Fibronectin Adherent Cell Populations Derived From Avascular and Vascular Regions of the Meniscus Have Enhanced Clonogenicity and Differentiation Potential Under Physioxia

Abstract

The meniscus is composed of an avascular inner region and vascular outer region. The vascular region has been shown to contain a progenitor population with multilineage differentiation capacity. Strategies facilitating the isolation and propagation of these progenitors can be used to develop cell-based meniscal therapies. Differential adhesion to fibronectin has been used to isolate progenitor populations from cartilage, while low oxygen or physioxia (2% oxygen) enhances the meniscal phenotype. This study aimed to isolate progenitor populations from the avascular and vascular meniscus using differential fibronectin adherence and examine their clonogenicity and differentiation potential under hyperoxia (20% oxygen) and physioxia (2% oxygen). Human vascular and avascular meniscus cells were seeded onto fibronectin-coated dishes for a short period and monitored for colony formation under either hyperoxia or physioxia. Non-fibronectin adherent meniscus cells were also expanded under both oxygen tension. Individual fibronectin adherent colonies were isolated and further expanded, until approximately ten population doublings (passage 3), whereby they underwent chondrogenic, osteogenic, and adipogenic differentiation. Physioxia enhances clonogenicity of vascular and avascular meniscus cells on plastic or fibronectin-coated plates. Combined differential fibronectin adhesion and physioxia isolated a progenitor population from both meniscus regions with trilineage differentiation potential compared to equivalent hyperoxia progenitors. Physioxia isolated progenitors had a significantly enhanced meniscus matrix content without the presence of collagen X. These results demonstrate that combined physioxia and fibronectin adherence can isolate and propagate a meniscus progenitor population that can potentially be used to treat meniscal tears or defects.

Keywords: chondrogenesis; hypoxia; meniscus; meniscus progenitor cells; tissue engineeering.

FIGURE 1

Schematic describing the experimental plan for non-fibronectin adherent (NFA) avascular and vascular meniscus cells and isolation of fibronectin adherent progenitors (avMPCs and vMPCs) expanded and differentiated under hyperoxia (HYP/20% oxygen) and physioxia (PHY/2% oxygen).

FIGURE 2

Representative photomicrographs of hyperoxia (HYP) and physioxia (PHY) expanded NFA (A) avascular and (B) vascular meniscus cells with corresponding population growth curves (n = 7; mean ± S.D.). (C) Representative photomicrographs of Oil-red-O staining for lipid droplet and alizarin red staining for calcium deposition for meniscus populations under hyperoxia (HYP) and physioxia (PHY). Pellet (D) wet weight (E) GAG content for avascular and vascular meniscus cells with representative (D) macroscopic and (E) DMMB-stained pellets.

FIGURE 3

(A) Representative crystal violet-stained colonies cultured under hyperoxia (HYP) and physioxia (HYP) on uncoated (PL) and fibronectin (FN)-coated dishes. (B) Number of colonies counted from conditions described in (A) from avascular and vascular meniscus cells (n = 6; data represent mean ± S.D.; *p < 0.05). (C) Representative photomicrographs of a fibronectin colony and avMPCs and vMPCs cultured under hyperoxia and physioxia with (D) population growth curves for expanded avMPC and vMPC population under hyperoxia and physioxia [n = 4 (3 clones/donor); data represent mean ± S.D.]. (E) Representative Oil-red-O staining for lipid droplet formation and alizarin red staining for calcium deposition for avMPCs and vMPCs.

FIGURE 4

Chondrogenic differentiation of avMPCs and vMPCs under hyperoxia and physioxia. Dot plot for pellet (A) wet weight and (B) GAG content for isolated clones (each dot represents mean of individual three clones) cultured under hyperoxia and physioxia with representative (A) macroscopic and (B) DMMB-stained pellets. (C) Gene expression data for meniscus matrix genes for (i) avascular and (ii) vascular MPCs. Data represent fold change for (i) avascular and (ii) vascular MPCs cultured under physioxia relative to corresponding clones under hyperoxic conditions (data represent mean ± S.D.; n = 5 donors; *p < 0.05). (D) Representative images of avMPC and vMPCs pellets stained with collagen I, collagen II, and collagen X cultured under hyperoxia and physioxia. Positive and negative controls are in Supplementary Figure S1.