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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I2 - Chondrogenesis of Mesenchymal Stem Cells Aggregates is Influenced by the Dynamism of Chondrogenic Factors

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Mesenchymal stem cells (MSC) have the capacity to differentiate to a variety of musculoskeletal cells including chondrocytes that form cartilage [1]. As such, MSC are of significant interest in biomedical research to develop novel approaches to regenerate and repair dysfunctional musculoskeletal tissues including cartilage which has little to no regenerative or reparative capacity post-injury. Early in vitro protocols of MSC differentiation to chondrocytes (i.e., chondrogenesis) were implemented under static conditions where forced or self-assembled MSC aggregates reminiscent of skeletal condensation in vivo are cultured in chondrogenic factors to form three-dimensional (3D) cartilage constructs1-4. However, over the past decade, it has become increasingly clear that mechanical signals imposed upon MSC by the extracellular microenvironment, play a central role in determining MSC fate, including chondrogenic differentiation5-7. The majority of the mechanical signal-related studies either involved cyclic deformation of MSC embedded in exogenous 3D scaffolds or direct agitation of self-assembled MSC aggregates8. In contrast, there has been no studies evaluating the effect of dynamic exchange of soluble chondrogenic factors on the chondrogenesis of static MSC aggregates. In this study, we hypothesized that the dynamic exchange of soluble chondrogenic factors during the in vitro chondrogenesis of human bone marrow MSC (hBMSC) aggregates will reveal distinct transcriptomic and tissue phenotypes relative to the same aggregates under static chondrogenic factors.

Materials and

Methods:  Human bone marrow aspirates were ethically sourced from the iliac crest of three male donors (age 20-40). The mononucleated cells (MNC) were harvested via fractionation using Histopaque (Sigma). MNC were plated at 5000 cells/cm2 in T175 flasks containing 𝛼-MEM,10% v/v heat-inactivated FBS,100 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin 0.29 mg/ml glutamine and basic fibroblast growth factor (FGF-2; 5 ng/ml) incubated at 37℃ under 21% O2, 5% CO2 and 95% humidity to allow the development of the plastic adherent hBMSC population. The hBMSC were used for experiments after two cycles of expansion and detachment by 0.05% w/v trypsin-EDTA. The hBMSC were assigned to three groups: pellet, Transwell and dynamic bioreactor. For chondrogenesis in the pellet, hBMSC were seeded at 5x105 in 1.5mL sterile tubes containing 600µl chondrogenic media and centrifuged at 200g for 5 minutes. For chondrogenesis in TransWell, 5x105 hBMSC were seeded unto 0.4µm pore size polyester (PET)membrane Transwell inserts in a 24-well plate with 100µl and 500 µl chondrogenic medium in the insert and well, respectively, which submerged the membrane and cells. For chondrogenesis in the dynamic bioreactor (BEST Inc.), 5x105 hBMSC were seeded unto the PET inserts then placed in the bioreactor with the equivalent volume of chondrogenic media in the insert and lower compartment as in the Transwell group but with micro-magnetic stir bar to mix the media at 100 rpm. The chondrogenic media contained TGFβ3 and ITS+1, and hBMSC aggregates were cultured for 3 weeks.

Results, Conclusions, and Discussions: The transcriptomic heatmap via RNA sequencing (Fig. 1A) revealed differences between the cartilage constructs via the pellet, Transwell and Bioreactor. The Venn diagram for the number of differentially expressed genes showed distinct numbers between the pellet, Transwell and Bioreactor generated constructs (Fig.1B). The gross appearance of the generated cartilage were unique to format of hBMSC culture (Fig. 1C). Safranin O staining to assess cartilage matrix formation in the constructs was distinct between pellet, Transwell and Bioreactor (Fig. 1D). Collagen I, II and X immunofluorescence detection to assess chondrogenesis of hBMSC was dependent on the method of hBMSC culture (Fig.1E). The micromechanical compression moduli of the cartilage constructs was also dependent on the modality of chondrogenic differentiation of the hBMSC aggregates (Fig. 1F and 1G).

The dynamic exchange of soluble chondrogenic factors during in vitro chondrogenesis of hBMSC aggregates revealed distinct transcriptomic and tissue phenotypes relative to hBMSC aggregates cultured under static soluble chondrogenic factors.

Acknowledgements (Optional):  [1].Pittenger et al. Science 284, 143-147 (1999); 2.Johnstone et al Exp Cell Res 238, 265-272 (1998); 3.Mackay et al. Tissue Eng 4, 415-428 (1998); 4.Murdoch et al Stem Cells 25, 2786-2796 (2007); 5.Kelly & Jacobs Birth Defects Research Part C: Embryo Today: Reviews 90, 75-85 (2010); 6. Mellor et al Aerosp Med Hum Perform 88, 377-384 (2017) ;7. Choi et al. Artif Organs 37, 648-655 (2013); 8. Sart et al Tissue Eng Part B Rev 20, 365-380 (2014).