(L-462) Enhancing stem cell treatment via biosponge encapsulation and hypoxic preconditioning

Introduction:: Volumetric muscle loss (VML) causes irreversible mass and function deficits and often results in permanent disability. Mesenchymal stromal cells (MSC) and their secretome can improve muscle regeneration. Placenta-derived stem cells (PDSCs), which share cell surface markers with MSCs, have better proliferation and differentiation capabilities due to their fetal origin. [1]. However, MSCs generally have a brief lifespan and do not persist in injured tissues beyond the first week following transplantation. We previously developed a biosponge composed of extracellular matrix proteins that enhanced muscle regeneration and function after VML [2]. We hypothesized that delivery of biosponge encapsulated human PDSCs (hPDSCs) to the VML defect will result in improved cell survival and engraftment [3-5]. Biosponges will offer adhesion sites, biomechanical support, and shielding from inflammatory cells while allowing nutrient diffusion [6]. Additionally, we implemented hypoxic preconditioning of PDSCs. This technique involves exposing cells to a controlled low-oxygen environment which is expected to enhance paracrine signaling, survival rate, and angiogenesis [7]. The ultimate goal of the study was to optimize PDSC culture through hypoxic conditioning and biosponge encapsulation in vitro, with the aim of promoting cell survival and engraftment post-transplantation in injured muscle tissue.

Materials and Methods:: Biosponges were prepared by combining gelatin, collagen, laminin-111, and chemical crosslinkers (EDC/NHS) in a 6 well plate as described previously [8, 9]. The biosponges were disinfected, cut into 10 mm disks, and seeded with hPDSCs (5 x 10⁵ cells) in a 48 well plate (n=6/group) and placed under standard culture conditions at 37ºC at 5% CO₂. The cell culture supernatants were collected on days 1, 3, and 5 and kept frozen at -80°C until needed for ELISA analysis (n=5/group). At each time-point, the DNA content of the biosponge encapsulated cells was determined using the CyQUANT cell proliferation assay. Additionally, a sponge disk was also cryopreserved for histological analysis. In a separate set of experiments, hPDSC seeded biosponges (5 x 10⁵ cells/sponge) were incubated under hypoxic (3% O₂) and normoxic (21% O₂) culture conditions in a 48 well-plate (n=6/group). Cell culture supernatants were collected on days 1, 3, and 6 of culture, and a CyQUANT assay was performed. hPDSCs seeded biosponges were implanted in a rat VML defect which was created by removing ~20% of the tibialis anterior muscle’s mass. The biodistribution of hPDSCs was assessed histologically on day 7 post-injury.

Results, Conclusions, and Discussions:: Results: Biosponge encapsulated PDSCs to a depth of ~300 µm (Fig 1A) and showed maintained viability for 5 days in culture. PDSCs released increasing amounts of myogenic factors over time, such as follistatin, hepatocyte growth factor (HGF), interleukin-6 (IL-6), and amphiregulin.

Next, biosponge encapsulated PDSCs were subjected to hypoxia and cellular viability was confirmed over 6 days of culture (Fig 1B). PDSCs released higher levels of angiogenic growth factors: hypoxia-inducing factor-1α (HIF-1α), fibroblast growth factor 2 (FGF2), and vascular endothelial growth factor (VEGF) (Fig 1C) compared to normoxic culture by day 3. Unexpectedly, myogenic factors: HGF (Figure 1D), and follistatin showed decreased production by day 6 under hypoxic conditions. Cellular spreading and actin stress fiber formation was only observed under normoxic but not hypoxic conditions. Biosponge encapsulated hPDSCs could be identified in the VML defect on day 7 post-injury (Fig 2).



Conclusion: Our findings indicate that biosponge encapsulation and hypoxic preconditioning can improve the therapeutic potential of PDSCs. We showed that PDSCs remained viable and functional when encapsulated in biosponges. Short-term hypoxia (i.e., for 3 days) increased the release of angiogenic factors, but long-term exposure (i.e., 6 days) decreased the release of myogenic factors.



Discussion: PDSCs remained functional and viable within the biosponges and released increasing levels of myogenic factors. For instance, follistatin acts as a potent myostatin antagonist [10, 11]. HGF influences both satellite cell proliferation and differentiation [12, 13]. IL-6 enhances cellular recruitment and myogenic cell proliferation[14]. Amphiregulin supports myogenesis while suppressing fibrosis [15].

Under hypoxia, biosponge encapsulated PDSCs released higher concentrations of angiogenic factors such as HIF-1α, FGF2, and VEGF up to day 3 of culture. VEGF and FGF2 can also stimulate muscle satellite cell activity and neurogenesis [16]. By day 6 of hypoxic culture, the concentration of myogenic factors such as HGF and follistatin decreased, while angiogenic factors remained similar to that of normoxia culture. These findings indicate that brief hypoxia (i.e., 3 days) can enhance the production of trophic factors, while prolonged exposure (i.e., 6 days) may have adverse consequences. Ongoing work is assessing if the biosponge encapsulated hPDSC promote functional muscle recovery post-VML.


Acknowledgements (Optional): : This work was supported by a grant through the Center for Defense Medicine at Biogenerator-STL.

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