Osteogenic Differentiation Of BMSCs Research Articles

Effect of Silent Information Regulator on the Survival and Osteogenic Differentiation of Inflammatory Bone Marrow Mesenchymal Stem Cells by Regulating NF-κB Signaling Pathway

Osteogenic differentiation of BMSCs is beneficial for osteoarthritis (OA) treatment. Silent information regulator (SIRT1) plays a role in endocrine diseases and aging-related diseases. However, the role of SIRT1 in OA has not yet been elucidated. Rat BMSCs were isolated and divided into control group, inflammation group (BMSCs were cultured with IL-6), SIRT1 group (SIRT1 agonist Resveratrol was added under the action of IL-6) followed by analysis of cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and OC and adipogenic differentiation gene PPARγ2 by Real time PCR, NF-κB expression by western blot and secretion of TNF-α and IL-6 by ELISA. In inflammation group, SIRT1 expression was significantly decreased, cell proliferation was significantly inhibited, and Caspase 3 activity was increased. Meanwhile, ALP activity, Runx2 and OC expression was decreased, PPARγ2 and NF-κB expression was increased, along with elevated TNF-α and IL-6 secretion compared to control (P < 0.05). Resveratrol can significantly promote the expression of SIRT1 in BMSCs of inflammation group, promote cell proliferation, decrease Caspase 3 activity, and increase Runx2 and OC expression. In addition, it decreased PPARγ2 and NF-κB expression and reduced the secretion of TNF-α and IL-6 (P < 0.05). The expression of SIRT1 was decreased in BMSCs under inflammation. SIRT1 overexpression in BMSCs under inflammation inhibits inflammation, promotes proliferation and osteogenic differentiation of BMSCs through regulating NF-κB signaling pathway.

Effects of carbon and nitrogen plasma immersion ion implantation on bioactivity of zirconia.

Zirconia is considered the most promising alternative material to titanium implants. However, zirconia is a biologically inert material and its surface modification is essential to obtain efficient osseointegration. Plasma immersion ion implantation (PIII) is a controllable and flexible approach that constructs functional groups on the surface of biomaterials and enhances osteogenic ability of host osteoclast cells. Zirconia disks were randomly divided into 4 groups (n = 50/group): (1) Blank, (2) C60N0, (3) C60N6, and (4) C60N18. Carbon and nitrogen plasma immersion ion implantation on zirconia (C and N2-PIII) surface modification was completed with the corresponding parameters. When zirconia was modified by carbon and nitrogen plasma implantation, a new chemical structure was formed on the material surface while the surface roughness of the material remained unaltered. The nitrogen-containing functional groups with high potential were introduced but the bulk crystal structure of zirconia was not changed, indicating that the stability of zirconia was not affected. In vitro data showed that zirconia with high surface potential promoted adhesion, proliferation, and osteogenic differentiation of BMSCs. C60N6 was found to be superior to the other groups. Our results demonstrate that a zirconia surface modified by C and N2-PIII can introduce desirable nitrogen functional groups and create a suitable extracellular environment to promote BMSCs biological activity. Taken together, these results suggest that C and N2-PIII modified zirconia is a promising material for use in the field of medical implantation.

A highly interweaved HA-SS-nHAp/collagen hybrid fibering hydrogel enhances osteoinductivity and mineralization.

The combination of bioactive hydroxyapatite (HAp) with biomimetic bone matrix biomaterials as bone filling scaffolds is a promising strategy for bone regeneration, but the undesirable dispersion of HAp and its interfacial interaction result in inefficient mineralization, mechanical instability, incomplete osteointegration, and even repair failure. Herein, the size dispersion and stabilization of nano-hydroxyapatite (nHAp) in aqueous media were obviously improved by hydrophilic solubilisation and strong negatively charged thiolated hyaluronic acid (HA-SH). Furthermore, the highly interweaved HA-SS-nHAp/collagen hybrid fibering hydrogel exhibited significantly improved mechanical properties and structural stability due to its thickened and densified interweaved fiber network, which ensured the homogeneous dispersion of nHAp in the matrix materials and its integration with the hydrogel network structure completely by covalent self-crosslinking among the sulfhydryl groups derived from the free HA-SH polymer and the mercapto functional groups on the surface of nHAp. Compared with the physically combined micro-hydroxyapatite (μHAp) (d≤25 μm) and nHAp (∼530 nm) with injectable bionic HA-SH and collagen type I biopolymers, HA-SS-nHAp/collagen achieved the maximum efficiency in facilitating rabbit bone marrow stromal cell (rBMSC) adhesion, proliferation and osteogenic differentiation in vitro. The in vivo murine dorsal subcutaneous implantation results further confirmed that the interweaved fiber network structure in HA-SS-nHAp/collagen significantly promoted osteoinductivity and mineralization. This work provides novel insights for the development of new low invasive bone filling biomaterials.

Tumor Necrosis Factor-α Inhibits Differentiation of Bone Marrow Mesenchymal Stem Cells into Osteoblasts by Inhibiting Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (AKT) Pathway

Bone marrow mesenchymal stem cells (BMSCs) have the potential for multi-directional differentiation. Tumor necrosis factor-α (TNF-α) plays a role in many diseases. However, the role of TNF-α in the differentiation of BMSCs into osteoblasts remains unclear. Rat-derived BMSCs were divided into normal control group and TNF-α group (5 ng/ml or 10 ng/ml) followed by analysis of BMSCs proliferation by MTT, Caspase3 activity, TNF-α mRNA and serum secretion by Real time PCR and ELISA, ALP activity, Runx2, OP and OC level by Real time PCR and PI3K/AKT signaling protein expression by Western blot. TNF-α significantly promoted the expression of TNF-α mRNA and serum secretion, inhibited cell proliferation, increased Caspase3 and ALP activity, decreased Runx2 and OP expression, and inhibited AKT phosphorylation compared to control (P < 0.05). The inhibition of osteogenic differentiation of BMSCs was more obvious with the increase of TNF-α concentration. TNF-α can inhibit the proliferation of BMSCs, promote apoptosis and inhibit BMSCs osteogenic differentiation via inhibiting the PI3K/AKT pathway.

Down-Regulation of Notch-2 Promotes the Osteogenic Differentiation of Bone Marrow Stromal Cells in High Glucose Environment by Targeting Fat Mass and Obesity-Associated Protein

BMSCs Differentiation into osteoblasts is beneficial for treating osteoporosis. Notch signaling pathway regulates the proliferation of BMSCs. However, Notch-2's effect on osteogenic differentiation of BMSCs in high glucose environment remains unclear. Rat BMSCs were isolated and divided into control group, high glucose group, Notch-2 siRNA group, in which BMSCs cells were transfected with Notch-2 siRNA under high glucose environment followed by analysis of Notch-2 mRNA expression by Real time PCR, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and BMP-2 by Real time PCR, TNF-α and IL-2 secretion by ELISA, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity and FTO protein expression by Western blot. In high glucose group, Notch-2 expression was increased with inhibited cell proliferation, increased Caspase 3 activity and decreased ALP activity. Meanwhile, high glucose significantly increased MPO content, decreased SOD activity, increased TNF-α and IL-2 secretion and FTO expression, and reduced Runx2 and BMP-2 expression (P < 0.05); Notch-2 siRNA transfection significantly down-regulated Notch-2 expression, promoted BMSCs cell proliferation, decreased Caspase 3 activity and increased ALP activity. In addition, it also significantly decreased MPO content, increased SOD activity, decreased TNF-α and IL-2 and FTO expression as well as increased Runx2 and BMP-2 expression (P < 0.05). Notch-2 is upregulated in BMSCs under high glucose. Down-regulation of Notch-2 in BMSCs under high glucose environment could promote BMSCs proliferation and osteogenic differentiation.

Effect of Long-Chain Non-Coding RNA GAS5 on Osteogenic/Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Under Oxidative Stress Through Targeting MiR-365

Oxidative stress affects BMSCs. LncRNA GAS5 regulates cell proliferation and apoptosis. However, the effect of LncRNA GAS5 on osteogenesis/adipogenic differentiation of BMSCs under oxidative stress has not been reported. Rat BMSCs were cultured and randomly divided into 4 groups, normal control group; oxidative stress group; GAS5 siRNA group; GAS5 siRNA+ miR-365 inhibitor group followed by analysis of LncRNA GAS5 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 activity, GAS5 and miR-365 targeting relationship by luciferase reporter assay, ALP activity, expression of Runx2, OP and PPAR 2 by Real time PCR, as well as ROS content and SOD activity. In oxidative stress group, GAS5 expression was significantly increased along with inhibited cell proliferation, increased Caspase3 activity, decreased ALP activity and the expression of Runx2 and OP, increased PPAR 2 expression and ROS content, and decreased SOD activity compared to control group (P < 0 05). miR-365 was the target miRNA of GAS5. GAS5 siRNA down-regulated GAS5 expression, significantly promoted cell proliferation, inhibited Caspase3 activity, increased ALP activity and Runx2 and OP expression, decreased PPAR 2 expression and ROS content, and increased SOD activity. (P < 0 05). However, GAS5 siRNA+ miR-365 inhibitor group reversed the effect of GAS5 siRNA. Oxidative stress promotes LncRNA GAS5 expression in BMSCs. LncRNA GAS5 regulates oxidative stress by targeting miR-365. Knockdown of GAS5 can promote BMSCs proliferation and osteogenic differentiation and inhibit adipogenic differentiation under oxidative stress.

Effect of Mir-663 on the Differentiation of Bone Marrow Mesenchymal Stem Cells in Inflammatory Environment Through Regulating Oxidative Stress and Transforming Growth Factor β1

BMSCs are beneficial for the treatment of Osteoarthritis (OA). Mir-663 involves in various diseases. However, the role of Mir-663 in the differentiation of BMSCs in an inflammatory environment remains unclear. Rat BMSCs were isolated and divided into control group, and inflammation group which was treated with 1 μg/ml lipopolysaccharide (LPS), Mir-663 group and Mir-663 siRNA groups which was respectively transfected with Mir-663 plasmid and Mir-663 siRNA into LPS-treated BMSCs followed by analysis of the survival rate of BMSC by MTT, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and OP by Real time PCR, ROS content and SOD activity, TGF-β1 level by Real time PCR and ELISA, TNF- and IL-1 secretion by ELISA. LPS treatment significantly increased Mir-663 expression, inhibited cell proliferation, increased Caspase 3 activity, and inhibited ALP activity. Meanwhile it also significantly decreased Runx2 and OP expression, increased ROS content, decreased SOD activity and TGF-β1 expression as well as elevated TNF-α and IL-1β secretion compared to control (P < 0.05); Mir-663 plasmid transfection can further promote the above changes (P < 0.05); whereas, Mir-663 siRNA could reverse the above changes (P < 0.05). Mir-663 expression was increased in BMSCs in inflammatory environment. Down-regulation of Mir663 expression can regulate oxidative stress, up-regulate TGF-β1 level, inhibit the secretion of TNF-α and IL-1β, and promote the proliferation and osteogenic differentiation of BMSCs in inflammatory environment.

Ebselen rescues high glucose suppressed osteogenic differentiation of BMSCs by the activation of PI3K AKT pathway

Ebselen rescues high glucose suppressed osteogenic differentiation of BMSCs by the activation of PI3K AKT pathway

The Regulatory Effect of Mir-149 on Bone Marrow Mesenchymal Stem Cells in Osteoporosis Rats and Its Related Mechanisms

BMSCs play an important role in osteoporosis (OP) and their differentiation can lead to OP progression. Mir-149 can participate in the regulation of BMSCs. However, the effect of Mir-149 on BMSCs in osteoporosis remains unclear. SD rats were divided control group and OP group. OP rat BMSCs were transfected with Mir-149 siRNA followed by analysis of Mir-149 expression by real time PCR, cell proliferation by MTT assay, apoptosis by flow cytometry, ERK/MAPK signaling protein expression by western blot, ALP activity, as well as expression of osteogenic genes Runx2 and OC by real time PCR. Mir-149 expression was significantly increased in BMSCs of OP rats, cell proliferation was inhibited, apoptosis was increased, as well as p-ERK1/2 expression, ALP activity and expression of Runx2 and OC was decreased compared to control group (P < 0.05). Transfection of Mir-149 siRNA into OP rat BMSCs reduced Mir-149 expression, promoted cell proliferation, decreased apoptotic rate, increased p-ERK1/2 expression, ALP activity and Runx2 and OC expression. Compared with OP group, the differences were statistically significant (P< 0.05). Mir-149 expression was increased in OP rat BMSCs. Down-regulation of Mir-149 promoted the activation of ERK/MAPK signaling pathway, inhibited apoptosis of BMSCs and promoted the proliferation and osteogenic differentiation of BMSCs in OP rats.

PLEXIN-B2 promotes the osteogenic differentiation of human bone marrow mesenchymal stem cells via activation of the RhoA signaling pathway

Plexin-B2 (PLXNB2), a transmembrane protein is found in various tissues. Recent studies have indicated the presence of PLXNB2 in large quantity in the growth plates of Sprague-Dawley rats and are believed to be potentially involved in their skeletal development. This study endeavored to analyze the effect of PLXNB2 on the osteogenic differentiation of BMSCs by using gene overexpression and knockdown assays. The results of our study revealed that PLXNB2 was upregulated during BMSCs differentiation into an osteoblastic lineage. By determining the expression levels of specific markers and mineral deposition, the study established that PLXNB2 promotes the osteogenic differentiation of human BMSCs through the activation of the RhoA signaling pathway. In conclusion, the study identified PLXNB2 as a novel regulator that enhanced the osteogenic differentiation of human BMSCs. The enhancing effect of PLXNB2 on osteogenesis of human BMSCs was mediated through activation of RhoA signaling. The results of our study imply that pharmacological targeting of PLXNB2 may initiate a possible improvement in bone formation.

Effect of zoladronate on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells

To evaluate the effect of zoledronate acid (ZA) on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (BMSCs). The BMSCs isolated from the SD rats were cultured with different concentrations of ZA (1, 5, 10, and 20 μmol·L), and the contro1 group received the same volume of culture medium but without ZA. Cell counting kit-8 was used to detect proliferation activity in each group. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability in each group. The gene expression levels of ALP, bone morphogenetic protein-2 (BMP-2), typeⅠcollagenase (COL-Ⅰ), runt-related transcription factor-2 (Runx-2), zinc finger structure transcription factor (Osx), osteocalcin (OCN), and osteopontin (OPN) were evaluated by real-time quantitative polymerase chain reaction (qRT-PCR). Zoledronate at 1 μmol·L⁻¹ concentration had no effect on the proliferation and osteogenic differentiation of BMSCs. No significant difference was observed between this group and the control group (P>0.05). When the ZA concentration was more than 1 μmol·L⁻¹, ZA inhibited the proliferation and osteogenic differentiation of BMSCs, and the effect was concentration dependent. The difference between each group and the control group was statistically significant (P<0.05). At ZA concentration of 5 μmol·L⁻¹, ZA enhanced the expression of ALP, BMP-2, COL-Ⅰ, Runx-2, Osx, OCN, and OPN (P<0.05). However, at ZA concentration of more than 5 μmol·L⁻¹, the expression levels of osteogenicrelated genes in each group was lower than those of the control group (P<0.05). Low ZA concentration has no effect on the proliferation and osteogenic differentiation of BMSCs. ZA at 5 μmol·L⁻¹ concentration inhibits the proliferation but promotes the osteogenic differentiation of BMSCs. High ZA concentration inhibits the proliferation and osteogenic differentiation of BMSCs.

MSC differentiation on two-photon polymerized, stiffness and BMP2 modified biological copolymers

Introduction. Bone tissue regeneration requires a three-dimensional biological setting. An ideal scaffold should enable cell proliferation and differentiation by mimicking structure and mechanical properties of the compromised defect as well as carrying growth factors. Two-photon polymerization (2PP) allows the preparation of 3D structures with a micrometric resolution. Methods. In this study, 2PP was applied to design scaffolds made from biocompatible methacrylated D,L-lactide-co-ε-caprolactone copolymers (LC) with a controlled porous architecture. Proliferation and differentiation of bone marrow mesenchymal stromal cells on LC was analyzed and compared to a standard inorganic urethane-dimethacrylate (UDMA) matrix. To functionalize LC and UDMA surfaces we analyzed a biomimetic, layer-by-layer coating, which could be modified in stiffness and integration of bone morphogenetic protein 2 (BMP2) and evaluated its effect on osteogenic differentiation. Results. On LC surfaces, BMSC demonstrated an optimal proliferation within pore sizes of 60–100 μm and showed a continuous expression of Vimentin. On the polyelectrolyte multilayer coating a significant increase in BMSC proliferation and differentiation as marked by Osteonectin expression was achieved using stiffness modification and BMP2 functionalization. Conclusion. Combining 3D-Design with biofunctionalization, LC offers a promising approach for future regenerative applications in osteogenic differentiation of BMSCs.

Promoting tendon to bone integration using graphene oxide-doped electrospun poly(lactic-co-glycolic acid) nanofibrous membrane.

BackgroundThese normal entheses are not reestablished after repair despite significant advances in surgical techniques. There is a significant need to develop integrative biomaterials, facilitating functional tendon-to-bone integration.Materials and methodsWe fabricated a highly interconnective graphene oxide-doped electrospun poly(lactide-co-glycolide acid) (GO-PLGA) nanofibrous membrane by electrospinning technique and evaluated them using in vitro cell assays. Then, we established rabbit models, the PLGA and GO-PLGA nanofibrous membranes were used to augment the rotator cuff repairs. The animals were killed postoperatively, which was followed by micro-computed tomography, histological and biomechanical evaluation.ResultsGO was easily mixed into PLGA filament without changing the three dimensional microstructure. An in vitro evaluation demonstrated that the PLGA membranes incorporated with GO accelerated the proliferation of BMSCs and furthered the Osteogenic differentiation of BMSCs. In addition, an in vivo assessment further revealed that the local application of GO-PLGA membrane to the gap between the tendon and the bone in a rabbit model promoted the healing enthesis, increased new bone and cartilage generation, and improved collagen arrangement and biomechanical properties in comparison with repair with PLGA only.ConclusionThe electrospun GO-PLGA fibrous membrane provides an effective approach for the regeneration of tendon to bone enthesis.

Osteogenic differentiation of BMSCs in collagen-based 3D scaffolds

Collagen-based scaffolds was fabricated through covalent crosslinking, and used as 3D scaffolds for promoting the osteogenic differentiation of BMSCs.

MiR-217 promotes cell proliferation and osteogenic differentiation of BMSCs by targeting DKK1 in steroid-associated osteonecrosis

MicroRNAs (miRNAs) have recently been recognized to play an important role in bone-associated diseases. This study aims to explore the expression profile and biological function of miR-217, which is known to be related to tumor cell proliferation and migration, to the proliferation and osteogenic differentiation of MSCs from the patients with steroid-associated osteonecrosis (ONFH). Bone marrow was obtained from the proximal femur of 10 patients with ONFH and 10 patients with femoral neck fractures. Bone marrow-derived mesenchymal stem cells (MSCs) were isolated and cultured. The expression profile, biological function of miR-217 and the interaction between miR-217 and DKK1 were assayed using cell viability measurement, western blot, Real-time PCR, luciferase reporter assay, Alizarin Red S (ARS) staining. We noted that the expression level of miR-217 was significantly decreased in the ONFH samples compared to the control samples (P < 0.0001). By targeting DKK1, miR-217 promoted nuclear translocation of β-catenin, increased expression of RUNX2, COL1A1 and obviously promoted the proliferation and differentiation of MSCs. Restoring the expression of DKK1 in the MSCs partially reversed the role of miR-217. These findings suggest that miR-217 promotes cell proliferation and osteogenic differentiation by inhibiting DKK1 during the development of steroid-associated osteonecrosis.

Influences of tantalum pentoxide and surface coarsening on surface roughness, hydrophilicity, surface energy, protein adsorption and cell responses to PEEK based biocomposite.

Polyetheretherketone (PEEK) biomaterial has become increasingly popular in orthopedic applications due to its favorable biocompatibility, biostability, mechanical strength and elastic modulus similar to natural bones. In this research, in order to improve the biological performances of PEEK, tantalum pentoxide (Ta2O5) was incorporated into PEEK to fabricate PEEK/Ta2O5 composites (PTC) using a method of cold press-sintering, and surface coarsening of PTC was prepared by sand blasting. The results showed that the Ta2O5 particles were uniformly disperse into PEEK, and thermal and mechanical properties of PTC were enhanced with the increase of Ta2O5 content. In addition, incorporating Ta2O5 into PEEK and surface coarsening could improve surface roughness, hydrophilicity, surface energy and protein absorption of PTC. Furthermore, the adhesion and proliferation as well as osteogenic differentiation of BMSCs on PTC were significantly promoted and regulated by Ta2O5 content and surface coarsening. The results indicated that surface coarsening of PTC (PTCS) with high surface roughness, hydrophilicity and surface energy could induce positive cellular responses, showing good cytocompatibility. PTCS might have a great potential as implants for bone repair.

Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 129. Plastrum testudinis treating glucocorticoid-induce osteoporosis by down-regulating TNFR2

BACKGROUND CONTEXT Glucocorticoid-induced osteoporosis (GIOP) is a systemic bone diseaseinduced by glucocorticoid (GC). The incidence of GIOP continues to rise with the widely use of GC, and GIOP has been identified as the most common type of secondary osteoporosis. Emerging evidence has demonstrated that bone fragility induced by GIOP is much more serious than that induced by postmenopausal osteoporosis, and GIOP has higher disability rate and mortality. Excessive or prolonged GC treatment has been considered to play a major role in inhibiting bone formation. However, the underlying mechanism of GIOP has not been confirmed. Thus, it is still an important and urgent project to find out how to research the mechanism and treat GIOP. PURPOSE To explore the effect of Plastrum Testudinis treating glucocorticoid-induce osteoporosis in Wnt/β-catenin pathway by down-regulating TNFR2.To explore the effect of Plastrum Testudinis treating glucocorticoid-induce osteoporosis in Wnt/β-catenin pathway by down-regulating TNFR2. STUDY DESIGN/SETTING Animal experiment:thirty 4-month-old SD rats were randomly divided into three groups: blank group, model group and Plastrum Testudinis group. The model group and the Plastrum Testudinis group were subcutaneously injected with dexamethasone, and were successfully gavaged with 0.9% sodium chloride solution and Plastrum Testudinis respectively. The lumbar spine (L1-6) were taken after 12 weeks. PATIENT SAMPLE No patients. Thirty 4-month-old SD rats were used in this study. OUTCOME MEASURES The mRNA expression of TNFR2, β-cantenin and GSK3β in the lumbar vertebra were detected by qPCR. The protein expressions of TNFR2, p-β-cantenin and p-GSK3β were detected by Western blot. Cell expeiment:CCK8 was used to detect the proliferation of BMSCs at different concentrations for 1,3,5,7 and 14 days. Osteogenesis was induced by selecting the best concentration. ALP and alizarin red staining were used to detect the effect of PTE on osteogenic differentiation of BMSCs. The expression of TNFR2, Β-cantenin, GSK3β mRNA expression, TNFR2, p-β-cantenin, p-GSK3β protein expression of turtle plate were detected by Werstern blot. METHODS Animal experiment: Thirty 4-month-old SD rats were randomly divided into three groups: blank group, model group and Plastrum Testudinis group. The model group and the Plastrum Testudinis group were subcutaneously injected with dexamethasone, and were successfully gavaged with 0.9% sodium chloride solution and Plastrum Testudinis respectively. The lumbar spine (L1-6) were taken after 12 weeks. The mRNA expression of TNFR2, β-cantenin and GSK3β in the lumbar vertebra were detected by qPCR. The protein expressions of TNFR2, p-β-cantenin and p-GSK3β were detected by Western blot. Cell expeiment:CCK8 was used to detect the proliferation of BMSCs at different concentrations for 1,3,5,7,14 days. Osteogenesis was induced by selecting the best concentration. ALP and alizarin red staining were used to detect the effect of PTE on osteogenic differentiation of BMSCs. The expression of TNFR2, Β-cantenin, GSK3β mRNA expression, TNFR2, p-β-cantenin, p-GSK3β protein expression of turtle plate were detected by Werstern blot. RESULTS Compared with the blank group, the level of β-catenin in the GIOP group was significantly decreased (P CONCLUSIONS Plastrum Testudinis can promote the proliferation, osteogenic differentiation and mineralization of BMSCs. It can effectively resist GIOP. The Plastrum Testudinis may resist the GIOP through down-regulating the Wnt /β-catenin pathway by inhibiting TNFR2. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Bone mesenchymal stem cell secretion of sRANKL/OPG/M-CSF in response to macrophage-mediated inflammatory response influences osteogenesis on nanostructured Ti surfaces

Although it has been well established that osteogenic differentiation of bone mesenchymal stem cells (bMSCs) as well as osteoclastic differentiation of macrophages can be manipulated by the nanostructure of biomaterial surfaces, the interactions among the effects of the surface on immune cells and bMSCs remained unknown. Therefore, in this study, the osteogenic behaviors and secretion of osteoclastogenesis-related cytokines of human bMSCs on TiO2 nanotubular (NT) surfaces in conditioned medium (CM) generated by macrophages cultured on the respective NT surfaces (NT-CM) were analyzed. Although bMSCs showed consistent osteogenic behaviors on the NT5 and NT20 surfaces in both standard culture medium and both types of NT-CM, collagen synthesis and extracellular matrix mineralization were partially impeded on the NT20 surface in NT20-CM and bMSC cytokine secretions on the NT20 surface in NT20-CM elicited remarkable multinuclear giant cell and osteoclast formation compared with that observed on the NT5 surface in NT5-CM. After implantation in vivo, mineralized bone formation was significantly delayed around the NT20 implant compared with the NT5 implant, but both surfaces contributed to good bone formation after 12 weeks. The results obtained in this study advance our understanding of the confounding influence of the implant surface nanostructure, macrophage inflammatory response, and osteogenic differentiation of bMSCs as well as the retro-regulative effects of bMSCs on the osteoclastic differentiation of macrophages, and the culture system based on different NT surfaces and CM generated on the respective surfaces may provide a systematic research model for evaluating the performance of endosseous implants as well as a prospective approach for improving implant osseointegration via immune-regulation.

Multilayered pore-closed PLGA microsphere delivering OGP and BMP-2 in sequential release patterns for the facilitation of BMSCs osteogenic differentiation.

Bone tissue regeneration may be more effectively administrated by controlled release of multiple biofactors, given that bone healing comprises a cascade of biological events controlled by numerous cytokines and growth factors (GFs). Here, we propose a novel microcarrier with the capability to sequentially deliver dual biofactors for better controlling the bone regeneration process. First, osteogenic growth peptide (OGP) was incorporated in porous poly(lactic-co-glycolic) acid (PLGA) microspheres by a simple solution dipping method and subsequent pore-closing treatment. Then, a multilayered polyelectrolyte coating ((HA-CS)2 -Hep-BMP-2-Hep-(CS-HA)2 ) was prepared on the surface of such OGP-loaded pore-closed PLGA microspheres by layer-by-layer assembly. Results showed that the OGP release was minimal (<17.1%) in the first 15 days but accelerated remarkably thereafter, while at least 60.3% of the bone morphogenetic protein-2 (BMP-2) load was released in the first 15 days and only very slow release was observed subsequently. Further in vitro cell experiments showed that the dual-biomolecule-loaded microspheres elicited more cells with extremely elongated cellular morphology, much higher alkaline phosphatase level and upregulated expression of osteocalcin. Such a dual loading of OGP and BMP-2 had a more positive impact on bone marrow mesenchymal stem cells proliferation and osteogenic differentiation compared with either OGP or BMP-2 alone, suggesting potential synergistic benefit of the sequential release of multiple peptide-based biofactors in a coordinated manner. Overall, this dual delivery system may provide a therapeutic strategy sequentially targeting multiple events (or mechanisms) during bone healing, which is believed to benefit the regenerative repair of bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 95-105, 2018.

Pharmacological inhibition of S6K1 impairs self-renewal and osteogenic differentiation of bone marrow stromal cells.

mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt-induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self-renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony-Forming Unit-Fibroblast (CFU-F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a-induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self-renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a-induced osteoblast differentiation and protein anabolism.