Tubular composite scaffolds produced via Diffusion Induced Phase Separation (DIPS) as a shaping strategy for anterior cruciate ligaments reconstruction

Injuries of tendons and ligaments are common, especially among the young population. Anterior cruciate ligament (ACL) injuries do not heal due to its limited vascularization and hence, surgical intervention is usually required. The ideal scaffold for ligament tissue engineering (TE) should be biocompatible and possess mechanical and functional characteristics comparable to the native ACL. The Diffusion Induced Phase Separation (DIPS) technique allows the preparation of homogenous porous tubular scaffold with micro-pores using a rather simple procedure. Composites based on biodegradable polymers and bioglass have attracted much attention in tissue reconstruction and repair because of their biological and physicochemical advantages. In this work a new approach in ACL TE will be proposed focussing on the development of a suitable technique for in vitro seeding of lapine ACL fibroblasts into tubular-shaped instructive Poly-lactic-acid (PLLA) scaffolds, supplemented or not with bioglass (BG) 1393, produced via DIPS. Tubular composite scaffold (diameters: 1.2 and 2 mm, +/- BG) were obtained through a dip coating around a cylindrical support followed by a DIPS. An 8%wt PLLA/dioxane solution was prepared with 5%wt of BG-1393 as filler. Preliminary in vitro cell culture trials were carried out by seeding lapine ACL fibroblasts inside the scaffolds (2 cm as length) employing different seeding strategies in order to find the best way that allows to obtain a homogeneous fibroblast distribution inside the tubes. (1) First trials consisted in the inoculating of the cell suspension inside the tubes and maintaining them in dynamical culture. (2) The second one was done by suspending the cells in a fibrin gel polymerized within the tubes by using of thrombin. (3) The third approach was carried out by using cell spheroids (three-dimensional self-assembled cell agglomerates). Cell attachment, viability and morphology were examined by live-death and Hematoxylin/Eosin stainings after 1, 7, 14 d and vimentin immunolabelings (7 d). ...