Injectable decellularized Wharton's jelly hydrogel containing CD56+ umbilical cord mesenchymal stem cell-derived exosomes for meniscus tear healing and cartilage protection

Abstract

Traditional meniscectomy or suture for meniscal tear usually leads to failed self-healing, cartilage degeneration and worse osteoarthritis. The strategies that facilitate the healing process of torn meniscus and safeguard knee cartilage against degeneration will be promising for clinical therapy. The CD56⁺ umbilical cord mesenchymal stem cells (UCSCs) (CD56⁺UCSCs) were sorted from Wharton's jelly using flow cytometer. Then, the modified decellularized Wharton's Jelly hydrogel (DWJH) was combined with isolated CD56⁺Exos from CD56⁺UCSCs to fabricate DWJH/CD56⁺Exos. The in vitro studies were performed to characterize the DWJ (decellularized Wharton's Jelly). The injectability and rheological properties were assessed by shear rate and frequency sweep analysis. The biocompatibility and chondrogenic differentiation inducibility of DWJH/CD56⁺Exos were performed on human bone marrow mesenchymal stem cells (hBMSCs) and RAW 264.7 cells. The release dynamics was evaluated in vitro and in vivo experiments. As for the in vivo experiments, the operated rats that subjected to a 2 mm full-thickness longitudinal tear in right medial anterior meniscus were injected a single dose of DWJH/CD56⁺Exos. At 4 and 8 weeks postoperatively, torn meniscus healing and articular cartilage degeneration were evaluated by hematoxylin and eosin (H&E), safranin O/fast green (SO&FG), and Sirius red staining. In in vitro experiments, the injectable DWJH/CD56⁺Exos demonstrated excellent biocompatibility, exosome releasing efficiency, injectable property and chondrogenic inducibility. The results of in vivo experiments revealed that DWJH/CD56⁺Exos degraded over time, promoted meniscal chondrogenesis, organized meniscal extracellular matrix remodeling, safeguard articular cartilage and inhibited secondary cartilage degeneration, which accelerated further facilitated torn meniscus healing. The novel injectable DWJH/CD56⁺Exos promoted meniscal tear healing by promoting meniscal chondrogenesis, safeguarding articular cartilage, and inhibiting secondary cartilage degeneration.

Keywords: CD56; Decellularized Wharton's jelly hydrogel; Exosome; Meniscal tear; Umbilical cord mesenchymal stem cell.

Graphical abstract of the experimental process.

Fig. 1

Preparation and characterization of CD56⁺UCSC-Exos. (A) Flow cytometry-based sorting of CD56⁺UCSC population. (B) Morphological assessment and evaluation of osteogenic, lipogenic, and chondrogenic differentiation potential in CD56⁺UCSCs. Scale bar: 100 μm. (C) Flow cytometric analysis of cellular markers expressed in CD56⁺UCSCs. (D) Identification of exosomes using transmission electron microscopy (TEM) and (E) nanoparticle tracking analysis (NTA). Scale bar: 100 nm. (F) Western blotting analysis to determine the presence of exosomal biomarkers. (G) Immunofluorescence imaging reveals internalization of CD56⁺UCSC-Exos or UCSC-Exos by hBMSCs. Scale bar: 50 μm.

Fig. 2

Evaluation of hBMSC differentiation induced by CD56⁺UCSC-Exos was conducted. (A) Histochemical identification using Alcian Blue staining was performed to assess chondrogenic induction of hBMSC by UCSC-Exos or CD56+UCSC-Exos. Scale bar: 100 μm. (E) Statistical analysis was carried out based on the results obtained in A. (B) Immunofluorescence staining for chondrogenic marker SOX9 was conducted and (F) quantification of positive cells was performed. (n = 3). Scale bar: 50 μm. (C) Transwell assay results demonstrated the cytocompatibility and chemotaxis differences between UCSC-Exos and CD56+UCSC-Exos (n = 3). Scale bar: 100 μm. (G) The graph illustrates disparities in cell migration between UCSC-Exos and CD56⁺UCSC-Exos. Scale bar: 100 μm. (D) qRT-PCR analysis revealed the expression levels of key markers, including SOX9, COL10, and ANCN during hBMSC differentiation (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Characterization of decellularized Wharton jelly. (A) Biophysical features were evaluated through macroscopic images, HE, DAPI, SEM, and SR-FTIR mappings. Scale bar: 100 μm. (B) Semi-quantitative analysis was performed to determine the collagen and proteoglycan contents in native or decellularized Wharton jelly samples. Comparative graph showing the quantitative analysis of proteoglycan (C), collagen (C) and DNA content (D) between native and decellularized Wharton jelly. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 4

Characterization and cytocompatibility of DWJH/CD56⁺Exo were assessed using various techniques. (A) Macroscopic observation and SEM imaging of DWJH/CD56⁺Exo. Scale bar: 10 μm. (B) The injectability of DWJH/CD56⁺Exo. (C) Cytotoxicity analysis of DWJH/CD56⁺Exo was assessed using the CCK-8 assay (n = 4). (D) Fluid dynamic characterization of DWJH/CD56⁺Exo. (E) Live/dead double staining and 3D reconstruction were performed to visualize cell viability (green: live cells, red: dead cells) (n = 4). (F) Statistical analysis of the proportion of live cells in E. (G) Pro-inflammatory cytokine (TNF-α, IL-6, and IL-1β) release between control, DWJH/CD56⁺Exo, and LPS groups after coculture for 3 days, Bar = 100 μm. (H) Representative RAW 264.7 morphology after coculture for 3 days in control, DWJH/CD56⁺Exo, and LPS groups. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Evaluation of hBMSC differentiation induced by DWJH/CD56⁺Exos and sustained release of CD56⁺Exos in DWJH/CD56⁺Exos. (A) Histochemical identification using Alcian Blue staining was performed to assess chondrogenic induction of hBMSC by DWJH/CD56⁺Exos. Scale bar: 100 μm. (E) Statistical analysis was carried out based on the results obtained in A. (B) Immunofluorescence staining for chondrogenic marker SOX9 and (F) quantification of positive cells. (n = 3). Scale bar: 50 μm. (C) Western blot of SOX9 in hBMSC treated with DWJH, CD56⁺Exos, or DWJH/CD56⁺Exos. (F) Quantification of SOX9, COL2, and COLX relative protein expression between different groups in control, DWJH, CD56⁺Exos, and DWJH/CD56⁺Exos groups. n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Sustained release of CD56⁺Exos and degradation of DWJH/CD56⁺Exos. (A) In vitro fluorescence of controlled release of CD56⁺Exos in DWJH/CD56⁺Exos. (B) In vivo fluorescence tracking analysis demonstrated that DiR-labeled CD56⁺Exos were delivered to the targeted area. (C) Semi-quantification analysis between the DWJH/CD56⁺Exos group and CD56⁺Exos group. (D) Release curves of CD56⁺Exos from DWJH/CD56⁺Exos at different time points. n = 5. (E) The gross view and (F) H&E staining of DWJH/CD56⁺Exos after implantation at 30 min, 3, 7, 14, and 21 days. Yellow circles denote hydrogel bumps after injection. T: tissue; H: hydrogel. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Histological evaluation of meniscal tear healing. Histological analysis of native meniscus and repaired meniscal at postoperative weeks 4 and 8, including (A) hematoxylin and eosin (H&E) staining as well as (B) safranin O/fast green (SO&FG), and (C) Sirius red staining. (D) Normalized tear area after meniscus tear repair. Data are presented as mean ± SD (n = 6). Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Histological assessment of joint cartilage degeneration was performed using hematoxylin and eosin (H&E) (A) staining as well as safranin O/fast green (SO&FG) (B) staining on sections obtained at 4 and 8 weeks postoperatively, with the femoral condyle and tibial plateau depicted above and below, respectively. Additionally, the articular cartilage surfaces in the femoral condyle and tibial plateau were evaluated using both International Cartilage Regeneration & Joint Preservation Society (ICRS) scores(C) and Mankin scores (D) (n = 6). Scale bar: 500 μm ∗P＜0.05; ∗∗P＜0.01; ∗∗∗P＜0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)