The Effects of Type Beta1 Transforming Growth Factor on Fibroblast

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					 Preliminary Studies on Engineering of Human Skin
Substitutes: Effect of Transforming Growth Factor-
          on Gene Expression by Skin Cells
                               Chris Folts1 and Craig D. Woodworth2
                          Department of Biology, Clarkson University

            Skin – the largest organ of the human body – serves multiple purposes: (1) protection against the
assault of injurious UV radiation; (2) retention of necessary fluids, electrolytes, and proteins and the
prevention against dessication; (3) an immunological barrier between the body and surrounding environ-
ment; (4) thermoregulation; and (5) sensory reception. A breach of this organ’s integrity translates into a
breach of its functions; that is to say, an incurred wound threatens its efficacy and, as a result, jeo-
pardizes the survival of the afflicted individual. For this reason, the immediate repair of damaged tissue
is required. Frequently in wound repair, function is placed above form in terms of importance; the phy-
sical appearance of repaired tissue is often compromised by the rapidity with which it is mended. This
compromise has generated an immense amount of interest in wound repair and tissue regeneration – spe-
cifically in a mechanism of repair that minimizes the fibrotic or hypertrophic scar which naturally results
from such rapid processes of restoration.
            The process of wound repair and tissue regeneration involves both the epidermis and dermis – the
two layers into which the skin is divided. The former is exposed to the environment in which the
organism lives and the latter is attached to subcutaneous tissues by a basement membrane. Each is
stratified into various layers and composed of different cells, each with diverse functions. The epidermis
is traditionally divided into five sublayers – stratum basale, stratum spinosa, stratum granulosa, stratum
lucidum, and stratum cornea – and is primarily composed of keratinocytes; other cells – i.e., melanocytes,
Merkel, and Langerhans cells – are present, but only constitute two or three percent of epidermal resident
cells. The dermis, by contrast, is divided into three sublayers – papillary dermis, reticular dermis, and
hypodermis – and its resident cell population is primarily composed of fibroblasts; adipocytes are also
present in small numbers. The structure and function of skin are maintained by macromolecular secre-
tions of keratinocytes and fibroblasts, which produce keratin and collagen, respectively. Keratin forms
the cornified upper epidermal layer that protects the lower layers of skin – and, as a result, the body –

    Class of 2005, Bio-Molecular Science, Clarkson University Honors Program; Oral Presentation
    Associate Professor of Biology, Clarkson University
from desiccation and the absorption of deleterious UV radiation. Collagen – acting in conjunction with
other ultrastructural molecules, e.g., laminin, fibronectin, and glycosaminoglycans – forms the extracel-
lular matrix to which cells adhere and on which the morphology of the tissue as a whole is dependent.
        The layers of this tissue are studded with three ectodermal appendages: hair follicles, sweat
glands, and sebaceous glands. Hair follicles or pilosebaceous units are not found in the soles of the feet
or in the palms of the hand; their functions include insulation and moderate protection from UVB
irradiation. Sweat glands are responsible for secreting a hypotonic saline solution that controls systemic
thermoregulation by evaporative cooling. Sebaceous glands are found at the follicular bulge region of the
hair and secrete sebum, an oil that covers the cornified epidermis and contributes to its antidesiccatory
properties.   While epithelial cells of the sebaceous gland (sebocytes) and follicular cells are of
keratinocytic lineage, the origins of sweat glands are less clear.
        Normal wound repair is a carefully orchestrated process that involves most dermal and epidermal
sublayers, and frequently these ectodermal appendages. It is traditionally divided into three overlapping
phases: (1) inflammation; (2) proliferation; and (3) regeneration. In the first phase, neutrophils and
eventually macrophages invade the wound bed; these consume any invading bacteria and other exogenous
material. The release of specific cytokines – namely, PDGF, TGF-β, and TNF-α – by macrophages has
been shown to facilitate the transistion from the first phase to the second.         The second phase is
characterized by the rapid proliferation of fibroblasts and the deposition of structural proteins, e.g.,
collagen, fibronectin, and α-actin, which compose the granulation tissue. This is responsible for the
contraction of the wound and the formation of a fibrotic sheath for temporary protection. Simultaneously,
a new extracellular matrix is produced, a highway by which keratinocytes on the wound’s marginal
regions are able to migrate and ultimately differentiate into necessary cell lineages. The final phase may
occur over a period of months or years; in it, the wound site returns to a prototypic, functional tissue by
the processes of neovascularization, degranulation, and by the reduction of cellular number – specifically,
those cells produced in phase two. This phase may – and frequently does – result in fibrotic scar tissue.
        A fibrotic scar is not always produced; it has been shown that scarless healing is common in
embryonic and fetal wound repair pathways. Vascularization, ectodermal appendages, and prototypic
function in general are restored after a wound is inflicted on an embryo or fetus. If the mechanisms by
which fetal tissue is restored in a scarless manner can be elucidated, the medical or therapeutic
repurcussions would be immeasurable. Several differences have been cited between fetal and adult
tissues, ranging from different dermal fibroblasts to the expression of various cytokines (e.g., TGF-β).
        The process of tissue repair becomes complicated when a wound is thermally induced, i.e., a
burn, especially if the affected area is large. One complication with skin replacements in burn and lesion
patients is the absence of the three epidermal appendages. Epidermal and dermal layers, i.e., keratino-
cytes and fibroblasts, can be replaced, but the re-installation of these appendages has been rarely accom-
plished in vivo. Methods of replacement are varied. For small and superficial burns (first-degree or
epidermal burns), autologous keratinocytic samples can be excised from a donor site, cultured, and placed
in the denuded wound. This is not effective or practical for larger or deeper burns (second- or third-
degree) for several reasons. First and foremost, a larger burn requires a larger grafted sample from the
donor site; this increases the risk of infection and exposes the patient to excessive pain and discomfort.
Second, deeper burns that involve the denudement of both epidermal and dermal layers cannot be as
easily repaired or replaced as easily as superficial burns, i.e., a combined dermis-epidermis cannot be
cultured as easily; in most cases, the dermis does not attach to subcutaneous membranes and as a result
the engrafted tissue ceases to be a viable replacement.
        For these reasons, artificial tissues have been developed. They are varied – using autogenic or
allogenic cell samples, or occasionally both – cultured on various substrates. In essence, these engineered
tissues consist of a polymeric scaffolding that attempts to mimic the complex extracellular matrix of the
dermis, which is seeded with fibroblasts, keratinocytes, and factors that aid in the processes of re-epitheli-
alization, revascularization, and fibrosis. The science of drug delivery by degradable polymeric systems
has been modified to form the necessary base analogs or substrates for these new tissue models. Some
polymers being assayed include poly(lactide-co-glycolide), tyrosine-derived polycarbonates, and poly-
(ethylene glycol).
        The lab of Dr. Anja Meuller intends to test novel polymer scaffoldings and the lab of Dr. Craig
Woodworth will provide biological/genetic data. The union of the two will hopefully be an improved
skin substitute that avoids the complications of current models. The long-term aims of this project
include (i) to provide a viable, polymeric delivery scaffolding seeded with chemokines or cytokines
needed for modulated keratinocyte differentiation into the desired ectodermal appendages (e.g., hair
follicles); and (ii) to provide a replacement or subsitute tissue that reduces hypertrophic scarring and the
associated morbidity. The aim of the current study is to assay the activity of a cytokine involved in the
process of fibrosis – TGF–β1. If fibrosis can be controlled by down regulating the production of
structural proteins (e.g., collagen I and III) by fibroblasts, the morbidity associated with hypotrophic
scarring may also be reduced. The effects of TGF–β1 on both fibroblasts and keratinocytes will be semi
quanititatively assayed with reverse transcriptase polymerase chain reaction (RT-PCR). Two keratinocyte
and two fibroblast sample populations were cultured according to standard protocol – keratinocytes in
Keratinocyte Serum Free Medium (KSFM) and fibroblasts in Dubrecco’s Minimum Eagle Medium
(DMEM/F12) – and grown to confluent monolayers at 37°C. Each experimental dish received 6 mL of
respective media treated with 3 μL of a 3 ng/mL recombinant TGF–β1; each control dish received 6 mL
of untreated, respective media. After 24 hours of exposure, RNA was extracted from all treated and
untreated sample dishes from each of the four sample populations; the RNA was later purified and reverse
transcribed into cDNA. The expression of various genes – including those believed to be affected by
TGF–β1 (e.g., collagen Iα) and those associated with housekeeping (e.g., β-actin) for comparison – was
assayed. This presentation will convey the data which were generated by these analyses of gene

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