Fracture Repair in Aging: Identifying networks by miRNA and mRNA co-sequencing

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There is an urgent clinical need to develop new bone regenerative therapeutics to treat delayed and non-union fractures, and critical sized bone defects. Moreover, aging results in severely impaired skeletal repair. A critical aspect of fracture healing begins with expansion of periosteal progenitors, which are activated immediately after injury and then differentiate into bone forming osteoblasts and chondrocytes. However, the molecular mechanisms controlling their activation, expansion, and differentiation in response to injury remains limited. microRNA (miRNA) mediated post-transcriptional regulation of gene expression plays a key role in the commitment and differentiation of stem cells toward the osteoblastic or chondrogenic lineages. Since a single miRNA can potentially target hundreds of distinct mRNAs, identifying the gene expression networks controlled by miRNAs continues to represent a significant gap in our understanding of skeletal biology and repair. Differences in miRNA expression can be observed between young and old bone, and likely mediate some of the impact of aging on fracture repair. Non-biased whole transcriptome analyses have revealed important information about gene expression programs active in the resting periosteum and in healing fractures. Studies utilizing RNA extracted from whole bone have documented changes in either mRNA or miRNAs in these tissues. However, single cell RNA sequencing studies readily demonstrate the cellular heterogeneity present in periosteum and in regenerating bone, making less clear the interpretation of bulk RNAseq studies in these complex tissues. The ability to observe coordinated mRNA-miRNA networks in individual cells during fracture repair will provide paradigm- shifting information that could be used to enhance repair. Further, local delivery of miRNA-based therapeutics represents an exciting novel strategy for facilitating bone healing in the elderly. A thorough understanding of the mRNA-miRNA networks active in each cell type in the healing bone will provide critical rationale for miRNAs to target in a therapeutic setting. We propose to perform single cell mRNA-miRNA co-sequencing on non-hematopoietic lineage cells isolated from resting periosteum and periosteum activated by fracture, comparing young and aged mice. This will allow us to identify expression-based cell subpopulations and mRNA-miRNA interaction networks within those populations, and how these are modulated during fracture repair and in aging. Protocols for single cell miRNA- mRNA co-sequencing were only recently developed. The complex micro-environment of fracture repair is an ideal application for this new technology, which will fill a significant void in our understanding of skeletal biology and provide a rationale for further investigation of miRNA-based therapeutics for bone repair.