UV fluorescence excitation spectroscopy as a noninvasive predictor of epidermal proliferation and clinical performance of cosmetic formulations.

BACKGROUND: The epidermis is the outermost layer of skin and is composed of cells primarily containing keratin. It consists of about ten layers of living cells (keratinocytes) and ten layers of dead cells (corneocytes). Thinning of the epidermis and decreased proliferation of its cells are associated with aging related changes in skin, including wrinkling and laxity. Fluorescence excitation spectroscopy is a noninvasive method of monitoring characteristic excitation-emission peaks in skin that have been related to the epidermal and dermal composition. The magnitude of the peak that occurs at 295nm excitation (F295) has been linked to changes in epidermal thickness, proliferation, and skin aging. AIM: The goal of this study is to correlate changes in the F295 signal with proliferation of cells and thickening of the epidermis induced by cosmetic formulations. We hypothesize that two commonly used cosmetic ingredients, retinol and glycolic acid, will increase these markers that have been implicated in skin anti-aging. METHODS: In a placebo-controlled study subjects' forearms were treated with formulations containing retinol or glycolic acid under occlusive patch for a period of 21 days. Skin fluorescence was measured at baseline and after treatment, and biopsies were taken following treatment for histological analysis of epidermal thickness and cell proliferation. RESULTS: After 21 days of treatment retinol and glycolic acid formulas significantly increased F295 (by 265.1±33.5% and 162.2±18.7% respectively), whereas the placebo control formula did not induce a change from baseline. Furthermore, retinol and glycolic acid treatments significantly increased epidermal thickness (by 63.1% and 7.8% respectively) and keratinocyte proliferation (by 236.9% and 62.8% respectively) versus placebo control. CONCLUSION: Increases in F295 were found to correlate with epidermal renewal, but more so with increased cell proliferation than epidermal thickness. We conclude that the F295 signal is a fast and reliable early indicator of epidermal remodeling in skin that can be used to distinguish between formulations with different cosmetic ingredients.

Technical approaches to select high-performance instant skin smoothing formulations: Correlation of in vitro and in vivo assessment methods.

BACKGROUND: Contractile films that smooth the surface of skin upon drying are popular among consumers due to their "instant" effect and perceivable smoothing benefits. The objective of our study was to correlate an in vitro measurement of contractile force with in vivo smoothing performance, thereby enabling rapid screening of film-forming technologies for impactful cosmetic results. METHODS: We introduce and characterize an in vitro method to measure drying stress of film-containing formulations. This method is used to measure the drying stresses of seven different cosmetic film formulations. We then evaluate these formulas in a blinded clinical study, measuring their effect on under-eye and Crow's Feet area smoothing through bioinstrumentation (3D PRIMOS imaging) and blinded expert grading of images. RESULTS: The in vitro drying stress measurement was found to be repeatable and sensitive enough to detect differences between formulations with typical amounts of film-forming agents. Significant correlation was found between the in vitro drying stress measurements and under-eye smoothing measured by 3D imaging (R2  = 0.71). Expert grading confirmed that film formulas deliver perceivable smoothing in the under-eye and Crow's Feet regions 15 minutes after application. CONCLUSION: The in vitro method described here can be used to predict the efficacy of formulations that deliver smoothing benefits to consumers. For consumer use, the esthetic properties of a formula should be balanced with film performance, guided by this model which predicts skin smoothing efficacy.

miR-146a is a critical target associated with multiple biological pathways of skin aging.

Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm-associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health.

Surface-patterned electrode bioreactor for electrical stimulation.

We present a microscale cell culture system with an interdigitated microarray of excimer-laser-ablated indium tin oxide electrodes for electrical stimulation of cultured cells. The system has been characterized in a range of geometeries and stimulation regimes via electrochemical impedance spectroscopy and used to culture primary cardiomyocytes and human adipose derived stem cells. Over 6 days of culture with electrical stimulation (2 ms duration, 1 Hz, 180 microm wide electrodes with 200 microm spacing), both cell types exhibited enhanced proliferation, elongation and alignment, and adipose derived stem cells exhibited higher numbers of Connexin-43-composed gap junctions.

Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair.

Cell-based therapies offer a promising approach to treat intervertebral disc (IVD) degeneration. The impact of the injury microenvironment on treatment efficacy has not been established. This study used a rat disc stab injury model with administration of mesenchymal stromal cells (MSCs) at 3, 14, or 30 days post injury (DPI) to evaluate the impact of interventional timing on IVD biochemistry and biomechanics. We also evaluated cellular localization within the disc with near infrared imaging to track the time and spatial profile of cellular migration after in vivo delivery. Results showed that upon injection into a healthy disc, MSCs began to gradually migrate outwards over the course of 14 days, as indicated by decreased signal intensity from the disc space and increased signal within the adjacent vertebrae. Cells administered 14 or 30 DPI also tended to migrate out 14 days after injection but cells injected 3 DPI were retained at a significantly higher amount versus the other groups (p < 0.05). Correspondingly the 3 DPI group, but not 14 or 30 DPI groups, had a higher GAG content in the MSC injected discs (p = 0.06). Enrichment of MSCs and increased GAG content in 3 DPI group did not lead to increased compressive biomechanical properties. Findings suggest that cell therapies administered at an early stage of injury/disease progression may have greater chances of mitigating matrix loss, possibly through promotion of MSC activity by the inflammatory microenvironment associated with injury. Future studies will evaluate the mode and driving factors that regulate cellular migration out of the disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:32-40, 2017.

Developments in intervertebral disc disease research: pathophysiology, mechanobiology, and therapeutics.

Low back pain is a leading cause of disability worldwide and the second most common cause of physician visits. There are many causes of back pain, and among them, disc herniation and intervertebral disc degeneration are the most common diagnoses and targets for intervention. Currently, clinical treatment outcomes are not strongly correlated with diagnoses, emphasizing the importance for characterizing more completely the mechanisms of degeneration and their relationships with symptoms. This review covers recent studies elucidating cellular and molecular changes associated with disc mechanobiology, as it relates to degeneration and regeneration. Specifically, we review findings on the biochemical changes in disc diseases, including cytokines, chemokines, and proteases; advancements in disc disease diagnostics using imaging modalities; updates on studies examining the response of the intervertebral disc to injury; and recent developments in repair strategies, including cell-based repair, biomaterials, and tissue engineering. Findings on the effects of the omega-6 fatty acid, linoleic acid, on nucleus pulposus tissue engineering are presented. Studies described in this review provide greater insights into the pathogenesis of disc degeneration and may define new paradigms for early or differential diagnostics of degeneration using new techniques such as systemic biomarkers. In addition, research on the mechanobiology of disease enriches the development of therapeutics for disc repair, with potential to diminish pain and disability associated with disc degeneration.

Inflammation induces irreversible biophysical changes in isolated nucleus pulposus cells.

Intervertebral disc degeneration is accompanied by elevated levels of inflammatory cytokines that have been implicated in disease etiology and matrix degradation. While the effects of inflammatory stimulation on disc cell metabolism have been well-studied, their effects on cell biophysical properties have not been investigated. The hypothesis of this study is that inflammatory stimulation alters the biomechanical properties of isolated disc cells and volume responses to step osmotic loading. Cells from the nucleus pulposus (NP) of bovine discs were isolated and treated with either lipopolysaccharide (LPS), an inflammatory ligand, or with the recombinant cytokine TNF-α for 24 hours. We measured cellular volume regulation responses to osmotic loading either immediately after stimulation or after a 1 week recovery period from the inflammatory stimuli. Cells from each group were tested under step osmotic loading and the transient volume-response was captured via time-lapse microscopy. Volume-responses were analyzed using mixture theory framework to investigate two biomechanical properties of the cell, the intracellular water content and the hydraulic permeability. Intracellular water content did not vary between treatment groups, but hydraulic permeability increased significantly with inflammatory treatment. In the 1 week recovery group, hydraulic permeability remained elevated relative to the untreated recovery control. Cell radius was also significantly increased both after 24 hours of treatment and after 1 week recovery. A significant linear correlation was observed between hydraulic permeability and cell radius in untreated cells at 24 hours and at 1-week recovery, though not in the inflammatory stimulated groups at either time point. This loss of correlation between cell size and hydraulic permeability suggests that regulation of volume change is disrupted irreversibly due to inflammatory stimulation. Inflammatory treated cells exhibited altered F-actin cytoskeleton expression relative to untreated cells. We also found a significant decrease in the expression of aquaporin-1, the predominant water channel in disc NP cells, with inflammatory stimulation. To our knowledge, this is the first study providing evidence that inflammatory stimulation directly alters the mechanobiology of NP cells. The cellular biophysical changes observed in this study are coincident with documented changes in the extracellular matrix induced by inflammation, and may be important in disease etiology.

Toll-Like Receptor 4 (TLR4) expression and stimulation in a model of intervertebral disc inflammation and degeneration.

STUDY DESIGN: We measured the expression and responses of Toll-Like Receptor 4 (TLR4) activation in the intervertebral disc (IVD) in vitro and in vivo. We hypothesize that stimulation of the IVD with the TLR4 ligand lipopolysaccharide (LPS) results in upregulation of a coordinated set of proinflammatory mediators and inhibition of matrix expression, both consistent with a molecular profile of degeneration. OBJECTIVE: To characterize early inflammatory and morphological changes induced by TLR4 activation in the IVD. SUMMARY OF BACKGROUND DATA: TLR4 is a pattern recognition receptor activated in innate immunity that has been implicated in disease mechanisms of inflammatory cartilaginous degeneration. However, no study to date has examined the expression and responses of TLR4 in the IVD. METHODS: IVD cells were stimulated with LPS in a dose-dependent manner, and inflammatory cytokine levels were measured by quantitative reverse transcription-polymerase chain reaction. Histological and inflammatory changes due to in vivo injection of LPS into the rat caudal IVD were measured by enzyme-linked immunosorbent assay and immunoblotting. RESULTS: Baseline TLR4 expression in IVD tissue varied according to cell type. LPS stimulation resulted in significant increases in tumor necrosis factor α (TNF)-α, interleukin (IL)-1ß, IL-6, and nitric oxide levels and significant inhibition in aggrecan and collagen-2. Intradiscal injection of LPS was found to cause moderate degenerative changes in the IVD, with increases in tissue levels of IL-1ß, TNF-α, high mobility group box 1 protein (HMGB1), and macrophage migration inhibitory factor (MIF). CONCLUSION: This study provides the first evidence that IVD cells express TLR4 and are responsive to TLR4 activation by upregulating a coordinated set of inflammatory cytokines. This study suggests that intradiscal injection of LPS offers a model for triggering inflammation of the IVD, demonstrating that inflammatory insults alone may potentially trigger degenerative changes of the IVD.

The effect of controlled expression of VEGF by transduced myoblasts in a cardiac patch on vascularization in a mouse model of myocardial infarction.

Key requirements for cardiac tissue engineering include the maintenance of cell viability and function and the establishment of a perfusable vascular network in millimeters thick and compact cardiac constructs upon implantation. We investigated if these requirements can be met by providing an intrinsic vascularization stimulus (via sustained action of VEGF secreted at a controlled rate by transduced myoblasts) to a cardiac patch engineered under conditions of effective oxygen supply (via medium flow through channeled elastomeric scaffolds seeded with neonatal cardiomyocytes). We demonstrate that this combined approach resulted in increased viability, vascularization and functionality of the cardiac patch. After implantation in a mouse model of myocardial infarction, VEGF-expressing patches displayed significantly improved engraftment, survival and differentiation of cardiomyocytes, leading to greatly enhanced contractility as compared to controls not expressing VEGF. Controlled VEGF expression also mediated the formation of mature vascular networks, both within the engineered patches and in the underlying ischemic myocardium. We propose that this combined cell-biomaterial approach can be a promising strategy to engineer cardiac patches with intrinsic and extrinsic vascularization potential.

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue.

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction, driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue but, due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation and unconstrained (i.e. not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate these three key factors in concert. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modelling studies. We then cultured cardiac cells obtained from neonatal rats in porous, channelled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After 8 days of culture, constructs grown with simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23 ± 0.10% vs 0.14 ± 0.05%, 0.13 ± 0.08% or 0.09 ± 0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization compared to control groups. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs.

Channelled scaffolds for engineering myocardium with mechanical stimulation.

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan-collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1-2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs.

Emerging trends in biological therapy for intervertebral disc degeneration.

Intervertebral disc disease is characterized by a series of deleterious changes in cellularity that lead to loss of extracellular matrix structure, altered biomechanical loading, and symptomatic pain. At present the "gold standard" of therapy is discectomy -- surgical removal of the diseased disc followed by fusion of the adjacent vertebral bodies. The procedure alleviates pain, but fusion limits range of motion and alters the mechanical loading at other spinal levels, hastening disease at previously unaffected sites. Biological therapeutics have the potential to repair damaged tissue by several means: (1) altering cell phenotype to regenerate matrix components, (2) augmenting tissue with reparative cells, (3) delivering bioactive materials to reestablish disc biomechanics and serve as a template for cell-based regeneration. Although research into biological treatments for disc degeneration has been ongoing for over a decade, few treatments have progressed to clinical testing and none are currently commercially available, primarily due to a limited understanding of disease etiology. Further work is needed to identify targets and interventional time points as disc degeneration progresses from early to later stages. This review focuses on emerging trends in biological treatments and identifies key obstacles to their clinical translation.

Optimization of electrical stimulation parameters for cardiac tissue engineering.

In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile engineered cardiac tissues. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge-injection capacity and producing cardiac tissues with the best structural and contractile properties, were thus used in tissue engineering studies. Engineered cardiac tissues stimulated at 3 V/cm amplitude and 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and connexin-43 and the best-developed contractile behaviour. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering.

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering.

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% +/- 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% +/- 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells.

Challenges in cardiac tissue engineering.

Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Engineered constructs can also serve as high-fidelity models for studies of cardiac development and disease. In a general case, the biological potential of the cell-the actual "tissue engineer"-is mobilized by providing highly controllable three-dimensional environments that can mediate cell differentiation and functional assembly. For cardiac regeneration, some of the key requirements that need to be met are the selection of a human cell source, establishment of cardiac tissue matrix, electromechanical cell coupling, robust and stable contractile function, and functional vascularization. We review here the potential and challenges of cardiac tissue engineering for developing therapies that could prevent or reverse heart failure.

Scaffold stiffness affects the contractile function of three-dimensional engineered cardiac constructs.

We investigated the effects of the initial stiffness of a three-dimensional elastomer scaffold--highly porous poly(glycerol sebacate)--on functional assembly of cardiomyocytes cultured with perfusion for 8 days. The polymer elasticity varied with the extent of polymer cross-links, resulting in three different stiffness groups, with compressive modulus of 2.35 ± 0.03 (low), 5.28 ± 0.36 (medium), and 5.99 ± 0.40 (high) kPa. Laminin coating improved the efficiency of cell seeding (from 59 ± 15 to 90 ± 21%), resulting in markedly increased final cell density, construct contractility, and matrix deposition, likely because of enhanced cell interaction and spreading on scaffold surfaces. Compact tissue was formed in the low and medium stiffness groups, but not in the high stiffness group. In particular, the low stiffness group exhibited the greatest contraction amplitude in response to electric field pacing, and had the highest compressive modulus at the end of culture. A mathematical model was developed to establish a correlation between the contractile amplitude and the cell distribution within the scaffold. Taken together, our findings suggest that the contractile function of engineered cardiac constructs positively correlates with low compressive stiffness of the scaffold.

Electrical stimulation systems for cardiac tissue engineering.

We describe a protocol for tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cells with the application of pulsatile electrical fields designed to mimic those present in the native heart. Tissue culture is conducted in a customized chamber built to allow for cultivation of (i) engineered three-dimensional (3D) cardiac tissue constructs, (ii) cell monolayers on flat substrates or (iii) cells on patterned substrates. This also allows for analysis of the individual and interactive effects of pulsatile electrical field stimulation and substrate topography on cell differentiation and assembly. The protocol is designed to allow for delivery of predictable electrical field stimuli to cells, monitoring environmental parameters, and assessment of cell and tissue responses. The duration of the protocol is 5 d for two-dimensional cultures and 10 d for 3D cultures.

Cardiac tissue engineering using perfusion bioreactor systems.

This protocol describes tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cell populations on porous scaffolds (in some cases with an array of channels) and bioreactors with perfusion of culture medium (in some cases supplemented with an oxygen carrier). The overall approach is 'biomimetic' in nature as it tends to provide in vivo-like oxygen supply to cultured cells and thereby overcome inherent limitations of diffusional transport in conventional culture systems. In order to mimic the capillary network, cells are cultured on channeled elastomer scaffolds that are perfused with culture medium that can contain oxygen carriers. The overall protocol takes 2-4 weeks, including assembly of the perfusion systems, preparation of scaffolds, cell seeding and cultivation, and on-line and end-point assessment methods. This model is well suited for a wide range of cardiac tissue engineering applications, including the use of human stem cells, and high-fidelity models for biological research.