Alla Danilkovitch, RN, MS, PhD discusses the various placental tissue products available for wound care and advantages and disadvantages. Dr. Danilkovitch outlines examples of wounds treated with nonviable tissue and those treated with living tissue.
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Alla Danilkovitch has disclosed that she is an employee of Osiris Therapeutics, Inc.
TAPE STARTS – [00:00]
Male Speaker: – introduce Alla – how do I say it? Trouble saying your Russian name, Danilkovitch. Alla Danilkovitch who is the Chief Scientific Officer of Osiris and she’s really on the cutting edge of tissue preservation and delivering products with her techniques to the marketplace. And she’s been very instrumental in bringing some very important cryopreserved products to the market.
So we’ve asked her to speak on viable cell and tissue products on the shelf and she’ll probably present a new revolutionary, new type of preservation technique that I think you’ll find very interesting. So Alla, where – there she is. Let’s welcome Alla Danilkovitch.
Alla Danilkovitch: Thank you very much. Pleasure to be here. Every year is – it’s just traditional really to be at the Desert Foot, so many things we can learn.
And I would talk about new cell and tissue preservation technology that we believe can change the face of cellular therapies and this is what is not very far away. We believe next year that you will get in your hands this type of product.
And as a model, I will be talking about placental membranes and about wounds and I'm very proud that the doctors who will get products in – based on new technology, will be you, podiatrists who treat chronic wounds.
My disclosure is, I'm a fulltime employee of Osiris Therapeutics. No other disclosures. So the objectives of this lecture are to overview benefits of placental membranes for wound treatment especially for difficult-to-treat wounds.
Understand what components are so important in the membranes that make this tissue to be so valuable and beneficial for wounds.
Understand when you do preservation of the tissue, what may happen to the components of the tissue and how it will affect functionality of the tissue.
I will discuss about viable placental tissue outcomes in the treatment of chronic wounds and of course talk about our new technology for preservation of living tissues. You know all that chronic wounds statistics is scary.
And it’s very unfortunate that despite of all our efforts, the number of chronic wounds is going up, not going down and we need to do something about it. And more and more good products are coming, but more we know, more questions we will ask and hopefully addressing fundamental questions of the wound healing, we should be able to have better products and help patients to close their wounds.
When you start treating wound, of course you apply all your knowledge and provide good wound care. You debride the wound, your foot wound, you use appropriate dressings, but unfortunately you’ll know that more than 50% of such wounds will not close.
What do you after that? After that you are trying to reach for advanced wound care modalities. And one example is skin substitutes. You can see here on the slide by composition, there are so many different types of skin substitutes from very simple like collagen matrixes to very complex containing bioengineered material like bioengineered matrix with growing on living cells. And even right now for you, living products containing mesenchymal stem cells are available.
So when to use simple collagen matrixes and when to reach advanced cellular modalities? Not a long time ago, a panel of experts and Dr. Friedberg was part of this project, they were sitting together around a round table and discussing when it’s appropriate to use cellular products for wound care.
And here is in one slide, in bullet points, the key conclusion of this meeting of among experts, you need to think about cellular therapies for wounds when you see a patient with a lot of comorbidities. Why? Because wounds in these patients, they have cellular deficiency. They don’t have sufficient number of stem cells, they don’t have functional cells, the cells that are coming from periphery over the wound to the central part becoming senescent and die.
They do not produce growth factors and matrix proteins that are required for undergoing all phases of wound healing to the wound closure.
So what is so unique about living cells? So what is different between, let’s say, tissue product, placental tissue product containing dead cells versus placental tissue product containing living cells. The unique property of living cells that they are responsive to local microenvironment. And when you are looking on the wound, your wound is not static. Your wound will require different types of growth factors and cytokines, different amounts of them at different time points.
And if you put in the wound a cellular product containing beneficial growth factors and matrix, yes it is helpful. However, you are not changing the composition and no one will produce other growth factors if wound will need different profile of growth factors tomorrow. Viable cells, as a source of matrix and growth factors, can respond and address this issue, can produce growth factors as needed.
Now, on the next slide, you can see a table containing a list of cellular skin substitutes that I will go to you today. If you ask me to create similar tables for every product in the category of skin substitutes without living cells, I probably will present, say, the slides only listing these products. So it’s good that something is available. However, all what is available is on one slide. I hope that in the nearest future, we will hear for more cellular products for treatment of difficult-to-close wounds.
So if you look on the table, you can recognize, you’re very familiar a long time on the market bioengineered skin substitutes. Plastic surgeons, they were using cellular skin allograft a long time ago. What is good right now that there is another category of cellular product is viable placental tissues.
So I am now going to talk a little bit more about viable placental tissues. So placental tissues were used for wound treatment long time ago. Doctor Darius from Johns Hopkins, he described in 1910 how he used amnion, fresh amniotic tissue to cover burn patients. So why this tissue is so beneficial? It has properties that are required for wound treatment. Placental membranes, they are anti-inflammatory, anti-microbial. They promote vascularization of the wound, they prevent fibrosis. So everything what you need for wound, you can find in placental tissue.
It’s all attributed to three key components in placental tissue. It’s variety of different types of extracellular matrix, nature will present growth factors and cytokines and what is very important that placental tissues are very resource of stem cells. And these stem cells, there are not belonging to the mom. They are belonging to the baby. This is the youngest, the most functional stem cells you can find. And then not embryonic, they consider it young adult or neonatal when you harvest this from the full-term placenta.
Ideally, you will use fresh placental tissue because what can replace fresh tissue. But it is not practical for several reasons. Number one, not always fresh placenta will be available to you. And number two, what we know that you need to test donors and tissues for safety. Otherwise, you can transmit infectious diseases to your patient. So by saying that, from long time ago, people stopped using fresh tissue because of the fear of transmitting infectious diseases.
So scientists are very smart. They started developing different types of tissue processing. And here you see different tissue processing what are available right now for different types of tissues including placental tissues. The primary goal of selling tissue preservation is to retain all components that are beneficial, at the same time prolong life or the tissue, so that you will have product sitting and available to you every time and to create windows sufficient enough to test donors and products for safety.
You can see here that every company who offer you placental-based products, they start from fresh tissue and fresh tissue has three components. However, every company developed own way how to process tissue and depending on the way how it is done, so it can result into different composition of the final product. You can see depending what you use, you can add with just collagen matrix up to routine all components in the placental membranes. And growing body of evidence saying that preservation of all component of placental tissue is very, very important. So here you can see least of all some of the studies, in vitro studies, animal models and now clinical studies support this statement.
Let’s look how viable placental tissue perform clinically. So this is a simple table combining together all clinical data, use of placental membranes specifically for chronic wounds. And you can see outcome so that whether it is retrospective studies, whether it’s complex wound or a randomized control multicenter clinical trial, you can see very consistent result across all these studies.
How about – again question, do I need to use – and a lot of people are saying it doesn’t work to preserve cells because anyway cells will die in the wound and there are no data supporting that cellular product perform better than products containing nonviable cells. So I dug deeper in the literature and on the next slide you can see, this is right now what is available. Not a lot, but again, not one study.
You can see that there are several studies where product containing living cells were compared clinically to product containing nonviable cells. Right now, we can say it’s five to one in favor of cellular product. Two studies that published more beneficial cellular product or, let’s say, devitalized placental tissue was better than bioengineered construct coming from one researcher and it’s actually the same study, it's just two publications.
So you can see – and I hope that next year when I will come, I will show not only one slide but several slides that we will have more evidence. And I encourage you to go back to your own data if you use different types of products and look on the clinical outcome and share it with the medical community because this is how we learn, this is how we can all understand what is what and how different products are performed.
Again, I am a cell biologist believer in the living cells. And living therapists, they carry a lot of promise to cure diseases that we cannot cure right now. So the key benefits – again, I wanted to emphasize key advantages of living cells that cells will provide dynamic response to changing environment, whether it’s wound or surgical settings or genetic disease. And cells, they are a source of extracellular matrix and growth factors.
However, cells cannot live forever outside of the body. That’s why there is a disadvantage. You cannot store long time living cells and tissue. So if you do it at room temperature or refrigerate, you have very short shelf life for your product.
The most common method of a long-term storage of cellular products is cryopreservation. It is good that you can store for years cryopreserved products. For example, who is banking cord blood? Right now, you know that you can store at liquid nitrogen cord blood cells for 25 to 30 years. This is good. However, there is a disadvantage that this storage requires special shipment conditions and you need to have liquid nitrogen or deep freezers meaning that cellular therapy is not available to a lot of physicians who is practicing in a regular office.
So ideally, for people who are working in the area of cellular therapies, we all wanted to have cellular products and living tissue that you can store on the shelf at room temperature. And is it possible? Yes, it is possible.
If you look in the literature, people started thinking about it and worked in this direction more than 30 years ago. So here is next shot for foundation that it is possible to dry living cells and tissue and keep them alive. After rehydration, everything will come back.
So it’s based on our knowledge about small animals that exists in the nature. There are several different types of animals that can survive dehydration. And then after rehydration, they will come back alive and function.
So what we know right now, these animals, they have a high concentration of specialized molecules, some will have increased amount of special proteins, some will have increased amount of sugars that will help them to survive dehydration. So we’re already using the same chemical for cryopreservation and it looks like the same molecules can be used in life preservation or freeze drying of living cells and tissues. And you can see a long list of different types of methods, what scientists use, and prove feasibility of the drying living cells and tissues. And different types of cells that scientists try to dehydrate and keep them alive. So that knowledge serves as a foundation how we found the way how to freeze dry living tissue and keep it alive.
Freeze drying is not new, so everyone knows that pharmaceutical industry use it very widely. So that’s why lyophilizers with very fancy programs that you can play around with all possible parameters, they exist and it gives you possibility to upscale the process from the left scale to the commercial manufacturing.
Let’s take a look how dehydrated tissue prepared by new method look like. So you can see it looks like a dry paper look papers. If you rehydrate, it’s very interesting. If you rehydrate dry tissue done by regular method, it will not come back a fresh tissue.
So if you use a special method, what we use with the lyopreservative agent. So after rehydration, you can’t distinguish a lyopreserved tissue from cryopreserved and from fresh.
If you look histologically, the structure of the matrix in the tissue look the same between lyo, cryo and fresh tissue. But what is most important is living cells.
You can see here, fluorescent staining for living and dead cells. Green dots represent living cells and red dots represent dead cells. So amnion has two layers, epithelial layer and stromal layer. And you can see that very viable, both layers.
What is very important that not only after lyopreservation you can see a high percent of living cells in the tissue, in the same way as after cryopreservation. But if you put lyopreserved tissue in the culture dish, even after three weeks you can still see living cells that they persist, they start coming from the tissue, they proliferate and they are still alive.
How about functionality? Whether these living are still functional that can respond to a wound environment?
So key experiments in vitro, when living lyopreserved tissue and cryopreserved tissue were placed together with activated immune cells – and activated immune cells, they produce higher level of inflammatory cytokines, for example TNF or tumor necrosis factor alpha. Tumor necrosis factor alpha is present in all chronic wounds. And what is known in the literature, that there is a correlation between the level of TNF in the chronic wound and the ability of this would to close.
So ideally you need to downgrade the expression of TNF. And you can see on the graph that in the presence of lyopreserved and cryopreserved tissue, so they are able to produce anti-inflammatory factor and shut down production of TNF.
And that example on the right is hypoxia. Your wound is very often hypoxic. What do we need to do? You need to attract endothelial stem cells that are circulating in the blood to the area of the wound that these endothelial stem cells will become mature endothelial cells and will form new blood vessels. So if you place lyopreserved than cryopreserved tissue in a hypoxic chamber, you can see that in the response to lack of oxygen, both tissues will produce more vascular endothelial growth factor trying to attract endothelial cells to the area.
So how about in vivo? This is a chronic wound model in diabetic animals. So if you wound such animal and once block anti-oxidative enzymes in such mice, wounds will become chronic.
And here, if you will treat just with debridement and regular dressing, wound will never close and will become larger overtime. But if you treat such wounds with placental tissue with a lyopreserved or a cryopreserved, you can see that, like in humans, it requires several applications on a weekly basis prior to wound closure. But in both cases, both groups, like a mice, will receive the amniotic tissue, they close their wound. And they close their wound with the same rate whether it was cryopreserved or a lyopreserved tissue.
Again, if you compare prepared tissue devitalized, meaning it has all benefits of matrix, growth factors but no living cells versus lyopreserved tissue containing viable cells, you can visually that after three weeks in a simple model, just diabetic wounds in mice, you can see that after three weeks, tissue containing living cells already close the wound. But in the case of lack living cells, it’s much closer to the control.
So, again, just to summarize that lyophilization is not new, but all current products that available to you prepared by a traditional lyophilization method will have no living cells. Why? Because at the time of the freezing, no lyopreservative has used and when you freeze water without cryo or lyopreservative, you form water crystals that are killing cells.
So with the normal lyopreservation, you use first special chemicals that will prevent with the crystal formation. And then you need just to take this water from the solid phase into vapor. And that in the end, you will have all three components in the living tissue exactly the same like in the fresh tissue.
In summary, what you supposed to remember after this lecture? That number one, the key difference between products containing no cells versus viable cells that viable cells are responsive to changing wound environment and it will give you power to self-regulate the environment in the presence of viable cells.
To overcome challenges of storage of product containing living cells, new technology was developed. And using this new technology in the nearest future, you will have cellular products on the shelf in your office. We hope that this will open new possibilities and new doors for the use of cellular therapies. And we also hope that a lot of other areas not only treatment of chronic wounds will benefit because it will be easier and cheaper to produce cellular therapies and easier to use that everyone will have access to it. As I mentioned at every lecture that my dream that in several years, you will go to the grocery store and you will buy a bandage containing lyophilized living stem cells.
Thank you very much.
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