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

Bone regeneration process driven to human periodontal ligament stem cells cultured onto corticocancellous scaffold

The aim of our research was to develop tissue-engineerized constructs composed by porcine cortico-cancellous scaffold (Osteobiol Dual Block) (DB) and xenofree ex vivo culture of human Periodontal Ligament Stem Cells (hPDLSCs) induced to osteogenic differentiation. hPDLSCs placed in xeno-free media formulation mantained the stem cells features, the expression of stemness and pluripotency markers, and the capacity to differentiate in different mesenchymal cell lines (1). Micrographs performed by transmission electron microscopy suggested that after one week of culture, both uninduced and osteogenic induced cells joined and grew on DB secreting extracellular matrix, hierarchically assembled in fibrils in osteogenic differentiation induced samples (2). Quantitative RT-PCR (qRT-PCR) of 92 osteogenesis-related assays of hPDLSCs seeded on the DB showed the upregulation of key genes involved in the osteogenic differentiation pathway such as RUNX2, collagens and SMAD. hPDLSCs induced to osteogenic differentiation in presence of DB expressed osteogenic- related transcripts such as BMP1-4-6, RUNX-2, collagens, MSX1-2, TGFβ3 and SMAD. Functional study revealed a significant increased response of calcium transients, in presence of the 3D-DB both in undifferentiated and differentiated cells stimulated with calcitonin and parathormone, suggesting that the biomaterial could drive the osteogenic differentiation process of hPDLSCs. These data were confirmed from the increase of gene expression of L-type voltage-dependent Ca2+ (VDCCL), subunits α1C and α2D1 in undifferentiated cells in presence of DB. Our results propose to consider DB a biocompatible, osteoinductive and osteoconductive biomaterial making it promising tools to regulate cell activities in biological environments and for a potential use for the development of new custom made tissue-engineering

Functional toll-like receptor 4, interleukin-6, -8 and CCL -20 release, and NF-kB translocation in human periodontal ligament mesenchymal stem cells stimulated with LPS- P. Gingivalis

Periodontal diseases, the major public health problem of the oral cavity, are clinically characterized by inflammation of the periodontal connective tissue that ultimately induces the destruction of periodontal tissue and the loss of alveolar bone. In chronic periodontitis, as well as aggressive periodontitis, the anaerobic gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) is implicated, and its pathogenicity is exerted by a wide variety of factors, among which the lipopolysaccharides (LPSs). LPSs activate the innate immune response during Gram-negative bacterial infections through the Toll-like receptor 4 (TLR-4)/myeloid differentiation protein 2 (MD- 2) complex. In this study, the expression of TLR-4, the cell growth, the cytokines release and the nuclear factor-KB (NF-kB) transcription factor expression in response to LPS-P. Gingivalis (LPS-G) were examined in Human Periodontal Ligament Mesenchymal Stem Cells (PDL-MSCs) [1]. The results obtained have demonstrated that, in basal conditions, human PDLMSCs express high levels of TLR-4. In inflammatory conditions mimicked by LPS-G challenge, the MTT assay carried out at different treatment times evidenced the decrease of the cell growth. Moreover, the recognition of P. gingivalis components by TLR-4 culminated with the activation of secretion of inflammatory mediators such as: IL-6, IL-8 and CCL-20, and with the up-regulation of NF-kB, which was translocated into the nucleus. Our data intended to specify that TLR-4 expressed by PDL-MSCs is functional and plays a key role in inflammation

Development of Xeno-free culture system for human Periodontal Ligament Stem Cells

The opportunity of transplanting adult stem cells into damaged organs has opened new prospectives for the treatment of several human pathologies. Aim of this study was to develop a culture system for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free media formulation ensuring the maintenance of the stem cells features comprising: the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype and genomic stability, essential elements for conforming to translation to cell therapy1. Somatic stem cells were isolated from the human periodontium using a minimally invasive periodontal access flap surgery. Expanded hPDLSCs in a xeno-free culture showed the morphological features of stem cells, expressed the markers associated with pluripotency, and a normal karyotype. Under appropriate culture conditions, hPDLSCs presented adipogenic and osteogenic potential; indeed, a very high accumulation of lipid droplets was evident in the cytoplasm of adipogenic induced cells, and indisputable evidence of osteogenic differentiation, investigated by transmission electron microscopy, and analyzed for gene expression analysis has been shown2. Our results prove that the novel xeno-free culture method might provide the basis for GMP culture of autologous stem cells, readily accessible from human periodontium, and can be a resource to facilitate their use in human clinical studies for potential therapeutic regeneration

631 rankl knock out mesenchymal stromal cells have an unexpected osteogenic differentiation defect which is improved by a rankl expressing lentiviral vector

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC

Current Evidence on Bisphenol A Exposure and the Molecular Mechanism Involved in Related Pathological Conditions

Bisphenol A (BPA) is one of the so-called endocrine disrupting chemicals (EDCs) and is thought to be involved in the pathogenesis of different morbid conditions: immune-mediated disorders, type-2 diabetes mellitus, cardiovascular diseases, and cancer. The purpose of this review is to analyze the mechanism of action of bisphenol A, with a special focus on mesenchymal stromal/stem cells (MSCs) and adipogenesis. Its uses will be assessed in various fields: dental, orthopedic, and industrial. The different pathological or physiological conditions altered by BPA and the related molecular pathways will be taken into consideration

Alternative source of stem cells derived from human periodontal ligament: a new treatment for experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is categorized as an autoimmune disease and is potentially one of the most common causes of neurological disability in young adults. Formation of the sclerotic plaques of which the disease gets its name represents the end stage of a process involving inflammation, demyelination and remyelination, oligodendrocytes depletion, and astrogliosis as well as neuronal and axonal degeneration (1). MS damages the central nervous system and leads to a disabling condition. Recently, the potential role of mesenchymal stem cells (MSCs), derived in promoting tissue repair and disease control has been investigated by using an experimental autoimmune encephalomyelitis (EAE) model (2). The objective of the research was to investigate the product effects by mesenchymal stem cells derived from human periodontal ligament (hPDLSCs) when administered in an experimental model of autoimmune encephalomyelitis (EAE). EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55 in C57BL/6 mice. Then, mice were observed every 48 hours for signs of EAE and weight loss. At the onset of disease, approximately 14 days after immunization, EAE mice were subjected to a single intravenous injection of hPDLSCs (10(6) cells/150 μl) into the tail vein. At the point of animal sacrifice on day 56 after EAE induction, spinal cord and brain tissues were collected in order to perform histological evaluation, immunohistochemistry and western blotting analysis. Obtained results reveal that treatment with hPDLSCs may produce neuroprotective effects against EAE, diminishing both clinical signs and histological score typical of the disease (lymphocytic infiltration and demyelination) probably through the production of neurotrophic factors (results focused on brain-derived neurotrophic factor and nerve growth factor expression). Furthermore, administration of hPDLSCs modulates expression of inflammatory key markers (tumor necrosis factor-α, interleukin (IL)-1β, IL-10, glial fibrillary acidic protein, Nrf2 and Foxp3), the release of CD4 and CD8α T cells, and the triggering of apoptotic death pathway (data shown for cleaved caspase 3, p53 and p21). In light of the achieved results, transplantation of hPDLSCs may represent a putative novel and helpful tool for multiple sclerosis treatment. These cells could have considerable implication for future therapies for multiple sclerosis and this study may represent the starting point for further investigations

Engineered vesiscles from gingival stem cells: a new approach in 3D printed bone tissue regeneration

In the bone regeneration field, properties of 3D scaffold could be improved using cellular and their released products. Even if previously documented, poly-(lactide) (PLA) scaffolds were not thoroughly evaluated for their design-related characteristics. The aim of the study was to investigate the properties of 3D printed PLA scaffolds for bone regeneration obtained through 3D printing, evaluating the differences in terms of structural properties, in vitro and in vivo cellular responses induced by different scaffold structures. Biofabrication is to generate a construct with biological function. In particular in our reserach we describe the fully process, including printing scaffold step, in vitro culture phase and subsequently in vivo transplantation. Five porous scaffold designs (A-B-C-D-E) were fabricated from a poly-(lactide) (PLA) filament. Scaffold structural parameters, such as porosity and pore size, were measured using scanning electron microscopy, and micro-computed tomography. Nano-topographic surface features were investigated by means of atomic force microscopy. Over a 112-day period, scaffolds were hydrolytically degraded and changes in weight, pH and mechanical properties were measured during degradation. Osteogenic differentiation of hPDLSCs on different scaffold designs after 21 days of culture was measured by means of RT-PCR and Western Blot. In vivo study was performed using C57BL/6 mice and was designed in 5 different groups: - Group1: Scaffold loaded with hPDLSCs - Group2: Scaffold loaded with conditioned medium (CM) derived from hPDLSCs - Group3: Scaffold loaded with exosomes (Exo) purified from CM - Group 4: Scaffold loaded with engineered exosomes (e-Exo), exosome treated with PEI (poly ether imide) - Group5: Scaffold, used as control. Histological analysis were performed after 60 days of in vivo transplantation and morphological evaluations revealed a high bone tissue formation and osteogenic cells commitment in group 3 and 4 when compared to other groups. From these results, the cell-laden PCAMSC scaffold offers a significant advantage in the TM regeneration in a rat subacute TM perforation model. It may offer attractive opportunities in the conservative clinical treatment. This study demonstrated that scaffold of group 3 and 4 significantly improved bone tissue regeneration in animal model and successfully showed new bone deposition in situ compared to the control scaffold (group 5) and group 1 and 2. Based on the results, we believe that the bioprinted scaffold may become a novel treatment for tissue regeneration approach therapy

identification of amino acid residues critical for the b cell growth promoting activity of hiv 1 matrix protein p17 variants

Abstract Background HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. Methods We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. Results By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. Conclusions The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. General significance Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL

Engineered Extracellular Vesicles From Human Periodontal-Ligament Stem Cells Increase VEGF/VEGFR2 Expression During Bone Regeneration

Bone regeneration represents still a challenge, in particular for calvarium defects. Recently, the development of biomaterials with the addiction of stem cells is giving promising results for the treatment of bone defects. In particular, it was demonstrated that scaffolds enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as conditioned medium (CM) and extracellular vesicles (EVs), may improve bone regeneration. Moreover, given the deep link between osteogenesis and angiogenesis, a successful approach must also take into consideration the development of vascularization. In this work we evaluated the bone regeneration capacity of a collagen membrane (3D-COL) enriched with human periodontal-ligament stem cells (hPDLSCs) and CM or EVs or EVs engineered with polyethylenimine (PEI-EVs) in rats subjected to a calvarial defect. We evaluated also their capacity to induce angiogenic factors. At first, in vitro results showed an increased expression of osteogenic markers in hPDLSCs cultured with the 3D-COL and PEI-EVs, associated also with the increased protein levels of Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). The increased expression of these proteins was confirmed also in vivo in rats implanted with the 3D-COL enriched with hPDLSCs and PEI-EVs. Moreover, histological examination evidenced in this group of rats the activation of bone regeneration and of the vascularization process. Also MicroCT imaging with morphometric analysis confirmed in rats transplanted with 3D-COL enriched with hPDLSCs and PEI-EVs an important regenerative process and a better integration level. All together, these results evidenced that the 3D-COL enriched with hPDLSCs and PEI-EVs may promote bone regeneration of calvaria defects, associated also with an increased vascularization