G13 - Synthetic-platelet fibrin scaffolds enhance exosome production of mesenchymal stem cells

Introduction: : Mesenchymal stem/stromal cells (MSCs) have been used in various clinical applications for healing. However, there is a need for improving therapeutic potential because MSCs do not persist in some parts of the body, such as the joints, post-injection. One issue is that low-stiffness biomaterials are needed for delivery, but do not sufficiently stimulate MSCs. To address this limitation, we developed an injectable biomaterial with synthetic platelet-like-particles (PLP) loaded fibrin gels embedded with MSCs that will polymerize post-injection to allow long term activity of MSCs. The properties of PLPs with fibrinogen and thrombin clotting factors have already been shown to enhance scaffold stability and local stiffness.
Exosome release from MSCs is indicative of bioactivity and has been associated with healing and repair. Additionally, exosomes have garnered significant attention for their ability to be used independently as an acellular therapy. Notably, exosomes derived from MSCs can accelerate regeneration, even in tissues with limited healing capacity. Fibrin-based scaffolds with fibrin-binding PLPs can induce a stable scaffold through fibrin retraction that provides mechanical stimulation to cells to induce enhanced production of exosomes and other healing factors, which could be injected as an acellular regenerative therapy in human and veterinary medicine. Thus, the objective of this study is to assess the potential of fibrin-PLP scaffolds to modulate the bioactivity of MSCs from different species.

Materials and

Methods: : Bone-marrow derived MSCs from horse, human, and porcine sources were seeded onto fibrin clots or tissue-culture treated plastic as a control. Fibrin clots were made with or without PLPs. For equine samples, to compare 2D and 3D environments, cells were either seeded on top of the fibrin clot or embedded within the fibrin clot mix, respectively. All groups were covered with DMEM culture media with 16.5% fetal bovine serum. After cells reached ~70% confluency, media was replaced with serum-free media to induce exosome release. After 3 days, exosomes were isolated from the resulting media. Exosomes were isolated through ultra-filtration. A Nanosight particle analyzer system was used to quantify the concentration and size of the isolated exosome samples. Western blot for CD81, CD9, and CD63 exosome surface markers was also performed for equine samples. MSC activity was assessed by the CellTiterGlo metabolic activity assay.
To further assess exosome potency, a scratch migration assay was performed where equine tenocyte cells were seeded onto 48 well-plates and kept in serum-free media after attachment. Cells were treated with 2x108 exosomes per well. Cells were stained with NucBlue live nuclear stain prior to imaging. Scratches were imaged at the same positions prior to treatment, on day 1, and day 6.

Results, Conclusions, and Discussions::

Results:
PLP-enhanced fibrin gels were able to maintain viable MSCs in serum-free conditions. Exosome sizes were in the typical range of 50-200 nm. For preliminary analysis, equine exosomes were confirmed by western blot for CD81, CD9, and CD63 surface markers and by TEM for size and morphology validation. For all species tested, PLP-laden scaffolds increased exosome concentration of MSCs significantly compared to MSCs on fibrin scaffolds alone and tissue culture plastic (Fig 1A). Additionally, the number of exosomes released from equine MSCs embedded in 3D within fibrin or PLP-scaffolds were not significantly different from scaffolds with MSCs grown in 2D (Fig. 1B). Tenocyte cells treated with PLP-scaffold derived exosomes showed indication of greater metabolism and migration than those treated with exosomes from fibrin-only and control groups (Fig. 1C-1D).

Discussion:
PLP-fibrin scaffolds promote higher and more bioactive exosome release. Maintaining activity in scaffolds over longer periods expands the application of MSCs to various therapies including injectables for areas with limited healing capacity. Higher exosome release from these PLP scaffolds occurred in both 2D (on top) and 3D (embedded), which can allow ease for exosome production in-vitro (2D) and injection of MSCs in-vivo (3D). The similar results between species highlight the robustness and versatility of the therapeutic effect for applications in human and veterinary medicine. Lastly, the modulation of exosome bioactivity through cell substrate stiffness can aid in development of more robust acellular therapeutics. Further studies will explore the mechanism for the differences in the activity of these exosomes observed and assess healing potential in-vivo.

Acknowledgements and/or References (Optional)::