The enteric neural crest progressively loses capacity to form enteric nervous system.

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS-donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (≤ QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2-3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce 'catch up' colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells.

Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation.

The aim of our investigations was to compare the effectiveness of two methods for cryopreservation of sheep ovarian tissue, slow freezing and vitrification. The quality of cryopreserved tissues was evaluated after 5 days of thawing and chorioallantoic membrane (CAM) transplantation. Follicular structure, stromal integrity and neovascularization were assessed. The areas of fibrosis and necrosis were measured using MICROVISIBLE software, and proliferation was assessed with Ki-67 immunostaning. After 5 days of culture, the proportion of primordial follicles decreased, whereas the primary and intermediary follicles increased insignificantly (p > .05). Only necrosis in the vitrified culture group increased significantly (p < .05). It was established also that 5 days CAM culture was not suitable methodology for detection of folliculogenesis. Follicular quality decreased after culture, but was better in fresh and slow frozen tissues than after vitrification (p < .05). Cellular proliferative activity fell, but it preserved to some extent in all groups. In conclusion, follicles was preserved better in grafted tissue after slow freezing than vitrification and stroma was more susceptible to ischemia in vitrified rather than conventional freezing in this view. Vitrification may not be a suitable alternative to the slow freezing.

Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken.

Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence of HSCs has never been formally proven in chicken embryos. Here, we have established a complete cartography and quantification of hematopoietic cells in the aorta during development. We demonstrate the existence of bona fide HSCs, originating from the chicken embryo aorta (and not the yolk sac, allantois or head), through an in vivo transplantation assay. Embryos transplanted in ovo with GFP embryonic tissues on the chorio-allantoic membrane provided multilineage reconstitution in adulthood. Historically, most breakthrough discoveries in the field of developmental hematopoiesis were first made in birds and later extended to mammals. Our study sheds new light on the avian model as a valuable system to study HSC production and regulation in vivo.

Human conchal cartilage and temporal fascia: an evidence-based roadmap from rhinoplasty to an in vivo study and beyond.

Conchal cartilage or cartilage/ temporal fascia composite grafting (DC-F) used for rhinoplasty is applied by plastic surgeons for reconstructive purposes. Previous studies on experimental models such as mice or rabbits have elucidated on the late events following grafting, with tissue specimens being harvested two months after implantation. Early microscopic and molecular events following DC-F grafting are completely unknown. We designed a chick embryo chorioallantoic membrane model for human grafts study, regarding the dynamic observation of graft survival and its mutual interrelation with the chick embryo chorioallantoic membrane microenvironment. The DC-F graft preserved its cartilage component in a normal state compared to cartilage graft-only because of protective factors provided by temporal fascia. Its strong adherence to the cartilage, lack of angiogenic factors and high content of collagen IV-derived fragments with anti-angiogenic effects make the temporal fascia a good protective tissue to prevent implanted cartilage degeneration. The cartilage graft produced high inflammation, stromal fibrosis and activated angiogenic cascade through VEGF-mediated pathways followed by cartilage degeneration. Also, high content of podoplanin from conchal cartilage chondrocytes exerted a major role in inflammation accompanying cartilage graft. The presently employed experimental model allowed us to characterize the early histological and molecular events triggered by temporal fascia, cartilage or composite graft DC-F implanted on chick embryo chorioallantoic membrane. Our microscopic and molecular observations may help explain some post-surgical complications generated after using cartilage alone as biomaterial for nasal augmentation, supporting the use of DC-F composite graft, with the aim to reduce unwanted post-surgical events.

Skeleton pattern and joint formation in chorioallantoic grafts containing the distal parts of the chick wing bud.

Skeleton pattern formation was examined in chick wing bud grafts using the chorioallantoic grafting method. The distal parts of the wing bud were excised from the donor wing and transplanted onto the chorioallantoic membrane (the experimental groups). Transplants with intact limb bud material served as the control group. The skeleton pattern formation in the grafts depended on the amount of transplanted material and donor's limb bud stage. The younger the donor's stage and the bigger the piece of the transplanted material the more proximal parts grafts had, more retarded growth and abnormal skeleton in the zeugopod and autopod was. The percentage of the signs of insufficient blood supply in the experimental groups was less than that in the control group. As the amount of the transplanted limb bud material decreased and donor's limb bud aged, post-axial polydactyly changed to the pre-axial one.

The effect of perfluorocarbon-based artificial oxygen carriers on tissue-engineered trachea.

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.

Animal models in endometriosis research.

Endometriosis is a common gynaecological disease, defined as the presence of endometrial tissue outside the uterus, causing pelvic pain and subfertility in approximately 10% of women of reproductive age. Current therapies lead to pain relief, however, do not address the causes and entail severe side effects. Still little is known about the pathogenic processes leading to the development and maintenance of endometriosis. Because endometriosis occurs spontaneously only in humans and some non-human primates, animal models of induced endometriosis have been developed and are of high value for the evaluation of pathophysiological mechanisms underlying the development of this disease. These experimental models include the autotransplantation of uterine fragments into the peritoneal cavity of rodents and non-human primates or the heterotransplantation of human endometrial or endometriotic tissue to immunodeficient mice or onto the chicken chorioallantoic membrane (CAM). This review describes the animal models for endometriosis and assesses their different potentials and limitations in regard to endometriosis research, with the aim of developing novel non-invasive diagnostic tools and improved strategies for the treatment of endometriosis in women.

Enhancement of implantable glucose sensor function in vivo using gene transfer-induced neovascularization.

The in vivo failure of implantable glucose sensors is thought to be largely the result of inflammation and fibrosis-induced vessel regression at sites of sensor implantation. To determine whether increased vessel density at sites of sensor implantation would enhance sensor function, cells genetically engineered to over-express the angiogenic factor (AF) vascular endothelial cell growth factor (VEGF) were incorporated into an ex ova chicken embryo chorioallantoic membrane (CAM)-glucose sensor model. The VEGF-producing cells were delivered to sites of glucose sensor implantation on the CAM using a tissue-interactive fibrin bio-hydrogel as a cell support and activation matrix. This VEGF-cell-fibrin system induced significant neovascularization surrounding the implanted sensor, and significantly enhanced the glucose sensor function in vivo. This model system, for the first time, provides the "proof of principle" that increasing vessel density at the sites of implantation can enhance glucose sensor function in vivo, and demonstrates the potential of gene transfer and tissue interactive fibrin bio-hydrogels in the development of successful implants.

Angiogenic response induced by acellular aortic matrix in vivo.

In this study, we investigated the angiogenic response induced by acellular aortic matrices implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. Results showed that acellular matrices were able to induce a strong angiogenic response comparable to that of fibroblast growth factor 2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta 1 (TGF-beta1) were added to the matrices and inhibited by the addition of an anti-FGF-2 or anti-TGF-beta1 antibodies. The response may be considered dependent on a direct angiogenic effect exerted by the matrices and in part also by the presence of FGF-2 and TGF-beta1 in the acellular matrices.