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.
Highlights
The present study compares bone morphogenetic protein (BMP)-4 and bone morphogenetic proteins (BMPs)-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures
Primary MSCs were infected with Ad.BMP-2, Ad.BMP-4 or Ad.green fluorescent protein (GFP) at 5 × 102 vp/cell, seeded into aggregates and analyzed biochemically during a three-week time course. (a, b) Values represent levels of (a) BMP-2 and (b) BMP-4 transgene product expressed in ng/mL in the conditioned media over a 24-hour period at days 3, 7, 14 and 21
GAG content and Alkaline phosphatase (ALP) activity As primary MSCs were shown to be capable of expressing the BMP-2 or the BMP-4 transgene in aggregate culture, we examined the effects of BMP-2 and BMP-4 gene delivery on cell proliferation using the WST1 cell proliferation assay
Summary
The 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. Inadequate delivery of the soluble factors necessary to drive the chondrogenic differentiation of the transplanted cells in vivo is a major impediment to effective chondrogenesis in situ [7]. To overcome this limitation, gene transfer approaches are being explored clinically [8] and experimentally [9,10,11,12] to enable the sustained delivery of chondrogenic and anti-inflammatory factors to cartilage defects
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