PCL-PEG-PCL film promotes cartilage regeneration in vivo

Abstract

Objective: Management of chondral defects has long been a challenge due to poor self-healing capacity of articular cartilage. Many approaches, ranging from symptomatic treatment to structural cartilage regeneration, have obtained very limited satisfactory results. Cartilage tissue engineering, which involves optimized combination of novel scaffolds, cell sources and growth factors, has emerged as a promising strategy for cartilage regeneration and repair. In this study, the aim was to investigate the role of poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) PCEC scaffold in cartilage repair.

Materials and methods: First, PCEC film was fabricated, and its characteristics were tested using SEM and AFM. Cell (rASC - rat adipose-derived stem cells, and mASCs - green fluorescent mouse adipose-derived stem cells) morphologies on PCEC film were observed using SEM and fluorescence microscopy, after cell seeding. Tests of cell viability on PCEC film were conducted using the CCK-8 assay. Furthermore, full cartilage defects in rats were created, and PCEC films were implanted, to evaluate their healing effects, over 8 weeks.

Results: It was found that PCEC film, as a biomaterial implant, possessed good in vitro properties for cell adhesion, migration and proliferation. Importantly, in the in vivo experiment, PCEC film exhibited desirable healing outcomes.

Conclusions: These results demonstrated that PCEC film was a good scaffold for cartilage tissue engineering for improving cell proliferation and adhesion and could lead to excellent repair of cartilage defects.

Figure 1

The morphologies of PCEC film. (a) Morphology of the PCEC film evaluated by SEM (n=3). The topological structure of the PCEC film had no fibres, and the surface was relatively smooth and featureless. (b) Morphology of the PCEC film evaluated by AFM (n=3). The topological structure of the PCEC film was detected randomly at 10 × 10 μm. The quantified surface roughness was 585.73 nm in height

Figure 2

Characterization of PCEC film. (a) Measurement of the water contact angle wetting behaviour of a water droplet on the PCEC film (n=3), the contact angle was determined at 69.23 ± 1.5°. (b) Mechanical parameters determined from slices of the PCEC film (10.0 × 5.0 × 0.7 m³) (n=3). The PCEC film could bear the tensile force up to 6 N, and the tensile displacement reached ~3 mm before its fragmentation and the Young's modulus was approximately 349.04519 MPa and the elongation at break was 3.07%

Figure 3

Architectural characteristics of the PCEC film elucidated by the morphology of ASCs from green fluorescent mice, as shown by microscope observation, green fluorescent protein‐positive mASCs were seeded on the PCEC film at different days. The Petri dish group showed the normal morphologies of cultured green fluorescent protein‐positive mASCs at different days (n=3). By 5 d after seeding, the cells attached to the PCEC film, significantly increased in numbers, in comparison to the other group

Figure 4

In vitro cell behaviour of the PCEC film. (a) Cell morphologies of mASCs on the PCEC film for implantation, as shown by SEM (n=3). mASCs were seeded, attached, spread and proliferated within 5 d. (b) Cell proliferation of mACSs on the PCEC film and Petri dishes (n=3). The results show that the proliferation rates within 5 d were significantly higher on the PCEC films than that found on the Petri dishes

Figure 5

HE staining of cross‐sections of repaired knee articular cartilage at 4 and 8 wk. HE staining showed the interfaces between the peri‐native tissues and new‐formed tissues in all the groups. The control group (8 wks) showed a reduced defect area but did not form repair morphology. Among the implant groups, the interfaces were more distinct at 4 wks than at 8 wks post‐surgery. At 8 wks, repaired newly formed tissue gradually covered the implanted films to form an integral cartilage‐like tissue. The experiments were repeated three times (n=3)

Figure 6

Safranin O staining of cross‐sections of repaired knee articular cartilage at 4 and 8 wk. One of the most important markers for evaluating cartilage repair is the assembly of proteoglycan. The internal repair integrity was shown in scaffold‐implanted groups by Safranin O staining and was especially clear in the implant group. We found that the control group (8 wk) showed a reduced defect area but did not form repair morphology. Among the implant groups, at 4 wk post‐surgery, the newly formed tissue showed a light colour relative to the original tissue, although the newly formed tissue showed the component of proteoglycan. At 8 wk, the newly formed tissue completely covered the implant material and formed an integral surface with a thickness of ~400 mm. However, the PCEC films were embedded in the deep defects and did not degrade. The experiments were repeated three times (n=3)

Figure 7

Type‐II collagen staining of cross‐sections of repaired knee articular cartilage at 4 and 8 wk. Type‐II collage is another important marker used to evaluate the cartilage repair. The interfaces between peri‐native and newly formed tissue were distinct among control and implant group. We found that the control group (8 wk) showed a reduced defectarea but did not form repair morphology. Among the implant groups, the interfaces between the peri‐native tissue and newly formed tissue were distinct at 4 wk. At the top of the indentation of the cartilage defects, a thin layer of type‐II collagen containing new tissue was formed and this newly formed tissue did not form an integral surface. At 8 wk, the original tissue and newly formed tissue were fused and an integral surface was formed. The experiments were repeated three times (n=3)