Poster BB12 - Mechano-Hypoxia Conditioning of Human Synovial Fluid Derived Mesenchymal Stem Cells

Introduction: Articular cartilage (AC) of the knee exists under a combined low oxygen and mechanically active environment. AC is made up of chondrocytes and a biomechanically functional extracellular matrix (ECM) which the chondrocytes produce and assemble. Mesenchymal stem cells (MSC) are a promising cell source for making cartilage, but current protocols can lead to undesirable hypertrophic differentiation that progress to bone. We have previously found that the combination of low oxygen and mechanical loading (or mechano-hypoxia conditioning) during chondrogenic stimulation of human bone marrow derived-MSC can reduce hypertrophic chondrogenesis. These conclusions have been made for human bone marrow-derived MSCs, but MSCs derived from other anatomical sites, such as synovial fluid, which is found in the cavities of synovial joints such as the knee, may have differing behaviours. This experiment explores the mechanical and biological effects of mechano-hypoxia conditioning on chondroinduced MSCs derived from synovial fluid.

Materials and

Methods: Human synovial fluid-derived mesenchymal stem cells (hSfMSC) were developed after plastic cell adherent culture (male, ages 32-40), chondroinduced, and conditioned using the following procedure (Figure 1). HSfMSC were expanded to passage 3 in 3% O2 (HYP) and seeded onto 10 mm diameter x 3.5mm type I collagen porous scaffolds. HsfMSC-scaffold constructs were pre-cultured for 3 weeks in HYP to generate tissue-engineered cartilage models. The engineered cartilage models were then mechanically loaded using a dynamic compression bioreactor in HYP for 10 min at 20-30% strain, 4 times per day, for 21 days. The static control models were cultured in HYP for 21 days with no mechanical loading. Lastly, the engineered cartilage models were mechanically tested and analyzed through histology, gene expression, and biochemical assays including glycosaminoglycan (GAG) ECM and DNA content.

Results, Conclusions, and Discussions: Static groups showed an increase in equilibrium modulus by an average of 35 kPa compared to the mechanically loaded group (Figure 2(A)). This was a statistically significant difference (p=0.020). Additionally, GAG measurements showed that mechanical loading decreased GAG production significantly (p=0.023) compared to the static group (Figure 2(B)). This finding was supported by a decreased expression of chondrogenic genes such as ACAN, an encoding gene for the cartilage-specific proteoglycan (aggrecan), which was found to decrease by a fold change of 0.34 (p=0.019), and an increased expression of cartilage catabolic gene ADAMTS5 (not significant) in the mechanically loaded group when compared to the static group (Figure 2(C,D)). These findings suggest mechanical loading decreased the chondrogenic capacity of hsfMSC-derived engineered cartilage. The mechanism underlying the decrease in chondrogenic capacity by mechanical loading of hsfMSC-derived engineered cartilage is unclear but may be associated with increased expression of cartilage catabolic genes and related proteins. Further investigation into this possibility is thus required. These results show the importance of MSC source and how this affects the mechanical and biological characteristics of MSC-derived chondrocytes and highlights the significance of the protocols used for cartilage engineering. Also, these results show that Synovial fluid MSC’s may not be optimal or use in applications involving significant mechanical loading such as cartilage engineering. Lastly, this research brings forward an opportunity for a new research topic; HsfMSC-derived cartilage has potential to be used to model the role of inflammation and mechanical loading in osteoarthritis development.

Acknowledgements (Optional): I gratefully acknowledge the funding and support provided by The Canadian Institutes of Health Research (MOP 125921), The Natural Sciences and Engineering Research Council of Canada (RGPIN2018-06290; RTI-2019-00310), The University of Alberta Pilot Seed Grant Program (UOFAB PSGP), The Alberta Cancer Foundation-Mickleborough Interfacial Biosciences Research Program (ACF-MIBRP 27128), and in-kind biomaterial support of Integra Life Sciences Inc., NJ, USA.