Collagen-Glycosaminoglycan Coprecipitate and Photochemically Cross-Linked Collagen for Disk Replacement
Authors: Choy A. T. H. , Chan B. P. , Leong K. W. 
Published: June 2012

Introduction

Traditional treatments of severe disk degeneration such as discectomy were not satisfactory. Recent research focuses on developing biological substitutes, using tissue engineering methods, for nucleus pulposus and annulus fibrosus.1,2 A few studies had demonstrated the fabrication of biphasic composite scaffold as nucleus and annulus replacements using alginate and polyglycolic acid and alginate and collagen.3,4 Being the prevalent matrix components in native disc, collagen and glycosaminoglycans (GAGs) are potentially suitable biomaterials for a more nature-simulating scaffold. Previously, we have demonstrated that chemical modifications of collagen by deamination, methylation, and amination were able to control the GAG binding to and retention from the collagen meshwork, resulting in controllable GAG:HYP ratio, while maintaining over 90% cell viability. In this study, the ultrastructure of the coprecipitates fabricated in acidic and neutral pH was evaluated. Moreover, aminated CG coprecipitate, with optimal GAG:HYP ratio, along with mechanically strengthened photochemically crosslinked collagen lamellae previously developed in our lab, were used to fabricate a biphasic composite scaffold. On-going work is underway to test the cytocompatibility and mechanical properties of the composite scaffold.

Materials and Methods

First, modified collagens (deaminated, aminated, and methylated) were coprecipitated with GAG to form CG coprecipitates, which were observed under SEM before and after neutralization. Second, aminated CG was entrapped as the core of a collagen shell, which was photochemically crosslinked in the presence of rose bengal and argon laser before partial dehydration of the whole biphasic scaffold. Rabbit mesenchymal stem cells would be cultured in the core of the scaffold for 7 days. Scaffolds would be evaluated by immunohistology and cell survival was visualized with live/dead staining. The scaffold would be tested for creep and dynamic responses.

Results

First, collagen fibers in modified CGs had distinctive ultrastructural features at different pH. In acidic conditions, fibers were compact with small meshes (∼50 nm) in aminated, methylated, and nontreated CGs. In physiological pH, observations in the three CGs were also distinctive. Meshsize increased in non-treated CG. Sheet-like substance wrapped around the fibers in methylated CG. Granule-like ground substances were found in aminated CG. Seoncd, the feasibility of fabricating a biphasic composite scaffold with aminated CG as the core and the photochemically cross-linked collagen lamellae as the shell has been demonstrated. Preliminary mechanical study demonstrated that a step strain of 72% (pre-strain included) resulted in resistance stress up to 1 MPa, which gradually decreased to 0.5 MPa over 20 minutes.

Conclusion

This study demonstrated that there were structural differences between different modified CGs and the fabrication of a biphasic collagen-GAG-based composite scaffold for IVD tissue engineering is feasible. The functional features of the composite scaffold have to be further evaluated. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared SebastineM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disk degeneration,” Conference proceedings?: Annual International Conference of the IEEE Engineering in Medicine and Biology Society.IEEE Engineering in Medicine and Biology Society. Conference 2007;2007:6401–6406 Nerurkar NL, Baker BM, Sen S, Wible EE, Elliott DM, Mauck RL. Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus. Nature materials 2009;8(12):986–992 Mizuno H, Roy AK, Zaporojan V, Vacanti CA, Ueda M, Bonassar LJ. Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials 2006;27(3):362–370 Bowles RD, Williams RM, Zipfel WR, Bonassar LJ. Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disk composite via collagen gel contraction. Tissue engineering. Part A 2010;16(4):1339–1348