Human Adipose-Derived Stem Cells (hASCs), an underappreciated source of stem cells, might consist... more Human Adipose-Derived Stem Cells (hASCs), an underappreciated source of stem cells, might consist in a new cell poll for tissue engineering (TE) applications, including bone TE. Furthermore, these cells can be harvested in a large number with low morbidity and their developmental plasticity was already proven both in vivo and in vitro. SSEA-4, an early stem cell marker is known to be expressed by hASCs though in a low percentage. Considering the pluripotency of SSEA-4 cells, we examined whether the SSEA-4 + hASCs could differentiate into the endothelial and the osteogenic lineages, aiming to develop a new strategy for promoting the vascularization of bone TE constructs. Analysis by immunocytochemistry and flow cytometry demonstrated the ability of SSEA-4 + hASCs cells to rapidly acquire stable endothelial features (CD31, CD34, CD105 and vWF expression), whilst formation of tubular structures resembling capillary like organization when seeded in Matrigel confirmed their in vitro functional competence. Also, the presence of scavenger receptors for Dil-ac-LDL is consistent with the characteristic of endothelial cells. Furthermore, SSEA-4 + hASCs subpopulation was found suitable for osteogenic differentiation. An upregulation of osteogenensis related genes (osteocalcin-OPN and osteopontin-OCN) was observed which was confirmed also by means on immunocytochemistry. Matrix mineralization was also revealed by Alizarin Red.
Proximal tubular damage is an important prognostic determinant in various chronic kidney diseases... more Proximal tubular damage is an important prognostic determinant in various chronic kidney diseases (CKDs). Currently available diagnostic methods do not allow for early disease detection and are neither efficient. Indoxyl sulfate (IS) is an endogenous metabolite and protein-bound uremic toxin that is eliminated via renal secretion, but accumulates in plasma during tubular dysfunction. Therefore, it may be suitable as a tubular function marker. To evaluate this, a fast bioanalytical method was developed and validated for IS in various species and a kidney cell line using LC-MS/MS. An isotope-labeled IS potassium salt as an internal standard and acetonitrile (ACN) as a protein precipitant were used for sample pretreatment. The analyte was separated on a Polaris 3 C18-A column by gradient elution using 0.1% formic acid in water and ACN, and detected by negative electrospray ionization in selected reaction monitoring mode. The within-day (≤ 4.0%) and between-day (≤ 4.3%) precisions and accuracies (97.7 to 107.3%) were within the acceptable range. The analyte showed sufficient stability at all conditions investigated. Finally, applying this assay, significantly higher plasma and lower urine concentrations of IS were observed in mice with diabetic nephropathy with tubular damage, which encourages validation toward its use as a biomarker.
The construction of scaffolds and subsequent incorporation of cells and biologics have been widel... more The construction of scaffolds and subsequent incorporation of cells and biologics have been widely investigated to regenerate damaged tissues. Scaffolds act as a template to guide tissue formation, and their characteristics have a considerable impact on the regenerative process. Whereas many technologies exist to induce specific twodimensional (2D) morphologies into biomaterials, the introduction of threedimensional (3D) micromorphologies into individual pore walls of scaffolds produced from biological molecules such as collagen poses a challenge. We here report the use of dicarboxylic acids to induce specific micromorphologies in collagen scaffolds and evaluate their effect on cellular migration and differentiation. Insoluble type I collagen fibrils were suspended in monocarboxylic and dicarboxylic acids of different concentrations, and unidirectional and random pore scaffolds were constructed by freezing and lyophilization. The application of various acids and concentrations resulted in variations in 3D micromorphologies, including wall structure, wall thickness, and pore size. The use of dicarboxylic acids resulted in acid-specific micromorphologies, whereas monocarboxylic acids did not. Dicarboxylic acids with an odd or even number of C-atoms resulted in frayed/fibrillar or smooth wall structures, respectively, with varying appearances. The formation of micromorphologies was concentration-dependent. In vitro analysis indicated the cytocompatibility of scaffolds, and micromorphology-related cell behavior was indicated by enhanced myosin staining and myosin heavy chain gene expression for C2C12 myoblasts cultured on scaffolds with frayedlike micromorphologies compared to those with smooth micromorphologies. In conclusion, porous collagen scaffolds with various intrawall 3D micromorphologies can be constructed by application of dicarboxylic acids, superimposing the second level of morphology to the overall scaffold structure. Acid crystal formation is key to the specific micromorphologies observed and can be explained by the odd/even theory for dicarboxylic acids. Scaffolds with a 3D micrometerdefined topography may be used as a screening platform to select optimal substrates for the regeneration of specific tissues.
Chronic kidney disease (CKD) is a major healthcare burden that takes a toll on the quality of lif... more Chronic kidney disease (CKD) is a major healthcare burden that takes a toll on the quality of life of many patients. Emerging evidence indicates that a substantial proportion of these patients carry a genetic defect that contributes to their disease. Any effort to reduce the percentage of patients with a diagnosis of nephropathy heading towards kidney replacement therapies should therefore be encouraged. Besides early genetic screenings and registries, in vitro systems that mimic the complexity and pathophysiological aspects of the disease could advance the screening for targeted and personalized therapies. In this regard, the use of patient-derived cell lines, as well as the generation of disease-specific cell lines via gene editing and stem cell technologies, have significantly improved our understanding of the molecular mechanisms underlying inherited kidney diseases. Furthermore, organs-on-chip technology holds great potential as it can emulate tissue and organ functions that are not found in other, more simple, in vitro models. The personalized nature of the chips, together with physiologically relevant read-outs, provide new opportunities for patient-specific assessment, as well as personalized strategies for treatment. In this review, we summarize the major kidney-on-chip (KOC) configurations and present the most recent studies on the in vitro representation of genetic kidney diseases using KOC-driven strategies. Keywords Genetic kidney disorders • Gene editing • In vitro models • Organ-on-chip • Kidney-on-chip
Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biolo... more Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue substitutes. One approach to achieve this aim is to develop osteoconductive scaffolds that facilitate the formation of functional bone tissue. We have fabricated nanoclay-enriched electrospun poly(e-caprolactone) (PCL) scaffolds for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface interactions between PCL and nanoclay. The effect of nanoclay on the mechanical properties of electrospun fibers is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the electrospun scaffolds as evident by an increase in alkaline phosphates activity of hMSCs and higher deposition of mineralized extracellular matrix compared to PCL scaffolds. Given its unique bioactive characteristics, nanoclayenriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering.
Chronic kidney disease (CKD) typically appears alongside other comorbidities, highlighting an und... more Chronic kidney disease (CKD) typically appears alongside other comorbidities, highlighting an underlying complex pathophysiology that is thought to be vastly modulated by the bidirectional gut-kidney crosstalk. By combining advances in tissue engineering, biofabrication, microfluidics, and biosensors, microphysiological systems (MPSs) have emerged as promising approaches for emulating the in vitro interconnection of multiple organs, while addressing the limitations of animal models. Mimicking the (patho)physiological states of the gut-kidney axis in vitro requires an MPS that can simulate not only this direct bidirectional crosstalk but also the contributions of other physiological participants such as the liver and the immune system. We discuss recent developments in the field that could potentially lead to in vitro modeling of the gut-kidney axis in CKD. Chronic Kidney Disease: A Metabolic Disorder with Disrupted Inter-Organ and Inter-Organismal Signaling Chronic kidney disease (CKD) is the most widespread kidney disease and is characterized by the gradual loss of organ function over time, which impairs the ability to filter metabolic waste products from the blood (Box 1). The kidneys have many highly specialized functions, such as blood filtration and active secretion for the removal of metabolic waste, reabsorption of essential nutrients, maintenance of blood volume and electrolyte homeostasis, and metabolic and endocrine activity [1]. Highlights Gut microbiota-derived metabolites are key molecular mediators of the microbiota-host axis. The excretory capacity of the kidney is an essential part of human gut microbial symbiosis. Chronic kidney disease (CKD) is a metabolic disease in which gut microbiotaderived metabolites accumulate in the blood and adversely affect host physiological functions. Intestine-on-a-chip models have been developed that recapitulate the 3D epithelial barrier, the gut-microbiome interaction, and intercellular crosstalk with remote organs. Components of the immune system are pivotal in remote communication between the gut and kidney. The combination of engineered microphysiological systems with highthroughput multiomic analysis will provide novel insights into organ intercommunication in CKD.
One of the promises of synthetic materials in cell culturing is that control over their molecular... more One of the promises of synthetic materials in cell culturing is that control over their molecular structures may ultimately be used to control their biological processes. Synthetic polymer hydrogels from polyisocyanides (PIC) are a new class of minimal synthetic biomaterials for threedimensional cell culturing. The macromolecular lengths and densities of biofunctional groups that decorate the polymer can be readily manipulated while preserving the intrinsic nonlinear mechanics, a feature commonly displayed by fibrous biological networks. In this work, we propose the use of PIC gels as cell culture platforms with decoupled mechanical inputs and biological cues. For this purpose, different types of cells were encapsulated in PIC gels of tailored compositions that systematically vary in adhesive peptide (GRGDS) density, polymer length, and concentration; with the last two parameters controlling the gel mechanics. Both cancer and smooth muscle cells grew into multicellular spheroids with proliferation rates that depend on the adhesive GRGDS density, regardless of the polymer length, suggesting that for these cells, the biological input prevails over the mechanical cues. In contrast, human adipose-derived stem cells do not form spheroids but rather spread out. We find that the morphological changes strongly depend on the adhesive ligand density and the network mechanics; gels with the highest GRGDS densities and the strongest stiffening response to stress show the strongest spreading. Our results highlight the role of the nonlinear mechanics of the extracellular matrix and its synthetic mimics in the regulation of cell functions.
Kappa carrageenan (κ-CA) is a natural-origin polymer that closely mimics the glycosaminoglycan st... more Kappa carrageenan (κ-CA) is a natural-origin polymer that closely mimics the glycosaminoglycan structure, one of the most important constituents of native tissues extracellular matrix. Previously, it has been shown that κ-CA can crosslink via ionic interactions rendering strong, but brittle hydrogels. In this study, we introduce photocrosslinkable methacrylate moieties on the κ-CA backbone to create physically and chemically crosslinked hydrogels highlighting their use in the context of tissue engineering. By varying the degree of methacrylation, the effect on hydrogel crosslinking was investigated in terms of hydration degree, dissolution profi les, morphological, mechanical, and rheological properties. Furthermore, the viability of fi broblast cells cultured inside the photocrosslinked hydrogels was investigated. The combination of chemical and physical crosslinking procedures enables the formation of hydrogels with highly versatile physical and chemical properties, while maintaining the viability of encapsulated cells. To our best knowledge, this is the fi rst study reporting the synthesis of photocrosslinkable κ-CA with controllable compressive moduli, swelling ratios and pore size distributions. Moreover, by micromolding approaches, spatially controlled geometries and cell distribution patterns could be obtained, thus enabling the development of cell-material platforms that can be applied and tailored to a broad range of tissue engineering strategies.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The design of constructs for tubular tissue engineering is challenging. Most biomaterials need to... more The design of constructs for tubular tissue engineering is challenging. Most biomaterials need to be reinforced with supporting structures such as knittings, meshes or electrospun material to comply with the mechanical demands of native tissues. In this study, coupled helical coils (CHCs) were manufactured to mimic collagen fiber orientation as found in nature. Monofilaments of different commercially available biodegradable polymers were wound and subsequently fused, resulting in right-handed and lefthanded polymer helices fused together in joints where the filaments cross. CHCs of different polymer composition were tested to determine the tensile strength, strain recovery, hysteresis, compressive strength and degradation of CHCs of different composition. Subsequently, seamless and stable hybrid constructs consisting of PDSII Ò USP 2-0 CHCs embedded in porous collagen type I were produced. Compared to collagen alone, this hybrid showed superior strain recovery (93.5 ± 0.9% vs 71.1 ± 12.6% in longitudinal direction; 87.1 ± 6.6% vs 57.2 ± 4.6% in circumferential direction) and hysteresis (18.9 ± 2.7% vs 51.1 ± 12.0% in longitudinal direction; 11.5 ± 4.6% vs 46.3 ± 6.3% in circumferential direction). Furthermore, this hybrid construct showed an improved Young's modulus in both longitudinal (0.5 ± 0.1 MPa vs 0.2 ± 0.1 MPa; 2.5-fold) and circumferential (1.65 ± 0.07 MPa vs (2.9 ± 0.3) Â 10 À2 MPa; 57-fold) direction, respectively, compared to templates created from collagen alone. Moreover, hybrid template characteristics could be modified by changing the CHC composition and CHCs were produced showing a mechanical behavior similar to the native ureter. CHC-enforced templates, which are easily tunable to meet different demands may be promising for tubular tissue engineering. Statement of Significance Most tubular constructs lack sufficient strength and tunability to comply with the mechanical demands of native tissues. Therefore, we embedded coupled helical coils (CHCs) produced from biodegradable polymers-to mimic collagen fiber orientation as found in nature-in collagen type I sponges. We show that the mechanical behavior of CHCs is very similar to native tissue and strengths structurally weak tubular constructs. The production procedure is relatively easy, reproducible and mechanical features can be controlled to meet different mechanical demands. This is promising in template manufacture, hence offering new opportunities in tissue engineering of tubular organs and preventing graft failure.
Application of nanoporous poly(vinyl alcohol)-based hydrogel matrices for the design of engineered controlled-release devices with various routes of administration
, A. (2014). Amphiphilic beads as depots for sustained drug release integrated into fibrillar sca... more , A. (2014). Amphiphilic beads as depots for sustained drug release integrated into fibrillar scaffolds.
The kidney is frequently involved in adverse effects caused by exposure to foreign compounds, inc... more The kidney is frequently involved in adverse effects caused by exposure to foreign compounds, including drugs. An early prediction of those effects is crucial for allowing novel, safe drugs entering the market. Yet, in current pharmacotherapy, drug-induced nephrotoxicity accounts for up to 25% of the reported serious adverse effects, of which one-third is attributed to antimicrobials use. Adverse drug effects can be due to direct toxicity, for instance as a result of kidney-specific determinants, or indirectly by, e.g., vascular effects or crystals deposition. Currently used in vitro assays do not adequately predict in vivo observed effects, predominantly due to an inadequate preservation of the organs' microenvironment in the models applied. The kidney is highly complex, composed of a filter unit and a tubular segment, together containing over 20 different cell types. The tubular epithelium is highly polarized, and the maintenance of this polarity is critical for optimal functioning and response to environmental signals. Cell polarity is dependent on communication between cells, which includes paracrine and autocrine signals, as well as biomechanic and chemotactic processes. These processes all influence kidney cell proliferation, migration, and differentiation. For drug disposition studies, this microenvironment is essential for prediction of toxic responses. This review provides an overview of drug-induced injuries to the kidney, details on relevant and translational biomarkers, and advances in 3D cultures of human renal cells, including organoids and kidney-on-a-chip platforms.
Co-axial printing of convoluted proximal tubule for kidney disease modeling To cite this article:... more Co-axial printing of convoluted proximal tubule for kidney disease modeling To cite this article: A M van Genderen et al 2022 Biofabrication 14 044102 View the article online for updates and enhancements.
Protein-bound uremic toxins (PBUTs) are associated with the progression of chronic kidney disease... more Protein-bound uremic toxins (PBUTs) are associated with the progression of chronic kidney disease (CKD) and its associated morbidity and mortality. The conventional dialysis techniques are unable to efficiently remove PBUTs due to their plasma protein binding. Therefore, novel approaches are being developed, but these require validation in animals before clinical trials can begin. We conducted a systematic review to document PBUT concentrations in various models and species. The search strategy returned 1163 results for which abstracts were screened, resulting in 65 full-text papers for data extraction (rats (n = 41), mice (n = 17), dogs (n = 3), cats (n = 4), goats (n = 1), and pigs (n = 1)). We performed descriptive and comparative analyses on indoxyl sulfate (IS) concentrations in rats and mice. The data on large animals and on other PBUTs were too heterogeneous for pooled analysis. Most rodent studies reported mean uremic concentrations of plasma IS close to or within the range ...
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