Margaret Nowicki

3D Bioprinting for Organ Regeneration.

Regenerative medicine holds the promise of engineering functional tissues or organs to heal or replace abnormal and necrotic tissues/organs, offering hope for filling the gap between organ shortage and transplantation needs. Three-dimensional (3D) bioprinting is evolving into an unparalleled biomanufacturing technology due to its high-integration potential for patient-specific designs, precise and rapid manufacturing capabilities with high resolution, and unprecedented versatility. It enables precise control over multiple compositions, spatial distributions, and architectural accuracy/complexity, therefore achieving effective recapitulation of microstructure, architecture, mechanical properties, and biological functions of target tissues and organs. Here we provide an overview of recent advances in 3D bioprinting technology, as well as design concepts of bioinks suitable for the bioprinting process. We focus on the applications of this technology for engineering living organs, focusing more specifically on vasculature, neural networks, the heart and liver. We conclude with current challenges and the technical perspective for further development of 3D organ bioprinting.

4D printing smart biomedical scaffolds with novel soybean oil epoxidized acrylate.

Photocurable, biocompatible liquid resins are highly desired for 3D stereolithography based bioprinting. Here we solidified a novel renewable soybean oil epoxidized acrylate, using a 3D laser printing technique, into smart and highly biocompatible scaffolds capable of supporting growth of multipotent human bone marrow mesenchymal stem cells (hMSCs). Porous scaffolds were readily fabricated by simply adjusting the printer infill density; superficial structures of the polymerized soybean oil epoxidized acrylate were significantly affected by laser frequency and printing speed. Shape memory tests confirmed that the scaffold fixed a temporary shape at −18 °C and fully recovered its original shape at human body temperature (37 °C), which indicated the great potential for 4D printing applications. Cytotoxicity analysis proved that the printed scaffolds had significant higher hMSC adhesion and proliferation than traditional polyethylene glycol diacrylate (PEGDA), and had no statistical difference from poly lactic acid (PLA) and polycaprolactone (PCL). This research is believed to significantly advance the development of biomedical scaffolds with renewable plant oils and advanced 3D fabrication techniques.

Development of Novel 3-D Printed Scaffolds With Core-Shell Nanoparticles for Nerve Regeneration

A traumatic injury of peripheral nerves is serious clinical problem that may lead to major loss of nerve function, affecting quality of patients life. Currently, nerve autograft is widely used to reconstruct the nerve gap. However, such surgical procedure suffers from many disadvantages including donor site morbidity and limited availability. In order to address these issues, neural tissue engineering has focused on the development of synthetic nerve scaffolds to support bridging a larger gap and improving nerve generation. For this purpose, we fabricated a novel 3-D biomimetic scaffold, which has tunable porous structure and embedded core-shell nanoparticles with sustained neurogenic factor delivery system, using stereolithography based 3-D printing and coaxial electrospraying techniques. Our results showed that scaffolds with larger porosity significantly improve PC-12 neural cell adhesion compared to ones with smaller porosity. Furthermore, scaffolds embedded with bovine serum albumin containing nanoparticles showed an enhancement in cell proliferation relative to bared control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with nerve growth factor nanoparticles greatly increased the length of neurites and directed neurite extension of PC-12 cells along the fiber. In addition, the 3-D printed nanocomposite scaffolds also improved the average neurite length of primary cortical neurons. The results in this study demonstrate the potential of this 3-D printed scaffold in improving neural cell function and nerve growth.

Fabrication of a Highly Aligned Neural Scaffold via a Table Top Stereolithography 3D Printing and Electrospinning.

Three-dimensional (3D) bioprinting is a rapidly emerging technique in the field of tissue engineering to fabricate extremely intricate and complex biomimetic scaffolds in the range of micrometers. Such customized 3D printed constructs can be used for the regeneration of complex tissues such as cartilage, vessels, and nerves. However, the 3D printing techniques often offer limited control over the resolution and compromised mechanical properties due to short selection of printable inks. To address these limitations, we combined stereolithography and electrospinning techniques to fabricate a novel 3D biomimetic neural scaffold with a tunable porous structure and embedded aligned fibers. By employing two different types of biofabrication methods, we successfully utilized both synthetic and natural materials with varying chemical composition as bioink to enhance biocompatibilities and mechanical properties of the scaffold. The resulting microfibers composed of polycaprolactone (PCL) polymer and PCL mixed with gelatin were embedded in 3D printed hydrogel scaffold. Our results showed that 3D printed scaffolds with electrospun fibers significantly improve neural stem cell adhesion when compared to those without the fibers. Furthermore, 3D scaffolds embedded with aligned fibers showed an enhancement in cell proliferation relative to bare control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with PCL/gelatin fibers greatly increased the average neurite length and directed neurite extension of primary cortical neurons along the fiber. The results of this study demonstrate the potential to create unique 3D neural tissue constructs by combining 3D bioprinting and electrospinning techniques.

4D printing of polymeric materials for tissue and organ regeneration

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex, stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applications. Although the 4D concept was first highlighted in 2013, extensive research has rapidly developed, along with more-in-depth understanding and assertions regarding the definition of 4D. In this review, we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in the field. Both transformation-preprogrammed 4D printing and 4D printing of shape memory polymers are intensively surveyed. Afterwards we will explore and discuss the applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissues and implantable scaffolds, as well as future perspectives and conclusions.

3D printing of novel osteochondral scaffolds with graded microstructure.

Osteochondral tissue has a complex graded structure where biological, physiological, and mechanical properties vary significantly over the full thickness spanning from the subchondral bone region beneath the joint surface to the hyaline cartilage region at the joint surface. This presents a significant challenge for tissue-engineered structures addressing osteochondral defects. Fused deposition modeling (FDM) 3D bioprinters present a unique solution to this problem. The objective of this study is to use FDM-based 3D bioprinting and nanocrystalline hydroxyapatite for improved bone marrow human mesenchymal stem cell (hMSC) adhesion, growth, and osteochondral differentiation. FDM printing parameters can be tuned through computer aided design and computer numerical control software to manipulate scaffold geometries in ways that are beneficial to mechanical performance without hindering cellular behavior. Additionally, the ability to fine-tune 3D printed scaffolds increases further through our investment casting procedure which facilitates the inclusion of nanoparticles with biochemical factors to further elicit desired hMSC differentiation. For this study, FDM was used to print investment-casting molds innovatively designed with varied pore distribution over the full thickness of the scaffold. The mechanical and biological impacts of the varied pore distributions were compared and evaluated to determine the benefits of this physical manipulation. The results indicate that both mechanical properties and cell performance improve in the graded pore structures when compared to homogeneously distributed porous and non-porous structures. Differentiation results indicated successful osteogenic and chondrogenic manipulation in engineered scaffolds.

3D printing nano conductive multi-walled carbon nanotube scaffolds for nerve regeneration

OBJECTIVE Nanomaterials, such as carbon nanotubes (CNTs), have been introduced to modify the surface properties of scaffolds, thus enhancing the interaction between the neural cells and biomaterials. In addition to superior electrical conductivity, CNTs can provide nanoscale structures similar to those present in the natural neural environment. The primary objective of this study is to investigate the proliferative capability and differential potential of neural stem cells (NSCs) seeded on a CNT incorporated scaffold. APPROACH Amine functionalized multi-walled carbon nanotubes (MWCNTs) were incorporated with a PEGDA polymer to provide enhanced electrical properties as well as nanofeatures on the surface of the scaffold. A stereolithography 3D printer was employed to fabricate a well-dispersed MWCNT-hydrogel composite neural scaffold with a tunable porous structure. 3D printing allows easy fabrication of complex 3D scaffolds with extremely intricate microarchitectures and controlled porosity. MAIN RESULTS Our results showed that MWCNT-incorporated scaffolds promoted neural stem cell proliferation and early neuronal differentiation when compared to those scaffolds without the MWCNTs. Furthermore, biphasic pulse stimulation with 500 µA current promoted neuronal maturity quantified through protein expression analysis by quantitative polymerase chain reaction. SIGNIFICANCE Results of this study demonstrated that an electroconductive MWCNT scaffold, coupled with electrical stimulation, may have a synergistic effect on promoting neurite outgrowth for therapeutic application in nerve regeneration.

Integrating Three-Dimensional Printing and Nanotechnology for Musculoskeletal Regeneration.

The field of tissue engineering is advancing steadily, partly due to advancements in rapid prototyping technology. Even with increasing focus, successful complex tissue regeneration of vascularized bone, cartilage and the osteochondral interface remains largely illusive. This review examines current three-dimensional printing techniques and their application towards bone, cartilage and osteochondral regeneration. The importance of, and benefit to, nanomaterial integration is also highlighted with recent published examples. Early-stage successes and challenges of recent studies are discussed, with an outlook to future research in the related areas.

Photolithographic-stereolithographic-tandem fabrication of 4D smart scaffolds for improved stem cell cardiomyogenic differentiation

4D printing is a highly innovative additive manufacturing process for fabricating smart structures with the ability to transform over time. Significantly different from regular 4D printing techniques, this study focuses on creating novel 4D hierarchical micropatterns using a unique photolithographic-stereolithographic-tandem strategy (PSTS) with smart soybean oil epoxidized acrylate (SOEA) inks for effectively regulating human bone marrow mesenchymal stem cell (hMSC) cardiomyogenic behaviors. The 4D effect refers to autonomous conversion of the surficial-patterned scaffold into a predesigned construct through an external stimulus delivered immediately after printing. Our results show that hMSCs actively grew and were highly aligned along the micropatterns, forming an uninterrupted cellular sheet. The generation of complex patterns was evident by triangular and circular outlines appearing in the scaffolds. This simple, yet efficient, technique was validated by rapid printing of scaffolds with well-defined and consistent micro-surface features. A 4D dynamic shape change transforming a 2-D design into flower-like structures was observed. The printed scaffolds possessed a shape memory effect beyond the 4D features. The advanced 4D dynamic feature may provide seamless integration with damaged tissues or organs, and a proof of concept 4D patch for cardiac regeneration was demonstrated for the first time. The 4D-fabricated cardiac patch showed significant cardiomyogenesis confirmed by immunofluorescence staining and qRT-PCR analysis, indicating its promising potential in future tissue and organ regeneration applications.

Stereolithographic 4D Bioprinting of Multiresponsive Architectures for Neural Engineering

4D printing represents one of the most advanced fabrication techniques for prospective applications in tissue engineering, biomedical devices, and soft robotics, among others. In this study, a novel multiresponsive architecture is developed through stereolithography‐based 4D printing, where a universal concept of stress‐induced shape transformation is applied to achieve the 4D reprogramming. The light‐induced graded internal stress followed by a subsequent solvent‐induced relaxation, driving an autonomous and reversible change of the programmed configuration after printing, is employed and investigated in depth and details. Moreover, the fabricated construct possesses shape memory property, offering a characteristic of multiple shape change. Using this novel multiple responsive 4D technique, a proof‐of‐concept smart nerve guidance conduit is demonstrated on a graphene hybrid 4D construct providing outstanding multifunctional characteristics for nerve regeneration including physical guidance, chemical cues, dynamic self‐entubulation, and seamless integration. By employing this fabrication technique, creating multiresponsive smart architectures, as well as demonstrating application potential, this work paves the way for truly initiation of 4D printing in various high‐value research fields.