ZAKß is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle.

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.

Mechanotransduction in tissue engineering: Insights into the interaction of stem cells with biomechanical cues.

Stem cells in their natural microenvironment are exposed to biochemical and biophysical cues emerging from the extracellular matrix (ECM) and neighboring cells. In particular, biomechanical forces modulate stem cell behavior, biological fate, and early developmental processes by sensing, interpreting, and responding through a series of biological processes known as mechanotransduction. Local structural changes in the ECM and mechanics are driven by reciprocal activation of the cell and the ECM itself, as the initial deposition of matrix proteins sequentially affects neighboring cells. Recent studies on stem cell mechanoregulation have provided insight into the importance of biomechanical signals on proper tissue regeneration and function and have shown that precise spatiotemporal control of these signals exists in stem cell niches. Against this background, the aim of this work is to review the current understanding of the molecular basis of mechanotransduction by analyzing how biomechanical forces are converted into biological responses via cellular signaling pathways. In addition, this work provides an overview of advanced strategies using stem cells and biomaterial scaffolds that enable precise spatial and temporal control of mechanical signals and offer great potential for the fields of tissue engineering and regenerative medicine will be presented.

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.

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.

Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells.

Chronic non-healing wounds are detrimental for the quality of life of the affected individuals and represent a major burden for the health care systems. Adipose-derived stem cells (ASCs) are being investigated for the development of novel treatments of chronic wounds, as they have shown several positive effects on wound healing. While these effects appear to be mediated by the release of soluble factors, it is has also become apparent that the extracellular matrix (ECM) deposited by ASCs is essential in several phases of the wound healing process. In this work, we describe an approach to produce ECM scaffolds derived from ASCs in culture. Upon growth of ASCs into an overconfluent cell layer, a detergent-based cell extraction approach is applied to remove the cellular components. The extraction is followed by an enzymatic treatment to remove the residual DNA. The resultant cell-derived scaffolds are depleted of cellular components, display low DNA remnant, and retain the native fibrillar organization of the ECM. Analysis of the molecular composition of the ECM scaffolds revealed that they are composed of collagens type I and III, and fibronectin. The decellularized scaffolds represent a substrate that supports adhesion and proliferation of primary human fibroblasts and dermal microvascular endothelial cells, indicating their potential as platforms for wound healing studies.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells.

Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

Biomaterials for Regenerative Medicine: Historical Perspectives and Current Trends.

Biomaterials are key components in tissue engineering and regenerative medicine applications, with the intended purpose of reducing the burden of disease and enhancing the quality of life of a large number of patients. The success of many regenerative medicine strategies, such as cell-based therapies, artificial organs, and engineered living tissues, is highly dependent on the ability to design or produce suitable biomaterials that can support and guide cells during tissue healing and remodelling processes. This chapter presents an overview about basic research concerning the use of different biomaterials for tissue engineering and regenerative medicine applications. Starting from a historical perspective, the chapter introduces the basic principles of designing biomaterials for tissue regeneration approaches. The main focus is set on describing the main classes of biomaterials that have been applied in regenerative medicine, including natural and synthetic polymers, bioactive ceramics, and composites. For each class of biomaterials, some of the most important physicochemical and biological properties are presented. Finally, some challenges and concerns that remain in this field are presented and discussed.

Bioengineered Scaffolds for Stem Cell Applications in Tissue Engineering and Regenerative Medicine.

Stem cell-based therapies, harnessing the ability of stem cells to regenerate damaged or diseased tissues, are under wide-ranging consideration for regenerative medicine applications. However, limitations concerning poor cell persistence and engraftment upon cell transplantation still remain. During the recent years, several types of biomaterials have been investigated to control the fate of the transplanted stem cells, aiming to increase their therapeutic efficiency. In the present chapter we focus on the general properties of some of these biomaterials, which include polymers, ceramics, and nano-biomaterials. In the first part of the chapter, a brief explanation about stem cell biology, sources, and their microenvironment is provided. The second part of the chapter presents some of the most recent studies investigating different types of biomaterials and approaches that aim to mimic the stem cell microenvironment for a more precise control of the stem cell fate.

Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype.

Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01). Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01). Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

Implications of Extracellular Matrix Production by Adipose Tissue-Derived Stem Cells for Development of Wound Healing Therapies.

The synthesis and deposition of extracellular matrix (ECM) plays an important role in the healing of acute and chronic wounds. Consequently, the use of ECM as treatment for chronic wounds has been of special interest-both in terms of inducing ECM production by resident cells and applying ex vivo produced ECM. For these purposes, using adipose tissue-derived stem cells (ASCs) could be of use. ASCs are recognized to promote wound healing of otherwise chronic wounds, possibly through the reduction of inflammation, induction of angiogenesis, and promotion of fibroblast and keratinocyte growth. However, little is known regarding the importance of ASC-produced ECM for wound healing. In this review, we describe the importance of ECM for wound healing, and how ECM production by ASCs may be exploited in developing new therapies for the treatment of chronic wounds.

Resistance to protein adsorption and adhesion of fibroblasts on nanocrystalline diamond films: the role of topography and boron doping.

Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes.

Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes.

Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

Exploring the Role of Hyaluronic Acid in Reproductive Biology and Beyond: Applications in Assisted Reproduction and Tissue Engineering.

Hyaluronic acid (HA) plays a prominent role in various aspects of reproductive biology and assisted reproductive technologies (ART). This review describes the multifaceted influence of HA, ranging from primordial germ cell migration, ovarian follicle development, and ovulation in females to sperm structure, physiology, motility, and capacitation in males. In addition, HA also plays an important role in fertilization and promotes embryo implantation by mediating cellular adhesion and communication within the uterus. Against this physiological background, the review examines the current applications of HA in the context of ART. In addition, the article addresses the emerging field of reproductive tissue engineering, where HA-based hydrogels offer promising perspectives as they can support the development of mature oocytes and spermatogenesis in vitro. Overall, this review highlights the integral role of HA in the intricate mechanisms of reproductive biology and its growing importance for improving ART outcomes and the field of tissue engineering of the reproductive system.

Engineering Photo-Cross-Linkable MXene-Based Hydrogels: Durable Conductive Biomaterials for Electroactive Tissues and Interfaces.

Light-cured conductive hydrogels have attracted immense interest in the regeneration of electroactive tissues and bioelectronic interfaces. Despite the unique properties of MXene (MX), its light-blocking effect in the range of 300-600 nm hinders the efficient cross-linking of photocurable hydrogels. In this study, we investigated the photo-cross-linking process of MX-gelatin methacrylate (GelMa) composites with different types of photoinitiators and MX concentrations to prepare biocompatible, injectable, conductive, and photocurable composite hydrogels. The examined photoinitiators were Eosin Y, Irgacure 2959 (Type I), and lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (Type II). The light-blocking effect of MX strongly affected the thickness, pore structure, swelling ratio, degradation, and mechanical properties of the light-cured hydrogels. Uniform distribution of MX in the hydrogel matrix was achieved at concentrations up to 0.04 wt % but the film thickness and curing times varied depending on the type of photoinitiator. It was feasible to prepare thin films (0.5 mm) by employing Type I photoinitiators under a relatively long light irradiation (4-5 min) while thick films with centimeter sizes could be rapidly cured by using Type II photoinitiator (<60 s). The mechanical properties, including elastic modulus, toughness, and stress to break for the Type II hydrogels were significantly superior (up to 300%) to those of Type I hydrogels depending on the MX concentration. The swelling ratio was also remarkably higher (648-1274%). A conductivity of about 1 mS/cm was attained at 0.1 mg/mL MX for the composite hydrogel cured by the Type I photoinitiator. In vitro cytocompatibility assays determined that the hydrogels promoted cell viability, metabolic activity, and robust proliferation of C2C12 myoblasts, which indicated their potential to support muscle cell growth during myogenesis. The developed photocurable GelMa-MX hydrogels have the potential to serve as bioactive and conductive scaffolds to modulate cellular functions and for tissue-device interfacing.

Enhancing volumetric muscle loss (VML) recovery in a rat model using super durable hydrogels derived from bacteria.

Bacteria can be programmed to deliver natural materials with defined biological and mechanical properties for controlling cell growth and differentiation. Here, we present an elastic, resilient and bioactive polysaccharide derived from the extracellular matrix of Pantoea sp. BCCS 001. Specifically, it was methacrylated to generate a new photo crosslinkable hydrogel that we coined Pantoan Methacrylate or put simply PAMA. We have used it for the first time as a tissue engineering hydrogel to treat VML injuries in rats. The crosslinked PAMA hydrogel was super elastic with a recovery nearing 100 %, while mimicking the mechanical stiffness of native muscle. After inclusion of thiolated gelatin via a Michaelis reaction with acrylate groups on PAMA we could also guide muscle progenitor cells into fused and aligned tubes - something reminiscent of mature muscle cells. These results were complemented by sarcomeric alpha-actinin immunostaining studies. Importantly, the implanted hydrogels exhibited almost 2-fold more muscle formation and 50 % less fibrous tissue formation compared to untreated rat groups. In vivo inflammation and toxicity assays likewise gave rise to positive results confirming the biocompatibility of this new biomaterial system. Overall, our results demonstrate that programmable polysaccharides derived from bacteria can be used to further advance the field of tissue engineering. In greater detail, they could in the foreseeable future be used in practical therapies against VML.

Electroconductive Nanofibrous Scaffolds Enable Neuronal Differentiation in Response to Electrical Stimulation without Exogenous Inducing Factors.

Among the various biochemical and biophysical inducers for neural regeneration, electrical stimulation (ES) has recently attracted considerable attention as an efficient means to induce neuronal differentiation in tissue engineering approaches. The aim of this in vitro study was to develop a nanofibrous scaffold that enables ES-mediated neuronal differentiation in the absence of exogenous soluble inducers. A nanofibrous scaffold composed of polycaprolactone (PCL), poly-L-lactic acid (PLLA), and single-walled nanotubes (SWNTs) was fabricated via electrospinning and its physicochemical properties were investigated. The cytocompatibility of the electrospun composite with the PC12 cell line and bone marrow-derived mesenchymal stem cells (BMSCs) was investigated. The results showed that the PCL/PLLA/SWNT nanofibrous scaffold did not exhibit cytotoxicity and supported cell attachment, spreading, and proliferation. ES was applied to cells cultured on the nanofibrous scaffolds at different intensities and the expression of the three neural markers (Nestin, Microtubule-associated protein 2, and ß tubulin-3) was evaluated using RT-qPCR analysis. The results showed that the highest expression of neural markers could be achieved at an electric field intensity of 200 mV/cm, suggesting that the scaffold in combination with ES can be an efficient tool to accelerate neural differentiation in the absence of exogenous soluble inducers. This has important implications for the regeneration of nerve injuries and may provide insights for further investigations of the mechanisms underlying ES-mediated neuronal commitment.

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.

Exploring the Potential of Ultrasound Therapy to Reduce Skin Scars: An In Vitro Study Using a Multi-Well Device Based on Printable Piezoelectric Transducers.

Excessive skin scarring affects over 100 million patients worldwide, with effects ranging from cosmetic to systemic problems, and an effective treatment is yet to be found. Ultrasound-based therapies have been used to treat a variety of skin disorders, but the exact mechanisms behind the observed effects are still unclear. The aim of this work was to demonstrate the potential of ultrasound for the treatment of abnormal scarring by developing a multi-well device based on printable piezoelectric material (PiezoPaint™). First, compatibility with cell cultures was evaluated using measurements of heat shock response and cell viability. Second, the multi-well device was used to treat human fibroblasts with ultrasound and quantify their proliferation, focal adhesions, and extracellular matrix (ECM) production. Ultrasound caused a significant reduction in fibroblast growth and ECM deposition without changes in cell viability or adhesion. The data suggest that these effects were mediated by nonthermal mechanisms. Interestingly, the overall results suggest that ultrasound treatment would a be beneficial therapy for scar reduction. In addition, it is expected that this device will be a useful tool for mapping the effects of ultrasound treatment on cultured cells.

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 [...].

Recent progress in the manipulation of biochemical and biophysical cues for engineering functional tissues.

Tissue engineering (TE) is currently considered a cutting-edge discipline that offers the potential for developing treatments for health conditions that negatively affect the quality of life. This interdisciplinary field typically involves the combination of cells, scaffolds, and appropriate induction factors for the regeneration and repair of damaged tissue. Cell fate decisions, such as survival, proliferation, or differentiation, critically depend on various biochemical and biophysical factors provided by the extracellular environment during developmental, physiological, and pathological processes. Therefore, understanding the mechanisms of action of these factors is critical to accurately mimic the complex architecture of the extracellular environment of living tissues and improve the efficiency of TE approaches. In this review, we recapitulate the effects that biochemical and biophysical induction factors have on various aspects of cell fate. While the role of biochemical factors, such as growth factors, small molecules, extracellular matrix (ECM) components, and cytokines, has been extensively studied in the context of TE applications, it is only recently that we have begun to understand the effects of biophysical signals such as surface topography, mechanical, and electrical signals. These biophysical cues could provide a more robust set of stimuli to manipulate cell signaling pathways during the formation of the engineered tissue. Furthermore, the simultaneous application of different types of signals appears to elicit synergistic responses that are likely to improve functional outcomes, which could help translate results into successful clinical therapies in the future.