ROCK Y-27632 Inhibitor, Ascorbic Acid, and Trehalose Increase Survival of Human Wharton Jelly Mesenchymal Stem Cells After Cryopreservation.

OBJECTIVES: Wharton jelly mesenchymal stem cells are good candidates for application in different aspects of regenerative medicine, and their long-time banking is important. In this study, the effects of trehalose, ascorbic acid, and Y-27632 on proliferation and survival rate of these cells after cryopreservation were investigated. MATERIALS AND METHODS: Mesenchymal stem cells were isolated from human umbilical cord Wharton jelly and frozen using a slow-rate cooling process. Different concentrations of trehalose (35, 75, and 125 mM), ascorbic acid (0.06, 0.125, 0.25, and 0.5 mM), and Y-27632 (10 µM) were used to treat culture medium and/or to supplement freezing medium. Assessment of cell viability after thawing was performed using Trypan blue staining, and MTT assay was performed to measure the cell proliferation rate. RESULTS: We observed significantly increased postthaw viability, increased cell proliferation, and decreased doubling time of cells when 75 mM trehalose, 0.25 and 0.5 mM ascorbic acid, and 10 mM Y-27632 were used. In addition, increased viability, proliferation, and attachment were observed after 24 hours of pretreatment with these cryoprotective agents and when they were added to conventional freezing medium. CONCLUSIONS: The use of different cryoprotective agents in culture and freezing media could be useful for long-term storage of Wharton jelly mesenchymal stem cells.

Microtubule destabilization caused by silicate via HDAC6 activation contributes to autophagic dysfunction in bone mesenchymal stem cells.

BACKGROUND: Silicon-modified biomaterials have been extensively studied in bone tissue engineering. In recent years, the toxicity of silicon-doped biomaterials has gradually attracted attention but requires further elucidation. This study was designed to explore whether high-dose silicate can induce a cytotoxicity effect in bone mesenchymal stem cells (BMSCs) and the role of autophagy in its cytotoxicity and mechanism. METHODS: Morphologic changes and cell viability of BMSCs were detected after different doses of silicate exposure. Autophagic proteins (LC3, p62), LC3 turnover assay, and RFP-GFP-LC3 assay were applied to detect the changes of autophagic flux following silicate treatment. Furthermore, to identify the potential mechanism of autophagic dysfunction, we tested the acetyl-α-tubulin protein level and histone deacetylase 6 (HDAC6) activity after high-dose silicate exposure as well as the changes in microtubule and autophagic activity after HDAC6 siRNA was applied. RESULTS: It was found that a high dose of silicate could induce a decrease in cell viability; LC3-II and p62 simultaneously increased after high-dose silicate exposure. A high concentration of silicate could induce autophagic dysfunction and cause autophagosomes to accumulate via microtubule destabilization. Results showed that acetyl-α-tubulin decreased significantly with high-dose silicate treatment, and inhibition of HDAC6 activity can restore microtubule structure and autophagic flux. CONCLUSIONS: Microtubule destabilization caused by a high concentration of silicate via HDAC6 activation contributed to autophagic dysfunction in BMSCs, and inhibition of HDAC6 exerted a cytoprotection effect through restoration of the microtubule structure and autophagic flux.

Ginkgo Biloba L. Extract Reduces H2O2-Induced Bone Marrow Mesenchymal Stem Cells Cytotoxicity by Regulating Mitogen-Activated Protein Kinase (MAPK) Signaling Pathways and Oxidative Stress.

BACKGROUND The oxidative stress environment of pathological tissue has an adverse effect on the survival of bone marrow mesenchymal stem cells (BMSCs) transplantation. Ginkgo biloba L. extract (EGB) has a potent antioxidant effect. In this research, we assessed the protective effects of EGB and EGB-Containing Serum (EGB CS) on BMSCs against injury induced by hydrogen peroxide (H2O2). MATERIAL AND METHODS BMSCs were pretreated with EGB or EGB CS and treated with H2O2. The cell counting kit-8 (CCK-8) method was utilized to detect cell viability. The DCFH-DA Fluorescent Kit method was used to detect intracellular ROS level. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and (CAT) were determined. The Hoechst staining assay and qRT-PCR assay were utilized to evaluate the effect of EGB on cell apoptosis. Mitogen-activated protein kinases (MAPKs) signaling pathway were detected by western blot analysis. RESULTS Compared to the H2O2 group, the number of apoptotic cells in the EGB and EGB CS pretreated groups significantly decreased. The mRNA expression ratio of Bax/Bcl-2 was also decreased. EGB and EGB CS can reduce the production of ROS in BMSCs exposed to H2O2. SOD, GSH-Px and CAT activities were significantly higher compared with those with H2O2 group. Furthermore, EGB or EGB CS pretreatment decreased the protein levels of p-p38MAPK and p-JNK in BMSCs compared to the H2O2 group. CONCLUSIONS Our findings suggested that EGB and EGB CS have protective effect on BMSCs against oxidative stress injury and increase the survival rate of BMSCs transplantation by regulating p38MAPK and JNK signaling.

Metformin Enhances Osteogenesis and Suppresses Adipogenesis of Human Chorionic Villous Mesenchymal Stem Cells.

Metformin is the first-line anti-hyperglycemic drugs commonly used to treat type 2 diabetes. Recent studies have shown that metformin can enhance bone formation through induction of endothelial nitric oxide synthase (eNOS). Human chorionic villous mesenchymal stem cells (CV-MSCs) are promising candidates for regenerative medicine. The present study aimed to investigate the effects of metformin on the osteogenic and adipocytic differentiation of human CV-MSCs, and to elucidate the underlying mechanism. CV-MSCs, prepared from human term placentae, were cultured with different concentrations of metformin. Treatment for 72 hours with 0.05 mM metformin had no noticeable effect on the proliferation of CV-MSCs. Consequently, CV-MSCs were cultured for seven or 14 days in the osteogenic medium supplemented with 0.05 mM metformin. Treatment for seven days with metformin increased the expression levels of osteogenic protein mRNAs, including alkaline phosphatase, runt-related transcription factor 2, and osteopontin. Metformin also enhanced the mineralization of CV-MSCs. Furthermore, metformin induced the expression of eNOS in CV-MSCs during osteogenic differentiation. By contrast, when CV-MSCs were cultured for 14 days in the adipogenic medium, 0.05 mM metformin inhibited the expression of adipogenic protein mRNAs, including proliferators-activated receptor-γ and CCAAT/enhancer binding protein-α. The lipid droplet accumulation was also reduced on 28 days after metformin treatment. These findings indicate that metformin can enhance osteogenic differentiation of CV-MSCs and reduce adipocyte formation. The effect of metformin on osteogenic differentiation of CV-MSCs may be associated with eNOS expression. Our findings will highlight the therapeutic potential of metformin in osteoporosis and bone fracture.

Melatonin reverses flow shear stress-induced injury in bone marrow mesenchymal stem cells via activation of AMP-activated protein kinase signaling.

Tissue-engineered heart valves (TEHVs) are a promising treatment for valvular heart disease, although their application is limited by high flow shear stress (FSS). Melatonin has a wide range of physiological functions and is currently under clinical investigation for expanded applications; moreover, extensive protective effects on the cardiovascular system have been reported. In this study, we investigated the protection conferred by melatonin supplementation against FSS-induced injury in bone marrow mesenchymal stem cells (BMSCs) and elucidated the potential mechanism in this process. Melatonin markedly reduced BMSC apoptotic death in a concentration-dependent manner while increasing the levels of transforming growth factor ß (TGF-ß), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and B-cell lymphoma 2 (Bcl2), and decreasing those of Bcl-2-associated X protein (Bax), p53 upregulated modulator of apoptosis (PUMA), and caspase 3. Notably, melatonin exerted its protective effects by upregulating the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), which promotes acetyl-CoA carboxylase (ACC) phosphorylation. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked the anti-FSS injury (anti-FSSI) effects of melatonin. Inhibition of AMPK by Compound C also counteracted the protective effects of melatonin, suggesting that melatonin reverses FSSI in BMSCs through the AMPK-dependent pathway. Overall, our findings indicate that melatonin contributes to the amelioration of FSS-induced BMSC injury by activating melatonin receptors and AMPK/ACC signaling. Our findings may provide a basis for the design of more effective strategies that promote the use of TEHCs in patients.

Proinflammatory Mediators Enhance the Osteogenesis of Human Mesenchymal Stem Cells after Lineage Commitment.

Several inflammatory processes underlie excessive bone formation, including chronic inflammation of the spine, acute infections, or periarticular ossifications after trauma. This suggests that local factors in these conditions have osteogenic properties. Mesenchymal stem cells (MSCs) and their differentiated progeny contribute to bone healing by synthesizing extracellular matrix and inducing mineralization. Due to the variation in experimental designs used in vitro, there is controversy about the osteogenic potential of proinflammatory factors on MSCs. Our goal was to determine the specific conditions allowing the pro-osteogenic effects of distinct inflammatory stimuli. Human bone marrow MSCs were exposed to tumor necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS). Cells were cultured in growth medium or osteogenic differentiation medium. Alternatively, bone morphogenetic protein 2 (BMP-2) was used as osteogenic supplement to simulate the conditions in vivo. Alkaline phosphatase activity and calcium deposition were indicators of osteogenicity. To elucidate lineage commitment-dependent effects, MSCs were pre-differentiated prior treatment. Our results show that TNF-α and LPS do not affect the expression of osteogenic markers by MSCs in the absence of an osteogenic supplement. In osteogenic differentiation medium or together with BMP-2 however, these mediators highly stimulated their alkaline phosphatase activity and subsequent matrix mineralization. In pre-osteoblasts, matrix mineralization was significantly increased by these mediators, but irrespective of the culture conditions. Our study shows that inflammatory factors potently enhance the osteogenic capacity of MSCs. These properties may be harnessed in bone regenerative strategies. Importantly, the commitment of MSCs to the osteogenic lineage greatly enhances their responsiveness to inflammatory signals.

Achyranthes bidentata saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK MAPK signaling pathway.

Achyranthes bidentata, is a herbal plant commonly used in the treatment of osteoporosis and bone nonunion in the Traditional Chinese Medicine. Saponins are the major compounds extracted from Achyranthes bidentata that have been shown to exert various pharmacological activities such as anti-inflammatory, antipyretic, antirheumatic, diuretic, and anti-osteoporosis. The Achyranthes bidentata saponins (ABS) were found to induce proliferation and differentiation in bone marrow stromal cells (BMSCs) as determined by the cell proliferation and alkaline phosphatase assays. Also, following the osteogenic induction, cells treated with ABS showed increased mRNA levels of rat bone morphogenetic protein-2, runt-related transcription factor 2, and osterix. Furthermore, ABS stimulated the activation of ERK as evidenced by increased phosphorylation of these proteins, which was blocked by an inhibitor of ERK (PD98059). Taken together, these results suggest that ABS stimulated osteogenic differentiation of BMSCs via activation of the ERK signaling pathway.

Myocardial protective effect of extracellular superoxide dismutase gene modified bone marrow mesenchymal stromal cells on infarcted mice hearts.

AIM: Extracellular superoxide dismutase (ecSOD) is a unique scavenger of superoxide anions and a promising target of gene therapy for ischemia/reperfusion injury (I/R). However, conventional gene therapies have limitation in effectiveness and efficiency. This study aimed to investigate the protective effects of ecSOD gene modified bone marrow mesenchymal stromal cells (BMSCs) on cardiac function improvement in mice infarcted heart. METHODS & RESULTS: BMSCs were isolated from Fluc(+) transgenic mice (Tg FVB[Fluc(+)]) and transfected by adenovirus combined with human ecSOD gene. ELISA was performed to determine ecSOD protein level. Female syngeneic FVB mice were randomized into 5 groups: (1) Sham group (sham); (2) MI group (MI); (3) MI+BMSCs group (BMSC); (4) MI+BMSCs-vector group (BMSC-vector); (5) MI+ BMSCs-ecSOD group (BMSC-ecSOD). MI was accomplished by ligation of the left anterior descending artery. BMSCs (2 x 10(6)) were injected into the border zone of infarction. In vivo bioluminescence imaging (BLI) was performed to monitor transplanted BMSCs viability. Echocardiography and histological staining revealed that BMSCs-ecSOD significantly reduced myocardial infarction size and improved cardiac function. Lucigenin chemiluminescence, DHE and TUNEL staining demonstrated that BMSCs-ecSOD delivery reduced ROS level and cell apoptosis both in vivo and in vitro. Western blot assay revealed that ecSOD supplementation increased FoxO3a phosphorylation in cardiomyocytes. Moreover, quantitative real-time PCR showed that pro-apoptotic factors (bim and bax) were decreased while the anti-apoptotic factor mir-21 expression was increased after ecSOD intervention. CONCLUSION: Intra-myocardial transplantation of adenovirus-ecSOD transfected BMSCs could exert potential cardiac protection against MI, which may be partly through reduction of oxidative stress and improvement of BMSCs survival.

Salvianolic acid B promotes osteogenesis of human mesenchymal stem cells through activating ERK signaling pathway.

Salvianolic acid B, a major bioactive component of Chinese medicine herb, Salvia miltiorrhiza, is widely used for treatment of cardiovascular diseases. Our recent studies have shown that Salvianolic acid B can prevent development of osteoporosis. However, the underlying mechanisms are still not clarified clearly. In the present study, we aim to investigate the effects of Salvianolic acid B on viability and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The results showed Salvianolic acid B (Sal B) had no obvious toxic effects on hMSCs, whereas Sal B supplementation (5µM) increased the alkaline phosphatase activity, osteopontin, Runx2 and osterix expression in hMSCs. Under osteogenic induction condition, Sal B (5µM) significantly promoted mineralization; and when the extracellular-signal-regulated kinases signaling (ERK) pathway was blocked, the anabolic effects of Sal B were diminished, indicating that Sal B promoted osteogenesis of hMSCs through activating ERK signaling pathway. The current study confirms that Sal B promotes osteogenesis of hMSCs with no cytotoxicity, and it may be used as a potential therapeutic agent for the management of osteoporosis.

Zinc in calcium phosphate mediates bone induction: in vitro and in vivo model.

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP - up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.

Osteoanabolic effect of alendronate and zoledronate on bone marrow stromal cells (BMSCs) isolated from aged female osteoporotic patients and its implications for their mode of action in the treatment of age-related bone loss.

SUMMARY: In the present study, we evaluated the potential for aminobisphosphonates to enhance the development of bone-forming osteoblasts from progenitor cells isolated from aged female osteoporotic patients. The aminobisphosphonates tested significantly enhanced osteoblast formation and thus lend further insights into their possible mode of action in the treatment of osteoporosis. INTRODUCTION: The primary aim of this study was to evaluate the influence of aminobisphosphonates on the osteogenesis of human bone marrow stromal cells (hBMSCs) and mineralization of differentiating bone-forming cells isolated from osteoporotic patients. METHODS: The influence of aminobisphosphonate treatment on hBMSC osteogenesis was assessed by the quantitative measurement of alkaline phosphatase (ALP) activity, in addition to quantitative reverse transcription polymerase chain reaction and Western blot analysis of known osteogenic markers. Mineralized matrix formation by hBMSC-derived osteoblasts was visualized and quantified using Alizarin red staining. RESULTS: hBMSC cultures treated with osteogenic medium supplemented with zoledronate demonstrated a significant increase in Alizarin red staining after 3 weeks as compared to cells cultured in osteogenic medium alone. Similarly, cultures of differentiating hBMSCs isolated from patients receiving alendronate treatment also demonstrated an increased propensity for mineralization, even in the absence of further in vitro stimulation by zoledronate. The stimulatory effects of aminobisphosphonate treatment on hBMSC-derived osteoblast-mediated mineralization were independent of any alterations in ALP activity, although significant decreases in the expression levels of osteopontin (SPP1) were evident in hBMSCs following exposure to aminobisphosphonates. Further analysis including Western blotting and loss-of-function studies revealed osteopontin as having a negative influence on the mineralization of differentiating osteoporotic bone-forming cells. CONCLUSIONS: The results presented here demonstrate for the first time that aminobisphosphonate treatment of osteoporotic hBMSCs enhances their capacity for osteoblast formation and subsequent mineral deposition, thus supporting the concept of aminobisphosphonates as having an osteoanabolic effect in osteoporosis.

Pigment epithelium-derived factor enhances differentiation and mineral deposition of human mesenchymal stem cells.

Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic factor found in a wide variety of tissues. Recent findings indicated that lack of PEDF leads to osteogenesis imperfecta type VI whose hallmark is a defect in mineralization. We investigated the effects of PEDF on human mesenchymal stem cells (hMSCs) and signaling pathways through which PEDF displays its activities in hMSCs. hMSCs incubated in a medium supplemented with PEDF induced expression of osteoblastic-related genes. In addition, PEDF induced alkaline phosphatase (ALP) activity in MSCs at 14 days of incubation in maintenance medium; hMSCs incubated in osteogenic medium in presence of PEDF expressed 19% more ALP activity (35.655 ± 1.827 U/mg protein, p = .041 than cells incubated in the same medium without PEDF supplementation (29.956 ± 2.100 U/µg protein). hMSCs incubated in osteogenic medium in presence of PEDF deposited 50% more mineral (2.108 ± 0.306 OD/ml per well per 1 × 10(4) cells per square centimeter, p = .017) than MSCs incubated in absence of the protein (1.398 ± 0.098 OD/ml per well per 1 × 10(4) cells per square centimeter) as determined by Alizarin Red quantitation. Reduction in PEDF expression in MSCs by siRNA led to decreased ALP activity (33.552 ± 2.009 U/ng protein of knockdown group vs. 39.269 ± 3.533 U/ng protein of scrambled siRNA group, p = .039) and significant reduction in mineral deposition (0.654 ± 0.050 OD/ml per well per 1 × 10(4) cells per square centimeter of knockdown group vs. 1.152 ± 0.132 OD/ml per well per 1 × 10(4) cells per square centimeter of wild-type group, p = .010). Decreased ALP activity and mineral deposition were restored by supplementation with exogenous PEDF protein. PEDF activated ERK and AKT signaling pathways in MSCs to induce expression of osteoblastic-related genes. These data suggest that PEDF is involved in MSCs osteoblastic differentiation.

The effects of Ca2SiO4-Ca3(PO4)2 ceramics on adult human mesenchymal stem cell viability, adhesion, proliferation, differentiation and function.

Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 wt.% or 3.0 wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate-Tricalcium Phosphate (C2S-TCP) were obtained by solid state reaction. All materials were composed of a single phase, αTCP in the case of a pure material, or solid solution of C2S in αTCP (αTCPss) for the doped αTCP. Viability, proliferation and in vitro osteoinductive capacity were investigated by seeding, adult mesenchymal stem cells of human origin (ahMSCs) which were CD73(+), CD90(+), CD105(+), CD34(-) and CD45(-) onto the 3 substrates for 30 days. Results show a non-cytotoxic effect after applying an indirect apoptosis test (Annexin V/7-AAD staining), so ahMSCs adhered, spread, proliferated and produced extracellular matrix (Heparan-sulfate proteoglycan (HS) and osteopontin (OP)) on all the ceramics studied. Finally, the cells lost the cluster differentiation marker expression CD73, CD90 y CD105 characteristic of ahMSCs and they showed an osteoblastic phenotype (Alkaline phosphatase activity (ALP), Osteocalcin production (OC), Collagen type I expression (Col-I), and production of mineralization nodules on the extracellular matrix). These observations were more evident in the αTCP ceramic doped with 1.5 wt.% C2S, indicating osteoblastic differentiation as a result of the increased concentration of solid solution of C2S in αTCP (αTCPss). Overall, these results suggest that the ceramics studied are cytocompatible and they are able to induce osteoblastic differentiation of undifferentiated ahMSCs.

Poly(L-lactide-co-glycolide) scaffolds coated with collagen and glycosaminoglycans: impact on proliferation and osteogenic differentiation of human mesenchymal stem cells.

In this study, we analyzed poly(L-lactide-co-glycolide) (PLGA) scaffolds modified with artificial extracellular matrices (aECM) consisting of collagen type I, chondroitin sulphate, and sulphated hyaluronan (sHya). We investigated the effect of these aECM coatings on proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. We found that scaffolds were homogeneously coated, and cross-linking of aECM did not significantly influence the amount of collagen immobilized. Cell proliferation was significantly increased on cross-linked surfaces in expansion medium (EM), but was retarded on cross-linked and non-cross-linked collagen/sHya coatings. The alkaline phosphatase activity was increased on sHya-containing coatings in EM even without the presence of differentiation supplements, but was six to ten times higher in differentiation medium (DM) and comparable for cross-linked and non-cross-linked collagen/sHya. The highest amount of calcium phosphate mineral was deposited on day 28 on cross-linked collagen/sHya. Therefore, coatings of PLGA scaffolds with collagen/sHya promoted the osteogenic differentiation of hMSCs in vitro and might be an interesting candidate for the modification of PLGA for bone reconstruction in vivo.

Osteogenic activity and antibacterial effects on titanium surfaces modified with Zn-incorporated nanotube arrays.

Titanium implants having enhanced osteogenic activity and antibacterial property are highly desirable for the prevention of implant associated infection and promotion of osseointegration. In this study, coatings containing titania nanotubes (NTs) incorporated with zinc (NT-Zn) are produced on Ti implants by anodization and hydrothermal treatment in Zn containing solutions. The amount of incorporated Zn can be adjusted by varying the structural parameters such as the nanotube diameter and length as well as hydrothermal treatment time. The suitable NT-Zn coatings with good intrinsic antibacterial properties can prevent post-operation infection. Excellent osteogenesis inducing ability in the absence of extraneous osteogenic supplements is demonstrated and the ERK1/2 signaling is found to be involved. The NT-Zn structure which is simple, stable, and easy to produce and scale up has immense potential in bone implant applications.

Caffeine regulates osteogenic differentiation and mineralization of primary adipose-derived stem cells and a bone marrow stromal cell line.

Caffeine consumption reportedly influences bone mineral density and body weight. However, the effects of caffeine on bone metabolism are still controversial, and whether the dosage of caffeine influences osteogenic differentiation is yet to be clarified. In the present study, we cultured primary adipose-derived stem cells (ADSCs) and a bone marrow stromal cell line (M2-10B4) in osteogenic differentiation media containing varying concentrations of caffeine. Caffeine had biphasic effects: 0.1 mM caffeine significantly enhanced mineralization and alkaline phosphatase (ALP) activity. Consistent with these observations, a caffeine concentration of 0.1 mM upregulated the osteogenic differentiation marker genes ALP and osteocalcin (OCN), and elevated osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2) and Sirtuin 1 (SIRT1) levels. However, a concentration of caffeine greater than 0.3 mM suppressed the differentiation of both the cell types. These findings indicate that caffeine has a beneficial effect on ADSCs and bone marrow stromal cells, enhancing differentiation to osteoblasts; this effect, which is mediated via RUNX2 activation at low doses is significantly suppressed at high doses.

Electroacupuncture promotes the differentiation of transplanted bone marrow mesenchymal stem cells overexpressing TrkC into neuron-like cells in transected spinal cord of rats.

Our previous study indicated that electroacupuncture (EA) could increase neurotrophin-3 (NT-3) levels in the injured spinal cord, stimulate the differentiation of transplanted bone marrow mesenchymal stem cells (MSCs), and improve functional recovery in the injured spinal cord of rats. However, the number of neuron-like cells derived from the MSCs is limited. It is known that NT-3 promotes the survival and differentiation of neurons by preferentially binding to its receptor TrkC. In this study, we attempted to transplant TrkC gene-modified MSCs (TrkC-MSCs) into the spinal cord with transection to investigate whether EA treatment could promote NT-3 secretion in the injured spinal cord and to determine whether increased NT-3 could further enhance transplanted MSCs overexpressing TrkC to differentiate into neuron-like cells, resulting in increased axonal regeneration and functional improvement in the injured spinal cord. Our results showed that EA increased NT-3 levels; furthermore, it promoted neuron-phenotype differentiation, synaptogenesis, and myelin formation of transplanted TrkC-MSCs. In addition, TrkC-MSC transplantation combined with EA (the TrkC-MSCs + EA group) treatment promoted the growth of the descending BDA-labeled corticospinal tracts (CSTs) and 5-HT-positive axonal regeneration across the lesion site into the caudal cord. In addition, the conduction of cortical motor-evoked potentials (MEPs) and hindlimb locomotor function increased as compared to controls (treated with the LacZ-MSCs, TrkC-MSCs, and LacZ-MSCs + EA groups). In the TrkC-MSCs + EA group, the injured spinal cord also showed upregulated expression of the proneurogenic factors laminin and GAP-43 and downregulated GFAP and chondroitin sulfate proteoglycans (CSPGs), major inhibitors of axonal growth. Together, our data suggest that TrkC-MSC transplantation combined with EA treatment spinal cord injury not only increased MSC survival and differentiation into neuron-like cells but also promoted CST regeneration across injured sites to the caudal cord and functional improvement, perhaps due to increase of NT-3 levels, upregulation of laminin and GAP-43, and downregulation of GFAP and CSPG proteins.

Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs.

Heterotypic cell interactions are essential for the homeostasis of bone tissue, in particular the widely studied interaction between osteoblasts and osteoclasts. Closely related with osteoclasts are monocytes/macrophages. These have been shown to produce osteogenic factors, e.g. BMP-2, which plays a key role in bone metabolism. However, the mechanisms through which monocytes/macrophages interact with osteoblasts are still elusive. The aim of this work was to assess the influence of human peripheral blood monocytes/macrophages over the early osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in the presence of dexamethasone-supplemented medium. The co-cultures were performed using porous transwells that allowed the interaction between both cell types through the production of paracrine factors. The potential effect of BMP-2 produced by monocytes/macrophages was addressed by adding an anti-BMP-2 antibody to the co-cultures. hBMSCs cultured in the presence of monocytes/macrophages had a higher proliferation rate than hBMSCs monocultures. The quantification of early osteogenic marker alkaline phosphatase (ALP) revealed higher activity of this enzyme in cells in the co-culture throughout the time of culture. Both of these effects were inhibited by adding an anti-BMP-2 antibody to the cultures. Moreover, qRT-PCR for osteocalcin and osteopontin transcripts showed overexpression of both markers. Once again, the effect of monocytes/macrophages over hBMSC osteogenic differentiation was completely inhibited in the co-cultures by blocking BMP-2. The present report confirmed that monocytes/macrophages produce BMP-2, which promotes osteogenic differentiation and proliferation of hBMSCs cumulatively to dexamethasone-supplemented medium. This potentially implies that monocyte/macrophages play a stronger role in bone homeostasis than so far supposed.

Sulfated hyaluronan/collagen I matrices enhance the osteogenic differentiation of human mesenchymal stromal cells in vitro even in the absence of dexamethasone.

Glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix (ECM) involved in different steps of the regulation of cellular differentiation. In this study artificial extracellular matrices (aECM) consisting of collagen (Col) I and different GAG derivatives were used as a substrate for human mesenchymal stromal cells (hMSC) to study osteogenic differentiation in vitro. hMSC were cultured on aECM containing col and hyaluronan sulfates (HyaS) with increasing degrees of sulfation (DS(S)) and were compared with aECM containing col and the natural GAG hyaluronan or chondroitin 4-sulfate. hMSC were analyzed for osteogenic differentiation markers such as calcium phosphate deposition, tissue non-specific alkaline phosphatase (TNAP) and expression of runt-related transcription factor 2 (runx2), osteocalcin (ocn) and bone sialoprotein II (bspII). Compared with aECM containing Col and natural GAG all Col/HyaS-containing aECM induced an increase in calcium phosphate deposition, TNAP activity and tnap expression. These effects were also seen in the absence of dexamethasone (an established osteogenic supplement). The expression of runx2 and ocn was not altered and the expression of bspII was diminished on the col/HyaS-containing aECM. The impact of the Col/HyaS-containing aECM on hMSC differentiation was independent of the DS(S) of the HyaS derivatives, indicating the importance of the primary (C-6) hydroxyl group of N-acetylglucosamine. These results suggest that Col/HyaS-containing aECM are able to stimulate hMSC to undergo osteogenic differentiation even in the absence of dexamethasone, which makes these matrices an interesting tool for hMSC-based tissue engineering applications and biomaterial functionalizations to enhance bone formation.

Silk ionomers for encapsulation and differentiation of human MSCs.

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.