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1.
Front Cardiovasc Med ; 9: 1013183, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36465472

RESUMO

The constantly increasing incidence of coronary artery disease worldwide makes necessary to set advanced therapies and tools such as tissue engineered vessel grafts (TEVGs) to surpass the autologous grafts [(i.e., mammary and internal thoracic arteries, saphenous vein (SV)] currently employed in coronary artery and vascular surgery. To this aim, in vitro cellularization of artificial tubular scaffolds still holds a good potential to overcome the unresolved problem of vessel conduits availability and the issues resulting from thrombosis, intima hyperplasia and matrix remodeling, occurring in autologous grafts especially with small caliber (<6 mm). The employment of silk-based tubular scaffolds has been proposed as a promising approach to engineer small caliber cellularized vascular constructs. The advantage of the silk material is the excellent manufacturability and the easiness of fiber deposition, mechanical properties, low immunogenicity and the extremely high in vivo biocompatibility. In the present work, we propose a method to optimize coverage of the luminal surface of silk electrospun tubular scaffold with endothelial cells. Our strategy is based on seeding endothelial cells (ECs) on the luminal surface of the scaffolds using a low-speed rolling. We show that this procedure allows the formation of a nearly complete EC monolayer suitable for flow-dependent studies and vascular maturation, as a step toward derivation of complete vascular constructs for transplantation and disease modeling.

2.
Front Bioeng Biotechnol ; 10: 918690, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36061430

RESUMO

3D-Bioprinting leads to the realization of tridimensional customized constructs to reproduce the biological structural complexity. The new technological challenge focuses on obtaining a 3D structure with several distinct layers to replicate the hierarchical organization of natural tissues. This work aims to reproduce large blood vessel substitutes compliant with the original tissue, combining the advantages of the 3D bioprinting, decellularization, and accounting for the presence of different cells. The decellularization process was performed on porcine aortas. Various decellularization protocols were tested and evaluated through DNA extraction, quantification, and amplification by PCR to define the adequate one. The decellularized extracellular matrix (dECM), lyophilized and solubilized, was combined with gelatin, alginate, and cells to obtain a novel bioink. Several solutions were tested, tuning the percentage of the components to obtain the adequate structural properties. The geometrical model of the large blood vessel constructs was designed with SolidWorks, and the construct slicing was done using the HeartWare software, which allowed generating the G-Code. The final constructs were 3D bioprinted with the Inkredible + using dual print heads. The composition of the bioink was tuned so that it could withstand the printing of a segment of a tubular construct up to 10 mm and reproduce the multicellular complexity. Among the several compositions tested, the suspension resulting from 8% w/v gelatin, 7% w/v alginate, and 3% w/v dECM, and cells successfully produced the designed structures. With this bioink, it was possible to print structures made up of 20 layers. The dimensions of the printed structures were consistent with the designed ones. We were able to avoid the double bioink overlap in the thickness, despite the increase in the number of layers during the printing process. The optimization of the parameters allowed the production of structures with a height of 20 layers corresponding to 9 mm. Theoretical and real structures were very close. The differences were 14% in height, 20% internal diameter, and 9% thickness. By tailoring the printing parameters and the amount of dECM, adequate mechanical properties could be met. In this study, we developed an innovative printable bioink able to finely reproduce the native complex structure of the large blood vessel.

3.
Bioengineering (Basel) ; 8(2)2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33499168

RESUMO

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly worldwide. So far, the etiology and the progression of AMD are not well known. Animal models have been developed to study the mechanisms involved in AMD; however, according to the "Three Rs" principle, alternative methods have been investigated. Here we present a strategy to develop a "Three Rs" compliant retinal three-dimensional (3D) in vitro model, including a Bruch's membrane model and retina pigment epithelium (RPE) layer. First, tensile testing was performed on porcine retina to set a reference for the in vitro model. The results of tensile testing showed a short linear region followed by a plastic region with peaks. Then, Bruch's membrane (BrM) was fabricated via electrospinning by using Bombyx mori silk fibroin (BMSF) and polycaprolactone (PCL). The BrM properties and ARPE-19 cell responses to BrM substrates were investigated. The BrM model displayed a thickness of 44 µm, with a high porosity and an average fiber diameter of 1217 ± 101 nm. ARPE-19 cells adhered and spread on the BMSF/PCL electrospun membranes. In conclusion, we are developing a novel 3D in vitro retinal model towards the replacement of animal models in AMD studies.

4.
Nanomaterials (Basel) ; 11(2)2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494337

RESUMO

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.

5.
Int J Artif Organs ; 43(10): 631-644, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32148174

RESUMO

Along with an increased incidence of cardiovascular diseases, there is a strong need for small-diameter vascular grafts. Silk has been investigated as a biomaterial to develop such grafts thanks to different processing options. Endothelialization was shown to be extremely important to ensure graft patency and there is ongoing research on the development and behavior of endothelial cells on vascular tissue-engineered scaffolds. This article reviews the endothelialization of silk-based scaffolds processed throughout the years as silk non-woven nets, films, gel spun, electrospun, or woven scaffolds. Encouraging results were reported with these scaffolds both in vitro and in vivo when implanted in small- to middle-sized animals. The use of coatings and heparin or sulfur to enhance, respectively, cell adhesion and scaffold hemocompatibility is further presented. Bioreactors also showed their interest to improve cell adhesion and thus promoting in vitro pre-endothelialization of grafts even though they are still not systematically used. Finally, the importance of the animal models used to study the right mechanism of endothelialization is discussed.


Assuntos
Materiais Biocompatíveis , Prótese Vascular , Células Endoteliais/citologia , Seda , Animais , Adesão Celular , Heparina , Modelos Animais
6.
Mater Sci Eng C Mater Biol Appl ; 105: 110035, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546369

RESUMO

Aiming to perfuse porous tubular scaffolds for vascular tissue engineering (VTE) with controlled flow rate, prevention of leakage through the scaffold lumen is required. A gel coating made of 8% w/v alginate and 6% w/v gelatin functionalized with fibronectin was produced using a custom-made bioreactor-based method. Different volumetric proportions of alginate and gelatin were tested (50/50, 70/30, and 90/10). Gel swelling and stability, and rheological, and uniaxial tensile tests reveal superior resistance to the aggressive biochemical microenvironment, and their ability to withstand physiological deformations (~10%) and wall shear stresses (5-20 dyne/cm2). These are prerequisites to maintain the physiologic phenotypes of vascular smooth muscle cells and endothelial cells (ECs), mimicking blood vessels microenvironment. Gels can induce ECs proliferation and colonization, especially in the presence of fibronectin and higher percentages of gelatin. The custom-designed bioreactor enables the development of reproducible and homogeneous tubular gel coating. The permeability tests show the effectiveness of tubular scaffolds coated with 70/30 alginate/gelatin gel to occlude wadding pores, and therefore prevent leakages. The synthesized double-layered tubular scaffolds coated with alginate/gelatin gel and fibronectin represent both promising substrate for ECs and effective leakproof scaffolds, when subjected to pulsatile perfusion, for VTE applications.


Assuntos
Vasos Sanguíneos/fisiologia , Hidrogéis/farmacologia , Resistência ao Cisalhamento , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Reatores Biológicos , Vasos Sanguíneos/efeitos dos fármacos , Linhagem Celular , Humanos , Permeabilidade , Porosidade , Resistência à Tração
7.
Nat Commun ; 9(1): 4286, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327457

RESUMO

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.


Assuntos
Esôfago/citologia , Esôfago/fisiologia , Músculo Esquelético/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Criança , Pré-Escolar , Criopreservação/métodos , Células Epiteliais , Matriz Extracelular/fisiologia , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Crista Neural/transplante , Ratos Sprague-Dawley
8.
Comput Methods Biomech Biomed Engin ; 20(10): 1077-1088, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28569086

RESUMO

The in vitro replication of physiological mechanical conditioning through bioreactors plays a crucial role in the development of functional Small-Caliber Tissue-Engineered Blood Vessels. An in silico scaffold-specific model under pulsatile perfusion provided by a bioreactor was implemented using a fluid-structure interaction (FSI) approach for viscoelastic tubular scaffolds (e.g. decellularized swine arteries, DSA). Results of working pressures, circumferential deformations, and wall shear stress on DSA fell within the desired physiological range and indicated the ability of this model to correctly predict the mechanical conditioning acting on the cells-scaffold system. Consequently, the FSI model allowed us to a priori define the stimulation pattern, driving in vitro physiological maturation of scaffolds, especially with viscoelastic properties.


Assuntos
Vasos Sanguíneos/fisiologia , Reologia , Engenharia Tecidual/métodos , Animais , Artérias/fisiologia , Fenômenos Biomecânicos , Reatores Biológicos , Modelos Teóricos , Pressão , Estresse Mecânico , Suínos , Alicerces Teciduais/química
9.
J Appl Biomater Funct Mater ; 15(2): e122-e132, 2017 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-28362040

RESUMO

BACKGROUND: In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. METHODS: A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. RESULTS: The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. CONCLUSIONS: The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.


Assuntos
Reatores Biológicos , Poliuretanos , Engenharia Tecidual , Alicerces Teciduais , Animais , Células Cultivadas , Fibroblastos/citologia , Camundongos
10.
Cells Tissues Organs ; 200(6): 363-73, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26562773

RESUMO

There is still an unmet clinical need for small-caliber artery substitution. Decellularized scaffolds in tissue engineering represent a promising solution. We have developed an innovative system for the automatic decellularization of blood vessels, used to process pig arteries. The system is able to automatically drive a decellularization process in a safe and reliable environment, with complex time patterns, using up to three different decellularization solutions, and providing at the same time a physical stress to improve the decellularization. The decellularization of pig arteries was evaluated by means of histology, DNA quantification and mechanical testing. Outcomes showed scaffolds with no cellular or nuclear remnants and a well-preserved tissue structure, corroborated by mechanical properties similar to native tissue. Decellularized scaffolds were seeded on the inner layer with human endothelial cells and implanted as iliac artery replacement in 4 pharmacologically immune-compromised pigs. This chimeric model was performed as a very preliminary evaluation to investigate the performances of these scaffolds in vivo, and to investigate the fate of seeded cells. Recipients were sacrificed on day 14 and day 70 after surgery, and vessels were found to be patent and with no evidence of thrombi formation. The inner layer was covered by endothelial cells, and the migration of cells positive for α-smooth-muscle actin was observed from the outer layer towards the tunica media. Intriguingly, the endothelial cells on explanted vessels were entirely derived from the host while the seeded cells were lost. In conclusion, this work presents a novel tool for a safe and controlled production of arterial scaffolds, with good decellularization outcomes and a good performance in a short-term, large-animal implantation.


Assuntos
Artérias/citologia , Prótese Vascular , Células Endoteliais/citologia , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Desenho de Equipamento , Humanos , Sus scrofa , Engenharia Tecidual/métodos
11.
Regen Med ; 10(4): 505-27, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26022767

RESUMO

The absence of successful solutions in treatments of small-caliber vessel diseases led to the Vascular Tissue Engineering approach to develop functional nonimmunogenic tissue engineered blood vessels. In this context, the choice of cells to be seeded and the microenvironment conditioning are pivotal. Biochemical and biomechanical stimuli seem to activate physiological regulatory pathways that induce the production of molecules and proteins stimulating stem cell differentiation toward vascular lineage and reproducing natural cross-talks among vascular cells to improve the maturation of tissue engineered blood vessels. Thus, this review focuses on (1) available cell sources, and (2) biochemical and biomechanical stimuli, with the final aim to obtain the long-term stability of the endothelium and mechanical properties suitable for withstanding physiological load.


Assuntos
Prótese Vascular , Vasos Sanguíneos/fisiologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Fenômenos Biomecânicos , Diferenciação Celular , Humanos
12.
Front Physiol ; 5: 362, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25295011

RESUMO

Skeletal muscle tissue is characterized by high metabolic requirements, defined structure and high regenerative potential. As such, it constitutes an appealing platform for tissue engineering to address volumetric defects, as proven by recent works in this field. Several issues common to all engineered constructs constrain the variety of tissues that can be realized in vitro, principal among them the lack of a vascular system and the absence of reliable cell sources; as it is, the only successful tissue engineering constructs are not characterized by active function, present limited cellular survival at implantation and possess low metabolic requirements. Recently, functionally competent constructs have been engineered, with vascular structures supporting their metabolic requirements. In addition to the use of biochemical cues, physical means, mechanical stimulation and the application of electric tension have proven effective in stimulating the differentiation of cells and the maturation of the constructs; while the use of co-cultures provided fine control of cellular developments through paracrine activity. This review will provide a brief analysis of some of the most promising improvements in the field, with particular attention to the techniques that could prove easily transferable to other branches of tissue engineering.

13.
Biomed Res Int ; 2013: 918753, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23865072

RESUMO

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at -80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Young's modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.


Assuntos
Artérias/citologia , Artérias/fisiologia , Desoxirribonuclease I/metabolismo , Detergentes/farmacologia , Engenharia Tecidual/métodos , Animais , Artérias/anatomia & histologia , Artérias/ultraestrutura , Fenômenos Biomecânicos/efeitos dos fármacos , DNA/metabolismo , Indóis/metabolismo , Teste de Materiais , Coloração e Rotulagem , Sus scrofa
15.
Biomaterials ; 33(20): 5085-93, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22510434

RESUMO

Materials based on synthetic polymers can be extensively tailored in their physical properties but often suffer from limited biological functionality. Here we tested the hypothesis that the biological performance of 3D synthetic polymer-based scaffolds can be enhanced by extracellular matrix (ECM) deposited by cells in vitro and subsequently decellularized. The hypothesis was tested in the context of bone graft substitutes, using polyesterurethane (PEU) foams and mineralized ECM laid by human mesenchymal stromal cells (hMSC). A perfusion-based bioreactor system was critically employed to uniformly seed and culture hMSC in the scaffolds and to efficiently decellularize (94% DNA reduction) the resulting ECM while preserving its main organic and inorganic components. As compared to plain PEU, the decellularized ECM-polymer hybrids supported the osteoblastic differentiation of newly seeded hMSC by up-regulating the mRNA expression of typical osteoblastic genes (6-fold higher bone sialoprotein; 4-fold higher osteocalcin and osteopontin) and increasing calcium deposition (6-fold higher), approaching the performance of ceramic-based materials. After ectopic implantation in nude mice, the decellularized hybrids induced the formation of a mineralized matrix positively immunostained for bone sialoprotein and resembling an immature osteoid tissue. Our findings consolidate the perspective of bioreactor-based production of ECM-decorated polymeric scaffolds as off-the-shelf materials combining tunable physical properties with the physiological presentation of instructive biological signals.


Assuntos
Materiais Biocompatíveis , Matriz Extracelular , Polímeros , Engenharia Tecidual , Animais , Diferenciação Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Osteoblastos/citologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Int J Artif Organs ; 35(3): 191-8, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22461114

RESUMO

OBJECTIVES: Decellularized biological scaffolds represent a promising solution for tissue engineering. They offer a good substrate for cells in terms of biochemical composition, ultrastructure and mechanical properties without generating an immunogenic response. The aim of this study was to design and develop a device for the automatic decellularization of biological tissues to overcome manual operation limits, toward a good manufacturing practice-compliant process. METHODS: A versatile, modular and easy-to-use device was designed, able to automatically exchange decellularization fluids and to provide mechanical shaking according to a user-defined protocol. Preliminary decellularization tests were made on porcine abdominal aortas comparing results between conventional process and device-operated process using water, sodium deoxycholate and DNase. Vessels were processed up to 4 cycles of the protocol and after each decellularization cycle histological analyses (hematoxylin-eosin, Movat pentachrome and DAPI stainings) were observed. Preliminary mechanical tests were also performed to compare the mechanical behavior of blood vessels processed with the 2 methods mentioned above. RESULTS: Briefly, the device consists of decellularization chambers, a shaking system and hydraulic modules for the exchange of fluids. The device was bench-tested for functionality and reliability with positive outcomes. The protocol used revealed to be effective, with a progressive tissue decellularization through repeated cycles. No difference between manual and automated operation was observed in histological or mechanical analyses. CONCLUSIONS: The developed device is able to automate the decellularization process lowering operator-related risks, and is a reliable and functional tool for clinical use.


Assuntos
Aorta/citologia , Engenharia Tecidual/instrumentação , Animais , Suínos , Alicerces Teciduais
17.
J Appl Biomater Biomech ; 9(2): 73-86, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22065385

RESUMO

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.


Assuntos
Engenharia Biomédica/métodos , Engenharia Biomédica/tendências , Medicina Regenerativa , Técnicas de Transferência de Genes/instrumentação , Técnicas de Transferência de Genes/tendências , Regeneração , Medicina Regenerativa/instrumentação , Medicina Regenerativa/métodos , Medicina Regenerativa/tendências , Células-Tronco
18.
J Appl Biomater Biomech ; 8(2): 68-75, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20740468

RESUMO

PURPOSE: Since stretching plays a key role in skeletal muscle tissue development in vivo, by making use of an innovative bioreactor and a biodegradable microfibrous scaffold (DegraPol(R)) previously developed by our group, we aimed to investigate the effect of mechanical conditioning on the development of skeletal muscle engineered constructs, obtained by seeding and culturing murine skeletal muscle cells on electrospun membranes. METHODS: Following 5 days of static culture, skeletal muscle constructs were transferred into the bioreactor and further cultured for 13 days, while experiencing a stretching pattern adapted from the literature to resemble mouse development and growth. Sample withdrawal occurred at the onset of cyclic stretching and after 7 and 10 days. Myosin heavy chain (MHC) accumulation in stretched constructs (D) was evaluated by Western blot analysis and immunofluorescence staining, using statically cultured samples (S) as controls. RESULTS: Western blot analysis of MHC on dynamically (D) and statically (S) cultured constructs at different time points showed that, at day 10, the applied stretching pattern led to an eight-fold increase in myosin accumulation in cyclically stretched constructs (D) with respect to the corresponding static controls (S). These results were confirmed by immunofluorescence staining of total sarcomeric MHC. CONCLUSIONS: Since previous attempts to reproduce skeletal myogenesis in vitro mainly suffered from the difficulty of driving myoblast development into an architecturally organized array of myosin expressing myotubes, the chance of inducing MHC accumulation via mechanical conditioning represents a significant step towards the generation of a functional muscle construct for skeletal muscle tissue engineering applications.


Assuntos
Reatores Biológicos , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/biossíntese , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células , Linhagem Celular , Camundongos , Músculo Esquelético/crescimento & desenvolvimento
19.
J Thorac Cardiovasc Surg ; 139(2): 437-43, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19995663

RESUMO

OBJECTIVE: We sought to determine the relative contributions of epithelial cells and mesenchymal stem cell-derived chondrocytes to the survival of tissue-engineered airway transplants in pigs. METHODS: Nonimmunogenic tracheal matrices were obtained by using a detergent-enzymatic method. Major histocompatibility complex-unmatched animals (weighing 65 +/- 4 kg) were divided into 4 groups (each n = 5), and 6 cm of their tracheas were orthotopically replaced with decellularized matrix only (group I), decellularized matrix with autologous mesenchymal stem cell-derived chondrocytes externally (group II), decellularized matrix with autologous epithelial cells internally (group III), or decellularized matrix with both cell types (group IV). Autologous cells were recovered, cultured, and expanded. Mesenchymal stem cells were differentiated into chondrocytes by using growth factors. Both cell types were seeded simultaneously with a dual-chamber bioreactor. Animals were not immunosuppressed during the entire study. Biopsy specimens and blood samples were taken from recipients continuously, and animals were observed for a maximum of 60 days. RESULTS: Matrices were completely covered with both cell types within 72 hours. Survival of the pigs was significantly affected by group (P < .05; group I, 11 +/- 2 days; group II, 29 +/- 4 days; group III, 34 +/- 4 days; and group IV, 60 +/- 1 days). Cause of death was a combination of airway obstruction and infection (group I), mainly infection (group II), or primarily stenosis (group III). However, pigs in group IV were alive, with no signs of airway collapse or ischemia and healthy epithelium. There were no clinical, immunologic, or histologic signs of rejection despite the lack of immunosuppression. CONCLUSIONS: We confirm the clinical potential of autologous cell- and tissue-engineered tracheal grafts, and suggest that the seeding of both epithelial and mesenchymal stem cell-derived chondrocytes is necessary for optimal graft survival.


Assuntos
Células-Tronco Mesenquimais/fisiologia , Traqueia/transplante , Animais , Reatores Biológicos , Condrócitos , Células Epiteliais , Sobrevivência de Enxerto , Suínos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Traqueia/citologia
20.
Int J Artif Organs ; 32(11): 763-8, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20020407

RESUMO

Autologous tissue transfer, allografts and prosthetic replacements have so far failed to offer functional solutions for the treatment of long circumferential tracheal defects. Because of the shortcomings related with these strategies, interest has turned increasingly to the field of tissue engineering which applies the principles of engineering and life sciences in an effort to develop in vitro biological substitutes able to restore, maintain, or improve tissue and organ function. The advances in this field during the past decade have thus provided a new attractive approach toward the concept of functional substitutes and may represent an alternative to the shortage of suitable grafts for reconstructive airway surgery. This article gives an overview of the tissue engineering approach and of the encouraging strategies attempted so far in trachea regeneration.


Assuntos
Órgãos Bioartificiais , Medicina Regenerativa , Engenharia Tecidual , Traqueia/transplante , Doenças da Traqueia/cirurgia , Animais , Humanos
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