RESUMO
This study describes the development of scaffolds based on carboxyethyl chitosan (CEC) and different oxidized cashew gums (CGOx) for tissue engineering (TE) applications. After the physico-chemical characterizations of CEC and CGOx (oxidation degree of 20, 35 and 50%), these macromolecules were used for producing the CGOx-CEC hydrogels through a Schiff base reaction, in the absence of any crosslinking agent. The CGOx-CEC scaffolds obtained after a freeze-drying process were characterized for their morphology, mechanical properties, swelling ability, degradation, and porosity. Those revealed to be highly porous (25-65%), and showed a stable swelling behavior, as well as degradation properties in the absence of enzymes. The use of the cashew gum with higher degree of oxidation led to scaffolds with higher crosslinking densities and increased compressive modulus. None of the hydrogels show cytotoxicity during the 14 days of incubation. Considering all the properties mentioned, these scaffolds are excellent candidates for soft tissue regeneration, owing to the use of eco-friendly starting materials and the easy tuning of their properties.
Assuntos
Quitosana/análogos & derivados , Gomas Vegetais/química , Alicerces Teciduais/química , Anacardium/química , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Fenômenos Biomecânicos , Linhagem Celular , Quitosana/síntese química , Quitosana/química , Força Compressiva , Reagentes de Ligações Cruzadas , Hidrogéis , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Estrutura Molecular , Oxirredução , Gomas Vegetais/síntese química , Gomas Vegetais/toxicidade , Porosidade , Engenharia Tecidual , Alicerces Teciduais/efeitos adversosRESUMO
Two scaffolds of chitosan/sodium alginate/hydroxyapatite (Ch/NaAlg/Hap) 1:1:0.2 and 1:1:0.6 were evaluated in the prevention of bisphosphonate-induced jaw osteonecrosis. Two groups of rats (n = 24, according to the euthanasia time: 15 or 30 days after the last Zoledronic acid (ZA) administration) were subdivided in four subgroups (n = 6): I - Control (saline + teeth extraction); II - ZA 0.6 mg/kg + teeth extraction; III - ZA + teeth extraction + scaffold 1:1:0.2; IV - ZA + teeth extraction + scaffold 1:1:0.6. Jaws were evaluated histologically and blood was evaluated for hematological and biochemical parameters. Histopathology showed significant osteonecrosis in AZ group. The scaffold's implantation, despite the inflammatory process, were able to prevent the osteonecrosis. In the 15-day euthanasia group, an increase in red blood cells and platelets was observed in the subgroup II. Hemoglobin and hematocrit decreased in subgroup IV compared to II. Hepatic transaminases and creatinine concentration increased significantly in subgroup II. Calcium concentration increased in subgroup IV compared to II. In the 30-day euthanasia group, no differences among the groups were observed for any parameter. Scaffolds proved to be efficient and safe to liver and kidney function. Some hematological parameters were altered by the scaffold, but returned to normal concentrations over time. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1799-1808, 2018.
Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/prevenção & controle , Durapatita/química , Durapatita/farmacologia , Alicerces Teciduais/química , Animais , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/metabolismo , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/patologia , Modelos Animais de Doenças , Durapatita/efeitos adversos , Masculino , Ratos , Ratos Wistar , Alicerces Teciduais/efeitos adversos , Extração Dentária , Ácido Zoledrônico/efeitos adversos , Ácido Zoledrônico/farmacologiaRESUMO
This study was designed to evaluate bone matrix gelatin (BMG)/fibrin glue and chitosan/gelatin composite scaffolds for cartilage tissue engineering. Chondrocytes were isolated from costal cartilage of Sprague-Dawley rats and seeded on BMG/fibrin glue or chitosan/gelatin composite scaffolds. After different in vitro culture durations, the scaffolds were subjected to hematoxylin and eosin, Masson's trichrome, and toluidine blue staining, anti-collagen II and anti-aggrecan immunohistochemistry, and scanning electronic microscopy (SEM) analysis. After 2 weeks of culture, chondrocytes were distributed evenly on the surfaces of both scaffolds. Cell numbers and the presence of extracellular matrix components were markedly increased after 8 weeks of culture, and to a greater extent on the chitosan/gelatin scaffold. The BMG/fibrin glue scaffold showed signs of degradation after 8 weeks. Immunofluorescence analysis confirmed higher levels of collagen II and aggrecan using the chitosan/gelatin scaffold. SEM revealed that the majority of cells on the surface of the BMG/fibrin glue scaffold demonstrated a round morphology, while those in the chitosan/gelatin group had a spindle-like shape, with pseudopodia. Chitosan/gelatin scaffolds appear to be superior to BMG/ fibrin glue constructs in supporting chondrocyte attachment, proliferation, and biosynthesis of cartilaginous matrix components.
Assuntos
Condrócitos/efeitos dos fármacos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Adesivos/efeitos adversos , Agrecanas/genética , Agrecanas/metabolismo , Animais , Matriz Óssea/química , Adesão Celular , Células Cultivadas , Quitosana/efeitos adversos , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Fibrina/efeitos adversos , Gelatina/efeitos adversos , Ratos , Ratos Sprague-Dawley , Alicerces Teciduais/efeitos adversosRESUMO
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
The aim of this study was to investigate the abilities of cartilage-derived morphogenetic protein 1 (CDMP1) transgenic cell sheets in repairing rabbit cartilage defects. Rabbit CDMP1 transgenic bone marrow mesenchymal stem cell (BMSC) sheets (CDMP1-BMSCs) were cultured on temperature-sensitive culture dishes, and CDMP1 expression and type II collagen protein in the cell sheets were detected. Tissue-engineered cell sheets were constructed and transplanted into defect rabbit thyroid cartilage, to investigate the expression of engineered cartilage collagen protein and proteoglycan (GAG). The experiment was divided into three groups; A) BMSC sheet, B) Ad-CMV-eGFP-transfected cell sheet, and C) Ad-CMV-hCDMP1-IRES-eGFP-transfected cell sheet. The expression of CDMP1 was detected in the transgenic cell sheets. The engineered cartilage exhibited positive immunohistochemical and Alcian blue staining. The expression levels of type II collagen protein and GAG in group A were positive, whereas those in group B and group C were negative (P < 0.05). The CDMP1-BMSC sheets had a good cartilage differentiation activity, and could effectively repair rabbit laryngeal cartilage defects.
Assuntos
Cartilagem/fisiologia , Fator 5 de Diferenciação de Crescimento/genética , Transplante de Células-Tronco Mesenquimais , Regeneração , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Cartilagem/citologia , Células Cultivadas , Feminino , Fator 5 de Diferenciação de Crescimento/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Coelhos , Alicerces Teciduais/efeitos adversosRESUMO
In recent years, there has been an increasing interest in the design of biomaterials for cartilage tissue engineering. This type of materials must meet several requirements. In this study, we apply ultrasound to prepare a compatibilized blend of polyelectrolyte complexes (PEC) based on carboxymethyl cellulose (CMC) and chitosan (CHI), in order to improve stability and mechanical properties through the inter-polymer macroradicals coupling produced by sonochemical reaction. We study the kinetic of the sonochemical degradation of each component in order to optimize the experimental conditions for PEC compatibilization. Scaffolds obtained applying this methodology and scaffolds without ultrasound processing were prepared and their morphology (by scanning electron microscopy), polyelectrolyte interactions (by FTIR), stability and mechanical properties were analyzed. The swelling kinetics was studied and interpreted based on the structural differences between the two kinds of scaffolds. In addition we evaluate the possible in vitro cytotoxicity of the scaffolds using macrophage cells in culture. Our results demonstrate that the ultrasound is a very efficient methodology to compatibilize PEC, exhibiting improved properties compared with the simple mixture of the two polysaccharides. The test with murine macrophage RAW 264.7 cells showed no evince of cytotoxicity, suggesting that PEC biomaterials obtained under ultrasound conditions could be useful in the cartilage tissue engineering field.
Assuntos
Materiais Biocompatíveis/química , Tecnologia Biomédica/métodos , Carboximetilcelulose Sódica/química , Quitosana/química , Alicerces Teciduais/química , Ondas Ultrassônicas , Animais , Materiais Biocompatíveis/toxicidade , Carboximetilcelulose Sódica/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quitosana/toxicidade , Força Compressiva , Elasticidade , Macrófagos/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Varredura , Propriedades de Superfície , Alicerces Teciduais/efeitos adversos , ViscosidadeRESUMO
We prepared a bone scaffold material comprising a PLGA/ß-TCP core and a Type I collagen cladding, and recombined it with bone marrow stroma stem cells (BMSCs) to evaluate its potential for use in bone tissue engineering by in vivo and in vitro experiments. PLGA/ß-TCP without a cladding was used for comparison. The adherence rate of the BMSCs to the scaffold was determined by cell counting. Cell proliferation rate was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The osteogenic capability was evaluated by alkaline phosphatase activity. The scaffold materials were recombined with the BMSCs and implanted into a large segmental rabbit radial defect model to evaluate defect repair. Osteogenesis was assessed in the scaffold materials by histological and double immunofluorescence labeling, etc. The adherence number, proliferation number, and alkaline phosphatase expression of the cells on the bone scaffold material with core-cladding structure were significantly higher than the corresponding values in the PLGA/ß-TCP composite scaffold material (P < 0.05). An in vivo test indicated that the bone scaffold material with core-cladding structure completely degraded at the bone defect site and bone formation was completed. The rabbit large sentimental radial defect was successfully repaired. The degradation and osteogenesis rates matched well. The bone scaffold with core-cladding structure exhibited better osteogenic activity and capacity to repair a large segmental bone defect compared to the PLGA/ß-TCP composite scaffold. The bone scaffold with core-cladding structure has excellent physical properties and biocompatibility. It is an ideal scaffold material for bone tissue engineering.
Assuntos
Regeneração Óssea , Células-Tronco Mesenquimais/citologia , Alicerces Teciduais/química , Animais , Fosfatos de Cálcio/efeitos adversos , Fosfatos de Cálcio/farmacologia , Proliferação de Células , Células Cultivadas , Ácido Láctico/efeitos adversos , Ácido Láctico/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteogênese , Ácido Poliglicólico/efeitos adversos , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Rádio (Anatomia)/citologia , Rádio (Anatomia)/fisiologia , Engenharia Tecidual , Alicerces Teciduais/efeitos adversosRESUMO
There is considerable interest in the design of polymeric biomaterials that can be used for the repair of bone defects. In this study, we used ultrasound to prepare a compatibilized blend of poly(epsilon-caprolactone) (PCL) and poly(diisopropyl fumarate) (PDIPF). The formation of post-sonication inter-polymer coupling products was verified by SEC analysis of a blend with azo-labeled PDIPF. We also analyzed the physicochemical and mechanical properties of the compatibilized blend. When compared to PCL alone, the PCL/PDIPF blend showed no difference in its resistance as evaluated by the elastic modulus, although it did show a 50% decrease in ultimate tensile stress (P < 0.05) and an 84% decrease in elongation-at-break (P < 0.05). However, the mechanical properties of this blend were comparable to those of trabecular bone. We next evaluated biocompatibility of the PCL/PDIPF blend, and of homo-polymeric PCL and PDIPF films for comparison, with UMR106 and MC3T3E1 osteoblastic cells. Osteoblasts plated on the compatibilized blend adhered and proliferated more than on either homo-polymer, showed a greater number of cellular processes with a better organized actin cytoskeleton and expressed more type-I collagen and mineral, both markers of osteoblast phenotype. These results support the hypothesis that this new compatibilized blend could be useful in future applications for bone regeneration.