Three-dimensional printed multiphasic scaffolds with stratified cell-laden gelatin methacrylate hydrogels for biomimetic tendon-to-bone interface engineering

Background
The anatomical properties of the enthesis of the rotator cuff are hardly regained during the process of healing. The tendon-to-bone interface is normally replaced by fibrovascular tissue instead of interposition fibrocartilage, which impairs biomechanics in the shoulder and causes dysfunction. Tissue engineering offers a promising strategy to regenerate a biomimetic interface. Here, we report heterogeneous tendon-to-bone interface engineering based on a 3D-printed multiphasic scaffold.

Methods
A multiphasic poly(ε-caprolactone) (PCL)–PCL/tricalcium phosphate–PCL/tricalcium phosphate porous scaffold was manufactured using 3D printing technology. The three phases of the scaffold were designed to mimic the graded tissue regions in the tendon-to-bone interface—tendon, fibrocartilage, and bone. Fibroblasts, bone marrow–derived mesenchymal stem cells, and osteoblasts were separately encapsulated in gelatin methacrylate (GelMA) and loaded seriatim on the relevant phases of the scaffold, by which a cells/GelMA-multiphasic scaffold (C/G-MS) construct, replicating the native interface, was fabricated. Cell proliferation, viability, and chondrogenic differentiation were evaluated in vitro. The C/G-MS constructs were further examined to determine the potential of regenerating a continuous interface with gradual transition of teno-, fibrocartilage- and osteo-like tissues in vivo.

Results
In vitro tests confirmed the good cytocompatibility of the constructs. After seven days in culture, cellular microfilament staining indicated that not only could cells well proliferate in GelMA hydrogels ​but also efficiently attach to and grow on scaffold fibres. Moreover, by immunolocalizing collagen type II, chondrogenesis was identified in the intermediate phase of the C/G-MS construct that had been treated with transforming growth factor β3 for 21 days. After subcutaneous implantation in mice, the C/G-MS construct exhibited a heterogeneous and graded structure up to eight weeks, with distinguished matrix distribution observed at one week. Overall, gene expression of tenogenic, chondrogenic, and osteogenic markers showed increasing patterns for eight weeks. Among them, expression of collagen type X gene was found drastically increasing during eight weeks, indicating progressive formation of calcifying cartilage within the constructs.

Conclusion
Our findings demonstrate that the stratified manner of fabrication based on the 3D-printed multiphasic scaffold is an effective strategy for tendon-to-bone interface engineering in terms of efficient cell seeding, chondrogenic potential, and distinct matrix deposition in varying phases.