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Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.

ABSTRACT: In vitro three-dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure-transmitting mode achieved by slight deformation of a flexible membrane in a completely sealed stainless steel device. The newly developed bioreactor efficiently avoided the potential risks of previously reported pressure-transmitting modes and simultaneously addressed a series of important issues, such as pressure scopes, culture chamber sizes, sealability, contamination control, and CO₂ balance. The whole bioreactor system realized stable long-term (8 weeks) culture under high HP (5-10 MPa) without the problems of medium leakage and contamination. Furthermore, the results of in vitro 3D tissue culture based on a cartilage regeneration model revealed that HP provided by the newly developed bioreactor efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, thickness, and homogeneity. Detailed analysis in cell proliferation, cartilage matrix production, and cross-linking level of collagen macromolecules, as well as density and alignment of collagen fibers, further revealed the possible mechanisms that HP regulated in vitro cartilage regeneration. The current study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation. Stem Cells Translational Medicine 2017;6:982-991.

SUBMITTER: Chen J

PROVIDER: S-EPMC5442788 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.

Chen Jie J Yuan Zhaoyuan Z Liu Yu Y Zheng Rui R Dai Yao Y Tao Ran R Xia Huitang H Liu Hairong H Zhang Zhiyong Z Zhang Wenjie W Liu Wei W Cao Yilin Y Zhou Guangdong G

Stem cells translational medicine 20161107 3

In vitro three-dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure-transmitting mode achieved by slight deformation of a flexible m ...[more]

PMID: 28297584

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Project description:Because of the superior characteristics of photocrosslinkable hydrogels suitable for 3D cell-laden bioprinting, tissue regeneration based on photocrosslinkable hydrogels has become an important research topic. However, due to nutrient permeation obstacles caused by the dense networks and static culture conditions, there have been no successful reports on in vitro cartilage regeneration with certain thicknesses based on photocrosslinkable hydrogels. To solve this problem, hydrostatic pressure (HP) provided by the bioreactor was used to regulate the in vitro cartilage regeneration based on hybrid photocrosslinkable (HPC) hydrogel. Chondrocyte laden HPC hydrogels (CHPC) were cultured under 5 MPa HP for 8 weeks and evaluated by various staining and quantitative methods. Results demonstrated that CHPC can maintain the characteristics of HPC hydrogels and is suitable for 3D cell-laden bioprinting. However, HPC hydrogels with concentrations over 3% wt% significantly influenced cell viability and in vitro cartilage regeneration due to nutrient permeation obstacles. Fortunately, HP completely reversed the negative influences of HPC hydrogels at 3% wt%, significantly enhanced cell viability, proliferation, and extracellular matrix (ECM) deposition by improving nutrient transportation and up-regulating the expression of cartilage-specific genes, and successfully regenerated homogeneous cartilage with a thickness over 3 mm. The transcriptome sequencing results demonstrated that HP regulated in vitro cartilage regeneration primarily by inhibiting cell senescence and apoptosis, promoting ECM synthesis, suppressing ECM catabolism, and ECM structure remodeling. Evaluation of in vivo fate indicated that in vitro regenerated cartilage in the HP group further developed after implantation and formed homogeneous and mature cartilage close to the native one, suggesting significant clinical potential. The current study outlines an efficient strategy for in vitro cartilage regeneration based on photocrosslinkable hydrogel scaffolds and its in vivo application.

Project description:While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, most tissue engineering strategies for cartilage regeneration encapsulate MSCs as single cells, partially due to the technical challenge to homogeneously encapsulate cell pellets in three-dimensional (3D) hydrogels. It remains unclear whether encapsulating MSCs as single cell suspension or cell aggregates in 3D hydrogels would enhance MSC-based cartilage formation. In this study, we determined that the optimal size of MSC micropellets (?Pellets) that can be homogeneously encapsulated in hydrogels with high cell viability is 100 cells/pellet. Using optimized ?Pellet size, MSCs were encapsulated either as single cell suspension or ?Pellets in four soft hydrogel formulations with stiffness ranging 3-6?kPa. Regardless of hydrogel formulations, single cell encapsulation resulted in more neocartilage deposition with improved mechanical functions over ?Pellet encapsulation. For single cell encapsulation, polyethylene glycol (PEG) hydrogels containing chondroitin sulfate led to the most cartilage matrix deposition, with compressive modulus reaching 211?kPa after only 21 days, a range approaching the stiffness of native cartilage. The findings from this study offer valuable insights on guiding optimal method design for MSCs and hydrogel-based cartilage regeneration. The optimized ?Pellet encapsulation method may be broadly applicable to encapsulate other stem cell types or cancer cells as aggregates in hydrogels. Impact Statement While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, it remains unclear whether encapsulating MSCs as cell pellets in three-dimensional hydrogels would enhance MSC-based cartilage formation. In this study, we determined the optimal size of MSC micropellet (?Pellet) that can be homogeneously encapsulated in hydrogels with high cell viability. Unexpectedly, single cell encapsulation resulted in more robust new cartilage formation than ?Pellet encapsulation. Furthermore, tuning hydrogel formulation led to rapid cartilage regeneration with stiffness approaching that of native cartilage. The findings from this study would facilitate clinical translation of MSCs and hydrogel-based therapies for cartilage regeneration with optimized parameters.

Dataset Information

Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.

Publications

Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.

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