Effect of photobiomodulation on the behaviour of mesenchymal stem cells in three-dimensional cultures.

Photobiomodulation (PBM) has been proposed as a strategy to improve the regenerative capacity of human adipose-derived stem cells (hASCs). Yet, this effect has been proved in 2D culture conditions. To analyze the effect of different doses of laser irradiation (660 nm) with different levels of energy (1 J, 2 J and 6 J) on hASCs cultured at 2D and 3D conditions. We used gellan gum spongy-like hydrogels as a biomaterial to 3D culture hASCs. Different doses (1-7 daily irradiations) and energy levels (1-6 J) of PBM were applied, and the metabolic activity, viability, proliferation, and release of ROS and IL-8 was evaluated up to 7 days. In 3D, cell proliferation increased at high energy (6 J) and after a single dose of irradiation, while in 2D, metabolic activity and proliferation was enhanced only after 3 doses and independently of the energy. More than 1 dose was needed to promote ROS secretion both in 2D and 3D culture conditions. Interestingly, a decrease of IL-8 secretion was detected only in 3D after 3-7 daily irradiations. Overall, hASCs response to PBM was not only dependent on the energy level and the number of applied stimuli, but also on the in vitro culture conditions.

Stem Cell-Containing Hyaluronic Acid-Based Spongy Hydrogels for Integrated Diabetic Wound Healing.

The detailed pathophysiology of diabetic foot ulcers is yet to be established and improved treatments are still required. We propose a strategy that directs inflammation, neovascularization, and neoinnervation of diabetic wounds. Aiming to potentiate a relevant secretome for nerve regeneration, stem cells were precultured in hyaluronic acid-based spongy hydrogels under neurogenic/standard media before transplantation into diabetic mice full-thickness wounds. Acellular spongy hydrogels and empty wounds were used as controls. Re-epithelialization was attained 4 weeks after transplantation independently of the test groups, whereas a thicker and more differentiated epidermis was observed for the cellular spongy hydrogels. A switch from the inflammatory to the proliferative phase of wound healing was revealed for all the experimental groups 2 weeks after injury, but a significantly higher M2(CD163+)/M1(CD86+) subtype ratio was observed in the neurogenic preconditioned group that also failed to promote neoinnervation. A higher number of intraepidermal nerve fibers were observed for the unconditioned group probably due to a more controlled transition from the inflammatory to the proliferative phase. Overall, stem cell-containing spongy hydrogels represent a promising approach to enhance diabetic wound healing by positively impacting re-epithelialization and by modulating the inflammatory response to promote a successful neoinnervation.

Gellan gum-hyaluronic acid spongy-like hydrogels and cells from adipose tissue synergize promoting neoskin vascularization.

Currently available substitutes for skin wound healing often result in the formation of nonfunctional neotissue. Thus, urgent care is still needed to promote an effective and complete regeneration. To meet this need, we proposed the assembling of a construct that takes advantage of cell-adhesive gellan gum-hyaluronic acid (GG-HA) spongy-like hydrogels and a powerful cell-machinery obtained from adipose tissue, human adipose stem cells (hASCs), and microvascular endothelial cells (hAMECs). In addition to a cell-adhesive character, GG-HA spongy-like hydrogels overpass limitations of traditional hydrogels, such as reduced physical stability and limited manipulation, due to improved microstructural arrangement characterized by pore wall thickening and increased mean pore size. The proposed constructs combining cellular mediators of the healing process within the spongy-like hydrogels that intend to recapitulate skin matrix aim to promote neoskin vascularization. Stable and off-the-shelf dried GG-HA polymeric networks, rapidly rehydrated at the time of cell seeding then depicting features of both sponges and hydrogels, enabled the natural cell entrapment/encapsulation and attachment supported by cell-polymer interactions. Upon transplantation into mice full-thickness excisional wounds, GG-HA spongy-like hydrogels absorbed the early inflammatory cell infiltrate and led to the formation of a dense granulation tissue. Consequently, spongy-like hydrogel degradation was observed, and progressive wound closure, re-epithelialization, and matrix remodelling was improved in relation to the control condition. More importantly, GG-HA spongy-like hydrogels promoted a superior neovascularization, which was enhanced in the presence of human hAMECs, also found in the formed neovessels. These observations highlight the successful integration of a valuable matrix and prevascularization cues to target angiogenesis/neovascularization in skin full-thickness excisional wounds.