Enhanced osteogenesis of adipose derived stem cells with Noggin suppression and delivery of BMP-2

Abstract

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 in vitro 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.

Figure 1. Expressional noggin gene in ASCs transduced with noggin shRNA lentiviral particles.

Noggin 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 (*p<0.05).

Figure 2. Alkaline phosphatase (ALP) expression of ASCs in monolayer culture.

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. (**p<0.01 compared to their corresponding controls transduced with control shRNA). Significantly more staining was observed after treatment with different doses of BMP-2 (§ p <0.05, §§ p <0.01).

Figure 3. Mineral formation of ASCs in monolayer culture.

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. (**p<0.01 compared to their corresponding controls transduced with control shRNA). Significantly more staining was observed after treatment with different doses of BMP-2 (§§ p <0.01).

Figure 4. Osteogenic gene expression of ASCs in monolayer culture.

Osteogenic gene markers including Runx2 (A), ALP (B), ON (C), Col1a (D) and OCN (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 (*p<0.05, ** p<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 (§ p <0.05, §§ p <0.01).

Figure 5. (A) SEM images of apatite-coated CH/CS scaffolds. Scale bar  =  200 µm; scale bar of the inset image  =  20 µm. (B) In vitro 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.

Figure 6. Osteogenic gene expression of ASCs cultured in apatite-coated CH/CS scaffolds releasing BMP-2.

Osteogenic gene markers including Runx2 (A), ALP (B), Col1a (C) and OCN (D) were evaluated in noggin suppressed ASCs cultured on CH/CS scaffolds for 14 days as assessed by quantitative real-time PCR. (*p<0.05, **p<0.01 compared to their corresponding controls transduced with control shRNA). Significant up-regulation of genes including Runx2, ALP and OCN was observed in ASCs cultured on CH/CS scaffolds releasing BMP-2 (§ p <0.05, §§ p <0.01).

Figure 7. Collagen deposition of ASCs cultured in apatite-coated CH/CS scaffolds releasing BMP-2.

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.