Deciphering Molecular Mechanisms of Calvarial Bone Regeneration with Oxygen Supplementation

Introduction: Tissue-engineered oxygen-releasing scaffolds have been shown to facilitate calvarial bone regeneration, but our understanding of the underlying mechanisms involved in remodeling of the bone healing microenvironment has been limited. Recently, we developed a multimodality in vivo imaging workflow to characterize angiogenesis and osteogenesis in real-time, which revealed that robust angiogenesis correlated with better bone healing outcomes in the presence of an oxygen-releasing scaffold. However, the underlying mechanisms via which oxygen-supplementation results in a denser vascular network, especially given that hypoxia is a known driver of angiogenesis, remain unresolved. Therefore, in this follow-up work, we report our most recent findings from quantifying the differential effects of 3D-printed control (PCL) or oxygen-releasing scaffolds (PCL-CPO) on bone regeneration, using a combination of molecular markers and quantitative microscopy. We hypothesized that PCL-CPO enhanced bone healing by promoting osteogenic cell proliferation and facilitating an early shift of the macrophage phenotype from M1 to M2.

Materials and

Methods: Twenty 10-week-old female C57BL/6 mice were anesthetized, and a 4-mm calvarial defect was created on the parietal bone of each mouse. PCL or PCL-CPO scaffolds (4-mm diameter × 0.4-mm thick) were implanted into the calvarial defects and harvested at the end of week (W) 1 and W8 post implantation. Prior to harvesting, the mice were intraperitoneally injected with 5 mg/kg EdU 24 hours before, and with 100 mg/kg hypoxyprobe™ 1 hour before. The harvested calvaria samples were frozen and sectioned at 15 μm thickness. Sections around the middle of each calvarial defect were saved for subsequent immunofluorescent analysis. For detecting cell proliferation, a click-reaction utilizing the Click-iT™ EdU kit was performed, with Alexa Fluor 488 azide bonding to the EdU alkyne. Slides underwent blocking and permeabilization before immunostaining. FITC-conjugated rat IgG1 monoclonal antibody was used for hypoxyprobe detection, along with DAPI counter-staining. For immune cell characterization, primary antibodies (CD45, F4/80, CD206, and iNOS) were applied, followed by appropriate secondary antibodies along with DAPI counter-staining. Slides were imaged on a Zeiss Z2 Axio Observer with Apotome. All staining was performed with n = 3–5 per group and 2-4 slides imaged per sample. Two-way ANOVA was used for statistical analysis with p-values < 0.05 considered statistically significant.

Results, Conclusions, and Discussions: At the end of the first week (i.e. W1), we observed significantly higher hypoxia near the bone defect in the control PCL group (black) compared to the oxygen-releasing PCL-CPO group (white), revealing the effectiveness of the PCL-CPO scaffold in reducing hypoxia by (Fig. 1a, b, e). At the end of W8, both groups exhibited similar hypoxia levels, with greater hypoxia observed in the bone marrow region, and no significant hypoxia detected in the defect region (Fig. 1c-e). Furthermore, we observed significantly higher cell proliferation in the oxygen-release group at early and late stages of healing (Fig. 1f-j). Additionally, we investigated immune cell infiltration. Quantitative analysis of the CD45 general immune cell marker did not reveal significant differences between groups at all time points (Fig. 2a-d, m). However, subsequent analysis focused on macrophages (Fig. 2e-l) showed significantly lower macrophage (F4/80) infiltration (Fig. 2n, q), reduced presence of M1 (iNOS) macrophages (Fig. 2o), and a lower fraction of M1 phenotype within the overall macrophage population (Fig. 2p) in the PCL-CPO group at early and late stages of bone healing. In contrast, the PCL-CPO group exhibited more M2 (CD206) macrophages at W1 and fewer at W8 (Fig. 2r), with the percentage of this phenotype within the overall macrophage population being higher at W1 in the PCL-CPO group and similar at W8 for both groups (Fig. 2s). Our results validate the hypothesis that oxygen-releasing scaffolds influence immune cell response and facilitate the early transition of proinflammatory M1 macrophages to the anti-inflammatory M2 phenotype. This shift toward M2 macrophages promotes angiogenesis and osteogenesis, enhancing bone healing. Motivated by these findings, our next steps involve employing flow cytometry to characterize a broader array of immune cells, thereby delving deeper into the impact of oxygen delivery on immune cell trafficking during healing. In conclusion, our study lays the crucial groundwork for understanding the mechanisms via which oxygen-releasing scaffolds enhance bone regeneration. By uncovering these mechanisms, we aim to advance strategies in regenerative orthopedic medicine and promote the therapeutic efficacy of oxygen-releasing scaffolds for treating bone defects.

Acknowledgements (Optional): This work was supported by NIH grant nos. 2R01CA196701-06A1, 5R01CA237597-04 and and 5R01DE027957-05.