RESUMO
The polarization of macrophages M0 to M1 or M2 using molecules embedded in matrices and hydrogels is an active field of study. The design of biomaterials capable of promoting polarization has become a paramount need nowadays, since in the healing process macrophages M1 and M2 modulate the inflammatory response. In this work, several immunocytochemistry and ELISA tests strongly suggest the achievement of polarization using collagen-based membranes crosslinked with tri-functionalized oligourethanes and coated with silica. Measuring the amount of TGF-ß1 secreted to culture media by macrophages growth on these materials, and quantifying the macrophage morphology, it is proved that it is possible to stimulate the anti-inflammatory pathway toward M2, having measurements with p ≤ 0.05 of statistical significance between the control and the collagen-based membranes. Furthermore, some physicochemical characteristics of the hybrid materials are tested envisaging future applications: collagenase degradation resistance, water uptake, collagen fiber diameter, and deformation resistance are increased for all the crosslinked biomaterials. It is considered that the biological and physicochemical properties make the material suitable for the modulation of the inflammatory response in the chronic wounds and promising for in vivo studies.
Assuntos
Materiais Biocompatíveis/química , Colágeno/química , Inflamação/patologia , Macrófagos/metabolismo , Membranas Artificiais , Animais , Polaridade Celular , Reagentes de Ligações Cruzadas/química , Citocinas/metabolismo , Isocianatos/química , Lisina/análogos & derivados , Lisina/química , Macrófagos/patologia , Camundongos , Poliuretanos/síntese química , Poliuretanos/química , Células RAW 264.7 , Ratos Wistar , Dióxido de Silício/químicaRESUMO
The hydrogels of natural extracellular matrix (ECM) are excellent biomaterials with promising applications in the physiological manufacture of three-dimensional (3D) constructs that replicate native tissue-like architectures and function as cargo-delivery, 3D bioprinting, or injectable systems. ECM hydrogels retain the bioactivity to trigger key cellular processes in the tissue engineering and regenerative medicine (TERM) strategies. However, they lack suitable physicochemical properties, which restricts their applications in vivo. This demand that mechanical and degradation properties of the ECM hydrogels must be balanced against biological properties. By incorporating poly(ethylene glycol) (PEG) into mammalian type I collagen-rich ECM substrates, this task can be accomplished. This review is focused on the use of PEG derivatives, widely used in formulations of pharmaceutical products or in synthesis of biomedical polyurethanes, as a strategy to modulate both physical and biological properties of natural ECM hydrogels. The processing-property relationship in decellularized ECM hydrogels, as well as the main results when used in TERM, are discussed. A comparison of the characteristics of PEG-ECM hydrogels is provided in terms of the improvement of structure, mechanics, and degradation behavior. Finally, the benefits of producing PEG-ECM hydrogels according to in vitro and in vivo performance in different proofs-of-concept of emergent biomedical technologies are overviewed.
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In this work, hydrolysates of extracellular matrix (hECM) were obtained from rat tail tendon (TR), bovine Achilles tendon (TAB), porcine small intestinal submucosa (SIS) and bovine pericardium (PB), and they were polymerized to generate ECM hydrogels. The composition of hECM was evaluated by quantifying the content of sulphated glycosaminoglycans (sGAG), fibronectin and laminin. The polymerization process, structure, physicochemical properties, in vitro degradation and biocompatibility were studied and related to their composition. The results indicated that the hECM derived from SIS and PB were significantly richer in sGAG, fibronectin and laminin, than those derived from TAB and TR. These differences in hECM composition influenced the polymerization and the structural characteristics of the fibrillar gel network. Consequently, the swelling, mechanics and degradation of the hydrogels showed a direct relationship with the remaining composition. Moreover, the cytocompatibility and the secretion of transforming growth factor beta-1 (TGF-ß1) by macrophages were enhanced in hydrogels with the highest residual content of ECM biomolecules. The results of this work evidenced the role of the ECM molecules remaining after both decellularization and hydrolysis steps to produce tissue derived hydrogels with structure and properties tailored to enhance their performance in tissue engineering and regenerative medicine applications.
Assuntos
Hidrogéis/química , Animais , Bovinos , Matriz Extracelular , Glicosaminoglicanos , Humanos , Laminina , Ratos , Engenharia TecidualRESUMO
The extracellular matrix molecules remaining in bioscaffolds derived from decellularized xenogeneic tissues appear to be important for inducing cell functions conducting tissue regeneration. Here, we studied whether decellularization methods, that is, detergent Triton X-100 (TX) alone and TX combined with reversible alkaline swelling (STX), applied to bovine pericardial tissue, could affect the bioscaffold components. The in vitro macrophage response, subdermal biodegradation, and cell infiltration were also studied. The results indicate a lower leaching of fibronectin, but a higher leaching of laminin and sulfated glycosaminoglycans from tissues decellularized with STX and TX, respectively. The in vitro secretion of interleukin-6 and monocyte chemoattractant protein by RAW264.7 macrophages is promoted by decellularized bioscaffold leachates. A lower polymorphonuclear cell density is observed around decellularized bioscaffolds at 1-day implantation; concurrently showing a higher cell infiltration in STX- than in TX-implant. Cells infiltrated into TX-implant show a fibroblastic morphology at 7-day implantation, concurrently the capillary formation is observed at 14-day. Pericardial bioscaffolds suffer biodegradation more pronounced in STX- than in TX-implant. Both TX and STX decellularization methods favor a high leaching of basal lamina components, which presumably promotes a faster macrophage stimulation compared to nondecellularized tissue, and appear to be associated with an increased host cell infiltration in a rat subdermal implantation. Meanwhile, the connective tissue components leaching from TX decellularized bioscaffolds, unlike the STX ones, appear to be associated with an enhanced angiogenesis accompanied by an early-promoted fibroblastic cell transition. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2810-2822, 2016.
Assuntos
Bioprótese , Macrófagos/imunologia , Pericárdio/química , Pericárdio/citologia , Alicerces Teciduais/química , Animais , Bioprótese/efeitos adversos , Bovinos , Citocinas/análise , Citocinas/imunologia , Detergentes/química , Macrófagos/citologia , Camundongos , Pericárdio/imunologia , Células RAW 264.7 , Ratos Wistar , Engenharia Tecidual , Alicerces Teciduais/efeitos adversosRESUMO
This paper reports a new method to modify hydrogels derived from the acellular extracellular matrix (ECM) and consequently to improve their properties. The method is comprised of the combination of liquid precursors derived from hydrolyzed acellular small intestinal submucosa (hECM) and water-soluble oligourethanes that bear protected isocyanate groups, synthesized from poly(ethylene glycol) (PEG) and hexamethylene diisocyanate (HDI). The results demonstrate that the reactivity of oligourethanes, along with their water solubility, properly induce simultaneously the polymerization of type I collagen and its crosslinking. The polymerization rate and the gel network parameters such as fiber diameter, porosity, crosslinking degree, mechanics, swelling, in vitro degradation and cell proliferation, keep a direct relationship with the oligourethane concentration. Consequently, the hybrid hydrogels formulated with 15 wt.% of oligourethane exhibit enhanced storage modulus and degradation resistance, while maintaining the cell viability and impeding the fibroblast-induced contraction in comparison with the hECM hydrogels without oligourethanes. Therefore, this method is adequate to prepare novel hydrogels where the adjustment of the crosslinking degree controls the materials structure and their properties. This new method offers advantages for regulating the features of ECM-derived templates, thereby extending their possibilities for tissue engineering (TE) applications.
Assuntos
Matriz Extracelular/química , Hidrogéis/química , Uretana/química , Animais , Proliferação de Células , Colágeno/química , Fibroblastos/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Isocianatos/química , Macrófagos/metabolismo , Camundongos , Ninidrina/química , Polietilenoglicóis/química , Reologia , Engenharia TecidualRESUMO
This paper reports the structure-property relationship of novel biomedical hydrogels derived from collagen, water-soluble oligourethanes, and silica. The molecular weight (MW) of oligourethanes, synthesized from polyoxyethylene diol and hexamethylene, l-lysine, isophorone or trimethylhexamethylene diisocyanates (P(HDI), P(LDI), P(IPDI) and P(TMDI), respectively), is determined by the chemical structure of the starting aliphatic diisocyanate. Thus, the collagen polymerization process and both the characteristics and mechanics of the formed three-dimensional (3D) network had a direct relation with the oligourethane MW. The crosslinking of collagen with oligourethanes was compatible with orthosilicate polycondensation to deposit silica particles on the fibrillar 3D network. A higher crosslinking index was found in hydrogels formulated with P(HDI) and P(LDI) in comparison with P(TMDI) and P(IPDI). In spite of similar crosslinking extensions, P(LDI) induced an enhanced water uptake and enhanced susceptibility to degradation, contrary to the impact of P(HDI). Fibroblasts and macrophages cultured for 3 days on hydrogels formulated with P(LDI) showed a metabolic activity similar to collagen only hydrogels. However, we observed the highest cell metabolic activity on hydrogels formulated with P(LDI) after 7 day culture. After this time lapse, an enhanced secretion of chemoattractant cytokines transforming growth factor-beta1 (TGF-ß1) and monocyte chemoattractant protein-1 (MCP-1 or CCL-2) was noted in macrophages cultured on hydrogels crosslinked with P(LDI). These tunable composite collagen hydrogels might be excellent candidates for holding and releasing bioactive molecules and nanomaterials intended to regulate cell behavior via their constituents and properties.
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This paper describes the preparation and characterization of water-soluble urethane oligomers bearing protected isocyanate groups. It also points out its ability to crosslink decellularized pericardium, as a model collagen scaffold, and to adjust their structural characteristics. A library of oligourethanes was synthesized by varying the molecular weight (Mw 400, 600, 1000 or 2000 g mol-1) of the poly(ethylene glycol) and the type of aliphatic diisocyanate (isophorone diisocyanate/IPDI or hexamethylene diisocyanate/HDI). 1H and 13C NMR, FTIR and mass spectrometry demonstrated that the crosslinkers are composed of chains with carbamoylsulfonate end groups that have trimeric and pentameric oligourethanes, and monomeric diisocyanate. The degree of crosslinking and hence the in vitro degradation susceptibility of the decellularized pericardium were inversely related to the Mw of the oligourethanes. The toxicity of the extractable products from oligourethane-collagen materials toward fibroblasts and macrophages was found to be lower for the crosslinker derived from IPDI than for those derived from HDI. On the other hand, the resistance to collagenase or oxidative degradation of the bovine pericardium crosslinked with HDI/oligourethane was higher than the one prepared with IPDI/oligourethane. As the Mw of the oligomers regulates the degree of crosslinking while the chemical composition influences the cytocompatibility and biodegradation of decellularized pericardium, these urethane oligomers can be used as safer crosslinkers for other protein-based biomaterials.
RESUMO
The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167±0.14 mmol/g of liberated amine groups vs. 598±60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Teste de Materiais , Fenômenos Mecânicos/efeitos dos fármacos , Pericárdio/efeitos dos fármacos , Poliuretanos/farmacologia , Água/química , Animais , Cálcio/metabolismo , Varredura Diferencial de Calorimetria , Bovinos , Módulo de Elasticidade/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Glutaral/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Pericárdio/ultraestrutura , Fósforo/metabolismo , Estresse Mecânico , Temperatura , Resistência à Tração/efeitos dos fármacos , Fatores de TempoRESUMO
Bovine pericardium is a collagenous tissue commonly used as a natural biomaterial in the fabrication of cardiovascular devices. For tissue engineering purposes, this xenogeneic biomaterial must be decellularized to remove cellular antigens. With this in mind, three decellularization protocols were compared in terms of their effectiveness to extract cellular materials, their effect on glycosaminoglycan (GAG) content and, finally, their effect on tensile biomechanical behavior. The tissue decellularization was achieved by treatment with t-octyl phenoxy polyethoxy ethanol (Triton X-100), tridecyl polyethoxy ethanol (ATE) and alkaline treatment and subsequent treatment with nucleases (DNase/RNase). The quantified residual DNA content (3.0±0.4%, 4.4±0.6% and 5.6±0.7% for Triton X-100, ATE and alkaline treatment, respectively) and the absence of nuclear structures (hematoxylin and eosin staining) were indicators of effective cell removal. In the same way, it was found that the native tissue GAG content decreased to 61.6±0.6%, 62.7±1.1% and 88.6±0.2% for Triton X-100, ATE and alkaline treatment, respectively. In addition, an alteration in the tissue stress relaxation characteristics was observed after alkaline treatment. We can conclude that the three decellularization agents preserved the collagen structural network, anisotropy and the tensile modulus, tensile strength and maximum strain at failure of native tissue.