Development of a multilayered palate substitute in rabbits: a histochemical ex vivo and in vivo analysis.

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.

Development of secretome-based strategies to improve cell culture protocols in tissue engineering.

Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration.

Immunocytochemical developmental patterns of the thoracolumbar sympathetic chain in the chick and a comparison with its adrenal counterpart.

The immunocytochemical development of the thoracolumbar sympathetic ganglion and its adrenal counterpart was studied in the chick from days 3.5 to 12 of incubation, using antibodies to 17 separate antigens, including antibodies to pan-neuroendocrine markers, catecholamine-synthesizing and proprotein-processing enzymes, and neuropeptides. Some of the antigens studied (Go protein-alpha subunit, thyrosine hydroxylase, and galanin) were strongly expressed from the first days of development, whereas others (chromogranin-A, chromogranin-B, 7B2 protein, and somatostatin) showed a diverse immunoreactive expression at different stages. Three different patterns were found in the development of both adrenal medulla and thoracolumbar sympathetic ganglion. In the first (chromogranin-A and B, Go protein-alpha subunit, tyrosine hydroxylase, HNK-1, and galanin), virtually all medullary and thoracolumbar sympathetic ganglion cells were strongly immunostained from day 4 onward. Except for HNK-1, chromogranin-A and B, there was a steady increase in immunoreactive cells for all the remaining antigens up to day 12. In the second (7B2 protein, proprotein convertase 2, and secretogranin II), full antigenic expression was reached in medullary and thoracolumbar sympathetic ganglion cells by day 10. In the third pattern (proprotein convertase 3, somatostatin, dopamine-beta-hydroxylase, neuron-specific enolase, vasoactive intestinal polypeptide, and met-enkephalin), differences in immunoreactivity were observed between the medullary and thoracolumbar sympathetic ganglion cells.

Relationships between the parotid gland and the facial nerve during human development.

A correlation was sought between the organization of the parotid gland and the formation of the large vessels and nerves that passed through the glands of 12 human embryos and 12 human fetuses. There was no evidence that the gland became a bilobate structure as a result of the course of the facial nerve, whose interglandular branches were surrounded during development by the multidirectional ramifications of the expanding parotid anlage.

Development of the human submandibular salivary gland.

The development and morphogenetic timetable of the submandibular gland was studied in 37 human embryos and human fetuses. The medial paralingual groove constituted the anlage of the submandibular gland: Its anterior three-quarters gave rise to Wharton's duct, and its posterior quarter to the submandibular gland proper. The sublingual process of the submandibular gland originated from a lateral ectodermal bud of the anlage of the submandibular gland, in the posterior quarter of the medial paralingual groove.

Modulation of HLA class I expression in multidrug-resistant human rhabdomyosarcoma cells.

An abnormal HLA expression has been detected in some tumors including rhabdomyosarcoma (RMS). Classical cytotoxic treatment of these tumors, the most common childhood soft tissue malignancy, may induce multidrug resistance (MDR) associated with the expression of a 170-kDa membrane-associated glycoprotein (P-glycoprotein). In order to analyse the connection between modulation of HLA expression and the development of the MDR phenotype mediated by P-glycoprotein in RMS, we used three resistant RMS cell lines; two of these resistant cell lines (TE.32.7.DAC and RD-DAC) were established by in vitro exposure to actinomycin D, a drug of choice in the treatment of RMS; the resistant RMS- GR cell line was established from an embryonal RMS tumor after polychemotherapy. Our results showed that all the resistant cell lines showed a significant increase in the expression of HLA class I surface antigens in comparison to drug-sensitive cells. Blockade of P-glycoprotein with verapamil led to a decrease in HLA class I expression in RMS resistant cell lines. However, no modulation of HLA class II expression was observed in any of the three analyzed cell lines. These findings support the hypothesis that the development of resistance mediated by mdr 1/P-glycoprotein, directly influences the expression of HLA class I in RMS cells, inducing to upregulation. This effect may be relevant to the application in RMS of immunotherapy against tumor-associated antigens presented by HLA class I molecules.

Factores asociados a las estancias anormalmente prolongadas en las hospitalizaciones por insuficiencia cardiaca en España / Associated factors with unusually long stays in heart failure hospitalizations in Spain

Fundamento. La insuficiencia cardiaca es un proceso de altaprevalencia que origina repetidos ingresos hospitalarios consobrecarga asistencial e incremento del gasto sanitario. Losobjetivos de este trabajo son describir y caracterizar los casoscon estancias prolongadas por este síndrome, detectandoposibles factores asociados a la misma.Método. Estudio de cohorte histórica de todos los episodiosde personas mayores de 45 años, ingresados por insuficienciacardiaca en el Sistema Sanitario Público Español en el período1997-2007. Fuente: 808.229 episodios clasificados como GruposRelacionados de Diagnóstico 127 y 544, según el Conjunto MínimoBásico de Datos del Instituto de Información Sanitaria. Seevaluaron variables sociodemográficas (edad, género, comunidadautónoma), clínicas (comorbilidades, complicaciones, tipode ingreso y alta) y de gestión (estancia, tipo de hospital, reingresos).Se definió estancia anormalmente prolongada aquellaque superó el percentil 90 (14 y 16 días, respectivamente),construyéndose un modelo de regresión logística para valorarsus posibles factores asociados.Resultados. Presentaron estancias anormalmente prolongadasel 11,4%, mostrando inferior edad media y mayor número dediagnósticos y procedimientos, reingresos y mortalidad que elgrupo sin estancias prolongadas. Padecer anemia, insuficienciarenal, TEP o ictus así como el reingreso y el ingreso programadose asociaron a mayor probabilidad de estancia anormalmenteprolongada.Conclusión. Es posible definir un perfil de comorbilidady sociodemográfico que valore la probabilidad de tener uningreso prolongado, si bien dadas las características de lasbases de datos administrativas la capacidad discriminativadel modelo es discreta(AU)

Background. Heart failure is a process of high prevalencethat causes repeated hospital admissions with increasedhealth care costs. The aim of this article is to describe andcharacterize the cases with long stays due to this syndrome,identifying associated factors wherever possible.Method. An historical cohort of all the episodes of peopleover 45 years with a diagnosis of heart failure admitted inthe Spanish Public Health System in the period 1997-2007.Source: 808,229 episodes classified as Diagnosis RelatedGroups 127 and 544 according to the Minimum Basic Dataprovided by the Institute for Health Information. We assessedsociodemographic variables (age, gender, region),clinical variables (comorbidities, complications, type of admissionand discharge) and management variables (lengthof stay, type of hospital readmissions). An abnormally prolongedstay (APS) was defined as one exceeding the 90thpercentile (14 and 16 days, respectively); we built a logisticregression model to assess their possible associated factors.Results. Eleven point four percent (11.4%) presented abnormallyprolonged stays, showing lower mean age and increasednumber of diagnoses and procedures, readmissions andmortality than the non-abnormally prolonged stay group.Anemia, kidney failure, pulmonary embolism or stroke aswell as readmission and scheduled admission were associatedwith increased likelihood of APS.Conclusion. It is possible to define a comorbidities andsociodemographic profile to assess the likelihood of a prolongedhospital stay, but given the nature of administrativedatabase the model’s discriminative ability is quite discreet(AU)

Origin of the styloglossus muscle in the human fetus.

The origin of the styloglossus muscle was histologically studied bilaterally in nine human fetuses (18 sides). In all cases, the muscle originated in Reichert's cartilage, which gives rise to the temporal styloid process. We identified three types of variation: type A, an accessory muscle fascicle originating from the mandibular angle, found in 7 cases (12 sides); type B, where the styloglossus muscle was attached to the mandibular angle by fibrous tracts, found in three cases (4 sides); and type C, where an accessory muscle fascicle arose from the fibrous tract connecting Reichert's cartilage to the mandibular angle; found in one case. In all cases (2 sides), the styloglossus muscle was innervated by the hypoglossal nerve. Relationships between the styloglossus muscle and vasculonervous elements of the prestyloid and retrostyloid spaces were analysed.

Morphogenesis of the second pharyngeal arch cartilage (Reichert's cartilage) in human embryos.

This study was performed on 50 human embryos and fetuses between 7 and 17 weeks of development. Reichert's cartilage is formed in the second pharyngeal arch in two segments. The longer cranial or styloid segment is continuous with the otic capsule; its inferior end is angulated and is situated very close to the oropharynx. The smaller caudal segment is in contact with the body and greater horn of the hyoid cartilaginous structure. No cartilage forms between these segments. The persistent angulation of the inferior end of the cranial or styloid segment of Reichert's cartilage and its important neurovascular relationships may help explain the symptomatology of Eagle's syndrome.

Morphogenesis of the juxtaoral organ in humans.

The juxtaoral organ was studied using light microscopy in 55 human embryos and 90 fetuses at different stages of development. The juxtaoral organ arises from the epithelium at the bottom of the transverse opening of the primitive mouth during O'Rahilly stage 16 and becomes detached from the epithelium after O'Rahilly stage 18. The juxtaoral organ is innervated by the buccal nerve from O'Rahilly stage 20 onward, and its connective tissue capsule is clearly visible after week 11 of development. This study enabled us to describe three main periods of juxtaoral organ development: (1) the period of epithelial condensation and invagination, at O'Rahilly stages 16-17; (2) the period during which the juxtaoral organ becomes detached from the oral epithelium and is innervated, at O'Rahilly stages 18-23; and (3) the period during which the connective tissue capsule is formed, after week 11 of development. We also analysed the juxtaoral organ of five additional fetuses by immunohistochemistry with anti-NF-200 to verify their innervation. The results show that the juxtaoral organ may have a function in the mechanical activity of the region.

Histogenesis of the spleen in the human embryo in O'Rahilly's stages 17 to 23.

A histological study of the spleen in 30 human embryos in O'Rahilly's stages 17-23 revealed that the splenic anlage is composed of mesenchymal elements. Furthermore, the spleen is formed by a cytoreticulum containing a number of cells similar in appearance to lymphoid elements. No evidence of a celomocapsular separation is seen in this period.

Development of the sympathoadrenal system in the chick embryo: an immunocytochemical study with antibodies to pan-neuroendocrine markers, catecholamine-synthesizing enzymes, proprotein-processing enzymes, and neuropeptides.

BACKGROUND: The adrenal chromaffin cells synthesize, store and secrete a complex mixture containing amines, structural proteins, enzymes, and neurohormonal polypeptides. Most of the studies dealing with the development of the avian sympathoadrenal system have been based on antibodies recognizing signal molecules like HNK-1, NC-1, and N-CAM. METHODS: The development of the chick sympathoadrenal system was studied from 3 1/2 to 21 days of incubation, both morphologically and immunocytochemically, using antibodies to 17 separate antigens, including antibodies to pan-neuroendocrine markers, catecholamine synthesizing enzymes, proprotein-processing enzymes, and neuropeptides. RESULTS: Some of the antigens studied were heavily expressed from the first days of development, e.g., chromogranin-A, chromogranin-B, Go protein-alpha subunit, tyrosine hydroxylase, and galanin, while for others a strong heterogeneity both in number of immunoreactive cells and intensity of immunostaining was recorded at the different stages, e.g., dopamine-beta-hydroxylase,, 7B2 protein, proprotein convertase 2, somatostatin, met-enkephalin, secretogranin II, proprotein convertase 3, neuropeptide Y, phenyl-N-methyl transferase, and neuron-specific enolase. The first immunoreactivities to appear at day 3 1/2 were those for HNK-1, tyrosine hydroxylase, chromogranin-A, and chromogranin-B. Except for HNK-1, immunoreactivity for all the remaining antigens showed a steady increase up to the hatching. CONCLUSIONS: Three expression patterns were found, in the developmental adrenal-gland: defining early permanent markers (chromogranin-A, chromogranin-B, Go protein-alpha subunit, tyrosine hydroxylase, and galanin), others that show a progressively increased expression until the day 10 of development (dopamine-beta-hydroxylase, 7B2 protein, proprotein convertase 2, somatostatin, met-enkephalin), and late-appearing antigens (secretogranin II, proprotein convertase 3, neuropeptide Y, phenyl-N-methyl transferase, and neuron-specific enolase).

Development of the human wrist joint ligaments.

BACKGROUND: Many studies have been published on the development of the human wrist joint, but scant attention has been given to the development of the wrist joint ligaments. Moreover, traditional description of wrist anatomy usually depict only the superficial capsular fibers of the wrist joint. The only ligamentous structure to receive much attention is the articular disc of the wrist joint, which has been described as a fibrocartilaginous structure extending from the medial edge of the lower end of the radius to the ulnar styloid process. METHODS: In the present report, we synthesize our observations in the wrist joint ligaments in 35 serially sectioned human embryonic and fetal hands (16 embryos and 19 fetuses). RESULTS: The interosseous intercarpal ligaments are organized from the mesenchyme, which, until O'Rahilly's stage 23, fills the intercarpal spaces. These ligaments are not individually distinguishable until the 9th week of development. The collateral ligaments begin to form in O'Rahillys's stage 22 and are completely formed by the end of week 10. The palmar radiocarpal and ulnocarpal ligaments (beginning with the palmar radiocarpal ligament) begin to form in O'Rahilly's stage 23 and are fully developed by the end of week 10. At this time, development of the dorsal radiocarpal ligament begins; this process is completed by the end of week 13. The articular disc which is initially formed of a single element, first appears in O'Rahilly's stage 23 and its organization is completed at week 10 of development. CONCLUSIONS: We establish the morphogenetic time-table of the wrist joint ligaments. Our descriptive findings may help explain carpal motion and the origin of wrist injuries.

Grafts of the third branchial arch in chick embryos.

The parathyroid glands have been classically considered derivatives of the third and fourth pharyngeal pouches in most species, including humans. The presence of neural crest-derived cells in parathyroid glands connective tissue has apparently been established. However, our previous studies have provided a new hypothesis on the origin of these glands in human and chick embryos. To determine the true origin of the third parathyroid (parathyroid III) gland in the chick embryo, pieces of the third branchial arch from donor chick embryos at Hamburger and Hamilton's stage 19 (embryonic day 3) were grafted to host chick embryos at the same stage of development. Starting from Hamburger and Hamilton's stage 27 (embryonic day 5), a structure identified as the parathyroid III appeared in the ectodermal (epipharyngeal) placode of the third branchial arch graft, from which it subsequently became separated at Hamburger and Hamilton's stage 28 (embryonic day 5.5) and continued to develop and mature. Our findings suggest the conclusion that the parathyroid III gland begins to develop from the epipharyngeal placode, so that this gland, from our point of view, could be considered ectodermal in nature.

Ectodermal ablation of the third branchial arch in chick embryos and the morphogenesis of the parathyroid III gland.

The parathyroid glands have been classically considered to be derivatives of the third and fourth pharyngeal pouches in most species, including humans. Furthermore, the presence of neural crest-derived cells in the parathyroid glands connective tissue has been apparently established. However, our previous studies have provided a new hypothesis on the origin of these glands in human and chick embryos. To determine the origin of the parathyroid III (P3) gland, ectoderm of the third branchial arch was cauterized in chick embryos at Hamburger and Hamilton's stage 19 (embryonic day 3). Cauterization of the ventral half of the ectoderm was followed by the non-formation, on the same side, of the P3 gland. When the dorsal half of the ectoderm was cauterized, both the right and left P3 glands formed. Our observations suggest that the ectoderm of the ventral half of the third branchial arch is necessary for the organization of the P3 gland.

Developmental differences in the ossification process of the human corpus and ramus mandibulae.

A correlation was sought between the organization of the dental crest and the ossification of the corpus mandibulae in 14 human embryos and 13 human fetuses. The different types of ossification between the corpus and the ramus mandibulae suggest that the cartilago mandibularis (meckeliensis) guides the formation of the mandibula, while the dental crest acts as a coorganizer. In the area of the foramen mentale, the lamina dentalis begins to invaginate (to give rise to the dental crest), and at this level intramembranous ossification of the corpus mandibulae commences. These findings, together with the presence of the cartilago mandibularis before the appearance of the dental crest, and the fact that the former is seen along the entire length of the mandibula (from the symphysis mandibulae to the capsula otica), support the hypothesis that the dental crest, rather than the cartilago mandibularis, acts as the coorganizer in the corpus mandibulae.

Contractile regulatory proteins tropomyosin and troponin-T as indicators of the modulatory role of retinoic acid.

Retinoic acid (RA), the active metabolite of vitamin A, plays a significant role in regulating cardiac form and function throughout the life of the organism. Both cardiac morphogenesis and myocardial differentiation are affected by alterations in RA homeostasis. In order to test the effect of all-trans RA and 13-cis RA on cardiomyocyte differentiation, we studied the level and the subcellular compartmentalization of alpha-tropomyosin and troponin-T proteins in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 22, 32 and 40 embryos. The retinoids increased the levels of alpha-tropomyosin and troponin-T in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The greatest increases in alpha-tropomyosin occurred in the cytoplasmic fraction in HH22 cells cultured for 24 h with all-trans RA or 13-cis RA, whereas the greatest increases in troponin-T were found in the cytoplasmic fraction of HH32 cells exposed to retinoids for 24 h. In cultures treated for 48 h with retinoids, the levels of alpha-tropomyosin and troponin-T showed significant increases in the cytoplasmic compartment of cells treated in HH32-with respect to the control values. These findings are further evidence that RA plays a modulating role in the formation and reorganization of sarcomeric proteins during the process of cardiomyocyte maturation.

Development of the human knee joint ligaments.

BACKGROUND: Many studies have been published on the development of the human knee joint, but scant attention has been given to the development of the knee joint ligaments. The only elements that have received much attention are the cruciate ligaments and their relationships with the synovial membrane. METHODS: We summarize our observations on the development of the knee joint ligaments in 50 serially sectioned human embryonic and fetal lower limbs (26 embryos and 24 fetuses). RESULTS: The patellar ligament begins to form in O'Rahilly stage 20, with the muscle fibers of the quadriceps muscle being attached inferiorly to the tibial tuberosity. The cruciate ligaments (beginning with the posterior) arise from the articular interzone in O'Rahilly stage 21. Subsequently, with the organization of the Wrisberg's meniscofemoral ligament, in week 10 of development, the cruciate ligament system is completed. The lateral collateral ligament begins to form in O'Rahilly stage 23, and from its first appearance it is independent of the knee joint capsule. At this time, development of the tendon of the popliteus muscle begins. The medial collateral ligament begins to develop in week 9 of development as a condensation of the joint capsule. Two weeks later, the intra-articular pad of fat begins to form from mesenchymal tissue below the patella and between the cruciate and the patellar ligaments. With the organization of the suprapatellar bursa in week 14 of development, knee joint development is complete. CONCLUSIONS: The morphogenetic time table of the knee joint ligaments was established.

Development of the human knee joint.

BACKGROUND: Many studies have been published on the development of the human knee joint, but different investigators disagree on its morphogenetic time table. Most discrepancies center on the cavitation of the knee joint and the participation of the superior tibiofibular joint in the joint knee system. METHODS: We summarize our observations of the development of the knee joint in 50 serially sectioned human embryonic and fetal lower limbs (26 embryos and 24 fetuses). RESULTS: The epiphysis of the femur and tibia become condryfied from O'Rahilly stage 18, and ossification begins during the 13th week of development. The patella appears as a dense blastema during O'Rahilly stage 19, becomes condryfied during O'Rahilly stage 22, and begins its ossification during the 14th week of development. The knee joint cavity appears during O'Rahilly stage 22, initially as the femoropatellar joint. This process begins at the periphery of the articular interzone. The superior tibiofibular joint communicates with the lateral meniscotibial joint between 10 and 11 weeks of development and becomes separated from the 13 week on. The menisci arise from the eccentric portions of the articular interzone during O'Rahilly stage 22; however, until week 9 of development, they are not easily distinguishable. CONCLUSIONS: We establish the morphogenetic time table of the human knee joint.

Development of Meckel's cartilage in the symphyseal region in man.

BACKGROUND: The aim of this work is to clarify the aspects which are at present most controversial about the development of the anterior segments of Meckel's cartilage, such as the role of and determination of the area that is incorporated in the development of the human mandible. METHODS: Light microscope studies were done on 25 embryos and human fetuses from the collection of the Institute of Embryology at the University Complutense of Madrid and the Department of Morphological Science from the University of Granada. Specimen length was between 18 and 125 mm crown-rump. RESULTS: During the embryonic period, Meckel's cartilages were placed in the midline of the mandibular arch but fusion was not observed between them. Ossification of Meckel's cartilage begins at the end of the embryonic period and is completed in the fetal period and the portion that participates in mandibular formation is determined. This segment extends from the mental foramen to near the midline of the mandible. In this region, on the dorsal surface of the symphysis, cartilaginous nodules that originate from Meckel's cartilage are isolated. CONCLUSIONS: The ventral portions of Meckel's cartilage do not fuse in the midline of the mandibular arch. These present endo- and perichondral ossification and the section from the mental foramen to near the midline (mandibular symphysis) participates in mandibular formation. The ventral ends of Meckel's cartilage, i.e., the ends nearest the midline, do not ossify and remain isolated on the dorsal surface of the fetal mandibular symphysis.