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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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Gene expression profile of Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions.The effect of oxygen tension on the matrix-forming capacity of differentially expanded MFCs prior to (monolayer cell culture; M) and after seeding and culture on Duragen® collagen scaffold (S) was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR). Total RNA isolated from monolayer cells culture (M) and cells-scaffold constructs (S) were analyzed for the mRNA expression of: the transcription factor, Sox9, and members of the collagen gene family and sulphated proteoglycans found in the extracellular matrix of meniscal fibrocartilage. All y-axes represent relative gene expression levels normalized to human RNA polymerase II (RPII). Data represents mean ± SD; one-way ANOVA with Tukey's post hoc test: n.s. (not significant), * = p<0.05, ** = p<0.001, n = 10, N = 3.
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pone-0039339-g003: Gene expression profile of Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions.The effect of oxygen tension on the matrix-forming capacity of differentially expanded MFCs prior to (monolayer cell culture; M) and after seeding and culture on Duragen® collagen scaffold (S) was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR). Total RNA isolated from monolayer cells culture (M) and cells-scaffold constructs (S) were analyzed for the mRNA expression of: the transcription factor, Sox9, and members of the collagen gene family and sulphated proteoglycans found in the extracellular matrix of meniscal fibrocartilage. All y-axes represent relative gene expression levels normalized to human RNA polymerase II (RPII). Data represents mean ± SD; one-way ANOVA with Tukey's post hoc test: n.s. (not significant), * = p<0.05, ** = p<0.001, n = 10, N = 3.

Mentions: In order to further characterize the ECM formed in the generated constructs, we examined gene expression in all constructs using quantitative real-time RT-PCR and in monolayer cell cultures prior to seeding and culture on scaffolds (Fig. 3). The expression of collagen I (Col1a2 transcript) was not statistically significant (p = 0.95) between constructs cultured under low and normal oxygen tensions as well as between monolayer cell cultures and cell-scaffold constructs. The expression of Col1a2 in monolayer MFC culture under normal oxygen tension was 2.3 fold higher than its expression in monolayer cultures of MFC under low oxygen tension. However, the fold difference was not statistically significant (p = 0.896; not shown in Fig. 3).


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)

Gene expression profile of Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions.The effect of oxygen tension on the matrix-forming capacity of differentially expanded MFCs prior to (monolayer cell culture; M) and after seeding and culture on Duragen® collagen scaffold (S) was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR). Total RNA isolated from monolayer cells culture (M) and cells-scaffold constructs (S) were analyzed for the mRNA expression of: the transcription factor, Sox9, and members of the collagen gene family and sulphated proteoglycans found in the extracellular matrix of meniscal fibrocartilage. All y-axes represent relative gene expression levels normalized to human RNA polymerase II (RPII). Data represents mean ± SD; one-way ANOVA with Tukey's post hoc test: n.s. (not significant), * = p<0.05, ** = p<0.001, n = 10, N = 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376130&req=5

pone-0039339-g003: Gene expression profile of Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions.The effect of oxygen tension on the matrix-forming capacity of differentially expanded MFCs prior to (monolayer cell culture; M) and after seeding and culture on Duragen® collagen scaffold (S) was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR). Total RNA isolated from monolayer cells culture (M) and cells-scaffold constructs (S) were analyzed for the mRNA expression of: the transcription factor, Sox9, and members of the collagen gene family and sulphated proteoglycans found in the extracellular matrix of meniscal fibrocartilage. All y-axes represent relative gene expression levels normalized to human RNA polymerase II (RPII). Data represents mean ± SD; one-way ANOVA with Tukey's post hoc test: n.s. (not significant), * = p<0.05, ** = p<0.001, n = 10, N = 3.
Mentions: In order to further characterize the ECM formed in the generated constructs, we examined gene expression in all constructs using quantitative real-time RT-PCR and in monolayer cell cultures prior to seeding and culture on scaffolds (Fig. 3). The expression of collagen I (Col1a2 transcript) was not statistically significant (p = 0.95) between constructs cultured under low and normal oxygen tensions as well as between monolayer cell cultures and cell-scaffold constructs. The expression of Col1a2 in monolayer MFC culture under normal oxygen tension was 2.3 fold higher than its expression in monolayer cultures of MFC under low oxygen tension. However, the fold difference was not statistically significant (p = 0.896; not shown in Fig. 3).

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