Experimental study on synergistic promotion of cartilage differentiation by microporous hydrogel combined with dual cytokines

doi:10.3877/cma.j.issn.1674-134X.2025.05.006

Abstract

Abstract:

Objective

To explore the effect of the combined application of transforming growth factor-β3 (TGF-β3) and stromal cell-derived factor 1α (SDF-1α) with microporous hydrogel on the repair of cartilage injury.

Methods

TGF-β3 was transfected into rat bone marrow mesenchymal stem cells (BMSCs) by adenovirus to obtain bone marrow mesenchymal stem cells carrying the TGF-β3 gene (Ad-BMSCs). Genipin crosslinked gelatin microspheres (GMs) were infiltrated into cytokine SDF-1α solution to obtain GMs containing SDF-1α (GMs-S). Then the Ad-BMSCs and BMSCs were suspending in alginate solution, respectively, and mixed with GMs-S, to obtain two groups of hydrogel microspheres (HMs) after calcium in crosslinking reaction as group S+T (containing SDF-1α and TGF-β3) and group S (containing only SDF-1α). Similarly, these two cells alginate solution mixed with GMs respectively to obtain two groups of gelatin microsphere hydrogel as group T (containing TGF-β3) and the blank group.The HMs of each group were cultured in cell incubators under the same conditions,and cell proliferation was detected by cell counting kit-8 assay (CCK-8), the release of SDF-1α and/or TGF-β3 were detected by enzyme-linked immunosorbent assay (ELISA). The gene expression of type I collagen, type Ⅱ collagen, type X collagen and aggrecan (ACAN) were detected by polymerase chain reaction assay (PCR), and expression of sex related Y-box transcription factor 9 (SOX9) protein was detected by western blotting analysis (WB). All the tests in this study were repeated for three times; the quantitative data were described as ±s, t test was used to compare the difference between two groups, and single factor variance analysis was applied for multipe groups comparison. P<0.05 indicated the difference was statistically significant.

Results

CCK-8 data indicated that group S+T had an outstanding ability of cell growth (F=6.904, R²=0.533, P<0.05). SDF-1α (P>0.05) and TGF-β3 (P>0.05) release curves of group S+T and group S were generally consistent. Other results demonstrated that group S+T had greater expression of typeⅡcollagen (comparing the blank group and the group S with the group S+T, P<0.05) and aggrecan (comparing the blank group and the group S with the group S+T, P<0.05) and more synthesized cartilaginous extracellular matrix. Moreover, the WB analysis suggested that the expression of SOX9 protein in group S+T was the highest (each group was compared with the group S+T, P<0.05).

Conclusion

Synergistic effect of SDF-1α and TGF-β3 on BMSCs cultured in microcavitary hydrogel can improve the effect of chondrogenesis.

Key words: Chemokine CXCL12, Transforming growth factor beta3, Chondrocytes, Hydrogels, Alginates, Gelatin

Yuyang Huang, Yongsheng Li, Cheng Luo, Yijun He, Qian Pan. Experimental study on synergistic promotion of cartilage differentiation by microporous hydrogel combined with dual cytokines[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2025, 19(05): 560-569.

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URL: https://zhgjwkzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-134X.2025.05.006

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Figures/Tables 10

Figure 1 Experimental microsphere culture groupsNote: S+T-containing the SDF-1α factor and the TGF-β3 factor; T- containing the TGF-β3 factor; S- containing the SDF-1αfactor

Figure 4 Views of BMSCs under the inverted phase-contrast microscope after adenovirus transfection (scale = 100 μm). A shows BMSCs under the bright field; B shows the fluorescence image of the cells 48 h after adenovirus transfection with the TGF-β3 gene

Figure 5 The survival status of BMSCs in microporous hydrogel. A shows the bright-field image of BMSCs in the microporous hydrogel; B shows the fluorescence image of BMSCs in the microporous hydrogel (scale 100 μm)

Figure 2 Primer sequences of target genes of BMSCs used for Q-PCRNote: COL I-type I collagen; COLⅡ-TypeⅡcollagen; COL X-type X collagen; ACAN-aggrecan; GAPDH- glyceraldehyde-3-phosphate dehydrogenase

Figure 3 Microscopic views of gelatin microspheres in dry state and swollen state(scale 100 μm). A is the cross-linked gelatin microspheres in the dry state; B is the cross-linked gelatin microspheres in the swollen state

Figure 6 Curves showing the proliferation activity of cells in each experimental groupNote: S+T- containing SDF-1α and TGF-β3; T- containing TGF-β3; S- containing SDF-1α

Figure 7 Release curves of SDF-1α in the group S+T and the group SNote: S+T- containing SDF-1α and TGF-β3; S- containing SDF-1α

Figure 8 Release curves of TGF-β3 in the group S+T and the group SNote: S+T- containing SDF-1α and TGF-β3; T- containing TGF-β3

Figure 9 Expressions of cartilage-related genes in different hydrogel-microsphere groups at day 28. A is the expression of type I collagen gene; B is the expression of type X collagen gene; C is the expression of type II collagen gene; D is the expression of ACAN geneNote: ^*-P<0.05; S+T- containing SDF-1α and TGF-β3; T- containing TGF-β3; S- containing SDF-1α

Figure 10 Expression of SOX9 protein in different hydrogel-microsphere groups at day 14. A represents the protein band; B is the bar graph obtained after Image J analysisNote: ^*-P<0.05; S+T- containing SDF-1α and TGF-β3; T- containing TGF-β3; S- containing SDF-1α

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