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

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.

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

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 â€‹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 â€‹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.

Structure-activity analysis of peptidic Chlamydia HtrA inhibitors.

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-ValP(OPh)2] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P1 and P3 yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P3 (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC50 = 0.68 ±â€¯0.02 µM against HNE, while valine at P1 retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.

Dual-Component Gelatinous Peptide/Reactive Oligomer Formulations as Conduit Material and Luminal Filler for Peripheral Nerve Regeneration.

Toward the next generation of nerve guidance conduits (NGCs), novel biomaterials and functionalization concepts are required to address clinical demands in peripheral nerve regeneration (PNR). As a biological polymer with bioactive motifs, gelatinous peptides are promising building blocks. In combination with an anhydride-containing oligomer, a dual-component hydrogel system (cGEL) was established. First, hollow cGEL tubes were fabricated by a continuous dosing and templating process. Conduits were characterized concerning their mechanical strength, in vitro and in vivo degradation and biocompatibility. Second, cGEL was reformulated as injectable shear thinning filler for established NGCs, here tyrosine-derived polycarbonate-based braided conduits. Thereby, the formulation contained the small molecule LM11A-31. The biofunctionalized cGEL filler was assessed regarding building block integration, mechanical properties, in vitro cytotoxicity, and growth permissive effects on human adipose tissue-derived stem cells. A positive in vitro evaluation motivated further application of the filler material in a sciatic nerve defect. Compared to the empty conduit and pristine cGEL, the functionalization performed superior, though the autologous nerve graft remains the gold standard. In conclusion, LM11A-31 functionalized cGEL filler with extracellular matrix (ECM)-like characteristics and specific biochemical cues holds great potential to support PNR.