U.S. patent application number 11/562703 was filed with the patent office on 2007-06-28 for hair follicle graft from tissue engineered skin.
This patent application is currently assigned to ADERANS RESEARCH INSTITUTE, INC.. Invention is credited to Thomas H. Barrows, Preeti MacIntyre, Kenneth Justin Washenik.
Application Number | 20070148138 11/562703 |
Document ID | / |
Family ID | 38068047 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148138 |
Kind Code |
A1 |
Barrows; Thomas H. ; et
al. |
June 28, 2007 |
HAIR FOLLICLE GRAFT FROM TISSUE ENGINEERED SKIN
Abstract
The present invention provides a hair graft comprising (a)
tissue engineered skin comprising a tissue engineered epidermal
layer, a tissue engineered dermal layer, and hair follicle
progenitor cells and (b) a scaffold. The invention also provides
methods of making and using the hair grafts of the present
invention.
Inventors: |
Barrows; Thomas H.;
(Austell, GA) ; MacIntyre; Preeti; (Marietta,
GA) ; Washenik; Kenneth Justin; (Beverly Hills,
CA) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH, LLP
ONE SOUTH PINCKNEY STREET
P O BOX 1806
MADISON
WI
53701
US
|
Assignee: |
ADERANS RESEARCH INSTITUTE,
INC.
9100 Wilshire Boulevard East Tower Penthouse
Beverly Hills
CA
90212
|
Family ID: |
38068047 |
Appl. No.: |
11/562703 |
Filed: |
November 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60738931 |
Nov 22, 2005 |
|
|
|
Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61L 27/58 20130101;
A61L 27/60 20130101; A61L 27/362 20130101; A61K 35/36 20130101;
A61L 27/3804 20130101; A61L 2430/18 20130101; A61L 27/3886
20130101; A61K 35/36 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/093.7 |
International
Class: |
A61K 35/36 20060101
A61K035/36 |
Claims
1. A hair graft comprising (a) tissue engineered skin having a
tissue engineered epidermal layer, a tissue engineered dermal
layer, and hair follicle progenitor cells; and (b) a scaffold,
wherein the graft is at least about 2 millimeters thick.
2. The graft of claim 1, wherein the hair follicle progenitor cells
are located between the dermal and epidermal layers.
3. The graft of claim 1, wherein a surface of the epidermal layer
of the graft is about 1 to about 9 square millimeters in area.
4. The graft of claim 1, wherein a moiety is associated with the
scaffold.
5. The graft of claim 4, wherein the moiety is selected from the
group consisting of a growth factor, an angiogenesis factor, a cell
attachment binding site moiety, a cell signaling molecule, a
polypeptide, a glycoprotein, and a bioactive molecule.
6. The graft of claim 1, wherein additional cells are associated
with the scaffold.
7. The graft of claim 6, wherein the additional cells are selected
from the group consisting of fat cells, pre-adipocytes, vascular
endothelial cells, and bone marrow cells.
8. The graft of claim 1, wherein the hair follicle progenitor cells
comprise dermal papilla cells.
9. The graft of claim 1, wherein the hair follicle progenitor cells
are aggregrated.
10. A method of making a hair graft, comprising: a) placing a
tissue engineered dermal layer on a bioabsorbable scaffold; b)
placing hair follicle progenitor cells on the tissue engineered
dermal layer; c) placing a tissue engineered epidermal layer on the
hair follicle progenitor cells to form a construct; d) allowing the
construct to mature in vitro; and e) cutting the construct into
implantable grafts.
11. A method of implanting a hair graft comprising creating a wound
in skin of a subject and implanting the graft of claim 1 into the
wound.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/738,931, filed Nov. 22, 2005, which is
incorporated by reference herein.
BACKGROUND
[0002] Hair loss may occur due to a variety of conditions and may
affect anyone: men, women and children. Hair loss conditions
include, but are not limited to, alopecia capitis totalis, i.e.,
loss of all scalp hair, alopecia universalis, i.e., loss of hair
over the whole body, alopecia areata, i.e., patchy hair loss, and
androgenetic alopecia, i.e., male pattern baldness. Medications are
available to treat alopecia including minoxidil, finasteride,
corticosteroids and anthralin. However, any new hair growth
resulting from the medication generally stops upon discontinuation
of the medication.
[0003] More aggressive hair restoration methods include hair
transplants and scalp reduction surgery. Hair transplantation
entails excision of a full-thickness strip of scalp tissue from the
back of the head, dissecting the excised scalp tissue into hundreds
of "follicular unit grafts", each containing from one to several
hairs, and implanting the grafts into recipient sites created by
making stab wounds in the bald sections of the scalp. Hair
transplantation creates no new hair follicles and often not all of
the explanted follicles successfully transplant. Scalp reduction
surgery, which is becoming less popular, aims to surgically reduce
the area of bald skin on subject's head. Both hair transplantation
and scalp reduction surgery are expensive and may be painful.
Moreover, both carry possible risks of infection and scarring.
[0004] It is well known that specific cells within the hair
follicle, including epidermal stem cells and dermal papilla cells,
have the capacity to induce follicle neogenesis. Attempts have been
made to exploit the inductive capabilities of these cells,
including injecting dermal papilla cells directly into the skin and
implanting plucked hairs carrying epithelial cells having various
proliferative and differentiative characteristics. Previous
attempts at producing follicle neogenesis have failed to produce
reliable, reproducible and cosmetically satisfactory results.
SUMMARY
[0005] In one embodiment, the present invention provides a hair
graft comprising (a) tissue engineered skin comprising a tissue
engineered epidermal layer, a tissue engineered dermal layer, and
hair follicle progenitor cells and (b) a scaffold, wherein the
scaffold is at least 2 millimeters thick.
[0006] In other embodiments, the invention provides methods of
making and using the hair grafts of the present invention.
DETAILED DESCRIPTION
[0007] The present invention provides a novel graft for hair
follicle formation comprising a layer of tissue engineered skin
comprising a tissue engineered epidermal layer and a tissue
engineered dermal layer and hair follicle progenitor cells on a
bioabsorbable scaffold. Tissue engineered skin by itself is
suitable for transplantation. However, it is extremely thin and
difficult to manipulate using traditional hair graft techniques.
The scaffold provides rigidity and stability to the tissue
engineered skin so that the graft can be easily manipulated.
[0008] The tissue engineered skin may be prepared by any suitable
method known to one skilled in the art. For example, human neonatal
foreskin tissue can be used as a source of human dermal fibroblasts
that are multiplied in culture and seeded onto a scaffold such as
collagen gel to provide a tissue engineered dermal layer. Epidermal
keratinocytes can be obtained from the same neonatal tissue or,
alternatively, obtained from plucked hair follicles. A tissue
engineered epidermal layer can be produced from plucked hair
follicles as disclosed in U.S. Pat. Nos. 6,730,513, 6,673,603,
6,548,058, 5,968,546, and references cited therein, the teachings
of which are incorporated by reference herein. The tissue
engineered dermal layer and tissue engineered epidermal layer can
be separately prepared and then assembled into tissue engineered
skin with hair follicle progenitor cells dispersed therein,
suitably sandwiched between the two assembled layers. The tissue
engineered dermal and epidermal layers can be prepared without the
use of a scaffold, for example by the method described by Pouliot,
et al. in Transplantation, 2002 Jun. 15; 73(11):1751-7, and
references cited therein, the teachings of which are incorporated
by reference herein.
[0009] In one embodiment, the tissue engineered skin containing
follicle progenitor cells is placed on a bioabsorbable scaffold of
suitable thickness to form a construct and is cultured further in
vitro until the living skin construct is firmly attached to the
bioabsorbable scaffold. In another embodiment, the tissue
engineered dermal layer is placed on a bioabsorbable scaffold. Hair
follicle progenitor cells are placed on the dermal layer and then a
tissue engineered epidermal layer is placed on top of the hair
follicle progenitor cells to form a construct. Alternatively, the
tissue engineered dermal layer can be formed on a bioabsorbable
scaffold. Hair follicle progenitor cells are then placed on the
dermal layer and a tissue engineered epidermal layer is placed on
top of the hair follicle progenitor cells.
[0010] In another embodiment, the tissue engineered dermal layer
can be prepared on a bilayer scaffold comprising a bottom layer of
an artificial skin implant, such as the Integra Dermal Regeneration
Template.TM. (Integra NeuroSciences, Plainsboro, N.J.) in which the
silicon rubber layer has been removed and replaced with a top layer
of collagen. The collagen coating is then seeded with dermal
fibroblasts and cultured in vitro.
[0011] Suitably, the hair follicle progenitor cells may be
mesenchymal stem cells, dermal papilla cells, dermal sheath cells,
follicular epidermal stem cells, also known as "bulge" cells, or
any combinations thereof. Suitably, the progenitor cells are
aggregated or clumped together prior to placement in the tissue
engineered skin. The size of the aggregates is suitably from about
10 to about 500 microns, or about 20 to about 200 microns or about
30 to about 60 microns.
[0012] The construct comprising the tissue engineered skin
containing hair follicle progenitor cells and the bioabsorbable
scaffold is then cut into grafts suitable for implantation into the
skin. The construct is cut such that grafts with the epidermal
layer facing one direction and the scaffold facing the opposite
direction are formed. The size and shape of the cuts is optimized
such that little or no construct is wasted and the longest
dimension is perpendicular to the tissue layers. The grafts are
suitably cut into a size that is equivalent to a single hair graft,
a follicular unit graft or modified follicular unit graft.
Typically, these grafts are about 1 to about 9 square millimeters
in surface area, or about 2 to about 8 square millimeters, or about
4 to about 6 square millimeters. The thickness is suitably about
the same as the thickness of scalp skin. Suitably, the graft is at
least about 2 millimeters thick, or at least about 5 millimeters
thick, or at least about 8 millimeters thick, or at least about 10
millimeters thick. As used herein, "thick" is used to describe the
height of the graft, i.e. the z-axis of the graft.
[0013] The bioabsorbable scaffold is a non-cytotoxic structure or
substance that is capable of containing or supporting living cells
and holding them in a desired configuration for a period of time.
The term "bioabsorbable" refers to any material the human body can
break down into non-toxic by-products that are excreted from the
body or metabolized therein. Suitable bioabsorbable materials for
the scaffold include, but are not limited to, poly(lactic acid),
poly(glycolic acid), poly(trimethylene carbonate),
poly(dimethyltrimethylene carbonate), poly(amino acids)s,
tyrosine-derived poly(carbonates)s, poly(carbonates)s,
poly(caprolactone), poly(para-dioxanone), poly(esters)s,
poly(ester-amides)s, poly(anhydrides)s, poly(ortho esters)s,
collagen, gelatin, serum albumin, proteins, polysaccharides,
mucopolysaccharides, carbohydrates, glycosaminoglycans,
poly(ethylene glycols)s, poly(propylene glycols)s, poly(acrylate
esters)s, poly(methacrylate esters)s, poly(vinyl alcohol),
hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate,
versican, copolymers, blends and mixtures of polymers, and
oligomers containing bioabsorbable linkages.
[0014] For example, hyaluronic acid may be converted into an
insoluble crosslinked material ("HAX") by treatment with a
condensing agent, suitably 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide ("EDC"). Alternatively, hyaluronic acid may be
converted into an insoluble material by esterification, e.g., the
benzyl ester of hyaluronic acid, and used to prepare the
bioabsorbable scaffold. Suitably, trans-esterification crosslinked
HAX is used because the resultant product is then converted back
into soluble hyaluronic acid upon hydrolysis of the ester linkages.
Hydrolysis of the ester linkages takes place within a few days in
vivo. Various cross linking agents may be employed including, but
not limited to, aliphatic diamines, diaminoacid esters such as
alkyl esters of lysine, and amine-terminated poly(ethylene
glycol).
[0015] Various molecular moieties may be associated with the
bioabsorbable scaffold using, for example, surface modifications,
graft polymerization, copolymerization of bioabsorbable materials
or blending of at least one moiety and the bioabsorbable
material(s) used in forming the bioabsorbable scaffold. Moieties
that may be associated with the bioabsorbable scaffold include, but
are not limited to, growth factors, cell attachment binding site
moieties, angiogenesis factors, cell signaling molecules, other
small molecules, e.g., drugs that enhance hair follicle regrowth
such as monoxidil, glycoproteins, e.g., chondroitin sulfate,
dermatan sulfate, and versican, other bioactive molecules or
combinations thereof.
[0016] Association of at least one moiety with the bioabsorbable
scaffold may suitably be advantageous for improved association
between various types of hair follicle progenitor cells and/or
improved cell function, cell aggregation or cell initiation of the
follicle neogenesis process. Attached moieties, such as growth
factors and angiogenesis factors, may be released during the
degradation of the bioabsorbable scaffold and encourage blood
vessel growth into the newly formed follicle. Attachment of higher
molecular weight moieties, such as proteins, glycoproteins, and
other biopolymers, such as collagen, laminin, and fibronectin, may
be physically or electro-statically bound to the bioabsorbable
scaffold to suitably provide greater physical integrity, cell
attachment capacity, or bioactivity.
[0017] For example, association of bioactive molecules to the HAX
structure suitably enhances the performance of the resultant
scaffold, e.g., association of peptides containing the cell
attachment domain sequence of amino acids Arg-Gly-Asp (RGD) may be
used to enhance dermal papilla cell attachment to the scaffold.
[0018] The term "growth factor" refers to a naturally occurring
protein capable of stimulating cellular proliferation and cellular
differentiation. Growth factors are important for regulating a
variety of cellular processes. Well-known growth factors suitable
for use in the present invention include, but are not limited to,
granulocyte-colony stimulating factor ("G-CSF"),
granulocyte-macrophage colony stimulating factor ("GM-CSF"), nerve
growth factor ("NGF"), neutrophins, platelet-derived growth factor
("PDGF"), erythropoietin ("EPO"), thrombopoietin ("TPO"), myostatin
("GDF-8"), growth differentiation factor-9 ("GDF9"), basic
fibroblast growth factor ("bFGF" or "FGF2"), epidermal growth
factor ("EGF"), placenta derived growth factor ("PLGDF"), and
hepatocyte growth factor ("HGF").
[0019] Similarly, the term "angiogenesis factor" refers to a
naturally occurring protein capable of stimulating angiogenesis.
Suitable angiogenesis factors for the present invention include,
but are not limited to, vascular endothelial growth factor
("VEGF"), endothelial cell stimulating angiogenesis factor ("ESAF")
and any nonmitogenic angiogenesis factors present in wound
fluid.
[0020] The term "cell attachment binding site moiety" refers to a
protein that plays a role in cell-cell/cell-matrix interaction and
cellular communications. Examples of suitable cell attachment
binding site moieties include, but are not limited to, integrins,
cadherins, cell adhesion molecules ("CAMs"), selectins, fibronectin
and fibronectin fragments including synthetic fribonectin-mimetic
binding sites such as the RGD amino acid sequence.
[0021] The term "cell signaling molecule" refers to a chemical
involved in transmitting information between cells. Such molecules
are released from the cell sending the signal by crossing over the
gap between cells, interacting with receptors in another cell, and
triggering a response in that cell. Cell signaling molecules
naturally are part of a complex system of communication that
governs basic cellular activities and coordinates cell actions.
These include nitric oxide and various steroids.
[0022] The term "bioactive molecule" refers to any molecule that
has pharmacological activity that is beneficial to hair follicle
neogenesis and survival. Suitable bioactive molecules may include,
but are not limited to, cell signaling agonists or antagonists.
[0023] In another embodiment of the present invention, the grafts
are manually implanted in wounds created in the desired
implantation site using techniques similar to those used with
traditional follicular units. Alternatively, the hair graft and
bioabsorbable scaffold combination may be implanted using the
"stick and place" method of grafting. In the "stick and place"
method, the skin is pierced with the sharp point of a hollow needle
or tube that also serves as a container for the bioabsorbable
scaffold ensheathed hair graft. The tube is then inserted into the
wound and withdrawn against a push rod that prevents the graft from
coming out of the tube and ensures correct placement of the graft.
A modification of the "stick and place" method may use a tool such
as the Choi implanter, which requires breaking the skin with a
pointed instrument prior to inserting the tube and depositing the
implant.
[0024] In a further embodiment of the present invention, the
scaffold used to provide sufficient thickness to the tissue
engineered follicle progenitor cell-seeded skin is the same
scaffold as that used to create the tissue engineered dermal layer.
Thus, the scaffold for this purpose may have an isotropic structure
such that the surface is designed for accepting dermal fibroblasts
and facilitating their multiplication and maturation into a tissue
engineered dermal layer, whereas the bulk of the scaffold is a
porous structure designed to allow rapid tissue ingrowth upon
implantation. A suitable scaffold for this purpose can be prepared,
for example, by coating the surface of a highly porous synthetic or
cross-linked biopolymer scaffold to be seeded with dermal cells
with a layer of collagen or other suitable biopolymer or
cell-compatible substance. After the dermal skin cells are
confluent with the surface of the scaffold the hair follicle
progenitor cells or aggregates thereof are placed on top of the
dermal tissue equivalent and allowed to attach. A tissue engineered
epidermal layer is placed on top of the hair follicle progenitor
cells to complete formation of the construct. The construct is
cultured further while submerged in culture medium and then brought
to the air interface to allow the epidermis to mature to complete
the formation of tissue engineered skin, as is standard practice in
the art of producing tissue engineered skin.
[0025] In a further embodiment of the present invention, the
scaffold that provides thickness to the tissue engineered follicle
progenitor cell-seeded skin can also serve as a vehicle to add
other cells to the graft that provide a beneficial effect. These
cells may include, for example, fat cells, pre-adipocytes,
endothelial cells, and bone marrow cells. These additional cells
suitably are autologous cells obtained from the patient in need of
the hair graft. Thus, the tissue engineered skin components of the
graft can be obtained from neonatoal foreskin, the follicle
progenitor cells can be obtained from a scalp biopsy of the patient
in need of hair restoration, and the supplemental cells optionally
also can be obtained from the patient. The use of these added
cells, which produce important beneficial growth factors, obviates
the need for or complements the effectiveness of the above
mentioned growth factors. The endothelial cells, fat cells, and
pre-adipocytes can be obtained from liposuction fat removed, for
example, from the patient's abdominal fat and the bone marrow cells
can be obtained, for example, by aspiration from the patient's hip
bone.
[0026] The wounds in the desired implantation site may be made by
any suitable technique. For example, the wounds may be made using a
sharp instrument, such as a scalpel, trochar or needle, or the
wounds may be made by a laser or by a punch. Suitably, the needle
is an 18 or 19 gauge needle. The depth of the wound can be
pre-determined by properly adjusting the protrusion length of a
spear-point blade attached to a handle equipped with a threaded
fastener made for this purpose. For example, SP90 and SP91 blades
(Swann-Morton Surgical, Sheffield, UK) with four sided spear-point
tips were specially designed to allow control over depth and angle
while creating recipient sites for hair follicle graft
implantation.
[0027] A protectant may be placed in the wound prior to, during or
following implantation of the hair graft. The term "protectant"
refers to any substance of temporary duration that serves to
protect the cells from trauma associated with implantation or
destruction by the inflammatory process of wound healing. Many
commercially and clinically available substances may be used as a
protectant. Suitable protectants include, but are not limited to,
collagen, hyaluronic acid, and chondroitin sulfate solutions. One
suitable protectant is autologous serum from the subject in which
the hair graft is implanted. Autologous serum may be obtained by
drawing a small amount of whole blood from the subject and removing
the cells by centrifugation. Advantages of using autologous serum
include providing an anchor for the hair graft via natural clotting
properties associated with the serum. Also, the autologous serum
may contain nutrient molecules and other native beneficial factors
to further nurture follicle neogenesis.
[0028] The grafts and methods of the present invention can be used
to create new hair follicles and new hair on any region of the
subject where new hair is desired. Suitably, the grafts and methods
of the present invention are utilized to develop new hair on the
scalp or eyebrow region of the subject. The subject may be any
mammal. Suitably, the subject is a human.
[0029] It is to be understood that the invention is not limited in
its application to the details of construction and the arrangement
of components set forth in the following description or illustrated
in the following drawings. The invention is capable of other
embodiments and of being practiced or of being carried out in
various ways. Also, it is to be understood that the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having" and variations thereof herein is meant to
encompass the items listed thereafter and equivalents thereof as
well as additional items.
[0030] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the content clearly dictates otherwise. It should also be noted
that the term "or" is generally employed in its sense including
"and/or" unless the content clearly dictates otherwise. All
publications, patents and patent applications referenced in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications, patents and
patent applications are herein expressly incorporated by reference
to the same extent as if each individual publication or patent
application was specifically and individually indicated by
reference. In case of conflict between the present disclosure and
the incorporated patents, publications and references, the present
disclosure should control.
[0031] It also is specifically understood that any numerical range
recited herein includes all values from the lower value to the
upper value, i.e., all possible combinations of numerical values
between the lowest value and the highest value enumerated are to be
considered to be expressly stated in this application. For example,
if a concentration range is stated as 1% to 50%, it is intended
that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are
expressly enumerated in this specification.
[0032] The following examples are provided to assist in a further
understanding of the invention. The particular materials, methods
and conditions employed are intended to be illustrative of the
invention and are not limiting upon the scope of the invention.
EXAMPLES
Example 1
[0033] Human dermal fibroblasts are obtained from newborn infant
foreskin tissue and cultured. The cultured cells are seed on a
bioabsorbable scaffold with a silicon rubber film backing. A piece
of dorsal skin is excised from a nu/nu mouse without disturbing the
underlying blood vessels. The seeded scaffold is implanted on the
wound and the in situ blood supply maintains the viability of the
seeded scaffold. After an appropriate healing time elapses, the
silicone rubber backing is removed and hair follicle progenitor
cells are delivered to the vascularized dermal tissue equivalent.
The hair follicle progenitor cells are covered with an epidermal
layer. The wound is allowed to heal and new hair follicles are
formed.
Example 2
[0034] Human dermal fibroblasts and keratinocytes are obtained from
newborn infant foreskin tissue and cultured according the
procedures developed by Professors Auger and Germain (see, Pouliot,
et al. in Transplantation, 2002 Jun. 15; 73(11):1751-7, and
referenced cited therein) to produce separate tissue engineered
dermal and epidermal layers. A porous dermal regeneration template
(5 mm thick) comprised of collagen and glycosaminoglycan, such as
that sold by Integra Life Sciences, Inc. (Plainsboro, N.J.), is
aseptically rinsed with sterile DMEM/F12 culture medium, and
combined with the tissue engineered dermal layer and incubated in a
cell culture incubator until the tissue engineered dermal layer and
dermal regeneration template are attached. Dermal papilla cells are
placed on top of the dermal layer and allowed to attach. The tissue
engineered epidermal layer comprised of a sheet of keratinocytes is
carefully transferred onto the papilla cell-seeded dermal tissue to
complete the assembly of tissue engineered dermal layer and
epidermal layer with dermal papilla cells sandwiched in between.
This construct is cultured further until it possesses sufficient
integrity, and then it is cultured with the epidermal layer exposed
to air to induce the formation of mature skin according to standard
procedure. The construct is than transported into the operating
room where the patient is anesthetized and the surgeon creates
recipient sites for the grafts with a spear-point blade, as is
traditionally done with current follicle transplantation.
Meanwhile, the technicians working with appropriate magnification
and surgical tools cut the tissue engineered construct into
"slivers" that are approximately 1 to 2 mm wide and are made by
slicing the epidermal surface down to the bottom of the scaffold.
The slivers are then cut into 1 to 2 mm wide pieces to produce
graft that are typically 1.times.1.times.5 mm. These grafts are
then loaded into the Choi implanter comprising a tube and push rod
assembly. The graft goes in "head first" such that the epidermal
surface is in contact with the push rod and the bottom of the
scaffold is at the open end of the tube. The technician then
inserts the tube into the recipient site and removes the tube while
pushing on the push rod to deposit the graft exactly as planned by
the surgeon. A properly implanted graft will have its epidermal
layer in contact with the surrounding epidermis and the bottom of
the scaffold will be at the level of the subcutaneous fat. The
follicle neogenesis process initiated in vitro will continue in
vivo such that new hairs will become visible at the implant sites
within 3 to 6 months.
Example 3
[0035] A tissue engineered graft is prepared and implanted as
described in Example 2 except that pre-adipocytes and vascular
endothelial cells are added to the porous scaffold just prior to
combination with the tissue engineered dermal layer. All other
steps are as described above.
Example 4
[0036] A tissue engineered graft is prepared and implanted as
described in Example 2 except that the scaffold is first coated
with a solution of human collagen dissolved in 0.005M acetic acid
and then is soaked and rinsed with buffered culture medium to
insolubilize the collagen. Human foreskin fibroblasts are then
seeded directly on the collagen coated scaffold and cultured until
a tissue engineered dermal layer forms. The subsequent steps of
seeding with papilla cells and layering with an epidermal sheet of
keratinocytes are performed as previously stated.
* * * * *