Trilobatin contributes to the improvement of myopathy in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to a deficiency in dystrophin production and consequent progressive degeneration of skeletal muscle fibres, through oxidative stress and an exacerbated inflammatory process. The flavonoid trilobatin (TLB) demonstrates antioxidant and anti-inflammatory potential. Its high safety profile and effective action make it a potent therapy for the process of dystrophic muscle myonecrosis. Thus, we sought to investigate the action of TLB on damage in a DMD model, the mdx mouse. Eight-week-old male animals were treated with 160 mg/kg/day of trilobatin for 8 weeks. Control animals were treated with saline. Following treatment, muscle strength, serum creatine kinase (CK) levels, histopathology (necrotic myofibres, regenerated fibres/central nuclei, Feret's diameter and inflammatory area) and the levels of catalase and NF-κB (western blotting) of the quadriceps (QUA), diaphragm (DIA) and tibialis anterior (TA) muscles were measured. TLB was able to significantly increase muscle strength and reduce serum CK levels in dystrophic animals. The QUA of mdx mice showed a reduction in catalase and the number of fibres with a centralized nucleus after treatment with TLB. In the DIA of dystrophic animals, TLB reduced the necrotic myofibres, inflammatory area and NF-κB and increased the number of regenerated fibres and the total fibre diameter. In TA, TLB increased the number of regenerated fibres and reduced catalase levels in these animals. It is concluded that in the mdx experimental model, treatment with TLB was beneficial in the treatment of DMD.

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.

The Tumor Microenvironment: Focus on Extracellular Matrix.

The extracellular matrix (ECM) regulates the development and maintains tissue homeostasis. The ECM is composed of a complex network of molecules presenting distinct biochemical properties to regulate cell growth, survival, motility, and differentiation. Among their components, proteoglycans (PGs) are considered one of the main components of ECM. Its composition, biomechanics, and anisotropy are exquisitely tuned to reflect the physiological state of the tissue. The loss of ECM's homeostasis is seen as one of the hallmarks of cancer and, typically, defines transitional events in tumor progression and metastasis. In this chapter, we discuss the types of proteoglycans and their roles in cancer. It has been observed that the amount of some ECM components is increased, while others are decreased, depending on the type of tumor. However, both conditions corroborate with tumor progression and malignancy. Therefore, ECM components have an increasingly important role in carcinogenesis and this leads us to believe that their understanding may be a key in the discovery of new anti-tumor therapies. In this book, the main ECM components will be discussed in more detail in each chapter.

Optimization of Canine Placenta Decellularization: An Alternative Source of Biological Scaffolds for Regenerative Medicine.

Due to the scarcity of tissues and organs for transplantation, the demand for bioengineered tissues is increasing with the advancement of technologies and new treatments in human and animal regenerative medicine. Thus, decellularized placental extracellular matrix (ECM) has emerged as a new tool for the production of biological scaffolds for subsequent recellularization and implantation for recovery of injured areas or even for replacement of organ and tissue fractions. To be classified as an ideal biological scaffold, the ECM must be acellular and preserve its proteins and physical features to be useful for cellular adhesion. In this context, we developed a process of decellularization of canine placentas with 35 and 40 days of gestation using dodecyl sulfate sodium under immersion and agitation in sterile conditions. Before use of this scaffold in recellularization processes, the decellularization efficiency needs to be confirmed by the absence of cellular content and an irrelevant amount of reminiscent DNA. Both vasculature architecture and ECM proteins, such as collagen types I, III, and IV, laminin, and fibronectin, were preserved with our method. In this way, we established a new biological scaffold model that could be used for recellularization in regenerative medicine of tissues.

Periurethral muscle-derived mononuclear cell injection improves urethral sphincter restoration in rats.

AIMS: Investigate the effect of a novel cell-based therapy with skeletal muscle-derived mononuclear cells (SMDMCs) in a rat model of stress urinary incontinence. METHODS: Male Wistar-Kyoto rats' hind limb muscles were enzymatically dissociated, and SMDMCs were isolated without needing expansion. The cell population was characterized. Twenty female rats underwent urethrolysis. One week later, 10 rats received periurethral injection of 106 cells (SMDMC group), and 10 rats received saline injections (Saline group). Ten rats underwent sham surgery (Sham group). Four weeks after injection, animals were euthanized and the urethra was removed. The incorporation of SMDMCs in the female urethra was evaluated with fluorescence in situ hybridization for the detection of Y-chromosomes. Hematoxylin and eosin, Masson's trichrome staining, and immunohistochemistry for actin and myosin were performed. The muscle/connective tissue, actin and myosin ratios were calculated. Morphological evaluation of the urethral diameters and fractional areas of the lumen, mucosa, and muscular layer was performed. RESULTS: SMDMCs population was consistent with the presence of muscle cells, muscle satellite cells, perivascular cells, muscle progenitor cells, and endothelial cells. SMDMCs were incorporated into the urethra. A significant decrease in the muscle/connective tissue ratio was observed in the Saline group compared with the SMDMC and Sham groups. The proportions of actin and myosin were significantly decreased in the Saline group. No differences were observed in the morphometric parameters. CONCLUSIONS: SDMSC were incorporated into the rat urethra and promoted histological recovery of the damaged urethral sphincter, resulting in decreased connective tissue deposition and increased muscle content.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells.

BACKGROUND: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-ß. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. RESULTS: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. CONCLUSION: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. STUDY DESIGN: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study.

Role of MSC-derived small extracellular vesicles in tissue repair and regeneration.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and repair, secreting vesicles to the extracellular environment. Isolated exosomes were shown to affect angiogenesis, immunomodulation and tissue regeneration. Numerous efforts have been dedicated to describe the mechanism of action of these extracellular vesicles (EVs) and guarantee their safety, since the final aim is their therapeutic application in the clinic. The major advantage of applying MSC-derived EVs is their low or inexistent immunogenicity, prompting their use as drug delivery or therapeutic agents, as well as wound healing, different cancer types, and inflammatory processes in the neurological and cardiovascular systems. MSC-derived EVs display no vascular obstruction effects or apparent adverse effects. Their nano-size ensures their passage through the blood-brain barrier, demonstrating no cytotoxic or immunogenic effects. Several in vitro tests have been conducted with EVs obtained from different sources to understand their biology, molecular content, signaling pathways, and mechanisms of action. Application of EVs to human therapies has recently become a reality, with clinical trials being conducted to treat Alzheimer's disease, retina degeneration, and COVID-19 patients. Herein, we describe and compare the different extracellular vesicles isolation methods and therapeutic applications regarding the tissue repair and regeneration process, presenting the latest clinical trial reports.

In vivo biocompatibility analysis of the recellularized canine tracheal scaffolds with canine epithelial and endothelial progenitor cells.

Decellularized extracellular matrix (ECM) has frequently been applied as a biomaterial for tissue engineering purposes. When implanted, their role can be essential for partial trachea replacement in patients that require a viable transplant solution. Acellular canine tracheal scaffolds with preserved ECM structure, flexibility, and proteins were obtained by high pressure vacuum decellularization. Here, we aimed to evaluate the cell adhesion and proliferation of canine tracheal epithelial cells (EpC) and canine yolk sac endothelial progenitor cells (YS) cultivated on canine decellularized tracheal scaffolds and test the in vivo biocompatibility of these recellularized scaffolds implanted in BALB-c nude mice. In order to evaluate the recellularization efficiency, scaffolds were evaluated by scanning electron microscopy (SEM), immunofluorescence, DNA quantification, mycoplasma test, and in vivo biocompatibility. The scaffolds sterility was confirmed, and EpC and YS cells were cultured by 7 and 14 days. We demonstrated by SEM, immunofluorescence, and genomic DNA analyzes cell adhesion to tracheal ECM. Then, recellularized scaffolds were in vivo subcutaneously implanted in mice and after 45 days, the fragments were collected and analyzed by Hematoxylin-Eosin and Gömori Trichrome staining and PCNA, CD4, CD8, and CD68 immunohistochemistry. In vivo results confirmed that the implanted tissue remains preserved and proliferative, and no fibrotic tissue process was observed in animals. Finally, our results showed the recellularization success due the preserved ECM proteins, and that these may be suitable to future preclinical studies applications for partial trachea replacement in tissue engineering.

Proteomic profile of extracellular matrix from native and decellularized chorionic canine placenta.

Placental plasticity, employing rapid growth and remodeling to supply the growing fetus, is majorly related to its extracellular matrix (ECM) components. Thus, we studied the proteome profiled of canine native and decellularized placenta to characterize the proteome related to maintenance of a microenvironment and structure suitable for tissue engineering applications. Protein was profiled from native (n=3) and decellularized (n=3) 35-days old canine placenta using the mass spectrometer Orbitrap Fusion Lumos. A total of 52 proteins were filtered and revealed ontologies connected to skeleton structuration, collagen processing, germ layers formation, cell adhesion, response to amino acids, and others. Also, the major enriched pathways were ECM-receptor interaction, focal adhesion, PI3K-Akt signaling, protein digestion and absorption. Aside, proteins related to structure (collagens), cell adhesion (laminin and fibronectin), ECM remodeling (MMP2 and TIMP3) and vascularization (VEGF and RLN) were present in decellularized condition. Our findings support the requirement of a proteomic profile to visualize the maintenance of essential protein groups for ECM structuring and physiology, that should support functions related to cell adhesion, vasculogenesis and as a reservoir of soluble molecules. Altogether, the 35-days old decellularized canine placenta can provide an adequate microenvironment for cell anchoring for further regenerative medicine application.

Biological Characterization of Polymeric Matrix and Graphene Oxide Biocomposites Filaments for Biomedical Implant Applications: A Preliminary Report.

Carbon nanostructures application, such as graphene (Gr) and graphene oxide (GO), provides suitable efforts for new material acquirement in biomedical areas. By aiming to combine the unique physicochemical properties of GO to Poly L-lactic acid (PLLA), PLLA-GO filaments were produced and characterized by X-ray diffraction (XRD). The in vivo biocompatibility of these nanocomposites was performed by subcutaneous and intramuscular implantation in adult Wistar rats. Evaluation of the implantation inflammatory response (21 days) and mesenchymal stem cells (MSCs) with PLLA-GO took place in culture for 7 days. Through XRD, new crystallographic planes were formed by mixing GO with PLLA (PLLA-GO). Using macroscopic analysis, GO implanted in the subcutaneous region showed particles' organization, forming a structure similar to a ribbon, without tissue invasion. Histologically, no tissue architecture changes were observed, and PLLA-GO cell adhesion was demonstrated by scanning electron microscopy (SEM). Finally, PLLA-GO nanocomposites showed promising results due to the in vivo biocompatibility test, which demonstrated effective integration and absence of inflammation after 21 days of implantation. These results indicate the future use of PLLA-GO nanocomposites as a new effort for tissue engineering (TE) application, although further analysis is required to evaluate their proliferative capacity and viability.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6.

BACKGROUND: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. METHODS: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. RESULTS: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. CONCLUSIONS: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector.

BACKGROUND: Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. RESULTS: We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. CONCLUSIONS: These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

Mouse placental scaffolds: a three-dimensional environment model for recellularization.

The rich extracellular matrix (ECM) and availability make placenta eligible as alternative biomaterial source. Herein we produced placental mouse scaffolds by decellularization, and structure, composition, and cytocompatibility were evaluated to be considered as a biomaterial. We obtained a cell-free scaffold containing 9.42 ± 5.2 ng dsDNA per mg of ECM, presenting well-preserved structure and composition. Proteoglycans were widespread throughout ECM without cell nuclei and cell remnants. Collagen I, weak in native placenta, clearly appears in the scaffold after recellularization, opposite distribution was observed for collagen III. Fibronectin was well-observed in placental scaffolds whereas laminin and collagen IV were strong expressed. Placental scaffolds recellularization potential was confirmed after mouse embryonic fibroblasts 3D dynamic culture, resulting in massive scaffold repopulation with cell-cell interactions, cell-matrix adhesion, and maintenance of natural morphology. Our small size scaffolds provide a useful tool for tissue engineering to produce grafts and organ fragments, as well as for cellular biology purposes for tridimensional culture substrate.

Isolation and Characterization of Pancreatic Canine Fetal Cells at the Final Stage of Gestation.

The incidence of diabetes mellitus in dogs is increasing in recent years, mainly because of genetic and/or environmental factors, including endocrine disorders (like in humans); failure of suitable control of blood sugar levels, which triggers hyperglycemia; glycosuria and weight loss, which demands the development of innovative treatments to cure or treat this complex disease in dogs. The present study established for the first time a protocol to obtain and characterize cells derived from pancreas of canine fetuses. Those fetuses do not have a defined breed and were at the final stage of gestation. The protocol aims to provide morphological data to enable future applications of these cells for therapeutic approaches. In cell culture, pancreatic cells showed a fibroblast-like appearance with a mono-layered growth pattern and were not tumorigenic. They exhibited a positive expression for the pluripotent proliferation markers NANOG and PCNA and expressed PDX1, a transcription factor that is important for activation of the insulin gene promoter. In addition, Tyrosine Hydroxylase-positive (TH+) sympathetic nerve fibers were identified. Histologically, the pancreatic epithelium was developed, pancreatic glands in the fetuses were like those in the parenchyma of postconception dogs and pancreatic islets were unevenly distributed and organized in small clusters along the glands close to the vasculature. Staining with dithizone indicated the presence of insulin in the cells. A large number of beta cells were confirmed by immunofluorescence. In conclusion, the canine fetal pancreas cells could be an alternative and adequate source of cell lineages for stem cell therapies for diabetes treatment. Anat Rec, 302:1409-1418, 2019. © 2018 Wiley Periodicals, Inc.

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.

Canine Placenta Recellularized Using Yolk Sac Cells with Vascular Endothelial Growth Factor.

Regenerative medicine has been growing because of the emergent need for tissues/organs for transplants and restorative surgeries. Biological scaffolds are important tools to try to solve this problem. The one used in this reserach was developed by an acellular biological scaffold from canine placenta with a rich source of cellular matrix. After decellularization, the cellular matrix demonstrated structural preservation with the presence of important functional proteins such as collagen, fibronectin, and laminin. We used cells transduced with vascular endothelial growth factor (VEGF) to recellularize this scaffold. It was succeeded by seeding the cells in nonadherent plaques in the presence of the sterelized placenta scaffold. Cells were adhered to the scaffold when analyzed by immunocytochemistry and scanning electron microscopy, both showing sprouting of yolk sac VEGF (YSVEGF) cells. This recellularized scaffold is a promissory biomaterial for repairing injured areas where neovascularization is required.

Equine lung decellularization: a potential approach for in vitro modeling the role of the extracellular matrix in asthma.

Contrary to conventional research animals, horses naturally develop asthma, a disease in which the extracellular matrix of the lung plays a significant role. Hence, the horse lung extracellular matrix appears to be an ideal candidate model for in vitro studying the mechanisms and potential treatments for asthma. However, so far, such model to study cell-extracellular matrix interactions in asthma has not been developed. The aim of this study was to establish a protocol for equine lung decellularization that maintains the architecture of the extracellular matrix and could be used in the future as an in vitro model for therapeutic treatment in asthma. For this the equine lungs were decellularized by sodium dodecyl sulfate detergent perfusion at constant gravitational pressure of 30 cmH2O. Lung scaffolds were assessed by immunohistochemistry (collagen I, III, IV, laminin, and fibronectin), scanning electron microscopy, and DNA quantification. Their mechanical property was assessed by measuring lung compliance using the super-syringe technique. The optimized protocol of lung equine decellularization was effective to remove cells (19.8 ng/mg) and to preserve collagen I, III, IV, laminin, and fibronectin. Moreover, scanning electron microscopy analysis demonstrated maintained microscopic lung structures. The decellularized lungs presented lower compliance compared to native lung. In conclusion we described a reproducible decellularization protocol that can produce an acellular equine lung feasible for the future development of novel treatment strategies in asthma.

Vascularization and VEGF expression altered in bovine yolk sacs from IVF and NT technologies.

Reproductive technologies are widely used in cattle, although many are associated with high-embryonic mortality, especially during early gestation, when the yolk sac undergoes macroscopic changes in structure. We hypothesized that vasculogenesis and angiogenesis are affected, thereby affecting embryonic and placental differentiation. To test this, we studied yolk sac development and gene expression of the vascular endothelial growth factor system (VEGF-A, VEGFR-1/Flt-1, VEGFR-2/KDR). Samples from Days 25 to 40/41 of pregnancy from control cattle (n = 8) and from pregnancies established with IVF, (n = 7) or somatic cell nuclear transfer/clones (n = 5) were examined by histology, immunohistochemistry, and quantitative reverse transcriptase PCR. Yolk sacs in IVF- and nuclear transfer-derived pregnancies were immature. Development of villi was sparse in IVF yolk sacs, whereas vascularization was barely formed in clones and was associated, in part, with thin or interrupted endothelium. Transcript levels of the genes characterized exceed minimum detection limits for all groups, except in the mentioned clone with interrupted endothelium. Levels of mRNA for VEGF-A and VEGFR-2 were significantly higher in IVF yolk sacs. Clones had substantial individual variation in gene expression (both upregulation and downregulation). Our data confirmed the broad range in expression of VEGF genes. Furthermore, overexpression in IVF yolk sacs may compensate for an immature yolk sac structure, whereas in clones, patchy expression may cause structural alterations of blood vessels. In conclusion, we inferred that disturbances of yolk sac vasculature contributed to increased early embryonic mortality of bovine pregnancies established with reproductive technologies.

Effects of two different decellularization routes on the mechanical properties of decellularized lungs.

Considering the limited number of available lung donors, lung bioengineering using whole lung scaffolds has been proposed as an alternative approach to obtain lungs suitable for transplantation. However, some decellularization protocols can cause alterations on the structure, composition, or mechanical properties of the lung extracellular matrix. Therefore, the aim of this study was to compare the acellular lung mechanical properties when using two different routes through the trachea and pulmonary artery for the decellularization process. This study was performed by using the lungs excised from 30 healthy male C57BL/6 mice, which were divided into 3 groups: tracheal decellularization (TDG), perfusion decellularization (PDG), and control groups (CG). Both decellularized groups were subjected to decellularization protocol with a solution of 1% sodium dodecyl sulfate. The behaviour of mechanical properties of the acellular lungs was measured after decellularization process. Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. TDG and PDG showed reduced Est and Edyn elastances after lung decellularization. Scanning electron microscopy showed no structural changes after lung decellularization of the TDG and PDG. In conclusion, was demonstrated that there is no significant difference in the behaviour of mechanical properties and extracellular matrix of the decellularized lungs by using two different routes through the trachea and pulmonary artery.

Endothelial differentiation of canine yolk sac cells transduced with VEGF.

Yolk sac (YS) is the site of blood-cell production where primitive erythroid cells originate and complete their maturation. YS is a source of precursor cells, however its differentiation potential and suitability for cell therapies are not well described. YS can be a cell source when neovascularization is required. This study characterized YS canine cells, transduced with VEGF, to analyze then using Immunocytochemistry, flow cytometry and real time PCR. Immunocytochemistry: positive expression for CD105, PCNA, VEGF and vWF, flow cytometry for CD105, VEGF, PCNA, OCT-4 and RT-qPCR for VEGF, CD31, CD105, PCNA and FLT - 1, indicating that these cells have characteristics of endothelial progenitor and pluripotency. After transduction, the YS cells changed their morphology and showed endothelial-like cells. We suggest, because of their cell surface phenotype as well as their capacity to differentiate into endothelial-like cells, that canine YS represents a source of cells for neovascularization therapies.