Biofabricated Constructs Research Articles

Hybrid scaffolds for bone tissue engineering: Integration of composites and bioactive hydrogels loaded with hDPSCs

Bone tissue regeneration remains a significant challenge in clinical settings due to the complexity of replicating the mechanical and biological properties of bone environment. This study addresses this challenge by proposing a hybrid scaffold designed to enhance both bioactivity and physical stability for bone tissue regeneration. This research is the fisrt to develop a rigid 3D structure composed of polycaprolactone (PCL) and hydroxyapatite nanoparticles (nHA) integrated with a bioink containing human dental pulp stem/stromal cells (hDPSCs), alginate, nHA and collagen (Col). The biofabricated constructs were extensively characterized through cytocompatibility tests, osteogenic differentiation assessment, and biocompatibility evaluation in a rat model. In vitro results demontrated that the hybrid scaffolds presented significantly higher cell viability after 168 h compared to the control group. Furthermore, the hybrid scaffolds showed increased osteogenic differentiation relative to other groups. In vivo evaluation indicated good biocompatibility, characterized by minimal inflammatory response and successful tissue integration. These findings highlight the scaffold's potential to support bone tissue regeneration by combining the mechanical strength of PCL and nHA with the biological activity of the alginate-nHA-Col and hDPSCs bioink. The current study provides a promising foundation for the development of biomaterials aimed at improving clinical outcomes in bone repair and regeneration, particulary for the treatment of critical-size bone defects, targeted drug administration, and three-dimensional models for bone tissue engineering.

Bioprinted keratinocyte and stem cell laden constructs for skin tissue engineering

Treating large-scale skin wounds continues to be a significant therapeutic challenge. Oftentimes, insufficient autologous material is available to achieve full coverage. Since biofabrication offers the chance of reproducible and precise large-scale production, the aim was to develop a customized 3D bioprinted skin construct containing immortalized HaCaT keratinocytes and ADSC to test the principal feasibility of this approach. Keratinocytes in different hydrogels containing varying concentrations of alginate, fibrin, collagen, gelatin, gelatin methacryloyl (GelMA) and hyaluronic acid as well as on a prefabricated collagen-elastin-matrix were cultured for a period of seven days. The metabolic activity of cultured keratinocytes was then evaluated during the co-cultivation of HaCaT and ADSC in a transwell model. The metabolic activity in all groups increased over the test period. The hydrogels containing alginate/hyaluronic acid/gelatin (Alg/HA/Gel) and GelMA demonstrated a good printability and the highest diffusion rates. There was no significant difference in pore size when comparing all hydrogels with each other. On the basis of the performed printability and diffusion assays as well as on the SEM analysis and rheological measurements Alg/HA/Gel and GelMA were chosen as hydrogels for the bioprinted 3D model. Fibrin hydrogel was integrated into the biofabricated constructs due to its excellent metabolic activity in the transwell model. As a second step, hydrogel stability, cell survival and metabolic activity in bioprinted 3D models containing keratinocytes and ADSC were evaluated over a period of 14 days. On day 14 the metabolic activity as well as the live cell count within the bioprinted constructs of the cocultured groups was significantly higher compared with day 1. The biofabricated constructs made of GelMA showed a higher cell viability compared with those made of Alg/HA/Gel. Additionally, to evaluate the cell migration out of the constructs, the metabolic activity and viability of the cells on the well bottom, was examined. After 14 days, an average of 50% of the well bottom was covered by HaCaT, which were initially printed in coculture into the constructs. These findings indicate that constructs made of GelMA containing keratinocytes and ADSC may offer a promising therapeutic option in the treatment of large chronic wounds.

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

3D bioprinting of tyramine modified hydrogels under visible light for osteochondral interface

Recent advancements in tissue engineering have demonstrated a great potential for the fabrication of three-dimensional (3D) tissue structures such as cartilage and bone. However, achieving structural integrity between different tissues and fabricating tissue interfaces are still great challenges. In this study, an in situ crosslinked hybrid, multi-material 3D bioprinting approach was used for the fabrication of hydrogel structures based on an aspiration-extrusion microcapillary method. Different cell-laden hydrogels were aspirated in the same microcapillary glass and deposited in the desired geometrical and volumetric arrangement directly from a computer model. Alginate and carboxymethyl cellulose were modified with tyramine to enhance cell bioactivity and mechanical properties of human bone marrow mesenchymal stem cells-laden bioinks. Hydrogels were prepared for extrusion by gelling in microcapillary glass utilizing an in situ crosslink approach with ruthenium (Ru) and sodium persulfate photo-initiating mechanisms under visible light. The developed bioinks were then bioprinted in precise gradient composition for cartilage-bone tissue interface using microcapillary bioprinting technique. The biofabricated constructs were co-cultured in chondrogenic/osteogenic culture media for three weeks. After cell viability and morphology evaluations of the bioprinted structures, biochemical and histological analyses, and a gene expression analysis for the bioprinted structure were carried out. Analysis of cartilage and bone formation based on cell alignment and histological evaluation indicated that mechanical cues in conjunction with chemical cues successfully induced MSC differentiation into chondrogenic and osteogenic tissues with a controlled interface.

A computational model of cell viability and proliferation of extrusion-based 3D-bioprinted constructs during tissue maturation process.

3D bioprinting is a novel promising solution for living tissue fabrication, with several potential advantages in many different applicative sectors. However, the implementation of complex vascular networks remains as one of the limiting factors for the production of complex tissues and for bioprinting scale-up. In this work, a physics-based computational model is presented to describe nutrients diffusion and consumption phenomena in bioprinted constructs. The model-a system of partial differential equations that is approximated by means of the finite element method- allows for the description of cell viability and proliferation, and it can be easily adapted to different cell types, densities, biomaterials, and 3D-printed geometries, thus allowing a preassessment of cell viability within the bioprinted construct. The experimental validation is performed on bioprinted specimens to assess the ability of the model to predict changes in cell viability. The proposed model constitutes a proof of concept of digital twinning of biofabricated constructs that can be suitably included in the basic toolkit for tissue bioprinting.

Generation and integration of hair follicle-primed spheroids in bioengineered skin constructs

Skin is a complex organ made up of different cell layers, appendages, connective tissues, and immune repertoires. These different components interact extensively to maintain the overall functions of the integumentary system. In particular, appendages such as hair follicles critically contribute to the skin’s function in thermoregulation, sensory perception, and homeostatic regeneration. Despite a strong need for better skin regenerative therapeutics, efforts to bio-engineer highly functional appendage-containing human reconstituted skin in vitro have not yielded much success. Here, we report methods in generating and incorporating hair follicle-primed heterotypic spheroids into epidermal-dermal skin constructs that induced invaginating outgrowths with follicle-like organization and lineage gene expression. By co-culturing epithelial keratinocytes (KCs) with dermal papilla (DP) cells in low attachment plates, we established the media and culture conditions that best supported the viability, signalling and remodelling of the cell aggregates to form 3D KC-DP spheroids with the expression of both DP inductiveness and hair follicle lineage genes. We show that long-term growth and maturation of KC cells in these spheroids was supported by incorporation into epidermal-dermal constructs but not in scaffold-less media. When cultured, the bio-fabricated constructs developed invaginations from the integrated spheroids with follicle-forming potential. The generation of these constructs is a step towards the development of functional hair-bearing skin mimetics.

Investigating Autoregressive and Machine Learning-based Time Series Modeling with Dielectric Spectroscopy for Predicting Quality of Biofabricated Constructs

Advances in biofabrication processes need to be complemented with appropriate nondestructive quality engineering techniques that can be integrated into scalable engineered tissue manufacturing systems. Previous studies have demonstrated the feasibility of dielectric spectroscopy (DS) as a inline, real time biological quality monitoring alternative. Time series modeling can help improve the efficiency and accuracy of quality prediction by analyzing trends in DS data as the biofabricated constructs mature over time. These models can help forecast potential future deviations in quality attributes and provide opportunities to take preemptive, corrective actions, leading to better yields and higher quality of final products. In this study, we investigated time series modeling of DS data to characterize the effects of two critical biofabrication parameters on constructs of gelatin methacryloyl (GelMA) hydrogel containing human adipose-derived stem cells (hASC) over 11 days of in vitro culture. The performance of standard autoregressive time series models (Exponential Smoothing, ARMA, ARIMA, SARIMA) and conventional sequence-based machine learning (ML) models (SVM, ANN, CNN and LSTM) were analyzed to forecast trends in Δɛ, a key DS metric that directly correlates to the volume of viable cells in constructs. The ML-based time series models, in general, showed superior performance in predicting future trends in Δɛ, with LSTM providing the lowest least mean square errors (MSE) in Δɛ forecasts. The outcomes of this study highlight the benefits of using DS and time series modeling synergistically for efficient quality monitoring in biofabrication.

Development and Characterization of Gelatin‐Norbornene Bioink to Understand the Interplay between Physical Architecture and Micro‐Capillary Formation in Biofabricated Vascularized Constructs (Adv. Healthcare Mater. 2/2022)

3D Bioprinting The crosstalk between cells during microcapillary formation is highlighted in article 2101873 by Khoon S. Lim and co-workers. Mesenchymal stromal cells (red) building bridges to allow endothelial cells (green) to cross spatial gaps in biofabricated constructs for rapid vascularisation. Providing the right microenvironment is key to support cell-driven microcapillary formation.

Hierarchical biomaterials via photopatterning-enhanced direct ink writing

Recent advances in additive manufacturing (AM) technologies provide tools to fabricate biological structures with complex three-dimensional (3D) organization. Deposition-based approaches have been exploited to manufacture multimaterial constructs. Stimulus-triggered approaches have been used to fabricate scaffolds with high resolution. Both features are useful to produce biomaterials that mimic the hierarchical organization of human tissues. Recently, multitechnology biofabrication approaches have been introduced that integrate benefits from different AM techniques to enable more complex materials design. However, few methods allow for tunable properties at both micro- and macro-scale in materials that are conducive for cell growth. To improve the organization of biofabricated constructs, we integrated direct ink writing (DIW) with digital light processing (DLP) to form multimaterial constructs with improved spatial control over final scaffold mechanics. Polymer–nanoparticle hydrogels were combined with methacryloyl gelatin (GelMA) to engineer dual inks that were compatible with both DIW and DLP. The shear-thinning and self-healing properties of the dual inks enabled extrusion-based 3D printing. The inclusion of GelMA provided a handle for spatiotemporal control of cross-linking with DLP. Exploiting this technique, complex multimaterial constructs were printed with defined mechanical reinforcement. In addition, the multitechnology approach was used to print live cells for biofabrication applications. Overall, the combination of DIW and DLP is a simple and efficient strategy to fabricate hierarchical biomaterials with user-defined control over material properties at both micro- and macro-scale.

Controlling cellular organization in bioprinting through designed 3D microcompartmentalization.

Controlling cellular organization is crucial in the biofabrication of tissue-engineered scaffolds, as it affects cell behavior as well as the functionality of mature tissue. Thus far, incorporation of physiochemical cues with cell-size resolution in three-dimensional (3D) scaffolds has proven to be a challenging strategy to direct the desired cellular organization. In this work, a rapid, simple, and cost-effective approach is developed for continuous printing of multicompartmental hydrogel fibers with intrinsic 3D microfilaments to control cellular orientation. A static mixer integrated into a coaxial microfluidic device is utilized to print alginate/gelatin-methacryloyl (GelMA) hydrogel fibers with patterned internal microtopographies. In the engineered microstructure, GelMA compartments provide a cell-favorable environment, while alginate compartments offer morphological and mechanical cues that direct the cellular orientation. It is demonstrated that the organization of the microtopographies, and consequently the cellular alignment, can be tailored by controlling flow parameters in the printing process. Despite the large diameter of the fibers, the precisely tuned internal microtopographies induce excellent cell spreading and alignment, which facilitate rapid cell proliferation and differentiation toward mature biofabricated constructs. This strategy can advance the engineering of functional tissues.

Suturable elastomeric tubular grafts with patterned porosity for rapid vascularization of 3D constructs

Vascularization is considered to be one of the key challenges in engineering functional 3D tissues. Engineering suturable vascular grafts containing pores with diameter of several tens of microns in tissue engineered constructs may provide an instantaneous blood perfusion through the grafts improving cell infiltration and thus, allowing rapid vascularization and vascular branching. The aim of this work was to develop suturable tubular scaffolds to be integrated in biofabricated constructs, enabling the direct connection of the biofabricated construct with the host blood stream, providing an immediate blood flow inside the construct. Here, tubular grafts with customizable shapes (tubes, Y-shape capillaries) and controlled diameter ranging from several hundreds of microns to few mm are fabricated based on poly(glycerol sebacate) (PGS)/poly(vinyl alcohol) (PVA) electrospun scaffolds. Furthermore, a network of pore channels of diameter in the order of 100 μm was machined by laser femtosecond ablation in the tube wall. Both non-machined and laser machined tubular scaffolds elongated more than 100% of their original size have shown suture retention, being 5.85 and 3.96 N mm−2 respectively. To demonstrate the potential of application, the laser machined porous grafts were embedded in gelatin methacryloyl (GelMA) hydrogels, resulting in elastomeric porous tubular graft/GelMA 3D constructs. These constructs were then co-seeded with osteoblast-like cells (MG-63) at the external side of the graft and human umbilical vein endothelial cells inside, forming a bone osteon model. The laser machined pore network allowed an immediate endothelial cell flow towards the osteoblasts enabling the osteoblasts and endothelial cells to interact and form 3D structures. This rapid vascularization approach could be applied, not only for bone tissue regeneration, but also for a variety of tissues and organs.

An advanced optical clearing protocol allows label-free detection of tissue necrosis via multiphoton microscopy in injured whole muscle.

Rationale: Structural remodeling or damage as a result of disease or injury is often not evenly distributed throughout a tissue but strongly depends on localization and extent of damaging stimuli. Skeletal muscle as a mechanically active organ can express signs of local or even systemic myopathic damage, necrosis, or repair. Conventionally, muscle biopsies (patients) or whole muscles (animal models) are mechanically sliced and stained to assess structural alterations histologically. Three-dimensional tissue information can be obtained by applying deep imaging modalities, e.g. multiphoton or light-sheet microscopy. Chemical clearing approaches reduce scattering, e.g. through matching refractive tissue indices, to overcome optical penetration depth limits in thick tissues.Methods: Here, we optimized a range of different clearing protocols. We find aqueous solution-based protocols employing (20-80%) 2,2'-thiodiethanol (TDE) to be advantageous over organic solvents (dibenzyl ether, cinnamate) regarding the preservation of muscle morphology, ease-of-use, hazard level, and costs.Results: Applying TDE clearing to a mouse model of local cardiotoxin (CTX)-induced muscle necrosis, a complete loss of myosin-II signals was observed in necrotic areas with little change in fibrous collagen or autofluorescence (AF) signals. The 3D aspect of myofiber integrity could be assessed, and muscle necrosis in whole muscle was quantified locally via the ratios of detected AF, forward- and backward-scattered Second Harmonic Generation (fSHG, bSHG) signals.Conclusion: TDE optical clearing is a versatile tool to study muscle architecture in conjunction with label-free multiphoton imaging in 3D in injury/myopathy models and might also be useful in studying larger biofabricated constructs in regenerative medicine.

Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment.

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be of a specific diameter (~500 μm), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNFα stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

Bioprinting: From Tissue and Organ Development to in Vitro Models.

Bioprinting techniques have been flourishing in the field of biofabrication with pronounced and exponential developments in the past years. Novel biomaterial inks used for the formation of bioinks have been developed, allowing the manufacturing of in vitro models and implants tested preclinically with a certain degree of success. Furthermore, incredible advances in cell biology, namely, in pluripotent stem cells, have also contributed to the latest milestones where more relevant tissues or organ-like constructs with a certain degree of functionality can already be obtained. These incredible strides have been possible with a multitude of multidisciplinary teams around the world, working to make bioprinted tissues and organs more relevant and functional. Yet, there is still a long way to go until these biofabricated constructs will be able to reach the clinics. In this review, we summarize the main bioprinting activities linking them to tissue and organ development and physiology. Most bioprinting approaches focus on mimicking fully matured tissues. Future bioprinting strategies might pursue earlier developmental stages of tissues and organs. The continuous convergence of the experts in the fields of material sciences, cell biology, engineering, and many other disciplines will gradually allow us to overcome the barriers identified on the demanding path toward manufacturing and adoption of tissue and organ replacements.

Plant seed-inspired cell protection, dormancy, and growth for large-scale biofabrication

Biofabrication technologies have endowed us with the capability to fabricate complex biological constructs. However, cytotoxic biofabrication conditions have been a major challenge for their clinical application, leading to a trade-off between cell viability and scalability of biofabricated constructs. Taking inspiration from nature, we proposed a cell protection strategy which mimicks the protected and dormant state of plant seeds in adverse external conditions and their germination in response to appropriate environmental cues. Applying this bioinspired strategy to biofabrication, we successfully preserved cell viability and enhanced the seeding of cell-laden biofabricated constructs via a cytoprotective pyrogallol (PG)-alginate encapsulation system. Our cytoprotective encapsulation technology utilizes PG-triggered sporulation and germination processes to preserve cells, is mechanically robust, chemically resistant, and highly customizable to adequately match cell protectability with cytotoxicity of biofabrication conditions. More importantly, the facile and tunable decapsulation of our PG-alginate system allows for effective germination of dormant cells, under typical culture conditions. With this approach, we have successfully achieved a biofabrication process which is reproducible, scalable, and provided a practical solution for off-the-shelf availability, shipping and temporary storage of fabricated bio-constructs.

Control of Nanoparticle Release Kinetics from 3D Printed Hydrogel Scaffolds.

The convergence of biofabrication with nanotechnology is largely unexplored but enables geometrical control of cell‐biomaterial arrangement combined with controlled drug delivery and release. As a step towards integration of these two fields of research, this study demonstrates that modulation of electrostatic nanoparticle–polymer and nanoparticle–nanoparticle interactions can be used for tuning nanoparticle release kinetics from 3D printed hydrogel scaffolds. This generic strategy can be used for spatiotemporal control of the release kinetics of nanoparticulate drug vectors in biofabricated constructs.

Modified amphiphilic copolymer based hydrogels as reinforcement for 3D biofabrication

Modified amphiphilic copolymer based hydrogels as reinforcement for 3D biofabrication

Biofabricated constructs as tissue models: a short review.

Biofabrication is currently able to provide reliable models for studying the development of cells and tissues into multiple environments. As the complexity of biofabricated constructs is becoming increasingly higher their ability to closely mimic native tissues and organs is also increasing. Various biofabrication technologies currently allow to precisely build cell/tissue constructs at multiple dimension ranges with great accuracy. Such technologies are also able to assemble together multiple types of cells and/or materials and generate constructs closely mimicking various types of tissues. Furthermore, the high degree of automation involved in these technologies enables the study of large arrays of testing conditions within increasingly smaller and automated devices both in vitro and in vivo. Despite not yet being able to generate constructs similar to complex tissues and organs, biofabrication is rapidly evolving in that direction. One major hurdle to be overcome in order for such level of complex detail to be achieved is the ability to generate complex vascular structures within biofabricated constructs. This review describes several of the most relevant technologies and methodologies currently utilized within biofabrication and provides as well a brief overview of their current and future potential applications.

Biofabrication of multi-material anatomically shaped tissue constructs

Additive manufacturing in the field of regenerative medicine aims to fabricate organized tissue-equivalents. However, the control over shape and composition of biofabricated constructs is still a challenge and needs to be improved. The current research aims to improve shape, by converging a number of biocompatible, quality construction materials into a single three-dimensional fiber deposition process. To demonstrate this, several models of complex anatomically shaped constructs were fabricated by combined deposition of poly(vinyl alcohol), poly(ε-caprolactone), gelatin methacrylamide/gellan gum and alginate hydrogel. Sacrificial components were co-deposited as temporary support for overhang geometries and were removed after fabrication by immersion in aqueous solutions. Embedding of chondrocytes in the gelatin methacrylamide/gellan component demonstrated that the fabrication and the sacrificing procedure did not affect cell viability. Further, it was shown that anatomically shaped constructs can be successfully fabricated, yielding advanced porous thermoplastic polymer scaffolds, layered porous hydrogel constructs, as well as reinforced cell-laden hydrogel structures. In conclusion, anatomically shaped tissue constructs of clinically relevant sizes can be generated when employing multiple building and sacrificial materials in a single biofabrication session. The current techniques offer improved control over both internal and external construct architecture underscoring its potential to generate customized implants for human tissue regeneration.