Integrating spatial and single-cell transcriptomics to characterize mouse long bone fracture healing process
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ABSTRACT:
PROVIDER: PRJNA1262827 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR33558582_1.fastq.gz | Fastqsanger.gz | |||
| SRR33558582_2.fastq.gz | Fastqsanger.gz | |||
| SRR33558583_1.fastq.gz | Fastqsanger.gz | |||
| SRR33558583_2.fastq.gz | Fastqsanger.gz | |||
| SRR33558584_1.fastq.gz | Fastqsanger.gz |
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Project description:Bone fracture healing is a complex and dynamic processhat requires a detailed understanding of the cellular interactions crucial for successful tissue regeneration. We employs optimized spatial transcriptomics to precisely delineate the locations and interactions of these cells within a mouse femur fracture model at day 0 before fracture and days 5, 15 post-fracture. We improved RNA quality significantly by optimizing our decalcification method using Morse's solution, coupled with the use of the Visium CytAssist platform and integrated analyses through the Seurat, CARD, and Monocle frameworks. This approach allowed us to accurately localize critical cell populations, such as periosteum progenitor cells, and identify pivotal transcription factors that regulate their activation and differentiation into chondrocytes or osteogenic cells. We particularly focused on the transformation from mesenchymal progenitor cells (MPCs) to regenerative MPCs (rMPCs), revealing how these cells recruit macrophages near the fracture line during early healing stages and their involvement in fracture healing. Furthermore, using CellChat, we explored potential receptor-ligand pathways that mediate these cellular interactions. This spatial-temporal mapping and molecular characterization substantially deepen our understanding of the cellular and molecular processes involved in fracture healing, highlighting spatial transcriptomics as a robust approach for elucidating the fundamental mechanisms governing bone regeneration.
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Project description:Genome-wide comparative gene expression analysis of callus tissue of osteoporotic mice (Col1a1-Krm2 and Lrp5-/-) and wild-type were performed to identify candidate genes that might be responsible for the impaired fracture healing observed in Col1a1-Krm2 and Lrp5-/- mice. To investigate bone healing in osteoporosis, we performed fracture healing studies in wild-type mice (C57BL/6 genetic background) and the low bone mass strains Col1a1-Krm2 and Lrp5-/- (Schulze et al., 2010; Kato et al., 2002). Osteotomy was set in femora of female mice and stabilized by a semi-rigid fixator to allow fast bone healing (RM-CM-6ntgen et al., 2010). 21 days post surgery we analyzed the fracture calli by biochemical/histological methods, as well as micro-computed tomography, and observed impaired fracture healing in Col1a1-Krm2 and Lrp5-/- mice in comparison to wild-type. To identify genes that may be responsible for the impaired healing in osteoporotic mice, we performed microarray analysis of three independent callus samples of each genotype. The callus tissue was taken 10 days after surgery, because extensive bone formation took place at this point.
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Project description:Fracture non-union is a complex clinical condition affecting many patients worldwide; however, known risk factors alone cannot help in predicting individual risk of progressing towards healing complications. The identification of novel biomarkers in the serum of fracture patients is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) that could correlate to the process of fracture healing. Toward this aim, serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at different times after injury. The results were correlated to miRNA that were found to be differentially secreted in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenesis.
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Project description:Phosphate is essential for healthy bone growth and plays an essential role in fracture repair. Although phosphate deficiency has been shown to impair fracture healing, the mechanisms involved in impaired healing are unknown. More recently, studies have shown that the effect of phosphate deficiency on the repair process varied based on the genetic strain of mice, which is not characterized. We used data from microarrays to (1) determine the effects of phosphate restriction on the biologic functions identified from the gene expression in fracture calluses; and (2) examine whether there are genetic differences within the primary biologic functions.
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Project description:Traumatic brain injury (TBI) accelerates fracture healing, but the underlying mechanism remains largely unknown. Accumulating evidence indicates that the central nervous system plays a pivotal role in regulating immune system and skeleton, however, the impact of TBI on hematopoiesis commitment was overlooked. Here, we found that the dramatically elevated sympathetic tone accompanied with TBI-accelerated fracture healing; chemical sympathectomy blocks TBI-induced fracture healing. Importantly, the adrenergic hypersensitivity swiftly skews bone marrow hematopoietic lineage cells toward anti-inflammation myeloid cells within 14 days, which favor fracture healing. Knockout of β3- or β2-adrenergic receptors (ARs) eliminate TBI mediated anti-inflammation macrophage expansion and TBI-accelerated fracture healing. Moreover, β3- and β2-ARs agonists synergistically promote M2 macrophages infiltration in callus and accelerate bone healing process. Our results suggest that TBI shapes the anti-inflammation environment during early stage of fracture healing, implicating the sympathetic nerve system as a potential target that can be exploited to treat fracture.
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Project description:Sprague-Dawley rats were placed on an ethanol-containing or pair-fed Lieber and DeCarli diets for 4 wks prior to surgical fracture. Following insertion of a medullary pin, a closed mid-diaphyseal fracture was induced using a Bonnarens and Einhorn fracture device. At 3 days post-fracture, the region of the fracture calluses were harvested from the right hind-limb. RNA was extracted and microarray analysis was conducted against the entire rat genome to study the effects of alcohol-consumption on the fracture healing. The experiments were on four rat subjects, i.e., pair-fed rats with subsequent surgical fracture or no surgical fracture, and alcohol-fed rats with subsequent surgical fracture or no surgical fracture. Each rat subject described above has three replicates so 6 kinds of pairing can be made and each pairing has a dye-swap replicate (thus, a total 12 array experiments). The focus of this study is on the pair-fed fracture subject vs. alcohol-fed fracture subject.
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