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

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.

Figure 1. 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).

Figure 2. 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).

Figure 3. 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.

Figure 4. Ratios of mRNA expression levels of collagen type II (Col2a1) to collagen type I (Col1a2) and of aggrecan (AGG) to versican (VCN).

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.

Figure 5. 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.