Study on the role and mechanism of magnesium-calcium-mineralized collagen composite scaffolds in the adjuvant treatment of cervical cancer

Abstract

One of the most prevalent malignant tumors in women is cervical cancer. Conventional chemoradiotherapy was frequently limited by significant side effects and acquired drug resistance. Consequently, there is an urgent need for high-performance biomaterials that effectively suppress tumor growth while exhibiting minimal off-target toxicity. Magnesium alloys represented a promising platform for anti-tumor applications due to their bioactive degradation products. This study developed novel magnesium alloy-mineralized collagen composite scaffolds and systematically evaluated their surface properties. Comprehensive in vitro and in vivo experimental models were used to elucidate the scaffolds' anti-tumor mechanisms. The results of this study demonstrated that magnesium alloy-mineralized collagen composite scaffolds significantly inhibit tumor cell invasion and metastasis while promoting cancer cell death. Based on in vivo and in vitro studies, this study showed that the degradation products of magnesium alloy-mineralized collagen composite scaffolds target epithelial-mesenchymal transition through the Wnt/β-catenin/TCF7 signaling pathway. These findings established a robust experimental foundation for advancing magnesium alloy-mineralized collagen composite scaffolds as next-generation biodegradable adjunctive therapeutic materials for cervical cancer treatment. The synergistic combination of biocompatibility and tumor-targeted activity positions this material as an innovative platform for circumventing shortcomings in existing clinical regimens.

Keywords: Wnt signaling pathway; magnesium-based metal; mineralized collagen; tumor microenvironment.

Graphical abstract

Figure 1.

Preparation and characterization of Mg-Ca-nHAC composite scaffolds. (A–C) The representative SEM images of Mg-Ca scaffolds, nHAC scaffolds, Mg-Ca-nHAC composite scaffolds. (D–I) The representative EDS element mapping of Mg-Ca-nHAC composite scaffolds. (J) AFM test to check the surface flatness of the Mg-Ca-nHAC composite scaffolds. (K) XRD diffractogram of Mg-Ca-nHAC composite scaffolds (04-011-5938 Mg(OH)₂, 04-016-1185 hydroxyapatite).

Figure 2.

Material properties testing. (A) Detection of Mg²⁺ concentration changes in Mg-Ca-nHAC materials using ICP-MS method at different PH. (B) Detection of Ca²⁺ concentration changes in Mg-Ca-nHAC materials using ICP-MS method at different PH. (C) Determination of Mg²⁺ concentration changes in Mg-Ca-nHAC materials at 1, 3, 5 and 7 days under varying pH conditions using the ICP-MS method. (D) Determination of Ca²⁺ concentration changes in Mg-Ca-nHAC materials at 1, 3, 5 and 7 days under varying pH conditions using the ICP-MS method. (E) Photograph indicating H₂ generation in PBS from Mg-Ca-nHAC composite scaffolds. (F) Mg²⁺ release rate under different pH conditions. (G) H₂ gas chromatography experiments in 24 h, 48 h, 72 h (**P < 0.01). (H) H₂ generation of Mg-Ca-nHAC composite scaffolds in PBS solutions with different pH values measured by gas chromatography in 72 h (***P < 0.001 vs pH 4.5, *P < 0.05).

Figure 3.

In vitro anti-tumor assays. (A–D) In different cell lines (H8/HeLa/SiHa), the cell proliferation activity of different treatment groups was detected at 24, 48 and 72 h through the CCK8 experiment (**P < 0.01,***P < 0.001). (E–G) Detection of cytoskeletal extension in different treatment groups at 24 h by staining with ghost closed loop peptide and the fluorescence intensity was quantified (scale bar: 20 μm, **P < 0.01). (H) Mitochondrial membrane potential was detected and quantitatively analyzed (*P < 0.05,**P < 0.01).

Figure 4.

In vitro anti-tumor assays. (A, B) The number of live and dead cells in the different treatment groups was detected by live-dead cell staining and the fluorescence intensity was quantified (scale bar: 100 μm, green: live cells, red: dead cells). (C) Detection of cell cycle of HeLa cells in different treatment groups by flow cytometry. (D) Detection of cell cycle of SiHa cells in different treatment groups by flow cytometry. (E) Detection of apoptosis in HeLa cells/SiHa cells of different treatment groups by flow cytometry (*P < 0.05, **P < 0.01). (F) Detection of ROS levels in different treatment groups by flow cytometry (scale bar: 100 μm, **P < 0.01).

Figure 5.

In vitro anti-tumor assays. (A) The migration of HeLa and SiHa cells was observed by microscopic photography and quantitative analysis (scale bar: 50 μm, *P < 0.05, **P < 0.01). (B) The invasion of HeLa and SiHa cells was observed by microscopic photography and quantitative analysis (scale bar: 50 μm, **P < 0.01). (C, D) The migration of HeLa and SiHa cells was observed by microscopic photography and quantitative analysis through scratch experiments (*P < 0.05, **P < 0.01). (E) Plate cloning to further test the proliferative clonogenic capacity of cells (*P < 0.05, **P < 0.01).

Figure 6.

In vitro anti-tumor assays. Transcriptome analysis was exhibited by (A) volcano plot, (B) heatmap, (C) PPI chart, (D) pathways enrichment based on GO enrichment analysis was showed by bubble chart plot, (E) pathways enrichment of DEGs based on KEGG enrichment analysis was showed by bubble chart plot. (F) Pathways enrichment of DEGs based on KEGG enrichment analysis was showed by histogram.

Figure 7.

In vitro anti-tumor assays. (A, B) The expression of protein levels (Wnt1, β-catenin, TCF7, E-cadherin, N-cadherin, Vimentin, Snail, Twist) in different treatment groups was detected by Western blot assay (*P < 0.05, **P < 0.01,***P < 0.001). (C) The expression of gene levels (Wnt1, β-catenin, TCF7, E-cadherin, N-cadherin, Vimentin, Snail, Twist) in different treatment groups was detected by PCR experiment (*P < 0.05, **P < 0.01,***P < 0.001).

Figure 8.

In vitro anti-tumor assays. (A) In different cell lines (HeLa/SiHa), the cell proliferation activity of different treatment groups was detected at 24 h through the CCK8 experiment (*P < 0.05, **P < 0.01,****P < 0.0001). (B) The cell migration detection of HeLa and SiHa cells was observed by microscopic photography and quantitative analysis (scale bar: 100 μm, ****P < 0.0001). (C, D) The expression of protein levels (Wnt1, β-catenin, TCF7, E-cadherin, N-cadherin, Vimentin) in different treatment groups was detected by Western blot assay (*P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001). (E, F) The expression of gene levels (Wnt1, β-catenin, TCF7, E-cadherin, N-cadherin, Vimentin) in different treatment groups was detected by PCR experiment (*P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001).

Figure 9.

In vivo level anti-tumor assay. (A) Scheme of subcutaneous HeLa-Luc mouse tumor model for different treatment groups. (B) Pre-implantation, post-implantation course of Mg-Ca rods and nHAC materials after establishment of a mouse tumor model. (C) Routine blood tests for routine white blood cell, hemoglobin and platelet counts were performed on mice 0, 7 and 14 days after implantation of the materials. (D) Observe the changes in body weight of mice under the effect of different treatment groups at each time point. (E–G) Mice from different treatment groups were photographed and analyzed for fluorescence intensity in vivo imaging at 0, 7 and 14 days. (H–I) Tumors were sampled and photographed and tumor volumes were measured in mice from different treatment groups at 14 days (*P < 0.05, **P < 0.01). (J) HE staining microscopic images of vital organs in different treatment groups after tumor sampling (scale bar: 100 μm). (K) Microscopy images of HE, Tunel and Ki67 stained (HE and Ki67 scale bar: 100 μm, Tunel scale bar: 200 μm). (L) Immunohistochemical staining for Wnt1, β-catenin, TCF7 after sampling (scale bar: 50 μm). (M) Detection of E-cadherin, N-cadherin, Vimentin gene expression level by PCR (**P < 0.01).

Figure 10.

In vivo level anti-tumor assay. (A) Observe the changes in body weight of mice under the effect of different treatment groups at each time point. (B) Changes in mice tumor volume at each time point for the different treatment groups. (C) Observe photographs of mice in each group administered the drug sequentially, and perform quantitative analysis of the average tumor weight in different groups by removing tumor tissue after 14 days and taking photographs. (D–F) Perform HE, Ki67 and Tunel staining on tumor tissue (scale bar: 200 μm). (G–I) Immunohistochemistry staining of EMT markers (E-cadherin, N-cadherin and Vimentin, scale bar: 200 μm). (J–L) Immunohistochemistry staining of Wnt, β-catenin, TCF7 (scale bar: 200 μm).