Surface topography or curvature plays a crucial role in regulating cell behavior, influencing processes such as adhesion, proliferation, and gene expression. Recent advancements in nano- and micro-fabrication techniques have enabled the development of biomimetic systems that mimic native extracellular matrix (ECM) structures, providing new insights into cell-adhesion mechanisms, mechanotransduction, and cell-environment interactions. This review examines the diverse applications of engineered topographies across multiple domains, including antibacterial surfaces, immunomodulatory devices, tissue engineering scaffolds, and cancer therapies. It highlights how nanoscale features like nanopillars and nanospikes exhibit bactericidal properties, while many microscale patterns can direct stem cell differentiation and modulate immune cell responses. Furthermore, we discuss the interdisciplinary use of topography for combined applications, such as the simultaneous regulation of immune and tissue cells in 2D and 3D environments. Despite significant advances, key knowledge gaps remain, particularly regarding the effects of topographical cues on multicellular interactions and dynamic 3D contexts. This review summarizes current fabrication methods, explores specific and interdisciplinary applications, and proposes future research directions to enhance the design and utility of topographically patterned biomaterials in clinical and experimental settings.

Glycosaminoglycan (GAG) represents an important extracellular matrix (ECM), particularly in GAG-rich tissues such as nucleus pulposus and cartilage. The ratio of GAGs/hydroxyproline (HYP) is an indicator of the relative abundance of the space-filling GAG matrix to the fibrous collagen matrix in a particular tissue. Here, we hypothesize that ECM niche with different GAG/HYP ratios will affect the outcomes of multiple differentiation of human mesenchymal stem cells (hMSCs). Specifically, we fabricated collagen-based biomaterials with different GAG/HYP ratios, and differentiate hMSCs in these materials towards osteogenic, chondrogenic and discogenic lineages. In osteogenic differentiation, Collagen without GAG (GAG/HYP ratio 0) showed higher calcium (Ca) and phosphorus (P) deposition and Ca/P ratio, more biomimetic ultrastructure, and better osteogenic phenotypic expression. For chondrogenic differentiation, aminated collagen (aCol-GAG) with intermediate GAG content (GAG/HYP ratio 5.0:1) showed higher GAG deposition, more biomimetic ultrastructure, and better chondrogenic phenotype. In discogenic differentiation, aminated collagen-aminated hyaluronic acid (aHA)-GAG (aCol-aHA-GAG) with the highest GAG content (GAG/HYP ratio 19.8:1), showed intensive GAG deposition, biomimetic ultrastructure, and higher phenotypic marker expression. This study contributes to developing collagen-based biomimetic materials with different GAG/HYP ratios and suggests the use of tissue-specific GAG/HYP ratio as a scaffold design parameter for hMSCs-based musculoskeletal tissue engineering. (198 words).

Curcumin (Cur) and epigallocatechin gallate (EGCG), the primary active compounds in turmeric and green tea, respectively, have been investigated for their anti-aging potential. The Cur and EGCG combination was encapsulated in sustained-release nanostructured lipid carriers (NLCs) to enhance their bioactivities and pharmaceutical properties. A significant enhancement in the antioxidant activities of the Cur and EGCG combination was observed at an optimal ratio, as demonstrated by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay (118.83 ± 3.78 %), ferric ion reducing antioxidant power assay (217.25 ± 13.45 %), and lipid peroxidation inhibition assay (106.08 ± 12.93 %), compared to Cur alone without compromising the antioxidant activities and total phenolic content of EGCG. This is due to the enhancement of total phenolic content of the combination of 218.83 ± 10.57 %. For anti-aging activities, the combination exhibited stimulation of SIRT1 protein and inhibition of collagenase and elastase of 27.53 ± 0.73 %, 43.70 ± 1.05 % and 51.76 ± 6.52 % compared with that achieved with Cur alone, respectively. The incorporation of the Cur and EGCG combination into NLCs resulted in high entrapment efficiencies of 98.60 ± 0.05 % for Cur and 98.40 ± 0.08 % for EGCG, with corresponding loading capacities of 0.789 ± 0.001 % and 3.935 ± 0.003 %, respectively. When formulated NLCs into an emulgel base, the system demonstrated sustained release profiles over 48 h, with 12.82 ± 0.99 % release of Cur and 63.77 ± 5.76 % release of EGCG. Significant skin retention was also observed after 24 h, with 23.88 ± 1.71 % Cur and 22.79 ± 4.65 % EGCG retained in the skin. Therefore, Cur: EGCG-loaded NLCs in emulgel can deliver the active compounds into the dermis, enhancing skin penetration, sustained delivery, and anti-aging activity superior to each conventional single active compound in topical formulations.

Intervertebral disc degeneration (IVDD) is increasingly prevalent in aging societies and poses a significant health challenge. Due to the limited blood supply to the disc, oral medications and systemic treatments are often ineffective. Consequently, localized injection therapies, which deliver therapeutic agents directly to the degenerated disc, have emerged as more efficient. Self-healing injectable hydrogels are particularly promising due to their potential for minimally invasive delivery, precise implantation, and targeted drug release into hard-to-reach tissue sites, including those requiring prolonged healing. Their dynamic viscoelastic properties accurately replicate the mechanical environment of the natural nucleus pulposus, providing cells with an adaptive biomimetic microenvironment. This review will initially discuss the anatomy and pathophysiology of intervertebral discs, current treatments, and their limitations. Subsequently, we conduct bibliometric analysis to explore the research hotspots and trends in applying injectable hydrogel technology to treat IVDD. It will then explore the promising features of injectable hydrogels in biomedical applications such as drug, protein, cells and gene delivery, tissue engineering and regenerative medicine. We discuss the construction mechanisms of injectable hydrogels via physical interactions, chemical and biological crosslinkers, and discuss the selection of biomaterials and fabrication methods for developing novel hydrogels for IVD tissue engineering. The article concludes with future perspectives on the application of injectable hydrogels in this field.

High-throughput screening (HTS) three-dimensional (3D) tumor models are a promising approach for cancer drug discovery, as they more accurately replicate in vivo cell behavior than two-dimensional (2D) models. However, assessing and comparing current 3D models for drug efficacy remains essential, given the significant influence of cellular conditions on treatment response. To develop in vivo mimicking 3D models, we evaluated two HTS 3D models established in 96-well plates with 3D polycaprolactone (PCL) scaffolds fabricated using two distinct methods, resulting in scaffolds with either homogenous or non-homogenous fiber networks. These models, based on human HeLa cervical cancer cells and cancer-associated fibroblasts (CAFs) cultured as mono- or co-cultures within the 3D scaffolds, revealed that anticancer drug paclitaxel (PTX) exhibited consistently higher inhibitory concentration 50 (IC50) in 3D (≥ 1000 nM) compared to 2D (≥ 100 nM), indicating reduced toxicity on cells cultured in 3D. Interestingly, the toxicity of PTX was significantly lower on mini-tumors in non-homogenous 3D (IC50: 600 or 1000 nM) than in homogenous 3D cultures (IC50 exceeding 1000 nM). Microscopic studies revealed that the non-homogenous scaffolds closely resemble the tumor collagen network than their homogeneous counterpart. Both 3D scaffolds offer optimal pore size, facilitating efficient cell infiltration into the depth of 58.1 ± 1.2 µm (homogenous) and 86.4 ± 9.8 µm (non-homogenous) within 3D cultures. Cells cultured in the 3D non-homogenous systems exhibited drug treatment responses closer to in vivo conditions, highlighting the role of scaffold structure and design on cellular response to drug treatment. The PCL-based 3D models provide a robust, tunable, and efficient approach for the HTS of anti-cancer drugs compared to conventional 2D systems.

Proteoglycans are high molecular weight glycoproteins with potential benefits in preventing osteoarthritis, reducing inflammation, enhancing immune function, and promoting skin health. Aggrecan, a key proteoglycan with glycosaminoglycan (GAG) chains, poses challenges in accurate quantification due to its complex structure. We hypothesize that by selecting target peptides from core proteins that exclude post-translational modifications such as GAG attachment, proteoglycans can be analyzed with high sensitivity and accuracy. In this study, we identified aggrecan from salmon nasal cartilage (Oncorhynchus keta) using liquid chromatography-mass spectrometry (LC-MS). We developed a quantitative multiple reaction monitoring (MRM)-MS assay with stable-isotope-labeled peptides. This method demonstrated high precision and sensitivity, achieving a limit of detection (LOD) of 0.0008 μg/mL and a lower limit of quantification (LLOQ) of 0.0025 μg/mL. These findings validate that targeting core proteins enables accurate proteoglycan quantification and support its application in quality assessment and development of salmon nasal cartilage-based foods.

Understanding the biochemistry and pharmacodynamics of chronic wounds is of key importance, due to the millions of people in the UK affected and the significant cost to the NHS. Chronic wounds are characterised by elevated concentrations of matrix metalloproteinases (MMPs) that destroy the surrounding extracellular matrix (ECM). However, fibroblasts can produce tissue inhibitors of MMPs (TIMPs) in order to regulate wound healing. Therefore, the role of TIMPs in both acute and chronic wounds needs to be properly understood in order to develop therapeutic treatments. In this work, we propose a reaction-diffusion system of four partial differential equations that describe the interaction of the ECM, fibroblasts, MMPs, and TIMPs in a wound. We observe that, subject to parameter sets corresponding to both acute and chronic wound healing, this mathematical model gives rise to travelling wave solutions. Using bifurcation analysis, we demonstrate that excessive degradation of the ECM results in the emergence of chronic wounds, and the reversal of these chronic wounds is prohibited for lower TIMP production values. These results are replicated within a simplified model obtained via a parameter sensitivity analysis. This model is further extended to more realistic spatial domains where we demonstrate the effectiveness of a therapeutic hydrogel containing TIMPs as a treatment for chronic wounds.

Advances in tissue engineering are focused on devising improved therapeutics to reconstruct craniofacial bones. In cell-based strategies, biomaterials with specific physicochemical properties can mimic natural environments, supporting stem cell renewal, survivability, and cell fate. This study highlights the engineering of a 3D-printed (Melt Electrowritten, MEW) fluorinated‑calcium phosphate (F/CaP)-coated polymeric scaffold infused with collagen (COL) that boosts the performance of transplanted alveolar bone-derived mesenchymal stem cells (aBMSCs). Electron microscopy revealed micron-sized (2.7 μm) polymeric fibers forming a porous (500 μm fiber strand spacing) composite scaffold with a uniform F/CaP coating homogeneously infiltrated with collagen. In vitro, our findings underscored the cytocompatibility of the collagen-infused F/CaP-coated composite scaffold, fostering a suitable environment for aBMSCs proliferation and differentiation. Cells within the F/CaP-coated constructs exhibited upregulated osteogenic gene activity, and the addition of collagen augmented the expression of critical bone-forming genes (i.e., Runx2 and OCN). After in vivo implantation, the scaffolds integrated well with the surrounding host tissue, supporting extensive blood vessel infiltration. Notably, the collagen-infused F/CaP-coated composite scaffolds showed an increased CD31-positive vessel growth compared to the non-coated counterparts. At 8 weeks, aBMSCs-laden F/CaP-Coated+COL composite scaffolds exhibited robust bone formation, creating connecting bony bridges in calvarial defects. Importantly, F/CaP-Coated+COL composite scaffolds displayed pronounced OCN expression, indicating enhanced osteogenic potential. Thus, the engineered F/CaP-coated polymeric scaffold laden with aBMSCs and infused with collagen has proven effective in supporting cell growth, vascularization, and rapid bone regeneration, suggesting potential for future clinical use.

Collagen type I (COL1) is the most abundant protein in the human body and is a main component in the extracellular matrix. The COL1 structure vastly influences normal tissue homeostasis, and changes in the matrix drive progression in multiple diseases. Cardiovascular diseases (CVD) are the leading cause of mortality and morbidity in many Western countries; alterations in the extracellular matrix turnover processes, including COL1, are known to influence the pathophysiological processes leading to CVD outcome. Peptides reflecting COL1 formation and degradation have been established and explored for over two decades in CVD. This review aims to combine and assess the evidence for using COL1-derived circulating peptides as biomarkers in CVD. Secondly, the review identifies existing pitfalls, and evaluates future opportunities for improving the technical characteristics and performance of the biomarkers for implementation in the clinical setting.

This article provides a comprehensive review of current neuroimaging techniques for visualizing and quantifying extracellular matrix (ECM) components and interstitial fluid (ISF) dynamics in cerebrovascular disorders. It examines how alterations in ECM composition and ISF movement patterns correlate with various cerebrovascular pathologies, including ischemic stroke, frontotemporal dementia, cerebral small vessel disease, Alzhheimer's disease, and vascular dementia. The review emphasizes novel imaging markers specific to ECM/ISF alterations and their utility in differentiating various cerebrovascular pathologies. Special attention is given to the clinical applications of these imaging techniques for early disease detection, monitoring progression, and guiding therapeutic interventions.

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells, defined by apical-basal polarity and tight intercellular junctions, acquire migratory and invasive properties characteristic of mesenchymal cells. Under normal conditions, EMT directs essential morphogenetic events in embryogenesis and supports tissue repair. When dysregulated, EMT contributes to pathological processes such as organ fibrosis, chronic inflammation, and cancer progression and metastasis. Matrix metalloproteinases (MMPs)-a family of zinc-dependent proteases that degrade structural components of the extracellular matrix-sit at the nexus of this transition by dismantling basement membranes, activating pro-EMT signaling pathways, and cleaving adhesion molecules. When normally regulated, MMPs promote balanced ECM turnover and support the cyclical remodeling necessary for proper development, wound healing, and tissue homeostasis. When abnormally regulated, MMPs drive excessive ECM turnover, thereby promoting EMT-related pathologies, including tumor progression and fibrotic disease. This review provides an integrated overview of the molecular mechanisms by which MMPs both initiate and sustain EMT under physiological and disease conditions. It discusses how MMPs can potentiate EMT through TGF-β and Wnt/β-catenin signaling, disrupt cell-cell junction proteins, and potentiate the action of hypoxia-inducible factors in the tumor microenvironment. It discusses how these pathologic processes remodel tissues during fibrosis, and fuel cancer cell invasion, metastasis, and resistance to therapy. Finally, the review explores emerging therapeutic strategies that selectively target MMPs and EMT, ranging from CRISPR/Cas-mediated interventions to engineered tissue inhibitors of metalloproteinases (TIMPs), and demonstrates how such approaches may suppress pathological EMT without compromising its indispensable roles in normal biology.

Obtaining the traces and the characteristics of elongated structures is an important task in computer vision pipelines. In biomedical applications, the analysis of traces of vasculature, nerves or fibres of the extracellular matrix can help characterise processes like angiogenesis or the effect of a certain treatment. This paper presents an objective comparison of six existing methodologies (Edge detection, CT Fire, Scale Space, Twombli, U-Net and Graph Based) and one novel approach called Trace Ridges to trace biomedical images with fibre-like structures. Trace Ridges is a fully automatic and fast algorithm that combines a series of image-processing algorithms including filtering, watershed transform and edge detection to obtain an accurate delineation of the fibre-like structures in a rapid time. To compare the algorithms, four biomedical data sets with vastly distinctive characteristics were selected. Ground truth was obtained by manual delineation of the fibre-like structures. Three pre-processing filtering options were used as a first step: no filtering, Gaussian low-pass and DnCnn, a deep-learning filtering. Three distance error metrics (total, average and maximum distance from the obtained traces to the ground truth) and processing time were calculated. It was observed that no single algorithm outperformed the others in all metrics. For the total distance error, which was considered the most significative, Trace Ridges ranked first, followed by Graph Based, U-Net, Twombli, Scale Space, CT Fire and Edge Detection. In terms of speed, Trace Ridges ranked second, only slightly slower than Edge Detection. Code is freely available at github.com/youssefarafat/Trace_Ridges.

Impaired efficacy of in vitro expanded mesenchymal stem cells (MSCs) is a universal and thorny situation, which cast a shadow on further clinical translation of exogenous MSCs. Moreover, the relatively lengthy healing process, host metabolic heterogeneity and the sophisticated cell recognition and crosstalk pose rigorous challenges towards MSC-based bone regeneration strategies. Three-dimensional (3D) cell aggregates facilitate more robust intercellular communications and cell-extracellular matrix (ECM) interactions, providing a better mimicry of microarchitectures and biochemical milieus in vivo, which is conducive for stemness maintenance and downstream bone formation.

This review enunciates the phenotypic features of MSCs in aggregates, which deepens the knowledge of the MSC fate determination in 3D microenvironment. By summarizing current empowerment methods and biomaterial-combined techniques for establishing functionalized MSC aggregates, this review aims to spark innovative and promising solutions for exalting the translational value of MSCs and improve their therapeutic applications in bone tissue repair.

3D aggregates optimize regenerative behaviors of in vitro cultured MSCs including cell adhesion, viability, proliferation, pluripotency and immunoregulation capacity, etc. Biomaterials hybridization endows MSC aggregates with tailored mechanical and biological properties, which offers more possibilities in adapting various clinical scenarios.

For several decades, research has studied the influence of the extracellular matrix (ECM) mechanical properties in cell response, primarily emphasising its elasticity as the main determinant of cell and tissue behaviour. However, the ECM is not purely elastic; it is viscoelastic. ECM viscoelasticity has now emerged as a major regulator of collective cell dynamics. This review highlights recent findings on the role of ECM viscoelasticity in development and pathology.

High thrombogenicity and shortened lifespan have limited the application of mechanical valves and bioprosthetic valves, respectively. Tissue engineering heart valve (TEHV) holds significant potential as a favorable prosthetic valve to overcome the limitations of the current prosthetic valves, featuring the capabilities of self-pairing and adaptive remodeling. However, TEHVs, mainly fabricated from decellularized xenogeneic heart valves (DHV), still have challenges such as thrombosis, inferior endothelialization, and immune responses. Herein, a drug-loaded glycoprotein-like network hybrid TEHV (OHSC-V) was engineered through the one-pot hybridization of DHV, oxidized HA (OHA), phenylboronic acid grafted silk fibroin (SF-PBA), and curcumin (Cur), where OHA served as a biocompatible backbone to cross-link the DHV and the conjugate of SF-PBA and Cur. With the introduction of the multifunctional drug-loaded glycoprotein-like network, OHSC-V not only effectively inhibited the adsorption of plasma proteins, blood cells, platelets, and thrombosis but also facilitated the endothelialization of TEHV. Furthermore, the OHSC-V eliminated the reactive oxygen species and responsively released Cur to modulate the immune responses. Moreover, the calcification degree of hybrid TEHVs was markedly lower than that of glutaraldehyde cross-linked DHV after 90 days of implantation. Overall, OHSC-V demonstrated enhanced performance of antithrombosis, endothelialization, immunomodulation, and anticalcification, showcasing the further potential for application exploration.

Cell sheet (CS)-based approaches hold significant potential for tissue regeneration, relying on the extracellular matrix (ECM) for success. Like in native tissues, the ECM provides structural and biochemical support for cellular homeostasis and function. Effective preservation strategies that maintain ECM integrity are critical to enhance the therapeutic potential of CS-based approaches. While cryogenic and hypothermic preservation methods offer potential solutions, their impact on CS ECM structure is not fully understood. Therefore, a comprehensive analysis of the ECM of hASCs CS following cryogenic and hypothermic preservation for 3 and 7 days, was conducted. Although proteomic analysis indicated that cryopreservation had no significant effect on the overall composition of the ECM, it induced significant ECM structural alterations, particularly disrupting collagen organization, which was not observed following hypothermic preservation. These structural changes were accompanied by alterations in mechanical properties, including a reduction in elastic modulus. In contrast, hypothermic preservation maintained ECM integrity and mechanical properties similar to the control. The notable ECM structural changes following cryogenic preservation can potentially impact cellular behavior, including adhesion, proliferation, and differentiation, thereby affecting the efficacy of CS therapies in vivo. This suggests that hypothermia may offer a promising alternative to cryopreservation for preserving CS integrity and functionality.

Advances in understanding the mechanisms behind genetic diseases like Duchenne muscular dystrophy (DMD) underscore the critical role of the extracellular matrix (ECM) composition in disease progression. Effective in vitro models must replicate the intercellular relationships and physicochemical properties of native ECM to fully capture disease-specific characteristics. Although recent biomaterials support the in vitro biofabrication of pathophysiological environments, they often lack disease-specific ECM features. In this study, DystroGel, a hydrogel derived from the cardiac ECM of a porcine DMD model, replicates the distinct molecular composition of dystrophic cardiac tissue for the first time. The findings indicate that the dystrophic ECM matrix exhibits a unique protein profile, impacting cellular processes critical to DMD pathology. This work demonstrates the importance of using a 3D substrate that recreates intercellular dynamics within a defined pathological environment, enhancing the ability to model genetic disorders and providing a valuable tool for advancing personalized therapeutic strategies.

Most microorganisms as well as bacteria live in a community under natural conditions. Bacteria adopted to biofilm mode of life more than 3 billion years ago to survive extreme, harsh environments. They become harmful when they acquire resistance to antibiotics and overcome the standard therapies, which is most commonly found in hospitals. Therefore, many studies have been published regarding antimicrobial resistance (AMR). Staphylococcus aureus is a dangerous pathogen, ubiquitously prevalent as a commensal and opportunistic microorganism in human populations. Methicillin-resistant Staphylococcus aureus (MRSA) is considered one of the major medical problems worldwide since they are frequent colonizers of implanted medical devices causing a variety of hospital-acquired infections. For many years, vancomycin has been the drug of choice for MRSA whereas linezolid is considered the last resort drug. This comparative, cross-sectional study investigated the effects of linezolid on biofilm formation in vitro compared to vancomycin across 85 MRSA isolates. To our knowledge, this is the first study to report high levels of linezolid resistance in MRSA in Iraq. In this brief report, 5 MRSA strains showed resistance to linezolid, with minimum inhibitory concentration (MIC) values of 256 μg/ml. The exact same isolates exhibited vancomycin resistance with MIC values of 1024 μg/ml. All linezolid-resistant MRSA (LR-MRSA) strains demonstrated biofilm formation ability. Additionally, linezolid inhibited the expression of adhesion-related genes cna and clfB. The authors concluded that linezolid exerts a comparable effect to vancomycin in biofilm treatment.

The extracellular matrix (ECM) and its primary chemical components, including collagen, play a pivotal role in carcinogenesis and tumor progression. The ECM actively regulates cell proliferation, migration, and, importantly, resistance to various adverse factors. It is widely recognized as a key factor in modifying the resistance of tumor cells to various treatment modalities and cytotoxic compounds. However, the role of the ECM in tumor cell adaptation to nutritional deficiencies and hypoxic conditions remains significantly less studied. Since it is generally accepted that tumor cells resistance increases when cultured in a three-dimensional matrix, we sought to experimentally test the universality of this statement. In this work, we analyzed the responses of tumor cells with varying origins and proliferative activities, including human bladder carcinoma, epidermoid carcinoma, and ovarian carcinoma, to deprivation of serum, glucose and oxygen. We compared cell resistance to suboptimal conditions when cultured in a monolayer on tissue culture (TC)-treated polystyrene, on collagen-coated surfaces, or within a three-dimensional hydrogel composed of collagen type I. All three cell lines were stably transfected with fluorescent protein genes. To register the cell growth dynamics, we used a fluorescence-based technique that allows long-term quantitative observations without disrupting the hydrogel. The analyzed cell lines demonstrated different patterns of relative sensitivity to suboptimal conditions. We revealed that the direction and intensity of the collagen matrix effect depend on the cell type. Slowly proliferating ovarian carcinoma cells showed no noticeable changes in their behavior when cultured in a gel compared to a monolayer. In the case of bladder carcinoma, we registered predominantly resistance-stimulating effect of the collagen matrix, but it was significant only under serum deprivation. The most pronounced effect of collagen was registered for epidermoid carcinoma. Importantly, this effect was ambivalent: gel-embedded cells demonstrated significantly enhanced resistance to serum deprivation, but, at the same time, they were more responsive to glucose starvation and hypoxic conditions. We attribute the registered phenomenon to the individual characteristics of tumor cells with different origins and metabolic activities.

While previous research has demonstrated the therapeutic efficacy of telomerase reverse transcriptase (TERT) overexpression using adeno-associated virus and cytomegalovirus vectors to combat aging, the broader implications of TERT germline gene editing on the mammalian genome, proteomic composition, phenotypes, lifespan extension, and damage repair remain largely unexplored. In this study, we elucidate the functional properties of transgenic mice carrying the Tert transgene, guided by precise gene targeting into the Rosa26 locus via embryonic stem (ES) cells under the control of the elongation factor 1α (EF1α) promoter. The Tert knock-in (TertKI) mice harboring the EF1α-Tert gene displayed elevated telomerase activity, elongated telomeres, and extended lifespan, with no spontaneous genotoxicity or carcinogenicity. The TertKI mice showed also enhanced wound healing, characterized by significantly increased expression of Fgf7, Vegf, and collagen. Additionally, TertKI mice exhibited robust resistance to the progression of colitis induced by dextran sodium sulfate (DSS), accompanied by reduced expression of disease-deteriorating genes. These findings foreshadow the potential of TertKI as an extraordinary rejuvenation force, promising not only longevity but also rejuvenation in skin and intestinal aging.

The extracellular matrix (ECM) is the natural scaffold of all soft tissues in tissue engineering. Of special interest is the use of ECM as a hydrogel, which can be used to enclose cells and to be molded into any form by 3D bioprinting. Protocols for the preparation of ECM vary in the use of physical and chemical processing steps, the use of different detergents for decellularization, and the removal of DNA and RNA residues and show a different use of solvents and wash buffers. We have, therefore, compared seven different variations for the decellularization of a primary porcine isolate to manufacture decellularized adipose tissue (DAT) for their use in adipose tissue engineering and as a hydrogel in particular. Decellularization efficacy was assessed by DNA quantification while retention of ECM components was evaluated by measuring the content of hydroxyproline and glycosaminoglycan (GAGs). Depending on the decellularization protocol, the composition and DNA content of the resulting DAT were different. All DAT samples were processed into hydrogels to assess their mechanical properties as well as their influence on cellular metabolic activity and cell differentiation. The different compositions of the DAT and the resulting hydrogels had an effect on the stability and printability of the gels. Some DAT that were digested with hydrochloric acid (HCl) were more stable than those that were digested with acetic acid (AA). In addition, depending on the protocol, there was a clear effect on adipose-derived stem cells (ASC), endothelial cells and fibroblasts, cultured with the hydrogels. The differentiation of ASC to adipocytes could be achieved on most of the hydrogels. Human dermal microvascular endothelial cells (HDMEC) showed significantly better metabolic activity on hydrogels digested with HCl than digested with AA. HDMEC cultured on hydrogel #2 digested with HCl showed a 40% higher metabolic activity compared to collagen as a positive control, whereas culturing HDMEC on hydrogel #2 digested with AA resulted in a cellular metabolic activity loss of 60%. In a triculture of all three cell types, the formation of first tubular networks by HDMEC was achieved depending on the hydrogel used.

Acute respiratory distress syndrome (ARDS) is pathologically characterized by diffuse alveolar damage (DAD) and is associated with high morbidity and mortality rates. Although pulmonary injury initiates alveolar-capillary barrier damage in ARDS, remodeling of the extracellular matrix (ECM), which is pivotal for both tissue repair and organ recovery, may play a large role in persistent ARDS. This study investigated the compositional changes in the ECM in different DAD stages in ARDS. Paraffin-embedded lung sections collected during autopsy or from posttransplant lungs were obtained from patients with ARDS (n = 28) admitted to the University Medical Center Groningen between 2010 and 2020. Sections were stained histochemically, and immunohistochemically for collagen III α1 chain (Col IIIa1), IV α3 chain (Col IVa3), VI α1 chain (Col VIa1), periostin (PSTN), lumican (LUM), and fibronectin (FN). The sections were divided into 118 regions based on DAD stages (54 early vs. 64 advanced). The differences in the expression of selected proteins were compared between DAD stages or across ARDS duration (<7 days, 7-14 days, and >14 days). The fiber pattern of Col VIa1 was analyzed using CellProfiler. Higher tissue density, lower proportional areas of Col IIIa1, Col IVa3, and LUM, and more concentrated Col VIa1 fibers were observed in the advanced DAD stage than in the early DAD stage. Areas with higher proportions of total collagen and FN, and lower proportional areas of Col IIIa1, Col IVa3, and LUM were detected in lung regions from patients with ARDS >14-days duration. These findings revealed proportional changes in ECM components, strongly suggesting that dynamic changes in ECM proteins play a role in pathophysiology in ARDS during progression.NEW & NOTEWORTHY Our study revealed ECM protein compositional differences in lung parenchyma between stages of DAD. In advanced DAD, tissue density was higher, but collagen type III, type IV, and lumican were proportionally lower compared with early DAD. The organization of collagen type VI fibers was highly concentrated in advanced DAD. Our results indicate that both composition and organization of ECM were remodeled in advanced DAD, suggesting a role in manifesting acute respiratory distress syndrome.

Soft biopolymer networks play pivotal roles in governing cellular mechanics, tissue structure, and physiological processes such as blood coagulation. Understanding their permeability and mechanical responses under compression is crucial for elucidating mass transport phenomena and their impact on extra- and intra-cellular behavior as well as processes affecting functionality of blood clots, cartilage and other fibrous tissues. The nonlinear responses of these networks to mechanical stresses prevent application of established linear poro-elasticity models. Despite extensive studies of fibrous network viscoelastic properties under shear deformations, their dynamic responses to compressive deformations remain poorly understood, particularly in physiological contexts of growth and collective migration of solid bodies. Conventional experimental techniques face challenges in accurately evaluating the permeability of these networks, hindering comprehensive understanding of their poromechanical behavior. In this study, we employ a novel poroelastic hybrid approach combining rheometer-based compression rheology with camera-facilitated sample shape detection to directly measure fluid flux and network permeability under controlled compressive strains. Accompanying experimental investigations, a continuum model implemented in finite elements, and an analytical model are developed to interpret the findings. The experimental data align well with the analytical model, revealing the emergence and disappearance of distinct densification regimes within the gel under mechanical stress. This study advances our understanding of the intricate interplay between mechanical forces, fluid flow, and structural properties in soft biopolymer networks, with a specific focus on fibrin- and collagen-based gels which represent the most abundant protein networks in the extracellular environment.

The fibroblast (FB), cardiomyocyte (CM), and vascular endothelial cell (Vas_EC) are the three major cell types in the heart, yet their relationships during development are largely unexplored. To address this gap, we employed RNA staining of the FB marker gene Col1a1 together with the CM marker gene Actn2 and the Vas_EC marker gene Cdh5 at different stages. This approach enabled us to discern the anatomical pattern of cardiac FBs and identify approximately one EC and four CMs directly interacting with each FB. Molecularly, through the analysis of single-cell mRNA sequencing (scRNA-seq) data, we unveiled collagen as the top signaling molecule derived from FBs influencing CM and Vas_EC development. Subsequently, we used a Pdgfra-CreER controlled diphtheria toxin A (DTA) system to ablate the FBs at different stages. We found that the ablation of FBs disrupted myocardium and vasculature development and led to embryonic heart defects. Using scRNA-seq, we further profiled the ablated hearts and identified molecular defects in their ventricular CMs and Vas_ECs compared to control hearts. Moreover, we identified a reduction of collagen in the ablated hearts and predicted collagen as the major signaling pathway regulating the differentially expressed genes in the ablated ventricular CMs. Finally, we performed both short-term and long-term fibroblast ablation at the neonatal stage. We found that short-term ablation caused a reduction in collagen and Vas_EC density, while long-term ablation may induce compensatory collagen expression without causing heart function reduction. In summary, our study has identified the function of fibroblasts in regulating myocardium and vasculature development and implicated an important role for the collagen pathway in this process.

The extracellular matrix (ECM), present in nearly all tissues, provides extensive support to resident cells through structural, biomechanical, and biochemical means, and in return the ECM undergoes constant remodeling from interacting cells to adapt to the evolving tissue states. Bioengineered 3D tissues, commonly known as cell-ECM composites, are robust model systems to recapitulate and investigate native pathophysiology. Key to this engineered morphogenesis process are the intricate cell-ECM interactions reflected by how cells respond to and thereby modulate their surrounding microenvironments through their ongoing ECM secretome. However, investigating ECM-regulated new ECM production has been challenging due to the proteomic background from the pre-existing biomaterial ECM. To address this hindrance, here we present a chemoselective strategy to label, enrich, and characterize newly synthesized ECM (newsECM) proteins produced by resident cells, allowing distinction from the pre-existing ECM background. Applying our analytical pipeline to bioengineered tumor tissues, either built upon decellularized ECM (dECM-tumors) or as ECM-free tumor spheroids (tumoroids), we observed distinct ECM synthesis patterns that were linked to their extracellular environments. Tumor cells responded to the dECM presence with elevated ECM remodeling activities, mediated by augmented digestion of pre-existing ECM coupled with upregulated synthesis of tumor-associated ECM. Our findings highlight the sensitivity of newsECM profiling to capture remodeling events that are otherwise under-represented by bulk proteomics and underscore the significance of dECM support for enabling native-like tumor cell behaviors. We anticipate the described newsECM analytical pipeline to be broadly applicable to other tissue-engineered systems to probe ECM-regulated ECM synthesis and remodeling, both fundamental aspects of cell-ECM crosstalk in engineered tissue morphogenesis.

The year 2024 marks the 50th anniversary of the discovery of surface-enhanced Raman spectroscopy (SERS). Over recent years, SERS has experienced rapid development and became a critical tool in biomedicine with its unparalleled sensitivity and molecular specificity. This review summarizes the advancements and challenges in SERS substrates, nanotags, instrumentation, and spectral analysis for biomedical applications. We highlight the key developments in colloidal and solid SERS substrates, with an emphasis on surface chemistry, hotspot design, and 3D hydrogel plasmonic architectures. Additionally, we introduce recent innovations in SERS nanotags, including those with interior gaps, orthogonal Raman reporters, and near-infrared-II-responsive properties, along with biomimetic coatings. Emerging technologies such as optical tweezers, plasmonic nanopores, and wearable sensors have expanded SERS capabilities for single-cell and single-molecule analysis. Advances in spectral analysis, including signal digitalization, denoising, and deep learning algorithms, have improved the quantification of complex biological data. Finally, this review discusses SERS biomedical applications in nucleic acid detection, protein characterization, metabolite analysis, single-cell monitoring, and in vivo deep Raman spectroscopy, emphasizing its potential for liquid biopsy, metabolic phenotyping, and extracellular vesicle diagnostics. The review concludes with a perspective on clinical translation of SERS, addressing commercialization potentials and the challenges in deep tissue in vivo sensing and imaging.

Resilient hydrogels are of great interest in soft tissue applications, such as soft tissue engineering and wound healing, with their biomimetic mechanical and hydration properties. A critical aspect in designing hydrogels for healthcare is their functionalities to control the surrounding biological environments to optimize the healing process. Herein, we have created an elastomer-clay nanocomposite hydrogel system with biomimetic mechanical behavior and sustained drug delivery of bioactive components and malodorous diamine-controlling properties. These hydrogels were prepared by a combined approach of melt intercalation of poly(ethylene glycol) and montmorillonite clay, followed by in situ cross-linking with a branched poly(glycerol sebacate) prepolymer. The hydration, vapor transmission, and surface wettability of the hydrogels were readily controlled by varying the clay content. Their mechanical properties were also modulated to mimic the Young's moduli (ranging between 12.6 and 105.2 kPa), as well as good flexibility and stretchability of soft tissues. A porous scaffold with interconnected pore structures as well as full and instant shape recovery was fabricated from a selected nanocomposite to demonstrate its potential applications as soft tissue scaffolds and wound healing materials. Biodegradability and biocompatibility were tested in vitro, showing controllable degradation kinetics with clay and no evidence of cytotoxicity. With the high surface area and absorption capacity of the clay, sustained drug delivery of a proangiogenic agent of 17β-estradiol as a model drug and the ability to control the malodorous diamines were both achieved. This elastomer-clay nanocomposite hydrogel system with a three-dimensional interconnected porous scaffold architecture and controllable hydration, mechanical, and biodegradable properties, as well as good biocompatibility and the ability to control the biological chemical species of the surrounding environments, has great potential in soft tissue engineering and wound healing.

The liver is the most common site of metastasis of colorectal cancer (CRC), and colorectal liver metastasis is one of the major causes of CRC-related deaths worldwide. The tumor microenvironment, particularly the extracellular matrix (ECM), plays a critical role in CRC metastasis and chemoresistance. Based on findings from clinical and basic research, this review attempts to offer a complete understanding of the role of the ECM in colorectal liver metastasis and to suggest potential ways for therapeutic intervention. First, the ECMs' role in regulating cancer cell fate is explored. We then discuss the hepatic ECM fingerprint and its influence on the metastatic behavior of CRC cells, highlighting key molecular interactions that promote metastasis. In addition, we examine how changes in the ECM within the metastatic niche contribute to chemoresistance, focusing on ECM remodeling by ECM stiffening and the activation of specific signaling pathways. Understanding these mechanisms is crucial for the development of novel strategies to overcome metastasis and improve outcomes for CRC patients.

The inherent carbohydrate-binding specificities of human galectins can serve as recognition elements in both biotechnological and biomedical applications. The combination of the carbohydrate-recognition domain (CRD) of galectins fused to peptides or proteins for purification, immobilization, and imaging enables multifunctional utilization within a single protein. We present here a library of color-coded galectin fusion proteins that incorporate a His6-tag, a fluorescent protein, and a SpyCatcher or SpyTag unit to enable immobilization procedures. These galectin fusion proteins exhibit similar binding properties to the non-fused galectins with micromolar apparent binding affinities. N- and C-terminal fusion partners do not interfere with the SpyCatcher/SpyTag immobilization. By applying SpyCatcher/SpyTag-mediated SC-ST-Gal-3 conjugates, we show the stepwise formation of a three-layer ECM-like structure in vitro. Additionally, we demonstrate the SpyCatcher/SpyTag-mediated immobilization of galectins in microgels, which can serve as a transport platform for localized targeting applications. The proof of concept is provided by the galectin-mediated binding of microgels to colorectal cancer cells.

The apical extracellular matrix (aECM), organized by polarized epithelial cells, exhibits complex structures. The tectorial membrane (TM), an aECM in the cochlea mediating auditory transduction, exhibits highly ordered domain-specific architecture. α-Tectorin (TECTA), a glycosylphosphatidylinositol (GPI)-anchored ECM protein, is essential for TM organization. Here, we identified that α-tectorin is released by distinct modes: proteolytic shedding by TMPRSS2 and GPI-anchor-dependent release from the microvillus tip in mice. In the medial/limbal domain, proteolytically shed α-tectorin forms dense fibers. In contrast, in the lateral/body domain, where supporting cells exhibit dense microvilli, shedding restricts α-tectorin to the microvillus tip, compartmentalizing collagen-binding sites. Tip-localized α-tectorin is released in a GPI-anchor-dependent manner to form collagen-crosslinking fibers, maintaining the spacing and parallel organization of collagen fibrils. Overall, these distinct release modes of α-tectorin determine domain-specific organization, with the microvillus coordinating release modes along its membrane to assemble the higher-order ECM architecture.

Ischemic stroke (IS) accounts for most stroke incidents and causes intractable damage to brain tissue. This condition manifests as diverse aftereffects, such as motor impairment, emotional disturbances, and dementia. However, a fundamental approach to curing IS remains unclear. This study proposes a novel approach for treating IS by employing minimally invasive and injectable jammed gelatin-norbornene nanofibrous hydrogels (GNF) infused with growth factors (GFs). The developed GNF/GF hydrogels are administered to the motor cortex of a rat IS model to evaluate their therapeutic effects on IS-induced motor dysfunction. GNFs mimic a natural fibrous extracellular matrix architecture and can be precisely injected into a targeted brain area. The syringe-injectable jammed nanofibrous hydrogel system increased angiogenesis, inflammation, and sensorimotor function in the IS-affected brain. For clinical applications, the biocompatible GNF hydrogel has the potential to efficiently load disease-specific drugs, enabling targeted therapy for treating a wide range of neurological diseases.

Despite progress, osteochondral (OC) tissue engineering strategies face limitations in terms of articular cartilage layer development and its integration with the underlying bone tissue. The main objective of this study is to develop a zonal OC scaffold with native biochemical signaling in the cartilage zone to promote articular cartilage development devoid of cells and growth factors. Herein, we report the development and in vivo assessment of a novel gradient and zonal-structured scaffold for OC defect regeneration. The scaffold system is composed of a mechanically supportive 3D-printed template containing decellularized cartilage extracellular matrix (ECM) biomaterial in the cartilage zone that possesses bioactive characteristics, such as chemotactic activity and native tissue biochemical composition. OC scaffolds with a bioactive cartilage zone were implanted in vivo in a rabbit osteochondral defect model and assessed for gross morphology, matrix deposition, cellular distribution, and overall tissue regeneration. The scaffold system supported recruitment and infiltration of host cells into the cartilage zone of the graft, which led to increased ECM deposition and physiologically relevant articular cartilage tissue formation. Semi-quantitative ICRS scoring (overall score double for OC scaffold with bioactive cartilage zone compared to PLA scaffold) further confirm the bioactive scaffold enhanced articular cartilage engineering. This strategy of designing bioactive scaffolds to promote endogenous cellular infiltration can be a much simpler and effective approach for OC tissue repair and regeneration.

With the continuous advancement of materials science, cell biology, and biotechnology, tissue engineering has introduced novel solutions to traditional wound healing approaches, particularly demonstrating significant potential in addressing complex or non-healing wounds. One of the key technologies in this field, dermal scaffolds, serve as wound coverage materials that mimic the structural framework of the dermis. They primarily assume the function of extracellular matrix, providing space for cell attachment, migration, and proliferation, thus supporting cellular growth and regulating multiple biological processes in healing. Tissue engineering utilizes combinations of natural or synthetic scaffolds, seeded cells, or growth factors to induce distinct effects in angiogenesis, extracellular matrix deposition, and functional recovery. Therefore, various bioengineered dermal scaffolds hold significant potential for clinical translation in wound healing. This review outlines various extracellular matrix molecules utilized in the development of dermal scaffolds, emphasizes recent progress in cell- and growth factor-modified scaffolds, and discusses the challenges and future perspectives in this evolving field.

Cells are highly responsive to changes in their mechanical environment, influencing processes such as stem cell differentiation and tumor progression. To meet the growing demand for materials used for high throughput mechanotransduction studies, simple means of dynamically adjusting the environmental viscoelasticity of cell cultures are needed. Here, a novel method is presented to dynamically and reversibly control the viscoelasticity of naturally derived polymer hydrogels through interactions with poly (ethylene glycol) (PEG). Interactions between PEG and hydrogel polymers, possibly involving hydrogen bonding, stiffen the hydrogel matrices. By dynamically changing the PEG concentration of the solution in which polymer hydrogels are incubated, their viscoelastic properties are adjusted, which in turn affects cell adhesion and cytoskeletal organization. Importantly, this effects is reversible, providing a cost-effective and simple strategy for dynamically adjusting the viscoelasticity of polymer hydrogels. This method holds promise for applications in mechanobiology, biomedicine, and the life sciences.

The musculoskeletal system relies on critical tissue interfaces for its function; however, these interfaces are often compromised by injuries and diseases. Restoration of these interfaces is complex by nature which renders traditional treatments inadequate. An emerging solution is three-dimensional printing, which allows for precise fabrication of biomimetic scaffolds to enhance tissue regeneration. This review summarizes the utility of 3D printing in creating scaffolds for musculoskeletal interfaces, mainly focusing on advanced techniques such as multi-material printing, bioprinting, and 4D printing. We emphasize the significance of mimicking natural tissue gradients and the selection of appropriate biomaterials to ensure scaffold success. The review outlines state-of-the-art 3D printing technologies, varying from extrusion, inkjet and laser-assisted bioprinting, which are crucial for producing scaffolds with tailored mechanical and biological properties. Applications in cartilage-bone, intervertebral disc, tendon/ligament-bone, and muscle-tendon junction engineering are discussed, highlighting the potential for improved integration and functionality. Furthermore, we address challenges in material development, printing resolution, and the in vivo performance of scaffolds, as well as the prospects for clinical translation. The review concludes by underscoring the transformative potential of 3D printing to advance orthopedic medicine, offering a roadmap for future research at the intersection of biomaterials, drug delivery, and tissue engineering.

Osteosarcoma, a highly aggressive bone cancer primarily affecting children and young adults, remains a significant challenge in clinical oncology. Metastasis stands as the primary cause of mortality in osteosarcoma patients. However, the mechanisms driving this process remain incompletely understood. Clarifying the molecular pathways involved in metastasis is essential for enhancing patient prognoses and facilitating the development of targeted therapeutic strategies.

RNA sequencing (RNA-Seq) analysis was employed to compare three conditions, hFOB1.19 versus Saos-2, hFOB1.19 versus SJSA-1, and Saos-2 versus SJSA-1, involving non-cancer osteoblasts (hFOB1.19) and highly metastatic osteosarcoma cell lines (Saos-2 and SJSA-1). Additionally, ENA datasets of RNA-Seq from osteosarcoma biopsies were included. Differentially expressed genes (DEGs) were identified and analyzed through enrichment pathway analysis and protein-protein interaction (PPI) networks. Additionally, for gene candidates, a biochemical evaluation was performed.

DEGs associated with biological functions pertinent to migration, invasion, and metastasis in osteosarcoma were identified. Notably, matrix metalloproteinase-2 (MMP-2) emerged as a promising candidate. Both canonical or full-length (FL-mmp-2) and N-terminal truncated (NTT-mmp-2) isoforms were discerned in biopsies. Moreover, MMP-2's activity was characterized in cell lines. Additionally, mRNA expression of voltage-gated sodium channels (NaVs) and voltage-gated potassium channels (KVs) was detected, and their functional expression was validated using patch clamp techniques. Evaluation of cell line migration and invasion capacities revealed their reduction in the presence of ion channel blockers (TTX and TEA) and MMP inhibitor (GM6001).

The gene functional enrichment analysis of DEGs enabled the identification of interaction networks in osteosarcoma, thereby revealing potential biomarkers. Moreover, the elucidated co-participation of TTX-sensitive NaVs and MMP-2 in facilitating migration and invasion suggests their suitability as novel prognostic biomarkers for osteosarcoma. Additionally, this study introduces a model delineating the potential interaction mechanism among ion channels, MMP-2, and other crucial factors in the metastatic cascade of osteosarcoma.

Bioengineering is applied in different areas, including women's infertility management. Among other approaches, decellularized tissues are being used to treat uterine disorders causing infertility. Biomaterials made from decellularized tissue consist of tissue-specific extracellular matrix and, as acellular scaffolds, are thought to be immune inert. Hence, they are good grafting candidates to replace and regenerate excised damaged uterine tissue to cure infertility. However, decellularization approaches differ among species and research groups, posing challenges for comparison and standardization. The diversity in data reporting and studied properties of the resulting decellularized scaffold make it even more difficult, especially when the ultimate goal is clinical translation. Thus, this review aims to critically assess whole uterus decellularization studies, extracting and comparing their main results and conclusions. After carefully evaluating the reviewed studies, we noticed that the vast majority base the uterus decellularization success and resulting scaffold efficacy on the DNA removal efficacy, while other crucial aspects, including the extracellular matrix integrity or immunogenicity, are underestimated. Thus, this review further proposes practical points for what should be considered and how results can be reported in studies involving whole uterus decellularization to facilitate comparison between studies and translational progress.