Diagnosis of induced cleft palate in rats from defective patterns of differentiation of isoenzymes.

Cleft palate was induced in 420 embryos of Sprague-Dawley rats with a single oral dose of 600 mg/kg beta-aminoproprionitrile (BAPN) on embryonal day 15, 7 hours. The cleft palate was accompanied by a pathological differentiation pattern of various isoenzymes in palatal shelves. These isoenzymes could be detected in amniotic fluid from the 16th to the 20th days of pregnancy when they also had a pathological differentiation pattern. We conclude that teratogenically induced cleft palate in rats is accompanied by a pathological differentiation pattern that can be traced by determination of isoenzymes in the palatal shelves as well as in amniotic fluid.     

Enzyme histochemical analysis of craniofacial malformations induced by retinoids

Craniofacial malformations were induced in 256 embryos from 32 pregnant rats by a single intraperitoneal injection of 10 mg/kg etretinate at 8.5 days gestation. The litters developed several malformations including microtia, low set and dorsally placed outer ears, defective middle ear ossicles, short cochleas, defectively differentiated Meckel's cartilages, micrognathia, rudimentary malar bones, lateral facial clefts, fistulas, and skin tags, all of which tissues are derivatives of the first and second branchial arches. The teratogenically induced syndrome shows similarities to the mandibulofacial dysostosis syndrome in man. The defects were accompanied by a change in the histochemical differentiation and location of various enzymes in the craniofacial tissues.     

Epithelio-mesenchymal transformation in the embryonic face: implications for craniofacial malformations

A high percentage of craniofacial malformations is ascribed to abnormalities in cell populations derived from the neural crest and ganglionic placodes. Both cell populations originate from the ectoderm of the head-neck area by means of the mechanism of epithelio-mesenchymal transformation (EMT). Neural crest cells are multipotent and form the majority of the mesenchymal compartment of the head-neck area. EMT of the neural crest will stop shortly after closure of the neural tube. The current opinion about ganglionic placodal cells is that they are derived from the surface ectodermal placodes after closure of the cranial neural tube, and transform into neurons of the sensory ganglia only. However, additional EMT sites of the cranial neural and surface ectoderm have also been found to produce multipotent cells. Because of the fact that most craniofacial malformations develop after closure of the cranial neural tube, in this study we focused on the EMT of the surface ectoderm of the head-neck area during the stages of outgrowth of facial swellings and branchial arches. The surface ectoderm was labeled by injecting various tracker dyes into the amniotic cavity of chick embryos after closure of the anterior neuropore, to exclude labeling of the neural ectoderm and the neural crest. After various incubation periods, ranging from 0–24 h, labeled cells were found in the mesenchymal compartment scattered over the embryonic face and neck, originating from non-placodal as well as placodal surface ectoderm. Thus, besides the neural ectoderm and neural crest, it could be shown that the entire surface ectoderm of the face and neck contributes cells to the underlying mesenchymal tissue, and that this phenomenon occurs in a higher frequency at sites of outgrowing swellings. As a consequence, the pathogenesis of congenital craniofacial malformations should be reconsidered. Received: 15 November 1999 / Accepted: 27 January 2000     

人羊水间充质干细胞的分离鉴定及其对抗Thy-1肾炎的治疗作用

探讨羊水间充质干细胞(amniotic fluid mesenchymal stem cells,AF-MSCs)对抗Thy-1肾炎模型的治疗作用。 方法采用差异性贴壁与机械性分离法从人孕中期羊水中分离出羊水间充质干细胞,流式细胞术鉴定其表面标志物,成脂、成骨诱导分化检测其分化能力。利用尾静脉注射抗Thy-1抗体建立SD大鼠抗Thy-1系膜增生性肾炎模型;将第5代羊水间充质干细胞通过尾静脉注射治疗抗Thy-1系膜增生性肾炎大鼠,检测大鼠尿蛋白的变化,PAS染色观察大鼠肾脏病理改变。 结果羊水间充质干细胞顺利分离,流式结果显示其表达间充质干细胞标志物(CD29、CD44、CD73、CD90、CD105)和胚胎干细胞标志物(SSEA-4)而不表达造血干细胞标志物(CD34、CD45、CD133),且在适当条件下能够被诱导分化为脂肪细胞和骨细胞。羊水间充质干细胞治疗后,与模型组相比,治疗组在第7、12天时24 h尿蛋白明显下降(P<0.01),且肾脏病理结果表明系膜细胞增殖减轻,系膜区细胞外基质积聚减少。 结论成功的从人孕中期羊水中分离出羊水间充质干细胞,羊水间充质干细胞治疗系膜增生性肾小球肾炎有效。     

Scanning and transmission electron microscope study of the amniotic epithelium in the rat at the end of gestation

The pattern of the amniotic epithelium during the eight last days of pregnancy is described with the aid of scanning and transmission microscopy. A flattening of the cells appears near the term. Their area becomes progressively greater increasing their contact surface with the amniotic fluid. Simultaneaously, their microvilli become more numerous. During the 20th, the 21st and the 22nd days of pregnancy, we observed cytoplasmic processes emanating from the base of the cell interrupting the basal membrane and entering the underlying connective substance. No evidence of cell degeneration was seen.     

Histological assessment of skin grafts stored in amniotic fluid and saline

We investigated the efficacy of amniotic fluid as a substance in which to store grafts; it is rich in nutrients, proteins, and growth factors, and has well-known antimicrobial features. We compared it with the widely-used and practical saline. Split-thickness grafts 4 × 4 cm were prepared from the back of 20 rats and divided into four groups (n = 5 each). The rolled grafts were wrapped in gauze dampened with saline or amniotic fluid and placed into refrigerators in sterile containers for storage. On days 7, 14, 21, and 28, histological examinations were made. A semiquantitative evaluation of the histological damage to the skin was made by scoring its degree of severity. Compared with saline, histological scores in the grafts stored in amniotic fluid were found to be significantly lower on the 14th, 21st, and 28th days (p values on days 14, 21, and 28; cell swelling: 0.014, 0.006, and 0.005, respectively; nuclear swelling: 0.003, 0.006, and 0.007, respectively; nuclear pleomorphism: 0.004, 0.005, and 0.003, respectively; nuclear haloes: 0.015, 0.005, and 0.005, respectively; nuclear pyknosis: 0.003, 0.005, and 0.003, respectively; dermo-epidermal clefting: 0.005, 0.003, and 0.003, respectively; eosinophilia and mitosis: 0.003, 0.006, and 0.004, respectively; dermal collagen: 0.003, 0.003, and 0.003, respectively). Amniotic fluid maintained preservation better for skin grafts than saline. Comparison with other modern storage media would be beneficial.     

Amniotic fluid and bone marrow derived mesenchymal stem cells can be converted to smooth muscle cells in the cryo-injured rat bladder and prevent compensatory hypertrophy of surviving smooth muscle cells

PURPOSE: Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. MATERIALS AND METHODS: The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. RESULTS: At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. CONCLUSIONS: In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism.     

Partial amniotic carbon dioxide insufflation (PACI) facilitates fetoscopic interventions in complicated monochorionic twin pregnancies

Background Adequate visualization of the placenta or umbilical cord during fetoscopic procedures in complicated monochorionic twin pregnancies may be difficult because of placental position and spatial constraints, as well as stained amniotic fluid. Partial amniotic carbon dioxide insufflation (PACI) has made it possible to overcome these obstacles in other fetoscopic procedures, but its value has not yet been reported in monochorionic twins. Methods Partial amniotic carbon dioxide insufflation was carried out in five expectant women with complicated monochorionic twin pregnancies between 19 + 6 to 29 + 4 weeks of gestation when adequate fetoscopic visualization of pathological placental surface vessels or the umbilical cord was impossible because of stained or too little amniotic fluid. In four cases, five fetoscopic laser ablations of pathological placental vessels in twin-to-twin transfusion syndrome (TTTS) were performed. In one discordant twin pregnancy with TTTS, PACI was carried out in order to achieve umbilical cord ligation in a recipient with omphalocele and cardiac malformation. Results Partial amniotic carbon dioxide insufflation offered superior visualization and did not result in any acute maternal or fetal complications. After fetoscopic laser coagulation, three women delivered one fetus at 27 + 5, two fetuses at 28 + 6, and two fetuses at 35 + 4 weeks of gestation, respectively. One set of twins with TTTS was lost. Following umbilical cord ligation, the surviving twin was delivered at 37 + 2 weeks of gestation. Conclusions Partial amniotic carbon dioxide insufflation may facilitate fetoscopic procedures in complicated monochorionic twin pregnancies when conventional fetoscopic approaches within amniotic fluid meet difficulties. Further studies are required to allow assessment of benefits, risks, and safety margins of the new approach before it can generally be recommended.     

Histological assessment of skin grafts stored in amniotic fluid and saline

Abstract

We investigated the efficacy of amniotic fluid as a substance in which to store grafts; it is rich in nutrients, proteins, and growth factors, and has well-known antimicrobial features. We compared it with the widely-used and practical saline. Split-thickness grafts 4 × 4 cm were prepared from the back of 20 rats and divided into four groups (n = 5 each). The rolled grafts were wrapped in gauze dampened with saline or amniotic fluid and placed into refrigerators in sterile containers for storage. On days 7, 14, 21, and 28, histological examinations were made. A semiquantitative evaluation of the histological damage to the skin was made by scoring its degree of severity. Compared with saline, histological scores in the grafts stored in amniotic fluid were found to be significantly lower on the 14th, 21st, and 28th days (p values on days 14, 21, and 28; cell swelling: 0.014, 0.006, and 0.005, respectively; nuclear swelling: 0.003, 0.006, and 0.007, respectively; nuclear pleomorphism: 0.004, 0.005, and 0.003, respectively; nuclear haloes: 0.015, 0.005, and 0.005, respectively; nuclear pyknosis: 0.003, 0.005, and 0.003, respectively; dermo-epidermal clefting: 0.005, 0.003, and 0.003, respectively; eosinophilia and mitosis: 0.003, 0.006, and 0.004, respectively; dermal collagen: 0.003, 0.003, and 0.003, respectively). Amniotic fluid maintained preservation better for skin grafts than saline. Comparison with other modern storage media would be beneficial.     

Case series: variations in the embryology of congenital midline cervical clefts

Congenital midline cervical clefts (CMCC) is a clinical diagnosis and represents a spectrum of rare developmental anomalies. Fewer than 100 cases have been reported overall, the first being described by Bailey in 1924 (1). It is not a true cleft because it does not include a gap between adjacent skin flaps. It is thought to represent a failure of midline fusion of the branchial arches although this is controversial. It is a clinical diagnosis and presents at birth with a ventral midline defect of the skin of the neck. This consists of a skin tag (nipple-like projection), an atrophic mucosal surface and a caudal sinus. It may be associated with a subcutaneous fibrous cord, which can cause a vertical midline tethering restricting cervical extension. Associated defects may be a median cleft of the mandible, tongue and lower lip. There may be an associated delay in mandibular development and hypoplasia or absence of neck structures such as the hyoid bone. Associated thyroglossal and bronchogenic cysts may occur as well as defects in other parts of the body such as a sternal cleft (2). CMCC has been previously reported in the literature as being of a branchial origin, however a review of the histology of previous cases suggests a combined branchial and bronchogenic component (2-6). We report on two cases that presented with the clinical picture of a classical CMCC. Our first case appears to have a bronchogenic origin with possible branchial components and the second case appears to be solely branchial in origin. There is much debate as to the embryology of this clinical entity; we shall endeavour to address the main theories.     

Trophic effect of amniotic fluid on cultured fetal gastric mucosal cells

This study examines the hypothesis that the growth and development of fetal gastric epithelial cells is critically dependent on the presence of trophic factors in amniotic fluid. Fetal gastric epithelial cells (predominantly parietal cells) were grown in culture in the presence of graded concentrations of rabbit amniotic fluid, fetal bovine serum, or a control solution containing the nutrients specific to amniotic fluid. After 7 days, cells were harvested and growth was assessed by cell counts and DNA contents. Amniotic fluid stimulated growth of these cultured cells with a potency similar to that of fetal bovine serum. Cell growth was significantly enhanced by amniotic fluid at each dose tested, when compared to the appropriate nutrient control group. Cell growth could not be maintained in the absence of amniotic fluid or fetal bovine serum. We conclude that amniotic fluid contains factors which are trophic to the growth of cultured fetal gastric epithelial cells. The nature of these factors remains to be determined.     

The role of amniotic fluid in fetal nutrition

The contribution of amniotic fluid to fetal growth and gastrointestinal tract development was studied in a rabbit model. In the fetal rabbit, at 23 days gestation, 3 conditions were surgically produced: (1) prevention of swallowing of amniotic fluid by esophageal ligation (n = 8); (2) esophageal ligation but insertion of an esophageal cannula distally to allow continuous infusion into the stomach of bovine amniotic fluid to mimic fetal swallowing (n = 7); and (3) sham operation (n = 7). Fetuses were delivered by Caesarean section at 28 days gestation. Esophageal ligation resulted in significant reductions of birth weight and crown-rump length and a trend to decreased liver weight when compared to sham operated controls. Additionally, marked reductions in gastric and intestinal tissue weight and gastric acidity were found following esophageal ligation. These reductions in both somatic and gastrointestinal tract growth and gastric function were reversed by infusion of amniotic fluid intragastrically. We conclude that amniotic fluid provides 10% to 14% of the nutritional requirements of the normal fetus, and that amniotic fluid contains a potent and as yet undefined gastrointestinal tract trophic factor.     

Composition of amniotic fluid with experimental jejuno-ileal atresia

The possibility that the composition of amniotic fluid is significantly altered with fetal intestinal obstruction was examined in 15 normal, 15 sham operated and 15 ewes in which jejuno-ileal atresia was created by devascularizing a 15 cm intestinal segment in the fetus at 90–100 days gestation. Samples of amniotic fluid forosmolality, urea N, creatinine, electrolytes, amylase, lipase, bilirubin, and proteins were obtained at 100 days gestation and at C-section performed 5 days prior to the anticipated date of delivery. One sham operated and four operated fetuses aborted prior to term for 83% overall fetal survival rate. All surviving operated animals had total intestinal obstruction with the classical appearance of jejuno-ileal atresia. The volume of amniotic fluid at term varied from gelatinous material to as much as 1.0 liter of clear yellow to dark orange fluid. Amniotic fluid composition in term animals was not significantly different from those at 100 days gestation when the creatinine was below 15 mg%. However, sodium and chloride were significantly elevated in all groups at term when the creatinine was above 15 mg%. The osmolality and urea N in the latter group was not significantly different from animals at 100 days gestation. The amylase, lipase, total proteins, albumin, total and direct, and bilirubin in control animals at term was not significantly different from sham operated and experimental subjects. This study suggests that routine amniocentesis in the “high risk” patient would not be diagnostic of fetal intestinal obstruction.     

Intrauterine tracheal obstruction, a new treatment for congenital diaphragmatic hernia, decreases amniotic fluid sodium and chloride concentrations in the fetal lamb.

OBJECTIVE: To evaluate the effect of fetal tracheal occlusion on sodium and chloride concentrations in amniotic and tracheal fluid. SUMMARY BACKGROUND DATA: Intrauterine tracheal occlusion has been proposed to reverse pulmonary hypoplasia, an important prognostic factor in congenital diaphragmatic hernia. In early human trials, technical failure of the obstructive device has been reported. METHODS: Eight fetal lambs (gestational age = 95 days) were subjected to fetal tracheoscopy, and amniotic and tracheal fluid samples were taken. In multiple pregnancies (n = 6), amniotic fluid was also sampled from the contralateral amniotic sac and used as a control. Subsequently, endotracheal obstruction, using a detachable balloon, was performed. After 14 days, all fetuses were delivered, and sodium and chloride concentrations in amniotic and tracheal fluid were measured again. Statistical analysis was done using a two-tailed Student's t test, paired or unpaired as appropriate. RESULTS: In controls, between 95 and 109 days gestational age, no significant changes occurred in sodium or chloride concentrations in amniotic or tracheal fluid. After 2 weeks of tracheal obstruction, however, chloride and sodium concentrations in amniotic fluid decreased (chloride = 76.7 mEq/L vs. 107.6 mEq/L, p = 0.0003; sodium = 109.6 mEq/L vs. 125.9 +/- 5.2 mEq/L, p = 0.019). A concomitant increase in chloride and sodium concentration was observed in tracheal fluid (chloride = 145.4 mEq/L vs. 130.0 mEq/L, p = 0.047; sodium = 153.1 mEq/L vs. 142.9 mEq/L, p = 0.051). When comparing groups at 109 days, chloride and sodium concentrations in amniotic fluid were markedly lower in the treated group versus controls (p = 0.0004 and p = 0.05 for chloride and sodium, respectively). CONCLUSION: Complete tracheal occlusion in ovine fetuses results in a significant decrease of amniotic fluid sodium and chloride concentrations.     

Cystic metastases of the neck from occult thyroid adenocarcinoma.

In 9 of 118 patients with differentiated thyroid carcinomas, a solitary cystic lateral cervical mass simulating a branchial cleft anomaly was the sole presenting sign of the disease. These masses were nodal metastases of occult papillary adenocarcinoma of the thyroid that underwent liquefaction necrosis. Sonographically, the masses presented a complex pattern. Accordingly, it is suggested that, in patients presenting with an asymptomatic solitary lateral cystic cervical mass, the possibility of metastasis from an occult thyroid malignancy should be considered, and consent for definitive surgery should be obtained. The ultrasonic pattern of the cystic mass is of importance in the differentiation of a cavitated lymph node from a branchial cleft cyst.     

Embryonic development of the head and neck: Part 2, the branchial region

The embryology of the branchial (pharyngeal) region is reviewed in terms of the embryonic pharynx, branchial apparatus and derivatives, tongue, larynx, and trachea. Derivatives of the branchial pouches, grooves (clefts), and arches are analyzed in relation to each other and to the first arch syndrome. Other common head and neck defects attributable to abnormal development of the branchial region are also discussed, particularly those defects involving the incomplete degeneration or the abnormal migration of branchial tissues.     

Fetal diaphragmatic wounds heal with scar formation

Fetal wound healing is fundamentally different from wound healing in the adult. Although experimental work in mice, rats, rabbits, monkeys, and sheep has demonstrated that fetal healing occurs without inflammation and scarring, all of these studies have been limited to fetal skin wounds. Whether all fetal tissues heal in a regenerative-like fashion is unknown. Amniotic fluid exposure may play an important role in scarless fetal skin wound healing, but the effect of amniotic fluid on fetal mesothelial wound healing has not been characterized. To investigate these questions we created bilateral linear diaphragmatic wounds in 100-day gestation fetal lambs (term = 145 days). The right thoracotomy was closed to exclude amniotic fluid. In contrast, the left thoracotomy was fashioned into an Eloesser flap which permitted the left diaphragmatic wound to be continually bathed in amniotic fluid. Wounds were harvested after 1, 2, 7, or 14 days and analyzed by light microscopy and immunohistochemistry with antibodies to collagen types I, III, IV, and VI. Whether bathed in or excluded from amniotic fluid, the mesothelial-lined diaphragm healed with scar formation and without evidence of muscle regeneration. Interestingly, diaphragmatic wounds exposed to amniotic fluid were covered by a thick fibrous collagen peel similar to that seen in gastroschisis bowel. These findings indicate that not all fetal tissues share the unique scarless healing properties of fetal skin.     

Midgestational excisional fetal lamb wounds contract in utero

Clinical observations and experimental data suggest that fetal wound healing is very different from adult wound healing. An understanding of the biology of scarless fetal wound healing has tremendous clinical potential for modulating postnatal wound problems. In this study, the fetal lamb model was used to assess excisional fetal skin wound contraction in utero. Full-thickness 9-mm punch biopsy wounds were created on fetal lambs at 100 days' gestation (term, 145 days). Half of the wounds remained exposed to amniotic fluid, whereas the other half were covered by a silastic patch to exclude amniotic fluid. Wounds were harvested 3, 7, or 14 days later and wound areas were calculated. Exposure to amniotic fluid retarded wound contraction significantly at 3 days, but by 14 days all wounds had completely contracted and reepithelialized. Myofibroblasts are an important cellular element of wound contraction. The presence of wound myofibroblasts was documented by both transmission electronmicroscopy and immunocytochemistry with antimuscle actin antibody. It is concluded that fetal lamb wounds contract in utero and exposure to amniotic fluid appears to retard fetal skin wound contraction only during the early healing process.     

Left sinus pyriform fistula treated by minimally invasive approach: A case report

IntroductionAbnormalities of the fourth branchial arch are less common than those of the second arch and usually present with inflammation of the left thyroid lobe.Case presentationWe report the case of a 10 years old girl who presented to our department with recurrent cervical cellulitis, and who was diagnosed, upon endoscopic exploration, with a left sinus pyriform fistula. The patient was treated using mini-invasive surgery by electrocoagulation, with good clinical outcome.ConclusionBranchial arch malformations are rare congenital malformations. The diagnosis is mainly based on clinical examination, imaging and, endoscopic investigations. The conservative attitude may be the treatment of choice, especially if the cervical mass is not well individualized.     

人羊膜间充质细胞向成骨细胞诱导分化的实验研究

目的 观察人羊膜间充质细胞(human amnion mesenchymal cells,hAMCs)体外诱导向成骨细胞分化,为骨组织工程提供种子细胞。方法 从剖宫产后废弃的人羊膜组织分离培养hAMCs,经成骨细胞诱导条件培养基诱导后,对细胞形态特征、碱性磷酸酶、骨桥素、骨钙素表达以及I型胶原分泌进行观察和检测。结果 原代培养的hAMCs形态呈长梭形或不规则形,呈均匀分布生长,传代后细胞体积略变大,约5～7d传代1次。经成骨细胞诱导培养15d后,hAMCs碱性磷酸酶、骨钙素、骨桥素的表达呈阳性,并且检测有I型胶原分泌。结论 hAMCs易于体外分离培养及扩增,体外成骨细胞定向诱导的hAMCs具有典型的成骨细胞的形态和功能性特征,是良好的骨组织工程种子细胞。