Cell Migration Chip (3DCM-chip): a New Tool Toward the Modelling of 3D Cellular Complex Systems.

3D hydrogel-based cell cultures provide models for studying cell behavior and can efficiently replicate the physiologic environment. Hydrogels can be tailored to mimic mechanical and biochemical properties of specific tissues and allow to produce gel-in-gel models. In this system, microspheres encapsulating cells are embedded in an outer hydrogel matrix, where cells are able to migrate. To enhance the efficiency of such studies, we design a lab-on-a-chip named 3D Cell Migration-chip (3DCM-chip) that offers substantial advantages over traditional methods. 3DCM-chip facilitates the analysis of biochemical and physical stimuli effects on cell migration/invasion in different cell types, including stem, normal and tumor cells. 3DCM-chip provides a smart platform for developing more complex cell co-cultures systems. Herein we investigate the impact of human fibroblasts on MDA-MB 231 breast cancer cells invasiveness. Moreover, how the presence of different cellular lines, including mesenchymal stem cells (hMSCs), normal human dermal fibroblasts (NHDFs), and human umbilical vein endothelial cells (HUVECs), affects the invasive behavior of cancer cells is investigated using 3DCM-chip and producing predictive tumoroid models with a more complex network of interactions between cells and microenvironment. 3DCM-chip moves us closer to creating in vitro systems that can potentially replicate key aspects of the physiological tumor microenvironment. This article is protected by copyright. All rights reserved.

Modelling the disease: H2S-sensitivity and drug-resistance of triple negative breast cancer cells can be modulated by embedding in isotropic micro-environment.

Three-dimensional (3D) cell culture systems provide more physiologically relevant information, representing more accurately the actual microenvironment where cells reside in tissues. However, the differences between the tissue culture plate (TCP) and 3D culture systems in terms of tumour cell growth, proliferation, migration, differentiation and response to the treatment have not been fully elucidated. Tumoroid microspheres containing the MDA-MB 231 breast cancer cell line were prepared using either tunable PEG-fibrinogen (PFs) or tunable PEG-silk fibroin (PSFs) hydrogels, respectively named MDAPFs and MDAPSFs. The cancer cells in the tumoroids showed changes both in globular morphology and at the protein expression level. A decrease of both Histone H3 acetylation and cyclin D1 expression in all 3D systems, compared to the 2D cell culture, was detected in parallel to changes of the matrix stiffness. The effects of a glutathionylated garlic extract (GSGa), a slow H2S-releasing donor, were investigated on both tumoroid systems. A pro-apoptotic effect of GSGa on tumour cell growth in 2D culture was observed as opposed to a pro-proliferative effect apparent in both MDAPFs and MDAPSFs. A dedicated ad hoc 3D cell migration chip was designed and optimized for studying tumour cell invasion in a gel-in-gel configuration. An anti-cell-invasion effect of the GSGa was observed in the 2D cell culture, whereas a pro-migratory effect in both MDAPFs and MDAPSFs was observed in the 3D cell migration chip assay. An increase of cyclin D1 expression after GSGa treatment was observed in agreement with an increase of the cell invasion index. Our results suggest that the "dimensionality" and the stiffness of the 3D cell culture milieu can change the response to both the gasotransmitter H2S and doxorubicin due to differences in both H2S diffusion and changes in protein expression. Moreover, we uncovered a direct relation between the cyclin D1 expression and the stiffness of the 3D cell culture milieu, suggesting the potential causal involvement of the cyclin D1 as a bio-marker for sensitivity of the tumour cells to their matrix stiffness. Therefore, our hydrogel-based tumoroids represent a valid tunable model for studying the physically induced transdifferentiation (PiT) of cancer cells and as a more reliable and predictive in vitro screening platform to investigate the effects of anti-tumour drugs.

Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behaviors in rats.

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing human nucleus pulposus cells (NPCs), generated by certain conditions in degenerating discs, that was associated with the onset of discogenic back pain. Single-cell transcriptomic analysis of human tissues showed a strong correlation between a specific cell subtype and the pain condition associated with the human degenerated disc, suggesting that they are pain-triggering. The application of IVD degeneration-associated exogenous stimuli to healthy NPCs in vitro recreated a pain-associated phenotype. These stimulated NPCs activated functional human iPSC-derived sensory neuron responses in an in vitro organ-chip model. Injection of stimulated NPCs into the healthy rat IVD induced local inflammatory responses and increased cold sensitivity and mechanical hypersensitivity. Our findings reveal a previously uncharacterized pain-inducing mechanism mediated by NPCs in degenerating IVDs. These findings could aid in the development of NPC-targeted therapeutic strategies for the clinically unmet need to attenuate discogenic LBP.

A 3D adipogenesis platform to study the fate of fibro/adipogenic progenitors in muscular dystrophies.

In human dystrophies, progressive muscle wasting is exacerbated by ectopic deposition of fat and fibrous tissue originating from fibro/adipogenic progenitors (FAPs). In degenerating muscles, the ability of these cells to promote successful healing is attenuated, and FAPs aberrantly expand and differentiate into adipocytes and fibroblasts. Thus, arresting the fibro/adipogenic fate of FAPs, without affecting their physiological role, represents a valuable therapeutic strategy for patients affected by muscle diseases. Here, using a panel of adipose progenitor cells, including human-derived FAPs, coupled with pharmacological perturbations and proteome profiling, we report that LY2090314 interferes with a genuine adipogenic program acting as WNT surrogate for the stabilization of a competent ß-catenin transcriptional complex. To predict the beneficial impact of LY2090314 in limiting ectopic deposition of fat in human muscles, we combined a poly-ethylene-glycol-fibrinogen biomimetic matrix with these progenitor cells to create a miniaturized 3D model of adipogenesis. Using this scalable system, we demonstrated that a two-digit nanomolar dose of this compound effectively represses adipogenesis at higher 3D scale, thus indicating the potential for LY2090314 to limit FAP-derived fat infiltrates in dystrophic muscles.

Modulus-dependent effects on neurogenic, myogenic, and chondrogenic differentiation of human mesenchymal stem cells in three-dimensional hydrogel cultures.

Human mesenchymal stromal cells (hMSCs) are of significant interest as a renewable source of therapeutically useful cells. In tissue engineering, hMSCs are implanted within a scaffold to provide enhanced capacity for tissue repair. The present study evaluates how mechanical properties of that scaffold can alter the phenotype and genotype of the cells, with the aim of augmenting hMSC differentiation along the myogenic, neurogenic or chondrogenic linages. The hMSCs were grown three-dimensionally (3D) in a hydrogel comprised of poly(ethylene glycol) (PEG)-conjugated to fibrinogen. The hydrogel's shear storage modulus (G'), which was controlled by increasing the amount of PEG-diacrylate cross-linker in the matrix, was varied in the range of 100-2000 Pascal (Pa). The differentiation into each lineage was initiated by a defined culture medium, and the hMSCs grown in the different modulus hydrogels were characterized using gene and protein expression. Materials having lower storage moduli (G' = 100 Pa) exhibited more hMSCs differentiating to neurogenic lineages. Myogenesis was favored in materials having intermediate modulus values (G' = 500 Pa), whereas chondrogenesis was favored in materials with a higher modulus (G' = 1000 Pa). Enhancing the differentiation pathway of hMSCs in 3D hydrogel scaffolds using simple modifications to mechanical properties represents an important achievement toward the effective application of these cells in tissue engineering.

Methacrylated fibrinogen hydrogels for 3D cell culture and delivery.

Methacrylation was performed on fibrinogen to design a new biomedical hydrogel for 3D cell culture or as a biodegradable delivery matrix for in vivo implantation. The methacrylation of denatured fibrinogen in solution was performed using methacrylic anhydride (MAA). The extent of fibrinogen methacrylation was quantified by proton NMR and controlled using stochiometric quantities of MAA during the reaction. The methacrylated fibrinogen (FibMA) hydrogels were formed by light-activated free-radical polymerization in the presence of macromolecular cross-linking polymers made from acrylated poly(ethylene glycol) (PEG). The biocompatibility and biodegradability of the FibMA hydrogels were characterized by in vitro assays and in vivo implantation experiments using quantitative magnetic resonance imaging (MRI) of the implant volume. The FibMA supported the growth and metabolic activity of human dermal fibroblasts in both 2D and 3D cultures. The methacrylation did not alter important biological attributes of the fibrinogen, including the ability to support cell adhesion and 3D cell culture, as well as to undergo proteolysis. Animal experiments confirmed the biodegradability of the FibMA for potential use as a scaffold in tissue engineering, as a bioink for 3D bioprinting, or as a biodegradable matrix for in vivo sustained delivery of bioactive factors. STATEMENT OF SIGNIFICANCE: This paper describes methacrylated fibrinogen (FibMA) and the formation of a biomedical hydrogel from FibMA for cell culture and other biomedical applications. Inspired from methacrylated gelatin (GelMA), the FibMA is made from blood-derived fibrinogen which is more suitable for clinical use. Sharing similar properties to other hydrogels made from methacrylated proteins, the FibMA has yet to be reported in the literature. In this manuscript, we provide the methodology to produce the FibMA hydrogels, we document the mechanical versatility of this new biomaterial, and we show the biocompatibility using 3D cell culture studies and in vivo implantations.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

In vivo restoration of dystrophin expression in mdx mice using intra-muscular and intra-arterial injections of hydrogel microsphere carriers of exon skipping antisense oligonucleotides.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the X-linked Dytrophin gene preventing the expression of the functional protein. Exon skipping therapy using antisense oligonucleotides (AONs) is a promising therapeutic strategy for DMD. While benefits of AON therapy have been demonstrated, some challenges remain before this strategy can be applied more comprehensively to DMD patients. These include instability of AONs due to low nuclease resistance and poor tissue uptake. Delivery systems have been examined to improve the availability and stability of oligonucleotide drugs, including polymeric carriers. Previously, we showed the potential of a hydrogel-based polymeric carrier in the form of injectable PEG-fibrinogen (PF) microspheres for delivery of chemically modified 2'-O-methyl phosphorothioate (2OMePs) AONs. The PF microspheres proved to be cytocompatible and provided sustained release of the AONs for several weeks, causing increased cellular uptake in mdx dystrophic mouse cells. Here, we further investigated this delivery strategy by examining in vivo efficacy of this approach. The 2OMePS/PEI polyplexes loaded in PF microspheres were delivered by intramuscular (IM) or intra-femoral (IF) injections. We examined the carrier biodegradation profiles, AON uptake efficiency, dystrophin restoration, and muscle histopathology. Both administration routes enhanced dystrophin restoration and improved the histopathology of the mdx mice muscles. The IF administration of the microspheres improved the efficacy of the 2OMePS AONs over the IM administration. This was demonstrated by a higher exon skipping percentage and a smaller percentage of centered nucleus fibers (CNF) found in H&E-stained muscles. The restoration of dystrophin expression found for both IM and IF treatments revealed a reduced dystrophic phenotype of the treated muscles. The study concludes that injectable PF microspheres can be used as a carrier system to improve the overall therapeutic outcomes of exon skipping-based therapy for treating DMD.

Effects of antibiotic treatment and phagocyte infiltration on development of Pseudomonas aeruginosa biofilm-Insights from the application of a novel PF hydrogel model in vitro and in vivo.

Background and purpose: Bacterial biofilm infections are major health issues as the infections are highly tolerant to antibiotics and host immune defenses. Appropriate biofilm models are important to develop and improve to make progress in future biofilm research. Here, we investigated the ability of PF hydrogel material to facilitate the development and study of Pseudomonas aeruginosa biofilms in vitro and in vivo. Methods: Wild-type P. aeruginosa PAO1 bacteria were embedded in PF hydrogel situated in vitro or in vivo, and the following aspects were investigated: 1) biofilm development; 2) host immune response and its effect on the bacteria; and 3) efficacy of antibiotic treatment. Results: Microscopy demonstrated that P. aeruginosa developed typical biofilms inside the PF hydrogels in vitro and in mouse peritoneal cavities where the PF hydrogels were infiltrated excessively by polymorphonuclear leukocytes (PMNs). The bacteria remained at a level of ~106 colony-forming unit (CFU)/hydrogel for 7 days, indicating that the PMNs could not eradicate the biofilm bacteria. ß-Lactam or aminoglycoside mono treatment at 64× minimal inhibitory concentration (MIC) killed all bacteria in day 0 in vitro biofilms, but not in day 1 and older biofilms, even at a concentration of 256× MIC. Combination treatment with the antibiotics at 256× MIC completely killed the bacteria in day 1 in vitro biofilms, and combination treatment in most of the cases showed significantly better bactericidal effects than monotherapies. However, in the case of the established in vivo biofilms, the mono and combination antibiotic treatments did not efficiently kill the bacteria. Conclusion: Our results indicate that the bacteria formed typical biofilms in PF hydrogel in vitro and in vivo and that the biofilm bacteria were tolerant against antibiotics and host immunity. The PF hydrogel biofilm model is simple and easy to fabricate and highly reproducible with various application possibilities. We conclude that the PF hydrogel biofilm model is a new platform that will facilitate progress in future biofilm investigations, as well as studies of the efficacy of new potential medicine against biofilm infections.

PEG-fibrinogen hydrogel microspheres as a scaffold for therapeutic delivery of immune cells.

Recent advances in the field of cell therapy have proposed new solutions for tissue repair and regeneration using various cell delivery approaches. Here we studied ex vivo a novel topical delivery system of encapsulated cells in hybrid polyethylene glycol-fibrinogen (PEG-Fb) hydrogel microspheres to respiratory tract models. We investigated basic parameters of cell encapsulation, delivery and release in conditions of inflamed and damaged lungs of bacterial-infected mice. The establishment of each step in the study was essential for the proof of concept. We demonstrated co-encapsulation of alveolar macrophages and epithelial cells that were highly viable and equally distributed inside the microspheres. We found that encapsulated macrophages exposed to bacterial endotoxin lipopolysaccharide preserved high viability and secreted moderate levels of TNFα, whereas non-encapsulated cells exhibited a burst TNFα secretion and reduced viability. LPS-exposed encapsulated macrophages exhibited elongated morphology and out-migration capability from microspheres. Microsphere degradation and cell release in inflamed lung environment was studied ex vivo by the incubation of encapsulated macrophages with lung extracts derived from intranasally infected mice with Yersinia pestis, demonstrating the potential in cell targeting and release in inflamed lungs. Finally, we demonstrated microsphere delivery to a multi-component airways-on-chip platform that mimic human nasal, bronchial and alveolar airways in serially connected compartments. This study demonstrates the feasibility in using hydrogel microspheres as an effective method for topical cell delivery to the lungs in the context of pulmonary damage and the need for tissue repair.

A combined cell and growth factor delivery for the repair of a critical size tibia defect using biodegradable hydrogel implants.

The ability to repair critical-sized long-bone injuries using growth factor and cell delivery was investigated using hydrogel biomaterials. Physiological doses of the recombinant human bone morphogenic protein-2 (rhBMP2) were delivered in a sustained manner from a biodegradable hydrogel containing peripheral human blood-derived endothelial progenitor cells (hEPCs). The biodegradable implants made from polyethylene glycol (PEG) and denatured fibrinogen (PEG-fibrinogen, PF) were loaded with 7.7 µg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs. The safety and efficacy of the implant were tested in a rodent model of a critical-size long-bone defect. The hydrogel implants were formed ex-situ and placed into defects in the tibia of athymic nude rats and analyzed for bone repair after 13 weeks following surgery. The hydrogels containing a combination of 7.7 µg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs were compared to control hydrogels containing 7.7 µg/ml of rhBMP2 only, 2.5 × 106 cells/ml hEPCs only, or bare hydrogels. Assessments of bone repair include histological analysis, bone formation at the site of implantation using quantitative microCT, and assessment of implant degradation. New bone formation was detected in all treated animals, with the highest amounts found in the treatments that included animals that combined the PF implant with rhBMP2. Moreover, statistically significant increases in the tissue mineral density (TMD), trabecular number and trabecular thickness were observed in defects treated with rhBMP2 compared to non-rhBMP2 defects. New bone formation was significantly higher in the hEPC-treated defects compared to bare hydrogel defects, but there were no significant differences in new bone formation, trabecular number, trabecular thickness or TMD at 13 weeks when comparing the rhBMP2 + hEPCs-treated defects to rhBMP2-treated defects. The study concludes that the bone regeneration using hydrogel implants containing hEPCs are overshadowed by enhanced osteogenesis associated with sustained delivery of rhBMP2.

3D Bioprinting of Engineered Tissue Flaps with Hierarchical Vessel Networks (VesselNet) for Direct Host-To-Implant Perfusion.

Engineering hierarchical vasculatures is critical for creating implantable functional thick tissues. Current approaches focus on fabricating mesoscale vessels for implantation or hierarchical microvascular in vitro models, but a combined approach is yet to be achieved to create engineered tissue flaps. Here, millimetric vessel-like scaffolds and 3D bioprinted vascularized tissues interconnect, creating fully engineered hierarchical vascular constructs for implantation. Endothelial and support cells spontaneously form microvascular networks in bioprinted tissues using a human collagen bioink. Sacrificial molds are used to create polymeric vessel-like scaffolds and endothelial cells seeded in their lumen form native-like endothelia. Assembling endothelialized scaffolds within vascularizing hydrogels incites the bioprinted vasculature and endothelium to cooperatively create vessels, enabling tissue perfusion through the scaffold lumen. Using a cuffing microsurgery approach, the engineered tissue is directly anastomosed with a rat femoral artery, promoting a rich host vasculature within the implanted tissue. After two weeks in vivo, contrast microcomputer tomography imaging and lectin perfusion of explanted engineered tissues verify the host ingrowth vasculature's functionality. Furthermore, the hierarchical vessel network (VesselNet) supports in vitro functionality of cardiomyocytes. Finally, the proposed approach is expanded to mimic complex structures with native-like millimetric vessels. This work presents a novel strategy aiming to create fully-engineered patient-specific thick tissue flaps.

Photo-Polymerization Damage Protection by Hydrogen Sulfide Donors for 3D-Cell Culture Systems Optimization.

Photo-polymerized hydrogels are ideally suited for stem-cell based tissue regeneration and three dimensional (3D) bioprinting because they can be highly biocompatible, injectable, easy to use, and their mechanical and physical properties can be controlled. However, photo-polymerization involves the use of potentially toxic photo-initiators, exposure to ultraviolet light radiation, formation of free radicals that trigger the cross-linking reaction, and other events whose effects on cells are not yet fully understood. The purpose of this study was to examine the effects of hydrogen sulfide (H2S) in mitigating cellular toxicity of photo-polymerization caused to resident cells during the process of hydrogel formation. H2S, which is the latest discovered member of the gasotransmitter family of gaseous signalling molecules, has a number of established beneficial properties, including cell protection from oxidative damage both directly (by acting as a scavenger molecule) and indirectly (by inducing the expression of anti-oxidant proteins in the cell). Cells were exposed to slow release H2S treatment using pre-conditioning with glutathione-conjugated-garlic extract in order to mitigate toxicity during the photo-polymerization process of hydrogel formation. The protective effects of the H2S treatment were evaluated in both an enzymatic model and a 3D cell culture system using cell viability as a quantitative indicator. The protective effect of H2S treatment of cells is a promising approach to enhance cell survival in tissue engineering applications requiring photo-polymerized hydrogel scaffolds.

Tumor Extracellular Matrix Stiffness Promptly Modulates the Phenotype and Gene Expression of Infiltrating T Lymphocytes.

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.

Alginate hydrogel beads embedded with drug-bearing polycaprolactone microspheres for sustained release of paclobutrazol.

In recent years there has been a growing demand for the development of agrochemical controlled release (CR) technologies. In the present study, we aimed to create a novel agricultural CR device using two polymeric systems that have been predominantly employed in biomedical applications: beads of alginate hydrogel embedded with drug-bearing Polycaprolactone (PCL) microspheres. The combined device utilizes the advantages of each polymer type for biodegradation and controlled release of Paclobutrazol (PBZ), a common growth retardant in plants. Surface morphology of the alginate beads was characterized by scanning electron microscopy (SEM) and water immersion tests were performed for stability and controlled release measurements. Bioassays were performed both in accelerated laboratory conditions and in field conditions. The results showed a capability to control the size of PBZ-loaded PCL microspheres through modification of homogenization speed and emulsifier concentration. Enlargement of PCL microsphere size had an adverse effect on release of PBZ from the alginate device. The growth of oatmeal plants as a model system was affected by the controlled release of PBZ. The preliminary field experiment observed growth retardation during two consecutive rainy seasons, with results indicating a substantial benefit of the sustained growth inhibition through the controlled release formulation. The final product has the potential to be used as a carrier for different substances in the agrochemical industry.

Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration.

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo-substitutes support the formation of new blood vessels and neuromuscular junctions-pivotal aspects for cell survival and muscle contractile functionalities-together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo-substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.

Photocurable Biopolymers for Coaxial Bioprinting.

Thanks to their unique advantages, additive manufacturing technologies are revolutionizing almost all sectors of the industrial and academic worlds, including tissue engineering and regenerative medicine. In particular, 3D bioprinting is rapidly emerging as a first-choice approach for the fabrication-in one step-of advanced cell-laden hydrogel constructs to be used for in vitro and in vivo studies. This technique consists in the precise deposition layer-by-layer of sub-millimetric hydrogel strands in which living cells are embedded. A key factor of this process consists in the proper formulation of the hydrogel precursor solution, the so-called bioink. Ideal bioinks should be able, on the one side, to support cell growth and differentiation and, on the other, to allow the high-resolution deposition of cell-laden hydrogel strands. The latter feature requires the extruded solution to instantaneously undergo a sol-gel transition to avoid its collapse after deposition.To address this challenge, researchers are recently focusing their attention on the synthesis of several derivatives of natural biopolymers to enhance their printability. Here, we present an approach for the synthesis of photocurable derivatives of natural biopolymers-namely, gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen-that can be used to formulate tailored innovative bioinks for coaxial-based 3D bioprinting applications.

Live reporting for hypoxia: Hypoxia sensor-modified mesenchymal stem cells as in vitro reporters.

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O2 ). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.

Growth Factor Delivery for the Repair of a Critical Size Tibia Defect Using an Acellular, Biodegradable Polyethylene Glycol-Albumin Hydrogel Implant.

Growth factor delivery using acellular matrices presents a promising alternative to current treatment options for bone repair in critical-size injuries. However, supra-physiological doses of the factors can introduce safety concerns that must be alleviated, mainly by sustaining delivery of smaller doses using the matrix as a depot. We developed an acellular, biodegradable hydrogel implant composed of poly(ethylene glycol) (PEG) and denatured albumin to be used for sustained delivery of bone morphogenic protein-2 (BMP2). In this study, poly(ethylene glycol)-albumin (PEG-Alb) hydrogels were produced and loaded with 7.7 µg/mL of recombinant human BMP2 (rhBMP2) to be tested for safety and performance in a critical-size long-bone defect, using a rodent model. The hydrogels were formed ex situ in a 5 mm long cylindrical mold of 3 mm diameter, implanted into defects made in the tibia of Sprague-Dawley rats and compared to non-rhBMP2 control hydrogels at 13 weeks following surgery. The hydrogels were also compared to the more established PEG-fibrinogen (PEG-Fib) hydrogels we have tested previously. Comprehensive in vitro characterization as well as in vivo assessments that include: histological analyses, including safety parameters (i.e., local tolerance and toxicity), assessment of implant degradation, bone formation, as well as repair tissue density using quantitative microCT analysis were performed. The in vitro assessments demonstrated similarities between the mechanical and release properties of the PEG-Alb hydrogels to those of the PEG-Fib hydrogels. Safety analysis presented good local tolerance in the bone defects and no signs of toxicity. A significantly larger amount of bone was detected at 13 weeks in the rhBMP2-treated defects as compared to non-rhBMP2 defects. However, no significant differences were noted in bone formation at 13 weeks when comparing the PEG-Alb-treated defects to PEG-Fib-treated defects (with or without BMP2). The study concludes that hydrogel scaffolds made from PEG-Alb containing 7.7 µg/mL of rhBMP2 are effective in accelerating the bridging of boney defects in the tibia.