The use of platelet-rich biopolymers in the enhancement of synthetic scaffolds for ligament tissue engineering applications

Introduction: Rupture of the anterior cruciate ligament (ACL) of the knee is a common injury among athletes, leading to instability of the knee. Due to the poor healing capacity of this ligament, reconstruction is currently the only suitable method of treatment. At present, material for reconstruction is most often harvested from the donor’s patellar tendon, which can lead to issues with donor site morbidity and further knee instability. This has led to the development of synthetic ligaments, which have demonstrated varying levels of success. Xiros Ltd. (Leeds) have developed a woven polyethylene terephthalate (PET) ligament prosthesis able to induce tissue formation in vivo. However, tissue induction is slow, leaving the material exposed to abrasive forces upon implantation, which eventually leads to graft failure. Objective: To assess whether a hybrid construct, composed of the Xiros PET ligament and a platelet-rich biopolymer coating, can enhance synovial cell proliferation, infiltration and expression of extracellular matrix genes, which may indicate promotion of tissue induction. Methods: Primary bovine synovial cell (bSC) cultures were established from the metatarsophalangeal joints of 12-18 month old bovines. Venous blood was taken from healthy human donors and platelet-rich and platelet-poor plasma fractions were extracted. 3D cell-seeded hybrid ligament constructs were fabricated by seeding bSCs onto sections of Xiros Ltd. PET scaffold before coating them with various combinations of plasma, collagen and alginate. Cells were monitored within coated constructs using confocal microscopy, DNA content analysis and Live/dead staining. Biopolymers were characterised using scanning electron microscopy (SEM) and rheology. Coating longevity was assessed using light microscopy image analysis. Cyclic tensile strain (5% strain at a frequency of 1 Hz) was applied to the three most promising coated constructs using an in-house house developed bioreactor, and qRT-PCR was used to measure changes in expression of ligament and non-ligament associated genes. The effect of cyclic tensile strain on tPA and PAI-1 gene expression was analysed using qRT-PCR and secreted PAI-1 protein was assessed using Western blot. Results: Alginate-based coatings demonstrated the greatest stability, but significantly inhibited cell infiltration and proliferation. In contrast both plasma and collagen coatings had poor longevity, but promoted cell infiltration, in comparison with the non-coated control scaffold. All constructs supported good cell viability. PDGF-AB and IGF-1 release from coatings occurred up to 3 days. Changes in expression of ECM and transcription factor genes were inconsistent between donors. Cyclic tensile strain increased cell proliferation in all constructs. The longevity of the 100% plasma (P100) coating was greatly enhanced by the application of cyclic tensile strain, leading to selection of this construct for final studies. Strain induced no change in tPA gene expression but caused a significant upregulation of PAI-1 gene expression and protein secretion, indicating a possible mechanism of enhanced coating longevity. Increasing the platelet density of the P100 coating had no significant effect on cell proliferation nor transcription of ECM genes. Discussion: This study has demonstrated that bovine synovial cell-seeded 3D hybrid ligament constructs composed of the Xiros Ltd. PET scaffold and a biopolymer coating, can be successfully fabricated, and are compatible with the cyclic strain bioreactor utilised for this work. The 100% plasma-coated construct was the most successful candidate due to its ability to promote cellular infiltration, as well as its superior longevity when subjected to cyclic tensile strain. Application of cyclic tensile strain to cell-seeded constructs resulted in upregulation of PAI-1, which may have contributed to this increase in coating longevity. Conclusion: These findings demonstrate the potential of a PET-fibrin construct for ligament regeneration purposes, and indicate the importance of regular exercise of the knee immediately following ACL reconstruction. Further studies should focus on the behaviour of this construct in vivo