Differential dynamics of bone graft transplantation and mesenchymal stem cell therapy during bone defect healing in a murine critical size defect

Abstract

Background: A critical size bone defect is a clinical scenario in which bone is lost or excised due to trauma, infection, tumor, or other causes, and cannot completely heal spontaneously. The most common treatment for this condition is autologous bone grafting to the defect site. However, autologous bone graft is often insufficient in quantity or quality for transplantation to these large defects. Recently, tissue engineering methods using mesenchymal stem cells (MSCs) have been proposed as an alternative treatment. However, the underlying biological principles and optimal techniques for tissue regeneration of bone using stem cell therapy have not been completely elucidated.

Methods: In this study, we compare the early cellular dynamics of healing between bone graft transplantation and MSC therapy in a murine chronic femoral critical-size bone defect. We employ high-dimensional mass cytometry to provide a comprehensive view of the differences in cell composition, stem cell functionality, and immunomodulatory activity between these two treatment methods one week after transplantation.

Results: We reveal distinct cell compositions among tissues from bone defect sites compared with original bone graft, show active recruitment of MSCs to the bone defect sites, and demonstrate the phenotypic diversity of macrophages and T cells in each group that may affect the clinical outcome.

Conclusion: Our results provide critical data and future directions on the use of MSCs for treating critical size defects to regenerate bone.Translational Potential of this article: This study showed systematic comparisons of the cellular and immunomodulatory profiles among different interventions to improve the healing of the critical-size bone defect. The results provided potential strategies for designing robust therapeutic interventions for the unmet clinical need of treating critical-size bone defects.

Keywords: Bone graft; Critical-size bone defect; CyTOF; Macrophages; Stem cells; T cells.

Fig. 1

Murine critical-size bone defect model and sample collections from bone defect sites. A) 2 ​mm critical-sized defect has been created in the mouse femur and stabilized with an external fixation device. B) Bone graft harvested from iliac crest (left) and microribbon (μRB) scaffold (middle) embedded with GFP-labeled MSCs (right) are ready for transplantation. C) Tissue samples containing bone graft (top panel) or μRB scaffold (bottom panel) collected from bone defect sites.

Fig. 2

Diagram of experimental design and bone defect model in mice. In each experiment, we first applied the external fixation device onto the left femur, created the 2 ​mm femoral midshaft diaphyseal bone defect, closed the wound, applied various treatments 4 weeks later after the nonunion was established, and then subsequently harvested the tissue in the defect 1 week later.

Fig. 3

Distinct cell compositions among tissues from bone defect sites compared with original bone graft. A) Frequency of major cell subsets. B) viSNE plots colored according to phenotypic marker expression. C) viSNE plots depicting major cell populations identified based on phenotypic marker expression. D) The frequency of cell populations depicted in C was quantified.

Fig. 4

Active Recruitment of MSCs to the Bone Defect Sites. A) Contour plots of MSCs population (Sca1+, CD44+) in 3 groups. B) Quantification of MSCs percentages versus total population and versus non-leukocytes. C) Validation of GFP and CD90.2 as biomarkers to distinguish recruited cells and implanted cells. D) Proportions of recruited MSCs versus implanted MSCs in 2 groups of bone defect tissues. E) Quantification of recruited MSCs percentages versus total population and versus non-leukocytes.

Fig. 5

Heterogeneity of macrophages. A) Different proportion of macrophage subtypes among three groups of samples. B) FlowSOM figures demonstrate the heterogeneity of macrophages among three groups. C) Activation of NF-κB signaling pathway in MSCs and M2 macrophages from the BD tissue containing bone graft.

Fig. 6

The Composition of T Cells is Distinctively Different among Bone Defect Tissues with Different Transplants. A) CD90.2 expression level shows the originality of the T cells. B) FlowSOM analysis demarcates distinct T cell subsets among groups. C) Comparison of T-cell subpopulation in percentage. D) Heatmaps of functional markers in T-cell subtypes among groups.