RESUMEN
Alkaline phosphatase (ALP) is an essential biomarker of osteoblastic activity. Currently, ALP activity has been used to study bone mineralization mechanisms and osteoactive biomaterials among others. The ALP quantification is usually performed by destructive methods either on growing cells or cells lysate in which the osteoconductive biomaterial is being assessed. This work addresses the evaluation of a non-destructive colorimetric approach for the determination of ALP activity on osteoblast-derived exosomes from culture supernatants. The efficiency of the method was evaluated on osteoconductive electrospun scaffolds of PCL compounded with ZnO as a reference biomaterial. The results demonstrated that the osteoblast cell line mineralization induced by osteoconductive scaffolds can be monitorized over time by the non-destructive measurement of ALP activity on osteoblast derived exosomes. Consequently, this non-destructive approach suggested to be a reliable alternative technique for the quantification of biomaterials osteoconductivity or even evaluation of osteoblastic response at stem cells.
Asunto(s)
Fosfatasa Alcalina/análisis , Medios de Cultivo/análisis , Exosomas/metabolismo , Osteoblastos/citología , Desarrollo Óseo , Regeneración Ósea , Calorimetría , Técnicas de Cultivo de Célula , Línea Celular , Humanos , Osteoblastos/metabolismoRESUMEN
Up to date, tissue regeneration of large bone defects is a clinical challenge under exhaustive study. Nowadays, the most common clinical solutions concerning bone regeneration involve systems based on human or bovine tissues, which suffer from drawbacks like antigenicity, complex processing, low osteoinductivity, rapid resorption and minimal acceleration of tissue regeneration. This work thus addresses the development of nanofibrous synthetic scaffolds of polycaprolactone (PCL) - a long-term degradation polyester - compounded with hydroxyapatite (HA) and variable concentrations of ZnO as alternative solutions for accelerated bone tissue regeneration in applications requiring mid- and long-term resorption. In vitro cell response of human fetal osteoblasts as well as antibacterial activity against Staphylococcus aureus of PCL:HA:ZnO and PCL:ZnO scaffolds were here evaluated. Furthermore, the effect of ZnO nanostructures at different concentrations on in vitro degradation of PCL electrospun scaffolds was analyzed. The results proved that higher concentrations ZnO may induce early mineralization, as indicated by high alkaline phosphatase activity levels, cell proliferation assays and positive Alizarin-Red-S-stained calcium deposits. Moreover, all PCL:ZnO scaffolds particularly showed antibacterial activity against S. aureus which may be attributed to release of Zn2+ ions. Additionally, results here obtained showed a variable PCL degradation rate as a function of ZnO concentration. Therefore, this work suggests that our PCL:ZnO scaffolds may be promising and competitive short-, mid- and long-term resorption systems against current clinical solutions for bone tissue regeneration.
Asunto(s)
Antibacterianos , Huesos/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Osteoblastos/metabolismo , Poliésteres/química , Staphylococcus aureus/crecimiento & desarrollo , Ingeniería de Tejidos , Andamios del Tejido/química , Óxido de Zinc , Antibacterianos/química , Antibacterianos/farmacología , Huesos/citología , Línea Celular , Humanos , Osteoblastos/citología , Óxido de Zinc/farmacologíaRESUMEN
Nowadays, soft tissue restoration techniques are mainly focused on volume regeneration instead of function recovering. So far, autologous fat transplant has been the most popular method although its multiple reported problems like volume and function loss. Adipose tissue engineering therefore emerges as a solution for development of biological substitutes for soft tissue which promotes not only volume regeneration but also function restoration with minimal consequences. Here we tested fibrous-structured atelocollagen (FSA) scaffolds and honeycomb atelocollagen (HCA) scaffolds for their ability to induce adipogenesis in vivo. Implants were subjected to histological and immunohistochemical assessment after 1, 2, and 4 weeks of implantation. Our studies showed that FSA scaffolds induced in vivo a markedly adipogenic response, whereas an acute inflammatory process was observed at HCA scaffolds without tissue regeneration detected within them. Our histological findings concerning FSA scaffolds clearly showed the presence of adipose-like tissue surprisingly composed by a mixture of brown-like and white-like adipocytes at week 2 whereas only white-like adipocytes at week 4. Subsequent positive Pax7 immunostaining at weeks 1 and 2 suggested the existence of a common myogenic progenitor shared by brown-like and white-like adipocytes observed. Then, in this work we present FSA scaffolds as a promising structure for brown and white adipose tissue engineering.
Asunto(s)
Colágeno/química , Andamios del Tejido/química , Adipogénesis , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Antígenos CD34/metabolismo , Diferenciación Celular , Colágeno/ultraestructura , Ratones , Ratones SCID , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Factor de Transcripción PAX7/metabolismo , Ingeniería de TejidosRESUMEN
Advanced engineering of materials for the development of drug delivery devices provides scope for novel and versatile strategies for treatment of various diseases. 'Electrospraying' was used to prepare PLGA microparticles and further encapsulate the drug, metronidazole (Met) within the particles to function as a drug delivery system. Two different solvents were utilized for the preparation of drug loaded PLGA particles, whereby the polymeric solution in dichloromethane (DCM) produced particles of bigger sizes than using trifluoroethanol (TFE). Scanning electron microscopy showed the spherical morphology of the particles, with sizes of 3946±407nm and 1774±167nm, respectively for PLGA-Met(DCM) and PLGA-Met(TFE). The FTIR spectroscopy proved the incorporation of metronidazole in the polymer, but without any specific drug-polymer interaction. The release of the drug from the particles was studied in phosphate buffered saline, where a sustained drug release was obtained for at least 41days. Cytotoxicity evaluation of the drug extract using mesenchymal stem cells (MSCs) showed not hindering the proliferation of MSCs, and the cell phenotype was retained after incubation in the drug containing media. Electrospraying is suggested as a cost-effective and single step process for the preparation of polymeric microparticles for prolonged and controlled release of drug.
Asunto(s)
Ácido Láctico/química , Metronidazol/química , Ácido Poliglicólico/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Cloruro de Metileno/química , Metronidazol/farmacología , Microscopía Electrónica de Rastreo/métodos , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Solventes/química , Propiedades de Superficie , Trifluoroetanol/químicaRESUMEN
Nanoengineered drug delivery systems (nDDS) have been successfully used as clinical tools for not only modulation of pharmacological drug release profile but also specific targeting of diseased tissues. Until now, encapsulation of anti-cancer molecules such as paclitaxel, vincristin and doxorubicin has been the main target of nDDS, whereby liposomes and polymer-drug conjugates remained as the most popular group of nDDS used for this purpose. The success reached by these nanocarriers can be imitated by careful selection and optimization of the different factors that affect drug release profile (i.e. type of biomaterial, size, system architecture, and biodegradability mechanisms) along with the selection of an appropriate manufacture technique that does not compromise the desired release profile, while it also offers possibilities to scale up for future industrialization. This review focuses from an engineering perspective on the different parameters that should be considered before and during the design of new nDDS, and the different manufacturing techniques available, in such a way to ensure success in clinical application.