Tutorial 3: Designing & testing immonumodulatory biomaterials

Question 1

A research group in Leiden is developing vascular grafts by implanting solid, non-degradable polymeric
rods under the skin. Implantation of the rod will trigger an inflammatory response, leading to
the formation of extracellular matrix. After development of the matrix, the cylinder + tissue is removed, after which the tissue tube is ready for implantation as a vascular graft.

Considering the different phases of the host response to a biomaterial, describe at which phase the researchers should take out the rod + tissue in order to develop a functional blood vessel graft, and briefly explain why this phase is the most appropriate in terms of matrix composition (consider the desired properties of a vascular graft). In your answer, include (at least) two dominant cell types present at this stage and briefly explain their function at this phase.

Answer 1

During the fibrous encapsulation phase. This is the most appropriate because the ECM that has formed by then is mainly fibrous collagen which provides the vessel with mechanical strength. Cell types in the graft at this stage will be:
- Activated myofibroblasts which produce the collagen
- And macrophages/ FBGCs which signal to and activate the myofibroblasts.

Question 2

List 4 different aspects in which the host response to the vascular graft will differ from the host response to the polymeric rod under the skin, and briefly explain (1-2 sentence) for each aspect how this will affect the host response.

Answer 2

• The vascular graft is exposed to a hemodynamic load. Macrophages are mechanosensitive so this may influence the macrophage polarization state. Shear stress may have an effect on cell recruitment/ adhesion from the bloodstream.
• Blood contact; the vascular graft is in direct contact with blood. This means that there is a risk of thrombosis.
• Different implant sites; the host response is known to be implant-site specific.
• The polymeric rod is solid, non-degradable vs the porous, degradable ECM; this will evoke a different inflammatory response.

Question 3

Describe 2 modifications that you would do to modify the vascular graft specifically for patients with chronic kidney disease and a reduced fibrogenic potential and explain why you would perform these 2 modifications.

Answer 3

Using a material with a longer degradation time to allow more time for sufficient tissue formation since the fibrogenic potential is reduced.

Using a more dense microstructure/ smaller pore size. This makes the graft less susceptible to degradation (because the cells will have trouble infiltrating) → typically leads to more fibrous encapsulation.

Question 4

What is the main motivation for chemical cross-linking of porcine heart valve prostheses?

Answer 4

The main reason is to mask the porcine antigens on the pig valve, in order to prevent immune rejection.

Question 5

One of the effects of chemical cross-linking is that it inhibits the fast degradation of the tissue. This can be both an advantage and a disadvantage. Make an argument for either case.

Answer 5

Advantage: Inhibiting the fast degradation rate is good for making a good connection in the body between the prosthesis and the native tissue. This is an advantage because it prevents the rapid degeneration of the valve, which is a common problem for heart valves because they function in such a demanding hemodynamic environment.

This is a disadvantage because it means that the cells cannot remodel the valve. So if there is a small damage of the hemodynamic loads change, the valve cannot adapt to this like a native valve can do. It also means that these valves cannot be sustainably used in children with congenital defects because they cannot grow with the patient.

Question 6

As an alternative to chemical cross-linking, a research group in Austria is developing decellularized porcine heart valves. During the development, the researchers are evaluating different decellularization methods. On deciding which method is the best, what would you advise the researchers to analyze their decellularized tissues for? Name at least three parameters and explain why.

Answer 6

DNA remnants and fragments. You want to have as little as possible in your tissue to prevent an immune reaction (rejection).

Measure the presence of matrix-bound antigens, such as alpha-Gal. Despite decellularization, there can be antigens left, typically bound to the matrix. This can be measured with assays if you know which antigen to look for. But there are also ways to check reactivity of decell. tissue with human antibodies.

ECM formation/ composition. There is a balance between the amount of DNA and the structure of a tissue. You would like to have the tissue as stable and good as possible. A Check by for example, measuring glycosaminoglycan content, collagen content, and structure or mechanical tests.

Question 7

What is the main downside of taking anti-thrombotic medication?

Answer 7

The risk of bleeding and hemorrhagic stroke is high: anti-thrombotic medication is systemic, affecting
the blood clotting capacity throughout the body.

Question 8

Warfarin and aspirin have a mechanistically different mode of action to prevent thrombus formation. Describe the anti-thombotic mode of action for warfarin and that for aspirin.

Answer 8

Thrombus formation involves the formation of a platelet plug by activate platelets and a fibrin clot via the coagulation cascade.

Warfarin is a Vitamin K antagonist. Vitamin K is essential for the activation of various factors in the coagulation cascade (e.g. prothrombin, factor II, and factor X). So warfarin blocks the formation of thrombin (and thus fibrin) in the blood plasma coagulation cascade.

Aspirin on the other hand is an anti-platelet drug, so it prevents the activation and aggregation of platelets into a platelet plug.

Question 9

Suppose the medical company St. Jude has developed a heart valve prosthesis from a new type of polymer. They give you some of the material and ask you to measure its thrombogenic potential. Based on your test, they would like to know for which patient cohort their new valve could be a good option and which medication (e.g. warfarin or aspirin, without considering any other negative side-effects of these specific drugs) would be most advisable. Describe how you would test this in an experiment. What would be the most important experimental parameters and which quantifiable would you measure as your read-out?

Answer 9

A The thrombogenicity is governed by platelet activation & thrombin/ fibrin generation. Consequently, it would be advisable to test both these wathways in vitro. Moreover, the wall shear stresses on a heart valve are of critical importance in thrombus formation. Taken together, it would be a good idea to use a simple bioreactor setup (suc as a parallel plate flow chamber or microfluidics chip) in which you can expose the material to a defined flow at various wall shear stresses. In the bioreactor you could perform 3 different experiments:
1. Expose the scaffold to platelets isolated from human blood and measure platelet activation on the material surface.
2. Expose the scaffold to blood plasma and perform a thrombin generation assay.
3. Expose the scaffold to whole blood and analyse the combination of both platelets and plasma proteins.

For whole blood experiments one should always consider appropriate use of anti-coagulation factors before the experiment to prevent that the blood immediately clots after it is taken from the donor. It would be advisable to include a sample from a known biomaterial as a control group, in order to compare the thrombogenicity of the new material to the known material that is already being used in the clinic.

Question 10

Medical devices such as urethral catheters, pacemaker leads and dental implants have a particularly high risk of biomaterial-associated infections. What is the cause of that increased risk?

Answer 10

The implanted devices have a high risk because it has a surgical wound and has, depending on the place, a porte d’entree. Therefore, bacteria can more easily acces the implant. .

Question 11

List 3 reasons why bacteria in a biofilm are difficult to treat.

Answer 11

1. Antibiotics cannot reach the bacteria because of the extracellular slime.
2. Antibiotics typically target the metabolism of bactetria. However, inside biofilm the bacteria
   can go into a dormant state, making them less susceptible for bacteria.
3. Bacteria in biofilm are present in a high local concentration; it is difficult to achieve a high
   local concentration of antimicrobial factors.

Question 12

Quorum sensing is an important mechanism in biofilm formation and the regulation of the bacterial transcriptome. Briefly describe (1-2 sentences) what is quorum sensing?

Answer 12

Quorum sensing is what the bacterias use to communicate with each other. One bacteria tells the other bacteria what to do, and they all follow. Quorum sensing is essential heard/ group behaviour. Bacteria use it to communicate to each other and act as a group, rather than as individual cells.

Question 13

List 3 clinically relevant reasons as to why antimicrobial therapy is often given in combinations of various antibiotics.

Answer 13

1. To broaden the antimicrobial spectrum; it is often not known which bacterium is active or bacteria are are active in combinations. By using a combination of antimicrobial therapy and antibiotics, several types of bacteria can be attacked instead of one specific type.
2. To prevent/delay that bacteria become immune to a specific, single antibiotic.
3. Antibiotics are much more effective when used in combinations.

Question 14

The risk of implant-associated infections increased with patient age. Explain why this is and what the underlying mechanisms are.

Answer 14

This is because the immune system goes into a senscent state with aging (=inflammaging). This means that (innate) immune cells will respond less fast and less effective in elderly patients. One of the reasons for this is a disturbed redux balance, which leads to a state of mild chronic inflammation.

Question 15

In the case of a synthetic material, various studies have shown that the pore size of the implant has a dominant effect on the foreign body response. List 2 cellular events that are important determinants of the foreign body response that are affected by the pore size.

Answer 15

The pore size affects many aspects of the host response. Important examples include, macrophage polarization, NETosis, ingrowth of microvasculature, influx of macrophages, degradability, etc..

Question 16

Our material indeed induces regeneration in Patient A, but it induces scar formation in Patient B. List 4 different host-related factors that may have caused this variability.

Answer 16

• Patient age: elderly have a reduced tissue forming capacity
• Patient sex: women are generally more prone to fibrosis than men.
• Lifestyle: patient lifestyle strongly affects the immune system
• Previous interventions or disease: this may lead to trained immunity
• Non-implant related co-morbidities such as obesity or diabetes; these strongly influence
  immunological and regenerative capacity

Question 17

In order to make our treatment suitable for Patient B, we decide to functionalize our matrix with an immunomodulatory factor. We have the choice of IFN-γ, IL-4, IL-10. Name which factor you would use, and briefly motivate why.

Answer 17

There is a case to be made for all three factors, but IL-10 is probably the best option here. Not only does IL-10 induce a favorable M2-type macrophage response, but it also inhibits myofibroblast activation, and thereby is a strong inhibitor of fibrosis.

IFN-y is a pro-inflammatory factor that is known to elicit strong anti-fibrotic functions. For that reason it is used to treat lung fibrosis (which is associated with plentiful presence of pro-fibrotic M2 macrophages). However, IFNy may stimulate prolonged chronic inflammation and a TH1/M1-dominated inflammatory response, which is likely to lead to a strong FBR in the long run.

IL-4 could be a good choice in the sense that it stimulated M2 polarization in macrophages, which is associated with functional regeneration. However, IL-4 stimulated M2 macrophages are also associated with fibrosis if not controlled properly, because they stimulate myofibroblast activation via the secretion of trophic factors, like TGF-b and PDGF. So it seems like a risky option in this case.