Biomechanically, structurally and functionally meticulously tailored polycaprolactone/silk fibroin scaffold for meniscus regeneration

Abstract

Meniscus deficiency, the most common and refractory disease in human knee joints, often progresses to osteoarthritis (OA) due to abnormal biomechanical distribution and articular cartilage abrasion. However, due to its anisotropic spatial architecture, complex biomechanical microenvironment, and limited vascularity, meniscus repair remains a challenge for clinicians and researchers worldwide. In this study, we developed a 3D printing-based biomimetic and composite tissue-engineered meniscus scaffold consisting of polycaprolactone (PCL)/silk fibroin (SF) with extraordinary biomechanical properties and biocompatibility. We hypothesized that the meticulously tailored composite scaffold could enhance meniscus regeneration and cartilage protection. Methods: The physical property of the scaffold was characterized by scanning electron microscopy (SEM) observation, degradation test, frictional force of interface assessment, biomechanical testing, and fourier transform infrared (FTIR) spectroscopy analysis. To verify the biocompatibility of the scaffold, the viability, morphology, proliferation, differentiation, and extracellular matrix (ECM) production of synovium-derived mesenchymal stem cell (SMSC) on the scaffolds were assessed by LIVE/DEAD staining, alamarBlue assay, ELISA analysis, and qRT-PCR. The recruitment ability of SMSC was tested by dual labeling with CD29 and CD90 by confocal microscope at 1 week after implantation. The functionalized hybrid scaffold was then implanted into the meniscus defects on rabbit knee joint for meniscus regeneration, comparing with the Blank group (no scaffold) and PS group. The regenerated meniscus tissue was evaluated by histological and immunohistochemistry staining, and biomechanical test. Macroscopic and histological scoring was performed to assess the outcome of meniscus regeneration and cartilage protection in vivo. Results: The combination of SF and PCL could greatly balance the biomechanical properties and degradation rate to match the native meniscus. SF sponge, characterized by fine elasticity and low interfacial shear force, enhanced energy absorption capacity of the meniscus and improved chondroprotection. The SMSC-specific affinity peptide (LTHPRWP; L7) was conjugated to the scaffold to further increase the recruitment and retention of endogenous SMSCs. This meticulously tailored scaffold displayed superior biomechanics, structure, and function, creating a favorable microenvironment for SMSC proliferation, differentiation, and extracellular matrix (ECM) production. After 24 weeks of implantation, the histological assessment, biochemical contents, and biomechanical properties demonstrated that the polycaprolactone/silk fibroin-L7 (PS-L7) group was close to the native meniscus group, showing significantly better cartilage protection than the PS group. Conclusion: This tissue engineering scaffold could greatly strengthen meniscus regeneration and chondroprotection. Compared with traditional cell-based therapies, the meniscus tissue engineering approach with advantages of one-step operation and reduced cost has a promising potential for future clinical and translational studies.

Keywords: 3D printing; SMSC-specific affinity peptide; meniscus repair; polycaprolactone; silk fibroin.

Figure 1

Schematic illustration. (A) Fabrication and crosslinking of the scaffolds. (B) Functional optimization of the scaffolds. (C) Biocompatibility assessment in vitro. (D) Implantation in vivo.

Figure 2

Scaffold characterization. (A) Macroscopic observation of the scaffolds. (B) SEM images of the scaffolds. (C) Degradation rate of the scaffolds. (D) Interfacial shear force of the scaffolds in PBS and HA. (E) Compressive modulus of the scaffolds in vitro. (F) Tensile modulus of the scaffolds in vitro (n = 4, *p < 0.05). (G) Elastic detection of the SF scaffold showing good memory-shape characteristic and elasticity: (i+ii) initial appearance of the SF scaffold before compression, (iii) scaffolds under a compressive strain of 80%, (iv+v) returning to the original shape.

Figure 3

Crosslinking procedures of the scaffolds. (A) Proposed cross-linking mechanism of SF under γ-irradiation. (B) Conjugation of L7 peptide onto the scaffolds. (C) Secondary structure changes of SF after ethanol treatment. (D) FTIR spectra of the samples: (1) Pure silk solution, (2) SF after γ-crosslinking and ethanol processing, (3) SF-PCL after γ-crosslinking and ethanol processing, (4) Untreated PCL.

Figure 4

Biocompatibility, recruitment, and chondrogenic differentiation of SMSCs in the scaffolds in vitro and in vivo. (A) i) SMSC recruitment was verified using immunofluorescence assay after 1-week of implantation with different scaffolds in vivo. ii) Viability of SMSCs was analyzed by Live/Dead staining 3 days after seeding on different scaffolds without chondrogenic incubation. iii) Morphology of SMSCs was observed via Phalloidin/Hoechst assay after 3 days of culturing with different scaffolds without chondrogenic induction. (B) Number of CD29+/CD90+ double-positive cells on different scaffolds in vivo at 1-week post-surgery. (C) Number of effluent cells at 12 and 24 hours after SMSCs were seeded on different scaffolds in vitro. (D) Viability of SMSCs in different groups was observed by alamarBlue assay, and the OD value at each point was normalized against the average of the first day in each group. No significant difference among different groups was observed at the same time point. (E-G) Cartilaginous matrix production in different scaffolds: (E-F) Col I and Col II production quantified by ELISA; (G) GAG assay. (H-K) cartilage-specific gene expression of Col I, Col II, Sox 9, and ACAN (n = 6, *p < 0.05).

Figure 5

Macroscopic observation, biomechanical and inflammation assessment of regenerated meniscus in vivo. (A-B) Macroscopic observation of joints at 12 weeks and 24 weeks after implantation. (scale bar = 10 mm) Medial meniscal excised from the tibial plateau is shown on the right. The Blank group received no implantation after total medial meniscectomy. (C-D) Biomechanical assay of implants at each time point (12 weeks and 24 weeks) (n = 4, *p < 0.05). (E) Histological evidence of the synovium at 1, 3, 6 weeks after surgery (scale bar = 200 µm). (F) Quantitative assay of Interleukin-1 in the synovial fluid at 1, 3, 6, 12, and 24 weeks after surgery. (G) Quantitative assay of tumor necrosis factor-α in the synovial fluid at 1, 3, 6, 12, and 24 weeks after surgery. (n = 6, **p < 0.01 vs 1 week)

Figure 6

Histological assessment of outer, intermediate, and inner zones of implants and native meniscus in vivo. (A) H&E staining; (B) PR staining; (C) Immunohistochemical staining for Col I; (D) SO staining; (E) TB staining, (F) Immunohistochemical staining for Col II. (G-H) Immunohistochemical semiquantitative analyses of native meniscus and implants at 12 and 24 weeks after surgery. Values for integrated optical density (IOD) per area of (G) Col I and (H) Col II were larger in the PS-L7 group compared with the PS group, similar to the native meniscus at 24 weeks. (scale bar = 200 µm) (n = 5, *p < 0.05)

Figure 7

Histological assessment of cartilage in vivo. (A) Histological assessment (H&E, TB, and SO staining) of the femoral condyle and tibial plateau cartilage in different groups at 12 weeks and 24 weeks. (B) International Cartilage Repair Society (ICRS) and Mankin scores of articular cartilage surfaces in the femoral condyle and tibial plateau; the PS-L7 group exhibited lower cartilage degeneration in both the femur and tibia compared with the PS group or Blank group. (scale bar = 200 µm) (n = 5, *p < 0.05)

Figure 8

SEM images of the femoral condyle and tibial plateau cartilage in different groups at 12 and 24 weeks. (scale bar = 5 µm)