Advancing Rotator Cuff Repair with a Multiphasic Scaffold System: Insights from Rabbit Model Evaluation

Introduction: Rotator cuff tears are prevalent, leading to pain and functional limitations [1-2]. While tissue engineering holds promise for improving rotator cuff enthesis repairs, current approaches lack essential bioactive and biophysical cues, structural complexity needed for effective regeneration [3-4]. To overcome these challenges, we introduce a novel biomimetic matrix-based multiphasic scaffold (BMS) for rotator cuff enthesis repair. Consisting of three distinct phases, the BMS mimics native tissue environments and supports zone-specific regeneration: Phase I integrates nanofibrous decellularized tendon extracellular matrix (dECM) with 'stiff' methacrylate hyaluronic acid (MeHA) for the tendon zone interface; Phase II utilizes nanofibrous dECM with 'soft' MeHA for the tendon-bone interface; Phase III employs porous poly(octamethylene citrate)/bioglass scaffolds for the calcified zone-bone interface. This innovative approach shows promise in addressing the current limitations of repair strategies [5]. To comprehensively assess the in vivo efficacy of the BMS in rotator cuff enthesis regeneration, in this study, we employed a preclinical rabbit model with a designed 4-week implantation period.

Materials and

Methods: Native and dECM juvenile bovine Achilles tendon tissues (4 different donors) underwent liquid chromatography with tandem MS (LC-MS/MS) analysis and protein-protein interaction analysis. Passage 1 bovine mesenchymal stem cells (P1 MSCs) were cultured for 3 days on four types of stiffness (Soft/Stiff)-and-alignment (Align/Non-align)-tunable nanofibrous scaffolds and primary anti-YAP and Alexa Fluor-546 antibodies were used to visualize YAP nuclear localization in MSCs. Cytoskeleton and cell nuclei were visualized by Phalloidin and DAP, respectively. Acetyl-H3K9 (a marker of epigenetic gene activation) was immunofluorescently visualized to evaluate transcriptional activation status in MSCs seeded on the scaffolds. All quantifications of immunofluorescent images were performed using Fiji software. Rabbit rotator cuff implantation surgeries were conducted with approval from the UPenn IACUC. For the in vivo tests, fibrocartilaginous tissues at the native enthesis were removed, and the BMS scaffolds were secured to the tendon (Fig. 2A). At 4 weeks post-surgery, rabbit shoulders underwent ex vivo µCT scanning (Scanco µCT45) to evaluate bone volume fraction and trabecular separation in a 2-mm-thick region of interest of each humerus bone (Fig.2B). Shoulder samples were then embedded in paraffin, sectioned into 6 mm slices, and subjected to H&E histological staining. Non-operated rabbit shoulders were used as controls for comparison.

Results, Conclusions, and Discussions: Proteomic analysis of dECM tissue confirmed preservation of main tendon ECM proteins, indicating the effectiveness of the decellularization process (Fig. 1A). A dense protein-protein interaction network comprising 18 proteins and 137 protein interaction relations was elucidated (Fig. 1B). P1 bMSCs seeded on stiff dECM scaffold exhibited increased YAP nuclear localization compared to soft dECM scaffold groups, indicating stiffness-dependent cellular responses (Fig. 1C-D). Additionally, bMSC nuclei seeded on stiff aligned scaffolds showed higher fluorescence intensity of Acetyl-H3K9 (H3K9AC) transcriptional activation markers, suggesting enhanced transcriptional activity in response to scaffold alignment and stiffness (Fig. 1E-F). Four weeks after surgery, μCT scanning of the anterior surgical sites showed comparable bone volume fraction between the native (17.4%±2.2%) and implanted (16.8%±0.9%) groups. Additionally, the implanted group exhibited a slightly higher trabecular separation value (0.52±0.08) compared to the native group (0.49±0.085) (Fig. 2B). Histological analysis at the early healing stage (Week 4) revealed distinct tendon, fibrocartilage, and bone layers at the surgical site in the implanted group (Fig. 2C). In summary, this study introduces a promising extracellular matrix-based multiphasic scaffold system that retains tendon-derived bioactive proteins and induces favorable cellular behavioral changes. This system demonstrates effectiveness in enhancing tendon-bone interface repairs in a rabbit model. Ongoing investigations aim to broaden the in vivo assessment of the repair efficacy of the biomimetic scaffold, including longer-term studies in large animals. Taken together, the integration of bioactive constituents with sophisticated scaffold fabrication methodologies represents a significant advancement in the field of rotator cuff enthesis repairs and musculoskeletal soft tissue regeneration, offering promising prospects for clinical translation.

Acknowledgements (Optional): Acknowledgment: NIH R21AR07770 and NSF CMMI 1548571.
Reference: [1] Sambandam+, World J Orthop 2015; [2] Kuroda+, Clin Orthop Relat Res 2013; [3] Newsham-West+, J Anat 2007; [4] Chainani+, Tech Orthop 2016; [5] Li+, ORS 2023.