Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

Abstract

IntroductionThe present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.MethodsAdenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.ResultsLevels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.ConclusionsBMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.

BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development

“…For example, both BMP2 and BMP4 induce mouse embryonic stem cell and human mesenchymal stem cell differentiation into chondrocytes (Kramer et al, 2000;Steinert et al, 2009). BMP2 and BMP4 also stimulate chondrocyte proliferation and hypertrophy (De Luca et al, 2001;Minina et al, 2001;Hatakeyama et al, 2004;Leboy et al, 1997).…”

Fibroblast Growth Factor-1 Is a Mesenchymal Stromal Cell-Secreted Factor Stimulating Proliferation of Osteoarthritic Chondrocytes in Co-Culture

“…In other studies, however, BMP-2 has been applied to promote the chondrogenic differentiation of MSCs as well [24,26]. Meanwhile, it is also believed that BMP-2-induced chondrogenic differentiation of MSCs will eventually lead to hypertrophy and endochondral ossification [36,37]. Besides Conditioned medium of co-culture and mono-culture pellets was collected at day 2 after seeding.…”

Chondrogenesis and Mineralization DuringIn VitroCulture of Human Mesenchymal Stem Cells on Three-Dimensional Woven Scaffolds

“…[34][35][36][37] Moreover, bone marker expression was amplified by prolonged culture in some studies. 38,39 Our use of type I collagen as the cell seeding gel may have played a role in committing some hMSC toward osteogenesis. 40,41 On the basis of previous studies associating ECM stiffness with MSC lineage commitment, 16 it is possible that mineralization was triggered by local mechanical cues imparted by the type I collagen gel and/or the PCL scaffold.…”