Wharton's jelly-derived mesenchymal cells as a new source for the generation of microtissues for tissue engineering applications.

Microtissues (MT) are currently considered as a promising alternative for the fabrication of natural, 3D biomimetic functional units for the construction of bio-artificial substitutes by tissue engineering (TE). The aim of this study was to evaluate the possibility of generating mesenchymal cell-based MT using human umbilical cord Wharton's jelly stromal cells (WJSC-MT). MT were generated using agarose microchips and evaluated ex vivo during 28 days. Fibroblasts MT (FIB-MT) were used as control. Morphometry, cell viability and metabolism, MT-formation process and ECM synthesis were assessed by phase-contrast microscopy, functional biochemical assays, and histological analyses. Morphometry revealed a time-course compaction process in both MT, but WJSC-MT resulted to be larger than FIB-MT in all days analyzed. Cell viability and functionality evaluation demonstrated that both MT were composed by viable and metabolically active cells, especially the WJSC during 4-21 days ex vivo. Histology showed that WJSC acquired a peripheral pattern and synthesized an extracellular matrix-rich core over the time, what differed from the homogeneous pattern observed in FIB-MT. This study demonstrates the possibility of using WJSC to create MT containing viable and functional cells and abundant extracellular matrix. We hypothesize that WJSC-MT could be a promising alternative in TE protocols. However, future cell differentiation and in vivo studies are still needed to demonstrate the potential usefulness of WJSC-MT in regenerative medicine.

[Adherence to the treatment of pre surgical nasoalveolar molding in cleft lip and palate]. / Adherencia al tratamiento de moldeado nasoalveolar en pacientes con fisura labiopalatina.

OBJECTIVES: The nasoalveolar molding device (NAM) designed to decrease the size of the defect and improve nasal asymmetry, facilitates surgical intervention improving the results. However, family involvement is highly necessary due to the fact that the treatment herein referred is long and tedious. The aim of this study is to determine adherence to treatment and possible variables that can determine their rejection. MATERIALS AND METHODS: Patients treated with NAM over the last 6 years (64 patients). Retrospective data collection and telephone surveys. 5 groups established depending on its respective adherence to the treatment (from rejection of the treatment to good adherence to it). Several aspects are herein analyzed related to the caregiver, the patient and the social background (degree of education, number of caregivers, distance to the hospital, etc.). Through the SPSS program, a descriptive study was carried out, analyzing the variables using chi2 with Fisher's exact correction. RESULTS: 46 surveys were completed (32 boys and 14 girls), 63% were unilateral, 91% had a cleft palate. Adherence to the treatment was good by 80%, and only 6% rejected the treatment. The adherence was significantly higher, if they had contacted with other families of patients (P < 0.01) and a higher tendency of abandonment of treatment in other nationalities than the Spanish one, although without significant differences. CONCLUSIONS: Adherence to treatment is very good in general, and we can not significantly determine the variables that can condition the rejection of treatment, but contacting with family members of patients or associations of patients makes the adherence greater.

OBJETIVOS: El dispositivo de moldeamiento nasoalveolar (NAM), diseñado para aproximar los segmentos fisurados y mejorar la asimetría nasal, facilita la intervención quirúrgica mejorando los resultados. Sin embargo, necesita gran colaboración por parte de la familia por ser largo, y a veces, tedioso. El objetivo de este trabajo es determinar la adherencia al tratamiento y las posibles variables que puedan determinar su rechazo. MATERIAL Y METODOS: Pacientes tratados con NAM en los últimos 6 años (64 pacientes). Recogida de datos retrospectiva (historias) y encuesta telefónica. Clasificación en 5 grupos según el seguimiento del tratamiento (rechazo del tratamiento hasta un cumplimiento correcto del tratamiento). Analizamos factores relacionados con el cuidador, el paciente y el contexto social (nivel de estudios, número de cuidadores, distancia al hospital, etc.). Mediante el programa SPSS se realizó un estudio descriptivo, analizando las variables mediante chi2 con corrección exacta de Fisher. RESULTADOS: Se completaron las 46 encuestas (32 niños y 14 niñas), el 63% fueron unilaterales, el 91% con fisura palatina. El cumplimiento terapéutico fue bueno en el 80%, y solo el 7% rechazaron el tratamiento. La adherencia fue mayor, de forma significativa, si habían contactado con otras familias de pacientes (P< 0,01) y tendencia a mayor abandono en otras nacionalidades distinta a la española, aunque sin significación estadística. CONCLUSIONES: El cumplimiento terapéutico de estos pacientes es bueno de forma general, y no podemos determinar de forma significativa las variables que pueden condicionar el rechazo al tratamiento, pero si, que el contacto con familiares de afectados o asociaciones de pacientes hacen que la adherencia sea mayor.

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.

[In vitro generation of a human bladder wall substitute by tissue engineering]. / Generación in vitro de un sustituto de pared vesical humana obtenido mediante ingeniería tisular.

INTRODUCTION: Certain urological congenital conditions, such as bladder exstrophy and acquired conditions such as trauma and tumors may require the use of different tissues like small bowel, sigmoid colon or stomach for bladder reconstruction. However, these tissues are often associated to important complications. The aim of this study is to develop a novel substitute of the human bladder wall by tissue engineering. MATERIAL AND METHODS: We first generated primary cell cultures of epithelial and stromal bladder mucosa cells from small tissue biopsies of human bladder by using enzymatic methods based on trypsin-EDTA and collagenase I. Then, a three-dimensional substitute of the bladder mucosa was generated using fibrin-agarose biomaterials. The analysis of the tissue substitutes was carried out at day 14th of development by histological examination of samples stained with hematoxylin-eosin. RESULTS: The use of enzymatic digestion methods allowed us to efficiently generate primary cell cultures of the human bladder epithelial and stromal cells. The proliferation rate was higher in stromal cells as compared to epithelial cells. Once the bladder mucosa substitutes were generated, a good biocompatibility of the stromal and epithelial cells into the biomaterial was found. The histological structure of the bladder wall substitutes was analogue to that of the native human bladder mucosa. CONCLUSIONS: The bladder mucosa substitute generated by tissue engineering showed structural and histological similarities with the native human bladder tissues and open the door to the future therapeutic use of these bioengineered tissues.

[Presurgical nasoalveolar molding a child with cleft lip and palate]. / Moldeamiento nasoalveolar prequirúrgico en el niño con fisura labiopalatina.

The cleft lip and palate are among the diseases that benefit most from the creation of multidisciplinary units (paediatric surgeons, maxillofacial and plastic, orthodontist, otolaryngologist, speech therapist, psychologist, etc.). The objectives of presurgical orthodontic and orthopedic treatment are: 1.--Align the alveolar segments and reduce the width of the cleft to facilitate cheiloplasty. 2.--To guide the growth of the segments in which the jaw is divided. 3.--Improve lingual function. 4.--Shaping the nasal cartilage and the columella. We review developments in the treatment results in presurgical NAM (PNAME) in 15 patients treated in cleft lip and palate Unit Hospital Virgen de las Nieves (10 unilateral and 5 bilateral). Three patients did not complete treatment because of lack of cooperation from parents who dropped by the baby's crying. In all patients who completed a significant improvement was achieved in the alignment and spacing of the alveolar segments and in the shape of the alar cartilage. The learning curve in shaping makes each time the results are better and communication between parents of patients causes dropouts are virtually zero.

[Generation of a substitute for human oral mucosa and verification of its viability by tissue-engineering]. / Generación de un sustituto de mucosa oral humana y comprobación de su viabilidad mediante ingeniería tisular.

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues. This way, tissue ingineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we have developed a new model for artificial oral mucosa generated by tissue engineering using a fibrin-agarosa scaffold. For that purpose, we have generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal mucosa oral using enzymatic treatments. Then, we have determined the viability of cultured cells by electron probe quantitative X-ray microanalysis, and we have demonstrated that most of the cells in the primary cultures were alive and hd high K/Na ratios. Once cell viability was determined, we used cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarosa extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and de oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

[Mandibular distraction osteogenesis in patients with craniofacial malformation]. / Distracción mandibular osteogénica en pacientes con malformaciones craneofaciales.

Craniofacial malformations (Pierre-Robin sequence, Treacher-Collins syndrome, Nager syndrome, etc.) are frequently associated to severe mandibular hypoplasia, which can cause upper airway obstruction by retroposition of the base of the tongue in the posterior pharyngeal space. Most of the patients respond to postural treatment. In prone decubitus position, it may be necessary to monitor oxygen saturation, insert a nasopharyngeal tube and even an endotracheal one. In more severe cases with prolonged and frequent pauses of apnea, tracheostomy may be necessary, but it is associated with high morbidity and sometimes mortality. In the last two years, in the Multidisciplinary Cleft Lip and Palate Unit of the Hospital Virgen de las Nieves, 4 children with severe obstructive apnea secondary to severe mandibular hypoplasia have been treated with mandibular distraction osteogenesis, this procedure being effective in the resolution of the condition. It has avoided tracheostomy, it has lengthened the jaw in a period of 2-3 weeks. During this time, the obstructive respiratory problems and also swallowing problems have disappeared. The esthetic results were excellent and the complications, for the moment, minimum.

Simultaneous maxillomandibular distraction in unilateral mandibular hypoplasia.

In craniomaxillofacial surgery we often deal with hypoplastic mandibles and mandibular asymmetries, the correction of which is critical to obtaining acceptable aesthetic results. In all of them we find common skeletal problems once growth has finished, such as an inclined occlusal plane and facial asymmetry with a stable dental occlusion. Simultaneous maxillomandibular distraction, which involves a Le Fort I osteotomy and a mandibular osteotomy with intermaxillary fixation during the period of active distraction, is an excellent technique to solve these problems. Virtual surgical planning, stereolithographic models, and surgical guides are supportive tools for obtaining excellent results. In this paper we present our experience with five cases of hypoplastic mandibles and mandibular asymmetries of different aetiologies. In all patients we achieved a considerable improvement in their physical appearance in the distance between the lateral canthus and oral commissure, the height of the mandibular ramus, the inclination of the occlusal plane, and the medial position of the chin. The benefits of virtual surgical planning in terms of choosing the optimal vector and the amount of distraction make it a promising technological tool to achieve excellent outcomes.

Bilateral auricular reconstruction with osseointegrated implant-retained prostheses. Optimization of aesthetic outcomes using virtual planning.

Auricular reconstruction in microtia patients is challenging, particularly in bilateral cases. The use of osseointegrated implants is a safe and effective way to retain the auricular prostheses. With the help of virtual planning we can produce more predictable results with better aesthetic outcomes. We present a case of an 8-year-old bilateral microtia patient who underwent auricular reconstruction with implant-retained prostheses, using virtual planning. Using stereolithographic models and surgical guides was also very helpful to achieve excellent results.

[VOT analysis in cleft palate patients after surgery]. / Estudio del VOT en pacientes intervenidos de fisura palatina.

OBJECTIVES: To determine the factors that modify the measures of voice onset time (VOT) as acoustic parameter in operated cleft palate children. The voice of cleft palate patient is defined by the generation of an abnormal intraoral pression. This doesn't allow the normal generation of voiceless plosives consonants and produces an increased VOT. Acoustic analysis of voice quantifies these parameters. In this study we observed an increased VOT if the patient hadn't got a normal teeth occlusion and a normal soft palate function. Also VOT measures were influenced by cleft lip and delay in beginning of ortodoncy. Hearing loss is associated with increased VOT. Our results are based in a correct sample and statistical analysis by multiple lineal regression.

Ex vivo and in vivo modulatory effects of umbilical cord Wharton's jelly stem cells on human oral mucosa stroma substitutes.

Novel oral mucosa substitutes have been developed in the laboratory using human umbilical cord Wharton's jelly stem cells -HWJSC- as an alternative cell source. In the present work, we have generated human oral mucosa substitutes with oral mucosa keratinocytes and HWJSC to determine the influence of these cell sources on stromal differentiation. First, acellular and cellular stroma substitutes and bilayered oral mucosa substitutes with an epithelial layer consisting of oral mucosa keratinocytes -OM samples- or HWJSC -hOM- were generated. Then, tissues were analyzed by light and electron microscopy, histochemistry and immunohistochemistry to quantify all major extracellular matrix components after 1, 2 and 3 weeks of ex vivo development, and OM and hOM were also analyzed after in vivo grafting. The results showed that bioengineered oral mucosa stromas displayed an adequate fibrillar mesh. Synthesis of abundant collagen fibers was detected in OM and hOM after 3 weeks, and in vivo grafting resulted in an increased collagen synthesis. No elastic or reticular fibers were found. Glycoprotein synthesis was found at the epithelial-stromal layer when samples were grafted in vivo. Finally, proteoglycans, decorin, versican and aggrecan were strongly dependent on the in vivo environment and the presence of a well-structured epithelium on top. The use of HWJSC was associated to an increased synthesis of versican. These results confirm the usefulness of fibrin-agarose biomaterials for the generation of an efficient human oral mucosa stroma substitute and the importance of the in vivo environment and the epithelial-mesenchymal interaction for the adequate differentiation of the bioengineered stroma.

Saccular aneurysm in arm secondary to accidental arterial puncture.

We present a rare case of saccular aneurysm localised in the arm of a breast-feeding baby, secondary to accidental arterial puncture. Colour Doppler echography showed a cystic lesion with turbulent arterial flow related to the humeral artery. Complete surgical resection of the aneurysm was achieved.

[Cancer-cryptorchism meta-analysis]. / Metaanálisis cáncer-criptorquidia.

INTRODUCTION: Testicular cancer in adult and young patients with cryptorchidism (operated or not operated on) has been reported previously, but its epidemiological impact (as concept to profile cancer and/or therapeutical result of cryptorchidism based on quantitative dates) is not defined actually. OBJECTIVE: Determination by quantitative meta-analysis of the strength of relation (relative risk = RR) cancer and cryptorchidism and by qualitative meta-analysis of factors of cryptorchidism (anatomical position of teste, age of intervention, testicular biopsy) associated to cancer. RESULTS: Case-control and cohort studies of medical literature have been included in this report. Relative risk of develop cancer in the patient with cryptorchidism is 7.75 more than poblational control without cryptorchidism (interval of confiance: 5.2-10.3). Qualitative meta-analysis of factors concluded that there is a significative relation cancer and abdominal position of teste, and also age of intervention after 10 and testicular biopsy. CONCLUSIONS: Significative relation between cancer and cryptorchidism with relative risk above control population is constated again, although with dates without epidemiological importance. But significative relation among cancer with abdominal testes and intervention after 10 years would recommend prospective studies, with strategy of multivariant analysis. Authors recommend not to make testicular biopsy, except if its indication is not questionable.

[Recto-sacral fixation in the treatment of rectal prolapse refractory to conservative treatment]. / La pexia recto-sacra en el tratamiento del prolapso rectal refractario a tratamiento conservador.

INTRODUCTION: The clinical cases of 20 patients submitted to Reifferscheid intervention (fixation of rectum to promontorium) between 1967 and 1997 are presented. PATIENTS AND METHODS: Twenty patients have been treated by means of the operation of Reifferscheid. They were divided in two groups: from 1967 to 1972, ten patients were submitted to this technique (group A). From 1974 to 1997, other ten patients (group B). In each case, the following items were registered: sex, eventual secondary diagnosis, incision, complications, middle-term evolution. RESULTS: Group A: Middle age: 2.15 years old. Sex: 7 girls (5 of them presented mielomeningocele) and 3 males; pararectal incision for babies, Pfannenstiel incision for children over 1 year-old. FOLLOW-UP: 2 years without recidives. Group B: Middle age: 2.5 years-old, 9 males (2 of them were diagnosed of mielomeningocele) and 1 girl. No recidives were detected. DISCUSSION: These excellent results obtained with Reifferscheid operation contrast with the poor results referred by other authors for other therapeutical approaches. That is why, in patients affected of serious rectal prolapse, especially when associated to other pathologies, rectal fixation to promontorium is an useful and long-term sure approach.

[Intralobar pulmonary sequestration: diagnosis by intravenous digital subtraction angiography]. / Secuestro pulmonar intralobar, diagnostico mediante divas (angiografia digital por inyeccion intravenosa con imagen por sustraccion).

We present a boy five years old who after suffering recurrent pneumonia was transferred to own center for study. A diagnosis of Intralobar Pulmonary Sequestration was made, before surgery, by non invasive method, digital intravenous angiographic subtraction (DIVAS) and surgical management by anomalous arterial ligation and lobectomy was performed.

[Macrostomia secondary to transverse facial cleft of Tessier No.4]. / Macrostomía secundaria a hendidura facial transversa de Tessier n-0 4.

A case of bilateral macrostomia type transverse facial cleft of Tessier n. 4 is reported, which was operated on with good results when had ten months of age, repaired using suture of three planes of three embryonic folds. A treatment plain precocious is recommended and pathogenic theories about this uncommon entity are exposed.

[The usefulness of ultrasonography in the diagnosis of acute appendicitis in children]. / Utilidad de la ultrasonografía para el diagnóstico de apendicitis aguda en la infancia.

The main objective of the present report was the evaluation of the utility of echography as a diagnostic method in abdominal pain suspicious of acute appendicitis. A control-case study was performed in 165 children hospitalized for abdominal pain. Appendicitis histologically confirmed were considered cases (n = 110) while control (n = 55) were the not-operated patients (46 children) or the not-confirmed appendicitis after laparotomy (n = 9). The variables considered here were: the results of echography (Eco+ when echography showed signs of acute appendicitis: Eco- when not), the age of the patient (randomized at three groups; < 6, 6-9, > 9 years), and the conclusions of fisical exploration (conclusive or not as acute appendicitis). The results were analyzed statistically with the SPSS program, calculating the following predictive values; sensibility, specificity, positive predictive value, negative predictive value. Odds Radio before and after test and the global value of echography. Authors conclude that ultrasonography as method of support to abdominal exploration for diagnostic in acute appendicitis is manifest and present a global value of 70.90%.

[Gonadal, hormonal and spermatic function in patients treated for cryptorchidism]. / Función gonadal hormonal y espermática en pacientes tratados por criptorquidia.

This paper studies the gonadal-hormonal and spermatic function of 48 adults (over 18 years old) who were treated in childhood for cryptorchidism by medical, surgical or both treatments. We measure plasmatic levels of FSH, LH, testosterone and prolactin of 34 patients and sperm samples of 31 patients. The testicular hormonal function was not disturbed except in four patients with high levels of prolactin (> 20 ng/ml) without symptoms. The spermatic function was disturbed in bilateral cryptorchidism mainly. The better sperm quality and testicular volume were at patients treated with hormonal treatment before surgical treatment.

[Alterations of nuclear maturity in spermatozoids of males with antecedents of cryptorchidism]. / Alteraciones de la madurez nuclear en espermatozoides de varones con antecedentes de criptorquidia.

The association between infertility and cryptorchidism is an accepted fact, usually attributed to the oligozoosperm, asthenozoosperm or teratozoosperm presented in ejaculation products of males with this antecedent. The nuclear maturity in a sample of men with antecedents of cryptorchidism have been studied and these results have been compared to those of a control group. The results of this work show the deficient transformation of nuclear proteins to protamines in males with antecedents of cryptorchidism compared to the control group, due to the remaining of immature histones. Alterations of nuclear maturity able to contribute to the subfertility of these men were found in spermatozoids of adult males with antecedents of cryptorchidism.

Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis.

INTRODUCTION: Human umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date. METHODS: We analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord -in situ analysis- and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence. RESULTS: Our results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers. CONCLUSIONS: These results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering.