Uticaj LPS i IL-33 na adultne matične tkiva periodoncijuma: funkcijske promene i aktivirani molekulski mehanizmi

Contemporary methods for treating various diseases of the oral cavity, caused by congenital deformities, mechanical damage or damage caused by inflammatory or tumor diseases can be a complex clinical task, given that the requirement for restoration of function and aesthetics of the orofacial region is imposed. The supporting tissue of the tooth in which the tissue of the periodontium represents the connection between the teeth and the alveolar bone, has an important role in maintaining the structure and function of the orofacial system. The tissue of the periodontium is very rich with mesenchymal stem cells (MSC) and can be a suitable source of MSC population for potential therapeutic use. Due to periodontium damage, the establishment of teeth structure and function, and consequently the orofacial system, is very difficult, even with the application of the most modern prosthetic treatments. However, functional features оf MSC from periodontium (PD-MSC) have not been fully revealed, which is a necessary requirement for their application. A particularly important aspect is understanding of PD-MSC’s behavior in the inflammatory microenvironment, since the periodontium destruction is most often caused by periodontal disease, chronic inflammatory disease of the supporting tissue of the teeth that can lead to tooth loss. In addition, inflammation of the teeth supportive tissue may be an accompanying complication after the dental implant, which also indicates the need to improve existing prosthetic treatments. The aim of the research covered by this dissertation was to obtain data about effects of factors frequently associated with the periodontium niche on the functional properties of PD-MSC. The influence of LPS, as a strong initiator of the inflammatory process, on the regenerative and immunomodulatory PD-MSC was examined. On the molecular level mechanisms mediating the effects of LPS were determined, wherein the activity of MAPK (Mitogen-Activated Protein Kinases) signaling pathways was determined. In addition, the effect of IL-33, as an endogenous alarm signal, on the regenerative properties of PD-MSC was examined...Savremeni načini lečenja različitih oboljenja usne duplje, nastalih usled urođenih deformiteta, mehaničkih oštećenja ili poremećaja uzrokovanim inflamatornim ili tumorskim oboljenjima mogu predstavljati kompleksan klinički zadatak s obzirom da se nameće zahtev obnove funkcije, ali i estetike orofacijalnog regiona. Značajnu ulogu u održanju strukture i funkcije orofacijalnog sistema ima potporno tkivo zuba u okviru koga tkivo periodoncijuma predstavlja vezu između zuba i alveolarne kosti. Tkivo periodoncijuma je bogato mezenhimskim matičnim ćelijama (MMĆ), te može biti pogodan izvor populacije MMĆ za potencijalnu terapijsku primenu. Usled oštećenja tkiva periodoncijuma uspostavljanje strukture i funkcije zuba, a time i orofacijalnog sistema je veoma otežano čak i uz primenu najsavremenijih protetskih tretmana. Ipak, funkcijske osobine MMĆ periodoncijuma (PD-MMĆ) nisu u potpunosti ispitane, što predstavlja neophodan uslov za njihovu primenu. Posebno važan aspekt predstavlja poznavanje ponašanja PD-MMĆ u inflamatornoj mikrosredini, budući da do razaranja tkiva periodoncijuma najčešće dolazi usled parodontopatije, hroničnog inflamatornog oboljenja potpornog tkiva zuba koje može dovesti do gubitka zuba. Pored toga, inflamacija potornog tkiva može biti prateća komplikacija nakon postavljanja dentalnih implantata što takođe ukazuje na potrebu za poboljšavanjem postojećih protetskih tretmana. Istraživanja obuhvaćena ovom disertacijom imala su za cilj dobijanje podataka o uticaju faktora koji se mogu naći u niši tkiva periodoncijuma na funkcijska svojstva PD-MMĆ. Ispitivan je uticaj LPS, kao snažnog inicijatora inflamatornog procesa, na regenerativna i imunomodulatorna svojstva PD-MMĆ..

Inflammatory niche: Mesenchymal stromal cell priming by soluble mediators

Mesenchymal stromal/stem cells (MSCs) are adult stem cells of stromal origin that possess self-renewal capacity and the ability to differentiate into multiple mesodermal cell lineages. They play a critical role in tissue homeostasis and wound healing, as well as in regulating the inflammatory microenvironment through interactions with immune cells. Hence, MSCs have garnered great attention as promising candidates for tissue regeneration and cell therapy. Because the inflammatory niche plays a key role in triggering the reparative and immunomodulatory functions of MSCs, priming of MSCs with bioactive molecules has been proposed as a way to foster the therapeutic potential of these cells. In this paper, we review how soluble mediators of the inflammatory niche (cytokines and alarmins) influence the regenerative and immunomodulatory capacity of MSCs, highlighting the major advantages and concerns regarding the therapeutic potential of these inflammatory primed MSCs. The data summarized in this review may provide a significant starting point for future research on priming MSCs and establishing standardized methods for the application of preconditioned MSCs in cell therapy

Adipogenesis in Different Body Depots and Tumor Development

Adipose tissue (AT) forms depots at different anatomical locations throughout the body, being in subcutaneous and visceral regions, as well as the bone marrow. These ATs differ in the adipocyte functional profile, their insulin sensitivity, adipokines' production, lipolysis, and response to pathologic conditions. Despite the recent advances in lineage tracing, which have demonstrated that individual adipose depots are composed of adipocytes derived from distinct progenitor populations, the cellular and molecular dissection of the adipose clonogenic stem cell niche is still a great challenge. Additional complexity in AT regulation is associated with tumor-induced changes that affect adipocyte phenotype. As an integrative unit of cell differentiation, AT microenvironment regulates various phenotype outcomes of differentiating adipogenic lineages, which consequently may contribute to the neoplastic phenotype manifestations. Particularly interesting is the capacity of AT to impose and support the aberrant potency of stem cells that accompanies tumor development. In this review, we summarize the current findings on the communication between adipocytes and their progenitors with tumor cells, pointing out to the co-existence of healthy and neoplastic stem cell niches developed during tumor evolution. We also discuss tumor-induced adaptations in mature adipocytes and the involvement of alternative differentiation programs

Dental mesenchymal stromal/stem cells in different microenvironments — implications in regenerative therapy

Current research data reveal microenvironment as a significant modifier of physical functions, pathologic changes, as well as the therapeutic effects of stem cells. When comparing regeneration potential of various stem cell types used for cytotherapy and tissue engineering, mesenchymal stem cells (MSCs) are currently the most attractive cell source for bone and tooth regeneration due to their differentiation and immunomodulatory potential and lack of ethical issues associated with their use. The microenvironment of donors and recipients selected in cytotherapy plays a crucial role in regenerative potential of transplanted MSCs, indicating interactions of cells with their microenvironment indispensable in MSC-mediated bone and dental regeneration. Since a variety of MSC populations have been procured from different parts of the tooth and tooth-supporting tissues, MSCs of dental origin and their achievements in capacity to reconstitute various dental tissues have gained attention of many research groups over the years. This review discusses recent advances in comparative analyses of dental MSC regeneration potential with regards to their tissue origin and specific microenvironmental conditions, giving additional insight into the current clinical application of these cells

Tumorigenic Aspects of MSC Senescence—Implication in Cancer Development and Therapy

As an organism ages, many physiological processes change, including the immune system. This process, called immunosenescence, characterized by abnormal activation and imbalance of innate and adaptive immunity, leads to a state of chronic low-grade systemic inflammation, termed inflammaging. Aging and inflammaging are considered to be the root of many diseases of the elderly, as infections, autoimmune and chronic inflammatory diseases, degenerative diseases, and cancer. The role of mesenchymal stromal/stem cells (MSCs) in the inflammaging process and the age-related diseases is not completely established, although numerous features of aging MSCs, including altered immunomodulatory properties, impeded MSC niche supporting functions, and senescent MSC secretory repertoire are consistent with inflammaging development. Although senescence has its physiological function and can represent a mechanism of tumor prevention, in most cases it eventually transforms into a deleterious (para-)inflammatory process that promotes tumor growth. In this review we are going through current literature, trying to explore the role of senescent MSCs in making and/or sustaining a microenvironment permissive to tumor development and to analyze the therapeutic options that could target this process

Mesenchymal stem cells isolated from human periodontal ligament

Mesenchymal stem cells (MSCs) were isolated from human periodontal ligament (hPDL-MSCs) and characterized by their morphology, clonogenic efficiency, proliferation and differentiation capabilities. hPDL-MSCs, derived from normal impacted third molars, possessed all of the properties of MSC, including clonogenic ability, high proliferation rate and multi-lineage (osteogenic, chondrogenic, adipogenic, myogenic) differentiation potential. Moreover, hPDL-MSCs expressed a typical MSC epitope profile, being positive for mesenchymal cell markers (CD44H, CD90, CD105, CD73, CD29, Stro-1, fibronectin, vimentin, alpha-SMA), and negative for hematopoietic stem cell markers (CD34, CD11b, CD45, Glycophorin-CD235a). Additionally, hPDL-MSCs, as primitive and highly multipotent cells, showed high expression of embryonic markers (Nanog, Sox2, SSEA4). The data obtained provided yet further proof that cells with mesenchymal properties can be obtained from periodontal ligament tissue. Although these cells should be further investigated to determine their clinical significance, hPDL-MSCs are believed to provide a renewable and promising cell source for new therapeutic strategies in the treatment of periodontal defects

Insight into the Biological Activity of Hennosides-Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead

Henna is the current name of the dye prepared from the dry leaf powder of Lawsonia inermis (Lythraceae). Several studies have focused on the chemistry and pharmacology of the henna dyeing active compound, lawsone, obtained from the main constituents of leaves, hennosides, during the processing of plant material. However, knowledge regarding the biological activity of hennosides is largely lacking. In this paper, the redox activity of three hennoside isomers is reported. The pro-oxidative activity was confirmed by their ability to induce mild lysis of erythrocytes and to increase the level of methemoglobin at the concentration gt = 500 mu g/mL. The antioxidant activity of hennosides (concentration gt = 100 mu g/mL) was determined by FRAP and ABTS assays. At concentration of 500 mu g/mL, antioxidant activity of hennoside isomers was equivalent to 0.46 +/- 0.08, 0.62 +/- 0.28 and 0.35 +/- 0.03 mM FeSO4 x 7H(2)O, and 0.15 +/- 0.01, 0.30 +/- 0.01 and 0.09 +/- 0.01 mM Trolox. Hennosides at 100 mu g/mL concentration did not influence viability of human breast cancer cell lines MDA231 and MCF-7 and primary human peripheral blood and periodontal ligament-mesenchymal stem cells, but produced a modest increase in concentration of antioxidants in the cell culture supernatants. The evidenced antioxidant and pro-oxidant activities indicate their potential to act as redox balance regulator, which opens up the possibility of using hennosides in commercial phytomedicines

Doxycycline Inhibits IL-17-Stimulated MMP-9 Expression by Downregulating ERK1/2 Activation: Implications in Myogenic Differentiation

Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 inmyoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulateMMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17's capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17

Inflammatory Cytokines Prime Adipose Tissue Mesenchymal Stem Cells to Enhance Malignancy of MCF-7 Breast Cancer Cells via Transforming Growth Factor-beta 1

Mesenchymal stem cells from human adipose tissue (hASCs) are proposed as suitable tools for soft tissue engineering and reconstruction. Although it is known that hASCs have the ability to home to sites of inflammation and tumor niche, the role of inflammatory cytokines in the hASCs-affected tumor development is not understood. We found that interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha) prime hASCs to produce soluble factors which enhance MCF-7 cell line malignancy in vitro. IFN-gamma and/or TNF-alpha-primed hASCs produced conditioned media (CM) which induced epithelial to mesenchymal transition (EMT) of MCF-7 cells by reducing E-Cadherin and increasing Vimentin expression. Induced EMT was accompanied by increased invasion, migration, and urokinase type-plasminogen activator (uPA) expression in MCF-7 cells. These effects were mediated by increased expression of transforming growth factor-beta 1(TGF-beta 1) in cytokines-primed hASCs, since inhibition of type I TGF-beta 1 receptor on MCF-7 cells and neutralization of TGF-beta 1 disabled the CM from primed hASCs to increase EMT, cell migration, and uPA expression in MCF-7 cells. Obtained data suggested that IFN-gamma and/or TNF-alpha primed hASCs might enhance the malignancy of MCF-7 cell line by inducing EMT, cell motility and uPA expression in these cells via TGF-beta 1-Smad3 signalization, with potentially important implications in breast cancer progression

Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

The biology of vitamin D3 is well defined, as are the effects of its active metabolites on various cells, including mesenchymal stromal/stem cells (MSCs). However, the biological potential of its precursor, cholecalciferol (VD3), has not been sufficiently investigated, although its significance in regenerative medicine—mainly in combination with various biomaterial matrices—has been recognized. Given that VD3 preconditioning might also contribute to the improvement of cellular regenerative potential, the aim of this study was to investigate its effects on bone marrow (BM) MSC functions and the signaling pathways involved. For that purpose, the influence of VD3 on BM-MSCs obtained from young human donors was determined via MTT test, flow cytometric analysis, immunocytochemistry, and qRT-PCR. Our results revealed that VD3, following a 5-day treatment, stimulated proliferation, expression of pluripotency markers (NANOG, SOX2, and Oct4), and osteogenic differentiation potential in BM-MSCs, while it reduced their senescence. Moreover, increased sirtuin 1 (SIRT1) expression was detected upon treatment with VD3, which mediated VD3-promoted osteogenesis and, partially, the stemness features through NANOG and SOX2 upregulation. In contrast, the effects of VD3 on proliferation, Oct4 expression, and senescence were SIRT1-independent. Altogether, these data indicate that VD3 has strong potential to modulate BM-MSCs’ features, partially through SIRT1 signaling, although the precise mechanisms merit further investigation