Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration

Abstract

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.

Mineralizing Gelatin Microparticles as Cell Carrier and Drug Delivery System for siRNA for Bone Tissue Engineering

“…Finally, we tested the effects of microtissues on bone homeostasis in a co-culture model of hMSC and human peripheral blood mononuclear cells (hPBMC). Here, we were able to show a high silencing effect due to the changed electrostatic properties of the particle surface in contrast to the previously published data [ 34 ].…”

“…In contrast to our previous study [34] where we used cGM that did not mineralize in the absence of cells, we could not measure any differences in the calcium content in the different groups after 14 or 21 days. Calcium content, however, was strongly increased in all groups compared to the previous study with cGM of a lower cross-linking degree [34]. This result supports our hypothesis that the electrostatic conditions associated with the amount of incorporated cross-linker are strongly related to the intensity and rate of mineralization.…”

“…These presumably support calcium phosphate nucleation, either directly or via the adsorption of mineralizing osteogenic proteins, such as osteopontin or bone sialoprotein [35]. In contrast to our previous study [34] where we used cGM that did not mineralize in the absence of cells, we could not measure any differences in the calcium content in the different groups after 14 or 21 days. Calcium content, however, was strongly increased in all groups compared to the previous study with cGM of a lower cross-linking degree [34].…”

“…The microparticles provide a substrate for cell adhesion and improve oxygen and nutrient supply in the microtissues [ 28 , 29 ]. In order to enhance bone regeneration by siRNA, we silenced chordin, an antagonist of the bone morphogenetic protein 2 (BMP-2) [ 30 , 31 ], and followed the hypothesis that the silencing of chordin in combination with BMP-2 supplementation may enhance osteogenic differentiation and accelerate mineralization in osteogenic cultures [ 10 , 13 , 32 , 33 , 34 ]. These microtissues may be applied similarly as autologous bone grafts.…”

“…In a previous study, we reported on non-mineralizing gelatin microparticles and showed improved mineralization of the chordin siRNA-releasing microparticles [ 34 ]. Here, we changed the protocol to greatly increase the cross-linking degree of cGMs from 37.2% to 50.7%.…”