Nanoparticles of cobalt-substituted hydroxyapatite in regeneration of mandibular osteoporotic bones.

Indications exist that paramagnetic calcium phosphates may be able to promote regeneration of bone faster than their regular, diamagnetic counterparts. In this study, analyzed was the influence of paramagnetic cobalt-substituted hydroxyapatite nanoparticles on osteoporotic alveolar bone regeneration in rats. Simultaneously, biocompatibility of the material was tested in vitro, on osteoblastic MC3T3-E1 and epithelial Caco-2 cells in culture. The material was shown to be biocompatible and nontoxic when added to epithelial monolayers in vitro, while it caused a substantial decrease in the cell viability as well as deformation of the cytoskeleton and cell morphology when incubated with the osteoblastic cells. In the course of 6 months after the implantation of the material containing different amounts of cobalt, ranging from 5 to 12 wt%, in the osteoporotic alveolar bone of the lower jaw, the following parameters were investigated: histopathological parameters, alkaline phosphatase and alveolar bone density. The best result in terms of osteoporotic bone tissue regeneration was observed for hydroxyapatite nanoparticles with the largest content of cobalt ions. The histological analysis showed a high level of reparatory ability of the nanoparticulate material implanted in the bone defect, paralleled by a corresponding increase in the alveolar bone density. The combined effect of growth factors from autologous plasma admixed to cobalt-substituted hydroxyapatite was furthermore shown to have a crucial effect on the augmented osteoporotic bone regeneration upon the implantation of the biomaterial investigated in this study.

Development of c-kit immunopositive interstitial cells of Cajal in the human stomach.

Interstitial cells of Cajal (ICC) include several types of specialized cells within the musculature of the gastrointestinal tract (GIT). Some types of ICC act as pacemakers in the GIT musculature, whereas others are implicated in the modulation of enteric neurotransmission. Kit immunohistochemistry reliably identifies the location of these cells and provides information on changes in ICC distribution and density. Human stomach specimens were obtained from 7 embryos and 28 foetuses without gastrointestinal disorders. The specimens were 7-27 weeks of gestational age, and both sexes are represented in the sample. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. Enteric plexuses were immunohistochemically examined by using anti-neuron specific enolase and the differentiation of smooth muscle cells (SMC) was studied with anti-alpha smooth muscle actin and anti-desmin antibodies. By week 7, c-kit-immunopositive precursors formed a layer in the outer stomach wall around myenteric plexus elements. Between 9 and 11 weeks some of these precursors differentiated into ICC. ICC at the myenteric plexus level differentiated first, followed by those within the muscle layer: between SMC, at the circular and longitudinal layers, and within connective tissue septa enveloping muscle bundles. In the fourth month, all subtypes of c-kit-immunoreactivity ICC which are necessary for the generation of slow waves and their transfer to SMC have been developed. These results may help elucidate the origin of ICC and the aetiology and pathogenesis of stomach motility disorders in neonates and young children that are associated with absence or decreased number of these cells.

Ultrastructure of the human palatine tonsil and its functional significance.

The human palatine tonsils represent a mucosa-associated lymphoid tissue with a significant function in mucosal protection against alimentary and airborne pathogens. The ultrastructure of different morphological compartments in the human palatine tonsil was studied in eighteen tonsils obtained from the patients who had undergone elective tonsillectomy due to chronic tonsillitis. The tonsillar specimens were analyzed by scanning and transmission electron microscopy. The results showed the presence of tight junctions between superficial epithelial cells of the oropharyngeal tonsillar surface. The crypt epithelium is a sponge-like structure infiltrated by non-epithelial cells, mostly lymphocytes, and is characterized by the presence of small pores - microcrypts occupied by large microvillus cells and/or lymphocytes. Antigen-presenting Langerhans cells with typical intracytoplasmic Birbeck granules were also found in the crypt epithelium. The lymphoid follicles are composed of lymphocytes and two types of non-lymphoid follicular cells: small fibroblast-like cells and large cells, morphologically consistent with antigen-bearing follicular dendritic cells or macrophages. The interfollicular areas consisted of a dense network of reticular cells and reticular fibers; many lymphocytes were interspersed between the reticular fibers. In addition to arterioles and high endothelial venules in the interfollicular lymphoid tissue, some fenestrated capillaries were seen intraepithelially and subepithelially. The complex ultrastructure of the human palatine tonsil provides a microenvironment necessary for antigen uptake, antigen processing and immune response.

Histomorphological and clinical study of primary and secondary glomerulopathies in Southeast Serbia (20-year period of analysis).

BACKGROUND/AIM: Epidemiological studies of renal biopsies have been performed to follow up the incidence of glomerular diseases on a specified territory and to compare the obtained results with results from other regions. The aim of this study was to analyze the frequency of certain histopathophysiological types of glomerular diseases on the territory of Southeast Serbia. METHODS: In a 20-year period (1986-2006), 316 kidney biopsies were performed in patients with clinical signs of impaired renal function, in Southeast Serbia. On average 1.6 biopsies were made per year per 100 000 inhabitants. RESULTS: Biopsies of adult patients represented 88% of all biopsies, biopsies in children (aged under 18 years) represented 8%, while biopsies of elderly patients (more than 60 years) represented 4% of all biopsies. The predominance of male patients was described with male/female ratio of 1.4. The most frequent clinical manifestation in patients at the time of biopsy were nephrotic syndrome (42.5%), and asymptomatic proteinuria and/or hemamuria (31.3%) and nephritic syndrome (14.9%). The most common glomerular disease was IgA nephropathy with an incidence of 21.5% of total biopsy diagnosed glomerulopathies, followed by: membranous glomerulonephritis (12.6%), focal segmental proliferative and sclerosing glomerulonephritis (10.7%), lupus nephritis (8.4%), nephroangiosclerosis (7.0%), mesangio-proliferative glomerulonephritis (6.1%), minimal change disease (2.8%), mesangiocapillary glomerulonephritis (2.3%). CONCLUSION: The frequency of certain histopathologic findings significantly correlated with data from studies that we used for comparison, with the exception of minimal change disease whose incidence in our study was smaller.

The clinical significance of antibody determination to cyclic citrullinated peptides in systemic sclerosis.

INTRODUCTION: Anti-citrullinated peptides antibodies (ACPA) are present in 80% of sera of rheumatoid arthritis (RA) patients with high specificity for diagnosis and prediction for the development of early erosive arthritis. A few studies have reported a low frequency ACPA in systemic sclerosis (SSc) patients with the presence of arthritis. OBJECTIVE: The aim of our study was to determine the frequency of ACPA in systemic sclerosis (SSc) patients, their correlation with clinical manifestations and radiographic features. METHODS: The study included 82 patients with SSc, mean age 54.4 years, 59 with the limited (ISSc) and 23 with the diffuse (dSSc) form of the disease. The control group included 28 healthy age and sex matched subjects. ACPA and rheumatoid factor (RF) were determined in all SSc patients and healthy subjects in whom standard radiography of hands and wrists was also done. RESULTS: The presence of ACPA was detected in 11 (13.4%) of SSc patients. Their level was not increased in any of the controls. Positive RF was found in 15.9% of SSc patients. Arthritis was present in 17.1%, as well as marginal bone erosions. There was a statistically significant association between positive ACPA and arthritis (p < 0.0001) and positive ACPA and marginal bone erosions (p = 0.0002). CONCLUSION: The research confirmed the correlation between ACPA with clinical signs of arthritis and radiographic damage of hand joints. ACPA is a useful diagnostic marker in the identification of SSc patients with arthritis and anatomic bone damage enabling the use of adequate therapy in order to prevent joint damage and poor quality of life.

[Tooth macromorphological and ultrastructural analysis of osteological material from the medieval locality of St. Panteleimon Church in Nis].

INTRODUCTION: Analysis of macromorphological and ultrastructural tooth characteristics of osteological material from the medieval site of St. Pantaleimon Church in Nis provides us with insight on the life, nutrition and habits of medieval population, as well as the structure and composition of their teeth. OBJECTIVE: The aim of this research, based on the tooth inspection of skeletal remains from the medieval site of St. Pantaleimon Church in Nis, was to analyze macromorphological characteristics, ultrastructure of the dental tissue of maxillary and mandibular molars, canines and incisors, as well as their chemical composition. METHODS: Macromorphological and ultrastructural analysis of the dental tissue of osteological material dating from the 12th century included 1312 teeth with advanced abrasion. Macromorphological changes were detected by using a dental mirror, probe and radiography. After irrigation, the teeth were prepared using the standard procedure and analyzed by scanning electronic microscopy (JEOL-JSM-5300). Chemical analysis was done by expanded downscaling (EDS) method for Mg, P, Ca. RESULTS: The analysis detected second degree abrasions of all teeth in individuals aged 20-25 years. Third and fourth degree abrasions of teeth were detected in individuals aged over 40 years. Ultrastructural analysis showed a complete obliteration of dentin tubules and pulp of the lower incisors, the apposition of intratubular dentin inside the tubules, as well as extensive deformity and loss of dentin structure on molars with preserved pulp volume and nerve fiber calcification. The calcification of nerve fibers showed that the formation of intratubular dentin was proportional with the biological potential of pulp and the degree of abrasion, and inversely proportional with the size of dentin surface. Chemical analysis showed that in the analyzed teeth Ca composition was slightly lower than that in the control group, P composition was almost identical, while Mg composition was multiply increased in comparison to the control group consisting of today's persons of matching age and examined teeth as the medieval individuals. CONCLUSION: Different ultrastructural tooth characteristics as a part of the complex masticatory system of the medieval man are the result of individual and the whole community adaptation to actual living conditions. Nerve fibers calcification of the pulp can explain the absence of pain despite the high level of dental abrasion.

Size effect of calcium phosphate coated with poly-DL-lactide- co-glycolide on healing processes in bone reconstruction.

In this article, synthesis and application of calcium phosphate/poly-DL-lactide-co-glycolide (CP/PLGA) composite biomaterial in particulate form, in which each CP granule/particle is coated with PLGA, are described. Two types of the particulate material having different particle sizes were synthesized: one with an average particle diameter between 150 and 250 mum (micron-sized particles, MPs) and the other with an average particle diameter smaller than 50 nm (nanoparticles, NPs). A comparative in vivo analysis was done by reconstructing defects in osteoporotic alveolar bones using both composites. The material, CP granules/particles covered with polymer, was characterized using X-ray structural analysis, scanning electron microscopy, and atomic force microscopy. Changes in reparatory functions of tissues affected by osteoporosis were examined in mice in vivo, using these two kinds of composite materials, with and without autologous plasma. Having defined the target segment, histomorphometric parameters-bone area fraction, area, and mean density-were determined. The best results in the regeneration and recuperation of alveolar bone damaged by osteoporosis were achieved with the implantation of a mixture of nanoparticulate CP/PLGA composite and autologous plasma. After the implantation of microparticulate CP/PLGA, in the form of granules, mixed with autologous plasma, into an artificial defect in alveolar bone, new bone formation was also observed, although its formation rate was slower.

[Investigation of Balkan endemic nephropathy in Serbia: how to proceed?].

Balkan endemic nephropathy (BEN) presents an unsolved puzzle despite fifty years of its investigation. Academy of Medical Sciences of the Serbian Medical Society organized a round table discussion on current unsolved problems related to BEN. The present paper summarizes presentations, discussion and conclusions of this meeting. During the last fifty years, the course of BEN prolonged and it shifted towards the older age in all endemic foci. Data on the incidence of BEN have been controversial and frequently based on the data on the number of BEN patients starting haemodialysis treatment. In Serbia, BEN patients present 6.5% of haemodialysis population and this percentage differs among different centres ranging from 5% (Leskovac) to 46% (Lazarevac). Maintenance of high prevalence of BEN patients on regular haemodialysis indicates that BEN is not an expiring disease. In addition, recent data have shown more frequent microalbuminuria and low-molecular weight proteinuria in children from endemic than from nonendemic families. Aetiology of BEN is still unknown despite numerous investigations of environmental and genetic factors. Today, there is a very current hypothesis on the aetiological role of aristolochic acid but the role of viruses, geochemical factors and genetic factors must not be neglected. Morphological features of BEN are nonspecific and characterized by acellular interstitial fibrosis, tubular atrophy and changes on pre- and postglomerular vessels. New immunohistochemical and molecular biology methods offer a new approach to BEN investigation. Association of BEN with high incidence of upper-urothelial tumours is well-known. Recent studies have shown significant changes of demographic characteristics of patients suffering upper-urothelial tumours, their prevalence in different endemic foci and characteristics of tumours. Further studies of BEN should be directed to determination of incidence and prevalence of disease in different endemic foci, investigations of different insufficiently examined aetiological factors as well as pathomorphological features of the disease by the use of modern methods.

[Efficiency of final irrigation of root canal in removal of smear layer].

INTRODUCTION: A smear layer forms on the root canal walls as the consequence of root instrumentation. The smear layer formed in such a way considerably influences the quality of root obturation and endodontic treatment outcome. OBJECTIVE: The aim of this study was to ultrastructurally analyse the surface of intracanal dentine after removal of the smear layer by the solution of doxycycline, citric acid and detergent Tween-80 (MTAD). METHODS: The study involved 60 single-rooted, extracted, human teeth divided into four groups. All samples were instrumented by a step-back technique and manual K-files, and rinsed during instrumentation by 2% CHX and H2O2. The first group of samples was exclusively rinsed by CHX and H2O2; in the second group, besides using CHX and H2O2, MTAD solution was used for the final irrigation. The samples which were rinsed by distilled water (+ control) and the samples rinsed by 5.25% NaOCI and 17% Na EDTA (-control) served as control groups. All samples were observed under the scanning electronic microscope JEOL-JSM-5300. The coronary, middle and apex thirds of the radix region were analysed. RESULTS: The obtained results of the SEM analysis showed that the application of 2% chlorhexidine and hydrogen peroxide did not give clear dentine walls, and the smear layer could not be removed completely. The application of the same combination (CHX and H2O2), added by the final MTAD irrigation solution, led to the efficient removal of the smear layer, while the morphological structure of dentine surface remained unchanged. Statistical analysis showed that canal walls in the experimental group with MTAD as the final irrigation were significantly clearer compared to the control group (p < 0.001). CONCLUSION: Based on the obtained results, it can be concluded that MTAD is an efficient solution for the final irrigation of the canal system.

[Alport's syndrome and benign familial haematuria: light and electron microscopic studies of the kidney].

INTRODUCTION: Hereditary nephropathy is clinically characterized by the familial occurrence in successive generations of progressive haematuric nephritis and neural hearing loss. Hereditary nephropathy of Alport's syndrome (AS) and benign familial (recurrent) haematuria (BFH) are morphologically characterized by specific and diagnostically important thickening and splitting of lamina densa of the glomerular basement membranes. Those lesions can be recognized only by electron microscopy. Hereditary nephritis is usually present clinically with haematuria, and new mutations without a family history of haematuria. It is therefore important to differentiate hereditary nephritis from BFH and no familial haematuria. Thus, electron microscopy is essential in diagnosis of haematuria. OBJECTIVE: The aim of this study was to describe, by light microscopy, constellation of renal alterations by which hereditary nephropathy can be recognized with high probability as well as to compare the diagnostic validity of the findings observed by light and electron microscopy in AS and BFH. METHOD: We examined 48 renal biopsies of the patients with hereditary nephoropathies by light and electron microscopy. Tissue samples were fixed in buffered paraformaldehyde and embedded in paraffin for long-term preservation. For the electron microscopy analysis, the following fixation in 4% glutaraldehyde tissue was postfixed in 1% osmium tetroxide. Thereafter, the following dehydration procedure tissue slices were embedded in epon. RESULTS: Our results demonstrated that the interstitial foam cells, foetal-like glomeruli, minimal glomerular abnormalities with stain less intense in basement membranes, mild irregular mesangial widening, focal thickening of Bowman's capsule, foci of dilatation tubules, tubular ectasia and atrophy, erythrocyte tubules casts were present in hereditary nephritis. Additionally, light microscopic biopsy findings in patients with BFH were either normal or revealed minor changes (e.g. increased mesangial matrix). All biopsies were reevaluated by electron microscopy and ultrastructural findings confirmed the diagnosis of hereditary nephropathies. CONCLUSION: The findings observed by light microscopy represent an important step that leads to a definitive diagnosis of AS and BFH.The definitive diagnosis, however, depends on electron microscopy.

Progression of kidney damage in Balkan endemic nephropathy: a 15-year follow-up of patients with kidney biopsy examination.

Progression of kidney damage was studied in 18 patients with Balkan endemic nephropathy (BEN), with a mean 15-year follow-up after renal biopsy. According to kidney function, estimated by 99mTc-DTPA clearance, patients were divided into three groups: with apparently normal kidney function (clearance 103.5+/-21.3 mL/min/1.73 m2), with incipient renal failure (clearance 65.5 +/- 11.3), and with advanced renal failure (clearance 28.0+/-6.2). The mean yearly decrease of glomerular filtration rate was 2.74 mL/min. In two patients, an increase of kidney function was recorded. Six patients become dialysis dependent, two from the group with incipient renal failure, but all four from the group with advanced renal failure. Three patients died after 8 to 12 years of follow-up, one from causes unrelated to kidney disease and two from end-stage renal failure. This study has shown that BEN is characterized by a slow course and prolonged evolution, modified by medical supervision and treatment.

[Effects of alfacalcidol therapy on serum cytokine levels in patients with multiple sclerosis].

Multiple sclerosis (MS) is a consequence of genetic and environmental factors. Geographic, genetic, and biological evidence suggests that an important immunopathogenic factor might be the insufficiency of vitamin D. The aim of our study was to investigate the immunomodulatory effect of alfacalcidol, a vitamin D analogue, on cytokine levels in RRMS patients in relapse. We investigated 15 patients suffering from RRMS relapse (an RRMS group) and two control groups: one control group of healthy subjects (n=10) and a NIND group, consisting of patients with non-inflammatory neurological diseases (n=10). All of the MS patients were treated with 5 microgr/day of oral alfacalcidol for a period of five days. The serum cytokine levels of TNF-alpha, IL-10, IL-4, and IL-12 were measured in all the MS patients one day prior to and one day after therapy, and in all the control subjects (ELISA, Quantikine human immunoassay, R&D Systems, UK). Our results showed significantly lower IL-4 and IL-12 levels in the RRMS patients group compared to the N group and the NIND group (p<0.001 Mann-Whitney U-test). No significant differences in TNF-alpha and IL-10 levels were found between the groups, and there was no influence of alfacalcidol on these cytokines in RRMS patients. High doses of oral alfacalcidol induced significant increases in IL-4 and IL-12 levels in RRMS patients (p<0.001, Wilcoxon rank signed test). Therefore, there were no differences in IL-4 and IL-12 levels compared to the N group and the NIND group. Alfacalcidol therapy in RRMS patients did not provoke any side effects. Vitamin D and its analogues, such as alfacalcidol, act as immunomodulatory agents, with potential therapeutic effects for patients with multiple sclerosis.

[Importance of breast-feeding in antioxidant defence].

We investigated the importance of two enzymes (superoxide dismutase--SOD and glutathione peroxidase--GSH-Px) in the antioxidant defence of newborns and analysed their activity in: human colostrum and milk (from 63 mothers, after normal delivery, without complications or signs of infection), gastric fluid (from 10 breast-fed newborns, 7-28 days after birth; and from 15 artificially-fed newborns, with no signs of infection, 7-28 days after birth), and plasma (from 10 newborns, 1-28 days old, with no signs of infection, and 10 newborns, 1-28 days old, with signs of neonatal sepsis). The results of the study showed that there was statistically significant increased activity of SOD (p<0.001) in colostrum compared to mature milk. There was no statistically important difference in the activity of GSH-Px between those two samples. The activity of SOD in the gastric fluid of the artificially-fed newborns was statistically significantly lower than in the breast-fed newborns (p<0.001). The same results were found for mature mother's milk. We discovered a significant increase of SOD plasma activity in the newborns with sepsis, compared to the breast-fed newborns, with no signs of infection. The negative correlation between the activities of SOD and GSH-Px in the gastric fluid samples of the breast-fed and the artificially-fed newborns and the newborns with sepsis, showed that the activities of both enzymes were important for adequate antioxidant defence during the neonatal period. Breast-feeding with both colostrum and mature human milk is probably very important for adequate antioxidant defence in newborns.

Possibilities and limitations of fibrin glue usage in nephron-sparing surgery: experimental study.

INTRODUCTION: The possibilities and limitations of fibrin glue (FG) usage in nephron-sparing surgery were studied. MATERIALS AND METHODS: A prospective experimental study was carried out in 50 pigs: 30 with polar resection, and 20 with mediorenal wedge resection of the kidney. Hemostatic sutures, FG, and FG with a muscle 'cup' in animals with polar resection of the kidney were compared. FG and sutures in animals with the wedge resection of the kidney were studied as well. Bleeding, hot ischemia time, complication rate, and additional scarring were also analyzed. RESULTS: Suture hemostasis is safe but with significant adverse effects in both polar and wedge resection of kidney. FG was not efficient as a sole hemostatic agent for polar resection. It was as efficient as hemostatic suture for wedge resection of the kidney. FG with a muscle 'cup' on a pole of the kidney achieved good results in animals with polar resection of the kidney. Histological analysis confirmed better results with FG because of both the less intense and smaller area of additional scarring. CONCLUSION: FG is a reliable and efficient hemostatic agent for nephron-sparing surgery whenever both sided gluing is possible.

Increased expression of glomerular mesangial cell 5'-nucleotidase in membranous nephropathy.

It has been demonstrated in anti-Thy1 glomerulonephritis that extracellular adenine nucleotides have a significant pro-inflammatory activity, however, glomerular ATP/ADPase, which in concert with 5'-nucleotidase converts ATP/ADP, and AMP to anti-inflammatory adenosine had an anti-inflammatory role. We have studied distribution of 5'-nucleotidase and divalent cation-activated ATPase in kidney biopsies of 15 patients with glomerulonephritis. The major finding was an overexpression of 5'-nucleotidase in the mesangium of kidney from patients with membranous nephropathy. No change in 5'-nucleotidase expression was observed in other common forms of glomerulonephritis: IgA nephropathy, mesangioproliferative and mesangiocapillary glomerulonephritis. The distribution of Mg(2+)-ATPase in investigated specimens was similar to control distribution. Results obtained in this study indicate increased mesangial expression of 5'-nucleotidase in non-proliferative form of glomerulonephritis consistent to a role of mesangial cells in inflammatory processes.

Effect of polyamines on mesangial cell ecto-5'-nucleotidase and ecto-ATPase activity.

Polyamines were found to modulate the activity of several membrane-bound enzymes, participating in cell growth and differentiation. We have studied the effect of polyamines (spermidine, spermine and putrescine) on rat mesangial cell ectoenzymes: 5'-nucleotidase, Mg(2+)-ATPase and Ca(2+)-ATPase. Ecto-5'-nucleotidase activity was significantly increased after 48 h treatment with spermine and spermidine. Mg(2+)-ATPase was increased only after treatment with spermidine; however, Ca(2+)-ATPase was significantly increased after both spermine and spermidine treatment of mesangial cells. Culture of mesangial cells with putrescine did not change the activity of these ectoenzymes. Increased expression of mesangial cell ecto-ATPase and ecto-5'-nucleotidase after spermine and spermidine treatment could result in an increased production of adenosine, a powerful autacoid interesting with respect to a role of mesangial cells in inflammatory processes.