Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids.

Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and ß-tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen type 1 (Col 1) 30-fold, osteopontin (OPN) 10-fold, and osteocalcin (OCN) 3-fold) after 14 days of culture with the incorporated CMPs, while no significant upregulation was observed with the incorporation of gelatin microparticles. The porous structure of the CMPs was exploited for loading and sustained release of an angiogenic small molecule. Dimethyloxaloylglycine (DMOG) was loaded efficiently onto the CMPs (loading efficiency: 65.32 ± 6%) and showed a sustained release profile over 12 days. Upon incorporation of the DMOG-loaded CMPs (DCMPs) within the mesenspheres, a similar osteogenic differentiation and an upregulation in angiogenic genes (VEGF 5-fold and kinase insert domain (KDR) 2-fold) were observed after 14 days of culture. These trends were also observed in immunostaining analysis. To evaluate scalable production of the osteogenic micro-tissues, the incorporation of microparticles was performed during cell aggregation in a spinner flask. The DCMPs were efficiently incorporated and directed the mesenspheres toward osteogenesis and angiogenesis. Finally, the DCMP mesenspheres were loaded within a three-dimensional printed cell trapper and transplanted into a critical-sized defect in a rat model. Computed tomography and histological analysis showed significant bone formation with blood vessel reconstruction after 8 weeks in this group. Taken together, we provide a scalable and cost-effective approach for fabrication of osteogenic micro-tissues, as building blocks of macro-tissues, that can address the large amounts of cells required for cell-based therapies.

Translating Biofabrication to the Market.

Biofabrication holds great potential to revolutionize important industries in the health, food, and textile sectors, but its translation to market is still challenging. I analyze the current state of innovation and commercialization in biofabrication and try to assess its limitations, strengths, and future progress.

Rapid 3D bioprinting of decellularized extracellular matrix with regionally varied mechanical properties and biomimetic microarchitecture.

Hepatocellular carcinoma (HCC), as the fifth most common malignant cancer, develops and progresses mostly in a cirrhotic liver where stiff nodules are separated by fibrous bands. Scaffolds that can provide a 3D cirrhotic mechanical environment with complex native composition and biomimetic architecture are necessary for the development of better predictive tissue models. Here, we developed photocrosslinkable liver decellularized extracellular matrix (dECM) and a rapid light-based 3D bioprinting process to pattern liver dECM with tailorable mechanical properties to serve as a platform for HCC progression study. 3D bioprinted liver dECM scaffolds were able to stably recapitulate the clinically relevant mechanical properties of cirrhotic liver tissue. When encapsulated in dECM scaffolds with cirrhotic stiffness, HepG2 cells demonstrated reduced growth along with an upregulation of invasion markers compared to healthy controls. Moreover, an engineered cancer tissue platform possessing tissue-scale organization and distinct regional stiffness enabled the visualization of HepG2 stromal invasion from the nodule with cirrhotic stiffness. This work demonstrates a significant advancement in rapid 3D patterning of complex ECM biomaterials with biomimetic architecture and tunable mechanical properties for in vitro disease modeling.

Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.

Printed organo-functionalized graphene for biosensing applications.

Graphene is a highly promising material for biosensors due to its excellent physical and chemical properties which facilitate electron transfer between the active locales of enzymes or other biomaterials and a transducer surface. Printing technology has recently emerged as a low-cost and practical method for fabrication of flexible and disposable electronics devices. The combination of these technologies is promising for the production and commercialization of low cost sensors. In this review, recent developments in organo-functionalized graphene and printed biosensor technologies are comprehensively covered. Firstly, various methods for printing graphene-based fluids on different substrates are discussed. Secondly, different graphene-based ink materials and preparation methods are described. Lastly, biosensing performances of printed or printable graphene-based electrochemical and field effect transistor sensors for some important analytes are elaborated. The reported printed graphene based sensors exhibit promising properties with good reliability suitable for commercial applications. Among most reports, only a few printed graphene-based biosensors including screen-printed oxidase-functionalized graphene biosensor have been demonstrated. The technology is still at early stage but rapidly growing and will earn great attention in the near future due to increasing demand of low-cost and disposable biosensors.

Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an accessible open-source 3D bioprinter capable of serving the needs of any laboratory interested in 3D cellular interactions and tissue engineering.

The Application of Ultrasound in 3D Bio-Printing.

Three-dimensional (3D) bioprinting is an emerging and promising technology in tissue engineering to construct tissues and organs for implantation. Alignment of self-assembly cell spheroids that are used as bioink could be very accurate after droplet ejection from bioprinter. Complex and heterogeneous tissue structures could be built using rapid additive manufacture technology and multiple cell lines. Effective vascularization in the engineered tissue samples is critical in any clinical application. In this review paper, the current technologies and processing steps (such as printing, preparation of bioink, cross-linking, tissue fusion and maturation) in 3D bio-printing are introduced, and their specifications are compared with each other. In addition, the application of ultrasound in this novel field is also introduced. Cells experience acoustic radiation force in ultrasound standing wave field (USWF) and then accumulate at the pressure node at low acoustic pressure. Formation of cell spheroids by this method is within minutes with uniform size and homogeneous cell distribution. Neovessel formation from USWF-induced endothelial cell spheroids is significant. Low-intensity ultrasound could enhance the proliferation and differentiation of stem cells. Its use is at low cost and compatible with current bioreactor. In summary, ultrasound application in 3D bio-printing may solve some challenges and enhance the outcomes.

A Review of 3D Printing Techniques and the Future in Biofabrication of Bioprinted Tissue.

3D printing has been around in the art, micro-engineering, and manufacturing worlds for decades. Similarly, research for traditionally engineered skin tissue has been in the works since the 1990s. As of recent years, the medical field also began to take advantage of the untapped potential of 3D printing for the biofabrication of tissue. To do so, researchers created a set of goals for fabricated tissues based on the characteristics of natural human tissues and organs. Fabricated tissue was then measured against this set of standards. Researchers were interested in not only creating tissue that functioned like natural tissues but in creating techniques for 3D printing that would print tissues quickly, efficiently, and ultimately result in the ability to mass produce fabricated tissues. Three promising methods of 3D printing emerged from their research: thermal inkjet printing with bioink, direct-write bioprinting, and organ printing using tissue spheroids. This review will discuss all three printing techniques, as well as their advantages, disadvantages, and the possibility of future advancements in the field of tissue fabrication.