Stimulation Of Mesenchymal Stem Cells Research Articles

Electrical Stimulation of Mesenchymal Stem Cells as a Tool for Proliferation and Differentiation in Cartilage Tissue Engineering: A Scaffold-Based Approach.

Electrical stimulation (ES) is a widely discussed topic in the field of cartilage tissue engineering due to its ability to induce chondrogenic differentiation (CD) and proliferation. It shows promise as a potential therapy for osteoarthritis (OA). In this study, we stimulated mesenchymal stem cells (MSCs) incorporated into collagen hydrogel (CH) scaffolds, consisting of approximately 500,000 cells each, for 1 h per day using a 2.5 Vpp (119 mV/mm) 8 Hz sinusoidal signal. We compared the cell count, morphology, and CD on days 4, 7, and 10. The results indicate proliferation, with an increase ranging from 1.86 to 9.5-fold, particularly on day 7. Additionally, signs of CD were observed. The stimulated cells had a higher volume, while the stimulated scaffolds showed shrinkage. In the ES groups, up-regulation of collagen type 2 and aggrecan was found. In contrast, SOX9 was up-regulated in the control group, and MMP13 showed a strong up-regulation, indicating cell stress. In addition to lower stress levels, the control groups also showed a more spheroidic shape. Overall, scaffold-based ES has the potential to achieve multiple outcomes. However, finding the appropriate stimulation pattern is crucial for achieving successful chondrogenesis.

Local delivery of stem cell spheroids with protein/polyphenol self-assembling armor to improve myocardial infarction treatment via immunoprotection and immunoregulation

Stem cell therapies have shown great potential for treating myocardial infarction (MI) but are limited by low cell survival and compromised functionality due to the harsh microenvironment at the disease site. Here, we presented a Mesenchymal stem cell (MSC) spheroid-based strategy for MI treatment by introducing a protein/polyphenol self-assembling armor coating on the surface of cell spheroids, which showed significantly enhanced therapeutic efficacy by actively manipulating the hostile pathological MI microenvironment and enabling versatile functionality, including protecting the donor cells from host immune clearance, remodeling the ROS microenvironment and stimulating MSC's pro-healing paracrine secretion. The underlying mechanism was elucidated, wherein the armor protected to prolong MSCs residence at MI site, and triggered paracrine stimulation of MSCs towards immunoregulation and angiogenesis through inducing hypoxia to provoke glycolysis in stem cells. Furthermore, local delivery of coated MSC spheroids in MI rat significantly alleviated local inflammation and subsequent fibrosis via mediation macrophage polarization towards pro-healing M2 phenotype and improved cardiac function. In general, this study provided critical insight into the enhanced therapeutic efficacy of stem cell spheroids coated with a multifunctional armor. It potentially opens up a new avenue for designing immunomodulatory treatment for MI via stem cell therapy empowered by functional biomaterials.

Retracted: Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Stimulated by Deferoxamine Accelerate Cutaneous Wound Healing by Promoting Angiogenesis.

[This retracts the article DOI: 10.1155/2019/9742765.].

Amelioration of DSS-induced colitis in mice by TNF-α-stimulated mesenchymal stem cells derived from feline adipose tissue via COX-2/PGE2 activation.

Mesenchymal stem cells (MSCs) have been investigated as therapeutic agents for inflammatory bowel disease (IBD). Stimulation of MSCs with pro-inflammatory cytokines is an approach to enhance their immunomodulatory effects. However, further investigation is required to support their application in immune-mediated disorders and companion animals. This study aimed to assess the therapeutic effect of tumor necrosis factor (TNF)-α-stimulated feline adipose tissue-derived MSCs (fAT-MSCs) in a dextran sulfate sodium (DSS)-induced colitis mouse model. Colitis mice was made by drinking water with 3% DSS and fAT-MSCs were injected intraperitoneally. Colons were collected on day 10. The severity of the disease was evaluated and compared. Raw 264.7 cells were cultured with the conditioned medium to determine the mechanism, using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. TNF-α-stimulated fAT-MSCs more improved severity of DSS-induced colitis in disease activity, colon length, histologic score, and inflammatory cytokine. In sectionized colon tissues, the group comprising TNF-α-stimulated fAT-MSCs had higher proportion of CD11b+CD206+ macrophages than in the other groups. In vitro, TNF-α-stimulation increased cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) secretion from fAT-MSCs. The conditioned medium from TNF-α-stimulated fAT-MSCs enhanced the expression of interleukin-10 and arginase-1 in LPS-activated Raw 264.7 cells. These results represent that TNF-α-stimulated fat-mscs ameliorate the inflamed colon more effectively. Furthermore, we demonstrated that the effectiveness was interlinked with the COX-2/PGE2 pathway.

Hypoxia-pretreated mesenchymal stem cell-derived exosomes-loaded low-temperature extrusion 3D-printed implants for neural regeneration after traumatic brain injury in canines.

Regenerating brain defects after traumatic brain injury (TBI) still remains a significant difficulty, which has motivated interest in 3D printing to design superior replacements for brain implantation. Collagen has been applied to deliver cells or certain neurotrophic factors for neuroregeneration. However, its fast degradation rate and poor mechanical strength prevent it from being an excellent implant material after TBI. In the present study, we prepared 3D-printed collagen/silk fibroin/hypoxia-pretreated human umbilical cord mesenchymal stem cells (HUCMSCs)-derived exosomes scaffolds (3D-CS-HMExos), which possessed favorable physical properties suitable biocompatibility and biodegradability and were attractive candidates for TBI treatment. Furthermore, inspired by exosomal alterations resulting from cells in different external microenvironments, exosomes were engineered through hypoxia stimulation of mesenchymal stem cells and were proposed as an alternative therapy for promoting neuroregeneration after TBI. We designed hypoxia-preconditioned (Hypo) exosomes derived from HUCMSCs (Hypo-MExos) and proposed them as a selective therapy to promote neuroregeneration after TBI. For the current study, 3D-CS-HMExos were prepared for implantation into the injured brains of beagle dogs. The addition of hypoxia-induced exosomes further exhibited better biocompatibility and neuroregeneration ability. Our results revealed that 3D-CS-HMExos could significantly promote neuroregeneration and angiogenesis due to the doping of hypoxia-induced exosomes. In addition, the 3D-CS-HMExos further inhibited nerve cell apoptosis and proinflammatory factor (TNF-α and IL-6) expression and promoted the expression of an anti-inflammatory factor (IL-10), ultimately enhancing the motor functional recovery of TBI. We proposed that the 3D-CS-loaded encapsulated hypoxia-induced exosomes allowed an adaptable environment for neuroregeneration, inhibition of inflammatory factors and promotion of motor function recovery in TBI beagle dogs. These beneficial effects implied that 3D-CS-HMExos implants could serve as a favorable strategy for defect cavity repair after TBI.

Anti-inflammatory effects of mesenchymal stem cell-conditioned media inhibited macrophages activation in vitro

The immunomodulatory effects of mesenchymal stem cells (MSCs) on macrophages have been reported, however, the underlying mechanism remains unknown. Therefore, this study aimed to investigate the anti-inflammatory effects of MSCs on lipopolysaccharide (LPS)-stimulated macrophages and the subsequent downregulation of their inflammatory mediators. Macrophages were treated with conditioned media from MSCs, without a subsequent change of MSCs responding to the inflammation state. This study also evaluated whether the interleukin (IL) 4 stimulation of MSCs can improve their anti-inflammatory effects. Results demonstrated that the MSC-conditioned medium (MSC-CM) stimulated with IL4 significantly inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression of LPS-activated macrophages. MSC-CM treatment inhibited the mRNA transcription of the cytokines IL1β and IL6, the chemokines C–C motif ligand (CCL) 2, CCL3, CCL4, and CCL5, and the chemokine receptors CCR2 and CCR5, in LPS-stimulated macrophages. As revealed through western blot and immunofluorescence analyses, the phosphorylation of p38, JNK, and ERK MAPKs, as well as phosphorylation of NF-κB in stimulated macrophages, were also inhibited by the MSC-CM. Further, more potent anti-inflammatory effects were observed with the IL4-stimulated cells, compared with those observed with the non-stimulated cells. The MSC-CM demonstrated a potent anti-inflammatory effect on LPS-activated macrophages, while the IL4 stimulation improved this effect. These findings indicate that MSCs could exert anti-inflammatory effects on macrophages, and may be considered as a therapeutic agent in inflammation treatment.

Mechanical Stimulation on Mesenchymal Stem Cells and Surrounding Microenvironments in Bone Regeneration: Regulations and Applications.

Treatment of bone defects remains a challenge in the clinic. Artificial bone grafts are the most promising alternative to autologous bone grafting. However, one of the limiting factors of artificial bone grafts is the limited means of regulating stem cell differentiation during bone regeneration. As a weight-bearing organ, bone is in a continuous mechanical environment. External mechanical force, a type of biophysical stimulation, plays an essential role in bone regeneration. It is generally accepted that osteocytes are mechanosensitive cells in bone. However, recent studies have shown that mesenchymal stem cells (MSCs) can also respond to mechanical signals. This article reviews the mechanotransduction mechanisms of MSCs, the regulation of mechanical stimulation on microenvironments surrounding MSCs by modulating the immune response, angiogenesis and osteogenesis, and the application of mechanical stimulation of MSCs in bone regeneration. The review provides a deep and extensive understanding of mechanical stimulation mechanisms, and prospects feasible designs of biomaterials for bone regeneration and the potential clinical applications of mechanical stimulation.

Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an invitro invasion of cancer cells.

IntroductionThe objective of this study is to design a co-culture system of cancer cells and three-dimensional (3D) mesenchymal stem cells (MSC) aggregates for the in vitro evaluation of cancer invasion.MethodsFirst, the MSC of an immunosuppressive phenotype (MSC2) were prepared by the MSC stimulation of polyriboinosinic polyribocytidylic acid. By simple mixing MSC2 and gelatin hydrogel microspheres (GM) in a U-bottomed well of 96 well plates which had been pre-coated with poly (vinyl alcohol), 3D MSC2 aggregates incorporating GM were obtained. The amount of chemokine (C–C motif) ligand 5 (CCL5) secreted from the MSC2 aggregates incorporating GM. Finally, an invasion assay was performed to evaluate the cancer invasion rate by co-cultured cancer cells and the 3D MSC2 incorporating GM.ResultsThe amount of CCL5 secreted for the 3D MSC2 aggregates incorporating GM was significantly higher than that of two-dimensional (2D) MSC, 2D MSC2, and 3D MSC aggregates incorporating GM. When MDA-MB-231 human breast cancer cells were co-cultured with the 3D MSC2 aggregates incorporating GM, the invasion rate of cancer cells was significantly high compared with that of 2D MSC or 2D MSC2 and 3D MSC aggregates incorporating GM. In addition, high secretion of matrix metalloproteinase-2 was observed for the 3D MSC2 aggregates/cancer cells system.ConclusionsIt is concluded that the co-culture system of 3D MSC2 aggregates incorporating GM and cancer cells is promising to evaluate the invasion of cancer cells in vitro.

Experimental-numerical analysis of cell adhesion-mediated electromechanical stimulation on piezoelectric nanofiber scaffolds

Electrospun nanofibers exhibiting piezoelectricity are a specific class of smart materials which could provide electric stimulation to cells in a noninvasive way and contribute to tissue regeneration. During cell-material interaction, the materials display electromechanical behavior by transforming cell adhesion force into surface charge. In the process, how the cell adhesion states and the electromechanical properties of scaffolds determine the actual piezoelectric potential implemented on a cell is still unclear. Herein, we fabricated piezoelectric poly(vinylidene fluoride) (PVDF) nanofiber scaffolds with different topographies, and investigated their influences on cell morphology and cell adhesion-mediated electromechanical stimulation of mesenchymal stem cell (MSC). Our results demonstrated that MSC seeded on aligned piezoelectric nanofibers exhibited elongated morphology combined with higher intracellular calcium activity than those adhered on random nanofibers with rounded shape. The underlying mechanism was further quantitatively analyzed using a three-dimensional (3D) finite element method with respect to cell adhesion states and architecture parameters of nanofiber scaffolds. The results suggested that cell morphology and cell adhesion force influenced the piezoelectric output through modulating the location and magnification of force implemented on the scaffolds. In addition, the change of alignment, pore size and diameter of the nanofiber network could alter the mechanical property of the scaffolds, and then bias the actual piezoelectric output experienced by a cell. These findings provide new insights for probing the mechanism of cell self-stimulation on piezoelectric scaffolds, and pave the way for rational design of piezoelectric scaffolds for cell regulation and tissue regeneration.

Bone mesenchymal stem cells stimulation by magnetic nanoparticles and a static magnetic field: release of exosomal miR-1260a improves osteogenesis and angiogenesis

BackgroundThe therapeutic potential of exosomes derived from stem cells has attracted increasing interest recently, because they can exert similar paracrine functions of stem cells and overcome the limitations of stem cells transplantation. Exosomes derived from bone mesenchymal stem cells (BMSC-Exos) have been confirmed to promote osteogenesis and angiogenesis. The magnetic nanoparticles (eg. Fe3O4, γ-Fe2O3) combined with a static magnetic field (SMF) has been commonly used to increase wound healing and bone regeneration. Hence, this study aims to evaluate whether exosomes derived from BMSCs preconditioned with a low dose of Fe3O4 nanoparticles with or without the SMF, exert superior pro-osteogenic and pro-angiogenic activities in bone regeneration and the underlying mechanisms involved.MethodsTwo novel types of exosomes derived from preconditioned BMSCs that fabricated by regulating the contents with the stimulation of magnetic nanoparticles and/or a SMF. Then, the new exosomes were isolated by ultracentrifugation and characterized. Afterwards, we conducted in vitro experiments in which we measured osteogenic differentiation, cell proliferation, cell migration, and tube formation, then established an in vivo critical-sized calvarial defect rat model. The miRNA expression profiles were compared among the exosomes to detect the potential mechanism of improving osteogenesis and angiogenesis. At last, the function of exosomal miRNA during bone regeneration was confirmed by utilizing a series of gain- and loss-of-function experiments in vitro.Results50 µg/mL Fe3O4 nanoparticles and a 100 mT SMF were chosen as the optimum magnetic conditions to fabricate two new exosomes, named BMSC-Fe3O4-Exos and BMSC-Fe3O4-SMF-Exos. They were both confirmed to enhance osteogenesis and angiogenesis in vitro and in vivo compared with BMSC-Exos, and BMSC-Fe3O4-SMF-Exos had the most marked effect. The promotion effect was found to be related to the highly riched miR-1260a in BMSC-Fe3O4-SMF-Exos. Furthermore, miR-1260a was verified to enhance osteogenesis and angiogenesis through inhibition of HDAC7 and COL4A2, respectively.ConclusionThese results suggest that low doses of Fe3O4 nanoparticles combined with a SMF trigger exosomes to exert enhanced osteogenesis and angiogenesis and that targeting of HDAC7 and COL4A2 by exosomal miR-1260a plays a crucial role in this process. This work could provide a new protocol to promote bone regeneration for tissue engineering in the future.Graphical abstract

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2.

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.

The effects of shock wave stimulation of mesenchymal stem cells on proliferation, migration, and differentiation in an injectable gelatin matrix for osteogenic regeneration.

The treatment of a variety of defects in bony sites could benefit from mitogenic stimulation of osteoprogenitor cells, including endogenous bone marrow-derived mesenchymal stem cells (bMSCs), and from provision of such cells with a matrix permissive of their migration, proliferation, and osteogenic differentiation. That such MSC stimulation could result from treatment with noninvasive (extracorporeal) shock waves (ESWs), and the matrix delivered by injection could enable this therapeutic approach to be employed for applications in which preformed scaffolds and growth factor therapy are difficult to deploy. The objectives of the present study were to investigate focused ESWs for their effects on proliferation, migration, and osteogenic differentiation in an injectable gelatin (Gtn) matrix capable of undergoing covalent cross-linking in vivo. Gtn was conjugated with hydroxyphenyl propionic acid (HPA) in order to enable it to be covalently cross-linked with minute amounts of horseradish peroxidase and hydrogen peroxide. The results demonstrated that 500 shocks of 0.4-mJ/mm2 energy flux density resulted in a twofold greater proliferation of bMSCs in the Gtn-HPA matrix after 14 days, compared with bMSCs grown with supplementation with platelet-derived growth factor (PDGF)-BB, a known mitogen for bMSCs. Moreover, SW treatment enhanced substantially osteogenic differentiation of bMSCs. The Gtn-HPA gel was permissive of MSC migration under the chemotactic influence of the growth factor, PDGF-BB, incorporated into and released by the gel. ESW treatment had no effect on the motility of the MSCs. The findings of the study warrant further investigation of this combined treatment modality for select bony defects.

Nanovibrational Stimulation of Mesenchymal Stem Cells Induces Therapeutic Reactive Oxygen Species and Inflammation for Three-Dimensional Bone Tissue Engineering.

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs’ responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells’ mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

FRI0375 VISFATIN EFFECTS ON MSCS DURING OD VIA DIFFERENTIAL REGULATION OF LNCRNA H19 AND MICRO RNA 675-3P

Background:Long non-coding (lnc-)RNA are regulatory molecules transcribed from DNA similar to mRNA and interact directly with DNA, RNA and proteins. Some lncRNAs have been shown to contain micro (mi-)RNAs in their sequence that can be released by splicing and lead to active miRNA molecules, e.g. lncRNA H19 includes two miRNAs 675-3p and -5p in its sequence.Adipose tissue derived factors (adipokines) are involved in inflammation processes and osteoarthritis (OA) development. The proinflammatory adipokine visfatin has been shown to alter osteogenic differentiation (OD) of pluripotent mesenchymal stem cells (MSCs) and reduces elastic fiber expression, increases matrix mineralization and proinflammatory cytokine and chemokine production(1).Objectives:We evaluated a novel effect of visfatin on lncRNA H19 in MSCs during OD. The goal was to explore the kinetics of the visfatin effect during OD with regard to H19 regulation and to investigate H19 downstream mechanisms leading to the observed altered MSC differentiation and osteoblast activity.Methods:MSCs isolated from OA hip or knee bone (phMSC) and commercially obtained healthy human (h-)MSCs were differentiated towards osteoblasts with/without visfatin, resistin, leptin, TNF and Wnt/TGFβ1 pathway inhibitors. Supernatants were collected at days 2, 7, 9, 14 and 21 of OD, cell lysates at day 2, 7, 9, 14 and matrix mineralization assays conducted at day 21. H19 and miRNA expression was evaluated by real-time PCR after mi-/RNA isolation. IL-6 was analyzed by ELISA.Results:H19 was continuously upregulated in unstimulated controls as expected during OD but also when stimulated with other adipokines. In contrast, stimulation with visfatin significantly decreased H19 (day 2 to 14 of OD, hip-phMSCs: p = 0.0097, knee-phMSCs: p=0.0075, h-MSC: p = 0.044). Visfatin increased matrix mineralization and IL-6 production as expected (hMSC: p = 0.03, phMSC: p = 0.013)(1). TNF stimulation during OD did not lead to a downregulation of H19 nor increased matrix mineralization, thus showing that the effects were visfatin-dependent. H19s endogenous miRNA 675-5p was changed in parallel with H19, increased during control OD and significantly down-regulated by visfatin (e.g. day 14 p = 0.015). However, H19s endogenous miRNA 675-3p was inversely regulated, downregulated during control OD while visfatin stimulation attenuated this effect (e.g. day 14 p = 0.025). Altered Wnt-signaling and involvement of the TGFβ1 pathway could not be observed.Conclusion:H19 is upregulated during OD and may therefore play a regulatory role in the process of osteogenesis. Visfatin stimulation of MSCs during OD showed pro-inflammatory effects, increased matrix mineralization while reducing elastic fiber production(1). These effects were associated with a downregulation of H19, a specific visfatin effect not triggered by other adipokines or TNF. The H19 sequence includes two endogenous micro-RNAs 675-3p and 5p. We demonstrated miRNA 675-5p to be regulated in parallel to H19, whereas miRNA 675-3p was inversely regulated and increased continuously upon visfatin stimulation. Based on these results, we hypothesize that visfatin provides a specific stimulus for the splicing of miRNA 675-3p from H19, in turn leading to H19 reduction. miRNA 675-3p thus represents an effector mechanism of visfatin that contributes to the observed functional effects in differentiating MSCs.

Physiologically Relevant Fluid-Induced Oscillatory Shear Stress Stimulation of Mesenchymal Stem Cells Enhances the Engineered Valve Matrix Phenotype.

Support of somatic growth is a fundamental requirement of tissue-engineered valves. However, efforts thus far have been unable to maintain this support long term. A key event that will determine the valve's long-term success is the extent to which healthy host tissue remodeling can occur on the valve soon after implantation. The construct's phenotypic-status plays a critical role in accelerating tissue remodeling and engineered valve integration with the host via chemotaxis. In the current study, human bone-marrow-derived mesenchymal stem cells were utilized to seed synthetic, biodegradable scaffolds for a period of 8 days in rotisserie culture. Subsequently, cell-seeded scaffolds were exposed to physiologically relevant oscillatory shear stresses (overall mean, time-averaged shear stress, ~7.9 dynes/cm2; overall mean, oscillatory shear index, ~0.18) for an additional 2 weeks. The constructs were found to exhibit relatively augmented endothelial cell expression (CD31; compared to static controls) but concomitantly served to restrict the level of the activated smooth muscle phenotype (α-SMA) and also produced very low stem cell secretion levels of fibronectin (p < 0.05 compared to static and rotisserie controls). These findings suggest that fluid-induced oscillatory shear stresses alone are important in regulating a healthy valve phenotype of the engineered tissue matrix. Moreover, as solid stresses could lead to increased α-SMA levels, they should be excluded from conditioning during the culture process owing to their associated potential risks with pathological tissue remodeling. In conclusion, engineered valve tissues derived from mesenchymal stem cells revealed both a relatively robust valvular phenotype after exposure to physiologically relevant scales of oscillatory shear stress and may thereby serve to accelerate healthy valve tissue remodeling in the host post-implantation.

Co-culture and Mechanical Stimulation on Mesenchymal Stem Cells and Chondrocytes for Cartilage Tissue Engineering.

Defects in articular cartilage injury and chronic osteoarthritis are very widespread and common, and the ability of injured cartilage to repair itself is limited. Stem cell-based cartilage tissue engineering provides a promising therapeutic option for articular cartilage damage. However, the application of the technique is limited by the number, source, proliferation, and differentiation of stem cells. The co-culture of mesenchymal stem cells and chondrocytes is available for cartilage tissue engineering, and mechanical stimulation is an important factor that should not be ignored. A combination of these two approaches, i.e., co-culture of mesenchymal stem cells and chondrocytes under mechanical stimulation, can provide sufficient quantity and quality of cells for cartilage tissue engineering, and when combined with scaffold materials and cytokines, this approach ultimately achieves the purpose of cartilage repair and reconstruction. In this review, we focus on the effects of co-culture and mechanical stimulation on mesenchymal stem cells and chondrocytes for articular cartilage tissue engineering. An in-depth understanding of the impact of co-culture and mechanical stimulation of mesenchymal stem cells and chondrocytes can facilitate the development of additional strategies for articular cartilage tissue engineering.

Generation of induced secretome from adipose-derived stem cells specialized for disease-specific treatment: An experimental mouse model.

Recently, the exclusive use of mesenchymal stem cell (MSC)-secreted molecules, named as the secretome, have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages. To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials. We collected the secretory materials (named as inducers) released from AML12 hepatocytes that had been pretreated with thioacetamide (TAA) and generated the TAA-induced secretome (TAA-isecretome) after stimulating adipose-derived stem cells with the inducers. The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy. TAA-isecretome infusion showed higher therapeutic potential in terms of (1) restoring disorganized hepatic tissue to normal tissue; (2) inhibiting proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α); and (3) reducing abnormally elevated liver enzymes (aspartate aminotransferase and alanine aminotransferase) compared to the naïve secretome infusion in mice with TAA-induced hepatic failure. However, the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice. Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome. In addition, peroxiredoxin-1, a potent antioxidant protein, was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury. Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins, especially peroxiredoxin-1, with higher antioxidant activity. Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen. This approach is expected to open a new way of developing various specific therapeutics based on the high plasticity and responsiveness of MSCs.

Design, construction and characterisation of a novel nanovibrational bioreactor and cultureware for osteogenesis

In regenerative medicine, techniques which control stem cell lineage commitment are a rapidly expanding field of interest. Recently, nanoscale mechanical stimulation of mesenchymal stem cells (MSCs) has been shown to activate mechanotransduction pathways stimulating osteogenesis in 2D and 3D culture. This has the potential to revolutionise bone graft procedures by creating cellular graft material from autologous or allogeneic sources of MSCs without using chemical induction. With the increased interest in mechanical stimulation of cells and huge potential for clinical use, it is apparent that researchers and clinicians require a scalable bioreactor system that provides consistently reproducible results with a simple turnkey approach. A novel bioreactor system is presented that consists of: a bioreactor vibration plate, calibrated and optimised for nanometre vibrations at 1 kHz, a power supply unit, which supplies a 1 kHz sine wave signal necessary to generate approximately 30 nm of vibration amplitude, and custom 6-well cultureware with toroidal shaped magnets incorporated in the base of each well for conformal attachment to the bioreactor’s magnetic vibration plate. The cultureware and vibration plate were designed using finite element analysis to determine the modal and harmonic responses, and validated by interferometric measurement. This helps ensure that the vibration plate and cultureware, and thus collagen and MSCs, all move as a rigid body, avoiding large deformations close to the resonant frequency of the vibration plate and vibration damping beyond the resonance. Assessment of osteogenic protein expression was performed to confirm differentiation of MSCs after initial biological experiments with the system, as well as atomic force microscopy of the 3D gel constructs during vibrational stimulation to verify that strain hardening of the gel did not occur. This shows that cell differentiation was the result of the nanovibrational stimulation provided by the bioreactor alone, and that other cell differentiating factors, such as stiffening of the collagen gel, did not contribute.

Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Stimulated by Deferoxamine Accelerate Cutaneous Wound Healing by Promoting Angiogenesis.

The exosomes are derived from mesenchymal stem cells (MSCs) and may be potentially used as an alternative for cell therapy, for treating diabetic wounds, and aid in angiogenesis. This study, aimed to investigate whether exosomes originated from bone marrow-derived MSCs (BMSCs) preconditioned by deferoxamine (DFO-Exos) exhibited superior proangiogenic property in wound repair and to explore the underlying mechanisms involved. Human umbilical vein endothelial cells (HUVECs) were used for assays involving cell proliferation, scratch wound healing, and tube formation. To test the effects in vivo, streptozotocin-induced diabetic rats were established. Two weeks after the procedure, histological analysis was used to measure wound-healing effects, and the neovascularization was evaluated as well. Our findings demonstrated that DFO-Exos activate the PI3K/AKT signaling pathway via miR-126 mediated PTEN downregulation to stimulate angiogenesis in vitro. This contributed to enhanced wound healing and angiogenesis in streptozotocin-induced diabetic rats in vivo. Our results suggest that, in cell-free therapies, exosomes derived from DFO preconditioned stem cells manifest increased proangiogenic ability.

Mesenchymal Stem Cells Stimulated by Growth Factors Release Exosomes with Potent Proangiogenic Activity

To explore the proangiogenic activity of exsomes released by human umbilical cord mesenchymal stem cells (MSCs) stimulated by erythropoietin and platelet-derived growth factor BB (PDGF-BB). Human umbilical cord-derived MSCs were seeded and maintained in culture overnight. The media were then replaced by alpha-MEM containing EPO (1 U/ml) and/or PDGF-BB (50 ng/ml), and the culture was maintained for 72 hours. The exosomes from the culture supernatants were isolated with a routine ultra-catrifagation method. Flow cytometric analysis was performed to identify the origin of the exosomes, and their morphological features were observed by using a transmission electron microscopy. The exosomes were added at a concentration of 10 µg/ml into the culture system of human umbilical cord vein endothelial cells. MTT assay was used to evaluate the proliferative status. The Matrigel assay was used to observe the formation of net-work structures which were calculated after culture for 12 hours. Flow cytometric analysis showed that microparticles released by human umbilical cord MSCs expressed CD9, CD63 and CD81, which was in accordance with the surface molecular features of exosomes. Under an electron microscope, the exosomes took the featured cystic shape. The protein contents of exosomes released by untreated, EPO-stimulated, PDGF-BB-stimulated and EPO plus PDGF-BB stimulated MSCs (108 cells) were 256±124 µg, 1021±392 µg, 830±265 µg and 2207±733 µg, respectively. The results revealed that MSCs treated by EPO and PDGF-BB released significantly higher amounts of exosomes (P<0.01). MTT assay proved that the exosomes from EPO and PDGF-BB treated MSCs had more potent proliferation-promoting activity on human umbilical cord vein endothelial cells than those from untreated MSCs. The Matrigel assay showed that the numbers of capillary-like structures in untreated, EPO-, PDGF-BB and EPO plus PDGF-BB-treated groups were 2.6±0.84, 4.6±1.57, 4.2±0.78 and 6.3±1.34 per high power objective. Treatment with EPO or PDGF-BB dramatically enhanced the numbers of capillary liue structure, compared with that of untreated group (P<0.01) and those in EPO and PDGF-BB combination group was significantly greater than those of EPO or PDGF-BB group (P<0.01). EPO and PDGF-BB can stimulate MSCs to release exosomes with more potent proangiogenic activity.