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Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels.

ABSTRACT: Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100 ?L/per minute) or perfusion and dynamic compressive loading (PL) culture (20% for 1 h, at 0.5 Hz), each day. Control FS alginate gels were maintained in six-well static culture. Gene expression analysis was conducted on days 7 and 14, while cell viability, immunostaining, and mechanical property testing were performed on day 14 only. Total glycosaminoglycan (GAG) content and GAG synthesis were assessed after 14 days. Col2a1 mRNA expression levels were significantly higher (at least threefold; p<0.05) in both bioreactor conditions compared with FS by days 7 and 14. For all gene studies, no significant differences were seen between P and PL treatments. Aggrecan mRNA levels were not significantly altered in any condition although both GAG/DNA and (35)S GAG incorporation studies indicated higher GAG retention and synthesis in the FS treatment. Collagen type II protein deposition was low in all samples, link protein distribution was more diffuse in FS condition, and aggrecan deposition was located in the outer regions of the alginate constructs in both bioreactor conditions, yet more uniformly in the FS condition. Catabolic gene expression (matrix metalloproteinase 3 [MMP3] and inducible nitric oxide synthase [iNOS]) was higher in bioreactor conditions compared with FS, although iNOS expression levels decreased to approximately fourfold less than the FS condition by day 14. Our data indicate that conditions created in the bioreactor enhanced both anabolic and catabolic responses, similar to other loading studies. Perfusion was sufficient alone to promote this dual response. PL increased the deposition of aggrecan surrounding cells compared with the other conditions; however, overall low GAG retention in the bioreactor system was likely due to both perfusion and catabolic conditions created. Optimal conditions, which permit appropriate anabolic and catabolic processes for accumulation of ECM and tissue remodeling for neocartilage development, specifically for humans, are needed.

SUBMITTER: Grogan SP

PROVIDER: S-EPMC3432904 | biostudies-other | 2012 Sep

REPOSITORIES: biostudies-other

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Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels.

Grogan Shawn P SP Sovani Sujata S Pauli Chantal C Chen Jianfen J Hartmann Andreas A Colwell Clifford W CW Lotz Martin K MK D'Lima Darryl D DD

Tissue engineering. Part A 20120612 17-18

Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100 μL/per minute) or perfusion and dynamic compressiv ...[more]

PMID: 22536910

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Project description:Hydrogels are effective platforms for use as artificial extracellular matrices, cell carriers, and to present bioactive cues. Two common natural polymers, fibrin and alginate, are broadly used to form hydrogels and have numerous advantages over synthetic materials. Fibrin is a provisional matrix containing native adhesion motifs for cell engagement, yet the interplay between mechanical properties, degradation, and gelation rate is difficult to decouple. Conversely, alginate is highly tunable yet bioinert and requires modification to present necessary adhesion ligands. To address these challenges, we developed a fibrin-alginate interpenetrating network (IPN) hydrogel to combine the desirable adhesion and stimulatory characteristics of fibrin with the tunable mechanical properties of alginate. We tested its efficacy by examining capillary network formation with entrapped co-cultures of mesenchymal stromal cells (MSCs) and endothelial cells (ECs). We manipulated thrombin concentration and alginate crosslinking density independently to modulate the fibrin structure, mesh size, degradation, and biomechanical properties of these constructs. In IPNs of lower stiffness, we observed a significant increase in total cell area (1.7 × 105 ± 7.9 × 104 µm2) and decrease in circularity (0.56 ± 0.03) compared to cells encapsulated in stiffer IPNs (4.0 × 104 ± 1.5 × 104 µm2 and 0.77 ± 0.09, respectively). Fibrinogen content did not influence capillary network formation. However, higher fibrinogen content led to greater retention of these networks confirmed via increased spreading and presence of F-actin at 7 days. This is an elegant platform to decouple cell adhesion and hydrogel bulk stiffness that will be broadly useful for cell instruction and delivery. STATEMENT OF SIGNIFICANCE: Hydrogels are widely used as drug and cell delivery vehicles and as artificial extracellular matrices to study cellular responses. However, there are limited opportunities to simultaneously control mechanical properties and degradation while mimicking the complex native adhesion motifs and ligands known to encourage cell engagement with the hydrogel. In this study, we describe a fibrin-alginate interpenetrating network (IPN) hydrogel designed to balance the compliance and provisional qualities of fibrin with the mechanical stability and tunability of alginate to interrogate these contributions on cell response. We used clinically relevant cell sources, a co-culture of endothelial cells and mesenchymal stromal cells, to test its efficacy in supporting capillary formation in vitro. These data demonstrate the promise of this IPN for use in tissue engineering.

Project description:Background 3D bioprinting cardiac patches for epicardial transplantation are a promising approach for myocardial regeneration. Challenges remain such as quantifying printability, determining the ideal moment to transplant, and promoting vascularisation within bioprinted patches. We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural self-organisation into networks. Methods We evaluated 3D-bioprinted double-layer patches using alginate/gelatine (AlgGel) hydrogels and three extrusion bioprinters (REGEMAT3D, INVIVO, BIO X). Bioink contained either neonatal mouse cardiac cell spheroids or free (not-in-spheroid) human coronary artery endothelial cells with fibroblasts, mixed with AlgGel. To test the effects on durability, some patches were bioprinted as a single layer only, cultured under minimal movement conditions or had added fibroblast-derived extracellular matrix hydrogel (AlloECM). Controls included acellular AlgGel and gelatin methacryloyl (GELMA) patches. Results Printability was similar across bioprinters. For AlgGel compared to GELMA: resolutions were similar (200–700 ?m line diameters), printing accuracy was 45 and 25%, respectively (AlgGel was 1.7x more accurate; p < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); p = 0.36. For durability, AlgGel patch median survival in culture was 14 days (IQR:10–27) overall which was not significantly affected by bioprinting system or cellular content in patches. We identified three factors which reduced durability in culture: (1) bioprinting one layer depth patches (instead of two layers); (2) movement disturbance to patches in media; and (3) the addition of AlloECM to AlgGel. Cells were viable after bioprinting followed by 28 days in culture, and all BIO X-bioprinted mouse cardiac cell spheroid patches presented contractile activity starting between day 7 and 13 after bioprinting. At day 28, endothelial cells in hydrogel displayed organisation into endothelial network-like structures. Conclusion AlgGel-based 3D bioprinted heart patches permit cardiomyocyte contractility and endothelial cell structural self-organisation. After bioprinting, a period of 2 weeks maturation in culture prior to transplantation may be optimal, allowing for a degree of tissue maturation but before many patches start to lose integrity. We quantify AlgGel printability and present novel factors which reduce AlgGel patch durability (layer number, movement, and the addition of AlloECM) and factors which had minimal effect on durability (bioprinting system and cellular patch content). Graphical Abstract

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Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels.

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Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels.

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