Role Of Long Pentraxin 3 (PTX3) In Wound Closure Induced By Bone Marrow-Derived Mesenchymal Stromal Cells

Although several studies have shown the capacity of mesenchymal stromal cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. Long Pentraxin 3 (PTX3) is a multifunctional protein produced by MSCs and other cell subsets upon activation with inflammatory cytokines. PTX3 is involved in innate immunity, inflammation and extracellular matrix deposition. In the present study we analyzed the potential role of PTX3 in wound repair process induced by MSCs.

PTX3 knockout MSCs (PTX3^-/-MSCs) were collected from bone marrow of PTX3^-/- mice. After 3-5 culture passages the expression of surface markers was analyzed by flow cytometry and their osteogenic and adipogenic differentiation capacity was detected by alizarin red O and oil red S staining, respectively. The ability of PTX3^-/-MSCs to abrogate T cell proliferation was evaluated by co-culturing MSCs and PBMCs previously activated with Phytohaemagglutinin. Finally, equal number of both PTX3^-/-MSCs and wild type (WT) MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3^-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histological analysis.

We demonstrated that PTX3^-/-MSCs, similarly to WT MSCs, displayed typical fibroblastoid morphology, they expressed common MSC markers and were able to differentiate into adipocytes and osteoblasts. In addition, they drastically decreased the mitogen-induced proliferation of lymphocyte. Importantly, in a mouse model of wound healing, PTX3^-/- MSCs showed a highly significant defect in wound closure compared to WT MSCs at each time point. Histological evaluation of skin samples treated with PTX3^-/- MSCs showed a reduction of the granulation tissue and a significant increase of neutrophils (GR-1⁺) in the wound bed. Moreover, wounds treated with PTX3^-/- MSCs were characterized by an excessive accumulation of fibrin at the 2^nd day after injury. Accordingly, PTX3^-/- MSCs showed a defective ability to degrade the fibrin matrix in vitro. Finally, PTX3^-/- MSCs failed to close the ulcers in PTX3^-/- mice.

In conclusion, we demonstrated that PTX3 deficiency does not alter the phenotype and the capacity of MSCs to differentiate into mesengetic lineages; however, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair.