Engineering natural based nanocomposite inks via interface interaction for extrusion 3D printing.

Nanocomposites and low-viscous materials lack translation in additive manufacturing technologies due to deficiency in rheological requirements and heterogeneity of their preparation. This work proposes the chemical crosslinking between composing phases as a universal approach for mitigating such issues. The model system is composed of amine-functionalized bioactive glass nanoparticles (BGNP) and light-responsive methacrylated bovine serum albumin (BSAMA) which further allows post-print photocrosslinking. The interfacial interaction was conducted by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking agent and N-Hydroxysuccinimide between BGNP-grafted amines and BSAMA's carboxylic groups. Different chemical crosslinking amounts and percentages of BGNP in the nanocomposites were tested. The improved interface interactions increased the elastic and viscous modulus of all formulations. More pronounced increases were found with the highest crosslinking agent amounts (4 % w/v) and BGNP concentrations (10 % w/w). This formulation also displayed the highest Young's modulus of the double-crosslinked construct. All composite formulations could effectively immobilize the BGNP and turn an extremely low viscous material into an appropriate inks for 3d printing technologies, attesting for the systems' tunability. Thus, we describe a versatile methodology which can successfully render tunable and light-responsive nanocomposite inks with homogeneously distributed bioactive fillers. This system can further reproducibly recapitulate phases of other natures, broadening applicability.

Decellularized kidney extracellular matrix-based hydrogels for renal tissue engineering.

Kidney regeneration is hindered by the limited pool of intrinsic reparative cells. Advanced therapies targeting renal regeneration have the potential to alleviate the clinical and financial burdens associated with kidney disease. Delivery systems for cells, extracellular vesicles, or growth factors aimed at enhancing regeneration can benefit from vehicles enabling targeted delivery and controlled release. Hydrogels, optimized to carry biological cargo while promoting regeneration, have emerged as promising candidates for this purpose. This study aims to develop a hydrogel from decellularized kidney extracellular matrix (DKECM) and explore its biocompatibility as a biomaterial for renal regeneration. The resulting hydrogel crosslinks with temperature and exhibits a high concentration of extracellular matrix. The decellularization process efficiently removes detergent residues, yielding a pathogen-free biomaterial that is non-hemolytic and devoid of α-gal epitope. Upon interaction with macrophages, the hydrogel induces differentiation into both pro-inflammatory and anti-inflammatory phenotypes, suggesting an adequate balance to promote biomaterial functionality in vivo. Renal progenitor cells encapsulated in the DKECM hydrogel demonstrate higher viability and proliferation than in commercial collagen-I hydrogels, while also expressing tubular cells and podocyte markers in long-term culture. Overall, the injectable biomaterial derived from porcine DKECM is anticipated to elicit minimal host reaction while fostering progenitor cell bioactivity, offering a potential avenue for enhancing renal regeneration in clinical settings. STATEMENT OF SIGNIFICANCE: The quest to improve treatments for kidney disease is crucial, given the challenges faced by patients on dialysis or waiting for transplants. Exciting new therapies combining biomaterials with cells can revolutionize kidney repair. In this study, researchers created a hydrogel from pig kidney. This gel could be used to deliver cells and other substances that help in kidney regeneration. Despite coming from pigs, it's safe for use in humans, with no harmful substances and reduced risk of immune reactions. Importantly, it promotes a balanced healing response in the body. This research not only advances our knowledge of kidney repair but also offers hope for more effective treatments for kidney diseases.

Renal Regeneration: The Role of Extracellular Matrix and Current ECM-Based Tissue Engineered Strategies.

Natural extracellular matrices (ECM) are currently being studied as an alternative source for organ transplantation or as new solutions to treat kidney injuries, which can evolve to end-stage renal disease, a life devastating condition. This paper provides an overview on the current knowledge in kidney ECM and its usefulness on future investigations. The composition and structure of kidney ECM is herein associated with its intrinsic capacity of remodeling and repair after insult. Moreover, it provides a deeper insight on altered ECM components during disease. The use of decellularized kidney matrices is discussed in the second part of the review, with emphasis on how these matrices contribute to tissue-specific differentiation of embryonic, pluripotent, and other stem cells. The evolution on the field toward different uses of xenogeneic ECM as a biological scaffold material is discussed, namely the major outcomes on whole kidney recellularization and its in vivo implantation. At last, the recent literature on the use of processed kidney decellularized ECM to produce diverse biomaterial substrates, such as hydrogels, membranes, and bioinks are reviewed, with emphasis on future perspectives of its translation into the clinic.

Retinoic Acid Benefits Glomerular Organotypic Differentiation from Adult Renal Progenitor Cells In Vitro.

When in certain culture conditions, organotypic cultures are able to mimic developmental stages of an organ, generating higher-order structures containing functional subunits and progenitor niches. Despite the major advances in the area, researchers have not been able to fully recapitulate the complexity of kidney tissue. Pluripotent stem cells are extensively used in the field, but very few studies make use of adult stem cells. Herein, we describe a simple and feasible method for achieving glomerular epithelial differentiation on an organotypic model comprising human renal progenitor cells from adult kidney (hRPCs). Their glomerular differentiative potential was studied using retinoic acid (RA), a fundamental molecule for intermediate mesoderm induction on early embryogenesis. Immunofluorescence, specific cell surface markers expression and gene expression analysis confirm the glomerular differentiative potential of RA in a short-term culture. We also compared the potential of RA with a potent WNT agonist, CHIR99021, on the differentiative capacity of hRPCs. Gene expression and immunofluorescence analysis confirmed that hRPCs are more sensitive to RA stimulation when compared to CHIR9901. Endothelial cells were also included on the spheroids, resulting in a higher organizational level. The assembly potential of these cells and their selective stimulation will give new insights on adult organotypic cell culture studies and will hopefully guide more works in this important area of research.

Particulate kidney extracellular matrix: bioactivity and proteomic analysis of a novel scaffold from porcine origin.

Decellularized matrices are attractive substrates, being able to retain growth factors and proteins present in the native tissue. Several biomaterials can be produced by processing these matrices. However, new substrates capable of being injected that reverse local kidney injuries are currently scarce. Herein, we hypothesized that the decellularized particulate kidney porcine ECM (pKECM) could support renal progenitor cell cultures for posterior implantation. Briefly, kidneys are cut into pieces, decellularized by immersion on detergent solutions, lyophilized and reduced into particles. Then, ECM particles are analyzed for nuclear material remaining by DNA quantification and histological examination, molecular conformation by FITR and structural morphology by SEM. Protein extraction is also optimized for posterior identification and quantification by mass spectrometry. The results obtained confirm the collagenous structure and composition of the ECM, the effective removal of nucleic material and the preservation of ECM proteins with great similarity to human kidneys. Human renal progenitor cells (hRPCs) are seeded in different ratios with pKECM, on 3D suspensions. The conducted assays for cell viability, proliferation and distribution over 7 days of culture suggest that these matrices as biocompatible and bioactive substrates for hRPCs. Also, by analyzing CD133 expression, an optimal ratio for specific phenotypic expression is revealed, demonstrating the potential of these substrates to modulate cellular behavior. The initial hypothesis of developing and characterizing a particulate ECM biomaterial as a consistent substrate for 3D cultures is successfully validated. The findings in this manuscript suggest these particles as valuable tools for regenerative nephrology by minimizing surgeries and locally reversing small injuries which can lead to chronic renal disfunction.

Decellularized kidney extracellular matrix bioinks recapitulate renal 3D microenvironmentin vitro.

Decellularized extracellular matrices (ECMs) are able to provide the necessary and specific cues for remodeling and maturation of tissue-specific cells. Nevertheless, their use for typical biofabrication applications requires chemical modification or mixing with other polymers, mainly due to the limited viscoelastic properties. In this study, we hypothesize that a bioink exclusively based on decellularized kidney ECM (dKECM) could be used to bioprint renal progenitor cells. To address these aims, porcine kidneys were decellularized, lyophilized and digested to yield a viscous solution. Then, the bioprinting process was optimized using an agarose microparticle support bath containing transglutaminase for enzymatic crosslinking of the dKECM. This methodology was highly effective to obtain constructs with good printing resolution and high structural integrity. Moreover, the encapsulation of primary renal progenitor cells resulted in high cell viability, with creation of 3D complex structures over time. More importantly, this tissue-specific matrix was also able to influence cellular growth and differentiation over time. Taken together, these results demonstrate that unmodified dKECM bioinks have great potential for bioengineering renal tissue analogs with promising translational applications and/or forin vitromodel systems. Ultimately, this strategy may have greater implications on the biomedical field for the development of bioengineered substitutes using decellularized matrices from other tissues.

Co-cultures of renal progenitors and endothelial cells on kidney decellularized matrices replicate the renal tubular environment in vitro.

AIM: Herein we propose creating a bilayer tubular kidney in-vitro model. It is hypothesized that membranes composed of decellularized porcine kidney extracellular matrix are valid substitutes of the tubular basement membrane by mimicking the physiological relevance of the in vivo environment and disease phenotypes. METHODS: Extracellular matrix was obtained from decellularized porcine kidneys. After processing by lyophilization and milling, it was dissolved in an organic solvent and blended with poly(caprolactone). Porous membranes were obtained by electrospinning and seeded with human primary renal progenitor cells to evaluate phenotypic alterations. To create a bilayer model of the in vivo tubule, the same cells were differentiated into epithelial tubular cells and co-cultured with endothelial cells in opposite sites. RESULTS: Our results demonstrate increasing metabolic activity, proliferation and total protein content of renal progenitors over time. We confirmed the expression of several genes encoding epithelial transport proteins and we could also detect tubular-specific proteins by immunofluorescence stainings. Functional and transport assays were performed trough the bilayer by quantifying both human serum albumin uptake and inulin leakage. Furthermore, we validated the chemical modulation of nephrotoxicity on this epithelium-endothelium model by cisplatin exposure. CONCLUSION: The use of decellularized matrices in combination with primary renal cells was shown to be a valuable tool for modelling renal function and disease in vitro. We successfully validated our hypothesis by replicating the physiological conditions of an in vitro tubular bilayer model. The developed system may contribute significantly for the future investigation of advanced therapies for kidney diseases.

Extracellular matrix electrospun membranes for mimicking natural renal filtration barriers.

Kidney diseases are recognized as a major health problem, which affect 10% of the population. Because currently available therapies have many limitations, some tissue engineering strategies have been emerging as promising approaches in this field. In this work, porcine kidneys were decellularized to obtain decellularized kidney extracellular matrix (dKECM).1 Our results demonstrate a successful protocol of decellularization characterized by the removal of nucleic acid material and preservation of collagen and glycosaminoglycans. Blends of polycaprolactone (PCL)2 and dKECM were prepared by electrospinning and characterized. The biological performance of the membranes was tested with a human kidney cell line (HK-2) for 7 days. It was observed that cellular metabolic activity, proliferation and protein content increased with an increase in dKECM concentrations (30, 50 and 70%). Additionally, the expression of zona occludens-1 was revealed on dKECM-containing membranes but not on pure PCL membranes. To the best of our knowledge this is the first time that natural extracellular matrix is used to mimic the kidney basement membrane as an in vitro model. This could be a valuable tool for regenerative nephrology and may have an impact on the development of kidney advanced therapies in the future.

Development of non-orthogonal 3D-printed scaffolds to enhance their osteogenic performance.

Three-dimensional (3D)-printed polycaprolactone (PCL)-based scaffolds have been extensively proposed for Tissue Engineering (TE) applications. Currently, the majority of the scaffolds produced are not representative of the complex arrangement of natural structures, since the internal morphologies follow an orthogonal and regular pattern. In order to produce scaffolds that more closely replicate the structure of the extracellular matrix (ECM) of tissues, herein both circular and sinusoidal scaffolds were fabricated and compared to their conventional orthogonal counterparts. This is an innovative, versatile and efficient strategy to 3D print PCL scaffolds with unique curved geometries. The morphology and the mechanical behavior of the scaffolds were assessed. The biological response was analyzed by evaluating the cell seeding efficiency, cell adhesion, proliferation, and osteogenic activity of an osteoblastic-like cell line seeded in these scaffolds. The scaffolds were designed and produced to have a similar porosity of about 56%. The non-orthogonal structures demonstrated lead to higher values of Young's modulus, both under dry conditions and when immersed in PBS. Moreover, the biological data corroborate that non-orthogonal scaffolds influence the cellular responses in a positive manner, namely in the osteogenic activity when compared with the orthogonal controls. These results suggest that introducing less orthogonal elements, which better mimic the tissue ECM and architecture, may have a positive influence on the cellular behavior, being a potential strategy to address bone tissue engineering applications.