Functionally graded scaffold with M2 macrophage-derived LncRNA-Encoded peptide: Mechanistic and therapeutic evaluation for rotator cuff repair

Figures

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  Fig. 1. Systematic illustration of the preparation and implantation of the MDP1&HA-loaded scaffold. (a) Structural prediction and biochemical coefficient of the peptide derived from lncRNA MM2P. (b) Preparation of corresponding nanofiber yarns from different electrospinning solutions, followed by woven into scaffolds. (c) Establishment of rotator cuff tear model and regulation of tendon-bone interface at cellular level by MDP1 and HA.

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  Fig. 2. Characterization of nanofiber yarns and scaffolds. (a) SEM image of nanofiber yarn: PLCL/SF (PS), PLCL/SF/MDP1 (PS-M) and PLCL/SF/HA (PS-HA). (b–c) Diameter distribution of nanofiber and comparison between groups. (d) Thermogravimetric analysis of yarns. (e) In vitro degradation of scaffolds for up to 12 weeks. (f) Cumulative release of MDP1 grafted with Rhodamine B for up to 28 days. (g) Cumulative release of HA for up to 28 days. (h) FTIR spectra of PLCL, SF, HA, and scaffolds. (i) Water contact angle of scaffolds. (j) Mechanical properties of nanofiber yarns. (k) Mechanical properties of scaffolds. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).

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  Fig. 3. Role of MDP1 in promoting macrophages polarization. (a–b) Flow cytometric analysis of RAW264.7 macrophages stimulated with the extract solution of PS-M scaffolds and lipopolysaccharide (LPS) containing medium. (c) Expression levels of IL-4 and TNF-α of RAW264.7 macrophages using qPCR. (d–e) Changes in the levels of macrophage-related transcription factors in RAW264.7 macrophages. (f–h) DCFH fluorescence staining assay, relative quantitative analysis and NO clearance rate of RAW264.7 macrophages. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).

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  Fig. 4. Cytocompatibility of scaffolds in vitro. (a) Proliferation of RAW264.7 macrophages, tenocytes, fibrochondrocytes, and rBMSCs seeded on scaffolds in vitro. (b) Live/dead staining of tenocytes cultured along with extract solution of different types of scaffolds for up to 5 days. (c) Preparation of conditional culture medium. (d–g) Immunofluorescence (IF) staining of COL I (green), nuclei (blue) and cytoskeletal staining (red) and quantitative analysis of tenocytes cultured for 10 days. (h) Relative expression levels of COL I, SCX, and TNC in tenocytes analyzed by qPCR. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).

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  Fig. 5. Effects of scaffolds loaded with different concentrations of MDP1 on fibrochondrocytes. (a) Preparation of conditional culture medium. (b) Relative expression levels of SOX9, AGG, and collagen type II (COL II) in fibrochondrocytes analyzed by qPCR. (c–e) Safranine O, Alcian blue staining and quantitative analysis of fibrochondrocytes cultured for 14 days in conditional culture medium of scaffolds. (f–i) IF staining of COL II (green), nuclei (blue), and cytoskeletal filaments (red) and quantitative analysis of fibrochondrocytes cultured for 14 days in conditional culture medium. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).

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  Fig. 6. Effect of MDP1 and HA on osteogenic differentiation of rBMSCs. (a) Preparation of conditional culture medium containing osteogenic induction components. (b) Relative expression levels of osteogenic-related genes, such as RUNX2, OPN, and OCN in rBMSCs, analyzed by qPCR. (c–d) Transwell migration assay of rBMSCs cultured for 12 h and its quantitative analysis. (e–f) ARS staining and relative quantification of rBMSCs. (g–i) Macroscopic image of ALP staining, magnified image (10x), and absolute quantification of rBMSCs cultured for 10 days. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).

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  Fig. 7. Whole transcriptome RNA sequencing analysis of tendon and bone tissue at the fourth month. (a–b) Heatmap of upregulated and downregulated muscle-tendon-related genes and volcano plot of differentially expressed genes in the control group and the MDP1&HA scaffold group. (c–e) Gene Ontology (GO) enrichment analysis showing top 15 upregulated and downregulated terms along with a partial GO chord plot. (f–g) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis presenting the top 5 upregulated and downregulated pathways (h–i) Heatmap of upregulated and downregulated bone-related genes and volcano plot of differentially expressed genes in the control group and the MDP1&HA scaffold group. (j–l) GO enrichment analysis displaying the top 15 upregulated and downregulated terms, accompanied by a partial GO chord plot. (m–n) KEGG enrichment analysis illustrating the top 6 upregulated and downregulated pathways.

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  Fig. 8. Histological analysis of tendon-bone interface repair in New Zealand rabbits. (a) Schematic illustration of rotator cuff tear model. (b) Hematoxylin and eosin (H&E) staining of different groups 2 months (2M) and 4 months (4M) post-operatively. (c) Masson's trichrome staining of all groups at 2M and 4M. (d) Safranin O/Fast Green staining of all groups at 2M and 4M and (e) Picro-Sirius Red staining under polarized light of all groups at 2M and 4M. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).

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  Fig. 9. Anti-Inflammatory efficacy and biomechanical evaluation of rotator cuff tear repair. (a) Quantitative analysis of COL I with Picro-Sirius Red staining. (b, h) Representative immunohistochemical (IHC) staining of CD206 and corresponding quantitative analysis of all groups at 2M and 4M. (c, i) IHC staining of IL-6 and quantitative analysis of all groups at 2M and 4M. (d, j) IF staining of COL II (green), COL I (red), and DAPI (blue) with quantitative evaluation of COL II. (e–f) Rotator cuff tissue with scaffold and biomechanical assay. (g) Biomechanical properties of rotator cuff tear model. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (n = 3).