Novel Therapy for Acquired Tracheomalacia with a Tissue-Engineered Extraluminal Tracheal Splint and Autologous Mesenchymal-Derived Chondrocytes

Abstract Introduction Acquired tracheomalacia (ATM) is characterized by a loss of structural strength of the tracheal framework, resulting in airway collapse during breathing. Near half of the patients undergoing prolonged invasive mechanical ventilation will suffer tracheal lesions. Treatment for ATM includes external splinting with rib grafts, prosthetic materials, and tracheal resection. Failure in the use of prosthetic materials has made reconsidering natural origin scaffolds and tissue engineering as a suitable alternative. Objective To restore adequate airway patency in an ovine model with surgicallyinduced ATM employing a tissue-engineered extraluminal tracheal splint (TE-ETS). Methods In the present prospective pilot study, tracheal rings were partially resected to induce airway collapse in 16 Suffolk sheep (Ovis aries). The TE-ETS was developed with autologous mesenchymal-derived chondrocytes and allogenic decellularized tracheal segments and was implanted above debilitated tracheal rings. The animals were followed-up at 8, 12, and 16 weeks and at 1-year postinsertion. Flexible tracheoscopies were performed at each stage. After sacrifice, a histopathological study of the trachea and the splint were performed. Results The TE-ETS prevented airway collapse for 16 weeks and up to 1-year postinsertion. Tracheoscopies revealed a noncollapsing airway during inspiration. Histopathological analyses showed the organization of mesenchymal-derived chondrocytes in lacunae, the proliferation of blood vessels, and recovery of epithelial tissue subjacent to the splint. Splints without autologous cells did not prevent airway collapse. Conclusion It is possible to treat acquired tracheomalacia with TE-ETS without further surgical removal since it undergoes physiological degradation. The present study supports the development of tissue-engineered tracheal substitutes for airway disease.

Gamma radiation-induced grafting of poly(2-aminoethyl methacrylate) onto chitosan: A comprehensive study of a polyurethane scaffold intended for skin tissue engineering.

A novel brush-like poly(2-aminoethyl methacrylate) (PAEMA) was grafted onto chitosan (CS) through gamma radiation-induced polymerization. The copolymer (CS-g-PAEMA) was used to prepare a sodium acetate leached poly(urethane-urea) scaffold. The above derivatives were developed, synthesized, and characterized to meet the specific characteristics of biomaterials. The results revealed that this method is an easy and successful route for grafting PAEMA onto CS. The feasibility of preparing a CS-g-PAEMA polyurethane foam was confirmed by mechanical, morphometric, spectroscopic, and cytotoxic studies. The scaffold showed high biocompatibility both in vitro and in vivo. The first experiment proved that CS-based polyurethane efficiently allows the dynamic culturing of human fibroblast cells. Additionally, an in vivo study in a murine model indicated a complete integration of the scaffold to surrounding subcutaneous tissue as supported by the histological and histochemical assessments. The aforementioned results support the use of CS-g-PAEMA poly(saccharide-urethane) as a model of in vitro-engineered skin.

Gamma radiation-induced grafting of n-hydroxyethyl acrylamide onto poly(3-hydroxybutyrate): A companion study on its polyurethane scaffolds meant for potential skin tissue engineering applications.

This study aimed at investigating the synthesis, characterization, and search for a biotechnological application proposal for poly [(R)-3-hydroxybutyric acid] (PHB) grafted with the n-hydroxyethyl acrylamide (HEAA) monomer. The novel copolymer was prepared by 60Co gamma radiation-induced-graft polymerization. The effect of different solvents in the graft polymerization; the degree of grafting, crystallinity, and hydrophilicity; the morphology and the thermal properties were evaluated. The polyurethane fabricated from the grafted PHB was suggested as a scaffold. The enzymatic degradation behavior and the spectroscopic, morphological, mechanical, and biological properties of the composites were assessed. According to the results, the successful grafting of HEAA onto PHB was verified. The grafting was significantly affected by the type of solvent employed. A decreased crystallinity and increased hydrophilicity of the graft copolymer, concerning the PHB, was found. An increased roughness was observed in the morphology of the polymer after grafting. The thermodynamic parameters, except for the glass transition temperature, also decreased for the synthetic biopolymer. The intended use of these scaffolds for skin tissue engineering was supported by a proper degradability and degree of porosity, improved mechanical properties, the optimal culture of human fibroblasts, and its transfection with a plasmid vector containing an enhanced green fluorescent protein.

Synthesis, characterization, and in vitro evaluation of gamma radiationinduced PEGylated isoniazid

Background: The search for innovative anti-tubercular agents has received increasing attention in tuberculosis chemotherapy because Mycobacterium tuberculosis infection has steadily increased over the years. This underlines the necessity for new methods of preparation for polymer-drug adducts to treat this important infectious disease. The use of poly(ethylene glycol)(PEG) is an alternative producing anti-tubercular derivatives. However, it is not yet known whether PEGylated isonicotinylhydrazide conjugates obtained by direct links with PEG are useful for therapeutic applications. Results: Here, we synthesized a PEGylated isoniazid (PEG-g-INH or PEG­INH) by gamma radiation-induced polymerization, for the first time. The new prodrugs were characterized using Raman and UV/Vis spectrometry. The mechanism of PEGylated INH synthesis was proposed. The in vitro evaluation of a PEGylated isonicotinylhydrazide macromolecular prodrug was also carried out. The results indicated that PEG­INH inhibited the bacterial growth above 95% as compared with INH, which showed a lower value (80%) at a concentration of 0.25 µM. Similar trends are observed for 0.1, 1, and 5 µM. Conclusions: In summary, the research suggests that it is possible to covalently attach the PEG onto INH by the proposed method and to obtain a slow-acting isoniazid derivative with little toxicity in vitro and higher antimycobacterial potency than the neat drug.

Silver-pig skin nanocomposites and mesenchymal stem cells: suitable antibiofilm cellular dressings for wound healing.

BACKGROUND: Treatment of severe or chronic skin wounds is an important challenge facing medicine and a significant health care burden. Proper wound healing is often affected by bacterial infection; where biofilm formation is one of the main risks and particularly problematic because it confers protection to microorganisms against antibiotics. One avenue to prevent bacterial colonization of wounds is the use of silver nanoparticles (AgNPs); which have proved to be effective against non-multidrug-resistant and multidrug-resistant bacteria. In addition, the use of mesenchymal stem cells (MSC) is an excellent option to improve wound healing due to their capability for differentiation and release of relevant growth factors. Finally, radiosterilized pig skin (RPS) is a biomatrix successfully used as wound dressing to avoid massive water loss, which represents an excellent carrier to deliver MSC into wound beds. Together, AgNPs, RPS and MSC represent a potential dressing to control massive water loss, prevent bacterial infection and enhance skin regeneration; three essential processes for appropriate wound healing with minimum scaring. RESULTS: We synthesized stable 10 nm-diameter spherical AgNPs that showed 21- and 16-fold increase in bacteria growth inhibition (in comparison to antibiotics) against clinical strains Staphylococcus aureus and Stenotrophomonas maltophilia, respectively. RPS samples were impregnated with different AgNPs suspensions to develop RPS-AgNPs nanocomposites with different AgNPs concentrations. Nanocomposites showed inhibition zones, in Kirby-Bauer assay, against both clinical bacteria tested. Nanocomposites also displayed antibiofilm properties against S. aureus and S. maltophilia from RPS samples impregnated with 250 and 1000 ppm AgNPs suspensions, respectively. MSC were isolated from adipose tissue and seeded on nanocomposites; cells survived on nanocomposites impregnated with up to 250 ppm AgNPs suspensions, showing 35% reduction in cell viability, in comparison to cells on RPS. Cells on nanocomposites proliferated with culture days, although the number of MSC on nanocomposites at 24 h of culture was lower than that on RPS. CONCLUSIONS: AgNPs with better bactericide activity than antibiotics were synthesized. RPS-AgNPs nanocomposites impregnated with 125 and 250 ppm AgNPs suspensions decreased bacterial growth, decreased biofilm formation and were permissive for survival and proliferation of MSC; constituting promising multi-functional dressings for successful treatment of skin wounds.

Cytoprotective effect of the enzyme-mediated polygallic acid on fibroblast cells under exposure of UV-irradiation.

The poly(gallic acid), produced by laccase-mediated oxidation of gallic acid in aqueous media (pH5.5) to attain a novel material with well-defined molecular structure and high water solubility (500mg/mL at 25°C), has been investigated to understand its potential biological activities. In this regard, a biomedical approach based on cytoprotective effect on human fibroblast cells exposed to UV-irradiation in the presence of the polymer has been demonstrated. The results also shows that 200µg/mL of poly(gallic acid) inhibits the growth and migration of dermal fibroblasts and cancer cell lines without affecting cell viability. Poly(gallic acid) pretreatment with 10µg/mL protects dermal fibroblasts from UV induced cell death and additionally, the cytoprotective effect reduce ROS presence in the cells. This property can be correlated with the antioxidant power (IC50 of 23.5µg/mL) of this novel material, which was ascertained by electronic paramagnetic resonance spectroscopy and spectrophotometrically. Additionally, the antimicrobial activity of this material was corroborated with the inhibition of Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212) strains (MIC=400mg/mL) common bacteria found in hospitals.

Generation of Two Biological Wound Dressings as a Potential Delivery System of Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (hADMSCs) are believed to be potential key factors for starting the regenerative process after tissue injury. However, an efficient method of delivering these regenerative cells to an external wound site is still lacking. Human amnion and pig skin have long been used as skin wound dressings for the treatment of burns and other skin lesions. Herein, we present the generation of two constructs using these two biomaterials as effective scaffolds for the culture of hADMSCs. It was found that hADMSCs seeded onto radiosterilized human amnion and pig skin are viable and proliferate. These cells are able to migrate over these scaffolds as demonstrated by using time-lapse microscopy. In addition, the scaffolds induce hADMSCs to secrete interleukin-10, an important negative regulator of inflammation, and interleukin-1ß, a proinflammatory protein. The interplay between these two proteins has been proven to be vital for a balanced restoration of all necessary tissues. Thus, radiosterilized human amnion and pig skin are likely suitable scaffolds for delivery of hADMSCs transplants that could promote tissue regeneration in skin injuries like patients with burn injuries.

Biocompatibility of human auricular chondrocytes cultured onto a chitosan/polyvynil alcohol/epichlorohydrin-based hydrogel for tissue engineering application / Biocompatibilidad de condrocitos humanos cultivados sobre un hidrogel a base de quitosano/alcohol polivnílico/epiclorhidrina para aplicación en ingeniería de tejidos

Tissue engineering (TE) has become an alternative for auricular reconstruction based on the combination of cells, molecular signals and biomaterials. Scaffolds are biomaterials that provide structural support for cell attachment and subsequent tissue development. Ideally, a scaffold should have characteristics such as biocompatibility and bioactivity to adequate support cell functions. Our purpose was to evaluate biocompatibility of microtic auricular chondrocytes seeded onto a chitosan-polyvinyl alcohol-epichlorohydrin (CS-PVA-ECH) hydrogel to propose this material as a scaffold for tissue engineering application. After being cultured onto CS-PVA-ECH hydrogels, auricular chondrocytes viability was up to 81%. SEM analysis showed cell attachment and extracellular matrix formation that was confirmed by IF detection of type II collagen and elastin, the main constituents of elastic cartilage. Expression of elastic cartilage molecular markers during in vitro expansion and during culture onto hydrogels allowed confirming auricular chondrocyte phenotype. In vivo assay of tissue formation revealed generation of neotissues with similar physical characteristics and protein composition to those found in elastic cartilage. According to our results, biocompatibility of the CS-PVA-ECH hydrogel makes it a suitable scaffold for tissue engineering application aimed to elastic cartilage regeneration.

La ingeniería de tejidos (TE) es una alternativa para la reconstrucción auricular basada en la combinación de células, señales moleculares y biomateriales. Los andamios fabricados con biomateriales brindan un soporte estructural que favorece la adhesión cellular y el desarrollo del tejido. Un andamio debe poseer características como biocompatibilidad y bioactividad para soportar adecuadamente funciones celulares. Nuestro objetivo fue evaluar la biocompatibilidad de condrocitos auriculares de microtia cultivados sobre un hidrogel a base de quitosano-alcohol polivinílico-epiclorhidrina (CS-PVA-ECH) y proponerlo como andamio con aplicaciones en ingeniería de tejidos. La viabilidad de los condrocitos auriculares es superior al 81% después de ser cultivados sobre el hidrogel. El análisis por SEM reveló la unión celular y formación de matriz extracellular sobre el hidrogel; confirmada mediante detección por IF de colágena tipo II y elastina. La expresión de marcadores moleculares durante la expansión in vitro y el cultivo sobre los hidrogeles confirmaron el fenotipo condral. El ensayo de formación de tejido in vivo demostró la generación de neotejidos con características físicas y composición similar al cartílago elástico. Nuestros resultados indican que la biocompatibilidad del hidrogel de CS-PVA-ECH lo hace un andamio adecuado para aplicaciones en ingeniería de tejidos enfocadas a regeneración de cartílago elástico.