Introduction of Three-dimensional Bioprinting and Additive Manufacturing

Definitions

Three-dimensional (3D) bioprinting is a tissue engineering fabrication method that uses spatial patterning of living cells and other biologic materials assembling them in a layer-by-layer deposition approach for construction of living tissues and organ analogs. In this way, precise control of the micro and microarchitecture can be controlled. Bioprinting is an additive manufacturing technique, as opposed to more common subtractive manufacturing whereby a large block of material is cut away until the desired shape or form is obtained. Although subtractive manufacturing is more efficient and economical for mass-produced, industrial scale manufacturing, subtractive manufacturing is unable to re-create the complex microarchitecture of biologic tissues. This architecture is critical to the function of biologic tissues and organs. Bioinks are the medium for printing and are available in a number of forms including hydrogels, microcarriers, tissue spheroids, cell pellet, tissue strands, and decellularized matrix components and are used with a variety of bioprinting processes including droplet-, extrusion-, and laser-based bioprinters. These different types of inks and techniques each have their own advantages and disadvantages and allow for the customization of a range of complex tissues required for orthopaedic applications.^1,2

Three-dimensional Bioprinting Methods

Extrusion-based Printing

Extrusion-based bioprinting is a pressure-based bioprinting method that has grown in use over the last decade (Table 1). Using a robotic system linked to a fluid-dispensing array, cells can be deposited in a 3D-shaped structures based on computer-aided design modeling. This process is done by using the shear-thinning behavior of the bioink and is generally plotted in cylindrical lines. This system is run by pneumatic, solenoid, or mechanical control. Each system has its own benefits and drawbacks depending on the spatial construct to be printed or bioink to be used.^1,2 Because of the flexible and larger diameter nozzle, and the ability to extrude bioink in a near solid state, bioinks, such as hydrogels, microcarriers, tissue spheroids, and tissue stands can all be used. This technique has demonstrated success with printing a number of different tissues including cartilage, lipid bilayers, lungs, and liver tissue deposition among others.^4–6 While comparing extrusion-based bioprinting to other methods, this printing method has the advantage of greater deposition and printing speed as well as anatomically correct porous construct generation. Fiscally, this printing method is commercially available and has high versatility allowing for the use of various different bioinks. Finally, the technology remains navigable by a novice user. Disadvantages include limited resolution which decreases the precision of patterning and organization of the cells, hydrogel use is complicated by the gelation and solidification requirements, and cells can be affected by the dehydration and the lack of nutrients.^1,2

Droplet/Inkjet-based Printing

Droplet-based bioprinting is an umbrella term that encompasses inkjet, acoustic-droplet-ejection, and microvalve bioprinting.¹ Development of this printing can be done by modification on a traditional printer by the addition of a controller to the print head for creation of a two-dimensional bioprinter.⁷ The bioink is then added to the printer via a storing cartridge. Inkjet printing is further subclassified into continuous, drop-on-demand, and electrohydrodynamic printing methods.¹ This method uses gravity, atmospheric pressure, and fluid mechanics to generate droplets that are delivered to a substrate. Continuous inkjet printing forces the bioink under pressure through a nozzle which then breaks into droplets as the potential energy is diminished. Drop-on-demand printing works to deliver single drops on demand by using a pressure pulses to push a drop through a nozzle that is held into place by surface tension. These pressure pulses are variable based on the system and can be piezoelectric, electrostatic, or thermal in nature. Electrohydrodynamic jet bioprinting uses an electric field to pull the bioink droplets onto the substrate, which again creates the droplet by disrupting the surface tension at the nozzle tip. When the generation of the electric field overcomes the surface tension, the droplet is ejected. Acoustic bioprinting eject droplets from a pool by generation of an acoustic field. This process helps to minimize exposure to excess pressure, voltage, heat, or shear. When the central focal point from a circular wave exceeds surface tension, the droplet is ejected. Finally, microwave bioprinting uses an electromechanical valve to control droplet release. This process occurs by the generation of a magnetic field with the use of a solenoid coil that ejects bioink droplets from the gated microvalve.

Laser-based Printing

Laser-based bioprinting is a form of bioprinting that uses laser-based modalities to initiate a droplet release. This process occurs by a two-layer approach. The top layer comprises an energy absorbing donor layer, whereas the bottom layer is the selected bioink (Figure 1). The release of the bioink droplet occurs when a laser pulse is emitted onto the top donor surface layer. This process creates a bubble at the interface when the top layer is vaporized, propelling the droplet onto the substrate. Laser-based bioprinting provides a number of advantages over other bioprinting methods. The first is the ability to avoid mechanical stress by eliminating the direct contact with the printer. Other advantages include high resolution and increased precision, which allows droplets to be printed more accurately to the template. Although resolution and precision are important factors in bioprinting, a number of disadvantages exist associated with the laser-based modality. The most formidable is the cost of laser-based systems. The equipment is large and complex, thus limiting its applicability in a standard research setting. Secondary to this, research applications have been somewhat more limited. Cellular effects on the viable components of the bioinks are also largely unknown.

Additive Manufacturing Concepts

Three fundamental principles of bioprinting exist, including biomimicry, biologic self-assembly, and mini-tissue units as building blocks.

Biomimicry

Hydrogels seeded with viable cells allow for patterning of cells. This cellular arrangement can recapitulate native anatomy. However, this alone is insufficient for successful bioprinting. The subsequent extracellular matrix (ECM) formation, the digestion and degradation of the hydrogel matrix, and the interactions and proliferation of encapsulated cells are all equally critical to the viability and functional success of the printed tissues. Limitations of tissue strand/spheroid formation using hydrogels include restricted cell interactions, proliferation, and colonization of immobilized cells within the hydrogel matrix, as well as the inability of cells to spread, stretch, and migrate to successfully generate the new tissue, particularly at high hydrogel concentrations. Bioinks fabricated with cell aggregates, without hydrogels, exhibit better biomimetic characteristics facilitating both homo- and heterocellular interactions because of the high cell densities and the lack of exogenous matrix immobilization seen when encapsulated in hydrogels. These tissue constructs closely resemble the native tissue and preserve cell phenotype and functionality for extended periods.

Biologic Self-assembly

Organ and complex tissue development evolution is fundamentally based on cellular self-assembly mechanisms. The tissue construct environment of the cells needs to resemble its native counterpart for cells to maintain their phenotype, establish appropriate cell-cell interactions, and express tissue-specific proteins along with ECM. Three-dimensional cell aggregate configurations allow for a more hospitable and more native anatomic environment for tissue self-assembly to occur compared with monolayer cell cultures. Tissue morphogenesis is dependent on the formation of multicellular aggregates. These aggregates are bound by cadherin molecules which facilitate strong intercellular adhesion. This cadherin mediated aggregates enable signal transduction, an increase in integrin expression as well as binding to arginyl-glycyl-aspartic acid motifs in the deposited ECM components.

Mini-tissue Units as Building Blocks

To bioprint scalable tissue, “mini-tissues” which represent the smallest composite tissue units can and should be used as building blocks. Such building blocks can be in spheroid or cylindrical form. Both the forms have been used in bioprinting.

Tissue spheroids represent a scaffold-free bioink-type, where the cells are organized spherically into 200- to 400-mm-diameter cell conglomerations. A number of different fabrication techniques have been used for fabrication of tissue spheroids including culturing cells in microwells with rounded ends on a cell adhesion inert mold made of hydrogels such as agarose, methacrylated hyaluronic acid, and alginate. In this approach, millions of cells are seeded into an array of microwells and cultured for 24 to 48 hours to facilitate cell aggregation. Cells will sediment to the bottom of the microwells and settle in close contact with each other, driving the cells to spontaneously adhere to one another to minimize free energy and develop into a neo-tissue.⁸ Because of intracellular cytoskeletal reorganization from cadherin mediated cell binding, tissue spheroids will diminish in size because of radial contraction.⁹ Other approaches which have also demonstrated success in tissue spheroid fabrication include the hanging drop method, microfluidic-assisted technology, and acoustic wave-assisted cell assembly.

Tissue strands are cylindrical neo-tissue building blocks that are used for bioprinting scale-up tissues like ink in an ink-jet printer.^9,10 To fabricate the tissue strands for bioprinting, cells at very high density are injected and packed into hollow alginate tubules.¹⁰ Semipermeable alginate tubules are used as these allow for exchange of nutrition and oxygen. The cells that are placed into the tubules will form into cylindrical neo-tissue strands as the cells self-adhere and pull away from the tubule walls. As with tissue spheroids, tissue strands will not bind to the alginate luminal surface. After cells have aggregated into the neotissue strand, the tube is dissolved using a decrosslinker solution. The formed tissue strand is then loaded into a custom-made bioprinter head and mechanically extrusion printed.

Cartilage Restoration and Reconstruction

Injury to the articular cartilage of joints is common. Current clinical restorative options for articular cartilage injury include marrow stimulating techniques, osteochondral grafting (auto and allogeneic), and cell-based techniques.^11–13 These options all have notable shortcomings to include cost, durability, potential disease transmission, and most notably the inability to re-create native articular cartilage architecture often resulting in a short-term solution that lacks durability.^11–13 The structure and composition of healthy articular cartilage is integral to its function.^12–14 This complex structure varies along the osteochondral axis, and many of the limitations of current techniques can be attributed to the lack of the native spatiotemporal control of biologic signals for guiding cell differentiation, hyaline cartilage formation, specific zonal biomechanical properties, and integration with the underlying bone.^11–14 The heterogeneous and anisotropic cartilage is composed of anatomic zones that possess zone-specific mechanical and biologic properties reflecting each zone's composition and architecture, which to date current clinical treatment technologies and tissue engineering strategies have been unable to recapitulate. Current cell-based techniques result in a disorganized repair tissue that has poor durability.¹³ Currently, osteoarticular allograft is the clinical strategy most often used for reconstruction of large osteochondral defects and injury.¹⁵ Though this can yield good results, there remains a limited supply of these live osteoarticular allografts, with wait times often of a year or longer for graft matching, during which time significant detriment to the adjacent joint surfaces and global environment of the knee occurs, not to mention additional pain and suffering, with lost work time/wages. Furthermore, as with all allograft tissues, disease transmission remains an issue and especially so with osteoarticular allografts as this involves live bone and cartilage transplants. Given the limited clinical success of current scaffold- and cell-based strategies, there remains an unmet need for chondral and osteochondral constructs that recapitulate anatomy, histology, and biology, which promote rapid integration, and provide a durable clinical solution to articular cartilage injury. The additive manufacturing strategy of 3D bioprinting provides a potential solution.

Bioinks

The foundation of biologic printing revolves around the use of bioinks. Bioinks are the combination of inert printing medium seeded with living cells. Together these components form the raw material which are deposited onto the collection substrate. The development has allowed manipulation of living cells to create biologic constructs. The ideal bioink is printable; has high mechanical integrity, high stability, insoluble in cell culture medium, nontoxic, and nonimmunogenic; and can promote cell adhesion. For the bioink to be effective, it must maintain its design strength and integrity for implementation in vivo. Several studies have evaluated various bioinks for their effectiveness in cartilage restoration. Alginate and agarose may better support hyaline-cartilage whereas gelatin methacryloyl- and poly(ethylene glycol) methacrylate-based bioinks may support more fibrocartilaginous tissues.¹⁶ These bioinks have also been shown to be biocompatible for cartilage growth.¹⁷ Two major types have been developed, which include scaffold and scaffold-free techniques. Their implications in cartilage restoration are discussed later.

Scaffold and Scaffold-free In Vitro Work

Bioprinting can be performed with or without a scaffold. Scaffolds used for fabrication of articular cartilage refers to biomaterials, either synthetic or naturally occurring, that are used to support the cartilage construct or may be used to assist in inducing repair from native host cells (Figure 2). Scaffold-based bioprinting involved the loading of cells into hydrogels or other carrier that can be deposited onto prior construct designs. These hydrogels can facilitate the generation of tissues via cell proliferation and growth. Hydrogels come in a wide variety of substrates and types, all with various advantages and disadvantages (Table 2). Scaffolds are attractive in tissue engineering as they allow for immediate structural integrity and can be used to control the spatiotemporal structure, development, and interactions of the cells and the developing ECM. Several criteria can be used in deciding the appropriate scaffold. Considerations include biocapability, porosity, pore size, mechanical strength, biodegradability, and ability to promote cartilage tissue formation.¹⁵ The scaffold also offers “Time Zero” mechanical load-bearing properties, a characteristic that bioprinted tissues often initially lack. Scaffolds can be fabricated with gradients in composition and/or architecture to mimic the mechanical properties of native cartilage and allow the distribution of appropriate mechanical and biologic cues to cells throughout the different zonal architecture. Total porosity and interconnectivity of the pores also play a large role in effectivity of a scaffold. This characteristic assists with cell adhesion and seeding as well as maintaining proximity to blood supply for efficient oxygen and nutrient delivery.¹⁸ A number of studies have looked at in vitro bioprinting using a scaffold. Abbadessa et al¹⁹ tested hydrogels containing polyethylene glycol and partially (10%) methacrylated poly(N-(2-hydroxypropyl) methacrylamide mono/dilactate) (M₁₀P₁₀) and methacrylated hyaluronic acid which increased storage modulus, slowed degradation, and improved printability versus M₁₀P₁₀ alone. These authors demonstrated chondrocyte growth at 42 days of culture. This knowledge base continues to grow as various laboratories examine the various components of the scaffolds to optimize cartilage cell growth.

Scaffold-free techniques use neo-tissues and deposit them in specific patterns on a substrate. These tissues are then fused and mature over time into larger functional tissues by the concept of mini-tissue units as building blocks. Scaffold-free techniques allow a high-density deposition of cells on initial print without the need for biomaterials, which was accomplished prior by the use of tissue spheroids that are printed in close proximity and fuse over time. Spheroids do have several issues, however. They require a delivery medium for extrusion which complicates the printing process. Since fusion occurs by proximity, premature fusion can cause nozzle clogging. Finally, gaps between printed spheroids have been shown from tissues with gaps which can leak.¹ Yu et al¹⁰ developed a novel technique which allowed the printing of biostrands (Figures 3 and4), which leads to a number of advantages which included facilitation of rapid fusion and maturation through self-assembly, bioprinting in solid form, removal of the liquid delivery medium and do not require a support molding structure during bioprinting for cell aggregation and fusion. In their study, the strands were deposited and cultured in vitro. Similar to in vitro, cells demonstrated good survivability. After 2 weeks, histological staining was completed demonstrating substantial proteoglycan deposition with positive staining for Safranin-O, similar to native cartilage control (Figure 4). Aggrecan and type II collagen were also present on immunohistological staining further characterizing the cartilage material. Self-assembly was tested and demonstrated as early as 12 hours post printing. On day 7, an almost complete tissue patch was present, allowing biomechanical testing of the product. The Young's modulus of the printed cartilage was tested in compression and found to be 1,094 ± 26.33 kPa similar to native cartilage. The authors postulated that the Young's modulus would be more similar if strands were cultured for longer.

The unique ability of bioprinting allows the development of precise patterning of cartilage cells with the complex structure of the cartilage layers to produce mimicry of the native structure of cartilage. The scaffold-free technique has allowed printing of near solid state tissue which does not require a liquid medium for cell facilitation, viability, or fusion. Current scaffold-based bioprinting mechanisms require a mold to host the cells which may limit the size of the printable constructs. Scaffold-free techniques may provide a way for printing of larger implantable cartilage patches with similar biomechanical and histological properties to that of native cartilage.

In Vivo Work

As we continue to work to find the ideal parameters and environment for the bioprinting of chondrocytes, a number of researchers have begun to work on in vivo transplantation of these cells. Both rat or mouse and rabbit models have been used with the transplantation of cartilage constructs to evaluate in vivo survivability.^20,21 Two different types of in vivo studies have been attempted thus far. Most work has been on survivability of the 3D bioprinted cells implanted subcutaneously in mice. These constructs were then retained for varying periods and the cells histologically evaluated upon removal. Work has been promising showing development of vascular membranes, chondrocyte proliferation, and lacunae development without loss of cell integrity.²¹ Shim et al²⁰ implanted 3D printed cartilage cells into rabbit knee joints. Upon evaluation, the test group showed evidence of neocartilage formation, osteochondral integration, lacuna formation, and a smooth cartilage cap in the area of the defect. Early evidence demonstrates both survivability and integration of bioprinted cartilage cells with the use of a scaffold hydrogel in vivo. This finding may aid in the development of future cartilage tissue engineering techniques and possible treatment alternatives.

Bone Restoration and Reconstruction

Notable shortcomings remain with bone grafting options for bone restoration and reconstruction. Autograft bone continues to be used frequently but has the major drawback of donor site morbidity. Allograft bone has been used extensively, but disease transmission, lack of osteogenicity, cost, and an already limited supply despite rapidly growing demand are all notable concerns. The field of bone tissue engineering seeks to reconcile these challenges and the growing unmet need for a viable bone grafting alternative by combining (1) a biocompatible scaffold that recapitulates the natural bone ECM niche, (2) inclusion of osteogenic cells to secrete the necessary ECM, (3) morphogenic signals that spatiotemporally biodirect the cells to the phenotypically desirable type, and (4) sufficient vascularization to meet the growing tissue nutrient supply and metabolic needs. Three-dimensional bioprinting allows for additive manufacturing of this dynamic tissue that includes a highly complex microarchitecture integral to its function. Though a number of tissue engineering strategies may be employed to tackle the challenges of bone tissue engineering, 3D bioprinting offers a better control over the structural and mechanical properties of the scaffold over other previously described techniques including gas foaming, salt leaching, and freeze drying. Furthermore, 3D bioprinting allows for better cell-cell interconnection, improved oxygen diffusion, and nutrient transportation and provides cells with the necessary attachment, proliferation, and tissue formation factors.²²

Bone grafts have been created using natural hydrogels such as fibrin or alginate.²³ However, the scaffolds created in vitro have poor compressive modulus making them inadequate for bone tissue engineering.²³ Alternatively, 3D bioprinting using synthetic polymeric polyethylene glycol dimethacrylate hydrogel provides compressive modulus that can exceed 500 kPa.^24,25 This process approximates human tissue compressive moduli.²⁶ Furthermore, the use of polyethylene glycol hydrogel will provide better cell viability and promote ECM production.^24,27,28 Addition of human mesenchymal stem cells (hMSCs) can regenerate bone tissues when stimulated by a ceramic scaffold.²⁹ Bioactive glass and hydroxyapatite (HA) have both been shown to promote bone tissue formation.^23,30 Collectively, HA in polyethylene glycol hydrogel is able to maintain hMSCs viability and promotes hMSC osteogenic differentiation and biosynthetic function.³¹ Qi et al³² demonstrated that using hBMSCs in conjunction with calcium sulfate hydrate/mesoporous bioactive glass scaffolds stimulated the adhesion, proliferation, alkaline phosphatase activity and osteogenesis-related gene expression of hBMSCs. The authors also demonstrated that calcium sulfate hydrate/mesoporous bioactive glass scaffolds could markedly enhance new bone formation in in vivo osseous defects compared with controls.³²

As in cartilage, bioprinting without a scaffold provides the advantage of avoiding cytotoxic or otherwise biologically deleterious breakdown byproducts of the scaffold. This approach leads to preservation of cell phenotypes and function through a facilitation of cell-cell interactions and a more robust deposition of ECM. Evinger et al³³ demonstrated the use of NovoGen bioprinting platform with adipose-derived mesenchymal stem cells and/or endothelial cells to produce osteopontin and alkaline phosphatase positive cells after 5 days post bioprinting. Osteogenesis was also confirmed by measuring bone modeling biochemical markers including interleulin (IL)-1α, IL-6, IL-8, C-C motif chemokine ligand 2, and chemokine (C-X-C motif) ligand 1.³³

Keriquel et al³⁴ have recently demonstrated that laser-assisted bioprinting is an attractive tool for the in situ printing of a bone substitute. These authors successfully used the laser-assisted bioprinting technique to bioprint mesenchymal stromal cells, associated with collagen and nano-HA in a calvarial defect model in mice.³⁴ Using droplet-based bioprinting, Herberg et al³⁵ added growth factors, including bone morphogenetic protein (BMP)-2, transforming growth factor-β1, and stromal cell-derived factor-1 beta (SDF-1β) on 5-mm-diameter acellular DermaMatrix disks, which the authors then implanted into osseous defects in mice. The co-delivery of BMP-2 and SDF-1 was superior to BMP-2-only and SDF-β-only groups in bone formation.³⁵ The addition of a bioink carrier to the mixture allows for solidification by polymerization to ensure the maintenance of the construct. MSCs co-printed with HA can create scaffolds with Young's modulus values of 1 to 2 MPa.³⁶ These constructs also were shown to produce substantial bone tissues. Another important element to the bone formation process was the incorporation of type I collagen with thermo-responsive agarose hydrogels bioinks. This combination with high-collagen ratios optimizes the mechanical stiffness required for differentiation and provides precise contours for the constructs.³⁶

Das et al³⁷ demonstrated osteogenic differentiation of mesenchymal stem cells encapsulated in silk fibroin–gelatin which was cross-linked either by the enzyme tyrosinase during bioprinting or by sonication post-bioprinting.³⁷ The authors also demonstrated that by changing the culture media, they were able to differentiate cells with a construct that varies in mechanical properties and function.³⁸ Raja and Yun³⁹ also used this technique by depositing in a coaxial manner calcium-deficient HA and alginate laden with pre-osteoblast MC3T3-E1 cells,³⁹ leading to the engineering of bone tissue composites with the compressive modulus of 7.01 ± 0.82 MPa. Additive manufacturing of bone using 3D bioprinting offers the advantage of creating composite tissues including the ability to integrate essential vascularity to the bone tissue fabrication (Figure 5). Kolesky et al³⁸ have demonstrated this with the ability to create a 1-cm-thick tissue with embedded vasculature for tissue perfusion.

Clinical Application and Future Direction

A key advancement in clinical bioprinting of orthopaedic tissues, whether bone or cartilage, will be integration with computer-aided design, where medical images acquired by CT, MRI, positron emission tomography, and ultrasonography can be directly fed into a processor, which can rapidly produce the design through image reconstruction algorithms. This step involves image segmentation and mesh generation followed by mesh optimization re-creating highly intricate irregular geometric models. The mesh is then converted into a path plan for 3D bioprinting which can be directly fed into a bioprinter. These designs would be patient-specific with respect to not only the geometry of implant but also the level of tissue insufficiency and the anatomy of the composite tissue as well as the vascular network. Several advances in tissue segmentation algorithms from different imaging modalities have recently emerged that may eventually be used for bioprinting of composite vascularized tissues.

Summary

Notable shortcomings exist in the currently available surgical options for reconstruction of bone and articular cartilage defects. Three-dimensional bioprinting as an additive manufacturing tissue engineering technique incorporates viable cells and ECM for layer-by-layer fabrication of highly complex tissues such as bone and cartilage. Because of the scalability of 3D bioprinting, this technology has the ability to fabricate tissues in clinically relevant volumes and addresses the defects of varying sizes and geometries. Adhering to the principles of biomimicry, biologic self-assembly, and the use of mini-tissues as building blocks, notable success has already been achieved with cartilage and bone tissue bioprinting using extrusion-based bioprinting using alginate carriers and scaffold-free bioinks. Fabrication of composite tissues has been achieved which includes vascularity, a necessary requisite to tissue viability. As this technology evolves, and we are able to integrate high-quality radiographic imaging, computer-assisted design, computer-assisted manufacturing, with real-time 3D bioprinting and ultimately in situ surgical printing, this additive manufacturing technique can be used to reconstruct both bone and articular cartilage and has the potential to succeed where our currently available clinical technologies and tissue manufacturing strategies fail.

References

Evidence-based Medicine: Levels of evidence are described in the table of contents. In this article, reference 13 is a level IV study. References 12 and 14 are level V reports or expert opinions.

References printed inbold type are those published within the past 5 years.