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.

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.

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.

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.

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.

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.

EWS/FLI-1 rearrangement in small round cell sarcomas of bone and soft tissue detected by reverse transcriptase polymerase chain reaction amplification.

Recent cloning of the t(11;22) region has led to the detection of a number of sequences involved in the breakpoints by substituting a sequence which encodes a putative RNA binding domain for that of the DNA binding domain of the human homologue of murine FLI-1. Several tumours display consistent translocation at t(11;22) (q24;q12), a finding that suggests these fusion transcripts could be expressed and detected by reverse transcriptase polymerase chain reaction amplification. To date, only a small number of Ewing's sarcomas (Es) and peripheral neuroectodermal tumours (pPNET) of bone have been tested with this novel molecular biology approach. In this study, we confirmed the presence of the three putative chimaeric transcripts on 7 cases of Es and pPNET sarcomas of bone and soft tissue, providing 100% positivity for the tested tumours. For comparative purposes, a number of other neuroectodermal tumours were analysed with negative results: esthesioneuroblastoma, retinoblastoma, Schwannoma. A primitive soft tissue sarcoma (ectomesenchymoma) with a 22 chromosome rearrangement did not express any transcript, nor did a number of non-neuroectodermal small round cell sarcomas of soft tissue (rhabdomyosarcomas) and bone (microcellular osteosarcoma), conventional bone sarcomas, leiomyosarcomas, malignant fibrous histiocytomas and synovial sarcomas. These results reinforce the value of molecular biology techniques for the correct assessment of histology difficult evaluable neoplasms, such as the group of small round cell tumours within the Es family.

Endometrial stromal sarcomas: immunohistochemical, electron microscopical and cytogenetic findings in two cases.

Uterine sarcomas are approximately 3% of all malignant uterine corpus tumours. Of these, the tumours that originate solely in the stromal elements of the uterine wall are infrequent and have not been well characterized cytogenetically. We report two cases of endometrial stromal sarcomas (ESS), one low grade and one high grade, diagnosed by conventional histology, immunocytochemistry, electron microscopy and cytogenetics. Morphologically clear-cut differential structures were seen at optical, immunohistochemical, and electron microscopic levels, permitting a clear differential diagnosis. The low-grade ESS expressed hormonal receptors and vimentin, whereas the high-grade ESS showed no hormone receptors, high Ki-67 activity, and occasional cytokeratin-positive cells. Ultrastructurally, no malignant epithelial differentiation was seen in the tumour cells, but cilia were found in both cases. Cytogenetic study of the low-grade ESS showed pseudodiploid karyotype with chromosomes 6 and 20 rearranged. The high-grade ESS showed a complex karyotype with clonal numerical and structural anomalies. The chromosomes involved in the structural rearrangements were 1, 3, 6, 7, 13, 14, 15, 17, 19, and 21.

Analysis of p53 and mdm2 proteins in malignant fibrous histiocytoma in absence of gene alteration: prognostic significance.

TP53 and MDM2 genes and their protein expression were evaluated in frozen and paraffin-embedded tissue from 27 patients with malignant fibrous histiocytoma to elucidate the relationship between them, their implication in tumor progression mechanisms and their possible diagnostic-prognostic value in malignant fibrous histiocytoma. Single-strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction-amplified DNA were used to establish two TP53 mutations (7.4%): a point mutation and a 63-bp duplication. Amplification of the MDM2 gene was observed in two tumors (7.4%) by means of Southern-blot analysis, one of them also carrying the TP53 point mutation. Immunohistochemical and Western-blot techniques were used to study nuclear accumulation of p53 and mdm2 proteins: 11 cases (40.7%) with p53 protein expression and thirteen cases (48.1%) with mdm2 protein expression were detected. We confirmed overexpression of mdm2 protein in eight of ten cases (80%) with p53 protein expression without TP53 gene mutation. Statistical analysis shows that simultaneous co-expression of p53 and mdm2 in malignant fibrous histiocytoma is significantly correlated with survival in absence of gene alteration in contrast to the lack of statistical correlation with survival of p53 protein expression alone.

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.

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.

Molecular alterations of the RB1, TP53, and MDM2 genes in primary and xenografted human osteosarcomas.

We report the status of the RB1, TP53, and MDM2 genes in human osteosarcomas and cell lines established from surgical specimens and transplanted into athymic naked mice. By using reverse transcriptase-polymerase chain reaction (RT-PCR) as a prescreening technique and posterior sequencing, we observe new mutations in the RB1 gene, notably a duplication in tandem of exons 3 through 6. TP53 mutations appear in codons most frequently mutated in osteosarcomas. We have not seen MDM2 gene amplification in any reported case. These molecular alterations appear in different osteosarcomas not simultaneously present in the same tumor sample. A link has been described between these three genes in the pathways that control the cell cycle and the tumoral progression, but their functions are probably independent in the development of osteosarcomas. TP53 mutations appear in adult patients, whereas RB1 alterations occur mostly in younger patients.

Near-haploidy in a malignant sacrococcygeal teratoma.

Cytogenetic analysis of a malignant sacrococcygeal teratoma in an adult patient revealed near-haploid (77%), near-diploid (19%), and polyploid (4%) cells. The near-haploid cells had a karyotype of 25,XX,der(5)t(5;7)(p15;p13),+7,der(9)t(6;9)(p21;q34),r(17)(p13q25) . In the near-diploid and polyploid cells identical copies of the structural chromosomal changes were found. Although some of the anomalies observed appear unique to this case, a common breakpoint in chromosome 6 was previously reported as specific in a subgroup of extragonadal germ cell tumors of adults.

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.

Value of nude mice xenografts in the expression of cell heterogeneity of human sarcomas of bone and soft tissue.

Nude mice xenotransplants have been performed on human primary sarcomas of bone and soft tissues in order to delve into the cell heterogeneity of these neoplasms. Particular emphasis has been given to the group of small round blue cell sarcomas (Ewing's sarcomas and peripheral neuroectodermal tumors). Out of 31 xenotransplanted sarcomas, 16 cases have grown positively, and many of them continue to be transferred into nude mice on a regular time basis, being presently considered as fully established nude lines. Here we report the results of such a system, which has been followed with optical, electron microscopical, immunohistochemical and cytogenetic techniques. Osteosarcomas make up the group with the highest number of positivities taken (6 out of 9 transplanted cases). Diversity in growth rate, positive tumors and morphology are taken into consideration. Epithelial foci were seen in one of the transplanted neoplasms. Malignant fibrous histiocytoma and other mesenchymal sarcomas of bone and soft tissues are reviewed. Changes in immunohistochemical reactivity (alpha-1AT and alpha-1AQT) were observed in the transplanted neoplasms. Cytogenetic analysis performed on an undifferentiated (primitive) soft-tissue sarcoma provided clues to its synovial origin. Analysis of Ewing's sarcoma performed on nude mice transplanted tumors shows heterogeneous immunohistochemical response, with enhancement of cytokeratin positive cells, not previously seen in the primary neoplasms.

Toxicological assessment of recombinant xylanase X(22) in wine.

Toxicological evaluation of xylanase X(22) from Aspergillus nidulans expressed in a wine yeast strain was carried out. The safety of the X(22) intake was assessed by digestibility, bioinformatic, and mouse short-term repeated dosing studies, although X(22) shows resistance to proteolytic degradation in the gastrointestinal system, is a minority protein component (<0.5 10(-)(6) %) of the produced wine, and shows no significant amino acid sequence homology to any known food allergens. The 4-week oral toxicity study was performed in Swiss mice at a dose level of 0.01, 0.1, or 1 mg/kg/day (these dosages correlate to 8, 80, and 800 times, respectively, the enzyme amount contained in 250 mL of wine). Body weight, food and fluid intake, urinalysis, and hematology data were obtained. Postmortem examinations and histopathology by both light and electron microscopy were performed. According to the results of this study, no adverse effects were detected by oral administration of X(22).

Clinical and ultrastructural correlations in nasal mucociliary function observed in children with recurrent airways infections.

A study was made of 106 children between 1 and 15 years of age (mean 6 years) with recurrent upper and lower airways infections since birth. Nasal mucociliary transport (NMT) velocity was determined in all subjects by the Tc99m-labeled seroalbumin technique. In 42 children, NMT was found to be altered. In this group of patients the technique was repeated in a period of between 1 and 2 years later. In 23 cases (55%) transport had normalized, while in 19 (45%) it remained altered. Recurrent pneumonia and constant rhinorrhea were more frequent in this group. Situs inversus was only detected in 2 of these patients. Pathology showed ciliary ultrastructure, the absence of dynein arms and microtubule alterations. The absence of cilia was observed in some patients. Normal cilia were also encountered in children with persistently altered nasal mucociliary transport.

Nasal mucociliary transport and ciliary ultrastructure in cystic fibrosis. A comparative study with healthy volunteers.

Cystic fibrosis (CF) is a deadly hereditary disease that produces an abnormally thick, viscous and abundant secretion in the respiratory tract. This secretion in turn leads to the development of recurrent respiratory infections and irreversible lung damage. We have studied nasal mucociliary transport by means of an isotopic technique in 12 patients with CF and in 12 healthy volunteers. Nasal mucociliary transport was repeated at 12-18 months in the patients. In five randomly selected patients ciliary ultrastructure was studied. The velocity of nasal mucociliary transport was significantly slower than in healthy persons (P < 0.001) and no significant differences were observed in both studies (P < 0.05). No significant differences were either observed in the CF group between the homo- and heterozygotes (P < 0.5), or in those six patients infected by Pseudomonas aeruginosa (P < 0.05). Ciliary ultrastructure was normal in one patient. In another patient the sample showed no cilia, while the remaining three exhibited changes similar to those observed in chronic respiratory infections: supernumerary peripheral tubules, ciliary disorientation and ciliary complexes.

Young's syndrome: a further cause of chronic rhinosinusitis.

Three males--aged 32, 35, and 27 years--presented Young's syndrome: a combination of obstructive azoospermia and chronic sinopulmonary infection. The evaluation of nasal mucociliary transport using an isotopic technique revealed mucociliary stasis in one case and decreased clearance in the others (< 2 mm/min). Ciliary ultrastructure was normal in two patients, while the other showed mucous hyperplasia and low ciliary density which made correct ciliary evaluation not possible. The clinical development of this syndrome is chronic, although less severe than in the other two syndromes that exhibit primary failure of mucociliary transport: cystic fibrosis and primary ciliary dyskinesia. Young's syndrome should be considered in the differential diagnosis of patients suffering from chronic rhinosinusitis, particularly with cystic fibrosis and primary ciliary dyskinesia syndrome.

[The primary ciliary dyskinesia syndrome. A frequent pathology]. / Síndrome de discinesia ciliar primaria. Una patología frecuente.

The prevalence of primary ciliary dyskinesia syndrome (PCDS) in Western countries is of 1/40,000 but is 13% in patients with bronchiectasis. The aim of this study was to determine the prevalence of PCDS in patients with bronchiectasis and sinusitis, including whether or not these patients present specific clinical signs. Eighteen patients with these two conditions from an area with 750,000 inhabitants in Valencia (Spain), were studied for 2 years. Radiologic and clinical information was recorded and mucociliary motility was measured with albumin marked with radioactive technetium. The structure of the nasal mucosa cilia was also studied. In 14 patients (77%) mucociliary motility was suppressed and in 13 ultrastructural changes typical of PCDS were observed. Only male infertility and situs inversus were more frequent in patients with PCDS; other clinical signs were equally severe and frequent in patients with PCDS and in those in whom no cause for bronchiectasis and sinusitis could be found. We conclude that 1) the prevalence of PCDS in patients with bronchiectasis and sinusitis is 77%; 2) in these patients a test of mucociliary motility is sufficient for diagnosis (structural study not being required); 3) the prevalence of PCDS in our population seems to be greater than that described; and 4) clinical signs are similar in patients with PCDS and in those with bronchiectasis of unknown genesis.