Structuring gelatin methacryloyl - dextran hydrogels and microgels under shear.

Gelatin methacryloyl (GelMA) is a widely used semi-synthetic polymer for a variety of bioapplications. However, the development of versatile GelMA hydrogels requires tuning of their microstructure. Herein, we report the possibility of preparing hydrogels with various microstructures under shear from an aqueous two-phase system (ATPS) consisting of GelMA and dextran. The influence of an applied preshear on dextran/GelMA droplets and bicontinuous systems is investigated by rheology that allows the application of a constant shear and is immediately followed by in situ UV-curing of the GelMA-rich phase. The microstructure of the resulting hydrogels is examined by confocal laser scanning microscopy (CLSM). The results show that the GelMA string phase and GelMA hydrogels with aligned bands can be formed depending on the concentration of dextran and the applied preshear. The influence of the pH of the ATPS is investigated and demonstrates the formation of multiple emulsions upon decreasing the charge density of GelMA. The preshearing of multiple emulsions, following gelation, leads to the formation of porous GelMA microgels. The diversity of the formed structures highlights the application potential of preshearing ATPS in the development of functional soft materials.

3D Printable Gelatin Methacryloyl (GelMA)-Dextran Aqueous Two-Phase System with Tunable Pores Structure and Size Enables Physiological Behavior of Embedded Cells In Vitro.

The restricted porosity of most hydrogels established for in vitro 3D tissue engineering applications limits embedded cells with regard to their physiological spreading, proliferation, and migration behavior. To overcome these confines, porous hydrogels derived from aqueous two-phase systems (ATPS) are an interesting alternative. However, while developing hydrogels with trapped pores is widespread, the design of bicontinuous hydrogels is still challenging. Herein, an ATPS consisting of photo-crosslinkable gelatin methacryloyl (GelMA) and dextran is presented. The phase behavior, monophasic or biphasic, is tuned via the pH and dextran concentration. This, in turn, allows the formation of hydrogels with three distinct microstructures: homogenous nonporous, regular disconnected-pores, and bicontinuous with interconnected-pores. The pore size of the latter two hydrogels can be tuned from ≈4 to 100 µm. Cytocompatibility of the generated ATPS hydrogels is confirmed by testing the viability of stromal and tumor cells. Their distribution and growth pattern are cell-type specific but are also strongly defined by the microstructure of the hydrogel. Finally, it is demonstrated that the unique porous structure is sustained when processing the bicontinuous system by inkjet and microextrusion techniques. The proposed ATPS hydrogels hold great potential for 3D tissue engineering applications due to their unique tunable interconnected porosity.

Dual-frequency modulation quartz crystal tuning fork-enhanced laser spectroscopy.

An innovative trace gas-sensing technique utilizing a single quartz crystal tuning fork (QCTF) based on a photoelectric detector and dual-frequency modulation technique was demonstrated for the first time for simultaneous multi-species detection. Instead of traditional semiconductor detectors and lock-in amplifier, we utilized the piezoelectric effect and resonant effect of the QCTF to measure the light intensity. A fast signal analysis method based on fast Fourier transform (FFT) algorithm is proposed for overlapping signal extraction. To explore the capabilities of this technique, a gas-sensing system based on two lasers having center emission wavelength of 1.653 µm (a DFB laser diode in the near-IR) and 7.66 µm (an EC QCL in the mid-IR) is successfully demonstrated for simultaneous CH4 spectroscopy measurements. The results indicate a normalized noise equivalent absorption (NNEA) coefficients of 1.33×10-9 cm-1W·Hz-1/2 at 1.653 µm and 2.20×10-10 cm-1W·Hz-1/2 at 7.66 µm, were achieved. This proposed sensor architecture has the advantages of easier optical alignment, lower cost, and a compactness compared to the design of a conventional TDLAS sensor using multiple semiconductor detectors for laser signal collection. The proposed technique can also be expanded to common QEPAS technique with multi-frequency modulation for multiple species detection simultaneously.

Hand-held bioprinting for de novo vascular formation applicable to dental pulp regeneration.

Aim of the study: Deep carious lesions may cause irreversible pulpitis and the current endodontic treatment typically removes the whole dental pulp tissue, which finally reduces lifespan of the teeth. Nowadays, the most frequent treatment is based on removing the infected tissue and filling the root canal with inert synthetic materials. Tissue engineering approaches are important alternatives to the current treatment, because they can potentially maintain the biological function of the tooth instead of sacrificing it.Materials and Methods: In this study, we propose a tissue engineering approach based on a hand-held in situ bioprinting strategy. Our approach enabled bioprinting of cell-loaded collagen-based bioinks with suitable rheological, structural and biological properties, which allowed for vasculogenesis in the root canal.Results: The rheological properties of the bioprintable bioink were measured by oscillatory amplitude sweep testing and were corroborated by macroscopic evaluation after in vitro culture, in which printed bioinks maintained their original form without contraction. Moreover, we showed evidence for successful vasculogenesis in bioprintable bioinks with comparable quality and quantity to control fibrin and collagen non-bioprintable hydrogels.Conclusions: We conclude that hand-held bioprinting holds potential for in situ treatment of dental diseases with successful evidence for vascular tube formation, as an asset for maintenance of the biological function of the tooth.

Objective computerised assessment of residual ridge resorption in the human maxilla and maxillary sinus pneumatisation.

OBJECTIVES: Atrophic resorption of the maxillary alveolar ridge is a complication that makes implantological rehabilitation critical. Our aim was to develop a novel computer aided procedure for the accurate quantitative assessment of maxillary residual ridge resorption including pneumatisation of the maxillary sinus that goes beyond previously described approaches and to apply it to a large dataset. MATERIALS AND METHODS: To develop and refine the method, we performed a retrospective analysis using computed tomography data from 405 patients to generate segmented, three-dimensional models of zygomaticomaxillary bones and maxillary sinuses. Using anatomical landmarks and orientation lines or planes, all models were aligned automatically to subsequently generate cross-sectional images (n = 2835), enabling the classification of atrophy as well as the quantification of volumes and caudal extensions of the maxillary sinuses. RESULTS: We developed and implemented an accurate and reproducible workflow for the semi-automated analysis of volumetric maxillary images. Comprehensive statistical analysis of the large quantitative dataset revealed various correlations of maxillary process heights and sinus volumes with atrophy class, age and region and identified conjectural trends over the patient group. CONCLUSIONS: The method was used successfully to process a large dataset to classify atrophy, to measure alveolar height parameters, and to quantify maxillary sinus volume, bottom volume and pneumatisation. CLINICAL RELEVANCE: Apart from the anthropometric value of the generated dataset, the method could be applied to provide additional and more accurate data to assess the necessity of bone augmentation in the context of three-dimensional planning before implantation.

Effects of Strontium-Doped ß-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

It was hypothesized that strontium (Sr)-doped ß-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

Role of Nrf2 in Fracture Healing: Clinical Aspects of Oxidative Stress.

Fracture healing is a natural process that recapitulates embryonic skeletal development. In the early phase after fracture, reactive oxygen species (ROS) are produced under inflammatory and ischemic conditions due to vessel injury and soft tissue damage, leading to cell death. Usually, such damage during the course of fracture healing can be largely prevented by protective mechanisms and functions of antioxidant enzymes. However, intrinsic oxidative stress can cause excessive toxic radicals, resulting in irreversible damage to cells associated with bone repair during the fracture healing process. Clinically, patients with type-2 diabetes mellitus, osteoporosis, habitual drinkers, or heavy smokers are at risk of impaired fracture healing due to elevated oxidative stress. Although increased levels of oxidative stress markers upon fracture and effects of antioxidants on fracture healing have been reported, a detailed understanding of what causes impaired fracture healing under intrinsic conditions of oxidative stress is lacking. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been identified as a key transcriptional regulator of the expression of antioxidants and detoxifying enzymes. It further not only plays a crucial role in preventing degenerative diseases in multiple organs, but also during fracture healing. This narrative review evaluates the influence of intrinsic oxidative stress on fracture healing and sheds new light on the intriguing role of Nrf2 during bone regeneration in pathological fractures.

Retrievability of implant-supported zirconia restorations cemented on zirconia abutments.

STATEMENT OF PROBLEM: Retrievability of implant-supported restorations is important. Data are lacking for cemented zirconia crowns on zirconia abutments. PURPOSE: The purpose of this in vitro study was to investigate the influence of different cements and marginal discrepancy on the retrievability of implant-supported zirconia crowns. Furthermore, the influence of thermocycling on retrievability was evaluated. MATERIAL AND METHODS: Thirty tapered Camlog zirconia abutments (6-degree taper, 6×4.3 mm) were used. Thirty zirconia crowns with 3 different marginal cementation discrepancies (70, 130, 190 µm) were fabricated by using computer-aided design and computer-aided manufacturing (CAD-CAM) technology. Five cements for interim or semidefinitive cementation were used: eugenol-free zinc oxide (Freegenol) and acrylurethane (ImProv) and 3 different composite resin cements (X-Pand Implant, Dyna Implant, Telio CS Cem Implant). Specimens underwent either 3-day storage in sodium chloride or thermocycling (10 000 cycles). Crowns were removed by using a universal testing machine (UTM) and a clinical removal device. Data were analyzed using 1-way ANOVA and the Scheffé test (α=.05). RESULTS: Thermocycling decreased the retention force significantly (P<.001). Marginal discrepancy (70 to 190 µm) was not significantly influential on retrievability (P>.05). Therefore, groups were pooled according to the factor of marginal discrepancy. The mean retention force using the UTM after 3-day storage and thermocycling was as follows: Freegenol, 235 ±42 N (thermocycling, 29 ±9 N); Improv, 110 ±50 N (thermocycling, 35 ±38 N); Telio CS, 104 ±17 N (thermocycling, 6 ±10 N); Dyna implant, 61 ±17 N (thermocycling, 1 ±1 N); and X-Pand, 50 ±16 N (thermocycling, 2 ±2 N). CONCLUSIONS: Retention forces of the tested cements were significantly different and decreased considerably after thermocycling. Marginal cementation discrepancy between 70 and 190 µm did not influence retrievability.

Functional MR Imaging Mapping of Human Articular Cartilage Response to Loading.

Purpose To determine if multiparametric magnetic resonance (MR) imaging mapping can be used to quantify the response to loading of histologically intact human knee cartilage. Materials and Methods Institutional review board approval and written informed consent were obtained. Twenty macroscopically intact cartilage-bone samples were obtained from the central lateral femoral condyles in 11 patients undergoing total knee replacement. A clinical 3.0-T MR imaging system was used to generate T1, T1ρ, T2, and T2* maps with inversion recovery, spin-lock multiple gradient-echo, multiple spin-echo, and multiple gradient-echo sequences. Serial mapping was performed at three defined strain levels (strain 0 [δ0], 0%; strain 1 [δ1/2], 19.8% ± 4.6 [standard deviation]; strain 2 [δ1], 39.5% ± 9.3) by using displacement-controlled static indentation loading. The entire sample and specific cartilage zones (superficial zone [SZ], transitional zone [TZ], and deep zone [DZ]) and regions (subpistonal area [SPA] and peripistonal area [PPA]) were defined as regions of interest. Upon log transformation, repeated measures analysis of variance was used to detect groupwise regional and zonal differences. Load-induced relative changes were determined and analyzed by using paired Student t test and Spearman correlation. Biomechanical testing (unconfined compression) and histologic assessment (Mankin score) served as the reference standard. Results All samples were histologically intact. Strain-related decreases were found at the SZ and TZ for T1 and T2*; for T1ρ, increases were seen in all zones; and for T2, increases were seen at the SZ and PPA only. Significant parameter changes in the entire sample depth of SPA versus PPA were found for δ1/2 (T1ρ, 14% ± 12 vs 6% ± 9) and δ1 (T1, -4% ± 5 vs -1% ± 3; T1ρ, 13% ± 12 vs 7% ± 7; T2*, -9% ± 12 vs -2% ± 8). SPA versus PPA changes were significant at the SZ and TZ (T1), TZ and DZ (T1ρ), and SZ (T2*). No significant correlations were found between relative changes and biomechanical or histologic parameters. Conclusion Serial multiparametric MR imaging mapping can be used to evaluate cartilage beyond mere static analysis and may provide the basis for more refined graduation strategies of cartilage degeneration. © RSNA, 2016 Online supplemental material is available for this article.

Cellulose Nanofibril Hydrogel Tubes as Sacrificial Templates for Freestanding Tubular Cell Constructs.

The merging of defined nanoscale building blocks with advanced additive manufacturing techniques is of eminent importance for the preparation of multiscale and highly functional materials with de novo designed hierarchical architectures. Here, we demonstrate that hydrogels of cellulose nanofibrils (CNF) can be processed into complex shapes, and used as a sacrificial template to prepare freestanding cell constructs. We showcase our approach for the fabrication of hollow fibers using a controlled extrusion through a circular die into a coagulation bath. The dimensions of the hollow fibers are tunable, and the final tubes combine the nanofibrillar porosity of the CNF hydrogel with a submillimeter wall thickness and centimeter-scale length provided by the additive manufacturing technique. We demonstrate that covalent and supramolecular cross-linking of the CNFs can be used to tailor the mechanical properties of the hydrogel tubes within 1 order of magnitude and in an attractive range for the mechanosensation of cells. The resulting tubes are highly biocompatible and allow for the growth of mouse fibroblasts into confluent cell layers in their inner lumen. A detailed screening of several cellulases enables degradation of the scaffolding, temporary CNF hydrogel tube in a quick and highly cell-friendly way, and allows the isolation of coherent cell tubes. We foresee that the growing capabilities of hydrogel printing techniques in combination with the attractive features of CNFs-sustainable, globally abundant, biocompatible and enzymatically degradable-will allow making plant-based biomaterials with hierarchical structures and on-demand degradation useful, for instance, to engineer complex tissue structures to replace animal models, and for implants.

Effect of platelet mediator concentrate (PMC) on Achilles tenocytes: an in vitro study.

BACKGROUND: Although there are many studies discussing the etiological and pathological factors leading to both, acute and chronic tendon injuries, the pathophysiology of tendon injuries is still not clearly understood. Although most lesions are uncomplicated, treatment is long and unsatisfactory due to the poor vascularity of tendon tissue. Platelet mediator concentrate (PMC) contains many growth factors derived from platelets, which can promote wound healing. In this study we investigate the effects of PMC on tenocyte proliferation and differentiation in order to provide an experimental basis for tissue regeneration strategies and to develop new treatment concepts. METHODS: Using enzyme linked immunosorbent assay (ELISA) we were able to quantify the several growth factors and cytokines found in PMC. Tenocytes were isolated both from human and from mouse Achilles tendons and stimulated with PMC. CyQuant® and Cell Titer Blue® assays were carried out to analyze tendon growth and viability at different concentrations of PMC. Real time RT-PCR was used to analyze tenocyte gene expression with or without PMC treatment. Immunohistochemistry was carried out to detect the tenocyte-specific antibody tenomodulin (TNMD) and scleraxis (SCX). RESULTS: We were able to detect numerous mediators such as platelet derived growth factor BB (PDGF-BB), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF-α), transforming growth factor beta 1 (TGF-ß1), and bone morphogenetic proteins 2, 4 and 7 (BMP-4, BMP-2, BMP-7) in PMC. It was possible to show a positive effect of PMC on human tendon cell growth and viability in a dose-dependent manner. Furthermore, PMC treatment led to induction of gene expression of scleraxis (SCX), type I collagen A 1 (Col1A1) and TNMD by tenocytes. CONCLUSIONS: We suggest that the use of autologous PMC may be a suitable addition to conventional tendon therapy that is capable of increasing and optimizing tendon healing and reducing the risk of recurrence.

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

Impaired Fracture Healing after Hemorrhagic Shock.

Impaired fracture healing can occur in severely injured patients with hemorrhagic shock due to decreased soft tissue perfusion after trauma. We investigated the effects of fracture healing in a standardized pressure controlled hemorrhagic shock model in mice, to test the hypothesis that bleeding is relevant in the bone healing response. Male C57/BL6 mice were subjected to a closed femoral shaft fracture stabilized by intramedullary nailing. One group was additionally subjected to pressure controlled hemorrhagic shock (HS, mean arterial pressure (MAP) of 35 mmHg for 90 minutes). Serum cytokines (IL-6, KC, MCP-1, and TNF-α) were analyzed 6 hours after shock. Fracture healing was assessed 21 days after fracture. Hemorrhagic shock is associated with a significant increase in serum inflammatory cytokines in the early phase. Histologic analysis demonstrated a significantly decreased number of osteoclasts, a decrease in bone quality, and more cartilage islands after hemorrhagic shock. µCT analysis showed a trend towards decreased bone tissue mineral density in the HS group. Mechanical testing revealed no difference in tensile failure. Our results suggest a delay in fracture healing after hemorrhagic shock. This may be due to significantly diminished osteoclast recruitment. The exact mechanisms should be studied further, particularly during earlier stages of fracture healing.

Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia.

Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 µM thapsigargin (Tg), an inhibitor of Ca(2+) uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca(2+) concentration (Cacyto)] or Ca(2+)-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca(2+). Because C12 and Tg reduced Ca(2+) concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca(2+) concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.

Development of a biofabricated 3Din vitrovessel model for investigating transendothelial migration in stem cell therapy.

Systemic stem cell therapies hold promise for treating severe diseases, but their efficiency is hampered by limited migration of injected stem cells across vascular endothelium towards diseased tissues. Understanding transendothelial migration is crucial for improving therapy outcomes. We propose a novel 3Din vitrovessel model that aids to unravel these mechanisms and thereby facilitates stem cell therapy development. Our model simulates inflammation through cytokine diffusion from the tissue site into the vessel. It consists of a biofabricated vessel embedded in a fibrin hydrogel, mimicking arterial wall composition with smooth muscle cells and fibroblasts. The perfusable channel is lined with a functional endothelium which expresses vascular endothelial cadherin, provides an active barrier function, aligns with flow direction and is reconstructed byin situtwo-photon-microscopy. Inflammatory cytokine release (tumor necrosis factorα, stromal-derived factor (1) is demonstrated in both a transwell assay and the 3D model. In proof-of-principle experiments, mesoangioblasts, known as a promising candidate for a stem cell therapy against muscular dystrophies, are injected into the vessel model, showing shear-resistant endothelial adhesion under capillary-like flow conditions. Our 3Din vitromodel offers significant potential to study transendothelial migration mechanisms of stem cells, facilitating the development of improved stem cell therapies.

Transglutaminase enables highly hydrolytically and proteolytically stable crosslinking of collagen on titanium surfaces and promotes osteogenic differentiation of human mesenchymal stem cells.

Collagen with its bioactive ligand motives would be predestined as coating on bone implant surfaces like titanium hip stems to facilitate receptor-mediated cell adhesion and thereby improve early osseointegration. Unfortunately, collagen as coating exhibits very low proteolytic resistance in vivo. To overcome this limitation, different crosslinking methods of collagen (transglutaminase, GTA, EDC/NHS, riboflavin, and lysyl oxidase) with silanized titanium alloy (Ti6Al4V) were investigated in terms of degradation resistance, hydrolysis stability, tensile strength, and metabolic cell activity. The in vitro osteogenic differentiation ability of human mesenchymal stem cells (hMSCs) induced by the surface modification was evaluated by immunofluorescence of early osteogenic markers, Alizarin red staining, and energy dispersive X-ray spectroscopy. The expression of the adhesion-related protein vinculin was analyzed on the different functionalized surfaces. The results revealed that the enzymatic crosslinker transglutaminase offered high degradation resistance, tensile strength, and hydrolysis stability compared to the other crosslinking reagents tested. Remarkably, the adhesion sequences within the collagen were accessible to the hMSCs despite the transglutaminase crosslinking procedure. In conclusion, the organochemical functionalization of Ti6Al4V surfaces with collagen using transglutaminase holds great potential to facilitate an enhanced interaction with attached bone cells and thereby could potentially improve and accelerate osseointegration of a titanium-based bone implant in vivo.

Porous collagen scaffolds enable endothelial lumen formation in vitro under both static and dynamic growth conditions.

Despite recent advances in the field of tissue engineering, the development of complex tissue-like structures in vitro is compromised by the lack of integration of a functioning vasculature. In this study, we propose a mesoscale three-dimensional (3D) in vitro vascularized connective tissue model and demonstrate its feasibility to prompt the self-assembly of endothelial cells into vessel-like structures. Moreover, we investigate the effect of perfusion on the organization of the cells. For this purpose, primary endothelial cells (HUVECs) and a cell line of human foreskin fibroblasts are cultivated in ECM-like matrices made up of freeze-dried collagen scaffolds permeated with collagen type I hydrogel. A tailored bioreactor is designed to investigate the effect of perfusion on self-organization of HUVECs. Immunofluorescent staining, two-photon microscopy, second-harmonic generation imaging, and scanning electron microscopy are applied to visualize the spatial arrangement of the cells. The analyses reveal the formation of hollow, vessel-like structures of HUVECs in hydrogel-permeated collagen scaffolds under both static and dynamic conditions. In conclusion, we demonstrate the feasibility of a 3D porous collagen scaffolding system that enables and maintains the self-organization of HUVECs into vessel-like structures independent of a dynamic flow.

A Fibrin-Based Human Multicellular Gingival 3D Model Provides Biomimicry and Enables Long-Term In Vitro Studies.

Collagen-type I gels are widely used for the fabrication of 3D in vitro gingival models. Unfortunately, their long-term stability is low, which limits the variety of in vitro applications. To overcome this problem and achieve better hydrolytic stability of 3D gingival models, fibrin-based hydrogel blends with increased long-term stability in vitro are investigated. Two different fibrin-based hydrogels are tested: fibrin 2.5% (w/v) and fibrin 1% (w/v)/gelatin 5% (w/v). Appropriate numbers of primary human gingival fibroblasts (HGFs) and OKG4/bmi1/TERT (OKG) keratinocytes are optimized to achieve a homogeneous distribution of cells under the assumed 3D conditions. Both hydrogels support the viability of HGFs and the stability of the hydrogel over 28 days. In vitro cultivation at the air-liquid interface triggers keratinization of the epithelium and increases its thickness, allowing the formation of multiple tissue-like layers. The presence of HGFs in the hydrogel further enhances epithelial differentiation. In conclusion, a fibrin-based 3D gingival model mimics the histology of native gingiva in vitro and ensures its long-term stability in comparison with the previously reported collagen paralogs. These results open new perspectives for extending the period within which specific biological or pathological conditions of artificial gingival tissue can be evaluated.

Three-dimensional in vitro model of bone metastases of neuroblastoma as a tool for pharmacological evaluations.

In vitro metastatic models are foreseen to introduce a breakthrough in the field of preclinical screening of more functional small-molecule pharmaceuticals and biologics. To achieve this goal, the complexity of current in vitro systems requests an appropriate upgrade to approach the three-dimensional (3D) in vivo metastatic disease. Here, we explored the potential of our 3D ß-tricalcium phosphate (ß-TCP) model of neuroblastoma bone metastasis for drug toxicity assessment. Tailor-made scaffolds with interconnected channels were produced by combining 3D printing and slip casting method. The organization of neuroblastoma cells into a mesenchymal stromal cell (MSC) network, cultured under bioactive conditions provided by ß-TCP, was monitored by two-photon microscopy. Deposition of extracellular matrix protein Collagen I by MSCs and persistent growth of tumor cells confirmed the cell-supportive performance of our 3D model. When different neuroblastoma cells were treated with conventional chemotherapeutics, the ß-TCP model provided the necessary reproducibility and accuracy of experimental readouts. Drug efficacy evaluation was done for 3D and 2D cell cultures, highlighting the need for a higher dose of chemotherapeutics under 3D conditions to achieve the expected cytotoxicity in tumor cells. Our results confirm the importance of 3D geometry in driving native connectivity between nonmalignant and tumor cells and sustain ß-TCP scaffolds as a reliable and affordable drug screening platform for use in the early stages of drug discovery.

Functionalization of a zirconia surface by covalently immobilized fibronectin and its effects on resistance to thermal, acid, and mechanical exposure.

Silane chemistry has emerged as a powerful tool for surface modification, offering a versatile means to enhance the properties of various substrates, such as dental implant abutment materials. In this study, we investigated the stability of the 3-aminopropyldiisopropylethoxysilane (APDS) layer on yttria-partially stabilized zirconia (Y-TZP) surfaces after mechanical, acid, and thermal treatment in order to simulate fluctuations within the oral cavity. To accomplish that, the viability of human gingival fibroblasts on APDS-modified surfaces after applied treatment strategies was assessed by live/dead staining. Moreover, the hydrolysis stability and enzymatic degradation resistance of crosslinked fibronectin to the APDS layer was examined by immunostaining and western blot. The results revealed that the applied modifications were not affected by the different treatment conditions and could withstand the fluctuations in the oral cavity. Furthermore, crosslinked fibronectin on silanized Y-TZP was stable against hydrolysis over 21 days and enzymatic degradation. We thus can conclude that the proposed functionalization method has high potential to tolerate harmful effects within the oral cavity and remains unchanged on the surface.