"<div>Endochondral bone regeneration (EBR) recapitulates natural development of long bones during embryogenesis and fracture repair through implantation of a cartilaginous template into a defect, which is eventually remodeled into bone. Typically, autologous multipotent mesenchymal stromal cells (MSCs) are exploited to generate such cartilage constructs. However, the use of patient-own cells is associated with donor-site morbidity and donor-to-donor variability with respect to chondrogenic differentiation potential. Non-autologous MSCs are a promising alternative and offer an off-the-shelf solution due to pre-selection of chondrogenically potent donors, but could possibly cause immune rejection . Previously, we demonstrated that by devitalization of allogeneic cartilage constructs, an adverse immune reaction was avoided, and full defect bridging of a critical size rat femur defect was achieved . The next step to bring this approach into the clinic is the transition from small animal research to pre-clinical studies in large animals. However, successful bone regeneration through EBR using allogeneic constructs has not been achieved in large animal models before. This study aimed at proof of concept of devitalized, allogeneic cartilage constructs for EBR in a critical size defect in a large animal model.
Goat MSCs (gMSCs) were isolated from the iliac crest of Dutch Milk goats, encapsulated within a collagen hydrogel and differentiated in chondrogenic medium for 28 days, followed by devitalization . Constructs were implanted into bilateral, critical size iliac crest bone defects. Each defect was divided into three equal compartments by a titanium spacer. Experimental groups consisted of allogeneic devitalized cartilage constructs. Controls contained gold standard bone autograft, empty carrier control and empty defect (n = 6 for each group). One and two months post-implantation, fluorochromes (calcein and oxytetracycline) were administered intravenously to mark sites of active bone formation at the time of administration. Animals were sacrificed 3 months post-implantation and explanted samples were evaluated for mineralization and new bone formation via microCT, histology, histomorphometry and presence of fluorochromes.
Preliminary results demonstrate the feasibility of EBR using allogeneic cartilage constructs in a large animal model for the first time. Moreover, a critical size defect at the cm3-scale was regenerated by a cell-based implant. Devitalized allogeneic constructs induced more bone formation than the empty (2x more) and carrier (1.5x more) control. Further, the constructs induced new bone formation comparable to the gold standard autograft, as shown by microCT and histological analysis. These findings indicate that our novel approach can achieve bone regeneration, at least comparable to bone autograft, without associated drawbacks such as donor-site morbidity and a second surgical intervention. As this is the first time an allogeneic EBR approach has performed successfully in a pre-clinical large animal model, these results contribute to the clinical translation of EBR in the form of an off-the-shelf product. Further, these results allow us to take the next steps towards clinical translation of our approach by evaluating its safety and efficacy in further pre-clinical models, before proceeding to first in-man trials.
1 Longoni, A. et al., Frontiers in bioengineering and biotechnology 8, 651 (2020).
2 Longoni, A. et al., Advanced science (2021).</div>"