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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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Histological and biochemical analysis of Duragen®-meniscus fibrochondrocytes (MFCs) constructs after culture for 21 days under normoxic (21% O2) and hypoxic (3%O2) conditions.a) Low (4×); c) medium (10×); e) high (40×) magnification photomicrographs of analysis of paraffin wax embedded sections (5 µm thickness) stained with alcian blue/neutral red stain (for sulphated GAG detection). Constructs were seeded with normoxia-expanded MFCs and were cultured under normoxia. b) Low (4×); d) medium (10×); f) high (40×) power magnification photomicrographs of alcian blue/neutral red (for sulphated GAG) stained constructs seeded with hypoxia-expanded MFCs and were cultured under hypoxia. Solid scale bar = 100 µm and dotted bar = 50 µm. (g) Biochemical analysis was used to evaluate the glycosaminoglycan (GAG) and DNA contents of constructs after 21 days culture in serum-free chondrogenic medium under normal (21%O2) and low oxygen tension (3%O2). Data is presented here as chondrogenic capacity (i.e. GAG levels normalized to DNA content) of constructs generated from 3 independent donors and it represents the mean ± SD (n = 10, N = 3). Student's t statistics; not significant (n.s.; p>0.05).
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pone-0039339-g002: Histological and biochemical analysis of Duragen®-meniscus fibrochondrocytes (MFCs) constructs after culture for 21 days under normoxic (21% O2) and hypoxic (3%O2) conditions.a) Low (4×); c) medium (10×); e) high (40×) magnification photomicrographs of analysis of paraffin wax embedded sections (5 µm thickness) stained with alcian blue/neutral red stain (for sulphated GAG detection). Constructs were seeded with normoxia-expanded MFCs and were cultured under normoxia. b) Low (4×); d) medium (10×); f) high (40×) power magnification photomicrographs of alcian blue/neutral red (for sulphated GAG) stained constructs seeded with hypoxia-expanded MFCs and were cultured under hypoxia. Solid scale bar = 100 µm and dotted bar = 50 µm. (g) Biochemical analysis was used to evaluate the glycosaminoglycan (GAG) and DNA contents of constructs after 21 days culture in serum-free chondrogenic medium under normal (21%O2) and low oxygen tension (3%O2). Data is presented here as chondrogenic capacity (i.e. GAG levels normalized to DNA content) of constructs generated from 3 independent donors and it represents the mean ± SD (n = 10, N = 3). Student's t statistics; not significant (n.s.; p>0.05).

Mentions: MFC-seeded Duragen® scaffold constructs were cultured in serum-free chondrogenic medium. Constructs generated from cells expanded under normal oxygen tension were subsequently cultured under same oxygen tension and constructs formed from hypoxia-expanded meniscus cells were subsequently cultured under the same low oxygen tension. Histological assessment of the constructs after 3 weeks of cultivation using Alcian blue staining for sulphated proteoglycan showed that there was positive deposition of proteoglycan (GAG)-rich matrix as indicated by greenish-blue color, and good migration of meniscus cells into the porous Duragen® collagen scaffold (Figs. 2a and 2c – normoxic conditions and Figs. 2b and 2d – hypoxic conditions). Furthermore, the majority of cells within the GAG-rich matrix had a rounded chondrocyte-like morphology as seen at higher magnifications (Figs. 2e – normoxic conditions and 2f – hypoxic conditions). Quantitative assessment of total GAG per cellular DNA content in the constructs showed no statistical significance (p>0.05) between constructs cultivated under normal and low oxygen tensions (Fig. 2g). Interestingly, there were negligible differences in the ratio of GAG/DNA amongst the different donors examined in this study. The ratios for the three donors ranged from 11.25–16.28 for constructs cultivated under normal oxygen tension and 12.41–16.84 for constructs cultured under low oxygen tension.


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)

Histological and biochemical analysis of Duragen®-meniscus fibrochondrocytes (MFCs) constructs after culture for 21 days under normoxic (21% O2) and hypoxic (3%O2) conditions.a) Low (4×); c) medium (10×); e) high (40×) magnification photomicrographs of analysis of paraffin wax embedded sections (5 µm thickness) stained with alcian blue/neutral red stain (for sulphated GAG detection). Constructs were seeded with normoxia-expanded MFCs and were cultured under normoxia. b) Low (4×); d) medium (10×); f) high (40×) power magnification photomicrographs of alcian blue/neutral red (for sulphated GAG) stained constructs seeded with hypoxia-expanded MFCs and were cultured under hypoxia. Solid scale bar = 100 µm and dotted bar = 50 µm. (g) Biochemical analysis was used to evaluate the glycosaminoglycan (GAG) and DNA contents of constructs after 21 days culture in serum-free chondrogenic medium under normal (21%O2) and low oxygen tension (3%O2). Data is presented here as chondrogenic capacity (i.e. GAG levels normalized to DNA content) of constructs generated from 3 independent donors and it represents the mean ± SD (n = 10, N = 3). 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-g002: Histological and biochemical analysis of Duragen®-meniscus fibrochondrocytes (MFCs) constructs after culture for 21 days under normoxic (21% O2) and hypoxic (3%O2) conditions.a) Low (4×); c) medium (10×); e) high (40×) magnification photomicrographs of analysis of paraffin wax embedded sections (5 µm thickness) stained with alcian blue/neutral red stain (for sulphated GAG detection). Constructs were seeded with normoxia-expanded MFCs and were cultured under normoxia. b) Low (4×); d) medium (10×); f) high (40×) power magnification photomicrographs of alcian blue/neutral red (for sulphated GAG) stained constructs seeded with hypoxia-expanded MFCs and were cultured under hypoxia. Solid scale bar = 100 µm and dotted bar = 50 µm. (g) Biochemical analysis was used to evaluate the glycosaminoglycan (GAG) and DNA contents of constructs after 21 days culture in serum-free chondrogenic medium under normal (21%O2) and low oxygen tension (3%O2). Data is presented here as chondrogenic capacity (i.e. GAG levels normalized to DNA content) of constructs generated from 3 independent donors and it represents the mean ± SD (n = 10, N = 3). Student's t statistics; not significant (n.s.; p>0.05).
Mentions: MFC-seeded Duragen® scaffold constructs were cultured in serum-free chondrogenic medium. Constructs generated from cells expanded under normal oxygen tension were subsequently cultured under same oxygen tension and constructs formed from hypoxia-expanded meniscus cells were subsequently cultured under the same low oxygen tension. Histological assessment of the constructs after 3 weeks of cultivation using Alcian blue staining for sulphated proteoglycan showed that there was positive deposition of proteoglycan (GAG)-rich matrix as indicated by greenish-blue color, and good migration of meniscus cells into the porous Duragen® collagen scaffold (Figs. 2a and 2c – normoxic conditions and Figs. 2b and 2d – hypoxic conditions). Furthermore, the majority of cells within the GAG-rich matrix had a rounded chondrocyte-like morphology as seen at higher magnifications (Figs. 2e – normoxic conditions and 2f – hypoxic conditions). Quantitative assessment of total GAG per cellular DNA content in the constructs showed no statistical significance (p>0.05) between constructs cultivated under normal and low oxygen tensions (Fig. 2g). Interestingly, there were negligible differences in the ratio of GAG/DNA amongst the different donors examined in this study. The ratios for the three donors ranged from 11.25–16.28 for constructs cultivated under normal oxygen tension and 12.41–16.84 for constructs cultured under low oxygen tension.

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