Nejvíce citovaný článek - PubMed ID 24375970
Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells
Modern tissue engineering requires not only degradable materials promoting cell growth and differentiation, but also vascularization of the engineered tissue. Porous polylactide/polycaprolactone (PLA/PCL, ratio 3/5) foam scaffolds were prepared by a combined porogen leaching and freeze-drying technique using NaCl (crystal size 250-500 µm) and a water-soluble cellulose derivative (KlucelTM E; 10-100% w/w relative to the total PLA/PCL concentration) as porogens. Scanning electron microscopy, micro-CT, and Brunauer-Emmett-Teller analysis showed that all scaffolds contained a trimodal range of pore sizes, i.e., macropores (average diameter 298-539 μm), micropores (100 nm to 10 μm), and nanopores (mostly around 3.0 nm). All scaffolds had an open porosity of about 90%, and the pores were interconnected. The size of the macropores and the nanoporosity were higher in the scaffolds prepared with Klucel. Nanoporosity increased water uptake by the scaffolds, while macroporosity promoted cell ingrowth, which was most evident in scaffolds prepared with 25% Klucel. Human adipose-derived stem cells co-cultured with endothelial cells formed pre-vascular structures in the scaffolds, which was further enhanced in a dynamic cell culture system. The scaffolds are promising for the engineering of pre-vascularized soft tissues (relatively pliable 10% Klucel scaffolds) and hard tissues (mechanically stronger 25% and 50% Klucel scaffolds).
- Klíčová slova
- compression stress and strain, degradable polyesters, dynamic cultivation, endothelial cells, macroporosity, mesenchymal stem cells, mineralization, nanoporosity, pre-vascularization, three-dimensional scaffolds,
- MeSH
- biokompatibilní materiály chemie MeSH
- kmenové buňky cytologie MeSH
- lidé MeSH
- polyestery * chemie MeSH
- poréznost MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury * chemie MeSH
- tuková tkáň cytologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biokompatibilní materiály MeSH
- poly(lactide) MeSH Prohlížeč
- polycaprolactone MeSH Prohlížeč
- polyestery * MeSH
Collagen, as the main component of connective tissue, is frequently used in various tissue engineering applications. In this study, porous sponge-like collagen scaffolds were prepared by freeze-drying and were then mineralized in a simulated body fluid. The mechanical stability was similar in both types of scaffolds, but the mineralized scaffolds (MCS) contained significantly more calcium, magnesium and phosphorus than the unmineralized scaffolds (UCS). Although the MCS contained a lower percentage (~32.5%) of pores suitable for cell ingrowth (113-357 μm in diameter) than the UCS (~70%), the number of human-osteoblast-like MG-63 cells on days 1, 3 and 7 after seeding was higher on MCS than on UCS, and the cells penetrated deeper into the MCS. The cell growth in extracts prepared by eluting the scaffolds for 7 days in a cell culture medium was also markedly higher in the MCS extracts, as indicated by real-time monitoring in the sensory xCELLigence system for 7 days. From this point of view, MCS are more promising for bone tissue engineering than UCS. However, MCS evoked a more pronounced inflammatory response than UCS, as indicated by the production of tumor necrosis factor-alpha (TNF-α) in macrophage-like RAW 264.7 cells in cultures on these scaffolds.
One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.
- Klíčová slova
- Bone tissue engineering, Collagen scaffolds, Dynamic seeding, Mesenchymal stem cells, Static cultivation,
- MeSH
- buněčná diferenciace MeSH
- fosforečnany vápenaté metabolismus MeSH
- kolagen chemie MeSH
- kosti a kostní tkáň chemie MeSH
- kultivované buňky MeSH
- mezenchymální kmenové buňky metabolismus MeSH
- nanočástice MeSH
- osteogeneze účinky léků MeSH
- prasata MeSH
- regenerativní lékařství MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- transplantace mezenchymálních kmenových buněk metody MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- calcium phosphate MeSH Prohlížeč
- fosforečnany vápenaté MeSH
- kolagen MeSH
Nanocellulose is cellulose in the form of nanostructures, i.e., features not exceeding 100 nm at least in one dimension. These nanostructures include nanofibrils, found in bacterial cellulose; nanofibers, present particularly in electrospun matrices; and nanowhiskers, nanocrystals, nanorods, and nanoballs. These structures can be further assembled into bigger two-dimensional (2D) and three-dimensional (3D) nano-, micro-, and macro-structures, such as nanoplatelets, membranes, films, microparticles, and porous macroscopic matrices. There are four main sources of nanocellulose: bacteria (Gluconacetobacter), plants (trees, shrubs, herbs), algae (Cladophora), and animals (Tunicata). Nanocellulose has emerged for a wide range of industrial, technology, and biomedical applications, namely for adsorption, ultrafiltration, packaging, conservation of historical artifacts, thermal insulation and fire retardation, energy extraction and storage, acoustics, sensorics, controlled drug delivery, and particularly for tissue engineering. Nanocellulose is promising for use in scaffolds for engineering of blood vessels, neural tissue, bone, cartilage, liver, adipose tissue, urethra and dura mater, for repairing connective tissue and congenital heart defects, and for constructing contact lenses and protective barriers. This review is focused on applications of nanocellulose in skin tissue engineering and wound healing as a scaffold for cell growth, for delivering cells into wounds, and as a material for advanced wound dressings coupled with drug delivery, transparency and sensorics. Potential cytotoxicity and immunogenicity of nanocellulose are also discussed.
BACKGROUND: Our study focuses on the fabrication of appropriate scaffolds for skin wound healing. This research brings valuable insights into the molecular mechanisms of adhesion, proliferation, and control of cell behavior through the extracellular matrix represented by synthetic biodegradable nanofibrous membranes coated by biomolecules. METHODS: Nanofibrous polylactic acid (PLA) membranes were prepared by a needle-less electrospinning technology. These membranes were coated with fibrin according to two preparation protocols, and additionally they were coated with fibronectin in order to increase the cell affinity for colonizing the PLA membranes. The adhesion, growth, and extracellular matrix protein production of neonatal human dermal fibroblasts were evaluated on the nanofibrous membranes. RESULTS: Our results showed that fibrin-coated membranes improved the adhesion and proliferation of human dermal fibroblasts. The morphology of the fibrin nanocoating seems to be crucial for the adhesion of fibroblasts, and consequently for their phenotypic maturation. Fibrin either covered the individual fibers in the membrane (F1 nanocoating), or covered the individual fibers and also formed a fine homogeneous nanofibrous mesh on the surface of the membrane (F2 nanocoating), depending on the mode of fibrin preparation. The fibroblasts on the membranes with the F1 nanocoating remained in their typical spindle-like shape. However, the cells on the F2 nanocoating were spread mostly in a polygon-like shape, and their proliferation was significantly higher. Fibronectin formed an additional mesh attached to the surface of the fibrin mesh, and further enhanced the cell adhesion and growth. The relative gene expression and protein production of collagen I and fibronectin were higher on the F2 nanocoating than on the F1 nanocoating. CONCLUSION: A PLA membrane coated with a homogeneous fibrin mesh seems to be promising for the construction of temporary full-thickness skin tissue substitutes.
- Klíčová slova
- dermal fibroblasts, extracellular matrix synthesis, fibrin, nanocoating, nanofibers, polylactic acid, skin substitute,
- MeSH
- buněčná adheze fyziologie MeSH
- buněčné kultury přístrojové vybavení metody MeSH
- extracelulární matrix metabolismus MeSH
- fibrin chemie farmakologie MeSH
- fibroblasty cytologie účinky léků MeSH
- fibronektiny metabolismus MeSH
- kolagen typu I metabolismus MeSH
- kultivované buňky MeSH
- kůže cytologie MeSH
- lidé MeSH
- membrány umělé MeSH
- nanostruktury chemie MeSH
- nanotechnologie metody MeSH
- polyestery chemie MeSH
- proliferace buněk fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fibrin MeSH
- fibronektiny MeSH
- kolagen typu I MeSH
- membrány umělé MeSH
- poly(lactide) MeSH Prohlížeč
- polyestery MeSH
Fibrin plays an important role during wound healing and skin regeneration. It is often applied in clinical practice for treatment of skin injuries or as a component of skin substitutes. We prepared electrospun nanofibrous membranes made from poly(l-lactide) modified with a thin fibrin nanocoating. Fibrin surrounded the individual fibers in the membrane and also formed a thin fibrous mesh on several places on the membrane surface. The cell-free fibrin nanocoating remained stable in the cell culture medium for 14 days and did not change its morphology. On membranes populated with human dermal fibroblasts, the rate of fibrin degradation correlated with the degree of cell proliferation. The cell spreading, mitochondrial activity, and cell population density were significantly higher on membranes coated with fibrin than on nonmodified membranes, and this cell performance was further improved by the addition of ascorbic acid in the cell culture medium. Similarly, fibrin stimulated the expression and synthesis of collagen I in human dermal fibroblasts, and this effect was further enhanced by ascorbic acid. The expression of beta1-integrins was also improved by fibrin, and on pure polylactide membranes, it was slightly enhanced by ascorbic acid. In addition, ascorbic acid promoted deposition of collagen I in the form of a fibrous extracellular matrix. Thus, the combination of nanofibrous membranes with a fibrin nanocoating and ascorbic acid seems to be particularly advantageous for skin tissue engineering.
- Klíčová slova
- ascorbic acid, beta1-integrins, collagen I synthesis, electrospun nanofibers, fibrin, fibroblasts, nanocoating, nanomedicine, nanotechnology, skin tissue engineering,
- MeSH
- buněčná diferenciace MeSH
- elektrochemie metody MeSH
- extracelulární matrix metabolismus MeSH
- fibrin chemie metabolismus MeSH
- fibroblasty cytologie metabolismus MeSH
- fluorescenční protilátková technika MeSH
- imunoenzymatické techniky MeSH
- kolagen genetika metabolismus MeSH
- kultivované buňky MeSH
- kůže cytologie metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- nanovlákna chemie MeSH
- polyestery chemie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proliferace buněk MeSH
- regenerace fyziologie MeSH
- tkáňové inženýrství metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fibrin MeSH
- kolagen MeSH
- messenger RNA MeSH
- poly(lactide) MeSH Prohlížeč
- polyestery MeSH
