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Objective:To prepare the hydrogel scaffolds with different concentrations of laponite and compare their osteogenic properties.Methods:The scaffolds of gelatin/sodium alginate hydrogel into which laponite was added according to the mass ratios of 0%, 1%, 2%, and 3% were assigned into groups T0, T1, T2, and T3. In each group, the compressive modulus was measured and the leaching solution for 24 h extracted to measure the ion release. Bone marrow mesenchymal stem cells (BMSCs) were cultured in the extract medium from each group and common medium (blank group) ( n=3) in the in vitro experiments to determine the expression of osteogenic genes Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and type I collagen after 7 days of culture. In the in vivo experiments, the scaffolds were implanted into the femoral condyle defects in rats, and a blank group with no scaffolds was set. The bone repair in each group was evaluated by hematoxylin-eosin(HE) staining and immunohistochemical staining. Results:The compressive modulus in group T2 [(139.05±6.43) kPa] was significantly higher than that in groups T0, T1 and T3 [(68.83±3.76) kPa, (101.18±3.68) kPa and (125.40±3.28) kPa] ( P<0.05). The ion contents of lithium, magnesium and silicon released from the 24 h leaching solution in group T2 were (0.031±0.005) μg/mL, (3.047±0.551) μg/mL and (5.243±0.785) μg/mL, insignificantly different from those in group T3 ( P> 0.05) but significantly larger than those in group T1 ( P>0.05). The in vitro experiments showed that the expression levels of Runx2, ALP and type I collagen in group T2 were 1.59±0.11, 2.02±0.08 and 1.06±0.17, significantly higher than those in the other groups ( P<0.05). HE staining showed that the implanted hydrogel was tightly bound to the bone tissue. Immunohistochemical staining showed that the numbers of Runx2 and osteocalcin positive cells in group T2 were significantly higher than those in the other groups. Conclusions:With ideal biocompatibility, hydrogel scaffolds with different concentrations of laponite can slowly release the decomposed ions of lithium, magnesium and silicon to promote the osteogenic differentiation of BMSCs and the repair of bone defects in vivo. A 2% concentration of laponite in the hydrogel scaffolds may result in the best results.
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Objective:To prepare biomimetic tissue engineering scaffolds of gelatin/sodium alginate/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique，and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing bone marrow mesenchymal stem cells（BMSCs）.Methods:BMSCs were routinely extracted and identified by flow cytometry. Gelatin，sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting. Non-printed scaffolds containing cells were prepared by injection molding method. In vitro，the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed，with 12 samples per group. Another simple cell culture group was set as control. Then，the internal structure of the composite hydrogel was observed by scanning electron microscope，and the expansion rate and water content of the scaffolds were measured by freeze-drying method. At day 3 after culture，the growth status of BMSCs was observed by phalloidine staining. cell counting kit（CCK）-8 assay was used to detect cell activity in scaffolds at days 1，3，and 7 after culture and RT-PCR to detect the expression of osteogenesis related genes Osterix，osteocalcin（OCN）and collagen I at days 7 and 14 ofter culture. In vivo，four groups were set according to printing or not and whether containing cells or not：printing implant group with cells，non-printing implant group with cells，printing implant group without cells and non-printing implant group without cells，with 9 samples per group. Scaffolds in four groups were implanted to the posterior gluteal muscle pouches（random on left or right）of 36 8-week-old SD rats，respectively. The samples were taken X-ray images at 2，4 and 8 weeks after operation，respectively. The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining. Results:The flow cytometry showed that the cells were BMSCs. Internal pores of hydrogels were obvious，and cells stretched freely in the pores. Differences of the swelling rate and water content were not statistically significant between printing group with cells［（1，039.37±30.66）%，（91.21±0.26）%］and non-printing group with cells［（1，032.38±35.05）%，（91.16±0.28）%］（ P>0.05）. At day 3 after culture in vitro，the cells grew well in the hydrogel. After culturing for 1 day in vitro，there was no significant difference in absorbance between printing group with cells and non-printing group with cells（ P>0.05）. At day 3 after culture，there was no significant difference in absorbance between printing group with cells and non-printing group with cells，but both groups showed a higher level than simple cell culture group（ P<0.05）. At day 7 after culture，the absorbance in printing group with cells（2.72±0.17）was higher than that in non-printing group with cells（2.35±0.11），and both of which were higher than that in simple cell culture group（1.95±0.12）（ P<0.05）. At day 7 after culture in vitro，there was no statistically significant difference in the expression of osteogenic differentiation-related genes between printing group with cells and the non-printing group with cells（ P>0.05），but they were all higher than those in simple cell culture group（ P<0.05）. At day 14 after culture in vitro，the expression of osteogenesis-related genes Osterix（1.650±0.095），OCN（2.725±0.091），collagen I（2.024±0.091）in printing group with cells were higher than those in non-printing group with cells（1.369±0.114，2.174±0.198，1.617±0.082，respectively）and those in simple cell culture group（1.031±0.094，1.116±0.092，0.736±0.140，respectively）（ P<0.05）. After implantation for 2 weeks in vivo，with no statistically significant difference in the gray values of X-ray films in each group（ P>0.05）. At weeks 4 and 8 after implantation，the gray values of X-ray films in printing implant group with cells and non-printing implant group with cells were higher than those in printing implant group without cells and non-printing implant group without cells（ P<0.01）. At 8 weeks after implantation，HE staining showed that the scaffolds were degraded in different degrees and immersed with cells，with collagen production seen in Masson staining as well. Conclusions:Composite hydrogel scaffolds can provide a good three-dimensional environment for BMSCs growth. 3D bioprinting can promote the proliferation and osteogenic differentiation of BMSCs in hydrogel scaffolds. In addition，BMSCs-loaded scaffolds can be degraded slowly in vivo with good ectopic osteogenic ability.
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Purpose Cortex is one of the frequently involved sites of multiple sclerosis (MS),and the cortex and corpus callosum lesions of MS are gradually concerned.The study aims to observe the changes of cerebral cortex and corpus callosum of MS in experimental autoimmune encephalomyelitis (EAE) model by using 7.0T MRI diffusion tensor imaging (DTI).Materials and Methods Twenty female C57BL/6 mice of 6-8 week old were enrolled in the study,10 of which were induced by MOG35-55 to make EAE models and the rest 10 of which were taken as control group.On the 20 days after model establishment,the head T2WI and DTI were performed on both control and EAE mice.DTI quantitative indicators such as fractional anisotropy (FA),mean diffusivity (MD),axial dispersion coefficient λ//,and radial dispersion coefficient λ ⊥ in region of interest including bilateral prefrontal cortex,bilateral cingulate cortex and corpus callosum were compared between the two groups.Results No obvious lesions were observed on the T2WI in both control and EAE groups.In the experimental group,the FA mapping suggested the integrity of the left side of the corpus callosum was destroyed.The FA,MD,λ// λ ⊥ of bilateral prefrontal cortex and corpus callosum showed significant difference between experimental group and control group (P＜0.05);the increase of λ ⊥ in bilateral cingulate was significantly different from that in the control group.Meanwhile,HE staining in the experimental group showed that inflammatory cells gathered around the cortical and subcortical vessels.The LFB staining in experimental group showed a bit paler than that in the control group,and the corpus callosum showed patchy demyelination.Conclusion The technique of 7.0T MRI DTI sequence can detect cortex and corpus callosum lesions which cannot be found by conventional MRI,so that it provides radiological evidence for the study of MS with cortex and corpus callosum lesions.
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Purpose Intravoxel incoherent motion (IVIM) MR is a method developed in recent years which can quantitatively evaluate the diffusion and perfusion characteristics of microenvironment.The aim of this study was to investigate the application value of IVIM in assessing relapsing-remitting multiple sclerosis (RRMS).Materials and Methods A retrospective analysis of the clinical data of 27 patients with RRMS confirmed clinically at the First Affiliated Hospital of Chongqing Medical University from Jun.2015 to Jan.2016 was carried out in the study.All the patients underwent the conventional MRI and IVIM MRI based on multi-b-factor (b values of 10,20,30,40,50,100,150,200,350,500,650,800,1000 s/mm2) with 3.0T MR scanner.The apparent diffusion coefficient (ADC),ADCslow,ADCfast and f values were evaluated since they could reflect the diffusion and perfusion status of RRMS lesions and normal-appearing white matter (NAWM) regions.Results The ADC,ADCslow,ADCfast and f values of the non-enhancing (NE) lesions were significantly higher than those in the NAWM regions both near and far from NE lesions (P＜0.05).However,the ADC,ADCslow,ADCfast and f values in the NAWM regions close to the NE lesions had no significant differences with those in the NAWM regions far from the lesions (P＞0.05).Conclusion The IVIM MRI can measure the diffusion and perfusion status of the lesions and NAWM in RRMS patients,which,therefore,is helpful in speculation of the pathological changes of RRMS lesions and in its injury classification and identification.
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PurposeMultiple sclerosis (MS) is characterized by time and spatial multiple, and it is the main reason for disabled young people. This paper aims to investigate the application of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with dual-compartment Tofts model in relapsing-remitting multiple sclerosis (RRMS) and its correlation with clinical scoring.Materials and MethodsThe clinical data of 25 patients with RRMS were retrospectively studied. The patients underwent the conventional MRI and the DCE-MRI examination. The result was processed by dual-compartment Tofts model and quantitative measurement was carried out in terms of volume transfer constant (Ktrans), rate constant between EES and blood plasma (Kep) and the volume of EES per unit volume of tissue (Ve), cerebral blood flow (CBF) and cerebral blood volume (CBV) of the lesions and normal-appearing white matter (NAWM) regions. The correlation between imaging biomarkers, expanded disability states scale (EDSS) and disease duration were also analyzed.Results ① The differences of MR imaging biomarkers Ktrans and Kep were significant between the regions of nonenhancing (NE) lesions, the NAWM regions near NE lesions and the NAWM regions far from NE lesions (χ2=6.777 and 22.343,P0.05).②The CBF and CBV among these three groups had no signiifcant differences (P>0.05).③The CBF of NE lesions was signiifcantly correlated with disease duration (r=0.518,P0.05).Conclusion DCE-MRI with Tofts model can quantitatively measure microvascular permeability and perfusion characteristics of lesions and NAWM regions, which thus reflects hemodynamic changes in patients with multiple sclerosis.
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Objective To evaluate dynamic contrast-enhanced MRI (DCE-MRI) with Patlak model for depicting the perfusion and permeability characteristics of lesions and normal-appearing white matter (NAWM) regions in patients with relapsing-remitting multiple sclerosis (RRMS). Methods Twenty-three patients with clinical confirmed RRMS were retrospectively analyzed, who had underwent conventional MRI and DCE-MRI using a 3.0 T MR scanner . The clinical characteristics and imaging data were collected. Post-processing was performed using the Patlak model. Volume transfer constant (Ktrans), fractional plasma volume (Vp) and perfusion parameters including cerebral blood flow (CBF) and cerebral blood volume (CBV) were represented as median and interquartile range(IQR). The four parameters of non-enhanced(NE) lesions, NAWM regions located close to NE lesions(NAWM close) and NAWM regions located far from NE lesions (NAWM far) were compared using the Kruskal-Wallis H rank sum test. Artificial color mappings were also proceeded. Results MR imaging biomarkers Ktrans was 0.132(0.064, 0.233) min-1 for NE lesions, 0.111 (0.060, 0.233) min-1 for NAWM close and 0.077(0.044, 0.185) min-1 for NAWM far, respectively. CBV was 10.660(5.555, 22.193) ml · 100 g-1 for NE lesions, 9.359(4.883, 16.290) ml · 100 g-1 for NAWM close, 6.814 (4.699, 13.623) ml·100 g-1 for NAWM far, respectively. Ktrans and CBV of NE lesions was significantly higher than that of NAWM far(χ2=7.582,P0.05). Conclusion DCE-MRI with Patlak model can measure perfusion and permeability characteristics and hemodynamic abnormalities of NE lesions and NAWM regions in patients with multiple sclerosis.