Fabricating Multiphasic Angiogenic Scaffolds Using Amyloid/Roxadustat-Assisted High-Temperature Protein Printing.

Repairing multiphasic defects is cumbersome. This study presents new soft and hard scaffold designs aimed at facilitating the regeneration of multiphasic defects by enhancing angiogenesis and improving cell attachment. Here, the nonimmunogenic, nontoxic, and cost-effective human serum albumin (HSA) fibril (HSA-F) was used to fabricate thermostable (up to 90 °C) and hard printable polymers. Additionally, using a 10.0 mg/mL HSA-F, an innovative hydrogel was synthesized in a mixture with 2.0% chitosan-conjugated arginine, which can gel in a cell-friendly and pH physiological environment (pH 7.4). The presence of HSA-F in both hard and soft scaffolds led to an increase in significant attachment of the scaffolds to the human periodontal ligament fibroblast (PDLF), human umbilical vein endothelial cell (HUVEC), and human osteoblast. Further studies showed that migration (up to 157%), proliferation (up to 400%), and metabolism (up to 210%) of these cells have also improved in the direction of tissue repair. By examining different in vitro and ex ovo experiments, we observed that the final multiphasic scaffold can increase blood vessel density in the process of per-vascularization as well as angiogenesis. By providing a coculture environment including PDLF and HUVEC, important cross-talk between these two cells prevails in the presence of roxadustat drug, a proangiogenic in this study. In vitro and ex ovo results demonstrated significant enhancements in the angiogenic response and cell attachment, indicating the effectiveness of the proposed design. This approach holds promise for the regeneration of complex tissue defects by providing a conducive environment for vascularization and cellular integration, thus promoting tissue healing.

Osteogenic Differentiation of Human Gingival Fibroblasts Inhibits Osteoclast Formation.

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Mechanisms underlying the direct programming of mouse embryonic fibroblasts to thymic epithelial cells by FOXN1.

Thymic epithelial cells (TECs) are crucial to the ability of the thymus to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T-cell differentiation in culture or after transplant. In this study, we have characterized iTEC programming at the cellular and molecular level in mouse to determine how it proceeds, and have identified mechanisms that can be targeted for improving this process. These data show that iTEC programming consists of discrete gene expression changes that differ early and late in the process, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows the induction of mTEC differentiation. Finally, we show that MHCII expression is the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.

Engineering programmable material-to-cell pathways via synthetic notch receptors to spatially control differentiation in multicellular constructs.

Synthetic Notch (synNotch) receptors are genetically encoded, modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control, applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus, we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally, we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues.

Roughness affects the response of human fibroblasts and macrophages to sandblasted abutments.

BACKGROUND: A strong seal of soft-tissue around dental implants is essential to block pathogens from entering the peri-implant interface and prevent infections. Therefore, the integration of soft-tissue poses a challenge in implant-prosthetic procedures, prompting a focus on the interface between peri-implant soft-tissues and the transmucosal component. The aim of this study was to analyse the effects of sandblasted roughness levels on in vitro soft-tissue healing around dental implant abutments. In parallel, proteomic techniques were applied to study the interaction of these surfaces with human serum proteins to evaluate their potential to promote soft-tissue regeneration. RESULTS: Grade-5 machined titanium discs (MC) underwent sandblasting with alumina particles of two sizes (4 and 8 µm), resulting in two different surface types: MC04 and MC08. Surface morphology and roughness were characterised employing scanning electron microscopy and optical profilometry. Cell adhesion and collagen synthesis, as well as immune responses, were assessed using human gingival fibroblasts (hGF) and macrophages (THP-1), respectively. The profiles of protein adsorption to the surfaces were characterised using proteomics; samples were incubated with human serum, and the adsorbed proteins analysed employing nLC-MS/MS. hGFs exposed to MC04 showed decreased cell area compared to MC, while no differences were found for MC08. hGF collagen synthesis increased after 7 days for MC08. THP-1 macrophages cultured on MC04 and MC08 showed a reduced TNF-α and increased IL-4 secretion. Thus, the sandblasted topography led a reduction in the immune/inflammatory response. One hundred seventy-six distinct proteins adsorbed on the surfaces were identified. Differentially adsorbed proteins were associated with immune response, blood coagulation, angiogenesis, fibrinolysis and tissue regeneration. CONCLUSIONS: Increased roughness through MC08 treatment resulted in increased collagen synthesis in hGF and resulted in a reduction in the surface immune response in human macrophages. These results correlate with the changes in protein adsorption on the surfaces observed through proteomics.

Dry and wet wrinkling of a silk fibroin biopolymer by a shape-memory material with insight into mechanical effects on secondary structures in the silk network.

Surface wrinkling provides an approach to modify the surfaces of biomedical devices to better mimic features of the extracellular matrix and guide cell attachment, proliferation, and differentiation. Biopolymer wrinkling on active materials holds promise but is poorly explored. Here we report a mechanically actuated assembly process to generate uniaxial micro-and nanosized silk fibroin (SF) wrinkles on a thermo-responsive shape-memory polymer (SMP) substrate, with wrinkling demonstrated under both dry and hydrated (cell compatible) conditions. By systematically investigating the influence of SMP programmed strain magnitude, film thickness, and aqueous media on wrinkle stability and morphology, we reveal how to control the wrinkle sizes on the micron and sub-micron length scale. Furthermore, as a parameter fundamental to SMPs, we demonstrate that the temperature during the recovery process can also affect the wrinkle characteristics and the secondary structures in the silk network. We find that with increasing SMP programmed strain magnitude, silk wrinkled topographies with increasing wavelengths and amplitudes are achieved. Furthermore, silk wrinkling is found to increase ß-sheet content, with spectroscopic analysis suggesting that the effect may be due primarily to tensile (e.g., Poisson effect and high-curvature wrinkle) loading modes in the SF, despite the compressive bulk deformation (uniaxial contraction) used to produce wrinkles. Silk wrinkles fabricated from sufficiently thick films (roughly 250 nm) persist after 24 h in cell culture medium. Using a fibroblast cell line, analysis of cellular response to the wrinkled topographies reveals high viability and attachment. These findings demonstrate use of wrinkled SF films under physiologically relevant conditions and suggest the potential for biopolymer wrinkles on biomaterials surfaces to find application in cell mechanobiology, wound healing, and tissue engineering.

New Skin-Relevant Cell Coculture Model With Stratum Corneum-Like Layer.

Two-dimensional (2D) cell culture is an important tool in the discovery of skin-active agents. Fibroblasts and keratinocytes, more rarely fibroblast-keratinocyte cocultures, are usually used for that purpose, where test compounds are added by mixing with the overlaying growth medium. However, such an approach is suboptimal because it lacks the stratum corneum component. The stratum corneum acts as a selective gatekeeper and opposes the intradermal permeation of many compounds that are bioactive when placed in direct contact with cells. One solution is to use reconstituted epidermis, but this approach is costly and time consuming. Here, a model is proposed, where the simplicity and convenience of the 2D cell culture is combined with the advantage of a hydrophobic barrier reminiscent of the skin horny layer. This model was tested with skin-relevant solvents, as well as with "naked" hydrophilic and encapsulated compounds. Cell viability and collagen stimulation were used as readouts. The results showed that the incorporation of a stratum corneum-substitute barrier on top of a 2D cell culture reduced the cytotoxicity of a common cosmetic solvent, dimethyl isosorbide (DMI), in cell culture and modified the bioactivity of the added actives (magnesium ascorbyl phosphate [MAP] and oligomeric proanthocyanidins [OPCs]/levan biopolymer), which became dependent on their ability to penetrate through a lipidic layer. Taken together, these results indicate a better physiological relevance of this cell culture model in workflows aimed at the discovery and analysis of skin-active compounds than conventional 2D systems.

3D Bioprinting of Artificial Skin Substitute with Improved Mechanical Property and Regulated Cell Behavior through Integrating Patterned Nanofibrous Films.

Three-dimensional (3D) bioprinting has advantages for constructing artificial skin tissues in replicating the structures and functions of native skin. Although many studies have presented improved effect of printing skin substitutes in wound healing, using hydrogel inks to fabricate 3D bioprinting architectures with complicated structures, mimicking mechanical properties, and appropriate cellular environments is still challenging. Inspired by collagen nanofibers withstanding stress and regulating cell behavior, a patterned nanofibrous film was introduced to the printed hydrogel scaffold to fabricate a composite artificial skin substitute (CASS). The artificial dermis was printed using gelatin-hyaluronan hybrid hydrogels containing human dermal fibroblasts with gradient porosity and integrated with patterned nanofibrous films simultaneously, while the artificial epidermis was formed by seeding human keratinocytes upon the dermis. The collagen-mimicking nanofibrous film effectively improved the tensile strength and fracture resistance of the CASS, making it sewable for firm implantation into skin defects. Meanwhile, the patterned nanofibrous film also provided the biological cues to guide cell behavior. Consequently, CASS could effectively accelerate the regeneration of large-area skin defects in mouse and pig models by promoting re-epithelialization and collagen deposition. This research developed an effective strategy to prepare composite bioprinting architectures for enhancing mechanical property and regulating cell behavior, and CASS could be a promising skin substitute for treating large-area skin defects.

Disodium Cromoglycate Templates Anisotropic Short-Chain PEG Hydrogels.

Anisotropic hydrogels have found widespread applications in biomedical engineering, particularly as scaffolds for tissue engineering. However, it remains a challenge to produce them using conventional fabrication methods, without specialized synthesis or equipment, such as 3D printing and unidirectional stretching. In this study, we explore the self-assembly behaviors of polyethylene glycol diacrylate (PEGDA), using disodium cromoglycate (DSCG), a lyotropic chromonic liquid crystal, as a removable template. The affinity between short-chain PEGDA (Mn = 250) and DSCG allows polymerization to take place at the DSCG surface, thereby forming anisotropic hydrogel networks with fibrin-like morphologies. This process requires considerable finesse as the phase behaviors of DSCG depend on a multitude of factors, including the weight percentage of PEGDA and DSCG, the chain length of PEGDA, and the concentration of ionic species. The key to modulating the microstructures of the all-PEG hydrogel networks is through precise control of the DSCG concentration, resulting in anisotropic mechanical properties. Using these anisotropic hydrogel networks, we demonstrate that human dermal fibroblasts are particularly sensitive to the alignment order. We find that cells exhibit a density-dependent activation pattern of a Yes-associated protein, a mechanotransducer, corroborating its role in enabling cells to translate external mechanical and morphological patterns to specific behaviors. The flexibility of modulating microstructure, along with PEG hydrogels' biocompatibility and biodegradability, underscores their potential use for tissue engineering to create functional structures with physiological morphologies.

Bottlenose dolphin (Tursiops truncatus) immortalized fibroblasts on novel 3D in vitro collagen-free scaffolds.

Dolphins, as apex predators, can be considered relevant sentinels of the health of marine ecosystems. The creation of 3D cell models to assess in vitro cell-to-cell and cell-to-matrix interactions in environmental-mimicking conditions, is of considerable interest. However, to date the establishment of cetacean 3D culture systems has not yet been accomplished. Thus, in this study, different 3D systems of bottlenose dolphin (Tursiops truncatus) skin fibroblasts have been analyzed. Particularly, novel scaffolds based on hyaluronic acid and ionic-complementary self-assembling peptides such as RGD-EAbuK and EAbuK-IKVAV have been compared to Matrigel. Histological and fluorescent staining, electron microscopy (TEM) analyses and viability assays have been performed and RT-PCR has been used to detect extracellular matrix (ECM) components produced by cells. Results showed that Matrigel induced cells to form aggregates with lower viability and no ECM production compared to the novel scaffolds. Moreover, scaffolds allowed dispersed cells to produce a collagenous ECM containing collagen1a1, laminin B1 and elastin. The HA-EAbuK-IKVAV scaffold resulted in the most suitable 3D model in terms of cell quantity and viability. The development of this innovative approach is the first step towards the possibility to create 3D in vitro models for this protected species.

3D-bioprinted GelMA/gelatin/amniotic membrane extract (AME) scaffold loaded with keratinocytes, fibroblasts, and endothelial cells for skin tissue engineering.

Gelatin-methacryloyl (GelMA) is a highly adaptable biomaterial extensively utilized in skin regeneration applications. However, it is frequently imperative to enhance its physical and biological qualities by including supplementary substances in its composition. The purpose of this study was to fabricate and characterize a bi-layered GelMA-gelatin scaffold using 3D bioprinting. The upper section of the scaffold was encompassed with keratinocytes to simulate the epidermis, while the lower section included fibroblasts and HUVEC cells to mimic the dermis. A further step involved the addition of amniotic membrane extract (AME) to the scaffold in order to promote angiogenesis. The incorporation of gelatin into GelMA was found to enhance its stability and mechanical qualities. While the Alamar blue test demonstrated that a high concentration of GelMA (20%) resulted in a decrease in cell viability, the live/dead cell staining revealed that incorporation of AME increased the quantity of viable HUVECs. Further, gelatin upregulated the expression of KRT10 in keratinocytes and VIM in fibroblasts. Additionally, the histological staining results demonstrated the formation of well-defined skin layers and the creation of extracellular matrix (ECM) in GelMA/gelatin hydrogels during a 14-day culture period. Our study showed that a 3D-bioprinted composite scaffold comprising GelMA, gelatin, and AME can be used to regenerate skin tissues.

Age and sex effects on histological features and in vitro culturing of Antillean manatee (Trichechus manatus manatus, Linnaeus 1758) dermis.

The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis can be influenced by the age and sex of the individual. Therefore, evaluating these factors is interesting for forming biobanks of Antillean manatees. These animals, representatives of marine fauna, have had their population reduced, and biobanks are essential for their conservation. Then, we evaluated the effects of age (3.5 years vs. 3.6-16 years vs. 23.6 years) and sex (males vs. females) on morphological and cellular parameters using histological and in vitro culture techniques. Regardless of age, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery. Nonetheless, fibroblast reduction was observed in groups aged 23.6 years compared to other animals (p < 0.05). Additionally, cells from animals aged 3.6-16 years showed more significant mitochondrial damage than the other groups (p < 0.05). Regardless of sex, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery; however, females had fewer fibroblasts than males (p < 0.05). Cells from females showed lower mitochondrial damage when compared to cells from males. In summary, although age and sex do not influence dermal thickness and cell recovery, variations in the number of fibroblasts and mitochondrial characteristics were observed among the groups. These differences may be significant for understanding the dermis aspects to be correlated to biobank systems.

Inhibition and reversal of a TGF-ß1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors.

BACKGROUND: Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. METHODS: The transforming growth factor-ß1 (TGF-ß1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. RESULTS: Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-ß1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. CONCLUSIONS: Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.

Hydrogel Formulation for Biomimetic Fibroblast Cell Culture: Exploring Effects of External Stresses and Cellular Responses.

In the process of tissue engineering, several types of stresses can influence the outcome of tissue regeneration. This outcome can be understood by designing hydrogels that mimic this process and studying how such hydrogel scaffolds and cells behave under a set of stresses. Here, a hydrogel formulation is proposed to create biomimetic scaffolds suitable for fibroblast cell culture. Subsequently, we examine the impact of external stresses on fibroblast cells cultured on both solid and porous hydrogels. These stresses included mechanical tension and altered-gravity conditions experienced during the 83rd parabolic flight campaign conducted by the European Space Agency. This study shows distinct cellular responses characterized by cell aggregation and redistribution in regions of intensified stress concentration. This paper presents a new biomimetic hydrogel that fulfills tissue-engineering requirements in terms of biocompatibility and mechanical stability. Moreover, it contributes to our comprehension of cellular biomechanics under diverse gravitational conditions, shedding light on the dynamic cellular adaptations versus varying stress environments.

Viscoelastic Supramolecular Hyaluronan-Peptide Cross-Linked Hydrogels.

Viscoelasticity plays a key role in hydrogel design. We designed a physically cross-linked hydrogel with tunable viscoelasticity, comprising supramolecular-assembled peptides coupled to hyaluronan (HA), a native extracellular matrix component. We then explored the structural and molecular mechanisms underlying the mechanical properties of a series of these HA-peptide hydrogels. By modifying the peptide sequence, we modulated both long- and short-time stress relaxation rates as a way to target viscoelasticity with limited impact on stiffness, leading to gels that relax up to 60% of stress in 10 min. Gels with the highest viscoelasticity exhibited large mesh sizes and ß-sheet secondary structures. The stiffness of the gel correlated with hydrogen bonding between the peptide chains. These gels are cytocompatible: highly viscoelastic gels that mimic the native skin microenvironment promote dermal fibroblast cell spreading. Moreover, HA-peptide gels enabled cell encapsulation, as shown with primary human T cells. Overall, these physically-cross-linked hydrogels enable tunable viscoelasticity that can be used to modulate cell morphology.

Vimentin supports cell polarization by enhancing centrosome function and microtubule acetylation.

Cell polarity is important for controlling cell shape, motility and cell division processes. Vimentin intermediate filaments are important for cell migration and cell polarization in mesenchymal cells and assembly of vimentin and microtubule networks is dynamically coordinated, but the precise details of how vimentin mediates cell polarity remain unclear. Here, we characterize the effects of vimentin on the structure and function of the centrosome and the stability of microtubule filaments in wild-type and vimentin-null mouse embryonic fibroblasts. We find that vimentin mediates the structure of the pericentriolar material, promotes centrosome-mediated microtubule regrowth and increases the level of stable acetylated microtubules in the cell. Loss of vimentin also impairs centrosome repositioning during cell polarization and migration processes that occur during wound closure. Our results suggest that vimentin modulates centrosome structure and function as well as microtubule network stability, which has important implications for how cells establish proper cell polarization and persistent migration.

Electrofluidic control for textile-based cell culture: Identification of appropriate conditions required to integrate cell culture with electrofluidics.

Electric field-driven microfluidics, known as electrofluidics, is a novel attractive analytical tool when it is integrated with low-cost textile substrate. Textile-based electrofluidics, primarily explored on yarn substrates, is in its early stages, with few studies on 3D structures. Further, textile structures have rarely been used in cellular analysis as a low-cost alternative. Herein, we investigated novel 3D textile structures and develop optimal electrophoretic designs and conditions that are favourable for direct 3D cell culture integration, developing an integrated cell culture textile-based electrofluidic platform that was optimised to balance electrokinetic performance and cell viability requirements. Significantly, there were contrasting electrolyte compositional conditions that were required to satisfy cell viability and electrophoretic mobility requiring the development of and electrolyte that satisfied the minimum requirements of both these components within the one platform. Human dermal fibroblast cell cultures were successfully integrated with gelatine methacryloyl (GelMA) hydrogel-coated electrofluidic platform and studied under different electric fields using 5 mM TRIS/HEPES/300 mM glucose. Higher analyte mobility was observed on 2.5% GelMA-coated textile which also facilitated excellent cell attachment, viability and proliferation. Cell viability also increased by decreasing the magnitude and time duration of applied electric field with good cell viability at field of up to 20 V cm-1.

Photoacoustic processing of decellularized extracellular matrix for biofabricating living constructs.

The diverse biomolecular landscape of tissue-specific decellularized extracellular matrix (dECM) biomaterials provides a multiplicity of bioinstructive cues to target cells, rendering them highly valuable for various biomedical applications. However, the isolation of dECM biomaterials entails cumbersome xenogeneic enzymatic digestions and also additional inactivation procedures. Such, increases processing time, increments costs and introduces residues of non-naturally present proteins in dECM formulations that remain present even after inactivation. To overcome these limitations, herein we report an innovative conjugation of light and ultrasound-mediated dECM biomaterial processing for fabricating dECM biomaterials. Such approach gathers on ultrasound waves to facilitate dECM-in-liquid processing and visible light photocrosslinking of tyrosine residues naturally present in dECM biomaterials. This dual step methodology unlocked the in-air production of cell laden dECM hydrogels or programmable dECM hydrogel spherical-like beads by using superhydrophobic surfaces. These in-air produced units do not require any additional solvents and successfully supported both fibroblasts and breast cancer cells viability upon encapsulation or surface seeding. In addition, the optimized photoacoustic methodology also enabled a rapid formulation of dECM biomaterial inks with suitable features for biofabricating volumetrically defined living constructs through embedded 3D bioprinting. The biofabricated dECM hydrogel constructs supported cell adhesion, spreading and viability for 7 days. Overall, the implemented photoacoustic processing methodology of dECM biomaterials offers a rapid and universal strategy for upgrading their processing from virtually any tissue. STATEMENT OF SIGNIFICANCE: Leveraging decellularized extracellular matrix (dECM) as cell instructive biomaterials has potential to open new avenues for tissue engineering and in vitro disease modelling. The processing of dECM remains however, lengthy, costly and introduces non-naturally present proteins in the final biomaterials formulations. In this regard, here we report an innovative light and ultrasound two-step methodology that enables rapid dECM-in-liquid processing and downstream photocrosslinking of dECM hydrogel beads and 3D bioprinted constructs. Such photoacoustic based processing constitutes a universally applicable method for processing any type of tissue-derived dECM biomaterials.

Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair.

Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1-4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1ß (IL-1ß). The dominant effect of IL-1ß in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1ß or IL-1α release (denoted Pyro-1), we identify unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.