Repair abilities of mouse autologous adipose-derived stem cells and ShakeGel™3D complex local injection with intrauterine adhesion by BMP7-Smad5 signaling pathway activation

Abstract

Background: The objective was to explore the therapeutic effect of autologous adipose-derived stem cells (ADSCs) combined with ShakeGel™3D transplantation to activate the BMP7-Smad5 signaling pathway to treat intrauterine adhesions (IUA).

Methods: Autologous ADSCs were isolated and then merged with ShakeGel™3D. The IUA model was established by mechanical injury. The third generation of autologous ADSCs was injected directly into the uterus in combination with ShakeGel™3D. After 7 days of treatment, endometrial morphology, number of endometrial glands, endometrial fibrosis area, and fibrosis biomarker analysis by RT-PCR and IHC were examined. BMP7 and phosphorylation of Smad5 were also detected, and the recovery of infertility function in treated mice was evaluated.

Results: Fluorescence-activated cell sorting (FACS) showed that autologous ADSCs expressed CD105 (99.1%), CD29 (99.6%), and CD73 (98.9%). Autologous ADSCs could still maintain a good growth state in ShakeGel™3D. Histological examination revealed that the number of endometrial glands increased significantly, and the area of fibrosis decreased. At the same time, the expression of BMP7 and Smad5 in the ADSCs + Gel group was significantly upregulated, and the final reproductive function of this group was partly recovered.

Conclusions: Autologous ADSCs can be used in combination with ShakeGel™3D to maintain functionality and create a viable three-dimensional growth environment. The combined transplantation of autologous ADSCs and ShakeGel™3D promotes the recovery of damaged endometrial tissue by increasing BMP7-Smad5 signal transduction, resulting in endometrium thickening, increased number of glands, and decreased fibrosis, leading to restoration of partial fertility.

Keywords: Autologous adipose-derived stem cells; BMP7-Smad5 signaling pathway; Intrauterine adhesion; ShakeGel™3D.

Fig. 1

Study design

Fig. 2

Assay of the activity of ADSCs combined with ShakeGel™3D. a, b, and c respectively were ADSCs combined with ShakeGel™3D under white light 4×, 10×, and 20×. d, e, and f respectively were ADSCs combined with ShakeGel™3D under active staining 4×, 10×, and 20× (scale bar = 100 μm)

Fig. 3

The results of CCK-8 in 1*10⁶ ADSCs and 5*10⁶ ADSCs co-culture with ShakeGel™3D

Fig. 4

Engraftment of the endometrium with ADSCs following local injection. 4.1 Fluorescence microscopy analysis of uterine tissue sections (scale bar = 50 μm). 4.2 The percentage GFP-positive cells of ADSCs + Gel group (9.215 ± 2.470%, n = 3) was higher than ADSCs group (2.175 ± 0.9816%, n = 3) (*P<0.05)

Fig. 5

Uterine morphological features and changes. 5.1 The macroscopic appearance of the uterus. 5.2-a H&E staining of mice uterine tissue (scale bar = 50 μm). 5.2-b The endometrial thickness at transplantation day seven. Sham group, 264.5 ± 31.75 μm; PBS group, 163.8 ± 6.128 μm; ADSCs group, 190.1 ± 8.389 μm; Gel group, 217.8 ± 5.839 μm; ADSCs + Gel, 228.8 ± 2.254 μm. 5.2-c The number of endometrial glands at transplantation day seven. Sham group, 32.73 ± 3.952/section; PBS group, 1.545 ± 0.9342/section; ADSCs group, 9.182 ± 2.857/section; Gel group, 14.55 ± 1.293/section; ADSCs + Gel, 19.36 ± 2.063/section. *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001. 5.3-a Masson staining of mice uterine tissue (Scale bar = 50 μm). 5.3-b The percent of fibrosis area at transplantation day seven. Sham group, 29.44 ± 5.2305%; PBS group, 56.89 ± 0.4425%; ADSCs group, 50.10 ± 1.280%; Gel group, 45.17 ± 1.196%; ADSCs + Gel, 39.55 ± 0.4125%. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P < 0.05

Fig. 6

Immunohistochemistry results 6.1 Representative immunostaining of α-SMA, TGF-β1, BMP7, and Smad5 (scale bar = 50 μm). 6.2-a Mean density of α-SMA, Sham group, 0.007813 ± 0.003258; PBS group, 0.1109 ± 0.04010; ADSCs group, 0.05746 ± 0.008630; Gel group, 0.03392 ± 0.003734; ADSCs + Gel, 0.02151 ± 0.005611. 6.2-b Mean density of TGF-β1, Sham group, 0.0004134 ± 0.0004932; PBS group, 0.03246 ± 0.007779; ADSCs group, 0.02180 ± 0.001778; Gel group, 0.009999 ± 0.005183; ADSCs + Gel, 0.001736 ± 0.001229. 6.2-c Sham group, 0.2119 ± 0.03328; PBS group, 0.002817 ± 0.002115; ADSCs group, 0.02330 ± 0.006284; Gel group, 0.09967 ± 0.02718; ADSCs + Gel, 0.1314 ± 0.01210. 6.2-d Mean density of Smad5, Sham group, 0.3536 ± 0.005790; PBS group, 0.1676 ± 0.06002; ADSCs group, 0.2391 ± 0.02442; Gel group, 0.2910 ± 0.009053; ADSCs + Gel, 0.3372 ± 0.004266. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P >0.05

Fig. 7

7.1-a Relative expression of α-SMA mRNA. Sham group, 1.008 ± 0.1544; PBS group, 0.1544 ± 0.9503; ADSCs group, 2.474 ± 0.1598; Gel group, 1.703 ± 0.2415; ADSCs + Gel, 1.398 ± 0.1797. 7.1-b Relative expression of TGF-β1 mRNA. Sham group, 1.001 ± 0.05932; PBS group, 1.951 ± 0.2980; ADSCs group,1.391 ± 0.1269; Gel group, 1.227 ± 0.1269; ADSCs + Gel, 1.068 ± 0.06639. 7.1-c Relative expression of BMP7 mRNA. Sham group, 1.001 ± 0.03872; PBS group, 0.05062 ± 0.01281; ADSCs group, 0.06124 ± 0.006388; Gel group, 0.08836 ± 0.005922; ADSCs + Gel, 0.5575 ± 0.06289. 7.1-d Relative expression of Smad5 mRNA. Sham group, 1.003 ± 0.08697; PBS group, 0.6843 ± 0.07691; ADSCs group, 0.7127 ± 0.05734; Gel group, 0.7891 ± 0.07409; ADSCs + Gel, 0.9066 ± 0.07593. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P >0.05. 7.2 Results of the mating experiment. Reproductive outcomes in the sham group (7.2-a), PBS group (7.2-b), ADSCs group (7.2-c), Gel group (7.2-d), and ADSCs + Gel group (7.2-e). The number of fetuses in each group was analyzed (7.2-f). Data are presented as mean ± SD. The pregnancy rate is equal to the number of pregnant uteri divided by the total number of uteri