ХИРУРГИЧЕСКОЕ ЛЕЧЕНИЕ НЕКРОЗА ГОЛОВКИ БЕДРЕННОЙ КОСТИ С ПРИМЕНЕНИЕМ ПРЕДДИФФЕРЕНЦИРОВАННЫХ МЕЗЕНХИМАЛЬНЫХ СТВОЛОВЫХ КЛЕТОК

Imaging the survival and utility of pre-differentiated allogeneic MSC in ischemic heart

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

Mesenchymal stem cells: A new strategy for the treatment of femoral head necrosis.

Objective To study curative effect of core decompression and autologous bone marrow stem cell transplantation combined with traditional Chinese medicine in the treatment of early femoral head necrosis (ANFH).Methods 30 patients with femoral head necrosis treated by different methods were divided into 3 groups: core decompression A group) 10 cases(12 hips) ;core decompression pus stem cells transplantation(B group) 10 cases(13 hips) ;core decompression pus stem cells transplantation combined with traditional Chinese medicine(C group) 10 ca-ses( 10 hips) ;The X-ray 、CT MRI、Harris score( HHS) .curative effect were observed.Results The phase of Ⅰ,Ⅱ,Ⅲ,in 3 groups didn't appear deformation and collapse at 3,6,12 months;The score of Harris after 12 months 93 points were higher than that preoperatively 57.5 points(χ 2= 5.81 ,P<0.05) ;The signal ratio of femoral head volume in MRI was 42% before treatment,disappearance of femoral head necrosis after treatment;Total curative effect of B,C group,werehighter than that Agroup(χ2 =3.81,χ2 =3.98,P<0.05).Conclusion The operative treatment of ANFH with core decompression and stem cells transplantation combined with traditional Chinese medicine had the advantage of minimal damage,simplicity,accuracy,and effectiveness. Key words: Femoral head necrosis; Decompression; Mesenchymal stem cells transplantation; Traditional Chinese Medicine; Curative effect

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model

Stem cell transplantation is one of the most promising yet enigmatic treatments for spinal cord injury (SCI), a common problem in dogs. As pre-differentiated mesenchymal stem cells (MSCs) can be expanded and differentiated into neurospheres in vitro, before being transplanted back, they may prove to be more beneficial for treating SCI. Therefore, we compared the endogenous differentiation potential, including the neuronal cell differentiation, of neurospheres from canine bone marrow MSCs (cBMMSCs) with that of the adipose tissue-derived MSCs (cADMSCs). Nestin-positive neurospheres were generated from MSCs derived from the bone marrow and adipose tissue. Neuronal cells were differentiated from the neurospheres derived from both these tissues. Gene expression analysis revealed that Nestin, βIII-tubulin, NCAM, OCT4 and SOX2 were expressed in MSCs and the corresponding neurospheres. Notably, cBMMSC-derived neuronal cells expressed higher levels of βIII-tubulin. The mRNA expressions of NANOG, Nestin, OCT4 and SOX2 were upregulated in neurospheres derived from both. Immunofluorescence analysis detected the expression of neuronal markers, namely, βIII-tubulin, GFAP, S100, NF200 and MAP2, in differentiated neuron-like cells. Our findings highlight that both cBMMSCs and cADMSCs could be differentiated into neurospheres and neuron-like cells, and therefore, these cells are suitable candidates for cell transplantation. Further, cADMSCs form a more suitable cell source, as larger number of cells could be harvested from cADMSC-derived neurospheres. Future studies employing in vivo transplantation models to investigate the effectiveness of MSCs for treating SCI are warranted.

Femoral head necrosis (FHN) is a clinically progressive disease that leads to overwhelming complications without an effective therapeutic approach. In recent decades, transplantation of mesenchymal stem cells (MSCs) has played a promising role in the treatment of FHN in the initial stage; however, the success rate is still low because of unsuitable cell carriers and abridged osteogenic differentiation of the transplanted MSCs. Biopolymeric-derived hydrogels have been extensively applied as effective cell carriers and drug vesicles; they provide the most promising contributions in the fields of tissue engineering and regenerative medicine. However, the clinical potential of hydrogels may be limited because of inappropriate gelation, swelling, mechanical characteristics, toxicity in the cross-linking process, and self-healing ability. Naturally, gelated commercial hydrogels are not suitable for cell injection and infiltration because of their static network structure. In this study, we designed a novel thermogelling injectable hydrogel using natural silk fibroin-blended chitosan (CS) incorporated with magnesium (Mg) substitutes to improve physical cross-linking, stability, and cell osteogenic compatibility. The presented observations demonstrate that the developed injectable hydrogels can facilitate the controlled delivery of immobilized recombinant human bone morphogenic protein-2 (rhBMP-2) and rat bone marrow-derived MSCs (rBMSCs) with greater cell encapsulation efficiency, compatibility, and osteogenic differentiation. In addition, outcomes of in vivo animal studies established promising osteoinductive, bone mineral density, and bone formation rate after implantation of the injectable hydrogel scaffolds. Therefore, the developed hydrogels have great potential for clinical applications of FHN therapy.

Introduction Fibrin gels are considered to be a promising biomaterial for tissue engineering because of its role as a “physiological scaffold” in tissue regeneration. It has been reported that fibrin gels could lead to cell migration into a wound/tissue-engineered construct and induce cell proliferation/growth.1-5 Due to the limited self-repair capability of degenerated intervertebral disc, cell-based therapy such as mesenchymal stem cell (MSC) application is becoming an attractive strategy in disc regeneration.6 However, interactions between fibrin and MSCs during disc regeneration are unknown. The objective of this study is to explore the potential value of fibrin gel as a delivery system for MSC and nucleus pulposus cell (NPC) co-culture therapy. We tested different fibrin formulations (fibrinogen and thrombin concentrations) and characterized fibrin gel structure and extracellular matrix production under hypoxic conditions in vitro. The findings will help to find an effective delivery system for cell-based therapy in disc regeneration. Materials and Methods Cell Isolation Commercially available HMSCs (Lonza, MD) were expanded to the fifth passage, and NPCs were isolated from bovine tail discs using a collagenase/protease cocktail digestion. Fibrin Gel Preparation Fibrin scaffolds were prepared by combining fibrinogen with thrombin as described previously.7 Briefly, bovine fibrinogen was dissolved in buffer A (sodium chloride 29 mM, sodium citrate 25 mM, and bovine serum albumin [BSA] 15 mg/mL, 3,000 KIU/mL aprotinin), and then load MSC/NPC (1:1) into fibrinogen solution followed by mixing with bovine thrombin in buffer B (sodium chloride 149 mM, BSA 50 mg/mL, and calcium chloride 80 mM). Various concentrations of fibrinogen and thrombin (9:50, 9:100, 18:50, and 18:100) were used to prepared fibrin scaffold (60 μL/fibrin gel), and then allowed to crosslink without touch for 1 hour at 37°C. Finally, fibrin gels were transferred into incubator with 5% CO2 (normoxia) or 2% O2 (hypoxia) at 37°C. Histology and RT-PCR After 7 day's culture, fibrin gels were collected for H&amp;E, Safranin-O staining. In addition, matrix genes, collagen II, aggrecan, and MMP13 expression of cells within gel were detected with qRT-PCR and normalized to universal GAPDH. Post hoc multiple pair-wise comparison tests (Dunn) were performed to determine differences between treatment groups with significance of p &lt; 0.05 Results After 7 days, fibrin gels demonstrated much more compact and condense network structure where cells entrapped and spread as compared with day 1 (Figs. A and B). Safranin O staining showed enriched proteoglycan around cells (Fig. C). Under hypoxia condition, 9 mg/mL fibrin gel increased type II collagen expression at day 7 by 2.9-fold as compared with day 1 ( p &lt; 0.05, Fig. D), whereas no significant difference between normoxia and hypoxia was observed. Similar expression differences were observed for aggrecan. Interestingly, MMP13 expression increased significantly at day 7 in normoxia group, while it decreased dramatically in hypoxia group ( p &lt; 0.01, Fig. D). Overall, the 9 mg/mL (9:50 and 9:100) fibrin gel displayed more desirable effects on matrix gene (collagen II, aggrecan) expression than on 18 mg/mL (18:50 and 18:100) fibrin gel (Fig. E). [Figure: see text] Conclusion Fibrin gel promoted collagen II and aggrecan gene expression as well as proteoglycan production under both normoxia and hypoxia conditions. Among the formulation studies, 9 mg/mL fibrin exerted more desirable effects on cell spreading and matrix production. The findings suggesting fibrin gel may be a good delivery system for cell-based therapy in disc regeneration by promoting matrix production. Acknowledgments Supported by NIH R21 AR063357-02. Disclosure of Interest None declared References Ikari Y, Yee KO, Schwartz SM. Role of alpha5beta1 and alphavbeta3 integrins on smooth muscle cell spreading and migration in fibrin gels. Thromb Haemost 2PO.072;84(4):701–705 Nomura H, Naito M, Iguchi A, Thompson WD, Smith EB. Fibrin gel induces the migration of smooth muscle cells from rabbit aortic explants. Thromb Haemost 1999;82(4):1347–1352 Liu J, Tan Y, Zhang H, et al. Soft fibrin gels promote selection and growth of tumorigenic cells. Nat Mater 2012;11(8):734–741 Eyrich D, Brandl F, Appel B, et al. Long-term stable fibrin gels for cartilage engineering. Biomaterials 2007;28(1):55–65 Man AJ, Davis HE, Itoh A, Leach JK, Bannerman P. Neurite outgrowth in fibrin gels is regulated by substrate stiffness. Tissue Eng Part A 2011;17(23-24):2931–2942 Sakai D, Mochida J, Yamamoto Y, et al. Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials 2003;24(20):3531–3541 Bensaïd W, Triffitt JT, Blanchat C, Oudina K, Sedel L, Petite H. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials 2003;24(14):2497–2502

3D modular bioceramic scaffolds for the investigation of the interaction between osteosarcoma cells and MSCs

Femoral head necrosis (FHN) is a clinically progressive disease that leads to overwhelming complications without an effective therapeutic approach. In recent decades, transplantation of mesenchymal stem cells (MSCs) has played a promising role in the treatment of FHN in the initial stage; however, the success rate is still low due to unsuitable cell carriers and abridged osteogenic differentiation of the transplanted MSCs. Biopolymeric-derived hydrogels have been extensively applied as effective cell carriers and drug vesicles; they provide the most promising contributions in the fields of tissue engineering and regenerative medicine. However, the clinical potential of hydrogels may be limited due to inappropriate gelation, swelling, mechanical characteristics, toxicity in the cross-linking process, and self-healing ability. Naturally, gelated commercial hydrogels are not suitable for cell injection and infiltration because of their static network structure. In this study, we designed a novel thermo-gelling injectable hydrogel using natural silk fibroin-blended chitosan incorporated with magnesium (Mg) substitutes to improve physical cross-linking, stability, and cell osteogenic compatibility. The presented observations demonstrate that the developed injectable hydrogels can facilitate the controlled delivery of immobilized rhBMP-2 and rBMSCs with greater cell encapsulation efficiency, compatibility, and osteogenic differentiation. In addition, outcomes of in vivo animal studies established promising osteoinductive, bone mineral density, and bone formation rate after implantation of the injectable hydrogel scaffolds. Therefore, the developed hydrogels have great potential for clinical applications of femoral head necrosis therapy.

Mesenchymal stem cells (MSCs) have the ability of differentiating into osteoblasts. Elucidating the molecular mechanisms of MSC differentiation into osteoblasts may provide novel therapeutic strategies for bone-related diseases. Increasing evidence has confirmed that Wnt signaling plays the key role in osteoblast differentiation; however, the role of individual Wnt proteins in osteogenesis needs to be investigated. The present study thus aimed to explore the role of Wnt7a in bone formation. For this purpose, human bone-derived MSCs were identified by flow cytometry and the cell differentiation potential, including osteogenic and adipogenic differentiation was examined. In order to explore the role of Wnt7a in MSC osteogenic differentiation, Wnt7a expression was measured at the mRNA and protein level following treatment with the osteogenic inducer, bone morphogenetic protein (BMP)4/7, and following the induction of osteogenic or adipogenic differentiation. The ectopic expression of Wnt7a in MSCs was confirmed and its influence on MSC osteogenic differentiation was detected using osteocyte markers and by Alizarin Red S staining. Mechanistically, the influence of Wnt7a on Runt-related transcription factor 2 (RUNX2) expression was examined at the mRNA and protein level. The regulatory effects of Wnt7a on RUNX2 promoter activities were examined by promoter reporter assay, and by examining the binding of TCF1, a downstream target of Wnt, to the RUNX2 promoter by ChIP assay. The results revealed that the knockdown of Wnt7a in MSCs decreased the expression of osteocyte markers and inhibited osteogenic differentiation. In accordance, the overexpression of Wnt7a in MSCs increased the expression of osteocyte markers and promoted osteogenic differentiation. Mechanistically, the knockdown of Wnt7a in MSCs reduced RUNX2 expression and the overexpression of Wnt7a in MSCs promoted RUNX2 expression. Furthermore, it was confirmed that Wnt7a regulated RUNX2 promoter activities by promoter report assay, and by examining the binding of TCF1 to the RUNX2 promoter by ChIP assay. On the whole, the present study demonstrates that Wnt7a plays a key role in MSC differentiation into osteoblasts and the findings presented herein may provide a promising therapy target for bone-related diseases.

Stem Cells Derived from Human Amniotic Fluid Contribute to Acute Kidney Injury Recovery

Mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into osteoblasts and provide an excellent cell source for bone regeneration and repair. Recently, the canonical Wnt/beta-catenin signaling pathway has been found to play a critical role in skeletal development and osteogenesis, implying that Wnts can be utilized to improve de novo bone formation mediated by MSCs. However, it is unknown whether noncanonical Wnt signaling regulates osteogenic differentiation. Here, we find that Wnt-4 enhanced in vitro osteogenic differentiation of MSCs isolated from human adult craniofacial tissues and promoted bone formation in vivo. Whereas Wnt-4 did not stabilize beta-catenin, it activated p38 MAPK in a novel noncanonical signaling pathway. The activation of p38 was dependent on Axin and was required for the enhancement of MSC differentiation by Wnt-4. Moreover, using two different models of craniofacial bone injury, we found that MSCs genetically engineered to express Wnt-4 enhanced osteogenesis and improved the repair of craniofacial defects in vivo. Taken together, our results reveal that noncanonical Wnt signaling could also play a role in osteogenic differentiation. Wnt-4 may have a potential use in improving bone regeneration and repair of craniofacial defects.