ABSTRACT Acellular liver scaffolds (ALS) have arisen as potential candidates for transplantation. Until now, all reports involving ALS transplantation failed in surgical method descriptions and do not offer support to scientists to reproduce the procedures used in experimental microsurgery to make the results comparable to literature. To overcome the lack of detail information, we described surgical steps details to perform heterotopic and partial orthotopic surgical models to promote ALS transplantation. After preservation and vessel cannulation steps, the liver grafts were decellularized. In addition, ex vivo blood perfusion tests were performed to obtain a successful anticoagulation treatment prior in vivo transplantation. Then, methods of partial liver resection, combination of hand-suture and cuff techniques to complete end-to-end anastomosis between the scaffold and the recipient animal were performed. These procedures which take 30–60 min and were efficient to allow acellular liver scaffold viability and recellularization of different types of cell post-surgery. In conclusion, our methods are practical and simple promising approach that provides the opportunity to investigate ways to achieve sufficient liver function post-transplantation in vivo.

ABSTRACTPrevious studies indicated that the elevated mesenchymal Wnt/β-catenin signaling deprived dental mesenchyme of odontogenic fate. By utilizing ex vivo or pharmacological approaches, Wnt/β-catenin signaling in the developing dental mesenchyme was suggested to suppress the odontogenic fate by disrupting the balance between Axin2 and Runx2. In our study, the Osr2-creKI; Ctnnb1ex3f mouse was used to explore how mesenchymal Wnt/β-catenin signaling suppressed the odontogenic fate in vivo. We found that all of the incisor and half of the molar germs of Osr2-creKI; Ctnnb1ex3fmice started to regress at E14.5 and almost disappeared at birth. The expression of Fgf3 and Msx1 was dramatically down-regulated in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor and molar mesenchyme, while Runx2transcription was only diminished in incisor mesenchyme. Intriguingly, in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor epithelium, the expression of Noggin was activated, while Shh was abrogated. Similarly, the Wnt and BMP antagonists, Ectodin and Noggin were also ectopically activated in the E14.5 Osr2-creKI; Ctnnb1ex3f molar epithelium. Recombination of E13.5 Osr2-creKI; Ctnnb1ex3f molar mesenchyme with E10.5 and E13.5 WT dental epithelia failed to develop tooth. Taken together, the mesenchymal Wnt/β-catenin signaling resulted in the loss of odontogenic fate in vivo not only by directly suppressing odontogenic genes expression but also by inducing Wnt and BMP antagonists in dental epithelium.

ABSTRACTAutophagy has a critical role in liver regeneration. However, no studies have demonstrated autophagic flux in the regenerating fatty liver. The aim of this study was to clarify the dynamics of autophagy in the regeneration of the fatty liver. Following 70% partial hepatectomy (PH) in db/db fatty mice, which is a non-alcoholic fatty liver disease (NAFLD) model, we investigated the survival rate and recovery of liver volume. Histological examination of the regenerating liver was examined using electron microscopy. The 7-day survival rate after PH in db/db mice was 20%, which was significantly lower than that in control mice (P< .01). Liver regeneration within 48 h after PH was significantly impaired in db/db mice (P< .05). The number of proliferating cell nuclear antigen (PCNA) positive cells and the expression levels of cell-cycle markers cyclins D, E, and A were lower in db/db mice compared with controls. In the regenerating liver, LC3-II level was higher in db/db mice, but p62 expression was increased and cathepsin D expression, a marker of autophagolysosome proteolysis, was decreased compared with controls. Additionally, electronic microscopy revealed that autophagosomes during liver regeneration in db/db mice were mainly located in lipid droplets. Our findings indicate that the different localization of autophagosomes in db/db mice compared with controls led to impairment of liver regeneration in the fatty liver.

The transplantation of living cells, tissues or organs from one species to another is termed xenotransplantation. The history of xenotransplantation is as old as allogeneic transplantation itself. Early attempts were made at a time when the immunologic basis of organ rejection were poorly understood. The advent of potent immunosuppressive medications along with the parallel advances in the field of genetic engineering has provided a fresh perspective on the role of xenotransplantation as a means to alleviate the disparity between the number of candidates on the waitlist and the available organs. As the science behind xenotransplantation advances, the transplantation community must take it upon themselves to educate the community at large regarding both the benefits and potential risks of this promising field.

ABSTRACTExtracellular matrix (ECM) materials have had remarkable success as scaffolds in tissue engineering (TE) and as therapies for tissue injury whereby the ECM microenvironment promotes constructive remodeling and tissue regeneration. ECM powder and solubilized derivatives thereof have novel applications in TE and RM afforded by the capacity of these constructs to be dynamically modulated. The powder form allows for effective incorporation and penetration of reagents; hence, ECM powder is an efficacious platform for 3D cell culture and vehicle for small molecule delivery. ECM powder offers minimally invasive therapy for tissue injury and successfully treatment for wounds refractory to first-line therapies. Comminution of ECM and fabrication of powder-derived constructs, however, may compromise the biological integrity of the ECM. The current lack of optimized fabrication protocols prevents a more extensive and effective clinical application of ECM powders. Further study on methods of ECM powder fabrication and modification is needed.

ABSTRACTMaintaining hepatic functional characteristics in-vitro is considered one of the main challenges in engineering liver tissue. As hepatocytes cultured ex-vivo are deprived of their native extracellular matrix (ECM) milieu, developing scaffolds that mimic the biomechanical and physicochemical properties of the native ECM is thought to be a promising approach for successful tissue engineering and regenerative medicine applications. On the basis that the decellularized liver matrix represents the ideal design template for engineering bioinspired hepatic scaffolds, to derive quantitative descriptors of liver ECM architecture, we characterised decellularised liver matrices in terms of their biochemical, viscoelastic and structural features along with porosity, permeability and wettability. Together, these data provide a unique set of quantitative design criteria which can be used to generate guidelines for fabricating biomaterial scaffolds for liver tissue engineering. As proof-of-concept, we investigated hepatic cell response to substrate viscoelasticity. On collagen hydrogels mimicking decellularised liver mechanics, cells showed superior morphology, higher viability and albumin secretion than on stiffer and less viscous substrates. Although scaffold properties are generally inspired by those of native tissues, our results indicate significant differences between the mechano-structural characteristics of untreated and decellularised hepatic tissue. Therefore, we suggest that design rules - such as mechanical properties and swelling behaviour - for engineering biomimetic scaffolds be re-examined through further studies on substrates matching the features of decellularized liver matrices.

ABSTRACTIsolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

ABSTRACTPatch grafts are widely used in various kind of vascular surgeries such as detect repair or dilation of vascular stenosis. Expanded polytetrafluoroethylene (ePTFE) patches are flexible and handle well, but have shown problems with calcification as they are non-bioabsorbable and therefore permanently remain in the body. It is important to develop an alternative biocompatible patch. Silk fibroin (SF) was developed as a biocompatible material, but it lacks of the elasticity required for surgery as a patch. Polyurethane (PU) is also a well-known elastomer so this study focused on the SF and the PU blend materials with a weight ratio of 5:5 (SF/PU). To evaluate the SF/PU patch, the patches were implanted into the abdominal aortas of rats, using the ePTFE patch in the control group. Because it was more flexible the SF/PU patch was easier to implant than the ePTFE patch. At 1 week after implantation, the SF/PU patch had been infiltrated with cells and collagen fiber. The ePTFE control patch did not accumulate collagen fiber until 3 months and calcification occurred at 4 weeks. The SF/PU patch did not present any signs of calcification for 3 months. This study addressed the problems associated with using SF in isolation and showed that the SF/PU patch can be considered as a useful alternative to the ePTFE to overcome the problem of calcification.

ABSTRACTTurner's syndrome (TS) is depicted as a total or partial absence of X chromosome, and occurs in approximately 1/2200 of live born females. Generally, mosaic patients are diagnosed following karyotype analysis due to recurrent pregnancy loss, repeated in vitro fertilization (IVF) failure, and a history of malformed babies. The purpose of this case report is to show that even a selection of normal karyotype embryos can result in abnormalities for those with mosaic TS. A 32-year old patient who underwent IVF after ICSI-PGD, and was diagnosed with 45X/46XX karyotype. At the 12-week scan, one of the fetuses had an upper limb hemimelia in one arm, and feticide was applied to that fetus. The patient delivered a healthy, 2980 g female baby at the thirty-eighth week. In mosaic TS pregnancies (even those obtained by ICSI-PGD), fetal anomaly risk is high. Therefore, careful prenatal scanning is needed for these pregnancies.

ABSTRACTEndometriosis is defined by the presence of endometrial ectopia. Multiple hypotheses have been postulated to explain the etiology of endometriosis to understand various clinical evidences. The etiology of endometriosis is still unclear.The primary question to understanding the etiology of endometrial ectopia (endometriosis) is determining the origin of eutopic (normally cited) endometrium.According to the new theory, primordial germ cells migrate from hypoblast (yolk sac close to the allantois) to the gonadal ridges. The gonadal ridges which composed of primordial germ cells derive to the: eutopic endometrium, ovary, ovarian ligament and ligamentum teres uteri.There are 2 principal processes in uterine organogenesis: the intersection of gonadal ridges with mesonephral ducts to form the uterine folds with an endometrial cavity and the fusion of the both uterine folds together to form the unicavital (normal) uterus. In the uterine folds there are closer cell-to-cell communications, polypotential germ cells differentiate and grow into myometrium and endometrial layers.Some of the polypotential germ cells fail to reach the ridges and stay in the peritoneal cavity, where they may be transforming into external endometrial heterotopies.The main insight in the etiology of endometriosis is polypotential germ cells origin, which may explain its potency, pathogenesis and expansion.