Browsing by Author "Ribeiro, Viviana P."
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- 3D printed bioactive interference screw and PCL bio-filler for ligament fixationPublication . Rodrigues, Mafalda; Moreira, Rui; Silva, Inês V.; Duarte, Marta M.; Ribeiro, Viviana P.; Oliveira, Ana L.; Costa, João B.Musculoskeletal injuries, widespread across all ages, genders and sociodemographic groups, are prevalent in the knee joint and require a range of treatments - from conservative methods to surgical interventions, such as meniscal resection, repair, reconstruction or tissue engineering (TE) approaches. To address one of the most significant challenges in orthopedic procedures – long-term implant fixation – an innovative solution is being developed for knee ligaments and meniscus fixation. PLA screws are being developed through the combination of 3D printing, supercritical CO2 (scCO2) foaming and impregnation technologies, aiming to achieve a biodegradable and bioactive screw with improve bone integration ability. Additionally, to improve the anchor and fixation of the ligament treatments, PCL Bio-Fillers are being developed through the use of 3D printing, electrospinning and dipping methods with the final goal to induce the bone cells to reproduce itself (osteogenesis) and create a better grip between the ligaments and the bone. PLA screws were manufactured by 3D printing and further process to induce porosity by scCO2 foaming, followed by EPS impregnation through scCO2. Scanning electron microscopy (SEM) was used to evaluate microporosity and the EPS impregnation. The CO2 concentration, density and expansion ratio of the PLA screws were evaluated. FTIR (Fourier-transform infrared spectroscopy) was performed to evaluate chemical composition changes of the samples. DSC (Differential scanning calorimetry) was applied to analyze thermal stability both before and after treatment. PCL Bio-Fillers were produced by 3D printing, coated with PCL using electrospinning and dipped with bruxite. Then, to study the PCL fibers and bruxite dispersion, we have used the scanning electron microscopy (SEM) method. Results: Several conditions of foaming were tested (pressure, time, temperature and controlled expansion measures) and then, analyzed through SEM imaging. Samples with greater porosity were selected for further testing and analysis. The CO2 concentration results revealed that the saturation increase is proportional to the increase in pressure and inversely proportional to the increase in infill density. The expansion ratio results demonstrated that it typically decreases with increasing infill density and batch pressure. To optimize the 3D printed Bio-Fillers coated with PCL several parameters were adjusted (PCL concentration, flow rate, distance, potential difference, and nozzle size). Firstly, samples with apparent macroscopy uneven coating were remove with further analysis being performed via SEM analysis. The SEM analysis showed that increasing the potential difference and decreasing the flow rate produced more dispersed and thinner fibers. It also revealed that increase PCL concentration led to higher fiber density and size. In the end, the parameters that resulted in the better PCL fibers dispersion were with a concentration of 7,5% w/v of PCL, 20 μL/min flow rate, 10 cm of distance between the nozzle and the Bio-Filler, 23 kV of potential difference and 20 G of nozzle size. Further work is being performed to optimize the dipping process with bruxite and achieve an even coating. Conclusion: The sCO2 methodologies implemented were efficient in terms of generating porosity and EPS impregnation. EPS-induced bioactivity will be studied in the future.
- Advancing diabetes treatment: from human beta cell technology to bioartificial pancreas developmentPublication . Sá, Joana; Sá, Simone; Leménager, Hélène; Costa, Raquel; Onteniente, Brigitte; Soares, Raquel; Ribeiro, Viviana P.; Oliveira, Ana L.In 2021, approximately 537 million people worldwide, primarily in low- and middle-income countries, were affected by diabetes, leading to approximately 6.7 million deaths annually or severe secondary complications including life-threatening hyperglycemia. For nearly 50 years, current therapeutic approaches include full pancreas transplantation and isolated pancreatic islets, more recently, cell therapy such as in vitro generated islets and stem cell derived. The transplantation of pancreatic islet cells can be less invasive than full organ transplantation, however, does not achieve the same rate of functional success due to the low survival of the engrafted cells. Tissue-engineered bioartificial pancreas has been designed to address such issues, improving cell engraftment, survival, and immune rejection problems, with the added advantage that the tissue produced in vitro has an unlimited source of material.
- Biohybrid dressings: integrating silk fibroin textiles with decellularized biological tissue for wound healingPublication . Sousa, Teresa; Vale, Inês; Rosadas, Marta; Silva, Inês V.; Ribeiro, Viviana P.; Oliveira, Ana L.Burns affect 11 million people globally each year with 180,000 associated fatalities. This study proposes a multilayer hybrid dressing combining a silk fibroin (SF) textile with decellularized porcine small intestine (dPSI) capable of simultaneously provide wound protection and tissue regeneration. A silk sericin (SS) hydrogel was embedded into the decellularized tissue ensuring the integration with the textile layer while promoting anti-inflammatory benefits, enhancing the hybrid dressing’s biological performance.
- Combinatory approach for developing silk fibroin scaffolds for cartilage regenerationPublication . Ribeiro, Viviana P.; Morais, Alain da Silva; Maia, F. Raquel; Canadas, R. F.; Costa, João B.; Oliveira, Ana L.; Oliveira, Joaquim M.; Reis, Rui L.Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ± 0.6%), wide pore distribution and high interconnectivity (95.9 ± 0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and β-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. Statement of Significance In cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration.
- Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applicationsPublication . Ribeiro, Viviana P.; Yan, Le-Ping; Oliveira, Ana; Oliveira, Joaquim M.; Reis, Rui L.Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from highconcentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications.Conclusion: This study proposes an innovative approach to produce fast-formed porous SF scaffolds using enzymatically crosslinked SF hydrogels structured by the combination of salt-leaching and freeze-drying methodologies. The obtained results can provide a valuable reference of SF as a tunable and versatile biomaterial with great potential for applications in cartilage TE scaffolding.
- Cutting-edge hybrid dressings: combining silk medical textiles and decellularized biological tissue for advanced burn wound carePublication . Sousa, Teresa; Vale, Inês; Rosadas, Marta; Silva, Inês V.; Ribeiro, Viviana P.; Oliveira, Ana L.Aim: Burns affect 11 million people globally each year, with 180,000 fatalities [1]. This study investigates a multilayer burn dressing combining silk fibroin (SF) fabric with highly- preserved decellularized porcine small intestine (dPSI) to support tissue regeneration and wound integration [2]. A silk sericin (SS) hydrogel is included as interface for structural integrity and anti-inflammatory benefits, enhancing the hybrid dressing’s biological performance. Methods: An innovative decellularization protocol was proposed to obtain dPSI, maintaining submucosa, serosa, and muscle layers, using cycles of decellularizing agents (SDS, SDC, DMSO), washing agents (upH₂O, PBS), and sterilization (PAA/ethanol). The serosa and submucosa of dPSI were integrated with SF-based textiles using HRP- crosslinked SS hydrogels. Decellularization and integrity were assessed via DNA quantification and histology, while hybrid dressings’ morphology and mechanics were evaluated by SEM and tensile tests. Degradation profile was tested in simulated wound fluid, and biological performance was assessed by culturing human dermal fibroblasts (hDFs) on the submucosa layer up to 10 days. Results: The dPSI was successfully achieved (<50 ng/mg dsDNA). SEM images confirmed the full integration of the dPSI with SF-based textiles, especially when serosa faced the textile. The presence of the textile structure resulted in an enhancement of the mechanical strength. dPSI was able to degrade first in the multilayer dressing, and hDFs adhered and proliferated on the submucosa over 10 days, supporting hybrid structural integrity. Conclusions: This study is pioneer in confirming promising results for the first multilayer hybrid dressing combining medical textiles and dPSI for burn wound applications.
- Decellularized dermal matrices: unleashing the potential in tissue engineering and regenerative medicinePublication . Rosadas, Marta; Silva, Inês V.; Costa, João B.; Ribeiro, Viviana P.; Oliveira, Ana L.Decellularized dermal matrices (dDMs) have emerged as effective biomaterials that can revolutionize regenerative medicine, particularly in the field of wound healing and tissue regeneration. Derived from animal or human skin, dDMs offer great biocompatibility, remarkable biochemistry, and a macromolecular architecture equivalent to the native tissue. Notably, among the biomimetic extracellular matrix (ECM)-based scaffolds, dDMs stand out due to their inherent dermal microenvironment, holding high value for skin regeneration and reconstructive surgery. The integration of dDMs as a biomaterial base for bioinks in advanced manufacturing technologies opens promising avenues for crafting precise, biomimetic tissue engineering (TE) constructs with optimized recellularization properties. This mini review outlines the main sources, differential decellularization techniques applied to dDMs, and their significance intissue engineering and regenerative medicine. It subsequently delves into the different categories of decellularized materials obtained, their unique physical and biochemical attributes, as well as their applications to promote wound healing and regenerating skin and soft tissues. Additionally, the currently available market products based on dDMs are examined and the main outcomes are compared. Finally, the article highlights current barriers in the field and anticipates the future challenges and applications of dDMs-based therapies.
- Detergent-free supercritical CO2–assisted protocol for the production of sustainable and highly preserved decellularized porcine meniscus for orthopedic applicationsPublication . Ho, Chou I.; Rodrigues, Francisco A. P.; Reis, Mariana S.; Ribeiro, Viviana P.; Oliveira, Ana Leite; Costa, João B.Introduction & Objectives Meniscal injuries occur approximately 66 to 70 per 100,000 individuals annually (Fig. 1), potentially leading to the development of osteoarthritis (OA) or other degenerative cartilage disease in 10 to 20 years. One of the conventional treatments is meniscal allograft transplantation. However, its limitations constrain its comprehensive application in the healthcare system.
- 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.; Costa, João; Vervenne, Thibault; Oliveira, Ana L.; Ribeiro, Viviana P.; Mignon, ArnCardiovascular diseases are the leading cause of adult mortality worldwide, according to the WHO [1]. An important surgical approach for treating diseased aortic valves is the Ross procedure, in which the affected aortic valve is replaced with an autograft from the patient’s own pulmonary valve. The main concern about this procedure is linked to wall dilatation, which can lead to valve leakage and reoperation. Dilatation occurs due to the fivefold increase in blood pressure when transitioning from pulmonary to aortic conditions. Current solutions rely on permanet and stiff synthetic materials to provide structural support, however, these lack biological functionality. Our approach aim to incorporate a decellularized extracellular matrix (dECM) in a semi-permanent textile wrapped around the autograft promoting benign biological adaptation. The decellularization process by removing cellular components, reduces the risk of inflammatory responses and immune rejection. Moreover, essential ECM components that regulate cellular behavior are mantained, which is crucial for effective decellularization outcomes [2]. This study is focused in the development and optimization of an efficient decellularization protocol for obtaining dECM from porcine aorta with mild effects on ECM components preservation. Decellularization of the porcine aortic tissue was performed using a detergent and enzymatic-based protocol combined with supercritical CO₂ (scCO₂). Additional steps of sonication, agitation, washing and freeze-thaw were performed to enhance decellularization efficiency. Graphic A shows a significant decrease in the DNA content after optimized decellularization processing, indicating the removal of approximatly 85% of the DNA from the native tissue, and suggest an effective reduction of potential immunogenic components. Further characterization of ECM components (i.e. glycosaminoglycans, total collagen and elastin) is required to evaluate its preservation and the biological potencial of the dECM when integrated in the Ross processure. To do so, aorta dECM powder will be combined with polycaprolactone and elastin and processed through electrospinning used to create a tubular device to enhance bio-mechanocompatibility, further improving current external supports for the Ross procedure and enhancing its general clinical outcomes. Since this support is biodegradable, it allows the new valve to take over the required strength. The dECM specifically offers essential biochemical cues to promote tissue integration and enable long-term functional repair after the Ross procedure.
- 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.
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