Multi-omics profiling of bone regeneration in diabetes reveals association of increased mast cell activity with healing impairment

Introduction Conventional treatments for critical-sized bone defects are often insufficient in individuals comorbid with Type 2 diabetes mellitus(T2DM) due to their increased risk of prolonged healing and complications. To optimize personalized treatments based on bioactive and resorbable biomaterial-guided bone regeneration, a comprehensive understanding of the molecular mechanisms involved is essential. This study investigates the progression of diabetic bone healing over 42 days through imaging and multi-omics analyses of tissue as well as plasma proteomics for systemic evaluation.

Methods We developed the first sequential multi-omics extraction protocol for bone tissue and regenerating callus. Explants of biomaterial-supported regenerated and contralateral tissue after 21- or 42-days post-implantation from a diabetic rat model with a critical-size femur defect were extracted successively for their metabolome and proteome. In addition to analysis of central carbon metabolites using GC-MS, quantitative targeted metabolomics was performed using a mixture of flow injection analysis and LC-MS applying the Biocrates Quant MxP500 kit. TMT-based LC-MS/MS proteomic analyses of the same explants were conducted on a quadrupole-Orbitrap MS. Differential expression profiles of proteins and metabolites were determined by pairwise comparisons, providing a holistic view of molecular changes occurring during the healing process. Finally, results were integrated with imaging information from µCT scans and histological data to correlate molecular findings with clinical outcomes.

Results Employing this novel multi-omics extraction protocol on regenerating bone callus enabled the quantification of about 4000 proteins and over 500 metabolites, thus providing an in-depth perspective on bone healing under diabetic conditions. Integration of multi-omics with histological data revealed several key insights, including decreased levels of structural proteins essential for soft callus formation, a disturbed metabolism considering main energy production pathways (glycolysis, TCA cycle), and extended inflammatory processes. Furthermore, we discovered important links highlighting an imbalanced population of mast cells (MC) within the regenerating tissue of diabetic individuals. This imbalance was characterized by clusters of MC proteases, elevated levels of the MC mediator histamine, and increased MC presence, rendering MCs a potential target population to address in compromised diabetic bone healing.

Conclusion This study demonstrates the performance of a new multi-omics extraction protocol for bone, providing critical insights into molecular disruptions in diabetic bone regeneration. It underscores the importance of multi-omics approaches for deep molecular profiling to understand T2DM's negative impact on bone healing. Our ongoing preclinical study will evaluate interventions targeting the observed dysregulated mast cell activity to enhance bone regeneration.