Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Abstract

Introduction: Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods: Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results: At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion: This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.

Figures

Figure 1

Xenogeneic transplantation of mesenchymal stem cells. (A to E) Photomicrographs showing frontal sections, 10 μm thick, of the whole knee joint stained with safranin O. (B' to E') Fluorescent microscopy for in vivo tracing of injected mesenchymal stem cells (MSCs). Bar = 500 μm (A), 200 μm (B to E, B' to E').

Figure 2

Macroscopic findings. Macroscopic photographs (bird's-eye view) of the articular surface of the tibial plateau 5 weeks after injection of each reagent in Hartley strain guinea pigs, stained with India ink: (A) PBS group, (B) hyaluronic acid (HA) group, (C) PBS + mesenchymal stem cell (MSC) group, (D) HA + MSC group. Arrowheads: stained lesions. (E) Macroscopic osteoarthritis (OA) scores in the same animal groups evaluated 5 weeks after injection. Data are mean ± standard deviation of five guinea pigs in each group. *P < 0.05.

Figure 3

Histological findings. Photomicrographs showing serial frontal sections 10 μm thick stained with (A to D) H & E and (E to H) safranin O. Bar = 100 μm in all sections. (I) Histological grade of knee osteoarthritis assessed by modified Mankin criteria 5 weeks after injection, according to the treatment modality. Data are mean ± standard deviation of five guinea pigs in each group. *P < 0.05. HA, hyaluronic acid; MSC, mesenchymal stem cell.

Figure 4

Immunohistochemical and immunoblot analyses. Immunohistochemistry, western blotting, and quantification of type II collagen, type I collagen, and matrix metalloproteinase (MMP)-13 expressions at week 5 after injection. (A) Immunohistochemical analysis for type II collagen. Bar = 50 μm. (B) Western blot analysis of type II collagen, type I collagen, and MMP-13 (pooled samples in each group, n = 5). (C) Quantitative analysis of the expressions of type II collagen, type I collagen, and MMP-13 relative to the intensity of β-actin. Data are mean ± standard deviation of five guinea pigs in each group. *P < 0.05. HA, hyaluronic acid; MSC, mesenchymal stem cell.

Figure 5

Fluorescent microscopic findings. Fluorescent microscopy for in vivo tracing of injected mesenchymal stem cells (MSCs) at 1, 3, and 5 weeks. (A to C) PBS + MSC group. (D to F) Hyaluronic acid (HA) + MSC group. Bar = 50 μm. (G) Quantification of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)-labeled MSCs. Data are mean ± standard deviation of five guinea pigs in each group. *P < 0.05.

Figure 6

Double immunofluorescence microscopic findings. Photomicrographs showing serial sections of articular cartilage 5 weeks after injection in the hyaluronic acid (HA) + mesenchymal stem cell (MSC) group stained with (A) H & E and (B to D) double immunofluorescence (red, type II collagen; green, MSCs; blue, 4',6-diamidino-2-phenylindole (DAPI)). (B) corresponds to (A). (C) and (D) are magnified views of the boxes marked in (B). Bar = 100 μm (A, B), 50 μm (C, D).