In vitro optimization and in vivo application of injectable freeze-dried chitosan–thrombin implants for orthopaedic regenerative medicine [English translation]

Musculoskeletal injuries affect 11 million Canadians each year, resulting in costs of 37 billion dollars. Despite advances in medicine, current standard treatments have a high postoperative failure rate, ranging from 20 to 60% for rotator cuff repairs, for example. This situation high- lights unmet medical needs that current technologies fail to meet. To address this challenge, the Laboratory of Biomaterials and Cartilage (LBC) developed a freeze-dried chitosan formu- lation designed to be solubilized in platelet-rich plasma (PRP). This injectable biomaterial has the advantage of coagulatingin situ, thus facilitating its application at site of injury. However, the resulting implant solidifies slowly, particularly with human-derived PRP. This slow coagulation can be problematic, especially for surgeries where a fluid is used to irrigate the work area (e.g., arthroscopic surgeries). The fluid present at the injury site can then prevent the biomaterial from adhering. The overall objective of the three studies described in this thesis is to advance the field of regenerative medicine by thein vitro development and optimization of a chitosan-based biomaterial, validate it for its homogeneity, and then test itin vivofor its efficacy, particularly for the regeneration of the supraspinatus tendon of the rotator cuff in rabbits. The specific objective of the first study is to propose an algorithm to quantify homogeneity in images. Homogeneity is an important parameter for chitosan-PRP implants, as it influences their degradation and the tissue response that follows their implantation. A precise quantifi- cation of homogeneity thus allows comparing different formulations of the biomaterial among themselves, as well as validating that for each batch of biomaterial produced, the chitosan mixes homogeneously with the blood components. The specific objective of the second study is to developin vitro a new formulation of the chitosan-PRPbiomaterialwithacceleratedcoagulation. Thechallengeistofindaformulation that coagulates quickly enough to adhere in a wet environment, but not too quickly, as this would make it difficult (if not impossible) to inject and handle. To accelerate the coagulation of the implants, the use of thrombin is explored. The specific objective of the third study is to confirm that implants composed of chitosan, thrombin, and PRP have the potential to improve the outcome of orthopedic surgeries. More specifically, the biomaterial was used for the repair of a rotator cuff tear in rabbits. The research is based on three main hypotheses: (1) a reliable and quantifiable measure of homogeneity can be established and used to evaluate chitosan-PRP biomaterials; (2) the coagulation of the biomaterial can be accelerated by adding thrombin to the chitosan solution before freeze-drying; (3) a freeze-dried formulation of chitosan-thrombin, solubilized in PRP, can significantly improve the regeneration of the supraspinatus tendon of the rotator cuff in rabbits, thereby improving the outcomes of surgical repairs. The first study introduces MASQH, a new algorithm for quantifying homogeneity in images, which is multi-scale, segmentation-free, and based on a statistical approach. This algo- rithm is applied to synthetic or experimental images (histological sections of biomaterials and nanocomposite microscopy), demonstrating its reliability and robustness. The second study analyzes several formulations of chitosan-thrombin, freeze-dried, and then solubilized in PRP. Various concentrations of thrombin were evaluated for their impact on coagulation time, ease of handling, and adhesion to tendon tissues in a wet environment, in accordance with the requirements of orthopedic surgeons. The MASQH algorithm is also used here to evaluate the homogeneity of implants with and without thrombin. Thethirdstudymovestoananimalmodel, wherethemostpromisingformulationofchitosan- thrombin-PRP is tested for its effectiveness in improving rotator cuff supra-spinatus tendon regeneration in rabbits. Tissue repair is evaluated through non-destructive mechanical test- ing, microtomography, and histology. Tendons repaired by standard surgery (sutures) are compared to tendons repaired in the presence of the biomaterial (standard surgery + im- plant). The MASQH algorithm was used here to validate that the produced batch of bioma- terial met expectations. Throughout the work carried out, the MASQH method successfully quantified homogeneity, enabling objective comparisons between studies and validating the homogeneity of experi- mental biomaterials. The optimized formulation of chitosan-thrombin with 0.5 NIH/mL of thrombin led to a significant increase in coagulation speed while maintaining ease of han- dling, essential for clinical applications. Finally, chitosan-thrombin-platelet-rich-plasma (CS- FIIa-PRP) implants improvedin vivo tendon regeneration, characterized by an accelerated recovery of tendon mechanical properties and the inhibition of heterotopic ossification. This body of work highlights the usefulness of homogeneity quantification in the validation of chitosan-(thrombin)-PRP biomaterials and introduces a promising chitosan-thrombin-PRP implant for orthopedic tissue repair. The results suggest that integrating such biomaterials with standard surgical techniques could lead to better clinical outcomes in the treatment of rotator cuff tears, thereby reducing the high failure rates associated with current surgical interventions.