RESUMEN
Biomaterials serve as an integral component of tissue engineering. They are designed to provide architectural framework of native extracellular matrix so as to encourage cell growth and eventual tissue regeneration. Naturally occurring biopolymers as scaffolds offer options for cartilage tissue engineering due to anti-inflammatory, biocompatibility, biodegradability, low toxicity of degradation by-products and plasticity in processing into a variety of material formats. Here we studied in vitro anti-inflammatory potential of marine macromolecules cross-linked bio-composite scaffold composed of hydroxyapatite, alginate, chitosan and fucoidan named as HACF on LPS stimulated RAW 264.7 macrophage cells. The effects of HACF on the viability of RAW264.7 cells, nitrite level, intracellular ROS as well as the mRNA levels of NF-κB, iNOS, COX-2, TNF-α, IL-1ß and IL-6 were examined in LPS induced RAW264.7 macrophage cells. The results revealed that HACF hydrogel scaffold exerts anti-inflammatory effect by inhibiting the production of ROS, suppress NF-kB translocation to the nucleus and thereby inhibiting the production of inflammatory mediators. Hence, our results confirm that HACF has a strong anti-oxidant capacity to inhibit inflammation associated gene expression by suppressing NF-kB signaling pathway. It clearly reveals the anti-oxidant and anti-inflammatory effect of HACF hydrogel scaffold on LPS induced RAW 264.7 cells.
Asunto(s)
Lipopolisacáridos , Ingeniería de Tejidos , Animales , Antiinflamatorios/farmacología , Cartílago , Macrófagos , Ratones , FN-kappa B , Óxido Nítrico , Células RAW 264.7RESUMEN
There is an intense interest in developing innovative biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Present study demonstrated the in vivo biocompatibility and toxicity of a macromolecules cross-linked biocomposite scaffold composed of hydroxyapatite, alginate, chitosan and fucoidan abbreviated as HACF. The in vivo biocompatibility and toxicity of HACF scaffold were tested by comparing them with those of a biocompatible surgical metal implant (SMI) in a subcutaneous rat model. Following the implantation, animals were sacrificed and the scaffolds were resected at 1st, 4th, and 8th weeks; the surrounding tissue along with the implant was removed to evaluate its biocompatibility. The effects of implanted biomaterial scaffolds on vital organ systems such as liver, kidney, etc., have been studied by hematology and serum biochemistry. The activities of pro-inflammatory marker enzymes such as COX, 5-LOX, 15-LOX, and NOS were normal in rats implanted with HACF scaffold. Hematological parameters, antioxidant and lipid peroxidation status were also found to be normal in implanted rats same as that of control and SMI. The modulatory effect of implanted scaffold over inflammatory and stress signaling cascades were confirmed by the normalized mRNA expressions of NF-κB, TNF-α and IL-6. The histopathological analysis of liver, kidney and tissue support our results. Taken together, these results demonstrated that HACF biocomposite scaffold signifies its suitability for further research as a scaffold material for cartilage tissue engineering applications.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Cartílago/citología , Ensayo de Materiales , Ingeniería de Tejidos , Alginatos/química , Animales , Antioxidantes/metabolismo , Cartílago/efectos de los fármacos , Quitosano/química , Durapatita/química , Glutatión/metabolismo , Mediadores de Inflamación/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Masculino , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Polisacáridos/química , RatasRESUMEN
Marine biopolymer composite materials provide a technological platform for launching biomedical applications. Biomaterials demand good biocompatibility without the possibility of inflammation or foreign body reactions. In this study, we prepared two biocomposite hydrogels namely; HAC (hydroxyapatite, alginate & chitosan) and HACF (hydroxyapatite, alginate, chitosan & fucoidan) followed by calcium chloride cross linking. The prepared scaffolds were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Porosity measurement, swelling, biodegradation, hemolysis, RBC aggregation, plasma protein adsorption and cytotoxicity studies were also done. The hydrogel scaffold HACF possessed a well-defined porous architecture, sufficient water holding capacity, better hemocompatibility and biodegradability. The biocompatibility was confirmed through in vitro cytotoxicity studies such as MTT assay, Neutral red uptake, DAPI staining, Trypan blue dye exclusion test and direct contact assay in L929 mouse fibroblast cells. In addition, immunomodulatory and anti-inflammatory properties of both of these scaffolds were revealed by the mRNA expressions of major inflammatory marker genes in cytotoxic condition such as TNF-α, IL-6 and NF-κB. The physiochemical characterization and biological responses of HACF hydrogel signifies its suitability for various tissue engineering applications.