Aucubin promotes bone-fracture healing via the dual effects of anti-oxidative damage and enhancing osteoblastogenesis of hBM-MSCs.

BACKGROUND: Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo. METHODS: CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site. RESULTS: Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration. CONCLUSIONS: Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway.

Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach.

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1ß (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture.

MTHFR C677T mutation affects adipogenic differentiation abilities of human bone marrow-derived mesenchymal stem cells.

The effects of 5,10-Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) viability, morphology, physiology and differentiation capacity were investigated in this study. For this purpose, primary hBM-MSCs with wild type (WT, C/C), heterozygote (HTZ, C/T) and homozygote (HMZ, T/T) for the MTHFR gene were obtained with ethical committee permission and donor informed. Mutations were detected using RFLP and Sanger sequencing methods from genomic DNA isolated from cells, colonization properties were investigated by CFU-F test and proliferative differences were investigated by MTT test. Adipogenic, osteogenic, and chondrogenic differentiation were induced to study changes in their differentiation potentials, and the results were statistically analyzed using one-way ANOVA with Graphpad Prism. A total of 13 donors were screened and there were no differences in the hBM-MSC markers and in vitro morphologies of the cells. While there were significant differences between WT and HTZ as a result of the CFU-F test, there were no significant differences in the MTT test after 24 and 48 h. As a result of differentiation tests, it was found that adipogenic differentiation was significantly more in HMZ cells than WT cells. Osteogenic and chondrogenic differentiation results did not give statistically significant results. As a result of these experiments, adipogenic differentiation was found to be affected by the MTHFR genotype in hBM-MSCs.

Biomimetic Cell-Substrate of Chitosan-Cross-linked Polyaniline Patterning on TiO2 Nanotubes Enables hBM-MSCs to Differentiate the Osteoblast Cell Type.

Titanium-based substrates are widely used in orthopedic treatments and hard tissue engineering. However, many of these titanium (Ti) substrates fail to interact properly between the cell-to-implant interface, which can lead to loosening and dislocation from the implant site. As a result, scaffold implant-associated complications and the need for multiple surgeries lead to an increased clinical burden. To address these challenges, we engineered osteoconductive and osteoinductive biosubstrates of chitosan (CS)-cross-linked polyaniline (PANI) nanonets coated on titanium nanotubes (TiO2NTs) in an attempt to mimic bone tissue's major extracellular matrix. Inspired by the architectural and tunable mechanical properties of such tissue, the TiO2NTs-PANI@CS-based biofilm conferred strong anticorrosion, the ability to nucleate hydroxyapatite nanoparticles, and excellent biocompatibility with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). An in vitro study showed that the substrate-supported cell activities induced greater cell proliferation and differentiation compared to cell-TiO2NTs alone. Notably, the bone-related genes (collagen-I, OPN, OCN, and RUNX 2) were highly expressed within TiO2NTs-PANI@CS over a period of 14 days, indicating greater bone cell differentiation. These findings demonstrate that the in vitro functionality of the cells on the osteoinductive-like platform of TiO2NTs-PANI@CS improves the efficiency for osteoblastic cell regeneration and that the substrate potentially has utility in bone tissue engineering applications.

Circular RNA Hsa_circ_0006766 targets microRNA miR-4739 to regulate osteogenic differentiation of human bone marrow mesenchymal stem cells.

Circular RNAs (circRNAs) are emerging as important regulators in bone metabolism, which is mediated by microRNA (miRNA) sponges. However, it is not clear how circRNA regulates osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).Therefore, based on the previous circRNA chip results, hsa_circ_0006766, which is differentially expressed in the osteogenic differentiation of hBM-MSCs, was screened out, and bioinformatics analysis was performed to predict potential target miRNAs. During osteogenic differentiation of hBM-MSCs, hsa_circ_0006766 and its target miRNAs (miR-4739, miR-619-5p, miR-5787, miR-7851-3p, and miR-3192-5p) were detected by quantitative Real Time-PCR (qRT-PCR). Target gene prediction for the differentially expressed target miRNAs was performed, and target genes were validated by dual-luciferase reporter gene assay and qRT-PCR. It is shown that hsa_circ_0006766 was up-regulated and miR-4739 was down-regulated during osteogenic differentiation of hBM-MSCs.Moreover, the target gene Notch2 was predicted to be highly expressed during osteogenic differentiation. And dual-luciferase assay proved that Notch2 was the gene targeting to miR-4739. Taken together, our finding confirmed that hsa_circ_0006766 may act as a major regulatory part in osteogenic differentiation of hBM-MSCs via an hsa_circ_0006766-miR-4739-Notch2 regulatory axis. Accordingly, hsa_circ_0006766 may affect the development of osteoporosis and may thus become a therapeutic target.

Platelet lysates-based hydrogels incorporating bioactive mesoporous silica nanoparticles for stem cell osteogenic differentiation.

Scaffolds for bone tissue regeneration should provide the right cues for stem cell adhesion and proliferation, but also lead to their osteogenic differentiation. Hydrogels of modified platelet lysates (PLMA) show the proper mechanical stability for cell encapsulation and contain essential bioactive molecules required for cell maintenance. We prepared a novel PLMA-based nanocomposite for bone repair and regeneration capable of releasing biofactors to induce osteogenic differentiation. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were encapsulated in PLMA hydrogels containing bioactive mesoporous silica nanoparticles previously loaded with dexamethasone and functionalized with calcium and phosphate ions. After 21 d of culture, hBM-MSCs remained viable, presented a stretched morphology, and showed signs of osteogenic differentiation, namely the presence of significant amounts of alkaline phosphatase, bone morphogenic protein-2 and osteopontin, hydroxyapatite, and calcium nodules. Developed for the first time, PLMA/MSNCaPDex nanocomposites were able to guide the differentiation of hBM-MSCs without any other osteogenic supplementation.

Novel Nanocomposite PLA Films with Lignin/Zinc Oxide Hybrids: Design, Characterization, Interaction with Mesenchymal Stem Cells.

Herein we present the production of novel nanocomposite films consisting of polylactic acid (PLA) polymer and the inclusion of nanoparticles of lignin (LNP), ZnO and hybrid ZnO@LNP (ZnO, 3.5% wt, ICP), characterized by similar regular shapes and different diameter distribution (30-70 nm and 100-150 nm, respectively). The obtained set of binary, ternary and quaternary systems were similar in surface wettability and morphology but different in the tensile performance: while the presence of LNP and ZnO in PLA caused a reduction of elastic modulus, stress and deformation at break, the inclusion of ZnO@LNP increased the stiffness and tensile strength (σb = 65.9 MPa and EYoung = 3030 MPa) with respect to neat PLA (σb = 37.4 MPa and EYoung = 2280 MPa). Neat and nanocomposite PLA-derived films were suitable for adult human bone marrow-mesenchymal stem cells and adipose stem cell cultures, as showed by their viability and behavior comparable to control conditions. Both stem cell types adhered to the films' surface by vinculin focal adhesion spots and responded to the films' mechanical properties by orchestrating the F-actin-filamin A interaction. Collectively, our results support the biomedical application of neat- and nanocomposite-PLA films and, based on the absence of toxicity in seeded stem cells, provide a proof of principle of their safety for food packaging purposes.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

Tendon and Cytokine Marker Expression by Human Bone Marrow Mesenchymal Stem Cells in a Hyaluronate/Poly-Lactic-Co-Glycolic Acid (PLGA)/Fibrin Three-Dimensional (3D) Scaffold.

We developed a (three-dimensional) 3D scaffold, we named HY-FIB, incorporating a force-transmission band of braided hyaluronate embedded in a cell localizing fibrin hydrogel and poly-lactic-co-glycolic acid (PLGA) nanocarriers as transient components for growth factor controlled delivery. The tenogenic supporting capacity of HY-FIB on human-Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) was explored under static conditions and under bioreactor-induced cyclic strain conditions. HY-FIB elasticity enabled to deliver a mean shear stress of 0.09 Pa for 4 h/day. Tendon and cytokine marker expression by hBM-MSCs were studied. Results: hBM-MSCs embedded in HY-FIB and subjected to mechanical stimulation, resulted in a typical tenogenic phenotype, as indicated by type 1 Collagen fiber immunofluorescence. RT-qPCR showed an increase of type 1 Collagen, scleraxis, and decorin gene expression (3-fold, 1600-fold, and 3-fold, respectively, at day 11) in dynamic conditions. Cells also showed pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokine gene expressions, with a significant increase of anti-inflammatory cytokines in dynamic conditions (IL-10 and TGF-ß1 300-fold and 4-fold, respectively, at day 11). Mechanical signaling, conveyed by HY-FIB to hBM-MSCs, promoted tenogenic gene markers expression and a pro-repair cytokine balance. The results provide strong evidence in support of the HY-FIB system and its interaction with cells and its potential for use as a predictive in vitro model.

Thermoresponsive Stiffness Softening of Hierarchically Porous Nanohybrid Membranes Promotes Niches for Mesenchymal Stem Cell Differentiation.

Despite the attention given to the development of novel responsive implants for regenerative medicine applications, the lack of integration with the surrounding tissues and the mismatch with the dynamic mechanobiological nature of native soft tissues remain in the current products. Hierarchical porous membranes based on a poly (urea-urethane) (PUU) nanohybrid have been fabricated by thermally induced phase separation (TIPS) of the polymer solution at different temperatures. Thermoresponsive stiffness softening of the membranes through phase transition from the semicrystalline phase to rubber phase and reverse self-assembly of the quasi-random nanophase structure is characterized at body temperature near the melting point of the crystalline domains of soft segments. The effects of the porous structure and stiffness softening on proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBM-MSCs) are investigated. The results of immunohistochemistry, histological, ELISA, and qPCR demonstrate that hBM-MSCs maintain their lineage commitment during stiffness relaxation; chondrogenic differentiation is favored on the soft and porous scaffold, while osteogenic differentiation is more prominent on the initial stiff one. Stiffness relaxation stimulates more osteogenic activity than chondrogenesis, the latter being more influenced by the synergetic coupling effect of softness and porosity.

Human bone marrow mesenchymal stem cells-derived exosomes alleviate liver fibrosis through the Wnt/ß-catenin pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) are increasingly being applied as a therapy for liver fibrosis. Exosomes possess similar functions to their parent cells; however, they are safe and effective cell-free reagents with controllable and predictable outcomes. In this study, we investigated the therapeutic potential and underlying molecular mechanism for human bone mesenchymal stem cells-derived exosomes (hBM-MSCs-Ex) in the treatment of liver fibrosis. METHODS: We established an 8-week CCl4-induced rat liver fibrosis model, after which, we administered hBM-MSCs-Ex in vivo for 4 weeks. The resulting histopathology, liver function, and inflammatory cytokines were analyzed. In addition, we investigated the anti-fibrotic mechanism of hBM-MSCs-Ex in hepatic stellate cells (HSCs) and liver fibrosis tissue, by western blotting for the expression of Wnt/ß-catenin signaling pathway-related genes. RESULTS: In vivo administration of hBM-MSCs-Ex effectively alleviated liver fibrosis, including a reduction in collagen accumulation, enhanced liver functionality, inhibition of inflammation, and increased hepatocyte regeneration. Moreover, based on measurement of the collagen area, Ishak fibrosis score, MDA levels, IL-1, and IL-6, the therapeutic effect of hBM-MSCs-Ex against liver fibrosis was significantly greater than that of hBM-MSCs. In addition, we found that hBM-MSCs-Ex inhibited the expression of Wnt/ß-catenin pathway components (PPARγ, Wnt3a, Wnt10b, ß-catenin, WISP1, Cyclin D1), α-SMA, and Collagen I, in both HSCs and liver fibrosis tissue. CONCLUSIONS: These results suggest that hBM-MSCs-Ex treatment could ameliorate CCl4-induced liver fibrosis via inhibition of HSC activation through the Wnt/ß-catenin pathway.

MicroRNA-499a-5p Promotes Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to Cardiomyocytes.

Since the adult mammalian heart has limited regenerative capacity, cardiac trauma, disease, and aging cause permanent loss of contractile tissue. This has fueled the development of stem cell-based strategies to provide the damaged heart with new cardiomyocytes. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of self-renewal and differentiation into cardiomyocytes, albeit inefficiently. MicroRNAs (miRNAs, miRs) are non-coding RNAs that have the potential to control stem cell fate decisions and are employed in cardiac regeneration and repair. In this study, we tested the hypothesis that overexpression of miR-499a induces cardiomyogenic differentiation in BM-MSCs. Human BM-MSCs (hBM-MSCs) were transduced with lentiviral vectors encoding miR-499a-3p or miR-499a-5p and analyzed by immunostaining and western blotting methods 14 days post-transduction. MiR-499a-5p-transduced cells adopted a polygonal/rod-shaped (myocyte-like) phenotype and showed an increase in the expression of the cardiomyocyte markers α-actinin and cTnI, as cardiogenic differentiation markers. These results indicate that miR-499a-5p overexpression promotes the cardiomyogenic differentiation of hBM-MSCs and may thereby increase their therapeutic efficiency in cardiac regeneration.

Extremely low-frequency electromagnetic field promotes astrocytic differentiation of human bone marrow mesenchymal stem cells by modulating SIRT1 expression.

It has been shown that extremely low-frequency electromagnetic fields (ELFMF) affect regulation of cell fate and differentiation. Thus, the aim of this study was to investigate the role of ELFMFs in the enhancement of astrocytic differentiation. ELFMF exposure reduced the rate of proliferation and enhanced astrocytic differentiation. The ELFMF-treated cells showed increased levels of the astrocyte marker (GFAP), while those of the early neuronal marker (Nestin) and stemness marker (OCT3/4) were downregulated. The reactive oxygen species (ROS) level was observed to be significantly elevated after ELFMF exposure, which strengthens the modulatory role of SIRT1 and SIRT1 downstream molecules (TLE1, HES1, and MASH1) during astrocytic differentiation. After nicotinamide (5 mM) mediated inhibition of SIRT1, levels of TLE1, HES1, and MASH1 were examined; TLE1 was significantly upregulated and MASH1 was downregulated. These results suggest that ELFMFs induce astrocytic differentiation through activation of SIRT1 and SIRT1 downstream molecules.

In vitro biocompatibility and proliferative effects of polar and non-polar extracts of cucurbita ficifolia on human mesenchymal stem cells.

Cucurbita ficifolia (C. ficifolia) has been traditionally known for its medicinal properties as an antioxidant, anti-diabetic and anti-inflammatory agent. However, there has been an enduring attention towards the identification of unique method, to isolate the natural components for therapeutic applications. Our study focuses on different polar and non-polar solvents (methanol, hexane and chloroform) to extract the bioactive components from C. ficifolia (pumpkin) and to study the biocompatibility and cytotoxicity effects on human bone marrow-mesenchymal stem cells (hBM-MSCs). The extracts were screened for their effects on cytotoxicity, cell proliferation and cell cycle on the hBM-MSCs cell line. The assays demonstrated that the chloroform extract was highly biocompatible, with less cytotoxic effect, and enhanced the cell proliferation. The methanol extract did not exhibit significant cytotoxicity when compare to the control. Concordantly, the cell cycle analysis confirmed that chloroform extract enhances the proliferation at lower concentrations. On the other hand, hexane extract showed high level of cytotoxicity with apoptotic and necrotic changes in hBM-MSCs. Collectively, our data revealed that chloroform is a good candidate to extract the bioactive components from C. ficifolia. Furthermore, our results suggest that specific gravity and density of the solvent might play a crucial role in the extraction process, which warrants further investigations.

The effect of ultrasound for increasing neural differentiation in hBM-MSCs and inducing neurogenesis in ischemic stroke model.

AIMS: This study's purpose was to evaluate the effect of ultrasound in air at a frequency of 0.04MHz and an intensity of 50mW/cm2 on neural differentiation of hBM-MSCs in vitro and on neurogenesis in an ischemic stroke model. MATERIALS AND METHODS: hBM-MSCs were exposed to 0.04MHz ultrasound and then compared with no exposed one in cell morphology, lactate dehydrogenase (LDH) activity, RT-PCR, and Western blot. In addition, we made stroke model mice by means of the photothrombosis (PT) method and these models were exposed to 0.04MHz ultrasound after hBM-MSCs injection. We compared with sham group in histological and immunohistochemical analysis and western blot. KEY FINDINGS: Ultrasound induced neural differentiation without cell death. In stroke models, inflammatory cells were observed around the infarct area in the Cell, Cell/Ultrasound group and the brain infarct volume in the Cell/Ultrasound group was smaller than in the sham group. Further, the expression of neural proteins in the Cell/Ultrasound group was increased relative to the sham group. SIGNIFICANCE: The present study showed that ultrasound promotes neural differentiation of hBM-MSC and neurogenesis in a mouse stroke model. This may be applicable as a therapeutic device with the aim of inducing neurogenesis following stroke.

Effect of human mesenchymal stem cell transplantation on cerebral ischemic volume-controlled photothrombotic mouse model.

Various animal models of stroke have been developed to simulate the human stroke with the development of the ischemic method facilitates preclinical stroke research. The photothrombotic ischemia model, based on the intravascular photochemical reaction, is widely used for in vivo studies. However, this study has limitations, which generated a relatively small-sized infarction model on superficial cortex compared to that of the MCAO stroke model. In this study, the photothorombosis mouse model is adapted and the optimum conditions for generation of cell death and deficits with high reproducibility is determined. The extent of damage within the cortex was assessed by infarct volume and cellular/behavioral analyses. In this model, the neural cell death and inflammatory responses is detected; moreover, the degree of behavioral impairment is correlated with the brain infarct volume. Further, to enhance the understanding of neural repair, the effect of neural differentiation by transplantation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is analyzed. The authors demonstrated that transplantation of BM-MSCs promoted the neural differentiation and behavioral performance in their photothrombosis model. Therefore, this research was meaningful to provide a stable animal model of stroke with low variability. Moreover, this model will facilitate development of novel MSC-based therapeutics for stroke.

Osteogenic differentiation of human bone marrow mesenchymal stem cells in hydrogel containing nacre powder.

Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials.

Role of stromal cell-mediated Notch signaling in CLL resistance to chemotherapy.

Stromal cells are essential components of the bone marrow (BM) microenvironment that regulate and support the survival of different tumors, including chronic lymphocytic leukemia (CLL). In this study, we investigated the role of Notch signaling in the promotion of survival and chemoresistance of human CLL cells in coculture with human BM-mesenchymal stromal cells (hBM-MSCs) of both autologous and allogeneic origin. The presence of BM-MSCs rescued CLL cells from apoptosis both spontaneously and following induction with various drugs, including Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone. The treatment with a combination of anti-Notch-1, Notch-2 and Notch-4 antibodies or γ-secretase inhibitor XII (GSI XII) reverted this protective effect by day 3, even in presence of the above-mentioned drugs. Overall, our findings show that stromal cell-mediated Notch-1, Notch-2 and Notch-4 signaling has a role in CLL survival and resistance to chemotherapy. Therefore, its blocking could be an additional tool to overcome drug resistance and improve the therapeutic strategies for CLL.

Long-term Angiogenesis Efficacy Using a Heparin-Conjugated Fibrin (HCF) Delivery System with HBM-MSCs

BACKGROUND AND OBJECTIVES: Heparin-conjugated fibrin (HCF) is suitable for the release and localization of bFGF. We analyzed the effects of a bFGF delivery system using HCF with human bone marrow-derived mesenchymal stem cells (HBM- MSCs) in a dog ischemic limb model. METHODS AND RESULTS: Animals were divided into HBM-MSCs, HBM-MSCs+HCF, bFGF-HCF, and HBM-MSCs+bFGF-HCF groups. A total of 1x10(7) HBM-MSCs were injected per animal, and the amount of bFGF was 1 mg per dog. Ischemic muscles were harvested at eight weeks and six months after injection of cells. The HBM-MSCs+bFGF-HCF group exhibited decreased proportions of capillaries and arterioles six months after transplantation. However, there were more cells positive for the angiogenic factors, VEGF and PDGF, in the eight-week specimens compared with those harvested six months after transplantation. CONCLUSIONS: Our results demonstrated that a single injection of HBM-MSCs did not have significant long-term angiogenic effects; however, a bFGF delivery system using HCF exerted prolonged angiogenic effects when combined with HBM-MSCs.

Long-term Angiogenesis Efficacy Using a Heparin-Conjugated Fibrin (HCF) Delivery System with HBM-MSCs.

BACKGROUND AND OBJECTIVES: Heparin-conjugated fibrin (HCF) is suitable for the release and localization of bFGF. We analyzed the effects of a bFGF delivery system using HCF with human bone marrow-derived mesenchymal stem cells (HBM- MSCs) in a dog ischemic limb model. METHODS AND RESULTS: Animals were divided into HBM-MSCs, HBM-MSCs＋HCF, bFGF-HCF, and HBM-MSCs＋ bFGF-HCF groups. A total of 1×10(7) HBM-MSCs were injected per animal, and the amount of bFGF was 1 mg per dog. Ischemic muscles were harvested at eight weeks and six months after injection of cells. The HBM-MSCs＋ bFGF-HCF group exhibited decreased proportions of capillaries and arterioles six months after transplantation. However, there were more cells positive for the angiogenic factors, VEGF and PDGF, in the eight-week specimens compared with those harvested six months after transplantation. CONCLUSIONS: Our results demonstrated that a single injection of HBM-MSCs did not have significant long-term angiogenic effects; however, a bFGF delivery system using HCF exerted prolonged angiogenic effects when combined with HBM-MSCs.