Silencing Smad7 potentiates BMP2-induced chondrogenic differentiation and inhibits endochondral ossification in human synovial-derived mesenchymal stromal cells

Abstract

Background Bone morphogenetic protein 2 (BMP2) is a promising chondrogenic growth factor for cartilage tissue-engineering, but it also induces robust endochondral ossification. Human synovial-derived mesenchymal stromal cells (hSMSCs) have attracted great interest due to their poor potential for differentiation into osteogenic lineages. Smad7 plays a significant in the endochondral ossification. In this study, we explored a new method to amplify the BMP2-induced chondrogenic differentiation of hSMSCs by downregulating Smad7 and applying a cellular scaffold. Methods hSMSCs were isolated from human knee joint synovium from 3 donors through adhesion growth. In vitro and in vivo models of the chondrogenic differentiation of hSMSCs were established. Transgenic expression of BMP2 and silencing of Smad7 and Smad7 was achieved by adenoviral vectors. The osteogenic differentiation was detected by alkaline phosphatase staining, alizarin red staining, and RT-PCR analysis of the osteogenic genes RUNX2, Osterix, and Osteocalcin. The chondrogenic differentiation was detected by Alcian blue staining and RT-PCR analysis of the chondrogenic genes SOX9, COL2, and aggrecan. Hypertrophic differentiation was detected by the markers COL10 and MMP13. A subcutaneous stem cell implantation model was established with polyethylene glycol citrate-co-N-isopropylacrylamide (PPCN) scaffolds and athymic nude mice (3/group, 4–6 week-old female) and evaluated by micro-CT, H&E staining, and Alcian blue staining. An immunohistochemistry assay was used to detected COL1 and COL2, and an immunofluorescence assay was used to detect COL10 and MMP13. Results These hSMSCs identified by flow cytometry. These hSMSCs exhibited lower osteo-differentiation potential than iMads and C3H10T1/2-cells. When Smad7 was silenced in BMP2-induced hSMSCs, the chondrogenic differentiation genes SOX9, COL2, and aggrecan were enhanced in vitro. Additionally, it silencing Smad7 led to a decrease in the hypertrophic differentiation genes COL10 and MMP13. In subcutaneous stem cell implantation assays, immunofluorescence and immunohistochemical staining demonstrated that silencing Smad7 increased the number of COL2-positive cells and decreased the expression of COL1, COL10, and MMP13. Conclusion This study suggests that the application of hSMSCs, cell scaffolds, and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification. Thus, inhibiting the expression of Smad7 in BMP2-induced hSMSC differentiation may be a new strategy for cartilage tissue-engineering.

Scrapie-Responsive Gene 1 Promotes Chondrogenic Differentiation of Umbilical Cord Mesenchymal Stem Cells via Wnt5a

“…Results show that SCRG1 positively regulated the expression of chondrogenic genes, including SOX9 , COL2A1 , and ACAN , similar to previous research [ 9 , 25 ], indicating that SCRG1 promoted chondrogenic differentiation of UCMSCs in vitro . Chondrogenic and osteogenic differentiation occurs in opposite directions in stem cells [ 26 , 27 ]. Various transcription factors, such as Wnt10b, SOX9, and RUNX2 [ 22 , 28 , 29 ], are upregulated or downregulated to determine the cell fate of MSCs during chondrogenic and osteogenic differentiation.…”

Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells

“…Previous studies have found that BMP2-induced high expression of Smad7 serves as the key inhibitor of chondrogenic differentiation [32,34]. In addition, the hypertrophic differentiation of chondrocytes, marked with COL10A1 [15,35], was caused by Smad7, at least in part, during chondrogenic differentiation [15]. Further research revealed that Smad7 inhibited the formation of cartilaginous tissue induced by BMP2 by suppressing the P38 and Smad1/5/8 pathways [32,36].…”

“…This molecule plays an important role in the production and protection of the extracellular matrix of articular cartilage [10][11][12][13]. A previous study by our team showed that overexpression of Sox9 enhances BMP-2-induced chondrogenic differentiation in MSCs by downregulating Smad7 expression [14][15][16]. However, the underlying mechanism of how Sox9 regulates Smad7 is not clear.…”

Human amniotic mesenchymal stem cells combined with PPCNg facilitate injured endometrial regeneration

“…With biocompatibility, PPCNg has been reported to mediate BMP9-transduced MSCs, including iMEFs [29], iMADs [30] and iCALs [31], which were located at bone defect sites to enhance bone formation and angiogenesis. A recent study has shown that PPCNg combined with hSMSCs transplantation can effectively promote chondrogenic differentiation of hSMSCs by improving nutrient supply at the injured site and providing regular space for cell growth [32].…”