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April 11, 2018

Ancient and Futuristic – What is the Role of the Cleanroom in Advanced Sericulture?

Silk Cocoons with WormWe have a riddle for you: what is both ancient and futuristic, biodegradable and naturally sustainable, is delicately engineered in a cleanroom with a tensile strength five times that of steel, is a simple formulation but has applications in materials science, medicine, and reforestation? Need some more clues? It is edible, reintegrating into the human body without causing immune response, but – when created in a contamination controlled environment – can also be used in micro-electronics and in photonics. Do you give up?

It’s a material that’s been around for more than five millennia and we are most accustomed to seeing it used in the fashion and textile industries. One final clue: most commonly, it is made from the larvae of mulberry worms. Got it yet? Yes, it’s silk. First developed in China, silk has been found in Neolithic tombs in Jiahu and legend has it that the material was first created for Chinese Empress Lei Zu. The secrets of sericulture – the production of silk – were closely guarded in China where it became a significant cottage industry, leading to the development of the Silk Road, a network of trade routes between Asia, the Middle East, and southern Europe. Beginning during the Han Dynasty of 207BC to 220BC, the protection of the trade routes led to the construction of the Great Wall of China and trade in silk spawned significant developments in the relations between countries as diverse as Japan, Arabia, Persia, and Korea. With parts of it now designated as World Heritage Sites by UNESCO, the Silk Road is the stuff of myth, legend, and powerful history.

The cocoon is formed from one continuous fine thread of 10µm in diameter which, when unraveled, will measure between 1000ft and 3000ft.

So, beyond being a shimmery, iridescent material used for neckties, now popularly resurgent in multiple shades of grey, what is silk and why is it so important? Ancient and modern sericulture both rely upon a diminutive and altogether less than attractive creature, the silkworm. Bombyx mori – Latin for the ‘silkworm of the mulberry tree’ – is an insect of the order Lepidoptera which, in its domesticated form, is now flightless, drained of natural pigmentation, and both tolerant of and reliant upon humans for survival. The adult moth most commonly used in silk production is bivoltine – meaning she lays eggs twice annually – and each mating cycle will produce around five hundred eggs. Depending upon environmental conditions such as temperature, eggs will take around two weeks to hatch into larvae which are then fed a diet of mulberry leaves. After the first molt, the worm moves into its instar phase which is characterized by an unengaging white appearance and protrusions on its back resembling horns. After four moltings, the larvae enter the pupal phase which is where silk production gets interesting. Using its salivary glands, the grub spins a cocoon of raw silk, composed of the protein fibroin and a gum, sericin. The cocoon is formed from one continuous fine thread of 10µm in diameter which, when unraveled, will measure between 1000ft and 3000ft. A pretty impressive achievement for this small, worm-like grub.

Traditionally, silk has been used for fine garment creation, parachutes, and fabrics for the home. It has a certain caché, an exotic – oftentimes erotic – feel that brings to mind the intoxicating mix of opulence and sensuality. So why are we discussing it in Cleanroom News? Although significant insofar as it has played a vital role in human economic and political history, our interest is not based on its use as a conventional textile. Instead we are more compelled by the polyversity of its physical properties, its ability – in other words – to shape shift into a whole gamut of forms such as gels, films, scaffolds, and particles.

As mentioned, silk is basically composed of two parts: the protein fibroin (70%-80%); and the adhesive sericin. Forming the structure of the thread, fibroin contains a light chain of molecular mass 25kDA and a heavier chain of 350kDA (Dalton units), linked via a disulfide bond. The glue-like sericin allows fibroin threads to stick together and is made up of a group of 18 amino acids, which together create three separate layers that are observable via gamma ray analysis. What is most intriguing is that these layers all run in different directions: the innermost fibers running longitudinally; the middle layer runs cross fiber; and the outermost layer of fibers runs directionally. And most importantly, each layer has its own specific type of sericin: for the outermost layer, there is sericin, A which contains amino acids such as aspartic acid and glycine and is water insoluble. The middle layer has sericin B that adds tryptophan to the mix, and the innermost layer – sericin C – also contains proline, a supplement frequently used in plant tissue culture growth medium.

…silk has been the material of choice due in part to its resilience, handleability, knot strength, and natural infection resistance.

Paralleling this triumvirate of sub-types are three critical properties which make the protein incredibly useful: elasticity; a tensile strength ten times that of polylactic acid and hundreds of times that of collagen; and an affinity for keratin. Described as ‘liquid Kevlar’ by Fiorenzo Omenetto in his 2011 TedTalk ‘Silk, the ancient material of the future,’ sericin (in the form of silk) has long been used as a suture material for closing wounds.(1) In fact, as far back as 1887, when Johnson & Johnson first developed the first modern sutures, silk has been the material of choice due in part to its resilience, handlability, knot strength, and natural infection resistance.

For Omemnetto, it points to an exciting marriage of this ancient natural material with ultra-advanced nanotechnology and microelectronics.

But as Omenetto – a biomedical engineer whose research interest include topics as diverse as nanostructured materials such as photonic crystals, nonlinear optics, and biopolymer-based photonics – points out, the 21st century applications for silk are so much more complex than simply wound closure. Silk proteins are natural self-assemblers and, when placed on a medium and allowed to self-assemble, can replicate its structure. For instance, when grown on the surface of a recorded DVD, a film made from proteins will replicate the exact surface of that medium. According to Omenetto, ‘the recipe is simple: you take the silk solution, you pour it, and you wait for the protein to self-assemble. And then you detach the protein and you get this film, as the proteins find each other as the water evaporates.’(2) So what does this mean? For Omemnetto, it points to an exciting marriage of this ancient natural material with ultra-advanced nanotechnology and microelectronics. The silk fibers can be engineered to follow the topography of the surface of a DVD to create an exact print, a replica not only of the medium but of the data encoded upon it. In other words, data that is written into the disk can now be stored on a silken film composed only of proteins and water.

If that is not tantalizing enough, what other uses could this material have? Beyond data encoding, there’s also the possibility of using this pure protein to guide light – in essence creating a sustainable, biologically-based optical fiber. Or there’s also the potential to create holograms on the silken film by adding a third dimensionality to the product and engineering the precise angle in which a 3D image can occur. And then, of course, with a few not insignificant tweaks to the properties of the material, there are the myriad additional medical uses. Let’s evaluate the possibilities…

If there is one common medical procedure that makes us uncomfortable – aside, of course, from the universally dreaded colonoscopy – it is the injection. Along with root canal surgery, getting shots ranks high on most patients’ list of things to avoid wherever and whenever possible. But whether it is the flu jab for seniors, routine childhood vaccinations, or Novocain at the dental surgery, ‘Hypodermic Hate’ is an inevitable part of our healthcare experience. Why? Apart from our perhaps natural reluctance to allow foreign matter to be introduced into our bodies, a metal needle piercing all too tender skin can be uncomfortable, even painful. But what if the so-called ‘liquid Kevlar’ could be used to create a microarray of needles that deliver the goods without the pain. For an analogy, consider the slender needles used in acupuncture treatments – with a skilled practitioner, most patients can barely even register the insertion of the needles and this could be a more common experience if all hypodermics were made of silk proteins instead of stainless steel.

Continuing in this medical vein (pun intended), the strength of the material means that silk protein could be used in place of surgical steel in bone replacement.

Providing, that is, it can be made biocompatible. The one challenge with natural silk proteins is the body’s innate response to sericin. According to a 2001 patent lodged by Allergan, a company based in Dublin, Ireland, sericin is ‘antigenic and elicits a strong immune, allergic or hyper T-cell type (versus the normal mild “foreign body” response) response.’(3) Removing the sericin allows biocompatibility but weakens the inherent strength of the product. However, using undisclosed and proprietary processes, Allergan creates natural silk fiber constructs that are substantially free (that is, have a sericin content of less than 20% by weight, and ideally less than 10%) of sericin. These constructs are not only non-immunologic but also promote ingrowth of cells at the wound site and are fully biodegradable within one year of continuous contact with bodily tissue.

So how are these ‘de-gummed’ fibroin fibers constructed? Stripping the fiber of its sericin coating weakens it and makes it vulnerable to breakage. To combat this, Allergan coats the silk fibers with either collagen or a peptide composition, which improves the rates of cell ingrowth and infiltration, along with promoting, cell attachment and spreading. And this new fiber can be ‘spun’ into a mesh or matrix, and ‘may be modified to comprise a drug associated or a cell-attachment factor associated with fabric (i.e. RGD). In one embodiment of the present invention, the fabric is treated with gas plasma or seeded with biological cells.’(4) With a fabric composed of engineered silk fibroin, tissue repairs to hernias, pelvic floor reconstruction, tendon, blood vessel or ligament replacement, and cartilage repair are possible, offering alternatives to the traditional materials used. But the possibilities are not limited to soft tissue issues. Take, for instance, the issue of angular limb deformities which can occur in young patients. In the case of trauma to the growth plate in the leg or arm, one bone will stop growing even while healthy cells in the other bone continue to develop. This uneven growth rate applies tension to the healthy bone, causing the limb to curve or bow. The only treatment for this condition is surgery – the breaking of the affected bone and insertion of a metal joining plate, effectively lengthening the bone. However, as the limb continues to grow, the plate may need to be removed and replaced, requiring multiple surgical interventions and increasing the trauma to the body. With a bridging plate composed not of surgical steel but of silk proteins, a strong connector could be created to link the two ends of bone which, when healed, would be reintegrated into the body. And what’s more, the formulation of such a putative alternative could be customized to include anti-rejection medication or antibiotics, delivered exactly where they need to be, released over time as the bone is healing.

And the research is on-going. Sofregen, a Boston, Massachusetts-based plastic surgery start-up, is using a proprietary form of liquid silk protein as the basis of its own surgical scaffold, a material that can be injected into the body to create a substrate upon which other natural tissues can build.

Sofregen is initially looking into applications that facilitate the re-growth of tissue for the vocal folds within the larynx, which would allow patients to regain lost speech. Over a 20 to 24 month course of treatment, the patient’s own tissues would grow over the web of sterile silk proteins to allow the folds to come together and enable the production of sound.

Having raised $6.2 million in venture funding in 2016, Sofregen went on to acquire the Seri surgical scaffold line from Allergan. Pending the success of additional fund-raising initiatives, Sofregen intends to further develop the technology to create new ways of correcting facial and other soft-tissue injuries, initially in combat veterans but then expanding into what is thought to be a very sizable market worldwide. According to Sofregen chairman Howard Weisman, interviewed for an article in MassDevice.com: ‘“The global market for products to address soft-tissue aesthetics is estimated to reach $5 billion next year. […] adding the Seri product line to our platform [means] continuing to help surgeons who are eager to restore confidence and improve the quality of life for patients around the world.”’(5)

Much of the engineering of bio-compatible silk takes place in cleanroom and contamination-controlled environments because, although the silk is ‘natural,’ contamination is a very real risk.

It’s clear that the market for injectable, silk fiber products is expanding but, as it does, it is critical that, as with any foreign material designed for use within the human body, standards of sterile manufacture are upheld. Much of the engineering of bio-compatible silk takes place in cleanroom and contamination-controlled environments because, although the silk is ‘natural,’ contamination is a very real risk. As discussed in our earlier article ‘How Mussel-Glue is Transforming the Field of Fetal Surgery,’ interest in the use of nature-inspired compounds in biomedical research is accelerating as we search for ever more bio-inspired, sustainable solutions. But ‘natural’ does not always equate to ‘safe.’(6) Anything that is either inserted into the body – micro needles, for instance – or which becomes a part of the body – bone grafts or hernia repair matrices – must be manufactured under the strictest SOPs, cGMPs, and codes of conduct. It is incumbent upon every research partner and manufacturer to ensure that all other regulatory protocols are strictly adhered to.

Ideally, we would expect that development facilities boast a dedicated cleanroom. In his 2011 paper ‘Cleanrooms and tissue banking how happy I could be with either GMP or GTP,’ J. Klykens of the Cell and Tissue Banks, University Hospital Leuven (Belgium) concludes

“A controlled environment is mandatory for tissue and cell processing. A monitoring program based on at rest measurements is feasible for all cell and tissue banks and it is a strong element in the control of the environment in which safe allografts have to be processed.”(7)

And while Klykens was speaking of facilities that specifically bank human tissues for transplant, this recommendation holds true also for silk fiber biomedical products intended for use within the body. So should we expect that this ideal would become the norm for silk fiber constructs? At this stage, it’s a matter of conjecture and waiting to see, although one thing is certain: the expanding market for these types of biosorbable products offers great spoils to the key industry players, and future market domination is at stake. In the interest of maintaining the galloping pace of development, at this early stage of its growth it behooves all manufacturers in the industry to promote the absolute sterility and safety of their products, to take far-reaching measures protect public health, and to give regulatory authorities no reason to slow the pace of innovation.

What are your thoughts on silk as an alternative to synthetics or collagen in medical procedures? Are you more interested in such surgical applications or in the data encoding possibilities presented by Fiorenzo Omenetto? Let us know in the comments!

References:

  1. https://www.ted.com/talks/fiorenzo_omenetto_silk_the_ancient_material_of_the_future#t-112497
  2. ibid
  3. https://patents.google.com/patent/US8628791
  4. ibid
  5. https://www.massdevice.com/sofregen-buys-allergans-seri-surgical-scaffold/
  6. https://cleanroom-news.com/2017/08/mussel-glue-fetal-surgery/
  7. http://orbi.ulg.ac.be/bitstream/2268/140648/1/Klijkens-Cleanrooms%20and%20tissue%20banking%20how%20happy%20I%20could%20be%20with%20either%20GMP%20or%20GTP.pdf
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