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Objective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1 foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1 PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg-1 of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the matrix of effect of time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra(Y-matrix).(4)After the data of X and Y matrices were standardized,partial least squares(PLS)was used to calculate and analyze the quantitative and qualitative effect relationship,and variable importance for projection(VIP)>1 was used as the threshold to screen the effect components related to the changes of cell surface molecules CD41a and CD61,and chemical composition identification,as the potential effector components in the total flavonoid extracts of Sarcandra glabra were used to promote the differentiation of megakaryocytes,and finally the regression evaluation system was used to verify the efficacy of its medicinal effect.Results(1)Compared with the Dami group,the expression level of CD41a on the surface of Dami cells in the control group was significantly increased(P＜0.05).Compared with the control group,the expression levels of CD41a and CD61 on the surface of Dami cells in the PMA group were significantly increased(P＜0.01).Compared with the PMA group,the expression levels of CD41a and CD61 on the surface of Dami cells in the model group were significantly reduced(P＜0.01).(2)Compared with the blank plasma group,the expression levels of the molecules CD41a and CD61 on the surface of Dami cells at each time point of 15,30,60,90,120,and 240 minutes were significantly increased(P＜0.01),and the expression levels of CD41a and CD61 were both highest in the 30-minute group.The potential effective components with VIP value greater than 1 were screened out in the positive and negative ion mode,and 540.3638@12.25 and 559.2991@11.53 were selected for pharmacodynamic verification.559.2991@11.53 was identified as daucosterol(Dau),540.3638@12.25 was identified as rosmarinic acid 4-O-β-D-glucoside(Ros).After Ros and Dau intervened in the megakaryocyte differentiation and maturation disorder model respectively,the expression levels of CD41a and CD61 on the surface of Dami cells in the low-,medium-and high-dose groups(40,60 and 80 μg·mL-1)of Ros and Dau were significantly increased compared with the model group(P＜0.05,P＜0.01).Conclusion Ros and Dau may be the active components of the total flavonoids extracts of Sarcandra glabra to promote the differentiation of megakaryocytes.

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Background: Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics. Methods: BM donors (age: 48-85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed. Results: The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs. Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.

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Biopolymer hydrogels have become an important group of biomaterials in experimental and clinical use. However, unlike metallic or mineral materials, they are quite sensitive to sterilization. The aim of this study was to compare the effects of gamma irradiation and supercritical carbon dioxide (scCO2) treatment on the physicochemical properties of different hyaluronan (HA)- and/or gelatin (GEL)-based hydrogels and the cellular response of human bone marrow-derived mesenchymal stem cells (hBMSC). Hydrogels were photo-polymerized from methacrylated HA, methacrylated GEL, or a mixture of GEL/HA. The composition and sterilization methods altered the dissolution behavior of the biopolymeric hydrogels. There were no significant differences in methacrylated GEL release but increased methacrylated HA degradation of gamma-irradiated samples. Pore size/form remained unchanged, while gamma irradiation decreased the elastic modulus from about 29 kPa to 19 kPa compared to aseptic samples. HBMSC proliferated and increased alkaline phosphatase activity (ALP) particularly in aseptic and gamma-irradiated methacrylated GEL/HA hydrogels alike, while scCO2 treatment had a negative effect on both proliferation and osteogenic differentiation. Thus, gamma-irradiated methacrylated GEL/HA hydrogels are a promising base for multi-component bone substitute materials.

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The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems.

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BACKGROUND/AIMS: Although several studies have demonstrated that mesenchymal stromal cells (MSCs) exhibit beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been controversial. Recent evidence has shown that MSCs modify their in vivo immunomodulatory actions depending on the specific inflammatory environment encountered. Accordingly, we assessed whether the therapeutic properties of human mesenchymal stromal cells (hMSCs) could be potentiated by conditioning these cells with serum (hMSC-serum) obtained from patients with asthma and then transplanted in an experimental model of house dust mite (HDM)-induced allergic asthma. METHODS: hMSC and hMSC-serum were administered intratracheally 24 h after the final HDM challenge. hMSC viability and inflammatory mediator production, lung mechanics and histology, bronchoalveolar lavage fluid (BALF) cellularity and biomarker levels, mitochondrial structure and function as well as macrophage polarization and phagocytic capacity were assessed. RESULTS: Serum preconditioning led to: (i) increased hMSC apoptosis and expression of transforming growth factor-ß, interleukin (IL)-10, tumor necrosis factor-α-stimulated gene 6 protein and indoleamine 2,3-dioxygenase-1; (ii) fission and reduction of the intrinsic respiratory capacity of mitochondria; and (iii) polarization of macrophages to M2 phenotype, which may be associated with a greater percentage of hMSCs phagocytosed by macrophages. Compared with mice receiving hMSCs, administration of hMSC-serum led to further reduction of collagen fiber content, eotaxin levels, total and differential cellularity and increased IL-10 levels in BALF, improving lung mechanics. hMSC-serum promoted greater M2 macrophage polarization as well as macrophage phagocytosis, mainly of apoptotic hMSCs. CONCLUSIONS: Serum from patients with asthma led to a greater percentage of hMSCs phagocytosed by macrophages and triggered immunomodulatory responses, resulting in further reductions in both inflammation and remodeling compared with non-preconditioned hMSCs.

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The bone cancer osteosarcoma, found mainly in adolescents, routinely forms around the growth plate/metaphysis of long bones. Bone marrow composition changes with age, shifting from a more hematopoietic to an adipocyte-rich tissue. This conversion occurs in the metaphysis during adolescence, implicating a link between bone marrow conversion and osteosarcoma initiation. To assess this, the tri-lineage differentiation potential of human bone marrow stromal cells (HBMSCs) isolated from the femoral diaphysis/metaphysis (FD) and epiphysis (FE) was characterized and compared to two osteosarcoma cell lines, Saos-2 and MG63. Compared to FE-cells, FD-cells showed an increase in tri-lineage differentiation. Additionally, differences were found between the Saos-2 cells exhibiting higher levels of osteogenic differentiation, lower adipogenic differentiation, and a more developed chondrogenic phenotype than MG63, with the Saos-2 being more comparable to FD-derived HBMSCs. The differences found between the FD and FE derived cells are consistent with the FD region containing more hematopoietic tissue compared to the FE. This may be related to the similarities between FD-derived cells and Saos-2 cells during osteogenic and chondrogenic differentiation. These studies reveal distinct differences in the tri-lineage differentiations of 'hematopoietic' and 'adipocyte rich' bone marrow, which correlate with specific characteristics of the two osteosarcoma cell lines.

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In human, bone loss is associated with increased marrow adipose tissue and recent data suggest that medullary adipocytes could play a role in osteoporosis by acting on neighboring bone-forming osteoblasts. Supporting this hypothesis, we previously showed, in a coculture model based on human bone marrow stromal cells, that factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this work, we employed an original integrative bioinformatics approach connecting proteomic and transcriptomic data from adipocytes and osteoblasts, respectively, to investigate the mechanisms underlying their crosstalk. Our analysis identified a total of 271 predicted physical interactions between adipocyte-secreted proteins and osteoblast membrane protein coding genes and proposed three pathways for their potential contribution to osteoblast transdifferentiation, the PI3K-AKT, the JAK2-STAT3 and the SMAD pathways. Our findings demonstrated the effectiveness of our integrative omics strategy to decipher cell-cell communication events.

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Human bone marrow stromal cell (hBMSC)-derived exosomes are promising therapeutics for inflammatory diseases due to their unique microRNA (miRNA) and protein cargos. Periodontal diseases often present with chronicity and corresponding exuberant inflammation, which leads to loss of tooth support. In this study, we explored whether hBMSC exosomes can affect periodontitis progression. hBMSC exosomes were isolated from cell culture medium through sequential ultracentrifugation. miRNAs and proteins that were enriched in hBMSC exosomes were characterized by RNA sequencing and protein array, respectively. hBMSC exosomes significantly suppressed periodontal keystone pathogen Porphyromonas gingivalis-triggered inflammatory response in macrophages in vitro. Transcriptomic analysis suggested that exosomes exerted their effects through regulating cell metabolism, differentiation, and inflammation resolution. In vivo, weekly exosome injection into the gingival tissues reduced the tissue destruction and immune cell infiltration in rat ligature-induced periodontitis model. Collectively, these findings suggest that hBMSC-derived exosomes can potentially be used as a host modulation agent in the management of periodontitis.

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In this work we dissected the osteoinductive properties of selected, PLGA-based scaffolds enriched with gel-derived bioactive glasses (SBGs) of either binary SiO2-CaO or ternary SiO2-CaO-P2O5 system, differing in CaO/SiO2 ratio (i.e. high -or low-calcium SBGs). To assess the inherent ability of the scaffolds to induce osteogenesis of human bone marrow stromal cells (BMSC), the study was designed to avoid any osteogenic stimuli beyond the putative osteogenic SBG component of the studied scaffolds. The bioactivity and porosity of scaffolds were confirmed by SBF test and porosimetry. Condition media (CM) from BMSC-loaded scaffolds exhibited increased Ca and decreased P content corresponding to SBGs CaO/SiO2 ratio, whereas Si content was relatively stable and overall lower in CM from scaffolds containing binary SBGs. CM from cell-loaded scaffolds containing high-calcium, binary SBGs promoted migration of BMSC and BMP-response in reporter osteoblast cell line. BMSC culture on these scaffolds or the ones containing ternary, low-calcium SBGs resulted in the activation of BMP-related signaling and expression of several osteogenic markers. Ectopic bone formation was induced by scaffolds containing binary SBGs, but high-calcium ones produced significantly more osteoid. Scaffolds containing ternary SBGs negatively influenced the expression of osteogenic transcription factors and Cx43, involved in cell-cell interactions. High-calcium scaffolds stimulated overall higher Cx43 expression. We believe the initial cell-cell communication may be crucial to induce and maintain osteogenesis and high BMP signaling on the studied scaffolds. The presented scaffolds' biological properties may also constitute new helpful markers to predict osteoinductive potential of other bioactive implant materials.

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This article aims to investigate the mechanism of behaviors of human bone marrow stromal cells (hBMSCs) affected by scaffold structure combining Monte Carlo feature selection (MFCS), incremental feature selection (IFS) and support vector machine (SVM). The specific differentially expressed genes (DEGs) of hBMSCs cultured on nanofiber (NF) scaffolds and freeform fabrication (FFF) scaffolds were obtained. Key genes were screened from common genes between osteogenic DEGs and NF specific DEGs with MFCS, IFS and SVM. The results demonstrated that NF scaffolds induced hBMSCs to express more genes related to osteogenic differentiation. Finally, 16 key genes were identified among the common genes. The common genes were significantly enriched in Rap1 signaling pathway, extracellular matrix and ossification. The results in this study suggested that the gene expression of hBMSCs was sensitive to NF scaffolds and FFF scaffolds, and the osteogenic differentiation of hBMSCs could be enhanced by NF scaffolds.

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Synthetically sulfated hyaluronan derivatives were shown to facilitate osteogenic differentiation of human bone marrow stromal cells (hBMSC) by application in solution or incorporated in thin collagen-based coatings. In the presented study, using a biomimetic three-dimensional (3D) cell culture model based on fibrillary collagen I (3D Col matrix), we asked on the impact of binding mode of low sulfated hyaluronan (sHA) in terms of adsorptive and covalent binding on osteogenic differentiation of hBMSC. Both binding modes of sHA induced osteogenic differentiation. Although for adsorptive binding of sHA a strong intracellular uptake of sHA was observed, implicating an intracellular mode of action, covalent binding of sHA to the 3D matrix induced also intense osteoinductive effects pointing towards an extracellular mode of action of sHA in osteogenic differentiation. In summary, the results emphasize the relevance of fibrillary 3D Col matrices as a model to study hBMSC differentiation in vitro in a physiological-like environment and that sHA can display dose-dependent osteoinductive effects in dependence on presentation mode in cell culture scaffolds.

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Glycyrrhizic acid (GA) is a major triterpene glycoside isolated from liquorice root that has been shown to inhibit osteoclastogenesis. However, there have been no reports regarding the effect of GA on osteogenic differentiation. Therefore, this study was performed to explore the effects and mechanism of action of GA on osteogenesis. A CCK-8 array was used to assess cell viability. The osteogenic capability was investigated by real-time quantitative PCR, western blotting and immunofluorescence analyses. ALP staining and ARS were used to evaluate ALP activity and mineralization, respectively. GA-GelMA hydrogels were designed to verify the therapeutic effects of GA in vivo by radiographic analysis and histological evaluation. Our results show that GA had no significant influence on the viability or proliferation of human bone marrow stromal cells (hBMSCs). GA promoted osteogenic differentiation and enhanced calcium deposition. Furthermore, ratio of active ß-catenin and total ß-catenin protein increased after treatment with GA. Wnt/catenin signaling inhibitor partially attenuated the effects of GA on osteogenic differentiation. In a mouse femoral fracture model, GA-GelMA hydrogels accelerated bone healing. Our results show that GA promotes the osteogenic differentiation of hBMSCs by modulating the Wnt/ß-catenin signaling pathway. GA-GelMA hydrogels promoted bone fracture healing. GA has potential as a cost-effective treatment of bone defects.

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OBJECTIVE: The antibacterial activity of copper (Cu)-alloy biomaterials has shown a great potential in clinical application. Here, we evaluated the osteogenesis and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy in an in vivo model of infected bone defects and determine their responsible proteins and pathways using proteomics. METHODS: After bone defects were filled with Ti6Al4V and Ti6Al4V-6.5wt%Cu implants for 6 week, the tissue and bone samples around the implants were harvested for radiographic, micro-CT, histological, and bone-related gene expression analyses. An iTRAQ-based protein identification/quantification approach was used to analyze the osteogenic and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy. RESULTS: Imaging and histological results showed Ti6Al4V alloy induced a stronger inflammatory response than Ti6Al4V-6.5wt%Cu alloy; imaging results and osteogenic protein levels showed Ti6Al4V-6.5wt%Cu alloy exerted a stronger osteogenic effect. In vitro experiment, we found the Ti6Al4V-6.5wt%Cu had significant antibacterial effects and inhibited the activity of Staphylococcus aureus in the early stage. In addition, the bacterial biofilm formed in Ti6Al4V-6.5wt%Cu group was significantly lower than that in Ti6Al4V group. Proteomic screening of 4279 proteins resulted in 35 differentially expressed proteins for further examination which were mainly associated with the cellular process, metabolic process, stimulus response, and cellular component organization. In further exploration of the mechanism of osteogenic mineralization of Ti6Al4V-6.5wt%Cu alloy, we found out SDC4 and AGRN were the top two target proteins associated with osteogenic differentiation and bone mineralization. CONCLUSION: Ti6Al4V-6.5wt%Cu alloy shows a great potential as a bone implant material due to its positive effects against bacterial infection and on bone formation. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: At present, titanium alloys and other non-antibacterial metal materials are used in orthopedic internal fixation operations. Our study demonstrates that Ti6Al4V-6.5wt%Cu alloy has good antibacterial and osteogenic effects in vivo and in vitro. This means that Ti6Al4V-6.5wt%Cu alloy may become a new kind of antimicrobial metallic material as internal fixation material to continuously exert its antimicrobial effects and reduce the infection rate after clinical internal fixation.

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The development and maintenance of a functioning vascular system is a critical function for many aspects of tissue growth and regeneration. Vascular endothelial cell in vitro co-culture spheroids are self-organized cell composites that have the capacity to recapitulate the three-dimensional tissue microenvironment. These spheroid testing platforms aim to better understand the mechanisms of functional tissue and how new therapeutic agents can drive these 3D co-culture processes. Here we describe direct cell-cell 3D endothelial co-culture spheroid methods, to examine the physiological spatial growth and cell-cell interaction of vascular cells and surrounding native tissue cells in the formation of vascular networks within spheroids and the potential to regenerate tissue.

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Expandable implants including shape memory alloy (SMA) elements have great potential to minimize the risk of implant loosening and to increase the primary stability of bone anchoring. Surface structuring of such elements may further improve these properties and support osteointegration and bone healing. In this given study, SMA sheets were processed by deploying additive and removal manufacturing technologies for 3D-printed surgical implants. The additive technology was realized by applying a new laser beam melting technology to print titanium structures on the SMA sheets. The removal step was realized as a standard process with an ultrashort-pulse laser. The morphology, metabolic activity, and mineralization patterns of human bone marrow stromal cells were examined to evaluate the biocompatibility of the new surface structures. It was shown that both surface structures support cell adhesion and the formation of a cytoskeleton. The examination of the metabolic activity of the marrow stromal cells on the samples showed that the number of cells on the laser-structured samples was lower when compared to the 3D-printed ones. The calcium phosphate accumulation, which was used to examine the mineralization of marrow stromal cells, was higher in the laser-structured samples than in the 3D-printed ones. These results indicate that the additive- and laser-structured SAM sheets seem biocompatible and that the macrostructure surface and manufacturing technology may have positive influences on the behavior of the bone formation. The use of the new additive technique and the resulting macrostructures seems to be a promising approach to combine increased anchorage stability with simultaneously enhanced osteointegration.

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P0-related protein (PZR), a Noonan and Leopard syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and Leopard syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with ß1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(α5ß1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.

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OBJECTIVES: Polydatin (PD), extracted from Polygonum cuspidatum, has shown potential therapeutic applications due to its antiosteoporotic and anti-inflammatory activities. Our previous study suggested that PD promotes the osteogenesis of human bone marrow stromal cells (hBMSCs) via the BMP2-Wnt/ß-catenin pathway. The aim of our present study was to further explore the role of PD-mediated regulation of Tafazzin (TAZ), a transcriptional coactivator with a PDZ-binding motif, in osteogenesis. MATERIALS AND METHODS: hBMSCs were isolated and treated with PD at various concentrations. Alizarin red staining and RT-qPCR were performed to identify calcium complex deposition in hBMSCs as well as the expression of specific osteoblast-related markers, respectively, in each group. Next, TAZ-silenced hBMSCs were generated by lentivirus-produced TAZ shRNA. After treatment with PD, the osteogenic abilities of the TAZ-silenced and control hBMSCs were estimated by ALP activity assay, and expression of the TAZ protein was detected by Western blot analysis and immunofluorescence staining. In vitro, an ovariectomized (OVX) mouse model was established and used to evaluate the effect of PD on bone destruction by micro-CT, immunohistochemistry, and ELISA. RESULTS: In vitro, 30 µM PD significantly improved the proliferation and calcium deposition of hBMSCs and markedly stimulated the expression of the mRNAs RUNX2, Osteopontin, DLX5, ß-catenin, TAZ, and Osteocalcin (OCN). Osteogenic differentiation induced by PD was blocked by lentivirus-mediated TAZ shRNA. Furthermore, Noggin (a regulator of bone morphogenic protein 2 (BMP2)) and DKK1 (an inhibitor of the Wnt/ß-catenin pathway) were found to inhibit the increase in TAZ expression induced by PD. In vivo, PD prevented estrogen deficiency-induced bone loss in the OVX mouse model. CONCLUSION: Taken together, our findings suggest that PD improved the osteogenic differentiation of hBMSCs and maintained the bone matrix in the OVX mouse model through the activation of TAZ, a potential target gene of the BMP2-Wnt/ß-catenin pathway.

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Osteoporosis is an age-related deterioration in bone health that is, at least in part, a stem cell disease. The different mechanisms and signaling pathways that change with age and contribute to the development of osteoporosis are being identified. One key upstream mechanism that appears to target a number of osteogenic pathways with age is kynurenine, a tryptophan metabolite and an endogenous Aryl hydrocarbon receptor (AhR) agonist. The AhR signaling pathway has been reported to promote aging phenotypes across species and in different tissues. We previously found that kynurenine accumulates with age in the plasma and various tissues including bone and induces bone loss and osteoporosis in mice. Bone marrow mesenchymal stem cells (BMSCs) are responsible for osteogenesis, adipogenesis, and overall bone regeneration. In the present study, we investigated the effect of kynurenine on BMSCs, with a focus on autophagy and senescence as two cellular processes that control BMSCs proliferation and differentiation capacity. We found that physiological levels of kynurenine (10 and 100 µM) disrupted autophagic flux as evidenced by the reduction of LC3B-II, and autophagolysosomal production, as well as a significant increase of p62 protein level. Additionally, kynurenine also induced a senescent phenotype in BMSCs as shown by the increased expression of several senescence markers including senescence associated ß-galactosidase in BMSCs. Additionally, western blotting reveals that levels of p21, another marker of senescence, also increased in kynurenine-treated BMSCs, while senescent-associated aggregation of nuclear H3K9me3 also showed a significant increase in response to kynurenine treatment. To validate that these effects are in fact due to AhR signaling pathway, we utilized two known AhR antagonists: CH-223191, and 3',4'-dimethoxyflavone to try to block AhR signaling and rescue kynurenine /AhR mediated effects. Indeed, AhR inhibition restored kynurenine-suppressed autophagy levels as shown by levels of LC3B-II, p62 and autophagolysosomal formation demonstrating a rescuing of autophagic flux. Furthermore, inhibition of AhR signaling prevented the kynurenine-induced increase in senescence associated ß-galactosidase and p21 levels, as well as blocking aggregation of nuclear H3K9me3. Taken together, our results suggest that kynurenine inhibits autophagy and induces senescence in BMSCs via AhR signaling, and that this may be a novel target to prevent or reduce age-associated bone loss and osteoporosis.

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Kynurenine, a metabolite of tryptophan breakdown, has been shown to increase with age, and plays a vital role in a number of age-related pathophysiological changes, including bone loss. Accumulation of kynurenine in bone marrow stromal cells (BMSCs) has been associated with a decrease in cell proliferation and differentiation, though the exact mechanism by which kynurenine mediates these changes is poorly understood. MiRNAs have been shown to regulate BMSC function, and accumulation of kynurenine may alter the miRNA expression profile of BMSCs. The aim of this study was to identify differentially expressed miRNAs in human BMSCs in response to treatment with kynurenine, and correlate miRNAs function in BMSCs biology through bioinformatics analysis. Human BMSCs were cultured and treated with and without kynurenine, and subsequent miRNA isolation was performed. MiRNA array was performed to identify differentially expressed miRNA. Microarray analysis identified 50 up-regulated, and 36 down-regulated miRNAs in kynurenine-treated BMSC cultures. Differentially expressed miRNA included miR-1281, miR-330-3p, let-7f-5p, and miR-493-5p, which are important for BMSC proliferation and differentiation. KEGG analysis found up-regulated miRNA targeting glutathione metabolism, a pathway critical for removing oxidative species. Our data support that the kynurenine dependent degenerative effect is partially due to changes in the miRNA profile of BMSCs.

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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.