The Potential Use of Enamel Matrix Derivative for In Situ Anterior Cruciate Ligament Tissue Engineering: A Translational In Vitro Investigation

Abstract

Polyester scaffolds have been used as an alternative to autogenous tissues for the reconstruction of the anterior cruciate ligament (ACL). They are biocompatible and encourage tissue infiltration, leading to neoligament formation. However, rupture can occur, caused by abrasion of the scaffold against the bone tunnels through which it is implanted. Good early tissue induction is therefore considered essential to protect the scaffold from this abrasion. Enamel matrix derivative (EMD) is used clinically in the treatment of periodontal disease. It is a complex mix of proteins with growth factor-like activity, which enhances periodontal ligament fibroblast attachment, proliferation, and differentiation, leading to the regeneration of periodontal bone and ligament tissues. We hypothesized that EMD might, in a similar manner, enhance tissue induction around scaffolds used in ACL reconstruction. This preliminary investigation adopted a translational approach, modelling in vitro 3 possible clinical modes of EMD administration, to ascertain the suitability of each protocol for application in an animal model or clinically. Preliminary investigations in monolayer culture indicated that EMD had a significant dose-dependent stimulatory effect (p < 0.05, n = 6) on the proliferation of bovine primary synovial cells. However, pre-treating culture plates with EMD significantly inhibited cell attachment (p < 0.01, n = 6). EMD's effects on synovial cells, seeded onto ligament scaffolds, were then investigated in several in vitro experiments modelling 3 possible modes for clinical EMD administration (pre-, intra-, and post-operative). In the pre-operative model, EMD was adsorbed onto scaffolds before the addition of cells. In the intra-operative model, EMD and cells were added simultaneously to scaffolds in the culture medium. In the post-operative model, cells were pre-seeded onto scaffolds before EMD was administered. EMD significantly inhibited cell adhesion in the pre-operative model (p < 0.05, n = 6) and had no significant benefit in the intra-operative model. In the post-operative model, the addition of EMD to previously cell-seeded scaffolds significantly increased their total deoxyribonucleic acid content (p < 0.01, n = 5). EMD's stimulative effect on cell proliferation in vitro suggests that it may accelerate scaffold colonization by cells (and in turn tissue induction) in situ. However, its inhibitory effect on synovial cell attachment in vitro implies that it may only be suited to post-operative administration.

Enamel matrix derivative enhances tissue formation around scaffolds used for tissue engineering of ligaments

“…The current work represents the second stage in a unique translational approach to investigating the application of EMD to ACL reconstruction. In an earlier short-term (16-72 h) study (Messenger et al, 2007), we reported that EMD enhanced the proliferation, but not attachment, of synovial cells in monolayer and 3D culture. A dose of 250 µg/ml was found to be optimal, as larger doses were found to be less effective at enhancing proliferation of the cells.…”

“…EMD has been previously reported to be optimal for enhancing synovial cell proliferation (Messenger et al, 2007). Separate vials of propylene glycol alginate (PGA; Institut Straumann AG, Basel, Switzerland) were also supplied.…”

Enamel matrix derivative inhibits proteoglycan production and articular cartilage repair, delays the restoration of the subchondral bone and induces changes of the synovial membrane in a lapine osteochondral defect modelin vivo

“…Intriguingly, EMD led to changes in the synovial membrane, including increased villus thickening and changes in the architecture of the synovial villi. In vitro , EMD stimulated the proliferation (Messenger et al ., ) and TGF β 1 synthesis of primary synovial cells (Messenger et al ., ) and fibroblasts (Grayson et al ., ). Clinically, the application of EMD for regenerative periodontal surgery often results in expedited wound healing and minimal post‐operative symptoms, such as pain or swelling (Hoang et al ., ; Rincon et al ., ; Yuan et al ., ).…”

“…Over the course of days and weeks, the amelogenin assemblies (matrix) are slowly digested by matrix metalloendoproteases and serine protease, and biologically active components are slowly released into exposed tissues. Potential applications of amelogenins have been proposed for bone regeneration (Kawana et al ., ; Vowden et al ., ; Messenger et al ., ; Romanelli et al ., ).…”

Stem Cells in Periodontal Regeneration

“…Commercially available emdogain have been suggested to stimulate the formation of root cementum [35] . It also acts a signalling molecule which can regulate the activity of follicle cells, periodontal ligament cells, odontoblasts, gingival fibroblasts and cementoblasts [36][37][38][39][40][41] .…”