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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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Immunohistochemistry (IHC) of extracellular matrix deposition of collagen type II: Collagen type II as indicated by brown staining was detected in Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions using paraffin-wax embedded sections (5 µm thickness). a) Low (4×); c) medium (10×); e) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing normoxia expanded MFCs followed by culture under normoxia. b) Low (4×); d) medium (10×); f) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing hypoxia expanded MFCs followed by culture under hypoxic conditions.
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pone-0039339-g005: Immunohistochemistry (IHC) of extracellular matrix deposition of collagen type II: Collagen type II as indicated by brown staining was detected in Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions using paraffin-wax embedded sections (5 µm thickness). a) Low (4×); c) medium (10×); e) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing normoxia expanded MFCs followed by culture under normoxia. b) Low (4×); d) medium (10×); f) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing hypoxia expanded MFCs followed by culture under hypoxic conditions.

Mentions: Immuno-histochemical assessment of the constructs with collagen II antibody revealed a more intense (brown stain) staining of collagen II in constructs generated from normal oxygen tension (Figs. 5a, 5c and 5e) compared to constructs from low oxygen tension (Figs. 5b, 5d and 5f), supporting the enhanced mRNA expression of type II collagen in constructs cultivated under normal oxygen tension (Fig. 4a). We also stained our constructs for type I collagen, however, MFC-free collagen scaffold (empty control scaffolds) cross-reacted with the collagen I antibody, therefore the data was excluded from this study.


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)

Immunohistochemistry (IHC) of extracellular matrix deposition of collagen type II: Collagen type II as indicated by brown staining was detected in Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions using paraffin-wax embedded sections (5 µm thickness). a) Low (4×); c) medium (10×); e) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing normoxia expanded MFCs followed by culture under normoxia. b) Low (4×); d) medium (10×); f) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing hypoxia expanded MFCs followed by culture under hypoxic conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376130&req=5

pone-0039339-g005: Immunohistochemistry (IHC) of extracellular matrix deposition of collagen type II: Collagen type II as indicated by brown staining was detected in Duragen®-meniscus fibrochondrocytes constructs cultured for 21 days under normoxic (21%O2) and hypoxic (3%O2) conditions using paraffin-wax embedded sections (5 µm thickness). a) Low (4×); c) medium (10×); e) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing normoxia expanded MFCs followed by culture under normoxia. b) Low (4×); d) medium (10×); f) high (40×) magnification of IHC photomicrographs of collagen II stained sections of constructs containing hypoxia expanded MFCs followed by culture under hypoxic conditions.
Mentions: Immuno-histochemical assessment of the constructs with collagen II antibody revealed a more intense (brown stain) staining of collagen II in constructs generated from normal oxygen tension (Figs. 5a, 5c and 5e) compared to constructs from low oxygen tension (Figs. 5b, 5d and 5f), supporting the enhanced mRNA expression of type II collagen in constructs cultivated under normal oxygen tension (Fig. 4a). We also stained our constructs for type I collagen, however, MFC-free collagen scaffold (empty control scaffolds) cross-reacted with the collagen I antibody, therefore the data was excluded from this study.

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