Establishment And Treatment Of A Novel Mouse Model Of Osteogenesis Imperfecta Type Ⅰ

Objective:Osteogenesis imperfecta is a heritable disorder characterized by bone fragility,and skeletal deformity.Currently,orthopaedic surgery is the main treatment,but a high reoperation rate causes great pain to patients and their families.Osteogenesis imperfecta type Ⅰ is the most common type in all osteogenesis imperfecta patients,which is caused by haploinsufficiency of COL1A1,resulting in decreased amount of normal collagen type Ⅰ molecules.The abnormal changes of collagen type Ⅰ will affect the relevant pathways involved in bone development,but the molecular mechanism is still unclear.Heterozygous Mov13 mouse is the only model stimulating the phenotype of osteogenesis imperfecta type Ⅰ,but the process of reduced synthesis of collagen type Ⅰ of the strain is different from clinical patients.Besides,the Mov 13 mice have a phenotypic decline with them growth.Thus,the present study designed a novel heterozygous mouse by CRISPR/Cas9 system to imitate osteogenesis imperfecta type Ⅰ from genotype to phenotype.And the effect and relative mechanism of reduced synthesis of collagen collagen type Ⅰ on bone formation and effective therapeutics were explored,and the results provided theoretical basis for clinical treatment of osteogenesis imperfecta.Methods:（1）The transgenic mouse simulating osteogenesis imperfecta type Ⅰ was produced in the present study by partial exons knockout（exon 2-exon 5）using CRISPR/Cas9 system.Genotype of transgenic mice were identified by Polymerase Chain Reaction（PCR）,and heterozygous mice were named col1a1^+/-365.（2）Femurs,dermas,kidneys and peripheral blood serums were collected from col1a1^+/-365 mice and their normal littermates.The transcriptional expression of Col1a1 from femurs,dermas and kidneys were tested by quantitative real-time PCR（q PCR）.And protein level of Col1a1 of femurs was detected by Western blot.Distal sections of femurs were stained with hematoxylin & eosin（H&E）.The collagen type Ⅰ of femurs and dermas were stained with picrosirius red.And the collagen type Ⅰ of kidneys was stained by immunohistochemistry.（3）The femoral microstructure was examined by Micro-CT.The mechanical properties of femurs were measured by three-point bending analysis.Bone mineral density（BMD）and bone mineral content（BMC）of femurs were acquired by dual energy X ray absorptiometry（DXA）equipment.The levels of procollagen type Ⅰ N-terminal propeptide（PINP）and C-terminal telopeptide of type Ⅰ collagen（CTX-I）were detected by ELISA.（4）Differential expression genes of adipose derived mesenchymal stem cells（ADSCs）from wt and col1a1^+/-365 mice were detected by RNA-seq.Functional classification,pathway annotation and enrichment analyses were based on the Gene Ontology Database and KEGG pathway database.（5）The migratory capacity of ADSCs was tested by Transwell and scratch assay.The transcriptional expression of Sdf-1 of ADSCs was tested by q PCR,and the SDF-1 concentration of peripheral blood was examined by ELISA.The osteogenic differentiation potential and adipogenic differentiation potential of ADSCs post culturing with adipogenic and osteogenic induction medium were evaluated.（6）The transcriptional expression of Runx2 and Osterix in femurs were tested by q PCR.The bone gla protein（Bglap）expression was tested by Western blot and immunohistochemistry.（7）The YAP protein,the key member in hippo signal pathway,in femurs was tested by Western blot.The transcriptional expression of YAP target genes Ctgf and Cyp61 in ADSCs were checked by q PCR.The nuclear localization of YAP was determined by cell immunofluorescence staining of ADSCs cultured in dishes with and without collagen pre-coated.,（8）The particle size and morphology of nano-hydroxyapatite（n HA）was observed by transmission electron microscope.The cytotoxicity of n HA was measured by MTS.The effect of n HA on osteogenic differentiation of ADSCs was tested by q PCR and ALP staining.The femurs of col1a1^+/-365 mice received intramedullary injection of n HA mixing with matrigel,and the therapeutic effect was evaluated by Micro-CT,DXA and histological analysis.Results:（1）PCR detection showed that the col1a1^+/-365 mice had a 594 bp band and a 1623 bp band,while the wt mice only produced a 1623 bp band.It suggested that the exon 2-exon 5 of col1a1 were successfully deleted.（2）The significantly reduced expression of Col1a1 in femurs,dermas and kidneys could be observed.The Col1a1 protein showed obvious decrease in the femurs.H&E stained sections revealed that the femurs section of col1a1^+/-365 mice showed less sparse trabeculae in spongy bones.Picrosirius red staining performed decreased collagen type Ⅰ fibers in col1a1^+/-365 femur and derma.Immunohistochemistry staining demonstrated that Col1a1 in the kidney significantly decreased in col1a1^+/-365 mice.These results all indicated that the mouse model was successfully constructed.（3）Micro-CT analysis of the distal femur metaphysis revealed that femoral structure in col1a1^+/-365 mice was obviously abnormal.Stiffness and ultimate load of femurs were significantly lower than that of wt mice.The femoral BMD and BMC were remarkably lower in col1a1^+/-365 mice.The serum level of the bone formation marker PINP got obviously reduced.In contrast,the serum concentration of CTX-I,a marker for bone resorption,was significantly higher in col1a1^+/-365 mice.（4）The result of RNA-seq showed that there were 1287 differential expression genes,witha total of 454 up-regulated genes and 833 down-regulated genes in ADSCs of col1a1^+/-365 mice.The differential expression genes belonged to the functional categories of the extracellular matrix,cell migration and differentiation.（5）ADSCs of col1a1^+/-365 mice showed significantly decreased migratory capacity compared to wild type ADSCs,and its expression of Sdf-1 was obviously declined.Also,the serum SDF-1 level of col1a1^+/-365 mice was evidently lower than wild type,which might affect the homing of MSCs.（6）The ADSCs from col1a1^+/-365 mice exhibited obvious lessened osteogenic differentiation capacity accompanied by strengthened adipogenesis differentiation potential.The levels of osteogenesis differentiation markers Runx2 and Osterix m RNA and Bglap protein all got significantly decreased in femurs of col1a1^+/-365 mice.（7）The level of YAP protein in the femurs of col1a1^+/-365 mice significantly decreased,and the expression levels of its target genes,Ctgf and Cyr61,were distinctly down-regulated.The nuclear localization of YAP in the ADSCs of col1a1^+/-365 mice was obviously decreased post osteogenic differentiation induction,,while nuclear level of YAP was increased after collagen type Ⅰ supplementation.（8）Nano-hydroxyapatite（n HA）did not affect ADSCs proliferation and 0.2 mg/m L n HA enhanced the capacity for osteogenesis differentiation.The BMC,BMD and Bglap expression all significantly increased 8 weeks following the col1a1^+/-365 mice received n HA mixed with matrigel injectionConclusion1.The col1a1^+/-365 mice designed in the present study showed similar phenotype to clinical characteristics of osteogenesis imperfecta type Ⅰ,including the obviously decreased of collagen type Ⅰ expression,bone mass and bone mechanical properties..2.The reduced collagen type Ⅰ was decreased,caused significantly weakened migratory capacity of ADSCs and serum SDF-1 level of col1a1^+/-365 mice,which might affect the migration of mesenchymal stem cells（MSCs）to bone tissue and bone formation.3.The decreased osteogenic differentiation capacity of ADSCs from col1a1^+/-365 mice might result from the reduction of nuclear localization of YAP.4.Intramedullary injection of n HA mixed with matrigel could provide a promising and effective method in osteogenesis imperfecta type Ⅰ treatment.