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Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes.

ABSTRACT: OBJECTIVES:? Chitosan is widely used as a scaffold for bone tissue engineering. However, up-to-date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan-coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow. MATERIALS AND METHODS:? Tissue culture plates coated with chitosan at different coating densities were used to evaluate the effects on hMSC adhesion and osteoblast differentiation. hMSCs were induced to differentiate into osteoblasts on the chitosan-coated plates and were evaluated using established techniques: alkaline phosphatase assay, demonstration of presence of calcium and real time PCR. RESULTS:? The cells adhered to plates of lower coating density of chitosan, but formed viable cell aggregates at higher coating density (100 ?g/sq.cm). Coating density of 25 ?g/sq.cm, supporting cell adhesion was chosen for osteoblast differentiation experiments. Differentiating hMSCs showed higher mineral deposition and calcium content on chitosan-coated plates. Chitosan upregulated genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin-binding sialoprotein, osteopontin, osteonectin and osteocalcin, significantly. CONCLUSIONS:? We demonstrate for the first time that chitosan enhanced mineralization by upregulating the associated genes. Thus, the study may help clinical situations promoting use of chitosan in bone mineralization, necessary for healing non-union fractures and more.

SUBMITTER: Mathews S

PROVIDER: S-EPMC6496487 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes.

Mathews S S Gupta P K PK Bhonde R R Totey S S

Cell proliferation 20111020 6

<h4>Objectives</h4> Chitosan is widely used as a scaffold for bone tissue engineering. However, up-to-date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan-coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow.<h4>Materials and methods</h4> Tissue culture plates coated with chitosan at diffe ...[more]

PMID: 22011046

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Project description:The authors' previous study demonstrated that miR‑128 may exert an inhibitory effect on the osteogenic differentiation of bone marrow‑derived mesenchymal stem cells (BM‑MSCs), but its downstream mechanisms remain to be elucidated. The aim of the present study was to investigate the microRNA (miRNA/miR) and mRNA profiles of differentiated and undifferentiated BM‑MSCs and explore new downstream targets for miR‑128. The sequencing datasets of GSE107279 (miRNA) and GSE112318 (mRNA) were downloaded from the Gene Expression Omnibus database. The differentially expressed miRNAs (DEMs) and genes (DEGs) were identified using the DESeq2 method. The target genes of DEMs were predicted by the miRwalk 2.0 database. The hub target genes of miR‑128 were screened by constructing the protein‑protein interaction (PPI) network and module analysis. The expression levels of miR‑128 and crucial target genes were validated by reverse transcription‑quantitative (RT‑q) PCR before or after transfection of miR‑128 mimics to BM‑MSCs. The miRNA expression profile analysis identified miR‑128 as one of the significantly downregulated DEMs (total 338) in differentiated BM‑MSCs compared with the undifferentiated control. A total of 103 predicted target genes of miR‑128‑3p were overlapped with upregulated DEGs. By calculating the topological properties of each protein in the PPI network, 6 upregulated genes (KIT, NTRK2, YWHAB, GAB1, AXIN1 and RUNX1; fold change was the highest for NTRK2) were considered to be hub genes. Of these, 4 were enriched in module 4 (RUNX1, KIT, GAB1 and AXIN1; RUNX1 was particularly crucial as it can interact with the others), while one was enriched in module 7 (YWHAB). The expression levels of miR‑128 and these 6 target genes during the osteogenic differentiation were experimentally confirmed by RT‑qPCR. In addition, the expression levels of these 6 genes were significantly reversed after transfection of miR‑128‑3p mimics into rat BM‑MSCs compared with the miR‑control group. These findings indicated that miR‑128‑3p may inhibit the osteoblast differentiation of BM‑MSCs by downregulation of these 6 genes, particularly RUNX1, YWHAB and NTRK2.

Project description:Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell-based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast-derived EVs for bone regeneration. TSA was found to substantially alter osteoblast epigenetic function through reduced HDAC activity and increased histone acetylation. Treatment with TSA also significantly enhanced osteoblast alkaline phosphatase activity (1.35-fold), collagen production (2.8-fold) and calcium deposition (1.55-fold) during osteogenic culture (P ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) exhibited reduced particle size (1-05-fold) (P > 0.05), concentration (1.4-fold) (P > 0.05) and protein content (1.16-fold) (P ≤ 0.001) when compared to untreated EVs. TSA-EVs significantly enhanced the proliferation (1.13-fold) and migration (1.3-fold) of human bone marrow stem cells (hBMSCs) when compared to untreated EVs (P ≤ 0.05). Moreover, TSA-EVs upregulated hBMSCs osteoblast-related gene and protein expression (ALP, Col1a, BSP1 and OCN) when compared to cells cultured with untreated EVs. Importantly, TSA-EVs elicited a time-dose dependent increase in hBMSCs extracellular matrix mineralisation. MicroRNA profiling revealed a set of differentially expressed microRNAs from TSA-EVs, which were osteogenic-related. Target prediction demonstrated these microRNAs were involved in regulating pathways such as 'endocytosis' and 'Wnt signalling pathway'. Moreover, proteomics analysis identified the enrichment of proteins involved in transcriptional regulation within TSA-EVs. Taken together, our findings suggest that altering osteoblasts' epigenome accelerates their mineralisation and promotes the osteoinductive potency of secreted EVs partly due to the delivery of pro-osteogenic microRNAs and transcriptional regulating proteins. As such, for the first time we demonstrate the potential to harness epigenetic regulation as a novel engineering approach to enhance EVs therapeutic efficacy for bone repair.

Dataset Information

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes.

Publications

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes.

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