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Extensively Expanded Auricular Chondrocytes Form Neocartilage In Vivo.

Tseng A, Pomerantseva I, Cronce MJ, Kimura AM, Neville CM, Randolph MA, Vacanti JP, Sundback CA - Cartilage (2014)

Bottom Line: With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated.Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks.Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Massachusetts General Hospital, Boston, MA, USA ; Center for Regenerative Medicine, Boston, MA, USA.

ABSTRACT

Objective: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice.

Design: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed.

Results: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks.

Conclusions: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

No MeSH data available.


Related in: MedlinePlus

Histological and biochemical assessment of neocartilage engineered from passaged P3 chondrocytes after in vitro incubation in medium conditioned by P0 chondrocytes for 2 weeks and implantation in nude mice for 6 weeks. (A) The quality of neocartilage formed from sheep cells was poor as evidenced by toluidine blue, safranin, collagen type II, and elastin stains. No neocartilage formed from human cells as confirmed by the absence of cartilage-specific glycosaminoglycans (GAG) staining and positive collagen type I staining. Collagen scaffold fibers stained red on hematoxylin–eosin (H&E) stained sections. Scale bar: 100 µm. (B) GAG and (C) GAG/DNA ratio quantification confirmed histological results. *P < 0.01.
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fig2-1947603514546740: Histological and biochemical assessment of neocartilage engineered from passaged P3 chondrocytes after in vitro incubation in medium conditioned by P0 chondrocytes for 2 weeks and implantation in nude mice for 6 weeks. (A) The quality of neocartilage formed from sheep cells was poor as evidenced by toluidine blue, safranin, collagen type II, and elastin stains. No neocartilage formed from human cells as confirmed by the absence of cartilage-specific glycosaminoglycans (GAG) staining and positive collagen type I staining. Collagen scaffold fibers stained red on hematoxylin–eosin (H&E) stained sections. Scale bar: 100 µm. (B) GAG and (C) GAG/DNA ratio quantification confirmed histological results. *P < 0.01.

Mentions: Sheep P3 auricular chondrocytes seeded onto collagen scaffolds and incubated in medium conditioned with P0 chondrocytes partially redifferentiated after 6 weeks implantation in mice. Histologically, neocartilage was of poor quality and not contiguous as evidenced by cartilage-specific stains (Fig. 2A). No elastin was observed histologically. Human chondrocytes failed to redifferentiate and form neocartilage. Histological data were confirmed by GAG quantification (Fig. 2B, C); GAG content was low in sheep samples and was negligible in human samples.


Extensively Expanded Auricular Chondrocytes Form Neocartilage In Vivo.

Tseng A, Pomerantseva I, Cronce MJ, Kimura AM, Neville CM, Randolph MA, Vacanti JP, Sundback CA - Cartilage (2014)

Histological and biochemical assessment of neocartilage engineered from passaged P3 chondrocytes after in vitro incubation in medium conditioned by P0 chondrocytes for 2 weeks and implantation in nude mice for 6 weeks. (A) The quality of neocartilage formed from sheep cells was poor as evidenced by toluidine blue, safranin, collagen type II, and elastin stains. No neocartilage formed from human cells as confirmed by the absence of cartilage-specific glycosaminoglycans (GAG) staining and positive collagen type I staining. Collagen scaffold fibers stained red on hematoxylin–eosin (H&E) stained sections. Scale bar: 100 µm. (B) GAG and (C) GAG/DNA ratio quantification confirmed histological results. *P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4335768&req=5

fig2-1947603514546740: Histological and biochemical assessment of neocartilage engineered from passaged P3 chondrocytes after in vitro incubation in medium conditioned by P0 chondrocytes for 2 weeks and implantation in nude mice for 6 weeks. (A) The quality of neocartilage formed from sheep cells was poor as evidenced by toluidine blue, safranin, collagen type II, and elastin stains. No neocartilage formed from human cells as confirmed by the absence of cartilage-specific glycosaminoglycans (GAG) staining and positive collagen type I staining. Collagen scaffold fibers stained red on hematoxylin–eosin (H&E) stained sections. Scale bar: 100 µm. (B) GAG and (C) GAG/DNA ratio quantification confirmed histological results. *P < 0.01.
Mentions: Sheep P3 auricular chondrocytes seeded onto collagen scaffolds and incubated in medium conditioned with P0 chondrocytes partially redifferentiated after 6 weeks implantation in mice. Histologically, neocartilage was of poor quality and not contiguous as evidenced by cartilage-specific stains (Fig. 2A). No elastin was observed histologically. Human chondrocytes failed to redifferentiate and form neocartilage. Histological data were confirmed by GAG quantification (Fig. 2B, C); GAG content was low in sheep samples and was negligible in human samples.

Bottom Line: With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated.Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks.Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Massachusetts General Hospital, Boston, MA, USA ; Center for Regenerative Medicine, Boston, MA, USA.

ABSTRACT

Objective: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice.

Design: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed.

Results: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks.

Conclusions: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

No MeSH data available.


Related in: MedlinePlus