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1.
J Vis Exp ; (168)2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33645584

RESUMEN

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.


Asunto(s)
Neovascularización Fisiológica , Piel Artificial , Piel/irrigación sanguínea , Ingeniería de Tejidos/métodos , Animales , Biomarcadores/metabolismo , Células Cultivadas , Colágeno/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Imagenología Tridimensional , Imagen Óptica , Permeabilidad , Ratas , Coloración y Etiquetado , Suspensiones
2.
Nat Commun ; 12(1): 1913, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772014

RESUMEN

Diffusion is a major molecular transport mechanism in biological systems. Quantifying direction-dependent (i.e., anisotropic) diffusion is vitally important to depicting how the three-dimensional (3D) tissue structure and composition affect the biochemical environment, and thus define tissue functions. However, a tool for noninvasively measuring the 3D anisotropic extracellular diffusion of biorelevant molecules is not yet available. Here, we present light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), which noninvasively determines 3D diffusion tensors of various biomolecules with diffusivities up to 51 µm2 s-1, reaching the physiological diffusivity range in most biological systems. Using cornea as an example, LiFT-FRAP reveals fundamental limitations of current invasive two-dimensional diffusion measurements, which have drawn controversial conclusions on extracellular diffusion in healthy and clinically treated tissues. Moreover, LiFT-FRAP demonstrates that tissue structural or compositional changes caused by diseases or scaffold fabrication yield direction-dependent diffusion changes. These results demonstrate LiFT-FRAP as a powerful platform technology for studying disease mechanisms, advancing clinical outcomes, and improving tissue engineering.


Asunto(s)
Córnea/metabolismo , Espacio Extracelular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Tendones/metabolismo , Animales , Anisotropía , Colágeno/química , Colágeno/metabolismo , Difusión , Análisis de Fourier , Microscopía Confocal/métodos , Microscopía Electrónica de Rastreo/métodos , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Porcinos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química
3.
Ann R Coll Surg Engl ; 103(4): 245-249, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33682428

RESUMEN

Soft tissue reconstruction remains a continuing challenge for plastic and reconstructive surgeons. Standard methods of reconstruction such as local tissue transfer and free autologous tissue transfer are successful in addressing soft tissue cover, yet they do not come without the additional morbidity of donor sites. Autologous fat transfer has been used in reconstruction of soft tissue defects in different branches of plastic surgery, specifically breast and facial defect reconstruction, while further maintaining a role in body contouring procedures. Current autologous fat transfer techniques come with the drawbacks of donor-site morbidity and, more significantly, resorption of large amounts of fat. Advancement in tissue engineering has led to the use of engineered adipose tissue structures based on adipose-derived stem cells. This enables a mechanically similar reconstruct that is abundantly available. Cosmetic and mechanical similarity with native tissue is the main clinical goal for engineered adipose tissue. Development of novel techniques in the availability of natural tissue is an exciting prospect; however, it is important to investigate the potential of cell sources and culture strategies for clinical applications. We review these techniques and their applications in plastic surgery.


Asunto(s)
Tejido Adiposo/trasplante , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Procedimientos Quirúrgicos Reconstructivos/métodos , Ingeniería de Tejidos/métodos , Tejido Adiposo/citología , Humanos
4.
Life Sci ; 276: 119373, 2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-33744324

RESUMEN

Development of novel technologies provides the best tissue constructs engineering and maximizes their therapeutic effects in regenerative therapy, especially for liver dysfunctions. Among the currently investigated approaches of tissue engineering, scaffold-based and scaffold-free tissues are widely suggested for liver regeneration. Analogs of liver acellular extracellular matrix (ECM) are utilized in native scaffolds to increase the self-repair and healing ability of organs. Native ECM analog could improve liver repairing through providing the supportive framework for cells and signaling molecules, exerting normal biomechanical, biochemical, and physiological signal complexes. Recently, innovative cell sheet technology is introduced as an alternative for conventional tissue engineering with the advantage of fewer scaffold restrictions and cell culture on a Thermo-Responsive Polymer Surface. These sheets release the layered cells through a temperature-controlled procedure without enzymatic digestion, while preserving the cell-ECM contacts and adhesive molecules on cell-cell junctions. In addition, several novelties have been introduced into the cell sheet and decellularization technologies to aid cell growth, instruct differentiation/angiogenesis, and promote cell migration. In this review, recent trends, advancements, and issues linked to translation into clinical practice are dissected and compared regarding the decellularization and cell sheet technologies for liver tissue engineering.


Asunto(s)
Matriz Extracelular/química , Hepatopatías/terapia , Regeneración Hepática , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Humanos
5.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33672027

RESUMEN

Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3-3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2-type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa.


Asunto(s)
Materiales Biocompatibles/química , Hidrogeles/química , Algas Marinas/química , Sefarosa/química , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/farmacología , Fenómenos Biomecánicos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Módulo de Elasticidad , Fibroblastos/metabolismo , Estudios de Seguimiento , Voluntarios Sanos , Humanos , Hidrogeles/farmacología , Ratas , Ratas Wistar , Sefarosa/farmacología , Piel/citología , Andamios del Tejido/química
6.
Nat Protoc ; 16(4): 2213-2256, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33772245

RESUMEN

Tissue-like structures from human pluripotent stem cells containing multiple cell types are transforming our ability to model and understand human development and disease. Here we describe a protocol to generate cardiomyocytes (CMs), cardiac fibroblasts (CFs) and cardiac endothelial cells (ECs), the three principal cell types in the heart, from human induced pluripotent stem cells (hiPSCs) and combine them in three-dimensional (3D) cardiac microtissues (MTs). We include details of how to differentiate, isolate, cryopreserve and thaw the component cells and how to construct and analyze the MTs. The protocol supports hiPSC-CM maturation and allows replacement of one or more of the three heart cell types in the MTs with isogenic variants bearing disease mutations. Differentiation of each cell type takes ~30 d, while MT formation and maturation requires another 20 d. No specialist equipment is needed and the method is inexpensive, requiring just 5,000 cells per MT.


Asunto(s)
Corazón/fisiología , Células Madre Pluripotentes Inducidas/citología , Ingeniería de Tejidos/métodos , Diferenciación Celular , Fenómenos Electrofisiológicos , Humanos , Modelos Biológicos , Miocitos Cardíacos/citología , Andamios del Tejido/química
7.
Carbohydr Polym ; 261: 117810, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33766329

RESUMEN

Chitosan-based hydrogels have been widely used for various biomedical applications due to their versatile properties such as biocompatibility, biodegradability, muco-adhesiveness, hemostatic effect and so on. However, the inherent rigidity and brittleness of pure chitosan hydrogels are still unmanageable, which has limited their potential use in biomaterial research. In this study, we developed in situ forming chitosan/PEG hydrogels with improved mechanical properties, using the enzymatic crosslinking reaction of horseradish peroxidase (HRP). The effect of PEG on physico-chemical properties of hybrid hydrogels was thoroughly elucidated by varying the content (0-100 %), molecular weight (4, 10 and 20 kDa) and geometry (linear, 4-arm) of the PEG derivatives. The resulting hydrogels demonstrated excellent hemostatic ability and are highly biocompatible in vivo, comparable to commercially available fibrin glue. We suggest these chitosan/PEG hybrid hydrogels with tunable physicochemical and tissue adhesive properties have great potential for a wide range of biomedical applications in the near future.


Asunto(s)
Quitosano/química , Hidrogeles/síntesis química , Adhesivos Tisulares , Adhesividad , Animales , Células Cultivadas , Dermis/citología , Dermis/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Hemostasis/efectos de los fármacos , Humanos , Hidrogeles/química , Hidrogeles/farmacología , Inyecciones , Masculino , Ensayo de Materiales , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Polimerizacion , Polímeros/administración & dosificación , Polímeros/síntesis química , Polímeros/química , Polímeros/farmacología , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Adhesivos Tisulares/administración & dosificación , Adhesivos Tisulares/síntesis química , Adhesivos Tisulares/química , Adhesivos Tisulares/farmacología , Ingeniería de Tejidos/métodos
8.
Methods Mol Biol ; 2235: 127-137, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576974

RESUMEN

Human pericytes are a perivascular cell population with mesenchymal stem cell properties, present in all vascularized tissues. Human pericytes have a distinct immunoprofile, which may be leveraged for purposes of cell purification. Adipose tissue is the most commonly used cell source for human pericyte derivation. Pericytes can be isolated by FACS (fluorescence-activated cell sorting), most commonly procured from liposuction aspirates. Pericytes have clonal multilineage differentiation potential, and their potential utility for bone regeneration has been described across multiple animal models. The following review will discuss in vivo methods for assessing the bone-forming potential of purified pericytes. Potential models include (1) mouse intramuscular implantation, (2) mouse calvarial defect implantation, and (3) rat spinal fusion models. In addition, the presented surgical protocols may be used for the in vivo analysis of other osteoprogenitor cell types.


Asunto(s)
Células de la Médula Ósea/metabolismo , Pericitos/metabolismo , Ingeniería de Tejidos/métodos , Tejido Adiposo/citología , Animales , Células de la Médula Ósea/citología , Regeneración Ósea/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Separación Celular/métodos , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Osteogénesis/fisiología , Pericitos/citología , Ratas
9.
Methods Mol Biol ; 2273: 111-129, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604848

RESUMEN

Tissue engineering provides unique opportunities for disease modeling, drug testing, and regenerative medicine applications. The use of cell-seeded scaffolds to promote tissue development is the hallmark of the tissue engineering. Among the different types of scaffolds (derived from either natural or synthetic polymers) used in the field, the use of decellularized tissues/organs is specifically attractive. The decellularization process involves the removal of native cells from the original tissue, allowing for the preservation of the three-dimensional (3D) macroscopic and microscopic structures of the tissue and extracellular matrix (ECM) composition. Following recellularization, the resulting scaffold provides the seeded cells with the appropriate biological signals and mechanical properties of the original tissue. Here, we describe different methods to create viable scaffolds from decellularized heart and liver as useful tools to study and exploit ECM biological key factors for the generation of engineered tissues with enhanced regenerative properties.


Asunto(s)
Dermis Acelular/metabolismo , Medicina Regenerativa/métodos , Ingeniería de Tejidos/métodos , Animales , Matriz Extracelular/química , Corazón/crecimiento & desarrollo , Hepatocitos/citología , Hígado/crecimiento & desarrollo , Miocitos Cardíacos/citología , Conejos
10.
Methods Mol Biol ; 2273: 139-149, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604850

RESUMEN

Ovarian failure is the most common cause of infertility and affects about 1% of young women. One innovative strategy to restore ovarian function may be represented by the development of a bioprosthetic ovary, obtained through the combination of tissue engineering and regenerative medicine.We here describe the two main steps required for bioengineering the ovary and for its ex vivo functional reassembling. The first step aims at producing a 3D bioscaffold, which mimics the natural ovarian milieu in vitro. This is obtained with a whole organ decellularization technique that allows the maintenance of microarchitecture and biological signals of the original tissue. The second step involves the use of magnetic activated cell sorting (MACS) to isolate purified female germline stem cells (FGSCs). These cells are able to differentiate in ovarian adult mature cells, when subjected to specific stimuli, and can be used them to repopulate ovarian decellularized bioscaffolds. The combination of the two techniques represents a powerful tool for in vitro recreation of a bioengineered ovary that may constitute a promising solution for hormone and fertility function restoring. In addition, the procedures here described allow for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.


Asunto(s)
Células Madre Oogoniales/trasplante , Ovario/crecimiento & desarrollo , Ingeniería de Tejidos/métodos , Animales , Bioingeniería/métodos , Ingeniería Biomédica , Técnicas de Cultivo de Célula/métodos , Matriz Extracelular/metabolismo , Femenino , Fertilidad , Humanos , Células Madre Oogoniales/metabolismo , Organoides/crecimiento & desarrollo , Medicina Regenerativa , Porcinos , Andamios del Tejido/química
11.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540895

RESUMEN

Tissue engineering (TE) is the approach to combine cells with scaffold materials and appropriate growth factors to regenerate or replace damaged or degenerated tissue or organs. The scaffold material as a template for tissue formation plays the most important role in TE. Among scaffold materials, silk fibroin (SF), a natural protein with outstanding mechanical properties, biodegradability, biocompatibility, and bioresorbability has attracted significant attention for TE applications. SF is commonly dissolved into an aqueous solution and can be easily reconstructed into different material formats, including films, mats, hydrogels, and sponges via various fabrication techniques. These include spin coating, electrospinning, freeze drying, physical, and chemical crosslinking techniques. Furthermore, to facilitate fabrication of more complex SF-based scaffolds with high precision techniques including micro-patterning and bio-printing have recently been explored. This review introduces the physicochemical and mechanical properties of SF and looks into a range of SF-based scaffolds that have been recently developed. The typical TE applications of SF-based scaffolds including bone, cartilage, ligament, tendon, skin, wound healing, and tympanic membrane, will be highlighted and discussed, followed by future prospects and challenges needing to be addressed.


Asunto(s)
Materiales Biocompatibles/química , Fibroínas/química , Implantes Absorbibles , Animales , Biopolímeros , Bioimpresión/métodos , Matriz Extracelular/química , Fibroínas/aislamiento & purificación , Humanos , Hidrogeles/química , Insectos/metabolismo , Ensayo de Materiales , Fenómenos Mecánicos , Especificidad de Órganos , Conformación Proteica , Regeneración , Especificidad de la Especie , Arañas/metabolismo , Tapones Quirúrgicos de Gaza , Ingeniería de Tejidos/métodos , Andamios del Tejido/química
12.
Methods Mol Biol ; 2273: 239-250, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604858

RESUMEN

Various approaches have been evaluated for developing three-dimensional (3D) scaffolds for modeling or engineering of the bone tissue. However, most of such attempts have come up short in mimicking the natural bone tissue extracellular matrix (ECM) microenvironment, especially its natural bioactive content. Here we describe the methodology for the preparation of a natural ECM-based multichannel construct as a biomimetic 3D bone tissue model. We elucidate the construction of the composite scaffold incorporating decellularized small intestinal submucosa ECM, synthetic hydroxyapatite and poly(ε-caprolactone), and the mechanical stimulation of the cell-seeded construct under bioreactor culture.


Asunto(s)
Sustitutos de Huesos/química , Durapatita/química , Matriz Extracelular/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biomiméticos/química , Biomimética/métodos , Matriz Ósea/química , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Poliésteres/química , Impresión Tridimensional , Ratas
13.
Methods Mol Biol ; 2273: 279-296, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604861

RESUMEN

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.


Asunto(s)
Células Epiteliales/citología , Poliestirenos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Células CACO-2 , Línea Celular , Fibroblastos/citología , Humanos , Queratinocitos/citología , Porosidad , Piel/citología
14.
Nat Commun ; 12(1): 753, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33531489

RESUMEN

Cellular models are needed to study human development and disease in vitro, and to screen drugs for toxicity and efficacy. Current approaches are limited in the engineering of functional tissue models with requisite cell densities and heterogeneity to appropriately model cell and tissue behaviors. Here, we develop a bioprinting approach to transfer spheroids into self-healing support hydrogels at high resolution, which enables their patterning and fusion into high-cell density microtissues of prescribed spatial organization. As an example application, we bioprint induced pluripotent stem cell-derived cardiac microtissue models with spatially controlled cardiomyocyte and fibroblast cell ratios to replicate the structural and functional features of scarred cardiac tissue that arise following myocardial infarction, including reduced contractility and irregular electrical activity. The bioprinted in vitro model is combined with functional readouts to probe how various pro-regenerative microRNA treatment regimes influence tissue regeneration and recovery of function as a result of cardiomyocyte proliferation. This method is useful for a range of biomedical applications, including the development of precision models to mimic diseases and the screening of drugs, particularly where high cell densities and heterogeneity are important.


Asunto(s)
Bioimpresión/métodos , Hidrogeles/química , Ingeniería de Tejidos/métodos , Ingeniería Biomédica/métodos , Enfermedades Cardiovasculares , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Esferoides Celulares/citología
15.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-33530458

RESUMEN

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients' CNS and serve as a platform for therapeutic development and personalized precision medicine.


Asunto(s)
Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ingeniería de Tejidos/métodos , Animales , /patología , Enfermedades del Sistema Nervioso Central/patología , Descubrimiento de Drogas/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Humanos , Células Madre Pluripotentes Inducidas/patología , Dispositivos Laboratorio en un Chip , Organoides/citología , Organoides/efectos de los fármacos , Organoides/patología , Ingeniería de Tejidos/instrumentación , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/patología
16.
J Vis Exp ; (167)2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33522507

RESUMEN

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.


Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Neovascularización Fisiológica , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Actinas/metabolismo , Biomarcadores/metabolismo , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Fibronectinas/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Impresión Tridimensional , Imagen de Lapso de Tiempo
17.
Nat Commun ; 12(1): 1031, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589620

RESUMEN

The application of physical stimuli to cell cultures has shown potential to modulate multiple cellular functions including migration, differentiation and survival. However, the relevance of these in vitro models to future potential extrapolation in vivo depends on whether stimuli can be applied "externally", without invasive procedures. Here, we report on the fabrication and exploitation of dynamic additive-manufactured Janus scaffolds that are activated on-command via external application of ultrasounds, resulting in a mechanical nanovibration that is transmitted to the surrounding cells. Janus scaffolds were spontaneously formed via phase-segregation of biodegradable polycaprolactone (PCL) and polylactide (PLA) blends during the manufacturing process and behave as ultrasound transducers (acoustic to mechanical) where the PLA and PCL phases represent the active and backing materials, respectively. Remote stimulation of Janus scaffolds led to enhanced cell proliferation, matrix deposition and osteogenic differentiation of seeded human bone marrow derived stromal cells (hBMSCs) via formation and activation of voltage-gated calcium ion channels.


Asunto(s)
Plásticos Biodegradables/farmacología , Mecanotransducción Celular , Células Madre Mesenquimatosas/efectos de los fármacos , Poliésteres/farmacología , Andamios del Tejido , Plásticos Biodegradables/química , Regeneración Ósea/genética , Huesos/citología , Huesos/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/fisiología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Poliésteres/química , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Ondas Ultrasónicas
18.
Int J Mol Sci ; 22(2)2021 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-33466904

RESUMEN

Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.


Asunto(s)
Células de la Médula Ósea/metabolismo , Trasplante Óseo/métodos , Huesos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Biosíntesis de Proteínas , Ingeniería de Tejidos/métodos , Adulto , Bioimpresión/métodos , Células de la Médula Ósea/citología , Huesos/citología , Diferenciación Celular/genética , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Osteogénesis/genética , Impresión Tridimensional , Andamios del Tejido , Trasplante Autólogo
19.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33467447

RESUMEN

Mesenchymal stem cells (MSCs) are the main cell players in tissue repair and thanks to their self-renewal and multi-lineage differentiation capabilities, they gained significant attention as cell source for tissue engineering (TE) approaches aimed at restoring bone and cartilage defects. Despite significant progress, their therapeutic application remains debated: the TE construct often fails to completely restore the biomechanical properties of the native tissue, leading to poor clinical outcomes in the long term. Pulsed electromagnetic fields (PEMFs) are currently used as a safe and non-invasive treatment to enhance bone healing and to provide joint protection. PEMFs enhance both osteogenic and chondrogenic differentiation of MSCs. Here, we provide extensive review of the signaling pathways modulated by PEMFs during MSCs osteogenic and chondrogenic differentiation. Particular attention has been given to the PEMF-mediated activation of the adenosine signaling and their regulation of the inflammatory response as key player in TE approaches. Overall, the application of PEMFs in tissue repair is foreseen: (1) in vitro: to improve the functional and mechanical properties of the engineered construct; (2) in vivo: (i) to favor graft integration, (ii) to control the local inflammatory response, and (iii) to foster tissue repair from both implanted and resident MSCs cells.


Asunto(s)
Diferenciación Celular/fisiología , Condrogénesis/fisiología , Campos Electromagnéticos , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Transducción de Señal/fisiología , Huesos/citología , Células Cultivadas , Humanos , Ingeniería de Tejidos/métodos
20.
J Mater Chem B ; 9(5): 1272-1276, 2021 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-33427277

RESUMEN

A heparin-specific binding peptide was conjugated to a cowpea chlorotic mottle virus (CCMV) capsid protein, which was subsequently allowed to encapsulate heparin and form capsid-like protein cages. The encapsulation is specific and the capsid-heparin assemblies display negligible hemolytic activity, indicating proper blood compatibility and promising possibilities for heparin antidote applications.


Asunto(s)
Proteínas de la Cápside/metabolismo , Heparina/metabolismo , Ingeniería de Tejidos/métodos
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