RESUMO
Since the success of bone regenerative medicine depends on scaffold morphological and mechanical properties, numerous scaffolds designs have been proposed in the last decade, including graded structures that are suited to enhance tissue ingrowth. Most of these structures are based either on foams with a random pore definition, or on the periodic repetition of a unit cell (UC). These approaches are limited by the range of target porosities and obtained effective mechanical properties, and do not permit to easily generate a pore size gradient from the core to the periphery of the scaffold. In opposition, the objective of the present contribution is to propose a flexible design framework to generate various three-dimensional (3D) scaffolds structures including cylindrical graded scaffolds from the definition of a UC by making use of a non-periodic mapping. Conformal mappings are firstly used to generate graded circular cross-sections, while 3D structures are then obtained by stacking the cross-sections with or without a twist between different scaffold layers. The effective mechanical properties of different scaffold configurations are presented and compared using an energy-based efficient numerical method, pointing out the versatility of the design procedure to separately govern longitudinal and transverse anisotropic scaffold properties. Among these configurations, a helical structure exhibiting couplings between transverse and longitudinal properties is proposed and permits to extend the adaptability of the proposed framework. In order to investigate the capacity of common additive manufacturing techniques to fabricate the proposed structures, a subset of these configurations is elaborated using a standard SLA setup, and subjected to experimental mechanical testing. Despite observed geometric differences between the initial design and the actual obtained structures, the effective properties are satisfyingly predicted by the proposed computational method. Promising perspectives are offered concerning the design of self-fitting scaffolds with on-demand properties depending on the clinical application.
Assuntos
Osso e Ossos , Alicerces Teciduais , Porosidade , Alicerces Teciduais/química , Medicina Regenerativa , Engenharia TecidualRESUMO
We investigated polysaccharide films obtained by simultaneous and alternate spraying of a chitosan (CHI) solution as polycation and hyaluronic acid (HA), alginate (ALG), and chondroitin sulfate (CS) solutions as polyanions. For simultaneous spraying, the film thickness increases linearly with the cumulative spraying time and passes through a maximum for polyanion/CHI molar charge ratios lying between 0.6 and 1.2. The size of polyanion/CHI complexes formed in solution was compared with the simultaneously sprayed film growth rate as a function of the polyanion/CHI molar charge ratio. A good correlation was found. This suggests the importance of polyanion/polycation complexation in the simultaneous spraying process. Depending on the system, the film topography is either liquid-like or granular. Film biocompatibility was evaluated using human gingival fibroblasts. A small or no difference is observed in cell viability and adhesion between the two deposition processes. The CHI/HA system appears to be the best for cell adhesion inducing the clustering of CD44, a cell surface HA receptor, at the membrane of cells. Simultaneous or alternate spraying of CHI/HA appears thus to be a convenient and fast procedure for biomaterial surface modifications.
Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Quitosana/química , Sulfatos de Condroitina/química , Ácido Hialurônico/química , Poliaminas/química , Polímeros/química , Adsorção , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Engenharia Química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Gengiva/efeitos dos fármacos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Receptores de Hialuronatos/biossíntese , Microscopia de Força Atômica , Polieletrólitos , Soluções , Propriedades de SuperfícieRESUMO
The field of regenerative medicine has recently seen an emerging trend toward decellularized extracellular matrix (ECM) as a biological scaffold for stem cell-delivery. Human umbilical cord represents a valuable opportunity from both technical and ethical point of view to obtain allogenic ECM. Herein, we established a protocol, allowing the full removal of cell membranes and nuclei moieties from Wharton's jelly (WJ) tissue. No alterations in the ECM components (i.e., collagen, GAG content, and growth factors), physical (i.e., porosity and swelling) and mechanical (i.e., linear tensile modulus) properties were noticed following WJ processing. Furthermore, no effect of the tissue processing on macromolecules and growth factors retention was observed, assuring thus a suitable bioactive matrix for cell maintenance upon recellularization. Based on the in vitro and in vivo biodegradability and stromal cell homing capabilities, decellularized WJ could provide an ideal substrate for stromal cells adhesion and colonization. Interestingly, the tissue processing increased the antibacterial and antiadhesive properties of WJ against Staphylococcus aureus and Staphylococcus epidermidis pathogens. Altogether, our results indicate that decellularized WJ matrix is able to limit Staphylococcus-related infections and to promote stromal cell homing, thus offering a versatile scaffold for tissue regenerative medicine.
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
Foreign Body Reaction (FBR) is a critical issue to be addressed when polyethylene terephthalate (PET) textile implants are considered in the medical field to treat pathologies involving hernia repair, revascularization strategies in arterial disease, and aneurysm or heart valve replacement. The natural porosity of textile materials tends to induce exaggerated tissue ingrowth which may prevent the implants from remaining flexible. One hypothesized way to limit the FBR process is to increase the material surface roughness at the yarn level. Supercritical N2 (ScN2) jet particle projection is a technique that provides enough velocity to particles in order to induce plastic deformation on the impacted surface. This work investigates the influence of ScN2 jet projection parameters like standoff distance or particle size on the roughness that can be obtained on medical polymer yarns of various diameters (100 and 400 µm) and woven textile surfaces obtained from a 100 µm yarn. Moreover, the mechanical and biological performances of the obtained modified textile material are assessed. Results bring out that with appropriate testing conditions (500 bars jet/500 mm distance between nozzle and PET textile) and particle size around 50 µm, it is possible to generate 20 µm large and 4 µm deep craters on a 100 µm monofilament PET yarn and fabric. Regarding the strength of the textile material, it is only slightly modified with the treatment process, as the tenacity of the yarns decreases by only 10%. Moreover, It is shown that the obtained structures tend to limit the adhesion and slow down the proliferation of human fibroblasts.
Assuntos
Materiais Biocompatíveis , Polímeros , Humanos , Porosidade , Próteses e Implantes , TêxteisRESUMO
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
Carbonate apatites are sought as a bone substitute due to their biocompatibility and excellent resorbability. The present study deals with Cowrie's shell derived powder (CSDP) as natural biomaterial for bone regenerative medicine. Structural and physicochemical analysis showed that Cowrie's shells, presenting brick and mortar microstructures, were mainly composed of aragonite crystals, which were converted into poorly crystalline B-type carbonate apatite once soaked, at 37⯰C, in simulated body fluid for 7â¯days, reflecting bioactive features. Cytotoxic assays showed that CSDP boosted human stem cell proliferation over the study time compared to nacre derived powder (NDP), used as positive control. Human stem cells adopted a flattened morphology and established physical contact with CSDP, signature of a good biocompatibility. Thus, these results suggested that CSDP presents a great interest for bone regenerative medicine, and could be a useful and versatile carrier/scaffold for bone tissue engineering or a raw material for 3D printed orthopedic devices.
Assuntos
Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Carbonato de Cálcio/farmacologia , Gastrópodes/química , Medicina Regenerativa , Exoesqueleto/ultraestrutura , Animais , Líquidos Corporais/química , Forma Celular , Humanos , Pós , Células-Tronco/citologia , Células-Tronco/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
Imaging thick and opaque tissue, like blood vessel, in a noninvasive mode with high resolution, is nowadays possible with multiphoton technology. A near-infrared excitation presents the advantage to be compatible with living specimens and allows a deep penetration into tissues. The nonlinear excitation process is followed by several deactivation ways, among which fluorescence emission can be represented with Spectral or Lifetime imaging. Applied to ex vivo blood vessel imaging, these techniques enabled us to discriminate cell structures (nucleus, cytoskeleton) by fluorescent labelling (Hoechst, QDots). Another method, based on 2-photon excitation and which doesn't need any exogenous dye has also been experimented on arteries: SHG (Second Harmonic Generation) is a diffusion process generated from organized structures. Collagen molecules give rise to a strong SHG signal, enabling us to image the arterial wall (3-dimensional extracellular matrix).
Assuntos
Vasos Sanguíneos/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Vasos Sanguíneos/citologia , Humanos , Imageamento Tridimensional , Raios InfravermelhosRESUMO
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
Titania-Hydroxyapatite (TiO2/HAP) reinforced coatings are proposed to enhance the bioactivity and corrosion resistance of 316L stainless steel (316L SS). Herein, spin- and dip-coating sol-gel processes were investigated to construct two kinds of coatings: TiO2/HAP composite and TiO2/HAP bilayer. Physicochemical characterization highlighted the bioactivity response of the TiO2/HAP composite once incubated in physiological conditions for 7days whereas the TiO2/HAP bilayer showed instability and dissolution. Biological analysis revealed a failure in human stem cells adhesion on TiO2/HAP bilayer whereas on TiO2/HAP composite the presence of polygonal shaped cells, possessing good behaviour attested a good biocompatibility of the composite coating. Finally, TiO2/HAP composite with hardness up to 0.6GPa and elastic modulus up to 18GPa, showed an increased corrosion resistance of 316L SS. In conclusion, the user-friendly sol-gel processes led to bioactive TiO2/HAP composite buildup suitable for biomedical applications.
Assuntos
Materiais Revestidos Biocompatíveis/química , Durapatita/química , Titânio/química , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Materiais Revestidos Biocompatíveis/farmacologia , Corrosão , Citoesqueleto/efeitos dos fármacos , Técnicas Eletroquímicas , Géis/química , Dureza , Humanos , Microscopia Eletrônica de Varredura , Espectrometria por Raios X , Aço Inoxidável/química , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Cordão Umbilical/citologia , Difração de Raios XRESUMO
An important aim of bone regenerative medicine is to design biomaterials with controlled chemical and topographical features to guide stem cell fate towards osteoblasts without addition of specific osteogenic factors. Herein, we find that sprayed bioactive and biocompatible calcium phosphate substrates (CaP) with controlled topography induce, in a well-orchestrated manner, Wharton's jelly stem cells (WJ-SCs) differentiation into osteoblastic lineage without any osteogenic supplements. The resulting WJ-SCs commitment exhibits features of native bone, through the formation of three-dimensional bone-like nodule with osteocyte-like cells embedded into a mineralized type I collagen. To our knowledge, these results present the first observation of a whole differentiation process from stem cell to osteocytes-like on a synthetic material. This suggests a great potential of sprayed CaP and WJ-SCs in bone tissue engineering. These unique features may facilitate the transition from bench to bedside and the development of successful engineered bone. STATEMENT OF SIGNIFICANCE: Designing materials to direct stem cell fate has a relevant impact on stem cell biology and provides insights facilitating their clinical application in regenerative medicine. Inspired by natural bone compositions, a friendly automated spray-assisted system was used to build calcium phosphate substrate (CaP). Sprayed biomimetic solutions using mild conditions led to the formation of CaP with controlled physical properties, good bioactivity and biocompatibility. Herein, we show that via optimization of physical properties, CaP substrate induce osteogenic differentiation of Wharton's jelly stem cells (WJ-SCs) without adding osteogenic supplement factors. These results suggest a great potential of sprayed CaP and WJ-SCs in bone tissue engineering and may facilitate the transition from bench to beside and the development of clinically successful engineered bone.
Assuntos
Osso e Ossos/citologia , Fosfatos de Cálcio/farmacologia , Diferenciação Celular , Osseointegração/efeitos dos fármacos , Células-Tronco/citologia , Geleia de Wharton/citologia , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Microscopia de Força Atômica , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Células-Tronco/ultraestrutura , Propriedades de SuperfícieRESUMO
The cartilage is a hydrated connective tissue in joints that withstands and distributes mechanical forces. The chondrocytes utilize mechanical signals to regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The aim of this work was to study the influence of mechanical stress on cells behavior cultured in 3D biosystems (alginate and alginate supplemented with hyaluronate). After mechanical stimulation, cell viability and cell death process were the main studied parameters. Our results indicated that viability and cell cycle progression were inhibited under mechanical stimulation, as far as the extracellular matrix was not yet synthesized. In contrast, on day 21, the mechanical stimulation had positive effect on these parameters.
Assuntos
Cartilagem Articular/citologia , Condrócitos/fisiologia , Mecanotransdução Celular/fisiologia , Alginatos , Animais , Apoptose/fisiologia , Cartilagem Articular/fisiologia , Técnicas de Cultura de Células , Ciclo Celular/fisiologia , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Condrócitos/citologia , Matriz Extracelular , Ácido Glucurônico , Ácidos Hexurônicos , Masculino , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
Polyelectrolyte multilayer films were recently investigated to favour attachment of Human Vein Umbilical Endothelial Cells (HUVECs) on non-adhesive surfaces. In this study, we evaluated the initial adhesion of HUVECs after 3 h of seeding on two polyelectrolyte multilayer films ending by poly(D-lysine) (PDL) or poly(allylamine hydrochloride) (PAH). In order to obtain information about initial adhesion of HUVECs, cell morphology as well as the expression of beta1 integrins, specific receptors of adhesion, were evaluated after 3 h of seeding on polyelectrolyte multilayer films. The data were also compared to PDL or PAH monolayers (polyelectrolytes terminating the multilayer architecture). The expression of beta1 integrins was not different, whatever are the studied surfaces. However, HUVECs spreading on polyelectrolyte multilayer films, in particular on PAH ending film, was more important as compared to polyelectrolyte monolayers or glass. In conclusion, the best initial adhesion conditions of HUVECs on polyelectrolyte films could not be elucidated, moreover the results suggested also that beta1 integrins could only play a limited role.
Assuntos
Materiais Biocompatíveis/química , Células Endoteliais/citologia , Actinas/metabolismo , Adesão Celular , Células Cultivadas , Eletrólitos , Endotélio Vascular/citologia , Humanos , Integrina beta1/metabolismo , Microscopia de Força Atômica , Poliaminas/química , Polilisina/química , Propriedades de Superfície , Veias Umbilicais/citologiaRESUMO
Decellularized allograft tissues have been identified as a potential extracellular matrix scaffold for tissue-engineered vascular substitutes. In order to improve the thromboresistance, it is necessary to pre-coat the intra-luminal vessel surface. Recently a new surface modification technique appeared, based on the alternate adsorption of positive and negative charged polyelectrolytes. Our objective was to develop an alternative vascular scaffold made of decellularized human umbilical arteries treated with a PAH/PSS polyelectrolyte multilayered film. The vessels luminal surfaces covered with the multilayer film were observed by electronic scanning microscopy. Our observations showed that the luminal surface is completely devoid of ECs following treatment with trypsin. A top view of the coated artery indicated that the multilayer uniformly covered internal surface of the vessels. The successful of the multilayer correct deposition and retention on the arterial wall were controlled by confocal microscopy using a fluorescent polyelectrolyte (rhodamine-PAH). The data suggest that decellularized cryopreserved arteries represent a potential scaffold for further vascular tissue engineering efforts. Moreover, the multilayer films can be used to coat biological surfaces and following the terminated layer (PAH or PSS), favour the cell adhesion or cell resistance.
Assuntos
Engenharia Tecidual/métodos , Artérias Umbilicais/citologia , Artérias Umbilicais/patologia , Artérias/patologia , Sistema Cardiovascular/patologia , Adesão Celular , Criopreservação , Eletrólitos , Humanos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Modelos Químicos , Propriedades de Superfície , Tripsina/farmacologia , Veias Umbilicais/citologiaRESUMO
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: To favor regeneration following critical bone defect, a combination of autologous bone graft and biomaterials is currently used. Major drawbacks of such techniques remain the availability of the autologous material and the second surgical site, inducing pain and morbidity. OBJECTIVE: Our aim was to investigate the biocompatibility in vitro of three dimensions hybrid biodegradable scaffolds combining osteoconductive properties of hydroxyapatite and anti-inflammatory properties of chitosan. METHODS: Hybrid scaffolds were characterized by microscopic observations, equilibrium swelling ratio and overtime weight loss measurements. In vitro studies were performed using primary human bone cells cultured for 7, 14 and 21 days. Cell viability, proliferation, morphology and differentiation through alkaline phosphatase (ALP) activity measurement were assessed. RESULTS: Characterization of our scaffolds demonstrated porous, hydrophilic and biodegradable characteristics. In vitro studies showed that these scaffolds have induced slight decrease in cell death and proliferation comparing to the culture plastic substrate control condition, as well as increased short term osteoinductive properties. CONCLUSIONS: In this study, we have provided evidence that our hybrid hydroxyapatite/chitosan scaffolds could be suitable for bone filling.
Assuntos
Osso e Ossos/química , Quitosana/química , Durapatita/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis/química , Biodegradação Ambiental , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
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.
Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células , Células-Tronco Mesenquimais/fisiologia , Poliaminas/química , Poliestirenos/química , 5'-Nucleotidase/análise , Antígenos CD/análise , Antígenos CD34/análise , Cátions/química , Adesão Celular , Proliferação de Células , Forma Celular , Materiais Revestidos Biocompatíveis/química , Eletroquímica , Endoglina , Citometria de Fluxo , Proteínas Ligadas por GPI/análise , Vidro/química , Humanos , Antígenos Comuns de Leucócito/análise , Fenótipo , Polieletrólitos , Polímeros/química , Receptores de Superfície Celular/análise , Propriedades de Superfície , Antígenos Thy-1/análiseRESUMO
BACKGROUND: In tissue engineering, the endothelialization of vascular scaffold can be a crucial step to improve graft patency. A functional cellularization requires coating surfaces. Since 2003, our group used polyelectrolyte multilayer films (PEMFs) made of poly(allylamine hydrochloride) and polystyren sulfonate to coat luminal surface of blood vessel. Previous results showed that PEMFs have remarkable effect on cellular behavior: adhesion, proliferation, differentiation. However, no method seems adapted for in vitro measurement of the viscoelastic shift after PEMFs buildup. OBJECTIVE: In this present work, we proposed to use a new analytical method based on Brillouin spectroscopy (BS) to investigate the influence PEMFs coating on vessel intrinsic viscoelasticy. METHODS: On human umbilical arteries and rabbit vessels, PEMFs were buildup and the luminal surfaces viscoelasticy were measuring by BS. RESULTS: It seems that these films do not alter dynamic functionality and BS could be an interesting method for understanding the role of the tissue architecture, the interrelation between the different structures constituting the wall and the influence of this architecture on the tissue behavior, especially with the characterized components of the different vascular wall. CONCLUSION: The ability of BS to characterize biological samples opens potential applications in tissue engineering field, especially as a tool for a better understanding of vascular diseases.