Articular fibrocartilage-targeted therapy by microtubule stabilization

Abstract

The fibrocartilage presented on the joint surface was caused by cartilage injury or degeneration. There is still a lack of effective strategies for fibrocartilage. Here, we hypothesized that the fibrocartilage could be viewed as a raw material for the renewal of hyaline cartilage and proposed a previously unidentified strategy of cartilage regeneration, namely, "fibrocartilage hyalinization." Cytoskeleton remodeling plays a vital role in modifying the cellular phenotype. We identified that microtubule stabilization by docetaxel repressed cartilage fibrosis and increased the hyaline cartilage extracellular matrix. We further designed a fibrocartilage-targeted negatively charged thermosensitive hydrogel for the sustained delivery of docetaxel, which promoted fibrocartilage hyalinization in the cartilage defect model. Moreover, the mechanism of fibrocartilage hyalinization by microtubule stabilization was verified as the inhibition of Sparc (secreted protein acidic and rich in cysteine). Together, our study suggested that articular fibrocartilage-targeted therapy in situ was a promising strategy for hyaline cartilage repair.

Fig. 1.. Schematic of fibrocartilage hyalinization and the role of fibrocartilage in synovial joint.

(A) Schematic illustration of fibrocartilage hyalinization. The key is the modification in situ of FCs and fibril ECM. (B) Representative hematoxylin and eosin staining (H&E), Safranin O (SO)–Fast Green, and immunohistochemical staining for Col I and Col II of the fibrocartilage in cartilage defect (2, 4, and 6 weeks after surgery) and the control (sham operation) groups. Scale bar, 500 μm. (C) Quantification of the ratio of the positive area of Col II to Col I based on (B) (n = 3). (D) Immunofluorescent staining for ace-tubulin of the fibrocartilage in cartilage defect (2, 4, and 6 weeks after surgery) and the control (sham operation) group. Scale bar, 200 μm. Enclosed areas are enlarged in below panels. (E) Quantification of the ace-tubulin–positive cells based on (D) (n = 3). (F) SO staining and immunohistochemical staining for COL I, COL II, and MMP3 of the human OA cartilage (uninjured and injured cartilage). Scale bar, 500 μm. Enclosed areas are enlarged in below panels. Data are presented as the means ± SD.

Fig. 2.. The identification of CTGF- and TGF-β1–induced fibrocartilage chondrocytes.

(A) Schematic illustration of the induction of FCs via CTGF and TGF-β1. The rat BMSCs were applied with CTGF (100 ng/ml) and TGF-β1 (10 ng/ml) for 1 week, respectively, to induce FCs. (B) Heatmap representing differential expression genes of cells (BMSC_C+T versus BMSC_Ctrl). Fibrosis-related genes were increased, including Col1a1, Col1a2, Col3a1, Fmod, and Fndc1, and cartilage protective genes were decreased, including Frzb, Fgfr3, and Fgf2. (C) GSEA showing the enrichment of pathways between the BMSC treatment with or without CTGF and TGF-β1 treatment. NES, normalized enrichment score. (D) Western blot analysis of ace-tubulin, Sox9, Col II, Col I, Col III, and α–smooth muscle actin (α-SMA) in BMSC treated with or without CTGF and TGF-β1 treatment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Co-immunofluorescent staining of α-SMA and F-actin in BMSC treated with or without CTGF and TGF-β1 treatment. Enclosed areas are enlarged in right panels. Scale bar, 100 μm. Quantification of the α-SMA–positive cells (n = 6). (F) Co-immunofluorescent staining of ace-tubulin and F-actin in BMSC treated with or without CTGF and TGF-β1 treatment. Enclosed areas are enlarged in right panels. Scale bar, 100 μm. Quantification of the ace-tubulin–positive cells (n = 6). (G) Peak graphs representing the flow cytometric analysis for CD44, CD34, CD90, and CD45 in BMSC treated with or without CTGF and TGF-β1 treatment. Data are represented as the means ± SD. ***P < 0.001.

Fig. 3.. Microtubule stabilization inhibits fibrosis and improves chondrogenesis of induced fibrocartilage chondrocytes.

(A) Schematic illustration of the treatment for induced fibrocartilage chondrocytes (iFCs) with docetaxel (D) (2.5 nM) to stabilize microtubule of cells. (B) Heatmap representing differential expression genes of cells (D versus vehicle). The fibrosis related genes, including Fmod, Col1a1, Col3a1, and Fndc1 were inhibited and the cytoskeleton related genes, including Acta2, Tuba1a, Tuba4a, and Tubb2b were increased by D treatment. (C) GSEA showing the enrichment of pathways between the iFCs with or not with D. (D) Western blot analysis of ace-tubulin, Sox9, Col II, Col I, Col III, and α-SMA in iFCs treated with or without D treatment. (E) Co-immunofluorescent staining of ace-tubulin and F-actin in iFCs treated with or without D treatment. Enclosed areas are enlarged in right panels. Scale bar, 100 μm. Quantification of the ace-tubulin–positive cells (n = 6). (F) Co-immunofluorescent staining of α-SMA and F-actin in iFCs treated with or without D treatment. Enclosed areas are enlarged in right panels. Scale bar, 100 μm. Quantification of the α-SMA–positive cells (n = 6). (G) SO staining and Alcian blue (AB) for the micro-mass culture of iFCs with or without D treatment. Scale bars, 200 μm. (H) Macroscopic image, SO staining, and toluidine blue (TB) staining for the pellet culture of iFCs with or not with D treatment. Scale bars, 200 μm. Microtubule stabilization improved the iFCs to produce the hyaline cartilage–like ECM. Data are represented as the means ± SD. ***P < 0.001.

Fig. 4.. Characterization of thermosensitive docetaxel loaded injectable hydrogel.

Schematic illustration showing (A) the synthesis of thermosensitive docetaxel loaded injectable hydrogel (D-PDP@MC-Gel) and (B) the application of D-PDP@MC-Gel for fibrocartilage modification in situ. PLGA, poly(lactic-co-glycolic acid). (C) Immunohistochemical staining for transferrin of the fibrocartilage in cartilage defect (2, 4, and 6 weeks after surgery) and the control (sham operation) group. Scale bar, 200 μm. (D) Photographs of D-PDP@MC-Gel and the gel without docetaxel (PDP@MC-Gel) at 4 and 37°C. (E and F) Rheological test on D-PDP@MC-Gel and PDP@MC-Gel under different temperature. (G) Zeta potentials of D-PDP@MC-Gel and PDP@MC-Gel. (H) Images of fluorescence captured from the cartilage after the injection of FITC-PDP@MC-Gel 1 and 7 days. (I) In vitro drug concentrations of D-PDP@MC-Gel and D-PDP in saline at 37°C. The amount of D remaining in the solution released from the D-PDP and D-PDP@MC-Gel.

Fig. 5.. The effect of thermosensitive docetaxel loaded injectable hydrogel in fibrocartilage in vivo.

(A) Overview of animal experiments. (B) Macroscopic appearance and micro-CT 3D reconstruction of the fibrocartilage in the cartilage defect treated with D-PDP@MC-Gel and PDP@MC-Gel. White scale bar, 2 mm. Enclosed areas are enlarged in below panels. (C) H&E and SO staining of the fibrocartilage in the cartilage defect treated with D-PDP@MC-Gel and PDP@MC-Gel. Enclosed areas represent the middle, left and right of cartilage defect and are enlarged in below panels. Scale bar, 100 μm. (D) Immunofluorescent staining of ace-tubulin of the fibrocartilage in the cartilage defect treated with D-PDP@MC-Gel and PDP@MC-Gel. Enclosed areas are enlarged in below panels. Scale bar, 100 μm. (E) Histological ICRS evaluation of cartilage regeneration based on (C) (n = 5). ICRS, International Cartilage Repair Society. (F) Quantification of ace-tubulin–positive cells in the fibrocartilage in the cartilage defect (n = 5). (G to K) Immunohistochemical staining for Col I (G), Col II (H), FMOD (J), and aggrecan (K) of the fibrocartilage in the cartilage defect treated with D-PDP@MC-Gel and PDP@MC-Gel. Enclosed areas are enlarged in below panels. Scale bars, 100 μm. (I to M) Quantification of the ratio of Col II to Col I of (G) and (H), the Fmod-positive cells of (J), and the aggrecan-positive cells of (K) (n = 5). Data are represented as the means ± SD. ***P < 0.001.

Fig. 6.. The effect of microtubule stabilization on the fibrocartilage in human OA.

(A) Schematic illustration showing the experiment for microtubule stabilization on the human OA fibrocartilage. The fibrocartilage was harvested from the femoral condyle of OA patients and then divided it into two parts and cultured ex vivo for D treatment for 1 weeks and control. (B) Immunofluorescent staining for ace-tubulin in the cartilage ex vivo with or without D treatment. Enclosed areas are enlarged in below panels. Scale bars, 200 μm. (C) Quantification of ace-tubulin–positive cells of (B) (n = 4). (D) Western blot analysis for ace-tubulin, SOX9 and COL II of the cartilage ex vivo with or not with D treatment. (E) Quantification of data D (n = 6). (F and H) Immunohistochemical staining for COL I, COL II, FMOD, and AGGRECAN in the cartilage ex vivo with or without D treatment. Enclosed areas are enlarged in right panels. Scale bars, 500 μm. (G to I) Quantification of COL I– and COL II– (G) positive cells of (F) and FMOD- and AGGRECAN-positive (I) cells of (H) (n = 5). Data are represented as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7.. The mechanism of Sparc in the fibrocartilage hyalinization.

(A) PPIs of differentially expressed genes. Left: Fibrocartilage chondrocytes introduction. Right: D treatment. (B) Western blot analysis for Sparc in the BMSC with or without CTGF and TGF-β1 treatment (B) and in the iFCs with or without D (C). (C) Quantification of (B) (n = 6). Immunofluorescent staining for Sparc in the BMSC with or without CTGF and TGF-β1 treatment (D) and in the iFCs with or without D (F). Enclosed areas are enlarged in right panels. Scale bar, 100 μm. Quantification of Sparc-positive cells of (D) (E) and (F) (G) (n = 6). (H) Western blot analysis for ace-tubulin, Sox9, Col II, Col I, Col III, α-SMA, and Sparc in the BMSCs under the induction of CTGF and TGF-β1 with or without the transfection of siRNA for Sparc knockdown. (I) Quantification of data H (n = 6). ns, not significant. (J) Immunofluorescent staining for Sparc of the fibrocartilage during the cartilage repair. Enclosed areas are enlarged in below panels. Scale bar, 100 μm. Quantification of Sparc-positive cells in the fibrocartilage in the cartilage defect (n = 3). (K) Immunofluorescent staining for Sparc of the fibrocartilage in the cartilage defect treated with D-PDP@MC-Gel and PDP@MC-Gel. Enclosed areas are enlarged in below panels. Scale bar, 100 μm. Quantification of Sparc-positive cells in the fibrocartilage in the cartilage defect (n = 5). (L) Western blot analysis for Sparc of the cartilage ex vivo with or without D treatment. (M) Quantification of the Western blot analysis (n = 6). Data are represented as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 8.. Schematic of articular fibrocartilage-targeted therapy by microtubule stabilization.

Fibrocartilage was modified in situ by microtubule stabilization via docetaxel loaded hydrogel. The transformation of fibrocartilage toward hyaline cartilage by microtubule stabilization was through regulating Sparc. HyC, Hyaline chondrocytes.