Osteal macrophages as therapeutic targets for fracture repair

Abstract

Osteoporotic fracture results in disability, increased morbidity and even mortality. There is currently no broadly applicable pro-regenerative therapy for osteoporotic fractures. A growing body of evidence implicates osteal tissue macrophages (osteomacs) as key contributors to physiological bone dynamics particularly through their support of osteoblast-mediated bone anabolism. This new paradigm exposed the possibility that osteomacs could be therapeutically targeted to enhance bone regeneration. To investigate this, it is imperative to improve understanding regarding osteomac function in bone growth, turnover, repair and pathology. Macrophage contribution to postnatal bone development was further elucidated in this thesis using Csf1r knockout (Csf1rko) rats. Mapping of skeletal changes postnatally through immunohistochemistry and micro-computed tomography (micro-CT) revealed contribution of osteomacs/macrophages to formation of secondary ossification centres and ossification of cranial vault and suture. However, while this rat model was informative, it did not fully delineate macrophage and osteoclast contributions given both cells are Csf1r-dependent. The inability of experimental approaches to specifically target macrophages without undesired impacts on osteoclasts has been a major limitation in studies investigating macrophage contributions to bone dynamics. This limitation was next addressed. Flow cytometry of enriched periosteal and endosteal cell suspensions revealed that CD169 (Siglec 1) is expressed by both periosteal and endosteal osteomacs. Targeted depletion using CD169-diphtheria toxin receptor (DTR) knock-in mouse model demonstrated significant loss of endosteal osteomacs and concomitant loss of osteoblasts but had no effect on osteoclasts. Preclinical bone injury and fracture models were employed to assess CD169+ osteomac function during intramembranous- and endochondral-mediated bone healing respectively. CD169+ osteomac/macrophage depletion during the early healing phase of intramembranous ossification resulted in significant reduction in new collagen type I+ woven bone deposited within the injury site. Similarly, CD169+ osteomac/macrophage depletion during the early inflammatory phase of fracture healing decreased callus size, representing a delayed healing response. These findings provide compelling evidence that CD169+ macrophages/osteomacs, independent of osteoclasts, have vital pro-anabolic contributions during bone repair mechanisms.Based on this new knowledge, whether CD169 expression by macrophages influenced bone homeostasis was next investigated. Micro-CT assessment of CD169-knockout mice showed that loss of CD169 resulted in a low bone mass phenotype. This was the first demonstration that loss of expression of a single macrophage-specific molecule was sufficient to cause osteopenia. This led to the hypothesis that macrophage dysfunction could have a role in osteoporosis-induced bone loss. To investigate this, an ovariectomy (OVX)-induced model of post-menopausal osteoporosis in C3H/HeJ mice, which recapitulated features of human post-menopausal osteoporosis, was validated. Interestingly, osteomac frequency was increased on both trabecular and endocortical bone post-OVX. Dual F4/80 antigen (pan-murine macrophage marker) and tartrate resistant acid phosphatase (TRAP) activity staining demonstrated TRAP+ vesicles present within F4/80+ osteomacs, suggesting a role for osteomacs in phagocytosing extracellular TRAP. Additionally, CD169+ osteomac/macrophage depletion in vivo using CD169-DTR mice was associated with elevated TRAP+ deposits in the bone marrow and serum TRAP, suggesting that in the absence of bone-associated macrophages, combined with unchanged osteoclast function (static measure of TRAP+ osteoclasts/bone surface, TRAP staining area and trabecular bone area), TRAP liberated into the bone marrow interstitium during bone resorption was leaching into the serum. These data demonstrate a role for osteomacs in supporting bone resorption through sequestering resorption by-products and the observed increase in osteomacs post-OVX was likely a compensatory mechanism associated with elevated bone resorption.The observations generated thus far provided justification for assessing whether enhancing osteomacs/macrophages function is a valid strategy for improving fracture healing in healthy and osteoporotic bones. Osteomacs/macrophages can be increased in vivo using macrophage colony stimulating factor 1 (CSF1). Therefore, the utility of CSF1 in promoting pro-regenerative responses was investigated using the chimeric CSF1 molecule, CSF1-Fc, which was previously engineered to achieve improved pharmacokinetics in vivo. Bone catabolic actions of CSF1 particularly in osteoporotic bone is a potential contraindication of CSF1-Fc use for promoting fracture repair. Here, an intermittent CSF1-Fc treatment regimen for 4 weeks that avoided potential dose-limiting indications was optimised. This regimen also enhanced regenerative mechanisms in an internally plated mid-diaphyseal fracture model in healthy bones, culminating in increased fracture strength. To determine whether this treatment strategy can also improve osteoporotic fracture healing, the OVX-operated C3H/HeJ osteoporosis model validated herein was used, and fracture was generated by blunt trauma and stabilised using the MouseScrew intramedullary screw fixation system. Micro-CT assessment of fracture sites showed a reduction in cortical bridging at 5 weeks post-fracture in OVX mice compared to SHAM operated controls, indicative of delayed bone repair. Importantly, weekly CSF1-Fc treatment of OVX mice post-fracture corrected this delayed healing phenotype by significantly improving fracture bridging and strength. Overall, this study considerably advanced understanding on the role of osteomacs during postnatal bone growth, homeostasis, repair and pathology. Notably, this study reveals that targeting macrophages using CSF1-Fc is a promising fracture therapeutic to promote regeneration in both healthy and osteoporotic fracture patients.