Hypoxia as a Stimulus for the Maturation of Meniscal Cells: Highway to Novel Tissue Engineering Strategies?

Valentina Rafaela Herrera Millar,Van Thi Nguyen,Laura Mangiavini,Luigi Anastasia,Silvia Clotilde Modina,Umberto Polito,Giuseppe Maria Peretti,Federica Cirillo,Barbara Canciani,Alessia Di Giancamillo

doi:10.3390/ijms22136905

Abstract

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape “fibro-chondrocytic” cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.

Highlights

Introduction published maps and institutional affilRegenerative medicine is trying to achieve the goal of replacement or regeneration of the meniscus, as it plays a fundamental role in the biomechanics of the knee joint [1].Despite great efforts during the last few years, the different techniques adopted to replace or regenerate meniscus still present some criticisms and pitfalls [2,3,4]
The neonatal meniscal cells presented a fibroblast-like phenotype without matrix deposition (Figure 1A inner zone)
These modifications modifications are are strictly strictly linked to the achievement of the different cellular phenotypes that characterize the mature linked to the achievement of the different cellular phenotypes that characterize the mameniscus: fibroblast-like cells incells the in outer fibrochondrocyte-like cells concenture meniscus: fibroblast-like thezone, outerand zone, and fibrochondrocyte-like cells trated in the inner zone

Summary

Introduction

Regenerative medicine is trying to achieve the goal of replacement or regeneration of the meniscus, as it plays a fundamental role in the biomechanics of the knee joint [1]. Despite great efforts during the last few years, the different techniques adopted to replace or regenerate meniscus still present some criticisms and pitfalls [2,3,4]. II and aggrecan), the poorer biomechanical properties of the meniscal substitutes and the lack of integration between the cultured cells and the scaffolds [2,3]. These side effects may be caused by an improper technique or by inadequate culture conditions of meniscal tissue or cells.

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