Autologous bone grafting is currently the gold standard treatment for bone defects. However, it is still associated with multiple drawbacks such as limited availability and donor site morbidity. Various approaches have been explored to overcome this, of which endochondral bone regeneration (EBR) has emerged as a promising approach. EBR aims to mimic the process where bone is remodeled from a cartilage template, which naturally takes place in a fracture callus. Clinical translation of EBR would benefit from creating an off-the-shelf product consisting of donor-derived (allogeneic) and devitalized tissues. We have previously shown that such allogeneic devitalized cartilage tissues display accelerated full-bridging of a femoral defect in immunocompetent rats . However, there is still a need to fully elucidate the interplay between the immune response and new bone formation. To explore this further, the early and late immune response elicited by vital and devitalized cartilage derived from autologous or allogeneic cells were compared in a subcutaneous rat model.
Rat multipotent mesenchymal stromal cells (rMSCs) were isolated from the bone marrow of Brown Norway (BN; autologous/syngeneic) and Dark Agouti (DA:allogeneic) rats. rMSCs were encapsulated within collagen hydrogel spheroids and chondrogenically differentiated for 28 days, followed by devitalization . Two chondrogenic spheroids per group were implanted into subcutaneous pockets in BN rats (n=7) for 3,7,14,28 and 84 days. New bone formation and immune response were analyzed via micro-CT, histology (H&E, histomorphometry) and immunohistological staining (CD68,iNOS,CD206,CD3,myeloperoxidase).
Results and Discussion
Histological analyses revealed that the vital autologous spheroids were remodeled into bone including marrow cavities within 28 days. At this time, remnants of non-remodeled cartilage were still observed for the vital allogeneic and both devitalized groups. After 84 days, 5/7 samples from the vital autologous group could not be retrieved possibly due to resorption. Histomorphometry analyses from day 28 and 84 revealed no significant differences in bone formation or cartilage between groups (excluding vital autologous due to reduced sample size). The onset of bone formation appears to positively correlate with the presence of osteoclasts on day 14 in the vital autologous and both devitalized groups. Presence of osteoclasts in the vital autologous and both devitalized groups on day 84 indicate that active remodeling is still taking place. Initial analyses of the immune response revealed no significant differences between groups in terms of presence of macrophages (CD68, CD206 or iNOS) or T lymphocytes in the tissue surrounding the implants on day 3, 7 or 14. Further analysis of later timepoints and different immune cells are still on-going.
No differences in bone formation was observed between all groups. However, the vital autologous group demonstrated the fastest bone formation, most of which were resorbed by 84 days. Early analysis indicate that the onset of bone formation coincides with the presence of osteoclasts from as early as 14 days. Additional analyses are currently ongoing to elucidate the interplay of immune cells and bone formation at later time points.
1. Longoni, A. et al (2021).Acceleration of Bone Regeneration Induced by a Soft-Callus Mimetic Material.Adv Sci.mDOI:10.1002/advs.202103284