Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen® and Hoechst 33258

McGowan, Kevin B.; Kurtis, Melissa S.; Lottman, Lisa M.; Watson, Deborah; Sah, Robert L.

doi:10.1053/joca.2002.0794

Cited by 132 publications

(122 citation statements)

References 21 publications

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“…GAG and DNA contents were measured using dimethylmethylene blue (DMMB; Sigma‐Aldrich)43 and PicoGreen (Invitrogen, Carlsbad, CA) assays, respectively 44…”

Section: Methodsmentioning

confidence: 99%

A Modular Strategy to Engineer Complex Tissues and Organs

et al. 2018

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Currently, there are no synthetic or biologic materials suitable for long‐term treatment of large tracheal defects. A successful tracheal replacement must (1) have radial rigidity to prevent airway collapse during respiration, (2) contain an immunoprotective respiratory epithelium, and (3) integrate with the host vasculature to support epithelium viability. Herein, biopolymer microspheres are used to deliver chondrogenic growth factors to human mesenchymal stem cells (hMSCs) seeded in a custom mold that self‐assemble into cartilage rings, which can be fused into tubes. These rings and tubes can be fabricated with tunable wall thicknesses and lumen diameters with promising mechanical properties for airway collapse prevention. Epithelialized cartilage is developed by establishing a spatially defined composite tissue composed of human epithelial cells on the surface of an hMSC‐derived cartilage sheet. Prevascular rings comprised of human umbilical vein endothelial cells and hMSCs are fused with cartilage rings to form prevascular–cartilage composite tubes, which are then coated with human epithelial cells, forming a tri‐tissue construct. When prevascular– cartilage tubes are implanted subcutaneously in mice, the prevascular structures anastomose with host vasculature, demonstrated by their ability to be perfused. This microparticle–cell self‐assembly strategy is promising for engineering complex tissues such as a multi‐tissue composite trachea.

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“…GAG and DNA contents were measured using dimethylmethylene blue (DMMB; Sigma‐Aldrich)43 and PicoGreen (Invitrogen, Carlsbad, CA) assays, respectively 44…”

Section: Methodsmentioning

confidence: 99%

A Modular Strategy to Engineer Complex Tissues and Organs

et al. 2018

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“…Aliquots of cells from two of the animals containing 4 Â 10 6 cells each (by hemacytometer count) were lyophilized, digested, and assayed for DNA content in the same manner as the cell-seeded constructs. While it has been recommended that a species-specific DNA standard be used for the PicoGreen assay 23 none was available for this study so the procedure was used according to the manufacturer's instructions.…”

Section: Cross-linking Affects Cellular Condensation and Chondrogenesismentioning

confidence: 99%

Cross‐linking affects cellular condensation and chondrogenesis in type II collagen‐GAG scaffolds seeded with bone marrow‐derived mesenchymal stem cells

Vickers

Gotterbarm

Spector

2010

Journal Orthopaedic Research

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The formation of cartilaginous tissue by chondroprogenitor cells, whether in vivo or in vitro, appears to require a critical initial stage of ''condensation'' in which intercellular space is reduced through an aggregation of cells, leading to development of cell-to-cell junctions followed by chondrocytic differentiation. The objective of this study was to investigate the association of aggregation (condensation) of mesenchymal stem cell (MSCs) and chondrogenesis in vitro. Previous work with chondrocytes indicated that the cross-link density and related cell-mediated contraction of collagen scaffolds significantly affects cartilaginous tissue formation within the cell-seeded construct. Based on this finding, we hypothesized that the cell-aggregating effect of the contraction of MSC-seeded collagen scaffolds of lower cross-link density favors chondrogenesis; scaffolds of higher cross-link density, which resist cell-mediated contraction, would demonstrate a lower cell number density (i.e., subcritical packing density) and less cartilage formation. Type II collagen-GAG scaffolds, chemically cross-linked to achieve a range of cross-link densities, were seeded with caprine MSCs and cultured for 4 weeks. Constructs with low cross-link densities experienced cell-mediated contraction, increased cell number densities, and a greater degree of chondrogenesis (indicated by the chondrocytic morphology of cells, and synthesis of GAG and type II collagen) compared to more highly cross-linked scaffolds that resisted cellular contraction. These results provide a foundation for further investigation of the mechanisms by which condensation of mesenchymal cells induces chondrogenesis in this in vitro model, and may inform cross-linking protocols for collagen scaffolds for use in cartilage tissue engineering. ß

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“…An aliquot of the digest was used to determine the total DNA per sample using the PicoGreen dsDNA assay [31] (Molecular Probes, Eugene, OR) following the protocol provided by the manufacturer. Fluorescence was measured with a microplate reader (Tecan, Maennedorf, Switzerland) with excitation and emission wave-lengths of 485nm and 535nm, respectively.…”

Section: Biochemical Assays and Histologymentioning

confidence: 99%

Calcium Concentration Effects on the Mechanical and Biochemical Properties of Chondrocyte-Alginate Constructs

et al. 2008

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Alginate gel crosslinked by calcium ions (Ca 2+ ) has been widely used in cartilage tissue engineering. However, most studies have been largely performed in vitro in medium with a calcium concentration ([Ca 2+ ]) of 1.8mM, while the calcium level in the synovial fluid of the human knee joints, for example, has been reported to be 4mM or even higher. To simulate the synovial environment, the two studies in this paper were designed to investigate how the alginate scaffold alone, as well as the chondrocytes seeded alginate gel responds to variations in medium [Ca 2+ ]. In Study A, the mechanical properties of 2% alginate hydrogel were tested in 0.15M NaCl and various [Ca 2+ ] (1.0mM, 1.8mM, and 4mM). In Study B, primary bovine chondrocytes was seeded in alginate gel, and biochemical contents and mechanical properties were determined after incubation for 28 days in three [Ca 2+ ] (1.8mM, 4mM, and 8mM). For both studies, it was found that the magnitude of the complex shear modulus (|G*|) at 1Hz doubled and the corresponding phase angle shift angle (δ) increased > 2° as a result of the approximate 4-fold change in [Ca 2+ ]. At high [Ca 2+ ], the chondrocyte glycosaminogylcan (GAG) production inside the chondrocyte-alginate constructs was suppressed significantly. This is likely due to a decrease in the porosity of the chondrocyte-alginate constructs as a result of compaction in structure caused by an increased crosslinking density with [Ca 2+ ]. These may be important considerations in the eventual successful implementation of cartilage tissueengineered constructs in the clinical setting.

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Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen® and Hoechst 33258

Abstract: PicoGreen can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage.

Cited by 132 publications

References 21 publications

A Modular Strategy to Engineer Complex Tissues and Organs

A Modular Strategy to Engineer Complex Tissues and Organs

Cross‐linking affects cellular condensation and chondrogenesis in type II collagen‐GAG scaffolds seeded with bone marrow‐derived mesenchymal stem cells

Calcium Concentration Effects on the Mechanical and Biochemical Properties of Chondrocyte-Alginate Constructs

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