The contributions made to the field of modern medicine by Hippocrates, a physician in Ancient Greece, are hard to underestimate. The ‘Father of Modern Medicine,’ Hippocrates is not only credited with defining the discipline as a profession distinct from philosophy and theurgy (magical practices to invoke a divine spirit), he was arguably the first to understand that disease had a physical cause and was not occasioned by superstition or the wrath of the gods. Born around 460BCE on the island of Kos in the Greek archipelago, Hippocrates is perhaps now best remembered for having crafted the still sworn Hippocratic Oath as a guideline to physicians on their roles and responsibilities to patients, and the oft – perhaps misquoted – verse: “Let food be thy medicine and medicine be thy food.”
And within the modern ‘foodie’ movement, few nutritive groups are as widely acclaimed as ‘superfoods’ as those belonging to the ‘leafy green’ category – kale, collard greens, and spinach, for example. Beloved of vegetarians, vegans, body builders, and yoga-moms alike, the humble spinach leaf is endowed with seemingly magical properties, from boosting muscular strength to preventing vision loss to protecting against inflammatory diseases such as arthritis. In traditional Ayurvedic medicine, it is used to treat headaches, improve skin conditions, and even help with insomnia. And in laboratory experiments, spinach extracts have demonstrated the ability to slow down the division of cancer cells.(1) An all-round nutritional powerhouse famously espoused by everyone’s favorite sailor, Popeye, spinach contains high levels of lutein, antioxidants, potassium, vitamin K, magnesium, and fiber which combine to offer the body a real bang for its nutritional buck. But in addition to the plethora of available nutrients, could spinach offer other ways to do a body good? Could it, in fact, not only keep your heart healthy, but also offer you a new one?
Adventure with us, if you will, into the world of cellular tissue regeneration…
According to the Organ Procurement and Transplantation Network, an agency of the U.S. Department of Health and Human Services, more than 100,000 patients find themselves on the waiting list for a donor organ transplant at any given time, with a new name added to the list every ten minutes.(2) And the tragic reality is that, despite public awareness campaigns and advances in bio-medical technology, the number of patients in need of donated organs far outstrips the available donors. Each day, an average of 22 of those potential recipients will die without ever having come close to receiving the life-saving organ.
And in some situations, even the receipt of a transplanted organ may not be the end of the story. Take, for example, heart transplants. According to the Mayo Clinic, a common after-effect of a heart transplant is a thickening of the coronary arteries, leading to cardiac allograft vasculopathy (CAV).(3) Patients with post-surgical CAV may suffer abnormal heart rhythms, heart failure, or even sudden death due to the slowing of arterial blood flow. So how can blood flow be improved? This is the question that motivated the research of a team at the Worcester Polytechnic Institute (WPI) in Massachusetts. Led by graduate student Joshua Gershlak, WPI’s team noted the similarities between the vascular system of the spinach leaf and that of the human heart, and are now pioneering a way to create heart tissue from a spinach leaf scaffold. Here’s how…
One of the biggest concerns when treating human heart disease is the problem of perfusing the organ to avoid tissue death. As already described, in the absence of adequate blood flow to ensure the transmission of oxygen and vital nutrients allograft transplant materials or the patient’s own tissues can quickly suffer necrosis, decreasing their viability for use. And although the technology behind organ transplantation and tissue engineering has progressed in astonishing ways, we do not yet have the technical ability to create a viable vascular network for transplanted organs. Put bluntly: even bio-medical additive printing – also known as 3D printing – cannot produce the microvasculature (smaller than 10µm in diameter) necessary to artificially perfuse an organ like a heart.(4)
To address this problem, the WPI research group has created a process that removes plant-based cellular material (cellulose) from a spinach leaf, leaving behind an acellular ‘scaffold’ that forms an extracellular matrix. This matrix is then covered in human cells that grow around the leaf veins to form a small vascular system. Although there are differences between plant and mammalian vasculature, plants such as spinach follow Murray’s Law – whereby branching veins taper as they lengthen, in much the same way as human veins and arteries do. And this is precisely what makes them ideal for this kind of research. Once the spinach leaf has been transformed into a human-like platform, the team introduces fluids and microbeads into the veins, observing the flow throughout the system. (To see a short video of the miniscule heart perfusing, click this link.)
Amazing as this might sound, there is a limit to the use of the technology. As the authors acknowledge in the paper published in the journal Biomaterials, there is still a lot of work to be done before plant-based engineered tissues could be used in medical applications.(5) One significant concern is that the residual chemicals used in the decellularization process could affect cellular viability. Also, there is a question of immune response. Used widely in regenerative medical applications such as the engineering of cartilage tissue, bone tissue, and wound healing, cellulose is both biocompatible and biodegradable.(6) But the ‘native response’ – that is, how we respond as biological organisms – of plant scaffolds within the body is currently uncharted territory. And then there is the issue of outflow. Unlike mammals, plants require no separate but complementary outflow systems so lack a venous system that returns fluids from the body back to the organ. So, in effect, even with its coating of human cells the plant-based scaffold is a closed system that allows – at best – one way perfusion.
Another problem is the potential for contamination…
According to reports in Food Safety News, an online repository for stories connected to food-borne illnesses to sustainability and food policy, some leafy greens – including spinach – have been vectors for myriad food-borne illnesses. A recall was enacted in California after elevated levels (10 times the average) of cadmium were uncovered in organic spinach originating in Salinas, CA. Cadmium is a heavy metal that naturally occurs in some soils and can impact the liver, immune system, and the kidneys. And then there were recalls of products containing spinach such as vegetable lasagna and spinach pizza (Amy’s Kitchen), dips (La Terra Fina), and ravioli (Rising Moon Organics).(7) Additional recalls of the erstwhile healthy vegetable have hit the news in recent times and part of the reason that spinach is a dangerous vector may be the topography of its leaves. In computational modeling at the University of California-Riverside, researchers tested the adhesion rates of E. coli bacteria to leaves after washing. Motivated by an E. coli outbreak in 2006 that sickened 199 people across 26 states, the team found that the industry standard for washing spinach – chlorine added to rinse water at 50 to 200 parts per million – was insufficient to dislodge and clean the vegetable.(8) The wavy, crinkled shape of the leaves effectively forms a barrier to the disinfectant, allowing the pathogens to remain on the leaves and forming a potential contamination hazard.
And then there’s the case of listeria.
More commonly associated with highly processed foods such as deli meats and hot dogs or with unpasteurized dairy products, Listeria monocytogenes is a food-borne pathogen that is most dangerous to vulnerable individuals such as seniors, children, those with compromised immune systems, diabetics, patients taking glucocorticosteroids, people with cancer or kidney disease, and women who are pregnant. Listeriosis manifests as nausea, diarrhea, headache, confusion, convulsions, and flu-like symptoms and a full diagnosis is made either by a simple blood test or by the somewhat more traumatic spinal tap – making it an illness best avoided where possible.
But, although it is a dangerous pathogen, it is not a contaminant we normally associate with salad greens, and those who avoid dairy and meats have traditionally considered themselves relatively safe. That is until last month when the results of a study conducted by Purdue University were published in Food Safety Magazine. According to the news alert, ‘Purdue Study: Listeria Can Thrive Inside Lettuce Tissue,’ the pathogen can survive attempts at sanitization by penetrating the inside of romaine lettuce leaves.(9) The bacteria make their way into the leaves by means of ‘cracked seed coats, tears in root tissue during germination, and by way of damaged plant tissue.’(10) And this contamination could lead to infection of the plant within as few as 30 minutes, with the potential danger to consumers lasting up to 2 months.
How can this happen?
Although listeria is commonly found in the soil and water used to grow and irrigate crops, it is also carried by farmed animals. The application of animal wastes as plant fertilizer may increase the risk of contamination, especially in ‘organic’ products where ‘natural’ animal fertilizers are used within the growing cycle. And given the relative dearth of ‘veganic’ crops (those grown organically and without the use of animal-based products) in the supply chain, this could be an important hurdle on the path to creating those extracellular plant matrices for human tissue engineering we described above. Although an incidence of food-borne listeriosis or E.coli poisoning is bad enough, contamination by either pathogen via a plant-based scaffold in vivo would be potentially disastrous.
Which is why we have to look extremely closely at the protocols and practices of those vendors who supply the raw materials – spinach leaves – for medical use in cleanroom environments.
Given that this is a brand new area of research, cGMPs and good agricultural practices (GAPs) are unlikely to have been fully established and the field is open for our industry to help shape future protocols. As contamination control leaders, we should be ready to help formulate best practices for cleanrooms, aseptic processing, sterile and pathogen-free environments, and contamination containment within this nascent intersection of agriculture and medical bio-technology.
And we need to be ready now.
Although there is a long way to go before the current WPI research can be utilized in a real-world medical scenario, its potential is already abundantly clear. With demand for transplant organs and tissues outstripping availability, any sustainable means of increasing successful donations is important. And key concept here is sustainability. As noted in Gershlak et al’s paper ‘Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds’ the use of animal-based tissues for transplant is not truly sustainable:
“Annually, 115 million animals are estimated to be used in research. Due to this large number, a lot of energy is necessary for the upkeep and feeding of such animals as well as to dispose of the large amount of waste that is generated. Along with this environmental impact, animal research also has a plethora of ethical considerations, which could be alleviated by forgoing animal models in favor of more biologically relevant in vitro human tissue models.”(11)
Human tissue models in combination with plant tissue scaffolds – the harmonious interlacing of two kingdoms in the search for enhanced human health. Cost-effective, ‘green’, and ethically-sourced, spinach – Popeye’s much vaunted superfood – may just be the secret ingredient in this recipe for regenerative medicine.
Love it or loathe it – we’re keen to hear your thoughts on that most polarizing of leafy greens. If you have strong opinions on spinach in medicine, perhaps it’s time to lettuce know!