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+.

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.

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.

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 molar mass and alkyl chain length on the surface properties and biocompatibility of poly(alkylene terephthalate)s for potential cardiovascular applications.

Cardiovascular diseases are the leading cause of death worldwide. Treatments for occluded arteries include balloon angioplasty with or without stenting and bypass grafting surgery. Poly(ethylene terephthalate) is frequently used as a vascular graft material, but its high stiffness leads to compliance mismatch with the human blood vessels, resulting in altered hemodynamics, thrombus formation and graft failure. Poly(alkylene terephthalate)s (PATs) with longer alkyl chain lengths hold great potential for improving the compliance. In this work, the effect of the polymer molar mass and the alkyl chain length on the surface roughness and wettability of spin-coated PAT films was investigated, as well as the endothelial cell adhesion and proliferation on these samples. We found that surface roughness generally increases with increasing molar mass and alkyl chain length, while no trend for the wettability could be observed. All investigated PATs are non-cytotoxic and support endothelial cell adhesion and growth. For some PATs, the endothelial cells even reorganized into a tubular-like structure, suggesting angiogenic maturation. In conclusion, this research demonstrates the biocompatibility of PATs and their potential to be applied as materials serving cardiovascular applications.

Nature-Inspired Dual Purpose Strategy toward Cell-Adhesive PCL Networks: C(-linker-)RGD Incorporation via Thiol-ene Crosslinking.

In an attempt to mimic nature's ability to adhere cells, PCL is often coated with nature-derived polymers or its surface is functionalized with a cell-binding motif. However, said surface modifications are limited to the material's surface, include multiple steps, and are mediated by harsh conditions. Here, we introduce a single-step strategy toward cell-adhesive polymer networks where thiol-ene chemistry serves a dual purpose. First, alkene-functionalized PCL is crosslinked by means of a multifunctional thiol. Second, by means of a cysteine coupling site, the cell-binding motif C(-linker-)RGD is covalently bound throughout the PCL networks during crosslinking. Moreover, the influence of various linkers (type and length), between the cysteine coupling site and the cell-binding motif RGD, is investigated and the functionalization is assessed by means of static contact angle measurements and X-ray photoelectron spectroscopy. Finally, successful introduction of cell adhesiveness is illustrated for the networks by seeding fibroblasts onto the functionalized PCL networks.

Solid-State Crosslinkable, Shape-Memory Polyesters Serving Tissue Engineering.

Acrylate-endcapped urethane-based precursors constituting a poly(D,L-lactide)/poly(ε-caprolactone) (PDLLA/PCL) random copolymer backbone are synthesized with linear and star-shaped architectures and various molar masses. It is shown that the glass transition and thus the actuation temperature could be tuned by varying the monomer content (0-8 wt% ε-caprolactone, Tg,crosslinked = 10-42 °C) in the polymers. The resulting polymers are analyzed for their physico-chemical properties and viscoelastic behavior (G'max = 9.6-750 kPa). The obtained polymers are subsequently crosslinked and their shape-memory properties are found to be excellent (Rr = 88-100%, Rf = 78-99.5%). Moreover, their potential toward processing via various additive manufacturing techniques (digital light processing, two-photon polymerization and direct powder extrusion) is evidenced with retention of their shape-memory effect. Additionally, all polymers are found to be biocompatible in direct contact in vitro cell assays using primary human foreskin fibroblasts (HFFs) through MTS assay (up to ≈100% metabolic activity relative to TCP) and live/dead staining (>70% viability).

Volumetric Printing of Thiol-Ene Photo-Cross-Linkable Poly(ε-caprolactone): A Tunable Material Platform Serving Biomedical Applications.

Current thoroughly described biodegradable and cross-linkable polymers mainly rely on acrylate cross-linking. However, despite the swift cross-linking kinetics of acrylates, the concomitant brittleness of the resulting materials limits their applicability. Here, photo-cross-linkable poly(ε-caprolactone) networks through orthogonal thiol-ene chemistry are introduced. The step-growth polymerized networks are tunable, predictable by means of the rubber elasticity theory and it is shown that their mechanical properties are significantly improved over their acrylate cross-linked counterparts. Tunability is introduced to the materials, by altering Mc (or the molar mass between cross-links), and its effect on the thermal properties, mechanical strength and degradability of the materials is evaluated. Moreover, excellent volumetric printability is illustrated and the smallest features obtained via volumetric 3D-printing to date are reported, for thiol-ene systems. Finally, by means of in vitro and in vivo characterization of 3D-printed constructs, it is illustrated that the volumetrically 3D-printed materials are biocompatible. This combination of mechanical stability, tunability, biocompatibility, and rapid fabrication by volumetric 3D-printing charts a new path toward bedside manufacturing of biodegradable patient-specific implants.

Fine-Tuning the Endcap Chemistry of Acrylated Poly(Ethylene Glycol)-Based Hydrogels for Efficient Burn Wound Exudate Management.

Most commercial dressings with moderate to high exudate uptake capacities are mechanically weaker and/or require a secondary dressing. The current research article focuses on the development of hydrogel-based wound dressings combining mechanical strength with high exudate absorption capacities using acrylate-endcapped urethane-based precursors (AUPs). AUPs with varying poly(ethylene glycol) backbone molar masses (10 and 20 kg mol-1 ) and endcap chemistries are successfully synthesized in toluene, subsequently processed into UV-cured hydrogel sheets and are benchmarked against several commercial wound dressings (Hydrosorb, Kaltostat, and Mepilex Ag). The AUP materials show high gel fractions (>90%) together with strong swelling degrees in water, phosphate buffered saline and simulated wound fluid (12.7-19.6 g g-1 ), as well as tunable mechanical properties (e.g., Young's modulus: 0.026-0.061 MPa). The AUPs have significantly (p < 0.05) higher swelling degrees than the tested commercial dressings, while also being mechanically resistant. The elasticity of the synthesized materials leads to an increased resistance against fatigue. The di- and hexa-acrylated AUPs show excellent in vitro biocompatibility against human foreskin fibroblasts, as evidenced by indirect MTS assays and live/dead cell assays. In conclusion, the processed AUP materials demonstrate high potential for wound healing application and can even compete with commercially available dressings.

Utilizing Osteocyte Derived Factors to Enhance Cell Viability and Osteogenic Matrix Deposition within IPN Hydrogels.

Many bone defects arising due to traumatic injury, disease, or surgery are unable to regenerate, requiring intervention. More than four million graft procedures are performed each year to treat these defects making bone the second most commonly transplanted tissue worldwide. However, these types of graft suffer from a limited supply, a second surgical site, donor site morbidity, and pain. Due to the unmet clinical need for new materials to promote skeletal repair, this study aimed to produce novel biomimetic materials to enhance stem/stromal cell osteogenesis and bone repair by recapitulating aspects of the biophysical and biochemical cues found within the bone microenvironment. Utilizing a collagen type I-alginate interpenetrating polymer network we fabricated a material which mirrors the mechanical and structural properties of unmineralized bone, consisting of a porous fibrous matrix with a young's modulus of 64 kPa, both of which have been shown to enhance mesenchymal stromal/stem cell (MSC) osteogenesis. Moreover, by combining this material with biochemical paracrine factors released by statically cultured and mechanically stimulated osteocytes, we further mirrored the biochemical environment of the bone niche, enhancing stromal/stem cell viability, differentiation, and matrix deposition. Therefore, this biomimetic material represents a novel approach to promote skeletal repair.