title: BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold.
abstract: Osteogenic differentiation of bone marrow stromal cells (BMSC) in a three-dimensional (3-D) scaffold has not been well studied. In this work, we studied expression of bone-related genes during differentiation of rabbit BMSCs in response to bone morphogenetic protein (BMP)-2 in both 2-D and 3-D culture systems. When BMSCs were cultured on films (2-D) of biodegradable poly(lactide-co-glycolide) (PLGA), increases in mRNA expression of type I collagen (Col I) and vascular endothelial growth factor (VEGF) became evident after 1 week. However, expression of both genes was only mildly stimulated by BMP-2. Expression of the osteopontin gene was highly stimulated by BMP-2 treatment. Expression of chordin, a BMP antagonist, increased significantly after 7 days. The increase was abrogated by BMP-2 treatment. BMP-2 was also able to stimulate mineralization of cultured BMSCs. After cells were switched to 3-D PLGA scaffolds for 24 h, expression of osteopontin and VEGF were markedly increased while expression of type I collagen and chordin remained unchanged. Expression of Col I did not increase with time in a 3-D culture as it did when cells were cultured on a 2-D film. We further explored the possibility of engineering bone tissue in vitro by seeding BMSCs into PLGA scaffolds. Cellular differentiation and bone formation in the scaffolds were analyzed histologically at 2 weeks and 2 months. Secretion of ECM by cells was evident at both 2-week and 2-month scaffolds, and was enhanced by rhBMP-2. Striking differences in 2-month scaffolds were observed between BMP-treated and untreated cells. A woven bone-like structure appeared in the scaffolds treated with BMP-2. The structure was verified to be bone-related by: (1) the presence of organized collagen fibrils; (2) the presence of mineral; and (3) morphological features of trabecular bone. Although collagen was abundant in the untreated 2-month scaffolds, it was disorganized. The untreated scaffolds also lacked mineral deposits, which were present in 2-D cultured cells even in the absence of BMP-2. Our results indicate that the requirement of osteo-inductive agents, such as BMP-2, is essential for bone tissue engineering.
authors.affiliation: Plastic Surgery Section, VA Greater LA Healthcare System, Los Angeles, CA 90073, USA.
authors.forename: Weibiao
authors.initials: W
authors.lastname: Huang
authors.forename: Brian
authors.initials: B
authors.lastname: Carlsen
authors.forename: Isabella
authors.initials: I
authors.lastname: Wulur
authors.forename: George
authors.initials: G
authors.lastname: Rudkin
authors.forename: Kenji
authors.initials: K
authors.lastname: Ishida
authors.forename: Benjamin
authors.initials: B
authors.lastname: Wu
authors.forename: Dean T
authors.initials: DT
authors.lastname: Yamaguchi
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Miller
mesh_terms: D000818:Animals; D001846:Bone Development; D001853:Bone Marrow / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D001842:Bone and Bones / Q000166:cytology*; D002454:Cell Differentiation; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers; D011817:Rabbits; D017154:Stromal Cells / Q000378:metabolism*; D023822:Tissue Engineering; D016212:Transforming Growth Factor beta; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D011108:Polymers; D016212:Transforming Growth Factor beta; D042461:Vascular Endothelial Growth Factor A; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid