Zone-specific collagen alignment in porous polymer scaffolds for articular cartilage tissue engineering

摘要

Introduction: Articular cartilage is organized into distinct functional layers characterized by varying stiffnesses, collagen alignments, and chondrocyte phenotypes. We have designed a poly(ε-caprolactone) scaffold, which incorporates the structural and mechanical anisotropy of the native tissue in a manner superior to our previous anisotropic designs'. The design integrates a fibrous superficial zone that provides tensile strength, an isotropic foam intermediate zone, and a stiff vertically aligned deep zone, while maintaining full interconnectivity, mechanical fusion, and high levels of porosity. Materials and Methods: Scaffolds were produced from 80kDa poly(ε-caprolactone) by a novel combination of scaffold production techniques, producing a 4-zone cartilage scaffold 2 mm in thickness. Porogen leaching and directional freezing were used in concert to produce a foam with 100-300 μm spherical pores and 50 μm diameter channels. Electrospun fibres were deposited on the upper and lower surfaces of the scaffold with zone-specific orientation. Osteochondral constructs were produced for in vivo implantation through the fusion of a 4-mm-thick 3D-melt electrospun bone scaffold to the underside of the cartilage scaffold. Scaffolds were validated in vitro with primary bovine chondrocytes (passage 2) up to 12 weeks under chondrogenic conditions (10 ng/ml TGF-β3). Results and Discussion: The anisotropic scaffolds were mechanically fused and interconnected at all interfaces. Scaffolds exhibited intermediate and deep zone Young's moduli of 38 ± 6 kPa and 2.0 ± 0.7 MPa, respectively, at 10% strain. The stiffnesses mimic those within the physiological range and result in a bulk modulus of 640 ± 40 kPa. Scaffolds were validated in vitro, exhibiting full cellular penetration from a single seeding injection. Seeded cells exhibited chondrocyte-specific gene expression and glycosaminoglycan and collagen Ⅱ production sufficient to fully infiltrate the scaffold pores. The ECM produced in vitro was analyzed by multi-photon microscopy and found to possess differential zonal alignment, Mature collagen fibrils with parallel alignment were produced at the superficial zone, random alignment in the intermediate zone, and perpendicular alignment to the articulating surface in the deep zone. A preliminary in vivo validation of 6 mm Ø acellular scaffolds in a skeletally mature porcine osteochondral defect model indicates integration and full tissue penetration at 3 months. Additional 6-month in vivo investigations of acellular and allogeneic-chondrocyte seeded scaffolds are ongoing. Conclusion: To our knowledge this is the first multi-layered porous polymer cartilage scaffold to possess an interconnected gradient of physiologically-relevant stiffnesses and induce zonal collagen alignment comparable to the native tissue.