The process of reattaching bone to tendon presents considerable difficulties, especially when the enthesis is injured. The enthesis is a graded structure that joins tendon/ligament to bone, enabling efficient force transfer. It has been proven that the spatial gradients within the enthesis have a major impact on both the regulation of local cell fate and the structure's overall functionality. Simulating the complex gradient structure present in the natural enthesis is a potential approach to creating novel joint repair therapies. This study uses microfabrication techniques to create scaffolds with graded mechanical and chemical properties in an attempt to replicate the tendon-to-bone interface.
Scaffolds were fabricated as single and multi-layered structures, incorporating graded concentrations of hydroxyapatite particles (HA; 1–20% w/w, particle median diameter Dv (50) = 3.39 μm) to mimic the mineral gradient found in native enthesis tissue. Physicochemical and mechanical characterisation including SEM, wettability, surface roughness, porosity, and tensile testing were performed to evaluate the effects of HA incorporation and scaffold architecture on material performance.
Biological responses were assessed using MG-63 osteoblast like cells and MC-3T3 pre-osteoblast cells to examine cell viability, differentiation, and spatial distribution across the scaffolds through DAPI-Phalloidin staining, PrestoBlue™ and MTT assays, quantitative PCR, and SEM imaging. The findings informed the design of biomimetic multilayered graded scaffolds that integrate mechanical, chemical, and biological cues to promote bone–enthesis regeneration.
Our results indicate that the optimal multilayer scaffold configuration was composed of 0% HA- electrospinning (ES), 10% HA-ES, 20% HA-ES, and 20% HA- spin-coating (SC). Within this structure, HA concentrations of 10-20% significantly enhanced cell adhesion, elongation, and osteogenic differentiation. These results demonstrate that the fabrication of graded, multi-layered scaffolds may provide an effective strategy for replicating the native organisational complexity of the enthesis.
