Developing an In Vitro Model for Regeneration of the Avascular Human Knee Meniscus with Sex-Biased Implications

Abstract

Fibrocartilaginous tissues are challenging to regenerate, characterized by complex extracellular matrix architecture and decreasing levels of vascularity. The human knee meniscus is a fibrocartilaginous tissue which is critical to knee function. Tears in the human knee meniscus are the most common intra-articular knee injury and repairs in the meniscus often fail long term, leading to a higher risk of post-traumatic osteoarthritis (PTOA). The gold standard of care for meniscal tears is suture repair follow, whereby the torn tissue is bridged back together in an attempt to promote native healing. In cases where the torn tissue is too damaged, partial or total meniscectomies (e.g., tissue removal) are performed. Partial/total meniscectomies, or having a suture-based repair both lead to higher rates of PTOA, highlighting the need for alternative treatment options for restoring the function of the meniscus. As with other fibrocartilaginous tissues, the meniscus exhibits a regional gradient in vascularization, with most irreparable tears being in the white-white, or avascular zone. These tears are sexually dimorphic in their incidence, with women1 being at a higher risk for meniscal tears than men. While there are many aspects to sex differences, 17-β-estradiol (E2) is known to cause joint laxity contributing to a higher risk for meniscal tear. As such, there are sex specific considerations for the regeneration of these complex fibrocartilaginous tissues. To study these sex specific considerations in vitro, basic culture conditions that impact biological differences based on sex, especially the presence of exogenous estrogens, need to be characterized. Fundamental studies have previously focused on investigating the endogenous human mesenchymal stromal cell-like cells (hMSCs) to repair these avascular tears. As such, there is a need to understand the sex-specific considerations implicated in the use of hMSCs for fibrocartilage repair and regeneration. Despite this, the differences in male and female hMSC response to estrogen and estrogen-mimetic compounds, namely phenol red and unfiltered fetal bovine serum in traditional cell culture media, are not well elucidated. Avascular regeneration is usually rare, but there are examples of avascular regeneration in the human knee meniscus. The human discoid meniscus is a variant of the human knee meniscus that represents an overgrowth of the white-white zone of the meniscus covering the tibial plateau. When symptomatic, this excess tissue is often treated in the form of saucerization, whereby the excess avascular tissue is removed to return the meniscal to the C-shape. However, there is evidence that after the removal of this avascular tissue, the discoid meniscus can spontaneously re-grow into the space where the tissue was removed without further surgical intervention. While currently this re-growth is pathogenic in its nature and requires re-operation, the growth of fibrocartilaginous meniscal tissue in the avascular region of the meniscus represents a fascinating case study for understanding potential regenerative mechanisms for fibrocartilaginous tissues. Despite this, no research to date has looked at the human discoid meniscus for its potential regenerative mechanisms. As such the work within focuses on developing tools to elucidate the potential for regeneration of avascular meniscal tears. First, through the investigation of the impact of exogenous estrogens that are ubiquitous in cell culture on male and female hMSCs proliferation, metabolism, senescence, and adipogenic and osteogenic differentiation potential from 8 total donors. Second, an in vitro model using mechanically constrained 3D electrospun fibers for the human meniscus was developed that can re-capitulate the native tissue collagen anisotropy as a tissue level structural hallmark for extracellular matrix deposition in vitro. This model was then shown to be able to demonstrate tissue level structural differences in collagen deposition for discoid meniscal cells when compared to non-discoid cells in vitro and can be used for future investigation of the impact of structural and regenerative motifs involved in discoid avascular regeneration. To understand discoid specific pathways to target in this in vitro model, targets from a single cell RNA sequencing dataset were used to investigate transcriptional activity involved in the human discoid lateral meniscus and determined a transcription factor that drives the discoid specific phenotype. The identified transcription factor was then shown in our model system to have a significant impact on the alignment and thickness of collagen deposition and have potential sex dependent effects on the deposition of proteoglycans in discoid meniscal cells. In culture conditions, phenol red was shown to impact cellular proliferation and decrease osteogenic staining. Fetal bovine serum containing exogenous hormones was shown to alter the metabolic profiles of hMSCs to become less stem like, have an increase in senescence associated β-galactosidase staining, and increased osteogenic staining and gene expression. These results were sex specific, with only the metabolic profile from Seahorse analysis and Sox9 gene expression not showing any sex dependent effects. A mechanically constrained 3D electrospun model for in vitro culture was shown to recreate significant collagen anisotropy using fibers with aligned and unaligned orientation as a substrate, but not in unaligned or aligned scaffolds alone. Discoid meniscal cells produced unaligned ECM in these scaffolds indicating it can serve as a model for discoid versus non-discoid collagen deposition. Finally, a novel transcription factor NR4A1 was identified that drove alterations in collagen deposition via shRNA knockdown in discoid cells on our scaffolds. Taken together this work provides basic culture conditions for regenerative cell types in the meniscus, a model for anisotropic collagen deposition in the meniscus, and a novel transcription factor that can drive collagen alignment in the meniscus in vitro.