Musculoskeletal diseases are a significant problem in orthopedic practice. Meniscal pathologies conditions are among the most frequent knee injuries of the femorotibial joint posing a unique set of challenges for orthopedic surgeons and are a leading source of disability worldwide. The meniscus is a fibrocartilage complex tissue that acts as shock absorbent for forces transmitted over the joint compartment. The rate of healing and success of current surgical meniscus repair are limited by the tear properties, chronic or acute state, patient profile, and joint stability. Current repair techniques and treatments are only effective for defects in outer vascular zones of meniscus while healing of the lesion in the inner avascular zones of menisci remains a significant and present challenge, especially in cases of complex or traumatic and degenerative tears. Arthroscopic meniscectomy is the most common knee surgery which often leads to long-term functional instability and eventually progresses to early knee osteoarthritis (OA). Meniscus repair and allografts have been gaining attention as alternatives to meniscectomy. The significant clinical and financial burden of OA drives scientists to investigate augmentation and regenerative strategies for stimulation of menisci tissue response.

All the product development process, safety, and efficacy of medical implants need to be tested in preclinical animal models before the application for clinical translation. Determining of the most suitable animal proxy for investigation of mechanisms and process of meniscus healing to the human joint is a necessity. Thereby, large animal models which duplicate human meniscus biomechanical function, anatomy, and composition are more translatable to clinical practice. Ovine is one of the well-established models for preclinical assessment of meniscus replacement and repair tools.

My initial objective was to review the literature and discuss the current clinical management guidelines for primary meniscus repair techniques as well as the most current augmentation strategies to enhance the rate of meniscus healing by using trephination, synovial rasping, abrasion, blood clot placement, platelet-rich plasma (PRP) injections, and wrapping with extracellular matrix materials. I also discussed the rationale for using chitosan polymer and autologous blood component implants to improve meniscus repair. Performing such a review covering treatment algorithms of meniscus lesions guided me to better design my experimental research using polymer-autologous blood component implants to improve meniscus repair during my Ph.D.

Autologous PRP is widely used as a source of growth factors in different medical fields. Chitosan (CS) has documented beneficial therapeutic effects in context of orthopedics pathologies. Further randomized and controlled comparative trials are necessary in the field of meniscus and cartilage repair for improving symptoms in patients, joint function, and quality of life. Freeze-dried formulations of CS that can be solubilized in PRP have been shown to improve repair of cartilage and rotator cuff in preclinical small and large animal models. This motivated and drove me to assess mechanisms of action and repair process by using of chitosan-PRP in the context of fibrocartilage menisci repair in sheep as a relevant large animal model.

Our in vivo studies allowed us to refine the sheep model in order to be relevant to the pathological condition of joint disease found in the humans. We discovered that augmenting meniscus repair by using chitosan-PRP hybrid implants in combination with sutures, trephination, and rasping is beneficial and safe for treatments of complex menisci defects which could have the potential to prevent early onset of OA in the long-term. However, future work is necessary to further enhance regenerative properties, understand the mechanisms involved, and evaluate the effect of the implants in the long-term. We performed clinical observation, assessed implant retention, microscopic and macroscopic evaluation of meniscus, synovium and cartilage and electromechanical mapping of articular cartilage surfaces, at 3 weeks, 6 weeks, and 3 months postsurgery. Our original hypotheses for these feasibility pilot studies were that I. Meniscus repair would be improved by the application of CS-PRP to the tears, but not by application of PRP alone, and II. That repair outcomes would be improved by using CS-PRP implants in conjunction with the wrapping technique over CS-PRP implants injected in the tear site alone or wrapping alone. We found that the unilateral tear model allowed the animals to protect the treated knee from weightbearing post-operative and improved success rate from 25 to 50% compared to the bilateral model. The sheep had some intermittent lameness for the a few weeks post- treatment which was not specific to any treatment. The chitosan-PRP implants were partly resident in the tears and trephination channels at 1-day post-surgery. The tears were macroscopically visible at the time of necropsy at 1 day, 3, 6 weeks and 3 months and the edges of the tears were usually well apposed. A reddish repair tissue and signs of neo-vascularization were visible in chitosan-PRP treated knee at 6 weeks and 3 months and hybrids induced cell recruitment from the vascularized periphery of the menisci towards the trephination channels. Partial and total integration between the repair tissue and the original meniscal tissue was achieved in these treated knees. In addition, CS-PRP implants were well-tolerated in the knee environment and evidences of adverse effect did not observe during the follow-up period. Addition of wrapping technique by using of collagen membrane matrix of Chondro-Gide in conjunction with CS-PRP implants or PRP alone did not improve repair. In summary, our data suggest that chitosan-PRP implants by themselves could be efficient in overcoming the current limitations of meniscus repair and have several regenerative features that reveal a greater potential than PRP alone to improve repair outcomes and restore meniscus function.

Our third aim was to assess I. The compatibility of freeze-dried chitosan formulations with different types of commercially marketed PRP systems, and II. Define a range of chitosan degree of deacetylation (DDA) and the number average molecular weight (Mn) that would yield freezedried formulations with satisfactory performance characteristics for clinical orthopedic conditions. Our starting hypothesis was that I. Chitosan formulations should be soluble and easily reconstituted in commercial PRPs, have paste-like properties and coagulate in a timely fashion to produce homogenous hybrid chitosan-PRP clots that resist retraction and are mechanically strong, and II. Although the different PRP preparation systems would yield PRPs with varying properties, all PRP preparations would be compatible with this technology. Formulations containing 1% w/v CS, 1% w/v trehalose as lyoprotectant, and 42.2 mM CaCl2 as a clot activator were prepared with five different chitosan, encompassing the low, mid, and high range of DDA and Mn product specifications were freeze-dried. Seven different PRP preparations were used to solubilize the formulations in vitro. Performance characteristics of chitosan-PRP formulations for clotting properties, runniness, liquid expression, paste-like properties, clot mechanical strength, and clot homogeneity were assessed. Also, solubility, pH, and osmolality of all the formulations were measured. Macroscopic assessment of cakes showed all the cakes were white, homogenous, and were slightly retracted from the vial walls following lyophilization. We found that freeze-dried formulations were solubilized with all PRP preparations. CS-PRP formulations were less runny than their corresponding PRP controls demonstrating its paste-like properties for in vivo injection. Assessment by thromboelastography revealed that all CS-PRP formulations had a clot reaction time below 9 minutes. In the static clotting assay, all PRP controls clotted, expressed serum, retracted, and lost their mass significantly, while the CS-PRP clots resisted platelet-mediated clot retraction. Histological results revealed that CS dispersion was homogeneous within CS-PRP clots.

Our fourth objective was to optimize and reduce the freeze-drying (FD) cycle which has been historically used in our lab from 3 days to 1 or 2 days and to assess performance of the product with benchtop human PRP and human commercial plasma. Our starting hypotheses were that I. Freeze-drying cycle of CS formulations can be optimized to minimize overall freeze-drying time from 3 days to 1-2 days to produce cakes that will not collapse, II. FD cakes prepared with the shorter freeze-drying cycles would be soluble in benchtop human PRP to yield chitosan-PRP formulations which are paste-like, coagulate rapidly, and produce mechanically strong homogenous hybrid clots, and III. Commercial citrated plasma can be used instead of PRP to assess formulation performance. Formulations containing 1% w/v chitosan DDA 82-84% Mn 45-55 kDa with 1% w/v trehalose and 42.2 mM CaCl2 were prepared for freeze-drying. The same methodology was used as described above to assess performance characteristics of the freeze-dried cakes. The cakes that were non-collapsed were solubilized either in citrated pooled normal plasma or benchtop human PRP to assess different performance characteristics. CS-PRP formulations were less runny than their PRP controls and citrated pooled normal plasma. Data from thromboelastography machine revealed clot reaction time was shorter for chitosan-PRP formulation. Also, chitosan-PRP formulations resisted platelet-mediated clot retraction while PRP controls lost up to 80 % of their original mass in the glass tubes. Histological findings showed that chitosan was homogeneously dispersed within CS-PRP clots.

In summary, our feasibility pilot studies in sheep showed promising results in terms of repair of complex defects by injection of chitosan-PRP that could be potentially translated to humans in the future. We refined the animal model and showed that animals could withstand implants for the duration of study with no deleterious effect to the other joint compartment, which suggests high safety. In addition, the chitosan formulations were shown to be compatible with several PRPs isolated with commercial systems. Lastly, optimization of freeze-drying cycles from 3 day to 1 day was successfully performed and formulations behaved as expected when solubilised in PRP or plasma.