Male and Female Meniscus Cells Exhibit Sex Differences in Response to Estrogen: Implications for Knee Osteoarthritis

Introduction:: Osteoarthritis is a chronic condition understudied among women. While there are multiple potential causes for OA development, meniscal injuries pose a significant risk. Epidemiological evidence indicates that sex plays a role with meniscus injury higher in female high school athletes and females requiring 2.2X more repeat surgery after initial repair. Further, women show worse outcomes after partial meniscectomy including delayed maximal recovery and worse knee function compared to males. Lastly, females are at a higher risk of developing OA after meniscectomy compared to age-matched men. Although these empirical sex differences exist, there is no clear reasoning for what is driving differences in males and female meniscus injury, repair/regeneration, and OA development. Previous results from our group illustrated the loss of estrogen signaling in an ovariectomized mouse model resulted in a reduction in collagen and glycosaminoglycan (GAG) levels and an increase in matrix metalloproteinases (MMPs) in the meniscus. Excitingly, estrogen (17β-estradiol, E2) treatment recovered this loss with an increase in collagen and GAG production and expression of protease inhibitors. Further, estrogen signaling was modulated via estrogen receptor alpha (ERα) in this process as mice lacking this receptor did not respond with the same anabolic response to E2 treatment. As such, we aim to determine the mechanism of estrogen signaling to produce new extracellular matrix in the meniscus in both male and female human meniscal fibrochondrocytes (MFCs). We hypothesize sex-based differences in estrogen receptor dimerization type and function of those dimers and downstream effects that impact transcription and ECM production and homeostasis.

Materials and Methods:: To obtain initial transcriptome data of sex differences in human MFCs, male and female human MFCs (47 years old) were isolated from cadaveric meniscal tissue obtained from the National Disease Research Interchange. Bulk RNA-Seq was used to study changes in gene expression in MFCs after estrogen treatment (1 and 10 µM, 72 hours). To further pursue the role of ERs in male and female MFCs, cells were harvested from resected inner meniscal tissue after tear at Children’s Mercy Hospital (KC, MO) (ages 12-17) and dosed with E2 (0.1 and 1 nM, 72 hours) and bulk RNA sequencing completed. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry (ICC) was used to detect ER and mechanosensitive protein expression in response to E2 treatment. The proximity ligation assay (PLA) was used to study estrogen receptor dimerization. Lastly, male and female MFCs were cultured on electrospun polycaprolactone (PCL) fibrous mesh with low, medium, and high degrees of alignment to mimic native meniscal collagen fiber alignment and determine the impact of ERs on strain sensing in male and female MFCs. ERα-specific agonist (PPT 10 nM) and ER antagonist (ICI 182,780 100 nM) was included to investigate the impact of ERs. ICC for ERα, actin, paxillin, and vinculin was conducted to assess the role of ERα in focal adhesion and actin polymerization in response to static strain. Further, dynamic strain was applied to MFCs on PCL meshes loaded in a Mechanoculture J1 at 5 and 10% strain at 1Hz for 2 hours.

Results, Conclusions, and Discussions:: Increase in meniscal collagen type 2 and sulfated GAGs after E2 treatment in a mouse OVX model is shown in Figure 1A. RNA-Seq analysis of all MFCs tested revealed that E2 treatment controls differential expression of ECM and cell-matrix interactions gene pathways (Figure 1B). qPCR follow up (n=3 donors of each sex) on mechanotransductive genes confirmed that E2 treatment increased the expression of YAP, TAZ, PIEZO1, and LATS1 in males but only of TAZ in females (Figure 1C). Preliminary results from PLA analysis indicate sex-based differences in the ratios of ER dimers that are formed under estrogen deprivation Specifically, the predominant dimer detected in male cells (n=2) is the beta homodimer, while in female cells (n=2) it is the heterodimer (Figure 1D). The full implications of this require further study but given the differing signaling and transcriptional actions of the two ER isomers, it is likely that these differences also contribute to sex-based differences in the cells. Further, perturbing ERα with PPT and ICI in male and female MFCs cultured on fibers with increasing degree of alignment resulted in differences in focal adhesion density and actin alignment on the low and medium degree of alignment fibers but not the high alignment in both male and female MFCs (Figure 1E). Also, male MFCs appeared less responsive to ERα agonism and antagonism based on focal adhesion and actin morphology visualization. Current studies are investigating the role of dynamic strain. Collectively, these results indicate that male and female cells sense and respond to their physical and biochemical environment differently. Knowledge gleaned from these studies will inform surgical repair and regeneration strategies to promote functional healing and reduce OA with a focus on biological sex. Historically, studies investigating cell response to sex hormones was conducted in solely male cells or the cell sex was not reported. Results presented here illustrate sex differences in estrogen via ER signaling in response to both physical and chemical stimuli and highlight the dearth of research in this area and a call for both biological sexes to be investigated.

Acknowledgements (Optional): : The authors thank Dr. Donna Pacicca for the resected meniscal tear tissue for cell harvest and the fruitful discussions around meniscal pathology and tear repair procedures. Also, thanks to Jenny Hackett and the KU Genome Sequencing Core for the bulk transcriptome sequencing data.

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