Percorrer por autor "Pazmino, Carlos"
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- Bio-based hydrogel for beta cell islet transplantation in type 1 diabetes therapyPublication . Oliveira, Ana L.; Pazmino, Carlos; Sá, Simone; Amorim, SaraObjectives: Type 1 Diabetes Mellitus (T1DM) is a chronic autoimmune disease that destroys insulin-producing pancreatic cells, affecting over 9 million people globally. The primary treatment involves insulin therapy, but improper delivery can lead to poor glucose control or hypoglycemia. Pancreatic islet transplantation offers a potential alternative, but long-term graft survival remains a challenge. This study aims to enhance cell-based therapies by developing a permeable, pro-angiogenic immune-isolation core- shell hydrogel using decellularized pancreatic extracellular matrix (dECM) for the encapsulation of beta cells. The hydrogel will be formed using Riboflavin (RB) a bio crosslinker also known as Vitamin B2, which is reported to ameliorate oxidative stress and inflammation, key factors in the pathophysiology of insulin resistance. Methods: Porcine pancreas was decellularized using a supercritical carbon dioxide (scCO2) protocol developed by our group, to effectively preserve the biochemical structure of the ECM, while removing the cellular components. After five cycles of decellularization (110-120 bar, 36-38ºC for 1h cycle), the tissue was thoroughly analyzed. The residual DNA content, a key indicator of the efficiency of decellularization, and the ECM components (glycosaminoglycans, collagen, and elastin) were quantified using Blyscan, Sircol and Fasin assay kits. The hydrogel was developed by digesting the dECM using pepsin/HCl and crosslinked with Riboflavin (RB) in combination with Horseradish peroxidase (HRP). Pancreatic islets from a pancreatic cell line were seeded into the hydrogel, by placing a beta cell line spheroid into the core of the dECM RB system. Also, cell viability will be assessed by live/dead assay, and insulin and PDX 1 expression will be characterized by Elisa and RT PCR. Results: The DNA quantification revealed a significant reduction in DNA content (?12.7 ng/mg) after five cycles, indicating effective removal of cellular material. Moreover, the results from the quantification of ECM components (GAGs, elastin and collagen) demonstrated that the decellularization process effectively preserved its ECM components. The crosslinked dECM-RB hydrogel exhibited mechanical stability, making it ideal for cell culture. The crosslinking process also helped to preserve the native structure of the ECM, which is crucial for maintaining the biological activity of the seeded cells. Ongoing studies are focused on regulating insulin secretion by stimulating the cells with glucose. The preliminary results revealed that beta cells within the hydrogel could produce insulin in response to glucose, showcasing its functional capability. Conclusion: The findings highlight the potential of the use of riboflavin as a bio crosslinker with an added activity to improve the stability and performance of pancreatic decellularized ECM. The dECM-RB hydrogel provided a favorable microenvironment for beta cell activity. Overall, a robust and functional cell delivery platform was created, and studies are presently ongoing to monitor cell longevity within the hydrogel and insulin production upon glucose stimulation.
- Decellularized cardiac tissue for the development of non-immunogenic EPSs-rich biofabricated patches for cardiac regenerationPublication . Pazmino, Carlos; Duarte, Marta M.; Oliveira, Ana L.; Amorim, SaraIntroduction: Cardiovascular diseases account for 32% of the global deaths, comprising different pathologies related to heart failure. The heart self-regeneration is limited, which is associated to the highest mortality and morbidity rates of cardiovascular diseases. To bridge this gap, we developed a decellularized extracellular matrix (dECM)-based cardiac patch functionalized with sulfated exopolysaccharides (EPSs) to synergistically mimic native myocardial architecture and improve cardiomyocyte electrophysiological function. In fact, the sulfated groups in EPSs have the potential to influence the conductive properties of cardiomyocytes, enhancing ion exchange and electrical coupling between cells, thus improving the propagation of electrical signals. Conclusions: The decellularization protocol using solvents and supercritical CO? effectively removed cellular components but requires optimization to meet the recommended DNA threshold (?50 ?g/mg tissue). Quantitative analysis confirmed the retention of key ECM components, particularly sulfated glycosaminoglycans (sGAGs), essential for preserving myocardial structural and biochemical properties. Exopolysaccharides (EPS) from Porphyridium cruentum were successfully extracted and purified, showing a sulfate content of 6%. Future work will focus on integrating EPS with hydrogels to develop bioengineered cardiac patches supporting cardiomyocyte culture and myocardial repair.
- Decellularized matrix-based hydrogel for targeted beta cell islet delivery in type I diabetes treatmentPublication . Pazmino, Carlos; Sá, Simone; Amorim, Sara; Oliveira, Ana L.Introduction: Type 1 Diabetes Mellitus (T1DM) affects over 9 million people worldwide and is marked by autoimmune destruction of pancreatic ?-cells, which produce insulin. Current treatment relies on exogenous insulin, but inaccurate delivery often causes poor glycemic control or hypoglycemia. Islet transplantation offers a promising alternative, though long-term graft survival remains a challenge. This work proposes a permeable, pro-angiogenic immune-isolation hydrogel to improve cell- based therapies. The design uses pancreatic decellularized extracellular matrix (dECM) to mimic native tissue, supporting cell growth and insulin release. Riboflavin (Vitamin B2), with antioxidant and anti-inflammatory properties, and Ruthenium, providing photophysical crosslinking, are employed as photocrosslinkers. Objectives: Development of a: permeable, pro-angiogenic, immune-isolation hydrogel using decellularized pancreatic extracellular matrix (dECM), crosslinked with Riboflavin or Ruthenium for beta cells encapsulation. Conclusions: This study established a supercritical CO?–based decellularization protocol as a robust and reproducible method for generating pancreatic dECM, achieving consistent DNA removal despite inter-organ variability. The process led to a reduction in glycosaminoglycans (GAGs) and soluble collagen while enriching insoluble collagen, and also revealed regional compositional differences across pancreatic zones. Two hydrogel formulations were investigated: riboflavin-mediated hydrogels exhibited limited stiffness, whereas ruthenium-mediated hydrogels provided enhanced crosslinking and mechanical stability. Preliminary cell studies demonstrated distinct metabolic responses, with ruthenium hydrogels derived from the pancreatic body supporting the highest activity. Future efforts will aim to optimize mechanical properties, minimize residual crosslinkers, and broaden cellular evaluation. Collectively, these results highlight pancreatic dECM hydrogels as a promising platform for ?-cell replacement strategies in diabetes therapy.
- Development of a decellularized extracelular matrix from porcine aorta for heart valve applications in the Ross procedurePublication . Reis, Mariana S.; Rosadas, Marta; Ho, Chou I.; Sousa, Teresa; Pazmino, Carlos; Costa, João; Vervenne, Thibault; Oliveira, Ana L.; Ribeiro, Viviana P.; Mignon, ArnCardiovascular diseases are the leading cause of adult mortality worldwide, according to the World Health Organization [1]. An important surgical approach for treating diseased aortic valves is the Ross procedure, in whic the affected aortic valve is replaced with an autograft from the patient’s own pulmonary valve.
- Supercritical CO decellularized-ECM derived coreshell hydrogel for functional β-cell encapsulation and immune protectionPublication . Pazmino, Carlos; Sá, Simone; Azevedo, Ana; Oliveira, Ana L.; Amorim, SaraIntroduction: Type 1 Diabetes Mellitus (T1DM) is an autoimmune disease that destroys insulinproducing pancreatic cells, affecting over 9 million people worldwide. While insulin therapy is standard, it often leads to poor glucose control or hypoglycemia. Pancreatic islet transplantation shows promise but faces graft survival challenges. This study develops a permeable, pro-angiogenic immune-isolation hydrogel using pancreatic decellularized extracellular matrix (dECM) to mimic native tissue and support insulin production, with ruthenium as crosslinker surrounded by an Alginate shell aiming to reduce oxidative stress and inflammation. Conclusions: This study validated a supercritical CO? decellularization protocol as a robust and effective method for pancreatic dECM preparation, achieving reliable DNA removal across organ variability. Decellularization reduced GAGs and soluble collagen while increasing insoluble collagen, results also highlighted regional differences within the pancreas. A core-shell hydrogel for B cell spheroids seeding was developed. The core, ruthenium-based hydrogels showed desired crosslinking but low stability after 7 days which will need to be improved. The core-shell construct showed no signs of cytotoxicity and allowed cell seeding before alginate crosslinking. Preliminary cell studies showed similar metabolic activity along the timepoints tested. Future work will focus on optimizing stiffness, reducing residual of crosslinkers in hydrogels, and expanding cellular assays. Overall, these findings establish pancreatic dECM hydrogels as promising scaffolds for ?-cell replacement therapies in diabetes.
- Supercritical CO2-assisted decellularization: advanced pancreatic tissue platform for diabetes treatmentPublication . Sá, Simone C.; Pazmino, Carlos; Sá, Joana; Amorim, Sara; Ribeiro, Viviana P.; Costa, Raquel; Oliveira, Ana L.Introduction: Decellularization is a procedure that aims to remove the cellular and antigenic material of a tissue while preserving its extracellular matrix (ECM) and biological properties. Decellularized ECM offers a natural microenvironment with significant potential for use in cell-based therapies and tissue regeneration [1]. Different protocols have been employed to decellularize tissues and organs. However, many rely on solvents and detergents that compromise the bioactive signals inherent to the native tissue [2]. Thus, it is crucial to establish a method that achieves effective decellularization while maintaining the ECM biochemical integrity. Objectives: In this study, we focus on the pancreatic tissue decellularization to create a native-like matrix suitable for the delivery of functional, insulin-producing β-cells as a therapeutic approach for diabetes treatment [3]. We propose a decellularization protocol based on Supercritical CO₂ (scCO₂) technology as an innovative alternative to solvent-based methods, offering high transfer rates, diffusivity, chemical inertness, and non-toxicity [4]. Conclusions: Both decellularization protocols efficiently removed DNA and SEM confirmed ECM ultrastructure maintenance in both groups. Biochemical analysis revealed that scCO₂ decellularization slightly improves GAG’s preservation, while insoluble collagen is protocol-independent and soluble collagen remains similar in both methods. Proteomic analysis showed that traditional treatment retained more proteoglycans and SLRPs, whereas scCO₂ better preserved some basal membrane and especially elastic fiber proteins. Biocompatibility assays demonstrated that both scaffolds supported cell adhesion, viability, and proliferation. In conclusion, both protocols generate biocompatible scaffolds, with distinct ECM preservation profiles relevant for recellularization, but scCO₂ stands out for being a green technology, with a shorter effective decellularization time and reduced waste.
- Unlocking the potential of supercritical CO₂ for an efficient pancreatic tissue decellularization: a step towards an advanced cell therapyPublication . Sá, Simone C.; Pazmino, Carlos; Sá, Joana; Amorim, Sara; Ribeiro, Viviana P.; Costa, Raquel; Oliveira, Ana L.Introduction: Tissue decellularization is a procedure that removes cellular and antigenic material from specific tissues, while preserving the extracellular matrix (ECM), with conditions tailored to the tissue's properties. The decellularized ECM is a promising material for cell-based therapies or for tissue defect repair through the development of scaffolds and membranes, among others. Pancreas is an organ that regulates the glucose levels in the bloodstream, primarily through insulin secretion by endocrine pancreatic β-cells. Diabetes type 2 settles on the insufficient secretion of insulin, and the consequent increase of glucose in the body. Extensive research is being conducted to find alternatives to overcome this condition, particularly on cell-based therapies, that deliver viable and functional β-cells and restore insulin production to regulate blood glucose levels effectively. Objectives: This study aims to develop and validate a decellularization protocol for porcine pancreatic tissue using Supercritical CO₂ (scCO2) technology as an innovative alternative to solvent-based processes, offering high transfer rates, diffusivity, chemical inertness, and non-toxicity [3]. Three protocols were tested: Traditional decellularization method using detergents; scCO₂-based decellularization protocol; Hybrid protocol combining both technologies. Conclusions: Results suggest that scCO₂ is the most efficient method for porcine pancreatic tissue decellularization, reducing DNA below 50 ng/mL in four cycles and achieving near-complete removal by the fifth. The traditional + scCO₂ approach also meets the threshold in four cycles but retains more residual DNA than scCO₂. This highlights scCO₂ as a faster and more effective method, fully decellularizing tissue in 20 hours, while the traditional method takes 27 hours 30 minutes and remains incomplete.