Comparison of intraoral scanning systems
\r\n\tNearly 25% - 30% of the world population is affected by neurological diseases exerting a hard financial strain on the healthcare system. The costs are estimated at around $800 billion annualy, expected to exponentially increase as the elders, at high risk of debilitating neurological diseases, will double by 2050. A varied spectrum of neuroprotective strategies has been suggested, including combined antioxidative-anti-inflammatory treatments, ozone autohemotherapy, hypothermia, cell therapy, the administration of neurotrophic factors, hemofiltration, and others. Distressingly, none of the currently available neuroprotective approaches has so far proven to prolong either life span or the cardinal symptoms of the patients suffering from brain injury. Last but not least, translational studies are still lacking.
\r\n\r\n\tThe book aims to revisit, discuss, and compile some promising current approaches in neuroprotection along with the current goals and prospects.
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De Robertis, School of Medicine (UBA), Argentina. Then he moved abroad to perform his postdoctoral studies at the University of California San Diego (UCSD-NCMIR) and the Karolinska Institute, Department of Neuroscience. Over an eight-year period, his research focused on synaptic organization, combining electron tomography, 3-D reconstruction, and correlative light and electron microscopy techniques. Upon his return to Argentina in 2006, he devoted to study the mechanisms involved in the pathophysiology of the perinatal asphyxia supported by his broad experience in electron microscopy. 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The use of digital technology meets the demand of multiple-doctor practices, multiple practice locations, patient volume growth, and allows efficient and convenient storage, retrieval, and sharing of information. Orthodontics is rapidly embracing new materials and advanced technologies, making the fully equipped 3D orthodontic office a reality. Recent developments and introduction of intraoral and facial scanners, digital radiology, cone-beam computed tomography (CBCT), and additive manufacturing improved the efficiency, accuracy, consistency, and predictability of the treatment outcomes. All those daily advances also led to a rapid growth of digital educational components and teaching tools, 3D video presentations and patient communication.
Computer-aided design and computer-aided manufacturing (CAD/CAM) systems were first used in the dental field in the mid-1980s. CAD/CAM consists of three key components: 1) data acquisition and digitizing; 2) data processing and design; and 3) manufacturing [1]. As computer software and dental materials evolved over time, the CAD/CAM technology became increasingly popular resulting in chairside design and milling of high-quality complete crowns and multiple-unit ceramic restorations. The advent of digital intraoral impression devices allowed high-resolution 3D virtual models to be captured. Intraoral mapping based on different non-contact optical principles and technologies is now possible without the negative aspects of dental impressions such as discomfort for the patient, imprecision, and lab work. In-office chairside or send to the lab, the digital models give the flexible options for design and manufacture of a large range of dental restorations, implants, study models, and orthodontic appliances such as customized indirect brackets, arch wires, expanders, aligners, retainers, etc. (Figure 1). The highly-accurate open file formats are incorporated in the patient electronic health record which can be remotely stored, accessed, and managed through a secure, cloud-based digital hub from basically anywhere. Most digital intraoral scanners work in conjunction with cloud based technology where raw images once scanned can be securely transmitted to the cloud storage facility and further processed/refined for diagnostic purposes.
Traditional versus digital workflow in the orthodontic office
Cloud computing has significantly influenced the healthcare industry in the past years. The concept provides massive amounts of storage and computing power without requiring end-user special knowledge about the physical location and the configuration of the system [2]. The information technology enables convenient, on-demand access at a greater speed in less time to a shared pool of computer resources (network, server, database, software, storage, and applications). Hospitals, medical practices, insurance companies, and research facilities are now transitioning certain infrastructures to cloud services and mobile apps in order to improve the management and administration efficiency at a reduced cost. The cloud healthcare system, named hCloud, was specifically developed to address the requirements for highest level of availability, security, and privacy protection in healthcare [2]. Facilitated by hClouds, medical records and image archiving services can be synchronized and shared between healthcare organizations, medical professionals, clinics, and patients in real time on a daily basis. The file systems and employed structures are easily adaptable to since they are open, industry-standard formats instead of proprietary, closed formats.
Plaster casts have a long and proven history as a routine dental record and have been the gold standard for dentition analysis for years. Nevertheless, plaster models have several disadvantages including labor-intensive work, demand on physical storage space, fragility, degradation, and problems of potential loss during transfer [3].
Digital study models offer a reliable alternative to traditional plaster models (Figure 2). Their advantages in orthodontic diagnosis and treatment planning include easy and fast electronic transfer of data, immediate access, and reduced storage requirement [4]. Digital models can be integrated into various patient management systems, digital records, along with the digital photographs, radiographs, and clinical notes. Digital models may be virtually manipulated to section and analyze specific teeth, arch form, amount of crowding or spacing, and type of malocclusion. Measurements such as overjet, overbite, tooth size, arch length, transverse distances, and Bolton discrepancy are achievable. The user can obtain a digital diagnostic set-up, simulate a proposed treatment plan, perform bracket placement, and indirect bonding [5]. CBCT and digital models can be merged to facilitate treatment planning of orthognathic cases, creation of surgical guides, placement of temporary anchorage devices (TADs), exposure of impacted teeth, or preparation for dental prostheses. Moreover, if a physical model of the dentition is required for the manufacture of an orthodontic appliance, digital models can be 3D printed with a rapid prototyping technology.
3D digital models of the upper dental arch in Geomagic® (3D Systems, Cary, NC, USA). The software is an aid in the CAD/CAM process, able to repair errors in the mesh prior to 3D printing, edit the models, and design appliances.
Digital models were commercially introduced in 1999 by OrthoCAD™ (Cadent, Carlstadt, NJ). The results from a recent survey conducted by the Journal of Clinical Orthodontics demonstrated a significant increase in the use of digital models for pre-treatment diagnosis and treatment from 6.6% in 2002 to 18% in 2008 [6]. Today, many orthodontists acquire digital models through the use of proprietary services. Traditional impressions, plaster models, or intraoral scans are submitted to the selected company so that they can generate the digital models and made them available for download in proprietary or stereolithography (STL) file format. STL is an open, industry-standard file format that is supported by most intraoral scanners and widely used for rapid prototyping, computer-aided manufacturing, and across different 3D modeling interfaces. Another open file format is PLY, polygon file format (also known as Stanford triangle format), which is used when color and/or transparency information is needed.
Commercially available digital models may be obtained by a direct or an indirect method [5]. The direct method makes dental impressions redundant by using an intraoral scanner to capture directly in the patient’s mouth. Indirectly, digital models can be produced by scanning alginate impressions and plaster models with a desktop scanner, intraoral scanner, or computed tomography imaging.
There are no universal standards for defining model accuracy. However, in orthodontics, it is generally accepted that measurement accuracy up to 0.1 mm is adequate for clinical purposes and does not compromise the diagnostic value of a model. Numerous studies have evaluated the reliability of stereolithographic models obtained by indirect methods by assessing the agreement of measurements on plaster and digital models [7-10]. It has been shown in the literature that digital models have clinically insignificant differences for reproducibility of orthodontic measurements. Tooth size has been found to be similar or slightly smaller in OrthoCAD™ compared to measurements made on plaster models. Overjet measurements were not significantly different in some studies however Quimby et al. (2004) found a significantly smaller overjet measurement when obtained with OrthoCAD™. Space available or arch length to be used in estimating crowding, demonstrated to be significantly different between OrthoCAD™ models and plaster models with differences ranging from 0.4 mm to 2.88 mm [9]. Laser-scanned models are suggested to be highly accurate in comparison to plaster models and CBCT scans and provide clinicians an alternative to physical models and CBCT reconstructions in diagnosis and treatment planning [10]. Both CBCT and intraoral scanning of alginate impressions were concluded to be valid and reliable methods to obtain measurements for orthodontic diagnostic purposes [11,12]. Furthermore, different digital model conversion techniques have shown no statistically mean differences when using 3D palatal rugae landmarks for comparison. 3D digital models are also proven as an effective tool in evaluating palatal rugae patterns for human verification and identification [13].
Three-dimensional digital impressions were first introduced in 1987 by CEREC 1 (Siemens, Munich, Germany) using infrared camera and optical powder on the teeth to create a virtual model. Over the years, computer hardware and software developments have dramatically improved the technologies completely replacing traditional alginate and polyvinyl siloxane (PVS) impressions in a large number of dental and orthodontic offices. In most instances, production of precisely fitting final dental restorations no longer requires the use of a powder [14-16].
New intraoral scanners for acquisition of digital impressions are continuously entering the clinical practice all over the world. Improvements in the scanning technologies have resulted in truly portable cart-free systems with a single, forked USB cable that can be plugged into any workstation. Ergonomic design and reduction in wand size and weight have resulted in a more comfortable experience for both patients and staff. The optical scanners can be used to capture both in vivo images of the dentition and in vitro images of the physical models to create a 3D digital representation. Intraoral scanner devices offer numerous applications in orthodontics such as digital storage of study models and advanced software for cast analysis, landmark identification, arch width and length measurements, tooth segmentation, and evaluation of the occlusion [15]. The platforms allow clinicians to obtain a digital diagnostic set-up, perform indirect bonding, and export the digital scans into open source file formats. The electronic files are shared with third-party providers and imported into a variety of digital workflows for advanced treatment planning of surgical cases, implants, and superimposition with CBCT data.
Traditional alginate and polyvinyl siloxane (PVS) impressions have been associated with numerous limitations which include complex workflow, lack of precise reproduction, lacerations on the margins, poor dimensional stability, limited working time, plaster pouring and solidification, and problems of transport and packaging. A general disadvantage of the conventional impressions is also the need to start over if an impression fails or take additional impressions (e.g. study models and appliance fabrication). In addition, the contact between the tray and the teeth could cause discomfort for the patient and trigger a bad gag reflex. Digital impressions eliminate all those negative aspects. They streamline and expedite the traditional workflow, reduce the number of patients’ visits, and maximize the efficiency and cost savings in the orthodontic office [16]. Besides the better control and improved accuracy of the directly obtained digital models, scanners add the plug-and-play capability of an automatic exchange of patient information within the office or outside laboratories. Lost or broken appliances could easily be refabricated using the digital files from a database in the Cloud [2].
Several scanning technologies using different optical components and structured light sources are currently employed in orthodontics:
Confocal laser scanning microscopy (CLSM or LSCM) is a technique for acquiring images with high-resolution and in-depth selectivity. Images are projected point-by-point, line-by-line, or multiple points at once and three-dimensionally reconstructed with a computer, rather than obtained through an eyepiece [18]. The key feature of confocal microscopy is its ability to produce optical slices of the objects at various depths with high resolution and contrast in the x, y, and z coordinates. Spatial filtering is employed to eliminate out-of-focus glare or light of background information. Change of display magnification and image spatial resolution, termed the zoom factor, is enabled by altering the scanning sampling period [17-19].
The basic principle of confocal microscopy was pioneered by Marvin Minsky in 1957 [17]. Advances in laser and computer technology coupled the new algorithms for digital manipulation of images in the late 1970s and 1980s, and led to an increasing interest in the technology which became a standard technique. Modern confocal microscopy systems integrate various components such as beam scanning mechanisms and wavelength selection devices which are often referred to as a video or digital imaging system [16]. It is now possible to employ these scanning technologies for multi-dimensional functional and structural analysis of molecules, living cells, and tissues.
Optical triangulation measures distance to objects without touching them with accuracy from a few millimeters to a few microns [20]. Triangulation sensors are particularly useful in acquiring high-speed data in inspecting delicate, soft, or wet materials where contacts are undesirable. The system uses a lens, a laser light source, and a linear light sensitive sensor [16]. The laser irradiates a point on a specimen forming a light spot image on the sensor surface. The distance from the sensor to the surface is then calculated by determining the position of the imaged spot and the baseline angles and length involved. The principle of triangulation has been used for centuries but practical sensors became available for industrial applications in 1971. Triangulation sensors are commonly used for monitoring vibrations, tire dimensions while rotating at high speed, and as a safety mechanism in automatic doors [20-22].
Optical coherence tomography (OCT) is an interferometric technique that performs cross-sectional high-resolution imaging of the internal morphology of biological materials and tissues [23]. It is equivalent to ultrasound imaging, except that it uses light instead of sound. Micron-scale measurements of distance and microstructure are obtained from backscattered or backreflected light waves in real time and in vivo. Although OCT imaging depths are not as deep as with ultrasound, resolution of 1 to 15 µm can be achieved, 10 to 100 times higher than standard clinical ultrasound. The relatively long wavelength light is able to penetrate into the scattering medium up to 2-3 mm deep in most tissues [16,24]. OCT has become a well-established medical diagnostic technique after being first demonstrated in 1991. It is now widely used in ophthalmology, gastroenterology, and cardiology and can be successfully applied where standard excisional biopsy is not possible or hazardous [25].
Accordion fringe interferometry (AFI) employs a revolutionary linear interferometry technology that traditionally projects to three dimensions [16]. AFI delivers the most precise laser fringe projection available which quickly digitizes the shapes of 3D objects with the highest accuracy of point cloud data. AFI employs laser beams from two point sources to illumine the objects and uses a charge-coupled device (CCD) camera to capture the curvature of the borders [26]. AFI is less sensitive to ambient light which gives the ability to capture and measure a wider variety of surface coatings, textures, and finishes than structured light. AFI is suitable for a wide range of applications that require high speed, portability, and infinite projector depth of field from a 3D digital system. The AFI approach has already been implemented in automotive and aviation industries, reverse engineering, tool inspection, analysis, and fabrication [27].
Active wavefront sampling (AWS) uses a 3D surface imaging technique, which requires only one optical path of an AWS module and a single camera to acquire depth information [28]. The optical wavefront traversing a lens is sampled at two or more off-axis locations and a single image is recorded and measured at each position. Target feature image rotation can be used to calculate the feature\'s distance to the camera [29]. The aperture sampling can be implemented mechanically or electronically and different components can be modified for better performance. Aperture size, target illumination, and sampling plane position can be optimized in order to maximize the captured image quality. AWS reduces system cost by eliminating the need for expensive laser based target illuminators and multiple cameras to acquire 3D images [16]. That allows the technique to be applied in a wide range of currently available 2D systems such as cameras, endoscopes, and microscopes [30].
Table 1 summarizes some of the characteristics of several intraoral scanning systems used in orthodontics.
The iTero® digital impression scanner was developed by Cadent Ltd. in 2006, and acquired by Align Technology, Inc. (San Jose, CA) in 2011 [15]. iTero® employs confocal laser scanner microscopy technique. The device projects 100,000 parallel beams of red laser light which pass through a probing face and a focusing optics to reach the teeth. The reflected light is then transformed into digital data through the use of analog-to-digital converters. The system scanning capability does not require coating the teeth in powder thereby allowing the wand rest directly on the teeth during scanning. One disadvantage is that the iTero® camera needs a color wheel attached to the acquisition unit, which results in a larger and bulky scanner head in comparison to other systems.
The iTero® Intraoral digital scanner [31]
iTero® comes as a mobile cart containing a central processing unit, large working surface with a touch screen display, a wireless mouse, a wireless foot pedal, a built-in keyboard, and a scanning wand (Figure 3) [31]. The intraoral scanner features continuous and click to capture scan mode; rendering in traditional stone color and true-color models; and optional voice guidance and visual commands. Digital images for orthodontic treatment are captured by proceeding through upper and lower quadrants [32]. Five different views of the scan area are required: buccal, occlusal, lingual, and the proximal surfaces between adjacent teeth. Overall, complete full mouth scan and a bite registration can take about 10 to 15 minutes. In a two-year study including 328 scans, it has been reported that the fastest intraoral scan can take 6 minutes and 22 seconds and the longest almost 18 minutes [33]. At the end of the scanning process, a line of diagnostic tools are available to assess and finalize the impression. Additional scans can be added to any areas of incomplete data. The final digital models are stored in the online MyAligntech database under the service provider profile and are available for export in the STL open file format.
The 3M True Definition scanner was officially launched in 2013 as an updated version of the Lava™ chairside oral scanner which has been widely used in general and restorative dentistry since 2006 (Figure 4) [34]. The True Definition scanner captures 3D images using active wavefront sampling on the principle of structured light projection. 3M ESPE named this scanning technique “3D-in-motion video technology” [15]. The system employs a rotating aperture element placed off-axis in the optical apparatus either in the imaging or the illumination path which measures the defocus blur diameter. The user should first dry and lightly dust with powder the entire arch so the scanner can locate the reference points.
The True Definition features an ergonomic scanning wand and a rolling card with a HP® high-performance CPU computer unit with 22” touch screen monitor. The lightweight, stainless steel wand has a dental handpiece design with an on/off tap switch with no moving parts. Video imagery is captured at 20 frames per second and a complete full mouth scan with a bite registration take about 5 to 8 minutes. Upon completion, digital models are available immediately for 3D setup review, analysis, superimposition, enhanced measurements, and treatment planning. Unlimited patient scans can be stored in the Unitek™ treatment management portal allowing direct communication between the provider and the company customer service [34]. Open STL files are available for download, importing into a variety of digital workflows, and sharing with third-party providers.
The 3M™ True Definition Scanner [34]
The Lythos™ Digital Impression System was introduced by Ormco Corporation (Orange, CA) in 2013. The intraoral scanner uses accordion fringe interferometry technology to capture and stitch together a 3D data in real time, acquiring high-definition details at all angulations of the tooth surface. Lythos™ provides 3D video imagery of 2.5 million points per second [35].
The Lythos™ scanner is portable, weighs around 13 kg, and can be positioned directly on the ground or on a chairside table (Figure 5). The device platform features an extendable touch screen monitor with wireless internet connectivity and a lightweight scanning wand.
The Lythos™ Digital Impression System [36]
The touch screen helps in guiding the ward. First, the lower dental arch is scanned followed by the upper arch by pointing the tip of the wand on the occlusal surface and moving it from left to right [15]. The instantaneously displayed data are captured until missing regions are scanned or the results are satisfactory to the person who is scanning. The software also offers a capability to erase unwanted or accidently scanned regions. A dual-arch high-resolution scan can be completed in approximately 7 minutes. Ormco allows users to own and store the digital impressions on the company online portal, or send treatment scans to anyone that accepts STL files.
Carestream Dental (Atlanta, GA) launched the portable digital impression system, CS 3500, at the end of 2013 (Figure 6). The scanner employs confocal laser scanner microscopy technique which allows capture of true-color 2D and high-angulation 3D scans of up to 45º with a depth of field of -2 to 13 mm [37]. The image resolution is 1024 x 728 pixels and the accuracy measures up to 30 microns. The system is trolley-free and uses a wand with a single, forked USB cable that can be plugged in any computer, eliminating the need for a dedicated workstation. Two scanning tip sizes are included to accommodate children and adults.
The CS 3500 Intraoral Scanner [37]
A built-in heater streamlines scanning by preventing mirror fogging. No external heater or powder is required. CS 3500 features a light system which guides the user during the data capture and the image acquisition process [15, 16]. The green light indicates a successful scan while the amber light shows that a rescanning of the area is needed. A full impression typically takes about 10 minutes. CS 3500 is compatible with open source software or it can work as part of the integrated Carestream CAD/CAM dental restorations system.
3Shape (Copenhagen, Denmark) announced the TRIOS® intraoral scanning solution in December 2010 (Figure 7). The system operates by the principle of confocal microscopy, with a fast-scan rate [38]. Hundreds to thousands of 3D pictures, corresponding to different time instances and to respective different positions of the focus plane of the illumination pattern, are combined to create the final 3D digital impression. The high-definition camera features teeth shade measurement and provides scans in enhanced natural colors or in a standard noncolor pattern [16]. The scanning wand does not require the use of powder, has an autoclavable tip and an anti-fog heater. It takes approximately 5 minutes for a full mouth scan.
The 3Shape Intraoral Scanner is available as TRIOS® Cart or TRIOS® Pod configurations [38]
The 3Shape digital impression system is available as TRIOS® Cart, TRIOS® Chair integration, or TRIOS® Rod configuration. The TRIOS® Cart consists of a smart multi-touch screen which provides 3D visualization, video tutorials, Wi-Fi and Bluetooth connections, and technical support. The TRIOS® Rod configuration provides mobility by using USB connection to any computer or display. 3Shape offers Ortho Analyzer™ software for study model analysis and treatment planning which also stores cone-beam scans, patient photographs, panoramic x-rays, and cephalometric tracings [38]. TRIOS® saves the digital impression scans in the standard STL file format.
Glidewell Laboratories’ IOS FastScan® intraoral camera and modelling system was commercialized in July 2010 (Figure 8). The digital impression system uses the principle of active triangulation with sheet of light projection [16]. Ego-motion technology is used to optimize image stabilization by having the camera moving automatically on a track within the housing wand. That built-in motion detection software eliminates hand-movement distortion, capturing high-resolution surface detail. The camera scans 40 mm per second throughout a depth of field that is greater than 15 mm [39]. An application of titanium dioxide powder is required.
The FastScan® intraoral scanner [39]
FastScan® device is portable and consists of a touchscreen display and a large scanning area wand. A live 3D digital preview of the captured area is shown almost immediately. This feedback provides information about the wand positioning and orientation in relation to the patient’s dentition and whether an adjustment is necessary. A full mouth scan and a bite registration can be obtained in about 4 minutes. The system is able to acquire color and translucent data along with the 3D anatomy of the dentition [39]. A single digital file combines the surface information, translucency, and color which can be stored or electronically sent to a CAD/CAM lab for an appliance fabrication. The output data is in the open source STL format which can be recognized, viewed, and manipulated by third party providers. Unfortunately, FastScan® is no longer available on the market and the company is not planning to manufacture a replacement soon.
The 3D Progress digital impression system is supplied by Medical Height Technology (MHT) company (Verona, Italy) and MHT Optic research (Zurich, Switzerland) (Figure 9). The technology beyond the product is a confocal scanning microscopy with a Moiré pattern detector [16]. A Smart pixel sensor supports precise and quick capture of up to 28 scans per second which are stitched in a single 3D image in less than one tenth of a second. 3D Progress allows a wide focus of acquisition, ranging between 0 to 18 mm depth of field [40]. The scanning process can be paused and re-started at any moment and parts of the scan can be modified or updated with new data acquisitions. A full mouth digital impression can be completed in approximately 4 minutes.
3D Progress intraoral scanner [40]
3D Progress is portable, consisting of power supply and a light-weight wand which can be interfaced to a computer via USB connection. The system will not usually require powdering with exclusion of exceedingly reflective surfaces such as implant abutments [40]. Complete scans are generated as a point cloud which can be saved as digital 3D models in the usual STL open file format, compatible with all CAD systems.
Formerly known as the E4D NEVO scan and design system, the Planmeca PlanScan®, driven by E4D Technologies (Richardson, TX), is an intraoral scanner widely utilized in restorative dentistry (Figure 10). PlanScan® uses optical coherent tomography with blue laser technology [16]. Point-and-stitch image reconstruction occurs at video-rate scanning speed [41]. The single smaller wavelength of 450 nm is more reflective, capturing sharper images of various hard and soft tissue translucencies, and dental restorations. Adjustable field-of-view software optimizes the target window while scanning in order to avoid capture of extraneous data such as lips, cheeks, and tongue. Intraoral fogging is prevented by the use of actively heated mirrors on the scanning tip [15].
Planmeca PlanScan® intraoral scanner [41]
The PlanScan® intraoral scanner comes with three removable tips, a cradle, and a power cable. Planmeca CAD software is used for data process, analysis, and editing by utilizing Thunderbolt connectivity to any computer or workstation. Integration and collaboration with other systems and third-party providers for scanning review, completion, or appliance fabrication is enabled through the STL open file format.
\n\t\t\t\tFeatures\n\t\t\t | \n\t\t\t\n\t\t\t\tiTero®\n\t\t\t | \n\t\t\t\n\t\t\t\tTrue Definition\n\t\t\t | \n\t\t\t\n\t\t\t\tLythos™\n\t\t\t | \n\t\t\t\n\t\t\t\tCS 3500\n\t\t\t | \n\t\t\t\n\t\t\t\tTRIOS®\n\t\t\t | \n\t\t\t\n\t\t\t\tFastScan®\n\t\t\t | \n\t\t\t\n\t\t\t\t3D Progress\n\t\t\t | \n\t\t\t\n\t\t\t\tPlanScan®\n\t\t\t | \n\t\t
\n\t\t\t\tCompany\n\t\t\t | \n\t\t\tAlign Technology, San Jose, CA | \n\t\t\t3M ESPE, Monrovia, CA | \n\t\t\tOrmco, Orange, CA | \n\t\t\tCarestream, Atlanta, GA | \n\t\t\t3Shape, Copenhagen, Denmark | \n\t\t\tIOS Technologies, San Diego, CA | \n\t\t\tMedical High Technologies and Optic Research, Verona, Italy | \n\t\t\tE4D Technologies, Richardson, TX | \n\t\t
\n\t\t\t\tOptical Technology\n\t\t\t | \n\t\t\tConfocal Laser Microscopy | \n\t\t\tActive Wavefront Sampling | \n\t\t\tAccordion Fringe Interferometry | \n\t\t\tConfocal Laser Microscopy | \n\t\t\tConfocal Laser Microscopy | \n\t\t\tOptical Triangulation | \n\t\t\tConfocal Laser Microscopy | \n\t\t\tOptical Coherent Tomography | \n\t\t
\n\t\t\t\tYear Launched\n\t\t\t | \n\t\t\t2006 | \n\t\t\tMay 2013/ updated version August 2014 | \n\t\t\tMay 2013 | \n\t\t\tNov 2013 | \n\t\t\tDec 2010 | \n\t\t\tJuly 2010 | \n\t\t\t2012 | \n\t\t\t2008 | \n\t\t
\n\t\t\t\tPowder-Free\n\t\t\t | \n\t\t\t√ | \n\t\t\t\n\t\t\t | √ | \n\t\t\t√ | \n\t\t\t√ | \n\t\t\t\n\t\t\t | √ | \n\t\t\t√ | \n\t\t
\n\t\t\t\tScan Time\n\t\t\t | \n\t\t\t10-15 min | \n\t\t\t5-6 min | \n\t\t\t7 min | \n\t\t\t10 min | \n\t\t\t5 min | \n\t\t\t4 min | \n\t\t\t4 min | \n\t\t\t8-10 min | \n\t\t
\n\t\t\t\tFile Export\n\t\t\t | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t\tOpen STL | \n\t\t
\n\t\t\t\tTrolley-Free\n\t\t\t | \n\t\t\t\n\t\t\t | \n\t\t\t | \n\t\t\t | √ | \n\t\t\tCart and Pod-only versions available | \n\t\t\t\n\t\t\t | √ | \n\t\t\t√ | \n\t\t
\n\t\t\t\tInvisalign\n\t\t\t | \n\t\t\tYes | \n\t\t\tYes | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t
\n\t\t\t\tSureSmile\n\t\t\t | \n\t\t\tYes | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tYes | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t
\n\t\t\t\tIncognito\n\t\t\t | \n\t\t\tYes | \n\t\t\tYes | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t
\n\t\t\t\tInsignia\n\t\t\t | \n\t\t\tYes | \n\t\t\tNo | \n\t\t\tYes | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t\tNo | \n\t\t
\n\t\t\t\tProduct Website\n\t\t\t | \n\t\t\twww.itero.com | \n\t\t\thttp://solutions.3m.com | \n\t\t\twww.ormco.com | \n\t\t\twww.carestreamdental.com | \n\t\t\twww.3shapedental.com | \n\t\t\twww.ios3d.com | \n\t\t\twww.3dprogress.it | \n\t\t\thttp://planmecacadcam.com | \n\t\t
Comparison of intraoral scanning systems
Increased accuracy and time efficiency of the intraoral scanners have contributed to their growing popularity in dental offices. Published studies have found a comparable or better general accuracy of intraoral scans compared with conventional impressions [3-5,42]. Reported absolute mean differences in tooth-width measurements between digital and plaster models vary from 0 to 0.384 mm in the literature [10]. In 2013, Naidu and Freer analyzed the reproducibility of the iOC intraoral scanner (Cadent, Carlstand, NJ; later acquired by Align Technology, Inc.) by comparing plaster models and intraoral scans obtained from thirty subjects. Tooth widths were measured with a digital caliper from the physical models and with the OrthoCAD™ software (Align Technology, Inc., San Jose, CA) from the virtual models. Although there were statistically significant mean differences between tooth widths and Bolton rations, the bias of 0.024 mm was judged to be not clinically significant when the clinical threshold for a tooth-width discrepancy of 0.5 mm was applied [43]. The intraoral scans were suggested to be used for tooth-width measurements and Bolton ration calculations with clinically acceptable accuracy and excellent reliability [7-10]. Tooth-size arch-length measurements obtained in dry skulls from CBCT scans and iTero models provided interchangeable results with manual measurements, making both methods sufficient for orthodontic diagnosis and treatment planning [3]. In a study on the Lava chairside oral scanner, Cuperus et al. (2012) found that generally tooth-width measurements made on virtual models had the tendency to be greater than measurements made on physical stereolithographic models [10].
Orthodontic treatment with Invisalign requires intraoral scans or PVS impressions at several time points [32]. The rejection rate reported for 328 submitted cases with the iTero® intraoral scanner was less than 1% [33]. Intraoral scans demonstrated more accurate digital information in certain types of malocclusion compared to the conventional PVS impressions: severe anterior crowding, overlapping incisors, ectopically positioned teeth, missing teeth, planned extractions, severe deep bite, and late mixed dentition cases. The scanned distal surface of upper second molars in Class II cases showed adequate detailing, eliminating the need for retaking the frequently distorted PVS impressions in the time-consuming two-step impression technique. Intraoral scans submitted for Invisalign cases instead of PVS impressions showed no difference in terms of aligners’ quality, durability, or function. However, it was noticed that orthodontists receive the first ClinCheck much sooner, sometimes within 24 hours of scanning, and consequently the aligners are manufactured sooner, allowing the treatment to start earlier [32,33].
Although an individual clinician’s speed and effort have a substantial influence on the efficiency of the impression technique, procedure durations for digital and conventional approaches have been studied. The time efficiency for single implant restorations has been reported with a total treatment time of 24 minutes 42 seconds for the conventional approach and 12 minutes 29 seconds for the digital impression. Longer impression material preparation, working time, and retakes were necessary to complete an acceptable conventional impression. In 2014, Patzelt et al. investigated in vitro the working times of three intraoral scanners in three different prosthodontic scenarios. Compared with the conventional approach, digital impressions showed up to 23 minutes faster obtain rate and accelerated workflow in all tested scenarios of the study [45].
Digital scans were recognized as the most favored impression technique by inexperienced second year dental students [46]. The perceived level of difficulty for digital and conventional impression techniques showed that 60% of the participants favored the intraoral scans, 7% favored the conventional impressions, and 33% preferred either technique. The learning process for working with an intraoral scanner was suggested to be simpler, requiring less experience and proficiency than conventional impressions.
Various extraoral 3D scanners have been designed to capture 3D images of both impressions and physical casts for the acquisition of digital study models. The scanning technology employs a non-destructive laser beam and several digital cameras to reproduce high resolution images of the target’s surfaces. Impressions, models, or bite registrations are positioned inside a chamber platform which is automatically rotated and inclined during scanning, ensuring complete multiple angle coverage of the model’s geometry. The laser light is projected onto the object, and the cameras acquire its mirror image from the surface [47]. Upon scanning completion, a rendered stereolithographic model is created and plaster models, impressions, and bite registration can be discarded, eliminating the need for storage.
Ortho Insight 3D™ (Motion View Software, LLC, Chattanooga TN) was introduced in 2012, offering a high-resolution, robotic scan with an accuracy of 40-200 microns (Figure 11). The automated laser scanner is designed to capture full arch impressions, plaster models, and bite registrations, and create 3D digital models [47]. A single cast scan and virtual model reconstruction can be completed in approximately 5 to 7 minutes. The Ortho Insight 3D™ software offers digital storage of patient records, cast analysis, and treatment planning features. The software also allows measurements and automated functions such as landmark identification, arch length analysis, tooth segmentation, and evaluation of the occlusion. The optional software modules include indirect bonding and cephalometrics.
The Motion View 3D Desktop Model Scanner [48]
3Shape company (Copenhagen, Denmark) offers three desktop 3D scanners with the capability to digitize both plaster models and impressions with different resolutions and speeds (Figure 12). The R500 and R700 series use red light laser technology with two 1.3-megapixel digital cameras which ensure 20 microns accuracy [38]. The advertised R500 series scanning time is 2 minutes and 20 seconds for a plaster model and 6 minutes and 40 seconds for an impression. The advertised R700 series scanning time is 1 minute and 30 seconds for a plaster model and 7 minutes for an impression which makes the scanner suitable for medium-sized orthodontic offices and labs. The 3Shape R900 series scanner uses blue LED laser technology and employs four 5-megapixel cameras which ensure 15 microns scanning accuracy with color texture. The advertised R900 scanning time is 1 minute and 20 seconds for a plaster model and 2 minutes and 10 seconds for an impression which makes the scanner suitable for large high-volume, productive-orientated labs. Ortho Analyzer™ is the 3Shape imaging and digital model software package which features sculpt and rebase applications with collision control, tooth movement simulation, superimposition of study models with photographs or DICOM data originating from CBCT scanners, and digital manufacture of appliances or dental restorations.
3Shape’s R500 and R7000 model scanners [38]
Maestro 3D (AGE Solutions, Piza, Italy) is another desktop scanning device which allows digital conversion and storage of physical models and impressions (Figure 13). The scanner system has a LED projector with two digital cameras which capture scans with 0.07 mm resolution and 10 microns accuracy [49]. The Maestro 3D extraoral scanner comes with several modules: Easy Dental Scan software for inspection and editing; Ortho Studio software for tooth, arch, overjet, and overbite measurements, cross sectioning, and occlusion inspection; Virtual Setup module for tooth movement, distance and collision evaluation, attachment management, modeling, and export for 3D printing.
The Maestro OrthoScan A50 LED (AGE Solutions, Piza, Italy) [49]
To date, the precision of 3D model scanners has been verified in several papers in the literature. A recent study evaluated the accuracy of Ortho Insight 3D™ plaster model scans for assessing tooth width, arch width, and arch length [11]. Significant differences were found in mesio-distal widths of maxillary molars, mandibular premolars and molars, arch widths of maxillary premolars, and arch lengths between Ortho Insight 3D™ and plaster model measurements however 90% of the mean differences were less than 0.20 mm. Similarly, the mean bias of Ortho Insight virtual models in comparison with caliper measurements (0.24 ± 0.67 mm) was found smaller than the mean biases of GeoDigm emodels and CBCT generated models [50]. In 2013, Hayashi et al. compared the in vitro reliability of three scanning systems (SureSmile OraScanner, Konica Minolta VIVID910, and 3Shape R700) to the gold standard SLP250 Laser Probe digital models. The authors found that the deviation values for each comparison were small (< 0.048 mm) and each scanning device generated sufficiently accurate digital models for use by clinicians [51]. Another study also documented slight, non-significant difference in the linear tooth size and arch length measurements between the 3Shape R700 digital models and plaster models [47]. With respect to intraoral scanners, certain desktop scanners demonstrated higher digitization precision in areas with strong changes of curvature and undercuts but lower accuracy in reproduction of interdental spaces [42].
Facial scanners provide three-dimensional topography of the facial surface anatomy, automatic facial landmark recognition, and analysis of the symmetry and proportions of the face. Practical applications further include quantitative and qualitative assessment of growth and development, ethnic variations, gender differences, and isolation of specific diagnostic traits in selected populations of patients with craniofacial anomalies [52,53]. In addition, facial phenotype associated with fetal alcohol syndrome, cleft lip and palate patients, and short- and long-term effects of nasoalveolar molding have been evaluated using three-dimensional surface imaging [54]. Volumetric results are also valuable clinical tools to assess primary palate reconstruction in infants with cleft lip and palate.
Clinical evaluation of facial morphology is still largely subjective and prevents accurate documentation of facial structure or the changes following various esthetic and reconstructive procedures [55]. Recent scanning technology innovations provided valuable methods for precise three-dimensional clinical documentation and objective qualitative and quantitative analysis of the human face. Several techniques such as laser scanning, ultrasound, computed tomography, magnetic resonance imaging, and electromagnetic digitization can analyze facial characteristics in three dimensions but stereophotogrammetric systems are becoming the instrument of choice in anthropometric research [54,56].
Stereophotogrammetry is a unique method which utilizes means of triangulation and camera pairs in stereo configuration to recover the 3D distance to features on the facial surface (Figure 15 and 16). As early as 1967 Burke and Beard discussed and introduced the concept [57]. 3D stereophotogrammetry has evolved and is now systematically used for anthropometric assessment instead of the direct sliding and spreading caliper-based measurements. Today, it is predominately used in plastic surgery, medical genetics, and research settings. 3D photogrammetry acquires a 180° high-resolution color representation of the human face from ear to ear without direct contact or risks to the patients [56]. A major advantage of the surface imaging system is a near-instantaneous image capture (on the order of 1.5 milliseconds) which reduces motion artifacts and makes it suitable for children, even babies. Upon acquisition, image quality can be immediately reviewed to determine whether repeat imaging is necessary due to blurring or absence of surface data. Furthermore, software tools are available to view and manipulate the image, facilitate landmark identification and calculate anthropometric linear, angular, and volumetric measurements. The disadvantages of 3D photogrammetry are its relative expense, limited availability, difficulties in recording shiny, shadowed, or transparent facial structures, and lack of ability to calculate interactive landmarks.
Facial Insight 3D Scanner [48]
VECTRA M3 Imaging System [59]
Three-dimensional surface imaging enables assessment of the spatial position of soft-tissue facial landmarks by assigning coordinates to each point. It could be difficult to identify a substantial number of landmarks and place them accurately in the three planes of space to gain a comprehensive understanding of the facial structure (Figure 16) [58-63]. In a reproducibility study of 3D facial landmarks, Hajeer et al. (2002) used C3D facial scans before and after orthognathic surgery of five patients to identify 24 landmarks on each scan. In addition, they defined four new landmarks by utilizing the Farkas’s anthropometric landmarks (1994) [63]. Three sessions of landmark digitization were performed within a week interval. Each landmark had x, y, and z coordinates given by the software and the mean differences were calculated from the three identification sessions by identifying the differences between the individual coordinate points. The results showed that 20 of the chosen landmarks had high reproducibility based on accepted 0.5 mm cut off point. The following landmarks whose localization depended on the underlying skeleton had problems of reproducibility: menton, left and right zygion, and left and right gonion (x-coordinate); left and right zygion, left and right gonion, left and right tragion, and glabelle (y-coordinate); menton, left and right otobasion inferius, left and right tragion, and left and right gonion (z-coordinate) [62]. In a different study, facial landmarks with distinct margins or borders also showed higher degree of consistent identification than those located on gently curved rounded surfaces. It is likely that digital measurements of the philtral length/upper prolabial width will require direct marking [64]. Several other studies also recommended direct labeling of certain landmarks in order to improve identification before image acquisition and/or direct measurements [54,65].
Digitized facial landmarks used in direct and indirect anthropometric measurements: Tr- trichion; G- glabella; N- nasion; Prn- pronasale; C’- columella; Sn- subnasale; Ls- labiale superius; Sl- sublabiale; Pg- pogonion; Me- menton; Ex- exocanthion; En- endocanthion; Os- orbitale superius; Or- orbitale; Ft- frontotemporale; Zy- zygion; Chk- cheek; T-tragion; Pra- preaurale; Sa- superaurale; Pa- postural; Sba- subaurale; Al- alare; Ac- nasal alar crest; Itn- inferior point of the nostril axis; Stn- superior point of the nostril axis; Cph- crista philtri; Ch- cheilion; Go-gonion [70].
Several types of 3D photogrammetric imaging systems have been described and evaluated in the literature, e.g. 3dMDface System (3dMD, Atlanta, GA, USA), C3D Imaging System (Ferranti, Birmingham, UK), Rainbow 3D Camera (Genex Technologies, Inc., Kensington, MD, USA), 3D Vectra (Canfield Imaging Systems, Fairfield, NJ, USA), and the Facial Insight 3D (Motion View Software, LLC, Chattanooga TN, USA) [57,65-68]. Aldridge et al. (2005) evaluated the precision and reproducibility of coordinate data and 190 distances on two sets of measurements made on 30 3dMDface images taken from 15 human subjects and found this system to be highly precise and reliable with a submillimeter average error in landmark placement. Weinberg et al. (2006) published a comparison of measurements of mannequin heads using two 3D surface imaging systems, 3dMDface and Rainbow 3D Camera, and direct anthropometry. These three techniques yielded a high degree of agreement among selected anthropometric variables, and the intraobserver precision was high for each method [65].
Manipulation of the scans and precise landmark identification requires proficiency with 3D computer software [53]. That’s why, in order to improve reproducibility, it is necessary to become accustomed to the selected software implemented to capture and process the images (Figure 17). Facial models captured with Facial Insight 3D were found to be affected by the angles of the subject’s face, hair interference, and head position, but in general, the precision of the digital images was within an acceptable range. It has been suggested that head position, projection, and stabilization should be consistently the same in order to achieve optimum standardized settings. Several internal module failures of the software while initiating a new series of scan, clearing the cache after scans, or modifying the location of already placed landmarks are still to be addressed by the manufacturer [69].
Virtual facial model [59]
Direct comparison of multiple faces is challenging due to the diverse size and orientation of each face. Facial average methods like the Generalized Procrustes Analysis (GPA) have been proposed where the scans are scaled and fitted into equal size reference frames [60]. This implies that transition and rotation are applied in order to eliminate size difference between facial scans and equilibrate the squared summed distances between corresponding facial landmarks. To obtain linear measurements, surface areas, and color mapping, a facial shells superimposition can be applied using the best-fitting alignment method or registration over relatively stable anatomical reference points and planes. A study evaluating 350 facial scans of children aged 15.5 years old identified the midendocanthion landmarks as the most stable facial structure for facial shell superimposition [52].
3D facial images easily integrate with study models, radiographs, and photographs and allow further simulations of orthodontic tooth movement and treatment results (Figure 18) [53, 70]. In order to validate and elaborate the correlation and matching process between facial images and dental casts, Rosati et al. (2010) merged dental digital models with 3D stereophotogrammetric open and closed lips images. The present anterior teeth in both facial and dental acquisitions were used as reference in the open-lips image superimpositions. In the closed lips image superimpositions the following facial soft-tissues landmarks were marked on the face before each acquisition and were used as fiducial points: Ftl, frontotemporale left; Ftr, frontotemporale right; and N, nasion. Seven linear measurements were virtually and directly performed between the facial landmarks and the occlusal plane on each subject. The results showed mean relative error smaller than 1.2%. The matching process was also found within a tolerable anthropometric and clinical context: the forehead mean distance in the open and closed lips image acquisitions was 0.4 mm, with a range between 0.04 mm and 1.1 mm [70].
Virtual surgical planning with the 3D Vectra photosystem (Canfield Imaging Systems, Fairfield, NJ) [59]
Over the past 20 years, a number of 3D facial databases containing static and time sequence of images have been built with the purpose of aid in automatic face recognition and analysis algorithms. Several publicly available databases have facilitated the research for tracking and superimposition of 3D facial shells and feature extraction methodologies. However, most of the existing 3D expression datasets are of small size comprising of deliberately posed or exaggerated expressions mostly of the six universal emotions (e.g. happiness, surprise, anger, sadness, fear, and disgust), obtained under directed settings. Spontaneous behaviors have been suggested to vary in timing and appearance from acted ones. For instance, deliberate smiles have faster onset and offset and larger amplitude than the velocity and amplitude of genuine smiles [71]. Additional databases of recorded micro-expressions and dynamic 3D faces captured in wider range of contexts, unprompted behaviors, and affective states will have to be designed.
Production sequential 3D surface imaging systems (4D Facial Dynamics) are commercially available to provide a quantifiable understanding of soft tissue mobility, true anatomical motion, and facial expression [72]. The 4D systems are used to assess facial function in conjunction with natural head movements, functional progress and outcomes for patients undergoing dental treatment and surgical interventions. Human face is capable of making unique microexpressions which can be of very low intensity and last less than 0.04 seconds. Therefore, the dynamic systems continuously track frame by frame the facial surface movements in order to achieve accuracy in understanding the tracking motions. The 4D technology acquires exact 3D surface information at approximately 60 frames per second from various coordinated standpoints for up to a 10 minute acquisition high resolution cycle. The video sequence of the area of interest is recorded with grey-scale cameras record while the surface texture is captured with a color camera [73]. A unified point cloud continuous image is displayed from the viewpoints of two or more stereo cameras, reducing the errors from the stitching process of different datasets. Motion capture systems with automatic facial landmark recognition software have been found practical objective solutions for the soft tissue quantification movements.
Assessment of facial animation could be an essential part for orthodontic diagnosis and craniofacial abnormality, virtual surgical planning, and treatment outcomes. Furthermore, various surgical interventions could affect the function of nerves and associated musculature which could influence the magnitude and the speed of the soft tissue motions. Shujaat et al. (2014) evaluated the dynamics of four facial movements (maximum smile, grimace, cheek puff, and lip purse) pre- and post- lip split mandibulotomy, by using six facial landmarks. The similarity of the facial animation pattern before and after the surgery was calculated after eliminating the head motion and aligning the movement curves using the right and left endocanthion and pronasale as stable landmarks unaffected by the surgery. The results showed that the velocity of all landmarks was lower after the surgery; the smile animation difference was the least (-0.1 mm/s), whereas the largest changed was found for the grimace animation (-5.8 mm/s). Mouth width maximum change after the surgery was found to be for lip purse (3.4 mm), whereas grimace showed the least difference after the surgery. Lip purse animation similarity was highest (0.78) while grimace had lowest similarity (0.71). The 4D dynamic devices have also been employed for interlandmark and vector deviations, and shape and gender comparisons [74]. Virtual and sound animations have been incorporated in some of the recent system improvements [75].
Additive manufacturing or 3D printing was founded in 1990 by Wilfried Vancraen, CEO and Director of Materialise NV, the first Rapid Prototyping sector company in the Benelux region [1]. 3D printing technology allows the user to create or “print” 3D physical objects, prototypes, and production parts of any shape from a virtual model in a growing range of materials including plastic, cobalt, nickel, steel, aluminum, titanium, etc. [76,77]. Those materials are joined in successive layers one on top of the other through additive processes under automated computer control. The 3D printing process usually begins with a 3D model, virtually designed or obtained through scanning of a physical object. Slicing software automatically transforms the point cloud into a stereolithographic file which is sent to the additive manufacturing machine for building the object (Figure 19).
The 3D Printing process [79]
Today, 3D printing has grown to be competitive with the traditional model of manufacturing in terms of reliability, speed, price, and cost of use. In comparison with other technologies, additive manufacturing is more effective due its ability to use readily available supplies, recycle waste material, and has no requirements for costly tools, molds, or punches, scrap, milling, or sanding. 3D printing technology is used for distributed manufacturing, rapid manufacturing, mass customization, and rapid prototyping with applications in engineering, civil engineering, automotive, architecture, construction, aerospace, military, human tissue replacement, dental and medical industries, industrial design, jewelry, fashion, eyewear, geographic information, education, footwear, and many other fields (Figure 20) [78,80]. Additive manufacturing is likely to continue rapid growth in conjunction with intraoral scanning technology as a more effective system for orthodontic practices and laboratories for automatic fabrication of high-resolution study models, retainers, metal appliances, aligners, and indirect bonding, accelerating the production time and increasing the capability [15,77].
3D Printing is revolutionizing medicine and dentistry by creating body parts: heart valves, ears, artificial bone, joints, soft tissue prostheses, and blood vessels [86-88]
Currently, there is a huge selection of available 3D printing technologies suitable for orthodontic use:
Fused depositing modelling (FDM) is frequently used for modelling, manufacture applications, and prototyping. The technology was introduced by S. Scott Crump towards the end of 1980s and was popularized by Stratasys, Ltd in 1990 [1]. FDM employs the "additive" method of laying down thermoplastic material in layers. In order to produce a part the material is supplied through a heated nozzle after a metal wire or a plastic filament wound in a coil are released. The melted material hardens immediately after extrusion, thus minimizing inaccuracies [81]. The nozzle can be directed in both vertical and horizontal lines by a numerically controlled software mechanism. Several materials such as acrylonitrile butadiene styrene (ABS) polymer, polyphenylsulfones and waxes, polycaprolactone, polycarbonates, polyamides, lignin, among many others, with diverse strength and thermal properties are available.
Another approach to produce a 3D structure is for the material to be supplied from a basin through a small nozzle such as in the case of the 3D Bioplotter (EnvisionTEC, Gladbeck, Germany). The device is mainly applied in prototyping porous scaffolds for medical tissue engineering and organ bio-printing [82]. With an accuracy of just a few micrometers, the bioplotter is able to build body parts with different microstructural patterns including blood vessels, bone, and soft tissue. FDM is the most widely used process of 3D printing today, although there are other almost identical technologies like MakerBot (Stratasys, Ltd., Eden Prairie, MN) known as Fused Filiment Fabrication (FFF).
Laser based additive manufacturing, such as selective laser melting (SLM) and selective laser sintering (SLS), uses power in the form of a high energy laser beam directed by scanning mirrors to build three-dimensional objects by melting metallic powder and fusing the fine particles together [83]. The laser energy is strong enough to allow full welding/melting of the particles to create a solid part. The process which can include partial and full melting or liquid-phase sintering is recurring layer after layer until the object is completed. The technology is commonly utilized due to its ability to form parts with complex geometries with very thin walls and hidden channels or voids directly from digital CAD data. Compared to other types of 3D printing, SLM/SLS have very high productivity and can build objects from a relatively big selection of commercial powder materials [1]. These include polyamides, polycaprolactone, hydroxyapatite, ultra high molecular weight polyethylene, polyethylene, ceramic, glass, stainless steel, titanium, and Co/Cr alloys. Although most of the initial applications of the laser based technologies were for manufacture of lightweight aerospace parts, the SLM/SLS have found an acceptance for production of orthopedic and dental implants, dental crowns and bridges, partial denture frameworks, and bone analogs [84].
Electron beam melting (EBM) is a type of additive manufacturing for laying down successive layers and creating near-net-shape or highly porous metal parts that are particularly strong, void-free, and fully dense. The EBM technology uses the energy source of an electron beam, as opposed to a laser [85]. Objects are manufactured layer by layer from fully melted metal powder utilizing a computer controlled electron beam in a high vacuum. The technology operates at higher temperatures of up to 1000 °C, which could result in differences of the phases formed through solidification. EBM is able to form extremely porous mesh or foam structures in a wide range of alloys including stainless steel, titanium, and copper. The technology is commonly used in orthopedic and oral and maxillofacial surgery for manufacturing customized implants. Their structure permits the ingrowth of bone, provides better fixation, and helps to prevent stress shielding [1].
The term “stereolithography” was first presented by Charles W. Hull in 1986 as a technique for producing solid items by consecutively printing thin layers material that is solidified by a concentrated ultraviolet laser light. SLA is the first so-called “rapid prototyping” process. The resolution of the built item is higher when more layers are used and the number of layers may range from 5 to 20 per millimeter [1, 77]. After being built, objects are immersed into a solvent bath for excess resin removal and are consequently placed in an UV oven to finish the curing process. Based on object complexity and size, stereolithography can take from a few hours to more than 24 hours to create a particular part.
Most of the SLA immediate use was in the automotive and aerospace industries, but medical and dental applications of this technology gradually emerged. SLA models are currently used for planning cranial, maxillofacial, and neurosurgical procedures and constructing highly accurate replicas of human anatomy, customized implants, cranioplasties, orbital floors, and onlays. Surgical guides for dental implant placement are routinely produced by stereolithography [80].
The inkjet printing technology employs a nozzle which “prints” a pattern on a thin layer of powder substrate by propelling a liquid binding agent (Figure 21) [1,77]. The small ink droplets are forced through the orifice by pressure, heat, or vibrations. The object is built through a recurring process layer by layer with each layer of material adhered to the last. Phase transformation from liquid to solid occurs immediately after droplets are deposited upon the substrate by UV curing light, drying, chemical reaction, or heat transfer [86-88]. The polyjet printers allow volumetric color objects to be built in simultaneous incorporation of multiple materials with quite distinct physical properties. As of 2014, manufacturers were able to combine sand and calcium carbonate, ceramic powder and liquid binder, acrylic powder and cyanoacrylate, water and sugar (for making candies), etc.
Representation of PolyJet photopolymer (PPP) 3D printing [77]
By combining rigid and rubberlike materials, it is possible to create a mouthguard with soft and hard regions in different colors. Further dental applications include reproduction of study models, surgical guides for implant placement, sleep apnea appliances, orthodontic bracket guides, and try-in veneers [87,89]. Extrusion rate, nozzle size, and droplet travelling speed are able to affect the dimensional accuracy of dental restorations [90]. Specially engineered dental materials for polyjet printing provide fine layers as thin as 16 microns, which render small features in great details with strength and durability. Biocompatible materials, which allow short-term mucosal membrane contact of up to 24 hours and prolong skin contact of more than 30 days, are used for manufacture of soft tissue prostheses and hearing aids.
In general, the inkjet printing technology is faster than other additive manufacturing processes such as fused depositing modelling. However, depending on the material and process, surface finish, object density, and accuracy may be inferior to stereolithography and selective laser sintering.
Digital Light Processing (DLP) is a type of nanotechnology that uses a digital micromirror device as a power source projector to cure liquid resin into solid 3D objects. DLP is similar to stereolithography as the method also employs light polymerization. One difference is that DLP creates a single layer as one digital image in tiny volumetric pixels as opposed to SLA’s laser process which must scan the vat with a single point. DLP printing is faster and can build objects with a higher resolution, typically able to reach a layer thickness of fewer than 30 microns [91]. Furthermore, DLP can produce objects with a wide variety of properties such as high clarity, spngness, flexibility, water resistance, thermal resistance, and durability. The photopolymers have been designed to mimic ABS, polypropylene, and wax, blending layers together much more smoothly than plastic filament is able to. However, photopolymer prints can become brittle with increased light exposure over time. Objects may begin to show cracks and become more susceptible to breaking.
The DLP process can only use one material at a time since the object is built out of a vat containing a singular photopolymer solution. Post-print processing involves washing away the remaining resin and removal of the supports by snapping or cutting. DLP-based technologies are found in such diverse applications as movie projectors, cell phones, video wall, digital cinema, medical, security, and industrial uses [92].
Laminated object manufacturing (LOM) is a process that combines additive and subtractive techniques to build an object. It works by successively layering sheets of material one on top of another and binding them together using adhesive, pressure, and heat application. Once the process is complete, objects are cut to desired dimensions with a knife, a laser, or additionally modified by machine drilling. The technology is able to produce relatively large parts since no chemical reaction is necessary. The most common materials used in LOM are plastics, paper, ceramics, composites, and metals which are widely available and yield comparatively inexpensive 3D printing method. Materials can be mixed in various layers throughout the printing process giving more flexibility in the final outcome of the objects. Paper models have a wood-like texture and characteristics and can be finished accordingly. Surface accuracy is slightly inferior to stereolithography and selective laser sintering. LOM systems are used in sand casting, investment casting, ceramics processing, for concept modelling, and architectural applications [93].
The global additive manufacturing industry has been dominated by three large companies: Stratasys, Ltd. (Eden Prairie, MN), 3D Systems (Rock Hill, SC), and EnvisionTEC (Gladbeck, Germany), with market shares of 57%, 18%, and 11%, respectively [94]. As of January 2014, Stratasys sells 3D printing systems that range from $2,200 to $600,000 in price and are employed in several industries: aerospace, automotive, architecture, defense, medical and dental, among many others (Figure 22). MakerBot and Objet are the 3D printers recently acquired by Stratasys and currently used in dentistry and orthodontics. For example, ClearCorrect employs Objet in the aligner manufacture process while Invisalign uses the 3D Systems\' SLA technology. Other companies like Concept Laser (Lichtenfels, Germany), Realizer (Borchen, Germany), and SLM Solutions (Lübeck, Germany) are also offering printing technologies and new materials to be used in dental 3D printing. Furthermore, a broad line of innovative professional 3D printers, orthodontic practical solutions, and price points exist for generating full-color parts, wax patterns, and investment castings. Table 2 summarizes some of the characteristics of several 3D printers used in orthodontics [86-88,95,96].
3D Printing end markets [97]
\n\t\t\t\tFeatures\n\t\t\t | \n\t\t\t\n\t\t\t\tObjet30 OrthoDesk\n\t\t\t | \n\t\t\t\n\t\t\t\tProJet® 3510 MP\n\t\t\t | \n\t\t\t\n\t\t\t\tULTRA® 3SP™ Ortho\n\t\t\t | \n\t\t\t\n\t\t\t\tPerfactory® Micro Ortho\n\t\t\t | \n\t\t\t\n\t\t\t\tMakerBot Replicator 2\n\t\t\t | \n\t\t\t\n\t\t\t\tFORMIGA P 110\n\t\t\t | \n\t\t
\n\t\t\t\tCompany\n\t\t\t | \n\t\t\tStratasys, Ltd., Eden Prairie, MI | \n\t\t\t3D Systems, Rock Hill, SC | \n\t\t\tEnvisionTEC, Gladbeck, Germany | \n\t\t\tEnvisionTEC, Gladbeck, Germany | \n\t\t\tStratasys, Ltd., Eden Prairie, MI | \n\t\t\tEOS, Munich, Germany | \n\t\t
\n\t\t\t\tTechnology\n\t\t\t | \n\t\t\tPolyJet Printing technology | \n\t\t\tPolyJet Printing technology | \n\t\t\tDigital Light Processing | \n\t\t\tDigital Light Processing | \n\t\t\tFused Depositing Modelling | \n\t\t\tSelective Laser Sintering | \n\t\t
\n\t\t\t\tBuild Volume\n\t\t\t | \n\t\t\t300 x 200 x 100 mm | \n\t\t\t298 x 185 x 203 mm | \n\t\t\t266 x 177.8 x 76 mm | \n\t\t\t100 x 75 x 100 mm | \n\t\t\t285 X 153 X 155 mm | \n\t\t\t200 x 250 x 330 mm | \n\t\t
\n\t\t\t\tLayer Thickness\n\t\t\t | \n\t\t\t0.0011 in | \n\t\t\t0.001-0.002 in | \n\t\t\t0.00098 in | \n\t\t\t0.0039 in, 0.002 in, 0.004 in | \n\t\t\t0.0039 - 0.0133 in | \n\t\t\t0.0024 in, 0.0039 in, 0.0047 in | \n\t\t
\n\t\t\t\tApplications\n\t\t\t | \n\t\t\tHigh quality orthodontic models, surgical guides, temporary intraoral appliances and restorations | \n\t\t\tDrill guides, jaw models, orthodontic thermoforming model | \n\t\t\tHigh quality orthodontic appliances | \n\t\t\tHigh quality models for the fabrication of orthodontic appliances | \n\t\t\tRetainers and aligners with less esthetic appearance due to stair-stepping | \n\t\t\tHigh quality retainers and orthodontic appliances | \n\t\t
\n\t\t\t\tWeight\n\t\t\t | \n\t\t\t93 kg | \n\t\t\t323 kg | \n\t\t\t90 Kg | \n\t\t\t13 kg | \n\t\t\t11.5 kg | \n\t\t\t600 kg | \n\t\t
\n\t\t\t\tProduct Website\n\t\t\t | \n\t\t\twww.stratasys.com | \n\t\t\twww.3dsystems.com | \n\t\t\thttp://envisiontec.com | \n\t\t\thttp://envisiontec.com | \n\t\t\twww.makerbot.com | \n\t\t\twww.eos.info | \n\t\t
Comparison of 3D printers currently used in orthodontics
Objet30 OrthoDesk (Stratasys, Ltd., Eden Prairie, MN) employs the PolyJet printing technology and is suitable for orthodontic offices and small- to medium-sized orthodontic labs (Figure 23). The 3D printer is able to fabricate durable orthodontic models with high feature detail and ultrafine layers of surface quality [77]. Every print run can create up to 20 models. Three dental materials, specially engineered for dentistry, come with the printer in sealed cartridges: VeroDentPlus (MED690), a dark beige, acrylic-based material prints layers as fine as 16 microns with accuracy as thin as 0.1mm used for most appliances; Clear biocompatible (MED610), a transparent material medically approved for temporary intraoral applications and surgical guides; and VeroGlaze (MED620), an acrylic-based material for veneer models or diagnostic wax-ups in A2-shade color match that can be used in the mouth as long as 24 hours [86].
The Objet30 OrthoDesk (Stratasys, Ltd., Eden Prairie, MN) 3D printer [86]
ProJet® 3510 MP (3D Systems, Rock Hill, SC) is one of the several healthcare printing solutions, used for uniformly accurate thin wax-ups of crown, bridges, and partial dentures. The system can also produce any size dental or jaw models with a choice of two materials in smooth or matte printing mode. Up to 24 quad cases can be built at one time (Figure 24).
The ProJet® 3510 MP (3D Systems, Rock Hill, SC) 3D printer [88]
The 3D Systems professional printers support the VisiJet® line of materials, specially engineered to meet a wide range of applications. ProJet® 3510 series come with three UV curable acrylic materials: Dentcast, a dark-green, wax-up material, which burns out cleanly for ash-free castings (Figure 25); PearlStone, a while material with a solid stone appearance; and Stoneplast for transparent, clear or stone finish dental models. VisiJet® S300 is the fourth material which is a non-toxic white wax material for hands-free melt-away supports [88].
Dental wax-up and casting manufactured with ProJet® 3510 MP [88]
ULTRA® 3SP™ Ortho (EnvisionTEC, Gladbeck, Germany) employs the Scan, Spin, and Selectively Photocure (3SP™) technology, a DLP variant, which utilizes a laser diode with an orthogonal mirror spinning at 20,000 rpm (Figure 26). The printer is able to produce highly accurate and stable dental models that could be used for orthodontic appliance fabrication. The models are resistant to high temperature and have negligible water absorption.
The EnvisionTEC (Gladbeck, Germany) 3D printers used in dentistry and orthodontics [87]
ULTRA® 3SP™ Ortho also comes with specially engineered photosensitive resins for dental and orthodontic applications: Press-E-Cast (WIC300), a wax-filled photopolymer for production of copings with extremely thin margins as well as up to 16 multiple unit bridge; E-Denstone (HTM140 Peach), a peach color material able to achieve the look and feel of traditional gypsum models with a high-accuracy detail; and D3 White, a fast-growing, tough material with similar characteristics to ABS plastic and the most common medium for dental model manufacturing for the production of orthodontic appliances [87].
A variety of low cost printers are also available for home use such as the MakerBot Replicator 2 (Stratasys, Ltd., Eden Prairie, MI). Some of those low cost devices have the ability to locally print objects in an astonishing number of materials, including ice, chocolate, rubber caulk, frosting, and ceramic clay. Low cost printers, however, still lack supports for overhanging geometry and their use in orthodontics could be problematic. The machines are often fragile with temperature, deposition, and position controls not accurate enough to make functional end-parts [95].
3D printing solutions are capable to achieve various products with high level of precision. The use of the technology to build dental models, removable appliances, customized brackets and archwires, and occlusal splints has been attempted and reported in the orthodontic literature (Figure 27) [98-101]. Currently, the most common application of the 3D printers is for clear retainers and aligner fabrication [77]. Practitioners can virtually move the teeth to a final ideal position, print a sequence of physical models in the office, and use a thermoplastic material to fabricate aligner trays, working on similar premise to ClearCorrect and Invisalign. Skipping the step of 3D printing a physical model, researchers have also used the technology to digitally design a retainer and consequently 3D print it in a fine while polyamide material [99]. Sophisticated software is further available for shaping and trimming the dental model base, for design of bracket pads, hooks angulations, and guiding jigs. Digital titanium Herbst, Andresen, and sleep apnea appliances have been made with smooth surfaces, no sharp edges, and excellent fit on the teeth, palatal and gingival tissues. Additive manufacturing enables features such as hinge production, building threads, and wire insertion to be completed in a single build without assembly [102,104].
Dental model, RPE, and an implant surgical guide 3D printed from a STL generated file [38, 86]
Construction of metal dental frameworks of Co-Cr alloy, dental prosthesis wax patterns, facial prosthesis shells, and zirconia restorations using 3D printed technology have been successfully reproduced for use in prosthodontics [104-106]. In contrast, certain orthodontic appliances with soldered parts might require the use of a stone model since some 3D-printed models would deform or melt from the high temperature [77]. A broader range of materials with greater strength and resistance to moisture and heat should be specifically developed to suit the dental and orthodontic industries. Digitization of the manufacture process and standardization of the material ingredients are important steps for achieving consistent results.
With the rapid development and advanced research of diverse technologies and compatible materials, it is possible to obtain single scan digital impressions, virtually design, and 3D print different types of orthodontic appliances. 3D facial imaging further provides comprehensive analysis as an aid in orthodontics, maxillofacial, plastic, and esthetic surgery. Software integration of digital models, 3D facial scans, and CBCT facilitate treatment simulations and establish a meaningful communication with patients. Elimination of traditional impressions and dental-cast production stages enhance practice efficiency, patient and staff satisfaction for a fully integrated digital and streamlined workflow. Patient digital impressions are stored in a more convenient way and can be easily transferred to any lab or an in-office milling machine for a simpler, faster, and more predictable appliance fabrication. New companies, scanner and printer models are emerging daily which result in significant decline of systems cost and enhancement of material qualities. From imaging to product design and manufacture, technologies will offer more affordable and feasible diagnostic and treatment applications beyond the current methods.
This material is based on research sponsored by the Air Force Surgeon General’s Office under agreement number FA7014-09-2-0003. The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the Air Force Surgeon General’s Office or the U.S. Government. We thank the cited companies and journals for granting us permission to reproduce images included in this chapter. Figure 16 is reprinted from Rosati R, De Menezes M, Rossetti A, Sforza C, Ferrario VF. Digital dental cast placement in 3-dimensional, full-face reconstruction: a technical evaluation. Am J Orthod Dentofacial Orthop 2010;138(1): 84-8 with permission from Elsevier. Figure 21 is reprinted from Groth C, Kravitz ND, Jones PE, Graham JW, Redmond WR: Three-Dimensional Printing Technology. J Clin Orthod 2004;48: 475-485. Illustration by Nancy McCullick, reprinted with permission from JCO, Inc.
The term “molecular pharming,” blend of pharmaceutical and farming, surfaced in the literature in the 1980s to refer to the production of high-value compounds in transgenic animals. Nowadays, the expression is mainly employed to the production of recombinant pharmaceutically relevant proteins or secondary products in plants [1–3].
The roots of molecular pharming can be traced back to the mid-1980s when plants started to be genetically engineered to act as bioreactors that produced pharmaceutically relevant proteins. Barta et al. [4] demonstrated that tobacco and sunflower callus tissues were capable of expressing transcripts of a human growth hormone fusion gene. Although no protein was detected, this was the first report of plants expressing human genes and established plants as a potential production system for recombinant therapeutic proteins. Later on, the expression of a full-sized IgG in tobacco [5] was a major breakthrough since it revealed the ability of plants to produce complex functional mammalian proteins of pharmaceutical relevance. In 1990, the “authenticity” of plant-derived recombinant proteins was proved even further with the production of the first human protein (serum albumin) with confirmed native structure in tobacco and potato [6].
After several studies that demonstrated the capacity of various plant species and systems to produce recombinant pharmaceutical proteins and peptides, during the 1990s, the field of molecular pharming gained support and interest from the plant biotechnology community. The scientific attention was followed by commercial interest, with many start-up companies being created to capitalize the advantages of plants in relation to the established platforms. These advantages include being a more cost-effective, scalable, and safer means of producing pharmaceutically relevant proteins and peptides. In opposition to the fermentation-based traditional platforms that require a massive investment in bioreactors, plant-based production systems can be established with minimal investment and offer a myriad of different hosts and platforms [7]. However, the expectation that plants could easily compete for the market share of some well-established biopharmaceutical platforms, such as Chinese hamster ovary (CHO) cells, and that they could motivate the mainstream pharmaceutical industry to switch platforms was overinflated. The CHO epithelial cell lines are the most commonly used mammalian hosts for industrial production of therapeutic recombinant proteins. The technical limitations of plants, especially their lower yields compared to mammalian cell lines, allied to the colossal existing investment in fermentation infrastructures, the unfavorable public opinion on OGMs, and regulatory uncertainty, lead the mainstream pharmaceutical industry to be cautious and to a consequent stagnation of the molecular pharming field in the 2000s [8, 9]. This situation induced a change of paradigm concerning molecular pharming: the initial vision of a highly scalable and low-cost production system, while still valid, was replaced by the idea of a production system for certain niche products that are not easily manufactured by conventional systems [8, 9].
Molecular pharming embraces several platforms and technologies with different advantages and limitations, related by their use of plant tissues. Conversely to conventional biopharmaceutical production systems that are based on few selected platforms, particularly the bacterium Escherichia coli, yeasts such as Pichia pastoris, and mammalian cell lines such as Chinese hamster ovary (CHO) cells [3], pharming platforms range from plant cells or unicellular plants growing in bioreactors to whole plants growing in soil or hydroponic environments. Further, the technologies include stable integration of DNA into the nuclear genome or plastid genome and transient expression by infiltrating leaves with expression vectors based on Agrobacterium tumefaciens, plant viruses, or hybrids [3, 8]. This great diversity of molecular pharming confers adaptability and flexibility, allowing the selection of the most suitable platform for each product, but has also conduced to fragmentation. This fragmentation meant that in the early days of molecular pharming there was no driving force to establish molecular pharming as a single competitive platform. Consequently, no actions were made to match the industry requirements for high yields, standardized procedures, and good manufacturing practices (GMP) [7, 9]. More recently, efforts have been made to mimic the mainstream biopharmaceutical industry and place a focus only on a small number of platforms, namely, plant cell cultures, nuclear transgenic plants, and leafy plants transiently transformed [3, 10]. Since 2010 the attention of the biopharmaceutical industry to molecular pharming has been renewed as result of its consolidation on a small number of platforms and some target products that meet industry demands [8, 9].
In 2012, the FDA approval of the first recombinant plant-derived therapeutic for human use, Protalix Biotherapeutics’ taliglucerase alfa (Elelyso™), was an important breakthrough for molecular pharming. The enzyme taliglucerase alfa is a carrot cell-expressed human recombinant β-glucocerebrosidase and is prescribed for the treatment of Gaucher’s disease, a lysosomal storage disorder [11]. Imiglucerase, a recombinant form of glucocerebrosidase commercialized under the name Cerezyme®, was already produced in CHO cells. In this production platform, the enzyme required subsequent in vitro exposure to mannose residues in order to have biological activity, resulting in a time-consuming and expensive manufacturing process. Besides, this platform also has potential safety problems, namely, the risk of viral contamination, allergies, and other adverse reactions. In comparison, the plant-based platform is safer and less time-consuming and has reduced production costs, since the mannose units are posttranslationally added in vivo [11]. Glucocerebrosidase is a clear example of a target product in which safety, cost, and downstream processing issues were solved by switching from a traditional platform to molecular pharming. Another example that gathered mediatic exposure was ZMapp, a cocktail of three chimeric monoclonal antibodies targeting the Ebola virus surface glycoprotein produced in Nicotiana benthamiana using a hybrid transient expression system, the magnICON system. ZMapp was developed during the Ebola outbreak of 2014 by Mapp Biopharmaceutical Inc. (San Diego, USA), following initial studies on nonhuman primates [12]. ZMapp has since been used in humans under emergency compassionate protocols [13] and randomized controlled trials [14].
Following these examples of success, there has been a continuous increase in clinical trial applications and manufacturing capacity, which has also been correlated with the conception of more tangible regulations concerning plant-derived pharmaceuticals.
Although plants are still unlikely to substitute the established platforms [8], the recent promising developments in the field of molecular pharming demonstrate that glucocerebrosidase was not a lone case of success and that plant-based platforms could provide countless opportunities for the biopharmaceutical market. Plants combine the advantage of a full posttranslational modification potential with simple growth requirements and theoretically unlimited scalability in the case of field-grown whole plants. Plant-based platforms are versatile and allow the targeting of recombinant proteins and peptides produced to different organs or subcellular compartments, which provides an additional protection against proteolysis. Finally, plants are a safe host for therapeutic protein and peptide production since they do not harbor human or animal pathogens [15]. Therefore, instead of facing the red ocean of established pharmaceutical industries [16], molecular pharming is now evolving as a disruptive technology that creates its own marketplace by offering rapid drug development and production, unparalleled scalability, unique quality attributes such as tailored glycan structures, individualized therapies, and oral or topical applications of minimally processed plant tissues, thus reducing downstream costs [17].
The continuous development of genetic engineering technologies for plants has resulted in an expansion of well-established plant-based platforms [18]. Molecular pharming encompasses platforms based on stably transformed whole-plants transgene insertion in the nuclear or plastid genome, transient expression using agroinfiltration, viral and hybrid vectors; microalgae and aquatic plants (e.g., duck-weed) stably transformed; and in vitro culture systems (e.g., cell suspensions, hairy roots, and moss protonema) [19]. Each platform has particular advantages and limitations; therefore its selection is done on a case-by-case basis, depending on economic considerations as well as on the product characteristics and intended use [20].
Transgenic plants have been the most widely used platforms for recombinant protein production. To obtain stable transgenic lines, the gene encoding the desired protein is cloned into an expression construct, which generally includes a promoter and regulatory elements that ensure efficient RNA processing and protein synthesis [21]. This expression construct is then stably integrated into the plant nuclear genome, resulting in the stable inheritance of the transgene and expression of stable pharmaceutical proteins over generations [22]. Two major transformation strategies have been employed to insert expression constructs into the nuclear genome: Agrobacterium-mediated transformation in dicotyledonous species (dicots) and particle bombardment of DNA-coated gold or tungsten beads in monocotyledonous species (monocots) [3]. Transgenic plant lines offer several advantages as platforms for molecular pharming: they are suitable for long-term production of recombinant pharmaceutical proteins and are highly scalable, as each line can be used to produce seeds, which increase the number of plants in every generation. Ultimately, the production capacity of recombinant pharmaceutical proteins in transgenic plants is practically unlimited, as it only depends on the number of hectares available for the plant culture. The major drawbacks of transgenic plants are the long development and scale-up timescales, the unreliable production yields, and the potential spread of pharmaceutical crops in the environment and into the food chain by outcrossing and seed dispersal [3].
The development of simple transformation technologies has expanded the number of host plants available for molecular pharming. Currently, the major molecular pharming transgenic platforms are based on leafy crops, seeds, fruits, and vegetable crops. Leafy crops are benefic in terms of biomass yield and high soluble protein levels. Additionally, leaf harvesting does not need flowering and thus considerably reduces contamination through pollen or seed dispersal [23]. One disadvantage of leafy crops is that proteins are synthesized in an aqueous environment, which is more prone to protein degradation, resulting in lower production yields [24]. In fact, the mature leaves possess very large extra cytoplasmic vacuolar compartments containing various active proteolytic enzymes that are involved in the degradation of native and foreign proteins. This is particularly problematic in the case of therapeutic peptide production because short heterologous peptides have an inherent instability in plant cells [25]. In addition to the protein instability, the harvested material has limited shelf life and needs to be processed immediately after harvest.
Tobacco has been the most widely used leafy crop for molecular pharming. The major advantages of using tobacco to express pharmaceutical proteins are its high biomass yield, well-established technology for gene transfer and expression, year-round growth and harvesting, and the existence of large-scale infrastructure for processing [23]. However, the natural production of nicotine and other alkaloids in tobacco poses some safety issues in its use as a host system for heterologous protein production. Therefore, tobacco varieties with low nicotine and alkaloid levels have been produced to diminish the toxicity and overcome those safety issues. Recent studies have led to the approval of the first monoclonal antibody produced in transgenic tobacco plants, in phase I clinical trial [26]. Additionally, a 2018 publication reported the stable expression of adalimumab (a monoclonal antibody against tumor necrosis factor-alpha (TNF-α)) in tobacco plants [27]. Other leafy crops commonly used in molecular pharming include alfalfa and clover [19].
As an alternative to leafy crops, plant seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins [28]. The expression of proteins in seeds can overcome the shortcomings of leafy crops in terms of protein stability and storage. Seeds possess specialized storage compartments, such as protein bodies and vacuoles, which provide the appropriate biochemical environment for protein accumulation, thus protecting the proteins expressed in seeds from proteolytic degradation [29]. Reports have demonstrated that antibodies expressed in seeds remain stable for at least 3 years at room temperature without detectable loss of activity [30]. Furthermore, the small size of most seeds permits to achieve a high recombinant protein concentration in a small volume, which facilitates extraction and downstream processing and reduces the costs of the overall manufacturing process [31]. One essential property of seeds is dormancy, which not only permits the stability of recombinant proteins but also allows a complete decoupling of the cycle of cultivation from the processing and purification of the protein [28]. Finally, proteins expressed in the seed do not normally interfere with vegetative plant growth, and this strategy also reduces exposure to herbivores and other nontarget organisms such as microbes in the biosphere [21]. Several crops have been studied for seed-based production, including cereals, such as maize, rice, barley, and wheat; legumes, such as pea and soybean; and oilseeds such as safflower and rapeseed. Maize has several advantages for seed-based expression of proteins; it has the highest biomass yield among food crops, and it is easy to transform, in vitro manipulate, and scale up [24]. These potentialities were explored by Prodigene Inc. for the production of the first commercially available plant-made protein, avidin (a protein with affinity for biotin used in biochemical assays). Other maize-derived protein products developed by this company include β-glucuronidase, aprotinin, laccase, and trypsin [32]. Prodigene was the first company to demonstrate the commercial benefits of plant-based platforms and was also a forerunner in the study of the economic impact of downstream processing in molecular pharming, having developed several successful approaches to recover intact and functional recombinant seeds from maize [3].
Maize has also been used to produce recombinant pharmaceutical proteins, including enzymes, vaccines, and antibodies [32, 33]. One of the most notable therapeutic proteins produced in maize is Meristem Therapeutics’ gastric lipase, an enzyme intended for the treatment of exocrine pancreatic insufficiency—a disease significantly affecting cystic fibrosis sufferers—that has completed phase II clinical trial. In addition to this enzyme, Meristem Therapeutics has developed two other maize-derived products, human lactoferrin (phase I clinical trial), whose intellectual property was later acquired by Ventria Bioscience (
Rice is another leading platform for recombinant protein and peptide production. Similar to maize, rice is easy to transform and scale up, but unlike maize, rice is self-pollinating, which reduces the risk of horizontal gene flow. Ventria Bioscience, in its ExpressTec platform, has used rice to produce recombinant pharmaceutical proteins, including human albumin, transferrin, lactoferrin, lysozyme, and vaccines against human rabies and Lyme disease. Its lead therapeutic candidate VEN100, whose active ingredient is lactoferrin, has been shown to reduce significantly antibiotic-associated diarrhea in high-risk patients and recently completed phase II clinical trial [34]. Rice has also been widely used as host for peptide expression, especially for the production of allergen peptides (e.g., pollen and mite allergies) [35, 36]. Recent studies report that rice has the potential to offer an oral delivery system for vaccine antigens and therapeutic proteins and peptides [25, 35, 37].
Barley seeds have also been developed as commercial platforms. In comparison to other cereal crops, barley is less widely grown. However, this fact added to the self-pollinating nature of barley can be viewed as an advantage since the risk of contamination and outcrossing with non-transgenic crops is minimized. Considering this benefit, an Iceland-based company, ORF Genetics (
The use of legume seeds, such as soybean and pea, for the production of recombinant pharmaceutical proteins, has been less explored than cereal-based platforms, with platforms based on legume seeds having yet to achieve commercial success. However, the fact that legume seeds have exceptionally high protein content (20–40%) can be exploited to achieve high yields of recombinant protein [39]. Soybean seeds have been used to express recombinant growth factors [40, 41], coagulation factors [42], and vaccine peptides [43]. Transgenic pea seeds have been previously used to produce a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy [44]. In another study, pea seeds were used to produce a vaccine that showed high immunogenicity and protection against rabbit hemorrhagic disease virus [45].
Safflower and rapeseed seeds are rich in oil and are, thus, referred as oilseeds. Oilseeds can provide useful recombinant pharmaceutical protein production systems. SemBioSys (
Finally, fruit and vegetable crops can also be employed for molecular pharming. A major advantage of protein expression in fruit and vegetable crops is that edible organs can be consumed uncooked, unprocessed, or partially processed, making them particularly suitable for the production of recombinant subunit vaccines, nutraceuticals, and antibodies designed for topical application [29]. The oral delivery of recombinant therapeutics is one of the differentiating factor of molecular pharming in comparison to mainstream biopharmaceutical production systems, with several pharmaceutical products being produced in tomato fruits, potato tubers, and lettuce leaves for this purpose [3]. Tomato fruits are particularly useful for protein expression because the fruits are palatable as raw tissue but can also be lyophilized and stored for a long time [25]. Recently, human coagulation factor IX (hFIX) was expressed specifically in tomato fruits, constituting the first report on the expression of hFIX in plant [48]. Another study described the expression in tomato fruits of a thymosin α1 concatemer [49], an immune booster that plays an important role in the maturation, differentiation, and function of T cells. The thymosin α1 concatemer derived from transgenic tomatoes exhibited biological activity and was proven to stimulate the proliferation of mice splenic lymphocytes in vitro. Moreover, thymosin α1 specific activity was higher when produced in tomato than in Escherichia coli, demonstrating the authenticity of the plant-made product. Other examples of tomato fruit expression include F1-V [50], a candidate subunit vaccine against pneumonic and bubonic plague, and β-secretase [51], to serve as a vaccine antigen against Alzheimer’s disease.
In conclusion, platforms based on transgenic plants are a promising alternative to the conventional biopharmaceutical production platforms since they provide a stable source of pharmaceutical proteins and are also the most scalable of all molecular pharming platforms. This scalability of transgenic plants ensures the production of recombinant pharmaceutical proteins at levels previously inaccessible, namely, the commodity bulk production of monoclonal antibodies. In the current scenario of growing pharmaceutical demand, especially in developing countries, the use of transgenic plants can be game changing since they provide a highly scalable and low-cost means of producing medicines.
Transplastomic plants are a valuable alternative to transgenic plants for the production of recombinant pharmaceutical proteins. Transplastomic plants are obtained by the insertion of expression constructs into the plastid genome by particle bombardment. Since the Agrobacterium T-DNA (transfer DNA) complex is targeted to the nucleus, it is unsuitable for gene transfer to chloroplasts [24, 52]. Following the transformation procedure, the bombarded leaf explants are regenerated, and transplastomic plants with homoplastomic transformation (in which every chloroplast carries the transgene) are finally selected, recurring to a selection medium containing spectinomycin or in combination with streptomycin [53].
Plastid transformation can result in high yields of heterologous proteins because multiple copies of the genome are present in each plastid, and photosynthetic cells may contain hundreds or thousands of plastids [54]. As an example, the expression of a proteinaceous antibiotic in tobacco chloroplasts has achieved up to 70% of the total soluble proteins, which is the highest recombinant protein accumulation accomplished so far in plants [55]. Furthermore, chloroplasts provide a natural biocontainment of transgene flow since genes in chloroplast genomes are maternally inherited and consequently not transmitted through pollen, thereby avoiding unwanted escape into the environment. Other advantages of chloroplast engineering include the ability to express several genes as operons, and the accumulation of recombinant proteins in the chloroplast, thus reducing toxicity to the host plant [24].
Finally, transplastomic production platforms offer the possibility of oral delivery [54, 56]. In fact, it has been demonstrated that chloroplast-derived therapeutic proteins, delivered orally via plant cells, are protected from degradation in the stomach, probably due to the bioencapsulation of the therapeutic protein by the plant cell wall. They are subsequently released into the gut lumen by microbes that digest the plant cell wall, where the large mucosal intestine area offers an ideal system for oral drug delivery [57].
A shortcoming of expressing proteins via the chloroplast genome is that routine plastid engineering is still limited to tobacco, a crop that is not edible and thus unsuitable for oral delivery of therapeutic proteins. In addition, the synthesis of glycoproteins is not possible in the chloroplast system, as plastids do not carry out glycosylation [24]. Nevertheless, the expression of human somatotropin [58] in tobacco established that chloroplasts are capable of properly folding human proteins with disulfide bonds. In another study, the production of native cholera toxin B subunit [59] demonstrated the capacity of chloroplasts to fold and assemble oligomeric proteins correctly. Other therapeutic proteins expressed in tobacco chloroplasts include interferons alpha-2a and alpha-2b [60, 61] and anti-cancer therapeutic agents such as human soluble tumor necrosis factor (TNF) [62] and azurin [63]. Recently, chloroplast transformation of lettuce has also been developed [64, 65] to provide oral delivery transplastomic systems [66, 67]. Several therapeutic proteins were produced in lettuce chloroplast, namely, proinsulin [66, 67], tuberculosis vaccine antigens [68], and human thioredoxin 1 protein [69]. The chloroplast production platform has yet to achieve commercial success, though the referred developments in this field augur a promising future for therapeutic protein production in chloroplasts.
Transient expression is a phenomenon that occurs when genes are introduced into plant tissues and are expressed for a short period without stable DNA integration into the genome [3]. Traditionally, transient expression was used to verify expression construct activity and to test recombinant protein stability. This strategy allowed the identification and elimination of initial transformation problems, and thus the prospect of regenerating the desired transgenic lines was significantly improved. Recently, there has been an emergence of transient expression for the commercial production of recombinant pharmaceutical proteins. The advantages of transient expression platforms include the ease of manipulation, speed, low cost, and high yield of proteins. In comparison to transgenic plants, transient expression permits to achieve higher recombinant protein yields because there are no position effects (suppression of transgene expression by the surrounding genomic DNA following integration) [70].
Transient expression systems utilize the beneficial properties of plant pathogens to infect plants, spread systemically, and express transgenes at high levels, causing the rapid accumulation of recombinant proteins [8]. Currently, the major transient expression platforms are based on Agrobacterium tumefaciens, plant viruses, or hybrid vectors that utilize components of both (magnICON® technology).
The agroinfiltration method involves the vacuum infiltration of a suspension of recombinant A. tumefaciens into the plant leaf tissue, with the transgenes being then expressed from the uninterrupted T-DNA [8, 71]. Using this method, milligram amounts of recombinant protein are produced within a few weeks without the need to select transgenic plants, a process that takes months to years to be completed. This system has been commercially developed in tobacco [72] and alfalfa [73] but is also applicable to other crops such as lettuce [74], potato [75], and Arabidopsis [76]. An advantage of Agrobacterium-mediated transient expression is the fact that it allows to produce in plants complex proteins assembled from subunits [70].
Another transient expression technology is based on the use of plant viruses. In this technology, the gene of interest is inserted among viral replicating elements, episomically amplified and subsequently translated in the plant cell cytosol [77]. To date, the most efficient and high-yielding platforms have been developed using RNA viruses [78]. These plant viruses include Tobacco mosaic virus (TMV), potato virus X (PVX), and Cowpea mosaic virus (CPMV) (reviewed in [8]). The advantages of virus-based production include the rapid recombinant protein expression, the systemic spread of the virus, and the fact that multimeric proteins such as antibodies can also be produced by coinfecting plants with noncompeting vectors derived from different viruses [79, 80]. Transient expression vectors based on virus have been used to express peptides and long polypeptides (at least 140 amino acids long) as fusions to the coat protein, resulting in the assembly of chimeric virus particles (CVPs) displaying multiple copies of the peptide or polypeptide on its surface [77, 81]. Transient expression based in plant viruses has been commercially adopted by the now-closed Large Scale Biology Corporation (Vacaville, USA) that used a TMV-based vector for the production of patient-specific idiotype vaccines for the treatment of B-cell non-Hodgkin’s lymphoma, which had successfully passed the phase I clinical trials [82].
Finally, the third transient expression strategy is based on hybrid systems that incorporate components of the T-DNA transfer and virus replication systems [3]. These hybrid systems use deconstructed viruses obtained by removing the coat protein (responsible for systemic movement) of the noncompeting virus strains and use Agrobacterium as the vehicle for the systemic delivery of the resulting viral vectors to the entire plant. These systems effectively address most of the major shortcomings of earlier plant-based technologies by providing the overall best combination of the following features: high expression level, high relative yield, low up- and downstream costs, very fast and low-cost R&D, and low biosafety concerns [83]. Consequently, there has been a commercial development based on several hybrid systems. One of most notable examples is the magnICON® system developed by Icon Genetics (
Examples of therapeutic recombinant proteins produced in these platforms have been generally reviewed in [3]. Recombinant protein production using transient expression is now being mobilized to a large scale with several companies developing scalable, automated plant-based GMP biomanufacturing facilities to efficiently produce large amounts of pharmaceuticals within weeks. Such facilities include the ones of the Fraunhofer Center for Molecular Biotechnology (Newark, DE) (
In conclusion, the ability of transient plant expression systems to produce large quantities of recombinant protein, coupled to the use of current technology to increase yields, and the many promising technical solutions seems to be favorable compared with mammalian- or insect cell-based systems in quality, cost, and scale [19]. In case of emerging threats, transient platforms are advantageous since they produce large amounts of recombinant proteins rapidly (milligram quantities per plant within a few days) and can be scaled up quickly, currently providing the only reliable platform for rapid response situations [9]. During the H1N1 pandemic, the first batches of H1N1 virus-like particles (VLPs) could be produced by Medicago Inc. as soon as 3 weeks after the Centers for Disease Control and Prevention released the new influenza hemagglutinin sequence [73]. Similar lead times were reported for the H5N1 VLP vaccine [84]. Recently, the application of tobacco plant-based transient production systems, at Kentucky BioProcessing (KBP), to produce antibody lots against Ebola, was shown to significantly decrease the amount of time required for production over traditional methods, increase the quantity of antibody produced, and reduce the cost of manufacturing. Finally, at the other end of the market scale, transient expression platforms are economical for the production of pharmaceuticals for very small markets, such as orphan diseases and individualized therapies.
Plant cell suspension cultures grow as individual cells or small aggregates and are usually derived from callus tissue by the disaggregation of friable callus pieces in shake bottles and are later scaled up for bioreactor-based production. Recombinant pharmaceutical protein production is achieved using transgenic explants to derive the cultures or by transforming the cells after disaggregation, usually by co-cultivation with A. tumefaciens. The co-cultivation of plant cell suspensions and recombinant A. tumefaciens has also been used for the transient expression of proteins [85]. Since these plant cell suspension cultures are grown in sterile contained environments, they provide a cGMP-compatible production environment that is more acceptable to the established pharmaceutical industry and regulatory authorities [3, 86]. These systems have added benefits of complex protein processing compared to bacteria and yeasts and increased safety compared to mammalian cell systems, which can harbor human pathogens. Another advantage of plant suspension cultures is the very low maintenance cost in comparison to other fermenter-based eukaryotic systems such as mammalian or insect cells. Moreover, the possible secretion of the target protein into the culture medium simplifies downstream processing and purification procedures [87, 88]. Nevertheless, plant cell cultures also have some limitations such as poor growth rates, somaclonal variation (particularly due to chromosomal rearrangements, common in plant cell cultures generated by calli), and gene silencing, together with the inhibition of product formation at high cell densities, formation of aggregates, cell wall growth, as well as shear-sensitivity for some species [89]. However, high levels of functional recombinant protein in plant cell suspension cultures were already obtained [87]. Besides, the previously mentioned first licensed recombinant pharmaceutical protein, Elelyso™, was produced in plant cell suspension cultures (reviewed in [88]). Tobacco has been the most popular source of suspension cells for recombinant protein production. Tobacco plants proliferate rapidly and are easy to transform, but other plant species have also been used to generate suspension cells, including rice and Arabidopsis thaliana, alfalfa, soybean, tomato, Medicago truncatula, and carrot [85, 88, 90]. Carrot suspension cells have been used by the aforementioned Protalix Biotherapeutics to produce a recombinant glucocerebrosidase. This case of commercial success shows that suspension cell cultures have potential as a viable system for large-scale protein production. Recently, carrot callus cultures, expressing epitopes from the cholesteryl ester transfer protein, were accessed for the potential of becoming an atherosclerosis oral vaccine [91].
The lower expression levels in comparison to the established biopharmaceutical platforms were one of the major obstacles for the commercialization of molecular pharming [9]. Therefore, numerous techniques have been developed to enhance protein expression, including codon optimization of protein sequences, to match the preferences of the host plant, targeting subcellular compartments that allow proteins to accumulate in a stable form; the use of strong, tissue-specific promoters; and the testing of different plant species and systems [25].
Protein synthesis can be increased by optimizing the components of the expression construct to maximize transcription, mRNA stability, and translation or by diminishing the impact of epigenetic phenomena that inhibit gene expression [92]. In this field, the general strategy is to use strong and constitutive promoters, such as the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and maize ubiquitin-1 promoter (ubi-1), for dicots and monocots, respectively. However, organ- and tissue-specific promoters are also being used to drive expression of the transgenes to a specific tissue or organ such as the tuber, the seed, and the fruit. Additionally, inducible promoters, whose activities are regulated by either chemical or external stimulus, may equally be used to prevent the lethality problem. Furthermore, transcription factors can also be used as boosters for the promoters to further enhance the expression level of the transgenes [53].
Protein stability can be increased by targeting proteins to cell compartments that reduce degradation. Protein targeting also affects the glycan structures added to proteins and the type of extraction and purification steps required to isolate the protein from the plant matrix. Proteins can be targeted to the secretory pathway by an N-terminal signal peptide, which is cleaved off for the release of the protein into the endoplasmic reticulum (ER). Proteins that do not require posttranslational modification, e.g., glycosylation, for their activity, can be targeted to the chloroplast using N-terminal transit peptides [93]. In addition, the target gene can be used to transform chloroplast directly, with highly enhanced protein accumulation. Moreover, posttranslational modifications of the ER lumen can also be avoided by expressing the protein as translational fusion with oleosin protein, which target the expression of the foreign protein to oil bodies of the seeds [28]. Other subcellular compartments like the protein-storing vacuoles are now being explored for recombinant protein accumulation, as it has been observed in rice seed endosperm [94].
In the early years of molecular pharming, scientific studies were focused on demonstrating that plants could produce adequate quantities of recombinant pharmaceutical proteins and confer an oral delivery means. This led to downstream processing and the costs associated to it being basically overlooked. Downstream processing is now known to be an economically critical part of biomanufacturing processes (it can account for up to 80% of the total cost in a therapeutic protein production line) and also to be a key component of the regulatory process for evaluating the safety of pharmaceutical products [7]. The goal and the general steps for downstream processing are similar between plant and other expression systems: to recover the maximal amount of highly purified target protein with the minimal number of steps and at the lowest cost. The basic steps for downstream processes include tissue harvesting, protein extraction, purification, and formulation [22]. However, since in molecular pharming the costs of downstream processing are product-specific rather than platform-specific, the evaluation of downstream processing strategies and costs associated to it has to be done on a case-by-case basis. Nevertheless, even if unit operations have to be developed based on the properties of the product, others have to be developed based on the properties of the expression host. Plants produce process-related contaminants that require specific processing steps to ensure removal of fibers, oils, superabundant plant proteins such as RuBisCO, and potentially toxic metabolites such as the alkaloid nicotine in tobacco [8]. These secondary metabolites can be recovered from plant cells or tissues using methods such as adsorption, precipitation, and chromatography, often requiring phase partitioning and the use of mixtures of organic solvents. Several approaches have been used to facilitate downstream processing, including secretion of recombinant proteins, eliminating the plant cell disruption step; targeting of proteins into the protein bodies, oil bodies, or plastoglobules; and the use of affinity tags such as poly-histidine tags with the target protein, allowing protein purification by affinity chromatography [25]. In addition, oral delivery of whole plants or crude extracts containing the pharmaceutical relevant proteins can also be a way to simplify downstream processing and to easily distribute medicines to those in need. Furthermore, the optimization of plant’s expression level can also ease downstream processing, with higher protein concentrations conducting to higher protein volumes [7].
Finally, several purification strategies have been investigated to separate target transgenic proteins from host plant proteins, which are tailored for each individual protein based on its solubility, size, pI, charge, hydrophobicity, or affinity to specific ligands, and the parallel characteristics of plant host proteins. Chromatographic methods, such as affinity chromatography, have been the most extensively used. However, recently increasing attention is being paid to non-chromatographic methods to provide alternatives for large-scale production [22].
In the broad range of known bioactive peptides, angiotensin I-converting enzyme inhibitory (ACEI) peptides derived from food proteins have attracted particular attention and have been studied the most comprehensively for their ability to prevent hypertension [95]. In this chapter we will further focus on the possibility to genetically engineer crop plants to produce and deliver antihypertensive ACEI peptides, therefore creating alternative sources to fight hypertension and prevent cardiovascular disease.
Cardiovascular disease (CVD) has been recognized as the leading cause of death in developed countries. Hypertension or high blood pressure is one of the major independent risk factors for CVD [96]. States of CVD include conditions such as coronary heart disease, peripheral artery disease, and stroke. Hypertension is a condition defined by a blood pressure measurement of 140/90 mmHg or above and is thought to affect up to 30% of the worldwide adult population [95]. The kinin-nitric oxide (KNO) system and the renin-angiotensin system (RAS), Figure 1, play a crucial role in the control of hypertension by the action of angiotensin I-converting key enzyme (EC 3.4.15.1; ACE) [96–99].
The kinin-nitric oxide (KNO) system and the renin-angiotensin system (RAS). The left side (KNO system) shows the mechanism of the action of ACEI on ACE that cleaves bradykinin, a nonapeptide acting as vasodilatory hormone, and causes the formation of an inactive heptapeptide. In the right side (RAS system), the inhibition of ACE activity plays an important physiological role in regulation of blood pressure by inhibiting the conversion of the hormone angiotensin I to angiotensin II, a potent vasoconstrictor (figure adapted from Erdmann et al. [96]).
Several synthetic ACE inhibitors such as captopril, enalapril, and lisinopril have been prescribed for the treatment of hypertension, congestive heart failure, and diabetic neuropathy [100]. However, their consumption is associated with various side effects including cough, skin rashes, hypotension, loss of taste, angioedema, reduced renal function, and fetal abnormalities [95]. The side effects associated to synthetic ACE inhibitors and the high prevalence of hypertension have led scientists to search for natural and safer therapies. Interestingly, the study of ACEI peptides has revealed that they do not have significant effects on blood pressure in normotensive subjects, suggesting a convenient mechanism that avoids acute hypotensive effects. Based on this finding, it is hypothesized that ACEI peptides could be used in initial treatment of mildly hypertensive individuals or even as supplemental treatments [101].
So far, several ACEI peptides have been identified in food proteins, mainly in milk, eggs, and plants, currently constituting the most well-known class of bioactive peptides [102–104]. These peptides are inactive within the sequence of parent proteins, but they can be released by enzymatic proteolysis in vivo or in vitro, for example, during gastrointestinal digestion or during food processing. A common feature shared by the majority of ACEI peptides is the generally short sequence, i.e., 2–12 amino acids in length. However, some larger inhibitory sequences have been identified in milk fermented with Enterococcus faecalis [105] and Lactobacillus casei Shirota [106], in koumiss [107], tuna [108], bonito [109], and rotifer [110]. Studies have also indicated that binding to ACE is strongly influenced by the substrate’s C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the C-terminal tripeptide region seems to positively influence a peptide’s ACE-inhibitory activity [95]. In general, the peptides showing higher activity against ACE have Tyr, Phe, Trp, or Pro at their C-terminus [95]. The peptides TQVY from rice [111], MRW from spinach [112], and YKYY from wakame [113] are some examples of this principle. Table 1 reviews some examples of ACEI activities of plant origin, whose peptides responsible for such activity may be potential sources for the heterologous production of ACEI peptides.
Source | ACEI activity (IC50; μM) | Antihypertensive activity (mmHg) | Dose (mg/kg) | Reference |
---|---|---|---|---|
Chlorella vulgaris | 29.6 | Not determined | — | [114] |
Chebulic myrobalan | 100 | Not determined | — | [115] |
Bitter melon | 8.64 | −31.5 to −36.3 | 2–10 | [116] |
Mung bean | 13.4 | Not determined | — | [117] |
Pea | 64 | Not determined | — | [118] |
Peanut | 72 | Not determined | — | [100] |
Potato | 18–86* | Not determined | — | [119] |
Rapeseed | 28 | −11.3 | 7.5 | [120] |
Rice | 18.2 | −40 | 30 | [111] |
Soybean | 14–39* | −17.5 | 2 | [121] |
Soybean | 21 | Not determined | — | [122] |
Soybean | 1.69 | Not determined | — | [123] |
Soybean | 17.2 | Not determined | — | [124] |
Spinach | 0.6–4.2* | −13.5 to −20* | 20–100 | [112] |
Wakame | 21–213* | −50 | 50 | [113] |
Walnut | 25.7 | Not determined | — | [125] |
Wheat | 20 | Not determined | — | [126] |
Examples of ACEI peptide activity from different plant origin.
Different values for the same plant product related to the ACEI peptide sequence.
The most common method to produce and identify ACEI peptides is through enzymatic hydrolysis of food proteins with gastrointestinal enzymes such as pepsin and trypsin or with commercial proteases such as Alcalase™ [127]. ACEI peptides have also been produced with Lactobacillus, Lactococcus lactis, and E. faecalis strains during milk fermentation [105, 106]. Nevertheless, there are problems associated to this type of industrial production of ACEI peptides, including the difficulty to isolate the peptide of interest from the complex mixture of compounds produced by enzymatic hydrolysis, the high cost, low recovery, and the low bioavailability. These disadvantages denote the need to develop new and alternative approaches for their production.
In recent years, the application of recombinant DNA technologies for the production of ACEI peptides at a large scale and low cost has gathered attention in the biotechnology community. Investigation has been focused on the development of expression methods for antihypertensive peptide production in different plant crops [128]; and here, we tried to provide some promising examples.
Thus far, the main strategies that have been adopted are as follows: the overexpression of ACEI peptide precursor proteins and the production of particular peptides as heterologous components [101], the modification of some storage proteins to produce chimeric proteins carrying ACEI peptides [101], and also the generation of multimer proteins containing tandem repeats of ACEI peptides, flanked by protease recognition sequences that allow the peptide release during gastrointestinal digestion.
Transgenic rice plants that accumulate novokinin (RPLKPW), a potent antihypertensive peptide designed according to the structure of ovokinin (2–7) (RADHPF), as a fusion with the rice storage protein glutelin, have been generated. The engineered peptide is expressed under the control of endosperm-specific glutelin promoters and specifically accumulates in seeds. Oral administration of either the RPLKPW-glutelin fraction or transgenic rice seeds to spontaneously hypertensive rats (SHRs)—the main model for assessing the in vivo activity of ACEI peptides (e.g., [108, 111, 122])—significantly reduced systolic blood pressures, suggesting the possible application of transgenic rice seed as a nutraceutical delivery system and particularly for administration of antihypertensive peptides [129].
Wakasa et al. [130] attempted the generation of transgenic rice seeds that would accumulate higher amounts of novokinin peptide by expressing 10 or 18 tandemly repeated novokinin sequences, with the KDEL endoplasmic reticulum retention signal at the C-terminus, and using the glutelin promoter along with its signal peptide. Although the chimeric protein was unexpectedly located in the nucleolus and the accumulation was low, a significant antihypertensive activity was detected after a single oral dose to SHRs. More importantly, this effect was observed over a relatively longer duration time, with intervals of 5 weeks between doses as low as 0.0625 g transgenic seeds per kg.
Soybean [Glycine max (L.) Merr.] is an attractive option for the production of ACEI peptides given that soybean seeds contain a large amount of total protein. Therefore, there has been an effort to generate soybean lines with improved ACEI properties foreseeing the creation of novel functional foods.
Matoba et al. [128], introduced novokinin (RPLKPW) into homologous sequences of a soybean β-conglycinin α’ subunit by site-directed mutagenesis. Founded on first achievements from an E. coli expressed protein, the muted β-conglycinin α’ subunit carrying novokinin repeats were also expressed in soybean. This chimeric protein accumulated at levels of up to 0.2% of extracted protein from transgenic soybean seeds [131]. Still, the levels of expression were too low, and it was not possible to assess the in vivo effects of these soybean seeds.
Novokinin has also been expressed in transgenic soybean seeds in a fusion form along with a β-conglycinin α’ subunit. Interestingly, a reduced systolic blood pressure was observed in SHRs after administering a dose of 0.15 g kg−1 of protein extracts. A similar effect was attained following administration of a 0.25 g kg−1 dose of defatted flour. Thus, it was concluded that this chimeric protein produced in soybean possessed an antihypertensive activity [132].
Additionally, a synthetic gene of His-His-Leu (HHL), an ACEI peptide derived from a Korean soybean paste, was tandemly multimerized to a 40-mer, ligated with ubiquitin as a fusion gene (UH40), and subsequently expressed in E. coli. Following digestion with leucine aminopeptidase, the 405-Da HHL monomer was recovered by reverse-phase high-performance liquid chromatography (HPLC). MALDITOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE-inhibiting activity confirmed that the resulting peptide was the HHL [133]. The potential use of this antihypertensive chimeric protein in soybean has yet to be assessed.
A modified version of amarantin, the main seed storage protein of Amaranthus hypochondriacus, carrying four tandem repeats of the ACEI dipeptide Val-Tyr into the acidic subunit of amarantin, was expressed in cell suspension cultures of Nicotiana tabacum L. NT1. Protein hydrolysates obtained from transgenic calli showed high levels of inhibition of the angiotensin-converting enzyme, with an IC50 value of 3.5 μg ml−1, and 10-fold lower levels than that of protein extracts of wild-type cells (IC50 of 29.0 μg ml−1) [134]. This was the first time that a chimeric protein comprising an ACEI peptide was produced in plant cell suspension cultures.
This modified version of amarantin was also expressed in the fruit of transgenic tomato plants. Protein hydrolysates from transgenic tomato fruits showed in vitro ACE inhibition, with IC50 values ranging from 0.376 to 3.241 μg ml−1; this represented an increase of up to 13-fold in the inhibitory activity when compared with the protein hydrolysates of non-transformed fruits [135]. These two results suggest the possible application of tobacco plant cell suspension cultures and transgenic tomato fruits for massive production of this engineered version of amarantin, which could be especially used as an alternative hypertension therapy [134, 135].
Although amaranth has not been genetically modified to produce ACEI peptides, the feasibility of developing a modified amarantin acidic subunit has been widely assessed [129, 134–139]. Recently, the in vivo effect of an E. coli-modified amarantin protein, four units of Val-Tyr dipeptides (VY) in tandem, and one of Ile-Pro-Pro tripeptides (IPP) incorporated in the amarantin acidic subunit (AMC3) was evaluated in SHRs in a one-time oral administration experiment. This study showed that enzymatic hydrolysates of AMC3-containing ACEI peptide (4xVY and IPP) sequences had significant in vivo antihypertensive action [138]. The positive reports of amarantin expression in E. coli [136, 138, 139] along with the sustained expression of amarantin-modified proteins in tobacco [134] and tomato [135] prospect the successful production of ACEI peptide fusion proteins in amaranth.
Lettuce (Lactuca sativa) is a commercially important crop belonging to the Asteraceae family. It is a diploid (2n = 18), autogamous species with a genome size of 2.7 Gb [140]. This crop is particularly suitable for oral delivery of therapeutics as its raw leaves are consumed by humans, and the time to obtain an edible product is only weeks, compared to the months needed for crops such as tomato or potato. Therefore, recently lettuce has been investigated as a production host for edible recombinant therapeutics [66, 67, 141]. Furthermore, the fact that stable transformation procedures for both nuclear [142] and plastid genomes [64], and transient expression [74], are widely available, is also an advantage. Lettuce has been used as production host for several recombinant therapeutics, virus-like particles (VLPs) and monoclonal antibodies [143], antigens [142, 144], and human therapeutic proteins [66, 69].
Medicago truncatula is a model plant from the legume family. It is a diploid (2n = 16), autogamous species, with a relatively small genome and short life cycle of 3–5 months. These characteristics enable this species to be used in molecular genetic studies and expression of foreign genes [145]. The phylogenetic distance to economically important crops is crucial in the choice of this plant by many researchers and funding agencies, since it allows comparative studies within the legume family. The methodologies for the establishment of long-term cell suspension culture are well recognized [146], and the potential of M. truncatula as expression host has also been established for the production of feed additives [20, 87], human hormones [90], and human enzymes [147].
The use of these two species in molecular pharming is at the center of a recent collaboration between the Plant Cell Biotechnology (PCB) Laboratory (ITQB UNL), the Cell Differentiation and Regeneration Laboratory (iBiMED UA), and the Institute of Plant Genetics (IPG PAS). This cooperation foresees the usage of these two species as exceptional hosts for the heterologous production and/or delivery of ACEI peptides, and a resume of this ongoing project is here schematically presented (Figure 2). This figure also provides an overview of the technologies involved in different plant platforms discussed in this chapter.
Schematic representation of the technologies involved in different plant platforms for the production of therapeutically important proteins and peptides. Plastid transformation by particle bombardment can result in regeneration of transplastomic plants, revealing high-yield heterologous production, with the possibility of protein/peptide oral delivery or purification. Nuclear transformation can be accomplished by particle bombardment or by Agrobacterium-mediated transformation, resulting in the regeneration of stable transgenic plants. Finally, the technology based on transient expression, here with the example of agroinfiltration. We present Medicago truncatula and lettuce as examples: (a) M. truncatula co-culture of leaf explants with Agrobacterium, (b) and (c) plant regeneration via somatic embryogenesis according to Araújo et al. [145], (d) and (e) establishment of a cell suspension culture from callus for protein/peptide production [146, 147], (f) lettuce leaf explant co-culture with Agrobacterium, (g) and (h) plant regeneration via shoot organogenesis at PCB lab, (i) lettuce transgenic plants which can be used for oral delivery, (j) and (k) agroinfiltration of lettuce leaf explants according to Negrouk et al. [74], and (l) example of a control explant (left) and transient expression of a 35S::GUS(int) cassette in lettuce leaves (right).
Molecular pharming has been recently and extensively reviewed, and the future of this technology has gathered some optimistic expectations. A myriad of studies have already demonstrated the capacity of various plant species and systems to produce recombinant pharmaceutical proteins and peptides. This technology has already been put to the test in case of emerging threats, where transient platforms proved to be strategic for rapid production of large amounts of recombinant proteins in response to pandemic situations. However, their usefulness for the production of functional foods still falls short of expectations, as well as the attainment of its full potential in bioactive peptide production. With the improvement of known plant platforms and development of new genetic engineering techniques and their exploration, it is forthcoming an evolution in the production of heterologous bioactive peptides, to which we hope to contribute with our ACEI pharming project. The advent of genome editing techniques (with the advantage of site- specific gene insertion), like the CRISPR/Cas9 methodology, will undoubtedly increase and democratize plant transformation events and will certainly contribute to the increase of genetically modified species for molecular pharming purposes.
We acknowledge financial support from Fundação para a Ciência e Tecnologia (FCT), Portugal, through the research Grant SFRH/BPD/74784/2010 (Duque AS) and funding, through the research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013) and FCT/COMPETE/QREN/EU-FEDER (for Institute of Biomedicine iBiMED: UID/BIM/ 04501/2013). We also acknowledge the Program for Scientific Cooperation FCT/Poland 2017/2018 and Angelini Pharmaceuticals and PREMIVALOR (for Angelini University Award 2012/2013).
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