Musculoskeletal tissue regeneration by human non-embryonic stem cells

The aim of this thesis was to investigate the regenerative potential of alternative sources of stem cells, derived from human dental pulp (hDPSCs) and amniotic fluid (hAFSCs) and, specifically, to evaluate their capability to be committed towards osteogenic and myogenic lineages, for the eventual applicability of these stem cells to translational strategies in regenerative medicine of bone and skeletal muscle tissues. The in vitro bone production by stem cells may represent a radical breakthrough in the treatment of pathologies and traumas characterized by critical bone mass defects, with no medical or surgical solution. Human DPSCs and AFSCs were seeded and pre-differentiated on different scaffolds to test their capability to subsequently reach the osteogenic differentiation in vivo, in order to recover critical size bone defects. Fibroin scaffold resulted to be the best scaffold promoting mature bone formation and defect correction when combined to both hDPSCs and hAFSCs. This study also described a culture condition that might allow human DPSCs to be used for human cell therapy in compliance with good manufacturing practices (GMPs): the use of human serum (HS) promoted the expansion and the osteogenic differentiation of hDPSCs in vitro and, furthermore, allowed pre-differentiated hDPSCs to regenerate critical size bone defects in vivo. This thesis also showed that hDPSCs and hAFSCs can be differentiated towards the myogenic lineage in vitro, either when co-cultured with murine myoblasts and when differentiated alone after DNA demethylation treatment. Interestingly, when injected into dystrophic muscles of SCID/mdx mice - animal model of Duchenne Muscular Dystrophy (DMD) - hDPSCs and hAFSCs pre-differentiated after demethylating treatment were able to regenerate the skeletal muscle tissue and, particularly, to restore dystrophin expression. These observations suggest that human DPSCs and AFSCs might be eventually applied to translational strategies, in order to enhance the repair of injured skeletal muscles in DMD patients

Immunomodulating Profile of Dental Mesenchymal Stromal Cells: A Comprehensive Overview

: Dental mesenchymal stromal cells (MSCs) are multipotent cells present in dental tissues, characterized by plastic adherence in culture and specific surface markers (CD105, CD73, CD90, STRO-1, CD106, and CD146), common to all other MSC subtypes. Dental pulp, periodontal ligament, apical papilla, human exfoliated deciduous teeth, alveolar bone, dental follicle, tooth germ, and gingiva are all different sources for isolation and expansion of MSCs. Dental MSCs have regenerative and immunomodulatory properties; they are scarcely immunogenic but actively modulate T cell reactivity. in vitro studies and animal models of autoimmune diseases have provided evidence for the suppressive effects of dental MSCs on peripheral blood mononuclear cell proliferation, clearance of apoptotic cells, and promotion of a shift in the Treg/Th17 cell ratio. Appropriately stimulated MSCs produce anti-inflammatory mediators, such as transforming growth factor-\u3b2 (TGF-\u3b2), prostaglandin E2, and interleukin (IL)-10. A particular mechanism through which MSCs exert their immunomodulatory action is via the production of extracellular vesicles containing such anti-inflammatory mediators. Recent studies demonstrated MSC-mediated inhibitory effects both on monocytes and activated macrophages, promoting their polarization to an anti-inflammatory M2-phenotype. A growing number of trials focusing on MSCs to treat autoimmune and inflammatory conditions are ongoing, but very few use dental tissue as a cellular source. Recent results suggest that dental MSCs are a promising therapeutic tool for immune-mediated disorders. However, the exact mechanisms responsible for dental MSC-mediated immunosuppression remain to be clarified, and impairment of dental MSCs immunosuppressive function in inflammatory conditions and aging must be assessed before considering autologous MSCs or their secreted vesicles for therapeutic purposes

Effectiveness of Catch-Up Vaccination Interventions Versus Standard or Usual Care Procedures in Increasing Adherence to Recommended Vaccinations Among Different Age Groups: Systematic Review and Meta-Analysis of Randomized Controlled Trials and Before-After Studies

Background: To address the global challenge of vaccine hesitancy, the Strategic Advisory Group of Experts on Immunization strongly promotes vaccination reminder and recall interventions. Coupled with the new opportunities presented by scientific advancements, these measures are crucial for successfully immunizing target population groups. Objective: This systematic review and meta-analysis aims to assess the effectiveness of various interventions in increasing vaccination coverage compared with standard or usual care. The review will cover all vaccinations recommended for different age groups. Methods: In February 2022, 2 databases were consulted, retrieving 1850 studies. Following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, 79 manuscripts were included after the assessment phase. These comprised 46 trials/randomized controlled trials (RCTs) and 33 before-after studies. A meta-analysis using a random-effects model was performed with STATA software (version 14.1.2). The selected outcome was the risk ratio (RR) of vaccination coverage improvement effectiveness. Additionally, meta-regression analyses were conducted for the included manuscripts. Results: The analyses showed an overall efficacy of RR 1.22 (95% CI 1.19-1.26) for RCTs and RR 1.70 (95% CI 1.54-1.87) for before-after studies when considering all interventions cumulatively. Subgroup analyses identified multicomponent interventions (RR 1.58, 95% CI 1.36-1.85) and recall clinical interventions (RR 1.24, 95% CI 1.17-1.32) as the most effective in increasing vaccination coverage for RCTs. By contrast, educational interventions (RR 2.13, 95% CI 1.60-2.83) and multicomponent interventions (RR 1.61, 95% CI 1.43-1.82) achieved the highest increases for before-after studies. Meta-regression analyses indicated that the middle-aged adult population was associated with a higher increase in vaccination coverage (RCT: coefficient 0.54, 95% CI 0.12-0.95; before-after: coefficient 1.27, 95% CI 0.70-1.84). Conclusions: Community, family, and health care-based multidimensional interventions, as well as education-based catch-up strategies, effectively improve vaccination coverage. Therefore, their systematic implementation is highly relevant for targeting undervaccinated population groups. This approach aligns with national vaccination schedules and aims to eliminate or eradicate vaccine-preventable diseases

Quality of Grapes Grown Inside Paper Bags in Mediterranean Area

The aim of this study was to evaluate the influence of paper bagging of grape bunches on the morphological, mechanical, and chemical characteristics of berries of three table grapes varieties as an environmentally‐friendly technique for protecting clusters from biotic and abiotic agents. Clusters of Italia, Autumn Royal, and Regal Seedless grape cultivars were bagged and compared to a not‐bagged control. Air temperature inside and outside the bags was monitored. Bunch weight and length, number of berries per bunch, berry longitudinal and transversal diameter, berry mass, number of seeds per berry (normal in size and aborted), soluble solid content, titratable acidity, and skin color by CIEL*a*b* parameters were determined on four points of each berry. Berries were evaluated using texture analysis, and the main texture profile analysis parameters were compared. The air temperatures around not‐bagged clusters were slightly higher than inside the bags. In all the cultivars under evaluation, bagged bunches were heavier compared with not‐bagged ones. In Autumn Royal and Regal Seedless, these differences were mainly owing to the higher number of berries and higher berry weight of the bagged bunches. Regarding mechanical properties, in seedless varieties, the bagging treatment increased berry hardness (related to the berry firmness) and decreased berry cohesiveness and resilience, whereas an opposite behavior was found in cv. Italia. Berry skin break force was lower in the bagging treatment in all the analyzed varieties, indicating a softer and easier‐to‐chew berry skin. The findings demonstrate that the bagging technique affected the three variety parameters to different extents. The main differences were found in the seedless varieties in terms of berry size and bunch characteristics. For all varieties, bagged bunches achieved the quality level required by the market, confirming the suitability of this technique. However, the bag industry is proposing many different bag types (differing in material, shape, color, and closing system); therefore, further studies are needed to obtain more complete and exhaustive technical information

Neural crest derived niche of human dental pulp stem cells promotes peripheral nerve regeneration and remyelination in animal model of critical sized sciatic nerve injury

ABSTRACT Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The use of stem cells, easily accessible, capable of rapid expansion in culture as well as fully integrate into the host tissue and capable to differentiate in myelinating cells of the peripheral nervous system, represent an attractive therapeutic approach for the treatment of nerve injuries. Farther, stem cells sources sharing the same embryological origin of Schwann cells, might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal sub-population of STRO-1+/c-Kit+/CD34+ hDPSCs (1, 2), most of which being positive for neural crest (P75NTR) and neural progenitor cells (nestin) markers, to differentiate into Schwann cells-like cells in vitro and to promote axonal regeneration in vivo. As a matter of fact, following culture in appropriate induction medium, STRO-1+/c-Kit+/CD34+ hDPSCs were able to commit towards Schwann cells express- ing P75NTR, GFAP and S100b. After transplantation in animal model of sciatic nerve defect, hDPSCs promoted axonal regeneration from proximal to distal stumps, providing guidance to newly formed myelinated nerve fibers, which led to functional recovery as measured by sustained gait improvement. Particularly, transplanted hDP- SCs engrafted into critical sized sciatic nerve defect, as revealed by the positive stain- ing against human nuclei, showed the expression of typical Schwann cells markers, S100b and GFAP. In conclusion this study demonstrates that STRO-1+/c-Kit+/CD34+ hDPSCs, associated to neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues

Osteogenic Differentiation of hDPSCs on Biogenic Bone Apatite Thin Films

A previous study reported the structural characterization of biogenic apatite (BAp) thin films realized by a pulsed electron deposition system by ablation of deproteinized bovine bone. Thin films annealed at 400 degrees C exhibited composition and crystallinity degree very close to those of biogenic apatite; this affinity is crucial for obtaining faster osseointegration compared to conventional, thick hydroxyapatite (HA) coatings, for both orthopedics and dentistry. Here, we investigated the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (hDPCS) on as-deposited and heat-treated BAp and stoichiometric HA. First, we showed that heat-treated BAp films can significantly promote hDPSC adhesion and proliferation. Moreover, hDPSCs, while initially maintaining the typical fibroblast-like morphology and stemness surface markers, later started expressing osteogenic markers such as Runx-2 and OSX. Noteworthy, when cultured in an osteogenic medium on annealed BAp films, hDPSCs were also able to reach a more mature and terminal commitment, with respect to HA and as-deposited films. Our findings suggest that annealed BAp films not only preserve the typical biological properties of stemness of, hDPSCs but also improve their ability of osteogenic commitment

Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations

Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency

Use of a 3D floating sphere culture system to maintain the neural crest-related properties of human dental pulp stem cells

Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties

Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization

INTRODUCTION: The main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells and amniotic fluid stem cells, combined with collagen scaffold to reconstruct critical-size cranial bone defects in an animal model. METHODS: We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks, cranial tissue samples were taken for histological and immunofluorescence analysis. RESULTS: We observed a new bone formation in all of the samples but the most relevant differences in defect correction were shown by stem cell–collagen samples 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold. CONCLUSION: These data confirmed the strong potential of bioengineered constructs of stem cell–collagen scaffold for correcting large cranial defects in an animal model and highlighting the role of stem cells in neovascularization during skeletal defect reconstruction