Study Of Targeting Bone Marrow Mesenchymal Stem Cells For Inflammatory Bowel Diseases-related Skeletal Disorder And Underlying Mechanisms

Background Inflammatory bowel diseases(IBD)are chronic intestinal diseases caused by inflammation and can affect other organs via the circulation of inflammatory factors.The skeletal disorder is one of the common systemic complications in which increased bone resorption is evidenced by excessive osteoclast activation and elevated serum markers,while studies on bone formation are sparse.Currently,anti-resorptive drugs are applied to ameliorate bone loss,while anabolic therapies are more suitable for alleviating decreased bone mass.Thus,studies on osteogenesis would facilitate the development of anabolic therapies for effective intervention in IBD-related skeletal disorder.Bone formation is mediated by osteoblasts,sharing bone marrow mesenchymal stem cells(BMSC)as the progenitors with adipocytes.Increased inflammatory factors can reduce bone formation by inhibiting BMSC proliferation and osteogenic differentiation,leading to reduced bone mass and quality and accumulation of adipose tissue.However,BMSC fate can be modulated by targeting drug delivery to favor differentiation into osteoblast lineage rather than adipocyte lineage to treat bone loss,which is a promising measure to intervene in IBD-related skeletal disorder.BMSC proliferation and differentiation are associated with angiogenesis,forming the BMSC-endothelium niche.Golgi glycoprotein 1(GLG1)can bind to E-selectin,the ligand expressed on vascular endothelial cells to promote the circulating cells to anchor in the bone.Exosomes are natural extracellular vesicles with ideal biocompatibility and stability and can be modified into targeting drug-loaded nanoparticles based on receptor-ligand interactions.In this project,we determined the changes in bone mass,bone quality,bone formation,and bone marrow fat accumulation in IBD in vivo.Furthermore,we explored the commitment of the osteogenic and adipogenic potential of BMSC in IBD and the underlying mechanism of how inflammation affects BMSC osteogenic differentiation in vitro.Finally,we evaluated the therapeutic effects of BMSC-targeted and drug-loaded nanoparticles derived from exosomes in treating IBD-related skeletal disorder.ObjectivesPart I: To clarify the effects of IBD on trabecular bone,cortical bone,bone mechanical properties,bone marrow adipose tissue,bone formation,osteoblast number,and adipocyte number in the animal model.Part II: To explore the proliferative capacity,osteogenic and adipogenic differentiation potential,and cell signaling pathway changes of BMSC of IBD mice in vitro.To explore the underlying molecular mechanisms of the affected BMSC osteogenic differentiation by simulating BMSC inflammatory microenvironment in vitro.Part III: To construct GLG1 drug-loaded and BMSC-targeted nanoparticles derived from exosomes and evaluate their therapeutic effects on IBD-related skeletal disorder.MethodsPart I:(1)Dextran sodium sulfate and piroxicam were used to establish IBD in mice.(2)Bone mass and bone marrow adipose tissue were examined by μCT.(3)The mechanical properties of the femurs were assessed by mechanical stress testing.(4)Calcein doublelabeling assay was performed to assess the bone formation rate(BFR)and mineral apposition rate(MAR)of the femurs.(5)H&E staining,TRAP staining,and Goldner’s trichrome staining were performed to assess the morphological changes in colons and femurs.(6)Immunofluorescence(IF)staining and immunohistochemical(IHC)staining were performed to evaluate the number of osteoblast and adipocyte in the femurs,respectively.(7)q RT-PCR and Western blotting(WB)were performed to detect the expression of osteogenic genes and adipogenic genes.Part II:(1)q RT-PCR was performed to detect the transcriptional levels of cytokines,osteogenic genes,and adipogenic genes.(2)BMSC was collected from mouse bone marrow by fluorescence-activated cell sorting.(3)Colony formation assay was performed to evaluate the proliferative capacity of BMSC.(4)Alizarin red staining,alkaline phosphatase staining,and Von Kossa staining were performed to evaluate the osteogenic potential of BMSC,while oil red staining was performed to evaluate the adipogenic potential of BMSC.(5)RNA sequencing(RNA-seq)was performed to investigate the differently expressed genes in the cell signaling pathway of the BMSC from mice.(6)Inflammatory microenvironment of BMSC in bone was simulated by TNF-α treatment in vitro.(7)Co-immunoprecipitation was performed to detect β-catenin-p50 binding.(8)Immunoblotting was performed to detect the level of β-catenin and downstream molecules of the NF-κB signaling pathway within BMSC.(9)Luciferase reporter gene assay was performed to assess β-catenin transcriptional activity.(10)Cycloheximide chasing assay was performed to assess the degradation rate ofβ-catenin.(11)Chromatin immunoprecipitation assay was performed to assess the transcriptional activity of the osteogenic genes regulated by β-catenin.Part III:(1)NIH-3T3 cell line overexpressing GLG1 was constructed by lentiviral transfection.(2)The isolation of exosomes was achieved by ultracentrifuge,followed by the method of cyclic extrusion to prepare GLG1 nanoparticles(NP)loading Wnt pathway activator(Wnt agonist 1).(3)Transmission electron microscopy,nanoparticle tracking analysis,and WB were performed to evaluate the exosome features of NP.(4)The organ distribution of NP in vivo was detected by the fluorescence imaging system.(5)IF staining was performed to assess the location of Cy5-labeled NP,vascular endothelium,and BMSC.(6)Bone mass,bone marrow adipose tissue,mechanical properties,BFR,MAR,and the number of osteoblast and adipocytes in the femurs and callus were assessed according to the aforementioned methods.(7)Safranin o staining was performed to evaluate the soft callus area and relative callus mineralization of the femurs.ResultsPart I:(1)The IBD mice manifested shortened colon,weight loss,and infiltration of inflammatory cells in colon tissue.(2)μCT examination showed IBD mice had decreased bone mass,thinner cortical bone,and a higher percentage of adipose tissue volume than control mice.(3)Mechanical stress testing revealed a significantly reduction of mechanical properties of the femurs in IBD mice.(4)Calcein double-labeling assay showed that the BFR and MAR of the femurs in IBD mice were significantly lower.(5)Morphological staining revealed decreased trabecular bone and osteoid but increased adipocytes and osteoclasts in the femurs of IBD mice.(6)IF staining and IHC staining demonstrated fewer osteoblasts but more adipocytes in the femurs of IBD mice,respectively.(7)The results of q RT-PCR and WB revealed the downregulated express of osteogenic genes but the upregulated expression of adipogenic genes in IBD mice.Part II:(1)The results of q RT-PCR revealed elevated transcriptional levels of inflammatory factors in the femurs of IBD mice.(2)BMSC from IBD mice formed fewer colonies,synthesized less alkaline phosphatase and calcium after osteogenic induction in vitro,but generated more cells containing lipid droplets after adipogenic induction.(3)GO analysis,KEGG analysis,and GSEA were performed to analyze the RNA-seq data,indicating the downregulated Wnt/β-catenin but upregulated PPARγ in the BMSC from the bone marrow of IBD mice(p<0.05).(4)In BMSC,TNF-α treatment decreased the binding of β-catenin and p50,the amount of β-catenin,and reduced the transcriptional activity of β-catenin,but increased total p-IKKβ and p-p50,promoted the ubiquitination and degradation of β-catenin.Then,the nuclear β-catenin was decreased to inhibit the transcriptional activity of downstream osteogenic genes,runx2 and osterix.Part III:(1)Transmission electron microscopy,nanoparticle tracking analysis,and WB results revealed that the drug-loaded NP had similar features of exosomes,but the expression of GLG1 on GLG1-NP was higher than that on CTRL-NP.(2)GLG1-NP was more enriched in bone and resided there more than CTRL-NP.(3)Significant alleviations were observed in reduced trabecular bone mass,thinned cortical bone,increased percentage of adipose tissue volume,and reduced mechanical properties in IBD mice treated by GLG1-NP,compared with Vehicle-treated IBD mice.Besides,the femurs of IBD mice treated by GLG1-NP demonstrated higher BFR and MAR,more osteoblasts,and fewer adipocytes than that of Vehicle-treated IBD mice.(4)IBD mice treated by GLG1-NP demonstrated more calcified callus,better mechanical properties,and higher relative callus mineralization than that of Vehicle-treated IBD mice at 21 days after the fracture.Conclusion(1)IBD mice have decreased bone mass and bone quality,reduced bone formation but increased bone marrow adipose tissue.(2)The inflammatory microenvironment of the bone marrow caused by IBD results in decreased proliferation,limited osteogenic differentiation but facilitated adipogenic differentiation of BMSC.The Wnt/β-catenin signaling pathway is significantly downregulated in BMSC,with the underlying mechanism that inflammation activates the NF-κB signaling pathway to increase ubiquitination and degradation of β-catenin.(3)GLG1-NP could target the bone and upregulate the Wnt/β-catenin signaling pathway of BMSC to promote osteogenic differentiation and bone formation,thereby increasing bone mass and bone quality and promoting fracture healing in IBD mice.In summary,we verified decreased bone mass,bone quality,and bone formation in IBD mice,whose BMSC presented suppressed osteogenic differentiation.Targeting BMSC to activate the downregulated Wnt/β-catenin signaling pathway could favor osteogenic differentiation of BMSC and thus promote bone formation to treat IBD-related skeletal disorder.