Investigating the impact of cyclic tensile loading and local strain on spatiotemporal cellular behavior within a multicompartment scaffold for interfacial tendon-to-bone rotator cuff repair

Introduction: Rotator cuff injuries affect 1 in 4 people over the age of 60, yet these injuries still represent a significant clinical challenge, with high rates of early re-failure stemming from failed regeneration of the native tendon-to-bone insertion. This insertion, a fibrocartilaginous region known as the enthesis, serves as a crucial transition between highly dissimilar tissues (tendon and bone) both biologically and mechanically, dissipating local interfacial strain concentrations and helping prevent damage. To address the limitations of current strategies for rotator cuff repair, next-generation techniques must consider and incorporate the importance of enthesis regeneration within their design. Considering this need, we are investigating spatiotemporally guided cellular behavior by developing a triphasic tendon-to-bone biomaterial comprising instructive tendinous and osseous collagen scaffold zones mechanically integrated via a thiolated gelatin hydrogel. We have demonstrated the importance of cellular crosstalk between these three zones, and we plan to further test this material under conditions of physiologically relevant cyclic tensile loading. Mechanical loading is crucial to the development of a mature enthesis, and it can also benefit the healing process. In addition, we have previously shown that cells are significantly impacted by cyclic tensile loading in similar scaffold-based materials. We hypothesize that loading-induced local strain distribution, which differs across the tendon-to-bone scaffold, will significantly affect local cellular response, particularly tendinous and fibrocartilaginous behavior. Interfacial materials require precise mechanical and biological tuning, and the results from this work will provide valuable insight into using these design parameters, and their interaction, for improved spatiotemporal guidance.

Materials and

Methods: Triphasic tendon-to-bone scaffolds were fabricated through lyophilization of precursor solutions loaded into custom molds that promote unidirectional heat transfer, which creates anisotropic pore structure within the designated tendinous zone. Osseous and tendinous scaffold precursors consisted of homogenous slurries containing collagen, chondroitin sulfate, salts, and minerals. The interfacial hydrogel region was formed via 3.5 wt% thiolated gelatin crosslinked by disulfide bonds via hydrogen peroxide activated horseradish peroxidase. These precursors were loaded into our custom mold, allowed adequate time to integrate, and lyophilized.
Mechanical loading will be induced under sterile conditions via a custom bioreactor. Based on previous findings, scaffolds will be embedded with 3D-printed endblocks and stretched at a bulk strain rate of 5% at a frequency of 1 Hz and a period of 10 minutes. A rest period of 6 hours will follow each active period. These samples will be compared to an unstretched control group.
For in vitro testing, scaffolds will first be rehydrated and crosslinked via EDAC carbodiimide chemistry before being seeded with human mesenchymal stem cells (hMSCs, RoosterBio) at a density of 600,000 cells distributed across the material. Cells will be incubated under normal culture conditions (37°C, 5% CO2) in low glucose DMEM containing L-Glutamine and supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic. Cells will be evaluated via a variety of assays and markers, including alamarBlue-based metabolic activity; gene expression analysis via trizol/chloroform-based RNA isolation followed by NanoString nCounter or RNA-sequencing; protein expression via cytokine array, ELISA, or Western Blot; and matrix remodeling via histology.

Results, Conclusions, and Discussions: We have investigated hMSCs in static culture on triphasic scaffolds over a culture period of 21 days. Over this period, hMSCs displayed 2-fold increased metabolic activity (n=6) relative to Day 0 behavior and secreted high early levels of TGF-β1, a growth factor involved in early enthesis development. After 21 days, hMSCs within the interfacial hydrogel region significantly upregulated several enthesis-relevant genes, including matrix proteins (COL1A1, COL10A1), growth factors (TGFB1, BMP4), and transcription factors (KLF2) (Fig 1). Between scaffold regions, most individual genes did not show significant difference. However, principal component analysis indicated yielded a first principal component with a high proportion of total variance (0.47) and separation of the three groups, suggesting spatiotemporal differences on a broad level (Fig 1). Histological analysis of the interfacial scaffold region did not indicate significant deposition of enthesis-relevant proteoglycans (Alcian blue) or mineralization (Alizarin red) (Fig 1). These results serve as a valuable baseline and show that, while this material has potential for enthesis tissue engineering, the current environment does not sufficiently promote distinct spatiotemporal gene expression or observable, functional fibrochondrogenic formation. Introducing bulk tensile loading and, by extension, localized strain profiles could create a more conducive environment for both metrics.
We have mapped the local strain profile of a triphasic scaffold associated with 5% bulk strain (Figure 2). Ongoing efforts are now focused on finalizing protocols for bioreactor in vitro use, and in coming months we plan to compare loaded and unloaded samples over 21 days in vitro. We expect the data from these experiments to show significantly increased tenogenic and fibrochondrogenic activity (both gene expression and proteoglycan deposition) in the respective scaffold zones when under cyclic tensile strain, as well as potentially higher metabolic activity and protein secretion. We will also investigate histology images for co-localization of matrix changes with high levels of localized strain. Through this work, we will provide the field of interfacial tissue engineering with valuable data and insight into the effects of mechanical loading, local strain, and the design of multicompartment materials for improved guidance of local cell behavior.

Acknowledgements (Optional):