RESUMEN
Tissue engineering has great potential to provide a functional de novo living valve replacement, capable of integration with host tissue and growth. Among various valve conduit fabrication techniques, three-dimensional (3-D) bioprinting enables deposition of cells and hydrogels into 3-D constructs with anatomical geometry and heterogeneous mechanical properties. Successful translation of this approach, however, is constrained by the dearth of printable and biocompatible hydrogel materials. Furthermore, it is not known how human valve cells respond to these printed environments. In this study, 3-D printable formulations of hybrid hydrogels are developed, based on methacrylated hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel), and used to bioprint heart valve conduits containing encapsulated human aortic valvular interstitial cells (HAVIC). Increasing Me-Gel concentration resulted in lower stiffness and higher viscosity, facilitated cell spreading, and better maintained HAVIC fibroblastic phenotype. Bioprinting accuracy was dependent upon the relative concentrations of Me-Gel and Me-HA, but when optimized enabled the fabrication of a trileaflet valve shape accurate to the original design. HAVIC encapsulated within bioprinted heart valves maintained high viability, and remodeled the initial matrix by depositing collagen and glyosaminoglycans. These findings represent the first rational design of bioprinted trileaflet valve hydrogels that regulate encapsulated human VIC behavior. The use of anatomically accurate living valve scaffolds through bioprinting may accelerate understanding of physiological valve cell interactions and progress towards de novo living valve replacements.
Asunto(s)
Válvula Aórtica/citología , Prótesis Vascular , Hidrogeles/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Válvula Aórtica/efectos de los fármacos , Bovinos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Niño , Regulación de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Humanos , Ácido Hialurónico/síntesis química , Ácido Hialurónico/química , Hidrogeles/síntesis química , Inmunohistoquímica , Metacrilatos/síntesis química , Metacrilatos/química , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reología/efectos de los fármacosRESUMEN
Many soft tissues exhibit complex anatomical geometry that is challenging to replicate for regenerative medicine applications. Solid freeform fabrication (SFF) has emerged as an attractive approach for creating 3D tissues, but a detailed understanding of how specific fabrication parameters affect accuracy and viability has not been established to date. In this study, we evaluate the effects of printing parameters of the Fab@Home 3D printing system on accuracy using alginate, photocrosslinkable polyethylene-glycol diacrylate (PEG-DA) and gelatin as commonly used model hydrogel materials. Print accuracy and resolution along the length, width and height were determined based on quantitative image analysis. The effects of extrusion parameters on cell viability were assessed using porcine aortic valve interstitial cells (PAVIC) as a model cell type. We observed that pressure, pathheight and pathspace all significantly affected print accuracy and resolution. Printing conditions did not affect PAVIC viability within the ranges applied. We predicted that optimal pressure, pathheight and pathspace values would be increased linearly with increasing nozzle diameter, and we confirmed that the predicted values generate accurate 3D geometries while poorly chosen parameters yield inaccurate, unpredictable geometries. This systematic optimization strategy therefore improves the accuracy of 3D printing platforms for biofabrication and tissue engineering applications.
Asunto(s)
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/química , Animales , Supervivencia Celular , Células/citología , Células Cultivadas , PorcinosRESUMEN
The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12-22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over tenfold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 min, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0 and 73.3±5.2% for 22, 17 and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6 and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.