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Biomimetic extracellular matrix-incorporated scaffold induces osteogenic gene expression in human marrow stromal cells.

ABSTRACT: Engineering biomaterials mimicking the biofunctionality of the extracellular matrix (ECM) is important in instructing and eliciting cell response. The native ECM is highly dynamic and has been shown to support cellular attachment, migration, and differentiation. The advantage of synthesizing an ECM-based biomaterial is that it mimics the native cellular environment. However, the ECM has tissue-specific composition and patterned arrangement. In this study, we have employed biomimetic strategies to develop a novel collagen/chitosan template that is embedded with the native ECM of differentiating human marrow stromal cells (HMSCs) to facilitate osteoblast differentiation. The scaffold was characterized for substrate stiffness by magnetic resonance imaging and nanoindentation and by immunohistochemical analysis for the presence of key ECM proteins. Gene expression analysis showed that the ECM scaffold supported osteogenic differentiation of undifferentiated HMSCs as significant changes were observed in the expression levels of growth factors, transcription factors, proteases, receptors, and ECM proteins. Finally, we demonstrate that the scaffold had the ability to nucleate calcium phosphate polymorphs to form a mineralized matrix. The results from this study suggest that the three-dimensional native ECM scaffold directly controls cell behavior and supports the osteogenic differentiation of mesenchymal stem cells.

SUBMITTER: Ravindran S

PROVIDER: S-EPMC3267968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:FK506 (also known as tacrolimus) is a potent immunosuppressive agent that is widely used in the treatment of graft-rejection and autoimmune diseases. FK506 has attracted additional attention owing to its potential role in osteogenic differentiation and bone formation. MicroRNAs (miRNAs) have been demonstrated to serve important roles in the regulation of osteogenic differentiation; however, identification of specific miRNAs and their roles in regulating FK506‑induced osteogenic differentiation have been poorly examined. In the present study, osteodifferentiation of rat bone marrow stromal cells (BMSCs) was induced with varying concentrations of FK506 (5‑5,000 nM) for 3, 7 and 14 days. Differentially expressed miRNAs were profiled using miRNA array, verified by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and subjected to gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results from the present study identified a subset of miRNAs that were differentially expressed, of which five upregulated miRNAs (miR‑106b‑5p, miR‑101b‑3p, miR‑193a‑3p, miR‑485‑3p and miR‑142‑3p) and four downregulated miRNAs (miR‑27a‑3p, miR‑207, miR‑218a‑2‑3p and let‑7a‑5p) were confirmed by RT‑qPCR. GO and KEGG analysis revealed that the predicted target genes of these miRNAs are involved in multiple biological processes and signaling pathways, including cell differentiation and the mitogen‑activated protein kinase (MAPK) signaling pathway. Verification of the miRNA‑target genes revealed that Smad5, Jagged 1 and MAPK9 were significantly upregulated, whereas Smad7, BMP and activin membrane‑bound inhibitor, and dual‑specificity phosphatase 2 were significantly downregulated during FK506‑induced osteodifferentiation. The present study may provide an experimental basis for further research on miRNA functions during FK506‑induced osteogenic differentiation in rat BMSCs.

Project description:Bone defects affect millions of people worldwide each year, leading to severe disabilities. Biomimetic scaffolds mediated tissue regeneration represents a promising alternative for bone repair. However, the major problem associated with most currently clinical available artificial bone substitutes (scaffolds) is that they mainly possess filling function but lack of osteo-induction abilities. Therefore, development of biomaterials with osteo-induction property for effective bone regeneration is highly desired. Methods: We report the design and fabrication of a photo-crosslinked sericin methacryloyl (SerMA)/ graphene oxide (GO) hydrogel (SMH/GO) as a biomimetic scaffold for the functional repair of the bone. The mechanical strength, degradation and biocompatibility behavior of SMH/GO hydrogel were measured in vitro. The effect of SMH/GO hydrogel on BMSCs proliferation, migration, osteogenesis differentiation was assessed. After that, SMH/GO-2 was used as an artificial bone substitute for bone regeneration after calvarial defects and effect on bone repair was evaluated by histological, X-Ray and microCT analysis. Furthermore, the potential mechanism of SMH/GO hydrogel regulating BMSCs migration and differentiation was investigated by RNA sequencing. Results: This scaffold has good biocompatibility, cell adhesive property, proliferation- and migration-promoting effects, and osteogenic induction property. After being implanted in a rat calvarial defect model, this SMH/GO scaffold effectively promotes new bone regeneration and achieves structural and functional repair within 12 weeks by inducing autologous bone marrow-derived mesenchymal stem cells (BMSCs) differentiation. By utilizing cell-biological assays and RNA sequencing, we reveal its possible regeneration mechanisms: the SMH/GO hydrogel regulates BMSCs migration and osteo-differentiation via activating MAPK, TNF, and chemokine signaling for bone regeneration. Conclusion: Aiming to meet clinical demands and overcome current limitations of existing artificial bones, we have developed a new type of sericin/ graphene oxide composite scaffold and provided histological, functional, and molecular evidence demonstrating that it is capable of effectively repairing defective bones by inducing autologous BMSCs directional migration and osteogenic differentiation.

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Biomimetic extracellular matrix-incorporated scaffold induces osteogenic gene expression in human marrow stromal cells.

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