BACKGROUND

Dermal fibroblasts with impaired migration

for in vitro models of aged wound healing

for testing new biomaterials-based therapies

for chronic wounds

Elena García-Gareta, Alexandra Levin, Lilian Hook.

1RAFT Institute of Plastic Surgery, Leopold Muller Building, Mount Vernon Hospital , Northwood HA6 2RN, UK.

E-mail: garciae@raft.ac.uk

RESULTS

MATERIALS AND METHODS

AIM

CONCLUSIONS

• The geriatric population is prone to chronic wounds, which cost the

UK National Health System £4 billion/year. Current treatments are

inefficient.

• In vitro models that mimic the in vivo scenario would be ideal to

test biomaterial-based therapies for chronic wounds, to predict and

understand how they would behave in vivo.

• Aged dermal fibroblasts attach normally but show deficits in

migration due to a significant reduction in α2β1 integrin function.

However, expression of α2 integrin is normal [Reed et al. 2001; Mech

Ageing Dev 122(11):1203-20].

• Synthetic RGD peptides bind to integrins and have been used to

study cell migration or in biomaterials to promote cell attachment.

• To the best of our knowledge, no studies have used RGD peptides

to impair the migration of dermal fibroblasts, thus mimicking the

behaviour of aged dermal fibroblasts, for their further use in in vitro

models of aged wound healing for testing new biomaterials-based

therapies that address the clinically challenging chronic wounds.

To generate primary normal human dermal fibroblasts

with impaired migration using RGD peptides for in vitro

models of aged wound healing.

Routine surgical excisions of normal skin

Primary normal human dermal fibroblasts (pnHDFs)

Screening of conditions by alamarBlue® assay on 3 different surfaces

(uncoated, fibrinogen coated and gelatin coated):

• RGD peptide concentration (0 to 10.67mM).

• +/- pre-incubation pnHDFs/RGD peptides for 30min at 37ºC/5% CO2.

• alamarBlue® assay at 2h, 24h or 48h.

Choose conditions showing reduced

alamarBlue® activity on all 3 surfaces compared to control (0mM RGD peptide)

• Visual analysis of microscopy

images.

• Statistical analysis of

migration, cell number and viability results.

3) INTEGRIN EXPRESSION

• Immunocytochemistry (α2,

α5 and actin) and confocal

microscopy.

Conditions that impair

migration of pnHDFs without

significantly affecting cell

attachment of the population or

integrin expression

pnHDFs with

impaired migration

for in vitro models of aged wound healing

1) CELL ATTACHMENT:

• Phase-contrast light microscopy.

• Trypan blue assay on both

attached and unattached cells.

2) CELL MIGRATION:

• Scratch assays.

Complete bridging=2 Partial bridging=1 No bridging=0

4X

25µm

10X Viable cell count

Seed RGD treated pnHDFs on 3D dermal scaffolds to study attachment and migration:

testing of scaffolds in an in vitro environment that mimics the in vivo scenario.

1) CELL ATTACHMENT:

Trypan blue assay: As the concentration of RGD increases fewer cells are attached (*p<0.05

compared to 0mM). Unattached cells are in the media. Viability ≥ 96.25%.

Phase-contrast light microscopy: After addition of 2.67mM RGD cells with a “rounded” morphology

indicating reduced attachment were observed (white arrows in figure below left). In agreement with

the cell counts, samples with 5.33mM and 10.67mM RGD displayed large areas with noticeable fewer

or no cells (*in figure below right).

10X magnification 4X magnification

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2) CELL MIGRATION:

Adding RGD after formation of a

confluent monolayer did not have

an effect on cell migration (Top).

Adding RGD peptides at the time

of cell seeding impaired cell

migration (Bottom).

On gelatin the effect of RGD

peptides on cell migration was

concentration dependent.

3) INTEGRIN EXPRESSION:

Expression of integrins in the

presence of RGD peptides was intact.

Expression of integrins is seen

surrounding the cell nucleus, in

adhesion points (yellow arrows) and

at the leading front of migration where

lamellopodia are seen (white arrows).

Results suggest that RGD peptides

do not alter the components of the

migration pathway.

- Attachment and migration of pnHDFs can be tuned with synthetic

RGD peptides without affecting integrin expression and cell viability.

- In our study, 2.67mM RGD impaired migration without significantly

affecting attachment, thus mimicking the behaviour of aged dermal

fibroblasts in vivo.

- We are currently using RGD-treated pnHDFs to study attachment and

migration in 3D dermal scaffolds, thus testing them in an in vitro

environment that mimics the in vivo scenario.

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