(M-493) Investigating patterns of spatial differentiation within a triphasic biomaterial for rotator cuff enthesis repair

Introduction:: Rotator cuff tears are a common musculoskeletal injury associated with physiological degeneration, affecting nearly a third of the population over 60. Treatment of these injuries is challenging, as the natural healing process is prone to scar tissue formation, and surgical repair is prone to high failure rates. A key reason for these failings stems from the inability of current techniques to regenerate the native tendon-to-bone interface, or enthesis. Interfacial tissues possess properties not found in neighboring tissue and are crucial both mechanically and biologically: the enthesis integrates the structures, compositions, and biologics of aligned, elastic tendon and stiff, mineralized bone tissue. Development of next-generation repair techniques must therefore account for this enthesis tissue in addition to bulk tendon and bone. Motivated by this need, we are designing a triphasic, instructive biomaterial for repair of osteotendinous junctions. This biomaterial is comprised of osseous and tendinous collagen-glycosaminoglycan (Col-GAG) scaffold zones linked by a continuous thiolated gelatin (Gel-SH) hydrogel zone. The individual scaffolds can drive osteogenic or tenogenic differentiation of human mesenchymal stem cells (hMSCs) without need for exogenous factors, while the Gel-SH hydrogel region provides greater mechanical integration between the Col-GAG scaffolds, increasing overall toughness. We have previously demonstrated the importance of paracrine signaling and cellular crosstalk between individual biomaterial compartments, but we hypothesize multicellular interactions within the triphasic environment may be more complex than the sum of an independent tendon and bone secretome signals. We are defining the potential for rich multi-cellular crosstalk to enable improved rotator cuff enthesis repair.


Materials and Methods:: Col-GAG scaffolds are fabricated via the lyophilization of a homogenized precursor slurry containing collagen, chondroitin sulfate, and additional salts and minerals. Isotropic (osseous) or anisotropic (tendinous) pore alignment is controlled via heat transfer rates promoted by different mold designs. The interfacial region consists of a 3.5wt% Gel-SH hydrogel crosslinked by disulfide bonds, which are formed via radical chemistry by horseradish peroxidase. A triphasic scaffold is fabricated by loading these three components (osseous slurry, Gel-SH, and tendinous slurry) into a custom Teflon-copper mold. These components are kept separate during the loading process, then allowed to integrate for an hour, and finally lyophilized to form a porous, continuous scaffold with anisotropy in the tendinous end and isotropy in the osseous end. A biphasic scaffold (osseous slurry and tendinous slurry only, no interfacial Gel-SH) can also be created using the same custom mold and method.

For in vitro evaluation, scaffolds are seeded with hMSCs (RoosterBio, Frederick, MD, USA) at a density of 150,000 cells per scaffold compartment and incubated in a growth media of FBS-supplemented low glucose DMEM at 37°C, 5% CO₂. Methods of biological evaluation include live/dead confocal imaging, metabolic activity analysis via alamarBlue assay, secreted factor assessment via cytokine array (RayBiotech), gene expression analysis via RT-qPCR or NanoString nCounter analysis, and histological staining. Statistical analysis is performed via ANOVA and Tukey post-hoc tests, with significance defined by a p-value less than 0.05. PCR and NanoString expression data are adjusted based on reference gene expression.


Results, Conclusions, and Discussions:: Using cytokine arrays, we compared normal growth media with secretomes of conditioned media from osseous, tendinous, and biphasic scaffolds after 15 days of culture. The results of a TGF Beta C2 Array are provided in Figure 1A, and we also identified the presence of HGF, PDGF-BB, and IGFBP-6 in the conditioned groups through a Human Growth Factor C1 Array. In both arrays, we observed that the secretome environments of individual tendon and bone scaffolds are distinct. Notably, the secretome created by the biphasic scaffold environment behaved differently than expected – rather than an average of the bone and tendon environment, the biphasic scaffold appears to promote an additive or synergistic effect. To further explore this observation, we assessed cell behavior in these scaffolds more directly via gene expression. Instead of the expected similarities, we noted significant differences in expression of osteogenic and tenogenic markers between osseous (Bone) and tendinous (Tendon) individual scaffolds and osseous (Biphasic B) and tendinous (Biphasic T) compartments of biphasic scaffolds, as seen in three representative gene profiles (Figure 1B). These differences suggest that the triphasic environment could be more complex than the sum of its individual parts. We are conducting a new study that compares cellular response to conditioned media from the biphasic with that from individual scaffolds to further investigate this.

Within the triphasic scaffold, we are currently assessing structure and biological development via several spatially sensitive methods, including electron microscopy, histology, and RNA isolation of thin scaffold sections (Figure 2). Ongoing work is assessing matrix deposition, gene expression, and secreted factors to robustly define spatial patterns of hMSC differentiation across the three regions as well as the overall signaling environment that the material promotes.

Ongoing efforts are also evaluating the triphasic material under physiologically relevant cyclic tensile loading, as mechanical loading is a crucial component of native enthesis development and healing. Future collaborative efforts will be assessing these materials in vivo using a rat rotator cuff model. With this knowledge and toolbox, we will work to tailor our current design to promote optimal cell behavior and, overall, improve functional regeneration of the native tissue.


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