The zone of calcified cartilage is the mineralized region of articular cartilage that anchors the hyaline cartilage to the subchondral bone and serves to disperse mechanical forces across this interface. In an attempt to mimic this zonal organization, we have developed the methodology to form biphasic constructs composed of cartilaginous tissue anchored to the top surface of a bone substitute (porous calcium polyphosphate, CPP) with a calcified interface. To accomplish this, chondrocytes were selectively isolated from the deep zone of bovine articular cartilage, placed on top of the CPP substrate, and grown in the presence of beta-glycerophosphate (10 mM, beta-GP). By 8 weeks, cartilage tissue had formed with two zones: a calcified region adjacent to the CPP substrate and a hyaline-like zone above. Little or no mineralization occurred in the absence of beta-GP. The mineral that formed in vitro was identified as hydroxyapatite, similar in composition and crystal size to that found in vivo. The tissue stiffness was seven times greater, and the interfacial shear properties at the cartilage-CPP interface were at least two times greater in the presence of this mineralized zone within the in vitro-formed cartilage than in tissue lacking a mineral zone. In conclusion, developing a biphasic construct with a calcified zone at the tissue-biomaterial interface resulted in significantly better cartilage load-bearing (compressive) properties and interfacial shear strength, emphasizing the importance of the presence of a mineralized zone in bioengineered cartilage. Because failure due to shear occurred at the cartilage-CPP interface instead of the tidemark, as occurs with osteochondral tissue, further study is required to optimize this system so that it more closely mimics the native tissue.

Epileptic encephalopathies are severe epilepsy disorders with strong genetic bases. We performed targeted next-generation sequencing (NGS) in 70 patients with epileptic encephalopathies. The likely pathogenicity of variants in candidate genes was evaluated by American College of Medical Genetics and Genomics (ACMG) scoring taken together with the accepted clinical presentation. Thirty-three candidate variants were detected after population filtration and computational prediction. According to ACMG, 21 candidate variants, including 18 de novo variants, were assessed to be pathogenic/likely pathogenic with clinical concordance. Twelve variants were initially assessed as uncertain significance by ACMG, among which 3 were considered causative and 3 others were considered possibly causative after analysis of clinical concordance. In total, 24 variants were identified as putatively causative, among which 19 were novel findings. SCN1A mutations were identified in 50% of patients with Dravet syndrome. TSC1/TSC2 mutations were detected in 66.7% of patients with tuberous sclerosis. STXBP1 mutations were the main findings in patients with West syndrome. Mutations in SCN2A, KCNT1, KCNQ2 and CLCN4 were identified in patients with epileptic infantile with migrating focal seizures; among them, KCNQ2 and CLCN4 were first identified as potential causative genes. Only one CHD2 mutation was detected in patients with Lennox-Gastaut syndrome. This study highlighted the utility of targeted NGS in genetic diagnoses of epileptic encephalopathies and a comprehensive evaluation of the pathogenicity of variants based on ACMG scoring and assessment of clinical concordance. Epileptic encephalopathies differ in genetic causes, and the genotype-phenotype correlations would provide insights into the underlying pathogenic mechanisms.

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Objective and designSeveral works in the setting of early experimental diabetic nephropathy using anti-inflammatory drugs, such as the calcineurin inhibitor FK506, have shown prevention of the development or amelioration of renal injury including proteinuria. The exact mechanisms by which anti-inflammatory drugs lower the albuminuria have not been still clarified well.MaterialsThe diabetic rats were induced by using streptozotocin.TreatmentThe diabetic rats were subjected to oral FK506 treatment at a dose of 0.5 or 1.0 mg/kg daily for 4 weeks.MethodsRenal histology for the ultrastructural evaluation was determined by electron microscope, followed by analyses of renal nephrin and podocin and detection of renal iNOS+ macrophages and NF-κB-p-p65+.ResultsElevated 24-h urinary albumin excretion rate was markedly attenuated by FK506 treatment. In diabetic model rats, FK506 treatment at a dose of 0.5 or 1.0 mg/kg significantly increased the expression of nephrin and podocin when compared to control. As expected, rats in control diabetic group had an increase in GBM thickening and foot process effacement when compared to normal rats; increased GBM thickening and foot process effacement were ameliorated by FK506 treatment with 0.5 and 1.0 mg/kg. Histologically, there was marked accumulation of ED-1+cells (macrophages) in diabetic kidneys, and FK506 treatment failed to inhibit it. In contrast, FK506 treatment at 0.5 and 1.0 mg/kg doses significantly inhibited the elevated ED-1+/iNOS+ cells in the kidneys of diabetic rats. ED-1+/NF-κB-p-p65+ cells were significantly increased in positive diabetic kidneys compared to those of normal rats. FK506 treatment at 0.5 and 1.0 mg/kg significantly attenuated the elevated ED-1+/NF-κB-p-p65+ cells in diabetic kidneys. Additionally, a positive correlation was observed between ED-1+/iNOS+ cells and albuminuria (r = 0.87, p < 0.05). Likewise, ED-1+/iNOS+ cells were correlated negatively with both nephrin and podocin protein (r = −0.70, p < 0.05; r = −0.68, p < 0.05, respectively).ConclusionOur results show that FK506 not only upregulates expression of nephrin and podocin but also inhibits macrophage activation to protect against podocyte injury.