WO2017100713A1 - Human nipple areolar complex extracellular matrix scaffold and methods relating thereto - Google Patents
Human nipple areolar complex extracellular matrix scaffold and methods relating thereto Download PDFInfo
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- WO2017100713A1 WO2017100713A1 PCT/US2016/066024 US2016066024W WO2017100713A1 WO 2017100713 A1 WO2017100713 A1 WO 2017100713A1 US 2016066024 W US2016066024 W US 2016066024W WO 2017100713 A1 WO2017100713 A1 WO 2017100713A1
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- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/12—Mammary prostheses and implants
-
- 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
- A61L27/3641—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 characterised by the site of application in the body
- A61L27/3666—Epithelial tissues other than skin
-
- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- 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
- A61L27/3839—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 the site of application in the body
- A61L27/3869—Epithelial tissues other than skin
-
- 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
- A61L2430/04—Materials or treatment for tissue regeneration for mammary reconstruction
Definitions
- the present invention is related to the creation of an extracellular matrix scaffold from autologous or allograft tissue for use in human nipple areola tissue regeneration, reconstruction, or replacement.
- Nipple areolar reconstruction is desired in individuals whom have lost a nipple due to cancer, trauma, or congenital absence.
- Current options for nipple reconstruction include recreating the appearance of a nipple by a prosthetic silicone nipple that is adhered to the skin of the breast mound, surgical reconstruction with or without tattoo to match natural nipple pigmentation, or a tattoo of a nipple.
- Surgical reconstruction may be performed using a composite nipple graft from the contralateral nipple, a local skin flap, skin flaps with autologous graft augmentation, skin flaps with alloplastic augmentation, or skin flaps with allograft augmentation.
- Composite nipple grafts use a portion of the contralateral nipple as a tissue graft for the nipple reconstruction. This type of reconstruction requires a contralateral nipple with adequate volume to accommodate a donation to the other side. There is also risk of donor site morbidity, nipple discoloration, and loss of sensation of the donor nipple.
- Local skin flaps are most commonly used to recreate the appearance of a nipple. Loss of projection occurs in all nipples created from surgical flaps, due to retraction forces of the underlying tissue and contraction of scar tissue. To address loss of projection, surgical flaps may be augmented with autologous tissue such as cartilage or fat.
- Surgical flaps may also be augmented with alloplastic substances such as silicone gel, hyaluronic acid, calcium hydroxyl apatite (RADIESSETM), hydroxyapatite and tricalcium phosphate (CERATITETM), and polytetrafluoroethylene (PTFE).
- alloplastic substances such as silicone gel, hyaluronic acid, calcium hydroxyl apatite (RADIESSETM), hydroxyapatite and tricalcium phosphate (CERATITETM), and polytetrafluoroethylene (PTFE).
- a major disadvantage of alloplastic augmentation is risk of infection and or extrusion of the substance.
- Xenograft tissue such as extracellular collagen matrix derived from porcine small intestinal mucosa (Cook medical nipple reconstruction cylinder) may also be used to augment surgical flaps.
- porcine small intestinal mucosa may not be used in individuals with sensitivity to porcine material or with irradiated skin.
- Acellular dermal allografts have been used to augment surgical flaps. Allograft tissue has a higher rate of incorporation with limited resorption, reducing risk of infection and or extrusion. Maintained structure and biochemistry of the tissue matrix allows for tissue integration into the graft, with native cells repopulating the matrix.
- nipple All current methods of nipple reconstruction only recreate the appearance of a nipple.
- the nipple is a site of specialized epidermis since it is glabrous, associated with a unique gland (mammary), has distinct patterns of epidermal stratification, and expresses unique differentiation markers that are not present in trunk epidermis.
- the nipple undergoes high mechanical strain as well as prolonged exposure to high moisture and digestive enzymes in saliva.
- the nipple has approximately 15-20 lactiferous ducts centrally located that are lined mainly by a two-layered stratified cuboidal epithelium.
- Tightly packed collagen bundles surround the ducts as well as smooth muscle that is oriented parallel and circular to the ducts.
- the nipple has deep infolding of the epidermis into an extensive papillary dermis with fine collagen bundles.
- the human areola has similar invaginations of the epidermis, although less dramatic.
- the human areola has large sebaceous glands called Montgomery's tubercles that are thought to prevent sore or chapped nipples and secrete pheromone substances that aid in nursing.
- the human nipple areolar complex is more pigmented than surrounding skin with melanocytes abundant not only in the epidermis, but also in the basal layer of the sebaceous gland and lactiferous ducts. It is thought that the growth factor environment of the nipple areolar complex (NAC) tissue promotes melanocyte survival in areas where they are typically not found.
- NAC growth factor environment of the nipple areolar
- a method for processing tissue comprises harvesting a human nipple areolar complex tissue, treating the tissue with a stabilizing medium, wherein the medium stabilizes the tissue during transport, decellularizing the tissue, and sterilizing the tissue to form a human nipple areolar complex extracellular matrix scaffold (ECMS).
- the harvesting step comprises harvesting the tissue from a cadaver.
- the harvesting step comprises harvesting the tissue from an individual during a surgery.
- a method of treatment comprises identifying an individual with a need for a nipple reconstruction, forming a human nipple areolar complex extracellular matrix scaffold (ECMS), and applying the ECMS to the individual.
- the forming comprises harvesting a human nipple areolar complex tissue, treating the tissue with a stabilizing medium, wherein the medium stabilizes the tissue during transport, decellularizing the tissue, and sterilizing the tissue to form an ECMS.
- the individual is an individual who has lost a nipple due to a cancer, a trauma, or a congenital absence.
- the individual is an individual seeking a new nipple for a cosmetic reason.
- the applying step comprises direct implantation of the ECMS onto the individual for in vivo cellular repopulation using the individual's cellular responses.
- the method further comprises, before the applying step, recellularizing the ECMS to yield a recellularized ECMS, wherein the applying step comprises applying the recellularized ECMS to the individual.
- a method for regenerating a nipple areolar complex comprising:harvesting a nipple areolar complex tissue;treating the tissue with a stabilizing medium, wherein the medium stabilizes the tissue during a transport; and decellularizing the tissue; wherein a nipple areolar complex extracellular matrix scaffold (ECMS) is produced comprised of at least dermal or subdermal layers.
- a further step comprises sterilizing the nipple areolar complex ECMS.
- the harvested nipple areolar complex is human and may be harvested tissue from a cadaver or from an individual during a surgery.
- the decellularizing step comprises decellularization using one or more decellularization methods selected from the group consisting of: detergents, enzymes, salts, electrophoresis, and combinations thereof.
- An additional step includes after application of the nipple areolar complex ECMS to an individual, the scaffold is repopulated by cells in vivo.
- the present invention comprises repopulating the nipple areolar complex ECMS with exogenous cells selected from the group consisting of: fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stem cells, adipose derived stem cells, induced pluripotent stem cells, or combinations thereof.
- exogenous cells selected from the group consisting of: fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stem cells, adipose derived stem cells, induced pluripotent stem cells, or combinations thereof.
- a method of treatment comprises: identifying an individual with a need for a nipple reconstruction; forming a nipple areolar complex extracellular matrix scaffold (ECMS), the forming comprising; harvesting a nipple areolar complex tissue; treating the tissue with a stabilizing medium, wherein the medium stabilizes the tissue during transport; decellularizing the tissue to yield a sterile nipple areolar complex ECMS comprising at least dermal or subdermal layers; and applying the nipple areolar complex ECMS to the individual.
- ECMS extracellular matrix scaffold
- the method of the present invention may be performed for an individual who has lost a nipple due to a cancer, a trauma, congenital absence, or a cosmetic reason.
- the harvested nipple areolar complex tissue is human nipple areolar complex tissue.
- direct implantation of the ECMS onto the individual is performed.
- the implanted decellularized ECMS may be repopulated naturally by the patient's native cells, or may be populated by recellularizing the ECMS to yield a recellularized ECMS, and wherein the applying step comprises applying the recellularized ECMS to the individual.
- Such a recellularization step may further comprise recellularizing the nipple areolar complex ECMS with exogenous cells selected from the group consisting of: fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stem cells, adipose derived stem cells, induced pluripotent stem cells, or combinations thereof.
- exogenous cells selected from the group consisting of: fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stem cells, adipose derived stem cells, induced pluripotent stem cells, or combinations thereof.
- a nipple areolar complex extracellular matrix scaffold comprising a decellularized nipple areolar complex ECMS, wherein the nipple areolar complex ECMS is capable of supporting regeneration of a nipple areolar complex when applied to a patient.
- the nipple areolar complex ECMS may be derived from human nipple areolar tissue and may further be capable of supporting growth of melanocytes.
- the nipple areolar complex ECMS of the present invention comprises at least dermal layers.
- the present invention has the potential for increased sensation of the nipple area complex occurring within 2 years of ECMS incorporation and may further exhibit maintained projection of the nipple, retained structure after processing.
- the nipple areolar complex ECMS does not illicit a negative immune response when exposed to a recipient's cells.
- the nipple areolar complex ECMS may be applied to an individual, and is capable of generating pigmentation of the nipple area complex.
- FIG. 1 depicts a flow chart of an embodiment of a method of fabricating a human nipple areolar complex extracellular matrix scaffold from human nipple areolar complex tissue;
- FIG. 2 depicts an embodiment of native human nipple areolar complex tissue
- FIG. 3 depicts an embodiment of an acellular human nipple areolar complex extracellular matrix scaffold (ECMS) originating from the same human native nipple areolar complex in FIG. 2.
- FIG. 4 depicts a graph showing Relative Fluorescent Units (RFU) measurement of native versus processed tissue to show decreased immune response when tissue is decellularized.
- REU Relative Fluorescent Units
- FIG. 5 A depicts an illustration of a reconstructed breast mound after mastectomy.
- FIG. 5B depicts an illustration of a prepared area for a desired nipple location on the breast.
- FIG. 5C depicts an illustration of an ECM scaffold of the present invention affixed in the desired location.
- FIG. 5D depicts an illustration of a breast having an ECM scaffold of the present invention having cells repopulated by the patient to create an anatomical human nipple areolar complex.
- FIG. 5E depicts an illustration of a breast having a fully regenerated nipple areolar complex with pigmentation and projection.
- FIG. 6A depicts a histological slice of native human nipple tissue at 200X magnification stained with hematoxylin and eosin.
- FIG. 6B depicts a histological slice of human nipple ECM scaffold prepared using Example 1 methods at 200x magnification stained with hematoxylin and eosin.
- FIG. 6C depicts a histological slice of human nipple ECM scaffold prepared using Example 2 methods at 200x magnification stained with hematoxylin and eosin.
- FIG. 6D depicts a histological slice of human nipple ECM scaffold prepared using Example 3 methods at 200x magnification stained with hematoxylin and eosin. .
- FIG.7A depicts a histological slice of native human nipple tissue at 200x magnification stained with Trichrome Stain.
- FIG. 7B depicts a histological slice of human nipple ECM scaffold prepared using Example 1 methods at 200x magnification stained with Trichrome Stain.
- FIG. 7C depicts a histological slice of human nipple ECM scaffold prepared using Example 2 methods at 200x magnification stained with Trichrome Stain.
- FIG. 7D depicts a histological slice of human nipple ECM scaffold prepared using Example 2 methods at 200x magnification stained with Trichrome Stain.
- FIG. 8 depicts histological stains of native human nipple areolar tissue (left) versus human nipple areolar ECMS (right) at 200X magnification. Trichrome stain was used in FIG. 8A, and FIG. 8E. Hematoxylin and eosin stain was used in FIG. 8B-D and FIG. F-H.
- NAC replacement by utilizing a tissue-engineered extracellular matrix scaffold created from human nipple areolar tissue to enable the patient to regenerate an anatomical nipple made from her own cells.
- NAC of the present invention maintains the unique structural and growth factor environment of the nipple.
- ECMS extracellular matrix scaffold
- proteins remain behind in the scaffold (that aid in cellular in growth) following the decellularization process.
- the reformed nipple areolar complex utilizing the nipple areolar complex ECMS of the present invention results in increased projection as compared to traditional nipple areolar flap creation, as well as increasing potential for pigmentation as compared to nipple areolar flap creation.
- the scaffolding network of the nipple areolar complex ECMS allows for formation of actual nipple versus scar/tattoo, including texture, projection and pigmentation.
- the method comprises the steps of harvesting human nipple areolar complex tissue 10, treating the tissue with a stabilizing medium 20, the stabilizing medium configured to prepare the tissue for transport to a processing center, decellularizing the tissue 30 to form a nascent scaffold, and sterilizing the scaffold 40 to yield a human nipple areolar complex extracellular matrix scaffold (ECMS) from the tissue.
- ECMS extracellular matrix scaffold
- Harvesting the tissue 10 may comprise harvesting human nipple tissue from cadavers under standard processes of tissue procurement. Harvesting may be done in coordination with a tissue procurement company or a tissue bank. A donor nipple may be harvested preferably under aseptic conditions less than 48 hours after death with a dermatome, and maintained at low temperature, preferably no warmer than 4°C.
- human nipple tissue is subject to minimum tissue requirements, including but not limited to: no previous indication of breast cancer or irradiation in breast as well as donor testing for infectious agents, such as HIV, Hepatitis C, and Hepatitis B, as recommended by the American Association of Tissue Banks.
- the stabilizing solution may (1) prevent osmotic, hypoxic, autolytic and proteolytic degradation to the tissue, (2) protect against microbial contamination of the tissue, and (3) reduce mechanical damage to the tissue.
- the stabilizing solution may comprise one or more of a buffer, antioxidants, oncotic agents, protease inhibitors, antibiotics, and optionally a smooth muscle relaxant.
- the tissue may stay in the stabilizing solution for no more than 7 days, but ideally the tissue is transported by overnight delivery on wet ice to a tissue processing center.
- the tissue is decellularized 30.
- decellularization may be accomplished using a number of chemical treatments, including incubation in certain salts, detergents or enzymes.
- decellularizing detergents include polyoxyethylene (20) sorbitan mono-oleate and polyoxyethylene (80) sorbitan mono-oleate (Tween 20 and 80), sodium deoxycholate, 3-[(3- chloramidopropyl)-dimethylammino]-l-propane-sulfonate, octyl-glucoside and sodium dodecyl sulfate.
- the foregoing decellularization process is exemplary and is non-limiting with regard to the decellularization phase of the present invention.
- enzymes may be used to accomplish decellularization, including but not limited to dispase II, trypsin, and thermolysin. Trypsin and other non xeno-free enzymes may be used in preclinical studies as a comparison tool to compare decellularization efficiency. These enzymes react with different components of collagen and intercellular connections in achieving their effects. Dispase II attacks Type IV collagen, which is a component of the lamina densa and anchoring fibrils of the basement membrane.
- Thermolysin attacks the bulbous phemphigoid antigen in the hemidesmosome of the basal layer of keratinocytes. Trypsin attacks the desmosome complex between cells. Due to the proteolytic nature of these enzymes, care must be taken that cellular removal occurs without significant damage to the extracellular matrix, including the basement membrane complex. This is a function of concentration, time and temperature. If used for too long a time or at too high a concentration, dispase II for example can completely remove the basement membrane complex from the dermis.
- the processing solution also contains certain protease inhibitors, to prevent degradation of the extracellular matrix.
- Collagen-based connective tissues contain proteases and collagenases as endogenous enzymes in the extracellular protein matrix.
- certain cell types including smooth muscle cells, fibroblasts and endothelial cells contain a number of these enzymes inside vesicles called lysosomes. When these cells are damaged by events such as hypoxia, the lysosomes are ruptured and their contents released. As a result, the extracellular matrix can undergo severe damage from protein, proteoglycan and collagen breakdown.
- the processing solution contains one or more protease inhibitors selected from the group of N-ethylmaleimide (NEM), phenylmethylsulfonylfluoride (PMSF) ethylenediamine tetraacetic acid (EDTA), ethylene glycol- bis-(2-aminoethyl(ether)NNN'N'-tetraacetic acid, ammonium chloride, elevated pH, apoprotinin and leupeptin to prevent such damage.
- NEM N-ethylmaleimide
- PMSF phenylmethylsulfonylfluoride
- EDTA ethylenediamine tetraacetic acid
- ammonium chloride elevated pH
- apoprotinin and leupeptin to prevent such damage.
- the processing solution generally contains an appropriate buffer. This may involve one of many different organic buffers which are described above.
- the inventors prefer to use an organic buffer selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), Tris (hydroxymethyl)aminomethane (TRIS) and (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES).
- MES 2-(N-morpholino)ethanesulfonic acid
- TMS Tris (hydroxymethyl)aminomethane
- HEPES N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]
- a low salt or physiological buffer including phosphate bicarbonate acetate citrate glutamate with or without glycine may be more appropriate in certain applications.
- Low salt or physiological buffers are more able to support the infiltration of the graft with viable cells and hence are more relevant when cellular infiltration including
- the processing solution may contain chemicals that would be irritating or inflammatory on transplantation, it is important that the processing solution be thoroughly rinsed from the tissue. In some embodiments, this washing occurs by rinsing in sufficient changes of appropriate buffer, until residues of the processing solution are reduced to levels compatible with transplantation.
- components of the processing solution may be neutralized by specific inhibitors, e.g., dispase II by ethylenediaminetetraacetic acid (EDTA) or trypsin by serum.
- EDTA ethylenediaminetetraacetic acid
- decellularizing human nipple tissue 30 yields a decellularized extracellular matrix (ECM) of an existing human nipple tissue, the decellularized matrix aiding in the long-term maintenance of a reconstructed human nipple architectural structure.
- ECM extracellular matrix
- the ECM is a compilation of secretions from various cell types that make up each respective tissue and has been shown to aid in the cell proliferation, migration, and differentiation.
- Previous in vitro data indicates that the removal of cellular components from native tissues leaves behind a complex protein matrix, designated extracellular matrix, which can provide cells a combination of cues that closely resemble the in vivo environment in vitro.
- FIG. 4 presents a graph showing Relative Fluorescent Units (RFU) measurement of native versus processed tissue, showing decreased immune response when tissue is decellulanzed.
- MCP-1 Monocyte Hemotactic Protein -1
- MTF is a brand of commercially available acellular skin.
- Native is native porcine tissue.
- Processed is tissue processed to decellularize the tissue using similar methods claimed herein. Processed tissue shows decrease in monocyte chemoattraction (or immune response) as compared to all other materials.
- the human nipple areolar complex ECMS of the present invention exhibits reduced immunogenicity by utilization of a fully decellularized graft versus native tissue.
- a monocyte chemotactic assay was performed. Chemotaxis (migration) of human monocytes/macrophages, which are the first responders to tissue injury, were measured in decellularized versus native tissue adipose tissue to evaluate the response of these cells in vitro (see FIG. 4). When looking at decellularized tissue, chemo-attraction values of human monocytes (first responders to tissue injury) were significantly lower than those measured in native tissue, showing the decellularization process reduces innate immunogenicity.
- decellularizing the tissue 30 comprises processing and preserving human nipple areolar tissue for transplantation, wherein the processing and preserving steps may comprise chemical pretreatment, cell removal, and sterilization, yielding a human nipple areolar scaffold.
- the processing step is designed to generate a transplantable biological tissue graft, wherein the graft (a) provides an extracellular matrix which can be regenerated and remodeled by the host, (b) does not elicit an negative immune response by the host, and (c) can be easily stored and transported.
- the preserving step may comprise incubating the tissue in a decellularizing solution configured to remove antigenic donor cells within the tissue.
- Decellularizing the tissue 30 may further comprise optimizing the concentration and time duration for cellular removal while preserving extracellular matrix integrity of the ECM. After the cells are thoroughly removed from the tissue, the tissue is washed several times to ensure removal of any remaining detergent or decellularizing solution. Decellularization of the tissue 30 may be verified by visualizing nuclear material in tissue sections stained with 4',6-diamidino-2- phenylindole (DAPI) or hematoxylin and eosin (H&E). Additionally, remaining double stranded DNA can be quantified. Decellularizing solutions may comprise buffers, antibiotics, detergents, protease inhibitors and/or enzymes.
- a nipple areolar complex ECMS may then be sterilized 40.
- the nipple areolar complex ECMS of the present invention is not sterilized, or lacks a sterilization step.
- the scaffold is sterilized 40 with a combination of peracetic acid and supercritical carbon dioxide or a combination of peracetic acid and ethanol.
- the scaffold may be sterilized using gamma irradiation, ethylene oxide, and/or electron beam irradiation. The sterilizing step 40 yields an ECMS that is ready for implantation.
- FIG. 5A-E various stages of the regeneration process for the nipple areolar complex comprising the human nipple areolar complex ECMS of the present invention are presented.
- FIG. 5A provides an illustration of a reconstructed breast mound after mastectomy.
- FIG. 5B provides a prepared portion of a female breast in a location desired for a nipple areolar complex ECMS of the present invention to be administered. In a preferred embodiment, this occurs by de-epitheliating the skin in the location where the nipple will be located.
- FIG. 5C presents the administration of a human nipple areolar complex ECMS of the present invention being sutured or adhesively applied to the patient directly.
- nipple areolar scaffold may be applied to the recipient directly, whether autologous or allogeneic.
- the nipple areolar scaffold is then secured in place for incorporation into the donor tissue. May be used alone or in combination with other treatments both cellular and non-cellular deemed appropriate by the physician.
- FIG. 5D cells from the patient repopulate the nipple areolar complex ECMS of the present invention to create an anatomically correct nipple areolar complex.
- stem cells within the scaffold may differentiate into the appropriate cell types, directed by signaling molecules within the scaffold.
- FIG. 5E presents the nipple areolar complex ECMS of the present invention wherein the growth factor environment within the ECMS promotes natural pigmentation and improved projection.
- nipple areolar complex ECMS of the present invention is capable of being implanted in a single procedure, and wherein the implantation will lead to cellular incorporation through the patient's innate cellular response.
- the nipple areolar scaffold may be re-cellularized before implantation using xeno-free culturing methods with cells from the recipient.
- the cells may include but are not limited to fibroblasts, epithelial cells, mammary endothelial cells, mammary epithelial cells, vascular smooth muscle cells, bone marrow mesenchymal stems cells, adipose derived stem cells, or induced pluripotent stem cells. If using epithelial cells, the cells are isolated from the patient through a skin scraping. The cells will be cultured and expanded in the laboratory using an appropriate medium such as but not limited to keratinocyte serum-free media or other relevant xeno-free medias.
- the cells may be lifted for collection by adding a xeno-free cell detachment solution, such as but not limited to ACCUTASE (Stemcell Technologies), TrypLE Select (ThermoFisher Scientific) or ACCUMAX (EMD Millipore), to the media until sufficient cell lifting has occurred.
- a xeno-free cell detachment solution such as but not limited to ACCUTASE (Stemcell Technologies), TrypLE Select (ThermoFisher Scientific) or ACCUMAX (EMD Millipore).
- the cells are removed from the tissue culture plate and placed in a conical tube for centrifugation. Total cell count is calculated and appropriate volume of cells and media is added to the scaffold to obtain sufficient cell density per area of scaffold for optimal cell repopulation.
- a donor nipple areolar complex (as seen in FIG. 2) is harvested under aseptic conditions with a dermatome, and maintained at low temperature, preferably no warmer than 4°C.
- the tissue is treated and stabilized for transport with a stabilizing solution.
- This solution may contain cell culture medium containing 1% penicillin, 1% streptomycin, 1% amphotericin b, 1% antibiotic/antimycotic, 5% heparin, and 2% melatonin.
- the tissue may stay in the stabilizing solution for no more than 7 days.
- the tissue is removed from stabilizing solution and the fat and muscle tissue that remains beneath the human nipple areolar tissue is removed. This may be done immediately or just before processing. The tissue is then placed in a vacuum bag to prevent freezer burn and the excess air is removed. [0073] The tissue is then placed in -30° Celsius and freeze-thawed up to 3 times. The tissue is then placed in a conical tube or Erlenmeyer flask and exposed to 1% sodium dodecyl sulfate (SDS) diluted in PBS or Hanks Balanced Salt Solution while agitated at 175 RPM. The solutions are changed every 12-24 hours. The solutions may also contain 1% antibiotic/antimycotic if deemed necessary.
- SDS sodium dodecyl sulfate
- the tissue will be exposed to the decellularization solution for 3-14 days depending on the age of the donor tissue was collected from.
- the tissue will be agitated in solution at 37 ⁇ 3 °C at 175 RPM.
- the solution may be changed every 12-24 hours or when solution saturation is reached, whichever comes first. Tissue under the age of 45 years may need to be exposed to decellularization solutions longer due to thicker collagen base of younger human tissue.
- the tissue After exposure to decellularizing solutions, the tissue is thoroughly rinsed by agitating in deionized or Millipore water at 175 RPM for 3 hours. This process may be repeated 3 times.
- Decellularized tissue may be stored in 4°C or -30°C either in phosphate buffered saline (PBS) or a vacuum bag until such time as sterilization occurs.
- PBS phosphate buffered saline
- Sterilization is performed by exposing the human nipple areolar ECMS to a mixture of 0.1% peracetic acid and 4% ethanol for up to two hours.
- a donor nipple areolar complex (as seen in FIG. 2) is harvested under aseptic conditions with a dermatome, and maintained at low temperature, preferably no warmer than 4°C.
- the tissue is treated and stabilized for transport with a stabilizing solution.
- This solution may contain cell culture medium containing 1% penicillin, 1% streptomycin, 1% amphotericin b, 1% antibiotic/antimycotic, 5% heparin, and 2% melatonin.
- the tissue may stay in the stabilizing solution for no more than 7 days.
- the tissue is removed from stabilizing solution and the fat and muscle tissue that remains beneath the human nipple areolar tissue is removed. This may be done immediately or just before processing. The tissue is then placed in a vacuum bag to prevent freezer burn and the excess air is removed.
- the tissue is then placed in -30° Celsius and freeze-thawed up to 3 times.
- the tissue is then placed in a conical tube or Erlenmeyer flask and exposed to 1% sodium dodecyl sulfate (SDS), 5% TRITON-X 100, and 1% Ammonium Hydroxide diluted in PBS or Hanks Balanced Salt Solution while agitated at 175 RPM.
- SDS sodium dodecyl sulfate
- TRITON-X 100 5%
- Ammonium Hydroxide diluted in PBS or Hanks Balanced Salt Solution while agitated at 175 RPM.
- the solutions are changed every 12-24 hours.
- the solutions may also contain 1% antibiotic/antimycotic if deemed necessary.
- the tissue will be exposed to the decellularization solution for 3-14 days depending on the age of the donor tissue was collected from.
- the tissue will be agitated in solution at 37 ⁇ 3 °C at 175 RPM.
- the solution may be changed
- the tissue After exposure to decellularizing solutions, the tissue is thoroughly rinsed by agitating in deionized or Millipore water at 175 RPM for 3 hours. This process may be repeated 3 times.
- Decellularized tissue may be stored in 4°C or -30°C either in phosphate buffered saline (PBS) or a vacuum bag until such time as sterilization occurs.
- PBS phosphate buffered saline
- Sterilization is performed by exposing the human nipple areolar ECMS to a mixture of 0.1% peracetic acid and 4% ethanol for up to two hours.
- a donor nipple areolar complex (as seen in FIG. 2) is harvested under aseptic conditions with a dermatome, and maintained at low temperature, preferably no warmer than 4°C.
- the tissue is treated and stabilized for transport with a stabilizing solution.
- This solution may contain cell culture medium containing 1% penicillin, 1% streptomycin, 1% amphotericin b, 1% antibiotic/antimycotic, 5% heparin, and 2% melatonin.
- the tissue may stay in the stabilizing solution for no more than 7 days.
- the tissue is removed from stabilizing solution and the fat and muscle tissue that remains beneath the human nipple areolar tissue is removed. This may be done immediately or just before processing. The tissue is then placed in a vacuum bag to prevent freezer burn and the excess air is removed.
- the tissue is then placed in -30° Celsius and freeze-thawed up to 3 times.
- the tissue is then placed in a conical tube or Erlenmeyer flask and exposed to 5% TRITON-X 100 and 1% Ammonium Hydroxide diluted in PBS or Hanks Balanced Salt Solution while agitated at 175 RPM.
- the solutions are changed every 12-24 hours.
- the solutions may also contain 1% antibiotic/antimycotic if deemed necessary.
- the tissue will be exposed to the decellularization solution for 3-14 days depending on the age of the donor tissue was collected from.
- the tissue will be agitated in solution at 37 ⁇ 3 ° C at 175 RPM.
- the solution may be changed every 12-24 hours or when solution saturation is reached, whichever comes first. Tissue under the age of 45 years may need to be exposed to decellularization solutions longer due to thicker collagen base of younger human tissue.
- Decellularized tissue After exposure to decellularizing solutions, the tissue is thoroughly rinsed by agitating in deionized or Millipore water at 175 RPM for 3 hours. This process may be repeated 3 times.
- Decellularized tissue may be stored in 4°C or -30°C either in phosphate buffered saline (PBS) or a vacuum bag until such time as sterilization occurs.
- PBS phosphate buffered saline
- Sterilization is performed by exposing the human nipple areolar ECMS to a mixture of 0.1% peracetic acid and 4% ethanol for up to two hours.
- sterilization is performed using a combination of peracetic acid and supercritical carbon dioxide (SCC0 2 ) at concentrations and durations optimized for NAC tissue.
- SCC0 2 supercritical carbon dioxide
- the sterilization of the NAC extracellular matrix tissue is carried out in a 20-liter pressure chamber which houses wire baskets for holding packaged samples as well as an additive pad to which poly acrylic acid (PAA) (Sigma, -24-50% vol ./vol. PAA in acetic acid) can be added if necessary.
- PAA poly acrylic acid
- the pressure and temperature of the SCC0 2 is held constant at 7000-10000 kPa and 30-37 °C, respectively, whereas the amount of PAA and the duration of exposure to SCC0 2 will be varied.
- An additive of peracetic acid may be used to ensure sterility if found appropriate from -10-40% vol/vol in concentration (Sigma Aldrich).
- the pressure may be held at 7000-10000 kPA at temperatures ranging from 30-37°C.
- the amount of peracetic acid may vary as well as the duration of tissue exposure (10 to 140 minutes).
- FIG. 6A presents a histological slice of native human nipple tissue at 200X magnification stained with hematoxylin and eosin. Nuclei are stained evident through purple staining, while the ECM is stained pink.
- native human nipple tissue the cornified keratinocyte layer is visible at the surface with stratified epithelial cells within the epidermis. Melanocytes are primarily localized to the basal layer of the epidermis, although, unique to nipple areolar tissue, some melanocytes are also present within the dermal layers.
- FIG. 6B presents a histological slice of human nipple ECM scaffold prepared using Example 1 methods at 200x magnification stained with hematoxylin and eosin. As compared to native human nipple tissue, the nuclei and epithelial cell layers have been removed and only the ECM remains.
- FIG. 6C presents a histological slice of human nipple ECM scaffold prepared using Example 2 methods at 200x magnification stained with hematoxylin and eosin. As compared to native human nipple tissue, the nuclei and epithelial cell layers have been removed and only the ECM remains.
- FIG. 6D presents a histological slice of human nipple ECM scaffold prepared using Example 3 methods at 200x magnification stained with hematoxylin and eosin. As compared to native human nipple tissue, the nuclei and epithelial cell layers have been removed and only the ECM remains.
- FIG.7A presents a histological slice of native human nipple tissue at 200x magnification stained with Trichrome Stain. Nuclei are stained black; cytoplasm, muscle, and erythrocytes stained red; and collagen stained blue. The epidermal layer of the tissue is intact, with the epithelial layer removed.
- FIG. 7B presents a histological slice of human nipple ECM scaffold prepared using Example 1 methods at 200x magnification stained with Trichrome Stain.
- FIG. 7C presents a histological slice of human nipple ECM scaffold prepared using Example 2 methods at 200x magnification stained with Trichrome Stain.
- FIG. 7D presents a histological slice of human nipple ECM scaffold prepared using Example 3 methods at 200x magnification stained with Trichrome Stain.
- FIG. 8 presents histological stains of native human nipple areolar tissue (left) versus human nipple areolar ECMS (right) at 200X magnification.
- Trichrome stain was used in FIG. 8A
- FIG. 8E Hematoxylin and eosin stain was used in FIG. 8B-D and FIG. F-H. Corresponding structures are evident in both the native and decellularized tissue. H&E staining shows unique microstructure of the human nipple is maintained despite cell removal.
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Priority Applications (4)
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US15/626,058 US10729812B2 (en) | 2015-12-10 | 2016-12-09 | Human nipple areolar complex extracellular matrix scaffold and methods relating thereto |
BR112018011635A BR112018011635A2 (en) | 2015-12-10 | 2016-12-09 | human aeromamilar complex extracellular matrix support and related methods |
EP16874013.2A EP3386436A4 (en) | 2015-12-10 | 2016-12-09 | Human nipple areolar complex extracellular matrix scaffold and methods relating thereto |
US16/984,069 US11534529B2 (en) | 2015-12-10 | 2020-08-03 | Human nipple areolar complex extracellular matrix scaffold and methods relating thereto |
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US201562265798P | 2015-12-10 | 2015-12-10 | |
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US15/626,058 A-371-Of-International US10729812B2 (en) | 2015-12-10 | 2016-12-09 | Human nipple areolar complex extracellular matrix scaffold and methods relating thereto |
US16/984,069 Continuation US11534529B2 (en) | 2015-12-10 | 2020-08-03 | Human nipple areolar complex extracellular matrix scaffold and methods relating thereto |
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WO2019195226A1 (en) | 2018-04-02 | 2019-10-10 | Bioaesthetics Corporation | Poland syndrome and methods of treatment |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20130013068A1 (en) * | 2009-08-11 | 2013-01-10 | James Forsell | Acellular Dermal Allografts And Method Of Preparation |
US20150157451A1 (en) * | 2009-07-21 | 2015-06-11 | Lifecell Corporation | Graft Materials for Surgical Breast Procedures |
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US9150318B1 (en) * | 2009-01-02 | 2015-10-06 | Lifecell Corporation | Method for sterilizing an acellular tissue matrix |
DK3212247T3 (en) * | 2014-10-31 | 2021-06-28 | The Administrators Of The Tulane Educational Fund | SURGICAL IMPLANTS TO REPLACE Nipple and AREOLA OR DAMAGED EPIDERMIS |
-
2016
- 2016-12-09 EP EP16874013.2A patent/EP3386436A4/en active Pending
- 2016-12-09 BR BR112018011635A patent/BR112018011635A2/en not_active Application Discontinuation
- 2016-12-09 WO PCT/US2016/066024 patent/WO2017100713A1/en active Application Filing
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US20150157451A1 (en) * | 2009-07-21 | 2015-06-11 | Lifecell Corporation | Graft Materials for Surgical Breast Procedures |
US20130013068A1 (en) * | 2009-08-11 | 2013-01-10 | James Forsell | Acellular Dermal Allografts And Method Of Preparation |
Non-Patent Citations (3)
Title |
---|
PASHOS ET AL.: "A Tissue Engineered Nipple and Areola Complex,", MOLECULAR THERAPY, vol. 23, no. 1, May 2015 (2015-05-01), pages S254 - S255, XP055391372, Retrieved from the Internet <URL:http://www.cell.com/molecular-therapy-family/molecular-therapy/pdf/S1525-0016(16)34249-6.pdf> [retrieved on 20170126] * |
See also references of EP3386436A4 * |
SIGGELKOW ET AL.: "Skin-reducing Mastectomy with Primary Implant Reconstruction", GEBURTSHILFE FRAUENHEILKD, vol. 72, no. 7, July 2012 (2012-07-01), pages 616 - 621, XP055279420, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168326> [retrieved on 20151214] * |
Cited By (2)
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WO2019195226A1 (en) | 2018-04-02 | 2019-10-10 | Bioaesthetics Corporation | Poland syndrome and methods of treatment |
EP3773328A4 (en) * | 2018-04-02 | 2021-12-15 | Bioaesthetics Corporation | Poland syndrome and methods of treatment |
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US10729812B2 (en) | 2020-08-04 |
EP3386436A1 (en) | 2018-10-17 |
US20210015973A1 (en) | 2021-01-21 |
US20180177917A1 (en) | 2018-06-28 |
BR112018011635A2 (en) | 2018-11-27 |
US11534529B2 (en) | 2022-12-27 |
EP3386436A4 (en) | 2019-08-21 |
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