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Philly Biotech Scene: Biobots and 3D Bioprinting (Now Called Allevi)
Reporter: Stephen J. Williams, Ph.D.
Biobots now known as Allevi, Inc.. Their new Biobots community has been renamed Allevi Academy.
The goal of BioBots has always been the same: Give laboratories the ability to create living things from scratch. Those things–such as pieces of tissue or bone–could then be studied with the hopes of finding cures and solving diseases.
That vision helped the company’s co-founders, Ricky Solorzano and Danny Cabrera, land on Inc.’s 30 Under 30 list in 2016. And while the original goal has remained, much has changed. In August, Cabrera, the company’s first CEO, left the Philadelphia-based startup. And in November, the company rebranded, changing its name to a more mature but far less memorable name, Allevi.
“People think running a startup is just a straight line, that you go in one direction,” Solorzano, who has since shifted from CTO to CEO, tells Inc. “You really go up, down, sideways, left, right, 45 degrees this way, 90 degrees that way.”
For Solorzano and Cabrera, the split represents the end of an era. The two Miami residents both attended the University of Pennsylvania, where they first discussed the idea of developing an affordable three-dimensional printer that could produce living tissue. They founded the company together in 2014.
The following is based on an interview back in 2016 I did with Biobots founders Danial Cabrera, Ricardo Solorzano, and Sohaib Hashimi.
A year ago (2014), we founded BioBots in a dorm room on top of a noisy college bar with the mission of conquering the largest mystery of our generation – life. Disillusioned with existing tools and technologies for engineering organisms, and inspired by the idea of biology as technology, we launched BioBots with a command: “Build with Life.”
It only took a few weeks for our first apostles to join us. Dr. Dan Huh and his student Yooni at Penn began working with a prototype that would become the first BioBot. With the help and unyielding support of our early clients and partners like Elliot Menschik at DreamIt Health, we began the journey of bringing biofabrication technology to people across the world.
Today hundreds of labs are turning to BioBots for tools that allow them to engineer biology. I am constantly inspired by our partners’ research projects, goals and progress; they consistently remind me that we are accelerating the pace not only of regenerative medicine, but of human evolution.
None of this would be possible without all of our BioBot employees, their families, our friends in the media, investors, and most importantly – our visionary clients, who continue to pour their passion, talents, energy and love into building this company. A year ago we were two guys in a bar. Today, hundreds of supporters have taken up the mantle of biofabricator.
Our vision at BioBots is to make tools that harness life as an engineering discipline and push the human race forward. We look forward to helping you do much more and test the boundary of what we can build with biology. Thank you for being a part of our journey!
“Life is the oldest and most efficient manufacturing technology that we as people know of. It’s become clear over the past several decades as scientists have engineered life to work for us, that biology is the next frontier for manufacturing. However, there is one thing missing. Doing biology today is the equivalent of computer programming 50 years ago – it’s inefficient, it’s slow, and the technology is only available to scientists at well-funded institutions, out of the hands of the ordinary people that could be leading this new revolution.” ~ BioBots CEO Danny Cabrera to 3DPrint.com
BioBots is a company launched by Daniel Cabrera, a recent graduate of University of Pennsylvania’s Engineering School, as well as Ricardo Solorzano and Sohaib Hashmi, who are staff research specialists in the Perelman School of Medicine (UPenn). The three got together to create a 3D bioprinter capable of printing in multiple body tissues. While this certainly isn’t the first ever bioprinter created, Cabrera tells us that it is not the same as others on the market today.
“Employing the tool that transformed traditional avenues of manufacturing, we at BioBots are using 3D printers to engineer biology,” Cabrera told 3DPrint.com. “Our 3D bioprinters employ the use of a novel extrusion process that addresses the previous technical hurdles of 3D bioprinting, as well as a biomaterials cartridge system that makes this revolutionary technology accessible to untrained users. Just imagine the kind of products that people will build now that they can plug and print living tissues. At BioBots, we are building this future, today.”
The BioBot 3D printer works with both “Blue Light” and UV light. The cell solution, which contains living, growing cells as well as vasculature for nourishment, is extruded from the 3D printer in a similar fashion to how at-home fused filament fabrication (FFF) 3D printers work. However, different from your typical FFF 3D printer, once a biological material has been extruded, an ultraviolet light (or Blue Light) cures and hardens it. This occurs one layer at a time until the desired object is printed. The objects printed can be living cell tissue or non-living scaffolds, and Cabrera tells us that over a dozen different cell types have been used with these printers so far. The unique cartridge system that BioBots’ bioprinter uses, enable users to easily switch between the printing of different biological materials, almost as easily as a normal desktop printer can switch between colors.
“We have won several innovation competitions and recently received funding from DreamIt Health, a start-up accelerator program based out of Philadelphia,” said Cabrera. “We are opening a Beta program with the goal of placing printers in the hands of the best experts and working with them to generate publishable data. The idea is to generate interest in this area and inform scientists about the tool we’re developing through published research. We currently have Beta tester relationships in place with Dr. Dan Huh’s lab at Penn, Dr. Kara Spiller’s lab in Drexel, and Dr. Kevin Costa’s lab in Mt. Sinai and are definitely looking to expand.”
The company is also open to accepting many new Beta testers into the program. That program costs a mere $5,000 and supplies the following benefits to the testers:
A 3D bioprinter (80um resolution) capable of extruding a variety of hydrogels (collagen, alginate, agarose, polyethylene glycol, hyaluronic acid, etc.)
1 Year service agreement & active development for your bioprinter
BioBots software package
Access to an online community of collaborators who are working together to solve tough tissue engineering, regenerative medicine, and biomaterials problems
Having your work showcased at a number of conferences that BioBots has been invited to speak at
For those interested in joining the Beta program, they are asked to email the company for more details.
The team behind BioBots is equally as impressive as the machine itself. Cabrera has recently graduated from UPenn, where he studied computer science and biology, and won first place in the North America International Genetically Engineered Machines competition for his work on automating genetic engineering work flows and making life easier to engineer. The company’s CTO has been working in the field of regenerative medicine for about 4 years, and has authored several papers on building 3D blood vessels. He actually built the first BioBots prototype from his dorm room at UPenn.
While the Beta program is meant as a way in which the company can build up their user base, solidify a community of doctors, engineers, designers, educators and students, and test out their latest version of their BioBots bioprinter, others can pre-order the printer for $25,000. The team isn’t only targeting Ph.D researchers. They want these machines to be used by educators and researchers everywhere. “Our 3D bioprinters enable users to easily print high resolution biological structures – whether you’re a researcher on the frontier of regenerative medicine or a high school biology teacher,” said Cabrera.
While we are still far away from 3D printing working organs, the fact that BioBots offers a 3D printer capable of printing in a vast array of biological materials at a price starting as low as $5,000, means that this technology can reach the hands of virtually any researchers interested in studying the potential that it holds for the future. Other bioprinters from larger companies can cost upwards of $250,000, severely limiting access. This is wear BioBots may become quite revolutionary.
Cabrera tells us that they are working on curriculum/lesson plans to go along with their printers, so that high school students can learn about bioprinting through the use of these relatively affordable machines.
When I asked Cabrera how long he thinks it will be, before we see fully printed working organs, he told me that it isn’t about the technology not being there, but rather its about researchers being able to come up with ways to use it. His guess is that within the next 10-15 years we may see the first 3D printed working organ.
What do you think? Will the BioBots 3D bioprinter lead the way in allowing researchers to fully investigate and innovate upon this technology? Discuss in theBioBots forum thread on 3DPB.com. Check out the videos below, including the first one, showing a demo of the BioBots printer using photocurable PEG.
Biobots offers, on their site at https://www.biobots.io/build-with-life/
Wikis: where one can browse through these pages to learn about established biotechnologies, tissue fabrication methods, foundational advances in biology and in our ability to design and engineer living things.
Protocols: where one can find information in a “Use the protocols section” to learn more about how to interact with your BioBot 1, different bioinks, and new emerging biofabrication techniques. This is the place to develop and share new methods.
BioReports: a collection of experimental logs with methodology used and results obtained from experiments using the BioBot systems
Advantages of the Biobots system
PRECISION
Our team of engineers has worked hard to ensure precision in every aspect of BioBot 1. We use linear rails over less expensive belt systems that slip and require adjustment, guaranteeing a consistent 10 micron precision on each axis.
Other Articles on this Open Access Journal on 3D Bioprinting Include:
Medical 3D Printing and Metals in use in Medical Devices,
Presentation by Danut Dragoi, PhD
Curator: Danut Dragoi, PhD
The main objective of medical 3D printing (M3DP) is to build solid / semi-solid / scaffolds / or gel structures from bio-compatible materials that can be utilized in medicine in order to correct, alleviate, support certain surgeries, or even cure some diseases based on medical / biological principles applied to human body.
Materials that replace bones are metals like Ti, Ti alloys, Tantalum, Gold, Silver, Zr and other. For replacement of teeth is traditionally used a combination of Ti-pivots and ceramic / polymers / or in some cases Hydroxylapatite (HA) coated Ti.
In order to produce a metallic object implantable in the human body, most useful technology is 3D printing of metals, commonly known as AT (addition manufacturing) technology. A definition of 3D printing is a process for making a physical object from a three-dimensional digital model, typically by laying down many successive thin layers of a material. If a printer system uses metal powders and binder instead of normal ink the printed layer by layer will develop a 3D object.
The printed object may be an orthopedic bone replacement, a tooth pivot or an artificial tooth. The picture on Slide 4 shows a Laser Sintering System (SLM) for Medical 3D Printing for metals, find specs in here.
Slide 4
The machine shown on Slide 5 is one of the three metal printers from SLM Solutions using the technology of Selective Laser Melting, find specs in here,
Slide 5
Feature highlight: for aerospace and medical orthopedics. Large build volume.
Material: Stainless steel, tool steel, aluminium, titanium, cobalt-chrome, inconel
Build capacity: 19.68 x 11.02 x 12.80 in. / (500 x 280 x 325 mm)
Build rate: 70 cm³/h
Resolution/Layer thickness: 20 – 200µm
Machine dimensions: 118 x 98 x 43 in.
An important aspect of metal source for M3DP is the shape of the particles, uniform size distribution and chemical purity. Using a new manufacturing approach, Zecotek, a company in Germany, link in here, developed metallic powders that can be successfully used in M3DP. Next Slide 6 shows some characteristics of this breakthrough technology.
Information on Slide 8 can be found in here .
Slide 9
Information on Slide 9 can be found in here, which is a novelty in terms of materials, the fusion for the first time between a Ti alloy and a ceramic.
Slide 10
The schematic on Slide 10 can be found in here . SLS technology is in wide use around the world due to its ability to easily make very complex geometries directly from digital CAD data. While it began as a way to build prototype parts early in the design cycle, it is increasingly being used in limited-run manufacturing to produce end-use parts. Here is how it is working. The powders are in a compartment controlled by a piston going one small step up, the roller swipes to the right a thin layer of metallic powder on the second compartment controlled by a piston that goes only one small step down, due to the fact that the printed model starts to grow up. The tip of the laser beam melts the powder or fusion the particles according with a real drawing section of the model. The process is repeated until the model is done. The key element of this technology is the laser scan device that follows exactly the drawing section of the model.
Slide 12
Slide 12 shows a 3D printed foot that is light and well manageable for the patient. The picture can be found at this link in here. This prosthetic introduces the traces concept on light-weighting of replaceable parts for human body.
Slide 13
Slide 13 shows a 3D printed light orthopedic pieces that are using the concept of light-weighting using traces. Their picture can be found here.
Slide 14
Slide 14 shows tiny parts obtained with 3D printing technology, details in here.
Slide 15
A second way to obtain solid parts is using a 3D Bioplotter, link in here .
EnvisionTec’s 3D-Bioplotter builds its products in much the same way as a traditional 3D printer. However, instead of using plastics, metals or resins, the Bioplotter uses biologic materials to form a scaffold that will be used to grow more advanced cellular cultures.
Just like a traditional 3D printer, the 3D-Bioplotter can be fed a 3D model generated in a CAD program or from a CT scan. Users can slice and hatch a 3D model to define how it will be printed. That information is then translated to code and shipped off to the Bioplotter where the real work begins.
While prototype objects in the mechanical, architectural and civil worlds can be built from a single material, in the biological world it’s rare that the desired objects have a uniform material. To meet that reality, the Bioplotter can print a model in 5 different materials making it suitable for more complex cellular assemblies.
This ability to jet different materials during a single build requires the 3D-Bioplotter to change print heads. It comes equipped with a CNC-like tool holder that can be programmed to change “print-heads” based on the material being extruded. Most bio-engineering builds favor porosity. This machine’s ability to change print heads can also help alter the flow and spacing of successive print layers to give users greater control of their models.
Slide 16
The scaffold on slide 16 obtained with a 3D Bioploter, is useful in dentistry to augment the base of the future implantable tooth. The fixation in the picture is made of Vivos Dental’s OsteoFlux product, link see in here.
Slide 17
Slide 17 Metals in medical dental implants, Ti becomes fused with the bone, and the tooth attached to one end of the Ti pivot, see link in here.
Slide 18
Slide 18, Hot plasma spray bio-ceramics is the solution that doctors used for biocompatibility of an artificial jaws, link in here.
Slide 20
On slide 20 the traditional Ti casting is compared with Ti 3D printing from the powders. The advantage of 3D method is low cost and high productivity. This link in here is for traditional method, and this link here for 3D printing method.
Slide 21
Slide 21 For 3D Bioploter made by EnvisionTec we notice the usage of materials such as metal followed by post-processing sintering, Hydroxylapatite, TCP, Titanium. Using a preciptation method the machine can handle Chitosan, Collagen, 2-component system of the two possible combination: Alginate, Fibrin, PU, and Silicone. More details in here.
Slide 26
Slide 26 shows two ultra-miniature medical pressure sensors in the eye of a needle, for details see the link in here.
Slide 27
Slide 27 The electrodes of the bio-mems implanted on the surface of the heart are made of Gold for the electrical contact and good bio-compatibility. Classes of materials and assembly approaches that enable electronic devices with features – area coverage, mechanical properties, or geometrical forms – that would be impossible to achieve using traditional, wafer-based technologies. Examples include ’tissue-like’ bio-integrated electronics for high resolution mapping of electrophysiology in the heart and brain. The research on bio-integrated electronics can be found here.
Slide 28
Slide 28 shows a polymeric material for determining pressure inside the eye, which is useful to monitor patients at risk from glaucoma. Again the circular electrode is made of Gold and its role is that of an antena to transmit data to a iPhone / receiver about the intraocula pressure data.
Slide 29
The device in slide 29 is a bio-MEMS implantable for drug dosage. It has multiple micro-needles that are equivalent to a needle of a normal syringe, but painless since theyr tips do not reach the pain receptors. This picture taken from here, shows a side size of the MEMS of about 25 mm.
Slide 30
Slide 30 lists some effects of metals in human body. Traces of heavy metals are dangerous for human body. Human body is made of light elements C,H,N,O. Heavy metals: Pb, Hg, accumulate in the body, they disrupt the metabolic processes since they are very toxic to humans. Therefore, heavy metals don’t have “+” physiological effects and Al as element is known to produce Alzheimer’s which has been implicated as a factor. According to the Alzheimer’s Society, the medical and scientific opinion is that studies have not convincingly demonstrated a causal relationship between aluminium and Alzheimer’s disease. Nevertheless, some studies, cite aluminium exposure as a risk factor for Alzheimer’s disease. Some brain plaques have been found to contain increased levels of the metal. Research in this area has been inconclusive; aluminium accumulation may be a consequence of the disease rather than a causal agent, see link in here.
Slide 31
Slide 31 shows percent distribution of elements in human bodies, It is interesting that Ti is not making the list, see link in here.
Slide 32
Slide 32 has Ti element circled on the Table of the elements, we notice that Zr as element was found to be a bio-compatible element too just like Ti. It is very possible from chemical point of view that all elements in Ti group have same property. The only inconvenient of elements bellow Ti is that they are heavier and their density should be adapted closer to that of human body.
Slide 33
Slide 33 is a plot of stress (MPa) of some human implantable materials as a function of Young modulus E (GPa), their principal mechanical characteristic. There are crystalline materials such as: MgZnCa, MgZr, etc.) as well as amorphous materials bio-compatible such as: MgZnCa BMG, Ca based BMG, Sr based BMG, etc.) that have important mechanical strength that can be used in various applications. The circle in green centered on the point (75GPa, 650 MPa) is that for HydroxylApatite, which is a component of teeth and bones. Further details on this plot can be found at this link here, .
Magnesium and its alloys are suitable materials for biomedical applications due to their low weight, high specific strength, stiffness close to bone and good biocompatibility. Specifically, because magnesium exhibits a fast biodegradability, it has attracted an increasing interest over the last years for its potential use as “biodegradable implants”. However, the main limitation is that Mg degrades too fast and that the corrosion process is accompanied by hydrogen evolution. In these conditions, magnesium implants lose their mechanical integrity before the bone heals and hydrogen gas accumulates inside the body. To overcome these limitations different methods have been pursued to decrease the corrosion rate of magnesium to acceptable levels, including the growth of coatings (conversion and deposited coatings), surface modification treatments (ion implantation, plasma surface modification, etc) or via the control of the composition and microstructure of Mg alloys themselves.
Slide 34
Slide 34 shows two types of three point bending tests, one in which the flexural stress is plotted against displacement and second in which the stress intensity factor is plotted against the length of the crack extended beyond the notch. It is interesting that both plots can differentiate between young and aged bones. The plots can be downloaded from here, where more experimental details and explanation can be found.
Slide 35
Slide 35 shows the geometry for 3 point bending for fracture toughness testing. in which the stress intensity factor can be considered as a function of delta a, the depth of the notch at various values of loads. The equation of stress intensity factor can be found here.
Slide 36
Slide 36 describes a family of stress-strain curves as function of composition for four Ti alloys. As we can see the mechanical strength of Ti alloys is well above 400 MPa, which is more than enough for replacement of bones that have a lower mechanical strength of about 175 MPa. The plot in this slide can be reviewed at this site.
Slide 37
Slide 37 Mechanical strength of cortical bone, see link in here, and mechanical strength of Ti alloys, seen in here.
The comparison shows a limit of elasticity of 160 MPa which is well below 400 MPa of Ti alloys or even simply Ti element which has a yield strength of 434 MPa, see link video here.
Slide 38
Slide 38 provides information about the oxide layer on Ti binding biological tissues. Rutile and Anatase, are the two crystalline species of TiO2 formation on Ti surface. Rutile is less bio-reactive than Anatase, info in here, http://cdn.intechopen.com/pdfs-wm/33623.pdf . The metal work function changes as a consequence of the formation of the passivisation layer (the oxide), but ΔΦ is positive for rutile and negative for anatase, info in here, http://pubs.acs.org/doi/abs/10.1021/jp309827u?journalCode=jpccck .
Slide 39
Slide 39 provides information about the crystal structures of three species of Titanium oxide: Rutile, Anatase, and Brookite. As seen from the slide, the density varies with the crystal structure. The valence of Ti in these structures is 4+, same as Carbon in many organic molecules.
Slide 40
Slide 40 provides information about the crystal structures of Titanium monoxide. As seen from the slide, the density is the highest among all Titanium oxides. The crystal structure of Titanium monoxide is shown in this slide. The valence of Ti in these structure is 2+, that makes this oxide special in applications.
Slide 41
Slide 41 provides information about two metals, Ti and Zr that are used in human body implantable. An explanation of why these two metals are bio-compatible is given in this slide. As we know not all metals are inert/not reactive in human body environment. As a fact bulk cubic structures of metals is less preferred such as Al, Cu, Nb, Pb, etc.. Based on a symmetry remark for living structures (carbohydrates, nucleic acids, lipids and proteins), the lower implantable metals symmetry the better. As an example Lysozyme (S.G. P43212, space group number 96) as a possible interface material with an implantable metal such as Au, Ti, Zr, admits lower space groups such as Ti ( P63/mmc. Space group number: 194). Gold is not preferred for multiple reasons too: it has a high symmetry S.G. 225 (Fm-3m) 96<225, it has has a high density 19.32 g/cc, and it is expensive.
Many metals have a degree of leachability in human body fluids except the rare/precious metals Au, Pt, Ir that are expensive as implants. The coatings of Ti with a tiny thin layer of oxide or laser coated organic ceramics, makes Ti as the best choice as human body implantable with extremely low leachability in human body fluids.
Slide 42
Slide 42 provides crystallographic information on Ti crystal structure, unit cell size and directions.
Slide 43
Slide 43 provides information on Zr metal as the second choice on human body implantables. The crystal structure of Zr is same as Ti, with hexagonal close packed (HCP) unit cell. The HCP cell is shown together with a body center cubic (BCC) unit and face close cubic (FCC) unit for comparison reason.
Slide 44
Slide 44 shows the Table of major biomedical metals and alloys and their applications. More details about materials in the Table can be found here.
Slide 45
The Table on Slide 45 shows a comparison of mechanical properties for three metal alloys. Notice the the increase of the ultimate tensile strength of Ti 64, from 434 MPa for Titanium (see slide 37) to 900 MPa for Ti 64. More data about other materials can be found here.
Slide 46
Slide 46 lists some medical devices as they were created by the inventor Alfred Mann’s companies. Such devices are:
-rechargeable pacemaker,
-an implant for deaf people,
-an insulin pump and a
-prosthetic retina. (Mel Melcon, Los Angeles Times)
Slide 47
Slide 47 As we imagine, the implanted devices should be coated with one of these Ti, Zr, ceramic coated Ti and Stainless Steel. Three example are given as: Ti-plates and rods, 3D printed Jaws + plasma coated HAp, Gold nano-wires.
Slide 48
In the example on slide Slide 48, the pacemaker casing is made of titanium or a titanium alloy, electrodes are made of metal alloy insulated with polyurethan polymers, more info in here.
Slide 49
The second device shown in slide 49 is an implant for deaf people, whose surface in contact with human body fluids is coated with Ti. More info on how this implant works can be found in here.
Slide 50The insulin pump shown in slide 50 is a schematic of the pump controlled electronically by a control algorithm device, a sensor, an electronic receiver that connects with an iPhone through an wireless channel.
Slide 51
The prosthetic retina on slide 51 is an example of a bio-MEMS based optical sensor that takes the outside image through a tiny camera, the electrical signal of the camera is sent to a receiver and then to an array of micro-electrodes tacked to the retina which send electrical impulses to the brain through the optical nerve. More details can be found in here.
Slide 52Slide 52 describes how easily available bio-compatible metal powders
can revolutionize 3D printing for medical implants. The surgical implants need to generate expected responses from neighboring cells and tissues. Cell behavior (adhesion, functional alteration, morphological changes, and proliferation) is strongly affected by the surgical implants’ surface properties. Surface topography, surface chemistry, and surface energy influence decisively the biological response to an implanted device.
The well controlled 3D printing atmosphere (neutral gases and restricted oxygen) guarantees the high purity of the 3D printed parts and preserves the materials’ properties.
The advantages of 3D printing for medical applications is thoroughly discussed in here.
Slide 53
Slide 53 shows five conclusions of the presentation, in which 1) many engineered metals are mechanically resistant in human body, but prone to certain corrosion if not coated,
2) Ti, Zr coated bio-ceramics are bio-compatible materials in human body, 3) medical devices implants and MEMS are useful as heart stent, orthopedic prosthetic, prosthetic retina, 3) M3DP has low costs, high quality, long life cycle and 4) Metal/bio-ceramic and Vivos dental’s synthetic bone for oral augmentation is a solution for today’s dental health care.
Slide 54Slide 54 shows conclusions regarding the hardware of the presentation, in which: 6) there are two types of metal 3D printing hardware for medical applications: Selective Laser Melting / Selective Laser Sintering, and 3D Bioploter (metal powder mixed with binder and further thermal treatment to remove binder and sinter the metallic matrix in a solid object that can be used as a replacement. Thank you for your attention!
Montana State University, Department of Mechanical and Industrial Engineering and Xtant Medical Holdings created a 3D printer capable of printing resorbable bone grafts. The grafts produced can be broken down and absorbed into the body. The personalized bone grafts are custom made and the material used for MSU can minimize the material limitations.
The ability to bioprint usable bone and joint material has seen progress from all over the world and now MSU has contributed their breakthrough research in the medical race to 3D print reconstructive parts for the human body.
Secondary caries due to biofilm acids is a primary cause of dental composite restoration failure. To date, there have been no reports of dental composites that can repel protein adsorption and inhibit bacteria attachment. The objectives of this study were to develop a protein-repellent dental composite by incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC) and to investigate for the first time the effects of MPC mass fraction on protein adsorption, bacteria attachment, biofilm growth, and mechanical properties. Composites were synthesized with 0 (control), 0.75%, 1.5%, 2.25%, 3%, 4.5% and 6% of MPC by mass. A commercial composite was also tested as a control. Mechanical properties were measured in three-point flexure. Protein adsorption onto the composite was determined by the microbicinchoninic acid method. A human saliva microcosm biofilm model was used. Early attachment at 4 h, biofilm at 2 days, live/dead staining and colony-forming units (CFUs) of biofilms grown on the composites were investigated. Composites with MPC of up to 3% had mechanical properties similar to those without MPC and those of the commercial control, whereas 4.5% and 6% MPC decreased the mechanical properties (P<0.05). Increasing MPC from 0 to 3% reduced the protein adsorption on composites (P<0.05). The composite with 3% MPC had protein adsorption that was 1/12 that of the control (P<0.05). Oral bacteria early attachment and biofilm growth were also greatly reduced on the composite with 3% MPC, compared to the control (P<0.05). In conclusion, incorporation of MPC into composites at 3% greatly reduced protein adsorption, bacteria attachment and biofilm CFUs, without compromising mechanical properties. Protein-repellent composites could help to repel bacteria attachment and plaque build-up to reduce secondary caries. The protein-repellent method might be applicable to other dental materials.
Dental caries, a dietary carbohydrate-modified bacterial infectious disease, is a common infection in humans.1 The basic mechanism of caries is demineralisation of the enamel and dentin via acid generated by a bacterial biofilm.2 Resin composites are increasingly being used for tooth cavity restorations because of their aesthetics and their direct-filling capability.3,4 Extensive efforts have improved resin compositions and curing conditions and have reduced polymerisation shrinkage.5,6,7,8,9,10 However, secondary caries and restorative material fractures represent more than 90% of recorded failures.11,12Approximately one-half of all dental restorations fail within 10 years.13 The replacement of failed restorations has accounted for 50%–70% of all restorations performed.13 In addition, resin composites not only have no antibacterial properties, but they also can even accumulate more biofilm in vivo than other restorative materials.14,15 Therefore, it is desirable to improve the longevity of composite restorations by incorporating bioactive agents to combat microbial destruction and secondary caries while sustaining their load-bearing capability.
To reduce biofilm and plaque build-up and to combat caries, novel quaternary ammonium methacrylates were developed and incorporated into dental resins.16,17,18 12-Methacryloyloxydodecylpyridinium bromide can be copolymerised with other dental monomers to form antibacterial polymer matrices to reduce bacterial growth.19,20,21,22,23,24 Other compositions, such as methacryloxylethylcetyl dimethyl ammonium chloride, have also been developed.25 Recently, quaternary ammonium dimethacrylate was synthesised and incorporated into bonding agents and composites to obtain antibacterial activity.26,27,28,29,30
One potential limitation of resins containing quaternary ammonium methacrylates is that the depositing of salivary proteins on composite surfaces could decrease the efficacy of ‘contact inhibition’, thereby reducing antibacterial potency.31,32 Salivary proteins in the mouth can adhere to dental restoration surfaces to provide anchor points for bacteria attachment, an initial step in biofilm formation.33,34 Biofilm formation is a source of infection and a prerequisite for the occurrence of dental caries and secondary caries.35Accordingly, there is a great need to develop a novel resin composite that can repel proteins. Such a composite could repel proteins and hence inhibit bacteria attachment, and it could further enhance the antibacterial potency of quaternary ammonium methacrylate-containing restorations by having a protein-repellent coating and enhancing the contact-killing efficacy. However, to date, there has been no report on dental composites that possess protein-repellent capability.
2-Methacryloyloxyethyl phosphorylcholine (MPC) is a methacrylate with a phospholipid polar group in the side chain, and it is a common biocompatible and hydrophilic biomedical polymer.36 MPC has been shown to have excellent ability to repel protein adsorption and prevent bacterial adhesion.37,38 Several medical devices containing MPC have been developed and used clinically, such as artificial blood vessels, implantable artificial hearts and artificial lungs.38,39,40However, there have been no reports of the incorporation of MPC into dental composites.
Therefore, in this study, MPC was incorporated into a dental resin, which was then filled with glass particles to develop a protein-repellent composite for the first time. The objectives were (i) to develop a protein-repellent and bacteria-repellent dental composite and (ii) to investigate the effects of MPC mass fraction on the mechanical properties, protein adsorption and biofilm activity of the composite. The following hypotheses were tested: (i) MPC-containing composites would have mechanical properties matching those of a commercial control composite; (ii) MPC-containing composites would have much less protein adsorption on the composite surface than that without MPC; and (iii) MPC-containing composites would significantly reduce bacterial attachment and biofilm growth on the composite surface.
Fabrication of MPC-containing resin composites
Bisphenol glycidyl dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) (Esstech, Essington, PA, USA) were mixed at a mass ratio of 1:1 and were rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate (mass fractions). MPC, a methacrylate with a phospholipid polar group in the side chain, was used as the protein-repellent agent. The chemical structure of MPC, according to a previous study,36 is shown in Figure 1a. MPC was obtained from Sigma-Aldrich (St Louis, MO, USA) and was synthesized via the method reported by Ishihara et al.36 The MPC powder was mixed with photo-activated BisGMA–TEGDMA resin (referred to as BT) at the following MPC/(BT+MPC) mass fractions: 0, 2.5%, 5%, 7.5%, 10%, 15% and 20%, yielding seven respective groups. The different mass fractions enabled the investigation of the relationship between the MPC mass fraction and mechanical properties of the composite. Barium boroaluminosilicate glass of a mean particle size of 1.4 µm (Caulk/Dentsply, Milford, DE, USA) was silanised with 4% 3-methacryloxypropyltrimethoxysilane and 2% n-propylamine.41 The glass particles were mixed into each resin at the same filler level of 70% by mass. Because the resin mass fraction was 30% in the composite, the MPC mass fractions in the composite were 0 (control), 0.75%, 1.5%, 2.25%, 3.0%, 4.5% and 6.0% for the respective groups. As another control, a commercial composite with nanofillers of 40–200 nm was used (Heliomolar, Ivoclar, Ontario, Canada). The fillers consisted of silica and ytterbium-trifluoride at a filler mass fraction of 66.7%. Heliomolar is indicated for Class I and II restorations in the posterior region, as well as Class III–V restorations. Eight composites were tested for mechanical properties:
commercial control (Heliomolar);
70% glass filler+30% BisGMA–TEGDMA resin (termed ‘Control without MPC’);
Mechanical properties of dental composites. (a) Chemical structure of MPC; (b) flexural strength and (c) elastic modulus of composites (mean±standard deviation; n=6). The composite specimens were immersed in distilled water at 37 °C for 24 h and were then fractured while wet, within a few minutes after being removed from the water. Different letters indicate values that are significantly different from each other (P<0.05). MPC, 2-methacryloyloxyethyl phosphorylcholine.
Mechanical properties
Each composite paste was placed into rectangular moulds of 2 mm×2 mm×25 mm and was photo-cured (Triad 2000; Dentsply, York, PA, USA) for 1 min on each open side.26,27 Six specimens per composite were made. A computer-controlled Universal Testing Machine (5500R; MTS, Cary, NC, USA) was used.26,27 Composite specimens were stored in distilled water at 37 °C for 24 h and then were fractured in three-point flexure with a 10 mm span at a crosshead speed of 1 mm⋅min−1.26,27 The specimens were wet and not dried, and they were fractured within a few minutes after being removed from the water. Flexural strength (S) was calculated as: S=3PmaxL/(2bh2), where P is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus (E) was calculated as: E=(P/d)(L3/[4bh3]), where load P divided by displacement d is the slope in the linear elastic region.26,27
Measurement of protein adsorption
The mechanical results showed that group 7 with 4.5% MPC and group 8 with 6%MPC had relatively lower strength and elastic modulus values. Therefore, only groups 1–6 were included in further protein and biofilm experiments. For the protein adsorption and biofilm experiments, each composite paste was placed into disc moulds of 9 mm in diameter and 2 mm in thickness. They were light-cured and stored in distilled water at 37 °C for 24 h as described above.
The amount of protein adsorbed on the composite discs was determined by the microbicinchoninic acid method.42,43 Each composite disc was immersed in phosphate-buffered saline (PBS) for 2 h before immersing in 4.5 g⋅L−1 bovine serum albumin (BSA) (Sigma-Aldrich, St Louis, MO, USA) solution at 37 °C for 2 h. The discs then were rinsed with fresh PBS by stirring at a speed of 300 r·min−1 for 5 min (Bellco Glass, Vineland, NJ, USA), and they were immersed in 1%(m/m) sodium dodecyl sulphate in PBS and sonicated at room temperature for 20 min to detach completely the BSA adsorbed onto the surface of the disc.42,43 A protein analysis kit (micro bicinchoninic acid protein assay kit; Fisher Scientific, Pittsburgh, PA, USA) was used to determine the BSA concentration in the sodium dodecyl sulphate solution. Briefly, 25 µL of the sodium dodecyl sulphate solution and 200 µL of the bicinchoninic acid working reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min.42,43 Then, the 96-well plate was cooled to room temperature, and the absorbance at 562 nm was measured via a microplate reader (SpectraMax M5; Molecular Devices, Sunnyvale, CA, USA). Standard curves were prepared using the BSA standard. From the concentration of protein, the amount of protein adsorbed on the disc surface was calculated.42,43
Dental plaque microcosm biofilm model
A dental plaque microcosm biofilm model was used.26,27 Saliva is ideal for growing microcosm biofilms in vitro, with the advantage of maintaining much of the complexity and heterogeneity of dental plaque in vivo.44 Saliva was collected from 10 healthy donors having natural dentition without active caries and not having used antibiotics within the preceding 3 months. The donors did not brush their teeth for 24 h, and they abstained from food and drink intake for 2 h prior to donating saliva.26,27 Stimulated saliva was collected during paraffin chewing and was kept on ice. An equal volume of saliva from each of the ten donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a concentration of 70% and was stored at −80 °C for subsequent use.45
The saliva-glycerol stock was added, at a 1:50 final dilution, to the growth medium as an inoculum. The growth medium contained mucin (type II, porcine, gastric) at a concentration of 2.5 g⋅L−1, bacteriological peptone at 2.0 g⋅L−1, tryptone at 2.0 g⋅L−1, yeast extract at 1.0 g⋅L−1, NaCl at 0.35 g⋅L−1, KCl at 0.2 g⋅L−1, CaCl2 at 0.2 g⋅L−1, cysteine hydrochloride at 0.1 g⋅L−1, haemin at 0.001 g⋅L−1 and vitamin K1 at 0.000 2 g⋅L−1, at pH 7.46 Composite discs were sterilised in ethylene oxide (Anprolene AN 74i; Andersen, Haw River, NC, USA). A volume of 1.5 mL of inoculum was added to each well of a 24-well plate with a disc followed by incubation at 37 °C in 5% CO2 for 8 h. Then, the discs were transferred to new 24-well plates filled with fresh medium and incubated. After 16 h, the discs were transferred to new 24-well plates with fresh medium and were incubated for 24 h. This process totalled 48 h of incubation, which was adequate to form plaque microcosm biofilms, as shown in previous studies.26,27
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Figure 1 plots the flexural strength (Figure 1b) and elastic modulus (Figure 1c) of the composites (mean±standard deviation; n=6). The composites with 0.75% to 3% MPC had flexural strengths (P=0.22) and elastic moduli (P=0.37) similar to those of the commercial control and the control without MPC. Composites with 4.5% MPC and 6% MPC had strengths and moduli that were significantly lower than those of the commercial control (P<0.05).
The amounts of protein adsorption on composite disc surfaces are plotted inFigure 2 (mean±standard deviation; n=6). Adding MPC to composites significantly decreased the protein adsorption (P<0.05). Protein adsorption was inversely proportional to the MPC mass fraction in the composite from 0 to 3% MPC. The resin composite with 3% MPC had the lowest amount of protein adsorption, which was nearly 1/12 that of the commercial control and the composite without MPC (P<0.05).
Protein adsorption onto composite surfaces. Mean±standard deviation; n=6. The composite with 3% MPC had the lowest amount of protein adsorption, which was approximately 1/12 those of the commercial composite control and the experimental composite without MPC (P<0.05). Different letters indicate values that are significantly different from each other (P<0.05). MPC, 2-methacryloyloxyethyl phosphorylcholine.
Bacterial early attachment onto composite surfaces was examined at 4 h. Representative live/dead staining images at 4 h are shown in Figure 3a–3d, and the area fraction of composite surface covered by live bacteria is plotted inFigure 3e (mean±standard deviation; n=6). Live bacteria were stained green, and dead bacteria were stained red. The composite discs had primarily live bacteria, with few dead bacteria. The commercial control composite and the composite without MPC had noticeably more bacteria coverage than composites containing MPC. This outcome was found to be true and consistent by examining all 18 of the images per group. The composite with 3% MPC had much less bacterial adhesion. For the quantification of live bacteria coverage in Figure 3e), values with different letters are significantly different from each other.
Oral microcosm bacteria earl attachment on composites at 4 h.(a–d) Representative live/dead staining images of bacteria on composite surfaces; (e) area fraction of green staining of live bacteria coverage on composite surfaces (mean±standard deviation; n=6). The composite control had much more bacteria attachment. Increasing the MPC content decreased the bacterial attachment. Different letters in (e) indicate values that are significantly different from each other (P<0.05).
Figure 4 shows the results for 2-day biofilms on the composites. Relatively mature biofilms were formed in 2 days, covering nearly the entire surface of commercial composite and that without MPC (Figure 4a and 4b). However, there was less biofilm coverage on composite discs containing MPC (Figure 4c and 4d). In the quantification of the area fraction of live bacteria in Figure 4e (mean±standard deviation; n=6), composites with 1.5%–3% MPC had significantly less biofilm coverage than the controls (P<0.05).
Oral microcosm biofilm growth on composites at 48 h. (a–d) Representative live/dead staining images of biofilms on composite surfaces; (e) area fraction of green staining of live bacteria coverage on composite surfaces (mean±standard deviation; n=6). Increasing the MPC mass fraction decreased the biofilm coverage on the composite. The composite with 3% MPC had the least biofilm coverage (P<0.05). Different letters in (e) indicate values that are significantly different from each other (P<0.05).
Figure 5 plots the CFU counts of biofilms grown for 2 days on composite discs: total microorganisms (Figure 5a), total Streptococci (Figure 5b) and S. mutans(Figure 5c) (mean±standard deviation; n=6). The commercial control composite and the experimental composite without MPC had similarly high CFU counts (P=0.89). All three CFU counts showed a decreasing trend with an increasing MPC mass fraction. All three CFU counts on the composite with 3% MPC were greatly reduced compared to those of the controls (P<0.05).
CFU counts of 2-day biofilms on composites. (a) Total microorganisms, (b) total Streptococci and (c) Streptococcus mutans(mean±standard deviation; n=6). Increasing the MPC mass fraction decreased the biofilm CFUs on the composites (P<0.05). All three CFU counts on the composite with 3% MPC were much lower than those of the commercial composite control and the experimental composite without MPC (P<0.05). In each plot, different letters indicate values that are significantly different from each other (P<0.05). CFU, colony-forming unit; MPC, 2-methacryloyloxyethyl phosphorylcholine.
The present study represents the first report on the development of protein-repellent dental composite and investigation of the effects of MPC incorporation on protein adsorption, biofilm activity, and the mechanical properties of the composite. MPC addition provided strong protein-repellent activity for the composite, and the microcosm bacteria early attachment (4 h) and mature biofilm (2 days) viability showed consistent and substantial decreases with increasing MPC mass fraction. The protein-repellent activity of the composite was achieved without compromising the mechanical properties of the composite from 0 to 3% of MPC. In addition, MPC had good biocompatibility, and it has already been used in several medical devices with approval of the Food and Drug Administration of the United States.39,40 Therefore, the novel protein-repellent approach is promising for developing dental composites to reduce bacteria attachment, biofilm and plaque build-up, and the occurrence of caries. Furthermore, this protein-repellent approach could be applied to develop a new class of protein-repellent dental resins, adhesives, cements, coatings and sealants.
There are two potential benefits of MPC-containing dental composite. First, salivary proteins adsorbed onto the resin composite surface in the oral environment provide a medium for the attachment of bacteria and microorganisms, thereby initiating the basis for biofilm formation.33,34,35 MPC has been shown to have excellent protein-repellent ability to diminish bacterial adhesion.37,38 Regarding the protein-repellent mechanism,37,49,50,51 it was suggested that MPC is highly hydrophilic,36 and there is an abundance of free water but no bound water in the hydrated MPC polymer. The presence of bound water causes protein adsorption.37,50,51 By contrast, the large amount of free water around the phosphorylcholine group is believed to detach proteins effectively, thereby repelling protein adsorption.37,51 Based on this mechanism, it could be assumed that increasing the mass fraction of MPC in the resin composite would increase the presence of MPC and hence its protein-repellent potency. Indeed, in the present study, gradually increasing the MPC mass fraction from 0 to 3% in the composite significantly and monotonically decreased the amount of protein adsorption (Figure 3). That the MPC-containing composite could repel proteins indicated that the composite could potentially also reduce biofilm attachment. Indeed, the results in Figures 4–6 confirmed that the incorporation of MPC at a mass fraction of 3% into the composite greatly reduced bacteria attachment, biofilm growth and CFU counts.
Second, previous studies have suggested that salivary proteins adsorbed from physiological fluids are able to attenuate significantly the antibacterial properties of the underlying surfaces.31,32 Protein adsorption onto the resin surface rendered the contact-killing mechanism less effective. Indeed, several studies have demonstrated that a saliva-derived protein film on the cationic antibacterial surface reduced the original bactericidal effect.52,53,54 For example, 12-methacryloyloxydodecylpyridinium bromide-immobilized fillers, which contained 12-methacryloyloxydodecylpyridinium bromide at a concentration of 15.8%, greatly inhibited bacterial growth; however, saliva pretreatment of the surface reduced its antibacterial potency.54 Therefore, the reduction in the antibacterial effect by the adsorption of salivary proteins on the composite has been considered a drawback of dental materials with contact-killing antibacterial activities. Because the MPC-containing composite greatly reduced protein adsorption, it could likely enhance the antibacterial effects of contact-killing dental resins. Further study is needed to combine MPC with contact-killing antibacterial agents in the resin composite in order to investigate their synergistic effects on the antibacterial potency.
The present study used an artificial saliva-like culture medium (the McBain medium) for the biofilm culture experiments. A major component of this medium, mucin, accounts for up to 26% of the natural salivary proteins; mucin is an important salivary protein in the salivary pellicle.55,56 A recent study investigated the effects of salivary pellicle pre-coating on resin surfaces on the antibacterial properties of the resin.53 When cultured in McBain medium, there was no significant difference in antibacterial activity whether the resin specimens were precoated with salivary pellicles or not. These results suggested that the McBain artificial saliva medium produced medium-derived pellicles on the resin surfaces, which provided attenuating effects on biofilms similar to natural salivary pellicles. Hence, the present study used the McBain artificial saliva medium to test the bacteria attachment and biofilm growth on the MPC-containing composite, which better simulated the oral environment than using a culture medium such as water.
In addition to secondary caries due to biofilm acids, bulk fracture is also a main challenge facing composite restorations.11,12 Therefore, the new protein-repellent composite must possess load-bearing capability for tooth cavity restorations. In the present study, composites with MPC at up to 3% did not significantly decrease the composite strength, whereas 4.5% and 6% MPC decreased the mechanical properties. Further studies should investigate composite water sorption as a function of MPC mass fraction and determine whether the decrease in mechanical properties at ≥4.5% MPC was due to an increase in the water sorption of the composite.57 Nonetheless, the present study showed the promise of using an optimal MPC mass fraction in the composite to achieve the maximal protein-repellent capability without significantly compromising the mechanical properties. In the present study, incorporating MPC into the composite at 0.75%–3% MPC resulted in mechanical properties similar to those of a commercial composite used for Class I–IV restorations. The strength and elastic modulus of the composite containing 3% MPC were also similar to those of the glass-filled composite without MPC. This finding indicated that significant protein-repellent ability could be achieved in dental composite without compromising the load-bearing capability compared to the counterpart composite without MPC. Therefore, the protein-repellent method with MPC incorporation into dental composite is promising in its potential to yield new composites for repelling protein adsorption and hence bacteria attachment.
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Conclusions
In this study, the first protein-repellent dental composite was reported, and the effects of MPC mass fraction on the protein adsorption, biofilm activity, and mechanical properties of the composite were determined. Incorporation of MPC up to 3% into the composite did not compromise the strength and elastic modulus, which matched those of a commercial composite without protein-repellent ability. Increasing the MPC mass fraction monotonically decreased protein adsorption, dental microcosm bacteria early attachment and biofilm growth. Biofilm CFU counts for total microorganisms, total streptococci, and mutans streptococci on composite with 3% MPC were greatly reduced compared to the controls. The MPC-containing composite is promising for the reduction of biofilm formation and the combatting of secondary caries. The protein-repellent method could have applicability to other composites, adhesives, sealants and cements to repel proteins and inhibit oral bacteria attachment.
Antibacterial adhesives are promising to inhibit biofilms and secondary caries. The objectives of this study were to synthesize and incorporate quaternary ammonium methacrylates into adhesives, and investigate the alkyl chain length effects on three-dimensional biofilms adherent on adhesives for the first time. Six quaternary ammonium methacrylates with chain lengths of 3, 6, 9, 12, 16 and 18 were synthesized and incorporated into Scotchbond Multi-Purpose. Streptococcus mutans bacteria were cultured on resin to form biofilms. Confocal laser scanning microscopy was used to measure biofilm thickness, live/dead volumes and live-bacteria percentage vs. distance from resin surface. Biofilm thickness was the greatest for Scotchbond control; it decreased with increasing chain length, reaching a minimum at chain length 16. Live-biofilm volume had a similar trend. Dead-biofilm volume increased with increasing chain length. The adhesive with chain length 9 had 37% live bacteria near resin surface, but close to 100% live bacteria in the biofilm top section. For chain length 16, there were nearly 0% live bacteria throughout the three-dimensional biofilm. In conclusion, strong antibacterial activity was achieved by adding quaternary ammonium into adhesive, with biofilm thickness and live-biofilm volume decreasing as chain length was increased from 3 to 16. Antibacterial adhesives typically only inhibited bacteria close to its surface; however, adhesive with chain length 16 had mostly dead bacteria in the entire three-dimensional biofilm. Antibacterial adhesive with chain length 16 is promising to inhibit biofilms at the margins and combat secondary caries.
Dental resin composites are the principal materials for tooth cavity restorations due to their excellent esthetics and direct-filling capabilities.1,2,3,4 Composite restorations are bonded to tooth structures via bonding agents.5,6,7 Studies have shown that secondary caries is one of the primary reasons for restoration failure.3,5,8,9 and replacing the failed restorations accounts for 50%–70% of all restorations performed.10,11 The cause of caries is biofilm acid production;12therefore, efforts were made to develop antibacterial resins.13,14 Quaternary ammonium methacrylates (QAMs) were synthesized and copolymerized in dental resins to obtain antibacterial functions.15,16,17,18,19,20,21,22,23 Because residual bacteria often exist in the prepared tooth cavity and microleakage could allow new bacteria to invade the tooth-restoration interface, it would be especially useful for the bonding agent to possess antibacterial functions.13,14,15,16,24,25
Quaternary ammonium salts (QAS) can cause bacteria lysis by binding to bacterial membranes.17,26 When the negatively charged bacterial cell contacts the positively charged sites of the quaternary ammonium, the electric balance of the cell membrane could be disturbed, and the bacterium could be damaged or killed.17,26 The long polymeric chains with positive charges appeared to contribute to the efficacy of bactericide.27 Increasing the alkyl chain length (CL) increased the hydrophobicity, which could enhance the propensity to penetrate the hydrophobic bacterial membrane.28 Therefore, cationic polymers with longer CL could be more effective in penetrating bacterial cells to disrupt membranes.28,29 However, to date, there has been no report on the effect of CL of QAMs in dental bonding agents, except a recent study.30 That study synthesized a series of new QAMs with CL from 3 to 18, incorporated them into dental adhesive and achieved strong antibacterial efficacy, without compromising the dentin bond strength.30 However, the three-dimensional (3D) biofilm structure and live/dead bacteria viability distribution along biofilm thickness vs.CL were not investigated.
Previous studies on dental biofilms were performed using light, scanning and transmission electron microscopy which provided two-dimensional (2D) view of the top surface of the biofilm or the structure of a single cell.31,32 Besides these established techniques, the application of confocal laser scanning microscopy (CLSM) was also introduced in dental research for assessment of oral biofilms.33,34,35,36,37 CLSM allows horizontal and vertical optical sectioning of the 3D biofilm. The 3D biofilm structure can be reconstructed from 2D images of thin sections throughout the biofilm. Image-processing techniques are used for quantitative analysis of biofilms to obtain a detailed visualization of thick biofilm samples,38 which cannot be obtained via conventional phase contrast or fluorescence microscopy. Previous studies used CLSM techniques to analyze the 3D viability distribution in dental biofilms.36,37,39,40 However, there has been no report on the effect of CL on 3D viability distribution of biofilms adherent on dental bonding agents. It would be interesting to know: (i) how CL would affect biofilm thickness and live/dead biofilm volumes on adhesive resins; and (ii) if there would be more dead bacteria near the antibacterial bonding agent surface, and less dead bacteria in the biofilm further away from bonding agent surface.
Therefore, the objectives of this study were to incorporate QAMs into dental bonding agent, and investigate the effects of CL on the 3D biofilm structure, live biofilm volume and viability distribution along biofilm thickness adherent on dental bonding agents for the first time. It was hypothesized that: (i) the viability of 3D biofilms growing on dental adhesive containing QAM will decrease with increasing CL; (ii) CL of QAM in adhesive resin will have a significant effect on biofilm thickness and live and dead biofilm volumes; (iii) there will be less live bacteria in the biofilm near antibacterial bonding agent surface, and the percentage of live bacteria in the biofilm will increase with increasing distance away from bonding agent surface.
Synthesis of antibacterial QAMs with different chain length CL
New QAMs were synthesized using a modified Menschutkin reaction via the addition reaction of a tertiary amine with an organohalide.20,21,22,23 A benefit of this reaction is that the reaction products are generated at virtually quantitative amounts and require minimal purification.20 The 2-(dimethylamino) ethyl methacrylate (DMAEMA; Sigma Aldrich, St Louis, MO, USA) was the methacrylate-containing tertiary amine. For example, to synthesize dimethylaminododecyl methacrylate (DMADDM) with CL=12, 10 mmol of DMAEMA, 10 mmol of 1-bromododecane (TCI America, Portland, OR, USA) and 3 g of ethanol were added to a vial, which was capped and stirred at 70 °C for 24 h.41 After the reaction was completed, the ethanol was removed via evaporation. This yielded DMADDM as a clear liquid, which was verified via Fourier transform infrared spectroscopy in a recent study.41 Using this method, six QAMs with CL of 3, 6, 9, 12, 16 and 18 were synthesized,30 namely:
DMAEMA was reacted with 1-bromopropane to form dimethylaminopropyl methacrylate (DMAPM, CL=3).
DMAEMA was reacted with 1-bromohexane to form dimethylaminohexyl methacrylate (DMAHM, CL=6).
DMAEMA was reacted with 1-bromononane to form dimethylaminononyl methacrylate (DMANM, CL=9).
DMAEMA was reacted with 1-bromododecane to form DMADDM (CL=12).
DMAEMA was reacted with 1-bromohexadecane to form dimethylaminohexadecyl methacrylate (DMAHDM, CL=16).
DMAEMA was reacted with 1-bromooctadecane to form dimethylaminooctadecyl methacrylate (DMAODM, CL=18).
Processing of antibacterial bonding agents
To formulate antibacterial bonding agents, Scotchbond multi-purpose bonding agent (SBMP; 3M, St Paul, MN, USA) was used as the parent system. According to the manufacturer, SBMP adhesive contained 60%–70% of bisphenol A diglycidyl methacrylate and 30%–40% of 2-hydroxyethyl methacrylate, tertiary amines and photo-initiator. SBMP primer contained 35%–45% of 2-hydroxyethyl methacrylate, 10%–20% of a copolymer of acrylic and itaconic acids and 40%–50% water. Each QAM was mixed SBMP primer at a QAM/(SBMP primer+QAM) mass fraction of 10%, following previous studies.22,23,30 SBMP adhesive was also incorporated with 10% QAM.22,23,30 This yielded six antibacterial bonding agents, corresponding to the six QAMs. They are designated, respectively, as:
SBMP+DMAPM (CL3);
SBMP+DMAHM (CL6);
SBMP+DMANM (CL9);
SBMP+DMADDM (CL12);
SBMP+DMAHDM (CL16);
SBMP+DMAODM (CL18).
Fabrication of resin specimens for biofilm culture
Resin disks for biofilm experiments were fabricated using the cover of a sterile 96-well plate as molds.16 Following previous studies,16,30 10 µL primer was brushed onto the bottom of each dent of approximately 8 mm in diameter. The primer was dried with a stream of air and then 20 µL of adhesive was applied. A Mylar strip was used to covered on the adhesive which was then light-cured for 20 s (Optilux VCL 401; Demetron Kerr, Danbury, CT, USA). This yielded a cured resin disk of approximately 8 mm in diameter and 0.5 mm in thickness.30 The disks were removed from the cover of the 96-well plate, immersed in 200 mL of distilled water and stirred via a magnetic stirrer (Bellco Glass, Vineland, NJ, USA) at a speed of 100 r·min−1 for 1 h to remove any uncured monomers, following previous studies.15 The disks were then dried, sterilized in an ethylene oxide sterilizer (Anprolene AN 74i; Andersen, Haw River, NC, USA) and then de-gassed for 7 days following manufacturer’s instructions.
The use of Streptococcus mutans (S. mutans) bacteria (ATCC700610; American Type, Manassas, VA, USA) was approved by the Institutional Review Board of the University of Maryland.21 A 15 µL of S. mutans stock bacteria was added to 15 mL of brain heart infusion broth (Becton, Sparks, MD, USA) and incubated at 37 °C with 5% CO2 for 16 h; 150 µL of this S. mutans suspension was then diluted by 10-fold in a growth medium which consisted of brain heart infusion supplemented with 0.2% sucrose to form S. mutans inoculation medium of 1.5 mL.
CLSM analysis of biofilms
Seven bonding agent groups were tested: the six bonding agents containing QAM with different CLs and the unmodified SBMP as control. Six disks (n=6) were used for each bonding agent, requiring a total of 42 disks. Each disk was placed in a well of a 24-well plate and inoculated with 1.5 mL of the S. mutans inoculation medium. The samples were incubated at 5% CO2 and 37 °C. The medium consisted of brain heart infusion supplemented with 0.2% sucrose. After incubation for 8 h, the disks were transferred to new 24-well plates with fresh medium.21,22,23 After 16 h, the disks were transferred to new 24-well plates and incubated for 24 h. This totaled 2 days which were shown previously to form biofilms on resin specimens.21,22,23 The biofilms on the disks were washed three times with phosphate-buffered saline to remove loose bacteria, and then stained using a BacLight live/dead kit (Molecular Probes, Eugene, OR, USA). Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence.21,22,23
The biofilms were investigated using a 3D model as previously described.42 The fluorescence was examined visualized using a CLSM (LSM510; Carl Zeiss, Thornwood, NY, USA). Green fluorescence was provided with an argon laser (488-nm laser excitation) and red fluorescence was given with a helium-neon laser (543 nm laser excitation). Images were taken from the bottom of the biofilm that was in contact with the resin disk surface, section by section to the top surface of the biofilm. For the purpose of illustration, an example of a biofilm on the SBMP control disk is shown in Figure 1. The biofilm section parallel to the resin surface was referred to as the x–y plane, and the direction perpendicular to the resin surface is called the z axis. For each biofilm, 10 planes at equal distances (indicated by the 10 white lines at the upper left corner in Figure 1) along the z axis were imaged to obtain an overall view of the biofilm volume. These 2D sections were stacked and reconstructed into a 3D image of the biofilm using the IMARIS software (Bitplane, Saint Paul, MN, USA). The biofilm images were analyzed using a software (bioImageL; Faculty of Odontology, Malmö University, Malmö, Sweden). The bioImageL software is based on color segmentation algorithms written in MATLAB (MathWorks, Natick, MA, USA) and is able to produce information of the structure and spatial differences in the biofilm. The biofilm is characterized by parameters including biofilm thickness, green-stained live bacteria volume, red-stained dead bacteria volume, as well as the live and dead bacteria coverage on each two-dimensional x–y section in the biofilm.
Representative CLSM image of biofilm on SBMP control. The biofilm section parallel to the resin surface is referred to as the x–y plane. The direction perpendicular to resin surface is termed the z axis. The 2-day biofilm on SBMP control had a thickness of approximately 41 µm. Ten planes at equal distances along the z axis of the biofilm (indicated by the 10 white lines at the upper left corner) were imaged by CLSM. These 2D images were stacked to reconstruct the 3D biofilm image. 2D, two-dimensional; 3D, three-dimensional; CLSM, confocal laser scanning microscopy; SBMP, Scotchbond multi-purpose bonding agent.
A typical CLSM image of a 3D biofilm on SBMP control is shown in Figure 1 as described in the section on ‘CLSM analysis of biofilms’. Figure 2 shows representative images of biofilms on the six bonding agents containing QAM with various CL. Live bacteria were stained green, and dead bacteria were stained red. SBMP control and that with CL3 had primarily live bacteria. The amount of dead bacteria gradually increased when CL was increased to 6, 9 and 12. When CL was increased to 16, the biofilms were primarily dead with red staining. When CL was further increased to 18, there was an increase in green staining of live bacteria. Furthermore, the 3D images highlighted the differences in biofilm thickness, with biofilms on SBMP control being the thickest and that of CL16 being the thinnest.
Typical 3D images of biofilms cultured for 2 days on bonding agents. (a) SBMP+DMAPM (CL3); (b) SBMP+DMAHM (CL6); (c) SBMP+DMANM (CL9); (d) SBMP+DMADDM (CL12); (e) SBMP+DMAHDM (CL16); (f) SBMP+DMAODM (CL18). Live bacteria were stained green and dead bacteria were stained red. CL, chain length; 3D, three-dimensional; DMADDM, dimethylaminododecyl methacrylate; DMAHDM, dimethylaminohexadecyl methacrylate; DMAHM, dimethylaminohexyl methacrylate; DMANM, dimethylaminononyl methacrylate; DMAODM, dimethylaminooctadecyl methacrylate; DMAPM, dimethylaminopropyl methacrylate; SBMP, Scotchbond multi-purpose bonding agent.
The biofilm thickness results are quantified in Figure 3 (mean±s.d.; n=6). Biofilm thickness steadily decreased from SBMP control to those containing QAM with increasing CL, reaching a minimum at CL16. When CL was further increased to 18, the biofilm thickness increased. Values indicated by dissimilar letters are significantly different from each other (P<0.05).
Biofilm thickness cultured for 2 days on bonding agents (mean±s.d.; n=6). Seven bonding agents were tested: SBMP control, SBMP+DMAPM (CL3), SBMP+DMAHM (CL6), SBMP+DMANM (CL9), SBMP+DMADDM (CL12), SBMP+DMAHDM (CL16) and SBMP+DMAODM (CL18). Values indicated by dissimilar letters are significantly different from each other (P<0.05). CL, chain length; 3D, three-dimensional; DMADDM, dimethylaminododecyl methacrylate; DMAHDM, dimethylaminohexadecyl methacrylate; DMAHM, dimethylaminohexyl methacrylate; DMANM, dimethylaminononyl methacrylate; DMAODM, dimethylaminooctadecyl methacrylate; DMAPM, dimethylaminopropyl methacrylate; SBMP, Scotchbond multi-purpose bonding agent; s.d., standard deviation.
The biofilm volume results are plotted in Figure 4: live biofilm volume (Figure 4a) and dead biofilm volume (mean±s.d.; n=6) (Figure 4b). SBMP control had the greatest live biofilm volume. The live biofilm volume gradually decreased with increasing CL, reaching a minimum at CL16, and then increased at CL18. The dead biofilm volume first increased with increasing CL and then reached a plateau. CL16 had the least live biofilm volume which was two orders of magnitude lower than that of SBMP control.
Biofilm volume cultured for 2 days on bonding agents. (a) Live biofilm volume; (b) dead biofilm volume (mean±s.d.; n=6). Seven bonding agents were tested: SBMP control, SBMP+DMAPM (CL3), SBMP+DMAHM (CL6), SBMP+DMANM (CL9), SBMP+DMADDM (CL12), SBMP+DMAHDM (CL16) and SBMP+DMAODM (CL18). In each plot, values with dissimilar letters are significantly different from each other (P<0.05). CL, chain length; 3D, three-dimensional; DMADDM, dimethylaminododecyl methacrylate; DMAHDM, dimethylaminohexadecyl methacrylate; DMAHM, dimethylaminohexyl methacrylate; DMANM, dimethylaminononyl methacrylate; DMAODM, dimethylaminooctadecyl methacrylate; DMAPM, dimethylaminopropyl methacrylate; SBMP, Scotchbond multi-purpose bonding agent; s.d., standard deviation.
Typical live/dead staining images of 2D x–y sections are shown in Figure 5 for the top surface, the middle section and the bottom section (near the resin surface) of the biofilm. Three materials are shown in Figure 5 as examples: SBMP control (Figure 5a), SBMP+DMANM (CL9) (Figure 5b) and SBMP+DMAHDM (CL16) (Figure 5c). The top surface of SBMP control biofilm had the most live bacteria, while its bottom surface had a slight increase in dead bacteria amount. For SBMP+DMANM (CL9), the bottom of biofilm had noticeably more dead bacteria and the top section of the biofilm had more live bacteria. For SBMP+DMAHDM (CL16), there appeared to be predominantly compromised bacteria throughout the biofilm thickness, although there appeared to be a small amount of live bacteria in the top section of the biofilm.
Representative 2D live/dead staining images of cross-sectioned biofilm in the x–y plane. All seven bonding agents were tested; shown here are three examples: control, QAM with an intermediate CL of the most potent QAM. The top labels indicate the materials: SBMP control, SBMP+DMANM (CL9) and SBMP+DMAHDM (CL16). The left labels indicate the location of the section in the biofilm: the top surface, the middle section and the bottom section (near the resin surface) of the biofilm. Live bacteria were stained green and dead bacteria were stained red. CL, chain length; 2D, two-dimensional; DMAHDM, dimethylaminohexadecyl methacrylate; DMAHM, dimethylaminohexyl methacrylate; DMANM, dimethylaminononyl methacrylate; QAM, quaternary ammonium methacrylate; SBMP, Scotchbond multi-purpose bonding agent.
The biofilm vitality distribution in the different layers of the biofilm vs. biofilm height is shown in Figure 6 (mean±s.d.; n=6). The vertical axis shows the percentage of live bacteria measured from 2D sections such as those in Figure 5. The horizontal axis indicates the biofilm thickness at which the 2D image was taken. SBMP control group and the group with CL3 had similar results; hence, the CL3 group was not included in Figure 6 to save space. SBMP control had a percentage of live bacteria of 63% at the biofilm bottom; it gradually increased and approached 100% near the top of the biofilm. At CL of 6 and 9, the percentage of live bacteria at the biofilm bottom decreased to 60% and 37%, respectively. However, the percentage of live bacteria was still nearly 100% at the top of the biofilm. At CL of 12, not only did the percentage of live bacteria decrease to 19% at the biofilm bottom, but the top only had 63%. At CL16, the percentage of live bacteria was close to 0% throughout the biofilm thickness. When CL was further increased to 18, biofilm percentage of live bacteria increased. These results show that: (i) most of the tested antibacterial bonding agents could only inhibit bacteria close to the resin surface and the antibacterial efficacy decreased in biofilm away from resin surface; and (ii) the bonding agent containing DMAHDM with CL16 maintained a low viability of nearly 0% throughout the biofilm.
Viability distribution in biofilm (mean±s.d.; n=6). The percentage of live bacteria was measured from 2D images (Figure 5). Percentage of live bacteria=live bacteria area/(live bacteria area+dead bacteria area). Percentage of live bacteria is plotted vs. location of 2D image in biofilm at distance from resin surface. (a) SBMP control; (b) SBMP+DMAHM (CL6); (c) SBMP+DMANM (CL9); (d) SBMP+DMADDM (CL12); (e) SBMP+DMAHDM (CL16); (f) SBMP+DMAODM (CL18). The curve for SBMP+DMAPM (CL3) is similar to SBMP control and is not included. CL, chain length; 2D, two-dimensional; DMADDM, dimethylaminododecyl methacrylate; DMAHDM, dimethylaminohexadecyl methacrylate; DMAHM, dimethylaminohexyl methacrylate; DMANM, dimethylaminononyl methacrylate; DMAODM, dimethylaminooctadecyl methacrylate; DMAPM, dimethylaminopropyl methacrylate; SBMP, Scotchbond multi-purpose bonding agent; s.d., standard deviation.
The present study investigated the 3D biofilm live/dead volume and viability variation in biofilm thickness on dental bonding agents as a function of alkyl chain length for the first time. S. mutans biofilms were examined using a 3D digital reconstruction technique combined with quantitative image analysis. QAMs with CL varying from 3 to 18 were incorporated into bonding agent which exerted a significant anti-biofilm activity. Increasing the CL of bonding agent achieved a stronger effect in reducing the biofilm viability, evidenced by changes in biofilm structure with decreases in biofilm thickness, live biofilm volume and percentage of live bacteria. Oral bacteria in vivo colonize on the tooth–restoration surfaces to form biofilms and cariogenic bacteria such as S. mutans in the biofilm can metabolize carbohydrates to produce organic acids. This plays an important role in the development of tooth decay and secondary caries at the tooth–restoration margins. Furthermore, within the biofilm, S. mutans display properties that are dramatically distinct from their planktonic counterparts, including much higher resistance to antibacterial agents, which makes the biofilm much more difficult to kill than planktonic bacteria. Therefore, the antibacterial bonding agent containing the new QAMs, especially that using CL16 with effective killing of the biofilm, could be beneficial in caries-inhibiting dental applications.
This study showed a strong CL dependence of anti-biofilm properties of bonding agent. The mechanism of QAS to kill bacteria is believed to involve the alteration of membrane permeability or surface electrostatic balance of bacteria, thus causing cytoplasmic leakage.17,26,27,28,29 It has also been noticed that the alkyl chain length has a significant effect on biocidal activity since long cationic polymers may interact more effectively with the cytoplasmic membranes.28,29The present study revealed that the 3D structural changes in biofilms were associated with the CL of QAM in bonding agent. With CL increasing from 3 to 16, the biofilm thickness, live bacteria volume and the percentage of live bacteria all decreased. However, after reaching the maximum antimicrobial ability with CL16, no further strengthening of bacteria-inhibition effect was detected with increasing the CL to 18. A similar phenomenon was observed in previous studies which were not on dental bonding agents.18,43,44 This was explained as a cutoff effect.45 Among the various assumptions proposed to explain the origin of the cutoff effect, the concept of free volume could be applied to QAS.44 Free volume is the unoccupied space between molecules. In solution or culture medium, the polar ammonium heads will interact with those of phospholipids of the bacteria and their hydrocarbon chains will orient parallel to the hydrocarbon chains of phospholipids.44 The hydrocarbon chains are parallel to those of phopholipids of the cell. In this case, the density of the bilayer hydrophobic region is necessarily altered and a free volume is formed. When the hydrocarbon chain of the QAS is shorter than that of phospholipids, the total free volume generated in the bilayer is small. When the hydrocarbon chain length of QAS becomes comparable to that of phospholipids, the free volume drops off and tends toward zero. Molecules containing chains between these two extremes lead to the most essential free volume inside the bilayer.44 The larger the free volume, the more the membrane of bacteria is expected to be disrupted and the bactericidal activity is enhanced. Hence, the present study indicates that CL16 with the maximum antibacterial activity may possess the largest free volume in the bilayer.
Furthermore, CLSM examination of the present study showed that for SBMP control, the bottom layer of the biofilm adjacent to resin contained a higher proportion of nonviable bacteria than the upper layer of biofilm in contact with culture medium. This was likely because the deeper layer of the biofilm had less access to oxygen, a lower availability of primary nutrients and more secondary metabolites accumulation than the outer layer of the biofilm. For bonding agents containing QAMs, the compromised bacteria were more concentrated in the lower layer of the biofilm, and the viability percentage increased with the biofilm height away from the resin surface. This was likely related to the contact-killing mechanism of QAM resin in which the QAM was copolymerized with and immobilized in the resin. Hence, due to contact-killing effect, the bactericidal efficacy is decreased away from the surface due to a lack of contact. For example, for CL 9 in Figure 6, the percentage of live bacteria was only 37% near the antibacterial resin surface, but close to 100% away from the resin surface near the biofilm top.
For the bonding agent containing DMAHDM with CL16, the biofilm consisted mainly of dead microorganisms throughout the biofilm thickness. This may suggest another possible antibacterial mechanism. Previous studies suggested that a stress condition or challenge in bacteria could trigger a built-in suicide program in the biofilm,46,47 which was also called programmed cell death.46Being challenged by bactericidal agents may serve as a trigger for programmed cell death in the surrounding bacteria.46,47 Indeed, the present study showed that the bonding agent at CL16 killed the entire biofilm. This is consistent with a previous in vivo study showing that the biofilm on a QAM composite intraorally in human participants was dead not only on the resin surface, but also in the outer, more remote parts of the biofilm.48 This was explained as due to an intracellularly mediated death program, in which the bacterial lysis by QAM on the resin surface may function as a stressful condition triggering programmed cell death to the bacteria further away in the biofilm.48 Further study is needed to investigate if there is significant leachout of QAM from the resin which might contribute to killing bacteria at a distance away from the resin surface. In the present study, the resin disks were agitated in 200 mL of water via magnetic stirring for 1 h to remove possible uncured monomers, following a previous study.15 Further study should water-age the disks for long periods of time such as 6 months,49 and then inoculate bacteria to determine if the antibacterial activity is durable and if the bonding agent with CL16 can still kill the entire 3D biofilm. Nonetheless, the results of the present study such as Figure 6 clearly show the effect of CL in bonding agent on 3D biofilm properties, as all the disks were prepared and treated in the same manner.
Regarding potential applications of the bonding agent containing DMAHDM with CL16, recurrent caries at the tooth–restoration margins is a primary reason for restoration failure. Hence, the antibacterial bonding agent in the uncured state could flow into dentinal tubules and kill residual bacteria in the tooth cavity.14,15,24,50 The antibacterial bonding agent in the cured state could inhibit bacteria invasion along the margins, which could be especially useful to inhibit bacteria when marginal microgaps occur during service.14,15,24,50 Furthermore, there has been an increased interest in the less removal of tooth structure and minimal intervention dentistry.51 While the treatment could preserve tooth structure, it could also leave behind more carious tissues with active bacteria. Atraumatic restorative treatment does not remove the carious tissues completely, leaving remnants of lesions and bacteria.52 Therefore, these applications could potentially benefit from the bonding agent containing DMAHDM. In addition, DMAHDM could also be promising for incorporation into composites for antibacterial restorations.53,54 The materials need to be biocompatible for clinical applications. A recent study showed that the fibroblast viability and odontoblast viability of DMAHDM were better than bisphenol-glycerolate dimethacrylate.30 The eluents from the cured resin containing DMAHDM caused fibroblast viability and odontoblast viability that were not significantly different from the resin control without DMAHDM.30 Further studies are needed to investigate the potential dental applications DMAHDM-containing resins and composites.
Conclusions
This study investigated the effect of CL on 3D biofilm structure and live/dead viability variation vs. biofilm thickness on dental bonding agent for the first time. The results showed that: (i) strong antibacterial function was achieved by adding QAM into bonding agent; (ii) biofilm thickness and live volume decreased with increasing CL from 3 to 16, but then increased at CL of 18; (iii) except for CL16, antibacterial bonding agents with all tested CL values could only inhibit bacteria close to resin surface and the antibacterial efficacy decreased in the biofilm away from resin surface; (iv) the bonding agent containing DMAHDM with CL 16 yielded a percentage of live bacteria of close to 0% throughout the biofilm thickness. The 3D biofilm analysis via CLSM and digital reconstruction method is useful for understanding biofilm–resin interactions and antibacterial resin effects on biofilm structure and 3D viability distribution. Based on the specific resin formulations tested in this study, antibacterial bonding agents with CL16 are useful for a wide range of dental applications to combat bacteria and biofilms at tooth–restoration margins to inhibit secondary caries.
New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue
Reporter: Stephen J. Williams, Ph.D.
The FDA recently came out with a Draft Guidance on use of human cells, tissues and cellular and tissue-based products (HCT/P) {defined in 21 CFR 1271.3(d)} and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.
In 21 CFR 1271.10, the regulations identify the criteria for regulation solely under section 361 of the PHS Act and 21 CFR Part 1271. An HCT/P is regulated solely under section 361 of the PHS Act and 21 CFR Part 1271 if it meets all of the following criteria (21 CFR 1271.10(a)):
The HCT/P is minimally manipulated;
The HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent;
The manufacture of the HCT/P does not involve the combination of the cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage agent, provided that the addition of water, crystalloids, or the sterilizing, preserving, or storage agent does not raise new clinical safety concerns with respect to the HCT/P; and
Either:
The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function; or
The HCT/P has a systemic effect or is dependent upon the metabolic activity of living cells for its primary function, and:
Is for autologous use;
Is for allogeneic use in a first-degree or second-degree blood relative; or
Is for reproductive use.
If an HCT/P does not meet all of the criteria in 21 CFR 1271.10(a), and the establishment that manufactures the HCT/P does not qualify for any of the exceptions in 21 CFR 1271.15, the HCT/P will be regulated as a drug, device, and/or biological product under the Federal Food, Drug and Cosmetic Act (FD&C Act), and/or section 351 of the PHS Act, and applicable regulations, including 21 CFR Part 1271, and pre-market review will be required.
1 Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under 21 CFR Parts 607 and 207, respectively; (3) Secreted or extracted human products, such as milk, collagen, and cell factors, except that semen is considered an HCT/P; (4) Minimally manipulated bone marrow for homologous use and not combined with another article (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the bone marrow); (5) Ancillary products used in the manufacture of HCT/P; (6) Cells, tissues, and organs derived from animals other than humans; (7) In vitro diagnostic products as defined in 21 CFR 809.3(a); and (8) Blood vessels recovered with an organ, as defined in 42 CFR 121.2 that are intended for use in organ transplantation and labeled “For use in organ transplantation only.” (21 CFR 1271.3(d))
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Section 1271.10(a)(2) (21 CFR 1271.10(a)(2)) provides that one of the criteria for an HCT/P to be regulated solely under section 361 of the PHS Act is that the “HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.” As defined in 21 CFR 1271.3(c), homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor. This criterion reflects the Agency’s conclusion that there would be increased safety and effectiveness concerns for HCT/Ps that are intended for a non-homologous use, because there is less basis on which to predict the product’s behavior, whereas HCT/Ps for homologous use can reasonably be expected to function appropriately (assuming all of the other criteria are also met).2 In applying the homologous use criterion, FDA will determine what the intended use of the HCT/P is, as reflected by the the labeling, advertising, and other indications of a manufacturer’s objective intent, and will then apply the homologous use definition.
FDA has received many inquiries from manufacturers about whether their HCT/Ps meet the homologous use criterion in 21 CFR 1271.10(a)(2). Additionally, transplant and healthcare providers often need to know this information about the HCT/Ps that they are considering for use in their patients. This guidance provides examples of different types of HCT/Ps and how the regulation in 21 CFR 1271.10(a)(2) applies to them, and provides general principles that can be applied to HCT/Ps that may be developed in the future. In some of the examples, the HCT/Ps may fail to meet more than one of the four criteria in 21 CFR 1271.10(a).
III. QUESTIONS AND ANSWERS
What is the definition of homologous use?
Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use. We generally consider an HCT/P to be for homologous use when it is used to repair, reconstruct, replace, or supplement:
Recipient cells or tissues that are identical (e.g., skin for skin) to the donor cells or tissues, and perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor; or,
Recipient cells that may not be identical to the donor’s cells, or recipient tissues that may not be identical to the donor’s tissues, but that perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor.3
3“Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue-Based Products” 63 FR 26744 at 26749 (May 14, 1998).
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1-1. A heart valve is transplanted to replace a dysfunctional heart valve. This is homologous use because the donor heart valve performs the same basic function in the donor as in the recipient of ensuring unidirectional blood flow within the heart.
1-2. Pericardium is intended to be used as a wound covering for dura mater defects. This is homologous use because the pericardium is intended to repair or reconstruct the dura mater and serve as a covering in the recipient, which is one of the basic functions it performs in the donor.
Generally, if an HCT/P is intended for use as an unproven treatment for a myriad of
diseases or conditions, the HCT/P is likely not intended for homologous use only.4
What does FDA mean by repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues?
Repair generally means the physical or mechanical restoration of tissues, including by covering or protecting. For example, FDA generally would consider skin removed from a donor and then transplanted to a recipient in order to cover a burn wound to be a homologous use. Reconstruction generally means surgical reassembling or re-forming. For example, reconstruction generally would include the reestablishment of the physical integrity of a damaged aorta.5 Replacement generally means substitution of a missing tissue or cell, for example, the replacement of a damaged or diseased cornea with a healthy cornea or the replacement of donor hematopoietic stem/progenitor cells in a recipient with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. Supplementation generally means to add to, or complete. For example, FDA generally would consider homologous uses to be the implantation of dermal matrix into the facial wrinkles to supplement a recipient’s tissues and the use of bone chips to supplement bony defects. Repair, reconstruction, replacement, and supplementation are not mutually exclusive functions and an HCT/P could perform more than one of these functions for a given intended use.
What does FDA mean by “the same basic function or functions” in the definition of homologous use?
For the purpose of applying the regulatory framework, the same basic function or functions of HCT/Ps are considered to be those basic functions the HCT/P performs in the body of the donor, which, when transplanted, implanted, infused, or transferred, the HCT/P would be expected to perform in the recipient. It is not necessary for the HCT/P in the recipient to perform all of the basic functions it performed in the donor, in order to
4 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).
5 “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement” 69 FR 68612 at 68643 (November 24, 2004) states, “HCT/Ps with claims for “reconstruction or repair” can be regulated solely under section 361 of the PHS Act, provided the HCT/P meets all the criteria in § 1271.10, including minimal manipulation and homologous use.”
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meet the definition of homologous use. However, to meet the definition of homologous use, any of the basic functions that the HCT/P is expected to perform in the recipient must be a basic function that the HCT/P performed in the donor.
A homologous use for a structural tissue would generally be to perform a structural function in the recipient, for example, to physically support or serve as a barrier or conduit, or connect, cover, or cushion.
A homologous use for a cellular or nonstructural tissue would generally be a metabolic or biochemical function in the recipient, such as, hematopoietic, immune, and endocrine functions.
3-1. The basic functions of hematopoietic stem/progenitor cells (HPCs) include to form and to replenish the hematopoietic system. Sources of HPCs include cord blood, peripheral blood, and bone marrow.6
HPCs derived from peripheral blood are intended for transplantation into an individual with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. This is homologous use because the peripheral blood product performs the same basic function of reconstituting the hematopoietic system in the recipient.
HPCs derived from bone marrow are infused into an artery with a balloon catheter for the purpose of limiting ventricular remodeling following acute myocardial infarction. This is not homologous use because limiting ventricular remodeling is not a basic function of bone marrow.
A manufacturer provides HPCs derived from cord blood with a package insert stating that cord blood may be infused intravenously to differentiate into neuronal cells for treatment of cerebral palsy. This is not homologous use because there is insufficient evidence to support that such differentiation is a basic function of these cells in the donor.
3-2. The basic functions of the cornea include protecting the eye by forming its outermost layer and serving as the refracting medium of the eye. A corneal graft is transplanted to restore sight in a patient with corneal blindness. This is homologous use because a corneal graft performs the same basic functions in the donor as in the recipient.
3-3. The basic functions of a vein or artery include serving as a conduit for blood flow throughout the body. A cryopreserved vein or artery is used for arteriovenous access during hemodialysis. This is homologous use because the vein or artery is supplementing the vessel as a conduit for blood flow.
3-4. The basic functions of amniotic membrane include covering, protecting, serving as a selective barrier for the movement of nutrients between the external and in utero
6 Bone marrow meets the definition of an HCT/P only if is it more than minimally manipulated; intended by the manufacturer for a non-homologous use, or combined with certain drugs or devices.
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environment, and to retain fluid in utero. Amniotic membrane is used for bone tissue replacement to support bone regeneration following surgery to repair or replace bone defects. This is not a homologous use because bone regeneration is not a basic function of amniotic membrane.
3-5. The basic functions of pericardium include covering, protecting against infection, fixing the heart to the mediastinum, and providing lubrication to allow normal heart movement within chest. Autologous pericardium is used to replace a dysfunctional heart valve in the same patient. This is not homologous use because facilitating unidirectional blood flow is not a basic function of pericardium.
Does my HCT/P have to be used in the same anatomic location to perform the same basic function or functions?
An HCT/P may perform the same basic function or functions even when it is not used in the same anatomic location where it existed in the donor.7 A transplanted HCT/P could replace missing tissue, or repair, reconstruct, or supplement tissue that is missing or damaged, either when placed in the same or different anatomic location, as long as it performs the same basic function(s) in the recipient as in the donor.
4-1. The basic functions of skin include covering, protecting the body from external force, and serving as a water-resistant barrier to pathogens or other damaging agents in the external environment. The dermis is the elastic connective tissue layer of the skin that provides a supportive layer of the integument and protects the body from mechanical stress.
An acellular dermal product is used for supplemental support, protection, reinforcement, or covering for a tendon. This is homologous use because in both anatomic locations, the dermis provides support and protects the soft tissue structure from mechanical stress.
An acellular dermal product is used for tendon replacement or repair. This is not homologous use because serving as a connection between muscle and bone is not a basic function of dermis.
4-2. The basic functions of amniotic membrane include serving as a selective barrier for the movement of nutrients between the external and in utero environment and to retain fluid in utero. An amniotic membrane product is used for wound healing of dermal ulcers and defects. This is not homologous use because wound healing of dermal lesions is not a basic function of amniotic membrane.
4-3. The basic functions of pancreatic islets include regulating glucose homeostasis within the body. Pancreatic islets are transplanted into the liver through the portal vein,
7 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).
6
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for preservation of endocrine function after pancreatectomy. This is homologous use because the regulation of glucose homeostasis is a basic function of pancreatic islets.
What does FDA mean by “intended for homologous use” in 21 CFR 1271.10(a)(2)?
The regulatory criterion in 21 CFR 1271.10(a)(2) states that the HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.
Labeling includes the HCT/P label and any written, printed, or graphic materials that supplement, explain, or are textually related to the product, and which are disseminated by or on behalf of its manufacturer.8 Advertising includes information, other than labeling, that originates from the same source as the product and that is intended to supplement, explain, or be textually related to the product (e.g., print advertising, broadcast advertising, electronic advertising (including the Internet), statements of company representatives).9
An HCT/P is intended for homologous use when its labeling, advertising, or other indications of the manufacturer’s objective intent refer to only homologous uses for the HCT/P. When an HCT/P’s labeling, advertising, or other indications of the manufacturer’s objective intent refer to non-homologous uses, the HCT/P would not meet the homologous use criterion in 21 CFR 1271.10(a)(2).
What does FDA mean by “manufacturer’s objective intent” in 21 CFR 1271.10(a)(2)?
A manufacturer’s objective intent is determined by the expressions of the manufacturer or its representatives, or may be shown by the circumstances surrounding the distribution of the article. A manufacturer’s objective intent may, for example, be shown by labeling claims, advertising matter, or oral or written statements by the manufacturer or its representatives. It may be shown by the circumstances that the HCT/P is, with the knowledge of the manufacturer or its representatives, offered for a purpose for which it is neither labeled nor advertised.
There are many companies dedicated to improving the use of 3D printing in the medical sector, and one of them Osteo3D, just released an amazing online repository of medical 3D models. Of course, patient information is private, so some of the “live models” have been made anonymous. But there are now over 100 3D models available to doctors and other registered medical professionals online, which allows them t0 compare their own patient’s 3D files with Osteo3D models. In fact, they can 3D print these models at home or wherever they have access to a 3D printer.
But, if a doctor or medical student doesn’t have a 3D printer, they can order a print directly from Osteo3D. The online platform is powered by cloud printing site df3d and is meant to increase awareness of the usefulness of 3D printing among healthcare professionals. Osteo3D hopes that, by collaborating with healthcare professionals, the tech industry, and academic institutions, they will help facilitate access and lower costs for the use of 3D printing in the healthcare field.
Currently the models are divided into the following categories:
Head and neck
Spine
Chest
Abdomen and pelvis
Extremities
When you click on 3D view, you don’t get quite the same level of control and depth asSketchfab, and some of the models look like pixels are growing out of them, but I didn’t pay to download any of the models, so I can’t say for sure. They are currently on the lookout for 3D printing partners who can help them deliver these 3D prints from their cloud platform to their customers around the world.
We’ve covered a number of stories that revolve around the combination of scanning/sensing technologies, great 3D modeling software, and 3D printing. This combination has arguably had the most impact on the quality of human life as it is seen in the medical sector. Diagnoses are made to a patient with a life-threatening condition. Doctors, then, use 3D scanning technology to capture the reality data of a patient’s affected area, whether it be an organ, vasculature, bone structure, or combination of all three. Next, a 3D model is created and manipulated using innovative software, and the model is 3D printed. Surgery is practiced and visualized on the model, and then performed on the patient. After doctors have all of this information, they use it to teach other doctors and nurse practitioners. So, why not spread this wealth of information? This is digital information, after all. Any 3D model that is successfully used to print a 3D model of a diagnosed patient can be transferred anywhere, to anyone.
If you have a 3D printer then you are likely overwhelmed by the sheer number of possible objects you can find online to print out. At the same time, the technology is somewhat limited, unless you have professional CAD skills or are incredibly creative. What, for instance, would you do if you wanted to take an everyday item such as a hot glue gun and print an attached stand for it, or turn your child’s favorite action figure into a magnet?
Four researchers at Carnegie Mellon University, Xiang ‘Anthony’ Chen, Stelian Coros, Jennifer Mankoff and Scott E. Hudson, believe that they can change this via a new WebGL-based tool under development called Encore.
Funded by the National Science Foundation under grant NSF IIS 1217929, Encore is a multifaceted tool which enables three different techniques to augment already existing objects. The researchers call these three techniques Print-Over, Print-to-Affix and Print-Through, all of which allow for the adherence of newly 3D printed attachments to other objects. Before we get into what each of these three techniques involves, one first should understand the computational pipeline involved in designing these attachments.
First a user is required to design a basic attachment, or perhaps use a design that they’d like to iterate upon. Once they have a basic model of their desired attachment the Encore system will geometrically analyze the model along with a 3D scan of a target object, that the attachment will adhere to, in order to determine its printability while also deciding if it will be durable enough and usable once attached to the target object. Next comes the interactive exploration phase, where the tool will visualize and explore various areas where the attachment can be affixed to the target object. The tool will also adjust the design of the attachment, if required, to better fit the target object. Once this is all settled, it’s now time for Encore to generate a model which not only will include the attachment itself, but also any connecting structures and even supports to hold the target item or attachment in place. Below you will find the three techniques that the Encore tool can use to attach 3D printed items to a target object:
Print-Over
This technique, in my opinion, is one of the coolest, as it allows for the printing of an attachment directly on a given object. Once Encore establishes the parameters required and sizes up the model to fit the target object, it will automatically tell the printer to print supports to hold the target item in place. Once the target item is in place, Encore will then tell the printer to begin printing the attachment in a particular spot on the target item, based on its geometric analysis of that object.
“It is also important to ensure that the existing object will not impede the motion of the print head while the attachment is being printed,” warn the researchers.
An example used by the researchers for this technique was a magnet holder which they directly attached to a teddy bear figurine. They also printed an LED light onto a 9V battery by first placing a small amount of glue on the battery prior to beginning to print on top.
Print-to-Affix
This approach is similar to the Print-Over technique, only that instead of printing an attachment directly onto the target object, Encore will analyze the geometry of the target object prior to printing in order to create an attachment which will fit perfectly on that object via glue, straps (zip-ties) or even snaps. Once the attachment is printed the user can then use hot-glue or another adhesive or attachment mechanism to affix the printed object onto the target.
Print-Through
This technique is perfect for items which you don’t want to physically attach, but instead can connect to an object. For instance a tag on the loop of a pair of scissors or a charm onto a bracelet. This process requires that the printer be paused while a user manually places the target object within the print field.
“Print-through has aesthetic qualities that distinguish it from print-to-affix and print-over – it typically creates a loose but permanent connection between two objects,” explained the researchers.
While the new Encore tool is still under development as researchers improve upon its analytical capabilities, it certainly seems to show promise to those of us wishing to do more than just fabricate new items. In fact, the researchers were able to show that via all three techniques they could save a substantial amount of time and material over printing an object in one single piece. As an example, they used Slic3r to estimate the print time and total material required for printing a typical Utah teapot with a torus-shaped handle, as well as just printing the handle onto an already fabricated Utah teapot. Their estimate showed that the time of fabricating the item could be cut by more than 80% and material use reduced by as much as 85% by using their techniques and the Encore tool.
There are many variables going into the tool’s decision making algorithms, such as determining where to place attachments for the best balance when holding an object, what placement of an attachment will result in the best adhesion, etc. More research is still required as the team continues to develop the tool, as well as new techniques to attach multiple parts to one object, but it certainly seems like something which could have a sizable impact on the industry in general. Let us know your thoughts on the Encore tool in the Augmenting 3D Prints forum thread on 3DPB.com.
Contrary to what some say, 3D printing is not going to revolutionize the manufacturing sector, rendering traditional factories obsolete. The simple fact of the matter is the economics of 3D printing now and for the foreseeable future make it an unfeasible way to produce the vast majority of parts manufactured today. So instead of looking at it as a substitute for existing manufacturing, we should look to new areas where it can exploit its unique capabilities to complement traditional manufacturing processes.
Additive manufacturing, or “3D printing” as it is commonly known, has understandably captured the popular imagination: New materials that can be “printed” are announced virtually every day, and the most recent generation of printers can even print several materials at the same time, opening up new opportunities. Exciting applications have already been demonstrated across all sectors — from aerospace and medical applications to biotechnology and food production.
Some predict that a day is coming when we’ll be able to make any part at the push of a button at a local printer, which might even render the global supply lines that dominate today’s world of manufacturing a thing of the past. Unfortunately, this vision does not stack up to economic reality. Early findings from a research project being conducted by the Additive Manufacturing and 3D Printing Research Group at the University of Nottingham and Saïd Business School at the University of Oxford show that there are both significant scale and learning effects inherent in the 3D printing process. (The project, in which I am a principal investigator, is focusing on industrial selective laser sintering (or more accurately, melting) processes and not fused deposition modelling or stereolithography processes that are more suited to rapid prototyping and home applications.)
Furthermore, the pre- and post-printing cost amount to a significant proportion of total cost per printed part. So even when the cost for printers materials come down, the labor-cost penalty will remain.
3D printing simply works best in areas where customization is key — from printing hearing aids and dental implants to printing a miniature of the happy couple for their wedding cake. Using a combination of 3D scanning and printing, implants can be customized to specific anatomic circumstances in a way that was simply not feasible beforehand. However, we also know that 99% of all manufactured parts are standard and do not require customization. In these cases, 3D printing has to compete with scale-driven manufacturing processes and rather efficient logistics operations. A good example is the wrench that NASA printed on the International Space Station last year. The cost of shipping it to the space station would have been at least $400 (assuming the unpackaged weight of 18 grams per wrench and using the most recent cost data given by NASA for transporting goods into lower-earth orbit); in comparison, shipping it from China to the United States would only cost $0.002 per unit. Thus, while it makes a lot of sense to print the wrench on the space station, printing it for local consumption in the United States wouldn’t.
The simple fact is that when customization isn’t important, 3D printing is not competitive. For one, printing costs per part are highly sensitive to the utilization of the “build room,” the three-dimensional area inside the 3D printer where the laser fuses the metal or plastic powder. Therefore, contract manufacturers that perform 3D printing such as Shapeways generally wait to fill a batch that uses the entire build room. Printing just one part raises unit cost considerably; so economies of scale do matter. Interestingly, the economic case for the most-cited standard part in 3D volume production today, the GE fuel nozzle for the CFM LEAP engine, is it is lighter and more fuel efficient, not a lower manufacturing cost per se.
A second point often overlooked is that the labor cost that remains. Counter to common perception, 3D printing does not happen “at the touch of a button”; it involves considerable pre- and post-processing, which incur non-trivial labor costs. The starting point for any 3D printing process is a 3D file that can be “printed.” Just having an electronic CAD drawing is not sufficient; currently, there is no way to automatically convert the CAD drawing into a 3D file.
Creating printable files involves two steps: creating a three-dimensional volume model that can be printed, and “slicing” that volume model in the best possible way to avoid material wastage and prevent printing errors. Both steps require tacit knowledge. Following the printing, the parts produced have to be recovered, cleaned, washed (or sanded and polished, in the case of metal prints), and inspected. This, in turn, means that using 3D printing for the aftermarket services — an application where it makes a lot of sense — requires making a significant upfront investment in generating the printable files of the spare parts that would likely be needed. This investment would have to outweigh the cost of keeping a lifetime supply of spare parts in inventory, which is a tough call for small bolts, brackets, and connectors that make up the bulk of aftermarket demand.
So while I, like many others, have fallen in love with the notion of the “ultimate lean supply chain” of having 3D printers at every other corner table to print single parts just in time where they are needed, I am afraid that this vision does not stack up against reality. 3D printing technology undoubtedly has great potential. However, it is unlikely to replace traditional manufacturing. Instead, we should see it as a complement, a new tool in the box, and exploit its unique capabilities — both in making existing products better as well as being able to manufacture entirely new ones that we previously could not make.
Medical implants and printable body parts to drive 3D printer growth
With 3D bio-printing in the pipeline, dental and medical applications could be worth $6bn by 2025
False teeth, hip joints and replacement knees – and potentially printable skin and organs – will drive growth in the burgeoning market for 3D printers over the next decade, according to new research.
The technology is better known to British households for its ability to replace broken crockery or produce awkward figurine “selfies.”
But a report by Cambridge-based market research firm IDTechEx says ceramic jaw or teeth implants and metal hip replacements will become increasingly common 3D fare.
The parts are created by nozzles laying down fine sedimentary layers of material that build a product indistinguishable from an item that has rolled off a factory conveyor belt.
The dental and medical market for 3D printers is expected to expand by 365% to $867m (£523m) by 2025, according to IDTechEx analysts, even before bio-printing technology is taken into account. If bio-printing becomes suitable for commercial use – which scientists hope will allow the printing of pieces of skin, liver or kidney using live cells – analysts estimate the medical market could reach a value of $6bn or more within 10 years.
While printing of complete organs for transplants may be decades away, the use of pieces of tissue for laboratory toxicology tests for cosmetics or drugs could be ready within five years, helping the medical market for 3D printers overtake all other sectors.
Dr Jon Harrop, a director of IDTechEx, said: “Bio printing is a bit unsure as it doesn’t exist commercially at the moment but all the medical professionals we interviewed thought it was highly likely to be commercial within 10 years.”
In the US, dental labs have invested in technology that can scan a patient’s teeth so new teeth can be produced by pressing the print button.
Harrop said there are a number of stumbling blocks in the way of the commercial application of bio printing, but even in the past year, scientists have been able to extend the life of a piece of skin tissue created in the lab from just a few hours to 40 days, taking it closer to the three months required for toxicology tests.
At present, 3D printers are most widely used in the automotive industry where they help produce prototypes for new cars or car parts. The next biggest market is aerospace, where manufacturers are using the technology to make lighter versions of complex parts for aeroplanes.
Already, 3D printers have been used by the medical industry to create a jaw, a pelvis and several customised hip replacements from metal. This year, surgeons in Newcastle upon Tyne created a titanium pelvis for a man who lost half his original one to a rare bone cancer, while in May doctors in Southampton completed Britain’s first hip replacement made using a 3D printer. Professor Richard Oreffo at the University of Southampton, who helped develop the hip replacement technique, said at the time: “The 3D printing of the implant in titanium, from CT scans of the patient and stem cell graft, is cutting edge and offers the possibility of improved outcomes for patients.”
Dentists have been using 3D printers to create exact replicas of jaws or teeth in order to aid complex procedures for a few years, but increasingly they are creating implants made of durable plastic or medical ceramics.
In his most recent State of the Union address, President Barack Obama stated, “Last year, we created our first manufacturing innovation institute in Youngstown, Ohio. A once-shuttered warehouse is now a state-of-the art lab where new workers are mastering the 3D printing that has the potential to revolutionize the way we make almost everything. There’s no reason this can’t happen in other towns. So tonight, I’m announcing the launch of three more of these manufacturing hubs, where businesses will partner with the Department of Defense and Energy to turn regions left behind by globalization into global centers of high-tech jobs. And I ask this Congress to help create a network of 15 of these hubs and guarantee that the next revolution in manufacturing is made right here in America.” [“Remarks by the President in the State of the Union Address,” White House, 12 February 2013]
Official White House Photo by Chuck Kennedy (not shown)
Clearly, the President believes that 3D printing marks a new era in manufacturing that could change the entire business landscape. Kinaxis analyst Andrew Bell agrees that “change is inevitable.” “The question that we need to answer,” he writes, “is how will it change?” [“What Could 3D Printing Mean for the Supply Chain? The 21st Century Supply Chain, 9 January 2013] Bell offers a few thoughts on the subject; he writes:
“One thing is for sure, the supply chain isn’t going away. As usual, it will likely just get more complicated. Here are some of the areas that I propose will influence the supply chain as 3D printing becomes more and more mainstream, and I’m sure there are many more.
Local Manufacturing – More things will be made closer to their final destination. This will have definite impact on the logistics industry, and will change the way business try and schedule their operations.
Customizability – It will be easier, faster, and more efficient for companies to provide made-to-order products to their end users.
Distribution of raw materials – There will need to be a dramatic shift in the way raw materials are distributed since these printers will require raw materials in order to produce the final product.
New replacement parts model – Business will be able to provide replacement parts as required instead of trying to predict the need and manufacture the stock well in advance (as they do today)
Blurred boundaries within businesses – A closer integration of the various departments of an organization will be mandatory. A siloed manufacturing department will no longer allow for a competitive business.
“My predictions may be right, or they may be wrong, but one thing I think all will agree on is that 3D printing will make the supply chain more complex and more difficult to manage.”
Jim Stockton notes that 3D printing (or additive manufacturing) has been around since the 1980s. It has only broken into the mainstream because of recent breakthroughs that now have everyone talking. [“Top Innovations in the World of 3D Printing,” BestDesignTuts, 12 February 2013] He writes:
“Some products produced with this technology that might already be a part of your daily life include shoes, jewellery, clothing accessories, and educational products. The field of engineering is also benefitting from the development of 3D printing, in terms of boosting geographic information systems, aerospace engineering, engineering projects, and construction processes. Furthermore, humans are already benefitting in the field of healthcare with dental and medical instruments and products being formulated with 3D printing. The automotive industry is able to make more reliable products at a quicker speed, and the industrial design and architecture fields are able to develop new products that were inconceivable only a decade ago.”
What is really exciting people, however, is not so much what is currently being manufactured but the potential of what could be manufactured using 3D printers. Stockton writes:
“Engineers and scientists around the world are looking forward to the near future, in which printers will be able to create equipment, tools and devices via open-source models. This kind of advancement will drastically change the ways in which research and practical medicine are performed. Chemists are even attempting to build chemical compounds using 3D printers, and scientists have already started to replicate fossils and other ancient materials to better understand their compositions and functions. Architects wonder if in the future, buildings themselves can be printed from the ground up. The future of 3D printing is very bright – both in small-scale products for individual users and for large-scale projects, such as printing meters of building materials in the course of an hour and making intricate parts of automobiles and planes.”
Vivek Srinivasan and Jarrod Bassan offer ten trends that will likely define the direction that will be taken by additive manufacturing in the years ahead. [“Manufacturing The Future: 10 Trends To Come In 3D Printing,” Forbes, 7 December 2012] They are:
3D printing becomes industrial strength. Once reserved for prototypes and toys, 3D printing will become industrial strength. You will take a flight on an airliner that includes 3D-printed components, making it lighter and more fuel efficient. In fact, there are aircrafts that already contain some 3D-printed components. The technology will also start to be adopted for the direct manufacture of specialist components in industries like defense and automotive. Overall, the number of 3D printed parts in planes, cars and even appliances will increase without you knowing.
3D printing starts saving lives. 3D-printed medical implants will improve the quality of life of someone close to you. Because 3D printing allows products to be custom-matched to an exact body shape, it is being used today for making better titanium bone implants, prosthetic limbs and orthodontic devices. Experiments in printing soft tissue are underway, and may soon allow printed veins and arteries to be used in operations. Today’s research into medical applications of 3D printing covers nano-medicine, pharmaceuticals and even printing of organs. Taken to the extreme, 3D printing could one day enable custom medicines and reduce if not eliminate the organ donor shortage.
Customization becomes the norm. You will buy a product, customized to your exact specifications, which is 3D-printed and delivered to your doorstep. Innovative companies will use 3D printing technologies to give themselves a competitive advantage by offering customization at the same price as their competitor’s standard products. At first this may range from novelty items like custom smartphone cases or ergonomic improvements to standard tools, but it will rapidly expand to new markets. The leaders will adjust their sales, distribution and marketing channels to take advantage of their capability to provide customization direct to the customer. Customization will also play a big role in healthcare devices such as 3D-printed hearing aids and artificial limbs.
Product innovation is faster. Everything from new car models to better home appliances will be designed more rapidly, bringing innovation to you faster. Because rapid prototyping using 3D printers reduces the time to turn a concept into a production-ready design, it allows designers to focus on the function of products. Although the use of 3D printing for rapid prototyping is not new, the rapidly decreasing cost, improved design software and increasing range of printable materials means designers will have more access to printers, allowing them to innovate faster by 3D printing an object early in the design phase, modifying it, re-printing it, and so on. The result will be better products, designed faster.
New companies develop innovative business models built on 3D printing. You will invest in a 3D printing company’s IPO. Start-up companies will flourish as a generation of innovators, hackers and “makers” take advantage of the capabilities of 3D printing to create new products or deliver services to the burgeoning 3D printer market. Some enterprises will fail, and there may be a boom-bust cycle, but 3D printing will spawn new and creative business models.
3D print shops open at the mall. 3D print shops will begin to appear, at first servicing local markets with high-quality 3D printing services. Initially designed to service rapid-prototyping and other niche capabilities, these shops will branch into the consumer marketplace. As retailers begin to “ship the design, not the product,” the local 3D print shop will one day be where you pick up your customized, locally manufactured products, just like you pick up your printed photos from the local Walmart today.
Heated debates on who owns the rights emerge. As manufacturers and designers start to grapple with the prospect of their copyrighted designs being replicated easily on 3D printers, there will be high-profile test cases over the intellectual property of physical object designs. Just like file-sharing sites shook the music industry because they made it easy to copy and share music, the ability to easily copy, share, modify and print 3D objects will ignite a new wave of intellectual property issues.
New products with magical properties will tantalize us. New products – that can only be created on 3D printers – will combine new materials, nano scale and printed electronics to exhibit features that seem magical compared to today’s manufactured products. These printed products will be desirable and have distinct competitive advantage. The secret sauce is that 3D printing can control material as it is printed, right down to the molecules and atoms. As today’s research is perfected into tomorrow’s commercially available printers, expect exciting and desirable new products with amazing capabilities. The question is: What are these products and who will be selling them?
New machines grace the factory floor. Expect to see 3D printing machines appearing in factories. Already some niche components are produced more economically on 3D printers, but this is only on a small scale. Many manufacturers will begin experimenting with 3D printing for applications outside of prototyping. As the capabilities of 3D printers develop and manufacturers gain experience in integrating them into production lines and supply chains, expect hybrid manufacturing processes that incorporate some 3D-printed components. This will be further fueled by consumers desiring products that require 3D printers for their manufacture.
“Look what I made!” Your children will bring home 3D printed projects from school. Digital literacy – including Web and app development, electronics, collaboration and 3D design – will be supported by 3D printers in schools. A number of middle schools and high schools already have 3D printers. As 3D printing costs continue to fall, more schools will sign on. Digital literacy will be about things as well as bits.
If, as President Obama believes, a manufacturing revolution, led by 3D printing, is coming, it behooves business leaders in every field to ask themselves how it could affect their business model and assumptions about the future.
Research Leads to the 3D Printing of Pure Graphene Nanostructures
Researchers in Korea have successfully 3D printed graphene nano-structures without the use of any other material. With the entire printed structure being composed of graphene, the strength, as well as full conductivity of the material can be taken advantage of.
There is no question that graphene, has enormous potential, from solar cell technology, to electronics to medicine. A key factor in developing practical and commercial applications of the one-atom thick carbon sheets is in aligning the material in the desired form depending on the application.
Now 3D printing of graphene is nearing a feasible stage and companies such as Graphene 3D Lab, are at the forefront of the technology.
However, there is a difference between 3D printing pure graphene, and 3D printing a graphene/thermoplastic composites like Graphene 3D has been doing.
While printing with composite materials, using a typical FDM/FFF or powder based laser sintering process, will keep some of graphene’s superior properties intact, most will be lost. The plastic will eventually break down leaving any prints weak, and not much different from a typical object you’d print with a MakerBot Replicator.
Their techniques mean that graphene nano-structures can be fabricated without the use of any other material. With the entire printed structure being composed of graphene, the strength, as well as full conductivity of the material can be realized.
“We are convinced that this approach will present a new paradigm for implementing 3D patterns in printed electronics.”
“We developed a nanoscale 3D printing approach that exploits a size-controllable liquid meniscus to fabricate 3D reduced graphene oxide (rGO) nanowires,” Seol told Nanowerk. “Different from typical 3D printing approaches which use filaments or powders as printing materials, our method uses the stretched liquid meniscus of ink. This enables us to realize finer printed structures than a nozzle aperture, resulting in the manufacturing of nanostructures.”
“So far, to the best of our knowledge, nobody has reported 3D printed nanostructures composed entirely of graphene,” says Seol. “Several results reported the 3D printing (millimeter- or centimeter-scale) of graphene or carbon nanotube/plastic composite materials by using a conventional 3D printer. In such composite system, the graphene (or CNT) plays an important role for improving the properties of plastic materials currently used in 3D printers. However, the plastic materials used for producing the composite structures deteriorate the intrinsic properties of graphene (or CNT).”
“We are convinced that this approach will present a new paradigm for implementing 3D patterns in printed electronics,” says Seol.
For their technique, the team grew graphene oxide (GO) wires at room temperature using the meniscus formed at the tip of a micropipette filled with a colloidal dispersion of GO sheets, then reduced it by thermal or chemical treatment (with hydrazine).
The deposition of GO was obtained by pulling the micropipette as the solvent rapidly evaporated, thus enabling the growth of GO wires. The researchers were able to accurately control the radius of the rGO wires by tuning the pulling rate of the pipette; they managed to reach a minimum value of ca. 150 nm.
Using this technique, they were able to produce arrays of different freestanding rGO architectures, grown directly at chosen sites and in different directions: straight wires, bridges, suspended junctions, and woven structures.
Seol points out that this 3D nanoprinting approach can be used for manufacturing 2D patterns and 3D geometry in diverse devices such as printed circuit boards, transistors, light emitting devices, solar cells, sensors and so on.
A lot of work remains to reduce the 3D printable size to below 10 nm and increase the production yield. A short video of Seol’s process is below:
University of Alberta researchers have found that abundant materials in the Earth’s crust can be used to make inexpensive and easily manufactured nanoparticle-based solar cells.
The discovery, several years in the making, is an important step forward in making solar power more accessible to parts of the world that are off the traditional electricity grid or face high power costs, such as the Canadian North, said researcher Jillian Buriak, a chemistry professor and senior research officer of the National Institute for Nanotechnology based on the U of A campus.
Buriak and her team have designed nanoparticles that absorb light and conduct electricity from two very common elements: phosphorus and zinc. Both materials are more plentiful than scarce materials such as cadmium and are free from manufacturing restrictions imposed on lead-based nanoparticles.
“Half the world already lives off the grid, and with demand for electrical power expected to double by the year 2050, it is important that renewable energy sources like solar power are made more affordable by lowering the costs of manufacturing,” Buriak said.
“My goal is that a store like Ikea could sell rolls of these things with simple instructions and baggies of screws and do-dads and you could install them yourself,” said Buriak
Her team’s research supports a promising approach of making solar cells cheaply using mass manufacturing methods like roll-to-roll printing (as with newspaper presses) or spray-coating (similar to automotive painting). “Nanoparticle-based ‘inks’ could be used to literally paint or print solar cells or precise compositions,” Buriak said.
Buriak collaborated with U of A post-doctoral fellows Erik Luber of the U of A Faculty of Engineering and Hosnay Mobarok of the Faculty of Science to create the nanoparticles. The team was able to develop a synthetic method to make zinc phosphide nanoparticles, and demonstrated that the particles can be dissolved to form an ink and processed to make thin films that are responsive to light.
Buriak and her team are now experimenting with the nanoparticles, spray-coating them onto large solar cells to test their efficiency. The team has applied for a provisional patent and has secured funding to enable the next step to scale up for manufacturing.
https://pharmaceuticalintelligence.com/wp-content/uploads/2015/09/25063-grapheneonsilicon.jpg
raphene has extreme conductivity and is completely transparent while being inexpensive and nontoxic. This makes it a perfect candidate material for transparent contact layers for use in solar cells to conduct electricity without reducing the amount of incoming light – at least in theory. Whether or not this holds true in a real world setting is questionable as there is no such thing as “ideal” graphene – a free floating, flat honeycomb structure consisting of a single layer of carbon atoms: interactions with adjacent layers can change graphene’s properties dramatically.
“We examined how graphene’s conductive properties change if it is incorporated into a stack of layers similar to a silicon based thin film solar cell and were surprised to find that these properties actually change very little,” Marc Gluba explains.
To this end, they grew graphene on a thin copper sheet, next transferred it to a glass substrate, and finally coated it with a thin film of silicon. They examined two different versions that are commonly used in conventional silicon thin-film technologies: onesample contained an amorphous silicon layer, in which the silicon atoms are in a disordered state similar to a hardened molten glass; the other sample contained poly-crystalline silicon to help them observe the effects of a standard crystallization process on graphene‘s properties.
Even though the morphology of the top layer changed completely as a result of being heated to a temperature of several hundred degrees Celcius, the graphene is still detectable. “That’s something we didn’t expect to find, but our results demonstrate that graphene remains graphene even if it is coated with silicon,” says Norbert Nickel.
Their measurements of carrier mobility using the Hall-effect showed that the mobility of charge carriers within the embedded graphene layer is roughly 30 times greater than that of conventional zinc oxide based contact layers.
Says Gluba: “Admittedly, it’s been a real challenge connecting this thin contact layer, which is but one atomic layer thick, to external contacts. We’re still having to work on that.” Adds Nickel: “Our thin film technology colleagues are already pricking up their ears and wanting to incorporate it.” The researchers obtained their measurements on one square centimeter samples, although in practice it is feasible to coat much larger areas than that with graphene.
The advent of 3D printing technologies has generated new ways of representing and conceptualizing health and illness, medical practice and the body. There are many social, cultural and political implications of 3D printing, but a critical sociology of 3D printing is only beginning to emerge. In this article I seek to contribute to this nascent literature by addressing some of the ways in which 3D printing technologies are being used to convert digital data collected on human bodies and fabricate them into tangible forms that can be touched and held. I focus in particular on the use of 3D printing to manufacture non-organic replicas of individuals’ bodies, body parts or bodily functions and activities. The article is also a reflection on a specific set of digital data practices and the meaning of such data to individuals. In analyzing these new forms of human bodies, I draw on sociomaterialist perspectives as well as the recent work of scholars who have sought to theorize selfhood, embodiment, place and space in digital society and the nature of people’s interactions with digital data. I argue that these objects incite intriguing ways of thinking about the ways in digital data on embodiment, health and illnesses are interpreted and used across a range of contexts. The article ends with some speculations about where these technologies may be headed and outlining future research directions.
Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants.
Rapid prototyping is a valuable implant production tool that enables the investigation of individual geometric parameters, such as shape, porosity, pore size and permeability, on the biological performance of synthetic bone graft substitutes. In the present study, we have employed low-temperature direct 3D printing to produce brushite and monetite implants with different geometries. Blocks predominantly consisting of brushite with channels either open or closed to the exterior were implanted on the decorticated lumbar transverse processes of goats for 12 weeks. In addition, similar blocks with closed channel geometry, consisting of either brushite or monetite were implanted intramuscularly. The design of the channels allowed investigation of the effect of macropore geometry (open and closed pores) and osteoinduction on bone formation orthotopically. Intramuscular implantation resulted in bone formation within the channels of both monetite and brushite, indicating osteoinductivity of these resorbable materials. Inside the blocks mounted on the transverse processes, initial channel shape did not seem to significantly influence the final amount of formed bone and osteoinduction was suggested to contribute to bone formation.
Join These Medical 3D Printing Groups on Twitter and LinkedIn for great up to date news
Curator: Stephen J. Williams, Ph.D.
Below is a list with links to great groups on Twitter and LinkedIn that focus on the Medical 3D Printing Industry and Breaking News. These are Great resources for news, information, investment opportunities, and conference announcements!
Artificial Dentures: The Revolution of 3D Printing – Printed via Stereolithography (SLA), the resin dentures can be made directly from 3D models, instead of through Casts
Curators: Gerard Loiseau, ESQ and Aviva Lev-Ari, PhD, RN
DENTCA receives FDA Clearance for the First 3D printable denture base material; able to be worn inside the mouth
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
Indications for Use Form Approved: OMB No. 0910-0120 Expiration Date: January 31, 2017 See PRA Statement below. 510(k) Number (if known) K143033
Device Name Dentca Denture Base Indications for Use (Describe)
Dentca Denture Base is a light-cured resin indicated for fabrication and repair of full and partial removable dentures and baseplates. The material is an alternative to traditional heat-cured and auto polymerizing resins. Fabrication of dental prosthetics with Dentca Denture Base requires a computer-aided design and manufacturing (CAD/ CAM) system that includes the following components not part of the device: oral casting impression, digital denture base file created in an optical impression system, stereolithographic additive printer, and curing light equipment.
For the First Time in Dental History, DENTCA, Inc. Receives FDA Clearance for the First 3D Printable Denture Base Material; Able to Be Worn Inside the Mouth.
The FDA has officially approved last week a 510(k) for the use of DENTCA’s new 3D printable material as a denture base that will revolutionize the denture world; making the denture production process quicker, more accurate and more predictable.
The new developments will create incredible possibilities for patients, as doctors will soon be able to 3D print final dentures at their office, allowing the manufacture process to be done in one day everywhere in the world.
LOS ANGELES, CA. (PRWEB) AUGUST 08, 2015
DENTCA, Inc. continues to revolutionize the denture world.
The FDA last week officially approved a 510(k) for the use of DENTCA’s new 3D printable material as a denture base that will revolutionize the denture making world.
Dr. Jason Lee is the proud creator of this technology and has shared excitingly his opinion:
“After several years in development DENTCA’s new 3D printable Denture Base is finally cleared for use. We are very excited to begin applying this technology to continue revolutionizing the denture world. The material is a light-cured resin indicated for fabrication and repair of full and partial removable dentures and baseplates; which will eventually replace traditional heat-cured and auto polymerizing conventional denture making methods. By improving the manufacture process with the help of precise 3D printers, we will be making the denture production process quicker, more accurate and more predictable. DENTCA already revolutionized the fit of dentures by directly scanning the impressions instead of the need to pour a stone; now it will further increase it by removing the need for a flask in the future market, thus significantly reducing distortion.”
Fabrication of dental prosthetics with DENTCA Denture Base utilizes a computer-aided design and manufacturing (CAD/CAM) software system, specialized tray impression systems, and a digital denture file database the system automates the denture making process almost to perfection; and now by also incorporating an optical impression system, a stereolithographic additive printer, and curing light equipment; it finally closes a cycle for success, giving the doctor a completely evolved denture manufacture process.
The material has passed al recent biocompatibility tests placed in evaluation for the DENTCA Denture Base in accordance with the FDA Blue Book Memorandum #G95-1 and International Standard ISO 10993-1, as recognized by FDA. The battery of testing included tests for Genotoxicity, Cytotoxicity, Sensitization, Irritation, Acute Toxicity & Material Characterization which the printable material passed with flying colors. Declaring the 3D printable material for considered tissue contacting for a period longer than 30 days (a removable prosthesis). DENTCA Denture Base was tested for conformity with the industry consensus standard ISO 20795-1.
When inquired about DENTCA’s recent breakthrough, the CEO of the company Mr. Sun Kwon shared the following: “This clearance completely revolutionizes the denture manufacture process, which has barely changed in over 100 years. The new developments will create incredible possibilities for patients, as doctors will soon be able to 3D print final dentures at their office, allowing the manufacture process to be done in one day everywhere in the world. Stay tuned for new and exciting updates.”
It is indeed exciting times for prosthodontics today.
The FDA granted 16 dental devices 510(k) clearance in July, one of them is Dentca Denture Base fromDentca. At the end of last month, the FDA officially approved a 510(k) for the use of DENTCA’s new 3D printable material as a denture base.
According to a press release, the material has passed all recent biocompatibility tests placed in evaluation for the DENTCA Denture Base in accordance with the FDA Blue Book Memorandum #G95-1 and International Standard ISO 10993-1, as recognized by FDA. The battery of testing included tests for Genotoxicity, Cytotoxicity, Sensitization, Irritation, Acute Toxicity & Material Characterization which the printable material passed with flying colors. Declaring the 3D printable material for considered tissue contacting for a period longer than 30 days (a removable prosthesis). DENTCA Denture Base was tested for conformity with the industry consensus standard ISO 20795-1.
Fabrication of dental prosthetics with DENTCA Denture Base utilizes a CAD/CAM software system, specialized tray impression systems, and a digital denture file database. Now by also incorporating an optical impression system, a stereolithographic additive printer, and curing light equipment, it gives the doctor a completely evolved denture manufacture process.
Dr. Jason Lee, the proud creator of this technology shared excitingly his opinion:
“After several years in development DENTCA’s new 3D printable Denture Base is finally cleared for use. We are very excited to begin applying this technology to continue revolutionizing the denture world. The material is a light-cured resin indicated for fabrication and repair of full and partial removable dentures and baseplates; which will eventually replace traditional heat-cured and auto polymerizing conventional denture making methods. By improving the manufacture process with the help of precise 3D printers, we will be making the denture production process quicker, more accurate and more predictable. DENTCA already revolutionized the fit of dentures by directly scanning the impressions instead of the need to pour a stone; now it will further increase it by removing the need for a flask in the future market, thus significantly reducing distortion.”
Orthodontic company DENTCA has received 510(k) approval of a material that will be used to 3D print the bases of dentures, allowing DENTCA to create perfectly tailored dentures and baseplates from patient scans. Printed via stereolithography (SLA), the resin dentures can be made directly from 3D models, instead of through casts, allowing the company to automate the manufacturing process.
Dr. Jason Lee, inventor of the process, explains, “After several years in development DENTCA’s new 3D printable Denture Base is finally cleared for use. We are very excited to begin applying this technology to continue revolutionizing the denture world. The material is a light-cured resin indicated for fabrication and repair of full and partial removable dentures and baseplates; which will eventually replace traditional heat-cured and auto polymerizing conventional denture making methods.” He continues, “By improving the manufacture process with the help of precise 3D printers, we will be making the denture production process quicker, more accurate and more predictable. DENTCA already revolutionized the fit of dentures by directly scanning the impressions instead of the need to pour a stone; now it will further increase it by removing the need for a flask in the future market, thus significantly reducing distortion.”
DENTCA’s Denture Base involves a unique system that combines the company’s denture file database with an optical impression system, the SLA printer, and the necessary light curing to create a complete, closed cycle for producing their dentures. Now that the material involved has received FDA approval, deemed suitable for contact with human tissue for longer than 30 days, the product can be brought to market. DENTCA envisions their systems installed in doctor’s offices, with CEO Sun Kwon saying, “This clearance completely revolutionizes the denture manufacture process, which has barely changed in over 100 years. The new developments will create incredible possibilities for patients, as doctors will soon be able to 3D print final dentures at their office, allowing the manufacture process to be done in one day everywhere in the world. Stay tuned for new and exciting updates.”
It’s exciting to see an increasing range of technologies and materials approved by the FDA so that 3D printing can more quickly improve the lives of those with access to it.
The schematic on Slide 10 can be found in 
On slide 20 the traditional Ti casting is compared with Ti 3D printing from the powders. The advantage of 3D method is low cost and high productivity. This link 
Slide 21 For 3D Bioploter made by EnvisionTec we notice the usage of materials such as metal followed by post-processing sintering, Hydroxylapatite, TCP, Titanium. Using a preciptation method the machine can handle Chitosan, Collagen, 2-component system of the two possible combination: Alginate, Fibrin, PU, and Silicone. More details in 
The insulin pump shown in slide 50 is a schematic of the pump controlled electronically by a control algorithm device, a sensor, an electronic receiver that connects with an iPhone through an wireless channel.
Slide 52 describes how easily available bio-compatible metal powders
Slide 54 shows conclusions regarding the hardware of the presentation, in which: 6) there are two types of metal 3D printing hardware for medical applications: Selective Laser Melting / Selective Laser Sintering, and 3D Bioploter (metal powder mixed with binder and further thermal treatment to remove binder and sinter the metallic matrix in a solid object that can be used as a replacement. Thank you for your attention!
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