(A) SEM images of apatite-coated CH/CS scaffolds. Scale bar = 200 µm; scale bar of the inset image = 20 µm. (B) <i>In vitro</i> release of BMP-2 from CH/CS scaffolds. (C) Live-dead fluorescent staining of ASCs seeded on CH/CS scaffolds after 14 days in culture. Scale bar = 200 µm.

<p>(A) SEM images of apatite-coated CH/CS scaffolds. Scale bar  =  200 µm; scale bar of the inset image  =  20 µm. (B) <i>In vitro</i> release of BMP-2 from CH/CS scaffolds. (C) Live-dead fluorescent staining of ASCs seeded on CH/CS scaffolds after 14 days in culture. Scale bar  =  200 µm.</p

Alkaline phosphatase (ALP) expression of ASCs in monolayer culture.

<p>ALP was increased in ASCs transduced with noggin shRNA in the presence or absence of BMP-2 as assessed by ALP staining (A) and quantification (B) at day 3. (**<i>p</i><0.01 compared to their corresponding controls transduced with control shRNA). Significantly more staining was observed after treatment with different doses of BMP-2 (§ <i>p</i> <0.05, §§ <i>p</i> <0.01).</p

Osteogenic gene expression of ASCs in monolayer culture.

<p>Osteogenic gene markers including <i>Runx2</i> (A), <i>ALP</i> (B), <i>ON</i> (C), <i>Col1a</i> (D) and <i>OCN</i> (E), were elevated in ASCs transduced with noggin shRNA in the presence or absence of BMP-2 as analyzed by quantitative real-time PCR at day 14 (*<i>p</i><0.05, ** <i>p</i><0.01 compared to their corresponding controls transduced with control shRNA). Significant up-regulation of all genes was observed after treatment with different doses of BMP-2 (§ <i>p</i> <0.05, §§ <i>p</i> <0.01).</p

Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

<div><p>Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D <i>in vitro</i> environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.</p></div

Collagen deposition of ASCs cultured in apatite-coated CH/CS scaffolds releasing BMP-2.

<p>Picrosirius red staining of ASCs cultured on CH/CS scaffolds for 21 days, which was assessed by bright field (A) and polarized light (B) microscopy. Scale bar  =  100 µm. Significantly higher collagen deposition was observed in noggin suppressed ASCs cultured on CH/CS scaffolds releasing BMP-2.</p

Expressional noggin gene in ASCs transduced with noggin shRNA lentiviral particles.

<p><i>Noggin</i> expression was downregulated in ASCs transduced with lentivirus particles targeting noggin shRNA in the presence or absence of BMP-2 (100 ng/ml) as evaluated by quantitative real-time PCR analysis. Significantly lower noggin expression was observed with noggin shRNA transduction (*<i>p</i><0.05).</p

Mineral formation of ASCs in monolayer culture.

<p>Mineralization was increased in ASCs transduced with noggin shRNA in the presence or absence of BMP-2 as assessed by Alizarin red staining (A) and quantification (B) at day 14. (**<i>p</i><0.01 compared to their corresponding controls transduced with control shRNA). Significantly more staining was observed after treatment with different doses of BMP-2 (§§ <i>p</i> <0.01).</p

mRNA expression of cultured ISMC and MS 14 for after neonatal harvest.

<p>There was no significant difference in mRNA expression for smooth muscle markers between cultured ISMC and MS, including SMA, DES, and MHC. There was a significant increase in mRNA expression of BTUB in cultured MS (p<0.05, *; n = 6). Samples were normalized to smooth muscle from the harvest with GAPDH as a control gene.</p

Calcium indicator intensity in cultured MS after 14 days.

<p>An increase in calcium indicator intensity is observed between images of the MS just prior to depolarization (A) and 0.7 seconds later (B), after depolarization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114850#pone.0114850.s002" target="_blank">S2 Video</a>). Muscle strips and the surrounding cells that formed a “ganglia” are labeled. Regions of black indicate depolarization. 100x magnification, 200-µm scale bar. A plot of calcium indicator intensity (C) within MS and the “ganglia” show the periodic depolarization waves in the MS culture.</p

Immunofluorescent imaging of ISMC- or MS-seeded implant.

<p>After two weeks in vivo, ISMC and MS implants on scaffolds were identified with anti-GFP immunofluorescence. Both ISMC and MS expressed SMA, but ISMC had less immunofluorescence for DES and MHC compared to MS. ISMC also showed reduced S100 immunofluorescence compared to MS. Merged images of SMA (green) and s100 (red) show no co-localization of immunofluorescence in either ISMC or MS. BTUB immunofluorescence was decreased in ISMC implants compared to MS implants. Nuclei were indicated with DAPI. 200x magnification, 100-µm scale bars.</p