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Osteogenic differentiation of mesenchymal stem cells cultured on PLLA scaffold coated with Wharton's Jelly.

ABSTRACT: Poly-L-lactic acid (PLLA) electrospun nanofiber scaffold is one of the most commonly used synthetic polymer scaffolds for bone tissue engineering application. However, PLLA is hydrophobic in nature, hence does not maintain proper cell adhesion and tissue formation, moreover, it cannot provide the osteo-inductive environment due to inappropriate surface characteristic and the lack of surface motives participating in the first cellular events. To modify these shortcomings different approaches have been used, among those the most commonly used one is coating of the surface of the electrospun nanofiber with natural materials. In this work Wharton's jelly (WJ), a tissue which surrounds the umbilical cord vessels, reaches in high amounts of extracellular matrix (ECM) components mainly; collagen, hyaluronic acid and several sulphated glycosaminoglycans (GAGs) were used to cover the surface of electrospun PLLA nanofiber scaffolds. The surface morphology of the nanofiber scaffold was evaluated via scanning electron microscope, and the in vitro osteogenic differentiation potential was determined by MTT assay and common osteogenic marker tests such as alkaline phosphatase (ALP) activity and calcium deposition tests. Coating of WJ could not change the surface morphology and diameter of the nanofibers. However, WJ-PLLA scaffolds showed higher proliferation of human mesenchymal stem cells (MSC) than tissue culture plate (TCP) and pristine PLLA scaffolds, moreover, WJ-PPLA scaffold demonstrated significant alkaline phosphatase activity and calcium mineralization than either TCP or PLLA nanofiber scaffolds.

SUBMITTER: Ahmadi M

PROVIDER: S-EPMC5547391 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Osteogenic differentiation of mesenchymal stem cells cultured on PLLA scaffold coated with Wharton's Jelly.

Ahmadi Marziehsadat M Seyedjafari Ehsan E Zargar Seyed Jalal SJ Birhanu Gebremariam G Zandi-Karimi Ali A Beiki Bahareh B Tuzlakoglu Kadriye K

EXCLI journal 20170523

Poly-L-lactic acid (PLLA) electrospun nanofiber scaffold is one of the most commonly used synthetic polymer scaffolds for bone tissue engineering application. However, PLLA is hydrophobic in nature, hence does not maintain proper cell adhesion and tissue formation, moreover, it cannot provide the osteo-inductive environment due to inappropriate surface characteristic and the lack of surface motives participating in the first cellular events. To modify these shortcomings different approaches have ...[more]

PMID: 28827995

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Project description:Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17?-estradiol and 8-Br-cAMP on the differentiation system.WJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17?-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17?-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells.17?-estradiol at 1 ?M downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected.17?-estradiol at 1 ?M is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

Project description:In this research, hWJ-MSCs were grown on silk scaffolds and induced towards chondrogenesis by supplementation with L-ascorbic acid (LAA) or platelet rich plasma (PRP). Silk scaffolds were fabricated with salt leaching method by mixing silk fibroin (SF) with silk spidroin (SS). The silk fibroin was obtained from Bombyx mori cocoon that had been degummed, and the silk spidroin was obtained from wild-type spider Argiope appensa. The effect of scaffold composition and inducer on cell proliferation was observed through MTT assay. The most optimal treatment then continued to be used to induce hWJ-MSC towards chondrogenic differentiation for 7 and 21 days. Scaffolds characterization showed that the scaffolds produced had 3D structure with interconnected pores, and all were biocompatible with hWJ-MSCs. Scaffold with the addition of 10% SS + 90% SF showed higher compressive strength and better pore interconnectivity in comparison to 100% silk fibroin scaffold. After 48 h, cells seeded on scaffold with spidroin and fibroin mix had flattened morphology in comparison to silk fibroin scaffold which appeared to be more rounded on the scaffold surface. Scaffold with 10% (w/w) of silk spidroin (SS) + 90% (w/w) of silk fibroin (SF) was the most optimal composition for cell proliferation. Immunocytochemistry of integrin β1 and RGD sequence, showed that scaffold with SS 10% provide better cell attachment with the presence of RGD sequence from the spidroin silk which could explain the higher cell proliferation than SF100% scaffold. Based on Alcian Blue staining and Collagen Type II immunocytochemistry (ICC), cells grown on 10% SS + 90% SF scaffold with 10% PRP supplementation were the most optimal to support chondrogenesis of hWJ-MSCs. These results showed that the addition of spidroin silk from A. appensa. had impact on scaffold compressive strength and chondrogenic differentiation of hWJ-MSC and had the potential for further development of bio-based material scaffold in cartilage tissue engineering.

Project description:BackgroundMesenchymal stem/stromal cells (MSCs) are multipotent cells with a promising application potential in regenerative medicine and immunomodulation. However, MSCs cultured in vitro exhibit functional heterogeneity. The underlying molecular mechanisms that define MSC heterogeneity remain unclear.MethodsWe investigated the gene expression profile via single-cell RNA sequencing (scRNA-seq) of human primary Wharton's jelly-derived MSCs (WJMSCs) cultured in vitro from three donors. We also isolated CD142+ and CD142- WJMSCs based on scRNA-seq data and compared their proliferation capacity and "wound healing" potential in vitro. Meanwhile, we analyzed publicly available adipose-derived MSC (ADMSCs) scRNA-seq data and performed transcriptome comparison between WJMSCs and ADMSCs at the single-cell level.ResultsGO enrichment analysis of highly variable genes (HVGs) obtained from WJMSCs revealed that these genes are significantly enriched in extracellular region with binding function, involved in developmental process, signal transduction, cell proliferation, etc. Pathway analysis showed that these HVGs are associated with functional characteristics of classic MSCs, such as inflammation mediated by chemokine and cytokine signaling, integrin signaling, and angiogenesis. After regressing out the batch and cell cycle effects, these HVGs were used for dimension reduction and clustering analysis to identify candidate subpopulations. Differentially expressed gene analysis revealed the existence of several distinct subpopulations of MSCs that exhibit diverse functional characteristics related to proliferation, development, and inflammation response. In line with our data, sorted CD142+ and CD142- WJMSCs showed distinct proliferation capacity as well as "wound healing" potential. Although WJMSCs and ADMSCs were derived from different tissues and were displaying different differentiation potencies, their HVGs were largely overlapped and had similar functional enrichment.ConclusionHVGs identified in MSCs are associated with classic MSC function. Regarding therapeutic potential, these genes are associated with functional characteristics, on which the MSC clinical application were theoretically based, such as development and inflammation response. Altogether, these HVGs hold the potential to be used as candidate markers for further potency association studies.

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Osteogenic differentiation of mesenchymal stem cells cultured on PLLA scaffold coated with Wharton's Jelly.

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Osteogenic differentiation of mesenchymal stem cells cultured on PLLA scaffold coated with Wharton's Jelly.

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