Chorioallantoic Membrane Culture Research Articles

Detection and Isolation of Camelpox Virus in Wasit Province, Iraq.

Camels are susceptible to a variety of infectious diseases, such as trypanosomiasis, anthrax, hemorrhagic septicemia, brucellosis, mange, and pox, which can also affect other farm animal species. Camelpox is one of the most infectious skin diseases, which is caused by the Camelpox virus (CPV), a member of the Orthopoxvirus genus in the Poxviridae family. This study mainly aimed to detect and isolate CPV affecting camels in Wasit province, Iraq. Initially, the study focused principally on clinical manifestations of the disease in affected animals. Afterward, 110 skin samples were collected from infected animals under strict aseptic conditions. They were to be subjected to inoculation into the local embryonated chicken eggs to isolate the virus from the chicken embryo fibroblast cell culture. Finally, the isolates were confirmed using the molecular polymerase chain reaction (PCR) assay. Camelpox virus isolates appeared as several necrotic pock lesions on the Chorioallantoic Membrane (CAM) with cell clustering and sloughing or detachment from the monolayer. Molecular testing was conducted using the PCR assay by targeting the ATIP gene at 881bp. The findings demonstrated that all the investigated isolates were poxvirus positive. In addition, it was found that Camelpox disease is significantly endemic in Wasit province, Iraq. The visualization of the characteristic features of the virus also revealed it can easily adapt to replication in the CAM and cell culture.

Angiogenesis and follicular development in ovarian tissue of cattle following vitrification and post-warming culture on chicken chorioallantoic membrane

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1−2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems

BackgroundA suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system.MethodsOvarian cortical tissues (1–2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined.ResultsIn the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001).ConclusionsThe CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.

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.

Serological and Molecular Identification of Infectious Bovine Rhinotrachetitis Virus Isolation and Adaptation in Embryonated Chicken Eggs

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in livestock animals and had led to significant economic losses to the industrial production worldwide. However, IBR symptoms are not life-threatening. The present study was achieved to make seroprevalence and isolation of bovine herpes virus type (BHV-1) from nasal and ocular swabs samples of suspected cows and buffaloes, identification by serological and polymerase chain reaction (PCR). Total of 380 blood samples were collected from suspected 287 cattle and 93 buffaloes in different districts at Beni-suef and El-Fayoum governorates in winter 2017. Nasal and ocular discharge swabs were collected from cattle and buffaloes with clinical respiratory signs (Nasal discharge, cough, lachrymal discharge with or without mild diarrhea and higher body temperature). A total of 106 (27.89%) samples were positive by indirect ELISA and the positivity of 80 (27.87%) samples from cattle and 26 (27.96%) samples from buffaloes located at different centers at Beni-Suif and El-Fayoum governorates. Virus was isolated from nasal and ocular discharges swabs samples and it was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cells. Virus-infected CAM showed congestion, edematous vacuole, thickening small foci ranged from 2 to 3 mm in diameter, scattered on CAM membrane and MDBK cell line inoculated blind serial passages at 3rd passage showed characteristic of cytopathic effect (CPE). Identification of virus isolated on CAM and infected cell culture fluid gave precipitation against positive specific anti-BHV-1 immune serum by AGPT and clear blue zone by Dot ELISA, absent of pock lesion in pock reduction test (PRT), and confirmed by PCR with product size of 175 bp. Finally BHV-1 virus was isolated from nasal and ocular discharges of cattle and buffaloes but the further extensive study still need for clear final classification by phylogenic analysis.

Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane

In vitro culture and transplantation procedures are essential protocols employed in the evaluation of ovarian follicle survival and development. Culture in the chorioallantoic membrane (CAM) of chick embryos is an intermediate method that provides important follicle development information and has not been tested for cat ovaries to date. The aim of this study was to investigate if in vitro and CAM culture could be used as short-term systems to study cat ovarian tissue development. The ovaries of eight cats were dissected into 3-mm3 cubes, cultured in vitro and in CAM for up to 5 days, and stained with hematoxylin-eosin and Gomori trichrome. Cell proliferation was analyzed using anti-Ki67. Possible differences among groups were investigated by analysis of variance or the Kruskal–Wallis test followed by Bonferroni correction. The T-test or Wilcoxon test was used to verify differences between the CAM and IVC. Results revealed that 87.5% of all follicles were primordial during culture. The percentage of primordial follicles in the morphologically normal follicles (MNF) pool was always higher than 80%, with the exception of Day 3 of CAM culture, but the number of MNF reduced significantly from Day 0 (600 out of 777 follicles) to Day 5 in the CAM (91 out of 171) and IVC (296 out of 686). The number of primordial follicles in 1 mm3 in Days 2, 3, and 5 in the CAM was significantly lower than that in the control (Day 0). No cellular proliferation was observed in culture. Vascularization occurred in the CAM culture, but with no association to follicular viability. In addition, both methods showed an increase in connective tissue during culture. Although no significant differences were observed in the percentage of MNF, there was a reduction in the total number of follicles, both for IVC and CAM-cultured ovarian tissue. Furthermore, anti-Ki67 did not stain any follicle after Day 0 in IVC or in CAM culture. Neither system was capable of promoting follicle growth and/or development. The results show that the CAM is not a suitable system for feline ovarian tissue and highlight the necessity to improve IVC systems in cats.

Three-Dimensional Imaging of the Developing Vasculature within Stem Cell-Seeded Scaffolds Cultured in ovo.

Successful tissue engineering requires functional vascularization of the three-dimensional constructs with the aim to serve as implants for tissue replacement and regeneration. The survival of the implant is only possible if the supply of oxygen and nutrients by developing capillaries from the host is established. The chorioallantoic membrane (CAM) assay is a valuable tool to study the ingrowth and distribution of vessels into scaffolds composed by appropriate biomaterials and stem cell populations that are used in cell-based regenerative approaches. The developing vasculature of chicken embryos within cell-seeded scaffolds can be visualized with microcomputed tomography after intravenous injection of MicroFil®, which is a radiopaque contrast agent. Here, we provide a step-by-step protocol for the seeding of stem cells into silk fibroin scaffolds, the CAM culture conditions, the procedure of MicroFil® perfusion, and finally the microcomputed tomography scanning. Three-dimensional imaging of the vascularized tissue engineered constructs provides an important analytical tool for studying the potential of cell seeded scaffolds to attract vessels and form vascular networks, as well as for analyzing the number, density, length, branching, and diameter of vessels. This in ovo method can greatly help to screen implants that will be used for tissue regeneration purposes before their in vivo testing, thereby reducing the amount of animals needed for pre-clinical studies.

Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin-Darby bovine kidney cell line.

Aim:Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test.Materials and Methods:Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test.Results:Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test.Conclusion:BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.

47 THE AVIAN CHORIO-ALLANTOIC MEMBRANE: A SUITABLE SHORT-TERM CULTURE SYSTEM FOR BOVINE OVARIAN TISSUE

Successful cryopreservation of bovine ovarian tissue holds enormous potential for long-term maintenance of female gametes to preserve genetic diversity by tissue banking. Traditionally, in vitro culture followed by histopathological examination has been used to assess the post-thaw viability of cryopreserved tissues. Recently, in ovo transplantation of mammalian tissues on the chorio-allantoic membrane (CAM) of a growing chicken embryo has emerged as an alternative method for short-term culture. The purpose of this experiment was to compare CAM culture of bovine ovarian tissue over a 5-day period with the in vitro culture system. Fertilized White Leghorn eggs were incubated at 37°C and 62% relative humidity. A window (1 × 2 cm) was cut into the eggshell on Day 3 of incubation. Ovaries were retrieved from a local abattoir and brought to the laboratory within 6 h. Ovarian cortex pieces (1–2 mm3) were randomly assigned to control, CAM-culture, or in vitro-culture groups. Control-group tissues were fixed immediately in 4% paraformaldehyde. The CAM was traumatized on Day 10 of incubation to expose the underlying blood vessels, and tissue pieces were grafted at the site (one graft per egg). For in vitro culture, the ovarian cortex pieces were placed on tissue culture inserts within 6-well plates containing TCM199 with 1% insulin-transferrin-selenium, 100 mIU mL–1 of FSH, 100 IU mL–1 of penicillin, and 50 μg mL–1 of streptomycin and incubated at 38°C in 5% CO2. Ovarian tissues from the CAM and in vitro culture group were removed on Day 1, 3, and 5 of grafting/culture, fixed, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin-eosin, and analysed under a light microscope. The numbers of normal and degenerated follicles (indicating follicle survival) and number of blood vessels containing bovine and avian red blood cells (indicating angiogenesis) were counted using standard stereological procedures. All ovarian cortex grafts from surviving chick embryos showed adhesion with the CAM and a marked neo-vascularization in the graft areas. Gross and histological examination revealed the circulation of avian blood cells in ovarian stromal vessels with a concomitant decrease in the number of bovine blood vessels over the incubation period. Total follicle densities (mean ± s.e.m.) on Day 1, 3, and 5 were 13.3 ± 5.9, 27.9 ± 6.7, and 36.9 ± 7.3 in the in vitro-cultured group and 36.7 ± 13.0, 73.6 ± 24.0, and 44.02 ± 12.67 per millimeter cubed in the CAM-cultured group, respectively. Overall, total follicle density was higher in the CAM-cultured group (P &lt; 0.05). Likewise, the normal follicle densities on Day 1, 3, and 5 were 10.4 ± 4.9, 15.5 ± 3.6, and 20.7 ± 6.3 in the in vitro-cultured group and 30.5 ± 8.5, 45.7 ± 18.4, and 22.7 ± 7.3 per millimeter cubed in the CAM-cultured group (P &gt; 0.05). In conclusion, in ovo CAM grafting system was as successful as the in vitro-culture system and may be considered an acceptable alternative to the traditional in vitro-culture system for bovine ovarian tissue.

Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture

To achieve optimal and uniform outcomes, slow cooling protocols for human ovarian tissues generally initiate ice formation at high sub-zero temperatures (−6 to −9°C). The aim of the study was to investigate the function of ovarian tissue that had unintentionally self seeded at −20°C during the freezing step, by examining its development following chicken embryonic chorioallantoic membrane (CAM) grafting and after transplantation back to the patient. Ovarian tissue was frozen in 6% (v/v) dimethyl sulfoxide, 6% (v/v) ethylene glycol and 0.15M sucrose which had self-seeded at −20°C. Five years after cryopreservation, 8 pieces were thawed and transplanted back to the patient. Two small (1×2×1mm) pieces of this thawed tissue were cultured in a CAM-system for 5days to assess the tissue viability. The autografted ovarian tissue re-established spontaneous menstrual bleeding within five months and raised serum 17-β Estradiol from 19 to 330pg/ml. Ultrasound revealed a dominant follicle at the site of the transplanted tissue in the follicular phase after the menstrual bleed. Analysis of the CAM cultured tissue established that 88% of the primordial follicles are degenerated and there was limited in growth of blood vessels. In conclusion, in spite of the damage caused by the cryopreservation with spontaneous ice-formation the viability could be confirmed by CAM culture and the restoration of ovarian function after auto-transplantation.

Epizootiology of lumpy skin disease outbreak in cattle in middle of Egypt, 2006

The current investigation studied an outbreak of lumpy skin disease of cattle in Beni-Suef and Al-Fayium governorates from March up to September 2006. Epidemiological data over a total of 5500 cattle from all ages, breeds and sexes were investigated. Prevalence of lumpy skin antibodies was screened by enzyme-linked immunosorbent assay (ELISA) that revealed high exposure rates; 57% and 51.42% in Beni-Suef and Al Fayium governorates respectively Virus isolation was conducted on chorioallantoic membrane (CAM) of specific pathogen free embryonated chicken egg (SPF-ECE) and MDBK cell culture. The virus identity was confirmed by passive haemagglutination and polymerase chain reaction (PCR) on the infected CAM and MDBK cell culture. Experimental infection of rabbits was successful, demonstrating their possible roles in the epidemiological process of the disease.

008 Xenobiotic Skin Culture System Supported by Surrogate Avian Vascular Circulation

Currently, skin cells or tissues intended for therapeutical or cosmetic applications are typically cultured using standard in vitro culture techniques. The avian chorioallantoic membrane (CAM) has been traditionally used for many years to assess angiogenesis of exogenous substances. Avian embryos have also been used to generate viruses for vaccine development. Most of the uses of the avian CAM cultures are typically conducted in ovo. However, xenobiotic cultures of ex ovo avian embryos and explanted organs, tissues or cells from human or other mammalian origin have not been used in the past with the explicit purpose of achieving the appropriate extent of expansion, differentiation or vascularization required for therapeutic or cosmetic implantation into humans or animals. Here, a novel method of xenobiotic skin culture was developed that can be employed for wound healing studies or for tissue engineering applications to sustain the culture of live skin explants through the benefit of a xenobiotic vascularization. The method uses fertile Japanese quail (Cortunix cortunix japonica) eggs. The eggs were incubated for at least 3 days in ovo using a static egg incubator. On day 3 the egg is opened under sterile conditions through the air chamber and placed in a cylindrical culture dish slightly larger than the overall diameter of the egg yolk. The embryo was then cultured at 37 °C for an additional 24 to 48 hours until a well-vascularized CAM covers the entire surface of the culture dish. Next, fragments of skin were placed directly over the CAM. The cultures were continued for 8 days and then the skin explants were transferred to new CAMs. These changes, every 8 days, were done at least five times allowing the skin to be maintained in culture for several weeks without apparent damage in viability. The skin fragments remained viable and capable of reepithelialization when wounded. Skin explants from both mouse and human were successfully cultured in this manner. Because immunological response cannot occur in the embryonic culture environment, it was possible to xenotransplant skin fragments without risk of rejection. The ensuing embryonic vessels developed into the xenotransplanted skin and generated a permeable vascular network that provided oxygen and nutrients necessary to ensure viability and promote proliferation and differentiation. Viability of the explants was evaluated by immunohisto chemical methods using proliferation and apoptosis markers (Caspase-3, BrdU, and PCNA). Vascularization was assessed using species-specific anti endothelial antibodies (anti-PECAM human and mouse and QH1 and QH2 monoclonal antibodies). The new method described herein has the distinct advantage of sustaining live, functional skin cultures by means of a rich vascular supply that provides cells with nutrients and oxygen.

Endochondral Ossification of Chick Embryonic Femora in vitro and on Chorioallantoic Membrane

We observed angiogenesis and endochondral ossification of the femora of chick embryos in vitro, on chorioallantoic membrane (CAM), and in vivo to clarify the cellular processes of avian endochondral ossification. We found that the inside of the in vitro femora was still filled with chondrocytes, despite being cultured for 10 days, while calcification of the diaphysis was not observed. As well, only the cartilage tissue of the epiphysis was enlarged. By contrast, blood vessels invaded into the diaphysis and the marrow cavity was formed in CAM-cultured femora. Cartilage canals extended from the marrow cavity and reached to the resting chondrocyte zone, with normal endochondral ossification occurring as in vivo. This study demonstrates that endochondral ossification occurs in femora in CAM culture similar to that in in vivo femora, but not in in vitro femora. The ossification is dependent on vascular invasion into the embryonic femora. In conclusion, for the endochondral ossification of long bones it is essential to supply the embryo with blood vessels. CAM culture system was found to be a superior endochondral ossification model of the embryonic femur.

Matrix Metalloproteinase-9 Induces the Formation of Cartilage Canals in the Chondroepiphysis of the Neonatal Rabbit

An investigation of matrix metalloproteinase-9 (MMP-9) and its influence on vascular invasion in the secondary ossification center at the chondroepiphysis of developing long bones was undertaken. The effect of MMP-9 was compared with that of basic fibroblast growth factor (b-FGF), a potent angiogenic factor, and we assessed the chorioallantoic membrane (CAM) culture as a model for angiogenesis in osteochondral tissue. Seventy-two femoral and seventy-two humeral heads of thirty-six four-day postnatal rabbits were dissected immediately after each animal was killed. Solutions of MMP-9, b-FGF, and phosphate-buffered saline solution were applied, and the femoral and humeral chondroepiphyseal explants were incubated for ten days in CAM culture. This was used as an in vivo model to investigate the growth of blood vessels into the femoral and humeral heads of the neonatal rabbit. The explants were harvested from the CAM culture and analyzed histologically. A three-day incubation was also performed to look for early signs of vascular ingrowth into the cartilage matrix. One hundred and twenty epiphyses from thirty rabbits were placed onto CAM culture successfully; of these, two were harvested at three days to assess early changes and 118 were harvested at ten days. Forty of the 118 cultures were still viable when harvested after ten days, giving a 33% yield. Both MMP-9 and b-FGF caused an increased vascular invasion into the chondroepiphysis. New blood vessels derived from the chorioallantoic membrane within cartilage canals were more numerous in MMP-9 treated epiphyses, and larger canals were more commonly seen when compared with a control group. These findings confirmed that b-FGF is angiogenic at the chondroepiphysis. Matrix metalloproteinase-9 appears to be implicated in vascular invasion and induces the formation of new cartilage canals at the chondroepiphysis. The CAM culture model was a useful model for investigating angiogenesis in osteochondral tissue. This study adds to the understanding of the complex biochemical interaction that occurs in cartilage when the advancing vasculature begins growing into the chondroepiphysis. A better knowledge of this angiogenic process will enable a better understanding of the pathological failure or disturbance of vasculogenesis, which results in dysplastic growth disorders and osteonecrosis.

Stimulation of human bone marrow stromal cells using growth factor encapsulated calcium carbonate porous microspheres

The fabrication of self-assembling biomineral-related complexes for cell growth and gene/biomolecule delivery offers important opportunities for skeletal repair. In this work, 5–30 µm-sized calcium carbonate (vaterite) spheroids with elaborate sponge-like macroporous architectures were prepared by passive evaporation of water-in-oil microemulsions, and investigated as potential vehicles for the delivery of skeletal growth factors to human bone marrow stromal cells maintained in culture. The spheroids spontaneously form within dispersed droplets of supersaturated calcium bicarbonate, and become patterned in situ by entrapped micro-bubbles of carbon dioxide. Water-soluble biomolecules such as proteins (collagen type I, haemoglobin), cell growth factors (dexamethasone, pleiotrophin), bone morphogenetic proteins (rhBMP-2, SaOS ‘retentate’), and plasmid DNA were incorporated into the microspheres during synthesis or by soaking pre-washed spheroids. Primary human bone marrow stromal cells labelled with cell tracker green and ethidium homodimer-1 or transfected with an adenoviral vector expressing green fluorescent protein were co-cultured with the vaterite microspheres over three weeks without loss of function and viability. At three weeks, microspheres were encapsulated and integrated with the human bone marrow stromal cells. Histological analysis confirmed the expression of alkaline phosphatase, synthesis of extracellular matrix, and capacity for extensive mineralization. Use of vaterite microspheres to adsorb and deliver active growth factors such as pleiotrophin and an admixture of bone morphogenetic proteins derived from an osteosarcoma (SaOS ‘retentate’) was demonstrated using human bone marrow stromal cells. Ex vivo chorioallantoic membrane culture of pelleted vaterite spheres and human bone marrow cells placed into a chick femur defect resulted in the secretion of an organised collagen matrix. Vaterite spheres containing bioactive proteins induced osteogenesis in promyoblast C2C12 cells. These studies demonstrate the development of facile techniques for the generation of inorganic scaffolds constructed from porous microspheres that are biocompatible, aid mineralization, and offer potential for growth factor delivery through entrapment of functional biomolecules.

Inhibitory effect of no-spa on parainfluenza virus replication

No-Spa (20 μg/ml) effectively inhibits replication of parainfluenza viruses in cultured chick chorioallantoic membrane culture andMacaca rhesus kidney cells. Mechanism of the antiviral effect of vasodilator drugs is discussed.

Initiation of the bony epiphysis in long bones: chronology of interactions between the vascular system and the chondrocytes.

Many events occur concurrently during the initiation of the secondary ossification center in the cartilaginous epiphyses of long bones. We have investigated the chronology of interactions between the vascular system and epiphyseal chondrocytes by culturing explanted heads of femurs and humeri from pre- and neonatal rabbits on the chorioallantoic membrane (CAM) of growing chick embryos. We confirmed that, on the whole, the epiphyseal cartilage was resistant to vascular invasion, whereas the physeal growth plate was resorbed. However, new CAM-derived cartilage canals occasionally penetrated through the articular surface. This caused death of those chondrocytes in the immediate vicinity of the canal but no further reaction. If explants already contained a bony epiphysis and were halved prior to culture, CAM-derived vessels were attracted to the spongiosa. From there they pushed into the uncalcified cartilage, indicating that calcification was not a prerequisite for vascular invasion. Where at least two vessels were in apposition, a new pseudo-ossification center was initiated: chondrocytes became hypertrophic and the matrix calcified. This suggests that cumulative release of diffusible factors from more than one vessel was the trigger for chondrocyte hypertrophy, which, in turn, led to the initiation of the bony epiphysis. CAM cultures thus provide an experimental model for both the quiescent angiogenesis of cartilage canal formation and the reactionary angiogenesis associated with chondrocyte hypertrophy. By exploiting the different anatomy of CAM-derived vascularity, events that occur concurrently in vivo can be specially separated in CAM culture.

Antiviral and antibacterial activities of substituted benzalacetone amidinohydrazones

N-Methyl amidinohydrazones (AH) of substituted benzalacetones were synthesized, and examined in their activities against influenza virus A2/Adachi strain, bacteria and fungi. These AHs exhibited 50% inhibitory concentrations of 4.5-70.5μg/ml and 50% virucidal concentrations of 0.01-2.3μg/ml against the virus in the chorio-allantoic membrane culture. Satisfactory quantitative structure-activity relationships were obtained for inhibitory and virucidal activities against the virus and toxicity to chorio-allantoic membrane.Almost all AHs showed minimum inhibitory concentrations of 6.25-100μg/mlagainst Mycoplasma gallisepticum and 3.13-100μg/ml against S. aureus, B. subtilis and Pasteurella. Satisfactory quantitative structure-activity relationships were obtained for S. aureus and B. subtilis.

Antiviral compounds. XIV. Activities of alkoxy-acetophenones and -benzalacetones N-substituted amidinohydrazones

N-Substituted amidinohydrazone (AH) of alkoxyacetophenones and alkoxybenzalacetones (BZ) were synthesized and examined mainly for activities against influenza viruses. These AHs exhibited 50% inhibitory concentrations of 1.4-90 μg/ml (Index 10-159) and 50% virucidal concentrations of 0.001-0.7 μg/ml (Index 103-106) against influenza viruses such as A0/PR-8, A1/FM-1, A1/Fukuoka, A2/Adachi, A2/Aichi, B/Lee, B/Kagoshima and Horse/Miami in the chorio-allantoic membrane culture. N-Methyl AHs of p-decyloxy BZ and m-methoxy-p-decyloxy BZ showed excellent inhibitory activities, and N-methyl AHs of p-decyloxy BZ and m-methoxy-p-octyloxy BZ remarkable virucidal activities. It is inferred that introduction of methyl group on amidino moiety of AHs increases the activity and decreases the toxicity against host-cell. These AHs showed minimum inhibitory concentration of 0.78-100 μg/ml against mycoplasma gallisepticum, fungi and bacteria.

Abnormal morphogenesis of feather structures and pattern in the chick embryo integument. II. Histological description.

The development of skin and feathers in highly feathered scaleless mutants and normal Single Comb White Leghorn chick embryos was analyzed histologically. In addition, the growth of mutant feathers in chorioallantoic membrane culture is reported as a verification of several inferences made from observations of serially staged fixed specimens. The most striking feature of scaleless high line feather development is the widespread appearance of condensed or nearly condensed dermis. The discrete arrangement of normal placodes with underlying condensed dermis is replaced in the mutant by a heterogeneously shaped group of extremely large islands of columnar ("placodized") epithelium as long as 3,000 micron. The shape and extent of the condensed areas of dermis reflect the shape and extent of the overlying "placodized" epithelium. The polarity of the epidermis in normal feather germs, i.e., thicker epidermis on the posterior surface, is absent in mutant feather germs. This absence of epidermal polarity is reflected in the aberrant outgrowth of the mutant feather primordial. In the mutant, the basal cell layer of the epidermis invades the dermal core of the aberrant feather germs and may form barb vane ridges or feather sheaths. This process had no counterpart in the development of normal down feathers.