WO2007047707A1 - Method of delivering hair follicle progenitor cells to the skin - Google Patents

Method of delivering hair follicle progenitor cells to the skin Download PDF

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Publication number
WO2007047707A1
WO2007047707A1 PCT/US2006/040608 US2006040608W WO2007047707A1 WO 2007047707 A1 WO2007047707 A1 WO 2007047707A1 US 2006040608 W US2006040608 W US 2006040608W WO 2007047707 A1 WO2007047707 A1 WO 2007047707A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
skin
wound
depth
hair
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2006/040608
Other languages
English (en)
French (fr)
Inventor
Ying Zheng
Xiaobing Du
Kenneth Justin Washenik
Kurt Stricker Stenn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aderans Research Institute Inc
Original Assignee
Aderans Research Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aderans Research Institute Inc filed Critical Aderans Research Institute Inc
Priority to JP2008536744A priority Critical patent/JP5153638B2/ja
Priority to AU2006304516A priority patent/AU2006304516A1/en
Priority to CA002626199A priority patent/CA2626199A1/en
Priority to ES06817074.5T priority patent/ES2655113T3/es
Priority to MX2008004928A priority patent/MX2008004928A/es
Priority to PL06817074T priority patent/PL1945757T3/pl
Priority to EP06817074.5A priority patent/EP1945757B1/en
Publication of WO2007047707A1 publication Critical patent/WO2007047707A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/18—Materials or treatment for tissue regeneration for hair reconstruction

Definitions

  • Developing appropriate cell delivery techniques is important for many applications. Not only must the cells be delivered to the appropriate part of the subject, but the morphology of the cells must be maintained.
  • the morphology of cells can be important in many contexts.
  • Cell morphology indicates the status of the cells, both in terms of the health of the cells and in terms of the differentiation state of the cell. Changes in cell shape, or morphogenesis, are central to cell function, development and disease. Physiological processes in cells are often accompanied by changes in cellular morphology.
  • Examples include changes in the intracellular location, arrangement and structure of cellular constituents, such as organelles, macromolecular clusters or the cytoskeleton, changes in the morphology of the entire cell, such as its shape and area, changes in the spacing and proximity between cells, and properties of multi-cellular colonies such as its shape, size and cell locations.
  • the present invention is a method of forming egressing hair shafts comprising making a wound in the skin and placing hair follicle progenitor cells on the wound, wherein an egressing hair shaft is formed from the hair follicle progenitor cells.
  • the present invention is also a method of forming new skin comprising making a wound in the skin and placing skin forming cells on the wound, wherein new skin is formed from the skin forming cells.
  • FIG. 1 shows a grid of superficial cuts on the surface of the skin of a nude mouse and hair out-growth (inset) observed at two weeks post-administration with black newborn mouse skin cells.
  • FIG. 2 shows a scalpel blade being used to make a superficial stab wound on the surface of the skin of a nude mouse and hair out-growth (inset) observed at two weeks post-administration of black newborn mouse skin cells into the wound.
  • cells can be delivered into the superficial skin without disturbing the inherent characteristics of the cells by making a wound in an area where the cells are desired and placing the cells on the wound. It is expected that the cells would orientate themselves appropriately to form the desired structure upon delivery with the method of the present invention.
  • the cells can be placed on the surface of the skin on top of the wound or in the wound itself.
  • the cell delivery method of the present invention can be used to form egressing hair shafts.
  • the cell delivery method of the present invention may also be used to form new skin to treat, repair or improve conditions including, but not limited to, skin ulcers, diabetic foot ulcers, bed sores, burn wounds, microbial infections, and scars such as those resulting from surgery, acne, or illnesses such as chicken pox. Delivery of appropriate cells by the methods of the present invention could also be used to form sweat glands, nail, eyebrow, eyelash and other hairs.
  • the method of the present invention could also be used to form the dermis or epidermis using genetically altered autologous or allogenic cells.
  • the cell delivery method of the present invention may be used to form both the dermal and epidermal layers of the skin. Alternatively, either the dermal or the epidermal layers may be formed by the method of the present invention.
  • the wound may be formed by any technique that disrupts the outer surface of the area in which the cells are desired such as stabbing, cutting or scratching the area with a sharp instrument, including, but not limited to, a scalpel blade or a needle.
  • the wound may also be formed by abrading the area with, e.g. needles such as microneedles or grooved needles.
  • the sharp instrument may be modified to form a wound with a measured depth.
  • the wound may be at least about 10 ⁇ m in depth, at least about 200 ⁇ m in depth, no more than about 500 ⁇ m in depth, or no more than about 250 ⁇ m in depth.
  • the wound may be from about 10 ⁇ m to about 500 ⁇ m in depth.
  • the wound may be unlimited in maximal length.
  • the depth of the wound may be adjusted to deliver the cells into the subepidermis, the papillary dermis, the upper reticular dermis, or the same levels of the nail bed of the acral skin (palmer-plantar skin).
  • the wound is shallow enough that the subject does not bleed.
  • the wound suitably heals without forming a scar.
  • the wound may be covered with a bandage or dressing; for example, a non-adhering dressing, or a transparent plastic dressing such as Tegaderm® (3M, St. Paul, MN) or a gel-based burn dressing. Petroleum jelly or the like may also be applied to the wound.
  • the dressing may be left in place for about 3 to about 7 days.
  • the dressing may be substantially water-impermeable.
  • the cells may comprise dermal cells, epidermal cells, epidermal stem cells, basal cells, keratinocytes, fibroblasts or combinations thereof.
  • the cells may be derived from follicular, eccrine or nail sources.
  • the cells are skin forming cells or hair follicle progenitor cells.
  • the ratio of epidermal to dermal cells is suitably about 10:1 to about 1:10.
  • the ratio may be about 1 : 1 to about 5:1.
  • the cells may suitably be provided in a suspension in a physiologically acceptable carrier, e.g., sterile saline solution. Additional components may also be added to the cell suspension. Suitable additional components include growth factors, nutrient molecules or stabilizing molecules. [0014]
  • a physiologically acceptable carrier e.g., sterile saline solution.
  • Additional components may also be added to the cell suspension. Suitable additional components include growth factors, nutrient molecules or stabilizing molecules.
  • Example 1 Hair outgrowth from follicle-inductive cells administered to superficial cuts on the surface of mouse skin.
  • An athymic nude (nu/nu) mouse (Charles River, Inc.) was anesthetized and a grid of superficial cuts (Figure 1) was made on the dorsal skin with the use of a number 11 scalpel blade. The cuts were shallow enough not to draw blood.
  • a mixture of freshly isolated newborn black mouse skin cells comprising 100,000 epidermal cells and 200,000 dermal cells was prepared as follows. Truncal skin was removed from newborn mice and rinsed in Ca 2+ and Mg 2+ free PBS. The skin was laid flat in PBS containing Dispase (2.5 mg per ml, Invitrogen, Carslbad, CA) at 4 0 C overnight or 37°C for 2 hours.
  • Inductive dermal and epidermal cells were isolated as described in Weinberg et al., J. Invest. Dermatol., 100:229-236 (1993), which is incorporated herein by reference.
  • a suspension of the cells in 2 microliters of sterile buffered saline solution was delivered to the grid-of- cuts by pipette. The skin was gently pulled apart for a few seconds to allow the fluid to wick throughout the grid.
  • a non-adherent, hydrophobic (petrolatum coated gauze) dressing was applied to the wound and the mouse was further bandaged with an elastic wrap to prevent removal of the dressing upon recovery from anesthesia. The dressing was removed after 10 days. After 2 weeks the growth of hair was observed (Figure 1, inset) primarily within the grid pattern. The mouse was then euthanized and the skin gently removed. Observation of the underside of the skin revealed almost no ingrown hairs and the presence of follicle bulbs corresponding to the visible hair seen on the skin surface.
  • Example 2 Hair outgrowth from follicle-inductive cells administered to superficial stab wound on the surface of mouse skin.
  • Athymic nude (nu/nu) mouse (Charles River, Inc.) was anesthetized and a stab wound was made in the skin by piercing with the tip of a number 11 scalpel blade held a low angle to the surface of the skin ( Figure 2).
  • a mixture of freshly isolated newborn black mouse skin cells comprising 100,000 epidermal cells and 200,000 dermal cells was prepared as described above.
  • a suspension of the cells in 2 microliters of sterile buffered saline solution was instilled into the cut by pipette.
  • a non-adherent, hydrophobic (petrolatum coated gauze) dressing was applied to the wound and the mouse was further bandaged with an elastic wrap to prevent removal of the dressing upon recovery from anesthesia.
  • the dressing was removed after 10 days. After 2 weeks the growth of hair was observed (Figure 2, inset) precisely at the site of the stab incision. • The mouse was then euthanized and the skin gently removed. Observation of the underside of the skin revealed almost no ingrown hairs and the presence of follicle bulbs corresponding to the visible hair seen on the skin surface.
  • Example 3 Use of different wound dressing materials.
  • Example 4 New hair growth in bald scalp by application of autologous cells into superficial incisions.
  • Superficial wounds are created on a human subject's head with a surgical blade or scalpel, as grids, multiple crosses, parallel lines or similar such pattern to achieve the desired cosmetic effect for hair growth.
  • Autologous human trichogenic dermal and epidermal cells (optionally mixed with hair follicle melanocytes if needed to restore hair pigmentation) in suspension are then spread on top of the superficial cuts with pipette tips or syringes and the wound is covered for 2-3 days to prevent the cells from drying out prior to being incorporated into the skin. Egressing hair follicles are then formed.
  • Example 5 New hair growth in bald scalp by application of dermal cells into superficial incisions.
  • Wounds are created on a human subject's head with a surgical blade or scalpel, as grids, multiple crosses, parallel lines or similar such pattern to achieve the desired cosmetic effect for hair growth.
  • Human trichogenic dermal cells alone (optionally mixed with hair follicle melanocytes if needed to restore hair pigmentation) in suspension are then spread on top of the superficial cuts with pipette tips or syringes and the wound is covered for 2-3 days to prevent the cells from drying out prior to being incorporated into the skin. Egressing hair follicles are then formed.
  • Example 6 Formation of new skin to treat a subject with a diabetic foot ulcer.
  • a subject with a diabetic foot ulcer is anesthetized under local or general anesthesia and a series of superficial cuts is made in the ulcer.
  • the cuts are shallow enough not to draw blood.
  • a suspension of basal cells in 2 microliters of sterile buffered saline solution is delivered to the cuts by pipette.
  • the skin is gently pulled apart for a few seconds to allow the fluid to wick throughout the grid.
  • a non-adherent, hydrophobic (petrolatum coated gauze) dressing is applied to the wound. The dressing is removed after 10 days. New skin forms and the ulcer is healed.
  • Example 7 Formation of new skin to treat a subject with bed sores.
  • a subject with bed sores is anesthetized under local or general anesthesia and a series of superficial cuts is made in the bed sore.
  • the cuts are shallow enough not to draw blood.
  • a suspension of basal cells in 2 microliters of sterile buffered saline solution is delivered to the cuts by pipette.
  • the skin is gently pulled apart for a few seconds to allow the fluid to wick throughout the grid.
  • a non-adherent, hydrophobic (petrolatum coated gauze) dressing is applied to the wound. The dressing is removed after 10 days. New skin forms and the bed sore is healed.
  • any numerical range recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. If a concentration range is "at least 5%, it is intended that all percentage values up to and including 100% are also expressly enumerated. These are only examples of what is specifically intended.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Dermatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Transplantation (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Botany (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Virology (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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PCT/US2006/040608 2005-10-17 2006-10-17 Method of delivering hair follicle progenitor cells to the skin Ceased WO2007047707A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2008536744A JP5153638B2 (ja) 2005-10-17 2006-10-17 毛包前駆細胞を皮膚に送達する方法
AU2006304516A AU2006304516A1 (en) 2005-10-17 2006-10-17 Method of delivering hair follicle progenitor cells to the skin
CA002626199A CA2626199A1 (en) 2005-10-17 2006-10-17 Method of delivering hair follicle progenitor cells to the skin
ES06817074.5T ES2655113T3 (es) 2005-10-17 2006-10-17 Método de suministro de células progenitoras del folículo piloso a la piel
MX2008004928A MX2008004928A (es) 2005-10-17 2006-10-17 Metodo para suministrar celulas progenitoras de foliculo piloso a la piel.
PL06817074T PL1945757T3 (pl) 2005-10-17 2006-10-17 Sposób dostarczania komórek progenitorowych mieszków włosowych do skóry
EP06817074.5A EP1945757B1 (en) 2005-10-17 2006-10-17 Method of delivering hair follicle progenitor cells to the skin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US72758805P 2005-10-17 2005-10-17
US60/727,588 2005-10-17

Publications (1)

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WO2007047707A1 true WO2007047707A1 (en) 2007-04-26

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PCT/US2006/040608 Ceased WO2007047707A1 (en) 2005-10-17 2006-10-17 Method of delivering hair follicle progenitor cells to the skin

Country Status (11)

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US (1) US20070092496A1 (enExample)
EP (1) EP1945757B1 (enExample)
JP (1) JP5153638B2 (enExample)
AR (1) AR057158A1 (enExample)
AU (1) AU2006304516A1 (enExample)
CA (1) CA2626199A1 (enExample)
ES (1) ES2655113T3 (enExample)
MX (1) MX2008004928A (enExample)
PL (1) PL1945757T3 (enExample)
TW (1) TW200731982A (enExample)
WO (1) WO2007047707A1 (enExample)

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US7597885B2 (en) 2004-03-26 2009-10-06 Aderans Research Institute, Inc. Tissue engineered biomimetic hair follicle graft
US7780635B2 (en) 2006-02-09 2010-08-24 Aderans Research Institute, Inc. Apparatus and methods for delivering fluid and material to a subject
JP2010540425A (ja) * 2007-09-21 2010-12-24 インターサイテックス リミテッド 毛包形成を増強するための表皮刺激
USD690004S1 (en) 2012-03-16 2013-09-17 Aderans Research Institute, Inc. Holder for a device for delivering cellular material and physiologic fluids
US9023380B2 (en) 2005-11-22 2015-05-05 Aderans Research Institute, Inc. Hair follicle graft from tissue engineered skin

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US20070276211A1 (en) * 2006-05-26 2007-11-29 Jose Mir Compact minimally invasive biomedical monitor
US20100100005A1 (en) * 2006-07-11 2010-04-22 Infotonics Technology Center, Inc. Minimally invasive allergy testing system with coated allergens
JP2010504974A (ja) * 2006-09-28 2010-02-18 フォリカ，インコーポレーテッド 新しい毛嚢を生成させ毛髪を成長させる方法、キット、及び組成物
US7985537B2 (en) * 2007-06-12 2011-07-26 Aderans Research Institute, Inc. Methods for determining the hair follicle inductive properties of a composition
US8328720B2 (en) * 2007-08-10 2012-12-11 Infotonics Technology Center, Inc. MEMS interstitial prothrombin time test
KR20170024148A (ko) 2008-11-12 2017-03-06 더 트러스티스 오브 더 유니버시티 오브 펜실베니아 손상된 모낭의 재생을 촉진하는 섬유모세포 성장 인자-9
US20110130711A1 (en) * 2009-11-19 2011-06-02 Follica, Inc. Hair growth treatment
CA2819859A1 (en) 2010-12-06 2012-06-14 Follica, Inc. Methods for treating baldness and promoting hair growth
US10244981B2 (en) 2011-03-30 2019-04-02 SensiVida Medical Technologies, Inc. Skin test image analysis apparatuses and methods thereof

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