WO2016180789A1 - Compositions comprising mesenchymal stem cells and uses thereof - Google Patents

Compositions comprising mesenchymal stem cells and uses thereof Download PDF

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Publication number
WO2016180789A1
WO2016180789A1 PCT/EP2016/060352 EP2016060352W WO2016180789A1 WO 2016180789 A1 WO2016180789 A1 WO 2016180789A1 EP 2016060352 W EP2016060352 W EP 2016060352W WO 2016180789 A1 WO2016180789 A1 WO 2016180789A1
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WO
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Prior art keywords
tissue
composition
stem cells
asc
msc
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
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PCT/EP2016/060352
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English (en)
French (fr)
Inventor
Denis Dufrane
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.)
Universite Catholique de Louvain UCL
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Universite Catholique de Louvain UCL
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
Priority to CA2985272A priority Critical patent/CA2985272C/en
Priority to US15/571,460 priority patent/US20180126039A1/en
Priority to KR1020177034751A priority patent/KR20180008526A/ko
Priority to ES16725382T priority patent/ES2892974T3/es
Priority to AU2016259737A priority patent/AU2016259737A1/en
Priority to EP16725382.2A priority patent/EP3294356B1/en
Priority to RU2017139096A priority patent/RU2758468C2/ru
Priority to MX2017014245A priority patent/MX390493B/es
Priority to JP2017557916A priority patent/JP6966332B2/ja
Application filed by Universite Catholique de Louvain UCL filed Critical Universite Catholique de Louvain UCL
Priority to BR112017024013-0A priority patent/BR112017024013A2/pt
Priority to CN201680037547.6A priority patent/CN107810014B/zh
Publication of WO2016180789A1 publication Critical patent/WO2016180789A1/en
Priority to ZA2017/07478A priority patent/ZA201707478B/en
Priority to IL255480A priority patent/IL255480B/en
Anticipated expiration legal-status Critical
Priority to AU2020207792A priority patent/AU2020207792A1/en
Priority to AU2022241581A priority patent/AU2022241581A1/en
Ceased legal-status Critical Current

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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
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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Definitions

  • the present invention relates to a biomedical product and its use for treating soft tissue injuries.
  • the present invention relates to compositions comprising a mesenchymal stem cells population and a biocompatible matrix.
  • BACKGROUND OF INVENTION Every year, millions of people seek medical treatments for acute or overuse injuries of soft tissue, for example, injury to the skin (such as non-healing skin wounds) or to muscles (such as traumatic or surgical wounds).
  • the process of wound healing follows a precise pattern including three phases: the inflammatory phase consisting of the release of cytokines for inflammatory response improvement; the repair or proliferation phase characterized by the activation and proliferation of progenitor cells under the influence of growth factors and cytokines and by angiogenesis; and the remodeling or maturation phase characterized by scar tissue and cross-links of collagen to other collagen and with protein molecules, increasing the tensile strength of the scar.
  • a substantially pure mesenchymal stem cells population may enhance repair and regeneration at the site of the injury in comparison to a mesenchymal stem cells population not substantially pure.
  • the present invention relates to a composition comprising a substantially pure mesenchymal stem cells population and a biocompatible matrix, and its use for treating soft tissue injuries.
  • This invention relates to a composition
  • a composition comprising a biocompatible matrix and a mesenchymal stem cells (MSC) population, wherein said mesenchymal stem cells population is substantially pure.
  • the mesenchymal stem cells population of the invention comprises less than 25% of fibroblasts.
  • the mesenchymal stem cells of the invention are undifferentiated.
  • the mesenchymal stem cells of the invention are derived from adipose tissue, bone-marrow, umbilical cord tissue, Wharton's jelly, amniotic fluid, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, skin, bone, dental tissue or fetal liver.
  • the mesenchymal stem cells of the composition are derived from adipose tissue, more preferably from subcutaneous adipose tissue.
  • the biocompatible matrix of the invention is acellular.
  • the biocompatible matrix of the invention comprises collagen.
  • the biocompatible matrix of the invention is human, porcine, bovine or equine.
  • mesenchymal stem cells of the invention originate from the subject to be treated.
  • the invention also relates to a composition comprising a biocompatible matrix and a mesenchymal stem cells population as described hereinabove for use for treating a soft tissue injury in a subject in need thereof.
  • the soft tissue to be treated in the invention is selected from the group comprising skin tissue, muscle tissue, dermal tissue, tendon tissue, ligament tissue, meniscus tissue and bladder tissue.
  • the soft tissue injury is an acute or a chronic wound.
  • the soft tissue injury to be treated is selected from the group comprising tears or ruptures of soft tissue, skin wounds, skin burns, skin ulcers, surgical wounds, vascular diseases, muscle disease, hernias and radiation wounds.
  • the skin wound is a diabetic wound.
  • the composition for use of the invention is administered topically or by surgical implantation.
  • the invention also relates to a method for the preparation of a composition comprising a biocompatible matrix and a mesenchymal stem cells population, comprising incubating a substantially pure mesenchymal stem cells population with a biocompatible matrix.
  • MSC Mesenchymal stem cells
  • Mesenchymal stem cells are multipotent stem cells which are capable of differentiating into more than one specific type of mesenchymal or connective tissue including osteogenic, chondrogenic, adipogenic, myelosupportive stroma, myogenic, or neurogenic lineages.
  • Mesenchymal stem cells can be isolated from tissues including, without limitation, adipose tissue, bone-marrow, umbilical cord tissue, Wharton's jelly, amniotic fluid, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, skin, bone, dental tissue, pre-menstrual fluid, foreskin and fetal liver, and the like.
  • “Late passaged mesenchymal stem cell” refers to a cell exhibiting a less immunogenic characteristic when compared to an earlier passaged cell.
  • the immunogenicity of a mesenchymal stem cell corresponds to the number of passages.
  • the cell has been passaged up to at least the fourth passage, more preferably, the cell has been passaged up to at least the sixth passage, and most preferably, the cell has been passaged up to at least the eight passage.
  • “Normoxia” refers to tissular or physiological oxygen levels. In one embodiment of the invention, cellular culture in normoxia, or in physiological oxygen levels, means at an oxygen level from about 3% to about 6%, preferably at an oxygen level of about 5%.
  • “Hypoxia” refers to reduced oxygen levels.
  • cellular culture in hypoxia means at an oxygen level from about 0% to about 1%, preferably at an oxygen level of about 0.1%.
  • Normalglycemia refers to normal levels of glucose.
  • cellular culture in normoglycemia means at a concentration of glucose of from about 0.5 g/1 to about 1.5 g/1, preferably at a concentration of glucose of about i g/i.
  • “Hyperglycemia” refers to elevated levels of glucose.
  • cellular culture in hyperglycemia means at a concentration of glucose from about 2 g/1 to about 10 g/1, preferably from about 3 g/1 to about 6 g/1, more preferably at a concentration of glucose of about 4.5 g/1.
  • Biocompatible refers to the quality of not having toxic or injurious effects on the body, in particular on a soft tissue.
  • the biocompatibility of a material refers to the ability of said material to perform its desired function without eliciting any undesirable local or systemic effects in the subject, but generating the most appropriate beneficial cellular or tissue response.
  • Biocompatible matrix also referred as matrix or scaffold, refers to a three- dimensional scaffold formed by biocompatible material.
  • Acellular refers to a matrix from which substantially all endogenous cells have been removed, such as, for example, at least about 60% of endogenous cells (wherein the percentages are relative to the number of endogenous cells), preferably about 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% of endogenous cells or more.
  • the decellularization of the matrix may be evaluated by counting viable cells, for example by DAPI staining.
  • Soft tissue refers to the tissues that connect, support, or surround other structures and organs of the body, not being bone.
  • soft tissue includes, without limitation, tendons, ligaments, fascia, skin, fibrous tissues, fat, and synovial membranes (which are connective tissue), muscles, nerves and blood vessels (which are not connective tissue).
  • soft tissues are body tissues except bone, teeth, nails, hair, and cartilage.
  • Subject refers to a mammal, preferably a human.
  • a subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development or the healing of a soft tissue injury.
  • the subject is an adult (for example a subject above the age of 18).
  • the subject is a child (for example a subject below the age of 18).
  • the subject is a male.
  • the subject is a female.
  • Treating” or “treatment” or “alleviation” refers to therapeutic action taken and action refrained from being taken for the purpose of improving the condition of the patient, wherein the object is to slow down (lessen) or to reverse the progress, or to alleviate one or more symptoms of the soft tissue injury such as, for example, unclosed wound, fibrosis development, lack of vascularization and inflammation.
  • a subject or mammal is successfully "treated” for a soft tissue injury if, after receiving a composition according to the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: relief to some extent, of one or more of the symptoms associated with the soft tissue injury; reduced morbidity; reduced mortality, or improvement in quality of life.
  • the above parameters for assessing successful treatment and improvement in the injury are readily measurable by routine procedures familiar to a physician.
  • Passaging also known as subculture or splitting cells, refers to transferring a small number of cells into a new vessel when cells are at confluence or almost, to prolong the life and/or expand the number of cells in the culture.
  • the passage 0 P0
  • the passage 0 is the point at which cells were initially placed in culture.
  • “About” and “Approximately” as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
  • “And/Or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
  • Derivative refers to any fraction of a substance, derivative, subfamily, etc., or mixtures thereof, alone or in combination with other derivatives or other ingredients.
  • mesenchymal stem cells comprise other types of cells than mesenchymal stem cells, in particular they may comprise fibroblasts, or are in part differentiated into fibroblasts.
  • the Applicant herein demonstrates that mesenchymal stem cells have the capacity to better survive and proliferate in hypoxia and/or hyperglycemia than fibroblasts.
  • mesenchymal stem cells have the capacity to secrete more VEGF than fibroblasts, in particular mesenchymal stem cells secrete more VEGF in hypoxia and hyperglycemia conditions (i.e. diabetic wounds conditions) than in normoxia and normoglycemia (see Example 2).
  • mesenchymal stem cells seeded on a biocompatible matrix may restore dermal tissue (see Example 3), and may be used for the treatment of injured muscle (see Example 4).
  • a MSC population seeded on a biocompatible matrix demonstrated the capacity to survive under hypoxia and/or hyperglycemia, to improve the release of pro- angiogenic factors by an oxygen-sensitive mechanism and to reduce fibrotic scar. These properties could promote soft tissue reconstruction, such as, for example, in diabetic conditions.
  • This invention thus relates to a composition comprising a biocompatible matrix and a mesenchymal stem cells (MSC) population.
  • the composition of the invention is used in tissue engineering and regeneration in animals.
  • the exact composition of the invention may vary according to the use desired. Modifications and other embodiments of the invention will become apparent to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and associated drawings. It is to be understood that the invention is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
  • the composition of the invention is a biomedical product.
  • the MSC population may comprise other types of cells than MSC, such as, for example, fibroblasts, macrophages, endothelial cells, dendritic cells, osteoblasts, hematopoietic stem cells, cord blood stem cells, lymphocytes, or natural killer cells.
  • fibroblasts such as, for example, fibroblasts, macrophages, endothelial cells, dendritic cells, osteoblasts, hematopoietic stem cells, cord blood stem cells, lymphocytes, or natural killer cells.
  • Cells may be obtained from a donor (either living or cadaveric) or derived from an established cell strain or cell line. For example, cells may be harvested from a donor using standard biopsy techniques known in the art.
  • MSC are obtained from a human donor.
  • MSC are obtained from a human donor, provided that they are not embryonic stem cells.
  • the MSC population is an isolated MSC population. In one embodiment of the invention, the MSC population is substantially pure. As used herein, the term "substantially pure MSC population" means that the MSC population comprises less than 25% of other types of living cells, in particular of fibroblasts. In one embodiment, the substantially pure MSC population of the invention comprises at least about 75%, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of MSC, wherein percentages are relative to the total number of living cells.
  • the substantially pure MSC population of the invention comprises less than about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of fibroblasts, wherein percentages are relative to the total number of living cells.
  • the substantially pure MSC population of the invention secretes at most 100 pg/ml of SDF- ⁇ , preferably at most 50, 40, 30, 25, 20, 19, 18, 17,
  • the substantially pure MSC population of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml, when the MSC population is cultured at least 24 hours at about 21% 0 2 .
  • the substantially pure MSC population of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18,
  • the substantially pure MSC population of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml, when the MSC population is cultured at least 24 hours in hypoxia, preferably at about 0.1% 0 2 .
  • the substantially pure MSC population of the invention secretes at most 100 pg/ml of SDF- ⁇ , preferably at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ , when the MSC population is cultured at least 24 hours in normoglycemia, preferably at a concentration of glucose of about 1 g/1 of glucose.
  • the substantially pure MSC population of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ , when the MSC population is cultured at least 24 hours in hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the substantially pure MSC population of the invention secretes at most of 100 pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ , when the MSC population is cultured at least 24 hours at about 21% 0 2 and at low concentration of glucose, preferably at about 1 g/1 of glucose.
  • the substantially pure MSC population of the invention secretes at most of 50 pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,
  • the substantially pure MSC population of the invention secretes at most of 100 pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
  • the substantially pure MSC population of the invention secretes at most of 50 pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,
  • the substantially pure MSC population of the invention secretes at most of 100 pg/ml, preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,
  • the substantially pure MSC population of the invention secretes at most of 50 pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ , when the MSC population is cultured at least 24 hours in hypoxia, preferably at about 0.1% O2, and at high concentration of glucose, preferably at about 4.5 g/1 of glucose.
  • the substantially pure MSC population of the invention secretes at least 200 pg/ml of VEGF, preferably at least about 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290 pg/ml of VEGF, when the MSC population is cultured at least 24 hours in hypoxia, preferably at about 0.1% O2, and hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the substantially pure MSC population of the invention secretes at least about 90 pg/ml of VEGF, preferably at least about 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 188, 119 or 120 pg/ml of VEGF, when the MSC population is cultured at least 24 hours in physiological oxygen levels, preferably at about 5% O2, and hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the substantially pure MSC population of the invention secretes at least about 160 pg/ml of VEGF, preferably at least about 161, 162, 163, 164, 165, 166, 167, 168, 169 or 170 pg/ml of VEGF, when the MSC population is cultured at least 24 hours in physiological oxygen levels, preferably at about 5% O2, and normoglycemia, preferably at a concentration of glucose of about 1 g/1.
  • the substantially pure MSC population of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ ; and at least about 200 pg/ml of VEGF, preferably at least about 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290 pg/ml of VEGF, when the MSC population is cultured at least 24 hours in hypoxia, preferably at about 0.1% O2, and hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the substantially pure MSC population of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ ; and at least about 90 pg/ml of VEGF, preferably at least about 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 188, 119 or 120 pg/ml of VEGF, when the MSC population is cultured at least 24 hours in physiological oxygen levels, preferably at about 5% 0 2 , and hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the substantially pure MSC population of the invention secretes at most 100 pg/ml of SDF- ⁇ , preferably at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ ; and at least about 160 pg/ml of VEGF, preferably at least about 161, 162, 163, 164, 165, 166, 167, 168, 169 or 170 pg/ml of VEGF, when the MSC population is cultured at least 24 hours in physiological oxygen levels, preferably at about 5% O2, and in normoglycemia, preferably at a concentration of glucose of about 1 g/1 of glucose.
  • the MSC population is cultured in normoxia, preferably at about 21% O2, and in normoglycemia, preferably at about 1 g/1 of glucose, up to at least 1, 2, 3 or 4 passages before measuring the SDF- ⁇ and/or VEGF expression level.
  • the MSC population is cultured up to confluence of the MSC before measuring the SDF- ⁇ and/or VEGF expression level.
  • the MSC population is cultured up to at least about 80, 85, 90, 95, 99, or 100% confluence of MSC before measuring the SDF- ⁇ and/or VEGF expression level.
  • the mesenchymal stem cells are undifferentiated, or non- differentiated, i.e. MSC are not differentiated into a cellular type, in particular into fibroblasts.
  • MSC are isolated from tissues selected from the group comprising adipose tissue, bone marrow, umbilical cord blood, Wharton's jelly (such as, for example, Wharton's jelly found within the umbilical cord), amniotic fluid, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, skin, bone, dental tissue and fetal liver, or the like.
  • MSC are isolated from adipose tissue.
  • MSC are adipose stem cells (referred to as ASC, or as AMSCs).
  • MSC are isolated from any warm-blooded animal tissues, preferably from human, porcine, bovine or equine tissues, more preferably from human tissues.
  • MSC are human ASC.
  • the cells are cells in culture, preferably are cell lines and/or are derived from primary cells, i.e. cells isolated straight from the tissue.
  • the cells are recovered from a sample from an individual, obtained for example by biopsy.
  • the step of recovering the sample from an individual is not part of the method of the present invention.
  • Isolation of mesenchymal stem cells may be accomplished by any acceptable method known to one of ordinary skill in the art.
  • methods for isolating MSC include, but are not limited to, digestion by collagenase, trypsinization, or explant culture.
  • mesenchymal stem cells are isolated from adipose tissue by digestion of the tissue, for example by collagenase.
  • the MSCs of the invention may be stably or transiently transfected or transduced with a nucleic acid of interest using suitable a plasmid, viral or alternative vector strategy.
  • Nucleic acids of interest include, but are not limited to, those encoding gene products which enhance the production of extracellular matrix components found in the tissue type to be repaired.
  • the MSC population is cultured in any culture medium designed to support the growth of the cells known to one of ordinary skill in the art.
  • culture medium is called "proliferation medium” or “growth medium”.
  • growth medium include, without limitation, MEM, DMEM, CMRL, EVIDM, RPMI 1640, FGM or FGM-2, 199/109 medium, HamF10/HamF12 or McCoy's 5A, preferably DMEM or RPMI.
  • the culture medium may further comprise any supplementary factors.
  • supplementary factors include, but are not limited to, FBS; platelet lysate; glycine; amino acids, such as glutamine, asparagine, glutamic acid, aspartic acid, serine, proline or alanine, preferably the L-configuration of amino acids; and antibiotics, such as streptomycin or penicillin.
  • the MSC population is cultured in standard culture conditions.
  • standard culture conditions means at a temperature of 37°C and in 5% C0 2 .
  • the MSC population of the invention is not differentiated into a specific tissue. Accordingly, in one embodiment, the MSC population of the invention is not cultured in conditions to induce differentiation, such as, for example, in a differentiation medium. In a particular embodiment, the MSC population of the invention is not cultured in osteogenic differentiation medium, muscle differentiation medium or dermal differentiation medium.
  • a substantially pure MSC population is obtained by culturing the MSC population up to at least 3 passages, preferably in normoxia and normoglycemia.
  • the MSC population may be frozen, preferably at least after the 3th passage.
  • the cells population may be frozen and stored in liquid nitrogen or at any temperature, preferably from about -0°C to -196°C, so long as the cells are able to be used as stem cells after thawing therefrom.
  • the MSC population may be thawed and expanded further to obtain fresh cells.
  • the biocompatible matrix is formed by bioresorbable biological material.
  • bioresorbable means that the biological material can be assimilated back, dissolved or absorbed in the body and does not require mechanical or manual removal.
  • the biocompatible matrix of the invention is formed by a material that provides a structural support for the growth and propagation of cells.
  • biocompatible matrix comprises a natural or synthetic material, or a chemical-derivative thereof, selected from the group comprising agar/agarose, alginates chitosan, chondroitin sulfate, collagen, elastin or elastin-like peptides (ELP) , fibrinogen, fibrin, fibronectin, gelatin, heparan sulfate proteoglycans, hyaluronic acid, polyanhydrides, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene oxide/ polyethylene glycol (PEO/PEG), poly(vinyl alcohol) (PVA), fumarate -based polymers such as, for example poly(propylene fumarate) (PPF) or poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG) (P(PF
  • the biocompatible matrix of the invention is derived from any collagenous tissues.
  • collagenous tissues include, but are not limited to, skin, dermis, tendon, ligament, muscle, amnion, meniscus, small intestine submucosa, cardiac valve, vessel and bladder.
  • the biocompatible matrix of the invention is derived from tendon, preferably from fascia lata.
  • the biocompatible matrix is recovered from an individual.
  • the biocompatible matrix is recovered from a human.
  • the biocompatible matrix is recovered from a non-human animal.
  • Such biocompatible matrix are commercially available. Examples of commercially available biocompatible matrix include, but are not limited to, Integra® (Integra LifeSciences Corporation), Matriderm® (Skin & Health Care), Alloderm® (Life Cell) and Puramatrix® (3-D Matrix Medical Technology).
  • the step of recovering the biocompatible matrix from an individual is not part of the method of the present invention.
  • the biocompatible matrix is derived from human fascia lata tendon.
  • the biocompatible matrix of the invention has low or no immunogenicity.
  • the biocompatible matrix is processed, by any biological, chemical and/or physical means to reduce its immunogenicity. After a process to reduce immunogenicity, the biocompatible matrix of the invention is less immunogenic in comparison to unprocessed biocompatible matrix of the same type.
  • the biocompatible matrix is processed to remove cellular membranes, nucleic acids, lipids and cytoplasmic components.
  • the biocompatible matrix comprises intact the components typically associated with the matrix, such as, for example, collagen, elastins, glycosaminoglycans, and proteoglycans.
  • the biocompatible matrix is processed to reduce immunogenicity.
  • the biocompatible matrix of the invention is at least about 50%, 60%, 70%, 80% or 90% less immunogenic than unprocessed biocompatible matrix of the same type. The parameters for assessing the immunogenicity of a matrix are readily measurable by routine procedures familiar to a physician.
  • Examples of processes to reduce immunogenicity include, but are not limited to, decellularization of the biocompatible matrix and cellular disruption of the biocompatible matrix.
  • the biocompatible matrix of the invention has been processed with a cellular disruption method.
  • cellular disruption methods include, but are not limited to, cryopreservation, freeze/thaw cycling, and exposure to radiation.
  • the biocompatible matrix of the invention has been processed with to a decellularization method.
  • a decellularization method means a method to remove endogenous cells from the biocompatible matrix by use of physical, chemical, or biochemical means. Examples of means include, but are not limited to, enzymes such as proteases and/or nucleases; chemicals such as acids, bases, ionic detergents, non-ionic detergents, surfactants, and/or zwitterionic detergents; defatting agents such as acetone; and/or freeze-drying.
  • the biocompatible matrix of the invention is acellular. In one embodiment, the biocompatible matrix comprises at most about 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less of endogenous cells, in comparison of a biocompatible matrix of the same type which has not been subjected to removal of endogenous cells.
  • Decellularization may be quantified according to any method known from the person skilled in the art. Examples of methods of quantifying decellularization include, but are not limited to, staining with 40,6-diamidino-phenylindole (DAPI), or measuring DNA concentration in the processed biocompatible matrix, in comparison with the biocompatible matrix before processing or with a biocompatible matrix of the same type which has not been processed.
  • DAPI 4,6-diamidino-phenylindole
  • the biocompatible matrix is an acellular collagen matrix, preferably a human acellular collagen matrix (HACM).
  • HACM human acellular collagen matrix
  • the biocompatible matrix is prepared to obtain a matrix comprising no detectable chemical residues, such as, for example, acetone or H2O2, and less than about 10% of residual moisture, preferably less than 8%.
  • Such preparation of the biocompatible matrix may be accomplished by any acceptable method known to one of ordinary skill in the art.
  • the biocompatible matrix comprises no chemical residues, such as, for example, acetone or H2O2, and less than about 10% of residual moisture, preferably less than 8%.
  • the biocompatible matrix is sterilized to avoid infectious disease transmission, such as, for example, HIV, hepatitis C and B viruses (HCV, HBV) and bacterial infections.
  • infectious disease transmission such as, for example, HIV, hepatitis C and B viruses (HCV, HBV) and bacterial infections.
  • sterilization methods include, but are not limited to, chemicals, heat, UV and ionizing radiation, such as gamma irradiation.
  • the biocompatible matrix and the MSC population of the invention are from same species. In another embodiment, the biocompatible matrix and the MSC population of the invention are from different species. In another embodiment, the biocompatible matrix and the MSC population of the invention are from the same donor subject, who may, or may not be the recipient subject. In another embodiment, the biocompatible matrix and the MSC population of the invention are from different subjects, either one or neither, may be the recipient subject. In one embodiment, the mesenchymal stem cells population is incubated with the biocompatible matrix. As used herein, the term "incubated" means that the mesenchymal stem cells population contacts the biocompatible matrix.
  • the mesenchymal stem cells population is seeded on the biocompatible matrix. In another embodiment, the mesenchymal stem cells population is seeded into the biocompatible matrix. In another embodiment, the mesenchymal stem cells population is seeded on and into the biocompatible matrix.
  • the cells in the composition are seeded in any arrangement.
  • the cells may be distributed homogeneously throughout the biocompatible matrix or distributed in defined zones, regions or layers within the biocompatible matrix.
  • Seeding of the cells is preferably performed under suitable conditions of temperature, pH, ionic strength and sheer to maintain cell viability.
  • the MSC population is cultured up to at least passage 2, 3, 4 or 5 before being incubated with the biocompatible matrix.
  • the term "cultured up to at least passage 4" means that the cell population is detached and transferred into a new vessel up to at least 4 times.
  • the MSC population is cultured up to passage 4 before being incubated with the biocompatible matrix.
  • the mesenchymal stem cells incubated with the biocompatible matrix are late passaged mesenchymal stem cells.
  • the MSC population is passaged when the MSC population reaches about 70, 75, 80, 85, 90, 95, 99, or 100% confluence.
  • the MSC population is cultured for at least 24 hours, preferably for at least 36, 48, 60 or 72 hours before being incubated with the biocompatible matrix. In another embodiment, the MSC population is cultured for at least 1 day, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 days before being incubated with the biocompatible matrix.
  • the biocompatible matrix is incubated with the MSC population in 12-well or 24-well plates, or in cell culture flasks of 25 cm 2 , 75 cm 2 or 150 cm 2 .
  • the MSC population is counted and initially incubated with the biocompatible matrix at a density of at least 0.5xl0 5 , lxlO 5 , 2xl0 5 or 3xl0 5 cells per well.
  • the MSC population is initially incubated with the biocompatible matrix at a density from about 0.5xl0 5 to about 5xl0 5 cells/well (per well), preferably from about lxlO 5 to about 4xl0 5 cells/well, more preferably from about 2xl0 5 to about 3xl0 5 cells/well. In a preferred embodiment, the MSC population is initially incubated with the biocompatible matrix at a density of about 2xl0 5 cells per well.
  • the MSC population is counted and initially incubated with the biocompatible matrix at a density of at least lxlO 4 , 2.5xl0 4 , 5xl0 4 or 8xl0 4 cells per cm 2 of culture dishes or plates.
  • the MSC population is initially incubated with the biocompatible matrix at a density from about lxlO 4 to about 15xl0 4 cells/cm 2 of culture dishes or plates, preferably from about 2.5xl0 4 to about 12.5xl0 4 cells/cm 2 , more preferably from about 5xl0 4 to about 8xl0 4 cells/cm 2 .
  • the MSC population is initially incubated with the biocompatible matrix at a density of about 5xl0 4 cells per cm 2 of culture dishes or plates.
  • the MSC population is counted and initially incubated with the biocompatible matrix at a density of at least 0.25xl0 5 , 0.5xl0 5 , lxlO 5 or 1.5xl0 5 cells per cm 2 of culture dishes or plates.
  • the MSC population is initially incubated with the biocompatible matrix at a density from about 0.25xl0 5 to about 2.5xl0 5 cells/cm 2 of culture dishes or plates, preferably from about 0.5xl0 5 to about 2xl0 5 cells/cm 2 , more preferably from about lxlO 5 to about 1.5xl0 5 cells/cm 2 .
  • the MSC population is initially incubated with the biocompatible matrix at a density of about lxlO 5 cells per cm 2 of culture dishes or plates.
  • the incubation of the biocompatible matrix with the mesenchymal stem cells population of the invention is performed in a culture medium as described hereinabove. In some embodiments, the incubation is performed at 37°C in 5% C0 2 .
  • the composition is cultured once the MSC population contacts the biological matrix. In one embodiment, the composition of the invention is cultured up to confluence of the MSC. In another embodiment, the composition of the invention is cultured up to at least 70, 75, 80, 85, 90, 95, 99, or 100% confluence of MSC.
  • the composition of the invention is cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after contacting the MSC population and the biological matrix. In another embodiment, the composition of the invention is cultured for more than 30 days. In another embodiment, the composition of the invention is cultured from 3 to 30 days, from 5 to 25 days, from 5 to 20 days, from 5 to 15 days or from 5 to 10 days.
  • the composition of the invention is washed to remove the culture medium.
  • the composition is washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition of the invention secretes at most 100 pg/ml of SDF- ⁇ , preferably at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml, when the composition is cultured at least 24 hours in hypoxia, preferably at about 0.1% 0 2 .
  • the composition of the invention secretes at most 100 pg/ml of SDF- ⁇ , preferably at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ , when the composition is cultured at least 24 hours in normoglycemia, preferably at a concentration of glucose of about 1 g/1 of glucose.
  • the composition of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ , when the composition is cultured at least 24 hours in hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the composition of the invention secretes at least 200 pg/ml of VEGF, preferably at least about 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 299 or 290 pg/ml of VEGF, when the composition is cultured at least 24 hours in hypoxia, preferably at about 0.1% 0 2 , and in hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the composition of the invention secretes at least about 90 pg/ml of VEGF, preferably at least about 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 pg/ml of VEGF, when the composition is cultured at least 24 hours in physiological oxygen levels, preferably at about 5% O2, and in hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • the composition of the invention secretes at least about 160 pg/ml of VEGF, preferably at least about 161, 162, 163, 164, 165, 166, 167, 168, 169 or 170 pg/ml of VEGF, when the composition is cultured at least 24 hours in physiological oxygen levels, preferably at about 5% O2, and in normoglycemia, preferably at a concentration of glucose of about 1 g/1.
  • the composition of the invention secretes at most 50 pg/ml of SDF- la, preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml of SDF- ⁇ ; and at least about 200 pg/ml of VEGF, preferably at least about 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 299 or 290 pg/ml of VEGF, when the composition is cultured at least 6 hours in hypoxia, preferably at about 0.1% O2, and in hyperglycemia, preferably at a concentration of glucose of about 4.5 g/1.
  • composition of the invention secretes at most 50 pg/ml of SDF- ⁇ , preferably at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,
  • VEGF vascular endothelial growth factor
  • physiological oxygen levels preferably at about 5% O2
  • hyperglycemia preferably at a concentration of glucose of about 4.5 g/1.
  • composition of the invention secretes at most 100 pg/ml of SDF- ⁇ , preferably at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
  • VEGF vascular endothelial growth factor
  • physiological oxygen levels preferably at about 5% 0 2
  • normoglycemia preferably at a concentration of glucose of about 1 g/1 of glucose.
  • the composition of the invention is cultured up to confluence of the MSC population before measuring the SDF- ⁇ and/or VEGF expression level. In another embodiment, the composition is cultured up to at least about 80, 85, 90, 95, 99, or 100% confluence of the MSC population before measuring the SDF- l and/or VEGF expression level.
  • the composition of the invention is a device (such as, for example, a medical device), a film, a three-dimensional structure, a dressing, a composite graft, a soft tissue substitute, a pharmaceutical composition and the like.
  • a device such as, for example, a medical device
  • a film such as, for example, a film, a three-dimensional structure, a dressing, a composite graft, a soft tissue substitute, a pharmaceutical composition and the like.
  • additional components being either hormone, protein, nucleic acid, lipid and/or carbohydrate in character, may be added to the composition of the invention in any amount that will allow the added component to be positive or negative effector of adherence, growth, differentiation, proliferation, vascularization, engraftment, and three-dimensional modelling of the MSC population in the composition of the invention.
  • Such components suitable for use in the composition include, but are not limited to naturally occurring, variants or fragments of immune- stimulators, immune-suppressors and/or immune-adjuvants such as cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSF), interferons (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, transforming growth factor (TGF), TGF-a, TGF- ⁇ , insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-a, TNF- ⁇ , mullerian-in
  • additional components may be present in the composition of the invention in any amount that will allow the added component to provide improved usability or handling characteristics, while not significantly altering the stability or form of the composition of the invention.
  • additional components suitable for use in the composition include, but are not limited to antiseptics, antibiotics, antivirals, antioxidants, anaesthetics, plasticizers, preservatives, cellular growth medium and/or cryoprotectants.
  • the present invention also relates to a method for the preparation of a composition comprising a biocompatible matrix and a mesenchymal stem cells population comprising:
  • MSC mesenchymal stem cells
  • the method for the preparation of a composition comprising a biocompatible matrix and a mesenchymal stem cells population comprises incubating a substantially pure MSC population with a biocompatible matrix.
  • the method for the preparation of a composition comprising a biocompatible matrix and a mesenchymal stem cells population comprises:
  • MSC mesenchymal stem cells
  • the method for the preparation of a composition comprising a biocompatible matrix and a mesenchymal stem cells population comprises incubating a MSC population comprising less than 25% of fibroblasts with a biocompatible matrix.
  • the method for the preparation of a composition comprising a biocompatible matrix and an adipose stem cells population comprises:
  • ASC a. isolating adipose stem cells
  • the method for the preparation of a composition comprising a biocompatible matrix and an adipose stem cells population comprises incubating a substantially pure ASC population with a collagen-containing biocompatible matrix.
  • the method for the preparation of a composition comprising a biocompatible matrix and an adipose stem cells population comprises:
  • ASC a. isolating adipose stem cells
  • the method for the preparation of a composition comprising a biocompatible matrix and an adipose stem cells population, comprises incubating an ASC population comprising less than 25% of fibroblasts with a collagen-containing biocompatible matrix.
  • the soft tissue injury may result, for example, from disease, trauma or failure of the tissue to develop normally.
  • the soft tissue that can be treated by the composition of the invention is selected from the group comprising skin tissue, muscle tissue, dermal tissue, tendon tissue, ligament tissue, meniscus tissue, cardiac valve tissue, vessel tissue, gastric mucosa, tympanic tissue, amnion and bladder tissue.
  • the composition of the invention is for treating, or for use for treating, a skin injury.
  • the composition of the invention is for treating, or for use for treating, a muscle injury.
  • the soft tissue injury that can be treated by the composition is selected from the group comprising skin wounds, skin burns, skin ulcers, muscle diseases (such as, for example, inflammatory muscle diseases, neurogenic muscle diseases, myogenic muscle diseases, muscular dystrophies, congenital myopathies, or myasthenia gravis), hernias, surgical wounds (such as, for example, post-operative wounds), or vascular diseases (such as, for example, peripheral arterial diseases, abdominal aortic aneurysms, carotid diseases, venous diseases, or vascular injuries).
  • muscle diseases such as, for example, inflammatory muscle diseases, neurogenic muscle diseases, myogenic muscle diseases, muscular dystrophies, congenital myopathies, or myasthenia gravis
  • hernias such as, for example, surgical wounds (such as, for example, post-operative wounds)
  • vascular diseases such as, for example, peripheral arterial diseases, abdominal aortic aneurysms, carotid diseases, venous diseases, or
  • the composition of the invention is for treating, or for use for treating, a skin wound, a skin burn or a skin ulcer.
  • the composition is for treating, or for use for treating, a skin ulcer due to drepanocytosis.
  • the composition is for treating, or for use for treating, a skin ulcer due to vasculitis.
  • the composition is for treating, or for use for treating, a diabetic wound, such as for example a diabetic ulcer.
  • the composition is for treating, or for use for treating, a radionecrosis.
  • the composition of the invention is for treating, or for use for treating, a muscle disease.
  • the soft injury is an acute wound (i.e. before about 2-4 days post- injury). In another embodiment, the soft injury is a sub-acute wound (i.e. between about 2-4 days to 6 weeks post-injury). In another embodiment, the soft injury is a wound at late stage (i.e. about 6 weeks post-injury). In another embodiment, the soft injury is a chronic wound. In one embodiment, a chronic wound is defined by a failure to achieve complete healing after 3 months. In another embodiment, the soft injury is a non-healing wound.
  • composition of the invention provides an ex vivo model for studying a soft-tissue injury in accordance with the preceding aspects.
  • the subject is affected by at least one soft tissue injury as described herein above.
  • the subject is diabetic.
  • the subject is diagnosed with diabetes, such as, for example, type I diabetes, type II diabetes, gestational diabetes, latent autoimmune diabetes, type 1.5 diabetes, lipoatrophic diabetes, maturity onset diabetes of the young, neonatal diabetes (e.g. permanent neonatal diabetes or transient neonatal diabetes), prediabetes, steroid-induced diabetes.
  • the subject to whom the composition is administered suffered of Type I Diabetes Mellitus or of Type II Diabetes Mellitus.
  • the subject was not treated previously with another treatment for soft tissue injury.
  • the subject previously received at least one treatment for soft tissue injury.
  • treatments for treating soft tissue injury include, but are not limited to, wound closure with sutures, staple or adhesive tape or glue, anti-inflammatory drugs, drainage of the infection, surgery, skin autografts, ultrasound therapy, hyperbaric oxygenotherapy, nursing care, topical growth factors application and keratinocyte cell spray.
  • the biocompatible matrix of the invention is xenogeneic to the subject to which the composition is administered.
  • a non-limiting example is a porcine biocompatible matrix and a human subject.
  • the biocompatible matrix of the invention is allogeneic to the subject to which the composition is administered.
  • a non-limiting example is a human biocompatible matrix and a human subject.
  • the MSC population of the invention is xenogeneic to the subject to which the composition is administered, i.e. from a member of a different species.
  • the MSC population of the invention is allogeneic to the subject to which the composition is administered, i.e. from a non-genetically identical member of the same species.
  • the MSC population of the invention is syngeneic to the subject to which the composition is administered, i.e. genetically identical or closely related, so as to minimize tissue transplant rejection.
  • Syngeneic MSC population include those that are autogeneic (i.e., from the subject to be treated) and isogeneic (i.e., a genetically identical but different subject, e.g., from an identical twin).
  • the biocompatible matrix and the MSC population of the invention are both xenogeneic to the subject to which the composition is administered.
  • at least one of the biocompatible matrix and the MSC population of the invention is allogeneic to the subject to which the composition is administered.
  • the biocompatible matrix and the MSC population of the invention are both allogeneic to the subject to which the composition is administered.
  • composition of the invention may be administered to the subject according to any method known in the art.
  • methods of administration include, but are not limited to, implantation (such as, for example, surgical implantation), topical application, injection, and transplantation with another tissue.
  • the composition of the invention is topically administered to the subject. In another embodiment, the composition of the invention is administered by surgical implantation to the subject. In another embodiment, the composition of the invention is injected to the subject. In one embodiment, the composition of the invention is administered to the subject at the soft tissue injury site. In some embodiments, the composition of the invention is configured to the shape and/or size of the tissue to be treated. In some embodiments, the composition of the invention is resized before being administered to the subject to the shape and/or size of the tissue to be treated.
  • the invention also relates to a method for treating a soft tissue injury, comprising administering a composition as described hereinabove. In one embodiment, the composition is administered topically or by surgical implantation. In one embodiment, the composition is administered at the site of the soft tissue injury.
  • the method for treating a soft tissue injury comprises administering the composition of the invention in a subject in need thereof.
  • the method for treating a soft tissue injury comprises administering the composition of the invention in a diabetic subject.
  • Another object of the present invention is a method for enhancing or improving closure of a wound, such as, for example, a non-healing skin wound.
  • the invention also relates to a method for promoting soft tissue regeneration, such as, for example, dermal regeneration or muscle regeneration.
  • Another aspect of the invention is a method for promoting or enhancing angiogenesis, preferably at the site of a soft tissue injury.
  • Another aspect of the invention is a method for promoting or enhancing synthesis of granulation tissue, preferably at the site of a soft tissue injury.
  • Still another aspect of the invention is a method for reducing fibrotic scar, preferably at the site of a soft tissue injury.
  • the invention also relates to a method for reducing soft tissue necrosis, such as, for example, skeletal muscle necros
  • Another object of the present invention is a method for improving the efficacy of an autograft, such as, for example, a skin autograft, comprising administering a composition of the invention before performing the autograft.
  • the improvement of the efficacy of an autograft is associated with a reduction of ulcerations (frequency and size).
  • Another object of the invention is a method for screening the effect of a compound on the composition of the invention, wherein said method comprises contacting the compound with the composition of the invention and determining the effect of the compound on the cells present in composition.
  • Examples of determination of the effect of the compound on the cells present in composition include, but are not limited to, secretion of KGF for epidermal remodeling and VEGF for dermal angiogenesis, both for skin regeneration; secretion of VEGF, IGF- 1, HGF, FGF, and TGF- ⁇ implied in the activation, proliferation and differentiation of progenitor cells, for skeletal muscular regeneration; and secretion of VEGF in function of the tissue oxygenation.
  • Another object of the invention is a method for screening the effect of a compound on the formation of the film, three-dimensional structure, dressing, composite graft, or soft tissue substitute of the invention, wherein said method comprises contacting the compound with the composition of the invention and determining the effect of the compound on the formation of the film, three-dimensional structure, dressing, composite graft, or soft tissue substitute of the invention.
  • determination of the effect of the compound on the formation of the film, three-dimensional structure, dressing, composite graft, or soft tissue substitute of the invention include, but are not limited to, pure stem cells in vitro recolonization of the a 3D scaffold; and in vitro stem cells spreading for scaffold surface recovering.
  • Another object of the invention is a method for screening the therapeutic effect of a compound on the soft tissue injury as described here above, wherein said method comprises:
  • determining the therapeutic effect of the compound on the composition of the invention to determine if the compound can be used for treating a soft tissue injury in a patient in need thereof.
  • Examples of determination of the therapeutic effect of the compound on the composition of the invention include, but are not limited to, selective growth factors profile secretion in function of the wound tissue, such as KGF for skin epidermal regeneration; and capacity of pure stem cells (obtained from diabetic or non-diabetic subjects) to cure a diabetic skin wound.
  • selective growth factors profile secretion in function of the wound tissue such as KGF for skin epidermal regeneration
  • pure stem cells obtained from diabetic or non-diabetic subjects
  • Another object of the invention is a test or a potency test to determine if the composition of the invention is suitable for being used in the treatment of a soft tissue injury, wherein: - the composition of the invention is prepared as described above,
  • the composition or film, three-dimensional structure, dressing, composite graft, or soft tissue substitute of the invention is analyzed to determine if it presents at least one characteristic of the soft tissue to be treated.
  • determination of at least one characteristic of the soft tissue to be treated include, but are not limited to, the increased secretion of VEGF from pure stem cells in a 3D composition in hypoxia.
  • Figure 1 is a graph showing the quantification of total protein extracted from fascia lata at the different stages of the decellularization method.
  • Figure 2 is a set of graphs showing the quantification of the growth factors SDF-1 alpha (A), VEGF (B), bFGF (C) and IGF (D) extracted from fascia lata at the different stages of the decellularization method.
  • Figure 3 is a graph showing the quantification of the total DNA extracted from fascia lata before and after the decellularization method.
  • Figure 4 is a graph showing the quantification of total protein extracted from dermal tissues at the different stages of the decellularization method.
  • Figure 5 is a set of graphs showing the quantification of the growth factors SDF-1 alpha (A), VEGF (B) and bFGF (C) extracted from dermal tissues at the different stages of the decellularization method.
  • Figure 6 is a graph showing the quantification of the total DNA extracted from dermal tissues before and after the decellularization method.
  • Figure 7 is a graph showing the quantification of total protein extracted from cancellous bone at the different stages of the decellularization method.
  • Figure 8 is a set of graphs showing the quantification of the growth factors SDF-1 alpha (A), VEGF (B), bFGF (C) and IGF (D) extracted from cancellous bone at the different stages of the decellularization method.
  • Figure 9 is a graph showing the quantification of the total DNA extracted from cancellous bone before and after the decellularization method.
  • Figure 10 is a graph showing the quantification of total protein extracted from cortical bone at the different stages of the decellularization method.
  • Figure 11 is a set of graphs showing the quantification of the growth factors SDF-1 alpha (A), VEGF (B) and bFGF (C) extracted from cortical bone at the different stages of the decellularization method.
  • Figure 12 is a graph showing the quantification of the total DNA extracted from cortical bone before and after the decellularization method.
  • Figure 13 is a photograph showing ASC and DF in proliferation medium (A) and in osteogenic differentiation medium (B).
  • Figure 14 is a graph showing the cell proliferation of ASC and DF according to the number of passages.
  • Figure 15 is a histogram showing the cell survival of ASC and DF in proliferation medium without FBS, at 0.1 or 5% 0 2 .
  • Figure 16 is a set of histograms showing KGF secretion (A), b-FGF secretion (B), IGF- 1 secretion (C), and HGF secretion (D) of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/1 glucose, at 0.1 or 5% 0 2 .
  • Figure 17 is a set of histograms showing VEGF secretion (A) and SDF-la secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/1 glucose, at 0.1% 0 2 .
  • Figure 18 is a set of histograms showing VEGF secretion (A) and SDF-la secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/1 glucose, at 5% 0 2 .
  • Figure 19 is a histogram showing SDF-la secretion of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/1 glucose, at 21% 0 2 .
  • Figure 20 is a set of histograms showing VEGF secretion (A) and SDF-la secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 1 g/1 glucose, at 0.1% 0 2 .
  • Figure 21 is a set of histograms showing VEGF secretion (A) and SDF- ⁇ secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 1 g/1 glucose, at 5% 0 2 .
  • Figure 22 is a histogram showing SDF- ⁇ secretion of 5 different ASC/DF dilutions in proliferation medium with 1 g/1 glucose, at 21% 0 2 .
  • Figure 23 is a set of graphs showing VEGF (A), SDF- ⁇ (B), and KGF (C) secretions from AMSC, dermal fibroblasts (FD) and keratinocytes (Kc) at 1 g/1 of glucose and at 5% 0 2 (light grey) or 0.1% 0 2 (dark grey).
  • Figure 24 is a set of graphs showing VEGF (A), SDF- ⁇ (B), and KGF (C) secretions from AMSC, dermal fibroblasts (FD) and keratinocytes (Kc) at 5% O2 and at a concentration of glucose of 1 g/1 (light grey) or 4.5 g/1 (dark grey).
  • Figure 25 is a set of graphs showing VEGF (A), SDF- ⁇ (B), and KGF (C) secretions from AMSC, dermal fibroblasts (FD) and keratinocytes (Kc) in physical conditions (5% O2 and 1 g/1 glucose, light grey) and in diabetic wounds conditions (0.1% O2 and 4.5 g/1 glucose, dark grey).
  • Figure 26 is a set of graphs showing VEGF secretions from AMSC (A) and dermal fibroblasts (B), from diabetic or non-diabetic human donors, in 0.1% O2 and 1 g/1 of glucose (0.1% nmglc), in 0.1% 0 2 and 4.5 g/1 of glucose (0.1% Hglc), in 5% 0 2 and 1 g/1 of glucose (5% nmglc), and in 5% O2 and 4.5 g/1 of glucose (5% Hglc).
  • Figure 27 is a set of graphs showing KGF secretions from AMSC (A) and dermal fibroblasts (B), from diabetic or non-diabetic human donors, in 0.1% O2 and 1 g/1 of glucose (0.1% nmglc), in 0.1% 0 2 and 4.5 g/1 of glucose (0.1% Hglc), in 5% 0 2 and 1 g/1 of glucose (5% nmglc), and in 5% O2 and 4.5 g/1 of glucose (5% Hglc).
  • Figure 28 is a photograph showing mesenchymal lineage differentiation of ASC: chondrogenic and adipogenic differentiation capacities were demonstrated by alizarin red staining (calcium deposition) (A), alcian blue staining (glycosaminoglycane deposition) (B) and Oil Red (intracellular lipid droplets) (C), respectively (magnification x20).
  • Figure 29 is a graph showing spreading (A) and adhesion (B) of ASC on HACM compared to ASC on plastic well plate.
  • Figure 30 is a photograph showing histological and macroscopic analyses 1 month and 3 months after implantation of the composite graft (HACM + ASC) and the scaffold alone (HACM, control) in nude rats.
  • Figure 31 is a graph showing VEGF secretion by ASC during normoxia (21% 0 2 ) or hypoxia (0.1% 0 2 ).
  • Figure 33 is a photograph showing clinical evolution of patient 1 suffering of radionecrosis.
  • Figure 34 is a photograph showing clinical evolution of patient 1 (A), 2 (B) and 3 (C) before the implantation (1), at the time of implantation (2), and after 22, 4 and 2 months (patient 1, 2 and 3 respectively) (3).
  • Figure 35 is a graph showing C-reactive protein (CRP) and fibrinogen concentrations at the time of implantation, and after 3, 13 and 28 days.
  • CRP C-reactive protein
  • Figure 36 is a photograph showing macrohistology with Masson trichome staining of the wound bed tissue at day 0 (A) and day 56 (B) after implantation in patient 1.
  • Figure 37 is a graph showing histomorphometric semi-quantitative analysis of VEGF and factor VIII (A) and of CD3 and CD68 (B) before (light grey) and after implantation (dark grey) in patient 1.
  • Figure 38 is a set of photographs showing the wound closure evolution of skin autograft alone (A), skin autograft after composite graft HACM+ASC implantation (B) and skin autograft after HACM implantation alone (C) in the patient 2; at day 0, macroscopically (1) and at dermis tissue level (2), and at the maximum duration of wound closure (28 days, 65 days and 16 days respectively) (3).
  • Figure 39 is a graph showing the BM-MSC and ASC survival in hypoxia (0.1% 0 2 ), relative to normoxia (21 O2).
  • Figure 40 is a set of graphs showing VEGF (A), FGF-beta (B), IGF- 1 (C), TGF-beta (D) and HGF (E) secretion of BM-MSC and ASC in normoxia (21% 0 2 ) and hypoxia (0.1% O2). Results are expressed as mean+SEM.
  • Figure 41 is a graph showing the cellular spreading of BM-MSC and ASC on the HACM (triangles and squares respectively) in comparison with the cellular spreading on plastic well (straight line) (*p ⁇ 0.05).
  • Figure 42 is a graph showing the cellular growth of BM-MSC and ASC on the HACM (triangles and squares respectively) in comparison with the cellular spreading on plastic well (straight line) (* ⁇ p ⁇ 0.05).
  • Figure 43 is a set of graphs angiogenesis (A) and fibrosis thickness (B) of muscular necrosis treated with HACM recellularized with ASC or BM-MSC, or with HACM alone. Results are presented as ratio in comparison to muscular necrosis untreated (sham).
  • Figure 44 is a graph showing the angiogenesis of explanted graft, HACM recellularized with ASC or with BM-MSC, or HACM alone, 30 days after implantation (p ⁇ 0.05).
  • Example 1 Bioactivity of decellularized matrices Material and methods
  • Human fascia lata was treated in absolute acetone for up to 24 hours, in ether for 15 hours, then in ethanol 70° for up to 8 hours. Between each step, the tissue was intensely washed in a continuous flow of demineralized water. Human fascia lata was then treated with a combination of NaOH IN and NaCl for 1 hour, and with H2O2 15% for up to 8 hours. The tissue was further intensely washed with demineralized water for up 68 hours.
  • Human dermal tissue was treated in ether for 8 hours, then in ethanol 70° for up to 16 hours and washed with demineralized water for 7 hours. Human dermal tissue was then treated with a combination of NaOH IN and NaCl for 1 hour, followed by a washing with demineralized water for 16 hours, and then treated with H2O2 15% for up to 15 hours. The tissue was further intensely washed with demineralized water for up 16 hours.
  • Human cancellous bone was procured from tibia and cortical bone from calcaneum.
  • the treatment of the bone tissues began with a centrifugation to eliminate the marrow and the blood. After the centrifugation, transplants were cut and cleaned with demineralized water. Bone tissues were then treated in acetone for up to 68 hours followed by a washing for 5 hours. Bone tissues were subsequently treated with a combination of NaOH IN and NaCl for 1 hour, followed by a washing with demineralized water for 3 hours, and then treated with H2O2 15% for up to 15 hours. The tissue was further intensely washed with demineralized water for up 72 hours. In each stage of the decellularization method, a sample was taken for proteins extraction. Moreover, total DNA was quantified on native tissues, i.e. before decellularization, and on decellularized tissues, i.e. at the end of the decellularization method. Protein extraction and purification
  • Proteins from human fascia lata and dermal tissues were extracted according to the protocol of Wolf et al. (Biomaterials. 2012, 33(10):2916-2925). In brief, 300 mg of tissue were incubated with 5 ml of urea-heparin buffer, pH 7.4 (urea 2M, tris 50 mM, heparin 5 mg/ml, N-ethylmaleimids 10 mM, benzamidine 5 mM and phenylmethylsulfonyl fluoride 1 mM) under stirring for up to 24 hours at a temperature of 4°C. After a centrifugation at a speed of 3000 g for 30 min, the supernatant was removed and treated according the same extraction protocol.
  • the second supernatant was then purified by exclusion chromatography and quantified by ELISA.
  • Proteins from human bone tissues were extracted according to the protocol of Pietrzal et al. (Radiat Oncol. 2007, 2(1):5). In brief, cancellous and cortical bones were mechanically crushed to obtain a bone powder. 300 mg wet weight of bone powder were incubated with 5 ml of a solution comprising 4M of guanidine and 5 mM of benzamidine for up to 24 hours at a temperature of 4°C. Then 5 ml of a Tris-HCl buffer were added and incubated for up 5 hours at 4°C. After a centrifugation for 10 min, the supernatant was removed purified by exclusion chromatography and quantified by ELISA.
  • decellularized matrices obtained by the method of the invention are inert material and could be administered to patients without fear of a rejection or emergence of a new pathology.
  • Example 2 ASC growth factors secretion Materials and Methods This study was performed according to the guidelines of the Belgian Ministry of Health. All procedures were approved by the Ethical Committee of the Medical Faculty (Universite Catholique de Louvain) for tissue procurement and clinical study (B40320108280). All materials were obtained from Lonza (Verviers, Switzerland), Sigma-Aldrich (St. Louis, MO, USA), or Invitrogen (Carlsbad, CA, USA) unless otherwise noted.
  • a combined harvesting of human adipose (mean: 7.4 g) and dermal (mean: 1.5 cm 2 ) tissues were performed in 8 patients (Table 1) undergoing elective plastic surgery after informed consent and serologic screening, by lipoaspiration using the Coleman technique, and skin biopsy, respectively.
  • Adipose tissue and skin samples were kept in sterile conditions for a maximum of 60 minutes at 4°C before adipose-derived stem cells (ASC) and dermal fibroblasts (DF) isolation.
  • ASC adipose-derived stem cells
  • DF dermal fibroblasts
  • the adipose tissue was digested with collagenase (1/2 w/v) in a water bath at 37°C for 60 minutes. Collagenase was inactivated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Collected tissue was centrifuged for 10 minutes at 1500 rpm at room temperature. The pellet was suspended in a proliferation medium made up of DMEM supplemented with 10% fetal bovine serum, L-glutamine (2 mM), and antibiotics (100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 1 ⁇ /ml amphotericin B) and filtered through a 500- ⁇ mesh screen.
  • DMEM Dulbecco's modified Eagle medium
  • DF were isolated by extraction from de-epidermized dermal biopsies, minced in 2 mm x 2 mm fragments and placed in plastic well. Small volume of the proliferation medium was added to avoid detachment from the plastic surface.
  • ASC and DF were characterized for standard cell surface markers (CD44, CD45, CD73, CD90, CD105, stro-1, CD106, CD146, CD166, CD19, CD31, CDl lb, CD79a, CD13, HLA-DR, CD14, CD34) [Dominici et al., Cytotherapy. 2006; 8(4):315- 317; Bourin et al. Cytotherapy. 2013; 15:641-648] by fluorescence-activated cell sorting (FACScan; BD Biosciences, San Jose, CA).
  • FACScan fluorescence-activated cell sorting
  • ASC were stained with saturating amounts of monoclonal antibodies: anti-Stro- 1, anti-CD90, anti-CD106, anti-CD105, anti-CD146, anti-CD166, anti-CD44, anti-CD 19, anti-CD45 (Human Mesenchymal Stem Cell marker antibody panel, R&D System, Minneapolis, MN, USA), anti-CD44 (PE mouse anti-human CD44, BD Bioscience, Franklin Lakes, NJ, USA), anti-CD73 (FITC mouse anti-human CD73, BD Bioscience), anti-CD31 (FITC, mouse anti-human, Abeam, Cambridge, UK), anti-CD l ib (FITC, mouse anti-human, Abeam, Cambridge, UK), anti-CD79a (PE, mouse anti-human, Abeam, Cambridge, UK), anti-CD 13 (FITC, mouse anti-human, Abeam, Cambridge, UK), anti-HLA-DR (FITC, mouse anti-human, Abeam, Cambridge, UK), anti-CD 14 (FITC, mouse, mouse
  • ASC and DF were tested at passage 4 in specific media to assess the capacity of differentiation toward osteogenic lineage.
  • the differentiation was evaluated by Alizarin red staining after culturing the cell during 3 weeks in specific differentiation medium (proliferation medium supplemented with dexamethasone (1 ⁇ ), sodium ascorbate (50 ⁇ g/ml), and sodium dihydrophosphate (36 mg/ml) [Qu et al., In Vitro Cell Dev Biol Anim. 2007; 43:95-100].
  • Osteogenic differentiation was confirmed by staining for calcium phosphate with Alizarin red after formalin fixation.
  • immunohistochemistry for osteocalcin was performed to confirm the bone phenotype. Impact of oxygen tension and fetal bovine serum (FBS) on cell proliferation: EdU assay
  • the cells were finally placed for 48 hours in the specific conditions: 0.1% 0 2 , 5% 0 2 and 21% 0 2 in proliferation medium supplemented with 1% FBS or 5% FBS and EdU (5-ethynyl-2'- deoxyuridine, a nucleoside analog of thymidine and incorporated into DNA during active DNA synthesis) was added. After revelation with Alexa Fluor® 488, positive cells were counted by flow cytometry (FACScan; BD Biosciences, San Jose, CA).
  • the cells were exposed (for each dilution and oxygen tension) to normoglycaemic (1 g/L) or hyperglycaemic (4.5 g/L) proliferation media. After incubation for 24 hours in these controlled conditions; cell culture supernatants were harvested individually and stored at -20°C for further growth factor quantification by enzyme-linked immunosorbent assay (VEGF, HGF, IGF-1, SDF-l and basic FGF by Quantikine ELISA kit; R&D System, Minneapolis, MN, USA).
  • VEGF enzyme-linked immunosorbent assay
  • the one- sample Kolmogorov test and Q-Q plots were used to assess the normal distribution of values. Statistically significant differences between groups (with normal distribution) were tested by paired t-test and one-way analysis of variance with the Bonferroni post hoc test. Statistical tests were performed with PASW 18 (SPSS; IBM, New York, NY, USA); p ⁇ 0.05 was considered significant.
  • ASC and DF were positive (>90 of positive cells) for mesenchymal cell markers (CD13, CD44, CD73, CD90, CD105, CD166), negative for endothelial (CD31), bone-marrow- derived stromal cells (CD106, Stro-1, CD146) and hematopoietic markers (CD14, CD45, CDl lb, CD34), and for HLA-DR, CD79a and CD19.
  • mesenchymal cell markers CD13, CD44, CD73, CD90, CD105, CD166
  • CD31 bone-marrow- derived stromal cells
  • CD106, Stro-1, CD146 bone-marrow- derived stromal cells
  • CD14, CD45, CDl lb, CD34 hematopoietic markers
  • ASC and DF had similar proliferation profile until passage 15 (Figure 14, NS).
  • Example 3 ASC comportment in hypoxia and hyperglycemia
  • a combined harvesting of human adipose (mean: 7.4 g) and dermal (mean: 1.5 cm 2 ) tissues were performed in 8 patients (Table 1) undergoing elective plastic surgery after informed consent and serologic screening, by lipoaspiration using the Coleman technique, and skin biopsy, respectively.
  • Adipose tissue and skin samples were kept in sterile conditions for a maximum of 60 minutes at 4°C before adipose-derived stem cells (ASC) and dermal fibroblasts (DF) isolation.
  • ASC adipose-derived stem cells
  • DF dermal fibroblasts
  • the adipose tissue was digested with collagenase (1/2 w/v) in a water bath at 37°C for 60 minutes. Collagenase was inactivated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Collected tissue was centrifuged for 10 minutes at 1500 rpm at room temperature. The pellet was suspended in a proliferation medium made up of DMEM supplemented with 10% fetal bovine serum, L-glutamine (2 mM), and antibiotics (100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 1 ⁇ /ml amphotericin B) and filtered through a 500- ⁇ mesh screen. The collected suspension was then seeded in 25 cm 2 culture flasks with proliferation medium.
  • DMEM Dulbecco's modified Eagle medium
  • antibiotics 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 1 ⁇ /ml amphotericin B
  • DF were isolated by extraction from de-epidermized dermal biopsies, minced in 2 mm x 2 mm fragments and placed in plastic well. Small volume of the proliferation medium was added to avoid detachment from the plastic surface.
  • Keratinocytes were purchased from ATCC (PCS-200-011) and cultured with a Keratinocyte Growth Kit (ATCC, PCS-200-040TM), in accordance with supplier's instructions.
  • passage 1 The proliferation media were replaced. This initial passage of the primary cells is referred to as passage 0. Dermal pieces were removed from the culture dish when adherent cells were visible on the plastic surface surrounding tissue fragments. Cells were maintained in proliferation medium (changed 2 times/week) up to passage 4, after sequential trypsinizations.
  • cells After trypsinization, cells (after passage 4) were seeded in 12-well culture plates with cells at a density leading to about 80 % to 95 % confluence in triplicate for incubation in hypoxic chambers (Modular Incubator Chamber MIC- 101 ; Billups-Rothenberg, Del Mar, CA, USA) at 0.1% 0 2 and 5% O2, corresponding to highly hypoxic environment and tissular oxygen tension, respectively.
  • the cells were exposed (for each dilution and oxygen tension) to normoglycaemic (1 g/L) or hyperglycaemic (4.5 g/L) proliferation media.
  • VEGF enzyme-linked immunosorbent assay
  • SDF-la enzyme-linked immunosorbent assay
  • KGF enzyme-linked immunosorbent assay
  • the one- sample Kolmogorov test and Q-Q plots were used to assess the normal distribution of values. Statistically significant differences between groups (with normal distribution) were tested by paired t-test and one-way analysis of variance with the Bonferroni post hoc test. Statistical tests were performed with PASW 18 (SPSS; IBM, New York, NY, USA); p ⁇ 0.05 was considered significant.
  • ASC secrete significantly more VEGF than in normoxia and normoglycemia (283.94 pg/ml at 0.1% 0 2 and 4.5 g/1 of glucose, compared to 171.13 pg/ml at 5% O2 and 1 g/1 of glucose; Figure 25A).
  • Fibroblasts in comparison, secrete lower levels of VEGF in hypoxia and hyperglycemia than in normoxia and normoglycemia.
  • ASC secrete more of the growth factor VEGF than fibroblasts in hypoxia, in hyperglycemia and in hypoxia and hyperglycemia.
  • diabetic wounds conditions lead to an increased secretion of VEGF from ASC in comparison to physiological conditions. Therefore, ASC can release VEGF to promote neoangiogenesis in a diabetic environment, and, consequently, may promote the repair of diabetic wounds.
  • VEGF secretion of ASC from diabetic human donors is as high or higher than VEGF secretion of ASC from non- diabetic human donors.
  • fibroblasts from diabetic human donors secrete less VEGF than fibroblasts from non-diabetic human donors ( Figure 26B).
  • KGF secretion of ASC from non-diabetic human donors is different to that of ASC from diabetic human donors ( Figure 27 A).
  • KGF secretion profiles are found for ASC and for fibroblasts, whether from non- diabetic human donors or from diabetic human donors ( Figure 27).
  • MSC from a diabetic subject may also release VEGF to promote neoangiogenesis, and, consequently, may promote the repair of a wound, such as a diabetic wound.
  • the MSC population of the invention may derived from a tissue of the subject to be treated.
  • Example 4 Dermal regeneration Materiel and methods
  • the adipose tissue was digested with GMP collagenase (0.075 g; Serva Electrophoresis GmbH, Heidelberg, Germany) in a water bath at 37°C for 60 minutes.
  • ASC were also seeded in 12-well culture plates for incubation in hypoxic chambers (Modular Incubator Chamber MIC- 101; Billups-Rothenberg, Del Mar, CA, USA) for 72 hours at 0.1% (highly hypoxic environment as seen in necrotic tissues) or 21% 0 2 levels (atmospheric normoxia, normal culture conditions) respectively.
  • hypoxic chambers Modular Incubator Chamber MIC- 101; Billups-Rothenberg, Del Mar, CA, USA
  • 0.1% highly hypoxic environment as seen in necrotic tissues
  • 21% 0 2 levels atmospheric normoxia, normal culture conditions
  • Cytogenetic stability was studied by karyotype and fluorescent in situ hybridization (FISH) analyses after different passages to assess the oncogenic safety of the cellular component of the biological dressing.
  • Metaphase chromosomes were obtained from ASC of five donors according to standard protocols. Briefly, cultured cells in the exponential growth phase after passages 1, 4, 10, 12, and 16 were processed for 4 hours with 0.02 ⁇ g/ml of Colcemid (Invitrogen, Carlsbad, CA). Harvested cells from the flasks after trypsinization were incubated for 30 minutes at 37°C in hypotonic 0.055 M KCl and fixed in 3: 1 methanol: glacial acetic acid.
  • Chromosome harvesting and metaphase slide preparation were performed according to standard procedures (Duhoux et al., PLoS ONE. 2011;6:e26311) Eleven to 20 reverse-trypsin-Wright G-banded (GTW) metaphases were analyzed and karyotypes were reported according to the 2013 International System for Human Cytogenetics Nomenclature (ISCN 2013). FISH analysis was performed according to standard protocols (Veriter et al., Biomaterials.
  • HACM acetoxymethyl esters
  • a triangular flap was elevated in each paravertebral area to create two subcutaneous pockets, allowing the placement of the grafts (HACM + ASC on the right side and HACM alone on the left side).
  • a thermic lesion was applied on the inner side of each flap to reproduce the hypoxic wound environment.
  • the biologic dressing (HACM + ASC) was implanted with the cells in contact with the burned dermis on the right side, whereas HACM alone was implanted on the left paravertebral area.
  • the flaps were closed with non-absorbable sutures after the placement of five to eight crystals of lithium phthalocyanine (LiPC crystals) to allow further measurement of the post-implantation intra-tissular oxygenation course.
  • Electron paramagnetic resonance oximetry (EPR spectrometer; Magnettech, Berlin, Germany) was used to follow the intra-tissular P0 2 course and assess the capacity of the composite graft to improve the tissue oxygenation in nude rats.
  • the biological dressing was proposed for three patients with non-healing wounds (Table 3).
  • Peri-umbilical fatty tissue 22 g, 8 g and 21 g, respectively
  • was harvested using the Coleman technique under local anesthesia) for ASC isolation and culture in line with Good Manufacturing Practices recommendations.
  • CMRL Mediatec, Manassas, VA, USA
  • wound debridement was performed by hydrosurgery before the implantation to ensure a minimally contaminated wound bed.
  • the composite graft was cut to an ideal size and oriented with the cell layer directly in contact with the wound surface and fixed with non-absorbable sutures.
  • Inflammatory parameters were followed (Creactive protein, fibrinogen), as were clinical and histological courses.
  • Vaselinated dressings were applied and changed daily. Biopsies were performed before and after implantation for immunohistochemistry and histomorphometry to study inflammatory reaction, angiogenesis, and tissue remodeling (CD3/CD68, VEGF/factor VIII, and Masson trichrome, respectively).
  • the one-sample Kolmogorov-Smirnov test and Q-Q plots were used to assess the normal distribution of values. Statistically significant differences between groups (with normal distribution) were tested by paired i-test and one-way analysis of variance with the Bonferroni post hoc test. Statistical tests were performed with Systat version 8.0 (Cranes Software International, Bangalore, India) or PASW 18 (SPSS; IBM, New York, NY, USA); p ⁇ 0.05 was considered significant.
  • ASC at passage 4th were characterized by mesenchymal stromal cell surface marker profile: CD44+ (>95% of cells), CD73+ (>90%), CD90+ (>95%), CD 105+ (>95%), CD45- ( ⁇ 5%), CD34- ( ⁇ 7%), CD14- ( ⁇ 7%), CDl lb- ( ⁇ 7%), CD79a- ( ⁇ 7%), CD19- ( ⁇ 5%) and HLA-DR- ( ⁇ 7%) (Table 4), and positive markings for mineralization, hyaline deposition and lipid vacuoles.
  • Table 4 Membrane marker phenotype characterized by flow cytometry of
  • CD90 >95% CD14 ⁇ 7%
  • ASC were also characterized in specific media to assess the mesenchymal differentiation capacity (adipogenesis, chondrogenesis, osteogenesis) (Figure 28). One day after seeding, most ASC appeared round (mean shape factor: 0.93+0.13) on both cellular supports, with a mean of 4.8% surface covering (not significant).
  • the ex vivo safety study revealed no clonal structural chromosomal aberrations on the karyotypes of ASC at passage 1, passage 4, and advanced passages (passages 10, 12, or 16). At all passages, borderline tetrasomies (1.5-5.5%) were detected for at least two tested chromosomes by FISH analysis on interphase cells (cut-off: 4.5%). This technique also revealed a clone with suspected monosomy 7 in 15% of interphase cells at passage 16. These aneuploid cells do not seem to have a proliferative advantage because they are not detected in metaphase cells. Macroscopic and microscopic analyses did not find any local tumor development in explanted tissue from immunocompromized rats (months 1 and 3 after implantation) ( Figure 30).
  • Dermal oxygenation at day 6 was considered as baseline after dermal injury.
  • ASC at the end of the Passage 3th are trypsinized and then seeded on the HACM to obtain the Passage 4th on the collagenic scaffold.
  • the implant was obtained when 90% of the HACM surface was covered with ASC (5.8 x 10 5 cells per cm 2 ).
  • the mononuclear cells were collected from the interface and washed in PBS at 450g for 10 min. The cells were placed in culture flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat- inactivated fetal bovine serum (FBS) and antibiotics (Veriter et al., Biomaterials. 2011; 32:5945-5956).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS heat- inactivated fetal bovine serum
  • antibiotics Veriter et al., Biomaterials. 2011; 32:5945-5956.
  • Adipose tissues (mean, 15 g) were washed three times with NaCl 9%, cut in a Petri dish to remove vessels and fibrous connective tissue, and placed in collagenase (0.075 g; Sigma- Aldrich) reconstituted in Hank's balanced salt solution (with calcium and magnesium ions) in a shaking water bath at 37°C with continuous agitation for 60 min. After digestion, the collagenase was inactivated in DMEM supplemented with 10% heat- inactivated FBS, L-glutamine (2 mM), and antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml). Collected tissue was centrifuged for 10 min at 450g at room temperature.
  • the pellet was then resuspended in proliferation medium (MP) made of DMEM supplemented with 10% FBS and antibiotics (penicillin 100 U/ml and streptomycin 100 mg/ml). After filtration through a 500-mm mesh screen, the tissue was centrifuged for 10 min at 450g at room temperature and then re-suspended in MP media. This initial passage of the primary cells was referred to as passage 0 (PO). After 24 to 48 h of incubation at 37°C in 5% C0 2 , the cultures were washed with PBS and maintained in MP media up to passage 4 (P4) and then differentiated in specific media (Schubert et al., Biomaterials. 2011; 32:8880-8891).
  • MP proliferation medium
  • FBS fetal bovine serum
  • Fluorescence-activated cell sorting (at least 10,000 events were analyzed by flow cytometry with CellquestPro software; FACScan, BD Biosciences, Franklin Lakes, NJ, USA) confirmed the mesenchymal stem cell lineage by revealing a positive shift of mean fluorescence intensity for CD44, CD73, CD90, and CD105 antibodies (BD Pharmigen, BD Biosciences) conjugated with phycoerythrin (PE), whereas CD45 antigen expression was negative.
  • peripheral blood mononucleated cells (negative control) demonstrated positive staining for CD45 and negative staining for CD44, CD73, CD90, and CD 105.
  • ASC and BM-MSC were seeded in 12- well culture plates and incubated in hypoxic chambers (Modular Incubator Chamber MIC- 101; Billups-Rothenberg, Del Mar, CA, USA) following a protocol described previously (Schubert et al., Biomaterials. 2011; 32:8880-8891).
  • Cells were incubated for 72 h at 0.1% and 21% 0 2 levels in relation to a highly hypoxic environment and atmospheric normoxia, respectively. After 72 h in each condition, cell culture supernatants were harvested individually, centrifuged, and stored at -20°C for subsequent growth factor quantification.
  • VEGF, IGF-1, FGF, HGF, and TGF- ⁇ quantification was achieved by enzyme-linked immunosorbent assay (ELISA Quantikine Kit; R&D System, Minneapolis, MN, USA). Samples were not diluted and the supplier's instructions were followed. Optical density of each well was measured by a Multiskan EX Labsystems spectrophotometer (Thermo Scientific, Breda, the Netherlands) set at 450 nm with a correction wavelength set at 690 nm. The growth factor releases were expressed by a ratio between hypoxia and normoxia.
  • MTS MTS cell proliferation assay
  • HACM Human acellular collagen matrix
  • Fascia lata from selected donors were procured according to European and Belgian legislation regarding human body materials after human tissue donor screening based on clinical history, serological tests, and microbiological testing.
  • the human fascia lata tendon was prepared as described by using a process developed by the Tissue Bank of the University Hospital Saint-Luc (Brussels, Belgium) to obtain an HACM containing no chemical residues (acetone/H 2 02) and containing less than 5% of residual moisture. It was sterilized by gamma irradiation at 25,000 Gy (Sterigenics, Fleurus, Belgium). Allografts were then stored at room temperature (Dufrane et al., Biomaterials. 2008, 29: 2237-2248).
  • grafts (cells plus HACM) were attached to a slide using cyanoacrylate glue and examined in the CLSM (Bio-Rad MRC 1024) using xlO air lens and the 488-nm excitation wavelength line from an argon ion laser.
  • Living cells were distinguished by the presence of ubiquitous intracellular esterase activity determined by the enzymatic conversion of the virtually nonfluorescent cell- permeable calcein AM to the intensely fluorescent calcein. Cell proliferation in the plastic well was observed on the same.
  • Anesthesia was induced and maintained by isoflurane (Abbvie, Wavre, Belgium) inhalation to perform a longitudinal abdominal incision to expose the skeletal musculature of the abdominal wall.
  • the skin was closed using a nonresorbable suture to cover the implantation site.
  • Nude rats were killed on postoperative day 30 by intracardiac injection of T61 (Intervet, Boxmeer, the Netherlands) under general anesthesia. Graft explantation was then performed and implants were processed for histomorphometry.
  • Graft integration was macroscopically assessed for inflammatory reaction, graft integration, tissue remodeling, adhesions to internal organs, and hernia occurrence in the full-thickness defect model.
  • Histomorphometry analysis was performed at 4 weeks after implantation to assess angiogenesis and tissue remodeling. Implants were immediately fixed overnight in 4% formaldehyde and paraffin-embedded. Serial sections (5 ⁇ thickness) were mounted on glass and dried for 12 h at 37°C. Hematoxylin and eosin staining and Masson trichrome staining were performed to assess the vascular proliferation and remodeling process. Additionally, muscular recolonization was studied by immunostaining for dystrophin (diluted at 1:450 and revealed by the En Vision anti -rabbit monoclonal antibody; Abeam, Cambridge, UK).
  • Tissue remodeling was histomorphologically quantified (in the model of electrocoagulation) by measuring the distance between the native intact muscle and the implant (HACM or skin for HACM without or with MSC and sham, respectively) after staining with Masson at xl2.5 magnification within a standard micrometer scale. A minimum of five regions of interest (ROIs) were analyzed on each slide. Tissue remodeling was calculated by a ratio between the thickness found with HACM (alone or with MSC) and sham. In the abdominal wall defect model, dystrophin staining was performed to study muscular recolonization of the implant. Vascular density was studied by counting the vessels at x25 magnification within a standard grid representing a surface of 0.16 mm 2 on Masson trichrome slides. A minimum of five ROIs were analyzed on each slide.
  • ROIs regions of interest
  • the one-sample Kolmogorov-Smirnov test and QQ-plots were used to ensure the normal distribution of values. Results were expressed as means+SD unless otherwise mentioned. Statistically significant differences between experimental groups were tested using Student i-test or one-way analysis of variance with a Bonferroni post hoc test. The statistical tests were performed with PASW 18 (SPSS; Westlands Centre, Quarry Bay, Hong Kong). Differences were considered to be significant at p ⁇ 0.05. Results
  • hypoxia in comparison with normoxia, a significantly better survival rate was found for ASC in comparison with BM-MSC with 119.5+6.1% vs. 86.8+1.5% of cellular viability, respectively (p ⁇ 0.05; Figure 39).
  • Significantly higher releases of FGF and VEGF were obtained by AMSC in comparison with BM-MSC at 0.1% and 21% 0 2 (p ⁇ 0.05) ( Figure 40, A and B).
  • hypoxia improved the VEGF release for ASC (+37%; p ⁇ 0.05) without any impact on BM-MSC.
  • HACM decellularization was first confirmed by the detection of rare fluorescent nuclei after DAPI staining on processed matrices in comparison with native matrices (data not shown). No DNA detection was measured by Qubit fluorometer ( ⁇ 0.01 ⁇ g/ml in comparison with a mean of 1.45 ⁇ g/ml found in four independent native fascia lata).
  • the total recovery of the plastic well was obtained within 2 weeks after incubation with both MSC origins, which was delayed on HACM at days 21 and 30 for ASC and BM- MSC, respectively (p ⁇ 0.05).
  • the percentage of HACM recovery area was significantly lower in comparison with that of plastic well recovery between day 3 and day 18 and between day 3 and day 27 for both ASC and BM-MSC, respectively (p ⁇ 0.05).
  • a significant delay of HACM recovery was obtained with BM-MSC in comparison with ASC between days 13 and 28 after incubation (p ⁇ 0.05) ( Figure 42).
  • a composite graft made of ASC demonstrated the capacity of adipose stem cells to survive under ex vivo hypoxia, the capacity to obtain optimal cellular delivery by a decellularized collagen matrix scaffold, the capacity to improve the release of pro- angiogenic factors by an oxygen-sensitive mechanism, the capacity of in vivo vascular recruitment during the early stress phase after transplantation, and, finally, the capacity to reduce the fibrotic scar in comparison with a cell-free scaffold. All these properties could promote skeletal muscle regeneration.
  • HACM is an ideal scaffold for MSC adhesion, spreading, and cell delivery, which remain mechanical requirements for skeletal muscular necrosis and critical size defects.

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