The aim of this study was the evaluation of cross-linked gelatin microparticles (cGM) as substrates for osteogenic cell culture to assemble 3D microtissues and their use as delivery system for siRNA to cells in these assemblies. In a 2D transwell cultivation system, we found that cGM are capable to accumulate calcium ions from the surrounding medium. Such a separation of cGM and SaOS-2 ​cells consequently led to a suppressed matrix mineral formation in the SaOS-2 culture on the well bottom of the transwell system. Thus, we decided to use cGM as component in 3D microtissues and get a close contact between calcium ion accumulating microparticles and cells to improve matrix mineralization. Gelatin microparticles were cross-linked with a N,N -diethylethylenediamine-derivatized (DEED) maleic anhydride (MA) containing oligo (pentaerythritol diacrylate monostearate- co - N -isopropylacrylamide- co -MA) (oPNMA) and aggregated with SaOS-2 or human mesenchymal stem cells (hMSC) to microtissue spheroids. We systematically varied the content of cGM in microtissues and observed cell differentiation and tissue formation. Microtissues were characterized by gene expression, ALP activity and matrix mineralization. Mineralization was detectable in microtissues with SaOS-2 ​cells after 7 days and with hMSC after 24–28 days in osteogenic culture. When we transfected hMSC via cGM loaded with Lipofectamine complexed chordin siRNA, we found increased ALP activity and accelerated mineral formation in microtissues in presence of BMP-2. As a model for positive paracrine effects that indicate promising in vivo effects of these microtissues, we incubated pre-differentiated microtissues with freshly seeded hMSC monolayers and found improved mineral formation all over the well in the co-culture model. These findings may support the concept of microtissues from hMSC and siRNA-loaded cGM for bone regeneration.

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8–9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.