Nejvíce citovaný článek - PubMed ID 22619532
Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering
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.
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
We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.
- MeSH
- chromatografie afinitní metody MeSH
- endoteliální buňky pupečníkové žíly (lidské) MeSH
- Escherichia coli metabolismus MeSH
- fibrin metabolismus MeSH
- klonování DNA MeSH
- kultivované buňky MeSH
- lidé MeSH
- molekulární chaperony metabolismus MeSH
- plazmidy metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- thioredoxiny metabolismus MeSH
- vaskulární endoteliální růstový faktor A metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fibrin MeSH
- molekulární chaperony MeSH
- rekombinantní fúzní proteiny MeSH
- thioredoxiny MeSH
- vaskulární endoteliální růstový faktor A MeSH
- VEGFA protein, human MeSH Prohlížeč
An investigation was made of the adhesion, growth and differentiation of osteoblast-like MG-63 and Saos-2 cells on titanium (Ti) and niobium (Nb) supports and on TiNb alloy with surfaces oxidized at 165°C under hydrothermal conditions and at 600°C in a stream of air. The oxidation mode and the chemical composition of the samples tuned the morphology, topography and distribution of the charge on their surfaces, which enabled us to evaluate the importance of these material characteristics in the interaction of the cells with the sample surface. Numbers of adhered MG-63 and Saos-2 cells correlated with the number of positively-charged (related with the Nb2O5 phase) and negatively-charged sites (related with the TiO2 phase) on the alloy surface. Proliferation of these cells is correlated with the presence of positively-charged (i.e. basic) sites of the Nb2O5 alloy phase, while cell differentiation is correlated with negatively-charged (acidic) sites of the TiO2 alloy phase. The number of charged sites and adhered cells was substantially higher on the alloy sample oxidized at 600°C than on the hydrothermally treated sample at 165°C. The expression values of osteoblast differentiation markers (collagen type I and osteocalcin) were higher for cells grown on the Ti samples than for those grown on the TiNb samples. This was more particularly apparent in the samples treated at 165°C. No considerable immune activation of murine macrophage-like RAW 264.7 cells on the tested samples was found. The secretion of TNF-α by these cells into the cell culture media was much lower than for either cells grown in the presence of bacterial lipopolysaccharide, or untreated control samples. Thus, oxidized Ti and TiNb are both promising materials for bone implantation; TiNb for applications where bone cell proliferation is desirable, and Ti for induction of osteogenic cell differentiation.
- MeSH
- biologické markery metabolismus MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné linie MeSH
- kolagen typu I metabolismus MeSH
- lidé MeSH
- lipopolysacharidy farmakologie MeSH
- makrofágy cytologie účinky léků metabolismus MeSH
- myši MeSH
- osteoblasty cytologie účinky léků metabolismus MeSH
- osteokalcin metabolismus MeSH
- oxidace-redukce MeSH
- povrchové vlastnosti MeSH
- proliferace buněk účinky léků MeSH
- slitiny chemie farmakologie MeSH
- statická elektřina MeSH
- tkáňové podpůrné struktury * MeSH
- TNF-alfa farmakologie MeSH
- vysoká teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- kolagen typu I MeSH
- lipopolysacharidy MeSH
- osteokalcin MeSH
- slitiny MeSH
- titanium-niobium alloy MeSH Prohlížeč
- TNF-alfa MeSH
