Mesenchymal Cells Retain the Specificity of Embryonal Origin During Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo.

Machine learning aided single cell image analysis improves understanding of morphometric heterogeneity of human mesenchymal stem cells.

The multipotent stem cells of our body have been largely harnessed in biotherapeutics. However, as they are derived from multiple anatomical sources, from different tissues, human mesenchymal stem cells (hMSCs) are a heterogeneous population showing ambiguity in their in vitro behavior. Intra-clonal population heterogeneity has also been identified and pre-clinical mechanistic studies suggest that these cumulatively depreciate the therapeutic effects of hMSC transplantation. Although various biomarkers identify these specific stem cell populations, recent artificial intelligence-based methods have capitalized on the cellular morphologies of hMSCs, opening a new approach to understand their attributes. A robust and rapid platform is required to accommodate and eliminate the heterogeneity observed in the cell population, to standardize the quality of hMSC therapeutics globally. Here, we report our primary findings of morphological heterogeneity observed within and across two sources of hMSCs namely, stem cells from human exfoliated deciduous teeth (SHEDs) and human Wharton jelly mesenchymal stem cells (hWJ MSCs), using real-time single-cell images generated on immunophenotyping by imaging flow cytometry (IFC). We used the ImageJ software for identification and comparison between the two types of hMSCs using statistically significant morphometric descriptors that are biologically relevant. To expand on these insights, we have further applied deep learning methods and successfully report the development of a Convolutional Neural Network-based image classifier. In our research, we introduced a machine learning methodology to streamline the entire procedure, utilizing convolutional neural networks and transfer learning for binary classification, achieving an accuracy rate of 97.54%. We have also critically discussed the challenges, comparisons between solutions and future directions of machine learning in hMSC classification in biotherapeutics.

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-ß1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-ß1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

MicroRNA-196a-5p overexpression in Wharton's jelly umbilical cord stem cells promotes their osteogenic differentiation and new bone formation in bone defects in the rat calvarium.

The peri-tooth root alveolar loss often does not have sufficient space for repair material transplantation and plasticity. Mesenchymal stem cell (MSC) sheets have an advantage in providing more extracellular matrix (ECM) and may prove to be a new therapeutic consideration for this bone defect repair. The identification of key regulators that stimulate MSCs' osteogenic potential and sheet-derived ECM deposition is the key to promoting its application. In this study, we found that inhibition or overexpression of miR-196a-5p led to a decline or enhancement, respectively, in the alkaline phosphatase (ALP) activity, mineralization, and the levels of osteogenic markers, Osteocalcin (OCN), Dentin Matrix Protein 1 (DMP1), Bone Sialoprotein (BSP), and Dentin Sialophosphoprotein (DSPP) of Wharton's jelly of umbilical cord stem cells (WJCMSCs) in vitro. Moreover, the 5,6-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) analysis revealed inhibition of the WJCMSCs' proliferative ability upon miR-196a-5p overexpression. Characterization of the sheet formation by picrosirius red and Masson staining indicated that miR-196a-5p overexpression significantly promoted the collagen content in whole WJCMSC sheet-derived ECM. Furthermore, micro-CT and histopathology results indicated that the miR-196a-5p-overexpressed WJCMSC sheets significantly promoted new bone regeneration and rat calvarial bone defect closure 12 weeks following transplantation. The mRNA microarray analysis of miR-196a-5p-overexpressed WJCMSCs revealed 959 differentially expressed genes (DEGs) (34 upregulated and 925 downregulated). Moreover, 241 genes targeted by miR-196a-5p were predicted by using miRNA function websites of which only 19 predicted genes were consistent with the microarray revealed DEGs. Hence, one unrevealed downregulated DEG Serpin Family B Member 2 (SERPINB2) was investigated. And the deletion of SERPINB2 enhanced the ALP activity and mineralization of WJCMSCs in vitro. In conclusion, our study found that miR-196a-5p, as a key regulator, could repress the proliferation tendency, while stimulating osteogenic ability and WJCMSC sheet-derived ECM deposition, thus promoting new bone formation and rat calvarial bone defect closure. Furthermore, SERPINB2 is a key downstream gene involved in the miR-196a-5p-promoted WJCMSC osteogenesis.

A novel hybrid polymer of PCL/fish gelatin nanofibrous scaffold improves proliferation and differentiation of Wharton's jelly-derived mesenchymal cells into islet-like cells.

BACKGROUND: Using a different source of stem cells to compensate for the lost beta cells is a promising way to cure diabetic patients. Besides, the best efficiency of insulin-producing cells (IPCs) will appear when we culture them in an environment similar to inside the body. Hence, three-dimensional (3D) culture ameliorates the differentiation of diverse kinds of stem cells into IPCs compared to those differentiated in two-dimensional (2D) culture. In this study, we aim to create an ideal differentiation environment by using PCL/Fish gelatin nanofibrous scaffolds to differentiate Wharton's jelly-derived mesenchymal cells (WJ-MSCs) to IPCs and compare them with a 2D cultured group. METHODS: The evaluation of cellular, molecular, and functional properties of differentiated cells on the 3D and 2D cultures was investigated by several assays such as electron microscopy, quantitative PCR, immunochemistry, western blotting, and ELISA. RESULTS: The in vitro studies showed that WJ-MSCs differentiated in the 3D culture have strong properties of IPCs such as islet-like cells. The expression of pancreatic-specific genes at both RNA and protein levels showed higher differentiation efficacy of 3D culture. Besides, the results of the ELISA tests demonstrate that in both groups the differentiated cells are functional and secreted C-peptide and insulin in glucose stimulation, but the secretion of C-peptide and insulin in the 3D culture group was higher than those cultured in 2D groups. CONCLUSION: Our findings showed the use of PCL/Fish gelatin nanofibrous scaffolds with optimized differentiation protocols can promote the differentiation of IPCs from WJ-MSCs compared to the 2D culture group.

Regenerative Medicine Application of Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC) has recently generated interest in regenerative medicine. For the definition of MSC, three criteria have been proposed - plastic adherent property, specific surface antigens, and multipotent differentiation capacity. MSC exists in almost all tissues such as synovium, fat, liver, dental pulp, cord blood, Wharton's jelly, and also differentiates into osteoblast, chondrocyte, adipocyte, epithelial, and neuron cells originating from three germ layers. The use of different MSCs for regenerative therapies has been studied over the years as a promising option for treatment of tissue damages and various diseases. Here, the most frequently applied and newly developed stem cell-based techniques are designated, and recent MSC applications knowledge for regenerative medicine in the field are explained.

Nanosheets Based Approach to Elevate the Proliferative and Differentiation Efficacy of Human Wharton's Jelly Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC)-based therapy and tissue repair necessitate the use of an ideal clinical biomaterial capable of increasing cell proliferation and differentiation. Recently, MXenes 2D nanomaterials have shown remarkable potential for improving the functional properties of MSCs. In the present study, we elucidated the potential of Ti2CTx MXene as a biomaterial through its primary biological response to human Wharton's Jelly MSCs (hWJ-MSCs). A Ti2CTx nanosheet was synthesized and thoroughly characterized using various microscopic and spectroscopic tools. Our findings suggest that Ti2CTx MXene nanosheet exposure does not alter the morphology of the hWJ-MSCs; however, it causes a dose-dependent (10-200 µg/mL) increase in cell proliferation, and upon using it with conditional media, it also enhanced its tri-lineage differentiation potential, which is a novel finding of our study. A two-fold increase in cell viability was also noticed at the highest tested dose of the nanosheet. The treated hWJ-MSCs showed no sign of cellular stress or toxicity. Taken together, these findings suggest that the Ti2CTx MXene nanosheet is capable of augmenting the proliferation and differentiation potential of the cells.

Fabrication of Hydroxyapatite with Bioglass Nanocomposite for Human Wharton's-Jelly-Derived Mesenchymal Stem Cell Growing Substrate.

Recently, composite scaffolding has found many applications in hard tissue engineering due to a number of desirable features. In this present study, hydroxyapatite/bioglass (HAp/BG) nanocomposite scaffolds were prepared in different ratios using a hydrothermal approach. The aim of this research was to evaluate the adhesion, growth, viability, and osteoblast differentiation behavior of human Wharton's-jelly-derived mesenchymal stem cells (hWJMSCs) on HAp/BG in vitro as a scaffold for application in bone tissue engineering. Particle size and morphology were investigated by TEM and bioactivity was assessed and proven using SEM analysis with hWJMSCs in contact with the HAp/BG nanocomposite. Viability was evaluated using PrestoBlueTM assay and early osteoblast differentiation and mineralization behaviors were investigated by ALP activity and EDX analysis simultaneously. TEM results showed that the prepared HAp/BG nanocomposite had dimensions of less than 40 nm. The morphology of hWJMSCs showed a fibroblast-like shape, with a clear filopodia structure. The viability of hWJMSCs was highest for the HAp/BG nanocomposite with a 70:30 ratio of HAp to BG (HAp70/BG30). The in vitro biological results confirmed that HAp/BG composite was not cytotoxic. It was also observed that the biological performance of HAp70/BG30 was higher than HAp scaffold alone. In summary, HAp/BG scaffold combined with mesenchymal stem cells showed significant potential for bone repair applications in tissue engineering.

Chondrogeneic Potential of MSC from Different Sources in Spheroid Culture.

We performed a comparative study of the proliferative potential of human mesenchymal stromal cells (MSC) from three sources (tooth pulp, adipose tissue, and Wharton's jelly) in spheroid culture; human chondroblasts served as the positive control. Histological examination revealed signs of chondrogenic differentiation in all studied cell cultures and the differences in the volume and composition of the extracellular matrix. Spheroids formed by MSC from the tooth pulp and Wharton's jelly were characterized by low content of extracellular matrix and glycosaminoglycans. Spheroids from adipose tissue MSC contained maximum amount of the extracellular matrix and high content of glycosaminoglycans. Chondrocytes produced glycosaminoglycan-enriched matrix. Type II collagen was produced by chondrocytes (to a greater extent) and adipose tissue MSC (to a lesser extent). The results of our study demonstrate that MSC from the adipose tissue under conditions of spheroid culturing exhibited maximum chondrogenic potential.

Multilineage Differentiation Potential of Human Dental Pulp Stem Cells-Impact of 3D and Hypoxic Environment on Osteogenesis In Vitro.

Human dental pulp harbours unique stem cell population exhibiting mesenchymal stem/stromal cell (MSC) characteristics. This study aimed to analyse the differentiation potential and other essential functional and morphological features of dental pulp stem cells (DPSCs) in comparison with Wharton's jelly-derived MSCs from the umbilical cord (UC-MSCs), and to evaluate the osteogenic differentiation of DPSCs in 3D culture with a hypoxic microenvironment resembling the stem cell niche. Human DPSCs as well as UC-MSCs were isolated from primary human tissues and were subjected to a series of experiments. We established a multiantigenic profile of DPSCs with CD45-/CD14-/CD34-/CD29+/CD44+/CD73+/CD90+/CD105+/Stro-1+/HLA-DR- (using flow cytometry) and confirmed their tri-lineage osteogenic, chondrogenic, and adipogenic differentiation potential (using qRT-PCR and histochemical staining) in comparison with the UC-MSCs. The results also demonstrated the potency of DPSCs to differentiate into osteoblasts in vitro. Moreover, we showed that the DPSCs exhibit limited cardiomyogenic and endothelial differentiation potential. Decreased proliferation and metabolic activity as well as increased osteogenic differentiation of DPSCs in vitro, attributed to 3D cell encapsulation and low oxygen concentration, were also observed. DPSCs exhibiting elevated osteogenic potential may serve as potential candidates for a cell-based product for advanced therapy, particularly for bone repair. Novel tissue engineering approaches combining DPSCs, 3D biomaterial scaffolds, and other stimulating chemical factors may represent innovative strategies for pro-regenerative therapies.

Gradient Chitosan Hydrogels Modified with Graphene Derivatives and Hydroxyapatite: Physiochemical Properties and Initial Cytocompatibility Evaluation.

In this study, we investigated preparation of gradient chitosan-matrix hydrogels through a novel freezing-gelling-thawing method. The influence of three types of graphene family materials (GFM), i.e., graphene oxide (GO), reduced graphene oxide (rGO), and poly(ethylene glycol) grafted graphene oxide (GO-PEG), as well as hydroxyapatite (HAp) on the physicochemical and biological properties of the composite hydrogels was examined in view of their potential applicability as tissue engineering scaffolds. The substrates and the hydrogel samples were thoroughly characterized by X-ray photoelectron spectroscopy, X-ray diffractometry, infrared spectroscopy, digital and scanning electron microscopy, rheological and mechanical analysis, in vitro chemical stability and bioactivity assays, as well as initial cytocompatibility evaluation with human umbilical cord Wharton's jelly mesenchymal stem cells (hUC-MSCs). We followed the green-chemistry approach and avoided toxic cross-linking agents, using instead specific interactions of our polymer matrix with tannic acid, non-toxic physical cross-linker, and graphene derivatives. It was shown that the most promising are the gradient hydrogels modified with GO-PEG and HAp.

Substitutions of strontium in bioactive calcium silicate bone cements stimulate osteogenic differentiation in human mesenchymal stem cells.

Calcium silicate cements have been considered as alternative bone substitutes owing to its extraordinary bioactivity and osteogenicity. Unfortunately, the major disadvantage of the cements was the slow degradation rate which may limit the efficiency of bone regeneration. In this study, we proposed a facile method to synthesize degradable calcium silicate cements by incorporating strontium into the cements through solid-state sintering. The effects of Sr incorporation on physicochemical and biological properties of the cements were evaluated. Although, our findings revealed that the incorporation of strontium retarded the hardening reaction of the cements, the setting time of different cements (11-19 min) were in the acceptable range for clinical use. The presence of Sr in the CS cements would hampered the precipitation of calcium phosphate products on the surface after immersion in SBF, however, a layer of precipitated calcium phosphate products can be formed on the surface of the Sr-CS cement within 1 day immersion in SBF. More importantly, the degradation rate of the cements increased with increasing content of strontium, consequentially raised the levels of released strontium and silicon ions. The elevated dissolving products may contribute to the enhancement of the cytocompatibility, alkaline phosphatase activity, osteocalcin secretion, and mineralization of human Wharton's jelly mesenchymal stem cells. Together, it is concluded that the strontium-incorporated calcium silicate cement might be a promising bone substitute that could accelerate the regeneration of irregularly shaped bone defects.

Impact of Graphene-Based Surfaces on the Basic Biological Properties of Human Umbilical Cord Mesenchymal Stem Cells: Implications for Ex Vivo Cell Expansion Aimed at Tissue Repair.

The potential therapeutic applications of mesenchymal stem/stromal cells (MSCs) and biomaterials have attracted a great amount of interest in the field of biomedical engineering. MSCs are multipotent adult stem cells characterized as cells with specific features, e.g., high differentiation potential, low immunogenicity, immunomodulatory properties, and efficient in vitro expansion ability. Human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs) are a new, important cell type that may be used for therapeutic purposes, i.e., for autologous and allogeneic transplantations. To improve the therapeutic efficiency of hUC-MSCs, novel biomaterials have been considered for use as scaffolds dedicated to the propagation and differentiation of these cells. Nowadays, some of the most promising materials for tissue engineering include graphene and its derivatives such as graphene oxide (GO) and reduced graphene oxide (rGO). Due to their physicochemical properties, they can be easily modified with biomolecules, which enable their interaction with different types of cells, including MSCs. In this study, we demonstrate the impact of graphene-based substrates (GO, rGO) on the biological properties of hUC-MSCs. The size of the GO flakes and the reduction level of GO have been considered as important factors determining the most favorable surface for hUC-MSCs growth. The obtained results revealed that GO and rGO are suitable scaffolds for hUC-MSCs. hUC-MSCs cultured on: (i) a thin layer of GO and (ii) an rGO surface with a low reduction level demonstrated a viability and proliferation rate comparable to those estimated under standard culture conditions. Interestingly, cell culture on a highly reduced GO substrate resulted in a decreased hUC-MSCs proliferation rate and induced cell apoptosis. Moreover, our analysis demonstrated that hUC-MSCs cultured on all the tested GO and rGO scaffolds showed no alterations of their typical mesenchymal phenotype, regardless of the reduction level and size of the GO flakes. Thus, GO scaffolds and rGO scaffolds with a low reduction level exhibit potential applicability as novel, safe, and biocompatible materials for utilization in regenerative medicine.

Bioactive calcium silicate/poly-ε-caprolactone composite scaffolds 3D printed under mild conditions for bone tissue engineering.

The present study provides a solvent-free processing method for establishing the ideal porous 3-dimension (3D) scaffold filled with different ratios of calcium silicate-based (CS) powder and polycaprolactone (PCL) for 3D bone substitute application. Characterization of hybrid scaffolds developed underwent assessments for physicochemical properties and biodegradation. Adhesion and growth of human Wharton's Jelly mesenchymal stem cells (WJMSCs) on the CS/PCL blended scaffold were investigated in vitro. Cell attachment and morphology were examined by scanning electron microscope (SEM) and confocal microscope observations. Colorimetric assay was tested for assessing cell metabolic activity. In addition, RT-qPCR was also performed for the osteogenic-related and angiogenesis-related gene expression. As a result, the hydrophilicity of the scaffolds was further significantly improved after we additive CS into PCL, as well as the compressive strength up to 5.8 MPa. SEM showed that a great amount of precipitated bone-like apatite formed on the scaffold surface after immersed in the simulated body fluid. The 3D-printed scaffolds were found to enhance cell adhesion, proliferation and differentiation. Additionally, results of osteogenesis and angiogenesis proteins were expressed obviously greater in the response of WJMSCs. These results indicate the CS/PCL composite exhibited a favorable bioactivity and osteoconductive properties that could be served as a promising biomaterial for bone tissue engineering scaffolds.

Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion.

BACKGROUND AIMS: Pooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. METHODS: Frozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. RESULTS: QSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. CONCLUSIONS: QSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications.

Changes in expression of cartilaginous genes during chondrogenesis of Wharton's jelly mesenchymal stem cells on three-dimensional biodegradable poly(L-lactide-co-glycolide) scaffolds.

BACKGROUND: In cartilage tissue regeneration, it is important to develop biodegradable scaffolds that provide a structural and logistic template for three-dimensional cultures of chondrocytes. In this study, we evaluated changes in expression of cartilaginous genes during in vitro chondrogenic differentiation of WJ-MSCs on PLGA scaffolds. METHODS: The biocompatibility of the PLGA material was investigated using WJ-MSCs by direct and indirect contact methods according to the ISO 10993-5 standard. PLGA scaffolds were fabricated by the solvent casting/salt-leaching technique. We analyzed expression of chondrogenic genes of WJ-MSCs after a 21-day culture. RESULTS: The results showed the biocompatibility of PLGA and confirmed the usefulness of PLGA as material for fabrication of 3D scaffolds that can be applied for WJ-MSC culture. The in vitro penetration and colonization of the scaffolds by WJ-MSCs were assessed by confocal microscopy. The increase in cell number demonstrated that scaffolds made of PLGA copolymers enabled WJ-MSC proliferation. The obtained data showed that as a result of chondrogenesis of WJ-MSCs on the PLGA scaffold the expression of the key markers collagen type II and aggrecan was increased. CONCLUSIONS: The observed changes in transcriptional activity of cartilaginous genes suggest that the PLGA scaffolds may be applied for WJ-MSC differentiation. This primary study suggests that chondrogenic capacity of WJ-MSCs cultured on the PLGA scaffolds can be useful for cell therapy of cartilage.

Effect of dynamic three-dimensional culture on osteogenic potential of human periodontal ligament-derived mesenchymal stem cells entrapped in alginate microbeads.

BACKGROUND AND OBJECTIVE: Bioreactors are devices that efficiently create an environment that enables cell cultures to grow in a three-dimensional (3D) context mimicking in vivo conditions. In this study, we investigate the effect of dynamic fluid flow on the osteogenic potential of human mesenchymal stem cells obtained from periodontal ligament and entrapped in alginate microbeads. MATERIAL AND METHODS: After proper immunophenotyping, cells were encapsulated in barium alginate, cultured in 3D static or 3D dynamic conditions represented by a bioreactor system. Calcein-AM/propidium iodide staining was used to assess cellular viability. Quantitative real-time polymerase chain reaction was used to analyze the expression of osteogenic markers (Runx2 and COL1). Alizarin Red S staining and the Fourier transform infrared spectroscopy were used to assess mineral matrix deposition. RESULTS: Optimal encapsulation procedure, in terms of polymer pumping rate, distance from droplet generator to the gelling bath and atomizing airflow was assessed. Cell viability was not affected by encapsulation in alginate microbeads. Bioreactor cell exposure was effective in anticipating osteogenic differentiation and improving mineral matrix deposition. CONCLUSION: For the first time human mesenchymal stem cells obtained from periodontal ligaments encapsulated in alginate microbeads were cultured in a bioreactor system. This combination could represent a promising strategy to create a cell-based smart system with enhanced osteogenic potential useful for many different dental applications.

Functional analysis reveals angiogenic potential of human mesenchymal stem cells from Wharton's jelly in dermal regeneration.

Disorders in skin wound healing are a major health problem that requires the development of innovative treatments. The use of biomaterials as an alternative of skin replacement has become relevant, but its use is still limited due to poor vascularization inside the scaffolds, resulting in insufficient oxygen and growth factors at the wound site. In this study, we have developed a cell-based wound therapy consisting of the application of collagen-based dermal scaffolds containing mesenchymal stem cells from Wharton's jelly (WJ-MSC) in an immunocompetent mouse model of angiogenesis. From our comparative study on the secretion profile between WJ-MSC and adipose tissue-derived MSC, we found a stronger expression of several well-characterized growth factors, such as VEGF-A, angiopoietin-1 and aFGF, which are directly linked to angiogenesis, in the culture supernatant of WJ-MSC, both on monolayer and 3D culture conditions. WJ-MSC proved to be angiogenic both in vitro and in vivo, through tubule formation and CAM assays, respectively. Moreover, WJ-MSC consistently improved the healing response in vivo in a mouse model of human-like dermal repair, by triggering angiogenesis and further providing a suitable matrix for wound repair, without altering the inflammatory response in the animals. Since these cells can be easily isolated, cultured with high expansion rates and cryopreserved, they represent an attractive stem cell source for their use in allogeneic cell transplant and tissue engineering.

Dielectric study of the adhesion of mesenchymal stem cells from human umbilical cord on a sugarcane biopolymer.

It is of current interest the identification of appropriate matrices for growing mesenchymal stem cells (MSC). These cells are able not only to regenerate themselves but also to differentiate into other type of functional cells, and so they have been extensively used in tissue engineering. In this work, we have evaluated the use of electric impedance spectroscopy (EIS) to follow the adhesion of MSC from Wharton's jelly of the human umbilical cord (hWJMSC) on sugarcane biopolymers (SCB). Impedance spectra of the systems were obtained in the frequency range of 10(2)-10(5) Hz. An EIS investigation showed that when deposited on a metallic electrode SCB films prevent the passage of electrons between the solution and the metallic interface. The impedance spectra of hWJMSCs adhered on SCB revealed that there is a significant increase in the magnitude of the impedance when compared to that of pure SCB. The corresponding resistance (real part of the impedance) was even higher for the SCB-hWJMSC system than for SCB without cells on their surface, in an indication of an increased blockage to the electron transfers. The resistance charge transfer is extracted by curve-fitting the impedance spectra to an equivalent circuit model. Also, a shift of the phase angle to higher frequencies was obtained for SCB-hWJMSC system as a result from hWJMSC adhesion. Our study demonstrates that EIS is an appropriate method to evaluate the adhesion of MSC. SCB can be considered as a promising biomaterial for tissue engineering.

Attachment and proliferation of human gingival fibroblasts seeded on barrier membranes using Wharton's jelly-derived stem cells conditioned medium: An in vitro study.

The effect of Wharton's jelly mesenchymal stem cells conditioned medium (WJMSCs-CM) and zinc oxide nanoparticles (ZnO-NPs) on cultured human gingival fibroblasts on various barrier membranes was investigated in this study. In this study, human gingival fibroblasts were prepared and cultured on three membranes: collagen membrane, acellular dermal matrix (ADM) with ZnO-NPs, and ADM without ZnO-NPs. WJMSCs-CM was given to the testing groups, while control groups received the same membranes without WJMSCs-CM. Following 48 and 72 h, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were performed to assess cell survival. Cell proliferation on the membranes was also evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining after 48 and 72 h. Field emission scanning electron microscopy was used to determine membrane surface structure and cell adhesion. Nanoparticles were also subjected to an energy-dispersive x-ray analysis to identify their chemical structure. Two-way analysis of variance was used to conduct the statistical analysis. The p-value ≤.05 was considered significant. When ADM-ZnO-NPs were combined with CM, fibroblast viability, and adhesion significantly differed from ADM-ZnO-NPs alone. DAPI results confirmed cell proliferation in all six groups on both experiment days. The abundance and concentrated distribution of cells during cell proliferation were found in CM-containing membranes, specifically the ADM-ZnO-NPs membrane, demonstrating the improved biocompatibility of the ADM-ZnO-NPs membrane for cell proliferation. The other groups did not significantly differ from one another. WJMSCs-CM positively affected the viability and proliferation of gingival fibroblasts, but only marginally. Under certain conditions, ZnO-NPs below a specific concentration increased the biocompatibility of the membranes.