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Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

ABSTRACT: The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by focusing on differentially supporting formation of cartilaginous and bony matrix by a single cell source in a spatially controlled manner within a single material system. A cell-polymer solution of bovine mesenchymal stem cells and agarose was cast against micromolds of a serpentine network and stacked to produce tissue constructs containing two independent microfluidic networks. Constructs were fluidically connected to two controlled flow loops and supplied with independently tuned differentiation parameters for chondrogenic and osteogenic induction, respectively. Constructs receiving inductive media showed differential gene expression of both chondrogenic and osteogenic markers in opposite directions along the thickness of the construct that was recapitulated at the protein level with respect to collagens I, II, and X. A control group receiving noninductive media showed homogeneous expression of these biomarkers measured in lower concentrations at both the mRNA and protein level. This work represents an important step in the rational design of engineered osteochondral units through establishment of an enabling technology for further optimization of scaffolding formulations and bioprocessing conditions toward the production of commercially viable osteochondral tissue products.

SUBMITTER: Goldman SM

PROVIDER: S-EPMC4854211 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

Goldman Stephen M SM Barabino Gilda A GA

BioResearch open access 20160401 1

The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by ...[more]

PMID: 27190700

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Project description:Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and "rosette-like structures" from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

Dataset Information

Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

Publications

Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

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