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Articular Chondroprogenitor Cells Maintain Chondrogenic Potential but Fail to Form a Functional Matrix When Implanted Into Muscles of SCID Mice.

Marcus P, De Bari C, Dell'Accio F, Archer CW - Cartilage (2014)

Bottom Line: After injection with clonal and enriched populations of chondroprogenitors, no distinct pellet was detected, but diffuse cartilage nodules were found with regions of safranin O staining and Sox9.The bovine articular cartilage progenitor cells were able to survive in vivo postimplantation, but failed to create a robust cartilage pellet, despite expressing sox9 and type II collagen.This suggests the cells require further signals for chondrogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cardiff School of Bioscience, Cardiff University, Cardiff, UK.

ABSTRACT

Objective: Articular cartilage is a complex tissue comprising phenotypically distinct zones. Research has identified the presence of a progenitor cell population in the surface zone of immature articular cartilage. The aim of the present study was to determine the in vivo plasticity of articular cartilage progenitor.

Design: Chondropogenitor cells were isolated from bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cells were labeled with PKH26 and injected into the thigh muscle of severe-combined immunodeficient (SCID) mice. After 2 weeks, the muscles were dissected and cryosectioned. Sections were stained with safranin O and labeled for sox9 and collagen type II. Polymerase chain reaction analysis was carried out to determine plasticity for a number of tissue-specific markers. Full-depth chondrocytes acted as a control.

Results: Fluorescent PKH26 labeled cells were detected after 2 weeks in all samples analyzed. A cartilage pellet was present after injection of freshly isolated chondrocytes. After injection with clonal and enriched populations of chondroprogenitors, no distinct pellet was detected, but diffuse cartilage nodules were found with regions of safranin O staining and Sox9. Low levels of collagen type II were also detected. Polymerase chain reaction analysis identified the presence of the endothelial cell marker PECAM-1 in one clonal cell line, demonstrating phenotypic plasticity into the phenotype of the surrounding host tissues.

Conclusions: The bovine articular cartilage progenitor cells were able to survive in vivo postimplantation, but failed to create a robust cartilage pellet, despite expressing sox9 and type II collagen. This suggests the cells require further signals for chondrogenic differentiation.

No MeSH data available.


Related in: MedlinePlus

Graph highlighting population doublings for the four clonal (PMC78, PMC79, PMC81, and PMC82) and three enriched (PME67, PME68, and PME69) cell lines used during in vivo experiments. Population doublings were similar between the different clonal and enriched cell lines used.
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fig1-1947603514541274: Graph highlighting population doublings for the four clonal (PMC78, PMC79, PMC81, and PMC82) and three enriched (PME67, PME68, and PME69) cell lines used during in vivo experiments. Population doublings were similar between the different clonal and enriched cell lines used.

Mentions: Clones were isolated from colonies comprising more than 32 cells derived from the differential adhesion assay. Colonies were defined as comprising over 32 cells, that is, more than 5 population doublings in order to negate mature chondrocytes or the transient amplifying population. Enriched populations were also used, where no colonies had been isolated. Both clonal and enriched cell lines were grown to between 30 and 40 population doublings. Sterile polystyrene 6.4 mm cloning rings (Sigma) were dipped into sterile “Vaseline” using sterile forceps. The ring was then placed over the marked colony with gentle pressure and 200 µL trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; GIBCO) added. The dish was then incubated at 37°C for 2 to 5 minutes. Cells were then aspirated by gentle pipetting and added to 1 mL DMEM/F12 plus 10% FCS in a 24-well plate. The cells were then expanded in culture. For comparison, some of the plates used for differential adhesion were allowed to grow until confluence. This procedure produced a chondroprogenitor-enriched population. When the cells in the 35 mm dishes were confluent, the cells were dissociated using TrypLE express trypsin with phenol red (Invitrogen), counted, and then placed into T75 flasks. All cells lines were fed every 2 days. In all cases, at each passage, population doublings were calculated (Fig. 1).


Articular Chondroprogenitor Cells Maintain Chondrogenic Potential but Fail to Form a Functional Matrix When Implanted Into Muscles of SCID Mice.

Marcus P, De Bari C, Dell'Accio F, Archer CW - Cartilage (2014)

Graph highlighting population doublings for the four clonal (PMC78, PMC79, PMC81, and PMC82) and three enriched (PME67, PME68, and PME69) cell lines used during in vivo experiments. Population doublings were similar between the different clonal and enriched cell lines used.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4335766&req=5

fig1-1947603514541274: Graph highlighting population doublings for the four clonal (PMC78, PMC79, PMC81, and PMC82) and three enriched (PME67, PME68, and PME69) cell lines used during in vivo experiments. Population doublings were similar between the different clonal and enriched cell lines used.
Mentions: Clones were isolated from colonies comprising more than 32 cells derived from the differential adhesion assay. Colonies were defined as comprising over 32 cells, that is, more than 5 population doublings in order to negate mature chondrocytes or the transient amplifying population. Enriched populations were also used, where no colonies had been isolated. Both clonal and enriched cell lines were grown to between 30 and 40 population doublings. Sterile polystyrene 6.4 mm cloning rings (Sigma) were dipped into sterile “Vaseline” using sterile forceps. The ring was then placed over the marked colony with gentle pressure and 200 µL trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; GIBCO) added. The dish was then incubated at 37°C for 2 to 5 minutes. Cells were then aspirated by gentle pipetting and added to 1 mL DMEM/F12 plus 10% FCS in a 24-well plate. The cells were then expanded in culture. For comparison, some of the plates used for differential adhesion were allowed to grow until confluence. This procedure produced a chondroprogenitor-enriched population. When the cells in the 35 mm dishes were confluent, the cells were dissociated using TrypLE express trypsin with phenol red (Invitrogen), counted, and then placed into T75 flasks. All cells lines were fed every 2 days. In all cases, at each passage, population doublings were calculated (Fig. 1).

Bottom Line: After injection with clonal and enriched populations of chondroprogenitors, no distinct pellet was detected, but diffuse cartilage nodules were found with regions of safranin O staining and Sox9.The bovine articular cartilage progenitor cells were able to survive in vivo postimplantation, but failed to create a robust cartilage pellet, despite expressing sox9 and type II collagen.This suggests the cells require further signals for chondrogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cardiff School of Bioscience, Cardiff University, Cardiff, UK.

ABSTRACT

Objective: Articular cartilage is a complex tissue comprising phenotypically distinct zones. Research has identified the presence of a progenitor cell population in the surface zone of immature articular cartilage. The aim of the present study was to determine the in vivo plasticity of articular cartilage progenitor.

Design: Chondropogenitor cells were isolated from bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cells were labeled with PKH26 and injected into the thigh muscle of severe-combined immunodeficient (SCID) mice. After 2 weeks, the muscles were dissected and cryosectioned. Sections were stained with safranin O and labeled for sox9 and collagen type II. Polymerase chain reaction analysis was carried out to determine plasticity for a number of tissue-specific markers. Full-depth chondrocytes acted as a control.

Results: Fluorescent PKH26 labeled cells were detected after 2 weeks in all samples analyzed. A cartilage pellet was present after injection of freshly isolated chondrocytes. After injection with clonal and enriched populations of chondroprogenitors, no distinct pellet was detected, but diffuse cartilage nodules were found with regions of safranin O staining and Sox9. Low levels of collagen type II were also detected. Polymerase chain reaction analysis identified the presence of the endothelial cell marker PECAM-1 in one clonal cell line, demonstrating phenotypic plasticity into the phenotype of the surrounding host tissues.

Conclusions: The bovine articular cartilage progenitor cells were able to survive in vivo postimplantation, but failed to create a robust cartilage pellet, despite expressing sox9 and type II collagen. This suggests the cells require further signals for chondrogenic differentiation.

No MeSH data available.


Related in: MedlinePlus