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.

Fibronectin‐coated polyurethane meniscal scaffolding supplemented with MSCs improves scaffold integration and proteoglycan production in a rabbit model

“…Recently, Kitahashi et al [10] identified several molecules deemed essential to promoting adhesion, proliferation, and PG production of MSCs injected into the knee joint. More recently, Pattappa et al [17] studied the importance of FN in the isolation of progenitor cells from the meniscus and its potential utility in cell‐based therapies for meniscal tears or defects. All these investigations, among others [11], lead us to think that MSCs cannot fix the PU scaffold on their own.…”

Bioprinting of scaled-up meniscal grafts by spatially patterning phenotypically distinct meniscus progenitor cells within melt electrowritten scaffolds

“…Based on differential adhesion to fibronectin [39,43,44,72], we were able to isolate progenitor cell populations from both the inner and outer zone of the meniscus. MPCs maintained their capacity to generate meniscal-like tissue upon serial passage, making them a particularly attractive cell source for meniscus TE strategies where large numbers of cells are required to fabricate human scale tissues.…”

“…Using a differential adhesion to fibronectin protocol that has been used to isolate progenitor cells from the articular cartilage [40][41][42], it is possible to isolate cellular subpopulations from within the meniscus which have a high proliferative capacity and are able to maintain their differentiation potential after serial subculture, unlike non-fibronectin selected meniscus cells [43]. Phenotypically distinct progenitor cells have been found in both the inner and outer regions of the meniscus, which retain distinct capabilities to differentiate into inner and outer meniscus fibroblasts with limited evidence of hypertrophy [44]. Therefore, integrating such meniscus progenitor cells (MPCs) into advanced biofabrication strategies could potentially enable the engineering of functional meniscus constructs that mimic the spatial composition of the native tissue.…”

“…The aim of the study is to combine the biological potential of MPCs, isolated from the inner and outer regions of the meniscus, with a multipletool biofabrication strategy to engineer large (5 mm tall) wedge-shaped meniscus-like tissues with spatially defined structure and composition. Building on previous work reported in the literature [43,44], we first sought to use a differential fibronectin adhesion protocol to isolate MPCs from the inner and outer zone of the meniscus, and then compare their capacity to generate phenotypically distinct meniscus tissue compared to meniscus fibrochondrocytes isolated from the inner and outer zone of the meniscus (figure 1). We then attempted to bioprint fibrocartilage tissue with preferentially aligned collagen fibers by depositing MPCs obtained from either the inner (iMPCs) or outer (oMPCs) region of the meniscus into MEW scaffolds with anisotropic pore networks.…”

Phenotypic characterization of regional human meniscus progenitor cells

“…The basic principles of the progenitor cells isolation protocol used in this study are based on the selection of cells that highly express β1 integrins and demonstrate rapid adhesion to extracellular matrix proteins ( Jones and Watt, 1993 ), which was originally used for chondroprogenitors isolation ( Williams et al, 2010 ). KorSimilar et al ( Korpershoek et al, 2021 ) and Pattappa et al ( Pattappa et al, 2022 ) also used the fibronectin selective protocol to isolate meniscus progenitor cells. However, we included a direct comparison of the protocol for the efficiency of donor-matched chondrocyte, avascular and vascular meniscus progenitor isolation, and present these results for the first time (to our knowledge).…”