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 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.

Approach to Design Loading Protocols for Cartilage Tissue Engineering: Hypothesis, Experiment and Model

2010 ◽  
Author(s):  
C. C. van Donkelaar ◽  
M. Khoshgoftar ◽  
L. M. Kock ◽  
K. Ito
Keyword(s):  
Tissue Engineering ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Native Cartilage ◽  
Native Collagen ◽  
Nutritional Effects ◽  
Proteoglycan Content ◽  
Gradual Variation ◽  
Engineered Cartilage

Tissue engineered cartilage has reached the level of maturity where the cells, either chondrocytes, BMSC’s or other cells are stimulated to produce a tissue of which the biochemical content qualitatively resembles that of native cartilage. Quantitatively, the proteoglycan content approaches that of native content in long term cultures, but to obtain native collagen fractions is still challenging. Engineered cartilage matrix is either homogeneously distributed, or shows gradual variation from the periphery to the center, caused by nutritional effects.

Porous Scaffolds Consisting of Collagen, Chondroitin Sulfate, and Hydroxyapatite with Enhanced Biodegradable Resistance for Cartilage Regeneration

2011 ◽  
Vol 1301 ◽  
Author(s):  
H. Kaneda ◽  
T. Ikoma ◽  
T. Yoshioka ◽  
M. Nishi ◽  
R. Matsumoto ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Porous Scaffolds ◽  
Collagen Scaffolds ◽  
Cross Linkage ◽  
Low Crystalline

ABSTRACTPorous scaffolds of alkaline-soluble collagen including nanocomposite particles of chondroitin sulfate and low crystalline hydroxyapatite for cartilage regeneration were fabricated by freeze-drying and thermal dehydration treatments; porous collagen scaffolds were also synthesized as a reference. The scaffolds were cross-linked using glutaraldehyde (GA) vapor treatment in order to enhance biodegradable resistance. Microstructural observation with scanning electron microscope indicated that the scaffolds with and without GA cross-linkage had open pores between 130 to 200 μm in diameter and well-interconnected pores of 10 to 30 μm even after cross-linkage. In vitro biodegradable resistance to collagenase was significantly enhanced by GA cross-linking of the scaffolds. All these results suggest that the GA cross-linked scaffolds consisting of collagen, chondroitin sulfate, and low crystalline hydroxyapatite have suitable microporous structures and long-term biochemical stability for cartilage tissue engineering.

scholarly journals Role of dexamethasone in the long-term functional maturation of MSC-laden hyaluronic acid hydrogels for cartilage tissue engineering

2017 ◽  
Vol 36 (6) ◽  
pp. 1717-1727 ◽  
Author(s):  
Minwook Kim ◽  
Sean T. Garrity ◽  
David R. Steinberg ◽  
George R. Dodge ◽  
Robert L. Mauck
Keyword(s):  
Tissue Engineering ◽  
Hyaluronic Acid ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Functional Maturation

scholarly journals 173 LONG-TERM STUDY OF STRATIFIED ALGINATE CONSTRUCT FOR CARTILAGE TISSUE ENGINEERING

2010 ◽  
Vol 18 ◽  
pp. S84
Author(s):  
J. Schiavi-Tritz ◽  
N. Charif ◽  
N. de Isla ◽  
R. Rahouadj ◽  
A. Pinzano ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Term Study ◽  
Long Term Study

Optimization of Protocol for Isolation of Chondrocytes from Human Articular Cartilage

Cartilage ◽  
2019 ◽  
pp. 194760351987633 ◽  
Author(s):  
Suleiman Alhaji Muhammad ◽  
Norshariza Nordin ◽  
Paisal Hussin ◽  
Muhammad Zulfadli Mehat ◽  
Sheau Wei Tan ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Collagen Type ◽  
Maximum Yield ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Human Articular Cartilage ◽  
Isolated Cells ◽  
Chondrocyte Phenotype

Objective Cartilage tissue engineering has evolved as one of the therapeutic strategies for cartilage defect, which relies on a large number of viable chondrocytes. Because of limited availability of cartilage and low chondrocytes yield from cartilage, the need for an improve isolation protocol for maximum yield of viable cells is a key to achieving successful clinical constructs. This study optimizes and compares different protocols for isolation of chondrocytes from cartilage. Design We employed enzymatic digestion of cartilage using collagenase II and trypsin. The chondrocytes yield, growth kinetics, aggrecan, and collagen type 2 (COL2) expression were evaluated. Collagen type 1 (COL1) mRNA expression was assessed to monitor the possibility of chondrocytes dedifferentiation. Results Chondrocyte yield per gram of cartilage was significantly higher ( P < 0.05) using collagenase II in Hank’s balanced salt solution (HBSS) compared with 0.25% trypsin. The number of chondrocyte yield per gram was higher in cartilage digested with collagenase in HBSS compared with Dulbecco’s modified Eagle medium/F12; however, the difference was not statistically significant. Chondrocytes seeded at lower densities had shorter population doubling time compared to those seeded at higher density. Protein and gene expression of chondrocyte phenotype indicates the expression of aggrecan and COL2. The expression of COL1 was significantly increased ( P < 0.05) in passage 3 compared with primary chondrocytes. The mRNA expression of chondrocyte phenotype was similar in primary and passaged one cells. Conclusions Collagenase in HBSS yield the highest number of viable chondrocytes and the isolated cells expressed chondrocyte phenotype. This protocol can be employed to generate large number of viable chondrocytes, particularly with limited cartilage biopsies.

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.

scholarly journals Prospective Isolation of Chondroprogenitors from Human iPSCs Based on Cell Surface Markers Identified using a CRISPR-Cas9-Generated Reporter

2019 ◽  
Author(s):  
Amanda Dicks ◽  
Chia-Lung Wu ◽  
Nancy Steward ◽  
Shaunak S. Adkar ◽  
Charles A. Gersbach ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Disease Modeling ◽  
Cartilage Tissue Engineering ◽  
Culture Conditions ◽  
Cartilage Tissue ◽  
Human Ipscs ◽  
Chondroprogenitor Cells ◽  
Cell Source ◽  
Induced Pluripotent

SUMMARYArticular cartilage shows little or no capacity for intrinsic repair, generating a critical need for regenerative therapies for joint injuries and diseases such as osteoarthritis. Human induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering andin vitrohuman disease modeling; however, heterogeneity and off-target differentiation remain a challenge. We used a CRISPR-Cas9-editedCOL2A1-GFPknock-in reporter hiPSC line, coupled with a surface marker screen, to identify a novel chondroprogenitor population expressing CD146, CD166, and PDGFRβ, but not CD45. Under chondrogenic culture conditions, these triple positive chondroprogenitor cells demonstrated decreased heterogeneity as measured by single cell RNA sequencing, as well as more robust and homogenous matrix production with significantly higher chondrogenic gene expression. Overall, this study has identified a unique hiPSC-derived subpopulation of chondroprogenitors that are CD146+/CD166+/PDGFRβ+/CD45-and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.

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