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

Cartilage ◽  
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.

scholarly journals A Synthetic, Closed-Looped Gene Circuit for the Autonomous Regulation of RUNX2 Activity during Chondrogenesis

2021 ◽  
Author(s):  
Biming Wu ◽  
Gurcharan Kaur ◽  
Thomas Lanigan ◽  
Rhima M Coleman
Keyword(s):  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Matrix Synthesis ◽  
Gene Circuit ◽  
Chondrocyte Phenotype ◽  
Chondrocyte Maturation ◽  
Autonomous Regulation ◽  
Chondroprogenitor Cells ◽  
Matrix Accumulation

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in 2D ATDC5 model. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis in ATDC5 cells. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Furthermore, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Long-term 3D cultures of reprogrammed ATDC5 cells had increased matrix accumulation compared to naive cells. Overall, our results demonstrate that the negative modulation of Runx2 activity with our autoregulatory gene circuit can reduce the effects of RUNX2 activity and enhance matrix synthesis in chondroprogenitor cells.

scholarly journals Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 962
Author(s):  
Monica Salamone ◽  
Salvatrice Rigogliuso ◽  
Aldo Nicosia ◽  
Marcello Tagliavia ◽  
Simona Campora ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cartilage Tissue Engineering ◽  
Enzymatic Digestion ◽  
Cartilage Tissue ◽  
Cartilage Defects ◽  
Nasal Cartilage ◽  
Tissue Dissociation ◽  
In Vitro Expansion ◽  
First Time

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

scholarly journals Dedifferentiation alters chondrocyte nuclear mechanics during in vitro culture and expansion

2021 ◽  
Author(s):  
Soham Ghosh ◽  
Adrienne K. Scott ◽  
Benjamin Seelbinder ◽  
Jeanne E. Barthold ◽  
Brittany M St. Martin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Engineering ◽  
Finite Element ◽  
Nuclear Envelope ◽  
Cell Shape ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Chondrocyte Phenotype ◽  
Nuclear Mechanics

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.

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

2007 ◽  
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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