to detect cell apoptosis. Sh-Circ_0000020 was used to study the function of Circ_0000020 in osteogenic differentiation of BMSCs. Luciferase assay and RNA immunoprecipitation were used to validate the interaction between Circ_0000020 and miR-142-5p, and BMP2 and miR-142-5p. Co-transfection of miR-142-5p and sh-Circ_0000020 was used to verify the downstream signaling pathway. Circ_0000020 expression was up-regulated during osteogenic differentiation, whereas miR-142-5p expression was significantly decreased. Silencing Circ_0000020 inhibited osteogenic differentiation and promoted apoptosis, and inhibited ALP activity and mineralization ability. Moreover, Circ_0000020 interacts directly with miR-142-5p which binds to the BMP2 3ʹUTR and inhibits its expression. Additionally, co-transfection of miR-142-5p inhibitors and sh-Circ_0000020 rescued down-regulated BMP2, increased the expression osteogenesis-related gene expressions, and thereby rescued the inhibition of osteogenic differentiation induced by Circ_0000020 silencing. Furthermore, co-transfection of miR-142-5p inhibitors and sh-Circ_0000020 reversed Circ_0000020 silencing-induced downregulation of p-Smad1/5/8, Runx2, and Osterix protein levels. Circ_0000020 regulates BMP2 expression through sponging miR-142-5p as ceRNA, thereby positively regulating BMSCs osteogenic differentiation through Circ_0000020/miR-142-5p/BMP2/SMAD-dependent signaling pathway.

Fluorine-incorporated TiO2 nanotopography enhances adhesion and differentiation through ERK/CREB pathway.

This study compared the topography of different titanium surface structures (TiO2 nanotube and grain) with similar elemental compositions (TiO2 and fluorine [F]) on the Ti surface. High magnification indicated that the surfaces of the control and etching groups were similar to each other in a flat, smooth form. The group anodized for 1 h was observed with TiO2 nanotubes organized very neatly and regularly. In the group anodized for 30 min after etching, uneven wave and nanopore structures were observed. In addition, MTT assay showed that the F of the surface did not adversely affect cell viability, and the initial cell adhesion was increased in the 2.8% F‐incorporated TiO2 nanograin. At the edge of adherent cells, filopodia were observed in spreading form on the surfaces of the anodizing and two‐step processing groups, and they were observed in a branch shape in the control and etching groups. Moreover, cell adhesion molecule and osteogenesis marker expression was increased at the F‐incorporated TiO2 nanostructure. In addition, it was found that the expression of p‐extracellular signal‐regulated kinase (ERK) and p‐cAMP response element‐binding protein (CREB) increased in the TiO2 nanograin with the nanopore surface compared to the micro rough and nanotube surfaces relative to the osteogenic‐related gene expression patterns. As a result, this study confirmed that the topographic structure of the surface is more affected by osteogenic differentiation than the pore size and that differentiation by specific surface composition components is by CREB. Thus, the synergy effect of osteogenic differentiation was confirmed by the simultaneous activation of CREB/ERK.

Endothelial progenitor cells promote osteogenic differentiation in co-cultured with mesenchymal stem cells via the MAPK-dependent pathway

BackgroundThe role of bone tissue engineering is to regenerate tissue using biomaterials and stem cell-based approaches. Combination of two or more cell types is one of the strategies to promote bone formation. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of mesenchymal stem cells (MSCs) and promote bone healing; this study aimed to investigate the possible mechanisms of EPCs on promoting osteogenic differentiation of MSCs.MethodsMSCs and EPCs were isolated and co-cultured in Transwell chambers, the effects of EPCs on the regulation of MSC biological properties were investigated. Real-time PCR array, and western blotting were performed to explore possible signaling pathways involved in osteogenesis. The expression of osteogenesis markers and calcium nodule formation was quantified by qRT-PCR, western blotting, and Alizarin Red staining.ResultsResults showed that MSCs exhibited greater alkaline phosphatase (ALP) activity and increased calcium mineral deposition significantly when co-cultured with EPCs. The mitogen-activated protein kinase (MAPK) signaling pathway was involved in this process. p38 gene expression and p38 protein phosphorylation levels showed significant upregulation in co-cultured MSCs. Silencing expression of p38 in co-cultured MSCs reduced osteogenic gene expression, protein synthesis, ALP activity, and calcium nodule formation.ConclusionsThese data suggest paracrine signaling from EPCs influences the biological function and promotes MSCs osteogenic differentiation. Activation of the p38MAPK pathway may be the key to enhancing MSCs osteogenic differentiation via indirect interactions with EPCs.

The synergistic effects of Xu Duan combined Sr-contained calcium silicate/poly-ε-caprolactone scaffolds for the promotion of osteogenesis marker expression and the induction of bone regeneration in osteoporosis

Osteoporosis and its related problems such as fractures are gradually becoming common due to an aging population. Current methods to treat osteoporosis include medical and surgical options such as bone implants. Recent developments in 3D printing and materials science technologies has allowed us to fabricate individualized scaffolds with desired properties. In this study, we mixed Xu Duan into strontium‑calcium silicate powder at 5% (XD5) and 10% (XD10) and fabricated 3D scaffolds with polycaprolactone. All scaffolds were assessed for its physical, mechanical, and biological properties to evaluated for its feasibility for bone tissue engineering in the osteoporosis model. Our results showed that such a scaffold could be fabricated using extrusion-based printing techniques and that addition of XD did not alter original structural properties of the SrCS. Furthermore, the XD5 and XD10 scaffolds were found to be non-toxic to cells and cells cultured on the scaffolds had significantly higher proliferation and secreted increased osteogenic-related proteins in in vitro studies as compared to the XD0 groups. Remarkably, the XD10 scaffolds could be used as substitutes for the critical-sized bone defect (7.0 mm diameter and 8.0 mm depth) in the osteoporotic rabbit model. The XD10 scaffolds can enhance bone ingrowth and accelerate new bone regeneration even in complex osteoporotic pathological environments. These results showed that such a Chinese medicine-contained scaffold had potential in osteoporosis bone tissue regeneration and could be considered as a promising tool for future clinical used applications.

Osteogenesis of adipose-derived stem cells from patients with glucose metabolism disorders

BackgroundAdipose derived stem cells (ADSCs) are clinically widely used somatic stem cells obtained from white adipose tissue. They are characterized by ability to differentiate e.g. into osteoblasts and might successfully regenerate bone tissue in fracture repair. However, the main problem of somatic stem cells is a documented influence of various diseases, drugs or age which can inhibit cells activity. Therefore, in the present study, we investigated the influence of insulin resistance (IR) and type 2 diabetes (T2D) on the proliferation and differentiation potential of ADSCs.MethodsThe fat from subcutaneous abdominal adipose tissue was acquired by lipoaspiration from 23 voluntary participants, divided into three groups: with diabetes type 2, with insulin resistance and control healthy donors. The proliferative potential was analyzed by cell cytotoxicity assays and by mRNA expression of genes connected with proliferation. Flow cytometry was done for identifying proteins characteristic for mesenchymal stem cells and an analysis of osteogenic differentiation potential based on the assessment of osteogenic markers by real time RT-qPCR, and the evaluation of calcium deposition were also performed.ResultsThe results showed that diabetes type 2 lowered the activity of ADSCs in proliferation assays and changed their phenotypical characteristics. Interestingly, we observed differences in the proliferation potential of ADSCs in patients with insulin resistance, which is often the first phase of diabetes, compared to the control. It might suggest that insulin resistance, early-stage T2D, alters the activity of cells. Moreover, expression of osteogenesis markers was higher in cells from T2D patients than in cells from patients with IR and control.ConclusionWe conclude that type 2 diabetes changes the activity of stem cells, and insulin resistance influences on the proliferation of ADSCs.

Spreading area and shape regulate the apoptosis and osteogenesis of mesenchymal stem cells on circular and branched micropatterned islands.

The topography of extracellular matrix regulates the differentiation of mesenchymal stem cells (MSCs). In particular, the effect of spreading shape or area on cellular differentiation and viability of individual MSCs cultured in the confined adhesive regions is an interesting fundamental issue. In this study, the adhesive patterns with the circularity of 0.1 or 1 and the areas of 314; 628; 1,256; or 2,512 μm2 were constructed using micropatterning technology. The expression of osteogenesis marker alkaline phosphatase and the apoptosis level of individual MSCs were measured using double fluorescent staining. Results indicated that individual MSCs confined in the small area showed an apoptotic tendency, and those in the large area might enter into osteogenesis. The branched shape with small circularity increased MSC viability but reduced their pluripotency compared with the circular shape. The expression of other osteogenesis markers, such as osteocalcin and Collagen I, confirmed that large and branched pattern promoted MSC osteogenesis. In addition, the transcriptional coactivator yes-associated protein (YAP) was transferred higher in the nuclei of the large and branched cells than other micropatterned groups. This study suggested that the spreading area and shape of individual MSCs regulate their viability and osteogenesis through the YAP pathway.

Synthesis and characterization of electrospun bioactive glass nanofibers-reinforced calcium sulfate bone cement and its cell biological response

In the present study, different weight ratios of electrospun bioactive glass nanofibers (3, 5, 10, 15, 25 and 35 wt%) (BGF) were added to calcium sulfate cement (CSC). Physical and mechanical properties of CSC/BGF composites were evaluated and compared with CSC. Initial and final setting times of CSC were reduced from 12 to 27 min to 6 and 21 min by increasing of BGF content, up to 15 wt%. BGFs with the average diameter of 80–100 nm were achieved by appropriate controlling of electrospinning parameters. SEM observations of nanocomposites revealed that BGFs filled the spaces between the crystals of the gypsum phase FTIR analysis and X-ray diffraction of the composites were confirmed the presence of BGF in the cement matrix. The best mechanical values (E~800 MPa and σ~30 MPa) were related to CSC containing 15 wt% of BGF (CSC/15%BGF). Apatite clusters were seen on the surface of CSC/15% BGF composite after 14 days of soaking in body fluid (SBF) solution. A.O. staining and MTT assay confirmed the absence of toxicity reactions for CSC/15%BGF nanocomposite. Alizarin red staining and calcium content were affirmed the biomineralization, and the expression of osteogenesis markers corroborated the differentiation potential of CSC/BGF. Collectively, our findings supported bioactivity and biocompatibility of CSC/15%BGF nanocomposite as a promising candidate for bone healing applications.

Lactoferrin ameliorates aging-suppressed osteogenesis via IGF1 signaling.

Lactoferrin (LF) is an iron-binding glycoprotein that plays an important role in promoting bone formation and inhibiting bone resorption; however, its effects on senile osteoporosis remain unknown. This study aimed to investigate the effects and mechanism of LF intervention using a senile osteoporosis model (SAMP6 mice) and senescent osteoblasts. Micro-CT and hematoxylin and eosin staining demonstrated that the intragastric administration (2 g/kg/day) of LF could improve the bone mass and microstructure of SAMP6 mice. Furthermore, LF treatment improved bone metabolism and increased insulin-like growth factor 1 (Igf1) mRNA expression and activated phosphorylation status of AKT. Using osteoblasts passaged for ten generations as an in vitro senescence model, various markers associated with osteoblast formation and differentiation, as well as related indices of oxidative stress were analyzed. Our results revealed that after multiple generations, osteoblasts entered senescence, in conjunction with increased oxidative stress damage, reduced bone metabolism and enhanced expression of aging-related markers. While inhibiting oxidative stress, LF improved osteoblast proliferation by promoting the expression of osteogenesis markers, including alkaline phosphatase (ALP) activity, Igf1, bone gla protein (Bglap) and osteoprotegerin/receptor activator of nuclear factor-kB ligand (Opg/Rankl) mRNA and delayed senescence by decreasing the level of p16 and p21 expression. RNAI-mediated downregulation of IGF1 attenuated the effect of LF on osteogenesis. Therefore, the findings of the present study indicate that LF may promote osteogenesis via IGF1 signaling, thereby preventing senile osteoporosis.

YAP1 influences differentiation of osteoblastic MC3T3-E1 cells through the regulation of ID1.

Yes-associated protein 1 (YAP1) transcriptional coactivator has recently been identified to regulate skeletal lineage cell differentiation and bone development. However, the role and molecular mechanisms of YAP1 in the regulation of osteoblastic differentiation remains to be elucidated. In this study, we demonstrated that YAP1 expression was increased during osteogenic differentiation of rat bone mesenchymal stem cells and MC3T3-E1. YAP1 overexpression MC3T3-E1 showed increased expression of osteogenesis markers, such as runt-related transcription factor 2, osteocalcin, and osteopontin, as well as alkaline phosphatase and alizarin red staining. Conversely, YAP1 knockdown significantly suppressed MC3T3-E1 osteoblastic differentiation. Mechanistically, we found that YAP1 overexpression upregulated the mRNA and protein expression of the inhibitor of differentiation/DNA binding 1 (ID1), which was contrary to the results of YAP1-knockdown group. Moreover, the early osteogenic differentiation of MC3T3-E1 cells was enhanced by ID1 overexpression. Furthermore, transient transfection with exogenous ID1 overexpression plasmid completely recaptured the decreased effects of YAP1 knockdown on MC3T3-E1 cell differentiation. In addition, β-catenin and AMP-activated protein kinase signaling pathways participated in YAP1 regulation processes. Taken together, our study suggests that YAP1 is a crucial modulator of osteoblast differentiation in vitro, and provides insight into the mechanism by which YAP1 regulates osteoblast differentiation.