179 DEMONSTRATION OF A NOVEL BIOSCAFFOLD SUITABLE FOR USE IN CARTILAGE TISSUE ENGINEERING THAT SUPPORTS CHONDROCYTE PHENOTYPE

ABSTRACTDedifferentiation of chondrocytes during in vitro passaging before implantation, and post implantation in vivo, is a critical limitation in cartilage tissue engineering. Several biophysical features define the dedifferentiated state including a flattened cell morphology and increased stress fiber formation. However, how dedifferentiation influences nuclear mechanics, and the possible long-term implications of this state, are unknown. In this study, we investigated how chondrocyte dedifferentiation affects the mechanics of the chromatin architecture inside the cell nucleus and the gene expression of the structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state suggesting a weaker nuclear envelope which can further intensify the intranuclear strain amplification. Our results indicate that dedifferentiation and altered nuclear strain could promote gene expression changes at the nuclear envelope, thus promoting further deviation from chondrocyte phenotype. This study highlights the role of cell shape on nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during expansion with a goal of successful cartilage tissue engineering.SIGNIFICANCEChondrocytes dedifferentiate into a fibroblast-like phenotype in a non-native biophysical environment. Using high resolution microscopy, intranuclear strain analysis, finite element method based computational modeling, and molecular biology techniques, we investigated how mechanical force causes abnormal intranuclear strain distribution in chondrocytes during the dedifferentiation process. Overall, our results suggest that the altered cell geometry aided by an altered or weakened nuclear envelope structure are responsible for abnormal intranuclear strain during chondrocyte dedifferentiation that can further deviate chondrocytes to a more dedifferentiated state.

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Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering

Cartilage ◽

10.1177/19476035211057242 ◽

2021 ◽

pp. 194760352110572

Author(s):

Yuxin Yan ◽

Rao Fu ◽

Chuanqi Liu ◽

Jing Yang ◽

Qingfeng Li ◽

...

Keyword(s):

Tissue Engineering ◽

High Efficiency ◽

Cartilage Tissue Engineering ◽

Enzymatic Digestion ◽

Cell Yield ◽

Cartilage Tissue ◽

Matrix Synthesis ◽

Chondrocyte Phenotype ◽

Yield Efficiency ◽

Rabbit Ear

Objective The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. Design Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity Results Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. Conclusions The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

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Scaffold Properties Play a Critical Role in the Retention of Synthesized Glycosaminoglycans in Tissue Engineered Cartilage

ASME 2007 Summer Bioengineering Conference ◽

10.1115/sbc2007-176558 ◽

2007 ◽

Cited By ~ 1

Author(s):

Lindsay E. Kugler ◽

Kenneth W. Ng ◽

Christopher J. O’Conor ◽

Gerard A. Ateshian ◽

Clark T. Hung

Keyword(s):

Tissue Engineering ◽

Critical Role ◽

Cartilage Tissue Engineering ◽

Three Dimensional ◽

Cartilage Tissue ◽

Mechanical Stimuli ◽

Chondrocyte Phenotype ◽

Engineering Research ◽

Engineered Cartilage ◽

Scaffold Properties

Agarose has been used as a model scaffold for cartilage tissue engineering research due to its maintenance of chondrocyte phenotype, support of cartilage tissue development, and ability to transmit mechanical stimuli [1–4]. In a previous study, the temporal application of TGF-β3 for only 2 weeks resulted in explosive growth in the functional properties of tissue engineered cartilage [5]. The role of scaffolds in tissue engineering includes providing a physiologic three-dimensional environment for cells, decreased path lengths for diffusion and retention of cell elaborated matrix. In past studies by our laboratory, it was hypothesized that the scaffold properties in engineered cartilage plays a crucial role in the retention of synthesized glycosaminoglycan (GAG) molecules, a major extracellular matrix constituent of articular cartilage [6, 7]. This study focuses on testing this hypothesis using 3%, 2%, and 1% (wt/vol) agarose as scaffolds for engineered cartilage.

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