Development of Injectable Polydactyly-Derived Chondrocyte Sheets

Abstract

We are conducting a clinical study of the use of allogeneic polydactyly-derived chondrocyte sheets (PD sheets) for the repair of articular cartilage damage caused by osteoarthritis. However, the transplantation of PD sheets requires highly invasive surgery. To establish a less invasive treatment, we are currently developing injectable fragments of PD sheets (PD sheets-mini). Polydactyly-derived chondrocytes were seeded in RepCell™ or conventional temperature-responsive inserts and cultured. Cell counts and viability, histology, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), and flow cytometry were used to characterize PD sheets-mini and PD sheets collected from each culture. To examine the effects of injection on cell viability, PD sheets-mini were tested in four experimental conditions: non-injection control, 18 gauge (G) needle, 23G needle, and syringe only. PD sheets-mini produced similar amounts of humoral factors as PD sheets. No histological differences were observed between PD sheets and PD sheets-mini. Except for COL2A1, expression of cartilage-related genes did not differ between the two types of PD sheet. No significant differences were observed between injection conditions. PD sheets-mini have characteristics that resemble PD sheets. The cell viability of PD sheets-mini was not significantly affected by needle gauge size. Intra-articular injection may be a feasible, less invasive method to transplant PD sheets-mini.

Keywords: cartilage regeneration; cell sheet; minimally invasive treatment; osteoarthritis.

Figure 1

Fabrication of polydactyly-derived chondrocyte (PD) sheets and PD sheets-mini. PD sheets and PD sheets-mini were fabricated in temperature-responsive culture inserts called UpCell® and temperature-responsive culture dishes called RepCell™ (CellSeed Inc.), respectively. RepCell™ is a unique culture dish with grid walls etched onto the culture surface creating 3 mm × 3 mm grids.

Figure 2

Comparison of cell count and cell viability for PD sheets and PD sheets-mini. Values are expressed as mean ± standard deviation. (a) Cell count of PD sheets and PD sheets-mini. (b) Cell viability of PD sheets and PD sheets-mini. The cell counts and cell viability did not differ between the two types of cell sheets.

Figure 3

Flow cytometric analysis comparing PD sheets and PD sheets-mini. Both sheets were positive for mesenchymal stem cell markers such as CD29, CD44, CD73, CD81, CD90, and CD105, and negative for vascular endothelium and blood cell markers such as CD31 and CD45.

Figure 4

Amounts of secreted humoral factors. The amounts of transforming growth factor beta-1 (TGF-β1), melanoma inhibitory activity (MIA), endothelial cell-specific marker 1 (ESM-1), Dickkopf-1 protein (DKK-1), monocyte chemoattractant protein 1 (MCP-1), matrix metalloproteinase (MMP)-3, and MMP-13, humoral factors related to cartilage, did not differ significantly between PD sheets-mini and PD sheets.

Figure 5

Cartilage-related gene expression analysis in PD sheets and PD sheets-mini. The relative gene expression of PD sheets was set at 1.0. The values are expressed as mean ± standard deviation. ** p < 0.01; ND, not detected. Only the expression of COL2A1 differed between PD sheets and PD sheets-mini.

Figure 6

Histological analysis of PD sheets and PD sheets-mini. Sections of both PD sheets and PD sheets-mini were stained with hematoxylin–eosin, safranin O, and toluidine blue (×10, scale bar = 100 μm).

Figure 7

Evaluation of the effect of injection on cell viability of PD sheets-mini. (a) To examine the effect of injection on cell viability, the PD sheets-mini suspension was examined under four conditions: non-injection control, 18 gauge (G) needle injection, 23G needle injection, and syringe injection, and cell viability was measured at 0, 4, and 24 h after injection. At 0 and 4 h after injection, cell viability of conventional PD sheets was compared. (b) Comparison of cell viability of PD sheets-mini between the four conditions immediately after, 4 h, and 24 h after injection. Cell viability decreased with time but did not differ significantly between the four conditions at any time.