A Study On Individualized Treatment Of Osteogenesis Imperfecta

Objective:Osteogenesis imperfecta(OI)is an autosomal dominant disease characterized by bone fragility,deformity and fracture susceptibility.OI is usually classified into types?to?according to the clinical manifestations.It is most commonly associated with mutations in COL1A1/COL1A2 genes encoding type?collagen.Type?OI patients perform decreased collagen quantity but normal collagen structure,with mild symptoms and the largest amount.Type?OI patients manifest abnormal collagen structure,with the most serious phenotype in survival patients.Currently,the single clinical treatment methods have large differences in the efficacy of various types of OI and certain side effects.In the present study,we applied two strategies for type?and?OI treatment according to their different causes and explored the related mechanism.Briefly,the approaches included replenishing the normal type?collagen for type?OI and removing the abnormal structure of type?collagen,which could provide reference for individualized treatment of OI.Methods:1.Combined use of NELL1 and adipose-derived mesenchymal stem cells(ADSCs)in OI type?treatment:(1)The expression level of osteogenic genes of MC3T3-E1 and ADSCs stimulated by recombinant mouse NELL1 protein(rm NELL1)were estimated by real-time PCR.ALP straining and activity were performed to evaluate the capacity of osteogenic differentiation.Lentiviral vector carrying mouse Nell1 gene was constructed.The expression of osteogenic genes and the osteogenic differentiation ability of early and late stage in vitro were explored in Nell1 gene modified ADSCs.(2)Adult male OI type I mice Col1a1^+/-365 with single Col1a1 gene knockout were randomly divided into five groups which received intravenously injected PBS,rm NELL1,ADSCs,rm NELL1 combined with ADSCs,or lenti-Nell1-ADSCs,respectively.Wildtype(WT)littermates served as normal control.Micro-CT was used to analyze the microstructures of the femurs.H&E and immunohistochemical staining were used to evaluate the bone formation in each group.Immunofluorescence staining was used to track the localization and differentiation of ADSCs settled in femurs to learn the therapeutic mechanism.2.Enhancing autophagy level to clear abnormal collagen to treat OI type?:(1)Mouse dermal fibroblast cells(MDFs)were isolated from Col1a2^oim/J and WT mice.MDC staining,immunofluorescence,western blot and transmission electron microscopy were used to detect the autophagy level and endoplasmic reticulum stress condition in MDFs of Col1a2^oim/J mice.The HSP47 inhibitor Col003 was used to construct a cell model with abnormal structure of type?collagen.Endoplasmic reticulum stress and autophagy level of Col003 treated NIH-3T3 cells were evaluated using MDC staining,immunofluorescence,western blot and transmission electron microscopy to verify the results of Col1a2^oim/J mouse model.(2)RNA-seq was used to detect differential gene expression of MDFs in WT and Col1a2^oim/J mice.The sequencing results were verified to determine the regulating targets of autophagy.The therapeutic drug was confirmed according to the relevant pathways and mechanisms affecting autophagy.Western blot and immunofluorescence were used to explore the clearance of type?collagen after intervening on the autophagy activity.Col003 model was used for further verification.(3)Osteoblasts from Col1a2^oim/J mouse cranial bones were isolated.The influence of the therapeutic drug on the early osteogenic differentiation capacity of osteoblasts after the intervention of autophagy level were studied in vitro.The drug was used to treat 4-weeks-old Col1a2^oim/J mice.The osteogenic indexes were detected by western blot and the therapeutic effects were evaluated by immunohistochemical staining.Results:(1)Medium and high concentrations of rm NELL1 could enhance the expression of osteogenic genes and early osteogenesis differentiation in MC3T3-E1 and ADSCs.The transcription level of osteogenic genes and mineralization capacity of ADSCs modified by Nell1 gene were significantly increased.(2)Systemic administration of rm NELL1 combined with ADSCs or lenti-Nell1-ADSCs markedly improved the femoral microstructure,promoted bone area increase and collagen deposition,much better than mice that received PBS.The results of GFP immunofluorescence staining demonstrated that a considerable amount of positive signals located in femurs of OI mice and differentiate into ALP-positive cells,indicating their osteogenic differentiation,which facilitated to the bone formation.NELL1 combined with ADSCs achieved much better outcomes than mice received single rm NELL1 or ADSCs.(3)Inhibition of the autophagy level in MDFs of Col1a2^oim/J mice was manifested by abnormal accumulation of autophagosomes and endoplasmic reticulum stress in cells.Col003 obviously induced the accumulation of autophagosomes,and caused a certain degree of endoplasmic reticulum stress,with a phenotype similar to MDFs of Col1a2^oim/J mice.(4)Both Col1a2^oim/J mice and Col003 model showed high expression level of FGFR4,which affected the activation of AKT3,AMPK,and JNK and led to the inhibition of the autophagy process.The FGFR4 inhibitor FGF401 could increase the phosphorylation of AKT3 and AMPK,activate autophagy-related pathways and accelerate the degradation of abnormal collagen.(5)FGF401 could promote the osteogenic differentiation of osteoblasts in Col1a2^oim/J and Col003 model in vitro.It could inhibit the expression of FGFR4 and increase autophagy level.The osteogenic ability of osteoblasts and bone mineralization were improved by clearing away the abnormal collagen in Col1a2^oim/J mice.Conclusions:1.Lentivirus-mediated Nell1 gene modification enhanced the osteogenic potential of ADSCs.Combined use of rm NELL1 with ADSCs or Nell1 gene modified ADSCs rather than single use of rm NELL1 or ADSC performed much better therapeutic effect on type?OI mouse model with bone mass decrease.2.Col1a2^oim/J mice and Col003 model showed obvious endoplasmic reticulum stress activation and autophagy level inhibition.Inhibition of FGFR4 could enhance autophagy through activating AKT3,AMPK,JNK,and accelerate the degradation of abnormal collagen.FGF401,an inhibitor of FGFR4,could regulate the autophagy level of Col1a2^oim/J mice to relieve the abnormal metabolism and improve osteogenesis of osteoblasts,which could serve as a candidate for OI type?treatment.