2.1 SVF cell sheet characteristics and angiogenic effects in in vitro experiments
After adherent growth, the rabbit adipose-derived SVF cells formed oblong fusiform or polygonal structures after approximately 10 days (Fig. 1a). HE staining showed that the SVF cells were arranged in a long fusiform and vortex shape and that the cell distribution was uniform (Fig. 1b). Flow cytometry revealed that the SVF included adipose stem cells (ADSCs) and EPCs[12]. At day 2 of culture in cell sheet-induction medium, observation under an inverted phase-contrast microscope showed that the cells in the temperature-sensitive culture dish had reached 100% confluence. When the induction culture was continued until day 14, a membranous tissue structure formed at the bottom of the culture dish. The culture dishes were incubated at 20-25 °C for approximately 40 min, and a white translucent membrane (SVF cell sheet) was obtained (Fig. 1c). SEM images of the SVF cell sheets showed that the SVF cells were connected and fused to each other and were tightly arranged, with a large amount of extracellular matrix deposited between the cells (Fig. 1d).
The expression of the proangiogenic factors Ang-1 and VEGF in the three groups of cells was assessed by IF (green fluorescence (positive signal) indicated Ang-1 and VEGF expression, and blue indicated nuclei). The results showed that in the SVF cell-sheet group, the number of cells with positive signals for Ang-1 and VEGF (green fluorescence) was significantly greater than that in the blank group (P <0.01) and the SVF cell group (P <0.05); moreover, the number of cells in the SVF cell group was greater than that in the blank group (P <0.01) (Fig. 2a-d). The qRT-PCR and WB results showed that, in the SVF cell-sheet group, the messenger RNA (mRNA) and protein expression levels of Ang-1 and VEGF were significantly higher than those in the blank group (P <0.01) and SVF cell group (P<0.05); the expression levels in the SVF cell group were higher than those in the blank group (P<0.01) (Fig. 2e- i). These findings suggest that compared with SVF cells, SVF cell sheets could further upregulate the gene and protein expression of angiogenic factors.
In vitro tube formation assays showed that cells in the blank group grew in cords rather than in tubes; in the SVF cell and SVF cell-sheet groups, after 6 h, the cells attached to the Matrigel and gradually extended their pseudopodia and made contact with the surrounding cells, forming a three-dimensional (3D) reticular lumen-like structure similar to blood vessels, with tube formation rates of 20.3% and 21.5%, respectively (Fig. 3). These results indicated that the SVF cell sheets had a greater proangiogenic effect than did the SVF cells.
2.2 An SVF cell sheet promotes cartilage regeneration in diced cartilage grafts
New Zealand rabbits were euthanized at 1, 3, and 6 months after surgery by intravenous injection of 1% sodium pentobarbital 100 mg/kg, the bilateral diced cartilage grafts on the back were removed, and the appearance and morphological characteristics of the grafts were observed. At 1 month after surgery, the diced cartilage grafts in the blank group were slightly soft when touching, with some unfused diced cartilage scattered around the incision; the texture of the grafts in the perichondrium group was slightly tough, and a small amount of unfused diced cartilage could be observed around the central incision; and the diced cartilage grafts in the SVF cell-sheet group were basically fused and slightly tough when touching. At 3 months after surgery, the diced cartilage grafts in the blank group and the perichondrium group were basically fused, with an irregular appearance, and were relatively tough when touching; in the SVF cell-sheet group, the diced cartilage grafts were completely fused and were tough when touching. At 6 months after surgery, the diced cartilage grafts in the three groups were completely fused and were slightly hard when touching, and connective tissue fibrous membrane wrapping and neovascular growth were seen along the periphery (Fig. 4a). The wet weight of the grafts in each group was measured before transplantation and at 1, 3, and 6 months after transplantation. The results showed that in the blank group, the wet weights of cartilage grafts at 1 month, 3 months and 6 months after surgery were not significantly different from those before transplantation (P>0.05); at 3 and 6 months after surgery, the wet weights of cartilage grafts in the perichondrium group and SVF cell-sheet group were greater than those of cartilage grafts in the blank group (P <0.05), and the wet weight of grafts in the SVF cell-sheet group was slightly greater than that in the perichondrium group (p <0.05) (Table 1).
To understand the regenerative activity of chondrocytes, specific staining of cartilage tissue blocks was performed. Safranin fast green staining results showed that at 3 months after surgery, cells at the edge of diced cartilage grafts in the SVF cell sheet and perichondrium groups were oblate and existed alone, indicating the presence of new chondrocytes; the chondrocytes near the center of the cartilage were round and oval, and two or three isogenic cell population aggregates were observed. At 6 months after surgery, the number of new chondrocytes in the perichondrium group gradually increased; in the SVF cell-sheet group, large amounts of new cartilage and red cartilage matrix were observed, multiple chondrocytes were present in the lacuna, and new cartilage had gradually matured. In the blank group, there was no significant increase in the number of new chondrocytes or red cartilage matrix at 3 months after surgery compared with 1 month after surgery, and the amount of new chondrocytes at 6 months after surgery was slightly lower than that at 1 month after surgery (Fig. 4b). After Masson staining, the collagen fibers in the cartilage matrix were stained blue. At 1 month after surgery, the differences in the amount of blue stained cartilage matrix in the 3 groups were not significant. At 3 and 6 months after surgery, the blue staining area and staining intensity of the cartilage matrix in the perichondrium group and the SVF cell-sheet group were significantly greater than those in the blank group; the blue staining area and staining intensity of the cartilage matrix in the SVF cell-sheet group were higher than those in the perichondrium group, suggesting that the SVF cell sheets can increase the secretion of cartilage collagen (Fig. 4c). After toluidine blue staining, proteoglycan, the main component of the cartilage matrix, was stained blue-purple, and the staining intensity was related to the amount of proteoglycans. At 1 month after surgery, the differences in the amount of blue-purple stained cartilage tissues in the 3 groups were not significant. At 3 and 6 months after surgery, the blue-purple staining area and staining intensity of the cartilage tissues in the perichondrium group and the SVF cell-sheet group were significantly greater than those in the blank group; the blue-purple staining area and staining intensity of the cartilage tissues in the SVF cell-sheet group were greater than those in the perichondrium group, suggesting that SVF cell sheets can increase cartilage matrix secretion (Fig. 4d). These cartilage tissue-specific staining results suggest that compared with autologous perichondrium, SVF cell sheets better promote the survival of and regeneration in grafted cartilage.
2.3 SVF cell sheets promote angiogenesis in diced cartilage grafts
To understand the effects of SVF cell sheets on vascularization around diced cartilage grafts, IHC was performed to assess the expression levels of Ang-1 and VEGF in cartilage tissues of the three groups, and the results showed that in the SVF cell-sheet group and perichondrium group, the chondrocytes surrounding the cartilage tissue and the tissue surrounding the cartilage block were intensely stained brownish yellow and dark brown. At 1, 3, and 6 months after surgery, the number of positive cells for both Ang-1 and VEGF was significantly higher in the perichondrium group than in the blank group, and the expression of Ang-1 and VEGF in the SVF cell-sheet group was further upregulated compared to that in the perichondrium group (Fig. 5a and 5b).
The qRT-PCR and WB results showed that at 1, 3, and 6 months after surgery, the mRNA and protein expression levels of Ang-1 and VEGF in the SVF cell-sheet group and the perichondrium group were greater than those in the blank group (P <0.01) and that the levels in the SVF cell-sheet group were greater than those in the perichondrium group (P <0.05) (Fig. 5c-g). These results indicate that SVF cell sheets upregulate the gene and protein expression of angiogenesis-related factors, thereby facilitating angiogenesis in cartilage grafts.
HE staining was used to observe the adipose tissue and vascularization around diced cartilage grafts. At 1 month after surgery, compared with those in the blank group, small amounts of connective and adipose tissues and blood vessel formation between diced cartilage grafts were observed in the perichondrium and SVF cell-sheet groups (P <0.05). At 3 and 6 months after surgery, in the blank group, small amounts of connective tissue and adipose tissue formed around the diced cartilage grafts, and a small amount of neovasculature could be observed. Compared with those in the blank group, in the perichondrium group, there was more connective and adipose tissue around the diced cartilage grafts, and more neovasculature could be observed around the cartilage (P<0.05). In the SVF cell-sheet group, a large amount of connective and adipose tissue and a large amount of neovasculature formed around the diced cartilage grafts (P <0.05) (Fig. 6a, b). The results indicate that the SVF cell sheets better promoted the formation of neovasculature in the cartilage grafts.
2.4 Effect of SVF cell sheets on pyroptosis in cartilage tissues
To understand the viability and survival of chondrocytes in grafts 6 months after diced cartilage transplantation, TUNEL staining was performed, and the results showed that in the blank group, the TUNEL-positive cells (green stained) were located at the center and surrounding area of the diced cartilage grafts; in the SVF cell-sheet group and the perichondrium group, most TUNEL-positive cells were located in the center of diced cartilage grafts, and the percentage of TUNEL-positive cells was significantly lower than that in the blank group (both P<0.01); moreover, the percentage of TUNEL-positive cells in the SVF cell-sheet group was lower than that in the perichondrium group (P <0.01), suggesting that SVF cell sheets can reduce the cell death of cartilage tissues (Fig. 7a, c).
To further elucidate the cause of cell death, the protein expression levels of NLRP3, Caspase1 and GSDMD, which are involved the canonical pyroptosis pathway, in the cartilage tissues of diced cartilage grafts were assessed by IHC, and the results confirmed that pyroptosis occurred in the chondrocytes. However, the protein expression of components of the pyroptosis signaling pathway was lower in the perichondrium group than in the blank group (P <0.05), and the expression in the SVF cell-sheet group was even lower (P <0.01). These findings suggest that SVF cell sheets reduced chondrocyte pyroptosis in the grafts, which is beneficial for cartilage regeneration (Fig. 7b, d).