Microencapsulation of parathyroid cells via electric field and non-surgical transplantation approach.

PURPOSE: Hypoparathyroidism is a rare disease with low PTH, mostly seen as a consequence of neck surgery. Current management is the prescription of calcium and vitamin D, but the definitive treatment is parathyroid allotransplantation, which frequently triggers an immune response, thus cannot achieve the expected success. To overcome this problem, encapsulation of allogeneic cells is the most promising method. By optimizing the standard alginate cell encapsulation technique with parathyroid cells under high-voltage application, the authors reduced the size of parathyroid-encapsulated beads and evaluated these samples in vitro and in vivo. METHODS: Parathyroid cells were isolated, and standard-sized alginate macrobeads were prepared without any electrical field application, while microbeads in smaller sizes (< 500 µm), by the application of 13 kV. Bead morphologies, cell viability, and PTH secretion were evaluated in vitro for four weeks. For the in vivo part, beads were transplanted into Sprague-Dawley rats, and after retrieval, immunohistochemistry and PTH release were evaluated in addition to the assessment of cytokine/chemokine levels. RESULTS: The viability of parathyroid cells in micro- and macrobeads did not differ significantly. However, the amount of in vitro PTH secretion from microencapsulated cells was significantly lower than that from macroencapsulated cells, although it increased throughout the incubation period. Immunohistochemistry of PTH staining in both of the encapsulated cells identified as positive after retrieval. CONCLUSION: Contrary to the literature, a minimal in vivo immune response was developed for alginate-encapsulated parathyroid cells, regardless of bead size. Our findings suggest that injectable, micro-sized beads obtained using high-voltage may be a promising method for a non-surgical transplantation approach.

Paracrine effects of living human bone particles on the osteogenic differentiation of mesenchymal stem cells.

Bone autografting remains the clinical model of choice for resolving problematic fractures. The precise mechanisms through which the autograft promotes bone healing are unknown. The present study examined the hypothesis that cells within the autograft secrete osteogenic factors promoting the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Particles of human bone ("chips") were recovered at the time of joint replacement surgery and placed in culture. Then, conditioned media were added to cultures of human, adipose-derived MSCs under both basal and osteogenic conditions. Contrary to expectation, medium conditioned by bone chips reduced the expression of alkaline phosphatase and strongly inhibited mineral deposition by MSCs cultured in osteogenic medium. Real time PCR revealed the inhibition of collagen type I alpha 1 chain (Col1A1) and osteopontin (OPN) expression. These data indicated that the factors secreted by bone chips inhibited the osteogenic differentiation of MSCs. However, in late cultures, bone morphogenetic protein-2 (BMP-2) expression was stimulated, suggesting the possibility of a delayed, secondary osteogenic effect.

The ovarian cancers in geriatric population: the validity of inflammatory markers, malignancy risk indices 1, 2, 3, 4, and CA-125 levels in malignancy discrimination of adnexal masses.

PURPOSE: To investigate the predictive value of the Risk of Malignancy Index (RMI), CA-125, and inflammatory markers in discriminating ovarian cancers (OCs). MATERIALS AND METHODS: The postmenopausal (PM) women (n= 139) with adnexal masses who un- derwent surgery were included. The predictive value of CA-125, RMI (1, 2,3, and 4) and inflammatory markers [neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR)] were calculated in geriatric (G) and non-geriatric women. RESULTS: OCs had significantly increased NLR and PLR. RMI models were highly reliable in PM (Kappa: 0.642-0.715; AUC: 0.907-0.934). CA-125 measurement alone had good accuracy and moderate reliability in PM (kappa: 0.507-0.587), excellent accuracy and moderate reliability in G, NLR, and PLR predicting OCs, showed fair agreement in the PM, while PLR had a moderate agreement with G. CONCLUSION: RMI algorithms were the best models for malignancy prediction. However, the rise of PLR and CA-125 levels in a G population may be used as refer- ring adnexal masses to gynecologic oncologists.

Design of a 3D aligned myocardial tissue construct from biodegradable polyesters.

The heart does not regenerate new functional tissue when myocardium dies following coronary artery occlusion, or if it is defective. Ventricular restoration involves excising the infarct and replacing it with a cardiac patch to restore the heart to a more healthy condition. The goal of this study was to design and develop a clinically applicable myocardial patch to replace myocardial infarcts and improve long-term heart function. A basic design composed of 3D microfibrous mats that house mesenchymal stem cells (MSCs) was developed from human umbilical cord matrix (Wharton's Jelly) cells aligned in parallel to each other mimicking the native myocardium. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(L-D,L-lactic acid) (P(L-D,L)LA) and poly(glycerol sebacate) (PGS) were blended and electrospun into aligned fiber mats with fiber diameter ranging between 1.10 and 1.25 microm. The micron-sized parallel fibers of the polymer blend were effective in cell alignment and cells have penetrated deep within the mat through the fiber interstices, occupying the whole structure; 8-9 cell layers were obtained. Biodegradable macroporous tubings were introduced to serve as nutrient delivery route. It was possible to create a thick myocardial patch with structure similar to the native tissue and with a capability to grow.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

A Feingold syndrome case with previously undescribed features and a new mutation.

Feingold syndrome (FS) is a dominantly inherited combination of microcephaly with or without learning disabilities, hand and foot abnormalities, short palpebral fissures and esophageal/duodenal atresia. The syndrome has autosomal dominant inheritance with full penetrance, and variable expressivity. Digital anomalies are almost always present. The gene for FS is localized to a 2.2 cM region in 2p23-p24. We report on the first Turkish family with Feingold syndrome. The propositus is a male infant with microcephaly, frontal balding, brachymesophalangy of the second and fifth fingers, bilateral syndactyly of toes 2-3, facial anomalies, choanal atresia and focal epilepsy. His father has microcephaly, and more severe hands and feet abnormalities. One of his brothers died because of eosofageal atresia. Clinical presentation of the family was suggestive of Feingold syndrome, and genetic testing of the MYCN gene confirmed the diagnosis. The missense mutation we report here has not been described previously. FS is an autosomal dominant condition, and therefore, the diagnosis has important implications for genetic counseling.

Giant axonal neuropathy: clinical and genetic study in six cases.

BACKGROUND: Giant axonal neuropathy (GAN) is a severe recessive disorder characterised by variable combination of progressive sensory motor neuropathy, central nervous system (CNS) involvement, and "frizzly" hair. The disease is caused by GAN gene mutations on chromosome 16q24.1. AIMS: To search for GAN gene mutations in Turkish patients with GAN and characterise the phenotype associated with them. METHODS: Linkage and mutation analyses were performed in six affected patients from three consanguineous families. These patients were also investigated by cranial magnetic resonance imaging (MRI) and electroencephalography (EEG). Electromyography (EMG) was performed in heterozygous carriers from family 1 and family 3. RESULTS: Linkage to 16q24.1 was confirmed by haplotype analysis. GAN mutations were identified in all families. Family 1 had the R293X mutation, previously reported in another Turkish family. Families 2 and 3, originating from close geographical areas, shared a novel mutation, 1502+1G>T, at the donor splice site of exon 9. All patients displayed a common phenotype, including peripheral neuropathy, cerebellar ataxia, and frizzly hair. Cranial MRI showed diffuse white matter abnormalities in two patients from family 1 and the patient from family 3, and minimal white matter involvement in the patient from family 2. EMG of a heterozygous R293X mutation carrier showed signs of mild axonal neuropathy, whereas a 1502+1G>T mutation carrier had normal EMG. EEG abnormalities were found in three patients. CONCLUSION: These findings highlight the association of CNS involvement, in particular white matter abnormalities, with peripheral neuropathy in GAN. The phenotypical consequences of both mutations (when homozygous) were similar.

Bone tissue engineering on patterned collagen films: an in vitro study.

This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane. Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27 microm, groove depth: 12 microm, ridge width: 2 microm) and macropatterned (groove width: 250 microm, groove depth: 250 microm, ridge width: 100 microm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation. Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used. This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.

Tumor necrosis factor-alpha gene polymorphisms in children with multiple sclerosis.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of infectious and autoimmune disorders including multiple sclerosis (MS). Childhood-onset MS presents some clinical and laboratory features differing from adult disease. We studied TNF-alpha - 238 G-->A and - 308 G-->A polymorphisms in 24 patients with childhood-onset MS using PCR-RFLP and compared them with healthy control subjects from the same population (n = 93). The genotypes and allele frequencies were not different between patient and control groups (p = 0.348, 95 %CI, 0.28 - 1.92, chi(2) = 0.10 and p = 0.797, 95 %CI, 0.312 - 2.285, chi(2) = 0.20 for - 238 and - 308 alleles, respectively). Although the size of the study group is small, these results do not support a role for TNF-alpha gene polymorphisms in susceptibility to MS in children.

Interleukin-1beta, interleukin-1 receptor antagonist levels in patients with subacute sclerosing panencephalitis and the effects of different treatment protocols.

Subacute sclerosing panencephalitis is a rare progressive inflammatory disease of the central nervous system caused by a persistent aberrant measles virus infection. Cytokines are polypeptides that regulate immune responses and inflammatory reactions. Interleukin-1beta has been implicated as a central mediator of tissue damage and destruction in a number of central nervous system diseases. Interleukin-1 receptor antagonist could function as an important anti-inflammatory cytokine. We studied interleukin-1beta and interleukin-1 receptor antagonist levels in the cerebrospinal fluids of patients with subacute sclerosing panencephalitis and evaluated the effects of different treatment protocols on these cytokines. Interleukin-1beta and interleukin-1 receptor antagonist levels were measured in 15 patients who had a recent diagnosis of subacute sclerosing panencephalitis (group 1), 6 patients who had been treated with isoprinosine (group 2), 5 patients with intraventricular interferon-alpha (group 3), and 6 patients with interferon-beta (group 4). The results were compared within the groups and also with the results of 10 patients with other neurologic disease (group 5). The interleukin-1beta concentrations in cerebrospinal fluid and sera were all below the detection limits (3.9 pg/mL). Interleukin-1 receptor antagonist levels were not statistically different, except for the group treated with intraventricular interferon-alpha. Interleukin-1 receptor antagonist levels were 170 +/- 52, 175 +/- 58, 1605 +/- 518, 77.5 +/- 24, and 108 +/- 18 pg/mL in groups 1 to 5, respectively. Interleukin-1 receptor antagonist levels and cerebrospinal fluid serum ratios were significantly increased during interferon-alpha treatment. In conclusion, interleukin-1 and interleukin-1 receptor antagonist levels were not elevated in the patients with subacute sclerosing panencephalitis. The only treatment protocol that affects interleukin-1 receptor antagonist levels in cerebrospinal fluid was intraventricular interferon-alpha. Further studies on higher numbers of patients may better document the immunologic status of patients with subacute sclerosing panencephalitis and the effects of different treatment modes.

Tissue inflammatory response in subacute sclerosing panencephalitis (SSPE).

The pattern of inflammatory infiltration was studied in the frontal brain biopsies of 28 cases with subacute sclerosing panencephalitis (SSPE) by immunohistochemistry. Lymphocytic infiltration and gliosis were common pathologic findings. CD4+ T lymphocytes were often observed in perivascular areas and CD8+ lymphocytes in the parenchyma. B lymphocytes were located in large perivascular cuffs associated with longer and slower disease. Major histocompatibility complex antigens, interferon-gamma, and tumor necrosis factor-alpha (TNF-alpha) were expressed in endothelial and glial cells. The inflammatory lesions in subacute sclerosing panencephalitis consist of various cell subtypes and cytokines localized in particular areas of the brain tissue and show certain associations with clinical course.

Apoptosis in brain biopsies of subacute sclerosing panencephalitis patients.

Subacute sclerosing panencephalitis (SSPE) is associated with inflammatory infiltration, neuronal loss, and demyelination. The pathogenesis of these changes is unclear. We examined DNA fragmentation and Bcl-2 expression in brain biopsies of nineteen SSPE patients to investigate the role of apoptosis in tissue damage. DNA fragmentation was present in oligodendroglia, and, in tissues with neuronal loss, in neurons. Reactive astrocytes had no DNA fragmentation, but strong Bcl-2 expression. These results suggest apoptosis as one of the mechanisms for oligodendroglial and neuronal death in SSPE.