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Leandro Teodoro,1,2 Ana Claudia O Carreira,1,3 Mari C Sogayar1,2 1Cell and Molecular Therapy NUCEL Group, School of Medicine, University of São Paulo, São Paulo, São Paulo, 01246-903, Brazil; 2Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, São Paulo, 05508-900, Brazil; 3Center of Human and Natural Sciences, Federal University of ABC, Santo André, São Paulo, 09280-560, BrazilCorrespondence: Mari C Sogayar, Institute of Chemistry – University of São Paulo, Prof. Lineu Prestes Avenue, 748 - Cidade Universitária, São Paulo, São Paulo, 05508-000, Brazil, Tel +5511992042844, Email mcsoga@iq.usp.brAbstract: Cancer is a complex and multifaceted group of diseases characterized by highly intricate mechanisms of tumorigenesis and tumor progression, which complicates diagnosis, prognosis, and treatment. In recent years, targeted therapies have gained prominence by focusing on specific mutations and molecular features unique to each tumor type, offering more effective and personalized treatment options. However, it is equally critical to explore the genetic commonalities across different types of cancer, which has led to the rise of pan-cancer studies. These approaches help identify shared therapeutic targets across various tumor types, enabling the development of broader and potentially more widely applicable treatment strategies. This review aims to provide a comprehensive overview of key concepts related to tumors, including tumorigenesis processes, the tumor microenvironment, and the role of extracellular vesicles in tumor biology. Additionally, we explore the molecular interactions and mechanisms driving tumor progression, with a particular focus on the pan-cancer perspective. To achieve this, we conducted an in silico analysis using publicly available datasets, which facilitated the identification of both common and divergent genetic and molecular patterns across different tumor types. By integrating these diverse areas, this review offers a clearer and deeper understanding of the factors influencing tumorigenesis and highlights potential therapeutic targets.Keywords: pan-cancer, tumor therapy, tumor biomarkers

Biomaterials derived from biological matrices have been widely investigated due to their great therapeutic potential in regenerative medicine, since they are able to induce cell proliferation, tissue remodeling, and angiogenesis in situ . In this context, highly vascularized and proliferative tissues, such as the uterine wall, present an interesting source to produce acellular matrices that can be used as bioactive materials to induce tissue regeneration. Therefore, this study aimed to establish an optimized protocol to generate decellularized uterine scaffolds (dUT), characterizing their structural, compositional, and biomechanical properties. In addition, in vitro performance and in vivo biocompatibility were also evaluated to verify their potential applications for tissue repair. Results showed that the protocol was efficient to promote cell removal, and dUT general structure and extracellular matrix composition remained preserved compared with native tissue. In addition, the scaffolds were cytocompatible, allowing cell growth and survival. In terms of biocompatibility, the matrices did not induce any signs of immune rejection in vivo in a model of subcutaneous implantation in immunocompetent rats, demonstrating an indication of tissue integration after 30 days of implantation. In summary, these findings suggest that dUT scaffolds could be explored as a biomaterial for regenerative purposes, which is beyond the studies in the reproductive field.

The uterine tube extracellular matrix is a key component that regulates tubal tissue physiology, and it has a region-specific structural distribution, which is directly associated to its functions. Considering this, the application of biological matrices in culture systems is an interesting strategy to develop biomimetic tubal microenvironments and enhance their complexity. However, there are no established protocols to produce tubal biological matrices that consider the organ morphophysiology for such applications. Therefore, this study aimed to establish region-specific protocols to obtain decellularized scaffolds derived from porcine infundibulum, ampulla, and isthmus to provide suitable sources of biomaterials for tissue-engineering approaches. Porcine uterine tubes were decellularized in solutions of 0.1% SDS and 0.5% Triton X-100. The decellularization efficiency was evaluated by DAPI staining and DNA quantification. We analyzed the ECM composition and structure by optical and scanning electronic microscopy, FTIR, and Raman spectroscopy. DNA and DAPI assays validated the decellularization, presenting a significative reduction in cellular content. Structural and spectroscopy analyses revealed that the produced scaffolds remained well structured and with the ECM composition preserved. YS and HEK293 cells were used to attest cytocompatibility, allowing high cell viability rates and successful interaction with the scaffolds. These results suggest that such matrices are applicable for future biotechnological approaches in the reproductive field.

Cleft palates are oral deformities that mostly affect puppies. They are frequently extensive and characterized by bone and palatal mucosa malformation. This deformity is a serious condition that may result in the death of the dog, therefore surgical treatment is recommended. Tissue bioengineering has emerged as a valuable option to treat cleft palates by applying acellular biological scaffolds as grafts. This case report proposed a new approach for surgical correction of canine cleft palate through a grafting technique using a decellularized scaffold. A decellularized portion of skin was implanted to correct a large cleft palate in a 3-month-old female Pug dog. The skin fragment was obtained from a dog cadaver and a decellularization protocol was performed. Under general anesthesia, a bilateral mucoperiosteal separation of the entire length of cleft margins was performed, and the scaffold was then positioned between the tissue and the bone palate. The interaction of the grafted scaffold with the oral mucosa and palatine layers resulted in total cleft closure, without postsurgical rejection or infection, indicating the applicability of this technique in dog's cleft palate correction. This is the first reported case demonstrating this new technique, which resulted in full cleft closure and healing., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Skeletal muscle degeneration is responsible for major mobility complications, and this muscle type has little regenerative capacity. Several biomaterials have been proposed to induce muscle regeneration and function restoration. Decellularized scaffolds present biological properties that allow efficient cell culture, providing a suitable microenvironment for artificial construct development and being an alternative for in vitro muscle culture. For translational purposes, biomaterials derived from large animals are an interesting and unexplored source for muscle scaffold production. Therefore, this study aimed to produce and characterize bovine muscle scaffolds to be applied to muscle cell 3D cultures. Bovine muscle fragments were immersed in decellularizing solutions for 7 days. Decellularization efficiency, structure, composition, and three-dimensionality were evaluated. Bovine fetal myoblasts were cultured on the scaffolds for 10 days to attest cytocompatibility. Decellularization was confirmed by DAPI staining and DNA quantification. Histological and immunohistochemical analysis attested to the preservation of main ECM components. SEM analysis demonstrated that the 3D structure was maintained. In addition, after 10 days, fetal myoblasts were able to adhere and proliferate on the scaffolds, attesting to their cytocompatibility. These data, even preliminary, infer that generated bovine muscular scaffolds were well structured, with preserved composition and allowed cell culture. This study demonstrated that biomaterials derived from bovine muscle could be used in tissue engineering.

Bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF) are known to regulate/stimulate osteogenesis, playing vital roles in bone homeostasis, rendering them strong candidates for osteoporosis treatment. We evaluated the effects of recombinant human BMP-7 (rhBMP7) and PDGF-BB (rhPDGF-BB) in an oophorectomy-induced osteoporosis rat model. Forty Sprague Dawley rats underwent oophorectomy surgery; treatments commenced on the 100th day post-surgery when all animals exhibited signs of osteoporosis. These peptide growth factors were administered intraocularly (iv) once or twice a week and the animals were monitored for a total of five weeks. Two weeks after the conclusion of the treatments, the animals were euthanized and tissues were collected for assessment of alkaline phosphatase, X-ray, micro-CT, and histology. The results indicate that the most promising treatments were 20 µg/kg rhPDGF-BB + 30 µg/kg rhBMP-7 twice a week and 30 µg/kg BMP-7 twice a week, showing significant increases of 15% ( p < 0.05) and 13% ( p < 0.05) in bone volume fraction and 21% ( p < 0.05) and 23% ( p < 0.05) in trabecular number, respectively. In conclusion, rhPDGF-BB and rhBMP-7 have demonstrated the ability to increase bone volume and density in this osteoporotic animal model, establishing them as potential candidates for osteoporosis treatment.

An essential step in the success of germ cell transplantation is the preparation of the recipient's testicular environment to increase the availability of stem cell niches. However, most methods for this purpose in birds face serious limitations such as partial germ cell depletion, high toxicity and mortality, or the need to use expensive technologies. Here, we validated a simple and practical technique of transferring quail testicular cells into chicken testes depleted of endogenous spermatozoa by fractioned chemotherapy (20 mg/kg/week busulfan for 5 weeks). This protocol resulted in a very low mortality of the treated day-old chicks and, despite maintenance of androgenic activity, sperm production was decreased by 84.3% at 25 weeks of age. NANOG immunostaining revealed that very few to no germ cells were present following treatment with 20 and 40 mg/kg, respectively. RT-qPCR data also showed that c-MYC and NANOG expression declined in these treatments, but GRFα1 and BID expressions remained unaltered among groups. After xenotransplantation, quail germ cells were immunodetected in chicken testes using a species-specific antibody (QCPN), and quail ovalbumin DNA was found in seminal samples collected from chicken recipients. Together, these data confirm that fractionated administration of busulfan in hatchlings is a practical, effective, and safe protocol to prepare recipient male birds capable of supporting xenogeneic spermatogenesis., (© 2024. The Author(s).)

Idiopathic Pulmonary Fibrosis (IPF) is a progressively debilitating lung condition characterized by oxidative stress, cell phenotype shifts, and excessive extracellular matrix (ECM) deposition. Recent studies have shown promising results using decellularized ECM-derived hydrogels produced through pepsin digestion in various lung injury models and even a human clinical trial for myocardial infarction. This study aimed to characterize the composition of ECM-derived hydrogels, assess their potential to prevent fibrosis in bleomycin-induced IPF models, and unravel their underlying molecular mechanisms of action. Porcine lungs were decellularized and pepsin-digested for 48 h. The hydrogel production process, including visualization of protein molecular weight distribution and hydrogel gelation, was characterized. Peptidomics analysis of ECM-derived hydrogel contained peptides from 224 proteins. Probable bioactive and cell-penetrating peptides, including collagen IV, laminin beta 2, and actin alpha 1, were identified. ECM-derived hydrogel treatment was administered as an early intervention to prevent fibrosis advancement in rat models of bleomycin-induced pulmonary fibrosis. ECM-derived hydrogel concentrations of 1 mg/mL and 2 mg/mL showed subtle but noticeable effects on reducing lung inflammation, oxidative damage, and protein markers related to fibrosis (e.g., alpha-smooth muscle actin, collagen I). Moreover, distinct changes were observed in macroscopic appearance, alveolar structure, collagen deposition, and protein expression between lungs that received ECM-derived hydrogel and control fibrotic lungs. Proteomic analyses revealed significant protein and gene expression changes related to cellular processes, pathways, and components involved in tissue remodeling, inflammation, and cytoskeleton regulation. RNA sequencing highlighted differentially expressed genes associated with various cellular processes, such as tissue remodeling, hormone secretion, cell chemotaxis, and cytoskeleton engagement. This study suggests that ECM-derived hydrogel treatment influence pathways associated with tissue repair, inflammation regulation, cytoskeleton reorganization, and cellular response to injury, potentially offering therapeutic benefits in preventing or mitigating lung fibrosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daniele Evangelista-Leite reports financial support provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001), Brazil. Maria Angelica Miglino reports financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 14/50844-3), Brazil. Daniele Evangelista-Leite reports financial support was provided by USP Vida Initiative in 2020 and 2021, University of São Paulo, Brazil., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Accurately printing customizable scaffolds is a challenging task because of the complexity of bone tissue composition, organization, and mechanical behavior. Graphene oxide (GO) and poly-L-lactic acid (PLLA) have drawn attention in the field of bone regeneration. However, as far as we know, the Fischer-Koch model of the GO/PLLA association for three-dimensional (3D) printing was not previously reported. This study characterizes the properties of GO/PLLA-printed scaffolds in order to achieve reproducibility of the trabecula, from virtual planning to the printed piece, as well as its response to a cell viability assay. Fourier-transform infrared and Raman spectroscopy were performed to evaluate the physicochemical properties of the nanocomposites. Cellular adhesion, proliferation, and growth on the nanocomposites were evaluated using scanning electron microscopy. Cell viability tests revealed no significant differences among different trabeculae and cell types, indicating that these nanocomposites were not cytotoxic. The Fischer Koch modeling yielded satisfactory results and can thus be used in studies directed at diverse medical applications, including bone tissue engineering and implants.

Hepatic microenvironment plays an essential role in liver regeneration, providing the necessary conditions for cell proliferation, differentiation and tissue rearrangement. One of the key factors for hepatic tissue reconstruction is the extracellular matrix (ECM), which through collagenous and non-collagenous proteins provide a three-dimensional structure that confers support for cell adhesion and assists on their survival and maintenance. In this scenario, placental ECM may be eligible for hepatic tissue reconstruction, once these scaffolds hold the major components required for cell support. Therefore, this preliminary study aimed to access the possibility of mouse embryonic stem cells differentiation into hepatocyte-like cells on placental scaffolds in a three-dimensional dynamic system using a Rotary Cell Culture System. Following a four-phase differentiation protocol that simulates liver embryonic development events, the preliminary results showed that a significant quantity of cells adhered and interacted with the scaffold through outer and inner surfaces. Positive immunolabelling for alpha fetus protein and CK7 suggest presence of hepatoblast phenotype cells, and CK18 and Albumin positive immunolabelling suggest the presence of hepatocyte-like phenotype cells, demonstrating the presence of a heterogeneous population into the recellularized scaffolds. Periodic Acid Schiff-Diastase staining confirmed the presence of glycogen storage, indicating that differentiate cells acquired a hepatic-like phenotype. In conclusion, these preliminary results suggested that mouse placental scaffolds might be used as a biological platform for stem cells differentiation into hepatic-like cells and their establishment, which may be a promissing biomaterial for hepatic tissue reconstruction., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest associated with this study., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries ( n = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.

Alpaca is a South American camelid, particularly present in Peruvian highlands, where oxygen concentration and atmospheric pressure are very low. Due to this fact, gestational physiology has adapted to preserve the conceptus' and mother's health. In this context, several cellular and molecular features play an essential role during and at the end of gestation. Structural carbohydrates act on maternal-fetal communication, recognize exogenous molecules, and contribute to placental barrier selectivity. Therefore, this study aimed to characterize the structural carbohydrate profiles that are present in the term alpaca placenta, kept in their natural habitat of around 4,000 m height. For this propose, 12 term alpaca placentas were collected, and the material was obtained at the time of birth from camelids raised naturally in the Peruvian highlands, in the Cusco region. All placenta samples were processed for histological analysis. A lectin histochemical investigation was performed using 13 biotinylated lectins, allowing us to determine the location of carbohydrates and their intensity on a semi-quantitative scale. Our results demonstrated that during term gestation, the epitheliochorial alpaca placenta shows a high presence of carbohydrates, particularly glucose, α-linked mannose, N-acetylglucosamine β (GlcNAc), galactose (αGal), and N-acetylgalactosamine α (GalNAc), present in the trophoblast, amnion epithelium, and mesenchyme, as well as the presence of sialic acid residues and low affinity for fucose. In fetal blood capillaries, the presence of bi- and tri-antennary complex structures and α-linked mannose was predominated. In conclusion, we characterized the glycosylation profile in the term alpaca placenta. Based on our data, compared to those reported in the bibliography, we suggest that these carbohydrates could participate in the labor of these animals that survive in Peruvian extreme environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Navarrete Zamora, Silva, Silva, Almeida, Silva-Júnior, Horvath-Pereira, Baracho Hill, Acuña, Carreira, Barreto, Sato and Miglino.)

Ovarian tissue has a unique microarchitecture and a complex cellular and molecular dynamics that are essential for follicular survival and development. Due to this great complexity, several factors may lead to ovarian insufficiency, and therefore to systemic metabolic disorders and female infertility. Techniques currently used in the reproductive clinic such as oocyte cryopreservation or even ovarian tissue transplant, although effective, have several limitations, which impair their wide application. In this scenario, mimetic ovarian tissue reconstruction comes as an innovative alternative to develop new methodologies for germ cells preservation and ovarian functions restoration. The ovarian extracellular matrix (ECM) is crucial for oocyte viability maintenance, once it acts actively in folliculogenesis. One of the key components of ovarian bioengineering is biomaterials application that mimics ECM and provides conditions for cell anchorage, proliferation, and differentiation. Therefore, this review aims at describing ovarian tissue engineering approaches and listing the main limitations of current methods for preservation and reestablishment of ovarian fertility. In addition, we describe the main elements that structure this study field, highlighting the main advances and the challenges to overcome to develop innovative methodologies to be applied in reproductive medicine. Impact Statement This review presents the main advances in the application of tissue bioengineering in the ovarian tissue reconstruction to develop innovative solutions for ovarian fertility reestablishment.

Extracellular matrix (ECM) proteins in the mammary gland provide structure and regulate its development and homeostasis. Alterations in its structure can regulate and support pathogenesis, like breast tumors. Aiming to identify the health and tumoral canine mammary ECM scaffold protein profile by immunohistochemistry, the decellularization process was carried out to remove the cellular content. Additionally, it was verified the influence of health and tumoral ECM on the attachment of health and tumoral cells. The types I, III, IV, and V structural collagens were scarce in the mammary tumor, and ECM fibers were disorganized. Vimentin and CD44 were more common in mammary tumor stroma, suggesting a role in cell migration that results in tumor progression. Elastin, fibronectin, laminin, vitronectin, and osteopontin were similarly detected under healthy and tumor conditions, providing the attachment of normal cells in healthy ECM, while tumoral cells were able to attach in tumoral ECM. The protein pattern demonstrates ECM alteration in canine mammary tumorigenesis, presenting new knowledge on mammary tumor ECM microenvironment., Competing Interests: Conflict of interest The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Traditional therapeutic interventions aim to restore male fertile potential or preserve sperm viability in severe cases, such as semen cryopreservation, testicular tissue, germ cell transplantation and testicular graft. However, these techniques demonstrate several methodological, clinical, and biological limitations, that impact in their results. In this scenario, reproductive medicine has sought biotechnological alternatives applied for infertility treatment, or to improve gamete preservation and thus increase reproductive rates in vitro and in vivo . One of the main approaches employed is the biomimetic testicular tissue reconstruction, which uses tissue-engineering principles and methodologies. This strategy pursues to mimic the testicular microenvironment, simulating physiological conditions. Such approach allows male gametes maintenance in culture or produce viable grafts that can be transplanted and restore reproductive functions. In this context, the application of several biomaterials have been proposed to be used in artificial biological systems. From synthetic polymers to decellularized matrixes, each biomaterial has advantages and disadvantages regarding its application in cell culture and tissue reconstruction. Therefore, the present review aims to list the progress that has been made and the continued challenges facing testicular regenerative medicine and the preservation of male reproductive capacity, based on the development of tissue bioengineering approaches for testicular tissue microenvironment reconstruction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Horvath-Pereira, Almeida, Silva Júnior, do Nascimento, Horvath Pereira, Fireman, Pereira, Carreira and Miglino.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aguiar Koga, Fernandes, Fratini, Sogayar and Carreira.)

Diabetes mellitus and pancreatitis are common pancreatic diseases in dogs, affecting the endocrine and exocrine portions of the organ. Dogs have a significant role in the history of research related to genetic diseases, being considered potential models for the study of human diseases. This review discusses the importance of using the extracellular matrix of the canine pancreas as a model for the study of diabetes mellitus and pancreatitis, in addition to focusing on the importance of using extracellular matrix in new regenerative techniques, such as decellularization and recellularization. Unlike humans, rabbits, mice, and pigs, there are no reports in the literature characterizing the healthy pancreatic extracellular matrix in dogs, in addition to the absence of studies related to matrix components that are involved in triggering diabetes melittus and pancreatitis. The extracellular matrix plays the role of physical support for the cells and allows the regulation of various cellular processes. In this context, it has already been demonstrated that physiologic and pathologic pancreatic changes lead to ECM remodeling, highlighting the importance of an in-depth study of the changes associated with pancreatic diseases.

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., (© 2022 S. Karger AG, Basel.)

Bioethical limitations impair deeper studies in human placental physiology, then most studies use human term placentas or murine models. To overcome these challenges, new models have been proposed to mimetize the placental three-dimensional microenvironment. The placental extracellular matrix plays an essential role in several processes, being a part of the establishment of materno-fetal interaction. Regarding these aspects, this study aimed to investigate term mice placental ECM components, highlighting its collagenous and non-collagenous content, and proposing a potential three-dimensional model to mimetize the placental microenvironment. For that, 18.5-day-old mice placenta, both control and decellularized ( n = 3 per group) were analyzed on Orbitrap Fusion Lumos spectrometer (ThermoScientific) and LFQ intensity generated on MaxQuant software. Proteomic analysis identified 2317 proteins. Using ECM and cell junction-related ontologies, 118 (5.1%) proteins were filtered. Control and decellularized conditions had no significant differential expression on 76 (64.4%) ECM and cell junction-related proteins. Enriched ontologies in the cellular component domain were related to cell junction, collagen and lipoprotein particles, biological process domain, cell adhesion, vasculature, proteolysis, ECM organization, and molecular function. Enriched pathways were clustered in cell adhesion and invasion, and labyrinthine vasculature regulation. These preserved ECM proteins are responsible for tissue stiffness and could support cell anchoring, modeling a three-dimensional structure that may allow placental microenvironment reconstruction.

Human adipose tissue-derived stem cells (hASC) secretome display various therapeutically relevant effects in regenerative medicine, such as induction of angiogenesis and tissue repair. The benefits of hASC secretome are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in host cells. However, the composition and the innate characteristics of hASC secretome can be highly variable depending on the culture conditions. Here, we evaluated the combined effect of serum-free media and hypoxia preconditioning on the hASCs secretome composition and biological effects on angiogenesis and wound healing. The hASCs were cultured in serum-free media under normoxic (NCM) or hypoxic (HCM) preconditioning. The proteomic profile showed that pro- and anti-antiangiogenic factors were detected in NCM and HCM secretomes. In vitro studies demonstrated that hASCs secretomes enhanced endothelial proliferation, survival, migration, in vitro tube formation, and in vivo Matrigel plug angiogenesis. In a full-thickness skin-wound mouse model, injection of either NCM or HCM significantly accelerated the wound healing. Finally, hASC secretomes were potent in increasing endothelial density and vascular coverage of resident pericytes expressing NG2 and nestin to the lesion site, potentially contributing to blood vessel maturation. Overall, our data suggest that serum-free media or hypoxic preconditioning enhances the vascular regenerative effects of hASC secretome in a preclinical wound healing model., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Silveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Bioceramic nanometer coatings have been regarded as potential substitutes for plasma-sprayed hydroxyapatite coatings, and the association with bone morphogenetic protein (BMP) is an attempt to achieve faster osseointegration to hasten oral rehabilitation. Objective: This study aimed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the osseointegration of titanium implants coated with a thin film surface of hydroxyapatite (HA). Methods: Two implants (n = 24) were placed in each white New Zealand rabbits’ femur (n = 6). Implants were placed in the right femur after standard instrumentation (A and B) and in the left femur after an over-instrumentation (C and D), preventing bone-implant contact. The distal implants were installed associated with rhBMP-7 (groups B [regular instrumentation] and D [over-instrumentation]) and, also, in the absence of without BMP (control groups A [regular instrumentation] and C [over-instrumentation]). After 4 weeks, the animals were euthanized. The bone blocks containing the implants were embedded in methyl methacrylate and sectioned parallel to the long axis of the implant, which were analyzed by image segmentation. The data were analyzed using a nonparametric statistical method. Results: We observed that Group A had a mean bone formation of 35.6% compared to Group B, which had 48.6% (p > 0.05). Moreover, this group showed 28.3% of connective tissue compared to Group A, with 39.3%. In the over-instrumented groups, rhBMP-7 (Group D) showed an enhanced and significant increase in bone formation when compared with the group without rhBMP-7 (Group C). Conclusion: We concluded that the association of rhBMP-7 to thin nanostructure HA-coated implants promoted greater new bone area than the same implants in the absence of rhBMP-7, mainly in cases of over-instrumented implant sites.

Complex wounds in dogs are a recurrent problem in veterinary clinical application and can compromise skin healing; in this sense, tissue bioengineering focused on regenerative medicine can be a great ally. Decellularized and recellularized skin scaffolds are produced to be applied in different and complex canine dermal wounds in the present investigation. Dog skin fragments are immersed in a 0.5% sodium dodecyl sulfate (SDS) solution at room temperature and overnight at 4 °C for 12 days. Decellularized samples are evaluated by histological analysis, scanning electron microscopy (SEM) and gDNA quantification. Some fragments are also recellularized using mesenchymal stem cells (MSCs). Eight adult dogs are divided into three groups for the application of the decellularized (Group I, n = 3) and recellularized scaffolds (Group II, n = 3) on injured areas, and a control group (Group III, n = 2). Wounds are evaluated and measured during healing, and comparisons among the three groups are described. In 30- and 60-day post-grafting, the histopathological analysis of patients from Groups I and II shows similar patterns, tissue architecture preservation, epithelial hyperplasia, hyperkeratosis, edema, and mononuclear inflammatory infiltrate. Perfect integration between scaffolds and wounds, without rejection or contamination, are observed in both treated groups. According to these results, decellularized skin grafts may constitute a potential innovative and functional tool to be adopted as a promising dog cutaneous wound treatment. This is the first study that applies decellularized and recellularized biological skin grafts to improve the healing process in several complex wounds in dogs, demonstrating great potential for regenerative veterinary medicine progress.

Tissue engineering is an innovative approach to develop allogeneic tissues and organs. The uterus is a very sensitive and complex organ, which requires refined techniques to properly regenerate and even, to rebuild itself. Many therapies were developed in 20th century to solve reproductive issues related to uterus failure and, more recently, tissue engineering techniques provided a significant evolution in this issue. Herein we aim to provide a broad overview and highlights of the general concepts involved in bioengineering to reconstruct the uterus and its tissues, focusing on strategies for tissue repair, production of uterine scaffolds, biomaterials and reproductive animal models, highlighting the most recent and effective tissue engineering protocols in literature and their application in regenerative medicine. In addition, we provide a discussion about what was achieved in uterine tissue engineering, the main limitations, the challenges to overcome, and future perspectives in this research field. Impact Statement This review presents the applications of tissue engineering in uterine reconstruction and in regenerative therapies of uterine wall injuries.

Advances in Artificial Reproductive Technologies (ARTs) in bovine embryos to produce cloned pregnancies have been developed in the last years, however high pregnancy losses rates still present. Those rates are associated to placental morphology alterations that are majorly focused on extracellular matrix (ECM) alterations and consequently placentome hyperplasia, increased trophoblast cell migration and vascular defects. Herein, we aimed to search, at protein level, pathways altered by ART that can modify the placental development harmony. For this, we used 4-month-old control (n = 3), SDS-decellularized (n = 3) and cloned (n = 3) cotyledons for proteomic analysis. Samples were grouped by condition and were washed, lysed, urea-reduced, acetone-precipitated, DTT-educed, iodoacetamide-alkylated, trypsin digested, and C-18 column purified. At the end, 3 μg protein were loaded in Orbitrap Fusion Lumos spectrometer (ThermoScientific). Generated spectra were exported to MaxQuant software (v1.6.10.43) to produce the protein list of each sample, and the LFQ intensity were statistically analyzed by Inferno software (v.1.1.6970). After this, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8. From 2577 identified protein sequences by MaxQuant software, 165 (7.1%) were filtered by selected ontologies. We found 10 proteins (B2M, COL6A6, FERMT3, LGALS3BP, NIBAN2, PDLIM5, PON1, PRP9, RASIP1 and SPARC) upregulated in clone, when compared to control condition. The ten pathways that enriched more proteins were: focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, protein digestion and absorption, amoebiasis, pathways in cancer, small cell lung cancer, platelet activation, regulation of actin cytoskeleton, and proteoglycans in cancer. Functionally, detected proteins, signaling pathways and ontologies are orchestrated to permit the binucleated trophoblastic cells migration and blood vessels modelling. In conclusion, the cloned condition presents the same mechanisms as control one, however overexpression of some specific ECM proteins could be responsible to exacerbate those mechanisms and can explain all morphophysiological alterations presented in cloned pregnancies associated to high pregnancies losses rates in this condition., (Copyright © 2022. Published by Elsevier Inc.)

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with significant gas exchange impairment owing to exaggerated extracellular matrix (ECM) deposition and myofibroblast activation. IPF has no cure, and although nintedanib and pirfenidone are two approved medications for symptom management, the total treatment cost is exuberant and prohibitive to a global uninsured patient population. New therapeutic alternatives with moderate costs are needed to treat IPF. ECM hydrogels derived from decellularized lungs are cost-effective therapeutic candidates to treat pulmonary fibrosis because of their reported antioxidant properties. Oxidative stress contributes to IPF pathophysiology by damaging macromolecules, interfering with tissue remodeling, and contributing to myofibroblast activation. Thus, preventing oxidative stress has beneficial outcomes in IPF. For this purpose, this review describes ECM hydrogel's properties to regulate oxidative stress and tissue remodeling in IPF. Impact statement Idiopathic pulmonary fibrosis (IPF) is a disease without a cure and with limited treatment options. At present, approved medications are expensive and pose a huge socioeconomic challenge to patients who depend on them. Affordability and effectiveness are desirable qualities for new therapeutic alternatives. Extracellular matrix hydrogels have properties that distinguish them other biomaterials, and it has been studied in the context of fibrosis-related molecular mechanisms. This review examines the biological processes involved in IPF and suggests developing a hydrogel-based treatment option for patients with IPF.

Cardiovascular diseases are considered the leading cause of death in the world, accounting for approximately 85% of sudden death cases. In dogs and cats, sudden cardiac death occurs commonly, despite the scarcity of available pathophysiological and prevalence data. Conventional treatments are not able to treat injured myocardium. Despite advances in cardiac therapy in recent decades, transplantation remains the gold standard treatment for most heart diseases in humans. In veterinary medicine, therapy seeks to control clinical signs, delay the evolution of the disease and provide a better quality of life, although transplantation is the ideal treatment. Both human and veterinary medicine face major challenges regarding the transplantation process, although each area presents different realities. In this context, it is necessary to search for alternative methods that overcome the recovery deficiency of injured myocardial tissue. Application of biomaterials is one of the most innovative treatments for heart regeneration, involving the use of hydrogels from decellularized extracellular matrix, and their association with nanomaterials, such as alginate, chitosan, hyaluronic acid and gelatin. A promising material is bacterial cellulose hydrogel, due to its nanostructure and morphology being similar to collagen. Cellulose provides support and immobilization of cells, which can result in better cell adhesion, growth and proliferation, making it a safe and innovative material for cardiovascular repair.

Mesenchymal stem cell (MSC) have immunomodulatory and anti-inflammatory effects, allowing its application in the therapy of different diseases, including articular cartilage injuries, which induce the establishment of a pro-regenerative microenvironment in the injured tissue. Therefore, our objective was to isolate, characterize and differentiate cartilage cells from different joints of New Zealand rabbit (Oryctolagus cuniculus), in order to verify their potential as MSC for future clinical use. For this, cartilage fragments were isolated from the humerus-radio-ulnar joints, humeral scapula, femoro-tibio-patellar, and lame femoris from rabbits. The results showed that the cells were rounded in the center of the plate and fibroblastoids in the periphery. After thawing, the cells did not change their growth time in culture, nor their morphology. The cells showed labeling for mesenchymal stem cell, cytoskeleton, pluripotency and cell proliferation, but not for hematopoiesis markers (CD105+ and CD34-). We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. After application of these cells in nude mice, no tumor growth was observed in spleen, kidney, liver, lung and heart. Therefore, we conclude that cells isolated from the articular cartilage of rabbits present characteristics of MSC with potential for future clinical applications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tissue engineering is gaining use to investigate the application of its techniques for infertility treatment. The use of pluripotent embryonic cells for in vitro production of viable spermatozoa in testicular scaffolds is a promising strategy that could solve male infertility. Due to cell-extracellular matrix (ECM) interactions, here we aim to investigate the differentiation of embryoid bodies (EBs) in cultured into decellularized rat testis scaffolds. Decellularized testis ( P = 0.019) with a low concentration of gDNA (30.58 mg/ng tissue) was obtained by sodium dodecyl sulfate perfusion. The structural proteins (collagens type I and III) and the adhesive glycoproteins of ECM (laminin and fibronectin) were preserved according to histological and scanning electron microscopy (SEM) analyses. Then, decellularized rat testis were cultured for 7 days with EB, and EB mixed with retinoic acid (RA) in non-adherent plates. By SEM, we observe that embryonic stem cells adhered in the decellularized testis ECM. By immunofluorescence, we verified the positive expression of HSD17B3, GDNF, ACRV-1, and TRIM-36, indicating their differentiation using RA in vitro , reinforcing the possibility of EB in male germ cell differentiation. Finally, recellularized testis ECM may be a promising tool for future new approaches for testicular cell differentiation applied to assisted reproduction techniques and infertility treatment. Abbreviations: ACRV-1: Acrosomal vesicle protein 1; ATB: Penicillin-streptomycin; DAPI: 4,6-Diamidino-2-phenylindole; EB: Embryoid bodies; ECM: Extracellular matrix; ESCs: Pluripotent embryonic stem cells; GAGs: Glycosaminoglycans; gDNA: Genomic DNA; GDNF: Glial cell line-derived neurotrophic factor; H&E: Hematoxylin and eosin; HSD17B3: 17-beta-Hydroxysteroid dehydrogenase type 3; PBS: Phosphate-buffered saline; PGCLCs: Primordial germ-cell-like cells; RA: Retinoic acid; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SSCs: Spermatogonial stem cells; TRIM-36: Tripartite Motif Containing 36.

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.

Introduction: Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years., Aims: Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice., Methods: 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC 50 ). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology., Results: In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE., Conclusion: CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The formation of hydrogels by photosensitized oxidation and crosslinking of histidine-derived polymers is demonstrated for the first time. The photooxidation of pendant His mediated by singlet oxygen was used to promote covalent coupling by its dimerization. As a proof-of-concept, two systems were studied: (i) chondroitin sulfate (CS) functionalized with His, and (ii) an elastin-like peptide (ELP) containing His produced by recombinant techniques. Both materials were crosslinked by irradiation at 425 nm in the presence of Zn-porphyrin derivatives yielding His-based hydrogels. The molecular structure and physicochemical properties of ELP-His and other 5 ELPs with photooxidizable amino acids were studied in silica by computer simulation. A correlation between the protein conformation and its elastic properties is discussed. CS-His hydrogels demonstrate larger storage moduli than ELPs with other amino acids. The obtained results show the potential use of photooxidation to create a new type of His-based hydrogels.

Our goal was to evaluate phytochemical characterization and the antitumor potential of Calotropis procera. The phytochemical constitution of the crude extract (CE) revealed the presence of flavonoids, glycosides and cardenolide. The MTT assay was used to evaluate the cytotoxicity of CE, methanolic (MF) and ethyl acetate fractions (EAF) of C. procera in canine osteosarcoma cells (OST), canine mammary tumor (CMT), and canine skin fibroblasts (non-tumor cell). Doxorubicin was also used as a positive control. Results showed that CE, MF and EAF promoted a decrease in the viability of OST and CMT cells and did not alter the fibroblasts viability. C. procera also decreased the number of cells, corroborating to the decrease in proliferation and the cell cycle arrest in the G0/G1 phase. It was also evaluated the cell morphology by light and fluorescence microscopy, being demonstrated a reduction in cytoplasmic and cell rounding characteristic of programmed cell death. Moreover, flow cytometry data demonstrated that CE treatment promoted increase of caspase-3 and p53, showing that the cell death was activated in OST cells. In addition, there was a decrease in CD31, VEGF, osteopontin and TGF-β after CE treatment, suggesting that CE exerts its antitumor effect by reducing angiogenesis and tumor progression in OST cells. Moreover, CMT cells showed a reduction in PCNA after treatment with MF and CE. Analyzing the data together, C. procera, especially CE, showed an antitumor potential in both OST and CMT cells, encouraging us to continue investigating its use in cancer therapy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Cardiovascular diseases are the leading cause of death in many countries. Advances in technology have been promoted in this regard, especially in tissue engineering, to meet the need for tissue or organ grafts. In this way, the porcine model has been used due to its morphophysiological similarity between the human species, mainly regarding the cardiovascular system. Tissue engineering is employed using biological scaffolds that are currently derived from porcine. These scaffolds are produced by decellularization, a process to remove cells aiming to maintain only its three-dimensional structure, formed by extracellular matrix (ECM). Its main objective is to produce organs through recellularized scaffolds that could eventually substitute the ones with impaired functions., Aim: In this way, the present study aimed to establish a new protocol for porcine heart decellularization with potential application on tissue engineering., Methods: A porcine heart aorta was cannulated with a silicon tube, and the organ was washed in 0.1% phosphate-buffered saline through a peristaltic pump (Harvard Peristaltic Pump - Harvard Apparatus). After that, deionized water was introduced in the same system. The decellularization procedure was carried out using ionic and non-ionic detergents, namely 4% sodium dodecyl sulfate (SDS) and 1% Triton X-100, respectively. SDS was perfused through myocardial circulation at 400 mL/min for 24 h for 6 days. Subsequently, the heart was infused with Triton X-100 and washed by PBS and water for 24 h. The heart volume was measured before and after the recellularization. After macroscopic evaluation, the heart samples were processed and stained by Hematoxylin and Eosin, Masson's Trichrome, Weigert-Van Gieson, Alcian Blue, and Pricrosirius Red techniques for microscopic analysis. To observe the cell adhesion, the recellularization was provided in this scaffold, which was analyzed under immunofluorescence and scanning electronic microscopy., Results: The protocol provided cells remotion, with adequate concentration of remaining DNA. ECM components as collagen type I, elastin, and glycosaminoglycans were successfully maintained. The scaffold showed a high cells adherence and proliferation in the recellularization process., Conclusion: According to results, the protocol described in this work preserved the ECM components and the organ architecture, minimizing ECM loss and being possible to state that it is a promising approach to tissue bioengineering., Relevance for Patients: This study provides a protocol for whole porcine heart decellularization, which will ultimately contribute to heart bioengineering and may support further studies on biocompatibility relationship of new cells with recellularized scaffolds., Competing Interests: The authors state no competing interest., (Copyright: © Whioce Publishing Pte. Ltd.)

Cells derived from the fetal liver have been shown to be a rich source of progenitor stem cells, constituting a promising source for Tissue Engineering and Regenerative Medicine. In this study, embryo and fetal liver-bud derived cells from Fischer 344 rats were obtained at E12.5, E14.5 and E16.5 gestational days and evaluated for cell phenotype, survival and proliferation. Liver transaminase (AST and ALT) and AFP levels were lower in embryo liver-bud-derived cells on day 12.5. Markers for stem cells, cell cycle progression and cell death were differentially expressed in E12.5 cell cultures. Analysis of mitochondrial electric potential on 14.5 and 16.5 days showed a tendency for cells with lower functional or metabolic ability, in comparison to cultures derived from day 12.5. The results demonstrated that the majority of the E16.5 cells were in the G0 / G1 phase. The capacity of synthesis (S) and cellular division (G2 / M) of embryo and fetal liver bud-derived cells was constant over all gestational periods. In conclusion, embryo and fetal liver-bud-derived cells during the periods of 12.5 and 14.5 days, showed expression profile of progenitor cells, cell activity and hematopoietic function in culture., (Copyright © 2021. Published by Elsevier Ltd.)

Background: R-spondins, including R-spondin 1 (RSPO1), are a family of Wnt ligands that help to activate the canonical Wnt/β-catenin pathway, which is critical for intestinal epithelial cell proliferation and maintenance of intestinal stem cells. This proliferation underpins the epithelial expansion, or intestinal adaptation (IA), that occurs following massive bowel resection and short bowel syndrome (SBS). The purpose of this study was to identify if recombinant human RSPO1 (rhRSPO1) could be serially administered to SBS zebrafish to enhance cellular proliferation and IA., Methods: Adult male zebrafish were assigned to four groups: sham + PBS, SBS + PBS, sham + rhRSPO1, and SBS + rhRSPO1. Sham fish had a laparotomy alone. SBS fish had a laparotomy with distal intestinal ligation and creation of a proximal stoma. Fish were weighed at initial surgery and then weekly. rhRSPO1 was administered post-operatively following either a one- or two-week dosing schedule with either 3 or 5 intraperitoneal injections, respectively. Fish were harvested at 7 or 14 days with intestinal segments collected for analysis., Results: Repeated intraperitoneal injection of rhRSPO1 was feasible and well tolerated. At 7 days, intestinal epithelial proliferation was increased by rhRSPO1. At 14 days, SBS + rhRSPO1 fish lost significantly less weight than SBS + PBS fish. Measurements of intestinal surface area were not increased by rhRSPO1 administration but immunofluorescent staining for β-catenin and gene expression for cyclin D1 was increased., Conclusions: Intraperitoneal injection of rhRSPO1 decreased weight loss in SBS zebrafish with increased β-catenin + cells and cyclin D1 expression at 14 days, indicating improved weight maintenance might result from increased activation of the canonical Wnt pathway., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Background: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366., Methods: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-β1 were used. For steroidogenic pathway hormones, an ELISA assay was performed., Results: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells., Conclusions: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Introduction: Cell therapy and tissue engineering has recently emerged as a new option for short bowel syndrome (SBS) treatment, generating tissue engineered small intestine (TESI) from organoid units (OU) and biodegradable scaffolds. The recombinant human R-Spondin 1 (rhRSPO1) protein may be a key player in this process due to its mitogenic activity in intestinal stem cells. Objective: Aiming at optimizing the TESI formation process and advancing this technology closer to the clinic, we evaluated the effects of rhRSPO1 protein on OU culture and TESI formation. Methods: Intestinal OU were isolated from C57BL/6 mice and cultured in Matrigel in the presence or absence of recombinant human rhRSPO1. Throughout the culture, OU growth and survival rates were evaluated, and cells were harvested on day 3. OU were seeded onto biodegradable scaffolds, in the presence or absence of 5 μg of rhRSPO1 and implanted into the omentum of NOD/SCID mice in order to generate TESI. The explants were harvested after 30 days, weighed, fixed in formalin and embedded in paraffin for histological analysis and immunofluorescence for different cell markers. Results: After 3 days, rhRSPO1-treated OU attained a larger size, when compared to the control group, becoming 5.7 times larger on day 6. Increased survival was observed from the second day in culture, with a 2-fold increase in OU survival between days 3 and 6. A 4.8-fold increase of non-phosphorylated β-catenin and increased relative expression of Lgr5 mRNA in the rhRSPO1-treated group confirms activation of the canonical Wnt pathway and suggests maintenance of the OU stem cell niche and associated stemness. After 30 days of in vivo maturation, rhRSPO1-treated TESI presented a larger mass than constructs treated with saline, developing a more mature intestinal epithelium with well-formed villi and crypts. In addition, the efficiency of OU-loaded rhRSPO1-treated scaffolds significantly increased, forming TESI in 100% of the samples ( N = 8), of which 40% presented maximum degree of development, as compared to 66.6% in the control group ( N = 9). Conclusion: rhRSPO1 treatment improves the culture of mouse intestinal OU, increasing its size and survival in vitro , and TESI formation in vivo , increasing its mass, degree of development and engraftment., (Copyright © 2020 Levin, Zuber, Squillaro, Sogayar, Grikscheit and Carreira.)

Background: The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygro r and subjected to cell clones isolation., Results: rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose., Conclusion: Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

Long non-coding RNAs (lncRNAs) serve critical roles in regulating cellular homeostasis, and their deregulated expression/activity is associated with neoplastic transformation. The maternally expressed gene 3 ( MEG3 ) has been extensively described as a tumor suppressor gene in different types of cancer, including breast cancer. Interestingly, using a panel of seven different breast cancer cell lines, the present study revealed that MEG3 is highly expressed in the triple negative metastatic human Hs578T breast cancer cell line, which is refractory to different therapeutic approaches. Therefore, the present study aimed to investigate the phenotypic impact of MEG3 deletion in this cell line. Using the CRISPR/Cas9 system, complete knockout (KO) of MEG3 was achieved. Deletion was confirmed by genomic PCR and reverse transcription-quantitative PCR. The MEG3&#95;KO cell population displaying the highest efficiency of genomic editing was selected for phenotypic in vitro assays, including wound scratch and Transwell assays, flow cytometry and immunofluorescence. The results demonstrated that MEG3 deletion increased cell proliferation, anchorage-independent cell growth and cell motility, which was consistent with its well-known tumor suppressor function. However, the present study revealed that MEG3&#95;KO also lead to decreased cell invasiveness ability, supporting previous evidence that MEG3 modulates epithelial-to-mesenchymal inducing factors. The present study demonstrated that deletion of MEG3 promoted an increase in transforming growth factor β and N-cadherin protein levels and significant reduction in matrix metallopeptidase 2, zinc-finger E-box binding homeobox 1 and collagen type III α1 chain gene expression levels. Additionally, MEG3&#95;KO cells displayed significant resistance to doxorubicin treatment, demonstrating the role of this lncRNA in cancer cell survival by regulating apoptosis. The present study highlighted the utility of CRISPR/Cas9 for anticancer studies of intergenic lncRNAs and demonstrated that, although Hs578T cells express MEG3 at high levels, these cells display mechanisms to escape the growth suppression effects of this lncRNA. Notably, the detailed pathological mechanisms of MEG3 concerning tumor metastasis remain to be elucidated prior to applying MEG3 expression/activation in future therapeutic approaches for breast cancer treatment., (Copyright: © Deocesano-Pereira et al.)

Sickle cell disease (SCD) is a monogenic red cell disorder associated with multiple vascular complications, microvessel injury and wound-healing deficiency. Although stem cell transplantation with bone marrow-derived mesenchymal stem cells (BMSC) can promote wound healing and tissue repair in SCD patients, therapeutic efficacy is largely dependent on the paracrine activity of the implanted BM stromal cells. Since in vitro expansion and culture conditions are known to modulate the innate characteristics of BMSCs, the present study investigated the effects of normoxic and hypoxic cell-culture preconditioning on the BMSC secretome, in addition to the expression of paracrine molecules that induce angiogenesis and skin regeneration. BMSCs derived from SCD patients were submitted to culturing under normoxic (norCM) and hypoxic (hypoCM) conditions. We found that hypoxically conditioned cells presented increased expression and secretion of several well-characterized trophic growth factors (VEGF, IL8, MCP-1, ANG) directly linked to angiogenesis and tissue repair. The hypoCM secretome presented stronger angiogenic potential than norCM, both in vitro and in vivo, as evidenced by HUVEC proliferation, survival, migration, sprouting formation and in vivo angiogenesis. After local application in a murine wound-healing model, HypoCM showed significantly improved wound closure, as well as enhanced neovascularization in comparison to untreated controls. In sum, the secretome of hypoxia-preconditioned BMSC has increased expression of trophic factors involved in angiogenesis and skin regeneration. Considering that these preconditioned media are easily obtainable, this strategy represents an alternative to stem cell transplantation and could form the basis of novel therapies for vascular regeneration and wound healing in individuals with sickle cell disease., Competing Interests: The authors have declared that no competing interests exist.

Background: Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting peritoneal membrane (PM) integrity and function. Understanding the mechanisms underlying PF development in an uremic environment aiming alternative therapeutic strategies for treating this process is of great interest. The aim of this study was to analyze the effects of tamoxifen (TAM) and recombinant BMP7 (rBMP7) in an experimental model of PF developed in uremic rats., Methods: To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and CKD with severe uremia, was developed in Wistar rats. PF was induced by intraperitoneal (IP) injections of chlorhexidine gluconate (CG), and CKD was induced by an adenine-rich diet. Uremia was confirmed by severe hypertension, increased blood urea nitrogen (BUN> 120 mg/dL) and serum creatinine levels (> 2 mg/dL). Uremic rats with PF were treated with TAM (10 mg/Kg by gavage) or BMP7 (30 μg/Kg, IP). Animals were followed up for 30 days., Results: CG administration in uremic rats induced a striking increase in PM thickness, neoangiogenesis, demonstrated by increased capillary density, and failure of ultrafiltration capacity. These morphological and functional changes were blocked by TAM or rBMP7 treatment. In parallel, TAM and rBMP7 significantly ameliorated the PM fibrotic response by reducing α-SMA, extracellular matrix proteins and TGF-ß expression. TAM or rBMP7 administration significantly inhibited peritoneal Smad3 expression in uremic rats with PF, prevented Smad3 phosphorylation, and induced a remarkable up-regulation of Smad7, an intracellular inhibitor of TGFβ/Smad signaling, contributing to a negative modulation of profibrotic genes. Both treatments were also effective in reducing local inflammation, possibly by upregulating IκB-α expression in the PM of uremic rats with PF. In vitro experiments using primary peritoneal fibroblasts activated by TGF-ß confirmed the capacity of TAM or rBMP7 in blocking inflammatory mediators, such as IL-1ß expression., Conclusions: In conclusion, these findings indicate important roles of TGF-ß/Smad signaling in PF aggravated by uremia, providing data regarding potential therapeutic approaches with TAM or rBMP7 to block this process.

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., (© 2018 Wiley Periodicals, Inc.)

Background: Matrix metalloproteinases (Mmps) and their tissue inhibitors (Timps) are widely recognized as crucial factors for extracellular matrix remodeling in the ovary and are involved in follicular growth, ovulation, luteinization, and luteolysis during the estrous cycle. Recently, several genes have been associated to the modulation of Mmps activity, including Basigin (Bsg), which induces the expression of Mmps in rat ovaries; Sparc, a TGF-β modulator that is related to increased expression of Mmps in cancer; and Reck, which is associated with Mmps inhibition. However, the expression pattern of Mmp modulators in ovary dynamics is still largely uncharacterized., Methods: To characterize the expression pattern of Mmps network members in ovary dynamics, we analyzed the spatio-temporal expression pattern of Reck and Sparc, as well as of Mmp2, Mmp9 and Mmp14 proteins, by immunohistochemistry (IHC), in pre-pubertal rat ovaries obtained from an artificial cycle induced by eCG/hCG, in the different phases of the hormone-induced estrous cycle. We also determined the gene expression profiles of Mmps (2, 9, 13 14), Timps (1, 2, 3), Sparc, Bsg, and Reck to complement this panel., Results: IHC analysis revealed that Mmp protein expression peaks at the early stages of folliculogenesis and ovulation, decreases during ovulation-luteogenesis transition and luteogenesis, increasing again during corpus luteum maintenance and luteolysis. The protein expression patterns of these metalloproteinases and Sparc were inverse relative to the pattern displayed by Reck. We observed that the gene expression peaks of Mmps inhibitors Reck and Timp2 were closely paraleled by Mmp2 and Mmp9 suppression. The opposite was also true: increased Mmp2 and Mmp9 expression was concomitant to reduced Reck and Timp2 levels., Conclusion: Therefore, our results generate a spatio-temporal expression profile panel of Mmps and their regulators, suggesting that Reck and Sparc seem to play a role during ovarian dynamics: Reck as a possible inhibitor and Sparc as an inducer of Mmps.

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., Competing Interests: No competing financial interests exist.

Breast cancer is the most prevalent cancer among women, with the basal-like triple negative (TNBC) being the most agressive one, displaying the poorest prognosis within the ductal carcinoma subtype. Due to the lack of adequate molecular targets, the diagnosis and treatment of patients with the TNBC phenotype has been a great challenge. In a previous work, we identified CD90/Thy-1 as being highly expressed in the aggressive high malignancy grade Hs578T basal-like breast tumor cell line, pointing to this molecule as a promising breast tumor marker, which should be further investigated. Here, CD90 expression was analyzed in human breast cancer samples and its functional role was investigated to better assess the oncogenic nature of CD90 in mammary cells. Quantification of CD90 expression in human breast cancer samples, by tissue microarray, showed that high CD90 positivity correlates with metastasis and poor patient survival in the basal-like subtype. The functional genetic approach, by overexpression in the CD90 cDNA in a basal-like normal mammary cell line (MCF10A) and knockdown in a highly malignant cell line (Hs578T), allowed us to demonstrate that CD90 is involved with several cellular processes that lead to malignant transformation, such as: morphological change, increased cell proliferation, invasiveness, metastasis and activation of the EGFR pathway. Therefore, our results reveal that CD90 is involved with malignant transformation in breast cancer cell lines and is correlated with metastasis and poor patient survival in the basal-like subtype, being considered as a promising new breast cancer target., Competing Interests: The authors have declared that no competing interests exist.

Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic ® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic ® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering., (© 2017 Wiley Periodicals, Inc.)

Background: FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in insulin-secreting beta-cells. Since in silico data revealed that FAM3B can be expressed in prostate tumors, we evaluated the putative role of this cytokine in prostate tumor progression., Methods: FAM3B expression was analyzed by quantitative PCR in tumor tissue clinical samples and prostate tumor cell lines. Culture growth and viability of DU145 cell line were evaluated after treatment with either exogenous FAM3B protein obtained from conditioned media (CM) of 293 T cells overexpressing FAM3B or a recombinant FAM3B protein produced in a bacterial host. DU145 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145/FAM3B cells after treatment with several cell death inducers, such as TNF-alpha, staurosporine, etoposide, camptothecin, and serum starvation conditions. Anchorage-independent growth in soft agarose assay was used to evaluate in vitro tumorigenicity. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice., Results: We observed an increase in FAM3B expression in prostate tumor samples when compared to normal tissues. DU145 cell viability and survival increased after exogenous treatment with recombinant FAM3B protein or FAM3B-secreted protein. Overexpression of FAM3B in DU145 cells promoted inhibition of DNA fragmentation and phosphatidylserine externalization in a time and dose-dependent fashion, upon apoptosis triggered by TNF-alpha. These events were accompanied by increased gene expression of anti-apoptotic Bcl-2 and Bcl-XL, decreased expression of pro-apoptotic Bax and diminished caspase-3, -8 and -9 proteolytic activities. Furthermore, inhibition of Bcl-2 anti-apoptotic family proteins with small molecules antagonists decreases protective effects of FAM3B in DU145 cells. When compared to the respective controls, cells overexpressing FAM3B displayed a decreased anchorage- independent growth in vitro and increased tumor growth in xenografted nude mice. The immunohistochemistry analysis of tumor xenografts revealed a similar anti-apoptotic phenotype displayed by FAM3B-overexpressing tumor cells., Conclusions: Taken together, by activating pro-survival mechanisms FAM3B overexpression contributes to increased resistance to cell death and tumor growth in nude mice, highlighting a putative role for this cytokine in prostate cancer progression.