Mechanisms of Tendon Repair: A Focus on Changes in Matrix Integrity

Tendon injuries are frequent and affect a wide range of people worldwide, including athletes, active working people, and the elderly population. Unfortunately, due to the poor intrinsic healing capacity of tendons, the mechanical and biochemical properties of post-injury tissue often do not restore to pre-injury levels. Tendon injuries result in structural changes that prevent full restoration of the original tendon integrity, often requiring surgical intervention or prolonged rehabilitation to regain function. This is because the highly anisotropic tendon transforms to a more disorganised, isotropic tissue, resulting in inferior mechanical properties. Understanding why the post-injury tendon structure fails to undergo effective remodelling to re-establish its original architecture and function during healing is a complex challenge, as it involves the interplay of numerous biological factors. Identifying the factors behind this aberrant healing process is crucial for developing treatments that ultimately promote functional remodelling. This thesis explores mechanisms and strategies influencing matrix integrity by examining how internal and external cues, including macrophages, nutrient deprivation, physical directional tools, and mechanical modulation of collagenase activity, affect the remodelling abilities of cells and collagen. In Chapter 2, the involvement of the immune system in tissue remodelling is studied. In the remodelling process of tendon healing, macrophages crucially support fibroblasts in the process of extracellular matrix degradation, synthesis, and rearrangement. Despite the relevance of macrophages in guiding fibroblast phenotype and function during tissue homeostasis, remarkably little is known about how the different types of macrophages can directly influence the remodelling properties of fibroblasts in terms of fibroblasts’ ability to reorganise collagen structures. Therefore, paracrine effects, i.e., factors secreted by pro- and anti-inflammatory macrophages, are tested on fibroblasts in their ability to remodel their surrounding collagen matrix. M1 conditioned medium, compared to M2 conditioned medium, was found to decrease the ability of fibroblasts to remodel their surrounding collagen environment. A prolonged presence of M1 macrophage secreted factors may therefore hamper the ability of fibroblast-driven functional tissue recovery after injury. In Chapter 3, the focus shifts to aberrant tendon remodelling in the context of Anterior Cruciate Ligament (ACL) reconstruction. ACL tears are often treated by reconstructive surgery using tendon autografts. Upon implantation, a tendon graft undergoes a remodelling response aimed at resembling a native ACL. However, this process is often aberrant, and the graft fails to fully restore the mechanical and structural properties of the original ligament. In the early remodelling phase, a drop in mechanical integrity is observed, believed to be driven by matrix metalloproteinases (MMPs), specifically collagenases. It is known that the susceptibility of collagen to degradation by collagenases is affected by the amount of static strain that is applied to the tissue. However, it is not known how this susceptibility changes when the tissue is subjected to dynamic strain, as is the case for these grafts in vivo. Therefore, degradation susceptibility of dynamically strained decellularised porcine tendons was assessed in the presence of bacterial collagenases. Static strain, as shown previously, was found to protect collagen from degradation, whereas dynamic strain increased the degradation rate relative to static strain at all strain values, and this effect trended to be less sensitive to strain levels. These results can support in creating optimal surgical strategies with sufficient pre-load and training regimens that prevent graft stress shielding for optimal patient recovery. In Chapter 4, maintaining the focus on ACL reconstruction surgeries, the role of the resident tenocytes on the graft remodelling process was studied. The graft environment is known to vary from the physiological tendon environment, i.e., the post-operative environment is highly nutrient deprived. It was hypothesised that as a consequence of nutrient deprivation, starved or dying cells adopt a catabolic phenotype and negatively affect matrix integrity. Therefore, the effect of nutrient deprivation on cell viability and structure-mechanics properties in reconstituted collagen tissues in-vitro was investigated. Strikingly, even though tenocyte viability was highly affected under nutrient deprivation conditions, structure-mechanics relations were not affected. Only under physiological conditions did the structure-mechanics relations improve. This may imply that cells in an environment similar to early graft healing conditions, do not affect the structure and mechanics of the collagen and are thus not key players in the drop in graft strength observed. Finally in Chapter 5, a direct approach to physically improve tissue anisotropy was explored. Matrix orientation in fibrous tissue is governed by reciprocity between the collagen matrix and contractile cells. Loss of cell polarity (the transition from spindle-shaped to stellate-shaped) promotes fibrotic scarring whereas recovery of polarity (from stellate-shaped to spindle-shaped) limits this process. It was hypothesised that the control of cell shape could dictate the type of tissue remodelling. Collagenous tissues populated with fibroblasts were created that inherently had an isotropic distribution of cells and collagen. Miniature magnetic rods were added to the cell-collagen mixture to create anisotropy by aligning the rods using a magnetic field. In this proof-of-concept study, it was shown that in the presence of magnetically aligned rods, the anisotropy of the cells could be improved. Further developing this tool may therefore become a strategy to support in improving cell and tissue anisotropy and thus function. To conclude, this dissertation explored factors that affect tendon structure-function relations. It was shown that a prolonged M1-macrophage immune response, may hinder functional remodelling. Dynamic strains increased the susceptibility to collagen degradation by collagenases. Adding to current knowledge on the poor reparative capacity of resident tenocytes, it was found that despite the reduced viability of tenocytes in nutrient deprived conditions, they did not affect the collagen structure and function. Lastly, physical directional cues can be used to override the inherent isotropy and promote cell anisotropy. These findings advance understanding of tendon healing and support the development of innovative tools and therapies