Antimicrobial and Regenerative Effects of Placental Multipotent Mesenchymal Stromal Cell Secretome-Based Chitosan Gel on Infected Burns in Rats

Background: There is a need for better strategies to promote burn wound healing and prevent infection. The aim of our study was to develop an easy-to-use placental multipotent mesenchymal stromal cell (MMSC) secretome-based chitosan hydrogel (MSC-Ch-gel) and estimate its antimicrobial and regenerative activity in Staphylococcus aureus-infected burn wounds in rats. Methods: Proteomic studies of the MMSC secretome revealed proteins involved in regeneration, angiogenesis, and defence responses. The MMSC secretome was collected from cultured cells and mixed with water-soluble chitosan to prepare the placental MSC-Ch-gel, which was stored in liquid phase at 4 °C. The wounds of rats with established II-IIIa-degree burns were then infected with S. aureus and externally covered with the MSC-Ch-gel. Three additional rat groups were treated with medical Vaseline oil, the antiseptic drug Miramistin®, or the drug Bepanthen® Plus. Skin wound samples were collected 4 and 8 days after burning for further microbiological and histological analysis. Blood samples were also collected for biochemical analysis. Results: Application of the MSC-Ch-gel cleared the wound of microorganisms (S. aureus wasn’t detected in the washings from the burned areas), decreased inflammation, enhanced re-epithelialisation, and promoted the formation of well-vascularised granulation tissue. Conclusions: MSC-Ch-gel effectively promotes infected wound healing in rats with third-degree burns. Gel preparation can be easily implemented into clinical practice.

Macroscopic, Histologic, and Immunomodulatory Response of Limb Wounds Following Intravenous Allogeneic Cord Blood-Derived Multipotent Mesenchymal Stromal Cell Therapy in Horses

Limb wounds are common in horses and often develop complications. Intravenous multipotent mesenchymal stromal cell (MSC) therapy is promising but has risks associated with intravenous administration and unknown potential to improve cutaneous wound healing. The objectives were to determine the clinical safety of administering large numbers of allogeneic cord blood-derived MSCs intravenously, and if therapy causes clinically adverse reactions, accelerates wound closure, improves histologic healing, and alters mRNA expression of common wound cytokines. Wounds were created on the metacarpus of 12 horses. Treatment horses were administered 1.51–2.46 × 108 cells suspended in 50% HypoThermosol FRS, and control horses were administered 50% HypoThermosol FRS alone. Epithelialization, contraction, and wound closure rates were determined using planimetric analysis. Wounds were biopsied and evaluated for histologic healing characteristics and cytokine mRNA expression. Days until wound closure was also determined. The results indicate that 3/6 of treatment horses and 1/6 of control horses experienced minor transient reactions. Treatment did not accelerate wound closure or improve histologic healing. Treatment decreased wound size and decreased all measured cytokines except transforming growth factor-β3. MSC intravenous therapy has the potential to decrease limb wound size; however, further work is needed to understand the clinical relevance of adverse reactions.

Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor β1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

Placental multipotent mesenchymal stromal cell-derived Slit2 may regulate macrophage motility during placental infection

Slit proteins have been reported to act as axonal repellents in Drosophila; however, their role in the placental microenvironment has not been explored. In this study, we found that human placental multipotent mesenchymal stromal cells (hPMSCs) constitutively express Slit2. Therefore, we hypothesized that Slit2 expressed by hPMSCs could be involved in macrophage migration during placental inflammation through membrane cognate Roundabout (Robo) receptor signaling. In order to develop a preclinical in vitro mouse model of hPMSCs in treatment of perinatal infection, RAW 264.7 cells were used in this study. Slit2 interacted with Robo4 that was highly expressed in RAW 264.7 macrophages: their interaction increased the adhesive ability of RAW 264.7 cells and inhibited migration. Lipopolysaccharide (LPS)-induced CD11bCD18 expression could be inhibited by Slit2 and by hPMSC-conditioned medium (CM). LPS-induced activation of p38 and Rap1 was also attenuated by Slit2 and by hPMSC-CM. Noticeably, these inhibitory effects of hPMSC-CM decreased after depletion of Slit2 from the CM. Furthermore, we found that p38 siRNA inhibited LPS-induced Rap1 expression in RAW 264.7 cells, indicating that Rap1 functions downstream of p38 signaling. p38 siRNA increased cell adhesion and inhibited migration through reducing LPS-stimulated CD11bCD18 expression in RAW 264.7 cells. Thus, hPMSC-derived Slit2 may inhibit LPS-induced CD11bCD18 expression to decrease cell migration and increase adhesion through modulating the activity and motility of inflammatory macrophages in placenta. This may represent a novel mechanism for LPS-induced placental infection.