Rapid and Large-Scale Formation of Chondrocyte Aggregates by Rotational Culture

2003 ◽  
Vol 12 (5) ◽  
pp. 475-479 ◽  
Author(s):  
Katsuko S. Furukawa ◽  
Hideyuki Suenaga ◽  
Kenshi Toita ◽  
Akiko Numata ◽  
Junzo Tanaka ◽  
...  
Keyword(s):  
Culture System ◽  
Large Scale ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Cell Interaction ◽  
Collagen Type I ◽  
Cell Contact ◽  
Type I ◽  
Pellet Culture ◽  
Cell Cell

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell–cell interactions and decrease chondrocyte–material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell–cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell–cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24–36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-β controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.

Collagen Expression in Tissue Engineered Cartilage of Aged Human Articular Chondrocytes in a Rotating Bioreactor

2003 ◽  
Vol 26 (4) ◽  
pp. 319-330 ◽  
Author(s):  
S. Marlovits ◽  
B. Tichy ◽  
M. Truppe ◽  
D. Gruber ◽  
W. Schlegel
Keyword(s):  
Electron Microscopy ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Three Dimensional ◽  
Collagen Type I ◽  
Human Articular Chondrocytes ◽  
Type I ◽  
Transmission Electron ◽  
Low Shear Stress ◽  
Engineered Cartilage

This study describes the culture and three-dimensional assembly of aged human articular chondrocytes under controlled oxygenation and low shear stress in a rotating-wall vessel. Chondrocytes cultured in monolayer were released and placed without any scaffold as a single cell suspension in a rotating bioreactor for 12 weeks. Samples were analyzed with immunohistochemistry, molecular biology and electron microscopy. During serial monolayer cultures chondrocytes dedifferentiated to a “fibroblast-like” structure and produced predominantly collagen type I. When these dedifferentiated cells were transferred to the rotating bioreactor, the cells showed a spontaneous aggregation and formation of solid tissue during the culture time. Expression of collagen type II and other components critical for the extracellular cartilage matrix could be detected. Transmission electron microscopy revealed a fine network of randomly distributed collagen fibrils. This rotating bioreactor proves to be a useful tool for providing an environment that enables dedifferentiated chondrocytes to redifferentiate and produce a cartilage-specific extracellular matrix.

scholarly journals Maintenance of chondrocyte phenotype during expansion on PLLA microtopographies

2018 ◽  
Vol 9 ◽  
pp. 204173141878982 ◽  
Author(s):  
Elisa Costa ◽  
Cristina González-García ◽  
José Luis Gómez Ribelles ◽  
Manuel Salmerón-Sánchez
Keyword(s):  
Lactic Acid ◽  
Type I Collagen ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Cell Phenotype ◽  
Type I ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Chondrocyte Phenotype

Articular chondrocytes are difficult to grow, as they lose their characteristic phenotype following expansion on standard tissue culture plates. Here, we show that culturing them on surfaces of poly(L-lactic acid) of well-defined microtopography allows expansion and maintenance of characteristic chondrogenic markers. We investigated the dynamics of human chondrocyte dedifferentiation on the different poly(L-lactic acid) microtopographies by the expression of collagen type I, collagen type II and aggrecan at different culture times. When seeded on poly(L-lactic acid), chondrocytes maintained their characteristic hyaline phenotype up to 7 days, which allowed to expand the initial cell population approximately six times without cell dedifferentiation. Maintenance of cell phenotype was afterwards correlated to cell adhesion on the different substrates. Chondrocytes adhesion occurs via the α5 β1 integrin on poly(L-lactic acid), suggesting cell–fibronectin interactions. However, α2 β1 integrin is mainly expressed on the control substrate after 1 day of culture, and the characteristic chondrocytic markers are lost (collagen type II expression is overcome by the synthesis of collagen type I). Expanding chondrocytes on poly(L-lactic acid) might be an effective solution to prevent dedifferentiation and improving the number of cells needed for autologous chondrocyte transplantation.

scholarly journals Pancreaticline Cell Differentiation of Bone Marrow Mesenchymal Stromal Cells in Acellular Pancreatic Scaffolds

2020 ◽  
Author(s):  
Zhao Li ◽  
Yue Du ◽  
Xin Wang
Keyword(s):  
Cell Differentiation ◽  
Treatment Group ◽  
Stromal Cells ◽  
Culture System ◽  
Collagen Type ◽  
Collagen Type I ◽  
In Vivo Study ◽  
Type I ◽  
Pancreatic Cell

Abstract Background: To evaluate the potential differentiation ability of bone mesenchymal stromal cells(BMSCs) to pancreatic line Cells on rat acellular pancreatic bioscaffold(APB) and the effect of differentiated BMSCs for chronic pancreatitis(CP) in vivo. Methods: After BMSCs were isolated and identified, they were dynamic cultured on the APB and static cultured in tissue culture flask(TCF),with or without the growth factors (GF) in both the culture system. The cytological behavior such as the proliferation and differentiation of BMSCs in all the above kinds of culture system were assessed by morphological observation, flow cytometry, ELASA analysis, qRT-PCR assay and western blot analysis. For the in vivo study, the pancreatic fibrosis and pathological score were evaluated. And also the expression of α-SMA, collagen type I and III, IL-10 in pancreas tissue were detected by ELASA. Results: 4ml/min was the most appropriate flow rate for the dynamic culture of BMSCs. The proliferation rate of BMSCs in the APB groups were significantly increased compared to TCF system. During the pancreatic line cell differentiation process, APB could induce BMSCs express markers such as PDX-1 and PTF-1 at higher mRNA levels. In contrast, the marker Oct 4 was expressed at a lower level in APB group. For the pancreatic functional cytoketatins including α-Amy, CK7, Flk-1, and C-peptide, they were all expressed at higher level in APB group. And metabolic enzymes secretion such as amylase and insulin were promoted significantly in APB system. By scanning electron microscope(SEM) and transmission electron microscopy(TEM), the ultrastructure of BMSCs in the APB group could further demonstrated the morphological characteristics of pancreatic-like cells. In vivo study,the expression of α-SMA, collagen type I and III in tissues were less in differentiated BMSCs treatment group, while the level of IL-10 in pancreatic tissue were higher in differentiated BMSCs treatment group with significant difference (P<0.05). In addition, in both in vitro and in vivo study, GF could significantly facilitate the function of proliferation, differentiation and pancreatic cell therapy. Conclusion: Together our data show the capacity of APB , 3D pancreatic biomatrix, promoting BMSCs differentiate toward pancreatic line phenotypes, and the considerable potential of using these cells for pancreatic cell therapies and tissue engineering.

Contraction-inducedMmp13and −14expression by goat articular chondrocytes in collagen type I but not type II gels

2011 ◽  
Vol 6 (9) ◽  
pp. 721-730 ◽  
Author(s):  
Agnes D. Berendsen ◽  
Lucienne A. Vonk ◽  
Behrouz Zandieh-Doulabi ◽  
Vincent Everts ◽  
Ruud A. Bank
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Type I ◽  
Type Ii

Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

2013 ◽  
Vol 19 (1-2) ◽  
pp. 59-65 ◽  
Author(s):  
Marijn Rutgers ◽  
Daniel B. Saris ◽  
Lucienne A. Vonk ◽  
Mattie H. van Rijen ◽  
Vanessa Akrum ◽  
...  
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Human Articular Chondrocytes ◽  
Type I ◽  
Type Ii

Effects of Controlled Released BMP-7 on Markers of Inflammation and Degradation During the Cultivation of Human Osteoarthritic Chondrocytes

2010 ◽  
Vol 26 (4) ◽  
pp. 419-433 ◽  
Author(s):  
Karsten Gavenis ◽  
Thomas Pufe ◽  
Lars Ove Brandenburg ◽  
Katharina Schiffl ◽  
Bernhard Schmidt-Rohlfing
Keyword(s):  
Gene Expression ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Type I ◽  
Matrix Synthesis ◽  
Gene Expressions ◽  
Markers Of Inflammation ◽  
Osteoarthritic Chondrocytes

The aim of the present study is to investigate the effects of BMP-7 released from polylactide microspheres on the appearance of various catabolic and inflammatory cytokines secreted by osteoarthritic chondrocytes cultivated in a collagen gel. Articular chondrocytes of 15 patients suffering from osteoarthritis are transferred to a collagen type-I gel. Additionally, BMP-7 encapsulated into polylactide microspheres (50 ng BMP-7/mL gel) is added. After 14 days, gene expression and protein appearance of various genes involved in matrix turnover and inflammation are investigated by immunohistochemical staining and RT-PCR and compared to untreated controls. TNF-α, MMP-13, IL-6, IL-1β, and VEGF gene expressions are decreased in the treatment group. In contrast, BMP-7-induced matrix synthesis is not affected, leaving collagen type-II (Col-II) gene expression to be elevated, while collagen type-I (Col-I) is decreased. In summary, controlled release of low concentrated BMP-7 from polylactide microspheres leads to a decrease in gene expression of the investigated inflammation and matrix degradation markers whereas matrix synthesis is induced.

scholarly journals Chondrogenic Gene Expression Differences between Chondrocytes from Osteoarthritic and Non-OA Trauma Joints in a 3D Collagen Type I Hydrogel

Cartilage ◽  
2016 ◽  
Vol 8 (2) ◽  
pp. 191-198 ◽  
Author(s):  
Vivek Jeyakumar ◽  
Florian Halbwirth ◽  
Eugenia Niculescu-Morzsa ◽  
Christoph Bauer ◽  
Hannes Zwickl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Specific Gene ◽  
Type I ◽  
Donor Age ◽  
Significant Difference ◽  
Age Variation ◽  
Oa Chondrocytes

Objective The purpose of the current study was to compare the donor age variation of chondrocytes from non-OA (osteoarthritic) trauma joints in patients of young to middle age (20.5 ± 3.7, 31.8 ± 1.9, 41.9 ± 4.1 years) embedded in matrix-associated autologous chondrocyte transplantation (MACT) grafts (CaReS). The chondrocyte-specific gene expression of CaReS grafts were then compared to chondrocytes from OA joints (in patients aged 63.8 ± 10 years) embedded in a collagen type I hydrogel. Design OA chondrocytes and articular chondrocyte-laden grafts were cultured over 14 days in chondrogenic growth medium. We performed reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression levels of chondrocyte-specific and hypertrophic markers. Results Gene expression analysis with RT-qPCR revealed no significant difference in chondrocyte-specific genes ( COL2A1, ACAN, SOX9, SOX5, SOX6) among 3 different age group of patients with CaReS grafts. In a comparative analysis of OA chondrocytes to articular chondrocytes, chondrogenic markers ( COL2A1, SOX6) exhibited higher expression in OA chondrocytes ( P < 0.05). Hypertrophic or OA cartilage pathogenesis marker ( MMP3, MMP13) expression was higher and COL1A1 had significantly lower expression ( P < 0.05) in OA chondrocytes than articular chondrocytes when cultivated in collagen type I hydrogels. Conclusion In summary, we identify that donor age variation does not influence the chondrogenic gene expression of the CaReS system. We also identified that freshly isolated OA chondrocytes embedded in collagen type I hydrogels can exhibit chondrogenic gene expression as observed in articular chondrocytes on the CaReS grafts. Transforming OA chondrocytes to articular chondrocytes can be regarded as an alternative option in the MACT technique.

A Novel Rat Tail Collagen Type-I Gel for the Cultivation of Human Articular Chondrocytes in Low Cell Density

2007 ◽  
Vol 30 (12) ◽  
pp. 1057-1067 ◽  
Author(s):  
R. Muller-Rath ◽  
K. Gavénis ◽  
S. Andereya ◽  
T. Mumme ◽  
B. Schmidt-Rohlfing ◽  
...  
Keyword(s):  
Cell Density ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Collagen Type I ◽  
Human Articular Chondrocytes ◽  
Type I ◽  
Low Cell Density ◽  
Rat Tail

scholarly journals Redifferentiation of Articular Chondrocytes by Hyperacute Serum and Platelet Rich Plasma in Collagen Type I Hydrogels

2019 ◽  
Vol 20 (2) ◽  
pp. 316 ◽  
Author(s):  
Vivek Jeyakumar ◽  
Eugenia Niculescu-Morzsa ◽  
Christoph Bauer ◽  
Zsombor Lacza ◽  
Stefan Nehrer
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Platelet Rich Plasma ◽  
Collagen Type I ◽  
Cartilage Defects ◽  
Type I ◽  
New Approach ◽  
Sgag Content ◽  
Col2a1 Expression

Matrix-assisted autologous chondrocyte transplantation (MACT) for focal articular cartilage defects often fails to produce adequate cartilage-specific extracellular matrix in vitro and upon transplantation results in fibrocartilage due to dedifferentiation during cell expansion. This study aimed to redifferentiate the chondrocytes through supplementation of blood-products, such as hyperacute serum (HAS) and platelet-rich plasma (PRP) in vitro. Dedifferentiated monolayer chondrocytes embedded onto collagen type I hydrogels were redifferentiated through supplementation of 10% HAS or 10% PRP for 14 days in vitro under normoxia (20% O2) and hypoxia (4% O2). Cell proliferation was increased by supplementing HAS for 14 days (p < 0.05) or by interchanging from HAS to PRP during Days 7–14 (p < 0.05). Sulfated glycosaminoglycan (sGAG) content was deposited under both HAS, and PRP for 14 days and an interchange during Days 7–14 depleted the sGAG content to a certain extent. PRP enhanced the gene expression of anabolic markers COL2A1 and SOX9 (p < 0.05), whereas HAS enhanced COL1A1 production. An interchange led to reduction of COL1A1 and COL2A1 expression marked by increased MMP13 expression (p < 0.05). Chondrocytes secreted less IL-6 and more PDGF-BB under PRP for 14 days (p < 0.0.5). Hypoxia enhanced TGF-β1 and BMP-2 release in both HAS and PRP. Our study demonstrates a new approach for chondrocyte redifferentiation.

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