Development of an in vitro keloid scar model for identifying and testing new anti-scar therapies.

Keloid formation is a result of adverse wound healing, in which a surplus of
scar tissue forms that exceeds the boundaries of the original wound. Currently
no adequate therapies exist and recurrences after treatment are common. In
order to develop better treatment strategies, superior scar models need to be
developed in order to test novel therapeutics, combinations of therapeutics and
to identify new drug targets. Since animals do not form keloids, relevant human
in vitro models need to be developed. Therefore we have developed a tissue
engineered basic keloid scar model from rest material isolated from excised
keloid scars. This model consisted of a reconstructed epidermis on a fibroblast
populated dermal matrix. Although this basic model did show abnormalities
indicative of a keloid scar developing, the strong keloid phenotype was
lacking. Recent literature suggests that an immune component (monocytes) is
essential for the development of keloids in addition to an intrinsic property
of the keratinocytes and fibroblasts. This finding therefore strongly indicates
that it is essential to include monocytes derived from keloid patients in the
keloid scar model. It stands to reason that other immune cells might also
demonstrate intrinsic abnormalities in keloid patients, therefore we aim also
to isolate T-cells in addition to PBMCs and bring these into co-culture with
the keloid model.

Of note, the objectives and end points described below all refer to in vitro
assessments. The only patient intervention is the removal of peripheral blood
in order to provide samples for the in vitro tests.

First Objective:
Development of a human, in vitro, immunocompetent full-thickness
tissue-engineered keloid scar model and identify robust and relevant parameters
for the assessment of the degree of keloid scar formation in vitro. The keloid
model will be compared with the healthy scar counterpart in order to identify
the parameters.
- In vitro keloid scar parameters: e.g.: increased epidermal and dermal
thickness; contraction, myofibroblasts (α-SMA expression), cytokine/ chemokine
secretion (ELISA).
- In vitro immune cell parameters: e.g.: changes in monocyte and T cell
phenotype after co-culture with keloid tissue compared to normal tissue. Flow
cytometric analysis to identify transition into e.g.: macrophages, Th1, Th2,
fibrocytes, endothelial cells etc.)

Second Objective(s):
Validate the in vitro keloid model with known therapeutics currently used in
the scar clinic of the Dept. of Plastic Surgery (e.g. 5FU, corticosteroids can
serve as positive controls) and therapeutics known to be ineffective in scar
treatment (e.g. vitamin D3 can serve as a negative control). The therapeutics
will be applied to the keloids during culture and at the time of harvesting,
the parameters identified in the first objective will be analysed. It is to be
expected that currently used scar therapeutics will (partially) normalize the
scar parameters whereas ineffective therapeutics will not. The model will then
be ready for testing new therapeutics and combinations of therapeutics in
preparation to clinical studies in the future.

Peripheral blood will be removed from keloid patients at the time when they are
scheduled for standard treatment in the Dept. Plastic Surgery. The blood will
be transported to the dermatology lab where PBMCs (e.g. monocytes and T-cells)
will be isolated for co-culture with skin equivalents. In parallel, peripheral
blood will be isolated from healthy volunteers with a known history of
developing normal scars.

Keloid skin equivalents and normal healthy skin equivalents will be constructed
from rest material obtained from standard surgical procedures. These will be
co-cultured with immune cells (monocytes, macrophages, T-lymphocytes, PBMCs)
isolated from peripheral blood with the aid of a transwell 2 compartmental
chamber. Hereafter, cultures will be harvested and scar forming parameters
assessed.

The total duration of the project is expected to take three years (Agentschap
NL financed project already underway). This includes the development of the in
vitro keloid scar model and its validation with therapeutics currently used to
treat scars and with therapeutics known to be ineffective in correcting scars.
Each independent experiment will require four independent runs each with
different donor material in order perform statistical analysis. Over a period
of three years, 140ml of peripheral blood will be drawn from a maximum of n=30
keloid patients and n=30 healthy volunteers in order to develop the
immunocompetent in vitro human keloid model and to validate it with known
therapeutics.

Patients with a history of keloid scar formation will be selected and
approached by plastic surgeons of the VUmc plastic surgery department during
their outpatient clinic sessions to participate in this study. Upon their
informed and signed consent, withdrawal of peripheral blood samples will be
done during regular clinic appointments.

Healthy controls known to form normal scars after trauma will also be selected
and approached by the plastic surgeons and researchers.

140 ml of peripheral blood will be withdrawn via standard procedures used in
venapuncture. This is not part of the medical treatment for the patient and
therefore is extra for this study. This blood will only be used for the
isolation of immune cells, no further laboratory tests (clinical/diagnostic or
otherwise) will be performed.

A questionnaire composed of eight questions is required.

Venapuncture itself is associated with slight risks such as excessive bleeding,
fainting or feeling light-headed, hematoma formation and infection (minimal
risk occurring whenever there is a break in the skin). However, venapuncture is
used on such a large scale without any problems worth mentioning, that there
are few people left who have not ever undergone the procedure. In addition to
these risks, there is a theoretical risk of keloid formation at the site of
venapuncture in keloid patients, although no such event has ever been described
in literature.
While there is no direct benefit for the patient group, our study may lead to
improved treatment strategies for keloid patients and could therefore certainly
be of benefit to them in the future. Given the limitations of current therapies
and their inability to effectively treat keloid scars, keloid patients are
often very willing participants because they know research is the only way
forward towards better management of their condition.