Simple modular bioreactors for tissue engineering: a system for characterization of oxygen gradients, human mesenchymal stem cell differentiation, and prevascularization

Abstract

Large-scale tissue engineering is limited by nutrient perfusion and mass transport limitations, especially oxygen diffusion, which restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. The goal of this work was to develop a modular approach to tissue engineering, where scaffold and tissue size, transport issues, and surgical implantation in vivo are considered from the outset. Human mesenchymal stem cells (hMSCs) were used as the model cell type, as their differentiation has been studied for several different cell lineages and often with conflicting results. Changes in the expression profiles of hMSCs differentiated under varied oxygen tensions are presented, demonstrating tissue-specific oxygen requirements for both adipogenic (20% O₂) and chondrogenic (5% O₂) differentiation. Oxygen and nutrient transport were enhanced by developing a bioreactor system for perfusing hMSC-seeded collagen gels using porous silk tubes, resulting in enhanced oxygen transport and cell viability within the gels. These systems are simple to use and scaled for versatility, to allow for the systematic study of relationships between cell content, oxygen, and cell function. The data may be combined with oxygen transport modeling to derive minimally sized modular units for construction of clinically relevant tissue-engineered constructs, a generic strategy that may be employed for vascularized target tissues.

FIG. 1.

Adipogenic differentiation at different oxygen tensions. Quantitative real-time reverse transcriptase–polymerase chain reaction results for adipogenic genes for hMSCs cultured at different oxygen tensions in collagen hydrogels. Results are given for both days 9 and 18 of culture. *p < 0.05 between groups differentiated at different oxygen tensions; ^#p < 0.05 between differentiated and control groups at the same oxygen tension (two-sample t-test). hMSCs, human mesenchymal stem cells.

FIG. 2.

Chondrogenic differentiation at different oxygen tensions. Quantitative real-time reverse transcriptase–polymerase chain reaction results for chondrogenic genes for hMSCs cultured at different oxygen tensions in collagen hydrogels. Results are given for both days 9 and 18 of culture. *p < 0.05 between groups differentiated at different oxygen tensions; ^#p < 0.05 between differentiated and control groups at the same oxygen tension (two-sample t-test).

FIG. 3.

Schematic of model system. (A) Schematic of bioreactor culture system indicating bioreactors (green arrow) and ports with Luer fittings for tubing for perfusion (red arrows) and gas exchange (blue arrow). (B) Syringe pump and assembled bioreactor setup for perfusion. (C) Close-up view of bioreactors within culture system. (D) Three-dimensional view of bioreactor and oxygen probe measurement system. The polydimethylsiloxane bioreactor system (1) is housed within an oxygen probe housing setup, which uses a micromanipulator (2) to allow precise positioning of the oxygen probe (3). Custom-made Delrin pieces serve as a bioreactor holder (4), probe mount (5), and probe holder (6), respectively. (E) Bottom view of system schematic showing oxygen probe within well of bioreactor. Blow-up of well (2 × 1.5 cm) demonstrates the silk tube within the collagen gel, which is under either static or perfusion conditions, with the oxygen probe entering from the side and measuring the oxygen gradient as a function of distance from the tube. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Cell viability as a function of distance from silk tube in the bioreactor system. Live/Dead images of hMSCs seeded within both perfused and static collagen gels in the bioreactor system for adipogenic and chondrogenic differentiation (20% and 5% O₂ tension, respectively) as well as cultured in control medium (20% O₂ tension). In each case, live cells are indicated by green fluorescence, whereas dead cells are red. Silk tubes are on the right side of each image (indicated by the dotted yellow line) with the edge of the gel to the left. Variations in gel size are due to differences in the amount of gel contraction. Scale bars are 150 μm. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Oxygen measurements in perfused and static gels. (A) System schematic. (B) Representative plots of oxygen tension versus distance from silk tube for hMSC-seeded perfused and static adipogenic and control gels at 20% O₂ tension on day 7 of culture. The plots span the entire gel, with data from next to the tube to the outer edge of the gel (greatest distance from tube). Variations in the distance from the tube are due to differences in the amount of gel contraction. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Oxygen modeling and cell viability. Comparison between cell viability results as determined by Live/Dead assay and modeling results. Higher magnification images of the Live/Dead assays highlight differences in cell morphology throughout the gels. Color images available online at www.liebertonline.com/ten.