title: Early cellular responses of BMSCs genetically modified with bFGF/BMP2 co-cultured with ligament fibroblasts in a three-dimensional model in vitro.
abstract: Currently, a number of strategies including the implantation of bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors have been developed to regenerate the tendon-to-bone interface after performing anterior cruciate ligament reconstruction. However, the mechanisms behind the interactions of the implanted BMSCs and tendon cells remain to be elucidated. The aim of this study was to evaluate the early cellular responses of BMSCs genetically modified with basic growth factor growth factor (bFGF)/bone morphogenic protein 2 (BMP2) and ligament fibroblasts in a three-dimensional co-culture model. BMSCs and ligament fibroblasts were both isolated from male Wistar rats. The BMSCs were then transfected with an adenoviral vector carrying bFGF or BMP2. The transfected BMSCs and ligament fibroblasts both encapsulated in alginate beads were co-cultured for 6 days in three-dimensional model. On days 0, 3 and 6, cell proliferation was assayed. On day 6, the expression of several tendon-bone related markers was evaluated. In the co-culture system, bFGF and BMP2 were highly expressed at the mRNA and protein level. During the process, bFGF significantly promoted cell proliferation, as well as the expression of scleraxis (SCX) and collagen (COL) type Ⅰ (COL1) in the BMSCs; however, it markedly decreased the expression of phenotype markers in the ligament fibroblasts, including COL1 and COL3. BMP2 markedly increased the expression of alkaline phosphatase and osteocalcin in the BMSCs and ligament fibroblasts, whereas it had no obvious effect on cell proliferation and collagen synthesis in the ligament fibroblasts. The combination of bFGF and BMP2 resulted in the similarly enhanced proliferation of BMSCs and ligament fibroblasts as observed with bFGF alone; however, this combination more potently promoted osteogenic differentiation than did BMP2 alone. The findings of our study demonstrate the superiority of the combined use of growth factors in inducing osteogenic differentiation and provide a theoretical foundation for the regeneration of the tendon-to-bone interface.
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital, Wuhan University, Wuhan, P.R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital, Wuhan University, Wuhan, P.R. China.
authors.forename: Ramesh Kumar
authors.initials: RK
authors.lastname: Jha
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital, Wuhan University, Wuhan, P.R. China.
authors.forename: Yong-Jian
authors.initials: YJ
authors.lastname: Qi
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital, Wuhan University, Wuhan, P.R. China.
authors.forename: Qu-Bo
authors.initials: QB
authors.lastname: Ni
authors.affiliation: Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital, Wuhan University, Wuhan, P.R. China.
authors.forename: Biao
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital, Wuhan University, Wuhan, P.R. China.
authors.forename: Liao-Bin
authors.initials: LB
authors.lastname: Chen
mesh_terms: D000256:Adenoviridae / Q000378:metabolism; D000257:Adenoviridae Infections / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology*; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation; D048430:Cell Shape; D018920:Coculture Techniques / Q000379:methods*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008022:Ligaments / Q000166:cytology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D008954:Models, Biological; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2