impact of calcium hydroxide on the osteoinductive capacity of demineralized freeze-dried bone allograft: an in-vitro study

Statement of the Problem: A great challenge in periodontal therapy is the regeneration enhancement of osseous defects through applying osteoinductive materials. Demineralized freeze-dried bone allograft [DFDBA] has already been introduced as an allograft with osteoconductive and variable osteoinductive properties. Calcium hydroxide [Ca[OH]2] is an available well-known material in dentistry, which induces hard tissue formation

Purpose: This study evaluated the efficiency of combination of DFDBA and Ca[OH]2 in improving the quality of osteoinduction of DFDBA

Materials and Method: Human bone marrow-derived mesenchymal stem cells were taken from volunteers' iliac crest. Cell proliferation was determined by MTT test at 18, 24 and 48 hours post-culture in 10 groups. The employed material were 0.5, 1.0 mg/ml Ca[OH]2 in two forms of suspension and pH-adjusted solution, 10mg/ml DFDBA per se and in combination with 0.5 and 1.0 mg/ml Ca[OH]2. Mineralization was assessed by Alizarin red staining in 10 mg/ml DFDBA, DFDBA+ 0.5 and 1 mg/ml Ca[OH]2 in solution and suspension forms. The data were statistically analyzed by using one-way ANOVA and Tukey's post-hoc test [p< 0.05]

Results: The pH-adjusted solutions exhibited better cell proliferation compared with the suspension groups. The combination of 0.5mg/ml Ca[OH]2 solution and DFDBA increased the cell proliferation and mineralization compared with DFDBA per se [p= 0.033]

Conclusion: The combination of Ca[OH]2 with DFDBA improved the osteoinductivity of DFDBA

in vitro model for hepatocyte-like cell differentiation from Wharton's jelly derived-mesenchymal stem cells by cell-base aggregates

The present study investigated the differentiation potential of human Umbilical Cord Mesenchymal Stem Cells [UCMSCs] into hepatic lineage through embryonic body-like aggregate formation in the presence of IGF-1. Cells derived from Wharton's jelly have been reported to display a wide multilineage differentiation potential, showing some similarities to both embryonic [ESC] and mesenchymal stem cells [MSCs]. Human MSCs isolated from the umbilical cord were plated in 20 microL micro drops. A two-step differentiation protocol was used and the cell aggregates were exposed to the media supplemented with IGF, HGF, oncostatin M, and dexamethasone for 21 days. Immunoperoxidase and immuno-fluorescence were performed for cyrokeratins 18, 19 and albumin. Functional assays were done by periodic acid Schiff [PAS] and indocyanine green. The expression of cytokeratin 19 was shown to be higher in the cells derived from 3D spheroids compared to those cultured in conventional protocol. They showed a polygonal shape after being exposed to hepatogenic media. Immunostaining demonstrated the expression of cytokeratin-18, 19 and albumin by the differentiated cells. Besides, PAS staining revealed glycogen storage in differentiated cells. Also, a greater number of large size differentiated cells were found at the periphery of the expanded cell aggregates. We established a protocol for UCMSC differentiation into hepatocytes and these cells were morphologically and functionally similar to hepatocytes. Thus, hepatocyte differentiation may be facilitated by the UCMSCs aggregate formation before administration of the differentiation protocols

Carrier screening and prenatal detection of chronic granulomatous disease in Iran

To develop an application that is simple and reliable using the nitroblue tetrazolium [NBT] method that clearly differentiates chronic granulomatous disease [CGD] patients with heterozygous carriers in groups suspected with CGD. This study was carried out in Shiraz University of Medical Sciences from October 2002 and March 2004. The study included 260 samples consisting of 123 children [2-24 months] and 106 neonates [<2 months], either suspected with bacterial infection or are immunodeficient, and 31 cord blood samples. Fifty healthy adult individuals were also diagnosed as normal control. Neutrophil reduction of NBT can be stimulated in vitro by protein kinase agonists such as phorbol myristate acetate [PMA], resulting to superoxide anion release. The PMA is an exceptionally powerful stimulant and when we used it in conjunction with adherence of glass slides, it causes transformation of nearly 100% of all normal neutrophils, and reduces NBT to formazan deposits. Of 260 blood samples, 12 unrelated CGD patients and 16 carriers of X-linked or autosomal recessive CGD patients were diagnosed. The carriers had a range of 15-75% stimulated neutrophils. We have established a PMA-stimulated NBT test for the detection of CGD patients, which clearly differentiate the CGD patients from heterozygote carriers. The results in the cord fetal blood samples indicate that this test may be used for antenatal diagnosis of affected boys, carrier females and autosomal recessive variants of CGD. The technique is simple, fast, inexpensive, and requires only a few microliters of blood

Maternal serum levels of transforming growth factor B1 [TGF-beta1] in normal and preeclamptic pregnancies

Successful pregnancy in allopregnant women depends upon the control of graft rejection mechanisms. It has been suggested that some immunosuppressive cytokines contribute to successful pregnancy and transplantation. Transforming growth factor beta [TGF-beta] exhibits potent immunoregulatory and anti-inflammatory properties which might prolong graft survival. Recent studies suggest a role for TGF-beta in the generation of T-regulatory lymphocytes which preserves the tolerance to peripheral self antigens and may control the response to allogenic tissues and thereby promote the transplantation tolerance. Also, the function of TGF-beta in trophoblast differentiation and hypertension is reported. To evaluate the maternal serum TGF-pl level in normal allopregnant women and in pregnancies complicated by preeclampcia [PE]. Sixty one pregnant preeclamptic women [32 cases with severe and 29 with mild PE], 22 normotensive healthy pregnant, and 20 non-pregnant controls constituted the studied groups. The active form of TGF-beta in serum from all cases was investigated by indirect ELISA technique. The results showed that TGF-beta1 level was higher in all three pregnant groups as compared with the non-pregnant controls. No significant changes in serum levels of TGF-pl were found in PE as compared with the normal pregnancy. TGF-beta may function as a regulatory factor in fetal allograft survival during pregnancy, and TGF-beta1 does not have a pathophysiological role in PE

Anticardiolipin antibody in patients with Behcet's disease with and without vascular thrombosis

The clinical value of IgG anticardiolipin antibody in patients with Behcet's disease with or without vascular thrombosis was evaluated. IgG isotype of anticardiolipin [aCL] antibody was assessed in 40 Behcet's disease [BD] patients with venous or arterial thrombosis, 40 BD patients without venous or arterial thrombosis and 80 healthy subjects as controls. The levels of IgG aCL were determined by an indirect ELISA method. Color Doppler Sonography, Magnetic Resonance Imaging and conventional angiography were the procedures used for other clinical evaluations. Out of 40 patients with vascular thrombosis, 20[50%] were positive for low to moderate level of IgG aCL. In patients without thrombosis 22[55%] were positive for low to moderate level of IgG aCL while in none [0%] of the healthy subjects the IgGaCL was positive, neither low nor moderate. The number of patients with headache but having a normal cerebral magnetic resonance imaging [MRI], was higher in anticardiolipin positive patients without vascular thrombosis as compared to those with vascular thrombosis, [P = 0.001].Arthritis was noticed in both patents groups. 15% of aCL positive patients without thrombosis had arthritis as compared to none in aCL negative patients without thrombosis [P = 0.02]. The results of this study indicate that although the frequency of IgG aCL was found to be higher in Iranian patients with BDin comparison with the previous reports, except in arthritis the observed elevated IgG aCL does not correlate with clinical disease manifestations, or vascular thrombotic complications

Anticardiolipin antibodies in juvenile rheumatoid arthritis and systemic lupus erythematosus

Antiphospholipid antibody syndrome [APS] can either occur as a primary syndrome or associated with other autoimmune diseases such as systemic lupus erythematosus [SLE]. Anticardiolipin antibody [aCL] of IgG and/or IgMisotype in blood, measured by a standardized ELISAis the most acceptable laboratory criteria. APS IgGisotype, particularly IgG2 subclass is more strongly associated with thrombosis. This study was done to determine the prevalence of IgG aCL and its subclasses in relation to APS symptoms, in a group of juvenile rheumatoid arthritis [JRA] and juvenile systemic lupus erythematosus [SLE] patients. In this prospective study, 28 JRAand 16 SLE patients, aged 3-18 years, were enrolled. IgG aCLwas assayed by standard aCL ELISA. IgG subclasses were also assayed by ELISA on sera with medium to high titers of aCL.ACL assay was performed on at least two occasions for each patient, over 3-6 months period of follow up. 29% [8/28] of JRApatients and 44% [7/16] of SLE patients had aCL. Six of SLE patients displayed APS related manifestations: hemolytic anemia, thrombocytopenia, arterial occlusion, valvular heart disease, livedo reticularis and pulmonary hypertension, but none of them had persistant medium or high titer of aCL. The lack of association of high titer of aCL with APS related symptoms was observed in two patients. The IgG subclasses were primarily IgG1 and IgG3. The prevalence of IgG aCL in this group of pediatric SLE and JRA is not uncommon but it's relation to clinical manifestations is not clear. IgG1 and IgG3 subclasses were not associated with thrombosis, which is in agreement with previous studies