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Abstract Background Using primary airway epithelial cells (AEC) is essential to mimic more closely different types and stages of lung disease in humans while reducing or even replacing animal experiments. Access to lung tissue remains limited because these samples are generally obtained from patients who undergo lung transplantation for end-stage lung disease or thoracic surgery for (mostly) lung cancer. We investigated whether forceps or cryo biopsies are a viable alternative source of AEC compared to the conventional technique. Methods AECs were obtained ex vivo from healthy donor lung tissue using the conventional method and two biopsy procedures (forceps, cryo). The influence of the isolation method on the quality and function of AEC was investigated at different time-points during expansion and differentiation in air-liquid interface cultures. In addition, fully-differentiated AECs were stimulated with house dust mite extract (HDM) to allow functional analyses in an allergic in vitro model. Vitality or differentiation capacity were determined using flow cytometry, scanning electron microscope, periodic acid-Schiff reaction, immunofluorescence staining, and proteomics. Results As anticipated, no significant differences between each of the sampling methods were detected for any of the measured outcomes. The proteome composition was comparable for each isolation method, while donor-dependent effects were observed. Treatment with HDM led to minor differences in mucociliary differentiation. Conclusions Our findings confirmed the adequacy of these alternative approaches for attaining primary AECs, which can now expand the research for a broader range of lung diseases and for studies at an earlier stage not requiring lung surgery.

Abstract Microbubbles (MB) are widely used as contrast agents for ultrasound (US) imaging and US‐enhanced drug delivery. Polymeric MB are highly suitable for these applications because of their acoustic responsiveness, high drug loading capability, and ease of surface functionalization. While many studies have focused on using polymeric MB for diagnostic and therapeutic purposes, relatively little attention has thus far been paid to improving their inherent imaging and drug delivery features. This study here shows that manipulating the polymer chemistry of poly(butyl cyanoacrylate) (PBCA) MB via temporarily mixing the monomer with the monomer‐mimetic butyl cyanoacetate (BCC) during the polymerization process improves the drug loading capacity of PBCA MB by more than twofold, and the in vitro and in vivo acoustic responses of PBCA MB by more than tenfold. Computer simulations and physisorption experiments show that BCC manipulates the growth of PBCA polymer chains and creates nanocavities in the MB shell, endowing PBCA MB with greater drug entrapment capability and stronger acoustic properties. Notably, because BCC can be readily and completely removed during MB purification, the resulting formulation does not include any residual reagent beyond the ones already present in current PBCA‐based MB products, facilitating the potential translation of next‐generation PBCA MB.

A novel scaffold design has been created to enhance tissue engineering and regenerative medicine by optimizing the controlled, prolonged release of Hepatocyte Growth Factor (HGF), a powerful chemoattractant for endogenous mesenchymal stem cells. We present a new stacked scaffold that is made up of three different fibrin gel layers, each of which has HGF integrated into the matrix. The design attempts to preserve HGF’s regenerative properties for long periods of time, which is necessary for complex tissue regeneration. These multi-layered fibrin gels have been mechanically evaluated using rheometry, and their degradation behavior has been studied using D-Dimer ELISA. Understanding the kinetics of HGF release from this novel scaffold configuration is essential for understanding HGF’s long-term sustained bioactivity. A range of cell-based tests were carried out to verify the functionality of HGF following extended incorporation. These tests included 2-photon microscopy using phalloidin staining to examine cellular morphology, SEM analysis for scaffold–cell interactions, and scratch and scatter assays to assess migration and motility. The analyses show that the novel stacking scaffold promotes vital cellular processes for tissue regeneration in addition to supporting HGF’s bioactivity. This scaffold design was developed for in situ tissue engineering. Using the body as a bioreactor, the scaffold should recruit mesenchymal stem cells from their niche, thus combining the regenerative abilities of HGF and MSCs to promote tissue remodeling and wound repair.

This study focuses on enhancing controllable fibrin-based hydrogels for tissue engineering, addressing existing weaknesses. By integrating a novel copolymer, we improved the foundation for cell-based angiogenesis with adaptable structural features. Tissue engineering often faces challenges like waste disposal and nutrient supply beyond the 200 µm diffusion limit. Angiogenesis breaks through this limitation, allowing the construction of larger constructs. Our innovative scaffold combination significantly boosts angiogenesis, resulting in longer branches and more capillary network junctions. The copolymer attached to fibrin fibers enables precise adjustment of hydrogel mechanical dynamic properties for specific applications. Our material proves effective for angiogenesis, even under suppression factors like suramin. In our study, we prepared fibrin-based hydrogels with and without the copolymer PVP12400-co-GMA10mol%. Using a co-culture system of human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), we analyzed angiogenetic behavior on and within the modified hydrogels. Capillary-like structures were reproducibly formed on different surfaces, demonstrating the general feasibility of three-dimensional endothelial cell networks in fibrin-based hydrogels. This highlights the biomaterial’s suitability for in vitro pre-vascularization of biohybrid implants.

Abstract Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.

A key challenge for the discovery of novel molecular targets and therapeutics against pediatric bone metastatic disease is the lack of bona fide in vitro cell models. Here, we show that a beta-tricalcium phosphate (β-TCP) multicellular 3D in vitro bone microtissue model reconstitutes key phenotypic and transcriptional patterns of native metastatic tumor cells while promoting their stemness and proinvasive features. Comparing planar with interconnected channeled scaffolds, we identified geometry as a dominant orchestrator of proangiogenic traits in neuroblastoma tumor cells. On the other hand, the β-TCP-determined gene signature was DNA replication related. Jointly, the geometry and chemical impact of β-TCP revealed a prometastatic landscape of the engineered tumor microenvironment. The proposed 3D multicellular in vitro model of pediatric bone metastatic disease may advance further analysis of the molecular, genetic and metabolic bases of the disease and allow more efficient preclinical target validations.

Abstract For 3D neural cultures durable hydrogels are required, which persist over a long differentiation period and thus enable the maturation of neuronal networks (NN). Here, 3D models based on human induced pluripotent stem cell‐derived neural progenitor cells that are embedded in hydrogels of either pure alginate or alginate functionalized with the extracellular matrix protein laminin 111 (L111) are established. This study analyzes material characteristics such as porosity, L111 distribution and shear viscosity, cell compatibility of hydrogels by measuring viability and cytotoxicity, and neural function by monitoring cell migration, differentiation as well as NN formation and activity on multielectrode arrays. The addition of L111 increases neural migration and enhances differentiation into neurons and astrocytes as well as synaptogenesis in alginate hydrogels. NN formed in hydrogels are electrically active for up to 206 d and L111‐supplementation further increases electrical activity, network maturation, and synchronicity compared to 2D controls and NN grown in pure alginate hydrogels. L111 addition to alginate gels further accelerates recovery of electrical activity after blockage of sodium channels with tetrodotoxin. In conclusion, NN grown in alginate‐L111 hydrogel blends are promising models for future long‐term applications in disease modeling, drug or chemical evaluation.

Chronic venous insufficiency (CVI) is a leading vascular disease whose clinical manifestations include varicose veins, edemas, venous ulcers, and venous hypertension, among others. Therapies targeting this medical issue are scarce, and so far, no single venous valve prosthesis is clinically available. Herein, we have designed a bi-leaflet transcatheter venous valve that consists of (i) elastin-like recombinamers, (ii) a textile mesh reinforcement, and (iii) a bioabsorbable magnesium stent structure. Mechanical characterization of the resulting biohybrid elastin-like venous valves (EVV) showed an anisotropic behavior equivalent to the native bovine saphenous vein valves and mechanical strength suitable for vascular implantation. The EVV also featured minimal hemolysis and platelet adhesion, besides actively supporting endothelialization in vitro, thus setting the basis for its application as an in situ tissue engineering implant. In addition, the hydrodynamic testing in a pulsatile bioreactor demonstrated excellent hemodynamic valve performance, with minimal regurgitation (

Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM–Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.

Biomaterial-driven modulation of cell adhesion and migration is a challenging aspect of tissue engineering. Here, we investigated the impact of surface-bound microgel arrays with variable geometry and adjustable cross-linking properties on cell adhesion and migration. We show that cell migration is inversely correlated with microgel array spacing, whereas directionality increases as array spacing increases. Focal adhesion dynamics is also modulated by microgel topography resulting in less dynamic focal adhesions on surface-bound microgels. Microgels also modulate the motility and adhesion of Sertoli cells used as a model for cell migration and adhesion. Both focal adhesion dynamics and speed are reduced on microgels. Interestingly, Gas2L1, a component of the cytoskeleton that mediates the interaction between microtubules and microfilaments, is dispensable for the regulation of cell adhesion and migration on microgels. Finally, increasing microgel cross-linking causes a clear reduction of focal adhesion turnover in Sertoli cells. These findings not only show that spacing and rigidity of surface-grafted microgels arrays can be effectively used to modulate cell adhesion and motility of diverse cellular systems, but they also form the basis for future developments in the fields of medicine and tissue engineering.

Vascular disease is a leading cause of death worldwide, but surgical options are restricted by the limited availability of autologous vessels, and the suboptimal performance of prosthetic vascular grafts. This is especially evident for coronary artery by-pass grafts, whose small caliber is associated with a high occlusion propensity. Despite the potential of tissue-engineered grafts, compliance mismatch, dilatation, thrombus formation, and the lack of functional elastin are still major limitations leading to graft failure. This calls for advanced materials and fabrication schemes to achieve improved control on the grafts' properties and performance. Here, bioinspired materials and technical textile components are combined to create biohybrid cell-free implants for endogenous tissue regeneration. Clickable elastin-like recombinamers are processed to form an open macroporous 3D architecture to favor cell ingrowth, while being endowed with the non-thrombogenicity and the elastic behavior of the native elastin. The textile components (i.e., warp-knitted and electrospun meshes) are designed to confer suture retention, long-term structural stability, burst strength, and compliance. Notably, by controlling the electrospun layer's thickness, the compliance can be modulated over a wide range of values encompassing those of native vessels. The grafts support cell ingrowth, extracellular matrix deposition and endothelium development in vitro. Overall, the fabrication strategy results in promising off-the-shelf hemocompatible vascular implants for in situ tissue engineering by addressing the known limitations of bioartificial vessel substitutes.

Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate—gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.

Abstract Background Bone marrow (BM) niches are often inaccessible for controlled experimentation due to their difficult accessibility, biological complexity, and three-dimensional (3D) geometry. Methods Here, we report the development and characterization of a BM model comprising of cellular and structural components with increased potential for hematopoietic recapitulation at ectopic transplantation sites. Cellular components included mesenchymal stromal cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs). Structural components included 3D β-tricalcium phosphate (β-TCP) scaffolds complemented with Matrigel or collagen I/III gels for the recreation of the osteogenic/extracellular character of native BM. Results In vitro, β-TCP/Matrigel combinations robustly maintained proliferation, osteogenic differentiation, and matrix remodeling capacities of MSCs and maintenance of HSPCs function over time. In vivo, scaffolds promoted strong and robust recruitment of hematopoietic cells to sites of ectopic transplantation, vascularization, and soft tissue formation. Conclusions Our tissue-engineered BM system is a powerful tool to explore the regulatory mechanisms of hematopoietic stem and progenitor cells for a better understanding of hematopoiesis in health and disease.

Poly(n-butyl cyanoacrylate) microbubbles (PBCA-MB) are extensively employed for functional and molecular ultrasound (US) imaging, as well as for US-mediated drug delivery. To facilitate the use of PBCA-MB as a commercial platform for biomedical applications, it is important to systematically study and improve their stability and shelf-life. In this context, lyophilization (freeze drying) is widely used to increase shelf-life and promote product development. Here, we set out to analyze the stability of standard and rhodamine-B loaded PBCA-MB at three different temperatures (4 °C, 25 °C, and 37 °C), for a period of time of up to 20 weeks. In addition, using sucrose, glucose, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG) as cryoprotectants, we investigated if PBCA-MB can be lyophilized without affecting their size, concentration, US signal generation properties, and dye retention. Stability assessment showed that PBCA-MB remain largely intact for three and four weeks at 4 °C and 25 °C, respectively, while they disintegrate within one to two weeks at 37 °C, thereby compromising their acoustic properties. Lyophilization analyses demonstrated that PBCA-MB can be efficiently freeze-dried with 5% sucrose and 5% PVP, without changing their size, concentration, and US signal generation properties. Experiments involving rhodamine-B loaded MB indicated that significant dye leakage from the polymeric shell takes place within two to four weeks in case of non-lyophilized PBCA-MB. Lyophilization of rhodamine-loaded PBCA-MB with sucrose and PVP showed that the presence of the dye does not affect the efficiency of freeze-drying, and that the dye is efficiently retained upon MB lyophilization. These findings contribute to the development of PBCA-MB as pharmaceutical products for preclinical and clinical applications.