Osteogenic Conditions Research Articles

Artemisinin protects DPSC from hypoxia and TNF-α mediated osteogenesis impairments through CA9 and Wnt signaling pathway

Dental pulp stem cells (DPSCs) possess the ability of multi-lineage differentiation, and are excellent sources of tissue engineering and regenerative medicine. Oxygen concentration and inflammation are two critical environmental factors that affect the osteogenic differentiation of DPSCs. We aimed to study the role of the antimalarial drug artemisinin on the osteogenic differentiation of human DPSCs under the hypoxia and inflammation conditions. We demonstrated that hypoxia (5% O2) and inflammation (20 ng/mL TNF-α), alone or in combination, significantly diminished in vitro cell survival and increased apoptotic rates. Notably, hypoxia and TNF-α exerted accumulative effect in suppressing the osteogenic differentiation of DPSCs, as evidenced by reduced expression levels of osteogenesis-associated genes including ALP, RUNX2 and OCN in osteogenic condition, as well as reduced mineral nodules formation as indicated by alizarin red staining. Artemisinin at the dose of 40 μM markedly reversed the suppression in cell survival caused by hypoxia or inflammation, and reduced apoptotic rates and the expressions of pro-apoptotic proteins. Additionally, artemisinin restored osteogenic differentiation of DPSCs under the hypoxia or/and inflammation conditions. Moreover, the beneficial effect of artemisinin was dependent on upregulated expression of CA9 and CA9-mediated antioxidant responses, as CA9 knockdown abolished the protective role of artemisinin on DPSC osteogenesis. Furthermore, while hypoxia or/and inflammation significantly inactivated the Wnt/β-catenin signaling in DPSCs, additional exposure to artemisinin re-activated this pathway to promote osteogenic differentiation of DPSCs. Our results provide novel insight on the link between artemisinin and DPSC osteogenesis, and suggest promising artemisinin-based strategies for better dentin/pulp tissue engineering.

The rise in expression and activity of 11β-HSD1 in human mesenchymal progenitor cells induces adipogenesis through increased local cortisol synthesis

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) plays an important role in pre-receptor glucocorticoid metabolism. This enzyme is expressed in bone, increases with age, and catalyzes the conversion of the inactive glucocorticoid cortisone into the active glucocorticoid cortisol and vice versa. Here we hypothesized that the physiological activity of 11β-HSD1 to produce cortisol in human mesenchymal progenitor cells (hMSC) is principally sufficient to shift the differentiation potential in the direction of adipogenic. We thus investigated differentiating hMSCs and the mesenchymal stem cell line SCP-1 cultured under osteogenic conditions and stimulated with supra-physiological cortisone levels. The release of active cortisol into the medium was monitored and the influence on cell differentiation analyzed. We revealed an increase in 11β-HSD1 expression followed by increased reductive activity of the enzyme, thereby inducing a more adipogenic phenotype of the cell models via cortisol with negative effects on osteogenesis. Through inhibition experiments with the specific inhibitor 10 j, we proved the enzyme specificity for cortisol synthesis and adipogenic differentiation. Increased expression of 11β-HSD1 followed by higher cortisol levels might thus explain bone marrow adiposity followed by reduced bone quality and stability in old age or in situations of supra-physiological glucocorticoid exposure.

Evaluation of stemness properties of cells derived from granulation tissue of peri-implantitis lesions.

ObjectivesPeri‐implantitis (PI) is an inflammatory disease associated with peri‐implant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri‐implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri‐implantitis derived mesenchymal stromal cells—PIMSCs).Material and MethodsPIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT‐PCR.ResultsPIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter‐individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri‐lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed.ConclusionsThis study provides evidence that MSC‐like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.

Coculture of Endothelial and Stromal Cells to Promote Concurrent Osteogenesis and Vasculogenesis.

A key challenge in the treatment of large bone defects is the need to provide an adequate and stable vascular supply as new tissue develops. Bone tissue engineering applies selected biomaterials and cell types to create an environment that promotes tissue formation, maturation, and remodeling. Mesenchymal stromal cells (MSCs) have been widely used in these strategies because of their established effects on bone formation, and their ability to act as stabilizing pericytes that support vascular regeneration by endothelial cells (ECs). However, the creation of vascularized bone tissue in vitro requires coupling of osteogenesis and vasculogenesis in a three-dimensional (3D) biomaterial environment. In the present study, 3D fibrin hydrogels containing MSCs and ECs were prevascularized in vitro for 7 days to create an endothelial network in the matrix, and were subsequently cultured for a further 14 days under either continued vasculogenic stimulus, a combination of vasculogenic and osteogenic (hybrid) stimulus, or only osteogenic stimulus. It was found that ECs produced robust vessel networks in 3D fibrin matrices over 7 days of culture, and these networks continued to expand over the 14-day treatment period under vasculogenic conditions. Culture in hybrid medium resulted in maintenance of vessel networks for 14 days, while osteogenic culture abrogated vessel formation. These trends were mirrored in data representing overall cell viability and cell number in the 3D fibrin constructs. MSCs were found to colocalize with EC networks under vasculogenic and hybrid conditions, suggesting pericyte-like function. The bone marker alkaline phosphatase increased over time in hybrid and osteogenic media, but mineral deposition was evident only under purely osteogenic conditions. These results suggest that hybrid media compositions can support some aspects of multiphase tissue formation, but that alternative strategies are needed to obtain robust, concomitant vascularization, and osteogenesis in engineered tissues in vitro. Impact statement The combined use of mesenchymal stromal cells (MSCs) and endothelial cells to concomitantly produce mature bone and a nourishing vasculature is a promising tissue engineering approach to treating large bone defects. However, it is challenging to create and maintain vascular networks in the presence of osteogenic cues. This study used a 3D fibrin matrix to demonstrate that prevascularization of the construct can lead to maintenance of vessel structures over time, but that osteogenesis is compromised under these conditions. This work illuminates the capacity of MSCs to serve as both supportive pericytes and as osteoprogenitor cells, and motivates new strategies for coupling osteogenesis and vasculogenesis in engineered bone tissues.

Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions.

ObjectivesVitamin D3 is known to activate osteogenic differentiation of human periodontal ligament stromal cells (hPDLSCs). Recently, inflammatory stimuli were shown to inhibit the transcriptional activity of hPDLSCs, but their effect on vitamin D3‐induced osteogenic differentiation is not known. The present study aimed to investigate whether the effects of 1,25‐dihydroxvitamin D3 (1,25(OH)2D3) and 25‐hydroxvitamin D3 (25(OH)D3) on the osteogenic differentiation of hPDLSCs are also altered under inflammatory conditions. Furthermore, the expression of osteogenesis‐related factors by hPDLSCs under osteogenic conditions was assessed in the presence of inflammatory stimuli.Materials and MethodsPrimary hPDLSCs of six donors were cultured in osteogenic induction medium containing either 1,25(OH)2D3 (0‐10 nM) or 25(OH)D3 (0‐100 nM) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) or Pam3CSK4 for 7, 14 and 21 days. Osteogenic differentiation of hPDLSCs was evaluated by analysis of mineralization as assessed by Alizarin Red S staining and gene expression levels of osteogenesis‐related factors osteocalcin, osteopontin and runt‐related transcription factor 2 (RUNX2) were analysed with qPCR.ResultsTreatment with 1,25(OH)2D3 significantly enhanced the osteogenic differentiation of hPDLSCs and their expression of osteocalcin and osteopontin. The 1,25(OH)2D3‐triggered expression of osteogenesis‐related factors was significantly lower in the presence of Pam3CSK4, but not P. gingivalis LPS. None of the inflammatory stimuli had significant effects on the 1,25(OH)2D3‐induced osteogenic differentiation. 25(OH)D3 neither affected gene expression levels nor osteogenic differentiation of hPDLSCs cultured in osteogenic induction medium.ConclusionThe results of this study indicate that inflammatory stimuli also diminish the 1,25(OH)2D3‐induced expression of osteogenesis‐related factors in hPDLSCs under osteogenic conditions, while having no effect on the osteogenic differentiation.

Menaquinone-7 Supplementation Improves Osteogenesis in Pluripotent Stem Cell Derived Mesenchymal Stem Cells.

Development of clinical stem cell interventions are hampered by immature cell progeny under current protocols. Human mesenchymal stem cells (hMSCs) are characterized by their ability to self-renew and differentiate into multiple lineages. Generating hMSCs from pluripotent stem cells (iPSCs) is an attractive avenue for cost-efficient and scalable production of cellular material. In this study we generate mature osteoblasts from iPSCs using a stable expandable MSC intermediate, refining established protocols. We investigated the timeframe and phenotype of cells under osteogenic conditions as well as the effect of menaquinone-7 (MK-7) on differentiation. From day 2 we noted a significant increase in RUNX2 expression under osteogenic conditions with MK-7, as well as decreases in ROS species production, increased cellular migration and changes to dynamics of collagen deposition when compared to differentiated cells that were not treated with MK-7. At day 21 OsteoMK-7 increased alkaline phosphatase activity and collagen deposition, as well as downregulated RUNX2 expression, suggesting to a mature cellular phenotype. Throughout we note no changes to expression of osteocalcin suggesting a non-canonical function of MK-7 in osteoblast differentiation. Together our data provide further mechanistic insight between basic and clinical studies on extrahepatic activity of MK-7. Our findings show that MK-7 promotes osteoblast maturation thereby increasing osteogenic differentiation.

Autologous micrografts from the palatal mucosa for bone regeneration in calvarial defects in rats: a radiological and histological analysis

BackgroundThe application of dental implants is often restricted by bone volume. In such cases, bone grafts are required, although bone graft materials have some disadvantages. Therefore, other effective approaches are needed. Our previous study showed that the autologous micrograft, a dissociated cell suspension made out of palatal connective tissue grafts, promoted bone-marrow cell proliferation and differentiation under osteogenic conditions. In this study, we aimed to evaluate the effects of dissociated soft-tissue suspensions relevant to bone regeneration in animal model.Material and methodsTwelve-week-old male Wistar rats were used in the study. Defects were created in rat calvaria, and were filled with hydrogel containing either dissociated soft-tissue suspension (test) or sucrose (control). The new bone formation was evaluated at 1 and 2 weeks after surgery (n = 16) by radiological and histological analysis.ResultsThe conducted radiological analysis showed that the new bone volume was significantly greater in the dissociated soft-tissue suspension group. This finding was further confirmed by the conducted histological analysis.ConclusionsThe dissociated mucosa tissue suspension enhanced bone regeneration in vivo; thus, it is a promising potential method to aid the successful application for bone augmentation in the implant practice.

Astragaloside IV Improves Tibial Defect in Rats and Promotes Proliferation and Osteogenic Differentiation of hBMSCs through MiR-124-3p.1/STAT3 Axis.

Astragaloside IV (AST-IV) facilitates the proliferation and migration of osteoblast-like cells. We sought to explore the effect and potential mechanism of AST-IV on regeneration of tibial defects. To reveal the effect of AST-IV on regeneration of tibial defects in rat, HE staining and microcomputed tomography (μCT) were performed on tibial bone. The binding relationship between miR-124-3p.1 and STAT3 was analyzed by TargetScan V7.2 and a dual-luciferase reporter assay. Human bone marrow mesenchymal stromal/stem cells (hBMSCs) were identified by morphological observation and flow-cytometric analysis. To reveal the effect and mechanism of AST-IV on phenotypes of hBMSCs, hBMSCs were treated with AST-IV, miR-124-3p.1 mimic, and pcDNA-STAT3, and cell viability, cell cycle, ALP activity, and calcium deposition of hBMSCs in vitro were determined by MTT, flow-cytometric analysis, ELISA, and Alizarin red staining, respectively. The expressions of osteoblast marker molecules (RUNX2, OCN, Smad4), miR-124-3p.1, and STAT3 were indicated by RT-qPCR and Western blot. AST-IV decreased miR-124-3p.1 expression, increased STAT3 expression in tibial bone defects, and promoted regeneration of tibial bone defects in a concentration-dependent manner. The hBMSCs appeared spindle-shaped and were positive for CD105, but negative for CD34. MiR-124-3p.1 negatively regulated STAT3 expression in hBMSCs under osteogenic conditions. AST-IV promoted viability, cell cycle, ALP activity, and osteogenic differentiation of hBMSCs along with increased expressions of osteoblast marker molecules, which was partially reversed by miR-124-3p.1 overexpression. However, the effect of miR-124-3p.1 overexpression on hBMSCs was also partially reversed by STAT3 overexpression. AST-IV improves tibial defects in rats and promotes proliferation and osteogenic differentiation of hBMSCs through the miR-124-3p.1/STAT3 axis.

Osteogenic Differentiation of Renal Interstitial Fibroblasts Promoted by lncRNA MALAT1 May Partially Contribute to Randall's Plaque Formation.

BackgroundThe current belief is that Randall’s plaques (RP) constitute a nidus for the formation of idiopathic calcium oxalate stones, but the upstream events in RP formation remain unclear. The present study aimed to investigate whether RP formation shares similarities with biomineralization and to illustrate the potential role played by the lncRNA MALAT1 in osteogenic differentiation of human renal interstitial fibroblasts (hRIFs).Materials and MethodsBiomineralization and MALAT1 expression were assessed in RP, and hRIFs were isolated and induced under osteogenic conditions for further experiments. The transcription initiation and termination sites in MALAT1 were identified by 5′ and 3′ RACE. RNA immunoprecipitation assays and luciferase assays were used to validate the interactions among MALAT1, Runx2 and miRNAs.ResultsUpregulated expression of osteogenic markers and MALAT1 was observed in RP and hRIFs induced with osteogenic medium. Biomineralization in RP and calcium phosphate (CaP) deposits in induced hRIFs were further verified by electron microscopy. Furthermore, overexpression of MALAT1 promoted the osteogenic phenotype of hRIFs, while treatment with a miR-320a-5p mimic and knockdown of Runx2 significantly suppressed the osteogenic phenotype. Further analysis showed that MALAT1 functioned as a competing endogenous RNA to sponge miR-320a-5p, leading to upregulation of Runx2 and thus promoting osteogenic differentiation of hRIFs.ConclusionEctopic calcification and MALAT1 partially contributed to the formation of RP, in which MALAT1 might promote Runx2 expression to regulate osteogenic differentiation of hRIFs by sponging miRNA-320a-5p. The current study sheds new light on the lncRNA-directed mechanism of RP formation via a process driven by osteogenic-like cells.

Collagen type I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media.

Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen.

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.

Metabolic Switch Under Glucose Deprivation Leading to Discovery of NR2F1 as a Stimulus of Osteoblast Differentiation.

Poor survival of grafted cells is the major impediment of successful cell-based therapies for bone regeneration. Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose-deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose-deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In the absence of glucose, expression levels of all metabolites involved in glycolysis were significantly decreased than those in a glucose-supplemented state. In glucose-deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)-predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose-deprived and glucose-supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. Small, interfering RNA (siRNA) for NR2F1 reduced cell viability and osteogenic differentiation of hBMSCs under glucose-supplemented conditions whereas NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose-deprived osteogenic conditions via the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) pathway. NR2F1-transfected hBMSCs significantly enhanced new bone formation in a critical size long-bone defect of rats compared with control vector-transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration. © 2022 American Society for Bone and Mineral Research (ASBMR).

Abstract 17021: A Novel Human Relaxin Receptor Agonist, ML290, Attenuates Atherosclerosis- and Chronic Kidney Disease-Induced Vascular Calcification in Mice

Introduction: Vascular calcification is the most significant predictor of cardiovascular morbidity and mortality, but therapeutic options are unavailable. Relaxin has emerged as a vasoprotective molecule, but several drawbacks prevent therapeutic translation. Targeting the relaxin receptor, RXFP1, is safe and well-tolerated in animal models of vascular disease and humans. We identified a biased allosteric agonist of human RXFP1, ML290, and aimed to test the hypothesis that ML290 arrests the progression of vascular calcification in mouse models of atherosclerosis and chronic kidney disease (CKD). Methods and Results: Recurrent treatment with ML290 significantly prevented ( P = 0.0422, n = 8) and reversed ( P = 0.0489, n = 6) atherosclerotic calcification in humanized ( hRXFP1/hRXFP1 ) Apoe -/- mice fed an atherogenic diet. Longitudinal tracing of mineral formation in the aortic arch of these mice revealed the presence of mineral in vehicle- but not ML290-treated mice after 15 weeks of diet. Accelerated mineral growth was observed in vehicle-treated mice after 20 weeks of the diet, which was reduced by ML290 treatment. In humanized mice with CKD, ML290 significantly prevented ( P = 0.0344, n = 9) medial calcification. In vitro , ML290 reduced ( P = 0.0005, n = 3) superoxide production under osteogenic conditions in vascular smooth muscle cells (VSMCs). Osteogenic changes in VSMC phenotype associate with a release of alkaline phosphatase (ALP) in extracellular vesicles (EVs), which promote mineralization. ML290 treatment significantly ( P = 0.0001, n = 3) suppressed the formation of ALP-loaded EVs in vitro . Bone morphogenetic protein-4, an inducer of osteogenic transitions, and caveolin-1, a scaffolding protein required for calcifying EV formation, were significantly ( P = 0.0059, n = 4) down-regulated after 24 h treatment with ML290 compared to vehicle-treated VSMCs under osteogenic conditions. Conclusions: We demonstrate the therapeutic potential for ML290 to mitigate atherosclerosis and CKD-induced vascular calcification in vivo . The actions of ML290 to prevent medial calcification are in part attributed to its ability to limit the release of calcifying EVs as a result of osteogenic differentiation, and to reduce vascular superoxide production.

Abstract 16123: In Vitro Valve-on-chip Platform to Assess the Effects of Hemodynamic and Mechanical Stimuli on Early Calcific Disease Progression

Introduction: Calcific aortic valve disease (CAVD) involves mechanical stress, endothelial dysfunction, matrix remodeling, and mineral deposition. Complex mechanical and hemodynamic environment around the valve closely affects its function, biology, and disease. However, the exact role of these forces in regulating cellular dysfunction during disease initiation is yet to be elucidated. Methods and Results: We developed a 3D valve-on-chip (VOC) model that encompasses cell-cell and cell-matrix interactions, flow shear stresses and cyclic strains, better mimicking the native valve. The multilayered VOC construct was built in house using photolithography and soft lithography and a silicon based polymer (Fig.1A). Valve endothelial cells (VECs) are seeded in a monolayer on a porous PDMS membrane (Fig.B) which separates the VECs from the a matrigel-collagen semi interpenetrating hybrid gel housing valve interstitial cells (VICs) (Fig.3C). We simulated either static or 20% (pathological) strain under quiescent or osteogenic conditions. Osteogenic VICs demonstrated nodule formation (Fig.1D) and had reduced redox ratios in 20% stretch conditions suggesting disease progression (Fig.1E,F). Effect of mechanical and hemodynamic forces on structure, function, and phenotypic changes in VECs and VICs particularly during disease initiation will be further assessed. Conclusions: This VOC model for VEC-VIC co-culture better mimics the native valve environment and can be used to study the early disease mechanisms. The VOC platform can be used as a drug testing tool and aid in development of treatment strategies through efficient employment in the laboratory. Further exploring the effect of mechanical and hemodynamic forces may reveal mechanosensitive signaling cascades involved in disease progression, which can be targeted for CAVD therapeutics.

Abstract 15843: A Novel Role for Telomerase in Calcific Aortic Valve Disease

Introduction: Calcific aortic valve disease (CAVD) is a disorder characterized by the slow, but a progressive thickening of the aortic valve leaflet that develops into severe calcification. The only current therapy is aortic valve replacement. Telomerase is an enzymatic complex best known for its telomere-extending activities on the ends of chromosomes yet, the catalytic subunit (TERT) has been implicated in multiple non-canonical transcriptional and epigenetic activities, including priming of mesenchymal stem cells (MSCs) to differentiate into osteoblasts, and transcriptional regulation of inflammatory genes. Hypothesis: We hypothesize that non-canonical TERT activity contributes to the progression of CAVD. Methods: We performed biochemical assays to study the role of TERT in the calcification process using primary tissues and valve interstitial cells (VICs) from control and CAVD patients, smooth muscle cells (SMCs) from WT and Tert knockout mice, and mesenchymal stem cells (MSCs). Results: We found that TERT protein is highly expressed in calcified aortic valves and VICs isolated from patients with calcified valves, compared to healthy valves. VICs can be induced to calcify under osteogenic differentiation conditions, and we found that TERT accumulated after fourteen days in this culture, with no effect on either telomere length, proliferation, or senescence. We expanded the scope of our approach by evaluating TERT's influence on calcification of mice aortic smooth muscle cells (mSMCs). We found WT mSMCs readily calcified in vitro, but mSMCs from Tert knockout mice did not, and Tert deletion also reduced valve calcification in a Ldlr / Tert double knockout mice model compared to Ldlr knockout alone. In VICs, shRNA mediated TERT downregulation reduced expression of RUNX2 . Finally, we found that inflammatory signals intensify in vitro calcification, induce TERT expression, and we show evidence that TERT interacts with STAT5. Conclusion: Our data suggest that TERT is required for valve calcification by stimulating transcriptional pathways promoting the osteogenic transition of quiescent VICs into calcifying VICs in the aortic valve. These results indicate that TERT is an active contributor to the calcification process of valve tissues.

Osteogenic differentiation potential of human bone marrow-derived mesenchymal stem cells enhanced by bacoside-A.

Stem cell therapy is growing rapidly to treat numerous diseases including bone-associated diseases. Mesenchymal stem cells (MSCs) are most commonly preferred to treat bone diseases because it possesses high osteogenic potency. Though, to obtain maximum osteogenic efficiency of MSCs is challenging. Therefore, this study was planned to evaluate the osteogenic efficiency of human bone marrow derived mesenchymal stem cells (hBMSCs) by bacoside-A. This study was investigated the activity of alkaline phosphatase (ALP) and expressions of the genes specific to osteogenic regulation mainly runt-related transcription factor 2 (Runx2), osterix (Osx), osteocalcin (OCN) and collagen type Iα1 (Col I α1) in hBMSCs cultured under osteogenic conditions at different concentrations of bacoside-A for 14 days. The results of this study depicted significant upregulation in the activity of ALP and expressions of osteogenic regulator genes in bacoside-A treated cells when compared with control cells. Besides, expressions of glycogen synthase kinase-3β (GSK-3β) and Wnt/β-catenin were evaluated; these expressions were also significantly increased in bacoside-A treated cells when compared with control cells. This result provides a further supporting evidence of bacoside-A role on osteogenesis in hBMSCs. The present study suggest that bacoside-A will be applied to ameliorate the process of osteogenesis in hBMSCs to repair damaged bone structure during MSC-based therapy; this will be an excellent and auspicious treatment for bone-associated disorders including osteoporosis. Significance of the study Osteoporosis is a bone metabolic disorder characterized by an imbalance between the activity of osteoblastic bone formation and osteoclastic bone resorption that disrupts the bone microarchitecture. Current anti-osteoporotic drugs are inhibiting bone resorption, but they are unable to restore the bone structure due to extreme bone remodelling process and causes numerous side effects. The finding of natural bioactive compounds with osteogenic property is very essential for osteoporosis treatment. This study was reported that bacoside-A ameliorated osteogenic differentiation of hBMSCs through upregulation of osteogenic differentiation genes and Wnt/β-catenin signalling pathway. This result is indicating that bacoside-A may be useful for osteoporosis treatments.

Bone Regeneration Potential of Human Dental Pulp Stem Cells Derived from Elderly Patients and Osteo-Induced by a Helioxanthin Derivative.

Human dental pulp stem cells (DPSCs) have high clonogenic and proliferative potential. We previously reported that a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2–b]pyridine-2-carboxamide (TH)) enhances osteogenic differentiation of DPSCs derived from young patients. However, in the clinical field, elderly patients more frequently require bone regenerative therapy than young patients. In this study, we examined and compared the osteogenic differentiation potential of TH-induced DPSCs from elderly patients and young patients to explore the potential clinical use of DPSCs for elderly patients. DPSCs were obtained from young and elderly patients and cultured in osteogenic medium with or without TH. We assessed the characteristics and osteogenic differentiation by means of specific staining and gene expression analyses. Moreover, DPSC sheets were transplanted into mouse calvarial defects to investigate osteogenesis of TH-induced DPSCs by performing micro-computed tomography (micro-CT). We demonstrated that osteogenic conditions with TH enhance the osteogenic differentiation marker of DPSCs from elderly patients as well as young patients in vitro. In vivo examination showed increased osteogenesis of DPSCs treated with TH from both elderly patients and young patients. Our results suggest that the osteogenic differentiation potential of DPSCs from elderly patients is as high as that of DPSCs from young patients. Moreover, TH-induced DPSCs showed increased osteogenic differentiation potential, and are thus a potentially useful cell source for bone regenerative therapy for elderly patients.

Extracellular matrix derived from allogenic decellularized bone marrow mesenchymal stem cell sheets for the reconstruction of osteochondral defects in rabbits

Bioactive scaffolds from synthetical polymers or decellularized cartilage matrices have been widely used in osteochondral regeneration. However, the risks of potential immunological reactions and the inevitable donor morbidity of these scaffolds have limited their practical applications. To address these issues, a biological extracellular matrix (ECM) scaffold derived from allogenic decellularized bone marrow mesenchymal stem cell (BMSC) sheets was established for osteochondral reconstruction. BMSCs were induced to form cell sheets. Three different concentrations of sodium dodecyl sulfate (SDS), namely, 0.5%, 1%, and 3%, were used to decellularize these BMSC sheets to prepare the ECM. Histological and microstructural observations were performed in vitro and then the ECM scaffolds were implanted into osteochondral defects in rabbits to evaluate the repair effect in vivo. Treatment with 0.5% SDS not only efficiently removed BMSCs but also successfully preserved the original structure and bioactive components of the ECM When compared with the 1% and 3% SDS groups, histological observations substantiated the superior repair effect of osteochondral defects, including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular cartilage integrated with native tissues in the 0.5% SDS group. Moreover, RT-PCR indicated that ECM scaffolds could promote the osteogenic differentiation potential of BMSCs under osteogenic conditions while increasing the chondrogenic differentiation potential of BMSCs under chondrogenic conditions. Allogenic BMSC sheets decellularized with 0.5% SDS treatment increased the recruitment of BMSCs and significantly improved the regeneration of osteochondral defects in rabbits, thus providing a prospective approach for both articular cartilage and subchondral bone reconstruction with cell-free transplantation.

Acquisition of cancer stem cell properties in osteosarcoma cells by defined factors

BackgroundCancer stem cells (CSCs) are considered to be responsible for tumor initiation, formation, and poor prognosis of cancer patients. However, the rarity of CSCs in clinical samples makes it difficult to elucidate characteristics of CSCs, especially in osteosarcoma (OS). The aim of this study is to verify whether it is possible to generate CSC-like cells by transducing defined factors into an OS cell line.MethodsWe retrovirally transduced the Octamer-binding transcription factor 3/4 (OCT3/4), Kruppel-like factor 4 (KLF4), and SRY-box transcription factor 2 (SOX2) genes into the MG-63 human OS cell line (MG-OKS). Parental and GFP-transduced MG-63 cells were used as negative control. We assessed the properties of the generated cells in vitro and in vivo. Multiple comparisons among groups were made using a one-way analysis of variance (ANOVA) followed by post hoc testing with Tukey’s procedure.ResultsMG-OKS cells in vitro exhibited the significantly increased mRNA expression levels of CSC markers (CD24, CD26, and CD133), decreased cell growth, increased chemoresistance and cell migration, and enhanced sphere formation. Notably, MG-OKS cells cultured under osteogenic differentiation conditions showed strongly positive staining for both Alizarin Red S and alkaline phosphatase, indicating osteogenesis of the cells. Gene ontology analysis of microarray data revealed significant upregulation of epidermal-related genes. Tumors derived from MG-OKS cells in vivo were significantly larger than those from other cells in μCT analysis, and immunohistochemical staining showed that Ki-67, osteocalcin, and HIF-1α-positive cells were more frequently detected in the MG-OKS-derived tumors.ConclusionsIn this study, we successfully generated OS CSC-like cells with significantly enhanced CSC properties following transduction of defined factors.

Remodeling of Three-Dimensional Collagen I Matrices by Human Bone Marrow Stromal Cells during Osteogenic Differentiation In Vitro

Cell fate is triggered by the characteristics of the surrounding extracellular matrix (ECM) including its composition and topological and mechanical properties. Human bone marrow stromal cells (hBMSC) are known to reside in a niche environment where they are maintained in a quiescent, multipotent state, also controlled by the ECM characteristics. In this in vitro study, three-dimensional (3D) fibrillary collagen I (Col)-based matrices with defined topological and mechanical characteristics were used (pore size of 3-4 μm, fibril diameter of ∼0.7 μm, ∼90 Pa (non-cross-linked), and ∼160 Pa (cross-linked)), mimicking conditions of the environment in the bone marrow. The performance of non-cross-linked and cross-linked scaffolds during osteogenic differentiation of hBMSC in terms of matrix stiffness and proteolytic degradability was investigated. Cell adhesion, morphology, and invasion as well as matrix remodeling were investigated on cross-linked and non-cross-linked Col matrices over 22 days. About 25% of the cells invaded the matrices and showed a spread morphology independent of cross-linking. Cellular proteolytic matrix degradation in terms of a decreased matrix layer thickness was only found for non-cross-linked matrices at constant pore size and fibril diameter. Osteogenic differentiation of hBMSC was examined by alkaline phosphatase staining and enzyme activity (early marker) and calcium phosphate deposition (late marker) and was similarly supported in both scaffolds. Furthermore, both matrices were strongly stiffened by about 10-fold because of high mineralization under osteogenic conditions. In summary, these results emphasize that fibrillary 3D Col matrices are a suitable model to study primary hBMSC behavior in terms of ECM remodeling during osteogenesis at defined in vitro conditions.