Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Bernstein, Peter S.; Dong, Meng; Corbeil, Denis; Gelinsky, Michael; Günther, Klaus‐Peter; Fickert, Stefan

doi:10.1002/btpr.186

Cited by 51 publications

(50 citation statements)

References 50 publications

(71 reference statements)

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“…We also noticed that the peak intensities for all metabolites are higher in the case of pellets as compared to chondrocytes growing in alginate beads confirming higher amount of macromolecule synthesis in pellets. This is in line with a previously published study showing that the pellet culture produces higher amounts of proteoglycans and collagen as compared to the alginate bead system [49].…”

Section: Representative Resultssupporting

confidence: 92%

Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy

Kotecha¹

2012

J Tissue Sci Eng

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In this article, based on the invited talk at the "Tissue Science 2012" meeting in Chicago on October 1-3, 2012, we describe some examples of characterization of engineered cartilage and bone tissue using magnetic resonance spectroscopy and imaging. Two different models of engineered cartilage and engineered bone tissue constructs were used for these studies: 1) chondrocyte based cartilage tissue engineering constructs: human and bovine chondrocytes seeded in alginate beads (Hydrogel scaffold model) or bovine chondrocytes grown as pellets (scaffold free model); 2) mesenchymal stem cell (MSC) based cartilage and bone tissue engineering constructs: human mesenchymal stem cell (HMSCs) seeded in cartilage biomimetic scaffolds (collagen/chitosan scaffold integrated with extracellular matrix of cartilage) or HMSCs seeded in collagen/chitosan scaffolds. Magnetic resonance spectroscopy and imaging experiments using 9.4 T (400 MHz proton frequency), 11.7 T (500 MHz proton frequency) or 14.1 T (600 MHz proton frequency) MR spectrometers/Imager were performed on these constructs over two to four weeks of tissue culture time. Specifically, water suppressed proton NMR spectroscopy; proton and sodium multi-quantum coherence spectroscopy and proton T1, T2 and ADC parametric MRI were used to study the chondrogenesis and osteogenesis of these tissues. We found that the change in MR relaxation and diffusion coefficient parameters correlate well with the growth of engineered tissues. We found that the MR parameters and the change in these parameters in growing tissue are strongly influenced by the choice of scaffolds. We also found as expected that the tissue-engineered cartilage lacked order or preference in collagen orientation. Further work is underway to elucidate these findings. We anticipate that in future, MRI will augment histological and immunohistochemical techniques by providing a complimentary and real time quantitative assessment of engineered tissue growth at all growth stages: (i) cell seeding to pre implantation; (ii) preclinical validation studies post implantation in small and large animal models; (iii) clinical studies of performance of engineered tissues.

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Section: Representative Resultssupporting

confidence: 92%

Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy

Kotecha¹

2012

J Tissue Sci Eng

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“…The micromass pellet culture is commonly used in studies investigating cell chondrogenesis in vitro and has been recently demonstrated as superior in comparison to encapsulation of chondrocytes into gel-like biomaterials (alginate-based systems) (Bernstein et al, 2009). Pellet culture allows for cell-cell interactions, cell-ECM interactions and exchange/ response to soluble factors that could occur during cellular cross-talk in a co-culture system.…”

Section: Discussionmentioning

confidence: 99%

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Giovannini

Diaz-Romero²,

Aigner³

et al. 2010

eCM

141

107

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Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-β1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilagespecific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

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“…Three-dimensional cell culture techniques offer the ability to control shape, structure, and biochemical environment, allowing researchers to conduct experiments at higher orders of complexity than those available in 2D. 13,14,17,18,20,21 Three-dimensional cell culture techniques generally depend on promoting direct cell-cell interactions, using cell aggregation techniques like spheroids and pellet cultures, 22,23 or cell-matrix interactions, either with protein gels [24][25][26] or synthetic polymer scaffolds. 27,28 When used for coculture, 3D approaches have varied, including the formation of spheroids with multiple cell types, 29 encapsulated protein gels seeded with multiple cell types, 30 or hybrid methods of 3D cultures and monolayers.…”

Section: Introductionmentioning

confidence: 99%

Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation

Tseng

Gage

Raphael

et al. 2013

Tissue Engineering Part C: Methods

112

110

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A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle a-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.

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Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Cited by 51 publications

References 50 publications

Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy

Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation

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