Initial Immune Response to New Biomaterials

Annually 60.000 patients in Europe only are born with congenital heart failure.
These young patients have to go trough multiple reoperations, since the current
available valve prostheses cannot accommodate for growth. This leads to
increasing risks of morbidity and mortality.
Tissue engineering holds great promise to overcome these limitations. Over more
than 15 years, the Eindhoven University of Technology and the Zurich University
Hospital made great progress in the development of decellularized tissue
engineered heart valves (DTEHVs) in order to develop living valvular prostheses
that allow for growth. These DTHEVs are based on vascular-derived cells seeded
into biodegradable synthetic polymer scaffolds. Recently, we completed our
first long term in vivo follow up in sheep, in which it was shown that these
valves show rapid host cell repopulation. In addition, the DTEHVs demonstrated
an increase in extracellular matrix content over time, indicative for
regenerative and growth potential. These results are very promising and have
never been observed with any other type of valve prosthesis.

The exact mechanism behind the observed fast recellularization of the DTEHV
constructs is still unknown. A reasonable explanation might be that scaffold
remnants inside the tissue-engineered constructs recruit the circulating blood
cells. In order to investigate this mechanism, an advanced in vitro flow setup
was developed to mimic native-like in vivo conditions in terms of flow and
pressure. Herein, a biomaterial of interest can be subjected to human
circulating blood-derived cells. Since it is not yet known whether the
interaction of the blood cells with the scaffold remnants is favorable, we will
investigate the response of these circulating cells to assess if their response
is targeted more towards degeneration or regeneration. For this first pilot
study we would like to address the following questions:

• Do the scaffold remnants in tissue-engineered constructs recruit
blood-derived cells?
• Is there a different infiltration profile between certain blood-derived cell
subsets?
• Is the initial immune response to the biomaterial representing a degenerative
or regenerative response?

In the absence of useful literature information on the subject to perform a
power size calculation, we will start with a pilot study to validate the setup
and to assess valuable initial outcomes. To minimize the load on the volunteers
and taking into account the limited availability of DTEHV biomaterial, we will
start with a total of n=4 volunteers. These will be healthy male volunteers in
the age between 25 and 35.

At the day of the experiment, 50 ml of blood will be collected from a volunteer
via a single venous puncture after the volunteer had given us his consent.
After blood collection, the volunteer will no longer participate in the study.
The collected blood will be used immediately for the in vitro experiments.
Subsets of blood cells will be separated via density gradient spinning. The two
obtained subsets (the granulocyte and agranulocyte cell fraction) will be
separated. These subsets will be stained with a live cell staining, after which
the subpopulations will be mixed again. These stained cells will be used in the
in vitro experimental set up.

A sample of the stained cell population will be taken prior to exposure to the
biomaterial as a control sample, representative for time point 0. Subsequently,
the stained cells are exposed to the biomaterials in the flow-setup. In total,
4 individual flow systems are available for simultaneous use. Each system will
contain a different type of biomaterial. We have a scaffold-based sample, a
scaffold-based sample with tissue, and a non-scaffold based sample with tissue.
As a control group we have a test setup without a biomaterial. Each of the
setups will contain the same amount of blood from the same donor (7.5 ml each).

The analyses that will be performed on the circulating cell samples will be
flow cytometry, ELISA and qPCR to assess the composition and response of the
circulating cells to the biomaterials. During the in vitro experiment, samples
will be collected for analyses every hour. After a total of 5 hours, the
experiment will be terminated. The biomaterials will be processed for qPCR,
fluorescence microscopy and SEM analyses to assess the response of the
infiltrated cells into the biomaterials.

The physical load on the volunteers is low. Just one single vena puncture.
The associated risks are low and the total amount of collected blood is small
(50 ml)