Computational model of articular cartilage regeneration induced by scaffold implantation in vivo.

Computational models allow to explain phenomena that cannot be observed through an animal model, such as the strain and stress states which can highly influence regeneration of the tissue. For this purpose, we have developed a simulation tool to determine the mechanical conditions provided by the polymeric scaffold. The computational model considered the articular cartilage, the subchondral bone, and the scaffold. All materials were modeled as poroelastic, and the cartilage had linear-elastic oriented collagen fibers. This model was able to explain the remodeling process that subchondral bone goes through, and how the scaffold allowed the conditions for cartilage regeneration. These results suggest that the use of scaffolds might lead the cartilaginous tissue growth in vivo by providing a better mechanical environment. Moreover, the developed computational model demonstrated to be useful as a tool prior experimental in vivo studies, by predicting the possible outcome of newly proposed treatments allowing to discard approaches that might not bring good results.

Bioactive potential of silica coatings and its effect on the adhesion of proteins to titanium implants.

There is an ever-increasing need to develop dental implants with ideal characteristics to achieve specific and desired biological response in the scope of improve the healing process post-implantation. Following that premise, enhancing and optimizing titanium implants through superficial treatments, like silica sol-gel hybrid coatings, are regarded as a route of future research in this area. These coatings change the physicochemical properties of the implant, ultimately affecting its biological characteristics. Sandblasted acid-etched titanium (SAE-Ti) and a silica hybrid sol-gel coating (35M35G30T) applied onto the Ti substrate were examined. The results of in vitro and in vivo tests and the analysis of the protein layer adsorbed to each surface were compared and discussed. In vitro analysis with MC3T3-E1 osteoblastic cells, showed that the sol-gel coating raised the osteogenic activity potential of the implants (the expression of osteogenic markers, the alkaline phosphatase (ALP) and IL-6 mRNAs, increased). In the in vivo experiments using as model rabbit tibiae, both types of surfaces promoted osseointegration. However, the coated implants demonstrated a clear increase in the inflammatory activity in comparison with SAE-Ti. Mass spectrometry (LC-MS/MS) analysis showed differences in the composition of protein layers formed on the two tested surfaces. Large quantities of apolipoproteins were found attached predominantly to SAE-Ti. The 35M35G30T coating adsorbed a significant quantity of complement proteins, which might be related to the material intrinsic bioactivity, following an associated, natural and controlled immune response. The correlation between the proteomic data and the in vitro and in vivo outcomes is discussed on this experimental work.

In vitro assessment of the biological response of Ti6Al4V implants coated with hydroxyapatite microdomains.

Dental implantology is still an expanding field of scientific study because of the number of people that receive dental therapies throughout their lives worldwide. Recovery times associated to dental surgery are still long and demand strategies to improve integration of metallic devices with hard tissues. In this work, an in vitro ceramic coating is proposed to improve and accelerate osseointegration of titanium surfaces conceived to be used as dental implants or hip or knee prosthesis, shaped either as dishes or screws. Such coating consists of hydroxyapatite microdomains on the implant surfaces obtained in vitro by immersion of titanium alloy samples (Ti6Al4V) in a simulated body fluid. This titanium alloy is highly used in implant dentistry and trauma surgery, among other fields. Once the immersion times under physiological conditions yielding to different ceramic topographies on this alloy were set, the acellular coating time of major interest so as to optimize its biological development was determined. For this purpose, dental pulp mesenchymal cells were cultured on titanium coated surfaces with different hydroxyapatite outline, and cell adhesion, proliferation and morphology were followed through histological techniques and scanning electron microscopy. It was found that 4 days of acellular hydroxyapatite coating led to a significant cell adhesion on the titanium alloys at an early stage (6 h). Cells tended although to detach from the surface of the coating over time, but those adhered on domains of intricated topography or hydroxyapatite cauliflowers proliferated on them, leading to isolated large cell clusters. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2723-2729, 2016.

Fibrin-chitosan composite substrate for in vitro culture of chondrocytes.

The aim of this study was to develop a biocompatible monolayer substrate based on fibrin and chitosan for in vitro culture of chondrocytes. Fibrin-chitosan composite substrates combined the proved cell adhesion properties of fibrin with the hydrophilicity and poor adhesion capacity of chitosan. Chitosan microspheres were produced by coacervation method, agglomerated within a fibrin network and subsequently crosslinked with genipin. The composite substrate was stable for 28 days of culture due to the high crosslinking density. Human chondrocytes cultured on the composite substrate were viable during the culture period. At the end of culture time (28 days) the composite substrate showed low cellular proliferation, 41% more collagen type II and 13% more production of sulfated glycosaminoglycans with respect to the amounts found at 14 days. The study revealed that dedifferentiated chondrocytes cultured in monolayer on the composite substrate can re-acquire characteristics of differentiated cells without using three-dimensional substrates or chondrogenic media.

N-Succinyl-chitosan systems for 5-aminosalicylic acid colon delivery: in vivo study with TNBS-induced colitis model in rats.

5-Aminosalicylic acid (5-ASA) loaded N-Succinyl-chitosan (SucCH) microparticle and freeze-dried system were prepared as potential delivery systems to the colon. Physicochemical characterization and in vitro release and swelling studies were previously assessed and showed that the two formulations appeared to be good candidates to deliver the drug to the colon. In this work the effectiveness of these two systems in the treatment of inflammatory bowel disease was evaluated. In vitro mucoadhesive studies showed excellent mucoadhesive properties of both the systems to the inflamed colonic mucosa. Experimental colitis was induced by rectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into male Wistar rats. Colon/body weight ratio, clinical activity score system, myeloperoxidase activity and histological evaluation were determined as inflammatory indices. The two formulations were compared with drug suspension and SucCH suspension. The results showed that the loading of 5-ASA into SucCH polymer markedly improved efficacy in the healing of induced colitis in rats.

Dietary administration of high doses of pterostilbene and quercetin to mice is not toxic.

The aim of this study is to evaluate possible harmful effects of high doses of t-pterostilbene (t-PTER) and quercetin (QUER) in Swiss mice. Mice were fed during 28 days at doses of 0, 30, 300, and 3000 mg/kg body weight/day of t-PTER, QUER, or a mixture of both, t-PTER + QUER, which are equivalent to 5, 50, and 500 times, respectively, the estimated mean human intake of these polyphenols (25 mg/day). Daily oral administration of QUER, t-PTER, or a mixture of both of them did not cause mortality during the experimental period. There were no differences in food and water consumption on sex. No significant body weight gain in the male or female groups was observed. Red blood cell number and the hematocrit increased after polyphenols administration compared to control groups. Biochemical parameters were not affected. Histopathological examination revealed no alterations in clinical signs or organ weight at any dose.

Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts.

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 degrees C/min) and stored in the liquid phase of a nitrogen tank for 9.1+/-1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me(2)SO. After thawing in a water bath at 42 degrees C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.

Pseudolinfoma B inducido por hidróxido de aluminio tras tratamiento hiposensibilizante / B-cell pseudolymphoma caused by aluminium hydroxide following hyposensitization therapy

El hidróxido de aluminio es una sustancia que se utiliza como adyuvante de las vacunas. Describimos el caso de una paciente que presenta una reacción adversa local y persistente a esta sustancia debido al tratamiento hiposensibilizante frente a los ácaros. Clínicamente se observan múltiples nódulos subcutáneos en ambos brazos, dolorosos, pruriginosos y con hipertricosis en el sitio de la inyección. El estudio histológico muestra una reacción pseudolinfomatosa de tipo B con predominio de células CD20 positivas, bcl-2 y t (14-18) negativos y una policlonalidad de los genes de cadenas pesadas de las inmunoglobulinas. El microanálisis de espectro de rayos X identificó depósitos inorgánicos de aluminio dentro de los histiocitos granulares entre los centros germinativos. Se establece el diagnóstico de pseudolinfoma B cutáneo por hidróxido de aluminio secundario a una inmunoterapia (AU)

Aluminium hydroxide is used as an adjuvant in vaccines. We describe the case of a patient who presented a persistent adverse local reaction to aluminium hydroxide due to hyposensitization therapy to dust mites. Multiple painful and pruriginous subcutaneous nodules were observed in both arms, along with hypertrichosis at the injection site. Histology revealed a pseudolymphomatous B cell reaction predominantly involving cells that were CD20 positive, did not express bcl-2, and did not display the t (14-18) translocation. The cells also exhibited polyclonal rearrangement of the immunoglobulin heavy chains. X-ray spectral microanalysis revealed deposits of inorganic aluminium in the granular histiocytes among the germinal centers. The patient was diagnosed with cutaneous B-cell pseudolymphoma due to aluminium hydroxide as a result of immunotherapy (AU)

[B-cell pseudolymphoma caused by aluminium hydroxide following hyposensitization therapy]. / Pseudolinfoma B inducido por hidróxido de aluminio tras tratamiento hiposensibilizante.

Aluminium hydroxide is used as an adjuvant in vaccines. We describe the case of a patient who presented a persistent adverse local reaction to aluminium hydroxide due to hyposensitization therapy to dust mites. Multiple painful and pruriginous subcutaneous nodules were observed in both arms, along with hypertrichosis at the injection site. Histology revealed a pseudolymphomatous B cell reaction predominantly involving cells that were CD20 positive, did not express bcl-2, and did not display the t(14-18) translocation. The cells also exhibited polyclonal rearrangement of the immunoglobulin heavy chains. X-ray spectral microanalysis revealed deposits of inorganic aluminium in the granular histiocytes among the germinal centers. The patient was diagnosed with cutaneous B-cell pseudolymphoma due to aluminium hydroxide as a result of immunotherapy.

Ultrastructural patterns of human dentinal tubules, odontoblasts processes and nerve fibres.

The structure of the dentin, consists of the following elements: the odontoblastic processes, dentinal tubules and their periodontoblastic spaces. The odontoblasts are aligned in a single layer in the periphery of the dental pulp and secrete the organic components of dentin. The vitality of dentin is mediated too by the nerve fibres. The ultrastructure of the trigeminal sensory nerves in dentin, especially in relation to odontoblasts remains to be clarified. We studied the third molars and young premolars. The specimens were fixed in glutaraldehyde immediately after extraction. Our investigations give evidence to prove that the distribution of the dentinary tubules is homogeneous, containing a principal odontoblastic prolongation in the regions of the inner dentine, and only in special cases more than one. The area of the dentinary tubules and the odontoblastic prolongations' area were studied. The nervous fibres appeared accompanying 30-70% of the odontoblastic prolongations and their synapsis-like relation with the odontotoblastic processes was demonstrated. The existence of very few periodontoblastic spaces, and intradentinal sensory axons, as well as the intercellular connections will allow us to discover more about the mechanisms of the dentinary permeability, and its significance in maintenance and repair of the human pulpodentinal complex.

Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint.

During embryogenesis the bone tissue of craniomandibular joint (CMJ) is formed through two pathways: intramembranous ossification and endochondral ossification. The development process is under the control of regulatory factors. The osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kappaB ligand are key regulators of osteoclastogenesis. The aim of this study is the localization of OPG and RANKL mRNA and protein in the foetal CMJ by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: OPG and RANKL mRNA and protein were co-localized in the same cell types; OPG and RANKL were specially immunolocated in osteogenic cells; immunolabeling was often seen in the nucleus and cytoplasm of otherwise negative hypertrophic chondrocytes; IHC and ISH labeling decreased from proliferative to hypertrophic chondrocytes; early osteocytes showed dual protein expression and some of the mature osteocytes were ISH-negative; periosteal osteoclasts and chondroclasts were mostly stained by IHC and variably labeled by ISH; the new bone matrix and trabecular borders showed intense immunolabeling. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism in the CMJ development and their extracellular presence in the new bone matrix and trabecular borders suggests a local regulatory role.

Clinicopathological and immunohistochemical analysis of 20 cases of Merkel cell carcinoma in search of prognostic markers.

AIMS: To evaluate the clinicopathological and immunohistochemical characteristics of Merkel cell carcinoma (MCC) in an attempt to find new, potentially significant, prognostic markers. METHODS AND RESULTS: Clinical data and follow-up, histopathological features (pattern, cell size, thickness, mitoses, vascular invasion, lymphocytic infiltration) and immunohistochemical detection [CK20, thyroid transcription factor (TTF-1), chromogranin A, synaptophysin, p53, Ki67, Fli-1, CD99, c-Kit] were evaluated in 20 cases of MCC. Fli-1 and CD99 were detected in 90% and 55% of cases, respectively. Tumour size>30 mm, stage II, 'absent' lymphocytic infiltration, and the presence of>50% of Ki67+ tumour cells, were found to be prognostic indicators of disease-free interval (DFI), but only 'absent' lymphocytic infiltration constituted an independent prognostic factor of DFI after multivariate analysis. For overall survival, the same variables, together with local recurrence and lymph node involvement, had prognostic significance, with only local recurrence as an independent prognostic factor after multivariate analysis. CONCLUSIONS: Absence of lymphocytic infiltration and Ki67 immunoreactivity in more than 50% of tumour cells should be evaluated in conjunction with other well-known prognostic markers in MCC. Furthermore, recognizing that Fli-1 and CD99 expression is commonly found in MCC by immunohistochemistry may avoid misinterpretation in the differential diagnosis of MCC with other small round cell tumours.

Carcinoma ductal micropapilar hipersecretor. Estudio inmunohistoquímico y ultraestructural / An atypical case of hypersecretory micropapillary ductal carcinoma. An immunohistochemical and ultrastructural study

El caso descrito corresponde a una variante inusual de carcinoma ductal de mama llamada Carcinoma Ductal Hipersecretor Quístico. Esta lesión esta caracterizada por la formación de ductos quísticos con mínima atipia celular, lo cual plantea el diagnostico diferencial con la contrapartida benigna llamada Hiperplasia Hipersecretora Quística. A la paciente se le detecta, en una revisión periodica mamográfica, multiples microcalcificaciones en el cuadrante superointerno de mama izquierda. Tras biopsia extemporánea se practica cuadrantectomía con linfadenectomía axilar. La paciente esta libre de enfermedad dos años después del tratamiento. De este caso se realizaron estudios ultraestructurales e inmunohistoquímicos complementarios (AU)

Ultrastructural and immunohistochemical characterization of the so-called giant multinucleate cells in cutaneous collagenomas.

AIMS: This study was undertaken to compare the histopathological, immunohistochemical and ultrastructural features of the so-called giant multi nucleate cells in cutaneous collagenomas: giant-cell collagenoma and solitary sclerotic fibroma. METHODS AND RESULTS: We studied four collagenomas: one giant-cell collagenoma and three solitary sclerotic fibromas. All the cases showed an indolent clinical presentation and were histologically constituted by a well-demarcated dome-shaped proliferation of coarse collagen bundles with a varying number of interspersed giant multinucleate cells and stellate mononuclear cells. The immunohistochemical study on paraffin sections revealed that neoplastic cells in both collagenomas were vimentin and CD34-positive, whereas FXIIIa was only expressed in solitary sclerotic fibromas. In regard to the so-called giant multinucleate cells, we have ultrastructurally found that these cells were 'real' multinucleate cells in giant-cell collagenoma, whereas in solitary sclerotic fibromas they consisted of closely packed aggregates of individual stellate mononuclear cells. Moreover, perinuclear cisternae focally containing finely textured material of moderate density were unexpectedly found in giant cells of giant-cell collagenoma, a finding which was not observed in solitary sclerotic fibromas. Additionally, a characteristic cell-cell interaction between tumour cells and mast cells was encountered in all collagenomas. CONCLUSIONS: This study supports a distinctive immunohistochemical and overall ultrastructural profile of giant multinucleate cells in giant-cell collagenoma and solitary sclerotic fibroma, which suggests a different pattern of differentiation for these two related cutaneous lesions.

Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors.

SUMMARY: The EWS-ETS rearrangements, and their respective fusion gene products, are specifically associated with histopathologically Ewing family tumors (EFT). These translocations are implicated in generating malignant transformation of EFT, but the presence of additional genetic alterations must be considered in the pathogenesis of such tumors. We analyzed 26 samples (biopsies and/or nude mice xenotransplants) collected from 19 patients with an EFT to determine whether molecular and cytogenetic alterations of the G(1)/S checkpoint genes are implicated in the pathogenesis of EFT. We found inactivating p53 mutations in three (16%) cases, which correlated with a loss of p21(WAF1/Cip1) expression and with a monosomy of chromosome 17 in two cases. Homozygous deletion of the p16(INK4A)/p14(ARF) gene was detected in four (21%) cases, three with codeletion of the p15(INK4B) gene and with chromosome 9 abnormalities. In all of these cases, expression of the implicated genes was absent. Hypermethylation of the p16(INK4A) and p15(INK4B) genes was detected in two (10%) and three (16%) cases, respectively, and was correlated with a low level of gene expression. Neither cyclin D1, nor MDM2 and CDK4 amplification was observed. Kaplan-Meier analysis showed that patients with tumors carrying homozygous deletion of the 9p21 locus, or point mutations of the p53 gene, had poorer outcomes than those without these molecular alterations (p = 0.005). In conclusion, 58% (11 of 19) of the analyzed patients showed genetic or epigenetic alterations in either the 9p21 locus or p53 tumor suppressor genes, defining a subgroup of patients with poor clinical outcome. This fact points to an important role of the G(1)/S cell cycle checkpoint dysregulation in the pathogenesis of EFT.

Translocation (10;11;22)(p14;q24;q12) characterized by fluorescence in situ hybridization in a case of Ewing's tumor.

It is well recognized that the identification by classic cytogenetics of t(11;22)(q24;q12) is a useful aid in the accurate diagnosis of Ewing's sarcoma and related tumors. This translocation induces the EWS/FLI-1 fusion transcript, which can be detected by reverse transcription-polymerase chain reaction. Recent studies have also used fluorescence in situ hybridization (FISH) to demonstrate the translocation. The authors coupled classic cytogenetics and FISH on tumor cells from the original specimen, the local recurrence, and the pulmonary metastasis as well as from the xenografted tumors in a case of extraosseous Ewing's sarcoma. FISH analysis not only confirmed the cytogenetic results but also allowed the identification of a tumor-specific chromosome change, consistent with a complex translocation, t(10;11;22), as well as revealed other chromosomal rearrangements on both metaphases and interphase nuclei of each material. In addition this technique served to identify, in the interphase nuclei of the original tumor, the clone that became dominant, from the cytogenetic point of view, in the lung metastasis and in the nude mice xenografted tumors. Current results indicate that the use of FISH on metaphases and interphase nuclei is an easy and reliable approach to complement or even to substitute classic cytogenetic studies for the detection of specific chromosomal rearrangements, especially for determining complex translocations and for describing tumoral clones with different cytogenetic markers.

Characterization of a new rat cell line established from 2'AAF-induced combined hepatocellular cholangiocellular carcinoma.

A rat cell line-nominated CC-62 derived from a combined hepatocellular and cholangiocellular carcinoma obtained by administration of 2-acetylaminofluorene to male Wistar rats, has been established. Using light and electron microscopy it was determined that morphologically the tumor consisted of a mixed population of hepatocytes and cholangiolar neoplastic cells, intermingled with small, undifferentiated oval-like cells. The CC-62 line has been maintained through 90 passages in culture adopting a paving stone arrangement. Doubling time at the 12th passage was 23 h. Immunostaining with a panel of antisera was performed to identify the cytological profiles of the cell line. There was no k-ras or p53 expression by immunohistochemistry, and molecular biology failed to detect mutations. Molecular analysis by reverse transcriptase-polymerase chain reaction revealed transcripts for c-met but no expression of HGF messenger ribonucleic acid. Three cell lines cloned from CC-62 showed the same immunohistochemical and molecular pattern as the parental line. Cytogenetic analysis revealed a chromosome number ranging from 74 to 82 with a modal number of 79 but no clonal structural abnormalities were found. Deoxyribonucleic acid ploidy analysis showed an aneuploid peak. CC-62 caused tumors 1 mo after subcutaneous transplantation into nude mice, with morphological patterns of mucosecretory solid and spindle-shaped carcinoma. This cell line is the first established from a primary rat combined hepatocellular and cholangiocellular neoplasm. The resulting cells expressed biological and morphological markers of hepatocytes and cholangiolar cells. Therefore this cell line may contribute to a better understanding of the histogenesis of liver cancer.

Generalized cutaneous depigmentation following sulfamide-induced drug eruption.

A 41-year-old male developed a generalized drug eruption following sulfamide therapy, with progressive albino-like generalized cutaneous depigmentation. Electron microscopy showed the absence of melanocytes, and clear cells with Langerhans cell characteristics were seen along the basal layer. The present case constitutes a unique reaction to sulfamides not previously reported in the literature.

Soft tissue Ewing sarcoma--peripheral primitive neuroectodermal tumor with atypical clear cell pattern shows a new type of EWS-FEV fusion transcript.

This study describes a new case of Ewing sarcoma (ES)-peripheral primitive neuroectodermal tumor (pPNET) with unusual phenotype and fusion gene structure. The tumor located in the inguinal area of a 15-year-old boy showed a highly aggressive behavior with hematogenous metastases after intensive chemotherapy and bone marrow transplant, causing death 28 months after diagnosis. The tumor displayed a clear cell pattern, and several neuroectodermal markers proved positive both in the original tumor and in xenografts. This neuroectodermal character was confirmed by electron microscopy. Moreover, cytogenetically the tumor has an unusual chromosomal rearrangement, t(2;22)(q13;q22,t(3;18)(p21;q23); representing a new EWS-FEV fusion type in which exon 7 of EWS gene is fused with exon 2 of FEV gene. This is the third published study of an ES-pPNET showing EWS-FEV fusion described, but it is the first study of a tumor with the aforementioned fusion points. These findings support the genetic and morphologic heterogeneity existing within the group of ES-pPNET tumors.