Browsing by Subject "Bioengineering"

Now showing 1 - 3 of 3
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    Open Access
    Development of decellularization protocols for female cat reproductive organs
    (Elsevier, 2024-06) Sanguansook, P; Martínez-López, Cristina; López-Orozco, Marina; Chatdarong, K; García-Vázquez, FA; Martínez Cáceres, Carlos Manuel; Izquierdo Rico, María José; Biología Celular e Histología
    Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2- cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns.
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    Open Access
    N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.
    (MDPI, 2021-06-06) Águila Martínez, Sonia; Noto, Rosina; Luengo-Gil, Ginés; Espín, Salvador; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Peñas, Julia; Rodenas, Maria Carmen; Vicente García, Vicente; Corral de la Calle, Javier; Manno, Mauro; Martínez-Martínez, Irene; Medicina Interna
    N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an Nglycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.
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    Open Access
    Salivary gland bioengineering - yesterday, today, tomorrow!
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2023) Iyer, Janaki; Pillai, Sangeeth; Munguia Lopez, Jose G.; Zhang, Yuli; Mielkozorova, Mariia; Tran, Simon D.
    Salivary glands are specialized structures developed as an extensively compact, arborized design through classical embryogenesis, accompanied by a cascade of events channelized by numerous growth factors and genetic regulatory pathways. Salivary secretions maintain oral homeostasis and, when diminished in certain conditions, present as xerostomia or salivary hypofunction, adversely impacting the patient’s quality of life. The current available treatments primarily aim at tackling the immediate symptoms providing temporary relief to the patient. Despite scientific efforts to develop permanent and effective solutions to restore salivation, a significant permanent treatment is yet to be established. Tissue engineering has proven as a promising remedial tool in several diseases, as well as in xerostomia, and aims to restore partial loss of organ function. Recapitulating the physiological cellular microenvironment to in vitro culture conditions is constantly evolving. Replicating the dynamic multicellular interactions, genetic pathways, and cytomorphogenic forces, as displayed during salivary gland development have experienced considerable barriers. Through this review, we endeavour to provide an outlook on the evolution of in vitro salivary gland research, highlighting the key bioengineering advances and the challenges faced with the current therapeutic strategies for salivary hypofunction, with an insight into our team’s scientific contributions.