Tag: Science Club

SC9: Challenges of Moving from Bench to Bedside in Tissue Engineering

Written by: Roshni Rainbow, PhD (Clerk), Nathan Holwell (Chair), and Laura McKiel (Editor)

A typical process of moving from bench to bedside includes developing a material and performing in vitro testing, followed by in vivo testing in animals, and finally human trials. In vitro analysis begins to test the biocompatibility of the material using cells outside the body. Animal models are used for in vivo testing to take a more detailed look at the biocompatibility within a living subject. Researchers usually explore the material’s role in tissue viability, inflammation, cellular infiltration, and fibrotic response. Finally, human trials explore the feasibility of the material for the intended application supported by clinical trials.

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Figure 1: The typical process for drug development, testing, and approval. Source: http://oirm.ca/sites/default/files/oirm-infographic-nov2016-final.pdf

In the first breakout session, we explores the next steps of  two different biomedical technologies in order to move them into a clinical application.

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A major challenge of clinical trials is to appropriately scale-up the manufacturing process. A significant factor in scale-up is the propagation of cells and automating device production. As an example, microencapsulated pancreatic islets for diabetes treatment needs 500 islets for a 100 g mouse. To scale up to a 20 kg dog, 1,000,000 islets would be needed. Due to diffusion limitations, implanted capsules cannot be larger than ~200 µm in one direction. For a 100 g mouse, 1 microcapsule is required, but for a 20 kg dog one would need a 15 foot long string.

For clinical trials to begin, there are a few conditions that must be met. Firstly, the trial benefit must outweigh the risk. Secondly, the researchers must obtain free and informed consent from the patients involved in the trial. Finally, the protocol must be reviewed in advance by an independent review board. With this knowledge in mind, another example of a clinical trial was explored.

The Hepatassist Circuit Therapy by Circe Biomedical Inc. was a device to provide bioartificial liver support. When they performed their clinical trials, they had a total patient size of 116. However, 85 of these patients were part of the control portion of the study. This demonstrates the need to fully consider the number patients being studied with the actual device. The study population is especially important to consider when determining whether the material in questions has a true effect or not. And if the device does not show an effect, it is possible that it either doesn’t work or wasn’t tested in enough patients to show its true effect.

Next, we discussed the limited success of tissue engineering companies or firms. It was agreed that these companies or firms are good at discovery but lack in product development and regulatory medicine. Tissue engineered products are generally required to follow phase I/II/III regulatory pathways for pharmaceutical compounds. These submissions can cost anywhere from 300-500 million dollars. Tissue engineered products are usually discovered by smaller companies with limited funds. Due to this funds limitation, they cannot afford submission and must rely on investors. If trials are performed, they are usually done in a cheap manner. Unfortunately, these trials typically fail or only provide a narrow indication as to the material’s effect.

At the end of the session, we discussed a recent paper that commented on the greatest regulatory challenges in the translation of biomaterials to the clinic.

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This article tackles the issue of clinical translation of biomaterials from a variety of viewpoints. Lawyers, regulatory officials, clinicians, and researchers comment on what they perceive to be the most difficult challenge of translating biomaterials from the bench to the clinic.

Among the arguments raised in this article by the various viewpoints, one question loomed at the end: Should scientists innovate or translate?

SC8: Tissue engineering beyond medical applications… What’s next?

Written by Gad Sabbatier, Ph.D (Chair), Ashley Clarke (Clerk), and Laura McKiel (Editor)

Tissue engineering principles were defined in 1993 by Langer and Vacanti with a great promise to produce laboratory-made organs, regenerate organs, or repair an organ function for everybody. In 2017, technologies developed with tissue engineering and regenerative medicine principles cure a small number of patients compared to the demand. Due to a long (and largely undefined) regulatory process and revamping standards to fit with this new market, companies work for decades between the proof of concept and the commercialization of a tissue engineered medical devices and spend millions of dollars of investments. In the meantime, academic scientists have gained a lot of expertise and knowledge towards refining this learning process and developing a wide range of principles, techniques, methods, materials, proof of concepts and possible applications in tissue engineering and regenerative medicine.

During this session, we imagined ourselves in a start up company trying to commercialize a tissue engineered product, developing short term products and considering the expertise required to finance longer-term products. Two focus questions included:

  • What else we can do with tissue engineering principles to avoid or decrease time of regulatory pathway?
  • What’s coming next to tissue engineering?

 

How tissue engineering and regenerative medicine can help the food industry

Cultured meat is the most popular example in the media for the past few years. It consists of highly proliferative progenitor cells that differentiate into myocytes. The first cultured beef burger was produced by Dr. Mark Post group from Maastricht University and eaten in London in 2013. Many start-up companies have since started to culture fish, chicken meat, duck or pork meat. Of course, cultured meat needs optimization and scale-up to decrease the cost, but is estimated to be commercialized in a decade at a reasonable price. Lab-grown meat solves many agricultural issues such as the large greenhouse gas emissions of farming and water needed for livestock. This application will potentially require lighter regulation processes compared to tissue engineered medical products, but to date this still remains unclear. Read more about this topic here.

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Figure 1: An overview of the process of making tissue engineered meat. Source: Z. F. Bhat and H. Bhat. Journal of Stored Products and Postharvest Research, 2011, vol. 2, no. 1, pp. 1 – 10.

 

 

How tissue engineering and regenerative medicine can be used in the fashion and sports industries

Several researchers are trying to push the boundaries of the fashion industry by producing ivory, leather, silk, or fur using tissue engineering principles. These techniques can be employed to make ethical jewelry or luxury clothes that do not need to kill animals. Another project based on protocell technology envisions self-repairing personalized shoes that can react to pressure, heat, and topography (Figure 2).

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Figure 2: Concept of protocell shoes. Source: https://www.dezeen.com/2014/02/19/movie-shamees-aden-protocells-regenerating-trainers/

 

How tissue engineering and regenerative medicine can be applied to robotics

Many scientists are interested in bridging the gap between humans and robots by building bio-bots from living human tissues. Engineers from the University of Illinois made a walking robot with skeletal muscle cells on a 3D printed polymeric matrix that can be stimulated electrically to move (Figure 3).

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Figure 3: The walking bio-bot. Source: http://engineering.illinois.edu/do-the-impossible/biobots/

During the science club, we discussed the problem of fabricating a vascularized artificial skin for robots. Living skin on a robot would require the presence of blood vessels and artificial blood (blood/ biofluid supply system) with oxygen and nutrients, in addition to finding a means to distribute stem cells to replace apoptotic skin or blood cells. We also thought about a way to stimulate skin growing on orthopedics or exoskeletons by grafting peptides onto metal, to promote migration and adhesion of epithelial cells.

 

How tissue engineering and regenerative medicine can help the aerospace industry

Finally, we discussed how organs may evolve on Mars. Recently, NASA performed study to understand DNA changes due to durations in space. The DNA of twin astronauts Scott and Mark Kelly were compared after one brother had been in the International Space Station (ISS) for one year while the other stayed on earth. After the trip to space, the twins no longer had identical genetic code, proving that the environment in the ISS affects DNA. We imagined a series of experiments to study the development of organs in space or on Mars using stem or mature cells in a bioreactor that reproduces the Mars environmental conditions. Also, we wondered how non-Earth environments will affect the bodies and genetics of future generations of humans after colonization

SC7: The ethics of using stem cells in tissue engineering

Written by Gad Sabbatier (Clerk), Hossein Riahinezhad (Chair), and Laura McKiel (Editor)

Two weeks ago, we watched a documentary “Supercells: from stem cells to artificial organ” about trachea transplantation to illustrate the topic of our weekly science club.

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The documentary was about a young Russian woman, Yulia Tuulik, a former dancer with the bolshoï ballet, who needed a tracheotomy because of a car accident in 2011. A multidisciplinary team of surgeons, scientists, and industrialists lead by Paolo Macchiarini, a world known surgeon, implanted a bioengineered synthetic nanofibrous trachea seeded with bone-marrow derived stem cells prior harvested on the patient. Cells were seeded on the scaffold using a rotational bioreactor for 48 hours, and then suspended and observed every two hours in aseptic conditions. The patient underwent this “first-in-man” transplantation surgery on June 2012 at Krasnodarsk in Russia. The documentary has shown how this surgery was publicized by the media, and the success of the surgery after implantation.

Yulia Tuulik unfortunately died on September 2014 because of chronic inflammation, chronic coughs, and choking; Paolo Miccharini was found guilty for misconduct in 2015. This documentary is impressive, very well explained, and continues to be very popular on the web (4.5 stars on Amazon – outstanding comments in 2017), but fails to explain the death of the patient.

At the end of the movie, we discussed the limitations of the documentary. In the following, there is a summary of our discussion.

  • Everyone agrees that the relationship between doctors and the patient was constructive. It was shown in the movie that team of doctors visited Yulia on a regular basis before surgery and Mr. Green, CEO of Harvard Bioscience (who designed the bioreactor for cell seeding on synthetic trachea), showed the bioreactor to the patient and explained the basic science of what they want to do. Jed Johnson, CTO of Nanofibers Solutions and material designer of the synthetic trachea, was not involved in the surgery.
  • We also discussed about differences between scientists and surgeons, and the need to teach surgeons how to work with biomaterials that are produced by scientists. Based on part of the movie, Dr. Macchiarini tested the stiffness of the synthetic trachea by putting a bottle on top of it and checking how easy it was to handle and suture. On the other hand, scientists are more careful and design different experiments to measure mechanical properties of an implant. We are wondering if scientists should design materials which completely fulfill the surgeon’s expectations, or surgeons should be properly trained to use that material. We agree that it is necessary to train surgeons regarding handling of the material, storage, and some of the characteristics of the material that could lead to improvement of the surgery and patient outcomes.
  • Since the surgery was done in Russia, the regulatory process that authorize the surgery using this synthetic trachea seeded with stem cells and transplant it on human might have been different from the United States (FDA) or the European Union.
  • The patient was young (34 years-old) and in very good physical health (dancer), which played a key role into the choice of the patient for a successful first-in-man surgery.

Overall, this procedure showed that tissue engineered trachea transplantation may be premature and need more investigation.

SC6: In vitro, in vivo, and in situ tissue engineering: pros and cons

Written by: Hossein Rhiahinezhad (Chair), Gad Sabbatier (Clerk), and Laura McKiel (editor)

Our last short science club addressed the expansive topic of tissue engineering strategies. Tissue engineering (TE) consists of multiple principles, techniques, and methods from engineering and life science to understand the relationships between structures and properties of normal and pathological tissues. This multidisciplinary approach allows us to design and develop biological substitutes to restore, maintain, or improve tissue functions. Traditional TE techniques take a processed material scaffold, seed it with mammalian cells, and add biological, chemical, or physical stimuli to support tissue growing and differentiation. Cells that are isolated for tissues and organs can be mature, embryonic, or induced pluripotent, or multipotent stem cells, or differentiated cell types.

These are some examples of the diverse scaffolding strategies that are currently available:

  • Pre-made porous scaffold can be processed and then cell-seeded (e.g. fiber scaffold, foams, films).
  • Human tissue can be decellularized using chemical agents and then cell-seeded with treated cells.
  • Cells can be cultured to produce ECM and cell sheets. Sheets are harvested and assembled layer-by-layer to produce tissues and organs.
  • Polymer solution can be directly mixed with cells and crosslink either physically or chemically and can be used as 3D culture environment.

 

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Figure 1. Scaffolding strategies. Source: Chan BP. European Spine Journal, 2008; 17(Suppl 4), 467-479.

Tissue engineering can use in vitro, in vivo, or in situ strategies to construct tissues and organs. In in vitro TE, cells are seeded in a bio-instructive scaffold and cultured in a static (incubator) or dynamic (bioreactor) environment. The culture media is generally supplemented with biomolecules such as growth factors to differentiate stem cells into the desired cell type. A typical example is illustrated in Figure 2 for using TE to create blood vessels. Autologous smooth muscle cells, fibroblasts, and endothelial cells are harvested from the patient, and the cells are seeded in a tubular scaffold such as collagen or nanofibers and cultured in a bioreactor for a certain amount of time.

 

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Figure 2. Blood vessel tissue engineering. Source: Seifu DG et al. Nat Rev Cardiol, 2013; 10(7), 410–21.

In In vivo TE, the scaffold is implanted usually with cells and an animal is used as an incubator to grow the tissue or the organ before being re-implanted in the same or another patient. Figure 3 shows the impressive example of a human ear scaffold seeded with cow cells implanted in an immune deprived mouse. (Editor’s note: There was a huge controversy when this image was released to the media. You can read more about it here.)

 

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Figure 3. The Vacanti mouse. Original article: Y. Cao et al. Plast Reconstr Surg, 1997; 100(2), 297-302.

In in situ TE, the scaffold is implanted or injected with or without cells into the patient’s (or animal’s) body. The tissue is expected to self-repair due to cell migration and cells growing directly in the body’s environment, such as in the example illustrated in Figure 4.

 

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Figure 4. Cell transplantation with a cell instructive ribbons structure. Source: Sahar Salehi, ACS Biomaterials Science & Engineering, 2017; 3(4), 579-589.

 

The science club attendees were then divided into two groups to discuss two different scenarios:

Group 1: You are asked to prepare safety data sheets for 20 different chemicals and you need to check if human skin cells survive or die in presence of chemicals.

  • Design an experiment (in vitro, in vivo, or both)
  • State your reasons why you prefer one to the other, or why you need both

 

Group 1 suggested to do a viability experiment (MTT, WST-1, Resazurin salt, etc) and put human cells in contact with chemicals at different concentration in a high throughput screening fashion experiment. The experiment could respect the ASTM standards F895 and ASTM F813.

In vitro testing for cell or tissue growth offers less animal testing, cheaper experiments than in vivo, is faster, and can be used extensively. However, in vitro testing is very simplified compared to the complexity of the body, leading to many inaccuracies.

 

Group 2: A patient is suffering from osteoarthritis (degeneration of cartilage in joint) and you are asked to use TE to regenerate his/her cartilage. You have two options:

  1. Using scaffold for in vitro TE followed by implantation (in vivo).
  2. Using in situ TE by injection of cells and polymer solution (using as scaffold).
  • Which one do you prefer and why?
  • What are the challenges of each method?

 

Group 2 preferred approach B. In vitro TE option offers a better control of cell invasion, but is made without considering the immune system. It must survive to severe mechanical stimulation of the joint, as well as be resistant to infection after an open surgery. Option B requires a highly cytocompatible hydrogel that is able to crosslink in seconds, in order to be properly injected though a minimally invasive surgery.

 

Following discussion of the advantages and disadvantages of different tissue engineering methods, here is a summary of our conclusions:

in vitro in vivo in situ
Pros • Able to perform extensive studies with many cell types and testing conditions

• Less animals sacrificed for studies

·       More reliable results compared to in vitro studies • Less invasive (injection)

• More accurate results compared to in vitro and in vivo studies

• Less possibility of infection

Cons •   Not an accurate simulation of the body’s environment

•   Usually using cell lines, which do not accurately portray primary cells

• Potential for contamination

• High costs

• Ethical issues (think about animal study)

• Potential for infection

• Animal cells (and body systems) are very different from humans

• No control of scaffold once injected

• Ethical issues related to trials in humans

• Difficult to monitor over time

 

SC5: Stimuli-Responsive Polymers

Written by: Fei Chen (Chair), Ashley Clarke (Clerk), and Laura McKiel (Editor)

 

Polymers that respond sharply to small changes in physical or chemical conditions with relative large phase or property changes are termed “stimuli-responsive”. Smart biomaterials may be:

  1. Dissolved in or phase-separated out of aqueous solutions
  2. Adsorbed onto aqueous-solid interfaces
  3. Chemically grafted onto aqueous-solid interfaces
  4. Chemically crosslinked, H-bonded, and/or physically entangled as hydrogels.

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Figure 1: Examples of stimuli triggers for polymers. Source: Journal of Controlled Release 194 (2014) 1–19

 

Temperature Responsive Polymers

Poly(N-isopropylacrylamide) (PNIPAAm) is one of the most popular and well-studied temperature responsive polymers. It is soluble in organic solvents, such as chloroform and acetone, as well as cold water. Heating an aqueous PNIPAAm solution past 32 °C instantly coverts the clear solution into a milky suspension. PNIPAAm remains the leader for biomedical applications because of the sharpness of the transition, a transition temperature that is close to physiological temperature, and the availability of information on the polymer and its phase transition. The mechanism of phase separation at lower critical solution temperature (LCST) is large entropy gain caused by release of the hydrophobically-bound water molecules around the isopropyl groups.

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Figure 2: Stimuli-responsive behaviour of PNIPAAm. Source: Material Matters 2010, 5.3, 56

One of the main applications of temperature responsive polymers is used as smart cell culture surfaces. Basically, using an electron beam to polymerize PNIPAAm from a solution of NIPAAm monomer in 2-propanol can coat a layer of PNIPAAm on TCPS surface. The thickness of the PNIPAAm layer can be adjusted by the intensity, length of irradiation of electron beam, or monomer concentration. Cells adhere to the hydrophobic PNIPAAm surface during cell culture, then you use a temperature change from 37oC to room temperature to cause a phase transition in PNIPAAm to free the cells, which then are floating in solution. The smart cell culture surface could be advantageous over requiring a degradable scaffold for cell culture.

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Figure 3: PNIPAAm for use as a smart culture surface. Source: Material Matters 2010, 5.3, 56

Another biomedical application of temperature responsive polymers is for drug delivery. Since high molecular weight PNIPAAm can’t be degraded or excreted, so there is research into other polymers such as poly(lactide co-glycoide) (PLGA)-polyethylene glycol (PEG)-PLGA block copolymers, the idea is that self-assembled micelles containing the drug collapse into gels (thermo-gelling behaviour) at 37 °C, resulting in a long-acting drug release.

 

pH Responsive Polymers

pH responsive polymers normally possess a functional group that can donate or accept protons, such as acidic groups (-COO, SO3H) or basic group (NH2). One example is poly(methyl methacrylate-methacrylic acid) (MMA-MAA), which can change its hydrophobicity in terms of change in pH. When pH is lower than pKa of carboxyl group, it is hydrophobic, and if pH is above pKa of carboxyl group it is hydrophilic. One of the clinical application is for enteric coatings being applied to drugs.

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Figure 4: Poly(MMA-MAA) chemical structure.

 

Enzyme responsive Polymers

Enzyme responsive polymers are a new class of smart materials that undergo macroscopic transitions triggered by selective catalytic actions of enzymes. The use of enzymes as stimuli to trigger properties change opens up many possible applications in biology and medicine. One example discussed during the session was that a matrix metalloprotease (MMP) cleavable crosslinker was used to tune the mechanical and chemical environment of a hyaluronic acid hydrogel, to elucidate the role of the microenvironment in cancer cell invasion.

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Figure 5: MMP cleavable crosslinker used to alter the chemical and mechanical environment of a hydrogel. Source: Adv. Funct. Mater. 2015 , 25, 7163 −7172

 

Photo-Responsive Polymer

Photo-responsive polymers can change their physicochemical properties or degrade in response to light irradiation of appropriate wavelength and intensity. Light is a readily available trigger and is often inexpensive (i.e. lamps), while being able to be remotely applied and allowing for spatiotemporal control.

One example was discussed based on presenter’s own work under the supervision of Dr. Amsden (Biomacromolecules 2016, 17, 208−214). By copolymerizing a trimethylene carbonate with a pendant acrylate group (AC) with L-lactide (LLA) and e-caprolactone (CL) to form a copolymer, the copolymer can be printed by Melt Electrospinning Writing (MEW) process and then crosslinked by UV irradiation. The crosslinked fibers had significantly improved mechanical properties compared to non-crosslinked fibers, with the crosslinked hydrated fibers possessing elastic moduli of 370 MPa compared to about 40 MPa for the noncrosslinked materials. This high modulus was retained even after 10 000 strain-relaxation cycles, with the samples remaining intact after 200 000 cycles. Such photo-crosslinked scaffold could avoid the creep effect upon hydration of commonly used thermoplastics, such as PCL.

 

Group Discussion:

Question 1: Why are there so few smart polymers which have made it into clinic?

  • Potential cellular toxicity of the smart polymers is a main concern especially for applications involving intracellular delivery of biomolecular drugs
  • Many of the smart polymers are not hydrolytically degradable
  • PNIPAAm and polyacrylic acid (PAAc) are not readily excreted by kidneys and tend to accumulate in the body
  • Smart polymer drug delivery systems have rarely been used in the clinic may be simple injection of biomolecular drug without a carrier is just a simple and well-accepted drug delivery method for now. Benefits from using a smart polymer device need to be proven to justify the time and money spent developing the materials
  • It used to be a lot easier to get materials approved – today there are more regulations to meet and tests to perform

 

Question 2: Which applications of smart polymers will likely succeed in clinic and why?

  • Greatly depends on application
    • Tissue Engineering
      • Harder to simulate bodily environment for tests
      • Also, difficult to assess performance of grafts/scaffolds/other devices in vivo
      • May be developed for longer-term applications, so in turn it will take longer to test materials
      • Depends on whether you want to implant polymer scaffold with cells or wait for scaffold to degrade before implantation
    • Drug delivery
      • Short-term operation, so it may take less time to complete testing
      • Simple methods available to study release rate in vitro and able to test drug concentration for in vivo tests
    • Smart diagnostic applications
      • Testing material, if not in body the regulations are much easier to meet/test (e.g. Immunoassay)