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WO2006110110A1 - Construction de tissus et méthode correspondante - Google Patents

Construction de tissus et méthode correspondante Download PDF

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Publication number
WO2006110110A1
WO2006110110A1 PCT/SG2006/000092 SG2006000092W WO2006110110A1 WO 2006110110 A1 WO2006110110 A1 WO 2006110110A1 SG 2006000092 W SG2006000092 W SG 2006000092W WO 2006110110 A1 WO2006110110 A1 WO 2006110110A1
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WO
WIPO (PCT)
Prior art keywords
agents
tissue
tissue construct
inhibitors
cells
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PCT/SG2006/000092
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English (en)
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WO2006110110A8 (fr
Inventor
Hongwei Ouyang
Cho Hong James Goh
Siew Lok Toh
Tong Earn Tay
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National University Of Singapore
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Publication of WO2006110110A1 publication Critical patent/WO2006110110A1/fr
Publication of WO2006110110A8 publication Critical patent/WO2006110110A8/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin

Definitions

  • the present invention is in the field of tissue engineering.
  • the invention generally relates to tissue constructs and to method(s) for the preparation thereof.
  • tissue constructs may be used as artificial grafts, for example, to replace or supplement natural tissue or parts of organs.
  • the distribution and attachment of cells in sponge or fiber structures are mainly determined by gravity (Sittinger et al, 1996).
  • the cell attachment of fiber structures can be improved by different pre-coating techniques and modifying the porosity and surface charge of the structure, 30 ⁇ 40% of the initially seeded cells do not attach to the structure, thus it will hinder further tissue development (Zund et al, 1999).
  • increasing the cell attachment surface of the structure would decrease the pore size.
  • large pore size is essential for promoting host fibrovascular in-growth into the structure (Mikos et al, 1993).
  • tissue grafts of the current art particularly those to replace connective tissue, include weak cell adhesion to scaffold, lack of mechanical strength, low cell seeding, low mass transport of nutrients and waste products, and lack of in-growth by host cells.
  • the present invention addresses the problems of the art and provides a novel tissue construct and a method thereof.
  • the present invention provides a tissue construct comprising at least one structure and multipotent cells.
  • the multipotent cells are in the form of a sheet before assembly with the at least one structure.
  • the structure is a structure of biocompatible material.
  • the multipotent cells may comprise mesenchymal stem cells.
  • the mesenchymal stem cells may be adult and/or embryonic stem cells.
  • the mesenchymal stem cells may be isolated from autologous and/or allogenic sources.
  • the tissue construct may further comprise at least one therapeutic agent.
  • the at least one therapeutic agent may be selected from the group consisting of angiogenic growth factors, angiogenic inhibitors, growth factors, thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, antimetabolites, antiproliferatives, anticancer chemotherapeutic agents, antiinflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents
  • the structure of the tissue construct may be in the form of a sponge, foam, mesh of fibers, sheet, woven or knitted materials and the structure may comprise natural and/or synthetic materials. Where a synthetic material is used, it may be ceramic material, biosorbable and/or biodegradeable material.
  • the biosorbable material may be poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(glaxanone), poly(orthoesters), poly(pyrolicacid), poly(phosphazenes and/or their derivatives.
  • the tissue construct may comprise at least one natural material such as collagen, gelatin, silkworm silk, spider silk, allogenic fascia tisues, autologous fascia tissues or alimentary canal/intestinal submucosa.
  • the tissue construct may be suitable for use as a graft, particularly for use as a graft for connective tissue or skin.
  • the tissue construct may be used as a graft for ligaments, tendons and/or skin.
  • the tissue construct may also be suitable for use in wound healing, urinary or gall bladder reinforcement patch or replacement, or as a vascular graft.
  • the present invention provides a kit comprising the tissue construct of the present invention.
  • the kit may further comprise packaging and information pertaining to use of the tissue construct.
  • the present invention provides a method of preparing a tissue construct, the method comprising: providing at least one structure; preparing at least one sheet of multipotent cells, and assembling the tissue construct from the at least one structure and at least one sheet of multipotent cells.
  • the structure is a structure of biocompatible material or comprises at least one biocompatible material.
  • the assembling may further comprise stacking, rolling, folding and/or wrapping the tissue construct.
  • the tissue construct may be further cultured after assembly.
  • the multipotent cells of the tissue construct may comprise mesenchymal stem cells and the mesenchymal stem cells may be adult and/or embryonic stem cells.
  • the mesenchymal stem cells may be isolated from autologous and/or allogenic sources.
  • the method may further comprise adding at least one therapeutic agent to the construct.
  • the at least one therapeutic agent may be selected from the group consisting of angiogenic growth factors, angiogenic inhibitors, growth factors, thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, anti-metabolites, antiproliferatives, anticancer chemotherapeutic agents, anti-inflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers,
  • the method may further comprise adding at least one natural material to the construct.
  • the natural material may be collagen, gelatin, silkworm silk, spider silk, allogenic fascia tissue, autologous fascia tissues, and alimentary canal/intestinal submucosa.
  • the structure of the tissue construct prepared by the method of the present invention may be in the form of a sponge, foam, mesh of fibers, sheet, knitted or woven materials and the structure may comprise natural and/or synthetic biocompatible materials. Where synthetic materials are used, they may be ceramic materials, biosorbable and/or biodegradable materials.
  • the biosorbable materials may be poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(glaxanone), poly(orthoesters), poly(pyrolicacid), poly(phosphazenes and/or their derivatives.
  • the tissue construct prepared by the method of the present invention may be suitable for use as a graft, particularly for use as a graft for connective tissue or skin.
  • the tissue construct may be used as a graft for ligaments, tendons and/or skin.
  • the tissue construct may also be suitable for use in wound healing, urinary or gall bladder reinforcement patch or replacement, or as a vascular graft.
  • Figure 1 shows the basic components of the present invention comprising a mesenchymal stem cell sheet and a structure.
  • the structure is a structure of biocompatible material.
  • the cell sheet and the structure in rectangular shape, they may also be in other shapes such as circles or triangles.
  • the tissue construct is prepared by assembling the stem cell sheet and the structure.
  • Figure 2 shows the tissue construct in a rolled form.
  • Figure 3 shows the tissue construct assembled with a single fold.
  • Figure 4 shows the tissue construct assembled with multiple folds.
  • Figure 5 shows the tissue construct assembled by wrapping the stem cell sheet around the structure.
  • Figure 6 shows the stacking of the components to have the structure sandwiched by the stem cell sheets.
  • Figure 7 shows a variation of the stacking of the components to obtain multiple layers of stem cell sheets and structures.
  • Figure 8 shows the histology of the longitudinal sections (a, b) and transverse section (c) of the engineered ligament analog tissue construct after 4 weeks of culture (H&E staining; original magnification, (a) 4OX and (b, c) 100X
  • Figure 9 shows the immunohistology of the engineered ligament analog tissue constructs after 4 weeks of culture showing the tissue constructs consisting primarily of collagen type I (a) and small amount of collage type III (b) and tenascin (c). lmmunohistofluorescent staining.
  • Figure 10 shows histograms comparing the mean ( ⁇ SD) tensile stiffness and maximum force of ligament analog tissue constructs with scaffold alone 4 weeks of culture after assembly.
  • Figure 11 shows macromorphology (a), transmission image (b), and viable cells as indicated by positive CFDA staining under confocal microscopy of bone Marrow Stroma Cell (bMSC) sheet after assembling with knitted PLLA scaffold
  • Sheet - A piece of material and/or collection of cohesive cells that is physically, chemically and/or biologically distinguishable from another piece of material and/or collection of cells.
  • a sheet may comprise more than one type of cells and the cells may be in more than one layer of cells and/or material.
  • Structure - A support, scaffold or substrate capable of supporting cells.
  • the structure may be in two-dimensional form such as a sheet or it may be in three- dimensional form and possess porosity by being sponge-like, in the form of foam or having a meshwork formed by fibers or it may be made of a knitted or woven material.
  • the structure may be a structure of biocompatible material.
  • Additive Any material that is added to the structure.
  • additives include hydroxyapatite or tricalcium phosphate to promote bone growth, and collagen and gelatin to encourage cell adhesion and/or growth.
  • Grafting The transplanting of a natural or artificial tissue (graft) from one place to another, for example, a piece of skin graft from a donor site to a recipient site.
  • the transplanting of tissue can be from one part of the patient to another (autologous graft), as in the case of a skin graft using the patient's own skin; or from one patient to another (allogenic graft), as in the case of transplanting a donor kidney into a recipient.
  • Biosorbable, biodegradable - A material is said to be biosorbable if it can be readily broken down and absorbed by the body over a period of time. This is in contrast to implanted biodegradable materials that break down in the body over time but are not necessarily absorbed by the body.
  • biosorbable may also be written as “bioresorabable” or “bioabsorbable”.
  • Biocompatible material Any material that will not elicit an adverse reaction in living cells or tissues such as an immunological or inflammatory response.
  • Therapeutic agent Any molecule, drug or compound that exerts a desired and/or beneficial effect.
  • Assemble - To place different components together to form an assembly or a structure.
  • the act of assembling may entail further working (stacking, rolling, folding or wrapping) the components of the construct to assume a desired shape.
  • fibers in this application refer to nanofibers.
  • a nanofiber structure or structure is a product comprising nanofibers.
  • the structure can further comprise cells and therapeutic agents and can be used as a structure and/or graft.
  • Natural materials - Materials derived from natural, non-synthetic sources such as collagen, gelatin, silk from silkworm or spider, allogenic or autologous fascia tissues, and alimentary canal/intestinal submucosa.
  • the present invention provides a tissue construct and method thereof.
  • the tissue construct comprises a sheet of cells and a structure such as a tissue engineered structure and may be useful as a graft. In particular, as a graft to supplement or replace connective tissue.
  • the structure may be a structure of biocompatible material.
  • MSCs mesenchymal stem cells
  • bMSCs bone marrow stromal cells
  • Multipotent cells may also be referred to pluripotent cells. They can differentiate into bone, cartilage, tendon, fat, and are able to grow well on various structures (Ouyang et al, 2002; Ouyang et al, 2003; Goh et al, 2003). Unlike other cells that display contact inhibition, the present inventors have discovered that these multipotent cells could surprisingly form cell sheets of one or more layers of cells and may thus be used in tissue engineered constructs.
  • the present invention aims to: to deliver the cells into structure efficiently and in a controlled manner; lessen the mass of structure and frees the design of structure from the concern of effective cell seeding; provide a large number of progenitor cells for connective tissue regeneration; preserve the matrix produced and the original cell-cell/cell-matrix interaction, thus accelerating the tissue formation after cell delivery; facilitate mass transport of nutrients and waste matter following construct assembly due to the interstitial space between cell layers; facilitate the vascularization of engineered tissue following implantation taking advantage of the interstitial space between cell layers.
  • VEGF vascular endothelial growth factor
  • a tissue construct comprising at least one structure and multipotent cells wherein the multipotent cells are in the form of a sheet before assembly with the at least one structure.
  • the structure is a structure of biocompatible material.
  • the multipotent cells may comprise mesenchymal stem cells and the the mesenchymal stem cells may be adult or embryonic stem cells.
  • the mesenchymal stem cells may be isolated from autologous and/or allogenic sources.
  • tissue construct of the present invention there is provided a method comprising providing at least one structure; preparing at least one sheet of multipotent cells, and assembling the tissue construct from the at least one structure and at least one sheet of multipotent cells.
  • the assembling may further comprise stacking, rolling, folding and/or wrapping the tissue construct (FIGS. 3-7).
  • the tissue construct may be further cultured after assembly.
  • the multipotent cells of the tissue construct may comprise mesenchymal stem cells and the mesenchymal stem cells may be adult and/or embryonic stem cells.
  • the mesenchymal stem cells may be isolated from autologous and/or allogenic sources.
  • the tissue construct according to the invention may be suitable for use as a graft, in particular as a connective tissue graft. More in particular, the present invention provides a three-dimensional tissue graft mimicking the host connective tissue such as ligaments, tendons, blood vessels and muscle fascia that the graft is meant to replace or supplement.
  • the tissue construct may also be suitable for use as a skin graft, in wound healing, or as a urinary or gall bladder reinforcement patch.
  • Natural organs and body structures usually possess layers of cells and tissue. Each layer is usually distinct from another, such as the different muscle layers of a limb and the tendon connecting the muscle to a bone. While each layer may have a predominant type of cell, each layer may also comprise other types of cells.
  • the present invention comprises at least one structure and at least one sheet of multipotent cells.
  • the construct of the invention may comprise at least one structure of one or more biocompatible materials.
  • the biocompatible material or cells may be provided, for example, in the form of a layer of biocompatible material and/or a sheet of cells.
  • the sheet of cell may have one or more layers of cells. The number of layers of biocompatible materials and/or sheets of cells depends on the application of the graft.
  • the present invention relates to a tissue construct for tendon and/or ligament repair. In another example, the present invention relates to a graft mimicking dermal or skin grafts
  • Suitable biocompatible and/or biosorbable polymer materials for use in tissue construct according to the present invention include, but are not limited to, synthetic polymers, such as poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL) and their co-polymers, poly(glaxanone), poly(orthoesters), poly(pyrolicacid) and poly(phosphazenes).
  • Other additives that can be incorporated into these materials include, but are not limited to, calcium phosphate based ceramics such as hydroxyapatite and tricalcium phosphateand natural polymers, such as collagen and gelatin. It is preferable but not necessary that the biodegradable and/or biosorbable materials be approved by food and drug authorities of specific countries for use in surgical applications.
  • the structure may be prepared by electrospinning of nanofibers.
  • the structure of the present invention may further comprise one or more additional sheets of a biodegradable and/or biosorbable (biodegradable/biosorbable) material formed by solution casting to confer other desirable physical, chemical and/or physiological characteristics to the nanofiber structure.
  • a biodegradable and/or biosorbable material formed by solution casting to confer other desirable physical, chemical and/or physiological characteristics to the nanofiber structure.
  • An example of such sheets is a solution cast sheet of PCL less than 100 ⁇ m thick, that is structurally homogenous and do not possess any nanofibrillary characteristics. Solution casting of such sheets is known in the art. For example, PCL is dissolved in a suitable solvent at low concentration and poured on top a substantially flat casting tray. After the sheet has formed, residual solvent is removed in a vacuum oven.
  • Such sheets may be further treated by addition of one or more therapeutic agents before or after assembly with the sheet of mesenchymal stem cells.
  • the mesenchymal stem cells may be obtained directly from a suitable donor, e.g. a patient's own cells; from a culture of cells from a donor; or from established cell culture lines. Using standard cell culture techniques and conditions well-known to a person skilled in the art, the cells are grown in culture until confluent and used when needed.
  • graft healing is important in determining the success or failure of a graft. All grafts, regardless of their composition and structure, evoke complex but predictable host responses. Blood-biomaterial surface reactions start immediately after restoration of circulation. The cellular and humoral responses to synthetic materials include the deposition of plasma proteins and platelets, the infiltration of neutrophils and monocytes, and the diffentiation, migration and proliferation of the mesenchymal stem cells, will determine graft failure and potential graft infection.
  • Angiogenesis is the sprouting of new capillaries from preexisting vasculature and represents a complex multistep process involving extensive interplay between cells, soluble factors, and ECM components (Steffens et al., 2004).
  • One of the prominent shortcomings of structures for tissue engineering is their poor ability to support angiogenesis. For this reason, the structure of the present invention may be enhanced by incorporating therapeutic agents such as angiogenic growth factors, peptides or proteins.
  • Angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor-1 (FGF-1), and fibroblast growth factor-2 (FGF-2), and angiogenic inhibitors such as platelet factor 4, interferons, and thrombospondin, are required to create a microenvironment in which angiogenesis is a function of balance between positive and negative regulators. Exogenous intervention such as controlled local delivery of exogenous angiogenic factors may change these balances so as to induce transmural capillary in-growth in vivo and stimulate endothelialization of prosthetic grafts.
  • VEGF vascular endothelial growth factor
  • FGF-1 fibroblast growth factor-1
  • FGF-2 fibroblast growth factor-2
  • angiogenic inhibitors such as platelet factor 4, interferons, and thrombospondin
  • Therapeutic agents can be incorporated into the structure by physically coating, chemically covalent-grafting or an advanced coaxial electrospinning technique (Zhang et al., 2004).
  • a coaxial electrospinning process two immiscible liquid solutions are injected at appropriate flow rates through two concentrically arranged capillary tubes.
  • a structured Taylor cone is formed at the exit of the coaxial tubes with an outer meniscus surrounding the inner one.
  • a liquid thread is issued from the vertex of each one of the two menisci, giving rise to a compound jet. After evaporation of the solvents during the course of jet flying, a "core-shell structured" bi-component composite nanofiber is produced.
  • Such core-shell structured nanofibers may be used to encapsulate one or more therapeutic agents by using an appropriated biodegradable polymer as a shell so that the encapsulated biomolecules may be released when the shell material degrades over time. This method provides longer term angiogenic support compared to simply coating the growth factors on the nanofibers.
  • other therapeutic agents may be used in the coating.
  • other therapeutic agents include thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, anti-metabolites, antiproliferatives, anticancer chemotherapeutic agents, anti-inflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents, photo
  • the tissue construct of the present invention may comprise natural materials such as collagen, gelatin, silk from silkworm or spider, allogenic or autologous fascia tissues, and alimentary canal/intestinal submucosa to confer certain desirable characteristics such as provision of a hospital environment for the graft or host cells.
  • Bone marrow was aspirated from the iliac crest of a 30-kg Yorkshire pig and collected into polypropylene tubes containing preservative-free heparin (1000
  • the knitted scaffold was fabricated using 3 yarns (20 filaments/yarn; diameter of filament is 25 ⁇ m) of PLLA fibers (Albany International Research Co.) on a knitting machine (Silver-reed SK270, Suzhou, China).
  • the plain knitted PLLA scaffolds were manufactured with 6 stitches per centimeter.
  • the pore size was 2 mm x 2 mm.
  • the length of PLLA sheet scaffold was standardized to 30 mm x 50 mm.
  • bMSCs were harvested, they were cultured in low glucose DMEM with 10% FBS and became confluent within 5 to 7 days. After confluence, bMSCs were cultured in high glucose DMEM with 20% FBS and 50 ⁇ g/mL ascorbic acid. Within 2 to 3 weeks a bMSC sheet was formed in a 200 cm 2 dish. It could be detached from the substratum by applying a small force. Then a 50-mm length of knitted scaffold was put on the cell sheet as shown in FIG. 1 and assembled by a rolling/wrapping technique to obtain the tissue construct shown in FIG. 2. The assembled cell-scaffold structure was then held in place in a 52-mm length frame device with 2 mm (4%) strain. Then the whole structure was put in a spinner flask with the speed of 6 rpm for 4 weeks and the ligament analog tissue construct thus prepared may be implanted into a recipient.
  • the PLLA ligament analog tissue constructs thus prepared and PLLA scaffolds without cells incubated with cell culture medium at the same condition were used.
  • CFDA molecule probe (5, 6-carboxyfluorescein diacetate) is well recognized as cell trace markers. Therefore, in this study it was used to investigate the survivability of bMSCs within the cell sheet after assembly.
  • the composite graft of bMSC sheet/knitted scaffold tissue construct was incubated for 20 min with 25 ⁇ M CFDA solution, and then changed with culture medium. The positive cells were observed under fluorescent microscopy on the second day. Histological and lmmunohistoflurorescent Examination
  • ligament analog tissue constructs were fixed in 4% buffered formaldehyde solution for histological and immunohistoflurorescent examination. They were then dehydrated through alcohol gradients, cleared, and embedded in paraffin blocks. Histological sections (10 ⁇ m) were prepared from these specimens using a microtome (Leica Instruments, Bensheim, Germany) and subsequently stained with hematoxylin-eosin (H&E). In addition, the streptavidin-biotin method was used to evaluate the different types of collagen in the regenerated tissue.
  • Histological sections were labeled with primary monoclonal antibodies (anticollagen type I, type III, and tenascin) at a 1 :200 dilution and left overnight at room temperature (ICN Biochemicals, Aurora, OH). Subsequently, fluorescence conjunct antimice secondary antibodies were administered at a 1 :100 dilution for 1 h.
  • primary monoclonal antibodies anticollagen type I, type III, and tenascin
  • the final cycle was checked for steady-state consistency and taken as the representative load displacement curve for the specimen.
  • the cyclic load of 12 N was chosen to keep the loading within the cell-scaffold tissue construct's elastic limit which was decided according to the pilot experiments of two specimens.
  • the test speed of 10 mm/min was chosen on the basis of earlier trial tests conducted on tendon samples, which showed consistency in the loading patterns.
  • the load-versus-elongation curves obtained for each specimen were compared to those reported in previous studies.
  • the tensile stiffness value was calculated from the linear region of the load- elongation curve.
  • FIG. 8(a,b) Histology of the longitudinal sections FIG.8(a,b) showed formation of fibrous matrix bundles with a large number of cells aligned in the matrix.
  • the transverse section FIG. 8(c) showed a large number of cells present in connective tissue that filled and wrapped the scaffold.
  • FIG. 9 shows the results of the immunohistochemical analysis which indicates that the matrix of neoligament tissues in ligament analog tissue constructs was composed primarily of collagen type I and small amount of collagen type III and tenascin.
  • This ligament analog tissue construct is completely composed of the cells and their synthesized matrix. It possesses more tissues as compared to the previously established in vitro ligament analogs which is composite of collagen gel and separated cells. (Altman et al, 2002).
  • the matrix components of the analogs include collagen type I and type III which are the major matrix components of the natural ligament and tenascin, one of the early marker for embryonic ligament. (Altman et al, 2002; Frank et al, 1988 and Amiel et al, 1984).
  • the mean tensile stiffness of ligament analog tissue construct group was significantly lower than that of scaffold group (p ⁇ 0.05).
  • the failure force of the ligament analog tissue construct group and the scaffold group were 46.68 ⁇ 2.29 N and 43.58 ⁇ 2.41 N, respectively.
  • the failure force of the ligament analog tissue construct group was higher than that of scaffold group (p ⁇ 0.05; FIG. 10). However, the failure force of the ligament analog tissue construct was lower than that of original scaffolds (51.45 ⁇ 3.2 N) without significance (p ⁇ 0.05).
  • the stiffness of the original scaffold was 28.4 ⁇ 1.812 N/mm which was not higher than that of scaffold group (p > 0.05).
  • the tissue construct of the present invention had an unexpected result: the bMSC sheet made the ligament analog tissue constructs stronger with a higher failure load with less stiffness.
  • the bone-marrow-derived adherent cells exhibited fibroblastlike colony-unit formation (CFU-F).
  • CFU-F fibroblastlike colony-unit formation
  • the bMSCs proliferated fast and formed cell sheet within 2 weeks after confluence in the presence of ascorbic acid.
  • the cell sheets could be detached from culture substratum, thus creating a sheet of cohesive living cells in a collagen matrix of endogenous origin.
  • the cell sheet was assembled onto the macroporous knitted PLLA scaffold, the cell sheets adhered to scaffold after several hours of culture as exhibited by the lack of flotation of cell sheet in the medium.
  • the cells within cell sheet were still alive after assembly as indicated by positive CFDA staining under confocal microscopy (FIG. 11).
  • Figure 11 shows the macromorphology (a), transmission image (b), and viable cells as indicated by positive CFDA staining under confocal microscopy of a bMSC sheet after assembling with knitted PLLA scaffold (c).
  • a tissue construct thus prepared may be used for dermal or skin grafts.
  • the method of the present invention may be characterized by providing at least one structure and preparing at least one sheet of mesenchymal stem cells and assembling the structure and sheet of cells to form a tissue construct.
  • the cells are then cultured and permitted to grow further on the structure for a period of time until desired characteristics are obtained.
  • the tissue constructs may then be arranged in a desired order for use as a graft mimicking the tissue to be supplemented or replaced.
  • the sheets can be arranged by contacting the sheets such that a sheet substantially covers another sheet below it, for example, by stacking the sheets one on another. Alternatively, the sheets can be arranged wherein a sheet only partially covers another sheet below it.
  • the constructs may be rolled around a mandrel to form a tube and further cultured for the cells to grow before being used as a graft.
  • the constructs may be staggered such that when rolled on the mandrel, the edges or seams of the layers do not coincide but are covered by at least one layer above them. This will allow cells to better proliferate over the edges and provides a stronger joining of the seams. While the use of a form such as a mandrel is taught, a person skilled in the art will appreciate that, depending on the graft prepared or fabricated, that a form such as a mandrel may not be necessary to roll the arranged sheets.
  • constructs in the examples were rectangular for ease of understanding the invention, the constructs fabricated under the present invention are not restricted to rectangular shapes.
  • the constructs may be cut into triangles and cones may be rolled from them as dictated by the application. Further square or rectangular constructs may be rolled alone their diagonals to form a spindle-like bundle wherein the middle of the rolled sheet is thicker and the ends thinner.
  • the invention may also be used to produce grafts wherein structures may also be rolled or twisted to form cords before being wrapped with at least one sheet of cells. Again, sheets of cells may be added at any stage of the process as needed to obtain a desired outcome. While the examples given above teach rolling of the constructs to form a tubular structure, a person skilled in the art will recognize that the structure of the present invention need not be rolled for grafting into muscle walls, or linings of other body parts, to replace or to reinforce these walls, linings or parts.
  • the graft of the present invention may be supplied as a kit comprising the construct, packaging and information and optionally instructions and/or illustrations pertaining to the use of the construct.

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  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Transplantation (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Developmental Biology & Embryology (AREA)
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  • Reproductive Health (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L’invention concerne une construction de tissus qui comprend au moins une structure et des cellules multipotentes et une méthode pour la préparation de la construction de tissus. Les cellules multipotentes peuvent être sous forme d'une feuille, avant l’assemblage avec ladite au moins une structure. La construction de tissus peut être utilisée comme greffon, en particulier comme greffon de tissu conjonctif.
PCT/SG2006/000092 2005-04-11 2006-04-10 Construction de tissus et méthode correspondante WO2006110110A1 (fr)

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US20110293685A1 (en) * 2008-10-03 2011-12-01 Trustees Of Tufts College Scaffolds for tissue engineering and regenerative medicine
US20120189587A1 (en) * 2011-01-26 2012-07-26 The Chinese University Of Hong Kong Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same
CN104436299A (zh) * 2014-11-11 2015-03-25 深圳市第二人民医院 一种三明治样多细胞片层的制备方法
CN106693059A (zh) * 2016-12-28 2017-05-24 广州迈普再生医学科技有限公司 复合组织修复补片及其制备方法和应用
CN109943519A (zh) * 2016-09-14 2019-06-28 四川蓝光英诺生物科技股份有限公司 人工组织前体及制备其的方法
CN111467576A (zh) * 2020-04-29 2020-07-31 上海市东方医院（同济大学附属东方医院） 仿真胆囊壁合成材料及其制备方法和应用以及仿真胆囊壁
CN112004922A (zh) * 2018-04-25 2020-11-27 北海道公立大学法人札幌医科大学 生物移植用细胞片及其制造方法

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110293685A1 (en) * 2008-10-03 2011-12-01 Trustees Of Tufts College Scaffolds for tissue engineering and regenerative medicine
US20120189587A1 (en) * 2011-01-26 2012-07-26 The Chinese University Of Hong Kong Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same
CN102614546A (zh) * 2011-01-26 2012-08-01 香港中文大学 用于组织修复和生物人造组织工程的细胞片及其制备方法
US8945536B2 (en) * 2011-01-26 2015-02-03 The Chinese University Of Hong Kong Stem cell sheet for tissue repair
CN104436299A (zh) * 2014-11-11 2015-03-25 深圳市第二人民医院 一种三明治样多细胞片层的制备方法
CN109943519A (zh) * 2016-09-14 2019-06-28 四川蓝光英诺生物科技股份有限公司 人工组织前体及制备其的方法
CN109943518A (zh) * 2016-09-14 2019-06-28 四川蓝光英诺生物科技股份有限公司 人工组织前体及制备其的方法
CN106693059A (zh) * 2016-12-28 2017-05-24 广州迈普再生医学科技有限公司 复合组织修复补片及其制备方法和应用
CN106693059B (zh) * 2016-12-28 2020-07-14 广州迈普再生医学科技股份有限公司 复合组织修复补片及其制备方法和应用
CN112004922A (zh) * 2018-04-25 2020-11-27 北海道公立大学法人札幌医科大学 生物移植用细胞片及其制造方法
TWI821280B (zh) * 2018-04-25 2023-11-11 北海道公立大學法人札幌醫科大學 生物體移植用細胞片及其製造方法
US12090251B2 (en) 2018-04-25 2024-09-17 Sapporo Medical University Cell sheet for transplantation into living body and method for producing same
CN111467576A (zh) * 2020-04-29 2020-07-31 上海市东方医院（同济大学附属东方医院） 仿真胆囊壁合成材料及其制备方法和应用以及仿真胆囊壁

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