Poster V6 - Exploring the therapeutic potential of primed hMSC extracellular vesicles for biomaterials applications in interfacial tendon-to-bone rotator cuff repair

Introduction: Rotator cuff tears represent a growing area of clinical need and concern, with reported surgery volume increases of over 200% and rates of re-failure as high as even 94% in large tears within the last two decades. A significant reason for surgical failure stems from an inability to regenerate the enthesis, a fibrocartilaginous insertion that joins stiff, mineralized bone to softer, highly aligned tendon, providing crucial mechanical support and dispersion of loading forces. Following injury, this region is replaced with fibrovascular scar tissue that is mechanically inferior and prone to painful inflammation, significantly reducing functional repair and patient quality of life.
Biomaterials-based approaches show exciting potential for functional enthesis regeneration, but precise and targeted signaling is crucial to this effort. A biomaterial able to promote or mimic a paracrine environment comparable to the developing enthesis, which incorporates signals from neighboring tendon and bone tissue, could thus be highly effective for regeneration. We have previously developed osseous and tendinous collagen-based scaffolds capable of guiding hMSC behavior toward these respective lineages and shown that conditioned media from hMSCs seeded on these scaffolds can be influential in enthesis tissue engineering. While growth factors play a significant role in paracrine signaling, hMSCs are also known to produce extracellular vesicles (EVs) that also guide cell behavior, proliferation, and immune responses. Using previously described EV isolation and self-formed hydrogel approaches, we hypothesize that EVs isolated from hMSCs primed on tendinous or osseous scaffolds could provide relevant and beneficial effects for enthesis tissue engineering and interfacial biomaterials.

Materials and

Methods: Collagen scaffolds are created by lyophilization of a homogenized precursor slurry containing collagen, glycosaminoglycans, and salts in an acidic buffer solution. Osseous scaffolds are formed with a mineralized buffer and lyophilized in aluminum wells that promote an isotropic pore structure. Tendinous scaffolds are formed with an acetic acid buffer, and they are lyophilized in Teflon/copper wells designed to promote unidirectional heat transfer during the freeze-drying process, resulting in an anisotropic pore structure beneficial for tendon tissue engineering. Prior to in vitro testing, scaffolds are sterilized with ethylene oxide, rehydrated using ethanol and phosphate buffered saline, and crosslinked via EDAC/NHS carbodiimide chemistry. Scaffolds are seeded with 150,000 hMSCs (P4-6, RoosterBio) and incubated under normal culture conditions (37°C, 5% CO2) in either basal hMSC media (low glucose DMEM with 10% FBS, 1% antibiotic-antimycotic), RoosterCollectTM-EV, or RoosterNourishTM-MSC. All media is supplemented with 5µM of an azido-sugar, Ac4ManAz, for EV metabolic labeling. Cell culture medium containing the secreted EVs is collected and concentrated via ultracentrifugation with an Amicon centrifugal filter (100 kDa). EVs are washed with PBS three times and resuspended in PBS. The size distribution and concentration of EVs is determined via a Nanoparticle Tracking Analysis instrument (NanoSight NS300, Malvern Panalytical, MA, USA).
Therapeutic effects will be evaluated based on hMSC behavioral changes, either in EV self-formed hydrogels or thiolated gelatin hydrogels supplemented with EVs. hMSCs will be evaluated based on proliferation (DNA isolation, alamarBlue assay), gene expression (RT-qPCR, nCounter NanoString panel), and functional protein expression (immunofluorescence).

Results, Conclusions, and Discussions: We have previously shown that conditioned media from hMSCs cultured on tendinous or osseous scaffolds results in significant changes to individual enthesis-relevant genes (COL10A1, KLF2, TGFB1, COL1A1) within hMSCs seeded on a thiolated gelatin hydrogel. Conditioned media from each individual scaffold type produced independent gene expression changes, highlighting the relevance of both tendon and bone signaling. In addition, conditioned media from the two sources combined resulted in non-significant but noticeable synergistic or antagonistic effects in some genes, further highlighting the relevance of these environments combined.
Using the methods described above, we have successfully isolated extracellular vesicles from hMSCs cultured in 2D flasks. These EVs will serve as a valuable baseline in comparing EVs from differently primed hMSCs (derived from osseous or tendinous scaffolds, possibly under different environmental conditions or stresses), collected via similar methods. We will assess hMSC behavior in response to biomaterials loaded with these differently primed EVs (self-formed EV hydrogels or supplemented thiolated gelatin hydrogels, depending on material capacity). This work will boost not only broad understanding of EV therapeutic capability in enthesis healing and interfacial tissue development but also the effects of differently primed EVs for greater regionally-targeted signaling. In the future, we hope to also explore the possibility and effects of matrix bound EVs generated within collagen scaffolds, as these might differ in therapeutic potential compared with secreted EVs, as well as exploring methods for EV-based spatiotemporal material guidance.

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