Bioprinted Scaffolds Research Articles

3D Bioprinted Scaffolds for Tissue Repair and Regeneration

Three-dimensional (3D) printing technology has emerged as a revolutionary manufacturing strategy that could realize rapid prototyping and customization. It has revolutionized the manufacturing process in the fields of electronics, energy, bioengineering and sensing. Based on digital model files, powdered metal, plastic and other materials were used to construct the required objects by printing layer by layer. In addition, 3D printing possesses remarkable advantages in realizing controllable compositions and complex structures, which could further produce 3D objects with anisotropic functions. In recent years, 3D bioprinting technology has been applied to manufacture functional tissue engineering scaffolds with its ability to assemble complicated construction under precise control, which has attracted great attention. Bioprinting creates 3D scaffolds by depositing and assembling biological and/or non-biological materials with an established tissue. Compared with traditional technology, it can create a structure tailored to the patient according to the medical images. This conception of 3D bioprinting draws on 3D printing technology, which could be utilized to produce personalized implants, thereby opening up a new way for bio-manufacturing methods. As a promising tool, 3D bioprinting can create complex and delicate biomimetic 3D structures, simulating extracellular matrix and preparing high precision multifunctional scaffolds with uniform cell distribution for tissue repair and regeneration. It can also be flexibly combined with other technologies such as electrospinning and thermally induced phase separation, suitable for tissue repair and regeneration. This article reviews the relevant research and progress of 3D bioprinting in tissue repair and regeneration in recent years. Firstly, we will introduce the physical, chemical and biological characteristics of biological scaffolds prepared by 3D bioprinting from several aspects. Secondly, the significant effects of 3D bioprinting on nerves, skin, blood vessels, bones and cartilage injury and regeneration are further expounded. Finally, some views on the clinical challenges and future opportunities of 3D bioprinting are put forward.

Characterisation of 3D Bioprinted Human Breast Cancer Model for In Vitro Drug and Metabolic Targeting.

Monolayer cultures, the less standard three-dimensional (3D) culturing systems, and xenografts are the main tools used in current basic and drug development studies of cancer research. The aim of biofabrication is to design and construct a more representative in vivo 3D environment, replacing two-dimensional (2D) cell cultures. Here, we aim to provide a complex comparative analysis of 2D and 3D spheroid culturing, and 3D bioprinted and xenografted breast cancer models. We established a protocol to produce alginate-based hydrogel bioink for 3D bioprinting and the long-term culturing of tumour cells in vitro. Cell proliferation and tumourigenicity were assessed with various tests. Additionally, the results of rapamycin, doxycycline and doxorubicin monotreatments and combinations were also compared. The sensitivity and protein expression profile of 3D bioprinted tissue-mimetic scaffolds showed the highest similarity to the less drug-sensitive xenograft models. Several metabolic protein expressions were examined, and the in situ tissue heterogeneity representing the characteristics of human breast cancers was also verified in 3D bioprinted and cultured tissue-mimetic structures. Our results provide additional steps in the direction of representing in vivo 3D situations in in vitro studies. Future use of these models could help to reduce the number of animal experiments and increase the success rate of clinical phase trials.

Alginate-Lysozyme Nanofibers Hydrogels with Improved Rheological Behavior, Printability and Biological Properties for 3D Bioprinting Applications.

In this study, alginate nanocomposite hydrogel bioinks reinforced with lysozyme nanofibers (LNFs) were developed. Alginate-LNF (A-LNF) suspensions with different LNF contents (1, 5 and 10 wt.%) were prepared and pre-crosslinked with 0.5% (w/v) CaCl2 to formulate A-LNF inks. These inks exhibit proper shear-thinning behavior and good recovery properties (~90%), with the pre-crosslinking step playing a crucial role. A-LNF fully crosslinked hydrogels (with 2% (w/v) CaCl2) that mimic 3D printing scaffolds were prepared, and it was observed that the addition of LNFs improved several properties of the hydrogels, such as the morphology, swelling and degradation profiles, and mechanical properties. All formulations are also noncytotoxic towards HaCaT cells. The printing parameters and 3D scaffold model were then optimized, with A-LNF inks showing improved printability. Selected A-LNF inks (A-LNF0 and A-LNF5) were loaded with HaCaT cells (cell density 2 × 106 cells mL−1), and the cell viability within the bioprinted scaffolds was evaluated for 1, 3 and 7 days, with scaffolds printed with the A-LNF5 bioink showing the highest values for 7 days (87.99 ± 1.28%). Hence, A-LNF bioinks exhibited improved rheological performance, printability and biological properties representing a good strategy to overcome the main limitations of alginate-based bioinks.

Methacrylate-Modified Gold Nanoparticles Enable Non-Invasive Monitoring of Photocrosslinked Hydrogel Scaffolds.

Photocrosslinked hydrogels, such as methacrylate-modified gelatin (gelMA) and hyaluronic acid (HAMA), are widely utilized as tissue engineering scaffolds and/or drug delivery vehicles, but lack a suitable means for non-invasive, longitudinal monitoring of surgical placement, biodegradation, and drug release. Therefore, we developed a novel photopolymerizable X-ray contrast agent, methacrylate-modified gold nanoparticles (AuMA NPs), to enable covalent-linking to methacrylate-modified hydrogels (gelMA and HAMA) in one-step during photocrosslinking and non-invasive monitoring by X-ray micro-computed tomography (micro-CT). Hydrogels exhibited a linear increase in X-ray attenuation with increased Au NP concentration to enable quantitative imaging by contrast-enhanced micro-CT. The enzymatic and hydrolytic degradation kinetics of gelMA-Au NP hydrogels were longitudinally monitored by micro-CT for up to one month in vitro, yielding results that were consistent with concurrent measurements by optical spectroscopy and gravimetric analysis. Importantly, AuMA NPs did not disrupt the hydrogel network, rheology, mechanical properties, and hydrolytic stability compared with gelMA alone. GelMA-Au NP hydrogels were thus able to be bioprinted into well-defined three-dimensional architectures supporting endothelial cell viability and growth. Overall, AuMA NPs enabled the preparation of both conventional photopolymerized hydrogels and bioprinted scaffolds with tunable X-ray contrast for noninvasive, longitudinal monitoring of placement, degradation, and NP release by micro-CT.

Long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids

Mechanical loading has been shown to influence various osteogenic responses of bone-derived cells and bone formation in vivo. However, the influence of mechanical stimulation on the formation of bone organoid in vitro is not clearly understood. Here, three-dimensional (3D) bioprinted human mesenchymal stem cells-laden graphene oxide composite scaffolds were cultured in a novel cyclic-loading bioreactors for up to 56 d. Our results showed that mechanical loading from day 1 (ML01) significantly increased organoid mineral density, organoid stiffness, and osteoblast differentiation compared with non-loading and mechanical loading from day 21. Importantly, ML01 stimulated collagen I maturation, osteocyte differentiation, lacunar-canalicular network formation and YAP expression on day 56. These finding are the first to reveal that long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids. Such 3D bone organoids may serve as a human-specific alternative to animal testing for the study of bone pathophysiology and drug screening.

3D Bioprinted Alginate-Silk-Based Smart Cell-Instructive Scaffolds for Dual Differentiation of Human Mesenchymal Stem Cells.

Designing smart bioinks, which can provide multifunctionality and instructive cues to cells, is a current need of the tissue engineering field. Addressing these parameters, this work aims at developing a smart dual 3D bioprinted scaffold that is capable of differentiating human mesenchymal stem cells into two different lineages within the same construct without providing any exogenous cues. Here, biocompatible alginate- and silk-based bioinks were developed to print self-standing structures with the ability of spatially controlled differentiation of the encapsulated hMSCs. We present this proof of concept and have demonstrated a smart design where the incorporation of phosphate groups enhanced the osteogenic differentiation, whereas the addition of silk promoted the chondrogenic differentiation. Altogether, the present work suggests the potential of the developed bioinks for use in creating clinically viable osteochondral grafts.

Towards 3D Bioprinted Spinal Cord Organoids.

Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.

Impact of oxygen-calcium-generating and bone morphogenetic protein-2 nanoparticles on survival and differentiation of bone marrow-derived mesenchymal stem cells in the 3D bio-printed scaffold

Although stem cell therapy is a major area of interest in tissue engineering, providing proper oxygen tension, good viability, and cell differentiation remain challenges in tissue-engineered scaffolds. In this study, an osteogenic scaffold was fabricated using the 3D bio-printing technique. The bio-ink contained alginate hydrogel, encapsulated human bone marrow-derived mesenchymal stem cells (hBM-MSCs), calcium peroxide nanoparticles (CPO NPs) as an oxygen generating biomaterial, and bone morphogenic protein-2 nanoparticles (BMP2 NPs) as an osteoinductive growth factor. CPO NPs were synthesized with the hydrolysis–precipitation method, and their concentrations in the bio-ink were optimized. Scaffolds containing CPO 3% (w/w) were preferred, because they generated sufficient oxygen gas for 20 days, increased mechanical strength after 20 days, and had sufficient stability. The CPO NPs effect on the viability of embedded hBM-MSCs under hypoxic conditions was analyzed. Live/Dead staining results represented a 22% improvement in CPO 3% scaffold viability on day 7. Therefore, CPO NPs constituted a promising survival factor. BMP2 NPs were prepared with the double emulsification technique. The incorporation of both BMP2 and CPO NPs resulted in the upregulation of Runt-related transcription factor 2, Collagen type I alpha 1, and the osteocalcin genes compared to internal references in osteogenic media. Overall, the proposed 3D bio-printed osteogenic scaffold in this study has moved scientific research one step forward toward successful stem cell therapy and helped improve host tissue healing by biological activity enhancement, especially for low oxygen pressure tissues.

Low-intensity pulsed ultrasound promotes cell viability and inhibits apoptosis of H9C2 cardiomyocytes in 3D bioprinting scaffolds via PI3K-Akt and ERK1/2 pathways.

The aim of this study was to investigate whether low-intensity pulsed ultrasound (LIPUS) promotes myocardial cell viability in three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. Cardiomyoblasts (H9C2s) were mixed in 6% (w/v) GelMA bio-inks and printed using an extrusion-based 3D bioprinter. These scaffolds were exposed to LIPUS with different parameters or sham-irradiated to optimize the LIPUS treatment. The viability of H9C2s was measured using Cell Counting Kit-8 (CCK8), cell cycle, and live and dead cell double-staining assays. Western blot analysis was performed to determine the protein expression levels. We successfully fabricated 3D bio-printed cell-laden GelMA scaffolds. CCK8 and live and dead cell double-staining assays indicated that the optimal conditions for LIPUS were a frequency of 0.5MHz and an exposure time of 10min. Cell cycle analysis showed that LIPUS promoted the entry of cells into the S and G2/M phases from the G0/G1 phase. Western blot analysis revealed that LIPUS promoted the phosphorylation and activation of ERK1/2 and PI3K-Akt. The ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt, respectively, which in turn reduced the LIPUS-induced viability of H9C2s in 3D bio-printed cell-laden GelMA scaffolds. A frequency of 0.5MHz and exposure time of 10min for LIPUS exposure can be adapted to achieve optimized culture effects on myocardial cells in 3D bio-printed cell-laden GelMA scaffolds via the ERK1/2 and PI3K-Akt signaling pathways.

3D Bioprinting of human Mesenchymal Stem Cells in a novel tunic decellularized ECM bioink for Cartilage Tissue Engineering

Tunicates are marine organisms renowned for their thick, leathery exoskeleton called tunic. This tunic is composed of an extracellular matrix packed with protein-cellulose complexes and sulfated polysaccharides, making it a charming biomaterial choice for cartilage tissue engineering. In this study, P.nigra tunicate was collected and processed to obtain its rich decellularized extracellular matrix (dECM). The dECM was either seeded with human mesenchymal stem cells (hMSCs) as is or underwent further processing to form a hydrogel for 3D bioprinting. The characterization of tunic dECM was achieved by FTIR, XRD, TGA, Raman spectroscopy, SEM and tensile mechanical analysis. Biological compatibility and staining were done by live/dead, alamar blue, alcian blue, safranin O and PCR gene expression. After decellularization, the tunic dECM scaffold preserved the natural honeycomb-shaped microstructure, as well as its functional cellulose and protein groups. Both the tunic dECM scaffolds and bioprinted scaffolds showed enhanced metabolic activity, cell proliferation and chondrogenic differentiation. Combining both the mechanical robustness and biocompatibility, the bioink is able to fill the elusive gap in cartilage regeneration. This study offers a new potential source of dECM scaffolds and bioinks which are both biologically compatible and mechanically stable, making it a one stop shop for cartilage tissue engineering.

3D Bioprinted GelMA/PEGDA Hybrid Scaffold for Establishing an In Vitro Model of Melanoma.

Due to the high incidence of malignant melanoma, the establishment of in vitro models that recapitulate the tumor microenvironment is of great biological and clinical importance for tumor treatment and drug research. In this study, 3D printing technology was used to prepare GelMA/PEGDA composite scaffolds that mimic the microenvironment of human malignant melanoma cell (A375) growth and construct in vitro melanoma micro-models. The GelMA/PEGDA hybrid scaffold was tested by the mechanical property, cell live/dead assay, cell proliferation assay, cytoskeleton staining and drug loading assay. The growth of tumor cells in two- and three-dimensional culture systems and the anti-cancer effect of luteolin were evaluated using the live/dead staining method and the Cell Counting Kit-8 (CCK-8) method. The results showed a high aggregation of tumor cells on the 3D scaffold, which was suitable for long-term culture. Cytoskeleton staining and immunofluorescent protein staining were used to evaluate the degree of differentiation of tumor cells under 2D and 3D culture systems. The results indicated that 3D bioprinted scaffolds were more suitable for tumor cell expansion and differentiation, and the tumor cells were more aggressive. In addition, luteolin was time- and dose-dependent on tumor cells, and tumor cells in the 3D culture system were more resistant to the drug.

CyMAD bioreactor: A cyclic magnetic actuation device for magnetically mediated mechanical stimulation of 3D bioprinted hydrogel scaffolds

Mechanical stimulation of bioprinted constructs can enhance the differentiation of cells within these scaffolds, such as driving chondrocytes towards cartilage tissue substitutes. In this study, a holistic approach is presented for designing and engineering a material-specific device based on a magnetic field setup using the Maxwell configuration for a touchless cyclic magnetic stimulation of (bioprinted) hydrogel scaffolds containing magnetic microparticles. We describe the entire development process, from the design of the magnetic field to the construction of the bioreactor and provide an evaluation of the calculation. Finally, an analysis of the distribution and orientation of the particles within the hydrogels and a cytocompatibility test after applying the intended stimulation regime were conducted. As a proof-of-principle, a model system in the shape of a cylindrical bending beam consisting of the established magnetisable bioink based on alginate, methylcellulose and magnetite microparticles (algMC + mag), was used instead of 3D printed, macroporous scaffolds of this material. Requirements for the dimensioning of the force, such as the Young's modulus, were determined experimentally. The magnetic field was calculated using the software Finite Element Method Magnetics (FEMM). The cyclic stimulation of the samples was performed over 14 days with a duration of 3 h per day. The aim was to achieve an elongation of up to 10%. The homogeneous particle distribution in stimulated and non-stimulated samples was proven via μCT and digital image processing (DIP). Even after applying a static magnetic field over 30 min, no structure formation such as chains or agglomeration of the magnetic particles were observed. The deformation behaviour defined as a shifted position of the neutral fibre (centre line of an object) during stimulation was measured via μCT and analysed using DIP. From these data, an elongation of approx. 9% was calculated for day 14. This elongation was achieved for both the stimulated samples and the control group without stimulation, which corresponds to the theoretically calculated value. The cytocompatibility of the bioink, scaffold environment and stimulation approach was demonstrated for bioprinted scaffolds with embedded human mesenchymal stem cells and chondrocytes. These findings proved the suitability and versatility of the bioreactor and the presented approach for stimulation experiments.

3D bioprinting of osteon-mimetic scaffolds with hierarchical microchannels for vascularized bone tissue regeneration

The integration of three-dimensional (3D) bioprinted scaffold’s structure and function for critical-size bone defect repair is of immense significance. Inspired by the basic component of innate cortical bone tissue—osteons, many studies focus on biomimetic strategy. However, the complexity of hierarchical microchannels in the osteon, the requirement of mechanical strength of bone, and the biological function of angiogenesis and osteogenesis remain challenges in the fabrication of osteon-mimetic scaffolds. Therefore, we successfully built mimetic scaffolds with vertically central medullary canals, peripheral Haversian canals, and transverse Volkmann canals structures simultaneously by 3D bioprinting technology using polycaprolactone and bioink loading with bone marrow mesenchymal stem cells and bone morphogenetic protein-4. Subsequently, endothelial progenitor cells were seeded into the canals to enhance angiogenesis. The porosity and compressive properties of bioprinted scaffolds could be well controlled by altering the structure and canal numbers of the scaffolds. The osteon-mimetic scaffolds showed satisfactory biocompatibility and promotion of angiogenesis and osteogenesis in vitro and prompted the new blood vessels and new bone formation in vivo. In summary, this study proposes a biomimetic strategy for fabricating structured and functionalized 3D bioprinted scaffolds for vascularized bone tissue regeneration.

3D bioprinted cancer cells are more tolerant to serum starvation than 2D cells due to autophagy

Cancer is a global issue and a serious threat to human health, one approach to treatment is starvation therapy. Recently, three-dimensional (3D) bioprinted tumor tissue models have been developed; however, whether 3D bioprinted models are good for in vitro study of starvation therapy is unclear. In this study, we studied the state of cells with serum-free medium in both 3D bioprinted scaffold and 2D cell cultures and found that 3D bioprinted cancer cells (3D cells) were more tolerant to serum starvation than 2D cells in terms of cell viability, cell proliferation, and M2 macrophage polarization. Moreover, the ratio of LC3II/I, an index of autophagy, increased much more in 3D cells, and 3D cells showed more autophagosomes than 2D cells after serum starvation, which indicated that the autophagy levels were higher in 3D cells. These results suggested that 3D cells are more tolerant to serum starvation than 2D cells, and autophagy may play an important role in this process.

Non-destructive mechanical assessment for optimization of 3D bioprinted soft tissue scaffolds

SummaryCharacterizing the mechanical properties of engineered tissue constructs provides powerful insight into the function of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogels used in tissue engineering are often destructive and ignore the effect of 3D bioprinting on the overall mechanical properties of a whole tissue construct. This work reports on using a non-destructive method of viscoelastic analysis to demonstrate the influence of bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include mechanical properties analysis during degradation, real-time monitoring of crosslinking, mechanical characterization of multi-material scaffolds, and monitoring the effect of encapsulated cell growth on the mechanical strength of 3D bioprinted scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering.

3D Bioprinted Scaffolds for Bone Tissue Engineering: State-Of-The-Art and Emerging Technologies.

Treating large bone defects, known as critical-sized defects (CSDs), is challenging because they are not spontaneously healed by the patient’s body. Due to the limitations associated with conventional bone grafts, bone tissue engineering (BTE), based on three-dimensional (3D) bioprinted scaffolds, has emerged as a promising approach for bone reconstitution and treatment. Bioprinting technology allows for incorporation of living cells and/or growth factors into scaffolds aiming to mimic the structure and properties of the native bone. To date, a wide range of biomaterials (either natural or synthetic polymers), as well as various cells and growth factors, have been explored for use in scaffold bioprinting. However, a key challenge that remains is the fabrication of scaffolds that meet structure, mechanical, and osteoconductive requirements of native bone and support vascularization. In this review, we briefly present the latest developments and discoveries of CSD treatment by means of bioprinted scaffolds, with a focus on the biomaterials, cells, and growth factors for formulating bioinks and their bioprinting techniques. Promising state-of-the-art pathways or strategies recently developed for bioprinting bone scaffolds are highlighted, including the incorporation of bioactive ceramics to create composite scaffolds, the use of advanced bioprinting technologies (e.g., core/shell bioprinting) to form hybrid scaffolds or systems, as well as the rigorous design of scaffolds by taking into account of the influence of such parameters as scaffold pore geometry and porosity. We also review in-vitro assays and in-vivo models to track bone regeneration, followed by a discussion of current limitations associated with 3D bioprinting technologies for BTE. We conclude this review with emerging approaches in this field, including the development of gradient scaffolds, four-dimensional (4D) printing technology via smart materials, organoids, and cell aggregates/spheroids along with future avenues for related BTE.

From 3D printed molds to bioprinted scaffolds: A hybrid material extrusion and vat polymerization bioprinting approach for soft matter constructs

Three-dimensional (3D) bioprinting methods vary in difficulty and complexity depending on the application desired and biomaterials used. 3D biofabrication is gaining increased traction with enhanced additive manufacturing technologies. Yet, high print resolution and efficiency for the fabrication of complex constructs still prove to be challenging. An intricate balance between biomaterial composition, machine maneuverability, and extrusion mechanism is required. While soft bioinks are highly desirable when used as a biodegradable scaffold material for tissue and organ fabrication, mechanical stiffness and shape fidelity are often compromised. Alternately, post-printing ultraviolet and chemical crosslinking processes improve fidelity but threaten cell viability. Herein, we propose a hybrid fabrication approach to facilitate 3D bioprinting using soft bioinks with instantaneous gelation properties while maintaining shape fidelity for tissue and organ structures of complex geometries. The approach entails a multi-step “3D Printed Molds to Scaffolds” method, which uses additive manufacturing to create accurate negative support structures for the desired construct. A tissue or organ model is first designed in computer-aided design (CAD) modeling software to create a negative mold structure of the desired tissue or organ. Using a Formlabs® SLA 3D printer, the negative mold is fabricated at desired scale using a biocompatible elastic resin. Then, a robotic 3D bioprinting system is loaded with a sliced g-code of the CAD model. The robot start position is aligned with the placement of the fabricated mold on the printbed. Microfluidic pumps deliver three solutions through a customized nozzle to extrude peptide bioink, which gels instantaneously. The initial layers of the structure are formed within the mold to create a solid foundation of the construct. The hybrid approach was found to enhance fidelity considerably and enabled the printing of a complex human ear structure. It is promising for tissue and organ fabrication as it offers a cost-effective support structure without increasing printing time. It could also be used as a rapid prototyping approach for researchers who do not have access to 3D bioprinting systems. Biofabrication, from printed molds to bioprinted scaffolds, will potentially enhance the printing experience with soft bioinks while preserving cell durability and viability. 

The 3D Bioprinted Scaffolds for Wound Healing.

Skin tissue engineering and regeneration aim at repairing defective skin injuries and progress in wound healing. Until now, even though several developments are made in this field, it is still challenging to face the complexity of the tissue with current methods of fabrication. In this review, short, state-of-the-art on developments made in skin tissue engineering using 3D bioprinting as a new tool are described. The current bioprinting methods and a summary of bioink formulations, parameters, and properties are discussed. Finally, a representative number of examples and advances made in the field together with limitations and future needs are provided.

Bioprinting of Chondrocyte Stem Cell Co-Cultures for Auricular Cartilage Regeneration.

Advances in 3D bioprinting allows not only controlled deposition of cells or cell-laden hydrogels but also flexibility in creating constructs that match the anatomical features of the patient. This is especially the case for reconstructing the pinna (ear), which is a large feature of the face and made from elastic cartilage that primarily relies on diffusion for nutrient transfer. The selection of cell lines for reconstructing this cartilage becomes a crucial step in clinical translation. Chondrocytes and mesenchymal stem cells are both studied extensively in the area of cartilage regeneration as they are capable of producing cartilage in vitro. However, such monoculture systems involve unfavorable processes and produce cartilage with suboptimal characteristics. Co-cultures of these cell types are known to alleviate these limitations to produce synergically active chondrocytes and cartilage. The current study utilized a 3D bioprinted scaffold made from combined gelatine methacryloyl and methacrylated hyaluronic acid (GelMA/HAMA) to interrogate monocultures and co-cultures of human septal chondrocytes (primary chondrocytes, PCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). This study is also the first to examine co-cultures of healthy human chondrocytes with human BM-hMSCs encapsulated in GelMA/HAMA bioprinted scaffolds. Findings revealed that the combination of MSCs and PCs not only yielded cell proliferation that mimicked MSCs but also produced chondrogenic expressions that mimicked PCs. These findings suggested that co-cultures of BM-hMSCs and healthy septal PCs can be employed to replace monocultures in chondrogenic studies for cartilage regeneration in this model. The opportunity for MSCs used to replace PCs alleviates the requirement of large cartilage biopsies that would otherwise be needed for sufficient cell numbers and therefore can be employed for clinical applications.

POS-459 EXAMINING THE EFFECT OF CALCULI SHAPE AND DIMENSION ON URETER OUTFLOW RATES USING AN ARTIFICIAL 3D PRINTED KIDNEY

Kidney stone disease is a urological disorder that affects about 12% of the world population. A deeper understanding of the underlying etiologies and effects which this condition generates, such as alterations in renal hemodynamics, is essential for developing models to study this disorder. Several experimental approaches have been employed, and recent developments in 3D printing offer an exciting opportunity to mimic impediments to urine flow characteristics caused by calculi. Here we present one such simplified model. A 3D artificial renal model was developed using SolidWorks 2019 software ( to provide the template (Figure 1a) for the bioprinted scaffolds (Figure 1b). Spherical and elliptical kidney stones, ranging from 0 to 6 mm, mimic calculi embedded in the artificial ureter designed with flexible plastic tubing (Figure 1C). A mechanical circulation pump was then used to induce urine flow (Figure 1D). Flow velocities were obtained using different geometries of 3D printed kidney stones. Flow magnitude and characteristics were compared to understand the effect of having different geometries on the distribution of urinary flow (Figure 1E). The minimum flow velocities for spherical and elliptical stones were 0 and 34.1 cm/sec for 5 and 4 mm stone sizes, respectively. Surprisingly, elliptical kidney stones showed an increase in the flow velocity at the maximum elliptical stone size of 6 mm (54.39 cm/sec) due to the nature of its geometry. For the case of the spherical shape, it converged to (0 cm/sec). Figure 1. The 3D artificial renal model outlining: SolidWorks 2019 organ template (a); (b) the bioprinter model; (c) spherical and elliptical artificial kidney stones embedded in the ureter; (d) mechanical circulation pump was then used to induce urine flow; and an outline of the variations in volumetric floe rate with calculi size. The results from this study provide insight on ways to create simplified artificial organ models that simulate hemodynamic observed in Kidney stone disease and can be used to develop more sophisticated reverse-engineered artificial 3D-printed kidney models.