Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering.

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.

Stereological analysis of the New Zealand rabbits (Oryctolagus cuniculus) placenta.

The onset of gestation is characterized by growth, morphological and functional changes of the placenta. We aim to evaluate the placental compartments in New Zealand rabbits by means of stereological methods. The fetal and maternal portion of placenta (12, 14, 18 and 20 gestational days) was randomly sampled for the stereological analysis. Histological sections were scanned to estimate fetal (labyrinth and junctional) and maternal (decidua) compartment volumes. The total volume of the placenta for the ages of 12, 14, 18 and 20 days was, respectively, 320 mm3, 340 mm3, 940 mm3 and 1300 mm3. The volume of the labyrinth was 56 mm3, 119 mm3, 231 mm3 and 481 mm3, respectively. The volume of junctional zone was 75 mm3, 76 cm3, 238 mm3 and 314 mm3, respectively. The volume of decidua was 174 mm3, 143 mm3, 469 mm3 and 504 mm3, respectively. We concluded that the rabbit´s placenta compartments varied according to the gestational period, increasing continuously over the 20 gestational days. However, on the onset of the development of the placenta the decidua presented faster growth, whereas after the 20 days of development, the labyrinth developed more quickly. This study represents an aid to the understanding of placentation in humans.

Macrophage-derived GPNMB accelerates skin healing.

Healing is a vital response important for the re-establishment of the skin integrity following injury. Delayed or aberrant dermal wound healing leads to morbidity in patients. The development of therapies to improve dermal healing would be useful. Currently, the design of efficient treatments is stalled by the lack of detailed knowledge about the cellular and molecular mechanisms involved in wound healing. Recently, using state-of-the-art technologies, it was revealed that macrophages signal via GPNMB to mesenchymal stem cells, accelerating skin healing. Strikingly, transplantation of macrophages expressing GPNMB improves skin healing in GPNMB-mutant mice. Additionally, topical treatment with recombinant GPNMB restored mesenchymal stem cells recruitment and accelerated wound closure in the diabetic skin. From a drug development perspective, this GPNMB is a new candidate for skin healing.

Placentation and fetal membrane development in the South American coati, Nasua nasua (Mammalia, Carnivora, Procyonidae).

BACKGROUND: Placental research in carnivores has concentrated on domestic species, which have zonary, labyrinthine placentas with an endotheliochorial barrier. Although the coati, Nasua nasua, is a widely distributed species in South America, data on the development of the placenta and the fetal membranes in this species are very sparse. FINDINGS: Four placentas from mid-gestation to near term were collected from wild individuals and were investigated based on gross morphology, histology, immunohistochemistry and electron microscopy. The available data support the concept that the ancestral condition of placentation in carnivores is phylogenetically characterized by a zonary and labyrinthine placental type with an endotheliochorial fetomaternal barrier, comprising extended epitheliochorial and haemochorial zones, such as hemophagous organs for iron supply and histiotrophe uptake and a yolk sac placenta. CONCLUSIONS: Because of the foundational mechanisms that lead to the considerable complexity of fetomaternal contact zones in carnivores have not been studied, carnivores are interesting animal models for interhaemal barrier differentiation.

Utilization of a Decellularized Skin Scaffold for Repair of a Cleft Palate in a Dog: A Case Report.

Cleft palates are oral deformities that mostly affect puppies. They are frequently extensive and characterized by bone and palatal mucosa malformation. This deformity is a serious condition that may result in the death of the dog, therefore surgical treatment is recommended. Tissue bioengineering has emerged as a valuable option to treat cleft palates by applying acellular biological scaffolds as grafts. This case report proposed a new approach for surgical correction of canine cleft palate through a grafting technique using a decellularized scaffold. A decellularized portion of skin was implanted to correct a large cleft palate in a 3-month-old female Pug dog. The skin fragment was obtained from a dog cadaver and a decellularization protocol was performed. Under general anesthesia, a bilateral mucoperiosteal separation of the entire length of cleft margins was performed, and the scaffold was then positioned between the tissue and the bone palate. The interaction of the grafted scaffold with the oral mucosa and palatine layers resulted in total cleft closure, without postsurgical rejection or infection, indicating the applicability of this technique in dog's cleft palate correction. This is the first reported case demonstrating this new technique, which resulted in full cleft closure and healing.

Morphometric analysis of the placenta in the New World mouse Necromys lasiurus (Rodentia, Cricetidae): a comparison of placental development in cricetids and murids.

BACKGROUND: Stereology is an established method to extrapolate three-dimensional quantities from two-dimensional images. It was applied to placentation in the mouse, but not yet for other rodents. Herein, we provide the first study on quantitative placental development in a sigmodontine rodent species with relatively similar gestational time. Placental structure was also compared to the mouse, in order to evaluate similarities and differences in developmental patterns at the end of gestation. METHODS: Fetal and placental tissues of Necromys lasiurus were collected and weighed at 3 different stages of gestation (early, mid and late gestation) for placental stereology. The total and relative volumes of placenta and of its main layers were investigated. Volume fractions of labyrinth components were quantified by the One Stop method in 31 placentae collected from different individuals, using the Mercator software. Data generated at the end of gestation from N. lasiurus placentae were compared to those of Mus musculus domesticus obtained at the same stage. RESULTS: A significant increase in the total absolute volumes of the placenta and its main layers occurred from early to mid-gestation, followed by a reduction near term, with the labyrinth layer becoming the most prominent area. Moreover, at the end of gestation, the total volume of the mouse placenta was significantly increased compared to that of N. lasiurus although the proportions of the labyrinth layer and junctional zones were similar. Analysis of the volume fractions of the components in the labyrinth indicated a significant increase in fetal vessels and sinusoidal giant cells, a decrease in labyrinthine trophoblast whereas the proportion of maternal blood space remained stable in the course of gestation. On the other hand, in the mouse, volume fractions of fetal vessels and sinusoidal giant cells decreased whereas the volume fraction of labyrinthine trophoblast increased compared to N. lasiurus placenta. CONCLUSIONS: Placental development differed between N. lasiurus and M. musculus domesticus. In particular, the low placental efficiency in N. lasiurus seemed to induce morphological optimization of fetomaternal exchanges. In conclusion, despite similar structural aspects of placentation in these species, the quantitative dynamics showed important differences.

Development of bovine embryos derived from reproductive techniques.

Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

Placental glycotype of the caviomorph rodent Lagostomus maximus and its evolution within Eutheria.

The main evolutionary milestone in the oviparity-viviparity transition is placentation. The placenta is an organ with great morphological diversity among eutherians. The expression of different glycosidic residues (Gr) in the near-term placenta constitutes its glycotype. In this study, the expression of different Gr was determined by lectin histochemistry in early, midterm, and near-term placentas of the plains viscacha (Lagostomus maximus), a caviomorph rodent with the highest poliovulatory rate and embryonic resorption rate among eutherians. Besides, a matrix with the expression of each Gr in the exchange trophoblast of viscacha and other eutherians was constructed to map and infer phylogenetic and evolutionary relationships. Between early, midterm, and near-term placentas, variations in the pattern expression of Gr were observed. The glycotype of the near-term placenta is composed of a high diversity of Gr. Reconstruction of the ancestral state for each Gr present in the near-term placenta showed a diverse scenario: some sugars were common to the species of Placentalia included in this study. In the analyzed species with synepitheliochorial and epitheliochorial placentas, no differential glycosylation patterns between them were observed. In species with invasive placentas, such as the endotheliochorial placentas of Carnivora, some common Gr were detected among them, while others were species-specific. In species with hemochorial placenta, the same Gr are shared. Particularly, in the viscacha greater differences with species of the Hominidae and even Muridae families were observed. Nevertheless, greater similarities with other caviomorph rodents were detected. Placental glycotype of each species constitutes an excellent tool to achieve phylogenetic and evolutionary inferences among eutherians.

The molecular mechanisms of extracellular matrix-derived hydrogel therapy in idiopathic pulmonary fibrosis models.

Idiopathic Pulmonary Fibrosis (IPF) is a progressively debilitating lung condition characterized by oxidative stress, cell phenotype shifts, and excessive extracellular matrix (ECM) deposition. Recent studies have shown promising results using decellularized ECM-derived hydrogels produced through pepsin digestion in various lung injury models and even a human clinical trial for myocardial infarction. This study aimed to characterize the composition of ECM-derived hydrogels, assess their potential to prevent fibrosis in bleomycin-induced IPF models, and unravel their underlying molecular mechanisms of action. Porcine lungs were decellularized and pepsin-digested for 48 h. The hydrogel production process, including visualization of protein molecular weight distribution and hydrogel gelation, was characterized. Peptidomics analysis of ECM-derived hydrogel contained peptides from 224 proteins. Probable bioactive and cell-penetrating peptides, including collagen IV, laminin beta 2, and actin alpha 1, were identified. ECM-derived hydrogel treatment was administered as an early intervention to prevent fibrosis advancement in rat models of bleomycin-induced pulmonary fibrosis. ECM-derived hydrogel concentrations of 1 mg/mL and 2 mg/mL showed subtle but noticeable effects on reducing lung inflammation, oxidative damage, and protein markers related to fibrosis (e.g., alpha-smooth muscle actin, collagen I). Moreover, distinct changes were observed in macroscopic appearance, alveolar structure, collagen deposition, and protein expression between lungs that received ECM-derived hydrogel and control fibrotic lungs. Proteomic analyses revealed significant protein and gene expression changes related to cellular processes, pathways, and components involved in tissue remodeling, inflammation, and cytoskeleton regulation. RNA sequencing highlighted differentially expressed genes associated with various cellular processes, such as tissue remodeling, hormone secretion, cell chemotaxis, and cytoskeleton engagement. This study suggests that ECM-derived hydrogel treatment influence pathways associated with tissue repair, inflammation regulation, cytoskeleton reorganization, and cellular response to injury, potentially offering therapeutic benefits in preventing or mitigating lung fibrosis.

The Anatomy of the Stomatognathic System in Different Skull Types in Dogs.

The anatomy of the stomatognathic system is important for both clinical evaluations and surgical approaches in all animal species. The aim of this study was to describe the innervation and vascularization of the stomatognathic system of the dog. Twelve dogs without a history of disease or cranial malformation were used: 4 brachycephalic, 4 mesocephalic, and 4 dolichocephalic. The dogs were dissected, and arteries, veins, and nerves related to the masticatory and swallowing components were identified. The distribution pattern of these structures in the 3 different skull types were observed. The entire blood supply of the stomatognathic system is derived from the external carotid artery, which originates from the common carotid artery, and terminates as it branches into the superficial temporal and maxillary arteries. The other main branches of the common carotid artery are the occipital, cranial laryngeal, ascending pharyngeal, lingual, facial, caudal auricular, and parotid arteries. Blood drainage was achieved via the external jugular vein, which originates from the union of the linguofacial and maxillary veins. Brachycephalic dogs had blood vessels with greater sinuosity (more deviations) when compared to dolichocephalic and mesocephalic dogs. The stomatognathic system innervation of brachycephalic skull dogs showed differences in the distribution of the facial nerve in the labial commissure and maxillary and mandibular regions. The cranial conformation of dogs demonstrated anatomical variations of the vascular and neural structures of the stomatognathic system. This data may be useful to improve clinical practice, surgical planning, and interpretation of clinical dysfunctions.

The fetomaternal interface in the placenta of three species of armadillos (Eutheria, Xenarthra, Dasypodidae).

BACKGROUND: Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria. Armadillos are known for villous, haemochorial placentas similar to humans. Only the nine-banded armadillo has been well studied so far. METHODS: Placentas of three species of armadillos were investigated by means of histology, immunohistochemistry including proliferation marker, and transmission and scanning electron microscopy. RESULTS: The gross anatomy differed: Euphractus sexcinctus and Chaetophractus villosus had extended, zonary placentas, whereas Chaetophractus vellerosus had a disk. All taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that the endothelium of the sinuses was largely intact. Tips of the villi and the columns entering the maternal tissue possessed trophoblast cell clusters with proliferation activity. Elsewhere, the feto-maternal barrier was syncytial haemochorial with fetal vessels near the surface. CONCLUSIONS: Differences among armadillos occurred in regard to the extension of the placenta, whereas the fine structure was similar. Parallels to the human suggest that armadillos are likely to be useful animal models for human placentation.

Placentation in the anteaters Myrmecophaga tridactyla and Tamandua tetradactyla (Eutheria, Xenarthra).

BACKGROUND: Since Xenarthra are serious candidates for being basal to Eutheria, their characteristics, e.g. the placental system, influence perceptions of evolution. However, in the subgroup containing the anteaters, data are very limited. The present study aims to elucidate the nature of the feto-maternal interface in the anteater placenta and to interpret these data within an evolutionary context. METHODS: Placentas of two species were investigated with histology, immunohistochemistry and transmission electron microscopy. RESULTS: Remnants of the maternal vessel endothelium were absent, resulting in a fully haemochorial barrier throughout the placenta. Two structurally different parts, the villous and trabecular areas were complex and intermingled. In particular, the trabeculae which consisted of cellular, proliferative trophoblast, associated with connective tissue, were attached to the decidua. The villi contained fetal capillaries and hypertrophied mesenchymal cells that occurred near the surface near the end of gestation. The surface of the villi consisted of flat, syncytial trophoblast, interspersed with proliferative trophoblast cells. CONCLUSIONS: Based on fundamental differences between anteaters and armadillos, we inferred that placental evolution was more complex than previously thought. The haemochorial pattern of anteaters was likely an ancient condition of xenarthrans. Consequently, villous placentation may be attributed, at least in part, by convergent evolution, but was also characterized by some features that were widespread among xenarthrans.

Placentation in Sigmodontinae: a rodent taxon native to South America.

BACKGROUND: Sigmodontinae, known as "New World rats and mice," is a large subfamily of Cricetidae for which we herein provide the first comprehensive investigation of the placenta. METHODS: Placentas of various gestational ages ranging from early pregnancy to near term were obtained for five genera, i.e. Necromys, Euryoryzomys, Cerradomys, Hylaeamys, and Oligoryzomys. They were investigated by means of histology, immunohistochemistry, a proliferation marker, DBA-lectin staining and transmission electron microscopy. RESULTS: The chorioallantoic placenta was organized in a labyrinthine zone, spongy zone and decidua and an inverted yolk sac persisted until term. The chorioallantoic placenta was hemotrichorial. The interhemal barrier comprised fetal capillary endothelium and three layers of trophoblast, an outermost, cellular layer and two syncytial ones, with interspersed trophoblast giant cells (TGC). In addition, accumulations of TGC occurred below Reichert's membrane. The junctional zone contained syncytial trophoblast, proliferative cellular trophoblast, glycogen cells and TGC that were situated near to the maternal blood channels. In three of the genera, TGC were also accumulated in distinct areas at the placental periphery. PAS-positive glycogen cells derived from the junctional zone invaded the decidua. Abundant maternal uNK cells with positive response to PAS, vimentin and DBA-lectin were found in the decidua. The visceral yolk sac was completely inverted and villous. CONCLUSION: The general aspect of the fetal membranes in Sigmodontinae resembled that found in other cricetid rodents. Compared to murid rodents there were larger numbers of giant cells and in some genera these were seen to congregate at the periphery of the placental disk. Glycogen cells were found to invade the decidua but we did not identify trophoblast in the walls of the deeper decidual arteries. In contrast these vessels were surrounded by large numbers of uNK cells. This survey of wild-trapped specimens from five genera is a useful starting point for the study of placentation in an important subfamily of South American rodents. We note, however, that some of these rodents can be captive bred and recommend that future studies focus on the study of time dated pregnancies.

Prenatal development in Lagostomus maximus (Rodentia, Chinchillidae): A unique case among eutherian mammals of physiological embryonic death.

Embryonic death followed by resorption is a conserved process in mammals. Among the polyovular species, Lagostomus maximus (plains viscacha) constitutes a model of early and physiological embryonic death, since out of a total of 10-12 implants, 8-10 are resorbed during early/intermediate gestation, surviving are only the most caudal implantations of each uterine horn. This regular reproductive event is unique to this species, but many characteristics of the implantations during the early gestation of L. maximus, when embryonic death processes begin are unknown. The aim of the present work was to analyze the implantation sites of this species using morphological, morphometric, histochemical, lectinhistochemical, and immunohistochemical techniques to infer the possible causes of this event. Macroscopically, the length and width of the implantation sites significantly increased in a craniocaudal direction. Histochemically, the implantation sites did not differ in the expression of glycoconjugates and glycosidic residues. Furthermore, no variations were observed in cell renewal, hormone receptor expression, and decidualization. Both the glandular and vascular areas of the implantation sites significantly increased in the craniocaudal axis. Some necrotic cells and an inflammatory response with a predominance of lymphocytes and fibrin were observed in the cranial and middle but not in the caudal implantation sites. We conclude that signs of embryonic death and resorption are already observed in the early gestation of L. maximus. Our results reaffirm the hypothesis that postulates the key potential role of uterine glands and blood vessels in the gestation of the species, with emphasis on embryonic death. This pattern of embryonic death in L. maximus makes this species an unconventional mammalian model, which adds to the peculiarities of polyovulation (200-800 oocytes/estrus) and hemochorial placentation.

Structural and ultrastructural characterization of the palatine epithelium of the Guinea pig: A new record of telocytes in the oral cavity.

The morphology of the oral cavity of mammals relates to diet, habitat, and function. The palate is an important region with adaptations for oral somatosensation and mechanical loads due to the pressure of the tongue with food. The research aimed to describe the structural and ultrastructural characteristics of the epithelium and the connective tissue cores of the guinea pig palate using macroscopic, light microscopy, scanning electron microscopy, and transmission electron microscopy analysis. The hard palate had conical and filiform papillae, and the soft palate had open salivary ducts. After the removal of the epithelium, the connective tissue cores revealed thin filaments and laminar projections in the hard palate, and opening ducts were evidenced in the soft palate. The palatine epithelium was keratinized and organized by layers, lamellated corpuscles were found in lamina propria of the hard palate. In contrast, the soft palate had glands clusters associated with nerve fibers, and in both regions were identified telocytes. We concluded that the hard palate presented conical and filiform papillae that differ from other mammals. Besides, it is a new description of the connective tissue cores morphology and the first record of the telocytes in this anatomical region for mammals.

Reproductive morphophysiology of the male scorpion mud turtle (Kinosternon scorpioides Linnaeus, 1766) in captivity.

Kinosternon scorpioides, popularly known as scorpion mud turtle (jurará in Brazil), is a fresh water species. There is little information about its reproduction and the present study aims to morphologically characterize the reproductive organs of male K.scorpioides bred in captivity in two seasons of the year. The reproductive tracts of adult animals under went macroscopic ultrastructural analysis of the lumen, as well as scanning electron and transmission microscopy. Macroscopically, the male genital organs consist of a pair of testicles, epididymis, the vas deferens and a penis. Testicles, epididymis and deferents ducts were characterized by reproductive activity during the rainy season and reproductive inactivity in the dry period. The morphometry regarding the tubular and luminal diameter and epithelial height of the testicles, epididymis and deferents ducts showed changes along the studied periods. The rainy season presented higher averages than the dry period. The penis did not show any changes during both periods. It was concluded that K. scorpioides exhibits reproductive seasonality.

Early and natural embryonic death in Lagostomus maximus: Association with the uterine glands, vasculature, and musculature.

The uterus is an organ with great plasticity due to the morphological and physiological changes it experiences during the estrous cycle and pregnancy. In mammals, pregnancy requires diverse sex hormones, growth factors and cytokines, among others, for promoting uterine remodeling to favor implantation, placentation, and embryo/fetus survival and growth. The hystricognathi rodent Lagostomus maximus (plains viscacha) has a high rate of embryonic resorption. The cranial and middle implants are reabsorbed 25-35 days after intercourse while the caudal embryos continue with their development until two precocial offspring are born. So far, no uterine studies of non-pregnant L. maximus females were performed to determine the possible existence of variations in the organ that could be related to the differential survival of the implants. We used ultrasonography, as well as morphological, morphometric, histochemical, lectinhistochemical, and immunohistochemical methods to study differences in the uterine glands (area), vasculature (area), and musculature (thickness) along the uterine horns in non-pregnant females. Along the uterus, all these structures were in more advanced developmental condition in the caudal region as compared to more anterior positions. These regional variations could be decisive in explaining the reason why only caudal implantations come to term. In contrast, no differences in the in the luminal and glandular epithelial cells, nor in the degree of cell proliferation and apoptosis, and hormonal receptor staining were found. These parameters could be related to implantation along the uterine horns, but not to the differential survival of the implants.

Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy.

BACKGROUND: Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities. METHODS: In this study, the proliferation activity and apoptosis in different regions of normal bovine placenta (central and boundary regions of placentomes, placentomal fusion, microplacentomes, and interplacentomal regions), from distinct gestation periods (Days 70 to 290 of pregnancy), were analyzed by flow cytometry. RESULTS: Our results indicated that microplacentomes presented a lower number of apoptotic cells throughout pregnancy, with a higher proliferative activity by the end of gestation, suggesting that such structures do not contribute significantly to normal of placental functions and conceptus development during pregnancy. The placentome edges revealed a higher number of apoptotic cells from Day 170 on, which suggests that placentome detachment may well initiate in this region. CONCLUSION: Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations seen in nature and after in vitro embryo manipulations.

Morphological and Molecular Analysis of In Vitro Tubular Structures from Bovine Yolk Sac-Derived MSCs.

The yolk sac is an extraembryonic membrane, of saccular form, connected to the ventral region of the embryo. It is the main source of nutrition for the embryo during the period when the placenta is not fully formed. The aim of this study was to generate tubular structures using mesenchymal stem cells from the bovine yolk sac (bYS-MSCs) and determine if these structures can be a model for in vitro vasculogenesis. The evaluation of this tissue by histochemistry revealed a strong marking of collagen fibers and PAS technique negativity. In transmission electron microscopy, cytoplasmic organelles with large nuclei were observed. The vessel formation assay on a Matrigel substrate showed that the mesenchymal cells of the yolk sac without growth factors (VEGF) are capable of forming branches, sprouting cells, and tubular structures similar to capillary blood. These tubular structures were xenotransplanted subcutaneously into the mesentery of BALB/c/nude mice; after 45 days, vascularized tissue and extensions of blood vessels around the tubular structures could be observed. Real-time PCR (qPCR) demonstrated an expression of the VEGF gene in different gestational age groups. No difference in distribution or expression was detected among groups. Our results suggest that the spontaneous formation of tubules from the yolk sac can be an experimental model to elucidate initial organogenesis and the possible formation of blood capillaries from in vitro mesenchymal cells and possible route of organoid production.

Combination of stem cells from deciduous teeth and electroacupuncture for therapy in dogs with chronic spinal cord injury: A pilot study.

Spinal cord injury (SCI) is a serious condition that causes profound economic and emotional impact in human patients and companion animal owners. It has been shown that the neurogenic effects of the stem cells are enhanced when combined with electroacupuncture (EA) in rodent models of SCI. To determine the safety and feasibility of combining transplantation of allogenic stem cells derived from canine exfoliated deciduous teeth (SCED) and EA in dogs with chronic spinal cord injury a canine pilot clinical study was conducted. A total of 16 individuals ranging from 5 to 11 years at 3 to 18 months of injury were investigated and randomly assigned to 4 experimental groups (SCED, EA, SCED + EA, control). Mild neurological and functional improvements were seen in all 4 groups. There was no clinical progression or mortality of the cases occurred in a follow up of 7 months after procedure. The study shows that SCED transplantation and electroacupuncture were feasible, safe and potentially beneficial. However Long-term patient monitoring is necessary to rule out any delayed side effects and assess any further improvements.