Composite material technology and advanced surface modifications are the two most substantial components in designing an effective bone scaffold. In this study, the extrusion of polycaprolactone (PCL)-45S5 Bioglass® (BG) composite filaments using a dry-mix hot-melt extrusion method was investigated. PCL-45S5 BG (PCL-BG) composite filaments of three different filler contents, 1, 2, and 5 wt%, and their three-dimensional (3D)-printed constructs were studied and compared to an industrial-grade PCL filament and its 3D-printed constructs. Following the composite material study, the PCL and PCL-BG composite scaffolds were treated with three surface modifications: subtractive chemical etching, conventional biomimetic calcium phosphate (CaP) coating, and bioinspired polydopamine (PDA) modification. A collective, comparative, and comprehensive analysis package comprising microstructure and morphology observation, topography characterization, mechanical performance, and a series of in vitro studies were conducted. The results showed considerable changes in mechanical performance and osteogenic properties across different combinations of substrate materials and surface modification strategies. Adding to the current general understanding that surface-modified scaffolds follow the substrate’s properties, varying filler contents of composite substrates in interaction with surface modification strategies certainly influenced the essential properties of bone scaffolds. These findings emphasize the considerable details involved in designing and engineering patient-specific bone scaffolds.

Polycaprolactone (PCL) is one of the most intensively studied biomaterials for three-dimensional (3D) printing in bone tissue engineering. However, without modification, it lacks bioactivity and osteogenic properties to function as a bone scaffold alone. Here, a facile single-step co-deposition to graft three different bioactive ceramics onto the surface of PCL scaffolds using polydopamine (PDA) is studied. The specific objective of this study was to conduct a comparative study on PDA-assisted bioactive ceramic coatings on 3D-printed PCL scaffolds, including conventional nanohydroxyapatite (nHA), industrial-grade 45S5 Bioglass (R) (BG), and synthesized mesoporous bioactive glass nanoparticles (MBGN), with a special interest in the application of MBGN for its applicability and expandability. First, PDA-assisted bioactive ceramic-coated PCL scaffolds were validated by observing and characterizing the microstructure, morphology, and chemistry. Thermogravimetric analysis (TGA), topographical characterization, and uniaxial compression test were performed to understand the effect of the PDA-assisted bioactive ceramic coatings on PCL scaffolds. An acellular bioactivity study was conducted using simulated body fluid (SBF), where the PDA-nHA/PCL and PDA-45S5 BG/PCL scaffolds were comparably superior in biomineralization ability. In vitro cell studies using osteoblast-like MG-63 cells allowed a general understanding of osteogenic properties, including cell adhesion, viability, differentiation, and proliferation, compared to PDA/PCL scaffolds without grafted bioactive ceramics. Osteogenic properties such as cell adhesion, viability, and differentiation were not distinctively different from those of PDA/PCL scaffolds, but PDA-MBGN/PCL scaffolds showed improved cell proliferation. Overall, the PDA-assisted bioactive ceramic coating enables convenient biofunctional surface modification of 3D-printed PCL scaffolds with potential for applicability, scalability, and expandability.

Extracellular matrix (ECM) is the main component of cartilage, making it an ideal environment to study cell-matrix interactions. Among ECM constituents, heparan sulfate (HS)-carrying proteoglycans (PGs) are of particular interest since they are not only structural components but are also involved in cell matrix adhesion and signalling processes. We previously demonstrated that transgenic mice with a clonal loss of HS synthesis in chondrocytes ( Col2-rtTA-Cre;Ext1e2fl/e2f l) develop clusters of enlarged cells in the articular cartilage (AC), which are surrounded by a glycosaminoglycan (GAG)-rich ECM. This led to the questions how HS regulate the molecular composition and mechanical properties of the ECM, how they sense alterations in the HS structure and how they respond to it. We stained tissue sections of Col2-rtTA-Cre;Ext1e2fl/e2f animals and detected increased levels of chondroitin sulfate (CS), Aggrecan (Acan), Perlecan (Pcan), Matrilin (Matn)-3 and-4, Collagen type II (Col2) and Col9, while Col12 was abolished in the HS-deficient clusters. We assessed the stiffness of the mutant matrix by Atomic Force Microscopy (AFM) and found that it was markedly softer than the surrounding, HS-containing tissue. Likely in response to this altered texture, HS-deficient clones showed increased protein levels of Integrin pathway components. To model a loss of HS-function in vitro , we treated murine embryonic fibroblasts (MEFs) with the HS-antagonist Surfen . Treatment during cell adhesion resulted in impaired cell-substrate adhesion, increased formation of filopodia-like membrane protrusions, decreased cell polarisation and migration, reduced formation of FA and SF, and a translocation of YAP into the cytoplasm. Similarly, we observed reduced cell polarisation in HS-deficient CHO pgsD-667 cells, which could not be rescued by external presentation of HS. When MEFs were treated with Surfen after the completion of the initial cell adhesion process, inhibition of HS-function led to an increased formation of FA and SF, in line with the increased levels of Integrin pathway components observed in HS-deficient chondrocytes in vivo . We detected high levels of Yes1-associated protein (YAP) in the HS-deficient clusters, and we investigated the effect of YAP modulation on high density micromass cultures from primary murine chondroprogenitors. YAP activation induced an increased GAG synthesis similar to Surfen, while YAP inactivation partially abolished the effect of Surfen, showing that YAP acts downstream of HS function and controls GAG synthesis. Taken together, we demonstrated that HS-function is essential for Integrin-dependent cell-matrix interactions. Information on the impaired cell matrix adhesion upon loss of HS is conveyed into the nucleus via YAP, which at least partially controls the synthesis of GAGs in chondrocytes. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Objective The Agc1 CreERT2 mouse line is a powerful tamoxifen-inducible genetic tool used for conditional gene manipulation specifically in cartilage. The aim of this study was to investigate the effects of aggrecan hypomorphism on the progression of post-traumatic osteoarthritis (PT-OA) in Agc1 CreERT2 mice. Methods Proteoglycan content in cartilage samples from the knees of E18.5 embryos were quantified by sulfated glycosaminoglycan (sGAG) assay. Destabilization of the medial meniscus (DMM) surgery was performed to induce PT-OA in 12-week-old wild-type, heterozygous Agc1 CreERT2/+ and homozygous Agc1 CreERT2/CreERT2 mice. Progression of OA was assessed at 4-, 8-, and 12-weeks post-DMM by OARSI, synovitis and osteophyte maturation histopathology scores and micro-computed tomography (µCT). Aggrecan deposition, cartilage matrix-degrading proteases, aggrecan and collagen II degradation neoepitopes were investigated by immunohistochemical staining, and serum C-terminal cross-linked telopeptide of type II collagen (CTX-II) levels by an enzyme-linked immunosorbent assay (ELISA). Chondrocyte apoptosis was analyzed with the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The biomechanical properties of articular cartilage (AC) were investigated with indentation-type atomic force microscopy (IT-AFM). Results Before DMM, homozygous Agc1 CreERT2/CreERT2 mice had reduced sGAG and aggrecan levels and increased cartilage stiffness. After DMM, they exhibited increased cartilage degradation, synovitis, osteophyte formation and meniscus mineralization, chondrocyte apoptosis and cartilage stiffness compared with wild-type mice. Immunohistochemistry demonstrated increased expression of the aggrecanase ADAMTS-5, the metalloproteinase MMP-13, the aggrecan degradation neoepitope NITEGE and the collagen degradation neoepitope C1,2C in AC. ELISA also revealed elevated serum CTX-II levels. Heterozygous Agc1 CreERT2/+ mice also exhibited accelerated PT-OA compared with wild-type mice, characterized by elevated CTX-II levels at 4-weeks, increased synovitis, osteophyte and soft tissue mineralization at 8-weeks, and more severe cartilage degeneration at 12-weeks post-DMM. Conclusion Both homozygous and heterozygous Agc1 CreERT2 mice exhibit increased susceptibility to PT-OA, underscoring the importance of physiological aggrecan expression in maintaining joint homeostasis and regulating joint pathophysiology. These findings indicate that Agc1 CreERT2/+ mice are not phenotypically neutral in the DMM model and that this intrinsic susceptibility should be considered when interpreting studies employing inducible, cartilage-specific gene deletion.

The fluoroquinolone (FQ) class of antibiotics includes the world’s most prescribed antibiotics such as ciprofloxacin, levofloxacin, and ofloxacin that are known for their low bacterial resistance. This is despite their potential to trigger severe side effects, such as myopathy, hearing loss, tendinopathy, and tendon rupture. Thus, healthcare organizations around the world have recommended limiting the prescription of FQs. Tendinopathy is a common name for maladies that cause pain and degeneration in the tendon tissue, which can result in tendon rupture. Whilst there are several identified effects of FQ on tendons, the exact molecular mechanisms behind FQ-mediated tendon rupture are unclear. Previous research studies indicated that FQ-mediated tendinopathy and tendon rupture can be induced by changes in gene expression, metabolism, and function of tendon resident cells, thus leading to alterations in the extracellular matrix. Hence, this review begins with an update on FQs, their mode of action, and their known side effects, as well as summary information on tendon tissue structure and cellular content. Next, how FQs affect the tendon tissue and trigger tendinopathy and tendon rupture is explored in detail. Lastly, possible preventative measures and promising areas for future research are also discussed. Specifically, follow-up studies should focus on understanding the FQ-mediated tendon changes in a more complex manner and integrating in vitro with in vivo models. With respect to in vitro systems, the field should move towards three-dimensional models that reflect the cellular diversity found in the tissue.

The mechanical properties of the extracellular matrix (ECM) are critical regulators of cell behavior. Native tissues span a broad mechanical spectrum, ranging from soft brain tissue to stiff bone and often exhibit a spatial heterogeneity in stiffness. To replicate these complex microenvironments in vitro, biomaterials must offer not only tunable stiffness but also spatial control over mechanical properties within three-dimensional (3D) constructs. Gelatin methacryloyl (GelMA) hydrogels are widely used in biofabrication. Due to their photopolymerizability, their stiffness can be modulated by altering the material composition and exposure to light. However, so far no bioprinting platform has integrated these tuning parameters to fabricate complex 3D constructs with spatially controlled mechanical properties. In this study, we demonstrate the use of a masked stereolithography bioprinter (mSLAb) to systematically tune the Young’s modulus (YM) of 3D printed GelMA hydrogel constructs. Systematic variations in YM are achieved by adjusting the material composition. Furthermore, the mSLAb allows for tuning of the YM during the printing process by varying the light exposure and intensity, enabling the creation of YM gradients within the 3D construct. The mSLAb thus allows for generating a predefined 3D structure which mechanical properties can be modified during the printing process. With this approach, we achieved a stiffness range, spanning from roughly 1 kPa to over 200 kPa. These results establish mSLAb as a powerful platform for engineering mechanically customizable and structurally complex hydrogel constructs, with cell-instructive YM gradients with high spatial precision.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage breakdown, subchondral bone remodeling, and inflammation. Mechanical stress, such as exercise, can influence OA progression, acting as either a therapeutic intervention or a risk factor depending on intensity. The sensory neuropeptide αCGRP plays a role in modulating cartilage, bone, and inflammatory responses, making it a potential mediator of exercise effects on OA. This study investigated the impact of αCGRP deficiency and exercise intensity on OA progression in a post-traumatic murine model. OA was induced in male αCGRP knockout (KO) and wild type (C57Bl/6J) mice via destabilization of the medial meniscus (DMM). Mice underwent moderate or intense treadmill exercise for up to 6 weeks (8 weeks post-surgery). Histological analyses were performed to assess cartilage degradation. Subchondral and metaphyseal bone morphology as well as cartilage stiffness were evaluated by nanoCT and atomic force microscopy (AFM), respectively. Serum inflammatory markers were analyzed using multiplex immunoassays. Serum levels of proinflammatory markers were elevated in αCGRP-deficient mice, particularly after intense exercise, independent of OA progression. DMM surgery induced significant cartilage degradation. Gross cartilage morphology was not influenced by exercise intensity or αCGRP deficiency, but αCGRP deficiency prevented articular cartilage extracellular matrix stiffening after DMM and intense exercise. Subchondral bone sclerosis was induced by αCGRP deficiency and DMM but mitigated by intense exercise. In metaphyseal bone, intense exercise induced trabecular loss in αCGRP-deficient mice. This study highlights αCGRP as an intrinsic regulator of joint and bone responses to mechanical loading during OA. While cartilage degradation after DMM and treadmill exercise was unaffected by lack of αCGRP, its deficiency altered ECM stiffness, bone remodeling, and inflammatory responses. These findings position αCGRP as a critical regulator of joint homeostasis, particularly for bone health during running exercise and OA progression.

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young's modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young's modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young's moduli, and generation of Young's modulus maps. Finally, it shows how to determine and isolate Young's modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

Facing the increasing popularity of 3D-cellular systems as alternative to classical 2D-culture models, Femtosecond Bioprinting (FSB) has been advanced, overcoming former geometrical constraints, now enabling the high precision transfer of individual mammalian cellular spheroids.

Osteoarthritis (OA) is a chronic degenerative joint disease driven by multifactorial causes, including aging, mechanical stress, and inflammation. Mechanical loading through exercise can either exacerbate or alleviate OA symptoms depending on intensity. Substance P (SP), a neuropeptide involved in inflammation and mechanotransduction, has been implicated in cartilage and bone remodeling. This study aimed to investigate how SP deficiency plus exercise intensity interact to influence disease progression in a surgical murine OA model. OA was induced in male wild-type (WT) and SP knockout (Tac1-/-) mice via destabilization of the medial meniscus (DMM). Mice were then exposed to moderate or intense treadmill exercise for up to eight weeks. Cartilage degeneration was assessed histologically using OARSI scoring. Cartilage stiffness was evaluated via atomic force microscopy (AFM), and subchondral and metaphyseal bone morphology was analyzed by high-resolution nanoCT. Serum cytokine levels were measured with multiplex ELISA. DMM surgery induced OA-like cartilage damage in most groups, and moderate exercise failed to prevent degeneration. However, SP-deficient mice subjected to intense exercise showed preserved cartilage matrix stiffness and morphology comparable to Sham controls. In contrast, SP deficiency as well as intense exercise promoted meniscal ossification and subchondral bone sclerosis, with increased bone volume fraction and trabecular thickness. These changes were consistent with prior findings in SP-deficient mice without exercise. Serum analysis revealed elevated levels of proinflammatory cytokines (e.g., CXCL10, VEGF-A, CCL2, CCL4) in SP-deficient mice after Sham surgery, although these did not correspond to the cartilage degradation timeline. SP plays a dual role in OA pathogenesis: its absence may protect cartilage from mechanical stress–induced stiffening but also promotes ectopic meniscal ossification and subchondral bone alterations. Additionally, SP appears to modulate systemic inflammatory responses independently of joint degeneration. These findings position SP as a key regulator of neuroimmune and mechanobiological processes in OA and highlight its potential as a therapeutic target for load-induced joint pathology.

Three-dimensional (3D) printing has paved the way for the precision manufacturing of patient-specific scaffolds. While personalized 3D-printed bone scaffolds are already in the clinic, further attempts to combine biofunctionalization and drug delivery with these scaffolds are of great interest to improve tissue regeneration and reduce recovery time. This study investigated the dextran-polydopamine (PDA) dual-coated 3D-printed polycaprolactone (PCL) scaffolds as a potential biofunctionalization platform, which will enable the design of more advanced coating systems. Despite PCL being one of the most well-established biomaterials used in manufacturing bone scaffolds, surface modification is essential for its application due to its hydrophobic surface and lack of osteogenic properties. PDA is a bioinspired synthetic polymer, known for its convenient coating strategy, superior osteogenicity, and ability to graft secondary biofunctionalization motifs. However, modifying the surface of PCL with PDA results in aggregates of PDA nanoparticles rather than forming a homogeneous coating layer. Here, dextran was introduced as a dual coating deposited as a thin layer, which further assists cell adhesion and proliferation. Dextran is a biomedical macromolecule with a long history in medicine, which can be used as a drug delivery carrier in various forms, and the focus of this study was to investigate the intricate interplay between dextran and PDA as a dual coating applied to 3D-printed PCL scaffolds, via microstructural, topographical, chemical, and mechanical validation. A series of cell studies using osteoblast-like MG-63 cells was conducted, and it has been confirmed that dextran can be introduced to the PDA-modified PCL scaffold while maintaining the maximum scaffold and cell interaction. Consequently, the present results suggest that the dextran-PDA dual coating offers a promising biofunctionalization platform for designing more complex systems involving dextran-based drug delivery, aimed at application in bone tissue engineering.

About 655 million persons worldwide are affected by osteoarthritis (OA). As no therapy modifies disease progression long-term, there is an immense clinical need for novel therapies. The joints are innervated by alpha calcitonin gene-related peptide (αCGRP)- and substance P (SP)-positive sensory nerve fibers. Both neuropeptides have trophic effects on target cells within the joints. The aim of this study was to examine the effects of SP- and αCGRP-expressing intra-articular (i.a.) applied rat(r)BMSC on cartilage and subchondral bone structural changes after OA induction. Mice were subjected to destabilization of the medial meniscus (DMM) surgery, followed by i.a. injections with rBMSC, transduced with lacZ, SP or αCGRP. 2, 8 and 16 weeks after DMM/Sham surgery, motion analysis and serum marker analysis were performed. Cartilage and subchondral bone properties were assessed by OA scoring, atomic force microscopy and nano-CT analysis. OARSI scores of the medial cartilage compartments indicated induction and progression of OA after DMM surgery in all groups. Differences between the treatment groups were mostly restricted to the lateral cartilage compartments, where αCGRP caused a decrease of structural changes. DMM-rBMSC-αCGRP or -SP mice displayed decreased cartilage stiffness in the cartilage middle zone. DMM-rBMSC-αCGRP mice revealed improved mobility, whereas Sham-rBMSC-SP mice revealed reduced mobility compared to rBMSC-lacZ. With respect to condyle length, subarticular bone and ephiphyseal bone morphology, DMM-rBMSC-SP mice had more alterations indicating either a more progressed OA stage or a more severe OA pathology compared to controls. In addition, DMM-rBMSC-SP mice developed osteophytes already 8 weeks after surgery. Adiponectin serum level was increased in DMM-rBMSC-αCGRP mice, and MIP1b level in DMM-rBMSC-SP mice. Notably, pain and inflammation markers increased over time in rBMSC-SP mice while rBMSC-αCGRP mice revealed a bell-shaped curve with a peak at 8 weeks. We conclude that i.a. injection of rBMSC in general have a beneficial effect on cartilage matrix structure, subchondral bone microarchitecture and inflammation. rBMSC-αCGRP have anabolic and possible analgesic properties and may attenuate the progression or severity of OA. In contrast, rBMSC-SP exert a more catabolic influence on knee joints of both, Sham and DMM mice, making it a potential candidate for inhibition studies.

3D bioprinting is a tissue engineering approach using additive manufacturing to fabricate tissue equivalents for regenerative medicine or medical drug testing. For this purpose, biomaterials that provide the essential microenvironment to support the viability of cells integrated directly or seeded after printing are processed into three-dimensional (3D) structures. Compared to extrusion-based 3D printing, which is most commonly used in bioprinting, stereolithography (SLA) offers a higher printing resolution and faster processing speeds with a wide range of cell-friendly materials such as gelatin- or collagen-based hydrogels and SLA is, therefore, well suited to generate 3D tissue constructs.While there have been numerous publications of conversions and upgrades for extrusion-based printers, this is not the case for state-of-the-art SLA technology in bioprinting. The high cost of proprietary printers severely limits teaching and research in SLA bioprinting. With mSLAb, we present a low-cost and open-source high-resolution 3D bioprinter based on masked SLA (mSLA). mSLAb is based on an entry-level (€350) desktop mSLA printer (Phrozen Sonic Mini 4 K), equipped with temperature control and humidification of the printing chamber to enable the processing of cell-friendly hydrogels. Additionally, the build platform was redesigned for easy sample handling and microscopic analysis of the printed constructs. All modifications were done with off-the-shelf hardware and in-house designed 3D printed components, printed with the same printer that was being modified.We validated the system by printing macroscopic porous scaffolds as well as hollow channels from gelatin-based hydrogels as representative structures needed in tissue engineering.

Purpose (the aim of the study): The development and homeostasis of cartilaginous tissues are regulated by diverse microenvironmental cues including integrin-mediated interactions between chondrocytes and the extracellular matrix (ECM). In this study, we investigated how integrin alpha 10 beta 1 (Itga10), the major collagen-binding integrin on chondrocytes, modulates chondrocytes and cartilage ECM functions.

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100–400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young’s modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young’s modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young’s modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young’s moduli and generation of Young’s modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1–2 d.

Purpose (the aim of the study): Cartilage matrix mainly consists of two components: Collagens, giving it tensile strength, and proteoglycans, conveying its elastic properties. Heparan sulfate (HS)-carrying proteoglycans are of particular interest since they are not only structural components but also regulate signalling processes. We demonstrated that transgenic mice with a clonal loss of HS synthesis in chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl) develop clusters of enlarged cells in the articular cartilage, which are surrounded by a matrix with increased glycosaminoglycan (GAG) content.

Interfacing with the peripheral nervous system is a powerful method for diagnosing and treating several diseases, such as drug‐resistant epilepsy and depression. In most clinical applications, large nerves such as the vagus and the hypoglossal nerve are targeted. Large nerves carry multiple nerve fibers, and maintaining selectivity of a specific target response demands complex stimulation strategies. As the large trunks bifurcate toward their distal ends, their diameter and number of comprised fibers reduce. Consequently, interfacing small nerves can provide increased fiber selectivity. However, their small size presents challenges to the fabrication and implantation of suitable electrodes due to their fragility and constrained environments. Here, a cuff electrode that combines two‐photon stereolithography and 3D inkjet printing techniques for the selective interfacing of small nerves in vivo is introduced. The device is easy to implant, and its size can be tailored for specific nerve dimensions. Its capability to record and selectively stimulate is demonstrated by targeting a locust's hind leg nerve.

A human-induced pluripotent stem cell (hiPSC)-3D artery mimicking the multi-layered anatomy of blood vessel has not yet been established. Here, we aimed to engineer arteries consisting of human endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts, correspondingly mimicking the main cellular components of the intima, media, and adventitia of a blood vessel. We differentiated hiPSC to obtain EC, VSMC, and fibroblasts. A 3D printing-based molding technique was used to engineer the circular structure of an artery. For the media-like layer, hiPSC-VSMCs mixed with collagen were seeded into 3D-printed polylactic acid molds with a centered needle serving as supporting structure. The adventitia-like layer was engineered around the VSMC-layer using the same method but with fibroblast-collagen mixture. After formation of the fibroblast layer, the needle was removed and hiPSC-ECs were coated on the VSMC lumen by rotational cell injection. Brightfield, fluorescence and two-photon microscopy were utilized to visualize cell arrangement, EC coverage, and collagen usage and organization. RNA sequencing (RNAseq) was harnessed to molecular profile cells in 2D (Petri dish) and 3D culture (engineered arteries). With 10M hiPSC-VSMCs, 10M hiPSC-fibroblasts, and 0.2M hiPSC-EC, we successfully engineered the three-layered “artery”. The built-up took 16 days. The condensation of cell-collagen mixture likely relied on the rearrangement of collagen fibers by the cells through enzymic reactions in the extracellular matrix. Immunostaining of alpha-SMC actin indicated rod-shaped hiPSC-VSMC aligned in the artificial tubes. Two-photon microscopy revealed over 95% coverage of hiPSC-EC of the luminal wall. Transcriptome data suggested that 3D culture promotes the expression of genes of the calcium signaling pathway and leads to downregulation of genes of the atherosclerosis pathway in SMC. Taken together, the human-induced pluripotent stem cell (hiPSC)-3D artery might serve as a model for functional studies and experimental models of vascular injury and inflammation. In addition, our three-layered tube - mimicking a human artery - seems to improve the molecular profile of vascular cells derived from hiPSC as compared to 2D culture.

The previously developed femtosecond (fs) laser bioprinting is a direct-printing technique, which applies an ultrashort laser source with a wavelength of 1030 nm. Unlike its predecessors, it no longer requires an absorbing layer. The ultrafast laser pulse steers the non-linear interaction within the volume of the transparent bioink. Optical breakdown allows for the strong absorption of the photons in a focal volume with dimensions of a few micrometres. The high energy density leads to the formation of an expanding cavitation bubble and a liquid jet from the surface. This advanced bioprinting technique allows a single cell transfer precision of approximately 15 μm [1]. Additionally, it shows promising results in terms of the survival rate of transferred mammalian cells. In recent studies, cell survival rates of up to 95% [2] could already be achieved, and since this technology is still considerably new, follow up studies are expected to further exceed these numbers. However, mastering the process in its entirety, requires a profound understanding of its physical background, particularly, the mechanisms involved in the jet formation and propagation. Recent studies already indicate that even slight variations of process parameters, drastically affect the transfer behaviour [1]–[3].

The desmoplastic reaction observed in many cancers is a hallmark of disease progression and prognosis, particularly in breast and pancreatic cancer. Stromal-derived extracellular matrix (ECM) is significantly altered in desmoplasia, and as such plays a critical role in driving cancer progression. Using fibroblast-derived matrices (FDMs), we show that cancer cells have increased growth on cancer associated FDMs, when compared to FDMs derived from non-malignant tissue (normal) fibroblasts. We assess the changes in ECM characteristics from normal to cancer-associated stroma at the primary tumor site. Compositional, structural, and mechanical analyses reveal significant differences, with an increase in abundance of core ECM proteins, coupled with an increase in stiffness and density in cancer-associated FDMs. From compositional changes of FDM, we derived a 36-ECM protein signature, which we show matches in large part with the changes in pancreatic ductal adenocarcinoma (PDAC) tumor and metastases progression. Additionally, this signature also matches at the transcriptomic level in multiple cancer types in patients, prognostic of their survival. Together, our results show relevance of FDMs for cancer modelling and identification of desmoplastic ECM components for further mechanistic studies.