Introduction
Allogeneic bone-marrow mesenchymal stem cells (BMSCs) can promote bone regeneration and substitute for autologous BMSC if autologous sources are unavailable, but the efficacy of bone regeneration by allogeneic BMSCs is still inconsistent. This study aims to investigate the potential application of allogeneic BMSCs in bone regeneration via a rat cranium critical-size defect model and compare the outcomes of bone regeneration between allogeneic and autologous BMSC seeded in gelatin-nHAP cryogels.

Materials & Methods
A Lewis rat cranium critical-size defect model was used to investigate the efficacy of bone regeneration between autologous and allogeneic BMSCs in gelatin-nanohydroxyapatite cryogel scaffolds. BMSCs from Wistar rats served as the allogeneic cell lineage. The full-thickness cranium defects were treated by either blank control, cryogel-only, allogeneic BMSC-seeded cryogel, or autologous BMSC-seeded cryogel (n=5). Bone regeneration was monitored by micro computer-tomography and examined histologically at week 12. In addition, we assessed the immune responses in vitro by mixed lymphocyte reaction (MLR) assay and CD4+ immunochemistry staining ex vivo.

Results
Micro-CT axial and sagittal views at week 12 show that both allogeneic and autologous BMSC-seeded cryogel scaffolds demonstrate higher radiopacity and bone regeneration than the cryogel group and control group. At week 4, there was no statistical difference in bone regeneration among groups. The bone regeneration in allogeneic BMSC was sustained throughout week 8 and 12. At week 8, the allogeneic BMSC (61.8 ± 3.9%) , autologous BMSC (62.2 ± 0.1%) and cryogel alone (56.3 ± 5.2%) showed substantially more regeneration than the control group (35.9 ± 8.2%). At week 12, both allogeneic (91.3 ± 1.1%) and autologous BMSC (93.5 ± 5.3%) showed statistically superior regeneration compared to the cryogel group (69.2 ± 2.7%) and control (42.50 ± 3.0). There was no statistical difference in bone regeneration between allogeneic and autologous BMSC at week 8 or 12. To further verify the immune response, we performed immunohistochemistry analysis using CD4 marker because CD4 + but not CD8+ cells are responsible for the initiation of allogeneic transplant rejection. We found no CD4+ lymphocyte infiltration along the regenerated bone at week 12 in either allogeneic or autologous BMSC group, suggesting the absence of CD4+-mediated immune response.

Conclusion
Our study demonstrated that allogeneic BMSC-seeded cryogel scaffolds could effectively induce bone regeneration in cranium defects after 12-weeks of implantation, comparable to autologous BMSC-seeded cryogel scaffold. Further, despite a transient early immune response in allogeneic transplantation, bone regeneration was sustained throughout the 12-week implantation period, while the CD4+-mediated immune response diminished by week 12. These results suggest that allogeneic BSMC is feasible in craniofacial bone regeneration and warrants further investigation for future clinical applications.

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