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

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.

Testing Hypoxia in Pig Meniscal Culture: Biological Role of the Vascular-Related Factors in the Differentiation and Viability of Neonatal Meniscus

“…In normoxia, cell death and hypertrophic phenomena were more pronounced. These findings differ from recent evidence, where the study of the entire tissue showed an onset of extracellular matrix production after 14 days of culture [38]. To explain these apparent conflicting data, we have to consider that the tissue cultured in the two experiments had different dimensions, and in the present study the smaller dimensions probably allowed for a better perfusion of the culture medium.…”

“…Thus, hypoxia can be used as a stimulus to overcome the problem related to the de-differentiation in human meniscal cells [44], and it can be applied to promote the maturation of neonatal piglet meniscal cells [38]. Furthermore, it has been observed that the change in cell phenotype coincides with a change in the vascularity of the tissue in the meniscus [29,45]; therefore, in this study we focused our attention on two particular vascular factors, the pro-angiogenic factor CD31 and the anti-angiogenic factor endostatin.…”

“…The choice to divide the meniscus into three portions was due to the slightly different and fully recognized micro-and macroscopic characteristics observed among the three zones [46]. In a recent study of our group, we found that hypoxia acted as boost for meniscal differentiation; however, the study was performed on the whole meniscus, and no regional differentiation has been evaluated [38]. The data presented in this manuscript showed that as concerns cell viability, hypoxia tended to retain the initial phenotype of cells, although some cells began to differentiate assuming a fibro-chondrocytic phenotype.…”

“…Our recent study revealed that hypoxia worked as a booster to achieve meniscal cell maturation on the whole meniscal tissue [38]. The aim of this novel study is to assess if hypoxia differently regulates meniscal cell proliferation and differentiation based on the anatomical position.…”

TEMPO-Oxidized Cellulose Nanofiber-Alginate Hydrogel as a Bioink for Human Meniscus Tissue Engineering

“…Collagen I, the major component of the meniscus fibrocartilage's ECM, served as a reference biomaterial. Hypoxia was used since it has been proven as a stimulus for the maturation of meniscus fibrochondrocytes (Herrera Millar et al, 2021). Our previous research has found that hypoxia and TGF-β3 synergistically mediated the inner meniscus-like tissue matrix formation (Szojka et al, 2019).…”