Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can be obtained from different tissues, including bone marrow, adipose tissue, umbilical blood, Wharton's jelly, and dental pulp. Due to their differentiation potential, regenerative and immunosuppressive properties, as well as ability to expand under in vitro conditions, these cells represent a promising therapeutic tool for regenerative medicine. However, the basic prerequisite for the therapeutic utilization of MSCs is obtaining a sufficient amount. While this may be achieved by prolonged cultivation, long-term culture of MSCs is associated with accumulation of morphological and functional changes. In our study, we focused on analyzing morphological and biological changes of cultured adipose tissue-derived stem cells over 30 passages. We performed morphological analysis using light and electron microscopy, as well as analysis of selected biological properties (expression of surface antigens and selected genes involved in cell regulation and apoptosis, cell cycle, and cell senescence) every 5 passages. Our results showed that long-term expansion leads to significant changes in morphology and affects proliferation kinetics and the cell cycle. On the other hand, the MSCs maintained a prototypical immunophenotype, normal cell cycle and apoptosis regulator function, and maintained a low level of telomerase activity during later passages.

[Results of gestational trophoblastic neoplasia treatment in the Slovak Republic in the years from 1993 to 2012]. / Výsledky liecby gestacnej trofoblastovej neoplázie v Slovenskej republike v rokoch 1993 az 2012.

OBJECTIVE: Analysis and epidemiology of gestational trophoblastic neoplasia treatment in the Slovak Republic in the years 1993-2012. DESIGN: Retrospective epidemiological national study. SETTING: Centre for gestational trophoblastic disease Ministry of Health the Slovak Republic, Bratislava. METHODS: Retrospective analysis results of gestational trophoblastic neoplasia treatment according to prognostic scoring and staging system FIGO/WHO in Centre for gestational trophoblastic disease Ministry of Health the Slovak Republic Bratislava in the years 1993-2012. RESULTS: The treatment of gestational trophoblastic neoplasia (GTN) in the Czech and Slovak Republics started in 1955 and lasted till 1993. After the split of the former Czechoslovakia the Centre for gestational trophoblastic disease was created in Slovakia. 75 patients were treated in this Centre in the years 1993-2012. According to prognostic scoring and staging system FIGO/WHO 56 (75%) patients had low-risk gestational trophoblastic neoplasia and 19 (25%) of patients had high-risk gestational trophoblastic neoplasia. There were 41 patients (55%), 2 (3%), 24 (32%) and 8 (11%) in stage I., II., III. and IV. respectively. Total curability rate was 94.7% and mortality rate was 5.3%. Curability rate 100% was achieved in stage I & II and all placental site trophoblastic tumours (PSTT), 98.3% in stage III and 50% stage IV. In the years 1993-2012 the incidence of choriocarcinoma was one in 76 273 pregnancies and one in 53 203 deliveries. The incidence of other gestational trophoblastic neoplasia in the same years was for PSTT one in 533 753 pregnancies and one in 372 422 deliveries, invasive mole one in 145 611 pregnancies and one in 101 569 deliveries, and persistent GTN one in 40 043 pregnancies and one in 27 932 deliveries. 225-241 patients were treated in the same period of time in the Czech Republic with curability rate 98.2-98. 3%. CONCLUSION: Early detection and treatment in the centre for trophoblastic disease are crucial points in the manage-ment of gestational trophoblastic neoplasia, because the effective therapy of gestational trophoblastic neoplasia with high curability rate is available.

Ultrasound monitoring of the treatment of clinically significant knee osteoarthritis.

OBJECTIVES: The study presented an ultrasound (US) monitoring of treatment as a new imaging US method with the results of therapy of clinically significant knee osteoarthritis. BACKGROUND: X-ray is widely used for knee osteoarthritis classification, which does not involve the evaluation of the soft tissue. High frequency and high resolution US of joints (arthrosonography, echoarthrography) assess not only morphologic but also functional changes in the knee joint. METHODS: In the prospective study, 110 patients with clinically significant knee osteoarthritis were treated non-operative. US examination and US monitoring of therapy was performed during 24 weeks therapy period. A remission of pathomorphologic (marginal osteofytes) and pathophysiologic (effusion in anterior knee and Baker´s cyst) attributes were evaluated according the US classification. RESULTS: Pathomorphologic attributes changes showed a static state, without remission or progression. Pathophysiologic attributes changes showed a remission during the study period. The highest remission was in the first three weeks, 60 % anterior knee effusion and 62 % Baker´s cyst. At the end of study, no changes from the initial US grade was observed in 16 % of effusion in anterior knee and 22 % of Baker´s cyst. Therapeutic resistant Baker´s cyst was present at the end of study in 36 %. CONCLUSION: We demonstrated a new method - US monitoring of therapy, which can objectivize the efficiency of treatment of clinically significant knee osteoarthritis. We would recommend US monitoring of therapy for the routine use in orthopedic clinical praxis (Tab. 6, Graph 3, Fig. 3, Ref. 15).

Mesenchymal stem cells - a promising perspective in the orofacial cleft surgery.

Autologous bone grafts provide the golden standard for closure of oronasal fistulas in the cleft palate. Augmentation may be performed also by homografts and various xenogenic or alloplastic materials to prevent morbidity at the donor site but they may cause many problems (transmission of infections, immune response etc.). All the mentioned approaches also often reveal recurrences of the fistulas and prolong suffering of the cleft patients. Combination of mesenchymal stem cells (MSCs) and so called "platelet gel" seems to be a perspective method in this way. The platelet gel contains hydroxyapatite particles mixed with platelet rich plasma coagulated under effect of the calcium ions. The MSCs from the pelvic bone marrow aspirate are cultivated on a scaffold (collagen membrane) for 3-4 weeks before placement into the cleft defect. The method provides promising results in the alveolar clefts. Authors document a successful case of the secondary surgery in 25-year-old man with the unilateral complete cleft (Fig. 5, Ref. 10).

[Expression of p57 marker in differential diagnosis of complete and partial mole - correlation with DNA analysis]. / Expresia markeru p57 v diferenciálnej diagnostike kompletnej a parciálnej moly - korelácia s DNA analýzou.

Nowadays valid classification of gestational trophoblastic disease, according to the World Health Organisation from the year 2003, divides gestational trophoblastic disease into three groups - molar pregnancies, non-neoplastic non-molar changes of trophoblast and tumours of trophoblast. To the molar pregnancies belong complete, partial, invasive and metastatic hydatidiform mole. In the differential diagnosis it is important to distinguish the complete hydatidiform mole from other forms of gestational trophoblastic disease, because there is an increased risk of malignant transformation of trophoblast cells in complete hydatidiform mole. 10 cases of genetically confirmed diploid complete mole and 10 cases of genetically confirmed triploid partial mole were included into our retrospective study. All cases were examined microscopically in the basic haematoxillin and eosin staining and immunohistochemically with the use of antibodies against human choriogonadotropin hormone, placental alkaline phosfatase and protein p57. Villous cytotrophoblast, stromal villous cells, extravillous trophoblast and decidual cells were p57 positive in all cases of partial hydatidiform mole. All 10 cases of complete hydatidiform mole were p57 negative in stromal villous cells and villous cytotrophoblast. P57 protein is a marker distinguishing complete hydatidiform moles from partial moles.

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells.

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterization showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.

Does cell therapy and tissue engineering represent a promising treatment of diabetic foot ulcers?

Diabetes mellitus is one of the most severe and costly chronic disease of our time. Approximately 2-3% of diabetics have an active foot ulcer, and 15% of all patients with diabetes will develop an ulcer during their lifetime. Treatment of foot complications is one of the main items in the absorption of enormous economic and health resources addressed to the diabetic patients. Advances in basic science, tissue culture techniques and cell therapy promise to improve the treatment of diabetes as well as its complications, i.e. also the ischemic ulcers of the foot. At present, the isolation of any specific type of cells, their in vitro expansion and biological characterization of acquired cell population are possible. For the healing process in ischemic diabetic ulcers, stem cells, endothelial progenitor cells and fibroblasts, both in suspension or placed on an extracellular scaffold are used. This process is focused on stimulating the new blood vessels formation. This is stimulated by the paracrine secretion of multiple growth factors and their receptors. Verified are the vascular endothelial growth factor and its receptor, fibroblast growth factor, interleukin-8 and proangiogenic cytokines (Ref. 62).

Diabetes--adult stem cells as an future alternative therapy?

In both types of the diabetes mellitus, the lack of functional beta-cells is crucial, leading to complications associated with development of hyperglycaemia. One way to achieve a constant normoglycemic state without hypoglycemic episodes is either whole pancreas transplantation, or transplantation of isolated islets of Langerhans. Another approach to correct the beta-cell deficit is the stimulation of beta-cells in pancreas to regeneration. The development of new diabetes therapy is the main goal for many scientists around the world. This article is focused on the stem cells and their potential for clinical applications (Ref. 47).

In vitro evaluation of the cytotoxicity and genotoxicity of resorcylidene aminoguanidine in human diploid cells B-HNF-1.

RAG belongs to appropriate inhibitors of protein glycation, i.e. formation of advanced glycation end products, which are thought to be responsible for some complications of DM, including neuropathy, angiopathy, retinopathy and nephropathy. In the present study authors have evaluated the genotoxic effect of RAG on the cell culture of human neonatal fibroblasts (B-HNF-1) in regard to its potential clinical application as inhibitor of advanced glycation end products in relationships to the pathogenesis of chronic diabetic complications. The direct contact cytotoxicity assay and micronucleus test were performed. The results showed that RAG in the concentration range of 1 x 10-4 to 1 x 10-6 mol.l-1 did not induce any changes in the morphology of exposed B-HNF-1 cells. The frequency of micronuclei was not significantly increased as well. The inhibitive effect of resorcylidene aminoguanidine was directly proportional to its concentration. It can be concluded that RAG at the selected concentrations has an inhibitive effect on proliferation of the treated cells and, at the same time, does not display any genotoxic effects on B-HNF-1 cells.

[Unusual infection complication of total hip arthroplasty]. / Neobvyklá infekcná komplikácia totálnej náhrady bedrového klbu.

Authors present the case history of a 66-year old patient after repeated reimplantations of the THA with a deep infect caused by a rare aetiological agent (Serratia marcescens) associated with a pyogenic sinus. They describe the disease history, therapeutic procedure, complications associated with the surgery as well as postoperative course after the reimplantation of a customized total hip replacement. In the conclusion they state that in case of an infected total hip arthroplasty the treatment is focused on the salvage of the infection process and preservation of the function of the affected limb. Of essential importance is surgical revision with a radical removal of necrotic tissues and hardware in combination with an intensive parenteral antibiotic administration.

[Incorporation of bone allografts with the use of Octopus revision system in total hip arthroplasty]. / Inkorporácia kostných alostepov pri pouzití revízneho systému Octopus.

PURPOSE OF THE STUDY: The study describes the revision arthroplasty of hip joints with grade III acetabular defects, as rated according to Paprosky, employing morsellized bone allografts and the Octopus revision acetabular system. MATERIAL: In the period from 1998 to 2002, a total of 8297 operations were performed in our Department; of these 1527 (18.4%) were total hip replacements. In this group, 267 (15.5%) revision arthroplasties were carried out, with the the Octopus revision system being used in 32 patients (age range, 30 to 79 years; average age, 60.3 years). The average period between the initial surgery and reimplantation was 6.5 (range, 2-14) years. METHODS: Acetabular defects were filled with morsellized cancellous bone allografts (one to three femoral heads per patient) obtained from the tissue bank. In nine patients, allografts were combined with hydroxypatite (Biovan). Osteointegration of the bone allografts and, if it occurred, loosening of the implant were checked by X-ray examination. RESULTS: The patients were examined at intervals of two months. The average Harris hip score was 76 points at 1 to 3 years. None of the patients had deep post-operative wound infection; in one patient, the distal hook of the Octopus system broke off. The complete incorporation of allografts was seen on X-ray images in 28 patients; at follow-up, a radiolucent zone between the acetabular bed and the allograft was still visible in four patients. No implant loosening was recorded. DISCUSSION: The Octopus system has an advantage of using morsellized allografts instead of solid ones that are associated with poorer incorporation and a higher risk of loosening. The remodeling of an allograft below the ring leads to the production of bone tissue, which is useful if revision arthroplasty were to be performed. CONCLUSION: Based on their experience, the authors regard the Octopus revision system with the use of morsellized bone allografts from the tissue bank as an effective surgical procedure for the treatment of Paprosky grade III acetabular defects. The allografts below the ring are fully incorporated and remodeled; as a result the initial defect is gradual filling with the patient's own tissue, which has very good clinical outcomes.

[Use of RT-PCR in the detection of circulating micrometastases]. / Vyuzitie metódy RT-PCR pri detekcii cirkulujúcich mikrometastáz.

During assessment of the progression of several types of carcinomas, such as cancer of the breast, lungs, prostate or urinary bladder world-wide the presence of circulating cells (micrometastases) in the circulation is followed up. These methods are gradually introduced also in Slovakia. First we tried in the Institute of Medical Biology and Genetics Medical Faculty Comenius University in collaboration with the Urological Clinic of Dérers the Faculty Hospital with policlinic to apply this method in carcinoma of the prostate (CaP). We detected the presence of epithelial prostate cells in the peripheral blood stream of patients with advanced prostate cancer where before secondaries were not detected.

Cytotoxicity test and cytogenetic analysis of effects of aminoguanidine in vitro.

The potential genotoxic activity of chemical substances in vitro is usually assessed by the micronucleus test and by karyological analysis. Use of the fluorescent plus Giemsa (FPG) technique is also recommended in the event that positive results are found in the micronucleus test, or if there is an increased rate of structural and numerical chromosome aberrations compared with controls. The tested substance, aminoguanidine (AG), has a marked ability to inhibit the toxic effects of carbonyl products (carbonyl stress) that arise during the end-phases of non-enzymatic protein glycation both in vitro and in vivo. The importance of this ability follows the finding that the production of advanced glycation end-products (AGE) is a part of the molecular mechanism of the pathogenesis of chronic diabetic complications. The aim of this study was to test the cytotoxic and clastogenic effects of AG on cells of the diploid cell line B-HEF-2, derived from a three-month-old male fetus. The results of the test did not reveal any induction of micronucleus production in the analyzed cells at AG concentrations ranging between 1 x 10(-2) and 1 x 10(-4) mol.L-1. Karyological analysis showed no clastogenic effect of the tested substance nor any increased rate of structural chromosome aberrations. The positive properties of AG and to its potential use as a glycoxidation inhibitor and AGE production are somewhat dimmed by its ionic nature, which hampers hydrophobic interaction with the nonpolar components of biological membranes. For this reason, the authors will further study the cytotoxicity and cytogenetic analysis of Schiff bases of AG synthesis on the basis of natural aldehydes (resorcine aldehyde, pyridoxal, etc.) in which antiglycation activity has been detected.

[DNA analysis in gestational trophoblastic disease]. / DNA analýza gestacnej trofoblastovej choroby.

OBJECTIVE: DNA analysis of different forms of gestational trophoblastic disease. DESIGN: Retrospective clinical study. SETTING: Slovak Center of Trophoblastic Disease, Bratislava, Slovak Republic. METHODS: In the period of September 1993 to April 2003, eighty-nine cases of gestational trophoblastic disease were analysed. There were 22 cases of partial hydatidiform moles, 58 cases of complete hydatidiform mole, 5 cases of invasive mole and 4 cases of gestational choriocarcinomas. Southern hybridization and polymerase chain reaction were used for DNA analysis. RESULTS: From 22 analyzed cases of partial hydatidiform moles 19 (86.4%) were triploid and 3 (13.6%) diploid ones. There were 58 cases of complete hydatidiform mole and out of them 29 (50%) were homozygous, 28 (48.3%) heterozygous, and in one case (1.7%) both paternal and maternal genome was detected. In 8 cases of heterozygous and in one case of homozygous complete hydatidiform mole occurred a malignant transformation to gestational choriocarcinoma. CONCLUSIONS: Molecular analysis can determine the nuclear DNA origin of complete hydatidiform mole and allow us to define the patients with higher risk of malignant transformation usually to gestational choriocarcinoma.

Results and perspectives of the treatment of skeletal neoplasms.

Improvements in the therapy of skeletal neoplasms at the end of the 20th century are the reason for a retrospective view on the development of the therapy of skeletal neoplasms, especially in the Slovak Republic. Up to the sixties years of the 20th century was amputation the imperative in the surgical treatment of malignant tumors, above all tumors of the extremities. Then chemotherapy was introduced into the treatment of these tumors, in Slovak Republic it was later. Bone transplantations were widely used in the 70 and 80 years of 20th century. Autotransplantations of bone grafts were used at the Orthopaedic department in Bratislava already in the year 1953, but low number of appropriate grafts limited their use in clinical practice. In the 70 and 80 years development of bone banks was observed. Results of the treatment of malignant diseases with this technique are considered poor. Only few patients survived more than 5 years after the surgery, mainly due to inappropriate techniques of detection, localization and determination of the size of the tumor. Biopsy played above all at this time an important role in the diagnostics of bone tumors. Apart from basic examinations of the fixed material, processed after it was imbedded in particular medium, perioperative biopsy is recently being performed with the technique of frozen sections from unfixed tissue obtained during the surgery. This type of material processing prolongs the period from obtaining the material till making the diagnosis, but the advantages of this type of examination are undoubted. (Ref. 17.).

High tibial osteotomy and debridement of the knee joint in treatment of varotic gonarthrosis.

UNLABELLED: BACKGROUND OF THE PROBLEM: We perform high tibial osteotomy with knee debridement and forage. GOAL: We evaluated the results of operations in 47 patients, who were operated from 1st January 1998 to 31st of December 2000. RESULTS: We examined the patients before operation and 2, 4, 6, 12, 24 and 30 months after it. The follow up was from 6 months to two and a half years after operation. The results after the operation were evaluated by both the patient (pain) and the doctor. The doctor evaluated the axial state of the extremity, the range of movement in the knee joint, present synovitis and the X-ray. The youngest woman was 52 years old, the oldest woman was 71 years old. The average age was 63.3 years. The youngest man was 25 years old, the oldest was 67 years old. The average age was 57.2 years. Patient's evaluation after the operation: 34 percent had no pain, 43 percent felt pain after excessive loading at the joint, 19 percent did not register any change and 4 reported increased pain. Doctor's evaluation: In 23 percent of patients, excellent results were achieved, 47 percent had good results, in 21 percent, they were satisfactory and in 9 percent, unsatisfactory results were recorded. CONCLUSION: After the theoretic analysis and evaluation of the group of operated patients, the authors indicate the possibility to perform high tibial osteotomy with debridement and forage as a joint saving knee operation in varotic gonarthrosis. (Fig. 1, Ref. 6.)

In vitro cytotoxicity testing of coladerm membrane.

At present, biodegradable and biocompatible membranes based on collagen and glycosaminoglycans play an important role in substitutive medicine. Modern biomaterials use a chemically modified collagen-based matrix for implants with programmable biodegradability as a substitute of buccal mucosa, skin, cartilage, etc. Besides the requirements for biocompatibility and biodegradability, the membranes must be also non-toxic. Therefore, cytotoxicity testing of these materials in vitro is an integral part of introducing newly developed types of membranes into clinical practice. As a biological model for the tested COLADERM membrane, cell cultures from human embryonic fibroblasts (B-HEF-2) were used for both cytotoxicity testing as well as in tests to assess the ability of cells to proliferate on this membrane. Along with the ability of cells to grow on the surface and inside the membrane, immunohistochemical examination and scanning electron microscopy (SEM) were performed as well. The obtained results have shown that the COLADERM membrane is non-toxic with suitable structural and biological properties for clinical application as a substitute of buccal mucosa following surgical ablation of malignant tissues from the oral cavity.

Transtrochanteric rotational osteotomy, section after Sugioka.

BACKGROUND: Femoral caput necrosis is an actual therapeutic problem, because it appears mainly in mid-aged people. The necrotic focus is most often localized in the proximal and ventral parts of the femoral bone capitulum, which, from the biomechanical point of view is the most loaded part. These cases can be possibly treated by transtrochanteric rotational osteotomy. By means of this operation we rotate the necrotic focus to the less loaded part of the joint in correlation with the acetabulum. PURPOSE: The aim of this paper is to inform about a rarely performed operation after Sugioka: transtrochanteric rotational osteotomy in coincidence with avascular necrosis of the femoral bone caput. We would like to point to the possibility of a joint-saving operation, which enables to postpone implantation of total endoprosthesis to older age. METHODS: We analyzed the operational therapy performed at our department in the period 1998-2000, focusing especially on joint-saving operations due to femoral caput necrosis. We turned our attention to transtrochanteric rotational osteotomy after Sugioka. In four casuistics, we describe the results of these operations. RESULTS: Transtrochanteric rotational osteotomy was performed in 12 patients. The treatment is described in four casuistics. The results after 1-3 years are good. The illness has not progressed in any of patients, pain has regressed, mobility improved and it was not necessary to implant total endoprosthesis. CONCLUSION: We consider the transtrochanteric rotational osteootomy, section after Sugioka, as one of the possible treatments in mid-aged patients. Our effort is to perform joint-saving operations in order to postpone the implantation of total endoprosthesis. (Fig. 10, Ref. 13.)

[DNA analysis of partial and complete hydatid mole]. / DNA analýza parciálnej a kompletnej moly hydatidózy.

DNA analysis enables an unambiquous recognition of partial and complete hydatiform moles and at the same time to distinguish the homozygous and heterozygous forms of KHM, the fact of which has its significance for target investigation of the course of the post-intervention period in mostly endangered patients. All investigated patients with gestational choriocarcinomas are proved to have molar pregnancy in their case histories.