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Analysis of siRNA silencing efficiency in osteogenic microtissues upon loading of cGM with siRNA‐carrying CaP‐NP and analysis of effects on osteogenic differentiation. a) Experimental setup: 1875 µL of siRNA‐loaded CaP‐NP were prepared and concentrated via ultrafiltration to a volume of 50 µL. Afterwards, 3.2 mg cGM (0.128 mg cGM/microtissue) were loaded with 100 µL of concentrated CaP‐NP. 10 000 hMSCs were then aggregated with 0.128 mg cGM, and osteogenic differentiation was induced by the addition of osteogenic supplements. Created in BioRender. Mitrach, F. (2025) b) siRNA‐mediated silencing efficiency of Chordin and WWP‐1 was quantified via gene expression levels over a period of 14 days of osteogenic differentiation and showed successful downregulation of both antagonists in osteogenic microtissues. c) Downstream effects of siRNA‐mediated antagonist silencing in microtissues were quantified via alkaline phosphatase activity on days 4 and 7, as well as d) calcium content as a measure of mineralization on day 18. e) Secretion of the important pro‐angiogenic <t>factor</t> <t>VEGF</t> by microtissues was quantified in cell culture supernatants using <t>ELISA</t> over 28 days of osteogenic differentiation. Data are presented as mean ± SD ( n = 4). Statistically significant differences are indicated with (∗) between the different groups ( p < 0.05), one‐way ANOVA with Tukey post hoc test. ALP : alkaline phosphatase; CaP‐NP : oligomer‐stabilized calcium phosphate nanoparticles; cGM : cross‐linked gelatin microparticles; DNA : deoxyribonucleic acid; hMSCs : human mesenchymal stem cells; mRNA : messenger RNA; n.d .: not detectable; pNP : para‐nitrophenyl; siRNA : small interfering RNA; VEGF : vascular endothelial growth factor.
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Analysis of siRNA silencing efficiency in osteogenic microtissues upon loading of cGM with siRNA‐carrying CaP‐NP and analysis of effects on osteogenic differentiation. a) Experimental setup: 1875 µL of siRNA‐loaded CaP‐NP were prepared and concentrated via ultrafiltration to a volume of 50 µL. Afterwards, 3.2 mg cGM (0.128 mg cGM/microtissue) were loaded with 100 µL of concentrated CaP‐NP. 10 000 hMSCs were then aggregated with 0.128 mg cGM, and osteogenic differentiation was induced by the addition of osteogenic supplements. Created in BioRender. Mitrach, F. (2025) b) siRNA‐mediated silencing efficiency of Chordin and WWP‐1 was quantified via gene expression levels over a period of 14 days of osteogenic differentiation and showed successful downregulation of both antagonists in osteogenic microtissues. c) Downstream effects of siRNA‐mediated antagonist silencing in microtissues were quantified via alkaline phosphatase activity on days 4 and 7, as well as d) calcium content as a measure of mineralization on day 18. e) Secretion of the important pro‐angiogenic factor VEGF by microtissues was quantified in cell culture supernatants using ELISA over 28 days of osteogenic differentiation. Data are presented as mean ± SD ( n = 4). Statistically significant differences are indicated with (∗) between the different groups ( p < 0.05), one‐way ANOVA with Tukey post hoc test. ALP : alkaline phosphatase; CaP‐NP : oligomer‐stabilized calcium phosphate nanoparticles; cGM : cross‐linked gelatin microparticles; DNA : deoxyribonucleic acid; hMSCs : human mesenchymal stem cells; mRNA : messenger RNA; n.d .: not detectable; pNP : para‐nitrophenyl; siRNA : small interfering RNA; VEGF : vascular endothelial growth factor.

Journal: Advanced Healthcare Materials

Article Title: siRNA Delivery via Cross‐Linked Gelatin Microparticles Enables Targeted Modulation of Osteogenic‐Vascular Cross‐Talk: An Advanced Human 3D in Vitro Test System for Therapeutic siRNA

doi: 10.1002/adhm.202504773

Figure Lengend Snippet: Analysis of siRNA silencing efficiency in osteogenic microtissues upon loading of cGM with siRNA‐carrying CaP‐NP and analysis of effects on osteogenic differentiation. a) Experimental setup: 1875 µL of siRNA‐loaded CaP‐NP were prepared and concentrated via ultrafiltration to a volume of 50 µL. Afterwards, 3.2 mg cGM (0.128 mg cGM/microtissue) were loaded with 100 µL of concentrated CaP‐NP. 10 000 hMSCs were then aggregated with 0.128 mg cGM, and osteogenic differentiation was induced by the addition of osteogenic supplements. Created in BioRender. Mitrach, F. (2025) b) siRNA‐mediated silencing efficiency of Chordin and WWP‐1 was quantified via gene expression levels over a period of 14 days of osteogenic differentiation and showed successful downregulation of both antagonists in osteogenic microtissues. c) Downstream effects of siRNA‐mediated antagonist silencing in microtissues were quantified via alkaline phosphatase activity on days 4 and 7, as well as d) calcium content as a measure of mineralization on day 18. e) Secretion of the important pro‐angiogenic factor VEGF by microtissues was quantified in cell culture supernatants using ELISA over 28 days of osteogenic differentiation. Data are presented as mean ± SD ( n = 4). Statistically significant differences are indicated with (∗) between the different groups ( p < 0.05), one‐way ANOVA with Tukey post hoc test. ALP : alkaline phosphatase; CaP‐NP : oligomer‐stabilized calcium phosphate nanoparticles; cGM : cross‐linked gelatin microparticles; DNA : deoxyribonucleic acid; hMSCs : human mesenchymal stem cells; mRNA : messenger RNA; n.d .: not detectable; pNP : para‐nitrophenyl; siRNA : small interfering RNA; VEGF : vascular endothelial growth factor.

Article Snippet: The amount of VEGF secreted was quantified by using a solid phase sandwich ELISA Kit (Human VEGF DuoSet ELISA, R&D Systems, Wiesbaden, Germany) according to the manufacturer's protocol.

Techniques: Gene Expression, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Small Interfering RNA