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. 2024 Jul;57(7):e13624.
doi: 10.1111/cpr.13624. Epub 2024 Feb 27.

MicroRNA-29c-tetrahedral framework nucleic acids: Towards osteogenic differentiation of mesenchymal stem cells and bone regeneration in critical-sized calvarial defects

Jiafei Sun  1   2 , Xingyu Chen  1   2 , Yunfeng Lin  1   2 , Xiaoxiao Cai  1   2
Affiliations

MicroRNA-29c-tetrahedral framework nucleic acids: Towards osteogenic differentiation of mesenchymal stem cells and bone regeneration in critical-sized calvarial defects

Jiafei Sun et al. Cell Prolif. 2024 Jul.

Abstract

Certain miRNAs, notably miR29c, demonstrate a remarkable capacity to regulate cellular osteogenic differentiation. However, their application in tissue regeneration is hampered by their inherent instability and susceptibility to degradation. In this study, we developed a novel miR29c delivery system utilising tetrahedral framework nucleic acids (tFNAs), aiming to enhance its stability and endocytosis capability, augment the efficacy of miR29c, foster osteogenesis in bone marrow mesenchymal stem cells (BMSCs), and significantly improve the repair of critical-sized bone defects (CSBDs). We confirmed the successful synthesis and biocompatibility of sticky ends-modified tFNAs (stFNAs) and miR29c-modified stFNAs (stFNAs-miR29c) through polyacrylamide gel electrophoresis, microscopy scanning, a cell counting kit-8 assay and so on. The mechanism and osteogenesis effects of stFNAs-miR29c were explored using immunofluorescence staining, western blotting, and reserve transcription quantitative real-time polymerase chain reaction. Additionally, the impact of stFNAs-miR29c on CSBD repair was assessed via micro-CT and histological staining. The nano-carrier, stFNAs-miR29c was successfully synthesised and exhibited exemplary biocompatibility. This nano-nucleic acid material significantly upregulated osteogenic differentiation-related markers in BMSCs. After 2 months, stFNAs-miR29c demonstrated significant bone regeneration and reconstruction in CSBDs. Mechanistically, stFNAs-miR29c enhanced osteogenesis of BMSCs by upregulating the Wnt signalling pathway, contributing to improved bone tissue regeneration. The development of this novel nucleic acid nano-carrier, stFNAs-miR29c, presents a potential new avenue for guided bone regeneration and bone tissue engineering research.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Synthesis and characterisation of stFNAs and stFNAs‐miR29c. (A) Schematic of the synthesis procedure. (B) PAGE results confirming successful synthesis (1: sS1; 2: sS1 + sS2; 3: sS1 + sS2 + sS3; 4: stFNAs; 5: miR29c and 6: stFNAs‐miR29c). (C) CE results indicating successful preparation. (D) Gel plots from CE. (E) TEM image displaying the tetrahedral structure of stFNAs. (F) AFM image showing miR29c structure. (G) Particle size and ζ potential of stFNAs. (H) Particle size and ζ potential of stFNAs‐miR29c. AFM, atomic force microscopy; TEM, transmission electron microscopy.
FIGURE 2
FIGURE 2
Biological characteristics, mechanism, and osteogenesis ability of stFNAs‐miR29c. (A) Uptake of stFNAs‐miR29c‐Cy5 and miR29c‐Cy5 by BMSCs after 12 h of treatment with stFNAs‐miR29c. (B) CCK‐8 assay results for various concentrations of stFNAs and stFNAs‐miR29c. (C) RT‐qPCR results for miR29c, Dkk1 and Beta‐catenin. (D) Western blot results for beta‐catenin and DKK1. (E) Statistical analysis of Western blot results is shown in Figure 2D. (F) Results and statistical analysis of alkaline phosphatase staining 7 days after osteogenic induction. (G) Results and statistical analysis of alizarin red staining 21 days after osteogenic induction. (H) Results and statistical analysis of oil red O staining 14 days following adipogenesis induction. ANOVA, analysis of variance; CCK‐8, cell counting kit‐8; RT‐qPCR, reserve transcription quantitative real‐time polymerase chain reaction. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 (ANOVA analysis, n ≥ 3).
FIGURE 3
FIGURE 3
Expression of osteogenesis‐specific markers in the presence of stFNAs‐miR29c. (A) Immunofluorescence staining results for ALP, OSX, and RUNX2 after 7 days of osteogenic induction. (B) Statistical analyses of fluorescence staining results for ALP. (C) Statistical analyses of fluorescence staining results for OSX. (D) Statistical analyses of fluorescence staining results for RUNX2. (E) Western blot results for ALP, OSX, RUNX2, OPN post‐osteogenic induction and PPAR‐GAMA post‐adipogenic induction. (F) Statistical analysis of Western blot results. (G) RT‐qPCR findings for Alp, Osx, Runx2, and Opn post‐osteogenic induction and Ppar‐gama following adipogenic induction. ANOVA, analysis of variance; RT‐qPCR, reserve transcription quantitative real‐time polymerase chain reaction. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, no significance (ANOVA analysis, n ≥ 3).
FIGURE 4
FIGURE 4
Micro CT analysis of CSBDs repair with stFNAs‐miR29c. (A) Three‐dimensional skull reconstruction and comparison of bone regeneration effects across groups. (B) Statistical analysis of BV/TV results. (C) Statistical analysis of Tb.N results. (D) Statistical analysis of Tb.Th results. (E) Statistical analysis of Tb.Sp results. ANOVA, analysis of variance; BV/TN, bone volume fraction; CSBD, critical‐sized bone defect; CT, computed tomography; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, no significance (ANOVA analysis, n ≥ 3).
FIGURE 5
FIGURE 5
Histologic results of CSBDs repairment under the action of stFNAs‐miR29c. (A) H&E and immunofluorescence staining images with statistical analysis results of 1‐month administrated samples. (B) H&E and immunofluorescence staining images with statistical analysis results of 2‐month administrated samples. ANOVA, analysis of variance; CSBD, critical‐sized bone defect; H&E, haematoxylin and eosin. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 (ANOVA analysis, n ≥ 3).
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