Slide 1:

Hello everyone! Welcome to module 3 of the basics of tissue engineering. I am Dario Rodriguez, and in this module, we will be talking about the aspects to consider for the clinical application of tissue engineering.

Slide 2:

In the previous modules we went over the key steps for engineering a tissue:

Biopsy

Cell Isolation

Proliferation

Differentiation

Tissue maturation

Implantation to patient

While this sounds straightforward, there are other important considerations in bringing engineered tissues to the clinic.

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The clinical application of tissue engineered products is a highly regulated and complex process. The FDA takes the accurate and verifiable data from past research and combines it with the testing needed to account for the patient’s safety before deeming a product usable in patients of a clinical trial. If all clinical trials are successful, we get a new FDA approved therapy. In the following slides we are going to discuss some of the aspects involving clinical use of engineered tissues.

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To work with these, first, we need Sterile environments to grow the tissues. These are specific areas or rooms free of microorganisms and toxins. Examples include: Biotechnology Manufacturing Facilities, Intensive care units, Operating rooms in hospitals, among others.

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Microbial contamination is found everywhere. This is a big issue because it can pose a threat to the tissue graft and the patient's safety. In order to protect the patient, the strictest aseptic techniques are implemented,

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Examples of these aseptic techniques include:

The Use of barriers: Clothing and Personal Protection equipment, incubators and biosafety hoods,

Patient and equipment preparation: aiming to prevent direct contamination of supplies and materials.

Environmental controls: working in a closed sterile area, proper cleaning, and air filtration systems.

Contact guidelines: allowing only sterile-to-sterile contact.

Aseptic techniques help us protect the patient from potential risks of contamination when growing our tissues for clinical applications.

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Following this line of thought for growing tissues, we have our second aspect to consider, which is minimal manipulation of cells and tissues. A crucial step in the process of tissue engineering is sourcing cells for the tissue graft. Cell isolation is a very involved process often utilizing complex chemical techniques to separate the desired cells from the tissue biopsy. The process by which cells are harvested from a biopsy must not alter the original characteristics of the cells. Remember, in clinical application we are dealing with human subjects and the patient’s safety most important above all.

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For our next aspect to consider, let us assume you have all previous steps right and you have managed to produce a sterile tissue following the aspects discussed so far, and now the FDA has approved your product for a small “first-in-human” clinical trial. Here, the surgeons are the designated personnel to do the implantation of your tissue-engineered product.

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In module #1, we mentioned that tissue engineering is a multidisciplinary effort of engineers, researchers and medical staff. When designing your tissue, it is important to have the input from all disciplines to tackle the clinical needs, tissue size, tissue strength, ease of surgeon use. Quite often, scientists create what they think are great products and are completely useless in the hands of the surgeon.

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For example, you managed to successfully create a tissue with YOUR desired characteristics, but it is missing the surgeon’s input in the design. Although you created what you thought was a functional tissue, when the surgeon tries to implant it using standard surgical procedures, your tissue falls apart. It was not strong enough to withstand the necessary stresses placed on it by the surgeon when placing it in the body. Bottomline, we want our tissue to resemble the native tissue inside the body and fabricate something that the doctors can easily implant into a patient.

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This brings us to our next aspect of the implantation. There are several things to consider beforehand in your tissue design: How is the graft going to be secured to the body? What are the potential stress points that may arise upon connecting the tissue to the body? To address this questions...

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...Let's look at abdominal Hernias for example. An abdominal hernia is when a tissue or organ exits through the abdominal wall cavity. It is caused by a combination of outward pressure and muscle weakness. Normally when repairing a hernia, after everything is put back in place, doctors use a special mesh to prevent the organ from exiting again while the tear in the abdominal wall heals. The design challenge for hernias is to develop a mesh that is stiff enough to hold the organ in a highly stressed area, and flexible enough so that it does not break nor damage the tissue it is attached to. When creating your tissue you may also face the same challenges as in the Hernia example. Our design must always take into consideration the necessary tools to implant the graft into the patient.

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Now, let’s assume we made it through the first few design aspects, and our product is successfully implanted.

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How do you keep the tissue alive after implantation? One important aspect that needs to be considered is tissue vascularization. In this example we can observe progressive damage and death of the tissue because it is lacking blood flow.

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Vascularization or blood flow is how tissues receive the oxygen and nutrients that keep them alive.. Engineering vascularized tissue is still one the biggest challenges of tissue engineering with the design complexity varying depending on the organ or tissue.

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When growing a tissue, you are supplying the nutrients in the media. These are absorbed by diffusion directly to the cells. When the tissue develops, depending on the type of tissue, it increases in size and can grow thicker which leads to a decreased access to nutrients and death of inner cells due to lack of nutrients. Vascularization helps to bring nutrients to all cells in a tissue regardless of location. Angiogenesis is the process through which new blood vessels form from other blood vessels. There are multiple strategies to promote vascularization of the tissue. Scientists have used growth factors, scaffolds, and genetically modified cells to trigger angiogenesis both in cell culture and in the body.

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However, the body can naturally regrow/grow new vascular tissue through the foreign body response mechanism. Foreign body response is the way our bodies protect themselves when foreign objects are inserted into the body. The body encapsulates the object in fibroblasts and other cells. But, more importantly it is capable of triggering angiogenesis around the encapsulated area. Scientists have capitalized on this natural defense response mechanism and use it to help vascularize the implanted tissues to supply nutrients during the repair process. However, a successfully vascularized tissue implant does not automatically restore functionality in the body. The repair is still missing one last component which is Stimuli.

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...Which brings us to our last aspect to discuss in this module, Tissue innervation. When the tissue is being cultured on a dish it can be artificially stimulated with electrodes and other mechanical forces. After implantation, these stimuli must be sourced from the body. Here, nerves are what drive electrical and mechanical stimuli of muscle and other tissues in the body. Through nerves we process all the motor functions and sensory data.

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To ensure reiinnervation, scientists have had to come up with different strategies to achieve the return of function of the tissue. Although many methods for reinnervation exist, when tested in clinical applications, the available options are narrowed down to very few techniques because of Patient Safety. The current techniques used are:

Neurorrhaphy: where the surgeon sutures the nerves back together,

Nerve Conduits: which are artificial grafts that facilitate nerve regeneration,

Neurotization: extension of a proximal motor nerve, and last but not least is

The Transplantation of full nerve sites.

In the end,the surgeon will help select the technique that fits better for your product since every case is a unique one and there is no perfect technique for all of them.

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In conclusion, testing for clinical applications must always have the patient's safety as its top priority. THIS is summary of some of the aspects to consider for clinical applications of tissue engineering. That's the end of this video! Thank you for watching and stay tuned for more Knowledge in Tissue Engineering.