Expanded Biological Characterization of Autograft-Derived Contributions to Spinal Fusion: Graft-Derived Progenitors Contribute via Endochondral Bone Formation.

Abstract

Background context: Autologous bone graft remains the clinical standard in spinal fusion due to its reliable performance. Characterization of the biological mechanism underlying the clinical superiority of autologous bone graft in spinal fusion remains incomplete. The field is divided between "creeping substitution," which posits the graft as a passive scaffold whose cells perish, and the "cellular hypothesis," which argues for an active contribution from viable donor cells.

Purpose: This study aimed to build upon prior work by further characterizing graft-derived cell survival after transplantation and identify the biological mechanisms by which these cells may contribute to spinal fusion.

Study design: We employed an immunocompetent "sibling-pair" murine posterolateral fusion model (L3-L4) of Iliac bone grafting for this study.

Methods: Autologous Iliac bone grafts from fluorescent reporter mice were transplanted into non-fluorescent siblings. A panel of inducible Cre-lox reporter lines (CAG, Col1a1, Sox9, and Aggrecan) was used to trace the fate of specific graft-derived cell populations. A pre-grafting induction strategy was used to label mature cells at harvest, while a post-grafting induction strategy identified donor progenitor cells that survived and differentiated in vivo. Fusion masses were analyzed at 2- and 6-weeks following grafting via microCT, histology, and fluorescence microscopy.

Results: Pan-cellular (CAG-Cre) tracing suggests robust donor cell survival and integration into the 2 and 6-week fusion masses. In contrast, pre-labeled grafted mature osteoblasts and osteocytes (Col1a1-Cre) were largely absent at all time points, suggesting this population does not meaningfully survive transplantation. Post-grafting induction of Col1a1-Cre mice revealed the emergence of numerous new, donor-derived osteoblasts at 2 and 6 weeks, suggesting their origin from an unlabeled progenitor pool. Additionally, post-grafting induction of chondrocyte-lineage reporters (Sox9-Cre, Aggrecan-Cre) demonstrated the appearance of donor-derived chondrocytes at 2 weeks, which subsequently transitioned into osteocytes within the mature bone by 6 weeks suggesting endochondral ossification.

Conclusions: These findings builds on prior work supporting the "cellular hypothesis" which argues that autologous bone grafts contribute viable cells. Furthermore, our findings suggest a model of " Skeletal Stem and Progenitor Cells (SSPC)-driven Adaptation," where the ICBG serves as a vehicle for resilient Skeletal Stem and Progenitor Cells (SSPCs). These progenitors, rather than mature osteoblasts, appear to survive transplantation and adapt to the avascular fusion bed by initiating endochondral ossification to form the fusion mass.

Clinical significance: This suggests that the clinical focus on mature "osteogenic" cells may be complemented by consideration of skeletal stem and progenitor cell (SSPC) populations. Autograft quality may be defined, in part, by its SSPC content. This provides an additional potential benchmark for evaluating autograft quality and developing cell-based biologics. Furthermore, these findings have potential implications for intraoperative decisions regarding graft handling and harvesting to preserve SSPC-rich regions such as periosteum.