Associate Professor FLORIDA INSTITUTE OF TECHNOLOGY Melbourne, Florida, United States
Introduction:: While viable graft choices for anterior cruciate ligament reconstruction (ACLR) are available, graft failure is reported in up to 20% of patients [1]. Poor integration at the bone-fibrocartilage-ligament (BFL) interface (i.e., enthesis) is attributed to be the key reason for suboptimal graft performance and failure [2]. Therefore, there is a need for a viable strategy to improve graft integration, permit appropriate interfacial load distribution, and promote more efficient healing post-ACLR. In this realm, we have previously developed a 3D printed bioceramic-gradient integrated collagen matrix (BioGIM) that mimics the compositional gradient of the native ACL enthesis [3]. While bioceramics can provide the osteostimulative cues to promote de novo bone formation on the mineral rich side of the enthesis, additional physicochemical factors may be essential to guide region-specific matrix remodeling along the BFL interface. Prior work has shown that topographical cues from anisotropically oriented collagen fibers can stimulate ligamentous cell differentiation [4]. The goal of the current study is to optimize the magnetic field parameters to introduce collagen fiber alignment in 3D constructs. Towards this goal, we employed a Helmholtz coil-based setup and investigated the effects of varying current densities, methacrylated collagen (CMA) concentration, and streptavidin-coated magnetic particle (SMP) concentration on collagen fiber alignment index. In addition, preliminary work on MSC response to aligned collagen topography was performed. Development of a novel continuous scaffold system that combines multiple tissue-specific biophysical and biochemical cues on a single platform can aid functional regeneration of ACL enthesis towards full reconstruction of the BFL interface.
Materials and Methods:: CMA (3, 6 mg/ml) was mixed with a photoinitiator (0.1% w/v), and SMP (0, 0.1, 0.5 mg/ml) and exposed to varying magnetic field (1,1.5 A) using a Helmholtz coil for 15 min to introduce collagen alignment, followed by incubation at 37 °C for 30 min to form a hydrogel (Figure 1). The hydrogel was then crosslinked with UV light (365 nm) for 90 seconds. Qualitative analysis of collagen fiber alignment was performed using scanning electron microscopy (SEM). Quantification of alignment index of collagen fibers was performed using ImageJ with an OrientationJ plug-in. Human MSCs were cultured on magnetically aligned CMA hydrogels for 7 days and the impact of SMP on cell viability was assessed using Alamar blue assay. Cell cytoskeleton staining was performed using Alexa Fluor 488 phalloidin to visualize the orientation of cells along the collagen fibers.
Results, Conclusions, and Discussions:: Results showed that application of higher current density (1.5 A) and incorporation of SMP at 0.5 mg/ml yielded CMA hydrogels with some evidence of collagen fiber alignment in both the 3 and 6 mg/ml collagen (Figure 2). Greater fiber alignment was observed when using 6 mg/ml compared to 3 mg/ml collagen in hydrogels containing 0.1 and 0.5 mg/ml SMP (Figure 3). When using 6 mg/ml collagen with 0.1 mg/ml SMP, no differences in collagen fiber alignment were observed between hydrogels exposed to 1 A and 1.5 A current density. Cell metabolic activity was maintained on all hydrogels indicating that that SMPs are cytocompatible at concentrations up to 0.5 mg/ml (Figure 4). Cell cytoskeleton staining showed that MSCs can sense the underlying aligned collagen topography – greater cell alignment index was observed in hydrogels with lower CMA concentration and higher SMP concentration suggesting that a less viscous collagen matrix is more conducive to cellular alignment (Figure 5). In conclusion, use of Helmholtz coil generated magnetic field together with optimization of process parameters can yield highly aligned collagen constructs. Future studies will entail: 1) quantifying collagen fiber alignment using confocal reflectance microscopy and Fast Fourier transform analyses to determine collagen alignment index and fiber orientation angle in magnetically aligned CMA hydrogels; 2) employing higher current densities and further optimizing magnetic field parameters to maximize collagen fiber alignment; and 3) assessing the printability of SMP incorporated CMA inks to deliver a highly aligned continuous biomimetic gradient towards developing a functional ACL enthesis for ACLR applications.
Acknowledgements (Optional): : This work was supported by a grant from the National Institute of Health (NIH 1R15AR071102).
References (Optional): : [1] Mather RC, The Journal of Bone and Joint Surgery American, 2013; [2] Vaquette C, Biomaterials, 2013 [3] Kajave N, Tissue Engineering - Part C, 2021; [4] Kishore V, Biomaterials, 2012.
