scholarly journals The Effects of Age and Cell Isolation on Collagen II Synthesis by Articular Chondrocytes: Evidence for Transcriptional and Posttranscriptional Regulation

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Vipin Asopa ◽  
Tonia Vincent ◽  
Jeremy Saklatvala
Keyword(s):  
Protein Synthesis ◽  
Articular Cartilage ◽  
Mrna Stability ◽  
Articular Chondrocytes ◽  
Cartilage Regeneration ◽  
Type Ii Collagen ◽  
Systematic Investigation ◽  
Mrna Levels ◽  
Age Related ◽  
Collagen Ii

Adult articular cartilage synthesises very little type II collagen in comparison to young cartilage. The age-related difference in collagen II synthesis is poorly understood. This is the first systematic investigation of age-related differences in extracellular matrix synthesis in fresh articular cartilage and following isolation of chondrocytes. A histological comparison of 3-year-old skeletally mature and 6-month-old juvenile porcine cartilage was made. Differences in collagen II, aggrecan, and Sox5, 6, and 9 mRNA and protein expression and mRNA stability were measured. Adult cartilage was found to be thinner than juvenile cartilage but with similar chondrocyte density. Procollagen α1(II) and Sox9 mRNA levels were 10-fold and 3-fold reduced in adult cartilage. Sox9 protein was halved and collagen II protein synthesis was almost undetectable and calculated to be at least 30-fold reduced. Aggrecan expression did not differ. Isolation of chondrocytes caused a drop in procollagen α1(II) and Sox9 mRNA in both adult and juvenile cells along with a marked reduction in Sox9 mRNA stability. Interestingly, juvenile chondrocytes continued to synthesise collagen II protein with mRNA levels similar to those seen in adult articular cartilage. Age-related differences in collagen II protein synthesis are due to both transcriptional and posttranscription regulation. A better understanding of these regulatory mechanisms would be an important step in improving current cartilage regeneration techniques.

scholarly journals Age-related decrease in the link-stability of proteoglycan aggregates formed by articular chondrocytes

1984 ◽  
Vol 220 (1) ◽  
pp. 337-340 ◽  
Author(s):  
A H K Plaas ◽  
J D Sandy
Keyword(s):  
Protein Synthesis ◽  
Articular Cartilage ◽  
Proliferative Activity ◽  
Monolayer Culture ◽  
Articular Chondrocytes ◽  
Link Protein ◽  
Age Related ◽  
Link Stability ◽  
Proteoglycan Aggregates

Chondrocytes were isolated from the articular cartilage of rabbits aged between 6 and 50 weeks and labelled with [35S]sulphate after 48 h in monolayer culture. The percentage of the total proteoglycan monomers synthesized by each culture that were present as link-stabilized aggregates was shown to be about 83% at 6, 9 and 12 weeks, 73% at 15 weeks, 48% at 30 weeks and 32% at 50 weeks. The proliferative activity of the cells in culture also decreased markedly with the age of the donor. The results suggest that aging of chondrocytes in vivo is accompanied by a decrease in their capacity for link-protein synthesis.

scholarly journals Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by coding sequence composition and mRNA localization

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Sarah L. Gillen ◽  
Chiara Giacomelli ◽  
Kelly Hodge ◽  
Sara Zanivan ◽  
Martin Bushell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Mrna Stability ◽  
Mrna Localization ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Mrna Fate

Abstract Background Regulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell’s requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Results This study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability, and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilization of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localized in p-bodies, contain disorder-promoting amino acids, and encode nuclear localized proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1. Conclusions We define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localization.

Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity

2007 ◽  
Vol 20 (03) ◽  
pp. 185-191 ◽  
Author(s):  
A. O. Oshin ◽  
E. Caporali ◽  
C. R. Byron ◽  
A. A. Stewart ◽  
M. C. Stewart
Keyword(s):  
Articular Cartilage ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Soluble Form ◽  
Mrna Levels ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Endogenous Expression ◽  
Chondrocytic Phenotype

SummaryArticular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.

Influence of 17β-estradiol and insulin on type II collagen and protein synthesis of articular chondrocytes

Bone ◽  
2006 ◽  
Vol 39 (2) ◽  
pp. 310-317 ◽  
Author(s):  
Horst Claassen ◽  
Matthias Schlüter ◽  
Michael Schünke ◽  
Bodo Kurz
Keyword(s):  
Protein Synthesis ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type Ii ◽  
17Β Estradiol

Facilitating In Vivo Articular Cartilage Repair by Tissue-Engineered Cartilage Grafts Produced From Auricular Chondrocytes

2017 ◽  
Vol 46 (3) ◽  
pp. 713-727 ◽  
Author(s):  
Chin-Chean Wong ◽  
Chih-Hwa Chen ◽  
Li-Hsuan Chiu ◽  
Yang-Hwei Tsuang ◽  
Meng-Yi Bai ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Cartilage Repair ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Articular Cartilage Repair ◽  
3 Dimensional ◽  
Cell Conversion

Background: Insufficient cell numbers still present a challenge for articular cartilage repair. Converting heterotopic auricular chondrocytes by extracellular matrix may be the solution. Hypothesis: Specific extracellular matrix may convert the phenotype of auricular chondrocytes toward articular cartilage for repair. Study Design: Controlled laboratory study. Methods: For in vitro study, rabbit auricular chondrocytes were cultured in monolayer for several passages until reaching status of dedifferentiation. Later, they were transferred to chondrogenic type II collagen (Col II)–coated plates for further cell conversion. Articular chondrogenic profiles, such as glycosaminoglycan deposition, articular chondrogenic gene, and protein expression, were evaluated after 14-day cultivation. Furthermore, 3-dimensional constructs were fabricated using Col II hydrogel-associated auricular chondrocytes, and their histological and biomechanical properties were analyzed. For in vivo study, focal osteochondral defects were created in the rabbit knee joints, and auricular Col II constructs were implanted for repair. Results: The auricular chondrocytes converted by a 2-step protocol expressed specific profiles of chondrogenic molecules associated with articular chondrocytes. The histological and biomechanical features of converted auricular chondrocytes became similar to those of articular chondrocytes when cultivated with Col II 3-dimensional scaffolds. In an in vivo animal model of osteochondral defects, the treated group (auricular Col II) showed better cartilage repair than did the control groups (sham, auricular cells, and Col II). Histological analyses revealed that cartilage repair was achieved in the treated groups with abundant type II collagen and glycosaminoglycans syntheses rather than elastin expression. Conclusion: The study confirmed the feasibility of applying heterotopic chondrocytes for cartilage repair via extracellular matrix–induced cell conversion. Clinical Relevance: This study proposes a feasible methodology to convert heterotopic auricular chondrocytes for articular cartilage repair, which may serve as potential alternative sources for cartilage repair.

scholarly journals High-resolution architecture of human epiphysis formation

2020 ◽  
Author(s):  
Heng Sun ◽  
Ya Wen ◽  
Weiliang Wu ◽  
Tian Qin ◽  
Chengrui An ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Articular Chondrocytes ◽  
Cartilage Regeneration ◽  
Hypertrophic Chondrocytes ◽  
Ossification Center ◽  
Articular Chondrocyte ◽  
Cartilage Growth ◽  
Proliferative Stage ◽  
Divergent Differentiation ◽  
Human Limb

SummaryHuman limb skeletal system consists of both bone and cartilage which originated from fetal cartilage. However, the roadmap of chondrocyte divergent differentiation to bone and articular cartilage has yet to be established. Epiphysis possesses articular cartilage, growth plate and the secondary ossification center (SOC), making it an ideal model to uncover the trajectory of chondrocyte divergent differentiation. Here, we mapped differentiation trajectory of human chondrocyte during postnatal finger epiphysis development by using single-cell RNA sequencing. Our results uncovered that chondroprogenitors have two differentiation pathways to hypertrophic chondrocytes during ossification, and one pathway to articular chondrocytes for formation of cartilages. Interestingly, we found that, as an addition to the known typical endochondral ossification path from resting, proliferative to hypertrophic chondrocytes, there was a bypass by which chondroprogenitors differentiate into hypertrophic chondrocytes without proliferative stage. Furthermore, our results revealed two new chondrocyte subpopulations (bypass chondrocytes as it appeared in the ossification bypass, and ID1+ chondroblasts in articular chondrocyte path) during postnatal epiphysis development in addition to six well-known subpopulations. Overall, our study provides a comprehensive roadmap of chondrocyte differentiation in human epiphysis thereby expanding the knowledge of bone and articular cartilage, which could be utilized to design biotherapeutics for bone and articular cartilage regeneration.

scholarly journals Coculture of hWJMSCs and pACs in Oriented Scaffold Enhances Hyaline Cartilage Regeneration In Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Yu Zhang ◽  
Shuyun Liu ◽  
Weimin Guo ◽  
Chunxiang Hao ◽  
Mingjie Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Articular Cartilage ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Human Umbilical Cord ◽  
Cartilage Cells ◽  
Collagen Ii ◽  
Differentiation Status

Seed cells of articular cartilage tissue engineering face many obstacles in their application because of the dedifferentiation of chondrocytes or unstable chondrogenic differentiation status of pluripotent stem cells. To overcome mentioned dilemmas, a simulation of the articular cartilage microenvironment was constructed by primary articular cartilage cells (pACs) and acellular cartilage extracellular matrix- (ACECM-) oriented scaffold cocultured with human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJMSCs) in vitro. The coculture groups showed more affluent cartilage special matrix ingredients including collagen II and aggrecan based on the results of histological staining and western blotting and cut down as many pACs as possible. The RT-PCR and cell viability experiments also demonstrated that hWJMSCs were successfully induced to differentiate into chondrocytes when cultured in the simulated cartilage microenvironment, as confirmed by the significant upregulation of collagen II and aggrecan, while the cell proliferation activity of pACs was significantly improved by cell-cell interactions. Therefore, compared with monoculture and chondrogenic induction of inducers, coculture providing a simulated native articular microenvironment was a potential and temperate way to regulate the biological behaviors of pACs and hWJMSCs to regenerate the hyaline articular cartilage.

scholarly journals Harnessing Biomechanics to Develop Cartilage Regeneration Strategies

2015 ◽  
Vol 137 (2) ◽  
Author(s):  
Kyriacos A. Athanasiou ◽  
Donald J. Responte ◽  
Wendy E. Brown ◽  
Jerry C. Hu
Keyword(s):  
Tissue Engineering ◽  
Articular Cartilage ◽  
Biochemical Characterization ◽  
Articular Chondrocytes ◽  
Lysyl Oxidase ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Main Function ◽  
Mechanical Stimuli

As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. This article provides a review of important steps toward regeneration of articular cartilage with suitable biomechanical properties. As a first step, biomechanical and biochemical characterization studies at the tissue level were used to provide design criteria for engineering neotissues. Extending this work to the single cell and subcellular levels has helped to develop biochemical and mechanical stimuli for tissue engineering studies. This strong mechanobiological foundation guided studies on regenerating hyaline articular cartilage, the knee meniscus, and temporomandibular joint (TMJ) fibrocartilage. Initial tissue engineering efforts centered on developing biodegradable scaffolds for cartilage regeneration. After many years of studying scaffold-based cartilage engineering, scaffoldless approaches were developed to address deficiencies of scaffold-based systems, resulting in the self-assembling process. This process was further improved by employing exogenous stimuli, such as hydrostatic pressure, growth factors, and matrix-modifying and catabolic agents, both singly and in synergistic combination to enhance neocartilage functional properties. Due to the high cell needs for tissue engineering and the limited supply of native articular chondrocytes, costochondral cells are emerging as a suitable cell source. Looking forward, additional cell sources are investigated to render these technologies more translatable. For example, dermis isolated adult stem (DIAS) cells show potential as a source of chondrogenic cells. The challenging problem of enhanced integration of engineered cartilage with native cartilage is approached with both familiar and novel methods, such as lysyl oxidase (LOX). These diverse tissue engineering strategies all aim to build upon thorough biomechanical characterizations to produce functional neotissue that ultimately will help combat the pressing problem of cartilage degeneration. As our prior research is reviewed, we look to establish new pathways to comprehensively and effectively address the complex problems of musculoskeletal cartilage regeneration.

scholarly journals Murine fibromodulin: cDNA and genomic structure, and age-related expression and distribution in the knee joint

2001 ◽  
Vol 355 (3) ◽  
pp. 577-585 ◽  
Author(s):  
Anna-Marja K. SÄÄMÄNEN ◽  
Heli J. SALMINEN ◽  
A. Juho RANTAKOKKO ◽  
Dick HEINEGÅRD ◽  
Eero I. VUORIO
Keyword(s):  
Genomic Structure ◽  
Type Ii Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Primary Role ◽  
Type Ii ◽  
Exon 2 ◽  
Collagen Fibrillogenesis ◽  
Calcified Cartilage ◽  
Age Related

The genomic structure of murine fibromodulin was determined, and its age-related expression and distribution were characterized in knee epiphyses, with decorin studied for reference. Fibromodulin, as well as decorin, have roles in collagen fibrillogenesis both in vitro and in vivo. The murine fibromodulin gene, Fmod, was similar with that in other species, with three exons and 86% of the translated sequence in exon 2. The 2.7kb long cDNA contains an open reading frame of 1131nt. Fibromodulin mRNA levels were highest in tissues rich in fibrillar collagens type I or type II. During growth, the distribution of fibromodulin mRNA was similar with that of type II collagen, with the highest levels between 5 days and 1 month of age. Thereafter, the expression of type II collagen declined to a level near the detection limit, whereas the fibromodulin expression decreased less markedly to a level of approx. 35% of maximum, and remained constant throughout the rest of the observation period. In contrast, decorin mRNA levels were the highest in old animals. Pericellular deposition of fibromodulin was strong around the late-hypertrophic chondrocytes of the secondary ossification centre and in the growth plate. In young epiphyses, both fibromodulin and decorin were found interterritorially, mainly in the uncalcified and deep-calcified cartilage. In the old mice, calcified cartilage became enriched with regard to fibromodulin, while, in contrast, decorin deposition diminished, particularly near the tidemark. In the subchondral bone trabeculae, decorin was found in the endosteum of growing, but not in the mature, epiphyses. Differences in the expression and distribution profiles suggest different roles for fibromodulin and decorin in the regulation of collagen fibrillogenesis, maintenance of the fibril organization and matrix mineralization. As fibromodulin is deposited closer to cells than decorin, it may have a primary role in collagen fibrillogenesis, whereas decorin might be involved in the maintenance of fibril structures in the interterritorial matrix.

scholarly journals Comparative Effectiveness of Structural versus Regulatory Protein Gene Transfer on Articular Chondrocyte Matrix Gene Expression

Cartilage ◽  
2017 ◽  
Vol 10 (1) ◽  
pp. 102-110 ◽  
Author(s):  
Shuiliang Shi ◽  
Congrong Wang ◽  
Albert Chan ◽  
Kashif Kirmani ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Gene Transfer ◽  
Cartilage Repair ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Matrix Proteins ◽  
Type Ii ◽  
Articular Cartilage Repair ◽  
Regulatory Factors ◽  
Igf I

Objective The production of extracellular matrix is a necessary component of articular cartilage repair. Gene transfer is a promising method to improve matrix biosynthesis by articular chondrocytes. Gene transfer may employ transgenes encoding regulatory factors that stimulate the production of matrix proteins, or may employ transgenes that encode the proteins themselves. The objective of this study was to determine which of these 2 approaches would be the better choice for further development. We compared these 2 approaches using the transgenes encoding the structural matrix proteins, aggrecan or type II collagen, and the transgene encoding the anabolic factor, insulin-like growth factor I (IGF-I). Methods We transfected adult bovine articular chondrocytes with constructs encoding type II collagen, aggrecan, or IGF-I, and measured the expression of type II collagen ( COL2A1) and aggrecan ( ACAN) from their native genes and from their transgenes. Results IGF-I gene ( IGF1) transfer increased the expression of the native chondrocyte COL2A1 and ACAN genes 2.4 and 2.9 times control, respectively. COL2A1 gene transfer did not significantly increase COL2A1 transcripts, even when the transgene included the genomic COL2A1 regulatory sequences stimulated by chondrogenic growth factors. In contrast, ACAN gene transfer increased ACAN transcripts up to 3.4 times control levels. IGF1, but not ACAN, gene transfer increased aggrecan protein production. Conclusion Taken together, these results suggest that the type II collagen and aggrecan production required for articular cartilage repair will be more effectively achieved by genes that encode anabolic regulatory factors than by genes that encode the matrix molecules themselves.

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