Critical size bone defects represent a significant challenge worldwide,often leading to persistent pain and physical disability that profoundly impact patients’quality of life and mental well-being.To address the int...Critical size bone defects represent a significant challenge worldwide,often leading to persistent pain and physical disability that profoundly impact patients’quality of life and mental well-being.To address the intricate and complex repair processes involved in these defects,we performed single-cell RNA sequencing and revealed notable shifts in cellular populations within regenerative tissue.Specifically,we observed a decrease in progenitor lineage cells and endothelial cells,coupled with an increase in fibrotic lineage cells and pro-inflammatory cells within regenerative tissue.Furthermore,our analysis of differentially expressed genes and associated signaling pathway at the single-cell level highlighted impaired angiogenesis as a central pathway in critical size bone defects,notably influenced by reduction of Spp1 and Cxcl12 expression.This deficiency was particularly pronounced in progenitor lineage cells and myeloid lineage cells,underscoring its significance in the regeneration process.In response to these findings,we developed an innovative approach to enhance bone regeneration in critical size bone defects.Our fabrication process involves the integration of electrospun PCL fibers with electrosprayed PLGA microspheres carrying Spp1 and Cxcl12.This design allows for the gradual release of Spp1 and Cxcl12 in vitro and in vivo.To evaluate the efficacy of our approach,we locally applied PCL scaffolds loaded with Spp1 and Cxcl12 in a murine model of critical size bone defects.Our results demonstrated restored angiogenesis,accelerated bone regeneration,alleviated pain responses and improved mobility in treated mice.展开更多
Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different gen...Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome.Based on Tn5,the Assay for Transposase-Accessible Chromatin using sequencing(ATAC-seq)and the Cleavage Under Targets and Tagmentation(CUT&Tag)were developed to measure chromatin accessibility and detect DNA–protein interactions.These methodologies can be applied to large amounts of biological samples with low-input levels,such as rare tissues,embryos,and sorted single cells.However,fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly.Furthermore,inappropriate data analysis can generate biased or misleading conclusions.Therefore,it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools,many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing(ChIP-seq)data.Here,we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data.We compared the sensitivity,specificity,and peak width distribution for both narrow-type and broad-type peak calling.We also tested the influence of the availability of control IgG input in CUT&Tag data analysis.Finally,we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data.Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies,which were implemented into Docker/Singularity images for streamlined data analysis.展开更多
基金supported by the following NIH grants:R01 grants(AR075860,AR077616,and AR083900 to JSHL138175,HL164062,and DK133949 to JG)and a R21 grant(AR077226 to JS)a P30 Core Center grant(AR074992 to the Musculoskeletal Research Center at Washington University in St.Louis).
文摘Critical size bone defects represent a significant challenge worldwide,often leading to persistent pain and physical disability that profoundly impact patients’quality of life and mental well-being.To address the intricate and complex repair processes involved in these defects,we performed single-cell RNA sequencing and revealed notable shifts in cellular populations within regenerative tissue.Specifically,we observed a decrease in progenitor lineage cells and endothelial cells,coupled with an increase in fibrotic lineage cells and pro-inflammatory cells within regenerative tissue.Furthermore,our analysis of differentially expressed genes and associated signaling pathway at the single-cell level highlighted impaired angiogenesis as a central pathway in critical size bone defects,notably influenced by reduction of Spp1 and Cxcl12 expression.This deficiency was particularly pronounced in progenitor lineage cells and myeloid lineage cells,underscoring its significance in the regeneration process.In response to these findings,we developed an innovative approach to enhance bone regeneration in critical size bone defects.Our fabrication process involves the integration of electrospun PCL fibers with electrosprayed PLGA microspheres carrying Spp1 and Cxcl12.This design allows for the gradual release of Spp1 and Cxcl12 in vitro and in vivo.To evaluate the efficacy of our approach,we locally applied PCL scaffolds loaded with Spp1 and Cxcl12 in a murine model of critical size bone defects.Our results demonstrated restored angiogenesis,accelerated bone regeneration,alleviated pain responses and improved mobility in treated mice.
基金supported by the National Institutes of Health,USA(Grant Nos.R35GM142917 to Bo Zhang,U24ES026699 to Bo Zhang,R25DA027995 to Bo Zhang,R03AG070474 to Guoyan Zhao,R01NS123571 to Guoyan Zhao,U19NS130607 to Guoyan Zhao,and U24HG012070 to Bo Zhang and Guoyan Zhao)National Institutes of Health,USA.
文摘Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome.Based on Tn5,the Assay for Transposase-Accessible Chromatin using sequencing(ATAC-seq)and the Cleavage Under Targets and Tagmentation(CUT&Tag)were developed to measure chromatin accessibility and detect DNA–protein interactions.These methodologies can be applied to large amounts of biological samples with low-input levels,such as rare tissues,embryos,and sorted single cells.However,fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly.Furthermore,inappropriate data analysis can generate biased or misleading conclusions.Therefore,it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools,many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing(ChIP-seq)data.Here,we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data.We compared the sensitivity,specificity,and peak width distribution for both narrow-type and broad-type peak calling.We also tested the influence of the availability of control IgG input in CUT&Tag data analysis.Finally,we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data.Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies,which were implemented into Docker/Singularity images for streamlined data analysis.
