Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.
OBJECTIVE
The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats.


METHODS
(Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery.


RESULTS
The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks.


CONCLUSIONS
These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.
Department of Orthopedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan; School of Health and Sports Science, Mukogawa Women's University, Nishinomiya, Japan.
Maki
M
Itokazu
School of Health and Sports Science, Mukogawa Women's University, Nishinomiya, Japan; Department of Artificial Joint & Biomaterials Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan.
Shigeyuki
S
Wakitani
School of Health and Sports Science, Mukogawa Women's University, Nishinomiya, Japan; International Medical Device Alliance, Foundation for Biomedical Research and Innovation, Kobe, Japan.
Hisashi
H
Mera
School of Health and Sports Science, Mukogawa Women's University, Nishinomiya, Japan.
Yoshihiro
Y
Tamamura
Laboratory of Cell Processing Engineering, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo, Japan.
Yasushi
Y
Sato
Laboratory of Cell Processing Engineering, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo, Japan.
Mutsumi
M
Takagi
Department of Orthopedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan.
Hiroaki
H
Nakamura
animal models; articular cartilage; knee; mesenchymal stem cells; rodent
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