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Oxygen tension is a determinant of the matrix-forming phenotype of cultured human meniscal fibrochondrocytes.

Adesida AB, Mulet-Sierra A, Laouar L, Jomha NM - PLoS ONE (2012)

Bottom Line: The results showed that constructs under normoxia produced a matrix with enhanced mRNA ratio (3.5-fold higher; p<0.001) of collagen type II to I.This was confirmed by enhanced deposition of collagen II using immuno-histochemistry.These findings suggest that the use of normal and low oxygen tension during MFC expansion and subsequent neo-tissue formation cultures may be important in engineering different regions of the meniscus.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Alberta, Edmonton, Alberta, Canada. adesida@ualberta.ca

ABSTRACT

Background: Meniscal cartilage displays a poor repair capacity, especially when injury is located in the avascular region of the tissue. Cell-based tissue engineering strategies to generate functional meniscus substitutes is a promising approach to treat meniscus injuries. Meniscus fibrochondrocytes (MFC) can be used in this approach. However, MFC are unable to retain their phenotype when expanded in culture. In this study, we explored the effect of oxygen tension on MFC expansion and on their matrix-forming phenotype.

Methodology/principal findings: MFC were isolated from human menisci followed by basic fibroblast growth factor (FGF-2) mediated cell expansion in monolayer culture under normoxia (21%O(2)) or hypoxia (3%O(2)). Normoxia and hypoxia expanded MFC were seeded on to a collagen scaffold. The MFC seeded scaffolds (constructs) were cultured in a serum free chondrogenic medium for 3 weeks under normoxia and hypoxia. Constructs containing normoxia-expanded MFC were subsequently cultured under normoxia while those formed from hypoxia-expanded MFC were subsequently cultured under hypoxia. After 3 weeks of in vitro culture, the constructs were assessed biochemically, histologically and for gene expression via real-time reverse transcription-PCR assays. The results showed that constructs under normoxia produced a matrix with enhanced mRNA ratio (3.5-fold higher; p<0.001) of collagen type II to I. This was confirmed by enhanced deposition of collagen II using immuno-histochemistry. Furthermore, the constructs under hypoxia produced a matrix with higher mRNA ratio of aggrecan to versican (3.5-fold, p<0.05). However, both constructs had the same capacity to produce a glycosaminoglycan (GAG) -specific extracellular matrix.

Conclusions: Our data provide evidence that oxygen tension is a key player in determining the matrix phenotype of cultured MFC. These findings suggest that the use of normal and low oxygen tension during MFC expansion and subsequent neo-tissue formation cultures may be important in engineering different regions of the meniscus.

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Proliferation rate of human meniscus fibrochondroytes under normal and low oxygen tension in the presence of FGF-2.a) Mean cell doubling rate for P1 and P2 human meniscus fibrochondrocytes from 3 donors in the presence of FGF-2. b) Mean total population doubling of P2 human meniscus fibrochondrocytes. Data is expressed as mean ± SD of 3 donors (n = 3, N = 3). FGF-2, fibroblast growth factor 2; P1, passage 1; P2, passage 2. Student's t statistics; not significant (n.s.; p>0.05).
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pone-0039339-g001: Proliferation rate of human meniscus fibrochondroytes under normal and low oxygen tension in the presence of FGF-2.a) Mean cell doubling rate for P1 and P2 human meniscus fibrochondrocytes from 3 donors in the presence of FGF-2. b) Mean total population doubling of P2 human meniscus fibrochondrocytes. Data is expressed as mean ± SD of 3 donors (n = 3, N = 3). FGF-2, fibroblast growth factor 2; P1, passage 1; P2, passage 2. Student's t statistics; not significant (n.s.; p>0.05).

Mentions: MFCs were isolated from human knee menisci and cultured in monolayer in the presence of FGF-2 under normal (21% O2) or low (3% O2) oxygen tension. The cells proliferated well with an elongated spindle-like morphology with rates of cell population doubling of: 0.1±0.02 per day at P1 and 0.21±0.04 per day at P2 under normal oxygen tension, and at population doubling rate of: 0.12±0.02 per day at P1 and 0.2±0.02 per day at P2 under low oxygen tension. There was no significant difference (p>0.05; p = 0.21 at P1 and p = 0.80 at P2) between cell population doubling rates under normal and low oxygen tension culture conditions (Fig. 1a). The mean total cell population doublings (i.e. P2 cells) prior to cell seeding on collagen scaffold was 3.61±0.18, for cells cultured under normal oxygen tension and 3.87±0.43 for cells cultured under low oxygen tension. There was no significant difference (p = 0.54) between the mean total population doublings at normal and low oxygen tension (Fig. 1b).


Oxygen tension is a determinant of the matrix-forming phenotype of cultured human meniscal fibrochondrocytes.

Adesida AB, Mulet-Sierra A, Laouar L, Jomha NM - PLoS ONE (2012)

Proliferation rate of human meniscus fibrochondroytes under normal and low oxygen tension in the presence of FGF-2.a) Mean cell doubling rate for P1 and P2 human meniscus fibrochondrocytes from 3 donors in the presence of FGF-2. b) Mean total population doubling of P2 human meniscus fibrochondrocytes. Data is expressed as mean ± SD of 3 donors (n = 3, N = 3). FGF-2, fibroblast growth factor 2; P1, passage 1; P2, passage 2. Student's t statistics; not significant (n.s.; p>0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376130&req=5

pone-0039339-g001: Proliferation rate of human meniscus fibrochondroytes under normal and low oxygen tension in the presence of FGF-2.a) Mean cell doubling rate for P1 and P2 human meniscus fibrochondrocytes from 3 donors in the presence of FGF-2. b) Mean total population doubling of P2 human meniscus fibrochondrocytes. Data is expressed as mean ± SD of 3 donors (n = 3, N = 3). FGF-2, fibroblast growth factor 2; P1, passage 1; P2, passage 2. Student's t statistics; not significant (n.s.; p>0.05).
Mentions: MFCs were isolated from human knee menisci and cultured in monolayer in the presence of FGF-2 under normal (21% O2) or low (3% O2) oxygen tension. The cells proliferated well with an elongated spindle-like morphology with rates of cell population doubling of: 0.1±0.02 per day at P1 and 0.21±0.04 per day at P2 under normal oxygen tension, and at population doubling rate of: 0.12±0.02 per day at P1 and 0.2±0.02 per day at P2 under low oxygen tension. There was no significant difference (p>0.05; p = 0.21 at P1 and p = 0.80 at P2) between cell population doubling rates under normal and low oxygen tension culture conditions (Fig. 1a). The mean total cell population doublings (i.e. P2 cells) prior to cell seeding on collagen scaffold was 3.61±0.18, for cells cultured under normal oxygen tension and 3.87±0.43 for cells cultured under low oxygen tension. There was no significant difference (p = 0.54) between the mean total population doublings at normal and low oxygen tension (Fig. 1b).

Bottom Line: The results showed that constructs under normoxia produced a matrix with enhanced mRNA ratio (3.5-fold higher; p<0.001) of collagen type II to I.This was confirmed by enhanced deposition of collagen II using immuno-histochemistry.These findings suggest that the use of normal and low oxygen tension during MFC expansion and subsequent neo-tissue formation cultures may be important in engineering different regions of the meniscus.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Alberta, Edmonton, Alberta, Canada. adesida@ualberta.ca

ABSTRACT

Background: Meniscal cartilage displays a poor repair capacity, especially when injury is located in the avascular region of the tissue. Cell-based tissue engineering strategies to generate functional meniscus substitutes is a promising approach to treat meniscus injuries. Meniscus fibrochondrocytes (MFC) can be used in this approach. However, MFC are unable to retain their phenotype when expanded in culture. In this study, we explored the effect of oxygen tension on MFC expansion and on their matrix-forming phenotype.

Methodology/principal findings: MFC were isolated from human menisci followed by basic fibroblast growth factor (FGF-2) mediated cell expansion in monolayer culture under normoxia (21%O(2)) or hypoxia (3%O(2)). Normoxia and hypoxia expanded MFC were seeded on to a collagen scaffold. The MFC seeded scaffolds (constructs) were cultured in a serum free chondrogenic medium for 3 weeks under normoxia and hypoxia. Constructs containing normoxia-expanded MFC were subsequently cultured under normoxia while those formed from hypoxia-expanded MFC were subsequently cultured under hypoxia. After 3 weeks of in vitro culture, the constructs were assessed biochemically, histologically and for gene expression via real-time reverse transcription-PCR assays. The results showed that constructs under normoxia produced a matrix with enhanced mRNA ratio (3.5-fold higher; p<0.001) of collagen type II to I. This was confirmed by enhanced deposition of collagen II using immuno-histochemistry. Furthermore, the constructs under hypoxia produced a matrix with higher mRNA ratio of aggrecan to versican (3.5-fold, p<0.05). However, both constructs had the same capacity to produce a glycosaminoglycan (GAG) -specific extracellular matrix.

Conclusions: Our data provide evidence that oxygen tension is a key player in determining the matrix phenotype of cultured MFC. These findings suggest that the use of normal and low oxygen tension during MFC expansion and subsequent neo-tissue formation cultures may be important in engineering different regions of the meniscus.

Show MeSH
Related in: MedlinePlus