RESUMO
Sufficient and controllable oxygen supply is essential for in vitro 3D cell and tissue culture at high cell densities, which calls for volumetric in situ oxygen analysis methods to quantitatively assess the oxygen distribution. This paper presents a general approach for accurate and precise non-contact 3D mapping of oxygen tension in high cell-density cultures via embedded commercially available oxygen microsensor beads read out by confocal phosphorescence lifetime microscopy (PLIM). Optimal acquisition conditions and data analysis procedures are established and implemented in a publicly available software package. The versatility of the established method is first demonstrated in model-assisted fluidic design of microperfused 3D printed hydrogel culture chips with the aim of full culture oxygenation, and subsequently for monitoring and maintenance of physiologically relevant spatial and temporal oxygen gradients in the 3D printed chips controlled by static or dynamic flow conditions during 3D culture.
Assuntos
Hidrogéis , Oxigênio , Microscopia ConfocalRESUMO
In this study, micelles composed of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) copolymer (mPEG-b-PCL), which has ionically conjugated lithocholic acid (LCA) and providing pH sensitive release of LCA in acidic media, were prepared as drug carrier devices for cancer therapy. Micelles were produced by co-solvent evaporation method at two different temperatures (60 °C and 25 °C) and coded as LCA60**M and LCA25**M, respectively). Hydrodynamic diameters were 86.9 nm and 228.2 nm, and zeta potentials were -7.54 mV and -18.83 mV for LCA60**M and LCA25**M, respectively. For all micelles, release of LCA was higher in acidic media (pH 5.0) compared to physiological media (pH 7.4). Micelles loaded with a fluorescent dye, coumarin 6, demonstrated effective internalization into triple negative MDA-MB-231 breast cancer cells in 4 h. LCA60**M (41.7 ± 1.5%) and LCA25**M (44.5 ± 2.2%) had higher inhibitory effect on the cell migration compared to free LCA (64.7 ± 1.3%). Both LCA conjugated micelles decreased lipogenic activity and increased expressions of Bax (1.3 fold) and p53 (1.2 fold) apoptotic genes. Annexin V-FITC results exhibited high apoptotic cell number after the treatment of MDA-MB-231 cells with micelles. Free LCA and LCA conjugated LCA60**M and LCA25**M micelles decreased mitochondrial transmembrane potential of the cells by 41.8 ± 3.0%, 30.4 ± 0.9%, and 57.1 ± 0.5, respectively. Micelles also caused an effective decrease in angiogenesis ability of HUVECs. The novelty of this study is the prepared micelles, which have ionic conjugation of LCA to mPEG-b-PCL, and pH responsive release of LCA demonstrating effective apoptosis on breast cancer cells. These micelles may have great potential for cancer treatment. However, further in vivo studies are needed before clinical translation.
Assuntos
Neoplasias da Mama , Micelas , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Ácido Litocólico , Poliésteres , Polietilenoglicóis/metabolismoRESUMO
We present a method for reproducible manufacture of multiassay platforms with tunable mechanical properties for muscle tissue strip analysis. The platforms result from stereolithographic 3D printing of low protein-binding poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Contractile microtissues have previously been engineered by immobilizing suspended cells in a confined hydrogel matrix with embedded anchoring cantilevers to facilitate muscle tissue strip formation. The 3D shape and mechanical properties of the confinement and the embedded cantilevers are critical for the tissue robustness. High-resolution 3D printing of PEGDA hydrogels offers full design freedom to engineer cantilever stiffness, while minimizing unwanted cell attachment. We demonstrate the applicability by generating suspended muscle tissue strips from C2C12 mouse myoblasts in a compliant fibrin-based hydrogel matrix. The full design freedom allows for new platform geometries that reduce local stress in the matrix and tissue, thus, reducing the risk of tissue fracture.
Assuntos
Hidrogéis/química , Impressão Tridimensional , Engenharia Tecidual/instrumentação , Animais , Fenômenos Biomecânicos , Desenho Assistido por Computador , Criopreservação/métodos , Camundongos , Músculos/citologia , Músculos/fisiologia , Mioblastos/citologia , Polietilenoglicóis/química , Impressão Tridimensional/instrumentação , Engenharia Tecidual/métodosRESUMO
BACKGROUND: A successful rhinoplasty procedure requires a well-defined and properly projected nasal tip; however, surgical control of the nasal tip is difficult. The aim of this investigation was to assess the efficacy and safety of a modified suture technique, which can be used to fix the caudal septal extension graft during primary rhinoplasty of the Asian population and revision septorhinoplasties of the Caucasian population, and to compare it with those of other commonly used techniques. METHODS: After peeling of perichondrium of scapular cartilages, cartilage pieces of 3â¯×â¯1â¯cm in size and 2â¯mm in thickness were divided into two from the midline. These pieces were repaired end-to-end using three different repair techniques: two simple interrupted in Group A (nâ¯=â¯40), vertical figure-of-eight in Group B (nâ¯=â¯40) and modified vertical figure-of-eight (transloop) in Group C (nâ¯=â¯40). All repaired cartilage specimens were subjected to a biomechanical analysis, in which four different forces were applied: tension, lateral bending, shearing and buckling. RESULTS: According to the tensile test, Group C had statistically significantly higher strength than Group A at 2â¯mm range. The lateral bending test similarly revealed that Group C had statistically significantly higher strength at 1.5â¯mm and 2â¯mm range than Group A. However, there was no statistically significant difference between the three groups in the assessment of shearing and buckling forces. CONCLUSION: The modified transloop suture technique provides a more stable repair, and we consider that it can be used as an alternative suture repair method.
Assuntos
Povo Asiático , Cartilagens Nasais/transplante , Septo Nasal/cirurgia , Rinoplastia/métodos , Técnicas de Sutura , População Branca , Humanos , Reoperação , Suturas , Resistência à Tração , Resultado do TratamentoRESUMO
Poly(ε-caprolactone) (PCL) is one of the most commonly used polymers in the production of tissue engineered scaffolds for hard tissue treatments. Incorporation of cells into these scaffolds significantly enhances the healing rate of the tissue. In this study, PCL scaffolds were prepared by wet spinning technique and modified by addition of fibrinogen in order to form a fibrin network between the PCL fibers. By this way, scaffolds would have micro- and nanofibers in their structures. Drying of the wet spun constructs was achieved by application of ethanol dehydration or freeze drying techniques. Fibrinogen solutions (as low: 2â¯mg/mL; or high: 10â¯mg/mL concentrations) were added onto the scaffolds and fibrin formation was achieved via fibrinogen crosslinking. Results showed that ethanol dehydration led to film-like coating on the fibers while freeze-drying led to nanofiber bridges between PCL fibers establishing an interconnected web in the structure. Mechanical properties of the scaffolds were improved in the presence of the fibrin net. After the seeding of Saos-2 cells, higher attachment and homogeneous distribution of the cells was achieved on the samples modified with high concentration of fibrinogen. These scaffolds can be good candidates for the treatment of problematic bone defects.
Assuntos
Fibrina/química , Géis/química , Nanofibras/química , Poliésteres/química , Polímeros/química , Alicerces Teciduais/química , Materiais Biocompatíveis/química , Células Cultivadas , Humanos , Teste de Materiais/métodos , Engenharia Tecidual/métodosRESUMO
Scaffolds produced for tissue engineering applications are proven to be promising alternatives to be used in healing and regeneration of injured tissues and organs. In this study, porous and fibrous poly(ε-caprolactone) (PCL) scaffolds were prepared by wet spinning technique and modified by addition of tricalcium phosphate (TCP) and by immobilizing gelatin onto fibers. Meanwhile, gelatin microspheres carrying Ceftriaxone sodium (CS), a model antibiotic, were added onto the scaffolds and antimicrobial activity of CS was investigated against Escherichia coli (E. coli), a model gram-negative bacterium. TCP and gelatin were added to enhance mechanical properties while directing the scaffold towards osteogenic infrastructure and to increase hydrophilicity by activating cell attachment via protein molecules, respectively. Modifications with TCP and gelatin enhanced the compression modulus by about 70%, and attachment of Saos-2 cells by 60%, respectively. Release of the antibiotic demonstrated effective antimicrobial activity against E. coli. The bioactive scaffolds were shown to be good candidates for bone tissue engineering applications.
Assuntos
Antibacterianos/química , Osso e Ossos/citologia , Fosfatos de Cálcio/química , Portadores de Fármacos/química , Microesferas , Poliésteres/química , Engenharia Tecidual , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Escherichia coli/efeitos dos fármacos , Humanos , Porosidade , Propriedades de Superfície , Alicerces Teciduais/químicaRESUMO
Biodegradable polymers have met with an increasing demand in medical usage over the last decades. One of such polymers is poly(ε-caprolactone) (PCL), which is a polyester that has been widely used in tissue engineering field for its availability, relatively inexpensive price and suitability for modification. Its chemical and biological properties, physicochemical state, degradability and mechanical strength can be adjusted, and therefore, it can be used under harsh mechanical, physical and chemical conditions without significant loss of its properties. Degradation time of PCL is quite long, thus it is used mainly in the replacement of hard tissues in the body where healing also takes an extended period of time. It is also used at load-bearing tissues of the body by enhancing its stiffness. However, due to its tailorability, use of PCL is not restricted to one type of tissue and it can be extended to engineering of soft tissues by decreasing its molecular weight and degradation time. This review outlines the basic properties of PCL, its composites, blends and copolymers. We report on various techniques for the production of different forms, and provide examples of medical applications such as tissue engineering and drug delivery systems covering the studies performed in the last decades.
Assuntos
Materiais Biocompatíveis/química , Portadores de Fármacos/química , Poliésteres/química , Eletricidade , Humanos , Microesferas , Engenharia TecidualRESUMO
The present study aimed to investigate the effect of structure (design and porosity) on the matrix stiffness and osteogenic activity of stem cells cultured on poly(ester-urethane) (PEU) scaffolds. Different three-dimensional (3D) forms of scaffold were prepared from lysine-based PEU using traditional salt-leaching and advanced bioplotting techniques. The resulting scaffolds were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), mercury porosimetry and mechanical testing. The scaffolds had various pore sizes with different designs, and all were thermally stable up to 300 °C. In vitro tests, carried out using rat bone marrow stem cells (BMSCs) for bone tissue engineering, demonstrated better viability and higher cell proliferation on bioplotted scaffolds compared to salt-leached ones, most probably due to their larger and interconnected pores and stiffer nature, as shown by higher compressive moduli, which were measured by compression testing. Similarly, SEM, von Kossa staining and EDX analyses indicated higher amounts of calcium deposition on bioplotted scaffolds during cell culture. It was concluded that the design with larger interconnected porosity and stiffness has an effect on the osteogenic activity of the stem cells.
Assuntos
Células da Medula Óssea/citologia , Osteogênese/efeitos dos fármacos , Poliésteres/química , Poliuretanos/química , Células-Tronco/citologia , Alicerces Teciduais/química , Animais , Osso e Ossos/patologia , Varredura Diferencial de Calorimetria , Bovinos , Proliferação de Células , Força Compressiva , Lisina/química , Masculino , Mercúrio , Microscopia Eletrônica de Varredura , Porosidade , Ratos , Ratos Sprague-Dawley , Sais/química , Soroalbumina Bovina/química , Estresse Mecânico , Termogravimetria , Engenharia Tecidual/métodosRESUMO
Previous studies have shown epidermal growth factor (EGF) facilitate peritoneal membrane healing by augmenting cell adhesion and migration. The objective of this study was to show the effect of sustained and local administration of EGF on peritoneal adhesion. Fourty-two rats were divided into six groups: control 7 and 14, gelatin 7 and 14, and EGF 7 and 14. Adhesions were created by scraping the cecum with mesh gause followed by application of absolute alcohol and placement of silk suture in the parietal peritoneum. The anterior walls of the intestines were covered with 5 x 5 cm unloaded, and EGF loaded gelatin films in the gelatin and EGF groups, respectively. The rats were killed on days 7 and 14 to assess the adhesion occurring, and for biochemical examination. The mean adhesion grades of EGF groups were significantly lower than in the other groups (P < 0.008). The mean adenosine deaminase (ADA) measurements of EGF 7 group were lower than in the gelatin 7 and control 7 groups but the difference was not significant (P > 0.008). The mean ADA measurements in the 14 days groups were as follows: control 14 < EGF 14 < gelatin 14 groups. The mean ADA measurements between 14 days groups did not significantly differ from each other (P > 0.008). The mean hydroxyproline measurements did not differ among the groups (P > 0.008). EGF decreased intestinal adhesion in our study. EGF has important roles in DNA synthesis and cell proliferation. Further studies are required to determine the exact mechanism by which EGF lowers the efficiency of intestinal adhesion.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Peritônio , Aderências Teciduais/prevenção & controle , Animais , Fator de Crescimento Epidérmico/administração & dosagem , Hidroxiprolina/metabolismo , Masculino , Ratos , Ratos Wistar , Estatísticas não ParamétricasRESUMO
Tranilast is an antiallergic drug that interferes with proliferation and migration of vascular smooth muscle cell induced by platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1). We investigated the local effect of tranilast on neointimal hyperplasia using tranilast-coated prosthetic grafts. The inner sides of the thin-walled polytetrafluoroethylene (PTFE) grafts were coated with chitosan and tranilast containing chitosan solution. Wistar albino rats (32) were used in the study. Patches (1 x 2 mm) for vascular grafts were prepared. Three groups were tested: group 1 (n = 12; tranilast coated), group 2 (n = 10; adhesive-only film-layer-coated), and group 3 (n = 10; normal ePTFE patch grafts sutured to the carotid arteries of the rats). Recipient sites of the carotid arteries were excised 4 weeks after surgery. All sections were examined histologically for graft patency, thrombus formation, and neointimal thickness. Expression of PDGF, fibroblast growth factor, and TGF-beta1 on cross-sections of the neointima were evaluated by immunohistochemistry. No significant differences were found regarding mean neointimal thicknesses. PDGF and TGF-beta-1 expressions were significantly lower in group 1. Although a decrease in local effect of tranilast was observed for growth factor expressions at a drug concentration of 0.05 mg/cm(2), a significant reduction in neointimal hyperplasia was not achieved. The coating concentration of 0.05 mg/cm(2) may have been too low to produce an antiproliferative effect. Given our promising results, further studies are recommended and planned using different drug concentrations and time intervals.
Assuntos
Antialérgicos/farmacologia , Prótese Vascular , Materiais Revestidos Biocompatíveis/farmacologia , Oclusão de Enxerto Vascular/prevenção & controle , ortoaminobenzoatos/farmacologia , Animais , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Oclusão de Enxerto Vascular/patologia , Sobrevivência de Enxerto , Hiperplasia , Politetrafluoretileno , Ratos , Ratos Wistar , Túnica Íntima/patologiaRESUMO
The conventional treatment with regular red-blood-cell transfusions and simultaneous chelation of excess iron with DFO (desferrioxamine) improves quality of life of thalassaemic patients while increasing their rate of survival considerably. Although DFO is the main iron- chelating drug currently utilized, it has various drawbacks, including high cost, poor oral effectiveness, toxicity and short plasma half-life. It has to be administered by slow, subcutaneous infusion during blood transfusion for 8-12 h at night, 5-7 nights a week, and this leads to a very poor patient compliance. In order to avoid frequent and uncomfortable infusions of DFO, application of controlled-release systems might be alternative routes in the supportive treatment of thalassaemia. In the present study, GMs (gelatin microspheres) and GFs (gelatin films) were prepared by coacervation and casting methods respectively to develop controlled DFO-release systems. Cross-linking by glutaraldehyde and carbodi-imide were performed to increase the stability of gelatin matrices. Microspheres and films prepared without the addition of cross-linker degraded completely in 4 h. On the other hand, addition of cross-linker extended this time from hours to weeks depending on the added amount. Therefore the amount of DFO released from microspheres in 7 days was found to be in the range 12-82%, whereas the amount permeated through the films in 5.0 h was found to be in the range 34-67%. GFs were elastic and demonstrated good mechanical properties. Films achieved 0.14-0.69 MPa tensile strength, with 0.12-1.29 MPa elastic modulus and 26.49-109.38% strain values at break point. These studies showed that gelatin-based controlled-release systems could be improved and could be good candidates for the production of long-term DFO-carrying systems.
Assuntos
Materiais Biocompatíveis/química , Desferroxamina/metabolismo , Sistemas de Liberação de Medicamentos , Gelatina/química , Reagentes de Ligações Cruzadas/farmacologia , Preparações de Ação Retardada , Diálise/métodos , Estabilidade de Medicamentos , Elasticidade , Gelatina/ultraestrutura , Glutaral/farmacologia , Humanos , Microesferas , Estresse Mecânico , Resistência à Tração , Talassemia beta/terapiaRESUMO
Epidermal growth factor (EGF) modulates Leydig cell proliferation, steroidogenesis, spermiogenesis, and Sertoli cell activity. It plays an important role in repairing ischemia-reperfusion injury in different tissues. The aim of this study was to evaluate the effects of sustained and local administration of EGF on improving bilateral testicular tissue after torsion. A total of 57 Wistar albino rats were used. For the EGF transport system, 1x2 cm gelatin films containing 2 microg EGF were used. Torsion was created by rotating the right testis 720 degrees in a clockwise direction for 4 h in all groups except the control group. Then, in the torsion group, bilateral orchiectomy was performed. After returning the torsioned ipsilateral testes to their normal state, the bilateral testes were wrapped by 1x2 cm unloaded gelatin films in the gelatin (G7 and G21) groups and, by 2 microg EGF loaded gelatin films in the EGF 7 and EGF 21 groups. The testes were removed on the seventh and 21st days, respectively, for biochemical and histological examination. Histologically, Johnsen's spermatogenesis criteria and mean seminiferous tubule diameter (MSTD) measurements were used. The EGF7 group did not show significant loss of Sertoli cells, while in the G7 group the number of these cells decreased. The ipsilateral ischemic testis of the EGF21 group showed Leydig cell hyperplasia, and the contralateral non-ischemic testes in this group were similar to the control group. In the G21 group, the bilateral testes showed Sertoli cell only syndrome in some sections, and most of the cells were undergoing apoptosis. The mean spermatogenesis scores and MSTD in the EGF7 and EGF21 groups were higher than in the G7 and G21 groups ( P<0.05). Malondialdehyde levels were significantly lower in the EGF groups than in the G groups ( P<0.05). Glutathione peroxidase (GSH-Px) levels in the G21 group were significantly higher than in the EGF21 group. Our study shows that local and sustained EGF release after testicular torsion improves bilateral testicular injury. EGF administration may be a new treatment choice for bilaterally injured testis after detorsion without removing the twisted testis.