RESUMEN
As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.
Asunto(s)
Tejido Adiposo/citología , Cartílago/crecimiento & desarrollo , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Tejido Adiposo/fisiología , Fenómenos Biomecánicos , Cartílago/citología , Cartílago/ultraestructura , Células Cultivadas , Humanos , Hipertrofia , Células Madre Mesenquimatosas/fisiología , Microscopía Electrónica de Transmisión , Esferoides Celulares/fisiología , Esferoides Celulares/ultraestructura , Células del Estroma/fisiologíaRESUMEN
Human adipose stem/stromal cell (ASC) spheroids were used as a serum-free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced-ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP-13) was upregulated at week 2 in induced-ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real-time PCR. In accordance, secreted levels of IL-6 (P < .0001), IL-8 (P < .0001), IL-10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP-1 was associated with the increase of hypertrophic genes expression at week 2 in induced-ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced-ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum-free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.
Asunto(s)
Cartílago/crecimiento & desarrollo , Condrogénesis/fisiología , Células Madre Mesenquimatosas/fisiología , Cultivo Primario de Células/métodos , Ingeniería de Tejidos/métodos , Tejido Adiposo/citología , Cartílago/citología , Cartílago/ultraestructura , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno Tipo X/metabolismo , Medio de Cultivo Libre de Suero , Matriz Extracelular/metabolismo , Humanos , Hipertrofia , Metaloproteinasa 13 de la Matriz/metabolismo , Microscopía Electrónica de Transmisión , Esferoides Celulares/fisiología , Esferoides Celulares/ultraestructura , Células del Estroma/fisiologíaRESUMEN
Two experiments were conducted to study the effect of standardized ileal digestible (SID) leucine and valine levels on tibiotarsus bone characteristics and the incidence of tibial dyschondroplasia of broilers from day 1 to 21 (Experiment I) and day 21 to 42 post-hatch (Experiment II). Each experimental phase was evaluated independently. In both experiments, a total of 1,500 one-day-old Cobb 500 male broiler chickens were distributed in a completely randomized design 5 × 5 factorial arrangement for a total of 25 treatments. The SID leucine and valine levels were ranged from 10.0 to 19.6 g/kg, and 6.0 to 12.0 g/kg from day 1 to 21 post-hatch, respectively, while day 21 to 42 post-hatch ranged from 10.0 to 18.0 g leucine/kg, and 5.2 to 11.2 g valine/kg. Serum calcium and phosphorus, bone concentrations of calcium, phosphorus and ash, diameter and Seedor index of the tibiotarsus were not affected (p > .05) by the treatments at 21 or 42 days of age. There was an interaction (p ≤.06) between the SID levels of leucine and valine on tibiotarsus breaking strength at 21 days, but not at 42 days of age (p > .05). Tibiotarsus breaking strength was maximized in broilers from day 1 to 21 with the dietary levels of leucine and valine at 14.2 and 9.0 g/kg respectively. Dietary leucine levels reduced linearly (p < .05) the hypertrophic zone of tibiotarsus cartilage at 21 days of age. Therefore, leucine and valine supplementation interact positively on bone strength of broilers from day 1 to 21 post-hatch. Leucine can be a useful amino acid for reducing the hypertrophic cartilage zone in broilers from day 1 to 21, but not from day 21 to 42 post-hatch.