RESUMEN
Scaffold contraction is a common but underestimated problem in the field of tissue engineering. It becomes particularly problematic when creating anatomically complex shapes such as the ear. The aim of this study was to develop a contraction-free biocompatible scaffold construct for ear cartilage tissue engineering. To address this aim, we used three constructs: (i) a fibrin/hyaluronic acid (FB/HA) hydrogel, (ii) a FB/HA hydrogel combined with a collagen I/III scaffold, and (iii) a cage construct containing (ii) surrounded by a 3D-printed poly-É-caprolactone mold. A wide range of different cell types were tested within these constructs, including chondrocytes, perichondrocytes, adipose-derived mesenchymal stem cells, and their combinations. After in vitro culturing for 1, 14, and 28 days, all constructs were analyzed. Macroscopic observation showed severe contraction of the cell-seeded hydrogel (i). This could be prevented, in part, by combining the hydrogel with the collagen scaffold (ii) and prevented in total using the 3D-printed cage construct (iii). (Immuno)histological analysis, multiphoton laser scanning microscopy, and biomechanical analysis showed extracellular matrix deposition and increased Young's modulus and thereby the feasibility of ear cartilage engineering. These results demonstrated that the 3D-printed cage construct is an adequate model for contraction-free ear cartilage engineering using a range of cell combinations.
Asunto(s)
Cartílago/citología , Matriz Extracelular/química , Polímeros/química , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tejido Adiposo/citología , Animales , Células Cultivadas , Condrocitos/citología , Condrogénesis , Cabras , Células Madre Mesenquimatosas/citología , Impresión TridimensionalRESUMEN
Reconstruction of a severely maimed ear is a major challenge. The ear is highly flexible yet tough, and has a very complex three-dimensional shape. Reconstruction of a patient's burned ear is even more complex due to surrounding tissue damage. Not only does this hamper reconstruction options, it also increases the likelihood of issues when using synthetic implant materials. In such cases, rib cartilage is the preferred option, but this tissue has practical limitations too. For these reasons, tissue engineering and 3D bioprinting may have the potential to create personalized cartilage implants for burns patients. However, 3D bioprinting is a tool to facilitate the reconstruction, and not by itself the Holy Grail. The clinical application of this technique is still at a very early stage. Nevertheless, we expect that 3D bioprinting can be utilised for facial reconstruction following burns come 2020.
Asunto(s)
Bioimpresión , Cartílago , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Quemaduras , Humanos , Prótesis e ImplantesRESUMEN
In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.