Polystyrene (PS) has brought in vitro cell culture from its humble beginnings to the modern era, propelling dozens of research fields along the way. This review discusses the development of the material, fabrication, and treatment approaches to create the culture material. However, native PS surfaces poorly facilitate cell adhesion and growth in vitro. To overcome this, liquid surface deposition, energetic plasma activation, and emerging functionalization methods transform the surface chemistry. This review seeks to highlight the many potential applications of the first widely accepted polymer growth surface. Although the majority of in vitro research occurs on two-dimensional surfaces, the importance of three-dimensional (3D) culture models cannot be overlooked. The methods to transition PS to specialized 3D culture surfaces are also reviewed. Specifically, casting, electrospinning, 3D printing, and microcarrier approaches to shift PS to a 3D culture surface are highlighted. The breadth of applications of the material makes it impossible to highlight every use, but the aim remains to demonstrate the versatility and potential as both a general and custom cell culture surface. The review concludes with emerging scaffolding approaches and, based on the findings, presents our insights on the future steps for PS as a tissue culture platform.

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Keratin, a naturally-derived polymer derived from human hair, is physiologically biodegradable, provides adequate cell support, and can self-assemble or be crosslinked to form hydrogels. Nevertheless, it has had limited use in tissue engineering and has been mainly used as casted scaffolds for drug or growth factor delivery applications. Here, we present and assess a novel method for the printed, sequential production of 3D keratin scaffolds. Using a riboflavin-SPS-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution we produced 3D keratin constructs via UV crosslinking in a lithography-based 3D printer. The hydrogels obtained have adequate printing resolution and result in compressive and dynamic mechanical properties, uptake and swelling capacities, cytotoxicity, and microstructural characteristics that are comparable or superior to those of casted keratin scaffolds previously reported. The novel keratin-based printing resin and printing methodology presented have the potential to impact future research by providing an avenue to rapidly and reproducibly manufacture patient-specific hydrogels for tissue engineering and regenerative medicine applications.

While articular cartilage defects affect millions of people worldwide from adolescents to adults, the repair of articular cartilage defects still remains challenging due to the limited endogenous regeneration of the tissue and poor integration with implants. In this study, we developed a 3D-printed scaffold functionalized with aggrecan that supports the cellular fraction of bone marrow released from microfracture, a widely used clinical procedure, and demonstrated tremendous improvement of regenerated cartilage tissue quality and joint function in a lapine model. Optical coherence tomography (OCT) revealed doubled thickness of the regenerated cartilage tissue in the group treated with our aggrecan functionalized scaffold compared to standard microfracture treatment. H&E staining showed 366 ± 95 chondrocytes present in the unit area of cartilage layer with the support of bioactive scaffold, while conventional microfracture group showed only 112 ± 26 chondrocytes. The expression of type II collagen appeared almost 10 times higher with our approach compared to normal microfracture, indicating the potential to overcome the fibro-cartilage formation associated with the current microfracture approach. The therapeutic effect was also evaluated at joint function level. The mobility was evaluated using a modified Basso, Beattie and Bresnahan (BBB) scale. While the defect control group showed no movement improvement over the course of study, all experimental groups showed a trend of increasing scores over time. The present work developed an effective method to regenerate critical articular defects by combining a 3D-printed therapeutic scaffold with the microfracture surgical procedure. This biofunctionalized acellular scaffold has great potential to be applied as a supplement for traditional microfracture to improve the quality of cartilage regeneration in a cost and labor effective way.

The layers in skin determine its protective and hemostasis functions. This layered microstructure cannot be naturally regenerated after severe burns; we aim to reconstruct it using guided tissue regeneration (GTR). In GTR, a membrane is used to regulate tissue growth by stopping fast-proliferating cells and allowing slower cells to migrate and reconstruct specialized microstructures. Here, we proposed the use of keratin membranes crosslinked via dityrosine bonding. Variables from the crosslinking process were grouped within an energy density (ED) parameter to manufacture and evaluate the membranes. Sol fraction, spectrographs, and thermograms were used to quantify the non-linear relation between ED and the resulting crosslinking degree (CD). Mechanical and swelling properties increased until an ED threshold was reached; at higher ED, the CD and properties of the membranes remained invariable indicating that all possible dityrosine bonds were formed. Transport assays showed that the membranes allow molecular diffusion; low ED membranes retain solutes within their structure while the high ED samples allow higher transport rates indicating that uncrosslinked proteins can be responsible of reducing transport. This was confirmed with lower transport of adipogenic growth factors to stem cells when using low ED membranes; high ED samples resulted in increased production of intracellular lipids. Overall, we can engineer keratin membranes with specific CD, a valuable tool to tune microstructural and transport properties.

Vascular grafts that can support total replacement and maintenance by the body of the injured vessel would improve outcomes of major surgical reconstructions. Building scaffolds using components of the native vessel can encourage biological recognition by native cells as well as mimic mechanical characteristics of the native vessel. Evidence is emerging that incorporating predetermined building-blocks into a tissue engineering scaffold may oversimplify the environment and ignore critical structures and binding sites essential to development at the implant. We propose the development of a 3D-printable and degradable hybrid scaffold by combining polyethylene glycol (PEG)acrylate and homogenized pericardium matrix (HPM) to achieve appropriate biological environment as well as structural support. It was hypothesized that incorporation of HPM into PEG hydrogels would affect modulus of the scaffold and that the modulus and biological component would reduce the inflammatory signals produced from arriving macrophages and nearby endothelial cells. HPM was found to provide a number of tissue specific structural proteins including collagen, fibronectin, and glycosaminoglycans. HPM and PEGacrylate formed a hybrid hydrogel with significantly distinct modulus depending on concentration of either component, which resulted in scaffolds with stiffness between 0.5 and 20 kPa. The formed hybrid hydrogel was confirmed through a reduction in primary amines post-cross-linking. Using these hybrid scaffolds, rat bone marrow derived macrophages developed an M2 phenotype in response to low amounts (0.03%, w/v) of HPM in culture but responded with inflammatory phenotypes to high concentrations (0.3%, w/v). When cultured together with endothelial cells, both M1 and M2 macrophages were detected, along with a combination of both inflammatory and healing cytokines. However, the expression of inflammatory cytokines TNFα and IL1β was significantly (p < 0.05) lower with hybrid hydrogels compared to single component PEG or HPM hydrogels. This reduction in inflammatory cytokines could impact the healing environment that persists at the implantation site. Finally, using this developed hybrid hydrogel, models of neonatal vasculature were manufactured using digital light projection (DLP) 3D printing. The structural control achieved with this novel biomaterial suggests a promising new tool in vascular graft development and research, with potential for complex structures for use in congenital heart defect reconstruction.

Stem cell cultures within perfusion bioreactors, while efficient in obtaining cell numbers, often lack the similarity to native tissues and consequently cell phenotype. We develop a three-dimensional (3D)-printed fluidic chamber for dynamic stem cell culture, with emphasis on control over flow and substrate curvature in a 3D environment, two physiologic features of native tissues. The chamber geometry, consisting of an array of vertical cylindrical pillars, facilitates actin-mediated localization of human mesenchymal stem cells (hMSCs) within ∼200 μm distance from the pillars, enabling spatial patterning of hMSCs and endothelial cells in cocultures and subsequent modulation of calcium signaling between these two essential cell types in the bone marrow microenvironment. Flow-enhanced osteogenic differentiation of hMSCs in growth media imposes spatial variations of alkaline phosphatase expression, which positively correlates with local shear stress. Proliferation of hMSCs is maintained within the chamber, exceeding the cell expansion in conventional static culture. The capability to manipulate cell spatial patterning, differentiation, and 3D tissue formation through geometry and flow demonstrates the culture chamber's relevant chemomechanical cues in stem cell microenvironments, thus providing an easy-to-implement tool to study interactions among substrate curvature, shear stress, and intracellular actin machinery in the tissue-engineered construct.

3D printing plays an important role in various biomedical research applications including, but not limited to, culture systems and implantable devices. In this review, we discuss recent development in the applications of 3D printing technologies for clinically motivated research, particularly focusing on the fabrication of constructs subsequently incorporated with cells. Applications of this technology include pharmaceutical delivery, bioreactor culture platforms, acellular scaffolds, imaging modalities, and organ-on-a chip systems. Emphasis is placed on technological developments not possible without 3D printing technologies: where traditional manufacturing approaches would be cumbersome to demonstrate research objectives. The clinical applications of 3D printing are rapidly moving from the research to production phases and will certainly continue to grow, with ever increasing numbers of therapies becoming commercialized. The work discussed here holds promise for various applications in structural improvements, drug delivery, and physiology research.