Title: Capillary Formation in Bioengineered Human Skin Constructs (BHSC)
Designed to Study Burn Injury
Authors: Sanjay Kumar, MD, Jean-Luc V.Tran, MS, Josef Hadeed, MD, Emily
Bellavance, MD, Louise Strande, MS, Riva Eydelman, BS, Martha Matthews, MD,
Steven W Marra, MD, Charles Hewitt, PhD
There is a great deal of morbidity and mortality associated with severe burns. It has
been estimated that there are over 50,000 moderate to severe burns every year in the
United States, all of which require hospitalization for treatment. This places a
significant burden on society to continually improve the medical and surgical
management of the burn patient. The majority of burn research in the past has been
solely dependent on the use of animals, subjecting them to a great deal of pain and
suffering. This pain and suffering for biomedical research has come under scrutiny in
recent years and has been considered unnecessary by various animal rights groups.
In response to these concerns, we have developed a non-animal alternative for burn
research.
This non-animal alternative involves the use of bioengineered human skin constructs
(BHSC). These BHSCs are bioartificially created from human elements so that thermal
injury can be reproduced at certain molecular, cellular and even gross levels without
the use of an animal. In essence, the model consists of the cellular components of
human skin introduced to and induced to grow within a matrix of collagen
(AlloDerm). This BHSC is thus similar to human skin and when exposed to scalding
temperatures, will mimic the injury responses exhibited by human skin.
Previously, we have reported on successfully infiltrating human fibroblasts and human
keratinocytes into the scaffold of AlloDerm, thus creating a BHSC that closely
resembles human skin. We were also able to grow an epidermal/dermal bi-layer
morphologically similar to human skin. When this BHSC was subjected to high
thermal energy, histopathologic changes within the BHSC were consistent and similar
to those seen in burned human skin. Specifically, the changes included the following:
separation of the dermal-epidermal junction, blister like changes in the epidermis,
necrosis of the human fibroblasts and keratinocytes, and swelling of dermal collagen
fibrils.
In this synopsis of our current undertaking with the BHSC, we report the introduction
of human endothelial cells into the BHSC. Furthermore, these endothelial cells were
induced to form capillary beds. Addition of the vascular component to the BHSC
allows it to model human skin more closely and thus greatly enhances its utility in
burn research without the use of animals.
Methods: Neonatal human microvascular endothelial cells of dermal origin (HMVEC)
and microvascular endothelial growth media (EGM2-MV) were purchased commercially
(Cambrex Bio Science, Walkersville, MD). The EGM2-MV is an endothelial cell basal
medium which is supplemented with the following: human epidermal growth factor
(hEGF), basic fibroblast growth factor (hFGF-b), vascular endothelial growth factor
(VEGF), ascorbic acid (Vitamin C), hydrocortisone, human recombinant insulin-like
growth factor (R3-IGF), heparin, and 2% human serum. Growth parameters of
HMVEC in EGM2-MV were compared with various other growth media to determine
the best media for bioengineering a skin construct containing human fibroblasts
(NHSF), human keratinocytes (NHEK), and HMVECs.
Pieces of freeze-dried AlloDerm (Life Cell Corp., Branchburg, NJ) were rehydrated in
sterile culture media according to the manufacturer’s instructions. These pieces were
oriented with the basement membrane (papillary) side facing down and the dermal
(reticular) side facing up. NHSF were seeded onto the dermal side in a small volume of
media. These pieces were then placed into an incubator at 37C for one hour to allow
the cells to attach. After the attachment period, a small amount of NHSF growth
media supplemented with FGF-b, TGF-, platelet derived growth factor (PDGF),
ascorbic acid, and amphotericin B was added to each plate. Media was changed every
other day.
After 14 days of growth, constructs (with NHSF infiltrates) were oriented with the
dermal side up. Growth media was removed and HMVECs (104 cells/cm2) were plated
in a small volume of media on the construct. Samples were returned to the 37C
incubator for 1 hour to allow the cells to attach and then were covered with the
combination media described above. Media was changed every other day. Samples
were taken once a week for three weeks, fixed, cut and stained with H&E for
microscopic evaluation.
Immunohistochemical staining was performed on samples of the BHSC at weeks 2 and
3 to assess for the presence of select cellular markers specific to endothelial cells.
Immunostaining was performed for the endothelial adhesion molecules ICAM
(intercellular adhesion molecule), VCAM (vascular cell adhesion molecule), and Tie-1.
ICAM and VCAM belong to the immunoglobulin family of adhesion molecules that are
expressed on endothelial cells in the presence of inflammatory mediators, ie, cytokines.
They allow leukocytes to transmigrate across blood vessels during tissue inflammation.
The tyrosine kinase receptor Tie-1 has been shown in the past to be expressed
predominantly on endothelial cells involved in angiogenesis.
For each immunostain, digital image analysis (DIA) was performed using Image-Pro
Plus 4.0 to determine the amount of immunostain present within each specimen. An
average intensity stain index (AISI) was computed for each specimen. AISI correlates
with the amount of cellular marker expressed within each digitally captured image of
every slide. Image-Pro Plus 4.0 determined the integrated optical density (IOD), total
stained area and total tissue area for each image. These parameters were used to
calculate the AISI for each immunostained marker at weeks 2 and 3.
Results: Progressive infiltration of HMVEC was noted from weeks one to three. In
areas with significant endothelial cell infiltration, capillary formation was dramatic.
Infiltration was best when cells were seeded on the dermal side of the AlloDerm.
When seeded on the basement membrane side, the HMVEC tended to form a layer
and did not infiltrate very well. Endothelial cells seemed to migrate into channels in
the AlloDerm.
Areas of significant infiltration of endothelial cells also exhibited marked
immunohistochemical staining for the VCAM, ICAM and Tie-1 biochemical markers.
DIA performed on these stains revealed a progressive increase in the expression of
these markers with time. For the VCAM immunostain, at week 2, the AISI was 26.4
and 49.0 at week 3. For ICAM, the AISI was 48.2 at week 2 and 36.3 at week 3. For
Tie-1, the AISI was 48.2 at week 2 and 66.8 at week 3.
Conclusion: HMVEC do infiltrate BHSCs and appear to form capillaries and
microvasculature. DIA and immunostaining confirmed the marked presence of
endothelial cells and capillaries. Expression of Tie-1 by the endothelial cells supports
the fact that the cells are undergoing organization and forming blood vessels.
Expression of the adhesion molecules VCAM and ICAM signifies that the cells are
actually functional and capable of responding to a stimulus in preparation for
margination and transmigration of potential inflammatory cells.
The addition of endothelial cells, the vascular component of tissues, to the burn model
greatly enhances the utility and relevance of the BHSC. Further studies can now be
undertaken to elucidate the effects of burn injury on bioartificial skin tissue at the
molecular and cellular level without the use of animals. In addition, the current model
can be eventually utilized to study the effects of inflammatory and growth mediators on
burned skin to help determine how the effects of burns can be minimized at the
cellular level. Wound healing accelerants and topical drugs could also be tested on the
BHSC to reveal how best to treat skin damaged from intense thermal energy.
Adding specific cellular components to these bioartificial constructs opens the door to
studies in other fields as well. For example, inhibition of angiogenesis, as it pertains to
tumor biology, could potentially be studied with the BHSC. BHSC treated with topical
immunosuppressants may even be able to be used to model responses similar to skin
grafted patients.
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