Misleading Pore Size Measurements in Gelatin and Alginate Hydrogels revealed by Confocal Microscopy.

It is a well-documented phenomenon that the porous structure of hydrogels observed with vacuum-based imaging techniques is generated during the freezing and drying process employed prior to observation. Nevertheless, vacuum-based techniques, such as scanning electron microscopy (SEM), are still being commonly used to measure pore sizes in hydrogels, which is often not representative of the actual pore size in hydrated conditions. The frequent underestimation of the impact of freezing and drying on hydrogel structures could stem from a lack of cross-fertilization between materials science and biomedical or food science communities, or from the simplicity and visually appealing nature of SEM imaging which may lead to an overemphasis on its use. Our study provides a straightforward and impactful way of pinpointing this phenomenon exploiting two hydrogels ubiquitously applied in tissue engineering, including gelatin methacryloyl (GelMA) and alginate as proof-of-concept hydrogels. By comparing images of the samples in the native hydrated state, followed by freezing, freeze-drying and rehydration using SEM and confocal microscopy, we highlight discrepancies between hydrogel pore sizes in the hydrated versus the dry state. To conclude, our study offers recommendations for researchers seeking insight in hydrogel properties and emphasizes key factors that require careful control when using SEM as a characterization tool.

Melt electrowriting of poly(ε-caprolactone) - poly(ethylene glycol) backbone polymer blend scaffolds with improved hydrophilicity and functionality.

Melt electrowriting (MEW) is an additive manufacturing technique that harnesses electro-hydrodynamic phenomena to produce 3D-printed fibres with diameters on the scale of 10's of microns. The ability to print at this small scale provides opportunities to create structures with incredibly fine resolution and highly defined morphology. The current gold standard material for MEW is poly(ε-caprolactone) (PCL), a polymer with excellent biocompatibility but lacking in chemical groups that can allow intrinsic additional functionality. To provide this functionality while maintaining PCL's positive attributes, blending was performed with a Poly(Ethylene Glycol) (PEG)-based Acrylate endcapped Urethane-based Precursor (AUP). AUPs are a group of polymers, built on a backbone of existing polymers, which introduce additional functionality by the addition of one or more acrylate groups that terminate the polymer chain of a backbone polymer. By blending with a 20kDa AUP-PEG in small amounts, it is shown that MEW attributes are preserved, producing high-quality meshes. Blends were produced in various PCL:AUP weight ratios (100:0, 90:10 and 0:100) and processed into both solvent-cast films and MEW meshes that were used to characterise the properties of the blends. It was found that the addition of AUP-PEG to PCL significantly increases the hydrophilicity of structures produced with these polymers, and adds swelling capability compared to the non-swelling PCL. The developed blend (90:10) is shown to be processable using MEW, and the quality of manufactured scaffolds is evaluated against pure PCL scaffolds by performing scanning electron microscopy image analysis, with the quality of the novel MEW blend scaffolds showing comparable quality to that of pure PCL. The presence of the functionalisable AUP material on the surface of the developed scaffolds is also confirmed using fluorescence labelling of the acrylate groups. Biocompatibility of the MEW-processable blend was confirmed through a cell viability study, which found a high degree of cytocompatibility.

Digital Light Processing of 19F MRI-Traceable Gelatin-Based Biomaterial Inks towards Bone Tissue Regeneration.

Gelatin-based photo-crosslinkable hydrogels are promising scaffold materials to serve regenerative medicine. They are widely applicable in additive manufacturing, which allows for the production of various scaffold microarchitectures in line with the anatomical requirements of the organ to be replaced or tissue defect to be treated. Upon their in vivo utilization, the main bottleneck is to monitor cell colonization along with their degradation (rate). In order to enable non-invasive visualization, labeling with MRI-active components like N-(2,2-difluoroethyl)acrylamide (DFEA) provides a promising approach. Herein, we report on the development of a gelatin-methacryloyl-aminoethyl-methacrylate-based biomaterial ink in combination with DFEA, applicable in digital light processing-based additive manufacturing towards bone tissue regeneration. The fabricated hydrogel constructs show excellent shape fidelity in line with the printing resolution, as DFEA acts as a small molecular crosslinker in the system. The constructs exhibit high stiffness (E = 36.9 ± 4.1 kPa, evaluated via oscillatory rheology), suitable to serve bone regeneration and excellent MRI visualization capacity. Moreover, in combination with adipose tissue-derived stem cells (ASCs), the 3D-printed constructs show biocompatibility, and upon 4 weeks of culture, the ASCs express the osteogenic differentiation marker Ca2+.

Tailorable acrylate-endcapped urethane-based polymers for precision in digital light processing: Versatile solutions for biomedical applications.

Bioengineering seeks to replicate biological tissues exploiting scaffolds often based on polymeric biomaterials. Digital light processing (DLP) has emerged as a potent technique to fabricate tissue engineering (TE) scaffolds. However, the scarcity of suitable biomaterials with desired physico-chemical properties along with processing capabilities limits DLP's potential. Herein, we introduce acrylate-endcapped urethane-based polymers (AUPs) for precise physico-chemical tuning while ensuring optimal computer-aided design/computer-aided manufacturing (CAD/CAM) mimicry. Varying the polymer backbone (i.e. poly(ethylene glycol) (PEG) versus poly(propylene glycol) (PPG)) and photo-crosslinkable endcap (i.e. di-acrylate versus hexa-acrylate), we synthesized a series of photo-crosslinkable materials labeled as UPEG2, UPEG6, UPPG2 and UPPG6. Comprehensive material characterization including physico-chemical and biological evaluations, was followed by a DLP processing parametric study for each material. The impact of the number of acrylate groups per polymer (2 to 6) on the physico-chemical properties was pronounced, as reflected by a reduced swelling, lower water contact angles, accelerated crosslinking kinetics, and increased Young's moduli upon increasing the acrylate content. Furthermore, the different polymer backbones also exerted a substantial effect on the properties, including the absence of crystallinity, remarkably reduced swelling behaviors, a slight reduction in Young's modulus, and slower crosslinking kinetics for UPPG vs UPEG. The mechanical characteristics of DLP-printed samples showcased the ability to tailor the materials' stiffness (ranging from 0.4 to 5.3 MPa) by varying endcap chemistry and/or backbone. The in vitro cell assays confirmed biocompatibility of the material as such and the DLP-printed discs. Furthermore, the structural integrity of 3D scaffolds was preserved both in dry and swollen state. By adjusting the backbone chemistry or acrylate content, the post-swelling dimensions could be customized towards the targeted application. This study showcases the potential of these materials offering tailorable properties to serve many biomedical applications such as cartilage TE.

Reconstructing Curves: A Bottom-Up Approach toward Adipose Tissue Regeneration with Recombinant Biomaterials.

The potential of recombinant materials in the field of adipose tissue engineering (ATE) is investigated using a bottom-up tissue engineering (TE) approach. This study explores the synthesis of different photo-crosslinkable gelatin derivatives, including both natural and recombinant materials, with a particular emphasis on chain growth and step growth polymerization. Gelatin type B (Gel-B) and a recombinant collagen peptide (RCPhC1) are used as starting materials. The gel fraction and mass swelling properties of 2D hydrogel films are evaluated, revealing high gel fractions exceeding 94% and high mass swelling ratios >15. In vitro experiments with encapsulated adipose-derived stem cells (ASCs) indicate viable cells (>85%) throughout the experiment with the RCPhC1-based hydrogels showing a higher number of stretched ASCs. Triglyceride assays show the enhanced differentiation potential of RCPhC1 materials. Moreover, the secretome analysis reveal the production of adipose tissue-specific proteins including adiponectin, adipsin, lipocalin-2/NGAL, and PAL-1. RCPhC1-based materials exhibit higher levels of adiponectin and adipsin production, indicating successful differentiation into the adipogenic lineage. Overall, this study highlights the potential of recombinant materials for ATE applications, providing insights into their physico-chemical properties, mechanical strength, and cellular interactions.

Cotton Fabric-Reinforced Hydrogels with Excellent Mechanical and Broad-Spectrum Photothermal Antibacterial Properties.

Antibacterial hydrogel wound dressings hold great potential in eliminating bacteria and accelerating the healing process. However, it remains a challenge to fabricate hydrogel wound dressings that simultaneously exhibit excellent mechanical and photothermal antibacterial properties. Here we report the development of polydopamine-functionalized graphene oxide (rGO@PDA)/calcium alginate (CA)/Polypyrrole (PPy) cotton fabric-reinforced hydrogels (abbreviated as rGO@PDA/CA/PPy FHs) for tackling bacterial infections. The mechanical properties of hydrogels were greatly enhanced by cotton fabric reinforcement and an interpenetrating structure, while excellent broad-spectrum photothermal antibacterial properties based on the photothermal effect were obtained by incorporating PPy and rGO@PDA. Results indicated that rGO@PDA/CA/PPy FHs exhibited superior tensile strength in both the warp (289 ± 62.1 N) and weft directions (142 ± 23.0 N), similarly to cotton fabric. By incorporating PPy and rGO@PDA, the swelling ratio was significantly decreased from 673.5% to 236.6%, while photothermal conversion performance was significantly enhanced with a temperature elevated to 45.0 °C. Due to the synergistic photothermal properties of rGO@PDA and PPy, rGO@PDA/CA/PPy FHs exhibited excellent bacteria-eliminating efficiency for S. aureus (0.57%) and E. coli (3.58%) after exposure to NIR for 20 min. We believe that the design of fabric-reinforced hydrogels could serve as a guideline for developing hydrogel wound dressings with improved mechanical properties and broad-spectrum photothermal antibacterial properties for infected-wound treatment.

Effect of Porosity on the Colonization of Digital Light-Processed 3D Hydrogel Constructs toward the Development of a Functional Intestinal Model.

With the rapid increase of the number of patients with gastrointestinal diseases in modern society, the need for the development of physiologically relevant in vitro intestinal models is key to improve the understanding of intestinal dysfunctions. This involves the development of a scaffold material exhibiting physiological stiffness and anatomical mimicry of the intestinal architecture. The current work focuses on evaluating the scaffold micromorphology of gelatin-methacryloyl-aminoethyl-methacrylate-based nonporous and porous intestinal 3D, intestine-like constructs, fabricated via digital light processing, on the cellular response. To this end, Caco-2 intestinal cells were utilized in combination with the constructs. Both porous and nonporous constructs promoted cell growth and differentiation toward enterocyte-like cells (VIL1, ALPI, SI, and OCLD expression showed via qPCR, ZO-1 via immunostaining). The porous constructs outperformed the nonporous ones regarding cell seeding efficiency and growth rate, confirmed by MTS assay, live/dead staining, and TEER measurements, due to the presence of surface roughness.

In vitro and in vivo evaluation of periosteum-derived cells and iPSC-derived chondrocytes encapsulated in GelMA for osteochondral tissue engineering.

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

The importance of mechanical and biological cues of tympanic membrane grafts to ensure optimal regeneration.

Chronic suppurative otitis media (CSOM) is often associated with permanent tympanic membrane (TM) perforation and conductive hearing loss. The current clinical gold standard, using autografts and allografts, suffers from several drawbacks. Artificial replacement materials can help to overcome these drawbacks. Therefore, scaffolds fabricated through digital light processing (DLP) were herein created to support TM regeneration. Various UV-curable printing inks, including gelatin methacryloyl (GelMA), gelatin-norbornene-norbornene (GelNBNB) (crosslinked with thiolated gelatin (GelSH)) and alkene-functionalized poly-ε-caprolactone (E-PCL) (crosslinked with pentaerythritol tetrakis(3-mercaptopropionate) (PETA4SH)) were optimized regarding photo-initiator (PI) and photo-absorber (PA) concentrations through viscosity characterization, photo-rheology and the establishment of working curves for DLP. Our material platform enabled the development of constructs with a range of mechanical properties (plateau storage modulus varying between 15 and 119 kPa). Excellent network connectivity for the GelNBNB and E-PCL constructs was demonstrated (gel fractions >95 %) whereas a post-crosslinking step was required for the GelMA constructs. All samples showed excellent biocompatibility (viability >93 % and metabolic activity >88 %). Finally, in vivo and ex vivo assessments, including histology, vibration and deformation responses measured through laser doppler vibrometry and digital image correlation respectively, were performed to investigate the effects of the scaffolds on the anatomical and physiological regeneration of acute TM perforations in rabbits. The data showed that the most efficient healing with the best functional quality was obtained when both mechanical (obtained with the PCL-based resin) and biological (obtained with the gelatin-based resins) material properties were taken into account.

Photo-crosslinkable polyester microneedles as sustained drug release systems toward hypertrophic scar treatment.

Burn injuries can result in a significant inflammatory response, often leading to hypertrophic scarring (HTS). Local drug therapies e.g. corticoid injections are advised to treat HTS, although they are invasive, operator-dependent, extremely painful and do not permit extended drug release. Polymer-based microneedle (MN) arrays can offer a viable alternative to standard care, while allowing for direct, painless dermal drug delivery with tailorable drug release profile. In the current study, we synthesized photo-crosslinkable, acrylate-endcapped urethane-based poly(ε-caprolactone) (AUP-PCL) toward the fabrication of MNs. Physico-chemical characterization (1H-NMR, evaluation of swelling, gel fraction) of the developed polymer was performed and confirmed successful acrylation of PCL-diol. Subsequently, AUP-PCL, and commercially available PCL-based microneedle arrays were fabricated for comparative evaluation of the constructs. Hydrocortisone was chosen as model drug. To enhance the drug release efficiency of the MNs, Brij®35, a nonionic surfactant was exploited. The thermal properties of the MNs were evaluated via differential scanning calorimetry. Compression testing of the arrays confirmed that the MNs stay intact upon applying a load of 7 N, which correlates to the standard dermal insertion force of MNs. The drug release profile of the arrays was evaluated, suggesting that the developed PCL arrays can offer efficient drug delivery for up to two days, while the AUP-PCL arrays can provide a release up to three weeks. Finally, the insertion of MN arrays into skin samples was performed, followed by histological analysis demonstrating the AUP-PCL MNs outperforming the PCL arrays upon providing pyramidical-shaped perforations through the epidermal layer of the skin.

AUP-PCL MN arrays provide long-term transdermal drug delivery of hydrocortisoneAUP-PCL-based MN arrays provide superior drug release profiles compared to PCL MNsEffective skin penetration AUP-PCL-based MNs on skin was achieved.

Powdered Cross-Linked Gelatin Methacryloyl as an Injectable Hydrogel for Adipose Tissue Engineering.

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

3D-Printed Shape Memory Poly(alkylene terephthalate) Scaffolds as Cardiovascular Stents Revealing Enhanced Endothelialization.

Cardiovascular diseases are the leading cause of death and current treatments such as stents still suffer from disadvantages. Balloon expansion causes damage to the arterial wall and limited and delayed endothelialization gives rise to restenosis and thrombosis. New more performing materials that circumvent these disadvantages are required to improve the success rate of interventions. To this end, the use of a novel polymer, poly(hexamethylene terephthalate), is investigated for this application. The synthesis to obtain polymers with high molar masses up to 126.5 kg mol-1 is optimized and a thorough chemical and thermal analysis is performed. The polymers are 3D-printed into personalized cardiovascular stents using the state-of-the-art solvent-cast direct-writing technique, the potential of these stents to expand using their shape memory behavior is established, and it is shown that the stents are more resistant to compression than the poly(l-lactide) benchmark. Furthermore, the polymer's hydrolytic stability is demonstrated in an accelerated degradation study of 6 months. Finally, the stents are subjected to an in vitro biological evaluation, revealing that the polymer is non-hemolytic and supports significant endothelialization after only 7 days, demonstrating the enormous potential of these polymers to serve cardiovascular applications.

Design of 3D-Photoprintable, Bio-, and Hemocompatible Nonisocyanate Polyurethane Elastomers for Biomedical Implants.

Polyurethanes (PUs) have adjustable mechanical properties, making them suitable for a wide range of applications, including in the biomedical field. Historically, these PUs have been synthesized from isocyanates, which are toxic compounds to handle. This has encouraged the search for safer and more environmentally friendly synthetic routes, leading today to the production of nonisocyanate polyurethanes (NIPUs). Among these NIPUs, polyhydroxyurethanes (PHUs) bear additional hydroxyl groups, which are particularly attractive for derivatizing and adjusting their physicochemical properties. In this paper, polyether-based NIPU elastomers with variable stiffness are designed by functionalizing the hydroxyl groups of a poly(propylene glycol)-PHU by a cyclic carbonate carrying a pendant unsaturation, enabling them to be post-photo-cross-linked with polythiols (thiol-ene). Elastomers with remarkable mechanical properties whose stiffness can be adjusted are obtained. Thanks to the unique viscous properties of these PHU derivatives and their short gel times observed by rheology experiments, formulations for light-based three-dimensional (3D) printing have been developed. Objects were 3D-printed by digital light processing with a resolution down to the micrometer scale, demonstrating their ability to target various designs of prime importance for personalized medicine. In vitro biocompatibility tests have confirmed the noncytotoxicity of these materials for human fibroblasts. In vitro hemocompatibility tests have revealed that they do not induce hemolytic effects, they do not increase platelet adhesion, nor activate coagulation, demonstrating their potential for future applications in the cardiovascular field.

Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Optimization of hybrid gelatin-polysaccharide bioinks exploiting thiol-norbornene chemistry using a reducing additive.

Thiol-norbornene chemistry offers great potential in the field of hydrogel development, given its step growth crosslinking mechanism. However, limitations exist with regard to deposition-based bioprinting of thiol-containing hydrogels, associated with premature crosslinking of thiolated (bio)polymers resulting from disulfide formation in the presence of oxygen. More specifically, disulfide formation can result in an increase in viscosity thereby impeding the printing process. In the present work, hydrogels constituting norbornene-modified dextran (DexNB) combined with thiolated gelatin (GelSH) are selected as case study to explore the potential of incorporating the reducing agent tris(2-carboxyethyl)phosphine (TCEP), to prevent the formation of disulfides. We observed that, in addition to preventing disulfide formation, TCEP also contributed to premature, spontaneous thiol-norbornene crosslinking without the use of UV light as evidenced via1H-NMR spectroscopy. Herein, an optimal concentration of 25 mol% TCEP with respect to the amount of thiols was found, thereby limiting auto-gelation by both minimizing disulfide formation and spontaneous thiol-norbornene reaction. This concentration results in a constant viscosity during at least 24 h, a more homogeneous network being formed as evidenced using atomic force microscopy while retaining bioink biocompatibility as evidenced by a cell viability of human foreskin fibroblasts exceeding 70% according to ISO 10993-6:2016.

2D fibrillar osteoid niche mimicry through inclusion of visco-elastic and topographical cues in gelatin-based networks.

Given the clinical need for osteoregenerative materials incorporating controlled biomimetic and biophysical cues, a novel highly-substituted norbornene-modified gelatin was developed enabling thiol-ene crosslinking exploiting thiolated gelatin as cell-interactive crosslinker. Comparing the number of physical crosslinks, the degree of hydrolytic degradation upon modification, the network density and the chemical crosslinking type, the osteogenic effect of visco-elastic and topographical properties was evaluated. This novel network outperformed conventional gelatin-based networks in terms of osteogenesis induction, as evidenced in 2D dental pulp stem cell seeding assays, resulting from the presentation of both a local (substrate elasticity, 25-40 kPa) and a bulk (compressive modulus, 25-45 kPa) osteogenic substrate modulus in combination with adequate fibrillar cell adhesion spacing to optimally transfer traction forces from the fibrillar ECM (as evidenced by mesh size determination with the rubber elasticity theory) and resulting in a 1.7-fold increase in calcium production (compared to the gold standard gelatin methacryloyl (GelMA)).

Bottom-Up Extrusion-Based Biofabrication of the Osteoid Niche.

Bone regeneration remains a clinical challenge given the transplantation incidence rate and the associated economic burden. Bottom-up osteoid tissue engineering has the potential to offer an alternative approach to current clinical solutions that suffer from various drawbacks. In this paper, deposition-based bioprinting is exploited while the effect is explored of both the crosslinking mechanism (gelatin methacryloyl (GelMA) versus gelatin norbornene (DS 91) crosslinked with thiolated gelatin (GelNBSH)) and the degree of substitution (GelNBSH versus norbornene-norbornene-modified gelatin (DS 169) crosslinked with thiolated gelatin (GelNBNBSH)) on the presented biophysical cues as well as on the osteogenic differentiation. The incorporation of tris(2-carboxyethyl)phosphine (TCEP) to the step-growth inks allows the production of reproducible and biocompatible scaffolds based on thiol-ene chemistry. Dental pulp stem cell encapsulation in GelNBNBSH biofabricated constructs shows a favorable response due to the combination of its stress relaxation and substrate rigidity (bulk compressive modulus of 11-30 kPa) as reflected by a sevenfold increase in calcium production compared to the tissue engineering standard GelMA. This work is the first to exploit a controlled biocompatible and cell-interactive thiolated macromolecular crosslinker (GelSH + TCEP) allowing the extrusion-based biofabrication of low concentration (5 w/v%) modified osteogenic gelatin-based inks (GelNBNBSH + TCEP).

Effect of Poly(Vinyl Pyrrolidone) on Iodine Release from Acrylate-Endcapped Urethane-Based Poly(Ethylene Glycol) Hydrogels as Antibacterial Wound Dressing.

Infections are still a major cause of morbidity in burn wounds. Although silver has been used strongly in past centuries as an anti-bacterial, it can lead to allergic reactions, bacterial resistance, and delayed wound healing. Iodine-based antibacterials are becoming an interesting alternative. In this work, the effect of complexation with poly(vinyl pyrrolidone) (PVP) and poly(ethylene oxide) (PEO)-based polymers is explored by using different acrylate-endcapped urethane-based poly(ethylene glycol) (AUP) polymers, varying the molar mass (MM) of the poly(ethylene glycol) (PEG) backbone, with possible addition of PVP. The higher MM AUP outperforms the swelling potential of commercial wound dressings such as Kaltostat, Aquacel Ag, and Hydrosorb and all MM show superior mechanical properties. The addition of iodine to the polymers is compared to Iso-Betadine Tulle (IBT). Interestingly, the addition of PVP does not lead to increased iodine complexation compared to the blank AUP polymers, while all have a prolonged iodine release compared to the IBT, which leads to a burst release. The observed prolonged release also leads to larger inhibition zones during antibacterial tests. Complexing iodine in AUP polymers with or without PVP leads to antimicrobial wound dressings which may hold potential for future application to treat infected wounds.

Polymeric reinforcements for cellularized collagen-based vascular wall models: influence of the scaffold architecture on the mechanical and biological properties.

A previously developed cellularized collagen-based vascular wall model showed promising results in mimicking the biological properties of a native vessel but lacked appropriate mechanical properties. In this work, we aim to improve this collagen-based model by reinforcing it using a tubular polymeric (reinforcement) scaffold. The polymeric reinforcements were fabricated exploiting commercial poly (ε-caprolactone) (PCL), a polymer already used to fabricate other FDA-approved and commercially available devices serving medical applications, through 1) solution electrospinning (SES), 2) 3D printing (3DP) and 3) melt electrowriting (MEW). The non-reinforced cellularized collagen-based model was used as a reference (COL). The effect of the scaffold's architecture on the resulting mechanical and biological properties of the reinforced collagen-based model were evaluated. SEM imaging showed the differences in scaffolds' architecture (fiber alignment, fiber diameter and pore size) at both the micro- and the macrolevel. The polymeric scaffold led to significantly improved mechanical properties for the reinforced collagen-based model (initial elastic moduli of 382.05 ± 132.01 kPa, 100.59 ± 31.15 kPa and 245.78 ± 33.54 kPa, respectively for SES, 3DP and MEW at day 7 of maturation) compared to the non-reinforced collagen-based model (16.63 ± 5.69 kPa). Moreover, on day 7, the developed collagen gels showed stresses (for strains between 20% and 55%) in the range of [5-15] kPa for COL, [80-350] kPa for SES, [20-70] kPa for 3DP and [100-190] kPa for MEW. In addition to the effect on the resulting mechanical properties, the polymeric tubes' architecture influenced cell behavior, in terms of proliferation and attachment, along with collagen gel compaction and extracellular matrix protein expression. The MEW reinforcement resulted in a collagen gel compaction similar to the COL reference, whereas 3DP and SES led to thinner and longer collagen gels. Overall, it can be concluded that 1) the selected processing technique influences the scaffolds' architecture, which in turn influences the resulting mechanical and biological properties, and 2) the incorporation of a polymeric reinforcement leads to mechanical properties closely matching those of native arteries.

A preclinical platform for assessing long-term drug efficacy exploiting mechanically tunable scaffolds colonized by a three-dimensional tumor microenvironment.

BACKGROUND: Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. METHODS: In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. RESULTS: The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. CONCLUSIONS: The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery.