Úvod: Vývoj ideálního krytí pro oblast hojení ran je stále nevyřešeným problémem. Díky rozvoji techniky elektrostatického zvlákňování se do popředí výzkumného zájmu dostaly polymery ve formě nanovláken. Moderním a velice slibným obvazovým materiálem je netkaný textil na bázi nanovláken syntetického polymeru polylaktidu (PLA). Cílem práce bylo posouzení regeneračních schopností PLA na standardizované ráně prasečího modelu a naše výsledky porovnat s literárními údaji. Metody: Na vytvořené standardizované rány na prasečím modelu jsme aplikovali nanovláknové obvazy na bázi PLA. Třetí, desátý, sedmnáctý a dvacátý čtvrtý den od tvorby defektu jsme ránu převázali za současného odběru tkáně k histopatologickému vyšetření. Průběžně jsme monitorovali hladiny sérových proteinů akutní fáze zánětu. Výsledky: PLA stimuloval zánětlivou reakci. Od třetího dne narůstala tloušťka fibrinové vrstvy s granulocyty. Maximálních hodnot dosáhla desátý převazový den (průměr 340 μm), zároveň kulminovala hladina sérového amyloidu A jakožto markeru zánětu. Jednotlivé fáze hojení se vzájemně prolínaly. Největších hodnot tloušťky granulační tkáně s buněčným vazivem (průměr 8463 μm) bylo docíleno sedmnáctý den. Dvacátý čtvrtý den byly defekty makroskopicky zhojeny s průměrnou vrstvou reepitelizace 9913 μm. Závěr: Nanovláknový obvaz na bázi PLA potencuje zánětlivou, proliferační i reepitelizační fázi hojení. Jeho struktura dokonale napodobuje stavbu a funkci extracelulární matrix a slouží jako 3D síť pro růst a interakci buněčných elementů. Mimo biokompatibilitu disponuje PLA unikátní schopností dvoufázové biodegradace. Je slibným materiálem pro výrobu převazových materiálů. Většina dostupných studií byla provedena in vitro. Dosud chybí in vivo srovnávací studie, přibližující použití PLA k denní praxi.
Introduction: The development of an ideal dressing for wound healing remains an unresolved issue. Thanks to the development of electrospinning technology, polymers in the form of nanofibers have come to the forefront of research interest. A modern and very promising dressing material is a “nonwoven” based on nanofibers of the synthetic polymer polylactide (PLA). The aim of this work was to assess the regenerative abilities of PLA in a standardized wound in a porcine model and compare our results to the literature data. Methods: We applied PLA-based nanofiber dressings to the standardized wounds created in the porcine model. On the third, tenth, seventeenth and twenty-fourth days after the formation of the defect, we changed the wound dressing while taking a tissue sample for histopathological examination. We continuously monitored serum levels of acute phase proteins. Results: PLA stimulated an inflammatory response. From the third day, the thickness of the fibrin layer with granulocytes increased. It reached its maximum values on the tenth day (mean 340 μm); at the same time the level of serum amyloid A, as a marker of inflammation, culminated. The individual phases of healing intertwined. The highest thickness values of the granulation tissue with cellular connective tissue (diameter 8463 μm) were reached on the seventeenth day. On the twenty-fourth day, the defects were healed macroscopically with a mean reepithelialization layer of 9913 μm. Conclusion: PLA-based nanofiber dressing potentiates the inflammatory, proliferative and reepithelialization phases of healing. Its structure perfectly mimics the extracellular matrix and serves as a 3D network for the growth and interaction of cellular elements. In addition to biocompatibility, PLA has a unique ability of two-phase biodegradation. It is a promising material for industrial production of dressing materials. Most of the available studies were performed in vitro. In vivo comparative studies approximating the use of PLA to daily practice are still missing.
- MeSH
- Wound Healing * MeSH
- Disease Models, Animal * MeSH
- Nanofibers * MeSH
- Bandages MeSH
- Polyesters MeSH
- Swine MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the architecture of tissue at the nanoscale. We evaluated the adhesion, growth, viability, and differentiation of human osteoblast-like MG 63 cells on polylactide (PLA) nanofibers prepared by needle-less electrospinning and loaded with 5 or 15 wt % of hydroxyapatite (HA) nanoparticles. On day 7 after seeding, the cell number was the highest on samples with 15 wt % of HA. This result was confirmed by the XTT test, especially after dynamic cultivation, when the number of metabolically active cells on these samples was even higher than on control polystyrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and comparable to that on control polystyrene dishes. An enzyme-linked immunosorbent assay revealed that the concentration of osteocalcin was also higher in cells on samples with 15 wt % of HA. There was no immune activation of cells (measured by production of TNF-alpha), associated with the incorporation of HA. Moreover, the addition of HA suppressed the creep behavior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particularly those with 15 wt % of HA.
- MeSH
- Cell Adhesion MeSH
- Cell Differentiation * MeSH
- Cell Line MeSH
- Durapatite chemistry MeSH
- Bone Substitutes MeSH
- Humans MeSH
- Nanofibers chemistry MeSH
- Osteoblasts cytology metabolism MeSH
- Osteocalcin biosynthesis MeSH
- Polyesters chemistry MeSH
- Tissue Engineering methods MeSH
- Cell Survival MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Cell Differentiation MeSH
- Electrochemistry methods MeSH
- Extracellular Matrix metabolism MeSH
- Fibrin chemistry metabolism MeSH
- Fibroblasts cytology metabolism MeSH
- Fluorescent Antibody Technique MeSH
- Immunoenzyme Techniques MeSH
- Collagen genetics metabolism MeSH
- Cells, Cultured MeSH
- Skin cytology metabolism MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- RNA, Messenger genetics MeSH
- Nanofibers chemistry MeSH
- Polyesters chemistry MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Cell Proliferation MeSH
- Regeneration physiology MeSH
- Tissue Engineering methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Micro- and nanostructures prepared from biodegradable homopolymers and amphiphilic block copolymers (AmBCs) have found application as drug-delivery systems (DDSs). The ability to accumulate a drug is a very important parameter characterizing a given DDS. This work focuses on the impact of DDS size, the packing of polymer chains in the DDS, and drug - polymer matrix compatibility on the hydrophobic drug - loading capacity (DLC) of nano/microcarriers prepared from a biodegradable polymer or its copolymer. Using experimental measurements in combination with atomistic molecular dynamics simulations, an analysis of curcumin encapsulation in microspheres (MSs) from polylactide (PLA) homopolymer and nanoparticles (NPs) from PLA-block-poly(2-methacryloyloxyethylphosphorylcholine) AmBC was performed. The results show that curcumin has good affinity for the PLA matrix due to its hydrophobic nature. However, the DLC value is limited by the fact that curcumin only accumulates in the peripheral part of these structures. Such uneven drug distribution in the PLA matrix results from the non-homogeneous density of MSs (non-uniform packing of the polymer chains in the coil). The results also indicate that the MSs can retain a greater amount of hydrophobic drug compared to the NPs, which is associated with the formation of drug aggregates inside the PLA microparticles.
- MeSH
- Financing, Organized MeSH
- Publication type
- Abstracts MeSH
Local application of anticancer agents prolongs the presence time and increases the concentration of drug in the target place and therefore may reduce serious side effects compared to drug systemic administration. The preparation of fibrous materials of polylactide (PLA) and polyethylene glycol (PEG) loaded with paclitaxel (PTX, 1 or 10 wt%) is presented. Scanning electron microscopy proves that PTX is homogeneously incorporated into the fibers. The addition of PEG of various molecular weights (6, 20, or 35 kDa) ensures the release of significantly higher amounts of hydrophobic PTX in a prolonged release time compared to the fibers containing PTX only. Present PLA-PEG fibrous carriers can serve as a drug depot for PTX since they exhibit significant toxicity for cancer cell lines in several-day experiment. They are promising for local recurrence therapy, where the initial release is efficient to kill tumor cells and continued release can prevent their subsequent proliferation.
- MeSH
- Delayed-Action Preparations chemistry pharmacokinetics pharmacology MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Neoplasms drug therapy metabolism pathology MeSH
- Drug Carriers * chemistry pharmacokinetics pharmacology MeSH
- Paclitaxel * chemistry pharmacokinetics pharmacology MeSH
- Polyesters * chemistry pharmacokinetics pharmacology MeSH
- Polyethylene Glycols * chemistry pharmacokinetics pharmacology MeSH
- Antineoplastic Agents * chemistry pharmacokinetics pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The replacement of pancreatic islets for the possible treatment of type 1 diabetes is limited by the extremely high oxygen demand of the islets. To this end, here we hypothesize to create a novel extra-hepatic highly-vascularized bioartificial cavity using a porous scaffold as a template and using the host body as a living bioreactor for subsequent islet transplantation. Polylactide-based capsular-shaped anisotropic channeled porous scaffolds were prepared by following the unidirectional thermally-induced phase separation technique, and were implanted under the skin and in the greater omentum of Brown Norway rats. Polyamide mesh-based isotropic regular porous capsules were used as the controls. After 4weeks, the implants were excised and analyzed by histology. The hematoxylin and eosin, as well as Masson's trichrome staining, revealed a) low or no infiltration of giant inflammatory cells in the implant, b) minor but insignificant fibrosis around the implant, c) guided infiltration of host cells in the test capsule in contrast to random cell infiltration in the control capsule, and d) relatively superior cell infiltration in the capsules implanted in the greater omentum than in the capsules implanted under the skin. Furthermore, the anti-CD31 immunohistochemistry staining revealed numerous vessels at the implant site, but mostly on the external surface of the capsules. Taken together, the current study, the first of its kind, is a significant step-forward towards engineering a bioartificial microenvironment for the transplantation of islets.
- MeSH
- Anisotropy MeSH
- Platelet Endothelial Cell Adhesion Molecule-1 MeSH
- Cellular Microenvironment MeSH
- Fibrosis MeSH
- Neovascularization, Physiologic drug effects MeSH
- Rats MeSH
- Lactic Acid chemistry MeSH
- Polyglycolic Acid chemistry MeSH
- Islets of Langerhans * MeSH
- Porosity MeSH
- Rats, Inbred BN MeSH
- Tissue Scaffolds MeSH
- Capsules MeSH
- Islets of Langerhans Transplantation methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The effectiveness of cell transplantation can be improved by optimization of the transplantation site. For some types of cells that form highly oxygen-demanding tissue, e.g., pancreatic islets, a successful engraftment depends on immediate and sufficient blood supply. This critical point can be avoided when cells are transplanted into a bioengineered pre-vascularized cavity which can be formed using a polymer scaffold. In our study, we tested surface-modified poly(lactide-co-caprolactone) (PLCL) capsular scaffolds containing the pro-angiogenic factor VEGF. After each modification step (i.e., amination and heparinization), the surface properties and morphology of scaffolds were characterized by ATR-FTIR and XPS spectroscopy, and by SEM and AFM. All modifications preserved the gross capsule morphology and maintained the open pore structure. Optimized aminolysis conditions decreased the Mw of PLCL only up to 10% while generating a sufficient number of NH2 groups required for the covalent immobilization of heparin. The heparin layer served as a VEGF reservoir with an in vitro VEGF release for at least four weeks. In vivo studies revealed that to obtain highly vascularized PLCL capsules (a) the optimal VEGF dose for the capsule was 50 μg and (b) the implantation time was four weeks when implanted into the greater omentum of Lewis rats; dense fibrous tissue accompanied by vessels completely infiltrated the scaffold and created sparse granulation tissue within the internal cavity of the capsule. The prepared pre-vascularized pouch enabled the islet graft survival and functioning for at least 50 days after islet transplantation. The proposed construct can be used to create a reliable pre-vascularized pouch for cell transplantation.
- MeSH
- Bioengineering * MeSH
- Diabetes Mellitus, Experimental chemically induced metabolism pathology MeSH
- Neovascularization, Physiologic * MeSH
- Injections, Intraperitoneal MeSH
- Blood Glucose analysis MeSH
- Rats MeSH
- Molecular Structure MeSH
- Polyesters chemistry metabolism MeSH
- Rats, Inbred Lew MeSH
- Streptozocin administration & dosage MeSH
- Capsules chemistry metabolism MeSH
- Islets of Langerhans Transplantation * MeSH
- Vascular Endothelial Growth Factors chemistry metabolism MeSH
- Particle Size MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.
- MeSH
- Fibrin pharmacology MeSH
- Fibroblasts cytology drug effects MeSH
- Hydrogels pharmacology MeSH
- Keratinocytes cytology drug effects MeSH
- Collagen pharmacology MeSH
- Rats MeSH
- Humans MeSH
- Membranes, Artificial * MeSH
- Mitochondria drug effects metabolism MeSH
- Nanofibers chemistry MeSH
- Infant, Newborn MeSH
- Cell Movement drug effects MeSH
- Polyesters pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Dermis cytology MeSH
- Skin, Artificial * MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Infant, Newborn MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
