3D printing in cell culture systems and medical applications

3D printing has rapidly moved from an innovation novelty to a widely accessible desktop fabrication method. With clinical applications in mind, rapid prototyping provides enormous flexibility and opportunity for engineers, physicians, and researchers to work collaboratively to rapidly solve medical problems. Currently, there are more than 20 active or recruiting clinical trials involving a 3D-printed component, ranging from metal bone implants, atrial fibrillation assistant devices, pulse oximeters, and sleep apnea masks.¹ This clinical work is proving fruitful, as many 3D printing-based technologies have received Food and Drug Administration (FDA) clearance.² However, these devices remain primarily acellular. (For a review of cellular based bioprinting, see Cui et al.³) 3D printing has wide appeal clinically to rapidly observe, design, and fix defects in a single day. The flexibility in acceptable designs and materials without sacrificing resolution or fabrication time has pushed clinical based research to embrace the technologies. In this review, we look at various 3D printing approaches (Table I) for pharmaceutical delivery, fabrication of acellular scaffolds, bioreactor systems, medical model devices, and organ-on-a-chip systems with practical applications in the clinic.

3D printing has shown promise as means of delivering therapeutics with controlled, patient-specific dosages by compounding polymer filaments with medications, and varying both the loading quantity and total mass of the tablet.⁹ Research into 3D-printed tablets has shown that drug release profiles can be controlled and designed to deliver active pharmaceutical ingredients (APIs) to intended therapeutic locations.^10,11 A number of medications have been compounded directly mechanically or absorbed into desktop extrusion 3D printing (e.g., Fab@home, MakerBot Replicator 2) filaments with material bases of hydroxypropyl methylcellulose, poly(acrylic acid), poly(vinyl alcohol), poly(lactic acid) (PLA), and/or Eudragit EPO. APIs include guaifensin,¹² aminosalicylate,¹³ nitrofurantoin,¹⁴ and theophylline.^15,16 Customizing the filament formulation based on the degradation mechanism, miscibility, and carrier geometry has been shown to improve compounding, extrusion, and release behavior,^16–19 and is easily explored using these extrusion 3D printing systems. Multiphase dosing,¹² multiple simultaneous drug combinations,¹⁷ and 3D-printed non-degradable casings²⁰ show promise for medications administered together frequently or of varying dose to maintain the therapeutic effect by fabricating API carriers to meet individual patient needs. Importantly, APIs appear to remain in their intended therapeutic form following printing.¹⁴ With the prevailing evidence and extensive testing, the Food and Drug Administration (FDA) approved the first oral prescription therapy using 3D printing technology in 2015, where the APIs are added directly onto a powder bed to create a patient-specific dose.^21,22 With Aprecia's ZipDose technology, a pharmaceutical containing layer is laid flat and then set with a binding liquid to control shape of the drug; dosing is controlled by repeating this process to the desired number of layers.²² Continuing this work would realize additional therapies beyond epilepsy treatment. Applications of controlled pharmaceutical delivery are not limited to ingestible tablets and pills. Using extrudable materials, such as ethylene vinyl acetate, polycaprolactone (PCL), and PLA on a MakerBot Replicator 2, custom drug-releasing polymers can provide pharmaceutical dosing and prevent biofilm formation on custom intrauterine implants^23,24 and potentially catheters.²⁵ Rapid prototyping of functionalized polymers could lead to novel devices such as coronary stent, gastric, and neurological implants where both technical design requirements and drug eluting or antimicrobial properties remain pertinent. The relatively little amount of time where a API-loaded filament is exposed to high heat, as compared to traditional injection molding techniques, likely contributes to the API's intended therapeutic effects following printing. 3D printing's greatest engineering impact remains in rapid modification of designs to address niche clinical issues, rather than mass produced components.

Applying 3D printing to the fabrication of culture systems may offer a more realistic biomimetic environment for cells, resulting in physiologically relevant cell phenotypes.²⁶ 3D printing is particularly useful for such cell culture applications due to the capability to achieve geometries that are otherwise difficult to fabricate using conventional techniques (e.g., casting and electrospinning). This technique can produce features with sizes that are relevant to cells, where parameters such as porosity, substrate roughness, and curvature can be tuned. Ultimately, the effect of these parameters on cellular behavior (growth, cell alignment, and differentiation) is investigated in these 3D culture platforms. The key to applying this technique for cell culture platforms relies on fabricating a geometry that provides the correct mechanical cues^27,28 (e.g., through perfusion flow that is guided by the geometry) and consequently the chemical cues,²⁹ which are needed for proper cellular signaling, specific tissue growth and development, and through cell-substrate interactions. The capability to fabricate application-specific geometries using 3D printing, which consequently dictates the substrate's mechanical properties and cell-cell organization, drives the development of culture platforms that can mimic various tissues.

Flat culture dishes, specifically tissue culture polystyrene, have been the foundation of biological based research, as they support efficient cell expansion. As various biomedical fields grow, vigorous research efforts related to tissue/organ development and disease modeling emerge, and therefore so does the need for a cell culture platform that provides biomimicry, which flat culture dishes fail to provide. Transitioning culture from 2D to 3D substrates could improve the biomimicry, thus improving cell-cell interactions and increasing the efficiency of in vitro cell culture, and has driven greater interest in complex topology and material choices for extended and directed cell growth.³⁰ For this application, 3D printing has been used to fabricate complex geometries with specific architecture, interconnected geometries, and microporous surfaces to facilitate tailored cell responses. Initial design of these cell culture platforms should utilize computational modeling to design the scaffold, in which one can study the fluid dynamics and understand the mechanical force transmission, therefore predicting the forces acting on the cells once these acellular constructs are seeded with cells. Successful integration of modeling and part design can yield scaffolds for cell culture which balance mechanical integrity with porous structures facilitating nutrient exchange and cell infiltration, and may direct cell behavior.^31–33 

3D printing allows for great flexibility in compounding multiple components to 3D-printing chemically complex homogenous materials unavailable in traditional manufacturing environments. This includes combinations of porogens, polymers, metals, and ceramics used to mimic the mechanical and/or chemical properties of native tissues and create complex, interconnected, topography.^34,35 Incorporating these chemically and topologically complex constructs into dynamic culture techniques generally improves cellular infiltration and media exchange, therefore better replicating the native environment.³⁶ Crucial aspects of cell culture applications within 3D-printed scaffolds include cell expansion and migration, attempting to improve construct models to evaluate the cell function ex vivo.³⁷ With all scaffolds, design approaches which verify study requirements ensure that anticipated cellular outcomes are achieved and research objectives are met, such as fluid flow dynamics, mechanical force transmission, elasticity, porosity, etc.³⁸ Without this design feedback, ultimate study results remain hard to support and justify.

The recapitulation of tissue models achieved through three-dimensionality, aided by 3D printing, typically provides an environment that better sustains cell proliferation and differentiation. These 3D-printed cell culture systems are often substitutes for existing commercial culture platforms, providing a more cost-effective, well-tailored solution for specific interests, and are therefore customizable for optimized specific tissue applications. One popular platform for cell culture is bioreactors for dynamic culture, which has been shown to enhance cell seeding efficiency,^39–41 proliferation,^42–45 and differentiation of various cell types.^46–54 For example, dynamic culture of human mesenchymal stem cells (MSCs) in a tubular perfusion bioreactor containing 3D-printed (with Eshell 300 as the resin on an EnvisionTEC Perfactory 4) vascular networks for an in vitro engineered bone tissue was found to increase the cell viability by 50% in the core of the construct compared to the static culture.⁵⁵ Through 3D printing, interpenetrating networks with various geometrical parameters (porosity and pore-to-pore distance) can be fabricated. Investigation of the effect of these parameters on the cellular response and mass transport within the tissue can be performed.⁵⁶ Such porous networks provide three-dimensionality to tissue models, allow nutrient diffusion and cell migration throughout the printed construct, and provide a high surface area per volume ratio for cell attachment and growth, increasing media efficiency. Additionally, the digital light processing (DLP) system selected balances the feature size, part size, and fabrication time/cost for this vascular application.

More recently, a 3D-printed miniaturized, modular spinning bioreactor was developed and used to generate forebrain-specific organoids from human induced pluripotent stem cells (iPSCs).⁵⁷ The design utilizes computer-aided design (CAD) and 3D printing to optimize designs that sustain organoids of varying sizes in suspension under moderate spinning speed and prevent aggregation. The bioreactor was 3D-printed using the Fortus 400mc printer (Stratasys), with the ULTEM 9085 heat-resistant plastic as the printing resin. The use of this bioreactor was shown to enhance cell viability and promote maintenance of the stem cell niche in the organoid compared to static cultures. Existing culture dish-based cerebral organoid models have limited applicability since they poorly mimic key features of the human brain development. Specifically, culture dish-based models contain progenitors that show morphological characteristics of outer radial glia cells, but not a well-developed outer sub-ventricular zone layer. The design of this culture platform, as enabled by 3D printing, allows custom, optimized design for the creation and maintenance of tissue models that resemble critical aspects of human cortical development in an affordable, high-throughout, and reproducible organoid platform. In addition to the enhanced biomimicry and functionality of the organoid, the custom bioreactor design also improves upon currently available spinning bioreactors, whose scalability is inhibited by the large required incubator space, frequent media exchanges, and large sample-to-sample variability [Fig. 1(a)].⁵⁷ 

Continued bioreactor system development seeks to scale-up cell culture for large-scale clinical applications. For various clinical applications, large-scale production of cells is necessary to meet the dosage requirements. It is important to first understand the scope of the clinical target and then work backwards to develop smaller scale culture platforms where studies can be done to investigate cellular mechanisms and scalable processes can be optimized at a much lower cost. This view has pushed research to scale-down. Integrating 3D printing presents a unique avenue to demonstrate small scale feasibility and perform extensive investigation using a much smaller footprint and working volume prior to migration to production scale or clinical applications.

The capability to spatially and temporally control cell growth and stimuli through substrate geometry and fluid transport, while simultaneously providing a platform for cell imaging, image-based analysis, and further biochemical analysis of single cells in tissues, makes microfluidics popular for biological applications.⁶¹ However, conventional poly(dimethyl siloxane) (PDMS)-on-glass microfluidic device fabrication starts with a complicated, time-consuming soft lithography process requiring expensive equipment in a cleanroom facility.⁶² This is followed by the assembly, which requires access to plasma treatment equipment.⁶² Alternatively, recent advances in 3D printing technologies support single-step and rapid fabrication of highly complex microfluidic devices, while reducing costs associated with institutional infrastructure, equipment, and physical space.⁶² Consequently, microfluidics are becoming widely accessible with the increasing availability of high-precision 3D printing.

One of the uses of 3D printing technology for the development of cell culture systems is the fabrication of microfluidic devices for high-throughput fabrication of hydrogel scaffold droplets for cell encapsulation. A microfluidic chip incorporating a coaxial flow device for co-extrusion has been fabricated using a DLP 3D-printer (Asiga pico27 with Asiga plat clear resin) to generate an extracellular matrix (ECM)-coated, hollow, sub-millimeter alginate capsules to encapsulate cells [Fig. 2(a)].⁶³ The device enabled the creation of an enclosed microenvironment within each sphere, which mimics the basal membrane of the cellular niche. Human neural stem cells derived from human induced pluripotent stem cells can be cultured and differentiated into neurons. A Lego-like modular 3D-printed microfluidic device has also been developed and used to encapsulate dental pulp stem cells within alginate droplets.⁶⁴ The devices were produced using fused filament fabrication in an Ultimaker 2 printer, with clear PLA as the printing material, resulting in a rapidly manufactured, low cost, transparent device that can be utilized for cell imaging. Furthermore, a combination of 3D printing methods can be applied to construct a low-cost microfluidic chip for long-term 3D cell culture and growth; this application combines a stereolithography-based desktop 3D printer and an industrial 3D printer based on polyjet technology.⁶⁵ In addition to 3D cell encapsulation, this platform's capabilities also include spatial patterning within gelatin methacryloyl (GelMA) hydrogels, as well as complex, predictable, and controllable fluid flow patterns inside the 3D channel.

Similarly, generation of multicellular spheroids has been demonstrated inside a microfluidic device fabricated with an Object260 Connex printer with VeroClear-RGD810 resin and with a commercial stereolithography-based contract manufacturer (Proto Labs) using a polycarbonate based resin.⁶⁶ The internal device geometry immobilized the spheroids, and gravity-driven flow perfused the cell-containing circuit. Viability and functionality of patient-derived parental and metastatic oral squamous cell carcinoma tumor and liver cell (HepG2) spheroids were maintained, presenting a simple device with possible applications in investigations of drug efficacy, metabolism, and toxicity.⁶⁶ Enabled by 3D printing approaches, the capability of fabricating geometrically defined micro-tissue models containing single cells and spheroids has allowed researchers to develop model cellular systems to study mechanistic interactions with their microenvironment, giving rise to understanding of various physiological processes and disease mechanisms.

In addition to the capability of fabricating microenvironments that mimic physiological tissues, the versatility of 3D printing in creating large, complex shapes also enables the development of culture constructs that capture interactions of multiple tissues. While the 3D printing applications in fabricating bioreactors and alternative culture systems and 3D printing of droplet-based microfluidic devices for cell encapsulation sections focus on the use of 3D printing for dynamic culture systems (bioreactor, microfluidic devices), this section emphasizes on utilizing 3D printing to mimic specific biological features of tissues to capture the cellular function and physiology inside a culture platform. These features allow for the study of diseases related to that particular tissue. For example, a dual-chamber bioreactor setup was fabricated and fitted into a microfluidic base as a representative model of the interactions between cartilage and the subchondral bone [Fig. 1(c)].⁵⁹ The bioreactor chamber and parts were fabricated using a stereolithography apparatus (EnvisionTec), with Eshell 300, a printing-material system capable of generating fine features with a non-cytotoxic resin. The geometry allows insertion of a biphasic osteochondral construct made from GelMA-encapsulated MSCs into the bioreactor chamber, which consequently exposes the chondral and osseous sides of the construct to chondrogenic and osteogenic medium, respectively. Such interaction between tissues in a controlled bioreactor environment provides a path to investigating the osteochondral tissue physiology, and possible pathogenic mechanisms of relevant diseases in the system such as osteoarthritis.

The use of 3D printing for cell culture platforms also enables the development of miniaturized systems due to the high achievable resolution of various printers. For example, stereolithography-based printers have typically achieved a high spatial resolution, giving structures with dimensions of less than 10 μm,⁶⁸ while microstereolithography systems incorporating two-photon polymerization have been reported to improve the spatial resolution of printed scaffolds down to <1 μm.⁶⁹ The fine features observed with this fabrication method enable mechanistic studies of cells in tissues. For example, in addition to mimicking the key features of human cortical development, the brain-region-specific organoids were also employed to model Zika virus exposure, where a decreased neuronal cell-layer volume, resembling microcephaly, was observed [Fig. 1(a)].⁵⁷ Similarly, a 3D-printed fluidic microscale bioreactor has been developed for the characterization of gastrointestinal epithelial cell physiology.⁷⁰ The fluidic bioreactor was 3D-printed using the Object30 Pro extrusion-based printer with VeroClear-RGD810 (Stratasys), where it houses a porous villous scaffold that mimics the topography of the small intestine in vitro. This 3D platform provides both accurately sized villus topography and fluid flow to improve the study of intestinal absorption, drug delivery, and barrier function. Mimicking the structure-function relationship extends across the body. For example, a 3D-printed placental model has been developed, where spiral artery geometry was successfully fabricated using an extrusion based EnvisionTEC Bioplotter with GelMA. With the incorporation of bioactive factors contained in placental ECM, this 3D-printed model was then used to study cellular migration in the development of preeclampsia, by placing growth factor loaded resin precisely.⁷¹ When the length scale of the fabricated models mimics relevant physiological dimensions, mechanistic studies can be performed on the cellular level, e.g., investigation of the impact of surface topography and fluid flow dynamics on cell growth, proliferation, and organ cell function.⁷⁰ Additional biomimicry is possible by combining manufacturing methods. For example, combining electrospinning and 3D printing has produced tympanic membrane analogues which successfully align fibers and facilitate cell growth, a promising start towards an implant and functional replacement of native tissue.⁷² Further investigation, such as sound transmission or in vivo implantation, would further demonstrate feasibility. Continued development of these and other 3D-printed medical technologies is likely to increase the understanding of cell behavior and interactions as test devices improve biomimicry.

While the use of 3D printing for culture systems is typically geared towards mimicry of tissues and enhanced cellular functions, other popular applications of 3D-printed culture platforms include cell/tissue imaging. For example, a 3D-printed, modular perfusion culture system was fabricated using the Object 260 Connex extrusion-based printer (Stratasys) and then integrated with miniature peristaltic pumps for live-cell imaging assays of easily incorporated microfabricated scaffolds.⁷³ Due to the modular design, possible studies with this platform included investigating the cellular response to chemical stimulants down to the single cell level using fluorescence imaging in long-term perfusion cultures. Another example of the use of 3D printing in cell-based imaging applications is the UniverSlide, a 3D-printed microscopy chamber for multidimensional imaging.⁷⁴ The frame of the UniverSlide is fabricated using the Micro Plus stereolithography 3D printer (EnvisionTEC) and is meant for use in long-term imaging experiments due to the chamber's controlled environment, compatibility with commercially available microscope stage holders, and possible perfusion. Due to the incorporation of a transparent agarose pads with imprinted microwells as the base, the platform is ideal for cell trapping and subsequently 3D visualization and tracking. Though it remains possible to image cells within 3D-printed constructs without sectioning, such as with confocal or fluorescence laminar optical tomography,⁷⁵ designing test objects from the start remains the easiest method to observe cellular interactions and mechanistic changes.⁷⁶ 

3D printing is being used to create tissue models and artificial organs inside microfluidic devices with the goal of providing the complexity, function, and physiological responses of real organs. The field of organ-on-a-chip engineering has integrated 3D printing technology by assembling tissues containing cells, ECMs, and other biomaterials with precisely controlled spatial distribution, creating organ models with 3D specific cellular arrangement within a microfluidic chip. The incorporation of other mechanical and electrical components into organ-on-a-chip systems is simplified in fully 3D-printed systems, enabling automated mass production and facilitating commercialization.⁷⁷ The ease and high-resolution of 3D printing for organ-on-a-chip applications provide a promising alternative to animal studies and conventional cell culture for investigating various biomedical research questions.⁷⁸ Review articles have been written where summary and advances in organs-on-a-chip technologies for tissue engineering applications are provided.^79–81 In this section, we will focus on recent developments that specifically utilize 3D printing for fabricating functional organ-like models and molds for use inside microfluidic devices.

Combining precise geometrical features possible with 3D printing, well-defined flow patterns, and imaging capabilities of microfluidic devices, a perfusion microfluidic platform has been developed to simulate the blood-brain barrier (BBB) environment. The BBB model is composed of a porous membrane where brain microvascular endothelial cells and rat primary astrocytes are cultured on each side of the membrane [Fig. 2(b)].⁶⁷ The modular chamber consists of sterilized parts fabricated using an Objet 30Pro printer with VeroClear (Stratasys) assembled with a cell insert accommodating two cell monolayers forming a complete closed-loop perfusion system. Characterization of the in vitro model reveals a high-fidelity solution to studying the BBB biology due to the fluid residence time, perfusion rates, permeability coefficients of model drugs (caffeine, cimetidine, and doxorubicin), and trans-endothelial electrical resistance (barrier integrity) that mimic in vivo values. Such functionality means that the BBB-on-a-chip could effectively screen candidate drugs targeting the brain, overcoming the limitations of typical BBB transwell culture, where controlled biochemical gradients are difficult to achieve in the large static fluid volume.

Similar to the BBB-on-a-chip model, cocultures relevant to other disease models are constantly being developed and optimized to capture biological interactions forming disease mechanisms. A 3D bone-on-a-chip model made of PDMS has been developed, consisting of a cell growth chamber and a media reservoir that are separated by a membrane to investigate metastasis of breast cancer cells to host bone marrow.⁸² The PDMS chambers were fabricated by casting over a 3D-printed (Rostock MAX V2 Desktop 3D-Printer) mold, resulting in a transparent growth chamber that allows for easy and frequent monitoring of the breast cancer model. Growth and phenotypic maturation of mineralized collagenous bone tissue was observed simply by modifying the geometrical features of the membrane and the culture chamber, optimizing for nutrient and waste transport and providing appropriate concentration of bone matrix building proteins. Through 3D-printing, fabrication of this bone-on-a-chip geometrical design was enabled, allowing maximized cancer cell interaction with bone matrix of a concentrated surface area in a high-throughput experimental fashion. This serves as a reliable in vitro model that captures the complexity of the native bone environment and mimics in vivo processes, thus eliminating the need to obtain bone metastasis samples from human patients, which has been one of the major limitations in studying breast cancer bone colonization.

A 3D printing approach for a perfused liver organoid model on a chip has been demonstrated where the model entails the sinusoidal structure of the liver lobule as enabled by 3D printing (Cellbricks Bioprinter), with gelatin and poly(ethylene glycol) (PEG)-based bioinks.⁸³ Characterization of HepaRG (a human hepatoma cell line) and human stellate cells cultured for two weeks within the liver organoid-on-a-chip revealed higher expression of albumin and CYP3A4 proteins in 3D-printed tissues compared to monolayer culture. Hepatocyte functionality was shown through tight junction formation and stable overall metabolism by glucose, lactate, lactate dehydrogenase, and liver-specific bile transporter multidrug resistance-associated protein 2 levels. This liver-on-a-chip model serves as an alternative platform for complex 3D liver model development, as opposed to 2D models, which are not as physiologically relevant, or the gold standard 3D spheroid culture, which are limited by diffusion of nutrient and oxygen. Utilizing 3D-printing capabilities, the geometry of the developed liver-on-a-chip can be tuned to ensure adequate nutritional supply within larger tissue models, therefore providing a new avenue to perform mechanistic studies in liver tissue engineering.

The organ-on-a-chip field exhibits an increasing demand to integrate other systems (e.g., electrochemical components, sensors and actuators, and imaging systems) to fully capture the functionality of the organ in interest. In particular, as 3D printing requires a CAD model to produce shapes, incorporating scanning/imaging techniques into the fabrication of organ models are often applied. In the fabrication of a 3D model of arterial thrombosis, computed tomography angiography scans were acquired, constructed, and processed into a printable 3D model.⁸⁴ The molds for microfluidic chips containing miniaturized healthy and stenotic vascular structures were fabricated using a Perfactory 3 stereolithography 3D printer with PIC100 resin with the resolution set as low as 25 μm. Taking advantage of the printers resolution limits, artery models within a microfluidic device successfully recapitulated vessel environments: confluent coating of the vessels with human umbilical vein endothelial cells, flow of human whole blood at physiologically relevant shear stresses, and thrombosis induced at and downstream of the stenotic region were observed.⁸⁴ Mimicry of the shape, cellular environment, and functional response of this 3D-printed organ-on-a-chip model emphasizes the superiority of 3D printing over typical microfluidic fabrication employing 2D soft lithography. Traditional microfluidic fabrication is limited to generating two-dimensional microstructures or 3D structures with very limited thickness. These design limitations coupled to the multiple complicated and time-consuming fabrication steps highlight the advantages of 3D printings. Structured-light scanning has been used to capture 3D topographical data of whole organs to generate a 3D-printed microfluidic device that directly interfaces with porcine kidney as a non-invasive platform to isolate and profile biomarkers from whole organs in real time.⁸⁵ The functionality of this conformal microfluidic device was shown by the transfer of relevant metabolic and pathophysiological biomarkers from the cortex of the kidney to the microfluidic device, while fluid flow is present in the microchannel. The use of this device could potentially overcome the limitations of whole-organ studies simply by facilitating transport of relevant markers of the corresponding organ to a much smaller platform and the subsequent analysis.

Highly complex organs require complex in vitro models, often incorporating high numbers of inputs and outputs within the corresponding organ-on-a-chip system. By integrating 3D printing, templating, sensors, and system automation, a 16-channel microfluidic perfusion chamber has been developed for investigating endocrine tissues and secretions.⁸⁶ This device is capable of precise temporal manipulation of nutrient inputs and hormone outputs, shown by the measurement of real-time fatty acid uptake by adipose tissue exposed to a temporal mimic of post-prandial insulin and glucose observed by fluorescence imaging. The demonstrated flexibility of the listed platforms suggests feasible use of 3D-printed microfluidics as building blocks for integrated, modular organ-on-a-chip microfluidic devices. Further validation of these models could lead to broadly accepted use, becoming the new gold-standard for studies investigating mechanistic interactions of cell populations.

Orthopedic reconstruction with 3D printing technologies has garnered the greatest interest, likely due to the relative ease in manufacturing stiff materials and replicating the rigid structures. Materials range from stereolithographically cured poly(propylene fumarate),⁸⁷ to sintered ceramic-polymer blends.⁸⁸ By matching material choices, fabrication systems, and patient defects, 3D printing technologies can serve to improve patient outcomes. For bone implants, the use of materials and structures which induce bone regeneration and cellular infiltration presents a viable path for custom implants. The resorption, integration, and osteoconduction of bioceramics such as brushite and monetite have survived 12 weeks in goats having undergone decortication of the lumbar transverse process, with significant bone formation seen in intramuscular implants.⁸⁹ Metal coatings (e.g., with niobium)⁹⁰ or select stable polymers [e.g., poly(ether ether ketone)]⁹¹ can improve porosity, osseointegration, and differentiation with surrounding tissue. With these stable materials capable of and inducing de novo bone regeneration, it remains critical to choose material systems which facilitate expected fabrication and can support cell growth, even if serving as a sacrificial material.⁹² Combining biphasic calcium and zirconia in an extrudable paste has successfully been used to fabricate biomimetic objects which induce differentiation.⁹³ Greater complexity to these implants can be achieved with extrusion 3D printing approaches. Interweaving multiple materials together such as a bone morphogenetic protein-2 (BMP-2) containing collagen solution in between PCL/poly(lactic-co-glycolic acid)/β-tricalcium phosphate fibers becomes possible with custom extrusion 3D-printers.⁹⁴ Directly compounding PLA and hydroxyapatite filaments for fused deposition modeling allows direct integration with commercial fused deposition modeling systems (i.e., 3DPRN LAB 3D).⁹⁵ 3D printing materials containing bioactive components has resulted in greater bone regeneration over multiple months as compared to non-bioactive implants.⁹⁴ Mimicking calcified bone with bioactive scaffolds has worked to guide MSC osteogenic differentiation in otherwise difficult regions where traditional implants may not succeed, such as the anterior cruciate ligament [Fig. 1(d)].⁶⁰ Additional coatings, such as dopamine, can be added to induce osteogenesis and angiogenesis, regulating cell behavior towards a functional implant.⁹⁶ 

Total reconstruction of the knee looks within reach; aligning collagen fibers with 3D-printed acrylonitrile butadiene styrene (ABS) fibers forces cells to align in hopes of encouraging tendogenesis.⁹⁷ A 3D-printed poly(carbonate urethane)—ultra-high molecular weight poly(ethylene) meniscus fabricated with a Lulzbot TAZ 6 extrusion printer shows some promise as a replacement device with appropriate wear properties demonstrated, with further investigation needed.⁹⁸ Continued developments of these technologies may yield custom 3D-printed total knee replacements in the near future. A 3D-printed casing made of MED610 on an Objet Connex 3D printer (Stratasys) held alginate beads loaded with MSCs have successfully been cultured in the shape of a human femur, showing potential to regenerate large bone sections.⁹⁹ Integrating a vascular network and rigid structure would increase the clinical relevance of such an implant, ultimately moving towards a load-bearing bone-regenerating implant. Varying the hydroxyapatite content¹⁰⁰ and fiber oritentation¹⁰¹ in 3D-printed objects could lead to zoned-structured objects where integration between tissues could help improve implant acceptance by mimicking the non-uniformity seen in biologic interfaces. These orthopedic structures benefit from the patient specificity available with 3D printing. 3D-printed structures tend to either maintain the structural stability demanded in orthopedic implants or tissue engineered approaches to induce active osteogenesis and osseointegration of the surrounding tissue. Further work to marry the mechanical and biological aspects of bone implants is warranted.

Dental implants and craniofacial repair lend itself readily to 3D printing, with the complex topology and need for cosmetically appealing repair schemes. Particularly in craniofacial injury, cosmetic restoration can greatly improve psychosocial abilities, but relies heavily on the surgeon's skill.¹⁰² 3D printing could provide a way to rapidly and accurately repair these cosmetic issues and relieve strain on the surgeon. With accurate representation of anatomic details with relative dimensional differences less than 3%,¹⁰³ mechanical stability,¹⁰⁴ and biocompatibility,¹⁰⁵ 3D-printed titanium implants show promise for custom dental repairs and implants.¹⁰⁶ Proper material and design selections aid in osseointegration and new bone formation, particularly by considering specific patient needs and anatomy of the implant site.¹⁰⁷ In instances of severe head trauma, a two-part technique has been proposed, where a custom mold is fabricated and filled with poly(methyl methacrylate), pressed into large defect areas and completely sterilized prior to surgery.¹⁰⁸ Whether as a guide for bridging-plates to pack with autograft¹⁰⁹ or a directly printed and sintered implant,¹¹⁰ osseointegration aided by a highly porous, a custom-material scaffold is achievable with 3D printing technologies. With the breadth of applications and methods still under evaluation, one can easily imagine a time where 3D-printed implants are available, and not just in extreme clinical instances. Rapidly analyzing the implant area with radiography, simulating the mechanical loading of the 3D-printed implant digitally to confirm integrity, performing the surgery, and following-up with the patients lays a logical framework for point-of-use implant fabrication, verification, and implantation.¹¹¹ Continued development and validation with patients could see wide adoption of custom bone implants, seen to decrease operational time, additional fixtures needed, and complications observed.¹¹² Layering techniques also allow for replication of human anatomy unachievable through traditional manufacturing, including the use of phase changing substrates.¹¹³ Where orthopedic and dental applications are developed near a point of clinical relevance, soft tissues remain challenging. The complexity of native tissues has led to numerous approaches in efforts to recapitulate native tissues.

Vascular implants and soft tissues present unique challenges, whereby combining strength, flexibility, and cellular compatibility have led to the use of materials which degrade and are replaced by native tissue overtime. A simple, non-degradable approach to mimic an externally visible soft tissue (i.e., non-functional ear) uses radiographs of human anatomy to develop desktop 3D-printed (3DTouch) ABS molds for a two-part cast silicone part.¹¹⁴ Parts fabricated in this manner were approximately 1/10th the cost of a similar 3D-printed and handmade replacement devices of similarly low functionality, providing a visually appealing device restoring cosmetic function.¹¹⁴ Building complexity, sacrificial layers can be used to hold the shape, where multicomponent inks can directly place cells or localize bioactivity to direct seeded cell populations.¹¹⁵ A basic example of this involves a selective laser sintered (EOS P100) PCL carrier seeded with a collagen gel laden with chondrocytes which was successfully implanted as model ears and noses in pigs.¹¹⁶ However, pairing the specificity of materials and application certainly remains pivotal, where proper material selection encourages neo-tissue formation. A silk bioink formulated with gelatin and glycerol and fabricated with a custom bio-printer was shown to degrade over 3 months, with cellular infiltration and collagen replacing the subcutaneously implanted material in murine models.¹¹⁷ Keratin patches maintain sufficient structural integrity and cytocompatibility to facilitate cell growth, and has great potential as a DLP (EnvisionTEC) fabricated dermal patch or skin regeneration tool.¹¹⁸ A Gelatin-GelMA system has successfully been used to repair tympanic membranes in a chinchilla model, where loading the 3D-printed (EnvisionTEC Bioplotter) bioink with an epidermal growth factor was seen to improve healing numbers and cell invasion.¹¹⁹ Using a highly flexible, multiple material extruding printer allowed for the complex model to be fabricated, without the need for post assembly processes other than removing the support material. Poly(ethylene glycol) (PEG) diacrylate—alginate co-hydrogels with calcium and UV crosslinking have been proposed to increase the toughness and recovery of hydrogel networks by forming a double crosslinking network, increasing the applicability of otherwise unstable hydrogels within the clinic [Fig. 1(b)].⁵⁸ Another approach to strengthen 3D-printed hydrogel fibers, without degrading the overall shape or individual fiber, involves a “guest-host” network with adamantane and β-cyclodextrin functionality on hyaluronic acid.¹²⁰ The hydrogel rapidly deforms and reforms noncovalent reversible bonds as one hydrogel is extruded into the other and can support further chemical modification to further improve the bioactivity or functionality.¹²⁰ However, the difficulty for these large soft tissues remains in encouraging vascularization to prevent necrosis within the bulk.

To avoid this pitfall, channeling or printing lumens appear as a reasonable approach to improve the diffusivity of the bulk implant and formed neotissue to facilitate nutrient transport.⁵⁵ Printing a filament network using a modified RepRap Mendel 3D printer to define vascular lumens with a carbohydrate glass, casting a cell containing ECM, and dissolving the network filaments has showed a sustained metabolic function of primary rat hepatocytes.¹²¹ Typically, the suppressed function would be exhibited in a bulk gel without network and the alignment of endothelial cells along the open lumens within the bulk demonstrates vascular formation.¹²¹ Drawing these individual filaments to define the location of the vasculature becomes significantly easier when using an extrusion based 3D printing system. Similar work with combinations of GelMA and Pluronic F127¹²² and gelatin and collagen^123,124 show the ability to print vascular networks directly, allowing perfusion of the circuit, improved expression of functional messenger ribonucleic acid (mRNA) markers over static culture, and cell budding. The challenge remains in creating these kinds of open networks which support complex shapes and networks,¹²⁵ and can translate to a wide range of clinical applications: translation from the lab to the clinic remains extremely challenging. Engineering vasculature poses the greatest clinical need in tissue engineering, as without adequate vascularization, any large cell-containing implant will fail from insufficient nutrient exchange.

In addition, vascular issues remain of immense importance with heart disease remaining the leading cause of death in the United States, according to the Centers for Disease Control and Prevention (CDC).¹²⁶ Engineered vessels could provide a viable path to help combat this ailment, and other vascular problems, by replacing diseased or damaged vessels with functional, anatomy-matched implants. To help address this, a pericardium-PEG 3D-printed (EnvisionTEC Perfactory 4) hydrogels have been shown to have sufficient strength to form the aortic arch, and reduce inflammatory signaling.¹²⁷ Aortic valves from PEG—alginate hydrogels have been also shown to support cell growth and reform tissue, as observed within porcine tissue models.¹²⁸ A 3D-printed (EnvisionTEC Perfactory 4) poly(propylene fumarate) vascular graft remained patent for up to 6 months in a mouse model, with initial mechanical properties comparable to the native replacement tissue.¹²⁹ For both of these applications, the highly liquidus resin would not have been well suited for an extrusion or sintering based printing. The appeal remains that 3D-printed object can easily replace individual physiology by imaging the defect ahead of the surgery. With promising results seen in animal models, continued work would translate these technologies to human clinical use. Engineered vessels have worked as interim solutions for hemodialysis patients with failing shunts until a transplant organ was available.¹³⁰ 3D printing provides a unique pairing of complex geometry with soft resorbable materials and appears to be a viable strategy to solve vascular challenges without permanent implants or xenografts, which cannot remodel as the patient ages and carry concerns of host immune response, infection, and rejection.^131–133 

Several 3D printing applications have moved from development to the clinic. Starting with training physicians, on demand, 3D-printed, anatomic models are becoming more available and increasingly accurate, becoming emphasized in medical school curriculums over anatomic dissection.¹³⁴ Using anatomically colored analogues helps alleviate ethical concerns and reduces stress on cadaveric supplies, storage, and medical school financial burden.¹³⁵ 3D-printed anatomies generated from computed tomography images are sufficiently accurate (with less than 2% dimensional variation) to serve as suitable replacements for cadavers for both large (arm) and small (inner ear) anatomies.¹³⁵ 

Training on combinations of 3D-printed models and cadaveric anatomy can be beneficial for both medical students and practicing physicians. Surgical planning for complex procedures has been revolutionized with 3D printing. Where projected 2D images may be sufficient for simple procedures, instances exist where a 3D model better serves clinicians in determining the most appropriate course of treatment. From complex neck aneurisms,¹³⁶ to pediatric cerebrovascular lesions,¹³⁷ and complex spine surgeries,¹³⁸ 3D printing improves visualization and tactile manipulation of the defects prior to surgery, improving assessments and, reducing surgical time (by approximately 12% based on matched operations¹³⁷). These guides and tools serve the patients well; 3D-printed fixing aids and packing tools have been shown to improve surgical intervention success.¹³⁸ 3D printed devices (Cube 2) have been used to plan the filling of soft tissue voids and design skin grafts, aiming to restore stability to a wound site.¹³⁹ Outside the operating theater, forensic models of bone fractures, vessels, and soft tissues for presentation in courtrooms and classrooms have begun to gain interest, where color choices are used to draw attention to regions of interest.¹⁴⁰ 

Certainly, the greatest area of interest for 3D printing related to medical applications remains the fabrication of medical devices and implants. With the FDA approval of software to convert radiographs to usable formats for 3D printing, medical device manufacture has begun.¹⁴¹ As of 2017, the distribution of 510k cleared medical devices is 75% orthopedic, 13% surgical, 6% dental, and the remaining for other applications.¹⁴² However, the great appeal of 3D printing remains the extreme flexibility. FDA approval limits the scope of individual technologies, for good reason, to specific areas of treatment. This does ultimately hamstring the technology and may be why 3D-printed devices remain most prevalent in the research realm and extreme clinical situations. Even with the rigors associated with FDA approval, the benefits of bringing patient-tailored devices to market should exceed the costs of narrowed applications. A likely niche that meets these requirements is on-demand surgical tools, which have already been shown to have sufficient mechanical integrity to compete with traditional stainless steel tools at 1/10th the cost.¹⁴³ Manufacturing surgical tools within a sterile environment would negate the need for secondary sterilization and help to provide rapid access to surgical tools in the developing world.¹⁴³ 

3D printing has rapidly moved from a fabrication novelty to a ubiquitous manufacturing technique for biomedical applications. Though there remain many challenges for general acceptance, such as validation and repeatability, milestones within the 3D printing revolution have been reached. A future where medical technology manufacturers work closely with additive manufacturing centers and hospitals to answer various biomedical questions and solve numerous clinical problems that are otherwise difficult to decipher. As an acellular technique, 3D-printed implants and scaffolds show promise to increase the biomimicry of culture surfaces, creating specific objects for targeted problems. As a technique to study single cells, large cell populations, and organoids, 3D printing is unparalleled in its proven flexibility to investigate cell interactions across size scales. Overall, continued investigation into 3D printing technology and applications will only see improvements in clinical and research objectives.

This work was supported by the Maryland Stem Cell Research Fund under Award No. 2015-MSCRFI1717 (J.P.F.), the Maryland Industrial Partnerships grant (J.P.F.), the Maryland Stem Cell Research Fund/TEDCO Postdoctoral Fellowship under Award No. 2017-MSCRF-9320 (J.L.), NIST fellowship program 2014-NIST-MSE-01 (M.J.L.), and the National Institute of Biomedical Imaging and Bioengineering/National Institutes of Health (NIBIB/NIH) Center for Engineering Complex Tissues P41 EB023833 (J.P.F.). Certain commercial equipment, instruments, or materials are identified in this paper. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the materials or equipment identified is necessarily the best available for the purpose. Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Institute of Standards and Technology.

No competing financial interests exist.