Team:Warwick/HP/Silver
Human Practices
Developing a Project Idea
At the start of the summer, whilst brainstorming our possible project ideas, we decided that we wanted to try and incorporate the expression of cellulose. As a natural biopolymer, it doesn't illicit an immune response and is almost completely resistant to both heat and mechanical stresses. We then weighed up the pros and cons of expressing the polysaccharide either intracellularly or extracellularly. We settled on extracellular expression, as this increased the chance of the cellulose produced by the different cells interacting and linking together to produce a more rigid material. With five engineers on the team, we wanted to design a project which utilized their skills and kept them busy in the workshop. With this in mind, we each set about finding an application which incorporated both biology and engineering. This led to the incorporation of our 3D biopolymer printer, which could then be used to produce surface coatings for medical implants.
Our plan seemed like a good idea on paper but we were unsure about how it could be put into practice in a clinical setting. In order to learn more about the issue of implant failure in the medical field, we interviewed both manufacturers of similar products and experts within the field of implantology and dental surgery. In addition to this, we also spent time researching current market leaders and looking at the limitations of their products.
Titanium is the most frequently used material for prosthetics and many bone implants thanks to its high degree of biocompatibility within the body. Titanium also has some inherent antimicrobial activity attributable to the oxide layer. Our system has the potential to be used in several different types of bone implant, but we will be focusing our attention on endosseous, or dental, implants. The success of dental implants greatly depends on the biocompatibility and microtopography of the implant surface; with the ultimate goal of achieving successful osteoconductivity.
Market Research
Surface roughness has been shown to be an important parameter for implants and their subsequent osseointegration. Many manufacturers of dental implants base their design upon a rough surface structure which allows ingrowth of osteoblasts and soft tissue. In the past few years, some implant manufacturers have experimented with the incorporation of a Hydroxyapatite (HA) surface coating. HA was designed to reduce healing time thanks to improved osseointegration, and at first the results of this revolutionary coating seemed promising. However, it was found that the initial success of the implants began to diminish. Reports on the clinical behaviour of HA-coated implants indicated a substantial decline in survival rate after 7 to 8 years. It is thought that this failure was due to both the in vivo degradation of the HA coating alongside the fact that HA doesn’t adhere well to the implant itself. Surrounding bone tissue adhered well to the HA coating but over time the titanium implants loosened from the HA coat and the metal implant itself loosened and subsequently failed. James C Taylor, Carl F Driscoll, Michael D Cunningham, Failure of a hydroxyapatite-coated endosteal dental implant: A clinical report, In The Journal of Prosthetic Dentistry, Volume 75, Issue 4, 1996, Pages 353-355. We spoke to Dr Rachel Sammons from the University of Birmingham, who is a senior lecturer in Biomedical Materials Science. She told us: “Hydroxyapatite has no inherent infection risk, however rough coatings including hydroxyapatite can “hide” microorganisms within the pores making then inaccessible to phagocytic cells and more difficult to clean.” Now, it seems that suppliers of dental implants focus their attention on modifying the surface of the titanium only. Here are some examples of the market leaders:
TiUnite® from Nobel Biocare
“TiUnite is a unique implant surface that enhances osseointegration – even under the most challenging conditions including soft bone and immediate loading. TiUnite is a thickened, moderately rough titanium oxide layer with high crystallinity and a high phosphorus content.”
The manufacturers of TiUnite titanium implants claim that their product maintains implant stability immediately after placement with enhanced osseointegration and anchorage in the surrounding bone. However, having spoken to dental clinicians, we have found that although these implants achieve high stability in the critical healing phase, i.e. the first 3 months after implantation, they can fail due to aseptic loosening. The high Ra value associated with titanium-only coatings does enhance initial osseointegration, but this rough, porous surface is more prone to bacterial infection. This is turn can result in implant failure.
Laser-Lok® from BioHorizons
“Laser-Lok microchannels is a proprietary dental implant surface treatment developed from over 25 years of research initiated to create the optimal implant surface. Through this research, the unique Laser-Lok surface has been shown to elicit a biologic response that includes the inhibition of epithelial downgrowth and the attachment of connective tissue. This physical attachment produces a biologic seal around the implant that protects and maintains crestal bone health.”
The manufacturer of Laser-Lok implants argue that these unique microchannels seal and allow the attachment of connective tissue and bone whilst inhibiting epithelial downgrowth (M. Nevins, D. M. Kim, S.-H. Jun, K. Guze, P. Schupbach, and M. L. Nevins, “Histologic evidence of a connective tissue attachment to laser microgrooved abutments: a canine study,” The International Journal of Periodontics & Restorative Dentistry, vol. 30, no. 3, pp. 245–255, 2010. ) . Although the Laser-Lok system achieves promising results in terms of osseointegration and fusion with the surrounding soft tissues, there is still a significant risk of osteomyelitis even after healing.
OsseoSpeed™ from Astra Tech
”OsseoSpeed is a unique fluoride-modified nanostructure implant surface that stimulates early bone formation and provides stronger bone-to-implant bonding. Factors such as enhanced osteoblast differentiation, biocompatibility and thrombogenic properties of the OsseoSpeed surface have been attributed to the improved and fastened osseointegration.”
OsseoSpeed is manufactured by blasting and treating the surface with fluoride ions in an attempt to facilitate more intensive BIC in the initial phase (2 weeks) after implantation Berglundh, T., Abrahamsson, I., Welander, M., Lang, N. P. and Lindhe, J. (2007), Morphogenesis of the peri-implant mucosa: an experimental study in dogs. Clinical Oral Implants Research, 18: 1–8. However, it is the long-term osseointegration which is most important, and Astra Tech do not address the issue of consequential infection.
http://www.dentsplyimplants.com/en/Implant%20systems/Implants/OsseoSpeed
SEM of untreated SLA machined Titanium surface.
Jia, Shengnan & Zhang, Yu & Ma, Ting & Chen, Haifeng & Lin, Ye. (2015). Enhanced Hydrophilicity and Protein Adsorption of Titanium Surface by Sodium Bicarbonate Solution. Journal of Nanomaterials. 2015. 1-12.
SEM of a TiUnite implant surface
Gedrange T, Gredes T, Gredes M, Allegrini MR, Borsos G, Vegh A, Salles MB, Heinemann F, Dominiak M, Allegrini S Jr. (2009) Comparative animal study on hard tissue integration and bone formation of different Nobel Biocare implants. J Physiol Pharmacol. 2009 Dec;60 Suppl 8:117-21.
SEM of a LaserLok titanium surface coating
Laser-Lok® technology from BioHorizons. http://www.biohorizons.com/laserloktechnology.aspx
Glossary
| Angiogenesis | The physiological process through which new blood vessels form from pre-existing vessels |
|---|---|
| Apoxia | Oxygen deficiency in the blood or tissues |
| BIC | Bone-to-Implant Contact |
| Fibroblast | A type of cell that synthesizes the extracellular matrix and collagen |
| Mastication | The process by which food is crushed and ground by teeth (chewing!) |
| Osseoinduction | The recruitment of immature cells and the stimulation of these cells to develop into preosteoblasts |
| Osseointegration | The direct structural and functional connection between living bone and the surface of a load-carrying implant |
| Osteoclast | A large multinucleate bone cell which absorbs bone tissue during growth and healing |
| Osteoconductive | Osteoconductive materials serve as a scaffold onto which bone cells can attach, migrate, grow and/or divide Osteomyelitis Infection within bone tissue |
| Osteonecrosis | Also known as vascular necrosis (AVN). Occurs when there is loss of blood to the bone which causes the bone to die |
| Otseoblast | A cell which secretes the substance of bone |
| Periodontal | Supporting structures of teeth |
| Preosteoblast | A mesenchymal cell that differentiates into an osteoblast |
| Ra/Sa value | Surface roughness is a measure of the texture of a surface - Rough (Ra) or Smooth (Sa) |
| SLA | Sandblasted and acid-etched |
Talking to the Experts
We gained invaluable advice from many individuals, from dental surgeons to antimicrobial specialists. We are incredibly grateful for their help and guidance, and couldn’t have developed our project without them.
Dr. Khalid Malik
Consultant in Restorative Dentistry
Birmingham & Peterborough Healthcare NHS Foundation Trust
Dr. Malik is based at hospitals in both Birmingham and Peterborough where he is involved with the rehabilitation of trauma, oncology, and patients who have a congenital defect which can use dental implants as part of the process. He is a GDC specialist for Endodontics, Periodontics, Prosthodontics and Restorative Dentistry. Dr. Malik undertook an MPhil thesis which examined the effects of dental implant microstructure on bone cell spreading. He also gives lectures on the field of implantology.
iGEM Warwick: Would the use of biopolymers in implants be preferable to metals in reducing traumas and poor osseointegration?
Dr. Malik: This depends on biocompatibility both short and long term. The implant will be going in to a dynamic tissue and so this is dependent on the type of implant. With dental implants the crucial time for osseointegration is 3 months. If the bone doesn’t like the implant for whatever reason, it may not integrate properly and the implants may even be avulsed when bone remodelling occurs. Hydroxyapatite surface coatings have a high failure rate; it integrates well initially but then fails due to poor osseointegration.
iGEM Warwick: Do you believe that cellulose could be successful as biocompatible scaffold for bone growth and osteoblast spreading?
Dr. Malik: [Opinion only] The cellulose would have to react with the bone in the short, medium and long term. Depending on how it reacts would define if it would be successful or not. Ideally, the biomaterial would need to be osseoinductive, osseoconductive and antibacterial. This would help to maintain the bone and avoid infection for its duration. If the cellulose has some flexibility, it may even act as a shock absorber to the forces placed upon the implant. Naturally, a periodontal ligament absorbs some of the impact of everyday mastication.
iGEM Warwick: Does apoxia near implants due to poor angiogenesis lead to cancers?
Dr. Malik: Not that we know of. Apoxia around implants is more likely to lead to osteonecrosis or osteomyelitis. Osteonecrosis can lead to implant failure. When cancer patients have radiotherapy, they can have hypobaric oxygen therapy to increase the oxygen concentration within the serum as an attempt to improve angiogenesis in a bid to avoid this.
iGEM Warwick: In your experience, what is the optimal micro-pore diameter for biocompatibility with bone cells?
Dr. Malik: The Ra/Sa value is important. You need to determine the ideal diameter or pore size for a bone cell to grow into and survive. The surface of the implant should have a Ra/Sa value similar to bone after osteoclasts have worked on it, but before the osteoblasts arrive. Ideally, you want to achieve something in the middle of the two Ra/Sa values; too rough and it may lead bacterial infection but too smooth and the bone doesn’t adhere as well to it and the implant may fail.
iGEM Warwick: Approximately what percentage of implants fail due to poor osseointegration?
Dr. Malik: This depends on the location and quality of bone. Approximately 96% of dental implants are successful over a 6 year period [1] but exact figures vary depending on whether in the mandible or maxilla as the quality of the bone differs between the two. However, post- implant complications such as peri-implantitis can affect up to 56% of patients [2]. (NB: Peri-implantitis is the destructive inflammatory process affecting the soft and hard tissues surrounding dental implants [3]. The bacterial pathogens found around failing implants similar to those found in other forms of periodontal disease.)
iGEM Warwick: We believe that by adding the cellulose surface coating, implant failure rates due to poor osseointegration and peri- implantitis could be greatly reduced, and this would reduce the failure for dental implants and need for repeated surgery. In theory, the cellulose surface should lower the risk of infection as the material is both sterile and biocompatible. Would this be an advantage?
Dr. Malik: A decreased failure rate would definitely reduce the need for repeated surgery of both the implant and bone augmentation and thus lowers the risk of bacterial infection. Strong analgesics, anti-inflammatories and antibiotics can be used for titanium dental implants or if a patient’s native bone is used as a frame work.
Implant Stability Dip Primary stability is a requirement of successful secondary stability. The latter, however, dictates the time of functional loading. Secondary stability has been shown to begin to increase at 4 weeks after implant placement. Osseointegration and implant stability is affected by various factors during healing process
Next, we spoke to ASP materials and asked if they could advise on a micropore size for our surface coating design. We learned that smaller pores disfavoured strong bone growth as they restricted blood vessel development and bone maturation. However more pores did help to increase bone adherence. Taking their advice on board, we compromised and aimed for a pore diameter of 600 micrometres.
Dr. Chandrika Nair
Project Manager
Warwick Antimicrobial Interdisciplinary Centre (WAMIC)
One of our biggest concerns was achieving an aseptic surface coating with long-lasting antimicrobial properties. We questioned whether incorporating antibiotics into the structure of our surface coating would be possible and if there would be any side effects. Dr. Nair warned that long-term exposure to sub-lethal levels of antibiotics could favour the emergence of antibiotic resistance. This meant that we would need to establish a continuous supply of high concentration antibiotic to be effective.
Professor David Roper
Warwick Antimicrobial Interdisciplinary Centre (WAMIC)
We asked Professor Roper how we would achieve a continuous, high supply of antibiotic. He advised that our two options would be either constant injections or antibiotics which are only activated in the presence of bacteria. From this, we decided that we would need antibiotics that are covalently attached to the surface coating and only released in the presence of bacteria.
Professor Andrew Dove
Professor of Chemistry
The University of Warwick
Professor Dove specialises in organic and biopolymer chemistry. We asked his which types of labile linkers could be used for attaching the antibiotics. We were told that disulphide bridges could be cleaved selectively by the reducing environment associated with bacterial periplasms. After learning this, we decided that we would utilise small amounts of artificial thioglucose that could be incorporated into the cellulose structure and used to attached antibiotics via disulphide bridges.
Aim
There is no doubt that genetically modified organisms have a bad reputation but is it justified? When some people hear “genetic engineering”, they immediately think of designer babies and mutant crops of killer cabbages. Genetically modified crops in particular are a topic of intense debate that have sparked a lot of controversy over the years. We believe that this is fueled largely by a lack of understanding and vast amounts of misinformation, often portrayed by the media as short, sensationalist news headlines.
The pace of genetic discovery has accelerated considerably over the past decade with the identification of many genetic alterations that contribute to animal, plant and human health and disease. The field of human and medical genetics are relatively new, yet it is evident that the public commonly hear terminology, such as “genetics”, ‘chromosomes’ and “CRISPR” without fully understanding what they mean. It is therefore understandable why there is a resistance to this new and developing technology, which can seem unnerving.
We designed a short survey with the goal of finding out how public opinions differ on the subject of genetic engineering, and if this varies with age and education. We suspected that those who knew more about scientific matters in general would be more likely to trust evidence from scientists and would therefore view new findings and research regarding genetic engineering in a more positive light.
Data Collection
We were very aware that we needed to approach our data collection in an ethical manner. We collected the vast majority of our surveys in person, with a small proportion using an online tool. We didn’t want participants to be influenced by the fact that they had two ‘genetic engineers’ stood over them whilst they filled in the survey. To overcome this, when we approached potential participants we didn’t explain who we were or what research we were working on. We merely told them that we were looking into public opinions, and did they have a few moments to spare? We surveyed just over 100 people in total, which is a reasonable sample size.
As we were partly looking at whether age and generational gaps may have a role in perceptions of genetic engineering, we recorded the age of each participant along with the rest of their responses. We also used the same survey to collect data but over two separate days. On the first day, Elsita and Amy spent a few hours on the streets of Leamington Spa, a town in Warwickshire. Here, they stopped passers by and asked if they would be happy to take part in a public opinions survey. It was a busy Sunday afternoon, and so many of the people approached politely declined as they had places to be and people to see! However, they still managed to collect a relatively even number of responses from each age group. The second survey was identical to the first, but this time participants were those attending the New Scientist Live exhibition at the ExCel Centre in London. It would be expected that as they have bought tickets to the show, these people have an interest in science and stay up-to-date with scientific developments.
Data Analysis
Figures 1 & 2. Here we can see that 32% of the members of the public who took part in our survey are worried by genetic engineering news stories, whereas only 15% of those asked the same question at NSLive were. Interestingly, if we look more closely at the age groups of the general public, we find that 69% of those aged between 41 and 65 are concerned, compared to just 26% of those aged between 17 and 25.
Figures 3 & 4. In this question, we asked the participants to grade themselves on how well the felt that they understood the science behind genetic engineering. This was purely based on their own perception and we asked them to be as honest and truthful as possible. Visitors at NSLive clearly felt that they had a better grasp on how genetic engineering is performed, whilst the public on the streets of Leamington very noticeably more unsure.
Figures 5 & 6. The majority of both survey groups agreed with the use of genetic engineering in the medical industry. Surprisingly, the NSLive group were almost unanimous (90%), with not a single person disagreeing with this notion. This suggests that people are more open to the concept of genetic engineering if it ‘directly’ benefits us in a healthcare sense.
Figures 7 & 8. A similar proportion of the general public in Leamington and the visitors at NSLive did not agree that the UK government should be promoting GM technology - 16% and 22% respectively. 72.5% of those aged 17-25 years agreed, as opposed to just 44.4% of those aged 41 and over.
Figures 9 & 10. Here, we can see that those with an interest in science were more likely to give an example of genetic engineering other than GM crops. 61% of the people surveyed on the streets of Leamington immediately thought of GM food, compared to 40% of those at NSLive.
Discussion
The fact that the younger generation is less likely to be apprehensive about the concept of genetic engineering is probably due to the teaching of ethical, social, and environmental issues associated with GM in secondary school biology classes. This suggests that general public opinions could be changed over time, if comprehensive education programs are put in place. It could be argued that current public resistance is due to the small amount of information which people hear on the news or read in the papers. Diederik van der Hoeven from the Genetic Literacy Project [4] argues that public judgment is largely determined by three dimensions: health, fashion and ethics. Either way, this growing acceptance of genetic engineering and its applications is a positive development, and researchers must do as much as they can to outreach to members of the general public in an accessible way to promote the benefits further. Not doing has the potential to hinder ongoing research, as sponsors of grants are not likely to invest in projects which may not secure a return due to an unresponsive market later down the line. It would be easy for the scientific community to dismiss public opinions, but they are not to be underestimated. Facts alone may not be enough to change a widely held opinions; we need to appeal emotionally and address the ethical issues too.
Conclusions & Recommendations
After analyzing our data, we were even more confident that we wanted to major on our public engagement and outreach activities. We recognize that our results are based on a relatively small sample size, and so we would recommend that future surveys are conducted on a larger scale.
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