RESUMO
Considered as some of the most devastating complications, Cutibacterium acnes (C. acnes)-related osteomyelitis are among the hardest infections to diagnose and treat. Mesenchymal stem cells (MSCs) secrete number of immunomodulatory and antimicrobial soluble factors, making them an attractive treatment for bacterial infection. In this study, we examined MSCs/C. acnes interaction and analyzed the subsequent MSCs and bacteria's behaviors. Human bone marrow-derived MSCs were infected by C. acnes clinical strain harvested from non-infected bone site. Following 3 h of interaction, around 4% of bacteria were found in the intracellular compartment. Infected MSCs increased the secretion of prostaglandin E2 and indolamine 2,3 dioxygenase immunomodulatory mediators. Viable intracellular bacteria analyzed by infrared spectroscopy and atomic force microscopy revealed deep modifications in the wall features. In comparison with unchallenged bacteria, the viable intracellular bacteria showed (i) an increase in biofilm formation on orthopaedical-based materials, (ii) an increase in the invasiveness of osteoblasts and (iii) persistence in macrophage, suggesting the acquisition of virulence factors. Overall, these results showed a direct impact of C. acnes on bone marrow-derived MSCs, suggesting that blocking the C. acnes/MSCs interactions may represent an important new approach to manage chronic osteomyelitis infections. STATEMENT OF SIGNIFICANCE: The interaction of bone commensal C. acnes with bone marrow mesenchymal stem cells induces modifications in C. acnes wall characteristics. These bacteria increased (i) the biofilm formation on orthopaedical-based materials, (ii) the invasiveness of bone forming cells and (iii) the resistance to macrophage clearance through the modification of the wall nano-features and/or the increase in catalase production.
Assuntos
Células-Tronco Mesenquimais , Osteomielite , Biofilmes , Células da Medula Óssea , Humanos , Propionibacterium acnes , Próteses e ImplantesRESUMO
Crosstalk between mesenchymal stem cells (MSCs) and bacteria plays an important role in regulating the regenerative capacities of MSCs, fighting infections, modulating immune responses and maintaining tissue homeostasis. Commensal Cutibacterium acnes (C. acnes) bacterium becomes an opportunistic pathogen causing implant-associated infections. Herein, we examined MSCs/C. acnes interaction and analysed the subsequent bacteria and MSCs behaviours following infection. Human bone marrow derived MSCs were infected by two clinical and one laboratory C. acnes strains. Following 3h of interaction, all bacterial strains were able to invade MSCs. Viable intracellular bacteria acquired virulence factors by increasing biofilm formation and/or by affecting macrophage phagocytosis. Although the direct and indirect (through neutrophil stimulation) antibacterial effects of the MSCs secretome were not enhanced following C. acnes infection, ELISA analysis revealed that C. acnes clinical strains are able to license MSCs to become immunosuppressive cell-like by increasing the secretion of IL-6, IL-8, PGE-2, VEGF, TGF-ß and HGF. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections. STATEMENT OF SIGNIFICANCE: The originality of this work relies on the study of relationship between human bone marrow derived mesenchymal stem cells (MSCs) phenotype and C. acnes clinical strains virulence following cell infection. Our major results showed that C. acnes are able to invade MSCs, inducing a transition of commensal to an opportunistic pathogen behaviour. Although the direct and indirect antibacterial effects were not enhanced following C. acnes infection, secretome analysis revealed that C. acnes clinical strains were able to license MSCs to become immunosuppressive and anti-fibrotic cell-like. These results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during associated implant infections.
Assuntos
Células da Medula Óssea/microbiologia , Osso e Ossos/patologia , Células-Tronco Mesenquimais/microbiologia , Propionibacteriaceae/fisiologia , Infecções Relacionadas à Prótese/microbiologia , Adulto , Idoso , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Células da Medula Óssea/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Imunomodulação/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Propionibacteriaceae/efeitos dos fármacos , Propionibacteriaceae/patogenicidade , Virulência/efeitos dos fármacosRESUMO
Full-scale cell penetration within porous scaffolds is required to obtain functional connective tissue components in tissue engineering applications. For this aim, we produced porous polyurethane structures with well-controlled pore and interconnection sizes. Although the influence of the pore size on cellular behavior is widely studied, we focused on the impact of the size of the interconnections on the colonization by NIH 3T3 fibroblasts and Wharton's jelly-derived mesenchymal stem cells (WJMSCs). To render the material hydrophilic and allow good material wettability, we treated the material either by plasma or by polydopamine (PDA) coating. We show that cells weakly adhere on these surfaces. Keeping the average pore diameter constant at 133 µm, we compare two structures, one with LARGE (52 µm) and one with SMALL (27 µm) interconnection diameters. DNA quantification and extracellular matrix (ECM) production reveal that larger interconnections is more suitable for cells to move across the scaffold and form a three-dimensional cellular network. We argue that LARGE interconnections favor cell communication between different pores, which then favors the production of the ECM. Moreover, PDA treatment shows a truly beneficial effect on fibroblast viability and on matrix production, whereas plasma treatment shows the same effect for WJMSCs. We, therefore, claim that both pore interconnection size and surface treatment play a significant role to improve the quality of integration of tissue engineering scaffolds.
Assuntos
Células-Tronco Mesenquimais/citologia , Poliuretanos/química , Animais , Células Cultivadas , Dopamina/química , Camundongos , Microscopia Confocal , Células NIH 3T3 , Porosidade , Propriedades de Superfície , Alicerces Teciduais/química , Geleia de Wharton/citologiaRESUMO
Bone mimicking coatings provide a complex microenvironment in which material, through its inherent properties (such as nanostructure and composition), affects the commitment of stem cells into bone lineage and the production of bone tissue regulating factors required for bone healing and regeneration. Herein, a bioactive mineral/biopolymer composite made of calcium phosphate/chitosan and hyaluronic acid (CaP-CHI-HA) was elaborated using a versatile simultaneous spray coating of interacting species. The resulting CaP-CHI-HA coating was mainly constituted of bioactive, carbonated and crystalline hydroxyapatite with 277 ± 98 nm of roughness, 1 µm of thickness, and 2.3 ± 1 GPa of stiffness. After five days of culture, CaP-CHI-HA suggested a synergistic effect of intrinsic biophysical features and biopolymers on stem cell mechanobiology and nuclear organization, leading to the expression of an early osteoblast-like phenotype and the production of bone tissue regulating factors such as osteoprotegerin and vascular endothelial growth factor. More interestingly, amalgamation with biopolymers conferred to the mineral a bacterial antiadhesive property. These significant data shed light on the potential regenerative application of CaP-CHI-HA bioinspired coating in providing a suitable environment for stem cell bone regeneration and an ideal strategy to prevent implant-associated infections.
Assuntos
Nanoestruturas , Regeneração Óssea , Materiais Revestidos Biocompatíveis , Durapatita , Osteoblastos , Osteogênese , Propriedades de Superfície , Fator A de Crescimento do Endotélio VascularRESUMO
Stem cells are the most powerful candidate for the treatment of various diseases. Suitable stem cell source should be harvested with minimal invasive procedure, found in great quantity, and transplanted with no risk of immune response and tumor formation. Fetal derived stem cells have been introduced as an excellent alternative to adult and embryonic stem cells use, but unfortunately, their degree of "stemness" and molecular characterization is still unclear. Several studies have been performed deciphering whether fetal stem cells meet the needs of regenerative medicine. We believe that a transcriptomic screening of Wharton's jelly stem cells will bring insights on cell population features.
Assuntos
Testes Genéticos/métodos , Células-Tronco Mesenquimais , Geleia de Wharton/citologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias , Matriz Extracelular/química , Humanos , Medicina Regenerativa , Transcriptoma , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Geleia de Wharton/metabolismoRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several lineages with valuable applications in regenerative medicine. MSCs differentiation is highly dependent on physicochemical properties of the culture substrate, cell density and on culture medium composition. OBJECTIVE: In this study, we assessed the influence of fetal bovine serum (FBS) level on Wharton's jelly (WJ)-MSCs behavior seeded on polyelectrolyte multilayer films (PEMF) made of four bilayers of poly-allylamine hydrochloride (PAH) as polycation and poly-styrene sulfonate (PSS) as polyanion. METHODS: MSCs isolated from WJ by explants method were amplified until the third passage. Their phenotypic characterization was performed by flow cytometry analyses. MSCs were seeded on PEMF, in Endothelial growth medium-2 (EGM-2) supplemented by either 5% or 2% FBS. Cell's behavior was monitored for 20 days by optical microscopy and immunofluorescence. RESULTS: Until 2 weeks on glass slides, no difference was observed whatever the FBS percentage. Then with 5% FBS, MSCs formed three-dimensional spheroids on PSS/PAH after 20 days of culture with a nuclear aggregate. Whereas, with 2% FBS, these spheroids did not appear and cells grown in 2D conserved the fibroblast-like morphology. CONCLUSIONS: The decrease of FBS percentage from 5% to 2% avoids 3D cell spheroids formation on PAH/PSS. Such results could guide bioengineering towards building 2D structures like cell layers or 3D structures by increasing the osteogenic or chondrogenic differentiation potential of MSCs.
Assuntos
Sangue , Técnicas de Cultura de Células/métodos , Meios de Cultura , Células-Tronco Mesenquimais/fisiologia , Materiais Biocompatíveis/química , Cátions/química , Agregação Celular/fisiologia , Contagem de Células , Forma Celular , Materiais Revestidos Biocompatíveis/química , Meios de Cultura/análise , Fator de Crescimento Epidérmico/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fibroblastos/citologia , Citometria de Fluxo , Humanos , Fator de Crescimento Insulin-Like I/administração & dosagem , Fenótipo , Poliaminas/química , Polieletrólitos , Polímeros/química , Poliestirenos/química , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
BACKGROUND: Polyelectrolyte multilayer (PEMs) films made of poly(allylamine hydrochloride) (PAH) as polycation and poly(styrene sulfonate) (PSS) as polyanion, with a PAH ending layer, can be used as a coating in order to improve the anti-thrombogenicity and patency of vascular grafts in vascular engineering field. They induce strong adhesion of mature endothelial cells on glass, expanded polytetrafluoroethylene and cryopreserved arteries. Despite their outstanding effect on mature and progenitor endothelial cells, PEMs ending with PAH showed a poor outcome on Wharton's jelly mesenchymal stem cells (WJ-MSCs) culture. OBJECTIVE: The aim of this work was to examine the influence of the ending charge of PEMs on WJ-MSCs behavior. METHODS: WJ-MSCs amplified until the 3rd passage were seeded and cultured on (PAH-PSS)3-PAH and on (PAH-PSS)4 coated glass for 10 days. Stem cell phenotype was checked by flow cytometry and cell morphology was followed by bright field microscopy. RESULTS: Flow cytometry analysis showed that WJ-MSCs were positive for MSC's markers CD73, CD90 and CD105 and negative for hematopoietic markers CD34 and CD45. Light microscopy showed development of nodule-like structures after 10 days of culture on (PAH-PSS)3-PAH, which resulted in a disturbance of cell monolayer. Whereas WJ-MSCs cultured on (PAH-PSS)4 ending with PSS showed a normal cell growth like on collagen and reached confluence after 10 days. CONCLUSION: The culture surface seems to have a determining role in WJ-MSC's "spatial" behavior, which could be considered in the field of tissue engineering.