Implantation of adipose-derived mesenchymal stem cell sheets promotes axonal regeneration and restores bladder function after spinal cord injury

Abstract

Background: Cell-based therapy using adipose-derived mesenchymal stem cells (ADSCs) is a promising treatment strategy for neurogenic bladder (NB) associated with spinal cord injury (SCI). However, therapeutic efficacy is low because of inefficient cell delivery. Cell sheets improve the efficacy of cell transplantation. Therefore, this study was conducted to investigate the therapeutic efficacy of transplanting ADSC sheets into an SCI rat model and focused on the function and pathological changes of the bladder.

Methods: ADSC sheets were prepared from adipose tissue of Sprague-Dawley (SD) rats using temperature-responsive cell culture dishes. Adult female SD rats were subjected to SCI by transection at the T10 level and administered ADSC sheets or gelatin sponge (the control group). Four and 8 weeks later, in vivo cystometrograms were obtained for voiding function assessment. Rats were sacrificed and the expression of various markers was analyzed in spinal and bladder tissues.

Results: The number of β-tubulin III-positive axons in the ADSC sheet transplantation group was higher than that in the control group. Conversely, expression of glial fibrillary acidic protein in the ADSC sheet transplantation group was lower than that in the control group. Cystometry showed impairment of the voiding function after SCI, which was improved after ADSC sheet transplantation with increased high-frequency oscillation activity. Furthermore, ADSC sheet transplantation prevented disruption of the bladder urothelium in SCI rats, thereby maintaining the intact barrier. Compared with fibrosis of the bladder wall in the control group, the ADSC sheet transplantation group had normal morphology of the bladder wall and reduced tissue fibrosis as shown by downregulation of type 1 collagen. ADSC sheet transplantation also resulted in strong upregulation of contractile smooth muscle cell (SMC) markers (α-smooth muscle actin and smoothelin) and downregulation of synthetic SMC markers (MYH10 and RBP1).

Conclusion: ADSC sheet transplantation significantly improved voiding function recovery in rats after SCI. ADSC sheet transplantation is a promising cell delivery and treatment option for NB related to SCI.

Keywords: Adipose-derived mesenchymal stem cells (ADSCs); Cell sheets; Cell-based therapy; Neurogenic bladder (NB); Spinal cord injury (SCI).

Fig. 1

Schematic diagram of the fabrication process of an ADSC sheet and transplantation of the ADSC sheet into the injury site of an SCI rat. The light blue lines represent peripheral efferent nerves, the deep blue lines represent peripheral afferent nerves, the red lines represent supraspinal afferent pathways and the purple lines represent supraspinal efferent pathways

Fig. 2

Preparation of ADSC sheets. a ADSCs were observed by light microscopy. b Oil Red staining revealed lipid droplet aggregation in the cytoplasm of ADSCs. c Alizarin Red staining revealed calcium deposition in ADSCs. d ADSC sheet formation after 14 days of culture. e Light microscopic view of ADSC sheet morphology. f SEM of an ADSC sheet. g TEM showing tight junctions in the ADSC sheet. h TEM showing the ECM in the ADSC sheet. i TPEF images showing collagen bundles in the ADSC sheet. Scale bars: 100 μm (a, b, c, e); 50 μm (f); 20 nm (g, h); 20 μm (i)

Fig. 3

HE and Immunofluorescence staining of spinal cord tissues in various groups. a Biomarkers for differentiation of stem cells to neurons (β-tubulin III, red) and the formation of glial scars (GFAP, green) were detected in regenerated spinal cord tissue. Scale bar: 1 mm (HE staining); 100 μm (immunofluorescence staining). b The quantitative analysis of cavity and β-tubulin III, GFAP-positive staining areas in spinal cord tissues. ^&p < 0.05 versus Sham, ^*p < 0.05 versus SCI 4 W, ^Δp < 0.05 versus SCI 8 W, ^#p < 0.05 versus SCI + ADSC 4 W

Fig. 4

Analyses of CMG parameters in various groups. Changes in MV (a), RV (b), BC (c), PBP (d), VE (e), and HFOs (f). ^&p < 0.05 versus Sham, ^*p < 0.05 versus SCI 4 W, ^Δp < 0.05 versus SCI 8 W, ^#p < 0.05 versus SCI + ADSC 4 W

Fig. 5

Histological and immunofluorescence analysis of the bladder urothelium in various groups. a The expression of markers krt20 (red), Upk (green), p63 (red), and krt5 (green) showed the distribution of umbrella, intermediate, and basal cells in the bladder urothelium. Scale bar: 100 μm. b mRNA expression levels of krt20, Upk, krt5, and p63 in the bladder wall. ^&p < 0.05 versus Sham; ^*p < 0.05 versus SCI 4 W; ^#p < 0.05 versus SCI + ADSC 4 W

Fig. 6

Histological evaluation of bladder smooth muscle tissue in various groups. a Masson’s trichrome and immunofluorescence staining of a contractile SMC marker (α-SMA) and collagen marker (collagen I) showed remolding of smooth muscle and collagen in the bladder wall. Scale bar: 100 μm. b mRNA expression levels of contractile SMC markers (α-SMA and smoothelin) and synthetic SMC markers (MYH10 and RBP1) in the bladder wall. ^&p < 0.05 versus Sham, ^*p < 0.05 versus SCI 4 W, ^Δp < 0.05 versus SCI 8 W, ^#p < 0.05 versus SCI + ADSC 4 W