Transplantation of miR-216a-5p-overexpressing mesenchymal stem cells encapsulated in a thermosensitive hydrogel promotes functional recovery in a rat model of spinal cord injury

Abstract

To evaluate the therapeutic efficacy of mesenchymal stem cells (MSCs) overexpressing miR-216a-5p delivered via a thermosensitive hydrogel in a rat model of spinal cord injury (SCI). A thermosensitive hydrogel was engineered to encapsulate MSCs overexpressing miR-216a-5p (MSC-miR-216a-5p). The hydrogel-cell construct was characterized for its physical properties and transplanted into rats with contusion SCI. Functional recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale, mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Inflammatory responses were evaluated by measuring pro-inflammatory cytokine levels. The engineered hydrogel demonstrated suitable mechanical properties, temperature-dependent swelling, and controlled degradation behavior. Rats treated with hydrogel-encapsulated MSC-miR-216a-5p showed significantly improved functional recovery, evidenced by higher BBB, MWT, and TWL scores than control groups. The treatment effectively modulated the inflammatory response by reducing pro-inflammatory cytokine levels. Mechanistic studies identified GPBP1 as a direct target of miR-216a-5p, mediating the observed neuroprotective and anti-inflammatory effects. The combination of miR-216a-5p-overexpressing MSCs with a thermosensitive hydrogel delivery system represents a promising therapeutic strategy for SCI treatment. This approach promotes functional recovery and modulates inflammatory responses through GPBP1 targeting, offering potential for clinical translation in SCI therapy.

Keywords: Mesenchymal stem cells; MiR-216a-5p; Spinal cord injury; Thermosensitive hydrogel.

Fig. 1

Characterization and differentiation assays for rUC-MSCs. A Flow cytometric analysis of rUC-MSCs. Cells were positive for the mesenchymal stem cell markers CD73, CD90, and CD105, and negative for the hematopoietic markers CD14, CD34, and CD45. B Differentiation potential of rUC-MSCs. Cells were cultured in osteogenic (left), adipogenic (middle), and chondrogenic (right) differentiation media for 21 days. Osteogenic differentiation was confirmed by Alizarin Red S staining, adipogenic differentiation by Oil Red O staining, and chondrogenic differentiation by Alcian Blue staining. Scale bars: 100 μm

Fig. 2

Behavioral characterization of the rat SCI model. A BBB locomotor rating scale scores of sham and SCI groups over 28 days post-injury. B MWT scores of sham and SCI groups at 28 days post-injury. C TWL scores of sham and SCI groups at 28 days post-injury. D Relative mRNA expression levels of TNF-α, IL-1β, and IL-6 in spinal cord tissues, as determined by qRT-PCR. E Protein concentrations of TNF-α, IL-1β, and IL-6 in spinal cord tissues, as measured by ELISA. Data are presented as mean ± SD (n = 5 per group). **P < 0.01, ***P < 0.001 vs. sham group

Fig. 3

Expression of miR-216a-5p in the rat SCI model. A Relative expression level of miR-216a-5p in spinal cord tissues, as determined by qRT-PCR. B Representative FISH images showing the expression of miR-216a-5p (green) in the L4-L6 spinal cord segments. Nuclei were counterstained with DAPI (blue). Data are presented as mean ± SD (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance vs. sham group

Fig. 4

Characterization of the hydrogel-based delivery system for MSC-miR-216a-5p. A Quantitative analysis of DNA, GAGs, and collagen content in primary and decellularized tissues. Data are presented as mean ± SD (n = 3). ***P < 0.001, **P < 0.01, ns = not significant. B Thermosensitive properties of the MSCs-gel, exhibiting a liquid state at room temperature and solidifying within 4–5 min at 37 °C. C SEM images of the hydrogel's interior structure, revealing an interconnected three-dimensional network with pore sizes ranging from 50 to 100 μm. Scale bar: 100 μm. D Rheological properties of the hydrogels, showing the storage modulus (G') and loss modulus (G") as a function of oscillation strain at 37 °C. E, F Temperature scanning of the hydrogels, revealing the phase transition temperatures of the Gel, Gel + Cells, Gel + NC, and Gel + agomir groups. G Swelling performance of the hydrogels in PBS solution at different time points. H, I Swelling rates of the hydrogels at different temperatures. J Degradation properties of the hydrogel at different temperatures, assessed by weight loss rate

Fig. 5

Gel/miR-216a-5p agomir improves functional recovery and reduces inflammation in SCI rats. A BBB scores. B MWT. C TWL. D, E Protein levels of TNF-α, IL-1β, and IL-6 by ELISA. Mean ± SD, n = 5/group. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Effect of gel/miR-216a-5p agomiR transplantation on neuronal apoptosis in SCI rats. A Representative images and quantification of TUNEL staining in the spinal cord sections. Scale bar: 50 μm. B Representative Western blots and quantification of cleaved caspase-3, Bcl-2, and Bax protein levels in the spinal cord tissues of sham, SCI, and gel/miR-216a-5p agomiR transplantation groups. β-actin was used as a loading control, n = 5 per group

Fig. 7

miR-216a-5p directly targets GPBP1 in neurons. A Venn diagram showing GPBP1 as the common target gene of miR-216a-5p across multiple databases. B Representative Western blots and quantification of GPBP1 protein levels in the spinal cord tissues of sham and SCI rats, n = 5. C GPBP1 protein and mRNA levels in the spinal cord tissues of sham, SCI, gel/NC agomiR, and gel/miR-216a-5p agomiR groups, n = 5. D Putative binding sites for miR-216a-5p in 3ʹUTR of GPBP1 by Targetscan. E Luciferase reporter assays in primary rat neurons co-transfected with WT or MUT GPBP1 3ʹUTR reporters and miR-216a-5p mimics or NC mimics, n = 5. F, G GPBP1 levels in primary rat neurons transfected with miR-216a-5p mimics, inhibitors, or respective controls. Data are presented as mean ± SD (n = 3 per group). **P < 0.01