Morphology Of Mesenchymal Stem Cells Research Articles

An Isotonic Drink Containing Pacific Cod (Gadus macrocephalus) Processing Waste Collagen Hydrolysate for Bone and Cartilage Health.

Malnutrition is one of the major factors of bone and cartilage disorders. Pacific cod (Gadus macrocephalus) processing waste is a cheap and highly promising source of bioactive substances, including collagen-derived peptides and amino acids, for bone and cartilage structure stabilization. The addition of these substances to a functional drink is one of the ways to achieve their fast intestinal absorption. Collagen hydrolysate was obtained via enzymatic hydrolysis, ultrafiltration, freeze-drying, and grinding to powder. The lyophilized hydrolysate was a light gray powder with high protein content (>90%), including collagen (about 85% of total protein) and a complete set of essential and non-essential amino acids. The hydrolysate had no observed adverse effect on human mesenchymal stem cell morphology, viability, or proliferation. The hydrolysate was applicable as a protein food supply or a structure-forming food component due to the presence of collagen fiber fragments. An isotonic fitness drink (osmolality 298.1 ± 2.1 mOsm/L) containing hydrolysate and vitamin C as a cofactor in collagen biosynthesis was prepared. The addition of the hydrolysate did not adversely affect its organoleptic parameters. The production of such functional foods and drinks is one of the beneficial ways of fish processing waste utilization.

Differentiation of Human-induced Pluripotent Stem Cell-derived Dental Stem Cells through Epithelial-Mesenchymal Interaction.

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Optimal technique for canine mesenchymal stem cells labeling with novel SPIO, MIRB™: for MRI detection of transplanted stem cells canine stroke model

ABSTRACT Background Cell-based therapy has emerged as a promising avenue for post-stroke recovery. A significant challenge lies in tracking the distribution and engraftment of transplanted cells within the target cerebral tissue. To address this, we turn to the potential of Brain MRI detection of mesenchymal stem cells (MSCs), achieved by labeling these cells with superparamagnetic iron oxide (SPIO). This is the first report of a technique to label canine MSCs using a commercially available SPIO, Molday ION Rhodamine B (MIRB), to optimize both viability and labeling efficacy for transplantation purposes.” Method Canine MSCs were incubated with addition of different MIRB concentration from 0, 10, 20, 30 μg Fe/ml. The cellular uptake of MIRB was confirmed through the analysis of fluorescent images and flow cytometry. The morphological characteristics of MSCs were assessed via microscopic visualization. Cellular viability was evaluated using both a cellometer and flow cytometry. Result Fluorescent microscopic images of all MIRB incubated MSCs groups show >70% labeled cells with homogenous signal intensity. Notably, the morphology of MSCs remained unaltered in the 10 μg Fe/ml group compared to the control group. Furthermore, among the labeled groups, the 10 μg Fe/ml concentration exhibited the highest viability when assessed using two different flow cytometry methods (95.3%, p < 0.05). Conclusion This study successfully labels canine MSCs with MIRB. The optimal concentration of 10 μg Fe/ml demonstrates optimal viability, labeling efficacy, and preserved cellular morphology.

The Mechanism of Setd2 in Mesenchymal Stromal Cell in MDS-Associated Ineffective Hematopoiety

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal, hematopoietic stem progenitor cell malignant disease. Bone marrow microenvironment participates in the occurrence and development of MDS and plays a key role. Histone methyltransferase Setd2 is the only enzyme that can catalyze the methylation of mammalian histone H3K36Me3. Previous studies have confirmed that Setd2 is involved in the the transformation of MDS to leukemia, and the expression of S100A9 decreases after Setd2 knockout. This study is based on NHD13 mice with mesenchymal stem cell (MSC) targeted knockout of Setd2. Through experimental methods such as flow cytometry, peripheral blood routine, Wright staining, HE staining, and Gomori staining, it finds that Setd2 targeted knockout mice undergo faster leukemia transformation compared to NHD13 mice; Peripheral blood white blood cells increase, while hemoglobin and platelets decrease; The differentiation of hematopoietic stem progenitor cells in bone marrow is abnormal, with abnormal hematopoiesis and increased blast cells; Spleen fibrosis aggravates and megakaryocyte infiltrates; and Extramedullary hematopoiesis in liver. Through flow cytometry,β- Galactosidase staining and other experimental methods, we finds that compared with NHD13 mice, the MSC morphology of knockout mice is irregular, and the support function for hematopoietic cells is weakened. This study demonstrates that MSC targeted knockout of Setd2 has a promoting effect on the progression of MDS. We used mass spectrometry to identify proteins that are significantly upregulated in the targeted knockout mouse bone marrow supernatant compared to the NHD13 mouse bone marrow supernatant, and and select the protein Uba5 related to hematopoiesis from them. The expression of Uba5 in the bone marrow supernatant of knockout mice is significantly higher than that in the NHD13 group through WB validation. It is speculated that the hematopoietic changes in Setd2 targeted knockout mice may be related to the downstream target protein Uba5 of Setd2. This study takes the change of bone marrow microenvironment as an entry point to explore the possible mechanism of MDS ineffective hematopoiesis, hoping to provide microenvironment regulation therapy for some MDS patients with genes that hematopoietic stem cell transplantation that cannot cure(such as TP53, NRAS, KRAS), and provide new ideas for MDS treatment. There are still many limitations in this study, further clinical validation and vivo and vitro rescue experiments on downstream target genes and proteins are still needed in the future.

COMPARATIVE STUDY ON THE EFFECT OF COLLAGEN AND FIBRIN HYDROGELS OF VARYING STIFFNESS ON MESENCHYMAL STEM CELL PARACRINE ACTIONS

Mesenchymal stem cells (MSC) have potent immunomodulatory and regenerative effects via soluble factors. One approach to improve stem cell-based therapies is encapsulation of MSC in hydrogels based on natural proteins such as collagen and fibrin, which play critical roles in bone healing. In this work, we comparatively studied the influence of collagen and fibrin hydrogels of varying stiffness on the paracrine interactions established by MSC with macrophages and osteoblasts.Type I collagen and fibrin hydrogels in a similar stiffness range loaded with MSC from donants were prepared by modifying the protein concentration. Viability and morphology of MSC in hydrogels as well as cell migration rate from the matrices were determined. Paracrine actions of MSC in hydrogels were evaluated in co-cultures with human macrophages from healthy blood donors or with osteoblasts from bone explants of patients with osteonecrosis of the femoral head.Lower matrix stiffness resulted in higher MSC viability and migration. Cell migration rate from collagen hydrogels was higher than from fibrin matrices. The secretion of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E2 (PGE2) by MSC in both collagen and fibrin hydrogels increased with increasing matrix stiffness. Tumor necrosis factor-α (TNF-α) secretion by macrophages cultured on collagen hydrogels was lower than on fibrin matrices. Interestingly, higher collagen matrix stiffness resulted in lower secreted TNF-α while the trend was opposite on fibrin hydrogels. In all cases, TNF-α levels were lower when macrophages were cultured on hydrogels containing MSC than on empty gels, an effect partially mediated by PGE2. Finally, mineralization capacity of osteoblasts co-cultured with MSC in hydrogels increased with increasing matrix stiffness, although this effect was more notably for collagen hydrogels.Paracrine interactions established by MSC in hydrogels with macrophages and osteoblasts are regulated by matrix composition and stiffness.

The expression of anti-aging protein Klotho is increased during neural differentiation of bone marrow-derived mesenchymal stem cells.

Klotho, as an antiaging protein, is involved in the maintenance and differentiation of neuronal or glial cells and, therefore, has been noticed as a potential therapeutic target for neurodegenerative disorders. Expression of Klotho has been examined in different cells and organs, however, our information about the developmental pattern of this protein during differentiation of mesenchymal stem cells (MSCs) into neuron-like cells is limited. In this study, we conducted neural differentiation of mouse bone marrow-derived-MSCs and monitored the expression of Klotho together with selected neuron-specific genes at messenger RNA (mRNA) on days 7 and 14 of differentiation using quantitative real-time PCR. In addition, Klotho status at protein level was evaluated by immunocytochemistry. The results showed a significant change in the morphology of MSCs towards neuron-like cells. These changes were observed with progressive growth and formation of cell connections towards the formation of a chain of neuron-like cells which occurred in the second week of differentiation. Morphological changes were associated with a significant increase in the expression of neuron-specific genes like pax-6, neuN and, neurofilaments (NfL). Likewise, there was an increased expression of Klotho mRNA, and accumulation of Klotho protein in neuronal cell bodies, during the cellular differentiation of MSCs. These findings provided new evidence that neuronal differentiation from the MSCs is associated with increased expression of Klotho. These data may provide insight into the importance of Klotho protein in stem cell differentiation and regeneration in response to cell death in the central nervous system.

Long Term Culture of Mesenchymal Stem Cells: No Evidence of Chromosomal Instability

Introduction: Mesenchymal stem cells (MSCs) technology has opened promising roads toward different aspects of medicine and biology. However, possible tumorgenic potential of MSCs has raised many concerns in using them in regenerative medicine. In the present study, we aimed to investigate the safety of MSCs by in-vitro culture and karyotype analysis. Materials and Methods: MSCs were successfully cultured in DMEM medium including 3.5 ng/ml recombinant human basic fibroblast growth factor (bFGF) and 20 % fetal bovine serum (FBS) within 25 passages. Results: No significant abnormality was observed in basic karyotype and morphology of MSCs in different passages of culture. Conclusions: Using of EGF free medium containing low bFGF concentration are possible explanations for observed integrity in karyotype and morphology of MSCs. Our findings would be further convincing evidence on the safety of MSCs under suggested conditions to guarantee using of them in regenerative medicine and disease treatment.

Favorable Biological Performance Regarding the Interaction between Gold Nanoparticles and Mesenchymal Stem Cells.

Gold nanoparticles (AuNPs) are well known to interact with cells, leading to different cell behaviors such as cell proliferation and differentiation capacity. Biocompatibility and biological functions enhanced by nanomedicine are the most concerning factors in clinical approaches. In the present research, AuNP solutions were prepared at concentrations of 1.25, 2.5, 5 and 10 ppm for biocompatibility investigations. Ultraviolet-visible spectroscopy was applied to identify the presence of AuNPs under the various concentrations. Dynamic Light Scattering assay was used for the characterization of the size of the AuNPs. The shape of the AuNPs was observed through a Scanning Electron Microscope. Afterward, the mesenchymal stem cells (MSCs) were treated with a differentiation concentration of AuNP solutions in order to measure the biocompatibility of the nanoparticles. Our results demonstrate that AuNPs at 1.25 and 2.5 ppm could significantly enhance MSC proliferation, decrease reactive oxygen species (ROS) generation and attenuate platelet/monocyte activation. Furthermore, the MSC morphology was observed in the presence of filopodia and lamellipodia while being incubated with 1.25 and 2.5 ppm AuNPs, indicating that the adhesion ability was enhanced by the nanoparticles. The expression of matrix metalloproteinase (MMP-2/9) in MSCs was found to be more highly expressed under 1.25 and 2.5 ppm AuNP treatment, relating to better cell migrating ability. Additionally, the cell apoptosis of MSCs investigated with Annexin-V/PI double staining assay and the Fluorescence Activated Cell Sorting (FACS) method demonstrated the lower population of apoptotic cells in 1.25 and 2.5 ppm AuNP treatments, as compared to high concentrations of AuNPs. Additionally, results from a Western blotting assay explored the possibility that the anti-apoptotic proteins Cyclin-D1 and Bcl-2 were remarkably expressed. Meanwhile, real-time PCR analysis demonstrated that the 1.25 and 2.5 ppm AuNP solutions induced a lower expression of inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6 and IL-8). According to the tests performed on an animal model, AuNP 1.25 and 2.5 ppm treatments exhibited the better biocompatibility performance, including anti-inflammation and endothelialization. In brief, 1.25 and 2.5 ppm of AuNP solution was verified to strengthen the biological functions of MSCs, and thus suggests that AuNPs become the biocompatibility nanomedicine for regeneration research.

Regulation of Mesenchymal Stem Cell Morphology Using Hydrogel Substrates with Tunable Topography and Photoswitchable Stiffness.

Cell function can be directly influenced by the mechanical and structural properties of the extracellular environment. In particular, cell morphology and phenotype can be regulated via the modulation of both the stiffness and surface topography of cell culture substrates. Previous studies have highlighted the ability to design cell culture substrates to optimise cell function. Many such examples, however, employ photo-crosslinkable polymers with a terminal stiffness or surface profile. This study presents a system of polyacrylamide hydrogels, where the surface topography can be tailored and the matrix stiffness can be altered in situ with photoirradiation. The process allows for the temporal regulation of the extracellular environment. Specifically, the surface topography can be tailored via reticulation parameters to include creased features with control over the periodicity, length and branching. The matrix stiffness can also be dynamically tuned via exposure to an appropriate dosage and wavelength of light, thus, allowing for the temporal regulation of the extracellular environment. When cultured on the surface of the hydrogels, the morphology and alignment of immortalised human mesenchymal stem cells can be directly influenced through the tailoring of surface creases, while cell size can be altered via changes in matrix stiffness. This system offers a new platform to study cellular mechanosensing and the influence of extracellular cues on cell phenotype and function.

Cinnamaldehyde regulates mitochondrial quality against hydrogen peroxide induced apoptosis in mouse lung mesenchymal stem cells via the PINK1/Parkin signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease without effective treatments. Mitochondrial dysfunction weakens the ability of mesenchymal stem cells (MSCs) to repair the distal lung epithelium, which is a probable pathogenesis of IPF. In previous research, we found that cinnamaldehyde (CA) can maintain the mitochondrial morphology of MSCs. This present study evaluated the effect and mechanism of CA on murine lung MSCs using the hydrogen peroxide model. Antioxidant effects and mitochondrial function were determined using flow cytometry. The mRNA levels of mitochondrial dynamics and the expressions of autophagy-related proteins were also detected. CA can increase the levels of SOD, MMP and ATP, decrease the rate of ROS and apoptosis, and restore the mitochondrial structure. CA can also improve the mRNA expression of MFN1, MFN2, FIS1, DRP1, OPA1, and PGC-1α, increase the expression of LC3 II and p62 and promote the PINK1/Parkin signaling pathway. Our results demonstrated that CA can control mitochondrial quality and avoid apoptosis, which may be associated with the regulation of the PINK1/Parkin signaling pathway.

High throughput screening of mesenchymal stem cell lines using deep learning

Mesenchymal stem cells (MSCs) are increasingly used as regenerative therapies for patients in the preclinical and clinical phases of various diseases. However, the main limitations of such therapies include functional heterogeneity and the lack of appropriate quality control (QC) methods for functional screening of MSC lines; thus, clinical outcomes are inconsistent. Recently, machine learning (ML)-based methods, in conjunction with single-cell morphological profiling, have been proposed as alternatives to conventional in vitro/vivo assays that evaluate MSC functions. Such methods perform in silico analyses of MSC functions by training ML algorithms to find highly nonlinear connections between MSC functions and morphology. Although such approaches are promising, they are limited in that extensive, high-content single-cell imaging is required; moreover, manually identified morphological features cannot be generalized to other experimental settings. To address these limitations, we propose an end-to-end deep learning (DL) framework for functional screening of MSC lines using live-cell microscopic images of MSC populations. We quantitatively evaluate various convolutional neural network (CNN) models and demonstrate that our method accurately classifies in vitro MSC lines to high/low multilineage differentiating stress-enduring (MUSE) cells markers from multiple donors. A total of 6,120 cell images were obtained from 8 MSC lines, and they were classified into two groups according to MUSE cell markers analyzed by immunofluorescence staining and FACS. The optimized DenseNet121 model showed area under the curve (AUC) 0.975, accuracy 0.922, F1 0.922, sensitivity 0.905, specificity 0.942, positive predictive value 0.940, and negative predictive value 0.908. Therefore, our DL-based framework is a convenient high-throughput method that could serve as an effective QC strategy in future clinical biomanufacturing processes.

The Structural Interactions of Molecular and Fibrillar Collagen Type I with Fibronectin and Its Role in the Regulation of Mesenchymal Stem Cell Morphology and Functional Activity.

The observed differences in the structure of native tissue and tissue formed in vitro cause the loss of functional activity of cells cultured in vitro. The lack of fundamental knowledge about the protein mechanism interactions limits the ability to effectively create in vitro native tissue. Collagen is able to spontaneously assemble into fibrils in vitro, but in vivo, other proteins, for example fibronectin, have a noticeable effect on this process. The molecular or fibrillar structure of collagen plays an equally important role. Therefore, we studied the interaction of the molecular and fibrillar structure of collagen with fibronectin. Atomic force and transmission electron microscopy showed that the presence of fibronectin does not affect the native structure and diameter of collagen fibrils. Confocal microscopy demonstrated that the collagen structure affects the cell morphology. Cells are better spread on molecular collagen compared with cells cultured on fibrillar collagen. Fibronectin promotes the formation of a large number of focal contacts, while in combination with collagen of both forms, its effect is leveled. Thus, understanding the mechanisms of the relationship between the protein structure and composition will effectively manage the creation in vitro of a new tissue with native properties.

Plant Tissue Parenchyma and Vascular Bundles Selectively Regulate Stem Cell Mechanosensing and Differentiation.

Plant tissues are plentiful, diverse, and due to convergent evolution are structurally similar to many animal tissues. Decellularized plant tissues feature microtopographies that resemble cancellous bone (porous parenchyma) and skeletal muscle (fibrous vascular bundles). However, the use of plant tissues as an inexpensive and abundant biomaterial for controlling stem cell behavior has not been widely explored. Celery plant tissues were cut cross-sectionally (porous parenchyma) or longitudinally (fibrous vascular bundles) and decellularized. Human mesenchymal stem cells (MSCs) were then cultured atop plant tissues and confocal imaging of single cells was used to evaluate the early effects of microtopography on MSC adhesion, morphology, cytoskeletal alignment, Yes-associated protein (YAP) signaling, and downstream lineage commitment to osteogenic or myogenic phenotypes. Microtopography was conserved post plant tissue decellularization and MSCs attached and proliferated on plant tissues. MSCs cultured on porous parenchyma spread isotropically along the periphery of plant tissue pores. In contrast, MSCs cultured on vascular bundles spread anisotropically and aligned in the direction of fibrous vascular bundles. Differences in microtopography also influenced MSC nuclear YAP localization and actin anisotropy, with higher values observed on fibrous tissues. When exposed to osteogenic or myogenic culture medium, MSCs on porous parenchyma had a higher percentage of cells stain positive for bone biomarker alkaline phosphatase, whereas myoblast determination protein 1 (MyoD) was significantly upregulated for MSCs on fibrous vascular bundles. Together, these results show that plant tissues are an abundant biomaterial with defined microarchitecture that can reproducibly regulate MSC morphology, mechanosensing, and differentiation. The online version of this article contains supplementary material available 10.1007/s12195-022-00737-9.

High resolution DLP stereolithography to fabricate biocompatible hydroxyapatite structures that support osteogenesis.

Lithography based additive manufacturing techniques, specifically digital light processing (DLP), are considered innovative manufacturing techniques for orthopaedic implants because of their potential for construction of complex geometries using polymers, metals, and ceramics. Hydroxyapatite (HA) coupons, printed using DLP, were evaluated for biological performance in supporting viability, proliferation, and osteogenic differentiation of the human cell line U2OS and human mesenchymal stem cells (MSCs) up to 35 days in culture to determine feasibility for future use in development of complex scaffold geometries. Contact angle, profilometry, and scanning electron microscopy (SEM) measurements showed the HA coupons to be hydrophilic, porous, and having micro size surface roughness, all within favourable cell culture ranges. The study found no impact of leachable and extractables form the DLP printing process. Cells seeded on coupons exhibited morphologies comparable to conventional tissue culture polystyrene plates. Cell proliferation rates, as determined by direct cell count and the RealTime-GloTM MT Cell Viability Assay, were similar on HA coupons and standard tissue culture polystyrene plates). Osteogenic differentiation of human MSCs on HA coupons was confirmed using alkaline phosphatase, Alizarin Red S and von Kossa staining. The morphology of MSCs cultured in osteogenic medium for 14 to 35 days was similar on HA coupons and tissue culture polystyrene plates, with osteogenic (geometric, cuboidal morphology with dark nodules) and adipogenic (lipid vesicles and deposits) features. We conclude that the DLP process and LithaBone HA400 slurry are biocompatible and are suitable for osteogenic applications. Coupons served as an effective evaluation design in the characterization and visualization of cell responses on DLP printed HA material. Results support the feasibility of future technical development for 3D printing of sophisticated scaffold designs, which can be constructed to meet the mechanical, chemical, and porosity requirements of an artificial bone scaffold.

The role of the aging microenvironment on the fate of PDGFRβ lineage cells in skeletal muscle repair

BackgroundDuring aging, perturbation of muscle progenitor cell (MPC) constituents leads to progressive loss of muscle mass and accumulation of adipose and fibrotic tissue. Mesenchymal stem cells (MSCs) give rise to adipocytes and fibroblasts that accumulate in injured and pathological skeletal muscle through constitutive activation of platelet-derived growth factor receptors (PDGFRs). Although the role of the PDGFRα has been widely explored, there is a paucity of evidence demonstrating the role of PDGFRβ in aged skeletal muscle.MethodsIn this study, we investigated the role of PDGFRβ lineage cells in skeletal muscle during aging by using Cre/loxP lineage tracing technology. The PDGFR-Cre mice were crossed with global double-fluorescent Cre reporter mice (mTmG) that indelibly marks PDGFRβ lineage cells. Those cells were analyzed and compared at different ages in the skeletal muscle of the mice.ResultsOur results demonstrated that PDGFRβ lineage cells isolated from the muscles of young mice are MPC-like cells that exhibited satellite cell morphology, expressed Pax7, and undergo myogenic differentiation producing myosin heavy chain expressing myotubes. Conversely, the PDGFRβ lineage cells isolated from muscles of old mice displayed MSC morphology with a reduced myogenic differentiation potential while expressing adipogenic and fibrotic differentiation markers. PDGFRβ lineage cells also gave rise to newly regenerated muscle fibers in young mice after muscle injury, but their muscle regenerative process is reduced in old mice.ConclusionsOur data suggest that PDGFRβ lineage cells function as MPCs in young mice, while the same PDGFRβ lineage cells from old mice undergo a fate switch participating in adipose and fibrotic tissue infiltration in aged muscle. The inhibition of fate-switching in PDGFRβ lineage cells may represent a potential approach to prevent fibrosis and fatty infiltration in skeletal muscle during the aging process.

Effects of nanofiber scaffolds coated with nanoparticulate and micro-particulate FDBA on the morphology, adhesion and proliferation of human mesenchymal stem cells.

Background:Freeze dried bone allograft nanoparticles on a nanofiber membrane may serve as an ideal scaffold for bone regeneration. This study aimed to assess the biological behavior of human MSCs in terms of proliferation and adhesion to nanoparticulate and microparticulate FDBA scaffolds on PLLA nanofiber membrane. Methods:In this experimental study, PLLA nanofiber scaffolds were synthesized by the electrospinning method. The FDBA nanoparticles were synthesized mechanically. The FDBA nanoparticles and microparticles were loaded on the surface of PLLA nanofiber membrane. A total of 64 scaffold samples in four groups of n-FDBA/PLLA, FDBA/PLLA, PLLA and control were placed in 24-well polystyrene tissue culture plates; 16 wells were allocated to each group. Data were analyzed using one-way ANOVA and Bonferroni test. Results:The proliferation rate of MSCs was significantly higher in the nanoparticulate group compared to the microparticulate group at five days (p = 0.034). Assessment of cell morphology at 24 hours revealed spindle-shaped cells with a higher number of appendages in the nanoparticulate group compared to other groups. Conclusion:MSCs on n-FDBA/PLLA scaffold were morphologically more active and flatter with a higher number of cellular appendages, as compared to FDBA/PLLA. It seems that the nanoparticulate scaffold is superior to the microparticulate scaffold in terms of proliferation, attachment, and morphology of MSCs in vitro.

Human Mesenchymal Stem Cells as a Carrier for a Cell-Mediated Drug Delivery.

A number of preclinical and clinical studies have demonstrated the efficiency of mesenchymal stromal cells to serve as an excellent base for a cell-mediated drug delivery system. Cell-based targeted drug delivery has received much attention as a system to facilitate the uptake a nd transfer of active substances to specific organs and tissues with high efficiency. Human mesenchymal stem cells (MSCs) are attracting increased interest as a promising tool for cell-based therapy due to their high proliferative capacity, multi-potency, and anti-inflammatory and immunomodulatory properties. In particular, these cells are potentially suitable for use as encapsulated drug transporters to sites of inflammation. Here, we studied the in vitro effects of incorporating synthetic polymer microcapsules at various microcapsule-to-cell ratios on the morphology, ultrastructure, cytokine profile, and migration ability of human adipose-derived MSCs at various time points post-phagocytosis. The data show that under appropriate conditions, human MSCs can be efficiently loaded with synthesized microcapsules without damaging the cell’s structural integrity with unexpressed cytokine secretion, retained motility, and ability to migrate through 8 μm pores. Thus, the strategy of using human MSCs as a delivery vehicle for transferring microcapsules, containing bioactive material, across the tissue–blood or tumor–blood barriers to facilitate the treatment of stroke, cancer, or inflammatory diseases may open a new therapeutic perspective.

Macromolecular modulation of a 3D hydrogel construct differentially regulates human stem cell tissue-to-tissue interface

The simultaneous generation of multiple tissues and their functional assembly into complex tissues remains a critical challenge for regenerative medicine. The tissue-to-tissue interface connecting two adjacent tissues is vital in effective tissue function. The presented worked hypothesize that differential functional property can be engineered by modulating the macromolecular composition of a 3D hydrogel construct and distinctively endow stem cell fate. Hence, it was possible to successfully generate macromolecular constructs by using the extracellular matrix (ECM)-based materials; type I collagen (Col I) and hyaluronic acid (HA); and natural-derived biomaterials as methacrylated gellan-gum (GGMA). The 3D hydrogel constructs consisted of two dissimilar layers: 1) Col I: HA hydrogel and 2) GGMA hydrogel. The tissue-to-tissue interface was created by seeding human mesenchymal stem cells (MSCs) between the two layers. Differential functional rheological and mechanical properties characterized the acellular 3D gradient hydrogel constructs. The cell-based 3D hydrogel constructs were assessed for MSCs viability by live/dead staining. Assessing apoptosis by flow cytometry, data showed the feasibility of the 3D hydrogel constructs in maintaining cell viability with no apoptosis induction onto MSCs. A homogeneous distribution was achieved in a successful cellular tissue-to-tissue interface. Human MSCs low proliferative rate and low ECM deposition were seen for all constructs; however, lower proliferative rate within the ECM microenvironment highlights controlled self-renewal of MSCs. The 3D hydrogel constructs maintained the human MSCs phenotype, yet the macromolecular modulation allowed tuning the human MSCs morphology from round to spindle-shaped phenotype. The intrinsic properties of the 3D cell-based hydrogel construct induced differential inflammatory and angiogenic paracrine secretory profiles owing to the dissimilar engineered biophysical milieu. Human MSCs sense the nearby macromolecular environment adjusting the cell-ECM interactions, which influence cell behaviour and fate. Beyond multi-tissue regeneration, the engineered cellular 3D hydrogel constructs may simultaneously address immune regeneration.

A Review on the Design of Hydrogels With Different Stiffness and Their Effects on Tissue Repair.

Tissue repair after trauma and infection has always been a difficult problem in regenerative medicine. Hydrogels have become one of the most important scaffolds for tissue engineering due to their biocompatibility, biodegradability and water solubility. Especially, the stiffness of hydrogels is a key factor, which influence the morphology of mesenchymal stem cells (MSCs) and their differentiation. The researches on this point are meaningful to the field of tissue engineering. Herein, this review focus on the design of hydrogels with different stiffness and their effects on the behavior of MSCs. In addition, the effect of hydrogel stiffness on the phenotype of macrophages is introduced, and then the relationship between the phenotype changes of macrophages on inflammatory response and tissue repair is discussed. Finally, the future application of hydrogels with a certain stiffness in regenerative medicine and tissue engineering has been prospected.

Two Hits for Bone Regeneration in Aged Patients: Vertebral Bone Marrow Clot as a Biological Scaffold and Powerful Source of Mesenchymal Stem Cells.

Recently, the use of a new formulation of bone marrow aspirate (BMA), the BMA clot, has been described. This product entails a naturally formed clot from the harvested bone marrow, which retains all the BMA components preserved in a matrix biologically molded by the clot. Even though its beneficial effects were demonstrated by some studies, the impact of aging and aging-associated processes on biological properties and the effect of BMA cell-based therapy are currently unknown. The purpose of our study was to compare selected parameters and properties of clotted BMA and BMA-derived mesenchymal stem cells (MSCs) from younger (<45 years) and older (>65 years) female donors. Clotted BMA growth factors (GFs) expression, MSCs morphology and viability, doubling time, surface marker expression, clonogenic potential, three-lineage differentiation, senescence-associated factors, and Klotho synthesis from younger and older donors were analyzed. Results indicated that donor age does not affect tissue-specific BMA clot regenerative properties such as GFs expression and MSCs morphology, viability, doubling time, surface antigens expression, colony-forming units, osteogenic and adipogenic differentiation, and Klotho and senescence-associated gene expression. Only few differences, i.e., increased platelet-derived growth factor-AB (PDGF-AB) synthesis and MSCs Aggrecan (ACAN) expression, were detected in younger donors in comparison with older ones. However, these differences do not interfere with all the other BMA clot biological properties. These results demonstrated that BMA clot can be applied easily, without any sample processing and avoiding potential contamination risks as well as losing cell viability, proliferation, and differentiation ability, for autologous transplantation in aged patients. The vertebral BMA clot showed two successful hits since it works as a biological scaffold and as a powerful source of mesenchymal stem cells, thus representing a novel and advanced therapeutic alternative for the treatment of orthopedic injuries.