Skin dilation generates "extra" skin tissue through mechanical traction, but its effectiveness is limited by the proliferation capacity of keratinocytes, fibroblasts and the level of angiogenesis. Cutaneous application of drug and subcutaneous injection are common interventions to promote skin dilation, but they have defects such as uneven drug distribution, high risk of infection and single targeting. Although Acellular adipose matrix (AAM) has the potential to promote cell proliferation and angiogenesis, its hydrogel/powder dosage forms still need frequent injection, which limits clinical application.

In this study, Acellular adipose matrix derived film (AAF) was successfully developed, and a flexible film was formed by acellular - lyophilized - enzymolysis - self-assembly process. In vitro experiments confirmed that AAF significantly promoted the activity of Human Immortalized Epidermal Cells (HaCaTs), Normal Skin Fibroblasts (NFbs) and Human Umbilical Endothelial Cells (HUVECs); It was also found that AAF can induce adipose mesenchymal stem cells (ASCs) to differentiate into adipocytes and promote subcutaneous fat regeneration. In vivo, the rat model showed that AAF wrapping expander could effectively improve the skin expansion efficiency, promote the skin thickness increase in the expanded area, and the density of new blood vessels was significantly increased compared with the comparative group, and there was no complication such as infection or skin collapse. It was found for the first time that AAF successfully formed new adipose tissue in the subcutaneous area.

AAF innovatively integrates the bionic structure of extracellular matrix and slow-release function, and solves the uneven drug distribution and associated infection risk of traditional intervention methods by regulating the synergistic regeneration of epidermodermis and vascular units. Its mechanical adaptability (dry toughness/wet plasticity) and the ability of inducing adipose regeneration provide a new strategy of both structural strengthening and metabolic support for skin dilation, also laying a mechanism and empirical foundation for clinical transformation of tissue engineering materials.

Fibro-adipogenic progenitor cells (FAPs) are mesenchymal stem cells that produce extracellular matrix (ECM) and intramuscular adipocytes in skeletal muscle. While FAPs have demonstrated responsiveness to their physical environment, there is limited knowledge of how the ECM substrate of FAPs impacts their differentiation, particularly in livestock animals. We hypothesized that the ECM substrate FAPs are cultured on will differentially impact their adherence, proliferation, and differentiation. Through an initial screen of 9 ECM proteins and their combinations, significant variation of bovine FAP attachment and differentiation across coatings was observed. The ECM substrates fibronectin, collagen 6, vitronectin, and a combination of fibronectin and collagen 6 were selected for further testing. Notably, fibronectin increased cell proliferation and attachment rates, without impairing FAP adipogenic or fibrogenic differentiation compared to the other coatings. Benefits of fibronectin were maintained at lower concentrations and when combined with less favorable coatings such as collagen 6. When assessed for their adipogenic potential on each coating at different substrate stiffnesses, lipid accumulation decreased with increasing substrate stiffness, while cell attachment increased on stiffer substrates. Overall, these results demonstrate the high responsiveness of FAPs to their ECM substrate, along with highlighting fibronectin as a preferred substrate for in vitro experiments with bovine FAPs.

Conditioned medium (CM), obtained by mechanical regulation of the paracrine activity of ADSCs, was fused with acellular adipose matrix (AAM) and methyl cellulose (MC) to synthesize a composite hydrogel which was grafted onto nude mice. The composite hydrogel could promote soft tissue regeneration by regulating the level of vascular regeneration and inflammation.

White adipose tissue (WAT) plays a crucial role in energy homeostasis and secretes numerous adipokines with far-reaching effects. WAT is linked to diseases such as diabetes, cardiovascular disease, and cancer. There is a high demand for suitable in vitro models to study diseases and tissue metabolism. Most of these models are covered by 2D-monolayer cultures. This study aims to evaluate the performance of different WAT models to better derive potential applications. The stability of adipocyte characteristics in spheroids and two 3D gellan gum hydrogels with ex situ lobules and 2D-monolayer culture is analyzed. First, the differentiation to achieve adipocyte-like characteristics is determined. Second, to evaluate the maintenance of differentiated ASC-based models, an adipocyte-based model, and explants over 3 weeks, viability, intracellular lipid content, perilipin A expression, adipokine, and gene expression are analyzed. Several advantages are supported using each of the models. Including, but not limited to, the strong differentiation in 2D-monolayers, the self-assembling within spheroids, the long-term stability of the stem cell-containing hydrogels, and the mature phenotype within adipocyte-containing hydrogels and the lobules. This study highlights the advantages of 3D models due to their more in vivo-like behavior and provides an overview of the different adipose cell models.

Cultured meat needs edible bio-scaffolds that provide not only a growth milieu for muscle and adipose cells, but also biomimetic stiffness and tissue-sculpting topography. Current meat-engineering technologies struggle to achieve scalable cell production, efficient cell differentiation, and tissue maturation in one single culture system. Here we propose an autoclaving strategy to transform common vegetables into muscle- and adipose-engineering scaffolds, without undergoing conventional plant decellularization. We selected vegetables with natural anisotropic and isotropic topology mimicking muscle and adipose microstructures respectively. We further adjusted vegetable stiffness by autoclaving, to emulate the mechanical properties of animal tissues. Autoclaved vegetables preserve rich cell-affinitive moieties, yielding a good cell culture effect with simplified processing. Autoclaved Chinese chive and Shiitake mushroom with anisotropic micro-patterns support the scalable expansion of muscle cells, improved cell alignment and myogenesis. Autoclaved isotropic loofah encourages adipocyte proliferation and lipid accumulation. Our engineered muscle- and fat-on-vegetables can further construct meat stuffing or layered meat chips. Autoclaved vegetables possess tissue-mimicking stiffness and topology, and bring biochemical benefits, operational ease, cost reduction and bioreactor compatibility. Without needing decellularization, these natural biomaterials may see scale-up applications in meat analog bio-fabrication.

In vitro embryo culture often falls short of mimicking the physiological dynamism occurring in the reproductive tract, prompting developmental plasticity in mammalian embryos with consequential genotypic and phenotypic deviations. Recent research highlights the potential of biological derivatives in in vitro culture to mitigate these effects, being the extracellular matrix (ECM) one of the most important components in retaining structural and biological signals derived from the native source tissue. Current bioengineering techniques could provide ECM-based biomaterials mimicking the native environment and offering optimal embryonic development. Rabbit oviducts (n = 24) were decellularized and solubilized to create tissue-specific ECM (OviECM) hydrogels. Following physicochemical characterization, these hydrogels were applied as coatings for the in vitro culture of two-cell embryos over 48 h, along with embryos cultured under In vitro control conditions (n = 218/group), which were subsequently transferred to recipient females. A subset of embryos was recovered on day 6 for transcriptomic analysis (n = 75-80/group), while the remaining embryos were used to assess implantation and birth rates. Rabbit weights were monitored over 20 weeks post-delivery, with blood tests conducted at weeks 8 and 20. Bayesian inference methods were used for statistical analysis. Differences were considered relevant if P ≥ 0.8 (80%). No differences in embryo development and morphology were detected between the OviECM coating and In vitro control conditions. However, embryos cultured on these coatings exhibited upregulation of pathways involved in antigen presentation and immune system activation, as well as, increased cellular response to external stimulus and intracellular protein transport. The implantation and live birth rates were significantly higher in the coating group than in the In vitro control group (30.8% vs. 26.1% and 21.2% vs. 18.1%, respectively). During the first 20 weeks of life, the animals from the coating group showed higher weights than the In vitro control group P0 > 0.8. The animals of both experimental groups showed normal blood parameters. Implementation of OviECM coatings allows for improving in vitro conditions and decreases postnatal phenotypic deviations after assisted reproductive technology (ART). This study could initiate a new embryo culture techniques era to guarantee that ART is utilized in the most efficient and safest possible practice.

The development of injectable hydrogels for soft tissue regeneration has gained significant attention due to their minimally invasive application and ability to conform precisely to the shape of irregular tissue cavities. This study presents a novel injectable porous scaffold based on natural polymers that undergoes in situ crosslinking, forming a highly resilient hydrogel with tailorable mechanical and physical properties to meet the specific demands of soft tissue repair. By adjusting the formulation, we achieved a range of stiffness values that closely mimic the mechanical characteristics of native tissues while maintaining very high resilience (>90%). The effects of gelatin, alginate, and crosslinker concentrations, as well as porosity, on the hydrogel's properties were elucidated. The main results indicated a compression modulus range of 2.7-89 kPa, which fits all soft tissues, and gelation times ranging from 5 to 30 s, which enable the scaffold to be successfully used in various operations. An increase in gelatin and crosslinker concentrations results in a higher modulus and lower gelation time, i.e., a stiffer hydrogel that is created in a shorter time. In vitro cell viability tests on human fibroblasts were performed and indicated high biocompatibility. Our findings demonstrate that these injectable hydrogel scaffolds offer a promising solution for enhancing soft tissue repair and regeneration, providing a customizable and resilient framework that is expected to support tissue integration and healing with minimal surgical intervention.

The adipogenic property of decellularized adipose-derived matrix (DAM) varies widely across reports, making it difficult to make a horizontal comparison between reports and posing challenges for the stable clinical translation of DAM. It is possibly due to differences in donor characteristics, but the exact relationship remains unclear. Despite extensive research on the differences between superficial and deep layers of abdominal subcutaneous fat, a main donor of DAM, little is known about their extracellular matrix (ECM) which is promising in regenerative medicine. In this study, we first confirmed the distinct compositional profiles and adipogenic potential between superficial and deep DAM (S-DAM and D-DAM). Both in vitro and in vivo assays confirmed superior adipogenic induction potential in S-DAM over D-DAM. Total amounts of ECM proteins like collagen and laminin were similar, however, the predominant types differed, with collagen I dominating S-DAM and collagen XIV prevailing in D-DAM. S-DAM was enriched with mitochondrial and immunological proteins, whereas D-DAM featured more neuronal, vascular, muscular, and endocrine-related proteins. More proteins involved in mRNA processing were found in D-DAM, with Protein-Protein Interaction (PPI) analysis revealing HNRNPA2B1, HNRNPA1, and HNRNPC as the most tightly interacting members. These findings not only deepen our comprehension of the structural and functional heterogeneity of adipose tissues but also become one of the reason for the large variability between batches of DAM products, providing guidance for constructing more efficient and stable bio-scaffolds.

Decellularised extracellular matrix (dECM) produced by mesenchymal stromal cells (MSCs) is a promising biomaterial for improving the ex vivo expansion of MSCs. The dECMs are often deposited on high modulus surfaces such as tissue culture plastic or glass, and subsequent differentiation assays often bias towards osteogenesis. We tested the hypothesis that dECM deposited on substrates of varying modulus will produce cell culture environments that are tailored to promote the proliferation and/or lineage-specific differentiation of MSCs. dECM was produced on type I collagen-functionalised polyacrylamide hydrogels with discrete moduli (∼4, 10, and 40 kPa) or in a linear gradient of modulus that spans the same range, and the substrates were used as culture surfaces for MSCs. Fluorescence spectroscopy and mass spectrometry characterization revealed structural compositional changes in the dECM as a function of substrate modulus. Softer substrates (4 kPa) with dECM supported the largest number of MSCs after 7 days (∼1.6-fold increase compared to glass). Additionally, osteogenic differentiation was greatest on high modulus substrates (40 kPa and glass) with dECM. Nuclear translocation of YAP1 was observed on all surfaces with a modulus of 10 kPa or greater and may be a driver for the increased osteogenesis on the high modulus surfaces. These data demonstrate that dECM technology can be integrated with environmental parameters such as substrate modulus to improve/tailor MSC proliferation and differentiation during ex vivo culture. These results have potential impact in the improved expansion of MSCs for tailored therapeutic applications and in the development of advanced tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are extensively used in tissue engineering and regenerative medicine due to their ability to proliferate, differentiate, and modulate the immune environment. Controlling MSC behavior is critical for advances in the field. Decellularised extracellular matrix (dECM) can maintain MSC properties in culture, increase their proliferation rate and capacity, and enhance their stimulated differentiation. Substrate stiffness is another key driver of cell function, and previous reports have primarily looked at dECM deposition and function on stiff substrates such as glass. Herein, we produce dECM on substrates of varying stiffness to create tailored environments that enhance desired MSC properties such as proliferation and differentiation. Additionally, we complete mechanistic studies including quantitative mass spec of the ECM to understand the biological function.

Along with a paradigm shift in looking at soft tissue fillers from space-filling to bioactive materials, decellularized extracellular matrix (DEM) fillers have gained more attention considering their superior bioactivity. However, the complex mechanisms that govern the interaction between host tissues and DEMs have been partially understood. This review first covers the mechanisms that determine immunogenicity, angiogenesis and vasculogenesis, and recellularization and remodeling after DEM implantation into host tissue, with a particular focus on related findings from filler materials. Accordingly, the review delves into the dual role of macrophages and their M1/M2 polarization paradigm to form both constructive and destructive immune responses to DEM implants. Moreover, the contribution of macrophages in angiogenesis has been elucidated, which includes but is not limited to the secretion of angiogenic growth factors and extracellular matrix (ECM) remodeling. The findings challenge the traditional view of immune cells as solely destructive entities in biomaterials and indicate their multifaceted roles in tissue regeneration. Furthermore, the review discusses how the compositional factors of DEMs, such as the presence of growth factors and matrikines, can influence angiogenesis, cell fate, and differentiation during the recellularization process. It is also shown that the biomechanical properties of DEMs, including tissue stiffness, modulate cell responses through mechanotransduction pathways, and the structural properties of DEMs, such as scaffold porosity, impact cell-cell and cell-ECM interactions. Finally, we pointed out the current clinical applications, the bottlenecks in the clinical translation of DEM biomaterials into soft tissue fillers, as well as the naïve research areas of the field.

Adipose tissue remodeling and plasticity are dynamically regulated by the coordinated functions of adipocytes, macrophages, and endothelial cells and extracellular matrix (ECM) that provides stiffness networks in adipose tissue component cells. Inflammation and fibrosis are crucial exogenous factors that dysregulate adipose tissue functions and drastically change the mechanical properties of the ECM. Therefore, communication among the ECM and adipose tissue component cells is necessary to understand the multifaceted functions of adipose tissues. To obtain in vivo stiffness, we used genipin as a crosslinker for collagen gels. Meanwhile, we isolated primary adipocytes, macrophages, and endothelial cells from C57BL/6J mice and incubated these cells in the differentiation media on temperature-responsive culture dishes. After the differentiation, these cell sheets were transferred onto genipin-crosslinked collagen gels with varying matrix stiffness. We found that inflammatory gene expressions were induced by hard matrix, whereas antiinflammatory gene expressions were promoted by soft matrix in all three types of cells. Interestingly, the coculture experiments of adipocytes, macrophages, and endothelial cells showed that the effects of soft or hard matrix stiffness stimulation on adipocytes were transmitted to the distant adipose tissue component cells, altering their gene expression profiles under normal matrix conditions. Finally, we identified that a hard matrix induces the secretion of CXCL13 from adipocytes, and CXCL13 is one of the important transmitters for stiffness communication with macrophages and endothelial cells. These findings provide insight into the mechanotransmission into distant cells and the application of stiffness control for chronic inflammation in adipose tissues with metabolic dysregulation.

Hydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by-products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non-toxic, xeno-free, and has good mechanical properties. A recombinant spider silk protein-derived hydrogel that rapidly forms at 37 °C is recently developed. The temperature and gelation times are well-suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE-19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days.

Biomaterials denoting self-healing and versatile structural integrity are highly curious in the biomedicine segment. The injectable and/or printable 3D printing technology is explored in a few decades back, which can alter their dimensions temporarily under shear stress, showing potential healing/recovery tendency with patient-specific intervention toward the development of personalized medicine. Thus, self-healing injectable hydrogels (IHs) are stunning toward developing a paradigm for tissue regeneration. This review comprises the designing of IHs, rheological characterization and stability, several benchmark consequences for self-healing IHs, their translation into tissue regeneration of specific types, applications of IHs in biomedical such as anticancer and immunomodulation, wound healing and tissue/bone regeneration, antimicrobial potentials, drugs, gene and vaccine delivery, ocular delivery, 3D printing, cosmeceuticals, and photothermal therapy as well as in other allied avenues like agriculture, aerospace, electronic/electrical industries, coating approaches, patents associated with therapeutic/nontherapeutic avenues, and numerous futuristic challenges and solutions.

Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that biomechanics, such as the microstructure of nanomaterials, can induce adipose-derived stem cells (ASCs) to differentiate into osteoblasts, mediating gene regulation at the epigenetic level. Therefore, in this study, transcriptome expression levels of histone demethylases in ASCs were screened after treatment with different matrix stiffnesses, and histone lysine demethylase 3B (KDM3B) was found to promote osteogenic differentiation of ASCs in response to matrix stiffness, indicating a positive modulatory effect on this biological process. ASCs exhibited widespread and polygonal shapes with a distinct bundle-like expression of vinculin parallel to the axial cytoskeleton along the cell margins on the stiff matrix rather than round shapes with a smeared and shorter expression on the soft matrix. Comparatively rigid polydimethylsiloxane material directed ASCs into an osteogenic phenotype in inductive culture media via the upregulation of osteocalcin, alkaline phosphatase, and runt-related transcription factor 2. Treatment with KDM3B-siRNA decreased the expression of osteogenic differentiation markers and impaired mitochondrial dynamics and mitochondrial membrane potential. These results illustrate the critical role of KDM3B in the biomechanics-induced osteogenic commitment of ASCs and provide new avenues for the further application of stem cells as potential therapeutics for bone regeneration.

The need for a long-term solution for filling the defects created during partial mastectomies due to breast cancer diagnosis has not been met to date. All available defect-filling methods are non-permanent and necessitate repeat procedures. Here, we report on novel injectable porous hydrogel structures based on the natural polymers gelatin and alginate, which are designed to serve for breast reconstruction and regeneration following partial mastectomy. The effects of the formulation parameters on the mechanical and physical properties were thoroughly studied. The modulus in compression and tension were in the range of native breast tissue. Both increased with the increase in the crosslinker concentration and the polymer-air ratio. Resilience was very high, above 93% for most studied formulations, allowing the scaffold to be continuously deformed without changing its shape. The combination of high resilience and low elastic modulus is favored for adipose tissue regeneration. The physical properties of gelation time and water uptake are controllable and are affected mainly by the alginate and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) concentrations and less by the polymer-air ratio. In vitro cell viability tests were performed on mouse preadipocytes and indicated high biocompatibility. The minimally invasive nature of this approach, along with the excellent properties of the scaffold, will enable the filling of complex voids while simultaneously decreasing surgical costs and greatly improving patient well-being.

The growing obesity epidemic necessitates increased research on adipocyte and adipose tissue function and disease mechanisms that progress obesity. Historically, adipocytes were viewed simply as storage for excess energy. However, recent studies have demonstrated that adipocytes play a critical role in whole-body homeostasis, are involved in cell communication, experience forces in vivo, and respond to mechanical stimuli. Changes to the adipocyte mechanical microenvironment can affect function and, in some cases, contribute to disease. The aim of this review is to summarize the current literature on the mechanobiology of adipocytes. We reviewed over 100 papers on how mechanical stress is sensed by the adipocyte, the effects on cell behavior, and the use of cell culture scaffolds, particularly those with tunable stiffness, to study adipocyte behavior, adipose cell and tissue mechanical properties, and computational models. From our review, we conclude that adipocytes are responsive to mechanical stimuli, cell function and adipogenesis can be dictated by the mechanical environment, the measurement of mechanical properties is highly dependent on testing methods, and current modeling practices use many different approaches to recapitulate the complex behavior of adipocytes and adipose tissue. This review is intended to aid future studies by summarizing the current literature on adipocyte mechanobiology.

Adipose tissue plays an essential role in systemic metabolism with white adipose tissue (WAT) making up most of the tissue and being involved in the regulation of energy homeostasis, and brown and beige adipose tissue (BAT) exhibiting thermogenic activity. There is promise in the conversion of white adipocytes into beige ones as a therapeutic potential to control and enhance systemic metabolism, but it is difficult to maintain this transformation in vivo because we do not fully understand the mechanism of conversion. In this study, we applied atomic force microscopy (AFM) to characterize beige or white adipocytes during the process of differentiation for morphology, roughness, adhesion, and elasticity at different time points. As cells differentiated to white and beige adipocytes, they exhibited morphological changes as they lipid loaded, transitioning from flattened elongated cells to a rounded shape indicating adipogenesis. While there was an initial decrease in elasticity for both beige and white adipocytes, white adipocytes exhibited a higher elasticity than beige adipocytes at all time points. Beige and white adipogenesis exhibited a decrease in adhesion energy compared to preadipocytes, yet at day 12, white adipocytes had a significant increase in adhesion energy compared to beige adipocytes. This work shows significant differences in the mechanical properties of white vs. beige adipocytes during differentiation. Results from this study contribute to a better understanding of the differentiation of adipocytes which are vital to the therapeutic induction, engineered models, and maintenance of beige adipocytes as a potential approach for enhancing systemic metabolism.

There is increasing consumer demand for alternative animal protein products that are delicious and sustainably produced to address concerns about the impacts of mass-produced meat on human and planetary health. Cultured meat has the potential to provide a source of nutritious dietary protein that both is palatable and has reduced environmental impact. However, strategies to support the production of cultured meats at the scale required for food consumption will be critical. In this review, we discuss the current challenges and opportunities of using edible scaffolds for scaling up the production of cultured meat. We provide an overview of different types of edible scaffolds, scaffold fabrication techniques, and common scaffold materials. Finally, we highlight potential advantages of using edible scaffolds to advance cultured meat production by accelerating cell growth and differentiation, providing structure to build complex 3D tissues, and enhancing the nutritional and sensory properties of cultured meat.

In the dynamic landscape of tissue engineering, the integration of tissue-engineered constructs (TECs) faces a dual challenge-initiating beneficial inflammation for regeneration while avoiding the perils of prolonged immune activation. As TECs encounter the immediate reaction of the immune system upon implantation, the unique immunomodulatory properties of mesenchymal stem/stromal cells (MSCs) emerge as key navigators. Harnessing the paracrine effects of MSCs, researchers aim to craft a localized microenvironment that not only enhances TEC integration but also holds therapeutic promise for inflammatory-driven pathologies. This review unravels the latest advancements, applications, obstacles, and future prospects surrounding the strategic alliance between MSCs and TECs, shedding light on the immunological symphony that guides the course of regenerative medicine.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. STATEMENT OF SIGNIFICANCE: Systematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.

V. carteri f. nagariensis constitutes, in its most simplified form, a cellularized spheroid built around and stabilised by a form of primitive extracellular matrix (ECM).

We developed a modular approach to soft tissue engineering, by compact stacking V. carteri-based building blocks. This approach is made possible by the structure and cell adhesive properties of these building blocks, which results from the composition of their algal ECM.

A primary biocompatibility assessment demonstrated the cytocompatibility of the algal suspension, its histogenesis-promoting properties, and that it did not induce an inflammatory response in vitro. These results allowed us to consider the use of this algal suspension for soft tissue augmentation, and to initiate an in vivo biocompatibility study. V. carteri exhibited cellular fate-directing properties, causing (i) fibroblasts to take on an alkaline phosphatase+ stem-cell-like phenotype and (ii) both human adipose-derived stem cells and mouse embryonic stem cells to differentiate into preadipocytes to adipocytes. The ability of V. carteri to support histogenesis and adipogenesis was also observed in vivo by subcutaneous tissue augmentation of athymic mice, highlighting the potential of V. carteri to support or influence tissue regeneration.

We present for the first time V. carteri as an innovative and inspiring biomaterial for tissue engineering and soft tissue regeneration. Its strategies in terms of shape, structure and composition can be central in the design of a new generation of bio-inspired heterogeneous biomaterials recapitulating more appropriately the complexity of body tissues when guiding their regeneration.

Breast cancer has become the most diagnosed cancer type, endangering the health of women. Patients with breast resection are likely to suffer serious physical and mental trauma. Therefore, breast reconstruction becomes an important means of postoperative patient rehabilitation. Polyvinyl alcohol hydrogel has great potential in adipose tissue engineering for breast reconstruction. However, its application is limited because of the lack of bioactive factors and poor structural stability. In this study, we prepared biodegradable polylactic acid-glycolic acid copolymer/polycaprolactone/gelatin (PPG) nanofibers. We then combined them with polyvinyl alcohol/collagen to create tissue engineering scaffolds to overcome limitations. We found that PPG fibers formed amide bonds with polyvinyl alcohol/collagen scaffolds. After chemical crosslinking, the number of amide bonds increased, leading to a significant improvement in their mechanical properties and thermal stability. The results showed that compared with pure PVA scaffolds, the maximum compressive stress of the scaffold doped with 0.9 g nanofibers increased by 500 %, and the stress loss rate decreased by 40.6 % after 10 cycles of compression. The presence of natural macromolecular gelatin and the changes in the pore structure caused by nanofibers provide cells with richer and more three-dimensional adsorption sites, allowing them to grow in three dimensions on the scaffold. So, the hydrogel scaffold by reinforcing polyvinyl alcohol hydrogel with PPG fibers is a promising breast reconstruction method.

This state-of-the-art review explores the emerging field of regenerative hydrogels and their profound impact on the treatment of skin wounds. Regenerative hydrogels, composed mainly of water-absorbing polymers, have garnered attention in wound healing, particularly for skin wounds. Their unique properties make them well suited for tissue regeneration. Notable benefits include excellent water retention, creating a crucially moist wound environment for optimal healing, and facilitating cell migration, and proliferation. Biocompatibility is a key feature, minimizing adverse reactions and promoting the natural healing process. Acting as a supportive scaffold for cell growth, hydrogels mimic the extracellular matrix, aiding the attachment and proliferation of cells like fibroblasts and keratinocytes. Engineered for controlled drug release, hydrogels enhance wound healing by promoting angiogenesis, reducing inflammation, and preventing infection. The demonstrated acceleration of the wound healing process, particularly beneficial for chronic or impaired healing wounds, adds to their appeal. Easy application and conformity to various wound shapes make hydrogels practical, including in irregular or challenging areas. Scar minimization through tissue regeneration is crucial, especially in cosmetic and functional regions. Hydrogels contribute to pain management by creating a protective barrier, reducing friction, and fostering a soothing environment. Some hydrogels, with inherent antimicrobial properties, aid in infection prevention, which is a crucial aspect of successful wound healing. Their flexibility and ability to conform to wound contours ensure optimal tissue contact, enhancing overall treatment effectiveness. In summary, regenerative hydrogels present a promising approach for improving skin wound healing outcomes across diverse clinical scenarios. This review provides a comprehensive analysis of the benefits, mechanisms, and challenges associated with the use of regenerative hydrogels in the treatment of skin wounds. In this review, the authors likely delve into the application of rational design principles to enhance the efficacy and performance of hydrogels in promoting wound healing. Through an exploration of various methodologies and approaches, this paper is poised to highlight how these principles have been instrumental in refining the design of hydrogels, potentially revolutionizing their therapeutic potential in addressing skin wounds. By synthesizing current knowledge and highlighting potential avenues for future research, this review aims to contribute to the advancement of regenerative medicine and ultimately improve clinical outcomes for patients with skin wounds.

Hydrazone-crosslinked hydrogels are attractive protein delivery vehicles for regenerative medicine. However, each regenerative medicine application requires unique hydrogel properties to achieve an ideal outcome. The properties of a hydrogel can be impacted by numerous factors involved in its fabrication. We used design of experiments (DoE) statistical modeling to efficiently optimize the physicochemical properties of a hyaluronic acid (HA) hydrazone-crosslinked hydrogel for protein delivery for bone regeneration. We modified HA with either adipic acid dihydrazide (HA-ADH) or aldehyde (HA-Ox) functional groups and used DoE to evaluate the interactions of three input variables, the molecular weight of HA (40 or 100 kDa), the concentration of HA-ADH (1-3% w/v), and the concentration of HA-Ox (1-3% w/v), on three output responses, gelation time, compressive modulus, and hydrogel stability over time. We identified 100 kDa HA-ADH3.00HA-Ox2.33 as an optimal hydrogel that met all of our design criteria, including displaying a gelation time of 3.7 minutes, compressive modulus of 62.1 Pa, and minimal mass change over 28 days. For protein delivery, we conjugated affinity proteins called affibodies that were specific to the osteogenic protein bone morphogenetic protein-2 (BMP-2) to HA hydrogels and demonstrated that our platform could control the release of BMP-2 over 28 days. Ultimately, our approach demonstrates the utility of DoE for optimizing hydrazone-crosslinked HA hydrogels for protein delivery.

The tissue engineering field is currently advancing towards minimally invasive procedures to reconstruct soft tissue defects. In this regard, injectable hydrogels are viewed as excellent scaffold candidates to support and promote the growth of encapsulated cells. Cross-linked gelatin methacryloyl (GelMA) gels have received substantial attention due to their extracellular matrix-mimicking properties. In particular, GelMA microgels were recently identified as interesting scaffold materials since the pores in between the microgel particles allow good cell movement and nutrient diffusion. The current work reports on a novel microgel preparation procedure in which a bulk GelMA hydrogel is ground into powder particles. These particles can be easily transformed into a microgel by swelling them in a suitable solvent. The rheological properties of the microgel are independent of the particle size and remain stable at body temperature, with only a minor reversible reduction in elastic modulus correlated to the unfolding of physical cross-links at elevated temperatures. Salts reduce the elastic modulus of the microgel network due to a deswelling of the particles, in addition to triple helix denaturation. The microgels are suited for clinical use, as proven by their excellent cytocompatibility. The latter is confirmed by the superior proliferation of encapsulated adipose tissue-derived stem cells in the microgel compared to the bulk hydrogel. Moreover, microgels made from the smallest particles are easily injected through a 20G needle, allowing a minimally invasive delivery. Hence, the current work reveals that powdered cross-linked GelMA is an excellent candidate to serve as an injectable hydrogel for adipose tissue engineering.

Regenerative medicine aims to identify new research strategies for the repair and restoration of tissues damaged by pathological or accidental events. Mesenchymal stem cells (MSCs) play a key role in regenerative medicine approaches due to their specific properties, such as the high rate of proliferation, the ability to differentiate into several cell lineages, the immunomodulatory potential, and their easy isolation with minimal ethical issues. One of the main goals of regenerative medicine is to modulate, both in vitro and in vivo, the differentiation potential of MSCs to improve their use in the repair of damaged tissues. Over the years, much evidence has been collected about the ability of cytochalasins, a large family of 60 metabolites isolated mainly from fungi, to modulate multiple properties of stem cells (SCs), such as proliferation, migration, and differentiation, by altering the organization of the cyto- and the nucleo-skeleton. In this review, we discussed the ability of two different cytochalasins, cytochalasins D and B, to influence specific SC differentiation programs modulated by several agents (chemical or physical) or intra- and extra-cellular factors, with particular attention to human MSCs (hMSCs).

Adipose tissue engineering plays a key role in the reconstruction of soft-tissue defects. The acellular adipose matrix (AAM) is a promising biomaterial for the construction of engineered adipose tissue. However, AAM lacks sufficient adipoinduction potency because of the abundant loss of matrix-bound adipokines during decellularization.

An adipose-derived extracellular matrix collagen scaffold, "adipose collagen fragment" (ACF), was prepared using a novel mechanical method that provides sustained release of adipokines. Here, the authors used label-free proteomics methods to detect the protein components in AAM and ACF. In vivo, ACF was incorporated into AAM or acellular dermal matrix and implanted into nude mice to evaluate adipogenesis. Neoadipocytes, neovessels, and corresponding gene expression were evaluated. The effects of ACF on adipogenic differentiation of human adipose-derived stem cells and tube formation by human umbilical vein endothelial cells were tested in vitro.

Proteomics analysis showed that ACF contains diverse adipogenic and angiogenic proteins. ACF can release diverse adipokines and induce highly vascularized, mature adipose tissue in AAM, and even in nonadipogenic acellular dermal matrix. Higher expression of adipogenic markers peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha and greater numbers of tubule structures were observed in ACF-treated groups in vitro.

The combination of ACF and AAM could serve as a novel and promising strategy to construct mature, vascularized adipose tissue for soft-tissue reconstruction.

The combined use of AAM and ACF has been proven to induce a highly vascularized, mature, engineered adipose tissue in the nude mouse model, which may serve as a promising strategy for soft-tissue reconstruction.

Scaffold materials that better support neurogenesis are still needed to improve cell therapy outcomes for neural tissue damage. We have used a modularly tunable, highly compliant, degradable hydrogel to explore the impacts of hydrogel compliance stiffness on neural differentiation. Here we implemented competitive matrix crosslinking mechanics to finely tune synthetic hydrogel moduli within soft tissue stiffnesses, a range much softer than typically achievable in synthetic crosslinked hydrogels, providing a modularly controlled and ultrasoft 3D culture model which supports and enhances neurogenic cell behavior.

Soluble competitive allyl monomers were mixed with proteolytically-degradable poly(ethylene glycol) diacrylate derivatives and crosslinked to form a matrix, and resultant hydrogel stiffness and diffusive properties were evaluated. Neural PC12 cells or primary rat fetal neural stem cells (NSCs) were encapsulated within the hydrogels, and cell morphology and phenotype were investigated to understand cell-matrix interactions and the effects of environmental stiffness on neural cell behavior within this model.

Addition of allyl monomers caused a concentration-dependent decrease in hydrogel compressive modulus from 4.40 kPa to 0.26 kPa (natural neural tissue stiffness) without influencing soluble protein diffusion kinetics through the gel matrix. PC12 cells encapsulated in the softest hydrogels showed significantly enhanced neurite extension in comparison to PC12s in all other hydrogel stiffnesses tested. Encapsulated neural stem cells demonstrated significantly greater spreading and elongation in 0.26 kPa alloc hydrogels than in 4.4 kPa hydrogels. When soluble growth factor deprivation (for promotion of neural differentiation) was evaluated within the neural stiffness gels (0.26 kPa), NSCs showed increased neuronal marker expression, indicating early enhancement of neurogenic differentiation.

Implementing allyl-acrylate crosslinking competition reduced synthetic hydrogel stiffness to provide a supportive environment for 3D neural tissue culture, resulting in enhanced neurogenic behavior of encapsulated cells. These results indicate the potential suitability of this ultrasoft hydrogel system as a model platform for further investigating environmental factors on neural cell behavior.

The online version contains supplementary material available at 10.1007/s12195-024-00794-2.

Electrical stimulation can effectively accelerate bone healing. However, the substantial size and weight of electrical stimulation devices result in reduced patient benefits and compliance. It remains a challenge to establish a flexible and lightweight implantable microelectronic stimulator for bone regeneration. Here, we use self-powered technology to develop an electric pulse stimulator without circuits and batteries, which removes the problems of weight, volume, and necessary rigid packaging. The fully implantable bone defect electrical stimulation (BD-ES) system combines a hybrid tribo/piezoelectric nanogenerator to provide biphasic electric pulses in response to rehabilitation exercise with a conductive bioactive hydrogel. BD-ES can enhance multiple osteogenesis-related biological processes, including calcium ion import and osteogenic differentiation. In a rat model of critical-sized femoral defects, the bone defect was reversed by electrical stimulation therapy with BD-ES and subsequent bone mineralization, and the femur completely healed within 6 weeks. This work is expected to advance the development of symbiotic electrical stimulation therapy devices without batteries and circuits.

Autologous fat grafting is the gold standard for treatment in patients with soft-tissue defects. However, the technique has a major limitation of unpredictable fat resorption due to insufficient blood supply in the initial phase after transplantation. To overcome this problem, we investigated the capability of a medical-grade poly L-lactide-co-poly ε-caprolactone (PLCL) scaffold to support adipose tissue and vascular regeneration. Deploying FDM 3D-printing, we produced a bioresorbable porous scaffold with interconnected pore networks to facilitate nutrient and oxygen diffusion. The compressive modulus of printed scaffold mimicked the mechanical properties of native adipose tissue. In vitro assays demonstrated that PLCL scaffolds or their degradation products supported differentiation of preadipocytes into viable mature adipocytes under appropriate induction. Interestingly, the chorioallantoic membrane assay revealed vascular invasion inside the porous scaffold, which represented a guiding structure for ingrowing blood vessels. Then, lipoaspirate-seeded scaffolds were transplanted subcutaneously into the dorsal region of immunocompetent rats (n = 16) for 1 or 2 months. The volume of adipose tissue was maintained inside the scaffold over time. Histomorphometric evaluation discovered small- and normal-sized perilipin+ adipocytes (no hypertrophy) classically organized into lobular structures inside the scaffold. Adipose tissue was surrounded by discrete layers of fibrous connective tissue associated with CD68+ macrophage patches around the scaffold filaments. Adipocyte viability, assessed via TUNEL staining, was sustained by the presence of a high number of CD31-positive vessels inside the scaffold, confirming the CAM results. Overall, our study provides proof that 3D-printed PLCL scaffolds can be used to improve fat graft volume preservation and vascularization, paving the way for new therapeutic options for soft-tissue defects.

The regeneration of adipose tissue in patients after breast cancer surgery would be desirable without the use of growth factors or cells to avoid potential recurrence and metastasis. We reported that prolate spheroidal-shaped poly-L-lactic acid (PLLA) mesh implants of approximately 18-mm polar diameter and 7.5-mm greatest equatorial diameter containing collagen sponge (CS) would be replaced by regenerated adipose tissue after implantation, thereby suggesting an innovative method for breast reconstruction. Our study aimed to evaluate the adipose tissue regeneration ability of implant aggregates in a porcine model.

We prepared implant aggregates consisting of thirty PLLA mesh implants containing CS packed in a woven poly (glycolic acid) bag. The implant aggregates were inserted under the mammary glands in the porcine abdomen for a year. Single and double groups were classified by inserting either one or two implant aggregates on each side of the abdomen, respectively.

In both groups, the volume of the implant aggregates decreased over time, and the formation of adipose tissue peaked between 6 and 9 months. Histologically, the formation of adipose tissue was confirmed in the area that was in contact with native adipose tissue.

Our implant aggregates could induce the autologous adipose tissue after long term implantation in vivo, without the use of any growth factor or cell treatment, presenting a potential novel method of breast reconstruction.

Alterations of the extracellular matrix contribute to adipose tissue dysfunction in metabolic disease. We studied the role of matrix density in regulating human adipocyte phenotype in a tunable hydrogel culture system. Lipid accumulation was maximal in intermediate hydrogel density of 5 weight %, relative to 3% and 10%. Adipogenesis and lipid and oxidative metabolic gene pathways were enriched in adipocytes in 5% relative to 3% hydrogels, while fibrotic gene pathways were enriched in 3% hydrogels. These data demonstrate that the intermediate density matrix promotes a more adipogenic, less fibrotic adipocyte phenotype geared towards increased lipid and aerobic metabolism. These observations contribute to a growing literature describing the role of matrix density in regulating adipose tissue function.

Volumetric bioprinting (VBP) is a light-based 3D printing platform, which recently prompted a paradigm shift for additive manufacturing (AM) techniques considering its capability to enable the fabrication of complex cell-laden geometries in tens of seconds with high spatiotemporal control and pattern accuracy. A flexible allyl-modified gelatin (gelAGE)-based photoclick resin is developed in this study to fabricate matrices with exceptionally soft polymer networks (0.2-1.0 kPa). The gelAGE-based resin formulations are designed to exploit the fast thiol-ene crosslinking in combination with a four-arm thiolated polyethylene glycol (PEG4SH) in the presence of a photoinitiator. The flexibility of the gelAGE biomaterial platform allows one to tailor its concentration spanning from 2.75% to 6% and to vary the allyl to thiol ratio without hampering the photocrosslinking efficiency. The thiol-ene crosslinking enables the production of viable cell-material constructs with a high throughput in tens of seconds. The suitability of the gelAGE-based resins is demonstrated by adipogenic differentiation of adipose-derived stromal cells (ASC) after VBP and by the printing of more fragile adipocytes as a proof-of-concept. Taken together, this study introduces a soft photoclick resin which paves the way for volumetric printing applications toward soft tissue engineering.

Matrix stiffness has been disclosed as an essential regulator of cell fate. However, it is barely studied how the matrix stiffness affects stem cell functions when cell morphology changes. Thus, in this study, the effect of hydrogel stiffness on adipogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) with controlled morphology was investigated. Micropatterns of different size and elongation were prepared by a photolithographical micropatterning technique. The hMSCs were cultured on the micropatterns and showed a different spreading area and elongation following the geometry of the underlying micropatterns. The cells with controlled morphology were embedded in agarose hydrogels of different stiffnesses. The cells showed a different level of adipogenic differentiation that was dependent on both hydrogel stiffness and cell morphology. Adipogenic differentiation became strong when the cell spreading area decreased and hydrogel stiffness increased. Adipogenic differentiation did not change with cell elongation. Therefore, cell spreading area and hydrogel stiffness could synergistically affect adipogenic differentiation of hMSCs, while cell elongation did not affect adipogenic differentiation. A change of cell morphology and hydrogel stiffness was accompanied by actin filament alignment that was strongly related to adipogenic differentiation. The results indicated that cell morphology could affect cellular sensitivity to hydrogel stiffness. The results will provide useful information for the elucidation of the interaction of stem cells and their microenvironmental biomechanical cues.

The extracellular matrix in tissue consists of complex heterogeneous soft materials with hierarchical structure and dynamic mechanical properties dictating cell and tissue level function. In many natural matrices, there are nanofibrous structures that serve to guide cell activity and dictate the form and function of tissue. Synthetic hydrogels with integrated nanofibers can mimic the structural properties of native tissue; however, model systems with dynamic mechanical properties remain elusive. Here we demonstrate modular nanofibrous hydrogels that can be reversibly stiffened in response to applied magnetic fields. Iron oxide nanoparticles were incorporated into gelatin nanofibers through electrospinning, followed by chemical stabilization and fragmentation. These magnetoactive nanofibers can be mixed with virtually any hydrogel material and reversibly stiffen the matrix at a low fiber content (≤3%). In contrast to previous work, where a large quantity of magnetic material disallowed cell encapsulation, the low nanofiber content allows matrix stiffening with cells in 3D. Using adipose derived stem cells, we show how nanofibrous matrices are beneficial for both osteogenesis and adipogenesis, where stiffening the hydrogel with applied magnetic fields enhances osteogenesis while discouraging adipogenesis. Skeletal myoblast progenitors were used as a model of tissue morphogenesis with matrix stiffening augmenting myogenesis and multinucleated myotube formation. The ability to reversibly stiffen fibrous hydrogels through magnetic stimulation provides a useful tool for studying nanotopography and dynamic mechanics in cell culture, with a scope for stimuli responsive materials for tissue engineering.

Cellular agriculture is an emerging research field of agribiotechnology that aims to produce agricultural products using stem cells, without sacrificing animals or cultivating crops. Cultivated meat, as a representative cellular product of cellular agriculture, is being actively researched due to global food insecurity, environmental, and ethical concerns. This review focuses on the application of stem cells, which are the seeds of cellular agriculture, for the production of cultivated meat, with emphasis on deriving and culturing muscle and adipose stem cells for imitating fresh meat. Establishing standards and safety regulations for culturing stem cells is crucial for the market entry of cultured muscle tissue-based biomaterials. Understanding stem cells is a prerequisite for creating reliable cultivated meat and other cellular agricultural biomaterials. The techniques and regulations from the cultivated meat industry could pave the way for new cellular agriculture industries in the future.

Hydrazone-crosslinked hydrogels are attractive protein delivery vehicles for regenerative medicine. However, each regenerative medicine application requires unique hydrogel properties to achieve an ideal outcome. The properties of a hydrogel can be impacted by numerous factors involved in its fabrication. We used design of experiments (DoE) statistical modeling to efficiently optimize the physicochemical properties of a hyaluronic acid (HA) hydrazone-crosslinked hydrogel for protein delivery for bone regeneration. We modified HA with either adipic acid dihydrazide (HA-ADH) or aldehyde (HA-Ox) functional groups and used DoE to evaluate the interactions of three input variables, the molecular weight of HA (40 or 100 kDa), the concentration of HA-ADH (1-3% w/v), and the concentration of HA-Ox (1-3% w/v), on three output responses, gelation time, compressive modulus, and hydrogel stability over time. We identified 100 kDa HA-ADH3.0HA-Ox2.33 as an optimal hydrogel that met all of our design criteria, including displaying a gelation time of 3.7 minutes, compressive modulus of 62.1 Pa, and minimal mass change over 28 days. For protein delivery, we conjugated affinity proteins called affibodies that were specific to the osteogenic protein bone morphogenetic protein-2 (BMP-2) to HA hydrogels and demonstrated that our platform could control the release of BMP-2 over 28 days. Ultimately, our approach demonstrates the utility of DoE for optimizing hydrazone-crosslinked HA hydrogels for protein delivery.

Skeletal stem and progenitor cells (SSPCs) are the multi-potent, self-renewing cell lineages that form the hematopoietic environment and adventitial structures of the skeletal tissues. Skeletal tissues are responsible for a diverse range of physiological functions because of the extensive differentiation potential of SSPCs. The differentiation fates of SSPCs are shaped by the physical properties of their surrounding microenvironment and the mechanical loading forces exerted on them within the skeletal system. In this context, the present review first highlights important biomolecules involved with the mechanobiology of how SSPCs sense and transduce these physical signals. The review then shifts focus towards how the static and dynamic physical properties of microenvironments direct the biological fates of SSPCs, specifically within biomaterial and tissue engineering systems. Biomaterial constructs possess designable, quantifiable physical properties that enable the growth of cells in controlled physical environments both in-vitro and in-vivo. The utilization of biomaterials in tissue engineering systems provides a valuable platform for controllably directing the fates of SSPCs with physical signals as a tool for mechanobiology investigations and as a template for guiding skeletal tissue regeneration. It is paramount to study this mechanobiology and account for these mechanics-mediated behaviors to develop next-generation tissue engineering therapies that synergistically combine physical and chemical signals to direct cell fate. Ultimately, taking advantage of the evolved mechanobiology of SSPCs with customizable biomaterial constructs presents a powerful method to predictably guide bone and skeletal organ regeneration.