Fabrication of polycaprolactone electrospun fibres with retinyl acetate for antioxidant delivery in a ROS-mimicking environment.

Background: Increased cancer rates denote that one in two people will be diagnosed with cancer in their lifetime. Over 60% of cancer patients receive radiotherapy, either as a stand-alone treatment or in combination with other treatments such as chemotherapy and surgery. Whilst radiotherapy is effective in destroying cancer cells, it also causes subsequent damage to healthy cells and surrounding tissue due to alterations in the tumor microenvironment and an increase in reactive oxygen species (ROS). This can cause extensive damage that impairs tissue function, and the likelihood of tissue regeneration and restoration of function is significantly reduced as new healthy cells cannot survive in the damaged environment. In the treatment of head and neck cancers, radiotherapy can cause salivary gland dysfunction. This significantly impairs the patient's quality of life and there is currently no cure, only palliative treatment options. Tissue engineering approaches are used to mimic the microenvironment of the tissue and can mediate the damaged microenvironment via bioactive compounds, to support the delivery, survival, and proliferation of new, healthy cells into the damaged environment. Methods: In this study, retinyl acetate, a derivative of vitamin A, was successfully incorporated into electrospun polycaprolactone fibres. Results: SEM images and characterization analyses showed that all scaffolds produced had similar characteristics, including fiber morphology and scaffold wettability. The vitamin scaffolds were shown to exert an antioxidant effect through scavenging activity of both DPPH and hydroxyl radicals in vitro. Critically, the antioxidant scaffolds supported the growth of human submandibular gland cells and significantly upregulated the expression of GPx1, an antioxidant enzyme, when cultured under both normal conditions and under a simulated oxidative stress environment. Discussion: These results suggest that incorporation of retinyl acetate into electrospun fibres has may mediate the damaged microenvironment post cancer radiation therapy.

A Unification of Nanotopography and Extracellular Matrix in Electrospun Scaffolds for Bioengineered Hepatic Models.

Donor liver shortage is a crucial global public health problem as whole-organ transplantation is the only definitive cure for liver disease. Liver tissue engineering aims to reproduce or restore function through in vitro tissue constructs, which may lead to alternative treatments for active and chronic liver disease. The formulation of a multifunctional scaffold that has the potential to mimic the complex extracellular matrix (ECM) and their influence on cellular behavior, are essential for culturing cells on a construct. The separate employment of topographic or biological cues on a scaffold has both shown influences on hepatocyte survival and growth. In this study, we investigate both of these synergistic effects and developed a new procedure to directly blend whole-organ vascular perfusion-decellularized rat liver ECM (dECM) into electrospun fibers with tailored surface nanotopography. Water contact angle, tensile test, and degradation studies were conducted to analyze scaffold hydrophilicity, mechanical properties, and stability. The results show that our novel hybrid scaffolds have enhanced hydrophilicity, and the nanotopography retained its original form after hydrolytic degradation for 14 days. Human hepatocytes (HepG2) were seeded to analyze the scaffold biocompatibility. Cell viability and DNA quantification imply steady cell proliferation over the culture period, with the highest albumin secretion observed on the hybrid scaffold. Scanning electron microscopy shows that cell morphology was distinctly different on hybrid scaffolds compared to control groups, where HepG2 began to form a monolayer toward the end of the culture period; meanwhile, typical hepatic markers and ECM genes were also influenced, such as an increasing trend of albumin appearing on the hybrid scaffolds. Taken together, our findings provide a reproducible approach and utilization of animal tissue-derived ECM and emphasize the synergism of topographical stimuli and biochemical cues on electrospun scaffolds in liver tissue engineering.

Recent Methods for Modifying Mechanical Properties of Tissue-Engineered Scaffolds for Clinical Applications.

The limited regenerative capacity of the human body, in conjunction with a shortage of healthy autologous tissue, has created an urgent need for alternative grafting materials. A potential solution is a tissue-engineered graft, a construct which supports and integrates with host tissue. One of the key challenges in fabricating a tissue-engineered graft is achieving mechanical compatibility with the graft site; a disparity in these properties can shape the behaviour of the surrounding native tissue, contributing to the likelihood of graft failure. The purpose of this review is to examine the means by which researchers have altered the mechanical properties of tissue-engineered constructs via hybrid material usage, multi-layer scaffold designs, and surface modifications. A subset of these studies which has investigated the function of their constructs in vivo is also presented, followed by an examination of various tissue-engineered designs which have been clinically translated.

A vertical additive-lathe printing system for the fabrication of tubular constructs using gelatin methacryloyl hydrogel.

Reproducing both the mechanical and biological performance of native blood vessels remains an ongoing challenge in vascular tissue engineering. Additive-lathe printing offers an attractive method of fabricating long tubular constructs as a potential vascular graft for the treatment of cardiovascular diseases. Printing hydrogels onto rotating horizontal mandrels often leads to sagging, resulting in poor and variable mechanical properties. In this study, an additive-lathe printing system with a vertical mandrel to fabricate tubular constructs is presented. Various concentrations of gelatin methacryloyl (gelMA) hydrogel were used to print grafts on the rotating mandrel in a helical pattern. The printing parameters were selected to achieve the bonding of consecutive gelMA filaments to improve the quality of the printed graft. The hydrogel filaments were fused properly under the action of gravity on the vertical mandrel. Thus, the vertical additive-lathe printing system was used to print uniform wall thickness grafts, eliminating the hydrogel sagging problem. Tensile testing performed in both circumferential and longitudinal direction revealed that the anisotropic properties of printed gelMA constructs were similar to those observed in the native blood vessels. In addition, no leakage was detected through the walls of the gelMA grafts during burst pressure measurement. Therefore, the current printing setup could be utilized to print vascular grafts for the treatment of cardiovascular diseases.

Rapid fabrication and screening of tailored functional 3D biomaterials: Validation in bone tissue repair - Part II.

Regenerative medicine strategies place increasingly sophisticated demands on 3D biomaterials to promote tissue formation at sites where tissue would otherwise not form. Ideally, the discovery/fabrication of the 3D scaffolds needs to be high-throughput and uniform to ensure quick and in-depth analysis in order to pinpoint appropriate chemical and mechanical properties of a biomaterial. Herein we present a versatile technique to screen new potential biocompatible acrylate-based 3D scaffolds with the ultimate aim of application in tissue repair. As part of this process, we identified an acrylate-based 3D porous scaffold that promoted cell proliferation followed by accelerated tissue formation, pre-requisites for tissue repair. Scaffolds were fabricated by a facile freeze-casting and an in-situ photo-polymerization route, embracing a high-throughput synthesis, screening and characterization protocol. The current studies demonstrate the dependence of cellular growth and vascularization on the porosity and intrinsic chemical nature of the scaffolds, with tuneable 3D scaffolds generated with large, interconnected pores suitable for cellular growth applied to skeletal reparation. Our studies showed increased cell proliferation, collagen and ALP expression, while chorioallantoic membrane assays indicated biocompatibility and demonstrated the angiogenic nature of the scaffolds. VEGRF2 expression in vivo observed throughout the 3D scaffolds in the absence of growth factor supplementation demonstrates a potential for angiogenesis. This novel platform provides an innovative approach to 3D scanning of synthetic biomaterials for tissue regeneration.

Effects of electrospun fibers containing ascorbic acid on oxidative stress reduction for cardiac tissue engineering.

Tissue engineering provides promise for regeneration of cardiac tissue following myocardial infarction. However, the harsh microenvironment of the infarct hampers the efficacy of regenerative therapies. Ischemia-reperfusion injury dramatically increases the levels of reactive oxygen species (ROS) within the infarcted area, causing a cascade of further cellular injury. Implantable tissue engineered grafts can target this oxidative stress by delivering pharmaceutical compounds directly into the diseased tissue. Herein, we successfully fabricated electrospun polycaprolactone (PCL) fibers containing varying concentrations of ascorbic acid, a potent antioxidant well known for its ROS-scavenging capabilities. The antioxidant scaffolds displayed significantly improved scavenging of DPPH radicals, superoxide anions and hydroxyl radicals, in a dose dependent manner. Mechanical properties testing indicated that incorporation of ascorbic acid enhanced the strength and Young's modulus of the material, correlating with a moderate but non-significant increase in the crystallinity. Moreover, the scaffolds supported adhesion and maintained survival of human umbilical vein endothelial cells in vitro, indicating good cytocompatibility. These results provide motivation for the use of ascorbic acid-containing fibrous scaffolds to regulate the highly oxidative microenvironment following myocardial infarction.

From hormone replacement therapy to regenerative scaffolds: A review of current and novel primary hypothyroidism therapeutics.

Primary hypothyroidism severely impacts the quality of life of patients through a decrease in the production of the thyroid hormones T3 and T4, leading to symptoms affecting cardiovascular, neurological, cognitive, and metabolic function. The incidence rate of primary hypothyroidism is expected to increase in the near future, partially due to increasing survival of patients that have undergone radiotherapy for head and neck cancer, which induces this disease in over half of those treated. The current standard of care encompasses thyroid hormone replacement therapy, traditionally in the form of synthetic T4. However, there is mounting evidence that this is unable to restore thyroid hormone signaling in all tissues due to often persistent symptoms. Additional complications are also present in the form of dosage difficulties, extensive drug interactions and poor patience compliance. The alternative therapeutic approach employed in the past is combination therapy, which consists of administration of both T3 and T4, either synthetic or in the form of desiccated thyroid extract. Here, issues are present regarding the lack of regulation concerning formulation and lack of data regarding safety and efficacy of these treatment methods. Tissue engineering and regenerative medicine have been applied in conjunction with each other to restore function of various tissues. Recently, these techniques have been adapted for thyroid tissue, primarily through the fabrication of regenerative scaffolds. Those currently under investigation are composed of either biopolymers or native decellularized extracellular matrix (dECM) in conjunction with either primary thyrocytes or stem cells which have undergone directed thyroid differentiation. Multiple of these scaffolds have successfully restored an athyroid phenotype in vivo. However, further work is needed until clinical translation can be achieved. This is proposed in the form of exploration and combination of materials used to fabricate these scaffolds, the addition of peptides which can aid restoration of tissue homeostasis and additional in vivo experimentation providing data on safety and efficacy of these implants.

Rat liver ECM incorporated into electrospun polycaprolactone scaffolds as a platform for hepatocyte culture.

Liver disease is expanding across the globe; however, health-care systems still lack approved pharmaceutical treatment strategies to mitigate potential liver failures. Organ transplantation is the only treatment for liver failure and with increasing cases of liver disease, transplant programs increasingly cannot provide timely transplant availability for all patients. The development of pharmaceutical mitigation strategies is clearly necessary and methods to improve drug development processes are considered vital for this purpose. Herein, we present a methodology for incorporating whole organ decellularised rat liver ECM (rLECM) into polycaprolactone (PCL) electrospun scaffolds with the aim of producing biologically relevant liver tissue models. rLECM PCL scaffolds have been produced with 5 w/w% and 10 w/w% rLECM:PCL and were analyzed by SEM imaging, tensile mechanical analyses and FTIR spectroscopy. The hepatocellular carcinoma cell line, HepG2, was cultured upon the scaffolds for 14 days and were analyzed through cell viability assay, DNA quantification, albumin quantification, immunohistochemistry, and RT-qPCR gene expression analysis. Results showed significant increases in proliferative activity of HepG2 on rLECM containing scaffolds alongside maintained key gene expression. This study confirms that rLECM can be utilized to modulate the bioactivity of electrospun PCL scaffolds and has the potential to produce electrospun scaffolds suitable for enhanced hepatocyte cultures and in-vitro liver tissue models.

Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

Electrospinning Fabrication Methods to Incorporate Laminin in Polycaprolactone for Kidney Tissue Engineering.

BACKGROUND: Today's treatment options for renal diseases fall behind the need, as the number of patients has increased considerably over the last few decades. Tissue engineering (TE) is one avenue which may provide a new approach for renal disease treatment. This involves creating a niche where seeded cells can function in an intended way. One approach to TE is combining natural extracellular matrix proteins with synthetic polymers, which has been shown to have many positives, yet a little is understood in kidney. Herein, we investigate the incorporation of laminin into polycaprolactone electrospun scaffolds. METHOD: The scaffolds were enriched with laminin via either direct blending with polymer solution or in a form of emulsion with a surfactant. Renal epithelial cells (RC-124) were cultured on scaffolds up to 21 days. RESULTS: Mechanical characterization demonstrated that the addition of the protein changed Young's modulus of polymeric fibres. Cell viability and DNA quantification tests revealed the capability of the scaffolds to maintain cell survival up to 3 weeks in culture. Gene expression analysis indicated healthy cells via three key markers. CONCLUSION: Our results show the importance of hybrid scaffolds for kidney tissue engineering.

Electrospun fibre diameter and its effects on vascular smooth muscle cells.

Bypass grafting is a technique used in the treatment of vascular disease, which is currently the leading cause of mortality worldwide. While technology has moved forward over the years, synthetic grafts still show significantly lower rates of patency in small diameter bypass operations compared to the gold standard (autologous vessel grafts). Scaffold morphology plays an important role in vascular smooth muscle cell (VSMC) performance, with studies showing how fibre alignment and surface roughness can modulate phenotypic and genotypic changes. Herein, this study has looked at how the fibre diameter of electrospun polymer scaffolds can affect the performance of seeded VSMCs. Four different scaffolds were electrospun with increasing fibre sizes ranging from 0.75 to 6 µm. Culturing VSMCs on the smallest fibre diameter (0.75 µm) lead to a significant increase in cell viability after 12 days of culture. Furthermore, interesting trends were noted in the expression of two key phenotypic genes associated with mature smooth muscle cell contractility (myocardin and smooth muscle alpha-actin 1), whereby reducing the fibre diameter lead to relative upregulations compared to the larger fibre diameters. These results showed that the smallest (0.75 µm) fibre diameter may be best suited for the culture of VSMCs with the aim of increasing cell proliferation and aiding cell maturity.

Influence of surface topography on PCL electrospun scaffolds for liver tissue engineering.

Severe liver disease is one of the most common causes of death globally. Currently, whole organ transplantation is the only therapeutic method for end-stage liver disease treatment, however, the need for donor organs far outweighs demand. Recently liver tissue engineering is starting to show promise for alleviating part of this problem. Electrospinning is a well-known method to fabricate a nanofibre scaffold which mimics the natural extracellular matrix that can support cell growth. This study aims to investigate liver cell responses to topographical features on electrospun fibres. Scaffolds with large surface depression (2 µm) (LSD), small surface depression (0.37 µm) (SSD), and no surface depression (NSD) were fabricated by using a solvent-nonsolvent system. A liver cell line (HepG2) was seeded onto the scaffolds for up to 14 days. The SSD group exhibited higher levels of cell viability and DNA content compared to the other groups. Additionally, the scaffolds promoted gene expression of albumin, with all cases having similar levels, while the cell growth rate was altered. Furthermore, the scaffold with depressions showed 0.8 MPa higher ultimate tensile strength compared to the other groups. These results suggest that small depressions might be preferred by HepG2 cells over smooth and large depression fibres and highlight the potential for tailoring liver cell responses.

A modified 3D printer as a hybrid bioprinting-electrospinning system for use in vascular tissue engineering applications.

There is a high demand for small diameter vascular grafts having mechanical and biological properties similar to that of living tissues. Tissue-engineered vascular grafts using current methods have often failed due to the mismatch of mechanical properties between the implanted graft and living tissues. To address this limitation, a hybrid bioprinting-electrospinning system is developed for vascular tissue engineering applications. The setup is capable of producing layered structure from electrospun fibres and cell-laden hydrogel. A Creality3D Ender 3D printer has been modified into a hybrid setup having one bioprinting head and two electrospinning heads. Fortus 250mc and Flashforge Creator Pro 3D printers were used to print parts using acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) polymers. An Arduino mega 2560 and a Ramps 1.4 controller board were selected to control the functions of the hybrid bioprinting setup. The setup was tested successfully to print a tubular construct around a rotating needle.

Architected fibrous scaffolds for engineering anisotropic tissues.

Mimicking the native three-dimensional microenvironment is of crucial importance when biofabricating a new healthcare material. One aspect of the native tissue that is often omitted when designing a suitable scaffold is its anisotropy. Not only is matching native mechanical properties important when designing implantable scaffolds or healthcare materials, but matching physiological structure is also important as many cell populations respond differently to fiber orientation. Therefore, novel aligned electrospun scaffolds with varying fiber angles and spacing of bundles were created and mechanically characterized. Through controlling the angle between the fibers in each layer of the scaffold, a range of different physiological anisotropic mechanical properties were achieved that encompasses values found in native tissues. Extrapolation of this mechanical data allowed for any native tissue's anisotropic Young's modulus to be mimicked by electrospinning fibers at a particular angle. These electrospun scaffolds were then incorporated with cell-laden hydrogels to create hybrid structures that contain the benefits of both scaffolding techniques with the ability to encapsulate cells in the hydrogel. To conclude, this study develops a novel bundled fiber scaffold that was architected to yield anisotropic properties matching native tissues.

Recent advancements in the bioprinting of vascular grafts.

Recent advancements in the bioinks and three-dimensional (3D) bioprinting methods used to fabricate vascular constructs are summarized herein. Critical biomechanical properties required to fabricate an ideal vascular graft are highlighted, as well as various testing methods have been outlined to evaluate the bio-fabricated grafts as per the Food and Drug Administration (FDA) and International Organization for Standardization (ISO) guidelines. Occlusive artery disease and cardiovascular disease are the major causes of death globally. These diseases are caused by the blockage in the arteries, which results in a decreased blood flow to the tissues of major organs in the body, such as the heart. Bypass surgery is often performed using a vascular graft to re-route the blood flow. Autologous grafts represent a gold standard for such bypass surgeries; however, these grafts may be unavailable due to the previous harvesting or possess a poor quality. Synthetic grafts serve well for medium to large-sized vessels, but they fail when used to replace small-diameter vessels, generally smaller than 6 mm. Various tissue engineering approaches have been used to address the urgent need for vascular graft that can withstand hemodynamic blood pressure and has the ability to grow and remodel. Among these approaches, 3D bioprinting offers an attractive solution to construct patient-specific vessel grafts with layered biomimetic structures.

Response differences of HepG2 and Primary Mouse Hepatocytes to morphological changes in electrospun PCL scaffolds.

Liver disease cases are rapidly expanding across the globe and the only effective cure for end-stage disease is a transplant. Transplant procedures are costly and current supply of donor livers does not satisfy demand. Potential drug treatments and regenerative therapies that are being developed to tackle these pressing issues require effective in-vitro culture platforms. Electrospun scaffolds provide bio-mimetic structures upon which cells are cultured to regulate function in-vitro. This study aims to shed light on the effects of electrospun PCL morphology on the culture of an immortalised hepatic cell line and mouse primary hepatocytes. Each cell type was cultured on large 4-5 µm fibres and small 1-2 µm fibres with random, aligned and highly porous cryogenically spun configurations. Cell attachment, proliferation, morphology and functional protein and gene expression was analysed. Results show that fibre morphology has a measurable influence on cellular morphology and function, with the alteration of key functional markers such as CYP1A2 expression.

Modulating electrospun polycaprolactone scaffold morphology and composition to alter endothelial cell proliferation and angiogenic gene response.

The aim of this study was to look at how the composition and morphology of polymer scaffolds could be altered to create an optimized environment for endothelial cells. Four polycaprolactone (PCL) scaffolds were electrospun with increasing fibre diameters ranging from 1.64 µm to 4.83 µm. The scaffolds were seeded with human umbilical vein endothelial cells (HUVEC) and cultured for 12 days. PCL scaffolds were then electrospun incorporating decellularized bovine aorta ECM and cultured in a hypoxic environment. We noted deeper cell infiltration on the largest fibre diameter compared to the other three scaffolds which resulted in an increase in the gene expression of CD31; a key angiogenic marker. Increased cell viability and cell proliferation were also noted on the largest fibre. Furthermore, we noted that the incorporation of extracellular matrix (ECM) had minimal effect on cell viability, both in normoxic and hypoxic culture conditions. Our results showed that these environments had limited influences on hypoxic gene expression. Interestingly, the major findings from this study was the production of excretory ECM components as shown in the scanning electron microscopy (SEM) images. The results from this study suggest that fibre diameter had a bigger impact on the seeded HUVECs than the incorporation of ECM or the culture conditions. The largest fibre dimeter (4.83 µm) is more suitable for seeding of HUVECs.

Integrational Technologies for the Development of Three-Dimensional Scaffolds as Platforms in Cartilage Tissue Engineering.

The prevalence of osteoarthritis is on the rise, and an effective treatment for cartilage defects is still being sought. Cartilage tissue in vivo encompasses complex structures and composition, both of which influence cells and many properties of the native cartilage. The extracellular matrix structure and components provides both morphological cues and the necessary signals to promote cell functions including metabolism, proliferation, and differentiation. In the present study, cryo-printing and electrospinning were combined to produce multizone scaffolds that consist of three distinctive zones. These scaffolds successfully mimic the collagen fiber orientation of the native cartilage. Moreover, in vitro analysis of chondrocyte-seeded scaffolds demonstrated the ability of multizone scaffolds to support long-term chondrocyte attachment and survival over a 5 week culture period. Moreover, multizone scaffolds were found to regulate the expression of key genes in comparison to the controls and allowed the detection of sulfated glycosaminoglycan. Evaluation of the compressive properties revealed that the multizone scaffolds possess more suitable mechanical properties, for the native cartilage, in comparison to the electrospun and phase-separated controls. Multizone scaffolds provide viable initial platforms that capture the complex structure and compressive properties of the native cartilage. They also maintain chondrocyte phenotype and function, highlighting their potential in cartilage tissue engineering applications.

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 µm and 5 µm diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

High-throughput production of silk fibroin-based electrospun fibers as biomaterial for skin tissue engineering applications.

In this work, a nozzle-free electrospinning device was built to obtain high-throughput production of silk fibroin-based biocompatible composite fibers with tunable wettability. Synthetic biomaterials tend to present suboptimal cell growth and proliferation, with many studies linking this phenomenon to the hydrophobicity of such surfaces. In this study, electrospun mats consisting of Poly(caprolactone) blended with variant forms of Poly(glycerol sebacate) (PGS) and regenerated silk fibroin were fabricated. The main aim of this work was the development of fiber mats with tunable hydrophobicity/hydrophilicity properties depending on the esterification degree and concentration of PGS. A variation of the conventional protocol used for the extraction of silk fibroin from Bombyx mori cocoons was employed, achieving significantly increased yields of the protein, in a third of the time required via the conventional extraction protocol. By altering the surface properties of the electrospun membranes, the trinary composite biomaterial presented good in vitro fibroblast attachment behavior and optimal growth, indicating the potential of such constructs towards the development of an artificial skin-like platform that can aid wound healing and skin regeneration.