The interplay between collagen content and organisation in enhancing the strength of tissue-engineered ligaments

摘要

Introduction: Anterior Cruciate Ligament rupture repair surgery can have a low success rate due to tendon pullout, or mechanical breakdown of synthetic grafts. Tissue engineered (TE) tendons and ligaments aim to produce viable tissue replacement grafts for implantation to an injured site, and there is a major focus on increasing construct strength to match the original tissue. Our previous work demonstrated that addition of ascorbic acid (AA) and proline (P), two factors important in collagen synthesis, was necessary for endogenous collagen production within the tissue-engineered ligament-like structures. In the current work we demonstrate that while AA + P concentrations can be optimised to maximise collagen production, collagen content alone does not exclusively increase the Young's modulus (YM) of the tissue construct and that organisation of collagen is key. Materials and Methods: TE constructs were formed as described in and briefly shown in fig1. Culture media with 250μM AA and 50μM P every 2 days in order to stimulate collagen production. wt% collagen was estimated using the hydroxproline assay and tensile mechanical properties characteriesed using an Instron microtester using a 0.4mm/min strain rate and 10N load cell. TEM imaging used 1μm thick resin-embedded tissue sections fixed in osmium tetroxide. Expression of matrix remodelling markers MMP-1,2,3,9, RhoA, ACTA2 and Ten-C were assessed by RT-PCR. Results and Discussion: After two weeks in culture without AA+P, the ligaments contained only 1.45%wt collagen (Fig1 top row). After two weeks with AA+P treatment, there was -6-fold increase in collagen to 8.34%wt and a ～10-fold increase in YM from 24kPa to 213kPa, suggesting that the increased levels of collagen stiffened the structure. Most interestingly, non-treated ligaments after 7 weeks contained 12%wt collagen, 1.4x that of the treated ligaments after 2 weeks, and yet both groups had a comparable YM. In this case collagen content did not appear to have an exclusive influence over achieving maximum YM of the tissue and suggested that collagen structuring played a large role in enhancing tissue mechanical properties. After 7 weeks in culture with supplementation, a the ligaments contained 32%wt collagen with a Young's modulus of 1.03MPa. TEM imaging of AA+P treated constructs confirmed that AA+P mediated formation of collagen fibres (Fig2e-h) whereas only fibrils were found in non-treated samples (Figure 2a-d). RT-PCR confirmed 6-fold upregulation of Ten-C, ACTA2 and RhoA which in combination represent markers of myofibroblast contraction and adhereance to the matrix, thus suggesting that AA+P has a secondary action of driving cell-mediated collagen remodelling and not just deposition. Conclusion: While initial anabolic stimulation of collagen within the construct is required, it is the concomitant hierarchical structuring of the collagen that is critical to maximising the mechanical strength of the tissue. Stimulation of cell-mediated remodelling of modest amounts of collagen can dramatically enagnce tissue mechanical properties. The implication on scale-up manufacture of TE ligament/tendon/muscle is the need to focus more on enhancing myofibroblast adhesio/activity rather than purely on collagen synthesis.