Enhanced Growth of Lapine Anterior Cruciate Ligament-Derived Fibroblasts on Scaffolds Embroidered from Poly(l-lactide-co-ε-caprolactone) and Polylactic Acid Threads Functionalized by Fluorination and Hexamethylene Diisocyanate Cross-Linked Collagen Foams.

Clemens Gögele,Michaela Schröpfer,Cindy Elschner,Ina Prade,Michael Meyer,Gundula Schulze-Tanzil,Annette Breier,Judith Hahn

doi:10.3390/ijms21031132

Abstract

Reconstruction of ruptured anterior cruciate ligaments (ACLs) is limited by the availability and donor site morbidity of autografts. Hence, a tissue engineered graft could present an alternative in the future. This study was undertaken to determine the performance of lapine (L) ACL-derived fibroblasts on embroidered poly(l-lactide-co-ε-caprolactone) (P(LA-CL)) and polylactic acid (PLA) scaffolds in regard to a tissue engineering approach for ACL reconstruction. Surface modifications of P(LA-CL)/PLA by gas-phase fluorination and cross-linking of a collagen foam using either ethylcarbodiimide (EDC) or hexamethylene diisocyanate (HMDI) were tested regarding their influence on cell adhesion, growth and gene expression. The experiments were performed using embroidered P(LA-CL)/PLA scaffolds that were seeded dynamically or statically with LACL-derived fibroblasts. Scaffold cytocompatibility, cell survival, numbers, metabolic activity, ultrastructure and sulfated glycosaminoglycan (sGAG) synthesis were evaluated. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of collagen type I (COL1A1), decorin (DCN), tenascin C (TNC), Mohawk (MKX) and tenomodulin (TNMD). All tested scaffolds were highly cytocompatible. A significantly higher cellularity and larger scaffold surface areas colonized by cells were detected in HMDI cross-linked and fluorinated scaffolds compared to those cross-linked with EDC or without any functionalization. By contrast, sGAG synthesis was higher in controls. Despite the fact that the significance level was not reached, gene expressions of ligament extracellular matrix components and differentiation markers were generally higher in fluorinated scaffolds with cross-linked collagen foams. LACL-derived fibroblasts maintained their differentiated phenotype on fluorinated scaffolds supplemented with a HMDI cross-linked collagen foam, making them a promising tool for ACL tissue engineering.

Highlights

The anterior cruciate ligament (ACL) is a hypocellular and hypovascular dense connective tissue
In order to prevent the development of osteoarthritis as a typical sequela of knee instability, resulting in chronic knee pain, autologous tendon grafts are currently used as a gold standard for ACL reconstruction [3]
Since lapine ACL (LACL)-derived fibroblasts were used for the scaffold seeding experiments, these cells were included in cytotoxicity testing experiments

Summary

Introduction

The anterior cruciate ligament (ACL) is a hypocellular and hypovascular dense connective tissue. It runs intraarticularly, directly connecting the inner surface of the lateral femur condyle with the anterior tibial plateau, thereby ensuring knee stability. Since self-healing of the ruptured ACL is unlikely, a reconstruction must be carried out [1,2]. In light of the very forceful anatomical double-bundle structure of the ACL, these tendon autografts may not be able to permanently withstand the mechanical loads [3]. Numerous experiments with various cell-free biomaterials to support ACL reconstruction have already been carried out, but with little success for clinical applications [4,5,6,7]. Since the ACL is a hypocellular tissue, a stable and transient cell carrier is required to provide stability until a sufficient extracellular matrix (ECM) is synthesized by the cells and novel ACL tissue is formed

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