IntroductionSilk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering and to improve tissue regeneration when cellularized with mesenchymal stromal cells (MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches cellularized with human adipose-derived MSCs (Ad-MSCs-SF), or decellularized (D-Ad-MSCs-SF), are effective in the treatment of skin wounds, improving skin regeneration in db/db diabetic mice.MethodsThe conformational and structural analyses of SF and D-Ad-MSCs-SF patches were performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5 mm punch biopsy tool on the mouse’s back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were transplanted and the efficacy of treatments was assessed by measuring the wound closure area, by histological examination and by gene expression profile. We further investigated the in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release of angiogenic factors.ResultsWe found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and D-Ad-MSCs-SF significantly improved tissue regeneration, reducing the wound area respectively by 40% and 35%, within three days, completing the process in around 10 days compared to 15–17 days of controls. RT2 gene profile analysis of the wounds treated with Ad-MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and matrix remodeling. Finally, Ad-MSCs-SF and D-Ad-MSCs-SF co-cultured with HUVECs, DFs and KCs, preferentially enhanced the HUVECs’ migration and the release of angiogenic factors stimulating microvessel outgrowth in the aortic ring assay.ConclusionsOur results highlight for the first time that D-Ad-MSCs-SF patches are almost as effective as Ad-MSCs-SF patches in the treatment of diabetic wounds, acting through a complex mechanism that involves stimulation of angiogenesis. Our data suggest a potential use of D-Ad-MSCs-SF patches in chronic diabetic ulcers in humans.

(2015) Mesenchymal stem cells: potential for therapy and treatment of chronic non-healing skin wounds, Organogenesis, 11:4, 183-206, DOI: 10.1080/15476278.2015 ABSTRACT. Wound healing is a complex physiological process including overlapping phases (hemostatic/inflammatory, proliferating and remodeling phases). Every alteration in this mechanism might lead to pathological conditions of different medical relevance. Treatments for chronic nonhealing wounds are expensive because reiterative treatments are needed. Regenerative medicine and in particular mesenchymal stem cells approach is emerging as new potential clinical application in wound healing. In the past decades, advance in the understanding of molecular mechanisms underlying wound healing process has led to extensive topical administration of growth factors as part of wound care. Currently, no definitive treatment is available and the research on optimal wound care depends upon the efficacy and cost-benefit of emerging therapies.Here we provide an overview on the novel approaches through stem cell therapy to improve cutaneous wound healing, with a focus on diabetic wounds and Systemic Sclerosis-associated ulcers, which are particularly challenging. Current and future treatment approaches are discussed with an emphasis on recent advances.

Ceramide represents an important sphingoid mediator involved in the signaling pathways that control cell proliferation, differentiation, and death. To determine whether ceramide levels correlate with the malignant progression of human astrocytomas, we investigated these levels in surgical specimens of glial tumors of low-grade and high-grade malignancy. Tumor samples obtained from 52 patients who underwent therapeutic removal of primary brain tumors were used. The tumors were classified according to standard morphologic criteria and were grouped into tumors of low-grade and high-grade malignancy. Sections of normal brain tissue adjacent to the tumor were also analyzed in 11 of the 52 patients. After extraction and partial purification, ceramide was measured by quantitative derivatization to ceramide-1-phosphate using diacylglycerol kinase and [gamma-(32)P]ATP. Ceramide levels were significantly lower in the combined high-grade tumors compared with low-grade tumors and in both tumor groups compared with peritumoral tissue. The results indicate an inverse correlation between the amount of ceramide and tumor malignancy as assessed by both the histological grading and ganglioside pattern. Moreover, overall survival analysis of 38 patients indicates that ceramide levels are significantly associated with patient survival. The present findings indicate that ceramide is inversely associated with malignant progression of human astrocytomas and poor prognosis. The downregulation of ceramide levels in human astrocytomas emerges as a novel alteration that may contribute to glial neoplastic transformation. The low ceramide levels in high-grade tumors may provide an advantage for their rapid growth and apoptotic resistant features. This study appears to support the rationale for the potential benefits of a ceramide-based chemotherapy.

The severity of coronavirus disease 2019 (COVID‐19) is a crucial problem in patient treatment and outcome. The aim of this study is to evaluate circulating level of sphingosine‐1‐phosphate (S1P) along with severity markers, in COVID‐19 patients. One hundred eleven COVID‐19 patients and forty‐seven healthy subjects were included. The severity of COVID‐19 was found significantly associated with anemia, lymphocytopenia, and significant increase of neutrophil‐to‐lymphocyte ratio, ferritin, fibrinogen, aminotransferases, lactate dehydrogenase (LDH), C‐reactive protein (CRP), and D‐dimer. Serum S1P level was inversely associated with COVID‐19 severity, being significantly correlated with CRP, LDH, ferritin, and D‐dimer. The decrease in S1P was strongly associated with the number of erythrocytes, the major source of plasma S1P, and both apolipoprotein M and albumin, the major transporters of blood S1P. Not last, S1P was found to be a relevant predictor of admission to an intensive care unit, and patient’s outcome. Circulating S1P emerged as negative biomarker of severity/mortality of COVID‐19 patients. Restoring abnormal S1P levels to a normal range may have the potential to be a therapeutic target in patients with COVID‐19.

Accumulating reports suggest that human glioblastoma contains glioma stem-like cells (GSCs) which act as key determinants driving tumor growth, angiogenesis, and contributing to therapeutic resistance. The proliferative signals involved in GSC proliferation and progression remain unclear. Using GSC lines derived from human glioblastoma specimens with different proliferative index and stemness marker expression, we assessed the hypothesis that sphingosine-1-phosphate (S1P) affects the proliferative and stemness properties of GSCs. The results of metabolic studies demonstrated that GSCs rapidly consume newly synthesized ceramide, and export S1P in the extracellular environment, both processes being enhanced in the cells exhibiting high proliferative index and stemness markers. Extracellular S1P levels reached nM concentrations in response to increased extracellular sphingosine. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to rapidly secrete newly synthesized S1P, suggesting that cooperation between S1P and these growth factors is of central importance in the maintenance and proliferation of GSCs. We also report for the first time that S1P is able to act as a proliferative and pro-stemness autocrine factor for GSCs, promoting both their cell cycle progression and stemness phenotypic profile. These results suggest for the first time that the GSC population is critically modulated by microenvironmental S1P, this bioactive lipid acting as an autocrine signal to maintain a pro-stemness environment and favoring GSC proliferation, survival and stem properties.

Glioblastoma multiforme (GBM) is the most common and fatal intracranial cancer in humans and exhibits intense and aberrant angiogenesis that sustains its malignancy and involves several angiogenic signals. Among them, vascular endothelial growth factor (VEGF) plays a key role and is overexpressed in GBM. Different cells appear to act as triggers of the aberrant angiogenesis, and, among them, platelets act as key participants. In order to provide further insights into the platelet features and angiogenic role in GBM, this study investigated the effects of platelet releasate on GBM-derived endothelial cells (GECs) and the levels of VEGF and endostatin, as pro- and anti-angiogenic components of platelet releasate from GBM patients. We demonstrate for the first time that: 1) platelet releasate exerts powerful pro-angiogenic effect on GECs, suggesting it might exert a role in the aberrant angiogenesis of GBM; 2) ADP and thrombin stimulation leads to significantly higher level of VEGF, but not of endostatin, in the releasate of platelets from GBM patients than those from healthy subjects; and 3) the intraplatelet concentrations of VEGF were significantly elevated in GBM patients as compared to controls. Moreover, we found a direct correlation between platelet-released VEGF and overall survival in our patient cohort. Although preliminary, these findings prompt further investigations to clarify the biologic relevance of platelet VEGF in GBM and prospective studies for screening GBM patients for anti-VEGF therapy and/or to optimize this treatment.

IntroductionBiglycan is an important proteoglycan of the extracellular matrix of intervertebral disc (IVD), and its decrease with aging has been correlated with IVD degeneration. Biglycan deficient (Bgn−/0) mice lack this protein and undergo spontaneous IVD degeneration with aging, thus representing a valuable in vivo model for preliminary studies on therapies for human progressive IVD degeneration. The purpose of the present study was to assess the possible beneficial effects of adipose-derived stromal cells (ADSCs) implants in the Bgn−/0 mouse model.MethodsTo evaluate ADSC implant efficacy, Bgn−/0 mice were intradiscally (L1-L2) injected with 8x104 ADSCs at 16 months old, when mice exhibit severe and complete IVD degeneration, evident on both 7Tesla Magnetic Resonance Imaging (7TMRI) and histology. Placebo and ADSCs treated Bgn−/0 mice were assessed by 7TMRI analysis up to 12 weeks post-transplantation. Mice were then sacrificed and implanted discs were analyzed by histology and immunohistochemistry for the presence of human cells and for the expression of biglycan and aggrecan in the IVD area.ResultsAfter in vivo treatment, 7TMRI revealed evident increase in signal intensity within the discs of mice that received ADSCs, while placebo treatment did not show any variation. Ultrastructural analyses demonstrated that human ADSC survival occurred in the injected discs up to 12 weeks after implant. These cells acquired a positive expression for biglycan, and this proteoglycan was specifically localized in human cells. Moreover, ADSC treatment resulted in a significant increase of aggrecan tissue levels.ConclusionOverall, this work demonstrates that ADSC implant into degenerated disc of Bgn−/0 mice ameliorates disc damage, promotes new expression of biglycan and increased levels of aggrecan. This suggests a potential benefit of ADSC implant in the treatment of chronic degenerative disc disease and prompts further studies in this field.

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