Menstrual blood-derived mesenchymal stem cells combined with collagen I gel as a regenerative therapeutic strategy for degenerated disc after discectomy in rats

Abstract

Background: Annulus fibrosis (AF) defects have been identified as the primary cause of disc herniation relapse and subsequent disc degeneration following discectomy. Stem cell-based tissue engineering offers a promising approach for structural repair. Menstrual blood-derived mesenchymal stem cells (MenSCs), a type of adult stem cell, have gained attention as an appealing source for clinical applications due to their potential for structure regeneration, with ease of acquisition and regardless of ethical issues.

Methods: The differential potential of MenSCs cocultured with AF cells was examined by the expression of collagen I, SCX, and CD146 using immunofluorescence. Western blot and ELISA were used to examine the expression of TGF-β and IGF-I in coculture system. An AF defect animal model was established in tail disc of Sprague-Dawley rats (males, 8 weeks old). An injectable gel containing MenSCs (about 1*10⁶/ml) was fabricated and transplanted into the AF defects immediately after the animal model establishment, to evaluate its repairment properties. Disc degeneration was assessed via magnetic resonance (MR) imaging and histological staining. Immunohistochemical analysis was performed to assess the expression of aggrecan, MMP13, TGF-β and IGF-I in discs with different treatments. Apoptosis in the discs was evaluated using TUNEL, caspase3, and caspase 8 immunofluorescence staining.

Results: Coculturing MenSCs with AF cells demonstrated ability to express collagen I and biomarkers of AF cells. Moreover, the coculture system presented upregulation of the growth factors TGF-β and IGF-I. After 12 weeks, discs treated with MenSCs gel exhibited significantly lower Pffirrmann scores (2.29 ± 0.18), compared to discs treated with MenSCs (3.43 ± 0.37, p < 0.05) or gel (3.71 ± 0.29, p < 0.01) alone. There is significant higher MR index in disc treated with MenSCs gel than that treated with MenSCs (0.51 ± 0.05 vs. 0.24 ± 0.04, p < 0.01) or gel (0.51 ± 0.05 vs. 0.26 ± 0.06, p < 0.01) alone. Additionally, MenSCs gel demonstrated preservation of the structure of degenerated discs, as indicated by histological scoring (5.43 ± 0.43 vs. 9.71 ± 1.04 in MenSCs group and 10.86 ± 0.63 in gel group, both p < 0.01), increased aggrecan expression, and decreased MMP13 expression in vivo. Furthermore, the percentage of TUNEL and caspase 3-positive cells in the disc treated with MenSCs Gel was significantly lower than those treated with gel alone and MenSCs alone. The expression of TGF-β and IGF-I was higher in discs treated with MenSCs gel or MenSCs alone than in those treated with gel alone.

Conclusion: MenSCs embedded in collagen I gel has the potential to preserve the disc structure and prevent disc degeneration after discectomy, which was probably attributed to the paracrine of growth factors of MenSCs.

Keywords: Annulus fibrosis defects; Disc degeneration; Disc repairment; Discectomy; Growth factors; Menstrual blood-derived mesenchymal stem cells; Paracrine; Tissue engineering.

Fig. 1

AF cells and MenSCs co-culture promote production of extra-cellular matrix and growth factors. (A): The coculture system of MenSCs and AF cells; (B) Alcian blue and safranine O staining; (C) collagen I immunofluorescence; (D): Scleraxis immunofluorescence;(E): CD146 immunofluorescence; (E) qPCR for collagen I, Scleraxis, CD146, Bpifa2f, Fibin, and Myoc, Igfbp5, MMP3, and IL-11. (F) Western blot for TGF-β and IGF-I; (G) ELISA for TGF-β and IGF-I. Values are presented as mean ± SEM. ** P < 0.01, *** P < 0.001. Col I, collagen I; SCX, scleraxis; MenSCs, menstrual blood-derived mesenchymal stem cells; AF, anulus pulposus. Scale bar = 20 μm for immunofluorescence, scale bar = 50 μm for safranine O staining

Fig. 2

MenSCs embedded in collagen gel preserved the water content of degenerated disc following discectomy. (A): Midsagittal MR images (T2- weighted) of degenerated disc at 4 and 12 weeks after various treatment. Images are representative of seven replicates. (B) MR imaging index for each group. (C): Pfirrmann grading of disc degeneration after various treatment. Values are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. MenSCs, menstrual blood-derived mesenchymal stem cells. MR, magnetic resonance

Fig. 3

MenSCs embedded in collagen gel preserved the structures of degenerated disc following discectomy. (A): Gross appearance of the disc in different groups. (B): Hematoxylin and eosin staining of disc in different groups. (C): Safranin O and fast green staining of disc in different groups. (D): Disc degeneration scores. Images are representative of seven replicates. Scale bar = 100 μm; Values are presented as mean ± SEM. ** P < 0.01, *** P < 0.001

Fig. 4

MenSCs embedded in collagen gel preserved extra-cellular matrix contents of degenerated disc following discectomy. (A): Immunohistochemical examination of aggrecan; (B): Immunohistochemical examination of MMP13; (C): Percentages of aggrecan and MMP13 positive cells relative to total cells in degenerated disc after discectomy. Images are representative of seven replicates. Scale bar = 50 μm; Values are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. MenSCs, menstrual blood-derived mesenchymal stem cells, MMP3, matrix metalloproteinase 3

Fig. 5

MenSCs embedded in collagen gel up-regulated TGF-β and IGF-I expression in degenerated disc following discectomy. (A): Immunohistochemical examination of TGF-β and IGF-I. (B): Percentages of TGF-β and IGF-I positive cells relative to total cells in degenerated disc after discectomy. Images are representative of seven replicates. Scale bar = 50 μm; Values are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. MenSCs, menstrual blood-derived mesenchymal stem cells, TGF-β, transforming growth factor-β. IGF-I, insulin-like growth factor-I

Fig. 6

MenSCs embedded in collagen gel reduced the cell apoptosis in degenerated disc following discectomy. (A): TUNEL staining of apoptotic cells in the discs after various treatments. (B): Caspase 3 staining in disc after various treatments. (C): Caspase 8 staining in the discs after various treatments. Percentages of TUNEL (D), caspase 3 (E), and caspase 8 (F) positive cells relative to total cells. Images are representative of seven replicates. Scale bar = 200 μm; Values are presented as mean ± SEM. ** P < 0.01, *** P < 0.001. MenSCs, menstrual blood-derived mesenchymal stem cells. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling