Engineering the Bone–Tendon Interface: A Composite Scaffold Approach

Introduction: : Musculoskeletal injuries affect approximately 32 million people in the United States each year(1), and self-repair mechanisms are often inefficient in restoring full function. Hard-to-soft tissue interfaces, e.g., the bone-tendon junction, are impacted in many musculoskeletal injuries. In the USA, over 300,000 shoulder, foot, and ankle surgeries are conducted to correct degeneration, costing over 30 billion dollars, and many cases require surgical revision(2). Physiologically, this interface facilitates critical load transfer from the compliant, unmineralized tendon to the mineralized bone (Fig. 1A). Following injury or overuse, an uncontrolled inflammatory response often results in excess scar tissue and a compromised bone-tendon interface, leading to insufficient healing. Therefore, there is an urgent need to develop improved models to elucidate properties that allow the interface to withstand complex mechanical loads while acting as a crosstalk hub for different cell populations, as the bone-tendon interface contains at least three major cell populations including osteoblasts/clasts, chondrocytes, and tenocytes. Combining tissue engineering fabrication methods is one way to replicate a bone-tendon interface in a single scaffold. We hypothesize that combining silk fibroin (SF) cryogelation and electrospinning will form a morphologically accurate bone-tendon interface model to support regeneration.

Materials and

Methods: : Bombyx mori SF was extracted using an established protocol(3). The cryogelation technique involved inducing a phase transformation of 6.5% (w/v) SF by sonication for 30 seconds, with/without pulse settings, followed by 24 hours at -20°C. This created porous, bone-like (CG) scaffolds (Fig. 1E). For analysis, bone scaffolds were cut to an 8:10 diameter:length ratio for 50% compression testing (Instron) and scanning electron microscopy (SEM) images were analyzed via ImageJ. For the tendon scaffold, 4, 6, and 8% polymer solutions of a 50:50 blend of SF and polyhydroxybutyrate (PHB) in solvent 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) was electrospun (Spinbox) onto a drum rotating at 1500 rpm, creating semi-aligned fibrous electrospun (ES) mats (Fig. 1C). For analysis, samples 0.1 mm thick were cut 6 x 2 mm and mounted on paper frame for tensile testing to failure(4). To create combined constructs (Fig. 1B), fibers were electrospun directly onto a CG attached to the drum, resulting in an overlapped interface (Fig. 1D). Before imaging and analysis of samples, they were frozen at -80°C for one hour and lyophilized. The combined construct and individual scaffolds (controls) were seeded with mouse-derived tenocytes on the ES segments and osteoblastic cells differentiated from MC3T3-E1-S4 on CG segments for 4, 8, 12 days. Endpoint studies include confocal microscopy with DAPI, Rhodamine-phalloidin-TRITC, and anti-Vinculin-FITC to understand cell infiltration and scaffold attachment. As our group previously demonstrated in vivo CG-mediated bone healing (data not shown), ongoing studies are focused on implanting the optimized ES scaffold in an injured mouse tendon (Fig. 3).

Results, Conclusions, and Discussions:: Our overall objective is to understand the relationship between morphological characteristics of the scaffold and cell morphology/behavior in the bone-tendon interface via a tissue engineered model (Fig. 1). Our preliminary scaffold mechanical characterization has identified ideal parameters for CG and ES scaffold development, and this will be used for developing optimized bone-tendon combination construct (Fig. 1B). Based on mechanical characterization, the optimal CG was found to be 6.5% SF sonicated with a 3 second on, 3 second off pulsed setting (3-3), which had an elastic modulus of about 0.4 MPa (native bone: ~0.1-10 GPa; Fig. 2). The optimal ES scaffold was found to be 6% polymer with a PHB:SF ratio of 50:50, which has an elastic modulus of about 40 MPa (native tendon: ~400 MPa). While neither value match native tissue, we hope the scaffold will encourage cells to replicate the extracellular matrix (ECM), which possess values closer to our results. Ongoing work is focused on the biological characterization of combined constructs with localized bone and tendon cell culture as well as integration into an animal model (Fig. 3). Study endpoints include confocal and SEM imaging across different timepoints to analyze cell morphology, ECM presence, changes in cellular interaction based on scaffold structural differences, and histology. The outcomes of this study include morphological and biological characterization of composite CG and ES scaffolds using SF to replicate the bone-tendon interface.

Acknowledgements and/or References (Optional):: (1) Ellis, I.; Schnabel, L. V.; Berglund, A. K. Defining the Profile: Characterizing Cytokines in Tendon Injury to Improve Clinical Therapy. J Immunol Regen Med 2022, 16, 100059. https://doi.org/10.1016/j.regen.2022.100059.
(2) Pugliese, E.; Rossoni, A.; Zeugolis, D. I. Enthesis Repair – State of Play. Biomaterials Advances 2024, 157, 213740. https://doi.org/10.1016/j.bioadv.2023.213740.
(3) Rockwood, D. N.; Preda, R. C.; Yücel, T.; Wang, X.; Lovett, M. L.; Kaplan, D. L. Materials Fabrication from Bombyx Mori Silk Fibroin. Nat Protoc 2011, 6 (10), 1612–1631. https://doi.org/10.1038/nprot.2011.379.
(4) Maccaferri, E.; Cocchi, D.; Mazzocchetti, L.; Benelli, T.; Brugo, T. M.; Giorgini, L.; Zucchelli, A. How Nanofibers Carry the Load: Toward a Universal and Reliable Approach for Tensile Testing of Polymeric Nanofibrous Membranes. Macromolecular Materials and Engineering 2021, 306 (7), 2100183. https://doi.org/10.1002/mame.202100183.