Gelatin microparticles aggregates as three-dimensional scaffolding system in cartilage engineering

A three-dimensional (3D) scaffolding system for chondrocytes culture has been produced by agglomeration of cells and gelatin microparticles with a mild centrifuging process. The diameter of the microparticles, around 10 μ, was selected to be in the order of magnitude of the chondrocytes. No gel was used to stabilize the construct that maintained consistency just because of cell and extracellular matrix (ECM) adhesion to the substrate. In one series of samples the microparticles were charged with transforming growth factor, TGF-β1. The kinetics of growth factor delivery was assessed. The initial delivery was approximately 48 % of the total amount delivered up to day 14. Chondrocytes that had been previously expanded in monolayer culture, and thus dedifferentiated, adopted in this 3D environment a round morphology, both with presence or absence of growth factor delivery, with production of ECM that intermingles with gelatin particles. The pellet was stable from the first day of culture. Cell viability was assessed by MTS assay, showing higher absorption values in the cell/unloaded gelatin microparticle pellets than in cell pellets up to day 7. Nevertheless the absorption drops in the following culture times. On the contrary the cell viability of cell/TGF-β1 loaded gelatin microparticle pellets was constant during the 21 days of culture. The formation of actin stress fibres in the cytoskeleton and type I collagen expression was significantly reduced in both cell/gelatin microparticle pellets (with and without TGF-β1) with respect to cell pellet controls. Total type II collagen and sulphated glycosaminoglycans quantification show an enhancement of the production of ECM when TGF-β1 is delivered, as expected because this growth factor stimulate the chondrocyte proliferation and improve the functionality of the tissue.JLGR acknowledge the support of the Spanish Ministry of Education through project No. MAT2010-21611-C03-01 (including the FEDER financial support). The support of the Instituto de Salud Carlos III (ISCIII) through the CIBER initiative of the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) is also acknowledged

Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration

Biomimetic bone tissue engineering strategies partially recapitulate development. We recently showed functional restoration of femoral defects using scaffold-free human mesenchymal stem cell (hMSC) condensates featuring localized morphogen presentation with delayed in vivo mechanical loading. Possible effects of construct geometry on healing outcome remain unclear. Here, we hypothesized that localized presentation of transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 to engineered hMSC tubes mimicking femoral diaphyses induces endochondral ossification, and that TGF-β1 + BMP-2-presenting hMSC tubes enhance defect healing with delayed in vivo loading vs. loosely packed hMSC sheets. Localized morphogen presentation stimulated chondrogenic priming/endochondral differentiation in vitro. Subcutaneously, hMSC tubes formed cartilage templates that underwent bony remodeling. Orthotopically, hMSC tubes stimulated more robust endochondral defect healing vs. hMSC sheets. Tissue resembling normal growth plate was observed with negligible ectopic bone. This study demonstrates interactions between hMSC condensation geometry, morphogen bioavailability, and mechanical cues to recapitulate development for biomimetic bone tissue engineering