Peripheral Blood Mesenchymal Stem Cells Research Articles

ICA/SDF-1α/PBMSCs loaded onto alginate and gelatin cross-linked scaffolds promote damaged cartilage repair.

A three-dimensional alginate-coated scaffold (GAIS) was constructed in the present study to showcase the multidifferentiation potential of peripheral blood mesenchymal stem cells (PBMSCs) and to investigate the role and mechanism by which Icariin (ICA)/stromal cell-derived factor (SDF-1α)/PBMSCs promote damaged articular repair. In addition, the ability of ICA, in combination with SDF-1α, to promote the migration and proliferation of stem cells was validated through the utilization of CCK-8 and migration experiments. The combination of ICA and SDF-1α inhibited the differentiation of PBMSCs into cartilage, as demonstrated by invivo experiments and histological staining. Both PCR and western blot experiments showed that GAIS could upregulate the expression of particular genes in chondrocytes. In comparison to scaffolds devoid of alginate (G0), PBMSCs seeded into GAIS scaffolds exhibited a greater rate of proliferation, and the conditioned medium derived from scaffolds containing SDF-1α enhanced the capacity for cell migration. Moreover, after a 12-week treatment period, GAIS, when successfully transplanted into osteochondral defects of mice, was found to promote cartilage regeneration and repair. The findings, therefore, demonstrate that GAIS enhanced the invitro capabilities of PBMSCs, including proliferation, migration, homing and chondrogenic differentiation. In addition, ICA and SDF-1α effectively collaborated to support cartilage formation invivo. Thus, the ICA/SDF-1α/PBMSC-loaded biodegradable alginate-gelatin scaffolds showcase considerable potential for use in cartilage repair.

Retracted: Effect of Recombinant Netrin-1 Protein Combined with Peripheral Blood Mesenchymal Stem Cells on Angiogenesis in Rats with Arteriosclerosis Obliterans.

[This retracts the article DOI: 10.1155/2022/3361605.].

Large-scale production of superparamagnetic iron oxide nanoparticles by flame spray pyrolysis: In vitro biological evaluation for biomedical applications

Despite the large number of synthesis methodologies described for superparamagnetic iron oxide nanoparticles (SPIONs), the search for their large-scale production for their widespread use in biomedical applications remains a mayor challenge. Flame Spray Pyrolysis (FSP) could be the solution to solve this limitation, since it allows the fabrication of metal oxide nanoparticles with high production yield and low manufacture costs. However, to our knowledge, to date such fabrication method has not been upgraded for biomedical purposes. Herein, SPIONs have been fabricated by FSP and their surface has been treated to be subsequently coated with dimercaptosuccinic acid (DMSA) to enhance their colloidal stability in aqueous media. The final material presents high quality in terms of nanoparticle size, homogeneous size distribution, long-term colloidal stability and magnetic properties. A thorough in vitro validation has been performed with peripheral blood cells and mesenchymal stem cells (hBM-MSCs). Specifically, hemocompatibility studies show that these functionalized FSP-SPIONs-DMSA nanoparticles do not cause platelet aggregation or impair basal monocyte function. Moreover, in vitro biocompatibility assays show a dose-dependent cellular uptake while maintaining high cell viability values and cell cycle progression without causing cellular oxidative stress. Taken together, the results suggest that the FSP-SPIONs-DMSA optimized in this work could be a worthy alternative with the benefit of a large-scale production aimed at industrialization for biomedical applications.

Immunomodulatory PEG-CRGD Hydrogels Promote Chondrogenic Differentiation of PBMSCs.

Cartilage damage is a common injury. Currently, tissue engineering scaffolds with composite seed cells have emerged as a promising approach for cartilage repair. Polyethylene glycol (PEG) hydrogels are attractive tissue engineering scaffold materials as they have high water absorption capacity as well as nontoxic and nutrient transport properties. However, PEG is fundamentally bio-inert and lacks intrinsic cell adhesion capability, which is critical for the maintenance of cell function. Cell adhesion peptides are usually added to improve the cell adhesion capability of PEG-based hydrogels. The suitable cell adhesion peptide can not only improve cell adhesion capability, but also promote chondrogenesis and regulate the immune microenvironment. To improve the interactions between cells and PEG hydrogels, we designed cysteine-arginine-glycine-aspartic acid (CRGD), a cell adhesion peptide covalently cross-linked with PEG hydrogels by a Michael addition reaction, and explored the tissue-engineering hydrogels with immunomodulatory effects and promoted chondrogenic differentiation of mesenchymal stem cells (MSCs). The results indicated that CRGD improved the interaction between peripheral blood mesenchymal stem cells (PBMSCs) and PEG hydrogels. PEG hydrogels modified with 1 mM CRGD had the optimal capacity to promote chondrogenic differentiation, and CRGD could induce macrophage polarization towards the M2 phenotype to promote tissue regeneration and repair. PEG-CRGD hydrogels combined with PBMSCs have the potential to be suitable scaffolds for cartilage tissue engineering.

β-Catenin promotes long-term survival and angiogenesis of peripheral blood mesenchymal stem cells via the Oct4 signaling pathway

Stem cell therapy has been extensively studied to improve heart function following myocardial infarction; however, its therapeutic potency is limited by low rates of engraftment, survival, and differentiation. Here, we aimed to determine the roles of the β-catenin/Oct4 signaling axis in the regulation of long-term survival and angiogenesis of peripheral blood mesenchymal stem cells (PBMSCs). These cells were obtained from rat abdominal aortic blood. We showed that β-catenin promotes the self-renewal, antiapoptotic effects, and long-term survival of PBMSCs by activating the Oct4 pathway through upregulation of the expression of the antiapoptotic factors Bcl2 and survivin and the proangiogenic cytokine bFGF and suppression of the levels of the proapoptotic factors Bax and cleaved caspase-3. β-Catenin overexpression increased Oct4 expression. β-Catenin knockdown suppressed Oct4 expression in PBMSCs. However, β-catenin levels were not affected by Oct4 overexpression or knockdown. Chromatin immunoprecipitation assays proved that β-catenin directly regulates Oct4 transcription in PBMSCs. In vivo, PBMSCs overexpressing β-catenin showed high survival in infarcted hearts and resulted in better myocardial repair. Further functional analysis identified Oct4 as the direct upstream regulator of Ang1, bFGF, HGF, VEGF, Bcl2, and survivin, which cooperatively drive antiapoptosis and angiogenesis of engrafted PBMSCs. These findings revealed the regulation of β-catenin in PBMSCs by the Oct4-mediated antiapoptotic/proangiogenic signaling axis and provide a breakthrough point for improving the long-term survival and therapeutic effects of PBMSCs.

Effect of Recombinant Netrin-1 Protein Combined with Peripheral Blood Mesenchymal Stem Cells on Angiogenesis in Rats with Arteriosclerosis Obliterans.

This work was aimed to explore the effect of recombinant netrin-1 protein and peripheral blood mesenchymal stem cells (MSCs) on the angiogenesis ability of atherosclerosis. 28 Sprague Dawley (SD) rats were taken as research models. The arterial occlusion models were created by surgery and then divided into the saline control group (n =7), netrin-1 treatment group (n =7), MSCs treatment group (n =7), and netrin-1 + MSCs combined treatment group (n =7). The peripheral blood MSCs were extracted from the peritoneal cavity of diseased SD rats and cultured alone or in combination with netrin-1. The individually cultured MSCs and netrin-1 were locally injected into the ischemic tissues of SD rats. The Tarlov scoring was performed at the first, second, and third week of treatment, respectively. The expression of vascular endothelial growth factor (VEGF) was also measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the capillary density was measured by immunofluorescence staining. The mean maximum contractility of the gastrocnemius muscle in each group was determined in the third week after treatment. The Tarlov score of the netrin-1 + MSCs group was significantly higher than that of the control group (P < 0.05) at the second week. To the 4th week of treatment, the Tarlov score of the netrin-1 + MSCs group was highly increased compared to the netrin-1 group and the MSCs group (P < 0.05). The expression of VEGF in the treatment groups was greatly increased each week compared to the control group (P < 0.05). Compared with the netrin-1 and the MSCs groups, the VEGF was also notably increased in the netrin-1 + MSCs group (P <0.05). The capillary densities of the treatment groups were observably greater than that of the control group in the second and third weeks (P <0.05), while the capillary density in the netrin-1 + MSCs group was also significantly increased than those in the netrin-1 group and the MSCs group (P < 0.05). The mean maximum contractility of the netrin-1 + MSCs group was remarkably higher than that of the other groups (P < 0.05). The netrin-1 + MSCs group achieved the higher Tarlov score, higher VEGF expression, higher capillary density, and better muscle recovery than netrin-1 and MSCs treatments.

A comparative evaluation of the effect of platelet rich fibrin matrix with and without peripheral blood mesenchymal stem cells on dental implant stability: A randomized controlled clinical trial

Technological advances in the field of implantology have led to the concept of surface modifications to enhance implant stability by utilization of current concepts of tissue engineering and materials such as platelet concentrates and stem cells. The purpose of the present randomized controlled clinical trial was to evaluate and compare the effect of platelet rich fibrin matrix (PRFM) with and without peripheral blood mesenchymal stem cells (PBMSCs) on implant stability; by assessing the bone to implant contact (BIC) using resonance frequency analysis (RFA), insertion torque and also to establish and correlate the same with implant stability quotient (ISQ). A total of 15 patients with 30 sites ensuring a minimum of two dental implants adjacently placed in an edentulous area; with the age group of 25-50years of both the sexes were categorized into Group 1 (dental implant with PRFM) and Group 2 (dental implant with PBMSCs embedded in PRFM). Insertion torque values at the time of dental implant placement and ISQ using RFA was recorded at 1week, 1month, and 3months post operatively. There was no significant difference (p=0.81) in Insertion torque values between both the groups (G1 and G2). Platelet rich fibrin matrix along with PBMSCs enhanced implant stability as higher and statistically significant ISQ values were noted at 1week (p=0.18), 1month (p≤0.001), and 3months (p≤0.001) intervals in the G2 group. Platelet rich fibrin matrix and PBMSCs showed promising results as a potential regenerative material for increasing and enhancing BIC and hence implant stability.

Understanding the Molecular Basis of Fragile X Syndrome Using Differentiated Mesenchymal Stem Cells.

ObjectivesFragile X syndrome (FXS) has been known as the most common cause of inherited intellectual disability and autism. This disease results from the loss of fragile X mental retardation protein expression due to the expansion of CGG repeats located on the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene. Materials & Methods In the present study, the peripheral blood-mesenchymal stem cells (PB-MSCs) of two female full mutation carriers were differentiated into neuronal cells by the suppression of bone morphogenesis pathway signaling. Then, the expression of genes adjacent to CGG repeats expansion, including SLIT and NTRK-like protein 2 (SLITRK2), SLIT and NTRK-like protein 4 (SLITRK4), methyl CpG binding protein 2 (MECP2), and gamma-aminobutyric acid receptor subunit alpha-3 (GABRA3), were evaluated in these cells using SYBR Green real-time polymerase chain reaction. ResultsThe obtained results indicated that the expression of SLITRK2 and SLITRK4 were upregulated and downregulated in the neuron-like cells differentiated from the PB-MSCs of females with FMR1 full mutation, compared to that of the normal females, respectively. Furthermore, the expression of MECP2 and GABRA3 genes were observed to be related to the phenotypic differences observed in the female FMR1 full mutation carriers. Conclusion The observed association of expression of genes located upstream of the FMR1 gene with phenotypic differences in the female carriers could increase the understanding of novel therapeutic targets for patients with mild symptoms of FXS and the patients affected by other FMR1-related disorders.

IMT504 blocks allodynia in rats with spared nerve injury by promoting the migration of mesenchymal stem cells and by favoring an anti-inflammatory milieu at the injured nerve.

IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1β and increased transforming growth factor-β1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-β1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1β transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

Abstract MP243: Detection Of Mesenchymal Stem Cells In Mobilized Autologous Peripheral Blood Transplantation From Patients With Ischemic Heart Disease

Background: Mesenchymal stem cells (MSCs) improve the cardiac function and remodeling in patients with ischemic heart disease. However, their presence in the circulating peripheral blood and post-transplantation has not been fully elucidated. We aimed to investigate the effects of intravenous transplantation of mobilized autologous peripheral blood on the number of MSCs in patients with ischemic heart disease. Methods: Granulocyte-colony stimulating factor (G-CSF, 5.0 μg/kg/day) was given subcutaneously once a day for five days to 7 patients (4 women and 3 men, aged 54-69 years) with ischemic heart disease. Leukapheresis procedure was started on the day 5 of G-CSF using the Spectra Optia cell separator. Circulating and intravenous transplantation of autologous MSCs after leukapheresis were analyzed by flow cytometry. MSCs were identified on the basis of dual positive cells (CD73 + /CD105 + or CD90 + /CD73 + or CD90 + /CD105 + ) and detected as MSCs if a cluster of at least 10 cells could be found. Results: MSCs in the circulating peripheral blood and after transplantation were detected in 2 (28%) and 6 (85%) patients, respectively. The frequency of intravenous peripheral blood MSCs increased significantly after transplantation (from 32.57 ± 22.76 x10 -4 % to 58.57 ± 28.49 x 10 -4 % , p&lt;0.001). Moreover, there were significant rise in the total white blood cells count (from 10.25 ± 4.86 x 10 3 /μl to 35.81 ± 7.07 x 10 3 /μl, p&lt;0.001) and the levels of CD34 + cells (from 1.17 ± 0.93 cells/μL to 138.30 ± 11.26 cells/μL, p&lt;0.001) after the infusion. Conclusions: The results show that intravenous transplantation of mobilized autologous peripheral blood increases the number of MSCs in patients with ischemic heart disease. Leukapheresis product of peripheral blood MSCs could therefore be a potential source for autologous transplantation in ischemic heart disease.

Cyasterone accelerates fracture healing by promoting MSCs migration and osteogenesis.

BackgroundMesenchymal Stem Cells (MSCs) therapy has become a new coming focus of clinical research in regenerative medicine. However, only a small number of implanted MSCs could successfully reach the injured areas. The previous studies have shown that fracture healing time is inversely proportional to concentration of MSCs in injured tissue.MethodsThe migration and osteogenesis of MSCs were assessed by transwell assay and Alizarin Red S staining. Levels of gene and protein expression were checked by qPCR and Western Blot. On the other hand, the enhanced migration ability of MSCs induced by Cyasterone was retarded by CXCR4 siRNA. In addition, the rat model of femoral fracture was established to evaluate the effect of Cyasterone on fracture healing. What's more, we also checked the effect of Cyasterone on mobilisation of MSCs in vivo.ResultsThe results showed that Cyasteron increased the number of MSCs in peripheral blood. The concentrations of SDF-1α in serum at different time points were determined by ELISA assay. Micro-CT and histological analysis were used to evaluate the fractured femurs.Our results showed that Cyasterone could promote the migration and osteogenesis capacities of MSCs. The fractured femurs healed faster with treatment of Cyasterone. Meanwhile, Cyasterone could significantly increase the level of SDF-1α in rats with femur fracture.ConclusionCyasterone could promote migration and osteogenesis of MSCs, and most importantly, it could accelerate bone fracture healing.Translational Potential statement: These findings provide evidence that Cyasterone could be used as a therapeutic reagent for MSCs mobilisation and osteogenesis. What's more, it could acclerate fracture healing.

Concentration Changes of Peripheral Blood Mesenchymal Stem Cells of Sprague Dawley Rats during Distraction Osteogenesis

ObjectivesTo observe the changes in the concentrations of circulating peripheral blood mesenchymal stem cells (PBMSCs) in Sprague Dawley (SD) rats and explore the pattern of changes in PBMSCs during the process of distraction osteogenesis.MethodsSD rats were randomly divided into the osteotomy with lengthening group (lengthening group), the osteotomy without lengthening group (osteotomy group), and the blank control group (control group). Each group included 24 rats. Percutaneous pinning with external fixation of the left femur was carried out in lengthening group and osteotomy group, but control group received no surgical treatment. On day 5 after operation, continuous traction was carried out at a rate of 0.25 mm/d in lengthening group, while no traction was carried out in osteotomy group. Peripheral blood was collected from all rats on days 1, 3, 7, and 16 after the start of traction. PBMSCs were isolated by density gradient centrifugation. CD105, CD34, and CD45 were selected as cell surface markers. The concentration of PBMSCs was detected by flow cytometry and compared between groups at different time points. X‐ray films were taken during and after the operation to observe whether the osteotomy end was pulled and the growth and mineralization of the new bone in the osteogenic area of the femur. Color ultrasound was used to monitor the width of the distraction space, the formation of new bone, and the blood supply of soft tissue around the distraction.ResultsAll rats were able to tolerate the operation well, and the external fixation was firm and reliable. X‐ray showed that, in lengthening group, the distraction space of femur gradually widened and new bone gradually formed in the distraction space; after 8 weeks, the samples were taken out, which showed that the new bone tissue in the lengthened area healed well. In osteotomy group, the average healing time of osteotomy was (7.12 ± 0.78) weeks. Ultrasonic examination showed that after the end of traction, the high echo callus shadow was seen in the traction space, and the blood flow signal was obviously rich at an earlier stage. In lengthening group and osteotomy group, the average concentrations of PBMSCs (3.02% ± 0.87% vs 2.95% ± 0.74%, respectively) were significantly increased in the early stage after osteotomy, and the average concentrations of PBMSCs on days 3, 7, and 16 after the start of traction were 5.34% ± 1.13% vs 3.28% ± 1.22%; 6.41% ± 1.05% vs 3.16% ± 0.92%; and 5.94% ± 1.23% vs 1.48% ± 0.52%, respectively. The concentration of PBMSCs in peripheral blood of lengthening group and osteotomy group was the same at osteotomy stage, and the difference between the two groups was not statistically significant (P > 0.05). After that, compared with lengthening group, the concentration of PBMSCs in osteotomy group gradually decreased and maintained at a certain level; the difference between the two groups was statistically significant (P < 0.05).ConclusionsDistraction osteogenesis of femur can significantly increase PBMSCs in SD rats and participate in the process of bone formation.

Chemokine-Induced PBMC and Subsequent MSC Migration Toward Decellularized Heart Valve Tissue.

Enhancing the recellularization of a decellularized heart valve in situ may lead to an improved or ideal heart valve replacement. A promising approach is leveraging the immune response for inflammation-mediated recellularization. However, this mechanism has not been previously demonstrated in vitro. This study investigated loading the chemokine MCP-1 into decellularized porcine heart valve tissue and measured the migration of human peripheral blood mononuclear cells (PBMCs) and mesenchymal stem cells (MSCs) toward the chemokine loaded valve tissue. The results of this study demonstrate that MCP-1-loaded tissues increase PBMC migration compared to non-loaded tissues. Additionally, we demonstrate MCP-1-loaded tissues that have recruited PBMCs lead to increased migration of MSCs compared to decellularized tissue alone. The results of this study provide evidence for the inflammation-mediated recellularization mechanism. Furthermore, the results support the use of such an approach for enhancing the recellularization of a decellularized heart valve.

Dimethyloxalyl Glycine Regulates the HIF-1 Signaling Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multidirectional differentiation capabilities. Dimethyloxalyl glycine (DMOG) mobilizes MSCs, and the hypoxia inducible factor-1 (HIF-1) signaling pathway plays an important role in MSC mobilization. We aimed to investigate the effect of DMOG on the HIF-1 pathway in MSCs. Rats were treated with DMOG, and the numbers of peripheral blood MSCs (PB-MSCs) and bone marrow MSCs (BM-MSCs) were detected by the Colony-forming unit fibroblastic (CFU-F) method. The growth curve, cell cycle and migration ability of PB-MSCs and BM-MSCs were detected by CCK-8, Flow cytometry and Transwell assays. Western blotting and real-time qPCR were used to detect the expression of the HIF-1 pathway. The number of bone marrow microvessels was detected by immunohistochemistry. DMOG significantly increased the numbers of PB-MSCs and BM-MSCs (P < 0.05). Further, the MSCs in peripheral blood and bone marrow still had the ability to proliferate and migrate after mobilization by DMOG. The expression levels of HIF-1α, stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) in MSCs were significantly regulated by DMOG (P < 0.05). The number of bone marrow microvessels decreased after the VEGF/VEGFR signaling pathway was blocked by SU5416 (P < 0.05). Therefore, these findings demonstrated that DMOG regulates the HIF-1α signaling pathway and promotes biological effects in MSCs.

Correlation of TCF4, GSK, TERT and TERC Expressions with Proliferation Potential of Early and Late Culture of Human Peripheral Blood Mesenchymal Stem Cells.

Objective In the recent years, mesenchymal stem cells (MSCs) were considered as the suitable source of cells for transplantation into the damaged tissues in regenerative medicine. There was low number of these cells in different organs and this characteristic was the main drawback to use them in treatment of diseases. Cellular senescence of the stem cells has been demonstrated to be dependent to the telomerase activity. The aim of present experimental study was to evaluate correlation of the expression of telomerase components and WNT signaling pathway in MSCs derived from human peripheral blood (PB-MSCs). Materials and Methods In this experimental study, following the isolation of MSCs from peripheral blood mononuclear cells, RNA was extracted from these cells in the early culture (8-9th days) and late culture (14-17th days). Then, expression of TERT, TERC, TCF4, GSK and CTNNB1 was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) based on SYBR Green. Results Our data indicated that there was a significantly reduced expression of TERT in the late culture of human MSCs derived from peripheral blood (P<0.05). Although a negative correlation was observed between GSK and TERC expression levels in the early culture of MSCs, spearman analysis showed that there was no significant correlation between the expression of telomerase components (TERC and TERT) and WNT signaling pathway (P>0.05). ConclusionThe obtained results suggested that WNT signaling pathway likely plays a minor role in the maintenance of telomere length and proliferation potential of MSCs.

Construction and preliminary study on biological characteristics of composite cell sheets of mesenchymal stem cells and endothelial progenitor cells derived from peripheral blood

To separate peripheral blood mesenchymal stem cells (PBMSC) and peripheral blood endothelial progenitor cells (PBEPC) from peripheral blood, and investigate the biological characteristics of composite cell sheets of PBMSC and PBEPC. The peripheral blood of healthy adult New Zealand white rabbits was extracted and PBMSC and PBEPC were separated by density gradient centrifugation. Morphological observation and identification of PBMSC and PBEPC were performed. The 3rd generation of PBMSC and PBEPC were used to construct a composite cell sheet at a ratio of 1∶1, and the 3rd generation of PBMSC was used to construct a single cell sheet as control. The distributions of cells in two kinds of cell sheets were observed by HE staining. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) in the supernatants of cell sheets were observed by ELISA at 1, 5, and 10 days after osteogenic induction. The morphology of PBMSC was spindle-shaped or polygonal, and PBMSC had good abilities of osteogenic and adipogenic differentiation. The morphology of PBEPC was paved stone-like, and the tube-forming test of PBEPC was positive. Two kinds of cell sheets were white translucent. The results of HE staining showed that the composite cell sheet had more cell layers and higher cell density than the single cell sheet. The expressions of ALP, OCN, and VEGF in the supernatant of the two groups of cell sheets increased with the time of induction. The expression of OCN in the group of composite cell sheet was significantly higher than that in the group of single cell sheet on the 5th and 10th day, ALP on the 10th day was significantly higher than that in the group of single cell sheet, VEGF expression on the 1st, 5th, and 10th day was significantly higher than that in the group of single cell sheet, all showing significant differences ( P<0.05), and there was no significant difference between the two groups at other time points ( P>0.05). PBMSC have stable differentiation ability, and they have good application prospects because of their minimally invasive access. Composite cell membranes constructed by co-culture of two kinds of cells and induction of membrane formation provides a new idea and exploration for tissue defect repair.

Low-Molecular-Weight Heparin-Functionalized Chitosan-Chondroitin Sulfate Hydrogels for Controlled Release of TGF-β3 and in vitro Neocartilage Formation.

Repair of hyaline cartilage remains a huge challenge in clinic because of the avascular and aneural characteristics and the paucity of endogenous repair cells. Recently, tissue engineering technique, possessing unique capacity of repairing large tissue defects, avoiding donor complications and two-stage invasive surgical procedures, has been developed a promising therapeutic strategy for cartilage injury. In this study, we incorporated low-molecular-weight heparin (LMWH) into carboxymethyl chitosan-oxidized chondroitin sulfate (CMC-OCS) hydrogel for loading transforming growth factor-β3 (TGF-β3) as matrix of peripheral blood mesenchymal stem cells (PB-MSCs) to construct tissue-engineered cartilage. Meanwhile, three control hydrogels with or without LMWH and/or TGF-β3 were also prepared. The gelling time, microstructures, mechanical properties, degradation rate, cytotoxicity, and the release of TGF-β3 of different hydrogels were investigated. In vitro experiments evaluated the tri-lineage differentiation potential of PB-MSCs, combined with the proliferation, distribution, viability, morphology, and chondrogenic differentiation. Compared with non-LMWH-hydrogels, LMWH-hydrogels (LMWH-CMC-OCS-TGF-β3) have shorter gelling time, higher mechanical strength, slower degradation rate and more stable and lasting release of TGF-β3. After two weeks of culture in vitro, expression of cartilage-specific genes collagen type-2 (COL-2) and aggrecan (AGC), and secretion of glycosaminoglycan (GAG), and COL-2 proteins in LMWH-CMC-OCS-TGF-β3 group were significantly higher than those in other groups. COL-2 immunofluorescence staining showed that the proportion of COL-2 positive cells and immunofluorescence intensity in LMWH-CMC-OCS-TGF-β3 hydrogel were significantly higher than those in other groups. The LMWH-CMC-OCS-TGF-β3 hydrogel can slowly release TGF-β3 in a long term, and meanwhile the hydrogel can provide a biocompatible microenvironment for the growth and chondrogenic differentiation of PB-MSCs. Thus, LMWH functionalized CMC-OCS hydrogels proposed in this work will be beneficial for constructing functional scaffolds for tissue-engineered cartilage.

Hypoxia reduces the osteogenic differentiation of peripheral blood mesenchymal stem cells by upregulating Notch-1 expression

ABSTRACTPurpose: Mesenchymal stem cells (MSCs) seeded on biocompatible scaffolds have therapeutic potential for bone defect repair. However, MSCs can be affected by hypoxia and nutritional deficiency due to a lack of blood vessels in the scaffolds. Here, we explored the effects of hypoxia on MSC differentiation to clarify these mechanisms.Methods: Peripheral blood mesenchymal stem cells (PBMSCs) were cultured in small individual chambers with oxygen concentrations of 1%, 9%, and 21%. Cell proliferation was evaluated by Cell Counting Kit 8 assays, and cell survival was determined using live/dead assays. Scratch assays were performed to evaluate cell migration. Ca2+ deposition/mineralization experiments, reverse transcription quantitative real-time polymerase chain reaction, and Western blotting were performed to assess the osteogenic differentiation of cells. Notch1 expression was downregulated by lentivirus-transfected PBMSCs to observe the effects of Notch1 knockdown on osteogenic gene and protein expression.Results: PBMSCs exposed to hypoxia (1% O2) demonstrated accelerated proliferation, increased migration, and reduced survival in the absence of serum. Although 9% oxygen promoted osteogenic differentiation, the osteogenic differentiation of PBMSCs was significantly reduced by 1% O2, and this effect was associated with increased Notch1 expression. Reducing Notch1 expression using small interfering RNA significantly restored the osteogenic differentiation of PBMSCs.Conclusions: Hypoxia accelerated proliferation, increased migration, and reduced PBMSC differentiation into osteoblasts by increasing Notch1 expression. These findings may contribute to the development of appropriate cell culture or in vivo transplantation conditions to maintain the full osteogenic potential of PBMSCs.

The relationship between mesenchymal stem cells, monocyte-macrophages, endothelial progenitor cells, and prognosis of hepatocellular carcinoma.

e14507 Background: This experiment is designed to investigate the clinical correlation between mesenchymal stem cells, macrophages, endothelial progenitor cells and hepatocellular carcinoma, furthermore to assess their diagnostic value. Methods: A prospective cohort study was conducted to collect patients who admitted to the Department of General Surgery, Second Affiliated Hospital of Xi'an Jiaotong University. The peripheral blood samples of patients with hepatocellular carcinoma (HCC) who diagnosed by pathology from September 2014 to August 2016 were collected. The mesenchymal stem cells, macrophages and endothelial progenitor cells (EPCs) were analyzed by flow cytometers. Results: There was no correlation between hepatocellular carcinoma and peripheral blood mesenchymal stem cells (p &gt; 0.05), and there was correlation between hepatocellular carcinoma and macrophages and endothelial progenitor cells (p &lt; 0.01). The positive correlated coefficient is 0.938 in macrophages, and the negative correlated coefficient is -0.835 in the endothelial progenitor cells. Conclusions: The differentiated pathways of mesenchymal stem cells in hepatocellular carcinoma play key roles in the progression and prognosis of hepatocellular carcinoma.

Peripheral Blood Mesenchymal Stem Cells Combined with Modified Demineralized Bone Matrix Promote Pig Cartilage Defect Repair

Objective: To investigate the mobilization of peripheral blood mesenchymal stem cells (PBMSCs) and whether a combination of PBMSCs and modified demineralized bone matrix (DBM) promoted the repair of cartilage lesions in a pig model. Methods: Pig PBMSCs were mobilized by the combined administration of granulocyte colony-stimulating factor (G-CSF) and the CXCR4 antagonist AMD3100. Colony formation was detected by the fibroblast colony-forming unit (CFU-F) count and the percentage of the CD45–CD90+ cell population by flow cytometry. The mobilized cells were identified as MSCs by their morphological characteristics, surface markers, and differentiation potentials. The composite scaffolds carrying BMP-2 and TGF-β₃ chitosan sustained-release microspheres/DBM were prepared by emulsion cross-linking and the Urist method, and scanning electron microscopy (SEM) observation was performed. The model of pig cartilage defect was prepared, and gross observation, histological examination, immunohistochemistry, and O’Driscoll scoring were performed 4, 8, and 12 weeks postoperation. Results: After mobilization, the number of CFU-Fs in the peripheral blood in the experimental group (G-CSF + AMD3100) was significantly increased compared with the control group (p < 0.05). The proportion and total number of CD45–CD90+ cells were increased (p < 0.05). The mobilized stem cells had MSC characteristics. SEM of the new tissue-engineered cartilage showed that PBMSCs were evenly grown on the surface of the scaffold and microsphere morphology had no obvious change. Gross observation, histological examination, immunohistochemistry, and O’Driscoll score were better in the experimental group than in the other groups (p < 0.05). Conclusion: G-CSF + AMD3100 is an effective mobilization agent for PBMSCs. The new tissue-engineering cartilage constructed by two-factor sustained-release microspheres/DBM composite PBMSCs effected good repair of the cartilage defect in pigs.