Bioprinting Based Tailoring of in Vitro Tissue Models

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Review

3D printing/bioprinting based tailoring of in vitro tissue models: Recent advances and challenges Shreya Mehrotra, Joseph Christakiran Moses, Ashutosh Bandyopadhyay, and Biman B. Mandal ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00073 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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ACS Applied Bio Materials

3D printing/bioprinting based tailoring of in vitro tissue models: Recent advances and challenges Shreya Mehrotra, Joseph Christakiran Moses, Ashutosh Bandyopadhyay, Biman B. Mandal* Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, Assam *Email id: [email protected]; [email protected] Abstract Prodigious progress in the last decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics the native human tissues. With the advent of technology, several improvements in printing techniques has facilitated the development of intricate strategies and designs which were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough co-ordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, yet much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyse and learn from several endeavours such that shortcomings can be understood and future efforts can be made towards negating them. Taking in account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements and the challenges in this field are also discussed. 1 ACS Paragon Plus Environment

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Keywords: Bioprinting, tissue engineering, organs-on-a–chip, bioinks, in vitro disease models, challenges

1.

INTRODUCTION

In less than a decade, latest developments from aerospace engineering to food industry are all resonating on the notes of 3D printing. This technique, also known as additive manufacturing acquires its name from the additive process that occurs by the successive deposition of materials layer-by-layer to form three dimensional shapes and objects.1 A particular implication of 3D printing that has intrigued several researches and already revealed promising leads is in the field of tissue engineering.

Tissue engineering as a

multidisciplinary field majorly centres on two broad facets (i) developing new approaches to repair and regenerate impaired tissues and organs and (ii) creating in vitro tissue models to either understand tissue and disease development or to help screen drugs.2-3 Much of the complex micro- and macro-architectures that are needed to achieve the above two goals, previously unachievable by the traditional scaffolding methods, have now come to existence by the 3D printing techniques. First instance of printing or cytoscribing biologicals and cells were demonstrated in 1988 by Klebe et al, where an inject printer was customized to place cells in specific position in two dimensional substrates4. Subsequently, Odde et al in 1999 utilised a direct write laser guided strategy to facilitate three-dimensional patterning of cells up to few micrometres in height 5. Though 3D printing inert ceramic/polymeric materials was demonstrated in 1990 by Sachs et al. 6, encapsulating viable cells in suitable bioinks and bioprinting intricate multi-layered structures was still in its infancy. With the dawn of the 20th century immense improvements in this field had taken place and a detailed account of this evolution is discussed in detail by 2 ACS Paragon Plus Environment

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Ozbolat et al 7. Pertinent to note, issues relating to (i) scaling up of bioprinted constructs; (ii) maintaining cellular viability and functionality within the bioink; (iii) wide array of smart bioinks developed from different biomaterials for tissue-specific use, bestowed with multifunctional traits and (iv) precision, spatiotemporal control and full-automation of bioprinting processes have been critically addressed over the years

8-9.

Thus, 3D bioprinting

has ushered in an era where rapid prototyping of human tissues is possible.3 With the advent of numerically controlled programming languages such as G-codes and computer-aided automation, construction of geometrically complex structures have been made fairly easy.10 High-resolution stepper motors and programmable microcontrollers, as a part of the printing machine, enable the precise modulation of geometries, porosities, mechanics and biological components for creation of microscopic and macroscopic structures that are physiologically relevant. These robotic deposition methods allow and ensure high degree of reproducibility and micron-level control in the whole fabrication process.11-12 To precisely mimic the complex biological tissues, 3D bioprinting technology utilizes a computer aided 3D design (CAD file) created by virtually breaking down the shape of the tissue, which may either be obtained from computed tomography or magnetic resonance imaging, into a series of 2D layers. The 3D bioprinter then deposits bioinks in a layer-by-layer based on the CAD file where each layer is bonded to the previous layer to fabricate 3D constructs. Small units such as cells and growth factors can be either printed along in a layer by layer fashion or may be decorated post printing onto the constructs to create viable and functional tissues. Taking clues from earlier lithographic methods and with the advent of breakthroughs in developmental biology, nanotechnology and electronics engineering, 3D printing technique has now carved a niche for itself in tissue engineering. In these lines, this review provides an overview of the several printing methods that are available to the researchers and their several applications from creating small sized constructs to micro-vascularized organ models. Taking 3 ACS Paragon Plus Environment


notes from the plethora of previous studies, the review also brings forth the bottlenecks that still exist for this technology and how with the advent of technological innovations these shortcomings can be met. 2. MODES OF 3D PRINTING IN TISSUE ENGINEERING 3D printing utilizes a bottom-up approach, in which the individual components of the tissue are patterned to allow for formation of complex tissue architecture.13 By utilizing CAD files , one can now cautiously control the placement of cells, materials and biomolecules to recapitulate the innate tissue organization found in the human body. In the recent years, this field has evolved immensely with the development of a variety of methods for creating 3D objects. These methods differ much in their capabilities according to the materials used from polymers to ceramics, cross-linking mechanisms and extrusion techniques.14 A complete control over matrix architecture, mechanical properties, rate of degradation and inclusion of biological components can be maintained depending upon the type of printing technique used.15 Exploitation of the above features in combination with the application-specific ink preparations generates a platform for formulating patient customized materials and devices. The most frequently used bioprinting and biofabrication technologies include the Microextrusion based, Inkjet based and Laser-guided based techniques. However, many nonconventional and upcoming specialized technologies have also been developed that have specific applications and can be highly convenient for developing up-scaled biologically functional tissue constructs with specialized features. 2.1 INKJET BIOPRINTING Inkjet bioprinting has its roots in the traditional office printing method.16 This technique has evolved from a slightly modified deskjet printer that could hold cell-laden bioink in its cartridge and fabricate multi-layered 3D constructs. The technique simply involved ejecting 4 ACS Paragon Plus Environment

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the bioink from its printer nozzle onto a printing surface, while moving in the X-Y-Z-axes. It is a non-contact bioprinting technique that can build 3D structures by depositing droplets of bioink containing cells in a layer-by-layer fashion. The two most common approaches that are applied in the dispensing process in this technology are piezoelectric and the thermalbased systems (Figure 1 I). 17-20 The piezoelectric mechanism involves the rapid actuation of a piezoelectric actuator that generates enough pressure momentarily to expel bioink from the cartridge onto the print bed. While, the thermal approach involves the use of a heating element to raise the temperature of the bioink at the nozzle to reach more than 300˚C for a few microseconds resulting in the formation of vapour bubbles that expand until the bioink is expelled from the nozzle. Since the rise in temperature is only for a few microseconds the overall rise in the temperature of the bioink is negligible and the viability of the cells and other biological components remain minimally affected.19 The inkjet dispensing method allows greater control over the volume of bioink to be extruded ranging from nanoliters to picoliters which not only produce higher printing resolution but also lower the requirement of greater volumes for patterning of cells and biologics. This makes this technique consistent in terms of 2D deposition and can reach up to 10000 droplets per second.20 However, the use of this technology is innately restricted by the narrow viscosity range (in the order of 0.1 Pa.s) for the bioinks and cell densities that are compatible with this printing method.16 High viscosity of the bioink tends to cause clogging of the nozzle as do very high cellular densities.

21

Also, the limitation of instantaneous

crosslinking of the low viscosity bioink, required for making self-standing constructs, poses a major challenge. Solutions such as the use of photo-crosslinking polymers that can be crosslinked on the go as well as use of chemical and pH based crosslinking polymers have been employed as corrective measures for the efficient fabrication of 3D structures. Addition of mild surfactants and other additives can be assistive for prevention of cell aggregation and 5 ACS Paragon Plus Environment


clogging of the nozzle, but however may lower cellular viability. Despite these corrective measures reproducibility and throughput of droplets, range of shear forces within the nozzle, cellular sedimentation in the cartridge reservoir, and the number of inks that canbe printed during a single tme experiment are some of the other limitations and challenges associated with this technique.16 2.2 MICRO-EXTRUSION BASED Extrusion based bioprinting has been the most used and promising technique for fabricating living constructs. It principally involves continuous layer-by-layer programmed deposition of bioink through a dispensing nozzle (printing head) using either a mechanical (screw driven or piston) or pneumatic dispensing system, onto a substrate in discrete volumes (Figure 1 II). Latest interventions for dispensing bioinks using this method include electrospinning and microfluidic based dispensing techniques for dispensing lower volumes and printing with better resolutions. The bioinks generally used in this fabrication method can comprise of a viscoelastic biocompatible polymer that doubles as a substitute for the extracellular matrix as well as plays a vital role in incorporating the other biological components such as growth factors and cells. For fabrication of 3D constructs, this method mainly relies on stepper motors and pneumatic actuators for the movement along the X, Y and Z axes. The discrete volumes of bioink that can be deposited fall in the range of microliters according to the specifications of the orifice that is employed for the deposition. Bioink deposition is mostly accompanied by its instant polymerization out of the nozzle leading to a self-standing structure in each layer. The crosslinking methods may be either physical or chemical in nature. A variety of biocompatible polymers such as PLGA, agar, polylactide, gelMA, sodium alginate, gelatin, silk can be used to bioprint using this approach.

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Micro-extrusion based techniques are the easiest approach for bioprinting in-terms of ease of programming, robust handling, widest range of bioink viscosities, easy polymerization approaches and highest cell densities that can be employed for the fabrication of self-standing structures. Nevertheless, this technique does have its limitations such as generation of shear stress and biomaterial selection due to the exigency for immediate encapsulation of cells via gelation.16 Also, the ability to deposit specific volumes and the precision offered by this printing method is limited by the size of the orifice and applied force which may lead to loss in the viability of cells and other biologics.

Figure 1. Printing mechanism behind (I) Inkjet based and (II) Micro-extrusion based techniques. 2.3 LASER BASED PRINTING Laser guided direct writing (LGDW) is one the latest techniques for bioprinting three dimensional tissues with microarchitectures. This technique involves the use of a low energy pulsed laser for the transfer of the cell-laden bioink from a donor layer onto a substrate by the principle of laser tweezers (Figure 2 I).22 Two widely used techniques under LDW are laser7 ACS Paragon Plus Environment


induced forward transfer (LIFT) and matrix-assisted pulsed laser evaporation direct writing (MAPLEDW). Although, the two techniques are schematically similar, MAPLEDW works on a lower powered pulsed laser in comparison to LIFT. These techniques allow precise deposition of cells in relatively small 3D structures. The optical forces generated from the laser beam controls the deposition of the low volumes of bioink in a programmed fashion with high resolution.16 The donor slide is moved in a pre-designed manner to generate intricate 2D layers that can be stacked to form 3D objects. Being a nozzle-free approach, this technology eliminates the problem of clogging of the nozzle on using high viscosity polymers. Providing high precision characteristic, lasers may offer the benefits of bioprinting the smallest features of an organ. Also, the distribution of cells can be regulated by the energy of the laser beam focussed onto the donor substrate and accurately distributed cells can be patterned on the receiving substrate with ease. Conversely, this technology also has a number of impediments. Using a powered laser light generates heat that may damage the cells or alter the capacity of cells to communicate and grow in the final tissue construct.16 Moreover, gravitational as well as arbitrary settling down of cells in the precursor ink solution, extended fabrication time, limitations of printing in the Z-dimension and the obvious need of photocrosslinkable biomaterials are some of the other limitations faced in laser-based bioprinting.23 2.4 ACOUSTIC DROPLET EJECTION Acoustic droplet ejection (ADE) is similar to inkjet printing but in an inverted manner making use of a nozzleless ejection technology (Figure 2 II).15 It is a droplet-based bioprinting method that encompasses the use of an acoustic transducer to generate surface acoustic waves. These waves when projected onto a focal point on a liquid surface result in formation of a mount of liquid that can be ejected onto the surface as a droplet. This nozzle8 ACS Paragon Plus Environment

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less contact-less method has dynamic control over the volume of fluid to be deposited as well as on the precise placement of the droplets.15 Droplet size can be manipulated by modulating the frequency of the acoustic wave. It is one of the most gentle processes for precise deposition of cells and biologics.24

Figure 2. Schematic illustration of (I) Laser based and (II) Acoustic Droplet ejection bioprinting techniques. 2.5 MICROVALVE BASED PRINTING Microvalve-based printing technique stands as a middle ground between droplet-ejection and micro-extrusion techniques. A representative microvalve bioprinter would consist of threeaxial robotic control platform attached to a printhead-array containing an electro-mechanical valve. Each of these printheads contain or are connected to a pneumatic regulator which helps with the positive displacement pressure as well as the valve opening and shut-off. Each microvalve set-up is controlled by the pneumatic pressure controlled plunger and the solenoid coil. The plunger is set to an ascending motion by the magnetic field of the solenoid coil and the orifice of the extruder is opened to deposit droplets of bioink.25 The process of bio-ink deposition is dependent upon the orifice diameter, bioink viscosity, the pneumatic positive pressure used and most importantly the valve opening duration.26 9 ACS Paragon Plus Environment


The major advantage offered by this printing technique is a blend of microextrusion and drop-ejection techniques. Henceforth, the precision of droplet deposition offered by the valve based system is added upon the vast range of bioink viscosities that can be used due to the pneumatic pressure and orifice based extrusion. This technique also provides the feasibility of very thin material layer depositions (1-2 μm), synchronous material and cell deposition from different printheads, precise positioning of cells13 with excellent viability (>86%)27-28 and a high-throughput deposition rate (~1000 droplets per second).26 Moreover, the affordability and user-friendly nature of these bioprinting systems serves as an addendum for its deployment.29 However, the converse factors that limit this printing modality are lower range of bioink viscosity supported by this technique as compared to the micro-extrusion modality and the difficulty of clogging orifice (100-250 μm)27, 30 at cellular densities higher than 106 cells/ml. Moreover, the challenge of cell-sedimentation over time leading to inhomogeneity of cellular deposition needs to be addressed. 2.6 3D CELL PRINTING 3D cell-printing is a direct cell deposition technology that uses solid cellular units as additive components for printing scaffolds free cellular constructs31. The cellular aggregates range between 250-500 µm

31

Cells are aggregated in moulds after shaking them in a suspension

obtained from 2D cell culture techniques.31-32 This technology employs positive displacement for directly extruding multiple cell types into complex geometries. While, the structures printed are fragile and lack strength initially, they mature over time and demonstrate resemblance to normal tissues. The advantage of this technique is the use of intrinsic property of cells for scaffold-free fabrication and avoiding any chemical or physical crosslinking.33. But, the process has its limitations of being lengthy, specific to only particular cell-types, occasionally detrimental to cell-viability and limited handling ability of constructs postfabrication. 10 ACS Paragon Plus Environment

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2.7 STEREOLITHOGRAPHY Stereolithography (STL) technology was a major breakthrough in the field of electronics in the 1980s and was eventually adapted in 3D bioprinting with the advent of photo-crosslinkable biopolymers and biomaterials such as methacrylates, epoxies and diacrylates. STL based bioprinting is a nozzle-free technology that involves the utilization of a photo-sensitive bioink that gets solidified in a layer-by-layer fashion according to the light falling on it.34-35 A digital micro-array of mirrors is used for controlling the pattern of light that falls on the substrate containing the photo-sensitive bioink. However, various limitations follow the use of this method such as the lack of biodegradability within the biopolymers, toxicity of the residual photo-cross-linking agents, reduction in cell viability in case of UV cross-linking and difficulty of producing horizontal gradients within the constructs. 2.8 SELECTIVE LASER SINTERING Selective Laser Sintering (SLS) is a prominent technology used in the formation of 3D ceramic, polymer and metal-based structures by the use of sintering process to selectively fuse layers on top of each other by a high powered laser scanning the surface of the material to be sintered.36-37 The mechanical properties of the sintered constructs can be dictated by the scanning speed of the laser. while the energy of the laser can be harvested and modulated to influence the nature of sintering from solid-state to liquid phase.

38-39

This technology has

been mainly employed for the fabrication of cell-free 3D constructs for bone and cartilage repair using polymers such PCL and cellulose, ceramics such as hydroxyapatite and bioglass 40-41,

metal

alloys

such

as

Ti6Al4V

and

CrCo42

(CP)/poly(hydroxybutyrate–co-hydroxyvalerate)(PHBV),

and

composites

carbonated

such

as

hydroxyapatite

(CHA)/poly(L-lactic acid) (PLLA) and HA/polyetheretherketone (PEEK).43 Though, these scaffolds have higher mechanical properties and durability but their slow degradation, lower 11 ACS Paragon Plus Environment


resorption and cell-free dry fabrication results in their limited applications for many tissue types. Each of the 3D printing approach described above entails its own advantages and limitations. Suitability of a particular approach can be decided on the basis of the application and the relevant parameters needed for printing a specific tissue. For instance, inkjet based biofabrication techniques can be used for precise placement of nano-liter volumes of cells but the construction of a bulk three dimensional tissue structure can be performed by microextrusion bioprinting more efficiently while compromising on the resolution of deposited bioink. Similarly LIFT and acoustic deposition can achieve higher cellular viabilities but fall short on the speed of fabrication with respect to physiological scale 3D structures. Hence, a synergistic balance created by the combination of more than one bioprinting technologies depending on the resolution needed and the advantages they offer (as described in Table 1) can be used for fabricating fully functional physiologically relevant multi-layered organs with complex micro-architecture of cells. Table 1. Overview of the existing printing techniques commonly used for fabricating 3D tissue consructs


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Printing Techniques

Resolution

Advantages

Disadvantages

Cell

Commercially

Viability

available bioprinters

Inkjet Bioprinting Piezoelectric /

100 μm

Thermal Inkjet Electro dynamic

10–20 μm

High speed

Low precision of

80-85%

JetLab 4

linear

droplet size and

(MicroFab),

fabrication

its placement, low

Aplha/Omega(3Dy

availability

viscosity bioink

namic Systems),

needed, slower

BioAssemblyBot

volumetric speed

(Advanced Life Sciences), Aether 1(Aether), Allevi 1,2,6(Allevi),), Inkredible+, BIO X (Cellink), Bioplotter (EnvisionTEC

Acoustic Droplet

37–150 μm

Ejection (ADE)

High precision

Low viscosity

and controlled

bioink needed

~ 95%

directionality Micro-Extrusion Bioprinting Mechanical/Pneumatic Extrusion

15–400 μm

Extrudes high

Distortion of cell

40-90%

BioX (CellLink),

viscosity

structure and

based on

3D-Bioplotter

bioinks and

chances of

the force

(EnvisonTEC),

prints with

reduction in cell

of

3DDiscovery

higher cell

viability

extrusion

(regenHU),



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density

BioScaffolder 3.1 (GeSiM

Laser Based Bioprinting BioLP / AFA-LIFT /

10–100 μm

MAPLE-DW Laser Guided Direct

100 nm –

Writing

10 μm

High resolution,

Very low

~ 95%

precision,

volumetric speed,

Regenova(Cyfuse

ability to use

time consuming,

biomedical),

medium to high

high cost

Regemat 3D

viscosity bioink

Poietis(France),

V1(Regemat 3D)

and print higher cell density

Stereolithography

∼1 mm

(SLA)

Highly accurate

UV light exposure ~ 90%

and quick

is potentially

fabrication

harmful for cells under long exposure, lengthy post-processing, limited number of compatible biomaterials

Direct 3D Cell

260-500

Printing (3DP)

μm

Scaffold-free

Structures are

printing method

initially weak and

and no physical

difficult to handle

or chemical

mechanically,

crosslinking

require time to

required

mature and


~ 95%

Regemat 3D V1(Regemat, Spain

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become selfstanding tissue and specific to limited cell types

20–200 μm

Selective Sintering (SLS)

Laser

High

Cell-free

mechanical

fabrication

strength

method, specific

scaffolds

to few materials

fabricated

that can be

-

sintered, slow degradation and resorption of scaffolds

3. APPLYING BIOPRINTING TOWARDS TISSUE AND ORGAN REGENRATION Based on Department of Health & Human Services’ (HSS, USA) Organ Procurement and Transplantation Network (OPTN) annual report (2018), every 10 minutes is added to the organ transplant waitlist and 20 people die each day awaiting a transplant. There is an enormous unmet demand for viable donor tissue which can be effectively addressed by biofabrication strategies. 3D printing offers the feasibility to fabricate clinically viable heterogeneously distinct tissue constructs all the while mimicking the structural and 15 ACS Paragon Plus Environment


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functional integrity of tissue. 3D printed constructs are commercially available and are being increasingly used in clinical trials (Table 2) for functional tissue reconstruction and cosmetic augmentation. Here we discuss in this section some of the recent advances made in different tissues listed below. Table 2. List of ongoing clinical trials obtained using the keyword “3D Printing” from ClinicalTrails.gov (Source: https://clinicaltrials.gov/, accessed 13.08.18) Identifier

Study Title

Intervention/

Sponsor

Phase

Treatment NCT03416387 Applicability of 3D Printing Liver diseases/ Hospital and

3D

Digital

Reconstruction

Image Surgery

in

the

Planning of Complex Liver

Recruiting

Universitario Virgen

de

la

Arrixaca, Spain

Surgery NCT03152916 Application of 3D Printing Ankle injuries Technology

in

Subtalar

Southwest

Recruiting

Hospital, China

Arthrodesis NCT03348293 Safety Study of 3D Printing Breast Personalized Biodegradable Reconstruction Implant

for

Xijing Hospital, Recruiting China

Breast / Breast Cancer

Reconstruction NCT03292679 Craniofacial Applications of Facial Fracture 3D Printing

of Recruiting

Maryland, USA

NCT03153332 Value of 3D Printing for Liver cancer Comprehension

University

of

Liver


Guangzhou Women

Observational and

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Surgical Anatomy

Children's Medical Center, China

NCT03057223 Three-Dimensional Printing Mandibular

The

of Patient-Specific Titanium Neoplasms

University Recruiting

of Hong Kong

Plates in Jaw Surgery: A Maxillary Pilot Study

Neoplasms Dentofacial Deformities Maxillofacial Injuries

NCT03166917 Clinical Personal

Application

of Internal

Shenzhen

Designed

3D Prosthetic

Hospital

Printing Implants in Bone Device, Defect Restoration

Implants

Recruiting of

Southern and Medical

Grafts,

University,

Orthopedic,

China

Bone Graft NCT03185286 3D-Printed

Personalized Bone Diseases

Metal Implant in Surgical

Southwest

Recruiting

Hospital, China

Treatment of Ankle Bone Defects NCT03365804 A New Spinal Brace Design Adolescent Concept for the Treatment Idiopathic of

Adolescent

Idiopathic Scoliosis 17 ACS Paragon Plus Environment

University

of Not

Alberta, Canada

recruiting

yet


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Scoliosis NCT03416387

Applicability of 3D Printing

Liver diseases/

Hospital

Recruiting

and 3D Digital Image

Surgery

Universitario

Reconstruction in the

Virgen de la

Planning of Complex Liver

Arrixaca, Spain

Surgery NCT03152916 Application of 3D Printing Ankle injuries Technology

in

Subtalar

Southwest

Recruiting

Hospital, China

Arthrodesis NCT03348293 Safety Study of 3D Printing Breast Personalized Biodegradable Reconstruction Implant

for

Xijing Hospital, Recruiting China

Breast / Breast Cancer

Reconstruction NCT03292679 Craniofacial Applications of Facial Fracture 3D Printing

University

of Recruiting

Maryland, USA

NCT03153332 Value of 3D Printing for Liver cancer Comprehension

of

Liver

Guangzhou Women

Surgical Anatomy

Observational and

Children's Medical Center, China

NCT03057223 Three-Dimensional Printing Mandibular of Patient-Specific Titanium Neoplasms Plates in Jaw Surgery: A Maxillary Pilot Study

Neoplasms Dentofacial 18 ACS Paragon Plus Environment

The

University Recruiting

of Hong Kong

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Deformities Maxillofacial Injuries NCT03166917 Clinical Personal

Application

of Internal

Shenzhen

Designed

3D Prosthetic

Hospital

Printing Implants in Bone Device, Defect Restoration

Implants

Recruiting of

Southern and Medical

Grafts,

University,

Orthopedic,

China

Bone Graft NCT03185286 3D-Printed

Personalized Bone Diseases

Metal Implant in Surgical

Southwest

Recruiting

Hospital, China

Treatment of Ankle Bone Defects NCT03365804 A New Spinal Brace Design Adolescent Concept for the Treatment Idiopathic of

Adolescent

University

of Not

Alberta, Canada

recruiting

Idiopathic Scoliosis

Scoliosis

3.1 SKIN Tissue engineered skin substitutes have resulted in bringing about a paradigm shift in wound care management and skin grafting. Although these skin substitutes have provided benefits in treating chronic or burn wounds, their commercial viability is crippled because of inability of their large-scale production44. Bioprinting overcomes this drawback through automated


yet


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fabrication processes and also helps in addressing some key concepts such as vascularization, hair follicle development, pigmentation which has been largely overlooked over the years 45. Chronologically, one could observe that the bioprinting strategy utilised for skin tissue engineering has progressed over the years. First generation 3D bioprinted skin substitutes were simple printed through laser assisted techniques

46-48

or solid free form printing

49

which involved the use of only keratinocytes and fibroblasts and was shown to form multilayered epidermis. The second generation of 3D bioprinted constructs emphasized on retaining the natural niche of skin during pre-printing and post-printing stages

50.

For

instance, mouse epithelial progenitor cells were encapsulated along with mouse plantar dermis, epidermal growth factor in 3D-extracellular matrix (ECM) hydrogel mimics to create an inductive niche. These bioprinted matrices helped in restoration of sweat glands successfully in burnt-paw models in mice

51.

The newer generation 3D-printed skin now

focuses on holistic development of skin tissues with cellular placement and structure similar to native skin. Scaffold free approaches to bioprint epidermal and dermal layers have been developed where the epithelium formed after 26 days of culture was morphologically similar to human skin but also expressed key functional markers such as cytokeratin 10, collagen I and V, vimentin, fibrillin, elastin and filaggrin 52. This matured 3D-printed constructs owing to the completely differentiated epithelium exhibit barrier function (loricrin expression) and also seen to exhibit remodelling with evidences of functional dermal-epidermal junction which was not seen in previous generations of bioprinted skins. In another approach, full thickness skin constructs containing melanocytes with insistence on pigmentation was investigated using a multi-step free-form bioprinting, utilising human dermal fibroblasts and keratinocytes

53.

The strategy involved printing melanocytes sequentially over layers of

keratinocytes and basal layers of fibroblasts, all encapsulated in collagen based bioinks, constituting the full thickness dermal layers. The constructs were matured in vitro under air-


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liquid interface for 9 days and was seen to express freckle-like pigmentation on the dermalepidermal junction. Similarly, a two-step drop-on-demand bioprinting strategy has been recently applied to recapitulate the melanin units using cocultures of keratinocytes, melanocytes and fibroblasts 54. The bioprinted constructs exhibited well developed epidermal layers with continuous basement membrane, post 39 days of culture in vitro with uniform pigmentation similar to the Caucasian donor’s pale pigmentation. 3.2 LIVER Despite the ability of the present tissue engineering strategies in maintaining the longevity of hepatocytes in culture, the lack of presentation of biomimetic environment with parenchymal and non-parenchymal cell types in case of hepatic tissue greatly limits the clinical viability of tissue engineered liver substitutes for transplantation. Addressing this, automated bioprinting platforms capable of precisely dispensing hepatocytes, CD31+ve endothelial cells and desmin+ve stellate cells in an accurate spatiotemporal manner have resulted in fabricating human liver tissues which actively secreted albumin, cholesterol, fibrinogen, and transferrin into the medium for 6 weeks

55.

Maintenance of the biomimetic niche enabled these liver

tissues to furthermore actively express important phase-I liver enzymes such as CYP3A4, 2D6, 2B6, 1A2, and 2C9 for over 28 days. Additionally, human induced pluripotent stem cells (hiPSCs) have been used for generation of mini-livers via gentle enough inkjet based printing to improve cell viability and pluripotency of hiPSCs 56. The technique resulted in a 40 layers of hiPSCs which could be differentiated into hepatocyte like cells in vitro and was also observed to maintain its functional phenotype as a consequence of higher albumin secretion noticed over 21 days of culture. As an advancement to this strategy, hiPSCs derived hepatic progenitor cells along with human umblical vein endothelial cells (HUVEC) and adipose derived stem cells were printed in microscale hexagonal architecture mimicking the exact liver lobule unit via digital light processing (DLP) based bioprinting (Figure 3 I) 57.The 21 ACS Paragon Plus Environment


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expression level of mature hepatocyte markers such as hepatocyte nuclear factor 4α, transthyretin and α-fetoprotein were significantly increased at day-20 of culture and also exhibited enhanced cytochrome P450 induction. The bioprinting technique utilised here bears an advantages of having superior speed and scalability to print in ease such complex structures hexagonal architectures noticed in liver tissues. In parallel lines, scaffold free bioprinting approaches where a xenotransplant model using porcine fetal derived fibroblast and liver cells aggregates have also been looked into with considerable success 58. These in vitro 3D bioprinted liver models that enable us to look forward towards a fully functional liver along with the recent encouraging findings in a chronic liver model in mice

59

has

vouched for further fruitful clinical translation of such bioprinted constructs. Stevens et al developed perfusable patterned liver seeds consisting of human hepatocytes, endothelial cells and stromal cells via 3D printed perfusable sacrificial moulds

60.

These developed liver

seeds were implanted at an ectopic site intraperitoneal fat of nude mice. The implanted tissue responded to regenerative cues from hepatectomized liver injury nude mice where the authors state the ectopic liver seeds was able to perform over 500 liver functions which was extensively studied for over 84 days. 3.3 LUNG Commendable improvements in respiratory tissue engineering have led to products that mimic the conducting airways such as trachea and bronchi

61-62;

no major development to

realistically recreate the air-blood barrier has been attempted 63. The only attempt made thus far to our knowledge is an extrusion based technique to engineer human air-blood barrier analogues using alveolar epithelial cells separated by thin layer of matrigelTM over a layer of endothelial cells, by adopting a layer-by-layer strategy. Though the attempt is far from realising a functional lung, it is one of the first stepping stone endeavours in recreating the


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alveolar 3D alveolar lung. The tightness of the barrier function was confirmed after 3 days post-culture by blue-dextran displacement from apical to basolateral compartment.



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Figure 3. Bioprinting strategies for I) Biomimetic hiPSCs derived liver exhibiting hexagonal lobule like structure as noticed in native liver (Copyright 2016. Reproduced with permission from National Accademy of Sciences) 57; II) Strategy for imparting vasculature in thick solid tissues 64; III) Perfusable 3D convoluted renal proximal tubules (Copyright 2016. Reproduced with permission from Nature Research, under creative common license) 65.

3.4 MYOCARDIUM Cardiac tissue engineering has gained so much attention over the years owing to the poor regenerative capacity of the myocardium. Among the early attempts of 3D printing cardiac tissues, Gaetani et al used human cardiomyocyte progenitor cells in alginate bioink to attest the printability of these sensitive cells through extrusion based printing

66.

Post printing, at

day-7 the cellular viability was determined to be 89 % and the progenitor cells expressed early cardiac transcription such as Nkx2.5, Gata-4 and Mef-2c showing the commitment of cells to maintain their phenotype. Encouraged by these early findings we have witnessed a surge in better bioinks and printing strategies targeting in reproducing the anisotropic myocardium effectively over the years

67-68.

For instance, 3D printed gelatin/hyaluronic acid

cardiac patches with encapsulated human cardiomyocyte progenitor cells were able to preserve the cardiac performance in a myocardial infarction model induced via ligation of left anterior descending coronary artery in NOD-SCID mice

69.

In order to further improve the

printed cardiomyocytes performance, endothelialized myocardium printed through co-axial extrusion system has been attempted

70.

Alginate- gelatin methacryloyl (GelMA) based

bioink bearing the endothelial cells were printed through co-axial extrusion nozzles with the crosslinkers delivered through the core of the nozzle. Post-curing the endothelial cells migrated outwards from the printed microfiber and formed confluent endothelium, now on to which cardiomyoctes were seeded on the 3D printed structure. Spontaneous beating of these cardiac 3D printed constructs was observed at 48 hours (55 – 75 beats per minute) and the


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beating frequency was considerably maintained even after 28 days (40 beats per minute) in culture, attesting the efficacy of endothelialisation of the myocardium. More recently, use of biomaterial or scaffold free 3D bioprinting of myocardium was investigated by using hiPSCs, fibroblasts and endothelial cells (HUVEC) cultured as spheroids (termed as cardiospheres) through a needle array spheroid dispensing 3D fabricator from Regenova, Cyfuse Biomedical K.K., Tokyo, Japan

71.

This unique and sophisticated

biofabricator platform selects and dispenses cardiosphere aggregates onto 96 well low adhesion cell culture plates in exact spatial coordinates according to the 3D design developed. The cardiac patches spontaneously started to beat and exhibited uniform ventricular-like action potential throughout the patch. Emphasis on recapitulating the submicron intricacy and hierarchical anisotropy noticed in myocardium has been the recent directive in cardiac bioprinting of late. In this regard, highly organized bioprinted cardiac patches with unique physiological, electrical and biomechanical properties were developed by printing neonatal primary rat cardiomycytes via 300 μm pressurized nozzle extrusion printer. The printed constructs exhibited spontaneous contraction post printing with progressive cardiac maturation

72.

Similar attempts using hiPSCs with submicron resolution via multiphoton

excited 3D printing were also investigated and were shown to alleviate myocardial infarction in mice

73.

Use of adult human mesenchymal stem cells (hMSCs) grown on aligned 3D

printed submicron gelatin microchannels

74

were also seen to enhance the contact guidance

and subsequently the cardiac contraction phenotype of stem cells post-printing in such printed cardiac patches. Biopolymers used in bioinks are now currently reinforced or included with conductive entities to facilitate electrical stimulation of cardiac patches triggering rhythmic contractions attuned with native tissues. For instance, methacrylated collagen with carboxyl functionalized carbon nanotubes used as a bioink helped in printing endotheliased cardiac patches with improved electrical conductivity 75. 25 ACS Paragon Plus Environment


3.5 VASCULATURE 3D bioprinting strategies innately help in developing cellularized blood vessels which are consistent with native blood vessels anatomically, physiologically and biochemically, whereby overcoming the shortcomings of autologous grafts.76 Moreover, fabrication of small diameter vascular grafts (< 6 mm) and vascularized tissues (Figure 3 II) 64 (few micron thick endotheliums) have now become feasible through different 3D printing strategies.77-78 Use of microfluiding bioprinting technique and low viscosity bioinks through a co-axial needle extrusion system have been employed to form matured endothelium (derived from HUVECs). The functionality of these microvasculatures was confirmed by expression of CD31, a key marker expressed during tube formation of endothelial cells.79 Hollow cell laden microchannels were developed through co-axial extrusion based printing using sodium alginate as the ink and crosslinker calcium chloride delivered in the core.80 Robust and flexible tubular structures with inner diameter of 892 μm and outer diameter of 1192 μm were formed with ultimate tensile strength upto 0.116 MPa and also maintaining the viability of fibroblasts (~ 67 %) 7 days post printing. Another approach to bioprint vasculature has been reported by Gao et al by adopting a multi-level fluidic channel extrusion based printing. The strategy involved printing hollow alginate filaments through coaxial extruders loaded with fibroblasts (mimicking tunic adventitia of blood vessel) on top of smooth muscle cells (mimicking tunia media) over a rotating rod template. The rod template was removed and endothelial cells were seeded on to the lumen of the macro-channel. The authors have reported that this multi-level channel efficiently mimicks the mechanical properties of blood vessel and micro-channel facilitate better cell survival and nutrient delivery 81. Scaffold free approaches for bioprinting blood vessels have also been evaluated. Selfassembled multicellular spheroids of smooth muscle cells and/or fibroblasts were concomitantly printed layer by layer over agarose rods which were used as moulding 26 ACS Paragon Plus Environment

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templates. The spheroids fused within 5-7 days post printing and the template was easily removed. Through this strategy tubular conduits of outer diameter 900 μm with wall thickness 300 μm were obtained 82. Utilising a similar approach, alginate support system was used to dispense endothelial cells (HUVEC) and human smooth muscle cells derived spheroids to form torrid shaped conduits

83.

3D bioprinting with impetus on cellular

patterning and thus improving the cellular phenotype is also a directive for enhancing vascular graft performance. Vascular smooth muscle cells bioprinted on to gelatin microchannels exhibited F-actin anisotropy along the channel direction and acquired a more contractile phenotype much similar to the native small diameter blood vessels

84.

More

recently, utilising a drop-on-demand bioprinting technique vascular channels with inner continuous endothelium, an elastic middle smooth muscle layer and outer collagenous fibroblast layer was developed

85.

These bioprinted conduits of outer diameter ~ 1 mm and

wall thickness 425 μm were perfusable and exhibited >83% viability of cells post-printing with expression of functional markers of all three cell types in culture post-printing. 3.6 KIDNEY Kidney remains to be one of the most sophisticated organs which is very difficult to engineer considering that it is made of 30 different cell types and manually seeding them into different location is impossible. The main prominence of 3D bioprinting was brought out by Atala et al at Wake Forest Institute for Regenerative Medicine, North Carolina, USA when they printed prototypes of various urolical tissues such as bladder, ureter and kidney

86.

Though these

strategies are clinically far from reality, studies are focussing on developing better printers with multiple printheads which can handle the delivery of different bioinks with different cell types to engineer such solid organs

87.

A biopitning method for developing renal proximal

tubules with perfusable lumen supporting the extratubular cellular heterogeneity was



developed (Figure 3 III)

65.

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These 3D printed renal tubules formed tissue-like polarised

epithelium and exhibited enhanced phenotypic traits similar to the in vivo microenvironemts. 3.7 PANCREAS The engineered endocrine pancreas’ success relies on its coordinated and interdependent action of various cell types, in particular the pancreatic islet cells (α, β, δ, γ, and ε cells) intertwined with vascular support for hormonal delivery and regulation. The deposition of these islet cells α, β, δ, γ, and ε cells in their respective ratios ~ 20%, ~70%, ~10%,

Bioprinting Based Tailoring of in Vitro Tissue Models

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