Inflammatory cell death of human macrophages induced by Aggregatibacter actinomycetemcomitans leukotoxin

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a bacterium mainly associated with aggressive forms of periodontitis. Among its virulence factors, a leukotoxin is suggested to play an important role in the pathogenicity. Periodontal infections with strains producing high levels of the leukotoxin are strongly associated with severe disease. Leukotoxin selectively kills human leukocytes and can disrupt the local defense mechanisms. Previous studies examining the role of the leukotoxin in host-parasite interactions have mainly focused on polymorphonuclear leukocytes (PMNs). In the inflamed periodontium, macrophages play a significant role in the regulation of the inflammatory reactions and the tissue breakdown and remodeling. Thus, the aim of this dissertation was to investigate death mechanisms of human macrophages exposed to leukotoxin. Human lymphocytes, PMNs, and monocytes/macrophages isolated from venous blood were exposed to purified leukotoxin or live A. actinomycetemcomitans strains producing variable levels or no leukotoxin. Different target cells were characterized by their expression of cell surface molecules. Cell death and viability were studied by examining cell membrane integrity and morphological alterations. Further, processes and cellular markers involved in apoptosis and necrosis were investigated. The expression and activation of pro-inflammatory cytokines of the leukotoxin-challenged leukocytes were examined at the mRNA and protein level. The biological activity of the secreted cytokines was investigated by testing the culture supernatants in a bone resorption assay. Additionally, different intracellular signaling pathways involved in the pro-inflammatory response from the macrophages were examined. Monocytes/macrophages were the most sensitive leukocytes for A. actinomycetemcomitans leukotoxin-induced lysis. This process in monocytes/ macrophages involved caspase-1 activation, and in addition, leukotoxin triggered abundant activation and secretion of IL-1β from these cells. The secreted IL-1β was mainly the 17 kDa bioactive protein and stimulated bone resorption. This activity could be blocked by an IL-1 receptor antagonist. When live bacteria were used, the A. actinomycetemcomitans-induced IL-1β secretion from human macrophages was mainly caused by the leukotoxin. Closer examination of the macrophages exposed to leukotoxin revealed that the induced cell death proceeded through a process that differed from classical apoptosis and necrosis. Interestingly, this process resembled a newly discovered death mechanism termed pyroptosis. The extensive leukotoxin induced IL-1β secretion did not correlate to increased levels of mRNA for IL-1β. It was mainly mediated by caspase-1 activation, since blocking it by a specific inhibitor also abolished the secretion of IL-1β. A similar pattern, but at much lower level, was seen for IL-18. In conclusion, these results show that A. actinomycetemcomitans leukotoxin induces a death process in human macrophages leading to a specific and excessive pro-inflammatory response. Our results indicate that this novel virulence mechanism of leukotoxin may play an important role in the pathogenic potential of A. actinomycetemcomitans

Aggregatibacter actinomycetemcomitans Leukotoxin Activates the NLRP3 Inflammasome and Cell-to-Cell Communication

Carriers of highly leukotoxic genotypes of Aggregatibacter actinomycetemcomitans are at high risk for rapid degradation of tooth-supporting tissues. The leukotoxin (LtxA) expressed by this bacterium induces a rapid pro-inflammatory response in leukocytes that results in cell death. The aim of the present study was to increase the understanding of LtxA-induced leukocyte activation mechanisms and of possible associated osteoclast differentiation. The effect of LtxA on activation of the inflammasome complex was studied in THP-1 wild type and in NLRP3- and ASC knockout cells. Cell-to-cell communication was assessed by fluorescent parachute assays, and THP-1 differentiation into osteoclast-like cells was investigated microscopically. The results showed that LtxA induced inflammatory cell death, which involved activation of the NLRP3 inflammasome and gap junction cell-to-cell communication. THP-1 cells treated with lipopolysaccharide (LPS) and LtxA together differentiated into an osteoclast-like phenotype. Here, LPS prevented LtxA-mediated cell death but failed to induce osteoclast differentiation on its own. However, pit formation was not significantly enhanced by LtxA. We conclude that A. actinomycetemcomitans leukotoxicity mediates activation of the NLRP3 inflammasome and cell-to-cell communication in the induced pro-inflammatory cell death. In addition, LtxA stimulated differentiation towards osteoclasts-like cells in LPS-treated THP-1 cell

Evaluation of growth, stemness, and angiogenic properties of dental pulp stem cells cultured in cGMP xeno-/serum-free medium

This study was aimed to investigate the effects of cGMP xeno-/serum-free medium (XSF, Irvine Scientific) on the properties of human dental pulp stem cells (DPSCs). DPSCs, from passage 2, were cultured in XSF or fetal bovine serum (FBS)-supplemented medium, and sub-cultured up to passage 8. Cumulative population doublings (PDs) and the number of colony-forming-units (CFUs) were determined. qRT-PCR, ELISA, and in vitro assays were used to assess angiogenic capacity. Flow cytometry was used to measure CD73, CD90, and CD105 expression. Differentiation into osteo-, adipo-, and chondrogenic cell lineages was performed. DPSCs showed more elongated morphology, a reduced rate of proliferation at later passages, and lower CFU counts in XSF compared with FBS. Expression of angiogenic factors at the gene and protein levels varied in the two media and with passage number, but cells grown in XSF had more in vitro angiogenic activity. The majority of early and late passage DPSCs cultured in XSF expressed CD73 and CD90. In contrast, the percentage of CD105 positive DPSCs in XSF medium was significantly lower with increased passage whereas the majority of cells cultured in FBS were CD105 positive. Switching XSF-cultured DPSCs to medium supplemented with human serum restored the expression of CD105. The tri-lineage differentiation of DPSCs cultured under XSF and FBS conditions was similar. We showed that despite reduced CD105 expression levels, DPSCs expanded in XSF medium maintained a functional MSC phenotype. Furthermore, restoration of CD105 expression is likely to occur upon in vivo transplantation, when cells are exposed to human serum

Platelet lysate for expansion or osteogenic differentiation of bone marrow mesenchymal stem cells for 3D tissue constructs

Background: The use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems. Methods: We evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining. Results: Cell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT. Conclusions: PLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs

Substance P and patterned silk biomaterial stimulate periodontal ligament stem cells to form corneal stroma in a bioengineered three-dimensional model

Background: We aimed to generate a bioengineered multi-lamellar human corneal stroma tissue in vitro by differentiating periodontal ligament stem cells (PDLSCs) towards keratocytes on an aligned silk membrane. Methods: Human PDLSCs were isolated and identified. The neuropeptide substance P (SP) was added in keratocyte differentiation medium (KDM) to evaluate its effect on keratocyte differentiation of PDLSCs. PDLSCs were then seeded on patterned silk membrane and cultured with KDM and SP. Cell alignment was evaluated and the expression of extracellular matrix (ECM) components of corneal stroma was detected. Finally, multi-lamellar tissue was constructed in vitro by PDLSCs seeded on patterned silk membranes, which were stacked orthogonally and stimulated by KDM supplemented with SP for 18 days. Sections were prepared and subsequently stained with hematoxylin and eosin or antibodies for immunofluorescence observation of human corneal stroma-related proteins. Results: SP promoted the expression of corneal stroma-related collagens (collagen types I, III, V, and VI) during the differentiation induced by KDM. Patterned silk membrane guided cell alignment of PDLSCs, and important ECM components of the corneal stroma were shown to be deposited by the cells. The constructed multi-lamellar tissue was found to support cells growing between every two layers and expressing the main type of collagens (collagen types I and V) and proteoglycans (lumican and keratocan) of normal human corneal stroma. Conclusions: Multi-lamellar human corneal stroma-like tissue can be constructed successfully in vitro by PDLSCs seeded on orthogonally aligned, multi-layered silk membranes with SP supplementation, which shows potential for future corneal tissue engineering

Mechanical stress potentiates the differentiation of periodontal ligament stem cells into keratocytes

Aims To explore the role of corneal-shaped static mechanical strain on the differentiation of human periodontal ligament stem cells (PDLSCs) into keratocytes and the possible synergistic effects of mechanics and inducing medium. Methods PDLSCs were exposed to 3% static dome-shaped mechanical strain in a Flexcell Tension System for 3 days and 7 days. Keratocyte phenotype was determined by gene expression of keratocyte markers. Keratocyte differentiation (inducing) medium was introduced in the Flexcell system, either continuously or intermittently combined with mechanical stimulation. The synergistic effects of mechanics and inducing medium on keratocyte differentiation was evaluated by gene and protein expression of keratocyte markers. Finally, a multilamellar cell sheet was assembled by seeding PDLSCs on a collagen membrane and inducing keratocyte differentiation. The transparency of the cell sheet was assessed, and typical markers of native human corneal stroma were evaluated by immunofluorescence staining. Results Dome-shaped mechanical stimulation promoted PDLSCs to differentiate into keratocytes, as shown by the upregulation of ALDH3A1, CD34, LUM, COL I and COL V. The expression of integrins were also upregulated after mechanical stimulation, including integrin alpha 1, alpha 2, beta 1 and non-muscle myosin II B. A synergistic effect of mechanics and inducing medium was found on keratocyte differentiation. The cell sheets were assembled under the treatment of mechanics and inducing medium simultaneously. The cell sheets were transparent, multilamellar and expressed typical markers of corneal stroma. Conclusion Dome-shaped mechanical stimulation promotes differentiation of PDLSCs into keratocytes and has synergistic effects with inducing medium. Multilamellar cell sheets that resemble native human corneal stroma show potential for future clinical applications

Three-dimensional osteogenic differentiation of bone marrow mesenchymal stem cells promotes matrix metallopeptidase 13 (Mmp13) expression in type i collagen hydrogels

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects

Cytokine Secretion, Viability, and Real-Time Proliferation of Apical-Papilla Stem Cells Upon Exposure to Oral Bacteria

The use of stem cells from the apical papilla (SCAPs) has been proposed as a means of promoting root maturation in permanent immature teeth, and plays a significant role in regenerative dental procedures. However, the role of SCAPs may be compromised by microenvironmental factors, such as hypoxic conditions and the presence of bacteria from infected dental root canals. We aim to investigate oral bacterial modulation of SCAP in terms of binding capacity using flow cytometry and imaging, real-time cell proliferation monitoring, and cytokine secretion (IL-6, IL-8, and TGF-β isoforms) under anaerobic conditions. SCAPs were exposed to key species in dental root canal infection, namely Actinomyces gerensceriae, Slackia exigua, Fusobacterium nucleatum, and Enterococcus faecalis, as well as two probiotic strains, Lactobacillus gasseri strain B6 and Lactobacillus reuteri (DSM 17938). We found that A. gerensceriae, S. exigua, F. nucleatum, and E. faecalis, but not the Lactobacillus probiotic strains bind to SCAPs on anaerobic conditions. Enterococcus faecalis and F. nucleatum exhibited the strongest binding capacity, resulting in significantly reduced SCAP proliferation. Notably, F. nucleatum, but not E. faecalis, induce production of the proinflammatory chemokine IL-8 and IL-10 from SCAPs. Production of TGF-β1 and TGF-β2 by SCAPs was dependent on species, cell line, and time, but secretion of TGF-β3 did not vary significantly over time. In conclusion, SCAP response is compromised when exposed to bacterial stimuli from infected dental root canals in anaerobic conditions. Thus, stem cell-mediated endodontic regenerative studies need to include microenvironmental conditions, such as the presence of microorganisms to promote further advantage in the field

Water jet-assisted lipoaspiration and Sepax cell separation system for the isolation of adipose stem cells with high adipogenic potential

Introduction: Water jet-assisted liposuction has gained popularity due to favourable fat grafting outcomes. In this study, we compared stem cells obtained from fat isolated with manual or the water jet-assisted procedure. Methods: Liposuction of abdominal fat was performed using the two methods on each donor (n = 10). Aspirate samples were collagenase digested and the isolated cells seeded in vitro prior to proliferation, adipogenic differentiation and angiogenic activity analyses. Results: Cells from either procedure proliferated at similar rates and exhibited a similar colony-forming ability. The cells expressed stem cell markers CD73, CD90 and CD105. In the water jet cell preparations, there were higher numbers of cells expressing CD146. Robust adipogenic differentiation was observed in cultures expanded from both manual and water jet lipoaspirates. Gene analysis showed higher expression of the adipocyte markers aP2 and GLUT4 in the adipocyte-differentiated water jet cell preparations, and ELISA indicated increased secretion of adiponectin from these cells. Both cell groups expressed vasculogenic factors and the water jet cells promoted the highest levels of in vitro angiogenesis. Given these positive results, we further characterised the water jet cells when prepared using an automated closed cell processing unit, the Sepax-2 system (Cytiva). The growth and stem cell properties of the Sepax-processed cells were similar to the standard centrifugation protocol, but there was evidence for greater adipogenic differentiation in the Sepax-processed cells. Conclusions: Water jet lipoaspirates yield cells with high adipogenic potential and angiogenic activity, which may be beneficial for use in cell-assisted lipotransfers

Bioprinted schwann and mesenchymal stem cell co-cultures for enhanced spatial control of neurite outgrowth

Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel’s highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration