Human Mesenchymal Stromal Cells Research Articles

A phase I/II clinical trial of ex-vivo expanded human bone marrow derived allogeneic mesenchymal stromal cells in adult patients with perianal fistulizing Crohn’s Disease

BackgroundPerianal fistulas (PF) affect one-third patients with Crohn’s disease (CD) with limited therapeutic options. There is dearth of literature on safety and efficacy of bone marrow-derived mesenchymal stromal cells (BMSCs) in this population.MethodsAn open-label, phase I/II, single-arm study was conducted involving local administration of human allogeneic bone marrow-derived mesenchymal stromal cells in perianal fistula of patients with Crohn’s disease refractory to standard therapies. Clinical severity and biomarkers were assessed at baseline and periodically until week 104 , and MRI at week 24 and 104. Primary and secondary objectives were to assess safety and efficacy respectively. Fistula remission was complete closure of fistula openings with < 2 cm perianal collection on MRI, and fistula response was decrease in drainage by ≥ 50%. Change in perianal disease activity index, quality-of-life and Van Assche index on MRI over time was assessed using mixed-effect linear regression model.ResultsTen patients (male:8, mean age:27.4 ± 12.0years) were recruited. Self-resolving procedure-related adverse events occurred in three patients, with no follow-up adverse events. In intention to treat analysis at week 24, two patients (20%) achieved fistula remission and seven (70%) had fistula response. At week 52, two (20%) patients were in remission and seven (70%) maintained response. At 104 weeks, two (20%) patients maintained response and one (10%) was in remission. Statistically significant decrease in perianal disease activity index (P = 0.008), Van Assche Index (P = 0.008) and improvement in quality-of-life (P = 0.001) were observed over time.ConclusionsAllogeneic BMSCs are safe and effective for the treatment of perianal fistulizing CD with significant improvement in clinical severity and radiological healing.Trial registrationThe study was prospectively registered on Clinical trials registry – India (CTRI), CTRI/2020/01/022743 on 14 January 2020, http://ctri.nic.in.

Proposal of Simplified Standardization of the Cell-Growth-Promoting Activity of Human Adipose Tissue Mesenchymal Stromal Cell Culture Supernatants

The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.

Effects of Ibuprofen and Diclofenac Pre-Treatment on Viability and Apoptosis Processes in Human Dental Pulp Stem Cells

Background and Objectives: Stem cell-based regeneration strategies have shown therapeutic efficacy in various fields of regenerative medicine. These include bone healing after bone augmentation, often complicated by pain, which is managed by using nonsteroidal anti-inflammatory drugs (NSAIDs). However, information is limited about how NSAIDs affect the therapeutic potential of stem cells. Materials and Methods: We investigated the effects of ibuprofen and diclofenac on the characteristics, morphology, and immunophenotype of human mesenchymal stromal cells isolated from the dental pulp (DPSCs) and cultured in vitro, as well as their effects on the expression of angiogenic growth factors (VEGFA and HGF) and selected genes in apoptosis signalling pathways (BAX, BAK, CASP3, CASP9, and BCL2). Results: Ibuprofen and diclofenac significantly reduced the viability of DPSCs, while the expression of mesenchymal stem cell surface markers was unaffected. Both ibuprofen and diclofenac treatment significantly upregulated the expression of HGF, while the expression of VEGFA remained unchanged. Ibuprofen significantly altered the expression of several apoptosis-related genes, including the upregulation of CASP9 and BCL2, with decreased CASP3 expression. BAK, CASP3, CASP9, and BCL2 expressions were significantly increased in the diclofenac-treated DPSCs, while no difference was demonstrated in BAX expression. Conclusions: Our results suggest that concomitant use of the NSAIDs ibuprofen or diclofenac with stem cell therapy may negatively impact cell viability and alter the expression of apoptosis-related genes, affecting the efficacy of stem cell therapy.

Nonlinear Elastic Bottlebrush Polymer Hydrogels Modulate Actomyosin Mediated Protrusion Formation in Mesenchymal Stromal Cells.

The nonlinear elasticity of many tissue-specific extracellular matrices is difficult to recapitulate without the use of fibrous architectures, which couple strain-stiffening with stress relaxation. Herein, bottlebrush polymers are synthesized and crosslinked to form poly(ethylene glycol)-based hydrogels and used to study how strain-stiffening behavior affects human mesenchymal stromal cells (hMSCs). By tailoring the bottlebrush polymer length, the critical stress associated with the onset of network stiffening is systematically varied, and a unique protrusion-rich hMSC morphology emerges only at critical stresses within a biologically accessible stress regime. Local cell-matrix interactions are quantified using 3D traction force microscopy and small molecule inhibitors are used to identify cellular machinery that plays a critical role in hMSC mechanosensing of the engineered, strain-stiffening microenvironment. Collectively, this study demonstrates how covalently crosslinked bottlebrush polymer hydrogels can recapitulate strain-stiffening biomechanical cues at biologically relevant stresses and be used to probe how nonlinear elastic matrix properties regulate cellular processes.

NATIVE CHARACTERIZATION AND QC PROFILING OF HUMAN AMNIOTIC MESENCHYMAL STROMAL CELL VESICULAR FRACTIONS FOR SECRETOME-BASED THERAPY

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension.This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities.This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Carbon Monoxide-induced Autophagy Enhances Human Mesenchymal Stromal Cell Function via Paracrine Actions in Murine Polymicrobial Sepsis

Sepsis is a life-threatening process due to organ dysfunction resulting from severe infections. Mesenchymal stromal cells (MSCs) are being investigated as therapy for sepsis, along with conditioning regimens to improve their function. Carbon monoxide (CO) gas, which is cytoprotective at low doses, induces autophagy and is a mediator of inflammation. We evaluated CO-induced autophagy in human(h) MSCs, and its impact on cell function in murine cecal ligation and puncture. Conditioning of hMSCs with CO ex vivo resulted in enhanced survival and bacterial clearance in vivo, and neutrophil phagocytosis of bacteria in vitro. Decreased neutrophil infiltration and less parenchymal cell death in organs were associated with increased macrophage efferocytosis of apoptotic neutrophils, promoting resolution of inflammation. These CO effects were lost when the cells were exposed to autophagy inhibition prior to gas exposure. When assessing paracrine actions of CO-induced autophagy, extracellular vesicles (EVs) were predominantly responsible. CO had no effect on EV production, but altered their miRNA cargo. Increased expression of miR-145-3p and miR-193a-3p by CO was blunted with disruption of autophagy, and inhibitors of these miRNAs led to a loss of neutrophil phagocytosis and macrophage efferocytosis. Collectively, CO-induced autophagy enhanced hMSC function during sepsis via paracrine actions of MSC-derived EVs.

Polycaprolactone/β-Tricalcium Phosphate Composite Scaffolds with Advanced Pore Geometries Promote Human Mesenchymal Stromal Cells' Osteogenic Differentiation.

Critical-sized mandibular bone defects, arising from, for example, resections after tumor surgeries, are currently treated with autogenous bone grafts. This treatment is considered very invasive and is associated with limitations such as morbidity and graft resorption. Tissue engineering approaches propose to use 3D scaffolds that combine structural features, biomaterial properties, cells, and biomolecules to create biomimetic constructs. However, mimicking the complex anatomy and composition of the mandible poses a challenge in scaffold design. In our study, we evaluated the dual effect of complex pore geometry and material composition on the osteogenic potential of 3D printed scaffolds. The scaffolds were made of polycaprolactone (PCL) alone (TCP0), or with a high concentration of β-tricalcium phosphate (β-TCP) up to 40% w/w (TCP40), with two complex pore geometries, namely a star- (S) and a diamond-like (D) shape. Scanning electron microscopy and microcomputed tomography images confirmed high fidelity during the printing process. The D-scaffolds displayed higher compressive moduli than the corresponding S-scaffolds. TCP40 scaffolds in simulated body fluid showed deposition of minerals on the surface after 28 days. Subsequently, we assessed the differentiation of seeded bone marrow-derived human mesenchymal stromal cells (hMSCs) over 28 days. The early expression of RUNX2 in the cell nuclei confirmed the commitment toward an osteogenic phenotype. Moreover, alkaline phosphatase (ALP) activity and collagen deposition displayed an increasing trend in the D-scaffolds. Collagen type I was mainly present in the deposited extracellular matrix (ECM), confirming deposition of bone matrix. Finally, Alizarin Red staining showed successful mineralization on all the TCP40 samples, with higher values for the S-shaped scaffolds. Taken together, our study demonstrated that the complex pore architectures of scaffolds comprised TCP40 stimulated osteogenic differentiation and mineralization of hMSCs in vitro. Future research will aim to validate these findings in vivo.

RhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals

BackgroundRecombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro.MethodsrhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes.ResultsThe data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo.ConclusionsAltogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity

Background: Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. Methods: The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Results: Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. Conclusions: The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.

Human mesenchymal stromal cells-laden crosslinked hyaluronic acid-alginate bioink for 3D bioprinting applications in tissue engineering.

Three-dimensional (3D) bioprinting is considered one of the most advanced tools to build up materials for tissue engineering. The aim of this work was the design, development and characterization of a bioink composed of human mesenchymal stromal cells (hMSC) for extrusion through nozzles to create these 3D structures that might potentially be apply to replace the function of damaged natural tissue. In this study, we focused on the advantages and the wide potential of biocompatible biomaterials, such as hyaluronic acid and alginate for the inclusion of hMSC. The bioink was characterized for its physical (pH, osmolality, degradation, swelling, porosity, surface electrical properties, conductivity, and surface structure), mechanical (rheology and printability) and biological (viability and proliferation) properties. The developed bioink showed high porosity and high swelling capacity, while the degradation rate was dependent on the temperature. The bioink also showed negative electrical surface and appropriate rheological properties required for bioprinting. Moreover, stress-stability studies did not show any sign of physical instability. The developed bioink provided an excellent environment for the promotion of the viability and growth of hMSC cells. Our work reports the first-time study of the effect of storage temperature on the cell viability of bioinks, besides showing that our bioink promoted a high cell viability after being extruded by the bioprinter. These results support the suggestion that the developed hMSC-composed bioink fulfills all the requirements for tissue engineering and can be proposed as a biological tool with potential applications in regenerative medicine and tissue engineering.

Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators.MethodsA male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators.ResultsSaline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1β) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks.ConclusionIntravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators.

Multifunctional platform for future applications in cell and tissue engineering based on silicate phosphate hydroxyapatite co-doped with Li+, Eu3+ and Gd3+ ions

Although hydroxyapatite has established itself as a promising biomaterial for the simultaneous diagnosis and treatment of bone defects, several physicochemical properties can be further refined to achieve a specialized theranostic platform for cell and tissue engineering. In this context, we synthesized silicate-substituted hydroxyapatites co-doped with Li+, Eu3+, and Gd3+ ions (abbr. as Si-HAp-LEG) using a microwave-assisted hydrothermal method. The aim was to create a combination of dopants to stimulate bone regeneration (Li+ ion) while enabling bioimaging (Eu3+ and Gd3+ ions). Structural and morphological assessments included, e.g., XRPD (X-ray Powder Diffraction), HR-TEM (High-resolution Transmission Electron Microscopy) imaging, and BET (Brauner-Emmet-Teller) analysis. Unit-cell parameters were estimated via Rietveld refinement. The luminescence properties (emission, excitation spectra, and emission kinetics) were recorded depending on the varying Li+-ion concentration. The cytocompatibility of biomaterials was tested using hBMSCs (multipotent human bone-marrow mesenchymal stromal cells). In vitro assays evaluated biomaterials’ effects on morphology, metabolic activity (Alamar blue assay), and transcriptomic activity (RT-qPCR). The results indicate that co-doping with Li+, Eu3+, and Gd3+ ions introduces distortions while preserving the crystal structure of hydroxyapatite. Additionally, the cells maintained proper morphology and ultrastructure, internalizing biomaterials without significant cytotoxicity. The biomaterials also triggered the expression of key transcriptional factors. Si-HAp-LEG biomaterials, particularly LEG-222 and LEG-221, exhibit desired physicochemical and biological features, rendering them highly bioactive and promising for cell and tissue engineering applications.

Remnant polarization and structural arrangement in P(VDF-TrFE) electrospun fiber meshes affect osteogenic differentiation of human mesenchymal stromal cells

Many tissues and cells are influenced by mechano-electric stimulation, thus the application of piezoelectric materials has recently received considerable attention in tissue engineering. This report investigated electrospun fiber meshes based on poly(vinylidenefluoride-co-trifluoroethylene) [P(VDF-TrFE)] as instructive biomaterials for osteogenic differentiation of human mesenchymal stromal cells (hMSCs). The influence played by methyl ethyl ketone (MEK), used as a solvent in place of dimethylformamide (DMF)/acetone mixture, and the effect of rotating velocity of the electrospinning collector on fiber morphology, mechanical and piezoelectric properties were studied. The solvent had noticeable effects on morphology and piezoelectric properties of electrospun fibers, with MEK outperforming DMF/acetone. By increasing the collector velocity up to 4000 rpm, the fiber diameter reduced and the mutual alignment of the fibers increased, corresponding to enhanced mechanical properties and piezo-active β-phase content. However, as a consequence of the diverse mechanical properties of random and aligned fibrous architectures, which ultimately affected the piezoelectric properties, randomly-oriented fibers exhibited higher remnant piezoelectric properties (Vout and d31 piezoelectric coefficient) than aligned ones. On these scaffolds, hMSCs showed an excellent capability of early osteogenic differentiation, leading to high calcium production. Fiber surface topology, fiber mesh morphology and remnant piezoelectric properties played a determinant role on hMSC osteogenic commitment.

Strontium and Copper Co-Doped Multifunctional Calcium Phosphates: Biomimetic and Antibacterial Materials for Bone Implants.

β-tricalcium phosphate (β-TCP) is a promising material in regenerative traumatology for the creation of bone implants. Previously, it was established that doping the structure with certain cations can reduce the growth of bacterial activity. Recently, much attention has been paid to co-doped β-TCP, that is explained by their ability, on the one hand, to reduce cytotoxicity for cells of the human organism, on the other hand, to achieve a successful antibacterial effect. Sr, Cu-co-doped solid solutions of the composition Ca9.5-xSrxCu(PO4)7 was obtained by the method of solid-phase reactions. The Rietveld method of structural refinement revealed the presence of Sr2+ ions in four crystal sites: M1, M2, M3, and M4. The M5 site is completely occupied by Cu2+. Isomorphic substitution of Ca2+ → (Sr2+and Cu2+) expands the concentration limits of the existence of the solid solution with the β-TCP structure. No additional phases were formed up to x = 4.5 in Ca9.5-xSrxCu(PO4)7. Biocompatibility tests were performed on cell lines of human bone marrow mesenchymal stromal cells (hMSC), human fibroblasts (MRC-5) and osteoblasts (U-2OS). It was demonstrated that cytotoxicity exhibited a concentration dependence, along with an increase in osteogenesis and cell proliferation. Ca9.5-xSrxCu(PO4)7 powders showed significant inhibitory activity against pathogenic strains Escherichia coli and Staphylococcus aureus. Piezoelectric properties of Ca9.5-xSrxCu(PO4)7 were investigated. Possible ways to achieve high piezoelectric response are discussed. The combination of bioactive properties of Ca9.5-xSrxCu(PO4)7 renders them multifunctional materials suitable for bone substitutes.

Retraction Note: miR-381 modulates human bone mesenchymal stromal cells (BMSCs) osteogenesis via suppressing Wnt signaling pathway during atrophic nonunion development

Retraction Note: miR-381 modulates human bone mesenchymal stromal cells (BMSCs) osteogenesis via suppressing Wnt signaling pathway during atrophic nonunion development

The IL-6 signaling pathway contributes critically to the immunomodulatory mechanism of human decidua-derived mesenchymal stromal cells

Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been proposed as a treatment for graft-versus-host disease (GVHD), which is a major complication following allogeneic hematopoietic cell transplantation. However, clinical trials have not yielded good results, and human decidua-derived mesenchymal stromal cells (DSCs) have been proposed as an alternative. In addition, the mechanism by which DSCs exert their immunomodulatory effects is still unknown. We found that knockdown of IL-6 in DSCs reduced the expression of PD-L1 and PD-L2, which are known as classical immune checkpoint inhibitors. Expression of PD-L1 and PD-L2 was restored by adding recombinant IL-6 to the DSCs. When DSCs and IL-6-knockdown DSCs were administered as treatment in a murine GVHD model, the group receiving IL-6-knockdown DSCs had significantly higher mortality and clinical scores compared to the group receiving DSCs. Taken together, these data suggest that the IL-6 signaling pathway is a crucial contributor to the immunosuppressive capacity of DSCs.

A comparative in vitro and in vivo analysis of the impact of copper substitution on the cytocompatibility, osteogenic, and angiogenic properties of a borosilicate bioactive glass.

The 0106-B1-bioactive glass (BG) composition (in wt %: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, and 12.5 B2O3) has demonstrated favorable processing properties and promising bone regeneration potential. The present study aimed to evaluate the biological effects of the incorporation of highly pro-angiogenic copper (Cu) in 0106-B1-BG in vitro using human bone marrow-derived mesenchymal stromal cells (BMSCs) as well as its in vivo potential for bone regeneration. CuO was added to 0106-B1-BG in exchange for CaO, resulting in Cu-doped BG compositions containing 1.0, 2.5 and 5.0 wt % CuO (composition in wt %: 37.5 SiO2, 21.6/ 20.1/17.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3, and 1.0/ 2.5/ 5.0 CuO). In vitro, the BGs' impact on the viability, proliferation, and growth patterns of BMSCs was evaluated. Analyses of protein secretion, matrix formation, and gene expression were used for the assessment of the BGs' influence on BMSCs regarding osteogenic differentiation and angiogenic stimulation. The presence of Cu improved cytocompatibility, osteogenic differentiation, and angiogenic response when compared with unmodified 0106-B1-BG in vitro. In vivo, a critical-size femoral defect in rats was filled with scaffolds made from BGs. Bone regeneration was evaluated by micro-computed tomography. Histological analysis was performed to assess bone maturation and angiogenesis. In vivo effects regarding defect closure, presence of osteoclastic cells or vascular structures in the defect were not significantly changed by the addition of Cu compared with undoped 0106-B1-BG scaffolds. Hence, while the in vitro properties of the 0106-B1-BG were significantly improved by the incorporation of Cu, further evaluation of the BG composition is necessary to transfer these effects to an in vivo setting.

Sustained adenosine release: Revealing its impact on osteogenic signalling pathways of human mesenchymal stromal cells

Non-healing fractures, a global health concern arising from trauma, osteoporosis, and tumours, can lead to severe disabilities. Adenosine, integral to cellular energy metabolism, gains prominence in bone regeneration via adenosine A2B receptor activation. This study introduces a controlled-release system for localized adenosine delivery, fostering human mesenchymal stromal cell (hMSC) differentiation into functional bone cells. The study investigates how the ratio of lactic acid to glycolic acid in microparticles can influence adenosine release and explores the downstream effects on gene expression and metabolic profiles of osteogenic differentiation in hMSCs cultured in growth and osteoinductive media. Insights into adenosine-modulated signalling pathways during MSC differentiation, with osteogenic factors, provide a comprehensive understanding of the pathways involved. Analysing gene expression and metabolic profiles unravels adenosine's regulatory mechanisms in MSC differentiation. Sustained adenosine release from microparticles induces mineralization, synergizing with osteogenic media supplements, showcasing the potential of adenosine for treating critical bone defects and metabolic disorders. This study highlights the efficacy of a polymeric microparticle-based delivery system, offering novel strategies for bone repair. Unveiling adenosine's roles and associated signalling pathways advances our comprehension of molecular mechanisms steering bone regeneration, propelling innovative biomaterial, combined with metabolites, approaches for clinical use.

Modelling Myeloma Dissemination in vitro with hMSC-Interacting Subpopulations of INA-6 Cells and their Aggregation/Detachment Dynamics.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSCs) stimulate myeloma cell expansion (e.g., IL-6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs co-cultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNAseq, cell viability and apoptosis. Results were correlated with gene expression data (n=837) and survival of myeloma patients (n=536). On dispersed hMSCs, INA-6 saturate hMSC-surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time-dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC-autonomy through superior viability after IL-6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance.