Platelet Lysate Activates Human Subcutaneous Adipose Tissue Cells by Promoting Cell Proliferation and Their Paracrine Activity Toward Epidermal Keratinocytes

Abstract

Skin chronic wounds are non-healing ulcerative defects, which arise in association with a morbidity state, such as diabetes and vascular insufficiency or as the consequence of systemic factors including advanced age. Platelet Rich Plasma, a platelet-rich blood fraction, can significantly improve the healing of human skin chronic ulcers. Given that the subcutaneous adipose tissue is located beneath the skin and plays a role in the skin homeostasis, in this study, we investigated the in vitro response of human subcutaneous adipose tissue cells to platelet content in a model mimicking in vitro the in situ milieu of a deep skin injury. Considering that, at the wound site, plasma turn to serum, platelets are activated and inflammation occurs, human adipose-derived stromal cells (hASC) were cultured with Human Serum (HS) supplemented or not with Platelet Lysate (PL) and/or IL-1α. We observed that HS sustained hASC proliferation more efficiently than FBS and induced a spontaneous adipogenic differentiation in the cells. PL added to HS enhanced hASC proliferation, regardless the presence of IL-1α. In the presence of PL, hASC progressively lessened the adipogenic phenotype, possibly because the proliferation of less committed cells was induced. However, these cells resumed adipogenesis in permissive conditions. Accordingly, PL induced in quiescent cells activation of the proliferation-related pathways ERK, Akt, and STAT-3 and expression of Cyclin D1. Moreover, PL induced an early and transient increase of the pro-inflammatory response triggered by IL-1α, by inducing COX-2 expression and secretion of a large amount of PGE2, IL-6, and IL-8. Media conditioned by PL-stimulated hASC exerted a chemotactic activity on human keratinocytes and favored the healing of an in vitro scratch wound. In order to bridge the gap between in vitro results and possible in vivo events, the stimuli were also tested in ex vivo cultures of in toto human adipose tissue biopsies (hAT). PL induced cell proliferation in hAT and outgrowth of committed progenitor cells able to differentiate in permissive conditions. In conclusion, we report that the adipose tissue responds to the wound microenvironment by activating the proliferation of adipose tissue progenitor cells and promoting the release of factors favoring wound healing.

Progenitor Cells Activated by Platelet Lysate in Human Articular Cartilage as a Tool for Future Cartilage Engineering and Reparative Strategies

“…Due to the high content in mitogenic growth factors, platelet products exert a strong proliferative stimulus on several in vitro cell cultures, as previously reported [14,15,30]. In this study, the proliferative trend demonstrated by doubling number and cyclin D1 protein levels were markedly higher in ACs-PL compared to ACs-FBS and similar between ACs-PL and CPCs-PL.…”

“…PL treatment induced increased release or de novo secretion of some pro-inflammatory cytokines and chemokines by ACs. Indeed, it is well known from previous works that PL exerts an initial strong pro-inflammatory activity resulting in NF-κB activation and secretion of pro-inflammatory cytokines, events that are transient in order to prime certain defensive and reparative tissue mechanisms, then it inhibits and promotes resolution of the inflammatory phase [14,15].…”

“…Scratch closure was monitored from t 0 = 0 h to t 1 = 24 h and t 2 = 48 h with inverted microscope (Leica DMi1, Leica Microsystems, Wetzlar, Germany). Analysis of acquired images was performed with TScratch software (https://github.com/cselab/TScratch) as reported by Romaldini et al [15]. Experiments were performed in triplicate on three different primary cultures (N = 3).…”

“…In particular, the first three were upregulated in CM of ACs-PL in comparison with ACs-FBS. In fact, the pro-inflammatory effect of PL is widely documented in triggering cascades of pro-resolving events [14,15,27] that could lead to regeneration in injured sites, just like it happens physiologically during wound healing. IL-6 and NGAL released by CPCs-PL was lower than that released by ACs-PL, while the release of IL-8 was similar in both cell populations.…”

A Comparative Analysis of Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative Potential

“…Additional "acquired" factors occur after cell isolation and following processing. The composition of culture medium and supplements, substrate properties and oxygen concentration would most likely affect the features of MSCs through different mechanisms 16,17 . Therefore, the ideal way to compare the sources of MSCs in view of further clinical application should be based on the assessment of the specific properties of candidate cell types expanded in similar conditions, starting from the initial stages of cell processing.…”