(19) United States
`(12) Patent Application Publication (10) Pub. No.: Us 2004/0101959 A1
`Marko et al.
`{43} Pub. Date:
`May 27, 2004
`
`US 20040101959A1
`
`(54) TREATMENT OF TISSUE WITH
`UNDIFFERENTIATED MESENCHYMAL
`CELLS
`
`(76)
`
`Inventors: Olga Marko, Paramus, NJ (US);
`William K. Boss JR., Essex Fells, NJ
`(US)
`
`Correspondence Addregq;
`WILLIAM J. HONE
`Flsll 3; Richardson EC.
`Suite 2300
`45 Rockefeller Plaza
`New York, NY 10111 (US)
`
`(21) Appl. N0;
`
`10/301,058
`
`(22)
`
`Filed:
`
`Nov. 21, 2002
`
`Publication Classification
`
`Int. Cl.1r
`(51)
`(52) US. Cl.
`
`C12N $08
`
`435,666
`
`.....
`
`(57)
`
`ABSTRACT
`
`The present invention provides compositions and methods
`for correcting cosmetic, aesthetic, and degenerative defects
`in the skin, soft tissue, and bone of a subject. In particular,
`methods of the invention involve the injection or implanta-
`tion of autologous UMC, fibroblasts, andfor keratinocytes
`into the tissue (e.g., subcutaneous tissue) adjacent or sub—
`adjacent to a defect or at the site of a defect. The cells that
`are injected, as provided herein, are histocompatible with the
`subject (e.g., are autologous) and have been expanded by
`passage in a cell culture system.
`
`1
`
`ALL 2004
`PROLLENIUM V. ALLERGAN
`|PR2020-00084
`
`1
`
`ALL 2004
`PROLLENIUM V. ALLERGAN
`IPR2020-00084
`
`

`

`US 2004/0101959 A1
`
`May 27, 2004
`
`TREATMENT OF TISSUE WITH
`UNDIFFERENTIATED MESENCHYMAL CELLS
`
`TECHNICAL FIELD
`
`[0001] This invention relates to compositions containing
`undifl‘erentiated mesenchymal cells and methods for treating
`dental defects, defects of skin and soft tissue, and bone
`defects with autologous undifl‘erentiated mesenchymal cells.
`
`BACKGROUND
`
`[0002] Defects of the skin, bone, soft tissue, and dental
`tissue can be caused by factors such as wounds, disease, and
`aging. Methods have been designed to repair andj'or aug-
`ment tissue that is affected by such conditions, but improved
`methods that avoid scarring and potentiate tissue growth
`would be useful.
`
`[0003] U.S. Pat. Nos. 5,591,444; 5,660,850; 5,665,372;
`and 5,858,390; and co-pending US. application Ser. No.
`093678347 are incorporated herein by reference in their
`entirety.
`
`SUMMARY
`
`[0004] The present invention provides compositions and
`methods for correcting cosmetic, aesthetic, and degenerative
`defects in the skin, soft
`tissue and bone of a subject. In
`particular, tnethods of the invention involve the injection or
`implantation of autologous, undifferentiated mesenchymal
`cells (UMC), fibroblasts, andfor keratinocytes into the tissue
`(e.g., intradermal tissue) adjacent or subadjacent to a defect
`or at the site of a defect. Defects that can be corrected by this
`method include,
`for example,
`rhytids,
`stretch marks,
`depressed scars, cutaneous depressions of non-traumatic
`origin, scarring from acne vulgaris, hypoplasia of the lip,
`periodontal defects, soft tissue defects such as velopharyn-
`geal incompetence and subcutaneous atrophy, bone defects,
`and burns. The cells that are injected, as provided herein, are
`histocompatible with the subject and have been expanded by
`passage in a cell culture system. The injected cells prefer-
`ably are autologous cells. The invention also features an in
`vitro method ofgenerating a population of cells that includes
`UMC.
`
`In one embodiment, the composition can include
`[0005]
`both passaged, autologous UMC and passaged, autologous
`fibroblasts. Such cells can be derived, for example, from the
`culture of one or more biopsy specimens taken from the
`subject.
`In another embodiment,
`the composition can
`include passaged, autologous UMC‘ with a biodegradable
`acellular matrix, with or without passaged, autologous fibro-
`blasts. In yet another embodiment,
`the composition can
`include passaged, autologous UMC together with a biode-
`gradable acellular filler, with or without passaged, auto
`logous fibroblasts. Other embodiments contain pass aged,
`auto logous keratinocytes and passaged, autologous fibro-
`blasts, with or without matrices or fillers.
`
`[0006] The invention further provides methods for render-
`ing the passaged autologous UMC, fibroblasts, and kerati-
`nocytes substantially free of immunogenic proteins present
`in the culture medium (i.e., culture medium serum-derived
`proteins), so that the cells can be used to correct defects in
`a subject without stimulating an immune response. The
`method involves incubating the expanded UMC‘, fibroblasts,
`
`keratinocytes, or biodegradable acellular matrices contain-
`ing one or more of these cell populations in serum-free
`medium for a period of time subsequent
`to culturing in
`serum (e.g., fetal bovine serum (F'BS)) containing medium.
`In another embodiment, the cells or biodegradable acellular
`matrices containing cells are cultured only in serum-free
`medium or in medium containing autologous serum.
`
`in part, on the
`invention is based,
`[000?] The present
`discovery that autologous cells are an ideal material with
`which to augment tissue such as the dermis, subcutaneous
`tissue, and bone, at sites that are at or near (cg, subadjacent
`to) a defect. The invention also is based on the recognition
`that an abundant supply of autologous cells can be obtained
`by culturing a biopsy specimen taken from a subject prior to
`treatment of the defect. The invention is further based on the
`recognition that, after expansion in xenogeneic serum-con-
`taining tissue culture medium, autologous cells contain a
`significant quantity of immunogenic proteins derived from
`the xenogeneic serum, but that the immunogenic proteins
`can be removed prior to injection into the subject.
`
`In one aspect, the invention features a method for
`[0008]
`making a cellular composition for
`repairing tissue;
`the
`method involves: (a) providing a biopsy of undilIerentiated
`mesenchymal cell (U MCJ-containing tissue to obtain start-
`ing cells; (b) separating starting cells from said biopsy; (c)
`culturing the starting cells; and (d) harvesting a population
`of non-adherent derivative cells frotn the culture, the non-
`adherent derivative cells containing UMC. The method can
`further include one or more (e.g., one, two, three, four, five,
`six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19,
`20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100,
`or more) rounds of derivatization that includes repeating
`steps (c) and (d) utilizing the population of harvested
`non-adherent derivative cells from the previous round as the
`starting cells. The one or more additional rounds of deriva—
`tization can include,
`for example,
`from one to twenty
`rounds. The UMC-containing tissue can be selected from the
`group consisting of: dermal tissue, adipose tissue, connec-
`tive tissue, fascia, lamina propria and bone marrow. The
`method can further involve culturing the non-adherent cells
`in the presence of acidic fibroblast growth factor (aFGF).
`
`In another aspect, the invention features a compo—
`[0009]
`sition for repairing tissue. The composition can contain
`autologous, passaged UMC, and can be substantially free of
`culture medium serum—derived proteins. The composition
`can further contain autologous, passaged fibroblasts. The
`autologous fibroblasts can be from gums, palate, skin,
`lamina propria, connective tissue, bone marrow, fascia, or
`adipose tissue of the subject. The UMC or the fibroblasts can
`be treated with an activating compound (e.g., ascorbyl
`palmitate, linoleic acid, C-MED 100® (Optigene-X LLC,
`Shrewsbury, N.J.), quinic acid, quinic acid salts, quinic
`lactones, Colinzyme Q-lU, L-hydroxy acid, L-lipoic acid,
`calcium monophosphate,
`calcium triphosphate, niacin,
`dehydroepiandrosterone {DIIEA}, dimethylamino ethanol
`(DMAE), ot-tocopherol, bone morphogenic proteins {BMP},
`vitamin E, vitamin C, carotenoids, glycolic acid, carboxy-
`alkyl esters, estriol, acetyl-L-eamitine, deprenyl, lycopene,
`nicotinamide adenine dinucleotide (NADH), cysteine, pro-
`cysteine, pikamilon, vinpocetine, retinoic acid, antineoplas-
`tons, or other stimulatory additives such as growth factors
`[e.g., human growth hormone,
`fibroblast growth factor
`(F'GF'), vascular endothelial growth factor (VEGF), insulin-
`
`2
`
`

`

`US 2004/0101959 A1
`
`May 27, 2004
`
`insulin, and long form R3
`like growth factor-I (IGF-I),
`IGF—[]). The UMC or the fibroblasts can also be exposed to
`a low energy laser light prior to administration to a subject.
`The composition can further contain a biodegradable acel-
`lular matrix, wherein the UMC andtor fibroblasts are inte-
`grated within or on the matrix. The matrix, prior to combi-
`nation with the UMC andr'or fibroblasts, can contain one or
`more substances selected from the group consisting of:
`collagen (e.g., bovine collagen, porcine collagen, human
`collagen, engineered collagen, or collagen and glycosami-
`noglycans cross-linked with glutaraldehyde), glycosami-
`noglyeans, gelatin, polyglycolic acid, cat gut, demineralized
`bone, hydroxyapatite, hyaluron, hyaluronic acid, coral, and
`anorganic bone. Sulficient UMC andr'or fibroblasts can inte—
`grate on and within the matrix to substantially fill the space
`on or within the matrix available for cells. The composition
`can also or alternatively contain a biodegradable acellular
`injectable liller. The biodegradable acellular injectable filler,
`prior to combination with the UMC andr'or fibroblasts, can
`include one or more substances selected from the group
`consisting of; (a) an injectable dispersion of autologous
`collagen fibers; (b) collagen (e.g., bovine collagen. recon-
`stituted bovine collagen fibers cross-linked with glutaralde-
`hyde, or autologous collagen); (c) solubilized gelatin; (d)
`solubilized polyglycolic acid; (e) solubilized cat gut; (f)
`porcine gelatin powder and amino caproic acid dispersed in
`sodium chloride solution and an aliquot of plasma from the
`subject; and {g} hyaluronic acid.
`
`In another aspect. the invention features a method
`[0010]
`for making a composition for repairing tissue that has
`degenerated in a subject as a result of a disease, disorder, or
`defect in the subject. The method can involve: (a) providing
`a biopsy of UMC-containing tissue frotn the subject; (b)
`separating autologous UMC from the biopsy; (c) culturing
`the autologous UMC under conditions that produce autolo-
`gous UMC that are substantially free of culture medium
`serum-derived proteins; and (d) exposing the cultured
`autologous UMC to conditions that result in suspension of
`the UMC. The UMC-containing tissue can be selected from
`the group consisting of: gums, palate, skin, lamina propria,
`connective tissue, fascia, bone marrow, and adipose tissue.
`Culturing of the UMC can involve: (I) incubation in a
`culture medium containing between 0.1% and about 20%
`human or non-human serum, followed by (2) incubation in
`a serum-free culture medium. Alternatively, the culturing of
`the UMC can be only in serum—free medium. The conditions
`that result in suspension of the UMC can include the use of
`a proteolytic enzyme. The method can further involve: (e)
`providing a biopsy of fibroblast-containing tissue from the
`subject;
`(I) separating autologous fibroblasts from the
`biopsy; (g) culturing the autologous fibroblasts under con-
`ditions that produce fibroblasts that are substantially free of
`culture medium serum-derived proteins; (h) exposing the
`cultured autologous fibroblasts to conditions that result in
`suspension of the fibroblasts; and (i) mixing the UMC
`suspension with the fibroblast suspension. The autologous
`fibroblast-containing tissue can be selected frotn the group
`consisting of: gums, palate, skin, lamina propria, connective
`tissue, fascia, bone marrow, and adipose tissue. Culturing of
`the fibroblasts can involve:
`(1} incubation in a culture
`medium containing between 0.1% and about 20% human or
`non-human serum, followed by (2) incubation in a serum-
`free culture medium. Alternatively,
`the culturing of the
`fibroblasts can be only in serum-free medium. The condi-
`
`tions that result in suspension of the fibroblasts can include
`the use of a proteolytic enzyme. Prior to combining, the
`UMC or the fibroblasts can be exposed to an activating
`compound (e.g., ascorbyl palmitate, linoleic acid, C-MED
`100®, quinic acid, quinic acid salts, quinic lactones, CoEn-
`zyme Q-10, L-hydroxy acid, L-lipoic acid, calcium mono-
`phosphate, calcium triphosphate, niacin, DHEA, DMAE,
`ct-tocopherol, BMP, vitamin E, vitamin C, carotenoids,
`glycolic acid, carboxyalkyl esters, estriol, acetyl-L;car-
`nitine, deprenyl, lycopene, NADH, cysteine, procysteine,
`pikamilon, vinpocetine, retinoic acid, antineoplastons, or
`other stimulatory additives such as growth factors [e.g.,
`human growth hormone, FGF, VEGF, IGF-I, insulin, and
`long form R3 IGF—I]). The UMC or the fibroblasts can be
`exposed to a low energy laser light.
`[0011]
`In yet another aspect,
`the invention features a
`method for making a composition for repairing tissue that
`has degenerated in a subject as a result of a disease, disorder,
`or defect
`in the subject. The method can involve:
`(a)
`providing autologous, passaged UMC; (1)) providing a bio-
`degradable acellular matrix; and (c) incubating the UMC
`with the biodegradable acellular matrix such that the UMC
`integrate on or within the biodegradable acellular matrix,
`wherein the incubation results in a composition for repairing
`tissue, and wherein the conditions of the incubation are such
`that the composition is substantially free of culture medium
`serum-derived proteins. The step of providing a suspension
`of autologous, passaged UMC can involve: (a) providing a
`biopsy of UMC-containing tissue from the subject; (b)
`separating autologous UMC from the biopsy; (c) culturing
`the UMC; and (d) exposing the cultured UMC to conditions
`that result in suspension of the UMC. The UMC-containing
`tissue can be selected from the group consisting of: gums,
`palate, skin, lamina propria, connective tissue, fascia, bone
`marrow, and adipose tissue. The biodegradable acellular
`matrix, prior to combination with the suspension of UMC,
`can contain one or more substances selected from the group
`consisting of: collagen (e.g., collagen and glycosaminogly—
`cans, cross-linked with glutaraldehyde, bovine collagen, or
`porcine collagen), glycosaminoglycans, gelatin. polygly-
`colic acid, cat gut, demineralized bone, hydroxyapatite,
`coral, and anorganic bone. The incubation conditions can
`include: (1} culturing in culture medium containing between
`0.1% and about 20% human or non—human serum, followed
`by (2) culturing in serum-free culture medium. Alternatively
`the culturing can be only in serum-free medium. Sufiicient
`UMC can integrate within the biodegradable acellular
`matrix to substantially [ill the space on or within the bio-
`degradable acellular matrix available for cells.
`
`[0012] The method can further involve providing autolo-
`gous, passaged fibroblasts, incubating the fibroblasts with
`the biodegradable acellular matrix such that the fibroblasts
`integrate on or within the biodegradable acellular matrix,
`wherein the incubation results in a composition for repairing
`tissue, and wherein the conditions of the incubation are such
`that the composition is substantially free of culture medium
`serum-derived proteins. The step of providing autologous,
`passaged fibroblasts can involve: (a) providing a biopsy of
`fibroblast-containing tissue from the subject; (b) separating
`autologous fibroblasts from the biopsy; (c) culturing the
`fibroblasts; and (d) suspending the fibroblasts. The fibro-
`blast-containing tissue can be selected from the group con-
`sisting of: gums, palate, skin,
`lamina propria, connective
`tissue, fascia, bone marrow, and adipose tissue. Sufiicient
`
`3
`
`

`

`US 2004/0101959 A1
`
`May 27, 2004
`
`UMC and fibroblasts can integrate on or within the biode-
`gradable acellular matrix to substantially fill the space on or
`within the biodegradable acellular matrix available for cells.
`Prior to the incubating with the biodegradable acellular
`matrix, the UMC andx’or the fibroblasts can be exposed to an
`activating compound {e.g., ascorbyl palmitate, linoleic acid,
`C-MED 100Gb, quinic acid, quinic acid salts, quinic lactones,
`CoEnzyme Q-lO, L-hydroxy acid, L-lipoic acid, calcium
`monophosphate,
`calcium triphosphate, niacin, DHEA,
`DMAE, u-tocopherol, BMP, vitamin E, vitamin C, caro-
`tenoids, glycolic acid, carboxyalkyl esters, estriol, acetyl-L-
`carnitine, deprenyl, lycopene, NADH, cysteine, procysteine,
`pikamilon, vinpocetine, retinoic acid, antineoplastons, or
`other stimulatory additives such as growth factors [e.g.,
`human growth hormone, FGF, VEGF, IGF—I, insulin, and
`long form R3 [GP-1]]. The UMC or the fibroblasts can be
`exposed to a low energy laser light. The UMC and the
`fibroblasts can be together incubated with the biodegradable
`acellular matrix, or the UMC and the fibroblasts are added
`separately to the biodegradable acellular matrix.
`[0013]
`In another aspect, the invention features a method
`for making a composition for repairing tissue that has
`degenerated in a subject as a result of a disease, disorder, or
`defect in the subject. The method can involve: (a) providing
`autologous, passaged UMC, wherein the UMC are substan-
`tially free of culture medium serum-derived proteins; (b)
`providing a biodegradable acellular filler; and (c) combining
`the autologous, passaged UMC and the biodegradable acel-
`lular filler. The step of providing a suspension of autologous,
`passaged UMC can involve:
`(a) providing a biopsy of
`UMC-containing tissue from the subject; (b) separating
`autologous UMC from the biopsy; (c) culturing the autolo-
`gous UMC under conditions that result in UMC that are
`substantially free of culture medium serum-derived proteins;
`and (d) exposing the cultured UMC to conditions that result
`in suspension of the UMC. The UMC-containing tissue can
`be selected from the group consisting of: gums, palate, skin,
`lamina propria, connective tissue, fascia, bone marrow, and
`adipose tissue. Culturing of the UMC can involve:
`(1)
`culturing in a medium containing between 0.1% and about
`20% human or non-human serum, followed by (2) culturing
`in a serum-free medium. Culturing of the UMC can involve
`culturing in serum-free medium. The conditions that result
`in suspension of the UMC can include the use of a pro—
`teolytic enzyme. The biodegradable acellular filler, prior to
`combination with the UMC, can contain one or more sub-
`stances selected from the group consisting of: (a) an inject-
`able dispersion of autologous collagen fibers; (b) collagen
`(e.g., bovine collagen, reconstituted bovine collagen fibers
`cross-linked with glutaraldehyde, or autologous collagen);
`(c) solubilized gelatin, (d) solubilized polyglycelic acid; (e)
`solubilized cat gut; (f) porcine gelatin powder and amino
`caproic acid dispersed in sodium chloride solution and an
`aliquot of plasma from the subject, and (g) hyaluronic acid.
`[0014] The method can further involve providing autolo-
`gous, passaged fibroblasts, wherein the autologous, pas-
`saged fibroblasts are substantially free of culture medium
`serum -derived proteins, and combining the autologous, pas-
`saged fibroblasts and the biodegradable acellular filler. The
`step of providing a suspension of autologous, passaged
`fibroblasts can involve: (a) providing a biopsy of fibroblast-
`containing tissue from the subject, (b) separating autologous
`fibroblasts from the biopsy; (c) culturing the autologous
`fibroblasts under conditions that result in fibroblasts that are
`
`substantially free of culture medium serum-derived proteins,
`and (d) exposing the incubated autologous fibroblasts to
`conditions that result in suspension of the fibroblasts. The
`fibroblast—containing tissue can be selected from the group
`consisting of: gums, palate, skin, lamina propria, connective
`tissue, fascia, bone marrow, and adipose tissue. Culturing of
`the fibroblasts can involve:
`(1) culturing in a medium
`containing between 0.1% and about 20% human or non-
`human serum, followed by (2) culturing in a serum-free
`medium. Alternatively the culturing of the libroblasts can be
`only in serum-free medium. The conditions that result in
`suspension of the fibroblasts can include a proteolytic
`enzyme. Prior to the combining with the UMC and the
`biodegradable acellular filler, the UMC or the fibroblasts can
`be exposed to an activating compound (e.g., ascorbyl palmi-
`tate, linoleic acid, C-MED 100®, quinic acid, quinic acid
`salts, quinic lactones, CoEnzyme 0-10, L-hydroxy acid,
`L-lipoic acid, calcium monophosphate, calcium triphos-
`phate, niacin, DHEA, DMAE, (L-tocopherol, BMP, vitamin
`E, vitamin C, carotenoids, glycolic acid, carboxyalkyl esters,
`estriol, acetyl-L-camitine, deprenyl, lycopene, NADH, cys-
`teine, procysteine, pikamilon, vinpocetine, retinoic acid,
`antineoplastons, or other stimulatory additives such as
`growth factors [e.g., human growth hormone, FGF, VEGF‘,
`IGF-I, insulin, and long fonn R3 IGF-I]). The UMC or the
`fibroblasts can be exposed to a low energy laser light.
`
`the invention features a
`In still another aspect,
`[0015]
`method for repairing tissue in a subject. The method can
`involve: (a) providing a composition containing autologous,
`passaged UMC, wherein the composition is substantially
`free of culture medium serum-derived proteins; (b) identi-
`fying a site of tissue defect or tissue degeneration in the
`subject; and (c) placing the composition at the site so that the
`tissue defect or degeneration is repaired. The tissue defect or
`tissue degeneration can include a soft tissue defect, a defect
`of an oral mucosa, trauma to an oral mucosa, periodontal
`disease, a bone defect, diabetes, a cutaneous ulcer, venous
`stasis, or a cosmetic defect of the skin. The soft tissue defect
`can be selected from the group consisting of a facial depres-
`sion, underdevelopment of the breast, absence of breast,
`breast reconstruction, a vocal cord defect, velopharyngeal
`incompetence, Poland’s syndrome, underdevelopment of
`male genitalia (e.g., penis), hypoplasia of the lips, and a
`subcutaneous atrophy or muscular atrophy. The trauma to an
`oral mucosa can be an extraction socket resulting from an
`extraction of a tooth. The periodontal disease can involve
`periodontal degeneration, gingivitis, or non-healing wounds
`of a palatal mucosa or a gingival mucosa. The bone defect
`can be selected from the group consisting of hemifacial
`microsomia, posttraumatic non-healing of a bone, enoph-
`thalrnos, Rornberg’s disease, cranial burr hole defects, loss
`of skull bone, a defect that result from a blunt trauma, and
`a defect that results from arthritis. The cosmetic defect can
`be selected from the group consisting of:
`a rhytid, a
`depressed scar, a cutaneous depression of non-traumatic
`origin, a laugh line, a stretch mark, a wrinkle, a wound scar,
`an acne scar, and subcutaneous atrophy. The tissue defect
`can be hypoplasia of a lip or a lip fold. The autologous UMC
`can be from gums, palate, skin, lamina propria, connective
`tissue, fascia, bone marrow, or adipose tissue of the subject.
`The composition can further comprise a biodegradable
`matrix, wherein the UMC‘ are integrated within or on the
`
`4
`
`

`

`US 2004/0101959 A1
`
`May 27, 2004
`
`matrix. The composition also can comprise a biodegradable
`acellular filler. The composition can further contain autolo-
`gous, passaged fibroblasts.
`
`In another aspect, the invention provides a method
`[0016]
`for repairing tissue in a subject. The method can involve: (a)
`providing a composition containing autologous, passaged
`UMC and autologous, passaged fibroblasts, wherein the
`composition is substantially free of culture medium serum-
`derived proteins; (b) identifying a site of tissue defect or
`tissue degeneration in the subject; and (c) placing the
`composition at the site so that the tissue defect or degen-
`eration is repaired.
`
`In another aspect, the invention features a method
`[0017]
`for repairing a tissue defect in a subject. The method can
`involve: (a) providing a pharmaceutical composition con-
`taining:
`(1) autologous, passaged UMC, (2) autologous,
`passaged fibroblasts, and (3) a pharmace utically acceptable
`carrier thereof; wherein the pharmaceutical composition is
`substantially free of culture medium serum—derived proteins;
`(b) identifying in the subject a site of tissue defect or tissue
`degeneration selected from the group consisting of: a dental
`or periodontal defect, a cosmetic defect of the skin, and a
`bone defect; and (c) injecting a therapeutically effective
`amount of the pharmaceutical composition adjacent to the
`site of tissue defect or degeneration, wherein the injecting
`results in repair of the tissue defect or degeneration. The
`UMC-containing and fibroblast-containing tissues can be
`selected from the group consisting of: gums, palate, skin,
`lamina propria, connective tissue, fascia, bone marrow, and
`adipose tissue. The UMC or the fibroblasts can have been:
`(1) cultured in a medium containing between 0.1% and
`about 20% human or non-human serum, and then (2) cul-
`tured in a serum-free medium. The UMC or the fibroblasts
`can have been cultured in serum-free medium.
`
`In another aspect, the invention features a method
`[0018]
`for repairing tissue that has degenerated in a subject as a
`result of a disease, disorder, or defect in the subject. The
`method can involve placing a composition into the subject at
`the site of degeneration so that the tissue is repaired. The
`composition can contain (1) autologous, passaged UMC, (2)
`autologous. passaged fibroblasts, and (3} a biodegradable
`matrix, wherein the UMC or the fibroblasts are integrated
`within or on the matrix, and wherein the composition is
`substantially free of culture medium serum-derived proteins.
`
`[0019] The invention also features a method for repairing
`tissue that has degenerated in a subject as a result of a
`disease, disorder, or defect in the subject. The method can
`involve injecting a composition containing {1} autologous,
`passaged UMC that are substantially free of culture medium
`serum-derived proteins and (2) a biodegradable acellular
`injectable filler into the subject at the site of the degeneration
`so that the tissue is repaired. The composition fiirther can
`further contain autologous, passaged fibroblasts.
`
`In another aspect, the invention features a method
`[0020]
`for repairing tissue that has degenerated in a subject as a
`result of a disease, disorder, or defect in the subject. The
`method can include the steps of: (a) injecting autologous,
`passaged UMC into the subject at a site of tissue degenera-
`tion, wherein the UMC are substantially free of culture
`medium serum-derived proteins; (b) injecting autologous,
`passaged fibroblasts into the subject at a site of a tissue
`defect or desired tissue augmentation, wherein the fibro-
`
`blasts are substantially free of culture medium serum-de-
`rived proteins; and (c) injecting a biodegradable, acellular
`filler into the site. The disease, disorder, or defect can be a
`defect selected from the group consisting of: a dental or
`periodontal defect, a cosmetic defect of the skin, and a bone
`defect. The autologous UMC and fibroblasts can be from
`gums, palate, skin, lamina propria, connective tissue, fascia,
`bone marrow, or adipose tissue ofthe subject. The UMC and
`the fibroblasts can be injected simultaneously. The UMC,
`the fibroblasts, and the biodegradable acellular filler can be
`injected simultaneously. The UMC and the fibroblasts can be
`injected separately. The UMC and the fibroblasts can be
`injected separately from the biodegradable acellular filler.
`The duration between injecting the UMC and the fibroblasts
`into the subject and injecting the biodegradable acellular
`filler into the subject can be about two weeks In another
`aspect, the invention features a method for repairing a burn
`wound in a subject. The method can involve providing a
`suspension of autologous, passaged keratinocytes and
`autologous, passaged fibroblasts, and injecting the suspen-
`sion into the burn wound. Providing a suspension of autolo—
`gous, passaged keratinocytes and autologous, passaged
`fibroblasts can involve: (a) providing a biopsy of kerati-
`nocyte-containing tissue from the subject; (b) separating
`autologous keratinocytes from the biopsy; (c) culturing the
`autologous keratinocytes under conditions that produce
`autologous keratinocytes that are substantially free of cul-
`ture medium serum-derived proteins; (d) exposing the kera-
`tinocytes to conditions that result
`in suspension of the
`keratinocytes; (e) providing a biopsy of fibroblast-contain-
`ing tissue from the subject; (D separating autologous fibro-
`blasts from the biopsy; (g) culturing the autologous fibro-
`blasts under conditions that produce autologous fibroblasts
`that are substantially free of culture medium serum-derived
`proteins; {h} exposing the fibroblasts to conditions that result
`in suspension of the fibroblasts; and (i) combining the
`suspended keratinocytes and the suspended fibroblasts. The
`keratinocyte-containing tissue can be selected from the
`group consisting of: skin, oral mucosa, gums, buccal tissue,
`esophageal tissue, exocervical tissue, and conjunctival tis-
`sue. Culturing of the keratinocytes can involve ( 1) incuba-
`tion in a culture medium containing between 0.1% and about
`20% human or non—human serum, followed by (2) incuba—
`tion in a serum-free culture medium. Alternatively the
`culturing of the keratinocytes can be only in serum-free
`medium. The fibroblast—containing tissue can be selected
`from the group consisting of: gums, palate, skin, lamina
`propria, connective tissue, fascia, bone marrow, and adipose
`tissue. Culturing of the fibroblasts can involve: (1) incuba-
`tion in a culture medium containing between 0.1% and about
`20% human or non-human serum, followed by (2) incuba-
`tion in a serum-free culture medium. Alternatively,
`the
`culturing of the fibroblasts can be only in serum-free
`medium. The conditions that result
`in suspension of the
`fibroblasts can include a proteolytic enzyme. Prior to com-
`bining with the keratinocytes, the fibroblasts can be exposed
`to an activating compound {e.g., ascorbyl palmitate, linoleic
`acid, C-MED [00®, quinic acid, quinic acid salts, quinic
`lactones, CoEnzyme 0-10, L-hydroxy acid, L-lipoic acid,
`calcium monophosphate,
`calcium triphosphate, niacin,
`DHEA, DMAE, rz-tocopherol, BMP, vitamin E, vitamin C,
`carotcnoids, glycolic acid, carboxyalkyl esters, cstriol,
`acetyl-L-carnitine, deprenyl,
`lycopene, NADH, cysteine,
`procysteine, pikamilon, vinpocetine, retinoic acid, antine-
`
`5
`
`

`

`US 2004/0101959 A1
`
`May 27, 2004
`
`oplastons, or other stimulatory additives such as growth
`factors [e.g., human growth hormone, FGF, VEGF, IGF-I,
`insulin, and long form R3 [GP-1]). 'l‘he fibroblasts can also
`be exposed to a low energy laser light prior to administration
`to a subject. The suspension can contain autologous kerati-
`nocytes and autologous fibroblasts in a ratio of about 1:3.
`The injecting can involve injecting the suspension into the
`central region of the burn wound. The suspension can further
`contain autologous, passaged UMC. The method can further
`include injecting an enhancing compound into the wound.
`
`[0021] Unless otherwise defined, all technical and scien-
`tific terms used herein have the same meaning as commonly
`understood by one of ordinary skill in the art to which this
`invention pertains. Although methods and materials similar
`or equivalent
`to those described herein can be used to
`practice the invention, suitable methods and materials are
`described below. All publications. patent applications. pat-
`ents, and other references mentioned herein are incorporated
`by reference in their entirety. In case of conflict, the present
`specification, including definitions, will control. In addition,
`the materials, methods, and examples are illustrative only
`and not intended to be limiting.
`
`[0022] The details of one or more embodiments of the
`invention are set forth in the accompanying drawings and
`the description below. Other features, objects, and advan-
`tages of the invention will be apparent from the description
`and from the claims.
`
`DETAILED DESCRIPTION
`
`[0023] The invention provides compositions and methods
`for treating disorders or defects that result in degeneration of
`tissue in a subject. Such disorders or defects include defects
`of the oral mucosa,
`trauma to the oral mucosa (e.g., an
`extraction socket that can result from extraction of a tooth),
`trauma to oral bones such as the maxillary or mandibular
`bones, periodontal disease, diabetes, cutaneous ulcers, or
`venous stasis. Examples of periodontal disease that can
`result in tissue degeneration include, but are not limited to,
`periodontal degeneration, gingivitis, or non-healing wounds
`of the palatal mucosa or gingival mucosa, or bone degen-
`eration. Other defects that can be treated using the compo-
`sitions and methods provided herein include, without limi-
`tation, skin defects such as scars, wrinkles,
`laugh lines,
`stretch marks, depressed scars, cutaneous d