Note: Descriptions are shown in the official language in which they were submitted.
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REPAIR OF AUTOLOGOUS TISSUE DEFECTS
FIELD OF INVENTION
The field of the present invention is the long-term augmentation and/or repair
of defects in dermal, subcutaneous, or vocal cord tissue.
BACKGROUND OF THE INVENTION
I. IN VITRO CELL CULTURE
The majority of in vitro vertebrate cell cultures are grown as monolayers on
an
artificial substrate which is continuously bathed in a nutrient medium. The
nature of the
substrate on which the monolayers may be grown may be either a solid (e.g.,
plastic) or a
semi-solid (e.g., collagen or agar). Currently, disposable plastics have
become a preferred
substrate for cell culture.
While the growth of cells in two-dimensions is frequently used for the
preparation and examination of cultured cells in vitro, it lacks the
characteristics of intact,
in vivo tissue which, for example, includes cell-cell and cell-matrix
interactions. Therefore, in
order to characterize these functional and morphological interactions, various
investigators
have examined the use of three-dimensional substrates in such forms as a
collagen gel
(Yang et al., Cancer Res. 41:1027 (1981); Douglas et al., In Vitro 16:306
(1980); Yang et al.,
Proc. Nat'l Acad. Sci. 2088 (1980), cellulose sponge (Leighton et al., J.
Nat'l Cancer Inst.
12:545 (1951)), collagen-coated cellulose sponge (Leighton et al., Cancer Res.
28:286
(1968)), and GELFOAMO (Sorour et al., J. Neurosurg. 43:742 (1975)). Typically,
these
aforementioned three-dimensional substrates are inoculated with the cells to
be cultured,
which subsequently penetrate the substrate and establish a
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"tissue-like" histology similar to that found in vivo. Several attempts to
regenerate "tissue-like" histology from dispe rsed monolayers of cells
utilizing three-dimensional substrates have been reported. For example,
three-dimensional collagen substrates have been utilized to culture a variety
of cells including breast epithelium (Yang, Cancer Res. 41:1021 (1981) ),
vascular epithelium (Folkman et al., Nature 288 :551 (1980) ), and
hepatocytes (Sirica et al., Cancer Res. 76:3259 (1980) ). However, long-
term culture and proliferation of cells in such systems has not yet been
achieved. Prior to the present invention., a three-dimensional substrate had
not been utilized in the autologous in vi tro culture of cells or tissues
derived
from the dermis, fascia, or lamina propria.
II. AUGMENTATION AND/OR REPAIR OF DERMAL AND
SUBCUTANEOUS TISSUES
In the practice of cosmetic and reconstructive plastic surgery, it is
frequently necessary to employ the use of various injectable materials to
augment and/or repair defects of the subcutaneous or dermal tissue, thus
effecting an aesthetic result. Non-biological injectable materials (e g.,
paraffin) were first utilized to correct facial contour defects as early as
the
late nineteenth century. However, numerous complications and the generally
unsatisfactory nature of long-term aesthetic results caused the procedure to
be rapidly abandoned. More recently, the use of injectable silicone became
prevalent in the 1960's for the correction of minor defects, although various
inherent complications also limited the use of this substance. Complications
associated with the utilization of injectable liquid silicone include local
and
systemic inflammatory reactions, formation of scar tissue around the silicone
droplets, rampant and frequently distant, unpredictable migration throughout
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the body, and localized tissue breakdown. Due to these potential
complications, silicone is not currently approved for general clinical use.
Although the original proponents of silicone injection have continued
experimental programs utilizing specially manufactured "Medical Grade"
silicone (e.g., Dow Corning's MDX 4.40116) with a limited number of
subjects, it appears highly unlikely that its use will be generally adopted by
the surgical community. See e.g., Spira and Rosen, Clin. Plastic Surgery
20:181 (1993); Matton et al., Aesthetic Plastic Surgery 9:133 (1985).
It has also been suggested to compound extremely small particulate
species in a lubricious material and inject such micro-particulate media
subcutaneously for both soft and hatd tissue augmentation and repair.
However, success has been heretofore limited. For example, bioreactive
materials such as hydroxyapatite or cordal granules (osteo conductive) have
been utilized for the repair of hard tissue defects. Subsequent undesirable
micro-particulate media migration and serious granulomatous reactions
frequently occur with the injection of this material. These undesirable
effects
are well-documented with the use of such materials as polytetrafluoro-
ethylene (TEFLON') spheres of small-diameter (¨ 90% of particles having
diameters of s30 m) in glycerin. See e.g., Malizia et al., JAMA 251:3277
(1984). Additionally, the use of very small-diameter particulate spheres
(-1-20 m) or small elongated fibrils (-1-301.1m in diameter) of various
materials in a biocompatible fluid lubricant as injectable implant composition
are disclosed in U.S. Patent No. 4,803,075. However. while these
aforementioned materials create immediate augmentation and/or repair of
defects, they also have a tendency to migrate and be reabsorbed from the
original injection site.
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The poor results initially obtained with the use of non-biological
injectable materials prompted the use of various non-immunogenic,
proteinaceous materials (e.g., bovine collagen and fibrin matrices). Prior to
human injection, however, the carboxyl- and amino-terminal peptides of
bovine collagen must first be enzymatically degraded, due to its highly
immunogenic nature. Enzymatic degradation of bovine collagen yields a
material, atelocollagen, which can be used in limited quantities in patients
pre-screened to exclude those who are immunoreactive to this substance.
The methodologies involved in the preparation and clinical utilization of
atelocollagen are disclosed in U.S. Patent Nos. 3,949,073; 4,424,208; and
4,488,911. Atelocollagen has been marketed as ZYDERM brand
atelocollagen solution in concentrations of 35 mg/ml and 65 mg/ml.
Although atelocollagen has been widely employed, the use of ZYDERM
solution has been associated with the development of antibovine antibodies
in approximately 90% of patients and with overt immunological complica-
tions in 1-3% of patients. See DeLustro et al., Plastic and Reconstructive
Surgery 79:581 (1987) .
Injectable atelocollagen solution also was shown to be absorbed
from the injection site, without replacement by host material, within a period
of weeks to months. Clinical protocols calling for repeated injections of
atelocollagen are, in practice, primarily limited by the development of
immunogenic reactions to the bovine collagen. In order to mitigate these
limitations, bovine atelocollagen was further processed by cross-linking
with 0.25% glutaraldehyde, followed by filtration and mechanical shearing
through fine mesh. The methodologies involved in the preparation and
clinical utilization of this material are disclosed in U.S. Patent
Nos. 4,582,640 and 4,642,117. The modified atelocollagen was marketed as
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ZYPLAS1'6 brand cross-linked bovine atelocollagen. The propertied
advantages of cross-linking were to provide increased resistance to host
degradation, however this was offset by an increase in solution viscosity. In
addition, cross-linking of the bovine atelocollagen was found to decrease the
5 number of host cells which infiltrated the injected collagen site. The
increased viscosity, and in particular irregular increased viscosity resulting
in
"lumpiness," not only rendered the material more difficult to utilize, but
also
made it unsuitable for use in certain circumstances. See e.g., U.S. Patent
No. 5,366,498. In addition, several investigators have reported that there is
no or marginally-increased resistance to host degradation of ZYPLAST
cross-linked bovine atelocollagen in comparison to that of the non-cross-
linked ZYDERM atelocollagen solution and that the overall longevity of the
injected material is, at best, only 4-6 months. See e.g., Ozgentas et al.,
Ann.
Plastic Surgery 33:171 (1994); and Matti and Nicolle, Aesthetic Plastic
Surgery 14:227 (1990).
Moreover, bovine atelocollagen cross-linked with glutaraldebyde
may retain this agent as a high molecular weight polymer which is
continuously hydrolyzed, thus facilitating the release of monomeric
glutaraldehyde. The monomeric form of glutaraldehyde is detectable in body
tissues for up to 6 weeks after the initial injection of the cross-linked
atelocollagen. The cytotoxic effect of glutaraldehyde on in vitro fibroblast
cultures is indicative of this substance's not being an ideal cross-linking
agent for a dermal equivalent which is eventually infiltrated host cells and
in
which the bovine atelocollagen matrix is rapidly degraded, thus resulting in
the release of monomeric glutaraldehyde into the bodily tissues and fluids.
Similarly,chondroitin-6-sulfate (GAG), which weakly binds to collagen at
neutral pH, has also been utilized to chemically modify bovine protein for
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tissue graft implantation. See Hansborough and Boyce, JAMA 136:2125
(1989). However, like glutaraldehyde, GAG may be released into the tissue
causing unforeseen long-term effects on human subjects. GAG has been
reported to increase scar tissue formation in wounds, which is to be avoided
in grafts. Additionally, a reduction of collagen blood clotting capacity may
also be deleterious in the application in bleeding wounds, as fibrin clot
contributes to an adhesion of the graft to the surrounding tissue.
The limitations which are imposed by the immunogenicity of both
modified and non-modified bovine atelocollagen have resulted in the isolation
of human collagen from placenta (see e.g., U.S. Patent No. 5,002,071); from
surgical specimens (see e.g., U.S. Patent Nos. 4,969,912 and 5,332,802);
and cadaver (see e.g., U.S. Patent No. 4,882,166). Moreover, processing of
human-derived collagen by cross-linking and similar chemical modifications
is also required, as human collagen is subject to analogous degradative
processes as is bovine collagen. Human collagen for injection, derived from
a sample of the patient's own tissue, is currently available and is marketed
as
AUTOLOGEM. It should be noted, however, that there is no quantitative
evidence which demonstrates that human collagen injection results in lower
levels of implant degradation than that which is found with bovine collagen
preparations. Furthermore, the utilization of autologous collagen preparation
and injection is limited to those individuals who have previously undergone
surgery, due to the fact that the initial culture from which the collagen is
produced is derived from the tissue removed during the surgical procedure.
Therefore, it is evident that, although human collagen circumvents the
potential for itnmtmogenicity exhibited by bovine collagen, it fails to
provide
long-term therapeutic benefits and is limited to those patients who have
undergone prior surgical procedures.
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An additional injectable material currently in use as an alternative to
atelocollagen augmentation of the subjacent dermis consists of a mixture of
gelatin powder, E-aminocaproic acid, and the patients plasma marketed as
FIBREL . See Multicenter Clinical Trial, J. Am. Acad. Dermatology
16:1155 (1987). The action of the FIBREL product appears to be dependent
upon the initial induction of a sclerogenic inflammatory response to the
augmentation of the soft tissue via the subcutaneous injection of the
material.
See e.g., Gold, J. Dermatologic Surg. Oncology, 20:586 (1994). Clinical
utilization of the FIBREL product has been reported to often result in an
overall lack of implant uniformity. (i.e., "lumpiness") and longevity, as well
as complaints of patient discomfort associated with its injection. See e.g.,
Millikan et al., J. Dermatoloqic. Surg. Oncology, 17:223 (1991). Therefore,
in conclusion, none of the currently utilized protein-based injectable
materials
appears to be totally satisfactory for the augmentation and/or repair of the
subjacent dermis and soft tissue.
The various complications associated with the utilization of
the aforementioned materials have prompted experimentation with the
implantation (grafting) of viable, living tissue to facilitate augmentation
and/or repair of the subjacent dermis and soft tissue. For example, surgical
correction of various defects has been accomplished by initial removal and
subsequent re-implantation of the excised adipose tissue either by injection
(see e.g., Davies et al., Arch. of Otolaryngology-Head and Neck Surgery
121:95 (1995); McKinney & Pandya, Aesthetic Plastic Surgery 18:383
(1994); and Lewis, Aesthetic Plastic Surgery,17:109 (1993)) or by the larger
scale surgical-implantation (see e.g., Ersck, Plastic & Reconstructive Surgery
87:219 (1991) ) . To perform both of the aforementioned techniques a
volume of adipose tissue equal or greater than is required for the subsequent
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augmentation or repair procedure must be removed from the patient. Thus,
for large scale repair procedures (e.g., breast reconstruction) the amount of
adipose tissue which can be surgically-excised from the patient may be
limiting. In addition, other frequently encountered difficulties with the
aforementioned methodologies include non-uniformity of the injectate,
unpredictable longevity of the aesthetic effects, and a 4-6 week period of
post-injection inflammation and swelling. In contrast, in a preferred
embodiment, the present invention utilizes the surgical engraftment of
autologous adipocytes which have been cultured on a solid support typically
derived from, but not limited to, collagen or isolated extracellular matrix.
The culture may be established from a simple skin biopsy specimen and
the amount of adipose tissue which can be subsequently cultured in vitro is
not limited by the amount of adipose tissue initially excised from the
patient.
Living skin equivalents have been examined as a methodology for
the repair and/or replacement of human skin. Split thickness autographs'
epidermal autographs (cultured autogenic keratinocytes), and epidermal
allographs (cultured allogenic keratinocytes) have been used with a varying
degree of success. However, unfortunately, these forms of treatment have all
exhibited numerous disadvantages. For example, split thickness autographs
generally show limited tissue expansion, require repeated surgical
operations, and give rise to unfavorable aesthetic results. E pidermal
autographs require long periods of time to be cultured, have a low success
("take") rate of approximately 30-48%, frequently form spontaneous blisters,
exhibit contraction to 60-70% of their original size, are vulnerable during
the
first 15 days of engraftment, and are of no use in situations where there is
both epidermal and dermal tissue involvement. Similarly, epidermal allografts
(cultured allogenic keratinocytes) exhibit many of the limitations which are
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inherent in the use of epidermal autographs. Additional methodologies have
been examined which involve the utilization of irradiated cadaver dennis.
However, this too has met with limited success due to, for example, graft
rejection and unfavorable aesthetic results. Living skin equivalents
comprising a dermal layer of rodent fibroblast cells cast in soluble collagen
and an epidermal layer of cultured rodent keratinocytes have been
successfully grafted as allografts onto Sprague Dawley rats by Bell et al., J.
Investigative Dermatology 81:2 (1983). Histological examination of the
engrafted tissue revealed that the epidermal layer had fully differentiated to
form desmonosomes, tonofilaments, keratohyalin, and a basement lamella.
However, subsequent attempts to reproduce the living skin equivalent using
human fibroblasts and keratinocytes has met with only limited success. In
general, the keratinocytes failed to fully differentiate to form a basement
lamella and the dermo-epidermal junction was a straight line.
The present invention includes the following methodologies for the
repair and/or augmentation of various skin defects: (1) the injection of
autologously cultured dermal or fascial fibroblasts into various layers of the
skin or injection directly into a "pocket" created in the region to be
repaired
or augmented, or (2) the surgical engraftment of "strands" derived from
autologous dermal and fascial fibroblasts which are cultured in such a manner
as to form a three-dimensional "tissue-like" structure similar to that which
is
found in vivo.
Moreover, the present invention also differs on a two-dimensional
level in that "true" autologous culture and preparation of the cells is
performed by utilization of the patient's own cells and serum for in vi tro
culture.
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According to one aspect of the present invention,
there is provided use, for corrective surgery in a subject
to repair a tissue defect, of a volume effective to treat
the defect, of a suspension of in vitro cultured autologous
cells that form a culture of cells and extracellular matrix,
wherein the in vitro cultured cells are adapted for
application to the subjacent tissue of the subject.
According to another aspect of the present
invention, there is provided a device for repairing a skin
defect in a subject comprising
(a) a hypodermic syringe having a syringe chamber, a piston
disposed therein, and an orifice communicating with the
chamber;
(b) a suspension comprising:
(1) cultured cells and extracellular matrix
produced by the cells, wherein the cells comprise lamina
propria fibroblasts, papillary fibroblasts, reticular
fibroblasts, dermal fibroblasts, fascia fibroblasts,
preadipocytes, adipocytes, smooth muscle cells, skeletal
muscle cells, non-dermal non-differentiated mesenchymal
cells, differentiated mesenchymal cells or a combination
thereof derived from the subject,
(2) a pharmaceutically acceptable carrier
solution,
said suspension being disposed in the chamber; and
(c) a hypodermic needle affixed to the orifice.
According to still another aspect of the present
invention, there is provide use of in vitro cultured
autologous cells and a carrier for preparing a composition
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for treating a defect in a subject, wherein the in vitro
cultured autologous cells have been cultured in vitro in a
medium that comprises autologous serum to expand the number
of cells.
According to yet another aspect of the present
invention, there is provided use of an in vitro cultured
cell composition for preparing a composition for correction
of a defect in a subject, wherein the composition comprises
a plurality of in vitro cultured viable fetal cells or in
vitro cultured juvenile cells cultured to form the in vitro
cultured cell composition and a carrier.
According to a further aspect of the present
invention, there is provided an in vitro produced
extracellular matrix composition, which is either
substantially pure or combined with cells embedded in the
matrix and is obtained from the process comprising the steps
of: a) culturing cells in vitro in a culture vessel for a
time sufficient for the cells to produce extracellular
matrix; b) separating the extracellular matrix from the
culture vessel and in addition, if the composition is
substantially pure, separating the extracellular matrix
produced by the cultured cells from such cells; and c)
collecting the extracellular matrix.
According to yet a further aspect of the present
invention, there is provided use, for corrective surgery in
a human subject of a defect rectified by augmentation of
tissue subjacent to the defect, of the extracellular matrix
as described above, wherein the extracellular matrix is
adapted for application to the subjacent tissue of the
subject.
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111. VOCAL CORD TISSUE AUGMENTATION AND/OR REPAIR
Phonation is accomplished in humans by the passage of air
past a pair of vocal cords located within the larynx. Striated muscle fibers
within the larynx, comprising the constrictor muscles, function so as to vary
5 the degree of tension in the vocal cords, thus regulating both their
overall
rigidity and proximity to one another to produce speech. However, when one
(or both) of the vocal cords becomes totally or partially immobile, there is a
diminution in the voice quality due to inability to regulate and maintain the
requisite tension and proximity of the damaged cord in relation to that of the
10 operable cord. Vocal cord paralysis may be caused by cancer, surgical or
mechanical trauma, or similar afflictions which render the vocal cord
incapable of being properly tensioned by the constrictor muscles.
One therapeutic approach which has been examined to allow
phonation involves the implantation or injection of biocompatible
materials. It has long been recognized that a paralyzed or damaged vocal
cord may be repositioned or supported so as to remain in a fixed location
relative to the operable cord such that the unilateral vibration of the
operable
cord produces an acceptable voice pattern. Hence, various surgical have
been developed which involve the formation of the thyroid cartilage and
subsequently providing a means for the support and/or repositioning of the
paralyzed vocal cord.
For example, injection of TEFLON into the paralyzed vocal cord to
increase its inherent "bulk" has been described. See e.g., von Leden et al.,
Phonosurgery 3:175 (1989). However, this procedure is now considered
unacceptable due to the inability of the injected TEFLON to close large
glottic gaps, as well as its tendency to induce inflammatory reactions
resulting in the formation of fibrous infiltration into the injected cord. See
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e.g., Mayes et al., Phonosurgery: Indications and Pitfalls 98:577 (1989).
Moreover, removal of the injected TEFLON may be quite difficult should it
subsequently be desired or become necessary.
Another methodology for supporting the paralyzed vocal cord
which has been employed involves the utilization of a custom-fitted block of
siliconized rubber (SILASTIC). In order to ensure the proper fit of the
implant, the surgeon hand carves SILASTIC block during the procedure in
order to maximize the ability of the patient to phonate. The patient is kept
under local anesthesia so that he or she can produce sounds to test the
positioning of the implant. Generally, the implanted blocks are formed into
the shape of a wedge which is totally implanted within the thyroid cartilage
or a flanged plug which can be moved back-and-forth within the opening in
the thyroid cartilage to fine-tune the voice of the patient.
Although SILASTIC implants have proved to be superior over
TEFLON injections, there are several areas of dissatisfaction with the
procedure including difficulty in the carving and insertion of the block, the
large amount of time required for the procedure, and a lack of an efficient
methodology for locking the block in place within the thyroid cartilage.
In addition, vocal-cord edema, due to the prolonged nature of the procedure
and repeated voice testing during the operation, may also prove problematic
in obtaining optimal voice quality.
Other methodologies which have been utilized in the treatment of
vocal cord paralysis and damage include GELFOAM hydroxyapatite, and
porous ceramic implants, as well as injections of silicone and collagen. See,
e.g., Kaufman, Laryngoplastic Phonosurqery (1988). However, these
materials have also proved to be less than ideal due to difficulties in the
sizing and shaping of the solid implants as well as the potential for
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subsequent immunogenic reactions. Therefore, there still remains a need for
the development of a methodology which allows the efficacious treatment of
vocal cord paralysis and/or damage.
SUMMARY OF THE INVENTION
The present invention discloses a methodology for the longterm
augmentation and/or repair of dermal, suboutaneous, or vocal cord tissue by
the injection or direct surgical placement/implantation of: (1) autologous
cultured fibroblasts derived from connective tissue, dermis, or fascia; (2)
lamina propria tissue; (3) fibroblasts derived from the lamina propria or (4)
adipocytes. The fibroblast cultures utilized for the augmentation and/or
repair of skin defects are derived from either connective tissue, dermal,
and/or fascial fibroblasts. Typical defects of the skin which can be corrected
with the injection or direct surgical placement of autologous fibroblasts or
adipocytes include rhytids, stretch marks, depressed scars, cutaneous
depressions of traumatic or non-traumatic origin, hypoplasia of the lip,
and/or
scarring from acne vulgaris. Typical defects of the vocal cord which can be
corrected by the injection or direct surgical placement of lamina propria or
autologous cultured fibroblasts from lamina propria include scarred,
paralyzed, surgically or traumatically injured, or congenitally underdeveloped
vocal cord(s).
The use of autologous cultured fibroblasts derived from
the derrais, fascia, connective tissue, or lamina propria mitigates the
possibility of an immunogenic reaction due to a lack of tissue
histocompatibility. This provides vastly superior post-surgical results. In a
preferred embodiment of the present invention, fibroblasts of connective
tissue, dermal, or fascial origin as well as adipocytes are derived from full
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biopsies of the skin. Similarly, lamina propria tissue or fibroblasts obtained
from the lamina propria are obtained from vocal cord biopsies. It should be
noted that the aforementioned from the individual who will subsequently
undergo the surgical procedure, thus mitigating the potential for an
immunogenic reaction. These tissues are then expanded in vitro utilizing
standard tissue culture methodologies.
Additionally, the present invention further provides a methodology
of rendering the cultured cells substantially free of potentially immunogenic
serum-derived proteins by late-stage passage of the cultured fibroblasts,
lamina propria tissue, or adipocytes in serum-free medium or in the patient's
own serum. In addition, immunogenic proteins may be markedly reduced or
eliminated by repeated washing in phosphate-buffered saline (PBS) or similar
physiologically-compatible buffers.
DESCRIPTION OF THE INVENTION
I. HISTOLOGY OF THE SKIN
The skin is composed of two distinct layers: the epidem a
specialized epithelium derived from the ectoderm, and beneath this, the
dermis, a vascular dense connective tissue, a derivative of mesoderm.
These two layers are firmly adherent to one another and form a region which
varies in overall thickness from approximately 0.5 to 4 mm in different areas
of the body. Beneath the dermis is a layer of loose connective tissue which
varies from areolar to adipose in character. This is the superficial fascia of
gross anatomy, and is sometimes referred as the hypodermis, but is not
considered to be part of the skin. The dermis is connected to the hypodermis
by connective tissue fibers which pass from one layer to the other.
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A. EPIDERMIS
The epidermis, a stratified squamous epithelium, is composed of
cells of two separate and distinct origins. The majority of the epithelium, of
ectodermal origin, undergoes a process of keratinization resulting in the
formation of the dead superficial layers of skin. The second component
comprises the melanocytes which are involved in the synthesis of
pigmentation via melanin. The latter cells do not undergo the process of
keratinization. The superficial keratanized cells are continuously lost from
the surface and must be replaced by cells that arise from the mitotic
activity of cells of the basal layers of the epidermis. Cells which result
from
this proliferation are displaced to higher levels, and as they move upward
they elaborate keratin, which eventually replaces the majority of the
cytoplasm. As the process of keratinization continues the cell dies and is
finally shed. Therefore, it should be appreciated that the structural
organization of the epidermis into layers reflects various stages in the
dynamic process of cellular proliferation and differentiation.
B. DERMIS
It is frequently difficult to quantitatively differentiate the limits of
the dermis as it merges into the underlying subcutaneous layer (hypodennis).
The average thickness of the dennis varies from 0.5 to 3 mm and is further
subdivided into two strata - the papillary layer superficially and the
reticular
layer beneath. The papillary layer is composed of thin collagenous, reticular,
and elastic fibers arranged in an extensive network. Just beneath the
epidermis, reticular fibers of the dennis form a close network into which the
basal processes of the cells of the stratum germinativum are anchored. This
region is referred to as the basal lamina.
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The reticular layer is the main fibrous bed of the derails. Generally,
the papillary layer contains more cells and smaller and finer connective
tissue
fibers than the reticular layer. It consists of coarse, dense, and interlacing
collagenous fibers, in which are intermingled a small number of reticular
5 fibers and a large number of elastic fibers. The predominant arrangement
of
these fibers is parallel to the surface of the skin. The predominant cellular
constituent of the dermis are fibroblasts and macrophages. In addition,
adipose cells may be present either singly or, more frequently, in clusters.
Owing to the direction of the fibers, lines of skin tension, Langer's lines,
10 are formed. The overall direction of these lines is of surgical
importance
since incisions made parallel with the lines tend to gape less and heal with
less scar tissue formation than incisions made at right-angles or obliquely
across the lines. Pigmented, branched connective tissue cells,
chromatophores, may also be present. These cells do not elaborate pigment
15 but, instead, apparently obtain it from melanocytes.
Smooth muscle fibers may also be found in the dermis. These fibers
are arranged in small bundles in connection with hair follicles (arrectores
pilonun muscles) and are scattered throughout the dermis in considerable
numbers in the skin of the nipple, penis, scrotum, and parts of the perineum.
Contraction of the muscle fibers gives the skin of these regions a wrinkled
appearance. In the face and neck, fibers of some skeletal muscles terminate
in delicate elastic fiber networks of the dermis.
C. ADIPOSE TISSUE/ADIPOCYTES
Fat cells, or adipocytes, are scattered in areolar connective tissue.
When adipocytes form large aggregates, and are the principle cell type, the
tissue is designated adipose tissue. Adipocytes are fully differentiated cells
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and are thus incapable of undergoing mitotic division. New adipocytes
therefore, which may develop at any time within the connective tissue, arise
as a result of differentiation of more primitive cells. Although adipocytes,
prior to the storage of lipid, resemble fibroblasts, it is likely that they
arise
directly from undifferentiated mesenchymal tissue.
Each adipocyte is surrounded by a web of fine reticular fibers; in the
spaces between are found fibroblasts, lymphoid cells, eosinophils, and some
mast cells. The closely spaced adipocytes form lobules, separated by fibrous
septa. In addition, there is a rich network of capillaries in and between
the lobules. The richness of the blood supply is indicative of the high rate
of
metabolic activity of adipose tissue.
It should be appreciated that adipose tissue is not static There is a
dynamic balance between lipid deposit and withdrawal. The lipid contained
within adipocytes may be derived from three sources. Adipocytes, under the
influence of the hormone insulin. can synthesize fat from carbohydrate. They
can also produce fat from various fatty acids which are derived from the
initial breakdown of dietary fat. Fatty acids may also be synthesized from
glucose in the liver and transported to adipocytes as serum lipoproteins. Fats
derived from different sources also differ chemically. Dietary fats may be
saturated or unsaturated, depending upon the individual diet. Fat which is
synthesized from carbohydrate is generally saturated. Withdrawals of fat
result from enzymatic hydrolysis of stored fat to release fatty acids into the
blood stream. However, if there is a continuous supply of exogenous glucose,
then fat hydrolysis is negligible. The normal homeostatic balance is affected
by hormones, principally insulin, and by the autonomic nervous system,
which is responsible for the mobilization of fat from adipose tissue.
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Adipose tissue may develop almost anywhere areolar tissue
is prevalent, but in humans the most common sites of adipose tissue
accumulation are the subcutaneous tissues (where it is referred to as the
panniculus adiposus), in the mesenteries and omenta, in the bone marrow,
and surrounding the kidneys. In addition to its primary function of storage
and metabolism of neutral fat, in the subautaneous tissue, adipose tissue also
acts as a shock absorber and insulator to prevent excessive heat loss or gain
through the skin.
IL HISTOLOGY OF THE LARYNX AND VOCAL CORDS
The larynx is that part of the respiratory system which connects the
pharynx and trachea. In addition to its function as part of the respiratory
system, it plays an important role in phonation (speech). The wall of the
larynx is composed of a "skeleton" of hyaline and elastic cartilages,
collagenous connective tissue, striated muscle, and mucous glands. The
major cartilages of the larynx (the thyroid, cricoid, and arytenoids) are
hyaline, whereas the smaller cartilages (the corniculates, cuneiforms, and the
tips of the arytenoids) are elastic, as is the cartilage of the epiglottis.
The
aforementioned cartilages, together with the hyoid bone, are connected by
three large, flat membranes: the thyrohyoid, the quadrates, and the
cricovocal. These are composed of dense fibroconnective tissue in which
many elastic fibers are present, particularly in the cricovocal membrane. The
true and false vocal cords (vocal-and vestibular ligaments) are, respectively,
the free upper boarders of the cricovocal (cricothyroid) and the free lower
boarders of the quadrate (aryspiglottic) membranes. Extending laterally on
each side between the true and false cords are the sinus and saccule
of the larynx, a small slit-like diverticulum. Behind the cricoid and
arytenoid
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cartilages, the posterior wall of the pharytut is formed. by the striated
muscle
of tb.e pharyngeal constrictor muscles.
The epithelium of the mucous membrane of the larynx
varies with location. For example, etver the -vocal RAds, the lamina propria
of
the stratified squamous epithelium is extremely dense and firmly bound to the
underlying connective tissue of the vocal ligament While there is no 'true
submucosa in the 1nryll2E, the Lumina propria othe mucous inelxibathe is thick
and contains large numbers of elastic fibers. =
ILL METTIODOLOGUES
A. IN vim CELL CULTURE OF FIBROBLASTS OR LAMINA
PROPItIA
While the present invention may be px-acticed by utilizing any type
of non-differentinted mesenchymal cell found in the akin whirlb can, be
expanded in. In vino cuhrtre, fibroblasts derived from dermal, connective
tissue, fascia, lamina propristl tissue adipocytes, andfor extraoellular
tiaaues
(matrix) derived from the cells which are differentiated or non-
differentiated, are utilized in a preferred embodiment due to their
relative of isolation and in vitro expansion in tissue culture. In general
tissue culture techniques which are suitable for the propagation of
non-differeniiated toesenchytual tells may be 'used to C21:paild tlae
aforementioned cells/tissue. and practice present invention as Birth=
discussed blow. See e.g.. Culture ofAnitnal Cells: A Manual of Basic
Techniques, Fresbney, R. L, ed., (Alan R. Liss & Co.õ New York 1987);
Animal Cell. Culture: A Pre.ctical Apprcrschõ Freshrsey, R.I. ed.., WU, Press,
Oxford, England (1986),
Th.e7rtilizpition of tunologous engraftinent is a preferred
therapeutic methodology due to the potential for graft rejection associated
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with the use of allograft-based engraftment. Autologous grafts (i.e., those
derived directly from the patient ensure histocompatibility by inilistlly
obtainin' g a tissue sample via biopsy directly from the patient who will be
undergoing the corrective surgical procedure and then subsequently culturing
fibroblasts derived from the dermal, connective tissue, fascia', or lamina
proptial regions contained therein.
While the following sections will primarily discuss the autologous
culture of fibroblasts of connective tissue, dennal, or fascia' origins, in
vino
culture of lamin' a. propria tissue may also be established irti1i7irig
analogous
methodologies. An autologous fibroblast culture is preferably initiated by the
following methodology. A full-thickness biopsy of the skin (--3x6 mrti) is
initially obtained through, for example, a punch biopsy procedure. The
specimen is repeatedly washed with antibiotic and anti-fungal agents prior to
culture. Through a process of sterile microscopic dissection, the keratinized
tissue-containing epidermis and subcutaneous adipocyte-containing tissue is
removed, thus ensuring that the resultant culture is substantially free of
non-fibroblast cells (e.g., adipoeytes and keratinocytes). The isolated
adipocres-containing tissue may then be utilized to establish arlipocyre
cultures. Alternately, whole tissue may be cultured and fibroblast-specific
growth medhim may be utilized to "select" for these cells.
Two methodologies are generally utilized for the autologous culture
of fibroblasts in the practice of the present invention - mechanical and
enzymatic. In the mechanical methodology, the fascia, dermis, or connective
tissue is intially dissected out and finely divided with scalpel or scissors.
The finely minced pieces of the tissue are initially placed in 1-2 ml of
medium in either a 5 mm peiri dish (Costar), a 24 multi-well culture plate
(Corning), or other appropriate tissue culture vessel_
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Incubation is preferably performed at 37 deg. C in a 5% CO2
atmosphere and the cells are incubated until a confluent monolayer of
fibroblasts has been obtained. This may require up to 3 weeks of incubation.
Following the establishment of confluence, the monolayer is trypsinized to
5 release the adherent fibroblasts from the walls of the culture vessel.
The
suspended cells are collected by centrifugation, washed in phosphate-
buffered saline, and resuspended in culture medium and placed into larger
culture vessels containing the appropriate complete growth medium.
In a preferred embodiment of the enzymatic culture methodology,
10 pieces of the finely minced tissue are digested with a protease for
varying
periods of time. The enzymatic concentration and incubation time are
variable depending upon t individual tissue source, and the initial isolation
of
the fibroblasts from the tissue as well as the degree of subsequent outgrowth
of the cultured cells are highly dependent upon these two factors. Effective
15 proteases include, but are not limited to, trypsin, chymotrypsin,
papain,
chymopapain, and similar proteolytic enzymes. Preferably, the tissue is
incubated with 200-1000 U/ml of collagenase type II for a time period
ranging from 30 minutes to 24 hours, as collagenase type H was found to be
highly efficacious in providing a high yield of viable fibroblasts. Following
20 enzymatic digestion, the cells are collected by centrifugation and
resuspended into fresh medium in culture flasks.
Various media may be used for the initial establishment
of an in vitro culture of human fibroblasts. Dulbecco's Modified
Eagle Medium (DMEM, Gibco/BRL Laboratories) with concentrations
of fetal bovine serum (FBS), cosmic calf serum (CCS) or the patient's own
serum varying from 5-20% (v/v) -- with higher concentrations resulting in
faster culture growth -- are readily utilized for fibroblast culture. It
should be
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noted that substantial reductions in the concentration of serum (i.e., O. 5%
v/v) results in a loss of cell viability in culture. In addition, the complete
culture medium typically contains Lglutamine, sodium bicarbonate,
pyridoxine hydrochloride, 1g/liter glucose, and gentamycin sulfate. The use
of the patient's own serum mitigates the possibility of subsequent
immunogenic reaction due to the presence of constituent antigenic proteins in
the other serums.
Establishment of a fibroblast cell line from an initial human biopsy
specimen generally requires 2 to 3. 5 weeks in total. Once the initial culture
has reached confluence, the cells may be passaged into new culture flasks
following trypsinization by standard methodologies known within the
relevant field. Preferably, for expansion, cultures are "split" 1:3 or 1:4
into
T-150 culture flasks (Corning) yielding ¨5x107 cells/culture vessel. The
capacity of the T-150 culture flask is typically reached following 5- 8 days
of
culture at which time the cultured cells are found to be confluent or near
confluent.
Cells are preferably removed for freezing and long-term storage
during the early passage stages of culture, rather thane the later stages due
to
the fact that human fibroblasts are capable of undergoing a finite numbers of
passages. Culture medium containing 70% DMEM growth medium, 10%
(v/v) serum, and 20% (v/v) tissue culture grade dimethyleulfcmide (DMSO,
Gibco/BRL) may be effectively utilized for freezing of fibroblast
cultures. Frozen cells can subsequently be used to inoculate secondary
cultures to obtain additional fibroblasts for use inthe original patient, thus
doing away with the requirement to obtain a second biopsy specimen. -
To rninimi7e the possibility of subsequent immunogenic reactions in
the engraftment patient, the removal of the various antigenic constituent
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proteins contained within the serum may be facilitated by collection of the
fibroblasts by centrifugation, washing the cells repeatedly in
phosphate-buffered saline (PBS) and then either re-suspending or culturing
the washed fibroblas for a period of 2-24 hours in serum-free medium
containing requisite growth factors which are well known in the field.
Culture media include, but are not limited to, Fibroblast Basal Medium
(FBM). Alternately, the fibroblasts may be cultured utilizing the patient's
own
serum in the appropriate growth medium.
After the culture has reached a state of confluence or sub-
confluence, the fibroblasts may either be processed for injection or further
cultured to facilitate the formation of a three-dimensional "tissue" for
subsequent surgical engraftment. Fibroblasts utilized for injection consist of
cells suspended in a collagen gel matrix or extracellular matrix. The collagen
gel matrix is preferably comprised of a mixture of 2 ml of a collagen solution
containing 0.5 to 1.5 mg/ml collagen in 0. 05% acetic acid, 1 ml of DMEM
medium, 270 I of 7.5% sodium bicarbonate, 48 microliters of 100
micrograms/ml solution of gentamycin sulfate, and up to 5x106 fibroblast
cell/nil of collagen gel. Following the suspension of the fibroblasts in the
collagen gel matrix, the suspension is allowed to solidify for approximately
15 minutes at room temperature or 37 deg C in a 5% CO2 atmosphere. The
collagen may be derived from human or bovine sources, or from the patient
and may be enzymatically- or chemically-modified (e.g., atelocollagen).
Three-dimensional "tissue" is formed by initially suspending the
fibroblasts in the collagen gel matrix as described above. Preferably, in the
culture of three-dimensional tissue, full-length collagen is utilized, rather
than truncated or modified collagen derivatives. The resulting suspension is
then placed into a proprietary "transwell" culture system which is typically
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comprised of culture well in whicii the lower growth medium is separated
from the upper region of the culture well by a inicroporoue illeMbraLle. The
tnieroporous membrane -typically possesses a pore size ranging *OM 0.4 to 8
tun ie diatneter and is constructed front materials including, but not limited
to, polyester, nylon, nitrocellulose, cellulose acetate, polyrterylamide,
cros.s-
linked dextrose, agarose, or other similar materials. The culture well
component of the trauswell culture system may be fabricated in any desired
abspe or size (e_g., square, round, ellipsoidal, et) to facilitate subsequent
surgieal tissue engrafbnent and typically holds a volume of culture medium
xaneng from 200 VI CO 5 ml. In general, a concentration ranging from 0,5 21,
10. to 10 x ltr cell.siad, and. preferably 5 x 106 cells/ml, are inoculated
into
the collagen/fibroblast-containing SUSpertlaisall aS described above, -
Utilizing a
preferred COMettlatiOli of cells (Le., 5 X 106 cellsiral), a total of
approximately 4-5 weeks is required for the formation of a tee-dimensional
tissue matrix. However, this time 'nay -vary with increasing or decreasing
concentrations of inoculated cells. Accordingly, the higher the concentration
of cells utilized the less time due to a higher overall rate of cell
proliferation
and =placement of the exogenous collagen with endogenous collagen an.d
other constituent materials which form. the mdracellular matrix synthesized by
the cultured fibroblasts. Constituent materials which Elarnzx the
extracellular
matrix (ECM) include, but are not limited to, collagen, elastin, fibrin,
fibrinogen, proteases, fibroneetin, laminin, fibrellins, and other
similar proteins. Constituent materials include glycosaminoglycans
and hyaluronic acid, that are in.tegral to the ECM and are intimately
associated with or part of the proteins in. the ECM. It
should be noted that tbe potential for immunogenic reaction_ in the engrafted
patient is markedly reduced due to the fact that the exogenous collagen used
in establishing the initial collagen/fibroblast-containing suspension is
gradually xvplaced during subsequent culture by endogenous collagen and
extracellular matrix materials aynthesized by the fibroblasts.
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. P7TRO CULTURE OF ADIPOCYTES
Adipoeytes require a "feeder-layer" or other type of solid support on
which to grow_ One potential. solid support may be provided by utilization of
the previously discussed collagen gel matrix- Alternately, the solid support
may be provided by eultUred toctmeellular matrix- In general, the in vitro
minim of adipocytes is performed by the mectuatical or enzymatic
disaggregation of the adipocytes Brom adipose tissue &Lived from a biopsy
specimen_ The adipoeytes are "seeded" onto the surface of the
aforementioned solid support and allowed to grow until near-confluence is
reached_ The adipocytes are 'removed by gentle stamping of the solid surface.
The isolated adipocytes are than alltiWad the some zummer as firilizmi For
fibroblasts as previously discussed in Section. HI A.
isoLATIoisi OF TIM EXTRA.CELLULAR MATRIX
The extraccliular matrix (ECM) may be isolated in either a cellular
or acellnlar form Constituent materials vvhich form the ECM include, but
are not limited to, collagen. elastin, fibrin, fibrinogen, professes,
fibronectin,
fibrellins, and other similar proteins. Constituent materials
include glycosaminoglycans and hyaluronic acid, that are integral to the
ECM and are intimately associated with or part of the proteins in the
ECM. These constituent materials singly, in combination or whole
represent extracellular matrix. ECM is typically isolated by
the initial culture of cells derived from skin, subcutaneous tissue, or vocal-
cord biopsy specimens as previously described. A.fter the cultured cells have
reached a rainin" mot of 25-50% sub-conftuence, the ECM may be obtained by
mecivirlical, enzymatic, ebemical, or denatunun treatment.. it/fecal:mica,
collection is performed by scraping the ECM off of the plastic culture vemsel
find re-suspending in phosphate-buffered saline ('ES) . f desired,. the
constituent cells are lysed or ruptured by incubation in hypownic saline
containing 5 mM EDTA. Pieferablyõ however, =aping followed by PBS re-
stopension is generally utilized. Voatzyucuttic treatment involves brief
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incubation, with a proteokytic enzyme such as trypsin. Additionally, the use
of
detergents such as sodium dodesyi sulfate (SDS) or treatment with
denaturants such as urea or dithicrtheritol (13TT) followed bydia' lysis
against
PBS, will also facilitate the release fibs ECM from %Mounding associated
5 tissue.,
The isolated ECM may then be utilized as a "ft.11er" material in the
various augmentation or repair procedures disclosed in the present
application. In addition, the ECM may possess c.extain cell growth- or
metabolism-promoting chaxactivistirs.
D. rN V17R0 CULTURE OE FETAL OR JUVENILE CELLS CR.
TISSUES
In another prefared embodiment.. rather dm utilizing the patient's
OWli tissue, all of the aforementioned cells, cell suspensions, or tissues may
be derived from fetal or juvenile sources or sources -that have beem exposed
to the sun little or not at all and, in any case, less than the tissue being
repaired. Allogenic or non-autologous sources are comprised of fetal or
juvenile sources. Juvenile sources include but are not limited to neonatal,
young or adult cells, that are preferably cells from a younger age than the
age of
the subject. Fetal cells lack the immunogenic determinants responsible for
eliciting the host graft-rejection reaction and this may be unliz' ed for
engraft:meat procedvaes witll little or no probability fa subseqeent
immunogenic reaction. An acellular ECM may also be obtained from fetal
- ECM by bypotonic lysing oftne constituent cells. The acelltdar ECM
derrip' ed from fetal orjuvemle or leas suu-expased sources sources or from in
vitro culture of early passage cells typically possesses cliff= in both
quantity
and chartacteristies from-that of -the ECM derived from senescent or late-
passage cells. The cellular or s.cellular ECM derived from fetal or juvenile
sources limy be used as a "filler' material in the vat-a). us augmentation or
repair procedures disclosed in the present application. In addition,- tbe
fetal
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or juvenile ECM may possess certain cell growth- or metabolism-promoting
characteristics.
E. INJECTION OF AUTOLOGOUS CULTURED
DERMAL/FASCIAL FIBROBLASTS
To augment or repair dermal defects, autologously cultured
fibroblasts are injected initially into the lower dermis, next in the upper
and
middle dermis, and finally in the subcutaneous regions of the skin as to form
raised areas or "wheals." The fibroblast suspension is injected via a syringe
with a needle ranging frog 30 to 18 gauge, with the gauge of the needle being
dependent upon such factors as the overall viscosity of the fibroblast
suspension and the type of anesthetic utilized. Preferably, needles ranging
from 22 to 18 gauge and 30 to 27 gauge are used with general and local
anesthesia, respectively.
To inject the fibroblast suspension into the lower dermis, the needle
is placed at approximately a 45 angle to the skin with the bevel of the
needle
directed downward. To place the fibroblast suspension into the middle
dermis the needle is placed at approximately a 20-30 angle. To place the
suspension into the upper dermis, the needle is placed almost horizontally
(i.e., 10-15 angle). Subcutaneous injection is accomplished by initial
placement of the needle into the subcutaneous tissue and injection of the
fibroblast suspension during subsequent needle withdrawal. In addition, it
should be noted that the needle is preferably inserted into the skin from
various directions such that the needle tract will be somewhat different with
each subsequent injection. This technique facilitates a greater amount of
total skin area receiving the injected fibroblast suspension.
Following the aforementioned injections, the skin should be
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expanded and possess a relatively taut feel. Care should be taken so as not
to produce an overly hard feel to the injected region. Preferably, depressions
or rhytids appear elevated following injection and should be "overcorrected"
by a slight degree of over-injection of the fibroblast suspension, as
typically
some degree of settling or shrinkage will occur post-operatively.
In some scenarios, the injections may pass into deeper tissue layers.
For example, in the case of lip augmentation or repair, a preferred manner of
injection is accomplished by initially injecting the fibroblast suspension
into
the dermal and subcutaneous layers as previously described, into the skin
above the lips at the vermillion border. In addition, the vertical philtrurn
may
also be injected. The suspension is subsequently injected into the deeper
tissues of the lip, including the muscle, in the manner described for
subcutaneous injection.
F. SURGICAL PLACEMENT OF AUTOLOGOUSLY CULTURED
DERMAL/FASCIAL FIBROBLAST STRANDS
In a preferred methodology utilized to augment or repair the skin
and/or lips by the surgical placement of autologously cultured dermal and/or
fascial fibroblast strands, a needle (the "passer needle") is selected which
is
larger in diameter and greater in length than the area to be repaired or
augmented. The passer needle is then placed into the skin and threaded
down the length of the area. Guide sutures are placed at both ends through
the dermal or fascial fibroblast strand. One end of the guide suture is fixed
to
a Keith needle which is subsequently placed through the passer needle. The
guide suture is brought out through the skin on the side furthest (distal
point)
from the initial entry point of the passer needle. The dermal or fascial
fibroblast graft is then pulled into the passer needle and its position may be
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adjusted by pulling on the distal point guide suture or, alternately, the
guide
suture closest to the passer needle entry point. While the dermal or fascial
strand is held in place by the distal point suture, the passer needle is
pulled
backward and removed, thus resulting in the final placement of the graft
following the fmal cutting of the remaining suture.
Generally, the fascial or dermal graft is placed into the subcutaneous
layer of the skin. However, in some situations, it may be placed either more
deeply or superficially.
If the area to be repaired or augmented is either smaller or larger
than would be practical to fill with the aforementioned needle method, a
subcutaneous "pocket" may be created with a myringotomy knife, scissors, or
other similar instrument. A piece of dermis or fascia is then threaded into
this area by use of guide sutures and passer needle, as described above.
G. INJECTION OF CELLS OR OTHER SUBSTANCES INTO THE
VOCAL CORDS OR LARYNX
Generally, it is not possible to inject cellular matter or other
substances directly into the vocal cord epithelium due to its extreme
thinness.
Accordingly, injections are usually made into the lamina propria layer or the
muscle itself.
Generally, lamina propria tissue (fmely minced if required for
injection), fibroblasts derived from lamina propria tissue, or gelatinous
substances are utilized for injection. The preferable methodology consists of
injection directly into the space containing the lamina propria, specifically
into Reinke's space. Injection is accomplished by use of laryngeal injection
needles of the smallest possible gauge which will accommodate the injectate
without the use of extraneous pressure during the actual injection process.
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This is a subjective process as to the overall "feel" and the use of too much
pressure may irreparably damage the injected cells. The material is injected
via a syringe with a needle ranging from 30 to 18 gauge, with the gauge of
the needle being dependent upon such factors as the overall viscosity of the
injectate and the type of anesthetic utilized. Preferably, needles ranging
from
22 to 18 gauge and 30 to 27 gauge are used with general and local
anesthesia, respectively. If required, several injections may be performed
along the length of the vocal cord.
To medialize a vocal cord with autologously cultured fascial or
dermal fibroblasts, the materials are preferably injected directly into the
tissue lateral or at the lateral edge of the vocal cord. The fibroblasts may
be
injected into scar, Reinke's space, or muscle, depending upon the specific
vocal cord pathology. Preferably, it would be injected into the muscle.
The procedure may be performed under general, local, topical,
monitored, or with no anesthesia, depending upon patient compliance and
tolerance, the amount of injected material, and the type of injection
performed.
If a greater degree of augmentation is required, a "pocket" may be
created by needle dissection. Alternately, laryngeal microdisection, using
knives and dissectors, may be performed. The desired material is then
placed into the pocket with laryngeal forceps, or directly injected, depending
upon the size of the pocket, the size of the graft material, the anesthesia,
and
the open access. If the pocket is left open after the procedure, it is
preferably
closed with sutures, adhesive, or a laser, depending upon the size and
availability of these materials and the individual preferences of the surgeon.
While embodiments and applications of the present invention have
been described in some detail by way of illustration and example for
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purposes of clarity and understanding, it would be apparent to those
individuals whom are skilled within the relevant art that many additional
modifications would be possible without departing from the inventive
concepts contained herein.
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