Stable hydrogel adhesion to polydimethylsiloxane enables cyclic mechanical stimulation of 3D-bioprinted smooth muscle constructs.

During normal urination, smooth muscle cells (SMCs) in the lower urinary tract (LUT) are exposed to mechanical signals that have a critical impact on tissue structure and function. Nevertheless, the mechanisms underlying the maintenance of the contractile phenotype of SMCs remain poorly understood. This is due, in part, to a lack of studies that have examined the effects of mechanical loading using three-dimensional (3D) models. In this study, surface modifications of polydimethylsiloxane (PDMS) membrane were evaluated to investigate the effects of cyclic mechanical stimulation on SMC maturation in 3D constructs. Commercially available cell stretching plates were modified with amino or methacrylate groups to promote adhesion of 3D constructs fabricated by bioprinting. After 6 days of stimulation, the effects of mechanical stimulation on the expression of contractile markers at the mRNA and protein levels were analyzed. Methacrylate-modified surfaces supported stable adhesion of the 3D constructs to the membrane and facilitated cyclic mechanical stimulation, which significantly increased the expression of contractile markers at the mRNA and protein levels. These effects were found to be mediated by activation of the p38 MAPK pathway, as inhibition of this pathway abolished the effects of stimulation in a dose-dependent manner. These results provide valuable insights into the role of mechanical signaling in maintaining the contractile phenotype of bladder SMCs, which has important implications for the development of future treatments for LUT diseases.

Correction: Peng et al. Distinct Dominant Lineage from In Vitro Expanded Adipose-Derived Stem Cells (ASCs) Exhibits Enhanced Wound Healing Properties. Cells 2022, 11, 1236.

In the original publication [...].

Tailoring Hydrogel Composition and Stiffness to Control Smooth Muscle Cell Differentiation in Bioprinted Constructs.

BACKGROUND: Reliable in vitro cellular models are needed to study the phenotypic modulation of smooth muscle cells (SMCs) in health and disease. The aim of this study was to optimize gelatin methacrylate (GelMA)/alginate hydrogels for bioprinting three-dimensional (3D) SMC constructs. METHODS: Four different hydrogel groups were prepared by mixing different concentrations (% w/v) of GelMA and alginate: G1 (5/1.5), G2 (5/3), G3 (7.5/1.5), and G4 (7.5/3). GelMA 10% was used as control (G5). A circular structure containing human bladder SMCs was fabricated by using an extrusion-based bioprinter. The effects of the mixing ratios on printability, viability, proliferation, and differentiation of the cells were investigated. RESULTS: Rheological analysis showed that the addition of alginate significantly stabilized the change in mechanical properties with temperature variations. The group with the highest GelMA and alginate concentrations (G4) exhibited the highest viscosity, resulting in better stability of the 3D construct after crosslinking. Compared to other hydrogel compositions, cells in G4 maintained high viability (> 80%), exhibited spindle-shaped morphology, and showed a significantly higher proliferation rate within an 8-day period. More importantly, G4 provided an optimal environment for the induction of a SMC contractile phenotype, as evidenced by significant changes in the expression of marker proteins and morphological parameters. CONCLUSION: Adjusting the composition of GelMA/alginate hydrogels is an effective means of controlling the SMC phenotype. These hydrogels support bioprinting of 3D models to study phenotypic smooth muscle adaptation, with the prospect of using the constructs in the study of therapies for the treatment of urethral strictures.

Sources, Selection, and Microenvironmental Preconditioning of Cells for Urethral Tissue Engineering.

Urethral stricture is a common urinary tract disorder in men that can be caused by iatrogenic causes, trauma, inflammation, or infection and often requires reconstructive surgery. The current therapeutic approach for complex urethral strictures usually involves reconstruction with autologous tissue from the oral mucosa. With the goal of overcoming the lack of sufficient autologous tissue and donor site morbidity, research over the past two decades has focused on cell-based tissue-engineered substitutes. While the main focus has been on autologous cells from the penile tissue, bladder, and oral cavity, stem cells from sources such as adipose tissue and urine are competing candidates for future urethral regeneration due to their ease of collection, high proliferative capacity, maturation potential, and paracrine function. This review addresses the sources, advantages, and limitations of cells for tissue engineering in the urethra and discusses recent approaches to improve cell survival, growth, and differentiation by mimicking the mechanical and biophysical properties of the extracellular environment.

Distinct Dominant Lineage from In Vitro Expanded Adipose-Derived Stem Cells (ASCs) Exhibits Enhanced Wound Healing Properties.

It has been suggested that immunophenotypically defined lineages within the in vitro expanded adipose-derived stem cell (ASC) may play a beneficial role from the perspective of a personalized intervention. Therefore, to better understand the implications of different surface marker profiles for the functionality, we set out to examine the evolution of ASC-variants based on the co-expression of five bright or eight dim epitopes. At passages P1, P4, and P8, the co-localization of five bright markers (CD73, CD90, CD105, CD166, and CD201), or eight dim markers (CD34, CD36, CD200, CD248, CD271, CD274, CD146, and the Stro-1), was investigated by flow cytometry. Selected subpopulations were isolated using the fluorescence-activated cells sorting from the cryopreserved P4 and analyzed in terms of proliferative and clonogenic properties, trilineage differentiation, and wound healing potential. Only two of the dim epitopes were found in representative subpopulations (SP), and from the P4 onwards, two major combinations featuring the CD274+ (SP1) or the CD274+ CD146+ (SP2) emerged. Upon sorting and growth, both subpopulations assumed new but highly similar clonal profiles, consisting of the CD274+ CD146+ and the CD274+ CD146+ CD248+ phenotypes. The functional analysis revealed that the SP2 surpassed SP1 and the unfractionated cells regarding the growth rate, clonogenic activity, and the wound closure and endothelial tube formation potential. The surface epitopes may be considered a tool to enrich specific functionality and thus improve therapeutic outcomes in dedicated circumstances.

Multiplex Analysis of Adipose-Derived Stem Cell (ASC) Immunophenotype Adaption to In Vitro Expansion.

In order to enhance the therapeutic potential, it is important that sufficient knowledge regarding the dynamic changes of adipose-derived stem cell (ASC) immunophenotypical and biological properties during in vitro growth is available. Consequently, we embarked on a study to follow the evolution of highly defined cell subsets from three unrelated donors in the course of eight passages on tissue culture polystyrene. The co-expression patterns were defined by panels encompassing seven and five cell surface markers, including CD34, CD146, CD166, CD200, CD248, CD271, and CD274 and CD29, CD31, CD36, CD201, and Stro-1, respectively. The analysis was performed using multichromatic flow cytometry. We observed a major paradigm shift, where the CD166-CD34+ combination which was found across all cell subsets early in the culture was replaced by the CD166+ phenotype as the population homogeneity increased with time. At all analysis points, the cultures were dominated by a few major clones that were highly prevalent in most of the donors. The selection process resulted in two predominant clones in the larger panel (CD166+CD34-CD146-CD271- CD274-CD248-CD200- and CD166+CD34+ CD146-CD271-CD274-CD248-CD200-) and one clone in the smaller panel (CD29+CD201+CD36- Stro-1- CD31-). The minor subsets, including CD166+CD34-CD146-CD271+CD274-CD248-CD200- and CD166+CD34+CD146+CD271-CD274-CD248-CD200-, and CD29+CD201-CD36-Stro-1-CD31-, CD29+CD201+CD36-Stro-1+CD31-, and CD29+CD201+CD36+Stro-1-CD31-, in the seven and five marker panels, respectively, were, on the other, hand highly fluctuating and donor-dependent. The results demonstrate that only a limited number of phenotypical repertoires are possible in ASC cultures. Marked differences in their relative occurrence between distinct individuals underscore the need for potency standardization of different ASC preparation to improve the clinical outcome.