Introduction

The meniscus plays an important role in knee function due to its roles in protecting the underlying articular cartilage from excessive high loads during motion and aiding in joint biomechanics and proprioception. It is composed of two distinct regions, an inner avascular region and an outer vascular region that has been extensively characterized in previous publications (1, 2). The avascular region has a matrix predominantly composed of collagen II and glycosaminoglycan similar to articular cartilage. In contrast, the vascular region is composed mainly of collagen I with vasculature throughout the outer half of the meniscus. Additionally, neurons are found at the horns of the meniscus that attach the tissue to the underlying bone (1, 2, 3). This unique structure is due to the loading mechanics during meniscus development, whereby the inner region is under compressive loading, whilst the outer region undergoes tensile loading, thus resulting in a heterogeneous tissue composition (3). However, inspite of this knowledge, the developmental, cellular and molecular aspects of the meniscus remain poorly understood compared to other musculoskeletal tissues (i.e. articular cartilage or bone). A greater understanding in the developmental and cellular aspects of the meniscus would help to form the basis of novel cell therapies and pharmacological strategies for meniscus treatment that preserve the tissue and prevent the onset of early osteoarthritis (4).

Content

This presentation will provide an overview of the current understanding of meniscus development and cellular function. Recent studies investigating joint development in mouse models have demonstrated that the meniscus derives from interzone cells during mesenchymal development and that hedgehog signaling is one pathway controlling its embryogenesis (5). We and other groups have begun to investigate meniscus-specific genes via microarray analysis, as previous studies have used a combination of cartilage and tendon specific genes to identify the meniscus phenotype. Identification of meniscus-specific genes could lead to the creation of meniscus induction protocols that can be applied to meniscus cells or to differentiate stem cells (e.g. bone marrow mesenchymal stem cells, embryonic, iPS) from different sources towards a meniscogenic lineage. We have also begun to study the presence of progenitor populations within both meniscus regions with the primary purpose of developing cell-based therapies (6). In parallel, we and other groups have begun to use different environmental conditions (e.g. oxygen tension, mechanical loading) to understand the optimal parameters for producing functional meniscus tissue (6). Our recent study has combined these approaches, whereby we have isolated a meniscus progenitor population that selectively adhered to fibronectin from both the avascular and vascular regions under a low oxygen tension or physioxia (6). Recent studies have used RNAseq to identify differential pathways between healthy and degenerative meniscus tissue that can be used for the establishment of pharmacological therapies and gain a greater understanding on meniscus development and degeneration (7). In summary, these recent publications show that the cellular and molecular aspects of the meniscus are critical in the design of novel therapeutic strategies that could lead a significant improvement in the quality of the life for the patient and prevent the onset of early osteoarthritis.

References

1) Verdonk PC, Forsyth RG, Wang J, Almqvist KF, Verdonk R, Veys EM, Verbruggen G: Characterisation of human knee meniscus cell phenotype. Osteoarthritis Cartilage. (2005) Jul;13(7):548-60.

2) Makris EA, Hadidi P, Athanasiou KA: The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration. Biomaterials. (2011) Oct;32(30):7411-31.

3) Gray JC: Neural and vascular anatomy of the menisci of the human knee. J Orthop Sports Phys Ther. (1999) Jan;29(1):23-30.

4) Verdonk R, Madry H, Shabshin N, Dirisamer F, Peretti GM, Pujol N, Spalding T, Verdonk P, Seil R, Condello V, Di Matteo B, Zellner J, Angele P: The role of meniscal tissue in joint protection in early osteoarthritis. Knee Surg Sports Traumatol Arthrosc. (2016) Jun;24(6):1763-74.

5) Wei Y, Sun H, Gui T, Yao L, Zhong L, Yu W, Heo SJ, Han L, Dyment NA, Liu XS, Zhang Y, Koyama E, Long F, Zgonis MH, Mauck RL, Ahn J, Qin L: The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair. Elife. (2021) Jun 4;10:e62917.

6) Pattappa G, Reischl F, Jahns J, Schewior R, Lang S, Zellner J, Johnstone B, Docheva D, Angele P: Fibronectin Adherent Cell Populations Derived From Avascular and Vascular Regions of the Meniscus Have Enhanced Clonogenicity and Differentiation Potential Under Physioxia. Front Bioeng Biotechnol. (2022) Jan 28;9:789621.

7) Sun H, Wen X, Li H, Wu P, Gu M, Zhao X, Zhang Z, Hu S, Mao G, Ma R, Liao W, Zhang Z: Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration. Ann Rheum Dis. (2020) Mar;79(3):408-417

Acknowledgments

The authors wish to acknowledge the funding support from European Union Horizon 2020 research and innovation programme (EU No.: 814444 (MEFISTO).

Purpose

The meniscus is a fibrocartilaginous tissue comprised of an inner avascular and an outer vascular region. To distinguish it from other mesenchymal tissues, studies have uncovered a set of genes to define the meniscus phenotype. Using the following genes - IGF-BP3, R-Spondin-2, CILP2, CHAD, CSRP2, ID2. Dermatopontin, Collagen XV - we investigated their specificity on meniscus tissue, and on avascular and vascular meniscus cells in monolayer and pellet culture.

Methods and Materials

Human meniscus tissue (n = 3) had a portion snap frozen, whilst remaining tissue was split into avascular and vascular regions for cell isolation. Avascular and vascular meniscus cells were cultured in either a 20% oxygen or 2% oxygen incubator. At passage 1, QIAzol was applied to monolayer cells at each oxygen tension and then frozen. At passage 2, avascular and vascular meniscus pellets were created and cultured for 21 days at each oxygen tension using previously described protocols. RNA was isolated from tissue, cells and pellets using QIAzol method and subsequent qPCR analysis for stated genes was performed using a Biorad CFX96 system.

Results

In meniscus tissue, there was elevated expression levels of all selected genes, with a significant upregulation in CLIP2, CSRP2, Dermatopontin and collagen XV (*p < 0.05). In monolayer, all genes were downregulated, whereas upon pellet redifferentiation, only IGF-BP3, CHAD (*p > 0.05) and Dermatopontin were upregulated in avascular and vascular meniscus cells. There was no significant difference in gene expression with respect to oxygen tension in monolayer and pellets for both meniscus cell types.

Conclusion

The selected genes were all upregulated in meniscus tissue. Monolayer expansion led to downregulation of all genes, whilst subsequent pellet redifferentiation resulted in upregulation of only IGF-BP3, CHAD and Dermatopontin. Future studies should identify optimal in vitro conditions to maintain expression of meniscus-specific genes, thus preventing phenotype drift during cell expansion.