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Donor Variation and Optimization of Human Mesenchymal Stem Cell Chondrogenesis in Hyaluronic Acid.

ABSTRACT: Mesenchymal stem cells (MSCs) are an attractive cell type for cartilage repair that can undergo chondrogenesis in a variety of three-dimensional (3D) scaffolds. Hyaluronic acid (HA) hydrogels provide a biologically relevant interface for cell encapsulation. While previous studies have shown that MSC-laden HA constructs can mature in vitro to match native mechanical properties using cells from animal sources, clinical application will depend on the successful translation of these findings to human cells. Though numerous studies have investigated chondrogenesis of human MSC (hMSC)-laden constructs, their functional outcomes were quite inferior to those using animal sources, and donor-specific responses to 3D HA hydrogels have not been fully investigated. To that end, hMSCs were derived from seven donors, and their ability to undergo chondrogenesis in pellet culture and HA hydrogels was evaluated. Given the initial observation of overt cell aggregation and/or gel contraction for some donors, the impact of variation in cell and HA macromer concentration on functional outcomes during chondrogenesis was evaluated using one young/healthy donor. The findings show marked differences in functional chondrogenesis of hMSCs in 3D HA hydrogels based on donor. Increasing cell density resulted in increased mechanical properties, but also promoted construct contraction. Increasing the macromer density generally stabilized construct dimensions and increased extracellular matrix production, but limited the distribution of formed matrix at the center of the construct and reduced mechanical properties. Collectively, these findings suggest that the use of hMSCs may require tuning of cell density and gel mechanics on a donor-by-donor basis to provide for the most robust tissue formation for clinical application.

SUBMITTER: Kim M

PROVIDER: S-EPMC6238652 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Donor Variation and Optimization of Human Mesenchymal Stem Cell Chondrogenesis in Hyaluronic Acid.

Kim Minwook M Erickson Isaac E IE Huang Alice H AH Garrity Sean T ST Mauck Robert L RL Steinberg David R DR

Tissue engineering. Part A 20180921 21-22

Mesenchymal stem cells (MSCs) are an attractive cell type for cartilage repair that can undergo chondrogenesis in a variety of three-dimensional (3D) scaffolds. Hyaluronic acid (HA) hydrogels provide a biologically relevant interface for cell encapsulation. While previous studies have shown that MSC-laden HA constructs can mature in vitro to match native mechanical properties using cells from animal sources, clinical application will depend on the successful translation of these findings to huma ...[more]

PMID: 29792383

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Project description:IntroductionThe quality of cartilaginous tissue derived from bone marrow mesenchymal stromal stem cell (BMSC) transplantation has been correlated with clinical outcome. Therefore, culture conditions capable of modulating tissue phenotype, such as oxygen tension and scaffold composition, are under investigation. The objective of this study was to assess the effect of hypoxia on in vitro BMSC chondrogenesis within clinically approved porous scaffolds composed of collagen and hyaluronic acid (HA). It was hypothesized that hypoxic isolation/expansion and differentiation would improve BMSC chondrogenesis in each construct.MethodsOvine BMSCs were isolated and expanded to passage 2 under hypoxia (3% oxygen) or normoxia (21% oxygen). Cell proliferation and colony-forming characteristics were assessed. BMSCs were seeded at 10 million cells per cubic centimeter on cylindrical scaffolds composed of either collagen I sponge or esterified HA non-woven mesh. Chondrogenic differentiation was performed in a defined medium under hypoxia or normoxia for 14 days. Cultured constructs were assessed for gene expression, proteoglycan staining, glycosaminoglycan (GAG) quantity, and diameter change.ResultsIsolation/expansion under hypoxia resulted in faster BMSC population doublings per day (P <0.05), whereas cell and colony counts were not significantly different (P = 0.60 and 0.30, respectively). Collagen and HA scaffolds seeded with BMSCs that were isolated, expanded, and differentiated under hypoxia exhibited superior aggrecan and collagen II mRNA expressions (P <0.05), GAG quantity (P <0.05), and proteoglycan staining in comparison with normoxia. GAG/DNA was augmented with hypoxic isolation/expansion in all constructs (P <0.01). Comparison by scaffold composition indicated increased mRNA expressions of hyaline cartilage-associated collagen II, aggrecan, and SOX9 in collagen scaffolds, although expression of collagen X, which is related to hypertrophic cartilage, was also elevated (P <0.05). Proteoglycan deposition was not significantly improved in collagen scaffolds unless culture involved normoxic isolation/expansion followed by hypoxic differentiation. During chondrogenesis, collagen-based constructs progressively contracted to 60.1% ± 8.9% of the initial diameter after 14 days, whereas HA-based construct size was maintained (109.7% ± 4.2%).ConclusionsHypoxic isolation/expansion and differentiation enhance in vitro BMSC chondrogenesis within porous scaffolds. Although both collagen I and HA scaffolds support the creation of hyaline-like cartilaginous tissue, variations in gene expression, extracellular matrix formation, and construct size occur during chondrogenesis.

Project description:BackgroundAlthough mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis.MethodsAggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-β1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence.ResultsReducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to β-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a β-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue.ConclusionsThis study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.

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Donor Variation and Optimization of Human Mesenchymal Stem Cell Chondrogenesis in Hyaluronic Acid.

Publications

Donor Variation and Optimization of Human Mesenchymal Stem Cell Chondrogenesis in Hyaluronic Acid.

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