Printability and Shape Fidelity of Bioinks in 3D Bioprinting.

Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance.

Hydrogel-Based Bioinks for Cell Electrowriting of Well-Organized Living Structures with Micrometer-Scale Resolution.

Bioprinting has become an important tool for fabricating regenerative implants and in vitro cell culture platforms. However, until today, extrusion-based bioprinting processes are limited to resolutions of hundreds of micrometers, which hamper the reproduction of intrinsic functions and morphologies of living tissues. This study describes novel hydrogel-based bioinks for cell electrowriting (CEW) of well-organized cell-laden fiber structures with diameters ranging from 5 to 40 µm. Two novel photoresponsive hydrogel bioinks, that is, based on gelatin and silk fibroin, which display distinctly different gelation chemistries, are introduced. The rapid photomediated cross-linking mechanisms, electrical conductivity, and viscosity of these two engineered bioinks allow the fabrication of 3D ordered fiber constructs with small pores (down to 100 µm) with different geometries (e.g., squares, hexagons, and curved patterns) of relevant thicknesses (up to 200 µm). Importantly, the biocompatibility of the gelatin- and silk fibroin-based bioinks enables the fabrication of cell-laden constructs, while maintaining high cell viability post printing. Taken together, CEW and the two hydrogel bioinks open up fascinating opportunities to manufacture microstructured constructs for applications in regenerative medicine and in vitro models that can better resemble cellular microenvironments.

Engineered Three-Dimensional Microenvironments with Starch Nanocrystals as Cell-Instructive Materials.

Naturally, cells reside in three-dimensional (3D) microenvironments composed of biopolymers that guide cellular behavior via topographical features as well as through mechanical and biochemical cues. However, most studies describing the influence of topography on cells' behavior are performed on rigid and synthetic two-dimensional substrates. To design systems that more closely resemble native microenvironments, herein we develop 3D nanocomposite hydrogels consisting of starch nanocrystals (SNCs) embedded in a gelatin matrix. The incorporation of different concentrations of SNCs (0.05, 0.2, and 0.5 wt %) results in an increase of compressive modulus when compared to hydrogels without SNCs, without affecting the swelling ratio, thus providing a tunable system. Confirming the cytocompatibility of the novel composites, the viability of encapsulated L929 fibroblasts is >90% in all hydrogels. The cellular metabolic activity and DNA content are similar for all formulations and increase over time, indicating that the fibroblasts proliferate within the hydrogels. After 4 d of culture, Live/Dead staining and F-actin/nuclei staining show that the encapsulated fibroblasts develop an elongated morphology in the hydrogels. On the other hand, encapsulated chondrogenic progenitor ATDC5 cells also maintain a viability around 90% but display a round morphology, especially in the hydrogels with SNCs, indicating a potential application of the materials for cartilage tissue engineering. We believe that topographical and mechanical cues within 3D microenvironments can be a powerful tool to instruct cells' behavior and that the developed gelatin/SNC nanocomposite warrants further study.

Design of Decorin-Based Peptides That Bind to Collagen†I and their Potential as Adhesion Moieties in Biomaterials.

Mimicking the binding epitopes of protein-protein interactions by using small peptides is important for generating modular biomimetic systems. A strategy is described for the design of such bioactive peptides without accessible structural data for the targeted interaction, and the effect of incorporating such adhesion peptides in complex biomaterial systems is demonstrated. The highly repetitive structure of decorin was analyzed to identify peptides that are representative of the inner and outer surface, and it was shown that only peptides based on the inner surface of decorin bind to collagen. The peptide with the highest binding affinity for collagen I, LHERHLNNN, served to slow down the diffusion of a conjugated dye in a collagen gel, while its dimer could physically crosslink collagen, thereby enhancing the elastic modulus of the gel by one order of magnitude. These results show the potential of the identified peptides for the design of biomaterials for applications in regenerative medicine.

Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool†Dedicated to the memory of Joachim H. G. Steinke.‡Electronic supplementary information (ESI) available: Further experimental data. See DOI: 10.1039/c3sm00131hClick here for additional data file.

The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheology studies and TEM of the corresponding hydrogels (including PNIPAAm-containing systems) showed no significant variation in properties and hydrogel morphology compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.

Robust gelatin hydrogels for local sustained release of bupivacaine following spinal surgery.

Adequate treatment of pain arising from spinal surgery is a major clinical challenge. Opioids are the mainstay of current treatment methods, but the frequency and severity of their side effects display a clear need for opioid-free analgesia. Local anesthetics have been encapsulated into sustained-release drug delivery systems to provide postoperative pain relief. However, these formulations are limited by rapid diffusion out of the surgical site. To overcome this limitation, we synthesized ring-shaped hydrogels incorporating bupivacaine, designed to be co-implanted with pedicle screws during spinal surgery. Hydrogels were prepared by riboflavin-mediated crosslinking of gelatin functionalized with tyramine moieties. Additionally, oxidized ß-cyclodextrin was introduced into the hydrogel formulation to form dynamic bonds with tyramine functionalities, which enables self-healing behavior and resistance to shear. Feasibility of hydrogel implantation combined with pedicle screws was qualitatively assessed in cadaveric sheep as a model for instrumented spinal surgery. The in-situ crystallization of bupivacaine within the hydrogel matrix provided a moderate burst decrease and sustained release that exceeded 72 hours in vitro. The use of bupivacaine crystals decreased drug-induced cytotoxicity in vitro compared to bupivacaine HCl. Thus, the presented robust hydrogel formulation provides promising properties to enable the stationary release of non-opioid analgesics following spinal surgery. STATEMENT OF SIGNIFICANCE: Currently, postoperative pain following spinal surgery is mainly treated with opioids. However, the use of opioids is associated with several side effects including addiction. Here we developed robust and cytocompatible gelatin hydrogels, prepared via riboflavin-mediated photocrosslinking, that can withstand orthopedic implantation. The implantability was confirmed in cadaveric instrumented spinal surgery. Further, hydrogels were loaded with bupivacaine crystals to provide sustained release beyond 72 hours in vitro. The use of crystallized bupivacaine decreased cytotoxicity compared to bupivacaine HCl. The present formulation can aid in enabling opioid-free analgesia following instrumented spinal surgery.

Comparison of in vitro and in vivo Toxicity of Bupivacaine in Musculoskeletal Applications.

The recent societal debate on opioid use in treating postoperative pain has sparked the development of long-acting, opioid-free analgesic alternatives, often using the amino-amide local anesthetic bupivacaine as active pharmaceutical ingredient. A potential application is musculoskeletal surgeries, as these interventions rank amongst the most painful overall. Current literature showed that bupivacaine induced dose-dependent myo-, chondro-, and neurotoxicity, as well as delayed osteogenesis and disturbed wound healing in vitro. These observations did not translate to animal and clinical research, where toxic phenomena were seldom reported. An exception was bupivacaine-induced chondrotoxicity, which can mainly occur during continuous joint infusion. To decrease opioid consumption and provide sustained pain relief following musculoskeletal surgery, new strategies incorporating high concentrations of bupivacaine in drug delivery carriers are currently being developed. Local toxicity of these high concentrations is an area of further research. This review appraises relevant in vitro, animal and clinical studies on musculoskeletal local toxicity of bupivacaine.

3D bioprinting of molecularly engineered PEG-based hydrogels utilizing gelatin fragments.

Three-dimensional (3D) bioprinting is an additive manufacturing process in which the combination of biomaterials and living cells, referred to as a bioink, is deposited layer-by-layer to form biologically active 3D tissue constructs. Recent advancements in the field show that the success of this technology requires the development of novel biomaterials or the improvement of existing bioinks. Polyethylene glycol (PEG) is one of the well-known synthetic biomaterials and has been commonly used as a photocrosslinkable bioink for bioprinting; however, other types of cell-friendly crosslinking mechanisms to form PEG hydrogels need to be explored for bioprinting and tissue engineering. In this work, we proposed micro-capillary based bioprinting of a novel molecularly engineered PEG-based bioink that transiently incorporates low molecular weight gelatin (LMWG) fragments. The rheological properties and release profile of the LMWG fragments were characterized, and their presence during hydrogel formation had no effect on the swelling ratio or sol fraction when compared to PEG hydrogels formed without the LMWG fragments. For bioprinting, PEG was first functionalized with cell-adhesive RGD ligands and was then crosslinked using protease-sensitive peptides via a Michael-type addition reaction inside the micro-capillary. The printability was assessed by the analysis of extrudability, shape fidelity, and printing accuracy of the hydrogel filaments after the optimization of the gelation conditions of the PEG-based bioink. The LMWG fragments supplemented into the bioink allowed the extrusion of smooth and uniform cylindrical strands of the hydrogel and improved shape fidelity and printing accuracy. Encapsulated cells in both bioprinted and non-bioprinted PEG-based hydrogels showed high viability and continued to proliferate over time in culture with a well-defined cell morphology depending on the presence of the cell adhesive peptide RGD. The presented micro-capillary based bioprinting process for a novel PEG-based bioink can be promising to construct complex 3D structures with micro-scale range and spatiotemporal variations without using any cytotoxic photoinitiator, UV light, or polymer support.

The Importance of Interfaces in Multi-Material Biofabricated Tissue Structures.

Biofabrication exploits additive manufacturing techniques for creating 3D structures with a precise geometry that aim to mimic a physiological cellular environment and to develop the growth of native tissues. The most recent approaches of 3D biofabrication integrate multiple technologies into a single biofabrication platform combining different materials within different length scales to achieve improved construct functionality. However, the importance of interfaces between the different material phases, has not been adequately explored. This is known to determine material's interaction and ultimately mechanical and biological performance of biofabricated parts. In this review, this gap is bridged by critically examining the interface between different material phases in (bio)fabricated structures, with a particular focus on how interfacial interactions can compromise or define the mechanical (and biological) properties of the engineered structures. It is believed that the importance of interfacial properties between the different constituents of a composite material, deserves particular attention in its role in modulating the final characteristics of 3D tissue-like structures.

Rapid and cytocompatible cell-laden silk hydrogel formation via riboflavin-mediated crosslinking.

Bioactive hydrogels based on naturally-derived polymers are of great interest for regenerative medicine applications. Among naturally-derived polymers, silk fibroin has been extensively explored as a biomaterial for tissue engineering due to its unique mechanical properties. Here, we demonstrate the rapid gelation of cell-laden silk fibroin hydrogels by visible light-induced crosslinking using riboflavin as a photo-initiator, in presence of an electron acceptor. The gelation kinetics were monitored by in situ photo-rheometry. Gelation was achieved in minutes and could be tuned owing to its direct proportionality to the electron acceptor concentration. The concentration of the electron acceptor did not affect the elastic modulus of the hydrogels, which could be altered by varying the polymer content. Further, the biocompatible riboflavin photo-initiator combined with sodium persulfate allowed for the encapsulation of cells within silk fibroin hydrogels. To confirm the cytocompatibility of the silk fibroin formulations, three cell types (articular cartilage-derived progenitor cells, mesenchymal stem cells and dental-pulp-derived stem cells) were encapsulated within the hydrogels, which associated with a viability >80% for all cell types. These results demonstrated that fast gelation of silk fibroin can be achieved by combining it with riboflavin and electron acceptors, which results in a hydrogel that can be used in tissue engineering and cell delivery applications.

Cytocompatible carbon nanotube reinforced polyethylene glycol composite hydrogels for tissue engineering.

Hydrogels are attractive materials for stimulating 3D cell growth and tissue regeneration, and they provide mechanical support and physical cues to guide cell behavior. Herein, we developed a robust methodology to increase the stiffness of polyethylene glycol (PEG) hydrogels by successfully incorporating carbon nanotubes (CNTs) within the polymer matrix. Interestingly, hydrogels containing pristine CNTs showed a higher stiffness (1915 ±â€¯102 Pa) than both hydrogels without CNTs (1197 ±â€¯125 Pa) and hydrogels incorporating PEG-grafted CNTs (867 ±â€¯103 Pa) (p < 0.005). The swelling ratio was lower for hydrogels with pristine CNTs (45.4 ±â€¯3.5) and hydrogels without CNTs (46.7 ±â€¯5.1) compared to the hydrogels with PEG-grafted CNTs (62.8 ±â€¯2.6). To confirm that the CNT-reinforced hydrogels were cytocompatible, the viability, proliferation, and morphology of encapsulated L929 fibroblasts was investigated. All hydrogel formulations supported cell proliferation, and the addition of pristine CNTs increased initial cell viability (83.3 ±â€¯10.7%) compared to both pure PEG hydrogels (51.9 ±â€¯8.3%) and hydrogels with PEG-CNTs (63.1 ±â€¯10.9%) (p < 0.005). Altogether, these results demonstrate that incorporation of CNTs could effectively reinforce PEG hydrogels and that the resulting cytocompatible nanocomposites are promising scaffolds for tissue engineering.

Mimicking the Articular Joint with In Vitro Models.

Treating joint diseases remains a significant clinical challenge. Conventional in vitro cultures and animal models have been helpful, but suffer from limited predictive power for the human response. Advanced models are therefore required to mimic the complex biological interactions within the human joint. However, the intricate structure of the joint microenvironment and the complex nature of joint diseases have challenged the development of in vitro models that can faithfully mimic the in vivo physiological and pathological environments. In this review, we discuss the current in vitro models of the joint and the progress achieved in the development of novel and potentially more predictive models, and highlight the application of new technologies to accurately emulate the articular joint.

Molecularly Engineered Polymer-Based Systems in Drug Delivery and Regenerative Medicine.

BACKGROUND: Polymer-based systems are attractive in drug delivery and regenerative medicine due to the possibility of tailoring their properties and functions to a specific application. METHODS: The present review provides several examples of molecularly engineered polymer systems, including stimuli responsive polymers and supramolecular polymers. RESULTS: The advent of controlled polymerization techniques has enabled the preparation of polymers with controlled molecular weight and well-defined architecture. By using these techniques coupled to orthogonal chemical modification reactions, polymers can be molecularly engineered to incorporate functional groups able to respond to small changes in the local environment or to a specific biological signal. This review highlights the properties and applications of stimuli-responsive systems and polymer therapeutics, such as polymer-drug conjugates, polymer-protein conjugates, polymersomes, and hyperbranched systems. The applications of polymeric membranes in regenerative medicine are also discussed. CONCLUSION: The examples presented in this review suggest that the combination of membranes with polymers that are molecularly engineered to respond to specific biological functions could be relevant in the field of regenerative medicine.

Hyaluronic acid-based hydrogels crosslinked by copper-catalyzed azide-alkyne cycloaddition with tailorable mechanical properties.

Biopolymers of the extracellular matrix are attractive starting materials for providing degradable and biocompatible biomaterials. In this study, hyaluronic acid-based hydrogels with tunable mechanical properties were prepared by the use of copper- catalyzed azide-alkyne cycloaddition (known as "click chemistry"). Alkyne-functionalized hyaluronic acid was crosslinked with linkers having two terminal azide functionalities, varying crosslinker density as well as the lengths and rigidity of the linker molecules. By variation of the crosslinker density and crosslinker type, hydrogels with elastic moduli in the range of 0.5-4 kPa were prepared. The washed materials contained a maximum of 6.8 mg copper per kg dry weight and the eluate of the gel crosslinked with diazidostilbene did not show toxic effects on L929 cells. The hyaluronic acid-based hydrogels have potential as biomaterials for cell culture or soft tissue regeneration applications.