Different types of cartilage neotissue fabricated from collagen hydrogels and mesenchymal stromal cells via SOX9, TGFB1 or BMP2 gene transfer

Abstract

Objective As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors. Design Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box ( SOX ) 9 , transforming growth factor beta (TGFB) 1 or bone morphogenetic protein ( BMP) 2 cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy. Results Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenes SOX9 , TGFB1 and BMP2 as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups ( BMP2 > TGFB1 > SOX9 ). Conclusions Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factors SOX9 , TGFB1 and BMP2 . This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.

Combinations of Hydrogels and Mesenchymal Stromal Cells (MSCs) for Cartilage Tissue Engineering—A Review of the Literature

“…We and others showed that COL type I hydrogels enhance chondrogenic differentiation and maintenance of chondrogenic phenotype in seeded MSCs in vitro, especially when paired with specific growth factors or gene transfer [ 19 , 53 , 55 ]. However, research has shown that both COL I-based scaffolds promote chondrogenesis in seeded MSCs with a possible superior synthesis of cartilage-specific ECM components but no significant differences in the expression of chondrogenic marker genes [ 55 , 56 , 57 ].…”

“…Further, we and others showed that gene transfer of different members of the TGF-ß superfamily led to enhanced chondrogenesis of BMSCs seeded in hydrogels in vitro and in vivo [ 19 , 99 , 100 , 101 , 103 , 104 , 105 , 106 ]. Xia et al found that adenoviral gene transfer of TGF-ß1 (encoded by TGFB1 ) to BMSCs encapsulated in PGA hydrogels led to enhanced chondrogenesis in vivo [ 99 ].…”

“…Wang et al showed that adenoviral gene transfer of TGF-ß3 (encoded by TGFB3 ) and BMP-2 (encoded by BMP2 ) to BMSCs encapsulated in demineralized bone matrix led to enhanced cartilage repair in pig models [ 100 ]. Different studies showed that the gene transfer of SRY-Box Transcription Factor (SOX)-9 (encoded by SOX9 ) may further enhance chondrogenesis in BMSC seeded in natural or synthetic hydrogels while limiting hypertrophic de-differentiation [ 19 , 102 , 107 ]. Cao et al showed that adenoviral gene transfer of SOX9 to BMSCs seeded in PGA hydrogels led to enhanced cartilage repair in rabbit models in comparison to conventional BMSC-laden PGA hydrogels [ 101 ].…”

“…Cell-laden hydrogels can be manufactured according to defect composition, can be applied via minimal-invasive approaches, and may promote the repair of full-thickness cartilage defects [ 18 ]. In addition, the combination of natural and synthetic hydrogels and growth factors has been shown to enhance chondrogenic differentiation, maintain chondrogenic phenotype, and limit chondrogenic hypertrophy in seeded MSCs [ 14 , 15 , 19 ].…”

Co-Modification of Beta-Catenin and Sex Determining Region Y-Box9 Genes Regulates the Chondrogenesis of Human Adipose Tissue Derived Stem Cells

“…A variety of genes have been identified as potential chondrogenic markers, among which sex determining region Y-box9 (Sox9) gene has been confirmed to be a key gene in cartilage formation [8,9] . The role of Sox9 gene in cartilage differentiation is achieved by inhibiting Beta catenin (β-catenin) gene and there is a complex relationship between them.…”