Enhanced regeneration of the ligament-bone interface using a poly(L-lactide-co-ε-caprolactone) scaffold with local delivery of cells/BMP-2 using a heparin-based hydrogel

Recently, the ligament-bone (LTB) junction has been emphasized for the effective transmission of mechanical force and the reduction in stress concentration between the soft ligament and hard bone tissue. The aim of this study was to regenerate an integrated LTB interface by inoculating LTB-relevant cells, isolated from fibrocartilage (FC) or ligament (LIG), separately into each designated region in a single porous cylindrical PLCL scaffold. An injectable, heparin-based hydrogel that has proved to be effective in the culture of chondrocytes as well as the sustained release of growth factor was employed to locally deliver fibrochondrocytes and osteoinductive bone morphogenetic protein-2 (BMP-2) into the FC region, to promote FC regeneration. In in vitro experiments the hydrogel-combined FC systems produced significantly larger amounts of calcium and glycosaminoglycans (GAGs), but less collagen and DNA than FC samples without the hydrogel and all LIG samples. After in vivo subcutaneous implantation in mice for 8 weeks the secreted calcium and GAG contents of the hydrogel-containing FC samples were superior or similar to those of the in vitro hydrogel-containing FC samples at 6 weeks. As a result of the enhanced production of calcium and GAG, the in vivo hydrogel-containing FC samples produced the highest compressive modulus among all samples. Histological and immunofluorescence analysis as well as elemental analysis also confirmed a denser and more homogeneous distribution of calcium, GAG, osteocalcin and neovascularization marker in the in vitro/in vivo hydrogel-containing FC systems than those without hydrogel. These results also show the beneficial effects of BMP-2 added using the hydrogel. In summary, the use of a heparin-based hydrogel for the local delivery of fibrochondrocytes and BMP-2 could accelerate the maturation and differentiation of LTB-specific FC tissues, and it was also possible to recreate the unique stratification of calcified FC and LIG tissues in a single porous PLCL scaffold in terms of both biochemical and biomechanical properties.