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The growing demand for personalized medicine requires innovation in drug manufacturing to combine versatility with automation. Here, three-dimensional (3D) printing was explored for the production of chitosan (CH)/alginate (ALG)-based hydrogels intended as active dressings for wound healing. ALG hydrogels were loaded with 0.75% w/v silver sulfadiazine (SSD), selected as a drug model commonly used for the therapeutic treatment of infected burn wounds, and four different 3D CH/ALG architectures were designed to modulate the release of this active compound. CH/ALG constructs were characterized by their water content, elasticity and porosity. ALG hydrogels (Young's modulus 0.582 ± 0.019 Mpa) were statistically different in terms of elasticity compared to CH (Young's modulus 0.365 ± 0.015 Mpa) but very similar in terms of swelling properties (water content in ALG: 93.18 ± 0.88% and in CH: 92.76 ± 1.17%). In vitro SSD release tests were performed by using vertical diffusion Franz cells, and statistically significant different behaviors in terms of the amount and kinetics of drugs released were observed as a function of the construct. Moreover, strong antimicrobial potency (100% of growth inhibition) against Staphylococcus aureus and Pseudomonas aeruginosa was demonstrated depending on the type of construct, offering a proof of concept that 3D printing techniques could be efficiently applied to the production of hydrogels for controlled drug delivery.
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Collagens, elastin, fibrillin, decorin, and laminin are key constituents of the extracellular matrix and basement membrane of mammalian organs. Thus, changes in their quantities may influence the mechanochemical regulation of resident cells. Since maintenance of a native stromal composition is a requirement for three-dimensional (3D) matrix-based recellularization techniques in tissue engineering, we studied the influence of the decellularization detergents on these proteins in porcine kidney, liver, pancreas, and skin. Using a quick thawing/quick microwave-assisted decellularization protocol and two different detergents, sodium dodecyl sulfate (SDS) vs Triton X-100 (TX100), at identical concentration, variations in matrix conservation of stromal proteins were detected by liquid chromatography-mass spectrometry coupled to light and scanning electron microscopies, in dependence on each detergent. In all organs tested except pancreas, collagens were retained to a statistically significant level using the TX100-based protocol. In contrast fibrillin, elastin (except in kidney), and decorin (only in liver) were better preserved with the SDS-dependent protocol. Irrespective of the detergent used, laminin always remained at an irrelevant level. Our results prompt attention to the type of detergent in organ decellularization, suggesting that its choice may influence morphoregulatory inputs peculiar to the type of 3D bioartificial mammalian organ to be reconstructed. Impact statement Simple change of the protocol's main detergent leads to a very substantial difference in the panel of the stromal proteins detected by qualitative and semiquantitative mass spectrometry in acellular porcine matrices. This remarkable methodological variable promises to yield proteomic reference panels in a number of different species-specific acellular matrices allowing for selective retainment of peculiar mechanochemical inputs, to differently address the development of the seeded cells in relation to the type of organ to be bioartificially reconstructed.
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Detergentes , Alicerces Teciduais , Animais , Colágeno/metabolismo , Decorina/metabolismo , Detergentes/química , Detergentes/metabolismo , Detergentes/farmacologia , Elastina/metabolismo , Matriz Extracelular/metabolismo , Fibrilinas/metabolismo , Laminina/metabolismo , Mamíferos , Espectrometria de Massas , Octoxinol/metabolismo , Proteômica , Suínos , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three-dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro- and micro-structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin-like polypeptides (HELPs) are stimuli-responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D-printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli-responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts.
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Quitosana , Alicerces Teciduais , Humanos , Alicerces Teciduais/química , Impressão Tridimensional , Quitosana/química , Alginatos , Engenharia TecidualRESUMO
Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.
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BACKGROUND: The investigation of the interactions between cells and active materials is pivotal in the emerging 3D printing-biomaterial application fields. Here, lipidomics has been used to explore the early impact of alginate (ALG) hydrogel architecture (2D films or 3D printed scaffolds) and the type of gelling agent (CaCl2 or FeCl3) on the lipid profile of human fibroblasts. METHODS: 2D and 3D ALG scaffolds were prepared and characterized in terms of water content, swelling, mechanical resistance and morphology before human fibroblast seeding (8 days). Using a liquid chromatography-triple quadrupole-tandem mass spectrometry approach, selected ceramides (CER), lysophosphatidylcholines (LPC), lysophosphatidic acids (LPA) and free fatty acids (FFA) were analyzed. RESULTS: The results showed a clear alteration in the CER expression profile depending of both the geometry and the gelling agent used to prepare the hydrogels. As for LPCs, the main parameter affecting their distribution is the scaffold architecture with a significant decrease in the relative expression levels of the species with higher chain length (C20 to C22) for 3D scaffolds compared to 2D films. In the case of FFAs and LPAs only slight differences were observed as a function of scaffold geometry or gelling agent. CONCLUSIONS: Variations in the cell membrane lipid profile were observed for 3D cell cultures compared to 2D and these data are consistent with activation processes occurring through the mutual interactions between fibroblasts and ALG support. These unknown physiologically relevant changes add insights into the discussion about the relationship between biomaterial and the variations of cell biological functions.
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Alginatos/química , Fibroblastos/metabolismo , Metabolismo dos Lipídeos , Alicerces Teciduais/química , Alginatos/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Células Cultivadas , Ceramidas/análise , Ceramidas/metabolismo , Fibroblastos/química , Humanos , Lipídeos/análise , Impressão TridimensionalRESUMO
In this contribution we describe the preparation and characterization of a series of cross-linked films based on the combination of an elastin-derived biomimetic polypeptide (Human Elastin-Like Polypeptide, HELP) with alginate (ALG) to obtain a composite with enhanced properties. ALG/HELP composite films loaded with the hydrophobic natural antioxidant curcumin were prepared by solvent casting method followed by the cross-linking with calcium chloride. The compatibility between the two components as well as the final properties was evaluated. The micro-morphological study of films showed a homogeneous structure, but the film tensile strength decrease with HELP content and elongation at break was adversely affected by biopolymer addition. Spectroscopic and thermal analyses confirmed an interaction between ALG and HELP which also causes a modification in swelling kinetics and faster degradation. Moreover, the study of curcumin release showed a controlled delivery up to 10â¯days with a faster release rate in the presence of HELP. Human Dermal Fibroblasts (hDF) were used to test the in vitro cytocompatibility. The antioxidant activity correlated to the increase of HELP content suggested the applicability of these composites to develop smart biomaterials. Overall, these features indicated how this composite material has considerable potential as customizable platforms for various biomedical applications.
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Alginatos/química , Antioxidantes/síntese química , Curcumina/síntese química , Derme/citologia , Elastina/química , Antioxidantes/química , Antioxidantes/farmacologia , Materiais Biomiméticos/química , Varredura Diferencial de Calorimetria , Células Cultivadas , Curcumina/química , Curcumina/farmacologia , Derme/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Estrutura Molecular , Termogravimetria , Cicatrização/efeitos dos fármacosRESUMO
Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.
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Materiais Biocompatíveis/química , Quitosana/química , Colágeno Tipo IV/química , Células Epiteliais/metabolismo , Impressão Tridimensional , Rafinose/química , Alicerces Teciduais/química , Células A549 , Adesão Celular , Materiais Revestidos Biocompatíveis/química , Colágeno/química , Fluoresceínas/química , Humanos , Teste de Materiais , Microscopia Confocal , Polímeros/química , Propídio/química , Regeneração , Temperatura , Engenharia TecidualRESUMO
Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against S. aureus and P. aeruginosa was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction.
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TiO2-supported chitosan scaffolds (TiO2/CS) are here proposed as promising material for wastewater treatment, in particular for the removal of pharmaceutical compounds. TiO2/CS are tested for the amoxicillin photodegradation under UV/Vis irradiation. Amoxicillin (AMX) is an antibiotic of the beta-lactam family. Due to the release of antibiotics in wastewater and their persistence in the environment, harmful effects can develop on the aquatic and terrestrial organisms. TiO2 chitosan scaffolds with photocatalytic activity for wastewater remediation have been prepared by 3D printing using commercial P25-TiO2. The formulation for the 3D printer was prepared by dispersion of chitosan and TiO2 in powder form at the concentration 6% w/v and 1% w/v, respectively. The TiO2 particles (crystalline anatase and rutile phases) embedded in the chitosan have a size of about 20â¯nm, like in the starting material, as verified by X-ray diffraction and Raman spectroscopy and are homogeneously distributed in the scaffold, also after repeated photocatalytic tests, as revealed by SEM-EDS. The mechanical properties of the 3D structures are suitable for the targeted application as they can be easily handled without breakage. The AMX photodegradation efficiency under light irradiation by TiO2/CS made with scaffolds of different thicknesses (3, 5, 15 layers), was assessed in water by means of UV-Vis absorption and HPLC/UV measurements, at two different AMX:TiO2 molar ratios: 1/100 and 1/10. The 3D printed TiO2/CS system, even after repeated cycles, shows a high photodegradation efficiency, compared to the direct AMX photolysis. A zero-order kinetics for TiO2 supported photodegradation was found, whereas a pseudo-first order was observed for water dispersed TiO2. Mass spectrometry analysis revealed the presence of AMX degradates such as penilloic and penicilloic acids and diketopiperazine. The proposed 3D printed chitosan scaffolds may be used as reusable substrate for the TiO2 photocatalytic degradation of antibiotic pollutants in wastewater.
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Quitosana , Poluentes Químicos da Água , Amoxicilina , Catálise , Fotólise , Impressão Tridimensional , Titânio , ÁguaRESUMO
A decisive step in cell-biomaterial interaction is represented by the adsorption of proteins at the interface, whose fine control may be useful to trigger proper cell response. To this purpose, we can selectively control protein adsorption on biomaterials by means of aptamers. Aptamers selected to recognize fibronectin dramatically enhance chitosan ability to promote cell proliferation and adhesion, but the underlying biological mechanism remains unknown. We supposed that aptamers contributed to ameliorate the adsorption of fibronectin in an advantageous geometrical conformation for cells, thus regulating their morphology by the proper activation of the integrin-mediated pathway. We investigated this possibility by culturing epithelial cells on chitosan enriched with increasing doses of aptamers in the presence or in the absence of cytoskeleton pharmacological inhibitors. Our results showed that aptamers control cell morphology in a dose dependent manner (p < 0.0001). Simultaneously, when the inhibition of actin polymerization was induced, the control of cell morphology was attenuated (p < 0.0001), while no differences were detected when cells contractility was challenged (p > 0.05). Altogether, our data provide evidence that aptamers contribute to control fibronectin adsorption on biomaterials by preserving its conformation and thus function. Furthermore, our work provides a new insight into a new way to accurately tailor material surface bioactivity.
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3D biomaterial manufacturing strategies show an extraordinary driving force for the development of innovative therapies in the tissue engineering field. Here, the behaviour of 3D printed chitosan (CH)-based scaffolds was explored as a function of the post-printing gelation process. To this purpose, gel forming properties of different media were tested on their capability to retain 3D structure, water content, mechanical resistance and surface/internal porosity. Three different gelation media (i.e. KOH 1.5 M, Na2CO3 1.5 M, ammonia vapours) were selected and the 3D CH scaffolds were tested in terms of biocompatibility toward fibroblast as skin associated human cell line.
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Aptâmeros de Nucleotídeos/química , Quitosana/química , Fibronectinas/química , Osteoblastos/citologia , Alicerces Teciduais/química , Animais , Aptâmeros de Nucleotídeos/metabolismo , Adesão Celular , Proliferação de Células , Fibronectinas/metabolismo , Espectrometria de Massas , Camundongos , Osteoblastos/química , Osteoblastos/metabolismoRESUMO
Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.
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Cromatografia Líquida/métodos , Matriz Extracelular/química , Colágenos Fibrilares/análise , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Glândula Tireoide/química , Animais , Colágenos Fibrilares/química , Colágenos Fibrilares/isolamento & purificação , Limite de Detecção , Masculino , Peptídeos/isolamento & purificação , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
The fabrication of porous 3D printed chitosan (CH) scaffolds for skin tissue regeneration and their behavior in terms of biocompatibility, cytocompatibility and toxicity toward human fibroblasts (Nhdf) and keratinocytes (HaCaT), are presented and discussed. 3D cell cultures achieved after 20 and 35 days of incubation showed significant in vitro qualitative and quantitative cell growth as measured by neutral red staining and MTT assays and confirmed by scanning electron microphotographs. The best cell growth was obtained after 35 days on 3D scaffolds when the Nhdf and HaCaT cells, seeded together, filled the pores in the scaffolds. An early skin-like layer consisting of a mass of fibroblast and keratinocyte cells growing together was observed. The tests of 3D printed scaffolds in wound healing carried out on streptozotocin-induced diabetic rats demonstrate that 3D printed scaffolds improve the quality of the restored tissue with respect to both commercial patch and spontaneous healing.
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Materiais Biocompatíveis/uso terapêutico , Quitosana/uso terapêutico , Diabetes Mellitus Experimental/metabolismo , Impressão Tridimensional , Alicerces Teciduais/química , Cicatrização/fisiologia , Animais , Bandagens , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Quitosana/toxicidade , Módulo de Elasticidade , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Porosidade , Ratos Wistar , Pele/efeitos dos fármacos , Técnicas de Fechamento de FerimentosRESUMO
The augmented demand for medical devices devoted to tissue regeneration and possessing a controlled micro-architecture means there is a need for industrial scale-up in the production of hydrogels. A new 3D printing technique was applied to the automation of a freeze-gelation method for the preparation of chitosan scaffolds with controlled porosity. For this aim, a dedicated 3D printer was built in-house: a preliminary effort has been necessary to explore the printing parameter space to optimize the printing results in terms of geometry, tolerances and mechanical properties of the product. Analysed parameters included viscosity of the starting chitosan solution, which was measured with a Brookfield viscometer, and temperature of deposition, which was determined by filming the process with a cryocooled sensor thermal camera. Optimized parameters were applied to the production of scaffolds from solutions of chitosan alone or with the addition of raffinose as a viscosity modifier. Resulting hydrogels were characterized in terms of morphology and porosity. In vitro cell culture studies comparing 3D printed scaffolds with their homologous produced by solution casting evidenced an improvement in biocompatibility deriving from the production technique as well as from the solid state modification of chitosan stemming from the addition of the viscosity modifier.
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Proliferação de Células/fisiologia , Quitosana/química , Hidrogéis/química , Engenharia Tecidual/métodos , Porosidade , Impressão Tridimensional , ViscosidadeRESUMO
INTRODUCTION: The main target of tissue engineering is the preparation and application of adequate materials for the design and production of scaffolds, that possess properties promoting cell adhesion, proliferation and differentiation. The use of natural polysaccharides, such as chitosan, to prepare hydrogels for wound healing and controlled drug delivery is a research topic of wide and increasing interest. Areas covered: This review presents the latest results and challenges in the preparation of chitosan and chitosan-based scaffold/hydrogel for wound healing applications. A detailed overview of their behavior in terms of controlled drug delivery, divided by drug categories, and efficacy was provided and critically discussed. Expert opinion: The need to establish and exploit the advantages of natural biomaterials in combination with active compounds is playing a pivotal role in the regenerative medicine fields. The challenges posed by the many variables affecting tissue repair and regeneration need to be standardized and adhere to recognized guidelines to improve the quality of evidence in the wound healing process. Currently, different methodologies are followed to prepare innovative scaffold formulations and structures. Innovative technologies such as 3D printing or bio-electrospray are promising to create chitosan-based scaffolds with finely controlled structures with customizable shape porosity and thickness. Chitosan scaffolds could be designed in combination with a variety of polysaccharides or active compounds with selected and reproducible spacial distribution, providing active wound dressing with highly tunable controlled drug delivery.
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Quitosana/administração & dosagem , Sistemas de Liberação de Medicamentos , Hidrogéis/administração & dosagem , Cicatrização , Animais , Quitosana/química , Humanos , Hidrogéis/químicaRESUMO
RATIONALE: Drug development efforts involving therapeutic peptides or proteins strongly lead optimization of drug delivery, drug stability, solubility and functionality. The key feature of controlled drug delivery is the use of biocompatible polymers able to interact via non-covalent bonds with an active principle through multiple functional groups. Here amide hydrogen/deuterium exchange (HDX) mass spectrometry was employed to localize insulin dynamics induced by interactions with three natural polysaccharides, i.e. chitosan (CH), sodium alginate (ALG) and chondroitin sulfate (CS). METHODS: LTQ-Orbitap continuous-labelling mass spectra were collected by diluting insulin stock solution (10 mM in 0.1% formic acid) to a final concentration of 0.1 mM in D2 O containing 1 mM deuterated ammonium acetate (final pH .6) (insulin:polysaccharide ratio 1:2, w/w). For peptide mapping, deuterated samples were quenched after 0.5, 30, 60, 120 minutes exchange by adding HCl (pH ) and digested with pepsin before LC-MS/MS analysis. RESULTS: Differences in the insulin backbone dynamics in the presence of the three polysaccharides were highlighted by monitoring peptic peptides at different time points. No significant differences were observed in the presence of CH, whereas the negatively charged ALG and CS were able to induce significant conformational variations at the B-chain level resulting in more protection against H/D exchange. The A-chain interacted only with CS reducing the protein mobility on a long time scale (120 min). HDX data evidenced heterogeneous insulin dynamics in the presence of ALG and CS. CONCLUSIONS: The studies reported here demonstrated the capabilities of mass spectrometry techniques and HDX methods to obtain useful information toward the flexibility and the behavior of native insulin in the presence of natural polysaccharides, and could provide insights to study the behavior of pharmaceutical formulations. Copyright © 2016 John Wiley & Sons, Ltd.