WO2007136936A2 - Cellules dérivées du derme pour des applications de génie histologique - Google Patents

Cellules dérivées du derme pour des applications de génie histologique Download PDF

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Publication number
WO2007136936A2
WO2007136936A2 PCT/US2007/066085 US2007066085W WO2007136936A2 WO 2007136936 A2 WO2007136936 A2 WO 2007136936A2 US 2007066085 W US2007066085 W US 2007066085W WO 2007136936 A2 WO2007136936 A2 WO 2007136936A2
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Prior art keywords
cells
aggrecan
asid
tissue
coated
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WO2007136936A3 (fr
Inventor
Kyriacos A. Athanasiou
Ying Deng
Jerry Hu
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William Marsh Rice University
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William Marsh Rice University
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Priority to AU2007254048A priority Critical patent/AU2007254048A1/en
Priority to EP07760205A priority patent/EP2007875A4/fr
Priority to CA002648648A priority patent/CA2648648A1/fr
Publication of WO2007136936A2 publication Critical patent/WO2007136936A2/fr
Anticipated expiration legal-status Critical
Priority to US12/246,367 priority patent/US20090142307A1/en
Priority to US12/246,320 priority patent/US8637065B2/en
Priority to US12/246,306 priority patent/US20090136559A1/en
Publication of WO2007136936A3 publication Critical patent/WO2007136936A3/fr
Priority to US13/029,325 priority patent/US20110212894A1/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
    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • 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
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • 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
    • A61L27/36—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
    • A61L27/3886—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 comprising two or more cell types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/76—Agarose, agar-agar

Definitions

  • sequence listing.txt created on March 15, 2007, with a size of 2,809 bytes, which is incorporated herein by reference.
  • the attached sequence descriptions and Sequence Listing comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.821-1.825.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUP AC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984).
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.822.
  • Tissue engineering is an area of intense effort today in the field of biomedical sciences. The development of methods of tissue engineering and replacement is of particular importance in tissues that are unable to heal or repair themselves, such as articular cartilage.
  • Articular cartilage is a unique avascular, aneural and alymphatic load-bearing live tissue, which is supported by the underlying subchondral bone plate. Articular cartilage damage is common and does not normally self-repair. Challenges related to the cellular component of an engineered tissue include cell sourcing, as well as expansion and differentiation.
  • Embryonic stem (ES) cells represent a valuable source for this purpose.
  • the application of ES cells in this area is still limited particularly because of ethical considerations.
  • a number of researchers have investigated various adult tissues including bone marrow, muscle, and adipose tissue as alternative cell sources for cartilage tissue engineering.
  • autologous procurement of these tissues has potential limitations.
  • Skin is the largest organ in the body and is relatively easily accessible with minimal insult to the donor.
  • the skin dermis is considered, therefore, one of the best autologous source organs to isolate stem/progenitor cells for future therapeutic applications not only in the replacement of skin, but also as an alternative cell source for several other organs outside of skin.
  • Recently accumulating evidence indicates that skin dermis contains cells that can generate multiple lineages including neurons, glia, smooth muscle cells and adipocytes.
  • cells from the skin dermis may prove to be a useful alternative cell source for articular cartilage tissue engineering.
  • human dermal fibroblasts cultured with demineralized bone powder acquire a chondroblast phenotype and express cartilage-specific matrix proteins.
  • tissue engineered construct refers to a three-dimensional mass having length, width, and thickness, and which comprises living mammalian tissue produced in vitro.
  • the present disclosure provides a modified rapid adhering process that involves isolating aggrecan sensitive isolated dermis (ASID) cells for chondrogenic differentiation and allowing differentiated cells to self-assemble into a tissue engineering construct.
  • ASID aggrecan sensitive isolated dermis
  • Dermis derived cells are attractive since they provide autologous cells without causing complications at the donor site, due to the high regenerative capacity of skin. These cells can also be harvested with a low degree of invasiveness.
  • the methods of the present disclosure are advantageous in preparing autologous cells to be transplanted to any patient for whom repair of damaged tissues by regeneration therapy will be needed.
  • ASID cells can be obtained with a low degree of invasiveness and without causing complications at the donor site due, to their high regenerative capacity.
  • the methods of the present disclosure also provide therapeutic strategy that uses the self-assembly of chondroinduced ASID cells to produce tissue in vitro for use as an autologous transplant in vivo.
  • Tissue engineered constructs formed by ASID cells may exhibit cartilage specific
  • the methods of the present disclosure provide substantially homogeneous tissue engineered constructs.
  • the methods of the present disclosure may reduce the likelihood of heterogeneous cell subpopulations spontaneously differentiating into divergent lineages and, in the case of fibroblasts, decreases the risk of fibrochondrocytic formation.
  • FIGURE 1 shows a photomicrograph image of fibroblasts grown on 2.5 ⁇ g/cm 2 aggrecan-coated TCP surface.
  • A Edge of the well (original magnification)
  • B Center of the well (original magnification 4 ⁇ ).
  • FIGURE 2 shows a photomicrograph image of eosin stained aggrecan-coated TCP surface.
  • A Schematic representation of a well. Panels B, C, D, and E show the center of the well. Panels F, G, H, and I show the edge of the well.
  • B, F control;
  • C, G 2.5 ⁇ g/cm 2 ;
  • D, H 5 ⁇ g/cm 2 ;
  • E, I 1 O ⁇ g/cm 2 .
  • FIGURE 3 shows a photomicrograph image of the morphology of aggrecan sensitive isolated dermis (ASID) cells and normal fibroblasts grown on a tissue culture treated polystyrene after 7 Days of culture.
  • ASID aggrecan sensitive isolated dermis
  • A ASID
  • B Fibroblasts.
  • FIGURE 4 is a graph of the effect of different aggrecan concentrations on the expression of collagen type I and II in ASID cells.
  • A Collagen type I
  • B Collagen type II.
  • FIGURE 5 is a photomicrograph image showing aggrecan induced morphological changes in chondrocytes, ASID cells and fibroblasts after 1 day in culture.
  • A chondrocytes with aggrecan
  • B chondrocytes without aggrecan
  • C ASID cells with aggrecan
  • D ASID cells without aggrecan
  • E fibroblasts with aggrecan
  • F fibroblasts without aggrecan.
  • FIGURE 6 is a photomicrograph image showing the detection of extracellular matrix of cartilage in ASID cells after 1 day in culture.
  • A, B Safranin-0 stain for proteoglycans
  • C, D Immunohistological stain for collagen type II protein
  • A, C Aggrecan treated surface
  • B Without aggrecan treated surface.
  • FIGURE 7 is a graph of the effect of aggrecan coated surfaces on aggrecan expression of ASID cells as a function of time in culture.
  • FIGURE 8 are graphs of the effect of aggrecan coated surfaces on collagen type I and type II expression of ASID cells cultured for a period of 2 weeks.
  • A) Collagen type I expression B) Collagen type II expression and C) Ratio of Collagen type II to collagen type I
  • FIGURE 9 shows fluorescent images illustrating organization of vinculin and F-actin in chondrocytes, ASID cells and fibroblasts after 36 hrs. Vinculin was stained with Alexa 488 (green), F-actin was stained with rhodamine phalloidin (red), Nucleus was stained with DAPI (blue). (A, B, C, D, E, F) vinculin, (a, b, c, d, e, f) F-actin, Original magnification, 63 x.
  • FIGURE 10 is a graph of the collagen type I and II expression of ASID cells cultured on tissue culture treated and non-tissue culture treated polystyrene, with or without aggrecan over a period of 14 days.
  • FIGURE 11 is a graph of the effect of aggrecan on aggrecan expression of ASID cells cultured on tissue culture and non-tissue culture treated polystyrene coated with or without aggrecan.
  • FIGURE 12 is a photomicrograph image of the detection of proteoglycans in ASID cells cultured in normal medium and chondrogenic medium at day 1.
  • A ASID cells cultured on non-tissue culture treated polystyrene with normal medium;
  • B ASID cells cultured on non-tissue culture treated polystyrene with chondrogenic medium;
  • C Fibroblasts cultured on non-tissue culture treated polystyrene with normal medium;
  • D Fibroblasts cultured on non- tissue culture treated polystyrene with chondrogenic medium;
  • E ASID cells cultured on aggrecan-coated non-tissue culture treated polystyrene with normal medium;
  • F ASID cells cultured on aggrecan-coated non-tissue culture treated polystyrene with chondrogenic medium;
  • G Fibroblasts cultured on aggrecan-coated non-tissue culture treated polystyrene with normal medium;
  • H Fibroblasts cultured on aggre
  • FIGURE 13 is a photomicrograph image of the detection of proteoglycans in ASID cells cultured in aggrecan-coated non-tissue culture treated polystyrene wells with normal medium and chondrogenic medium over a period of 14 days.
  • A Normal medium at day 1;
  • B Normal medium at day 7;
  • C Normal medium at day 14;
  • D Chondrogenic medium at day 1;
  • E Chondrogenic medium at day 7;
  • F Chondrogenic medium at day 14;
  • Original magnification 10x.
  • FIGURE 14 is a photomicrograph of the detection of type II collagen in ASID cells cultured on aggrecan-coated non-tissue culture treated polystyrene wells with normal medium and chondrogenic medium over a period ofl4 days.
  • A Normal medium at day 1;
  • B Normal medium at day 7;
  • C Normal medium at day 14;
  • D Chondrogenic medium at day 1;
  • E Chondrogenic medium at day 7;
  • F Chondrogenic medium at day 14;
  • Original magnification 1Ox.
  • FIGURE 15 is a graph of the effect of aggrecan on collagen type I gene expression of ASID cells and fibroblasts grown on non-tissue culture treated polystyrene with or without aggrecan coating over a period of 14 days.
  • FIGURE 16 is a graph of the effect of aggrecan on cartilage oligomeric protein gene expression of ASID cells and fibroblasts grown on non-tissue culture treated polystyrene with or without aggrecan coating over a period of 14 days.
  • FIGURE 17 is a graph of the effect of aggrecan on aggrecan abundance (A) and aggrecan gene expression (B) of ASID cells and fibroblasts grown on non-tissue culture treated polystyrene with or without aggrecan coating over a period of 14 days.
  • FIGURE 18 is a graph of the detection of cartilage matrix protein collagen type II in ASID cells and fibroblasts cultured on non-tissue culture treated polystyrene with or without aggrecan coating at day 1, 7 and 14.
  • FIGURE 19 is a photomicrograph image of oil red stain for differentiated ASID cells after four weeks of culture.
  • FIGURE 20 is a photomicrograph image of constructs formed using self-assembly of
  • Fibroblasts grown in an agarose well for 14 days (C) Construct formed by fibroblasts after culture for 14 days. (D) ASID cells grown in an agarose well for 1 day. (E) ASID cells grown in an agarose well for 14 days. (F) Construct formed by ASID cells after culture for 14 days.
  • FIGURE 21 is a photomicrograph image showing the detection of extracellular matrix of cartilage in constructs formed by ASID cells and fibroblasts.
  • A, B, C Fibroblasts;
  • D, E, F ASID cells;
  • A, D Collagen type I stain;
  • B, E Collagen type II stain;
  • FIGURE 22 is a photomicrograph image of constructs formed using self-assembly of ASID cells and fibroblast cells after culture on aggrecan-coated non-tissue culture treated polystyrene for a period of 14 days.
  • FIGURE 23 is a photomicrograph image showing the detection of cartilage specific extracellular matrix in constructs self-assembled by (A) chondrocytes, (B) ASID cells, and (C) fibroblasts. All were cultured on aggrecan-coated non-tissue culture treated polystyrene for 14 days.
  • FIGURE 24 shows detection of cartilage-specific extracellular matrix ASID cells cultured for 1-14 days on aggrecan-coated surfaces.
  • Safranin-O all nodules stained positively for glycosaminoglycans (GAGs) (A-C).
  • GAGs glycosaminoglycans
  • D-F type II collagen
  • FIGURE 25 shows expression and synthesis of cartilage specific markers in ASID cells compared with fibroblasts.
  • Reverse transcriptase-polymerase chain reaction results showed significant inhibition of type I collagen (Col I) gene expression for 1-7 days in both cell populations (A).
  • Enzyme linked immunosorbent assay showed that aggrecan coating of surfaces resulted in higher levels of type II collagen in ASID cell cultures than in fibroblast cultures (D) at every time point tested.
  • FIGURE 26 shows reorganization of filamentous actin (F-actin) and vinculin in chondrocytes, ASID cells, and fibroblasts after 36 hours of monolayer culture on aggrecan- coated surfaces.
  • F-actin was stained with rhodamine and phalloidin (red) (A-C). Vinculin was stained with Alexa Fluor 488 (green) (D-F). Nuclei were stained with 4', 6 diamidino-2- phenylindole (blue) (G-I).
  • a punctated distribution of F-actin was seen at the periphery of chondrocytes (A) and ASID cells (B), while a dense collection of F-actin was seen throughout the fibroblasts (C).
  • FIGURE 27 shows detection of cartilage specific extracellular matrix (ECM) in constructs self-assembled for 2 weeks using chondrocytes, ASID cells, and floating ASID (F- ASID) cells.
  • ECM extracellular matrix
  • GAGs glycosaminoglycans
  • Col II type II collagen
  • D chondroitin 4-sulfate
  • G chondroitin 6-sulfate
  • M type I collagen
  • Spherical chondrocytes were noted within a matrix containing GAGs, type II collagen, chondroitin 4-sulfate, and chondroitin 6-sulfate, indicative of cartilage formation.
  • ASID constructs also stained positively for the same cartilage specific ECM (B, E, H, and K). Type I collagen was not observed within chondrocyte or ASID constructs (M and N).
  • the methods of the present disclosure generally comprise providing aggrecan sensitive isolated dermis cells and seeding the cells onto an aggrecan coated surface.
  • aggrecan sensitive isolated dermis cells or “ASID cells” as used herein refers to any plastic rapidly adhering subpopulation of skin cells that are capable of chondrogenic differentiation when cultured on aggrecan.
  • chondrogenic differentiation refers to any process that would result in cells that produce glycosaminoglycans and collagen type II.
  • construct or "tissue engineered construct” as used herein refers to a three-dimensional mass having length, width, and thickness, and which comprises living mammalian tissue produced in vitro.
  • ASID cells used in conjunction with the methods of the present disclosure are fibroblastic cells.
  • ASID cells are a subpopulation of dermis derived fibroblastic cells that may be characterized by their fast attachment to the bottom surface of a culture flask and have the potential for chondrogenic differentiation when seeded on aggrecan-coated surfaces.
  • Aggrecan has been found to play an essential role in the chondrogenesis process and the subsequent maintenance of the chondroncyte phenotype in vivo.
  • Seeded or chondroinduced ASID cells are phenotypically, morphologically, and functionally similar to chondrocytes.
  • ASID may be derived from the dermis layer of the skin using methods known in the art.
  • the cells are generally derived from an autologous source so as to avoid biocompatibility issues. After isolation of the cells from the source, the cells may be cultured to form a homogenous culture of cells.
  • homogenous cultured ASID cells may be seeded on aggrecan coated surfaces (ACS).
  • the aggrecan may be coated on the ACS at a concentration of 10 ⁇ g/cm of well surface.
  • 2x10 cells in culture medium may be seeded per well in 24 well plates coated with aggrecan (bottom well area approximately 2 cm 2 ).
  • the cells may be cultured on the ACS for a period of about seven days.
  • differentiation assays may be performed to detect the presence of chondrocyte-specific extracellular matrix.
  • cartilage markers such as proteoglycans and collagen type II may be detected using methods known to those of ordinary skill in the art.
  • cartilage specific matrix gene expression may be evaluated using methods currently known in the art.
  • the cells may be assessed by semiquantitative RT-PCR analysis to determine the expression of cartilage specific matrix genes. Hvdrofiel coating of culture vessels
  • the culture vessels may be coated with hydrogel in conjunction with the methods of present disclosure.
  • Hydrogel refers to a colloid in which the particles are in the external or dispersion phase and water is in the internal or dispersed phase.
  • suitable hydrogels are non-toxic to the cells, are non-adhesive, do not induce chondrocyte attachment, allow for the diffusion of nutrients, do not degrade significantly during culture, and are firm enough to be handled.
  • the bottoms and sides of well plates may be coated with 2% agarose (w/v). While 2% agarose is used in certain embodiments, in other embodiments, the agarose concentration may be in the range of about 0.5% to about 4% (w/v). The use of lower concentrations of agarose offers the advantage of reduced costs; however, at concentrations below about 1% the agarose does not stiffen enough for optimal ease of handling. As an alternative to agarose, other types of suitable hydrogels may be used, such as, for example, alignate.
  • the chondrogenically induced ASID cells are seeded on hydrogel coated culture vessels and allowed to self-assemble.
  • hydrogel coated culture vessels For example, 4.8x10 chondrogenically induced ASID cells in medium may be seeded per well in 24 well plates (bottom well area approximately 2 cm 2 ).
  • the chondrogenically induced ASID cells are allowed to self-assemble on the hydrogel coated culture vessel.
  • the self-assembly may result in the formation of non-attached constructs on the hydrogel surfaces. It is preferable to use hydrogel coated culture vessels instead of tissue culture treated surfaces since articular chondrocytes seeded onto standard tissue culture treated plastic (TCP) readily attach, spread, and dedifferentiate.
  • TCP tissue culture treated plastic
  • the self-assembly process may occur in culture vessels that are shaken continuously on an orbital shaker and then pressurized.
  • the pressurization of the cells may occur in a pressure chamber. Pressurization of the samples during the self-assembly process may aid in increased extracellular matrix synthesis and enhanced mechanical properties.
  • the cells may be pressurized to 10 MPa at IHz using a sinusoidal waveform function. In other embodiments, the cells may be pressurized during self-assembly of the cells.
  • a loading regimen (e.g. compressive, tensile, shear forces) may be applied to the cells during self-assembly based on physiological conditions of the native tissue in vivo. Loading of the cells during self-assembly and/or construct development may cause enhanced cartilage specific gene expression and protein expression in the constructs.
  • the cells may be treated with staurosporine, a protein kinase C inhibitor and actin disrupting agent, during the self-assembly process to reduce synthesis of ⁇ SMA, a contractile protein. Reducing ⁇ SMA in the constructs via staurosporine treatment may reduce construct contraction and may also upregulate ECM synthesis.
  • the cells may be treated with growth factors to increase construct growth and matrix synthesis.
  • growth factors that may be used with the methods of the present disclosure include, but are not limited to, TGF- ⁇ l and IGF-I.
  • the dosing of the growth factors may be intermittent or continuous throughout the period of the self-assembly process.
  • One of ordinary skill in the art, with the benefit of this disclosure, will be able to determine the appropriate dosing regimen and amount and type of growth factor to provide to the developing constructs. Hydro ReI Molds
  • the chondrogenically induced ASID cells may be seeded on a hydrogel coated culture vessel, allowed to self-assemble into a tissue engineered construct, and molded into a desired shape.
  • the self-assembly of the cells into a construct may occur on hydrogel coated culture vessels for about 1 to about 7 days before being transferred to a shaped hydrogel negative mold for molding the construct into the desired shape.
  • the cells may be seeded directly onto a shaped hydrogel negative mold.
  • the shaped hydrogel negative mold may comprise agarose.
  • Other non-adhesive hydrogels, e.g. alignate, may be used in conjunction with the methods of the present disclosure.
  • the hydrogel mold may be a two piece structure comprising, a shaped hydrogel negative mold and a shaped hydrogel positive mold.
  • the shaped hydrogel negative and positive molds may comprise the same non-adhesive hydrogel or may be a comprised of different non-adhesive hydrogels.
  • the cells may be seeded on a hydrogel coated culture vessel and allowed to self-assemble into a construct.
  • the construct may be transferred to a shaped hydrogel negative mold.
  • a shaped hydrogel positive mold may be applied to the negative mold to form a mold-construct assembly.
  • the mold-construct assembly may then further be cultured.
  • the term "mold-construct assembly” refers to a system comprising a construct or cells within a custom-shaped positive and a shaped negative hydrogel mold.
  • the molds may be shaped from a 3-D scanning of a total joint to result in a mold fashioned in the shape of said joint.
  • the molds may be shaped from a 3-D scanning of the ear, nose, or other non-articular cartilage to form molds in the shapes of these cartilages.
  • the mold may be shaped to be the same size as the final product.
  • the molds may be shaped to be smaller than the final product, hi certain embodiments, the molds may be fashioned to a portion of a joint or cartilage so that it serves as a replacement for only a portion of said joint or cartilage.
  • constructs may be tested using any number of criteria including, but not limited to, morphological, biochemical, and biomechanical properties, which also may be compared to native tissue levels.
  • morphological examination includes histology using safranin-O and fast green staining for proteoglycan and GAG content, as well as picro-sirius red staining for total collagen, immunohistochemistry for collagens I and II, and confocal and scanning electron microscopies for assessing cell-matrix interactions.
  • Biochemical assessments includes picogreen for quantifying DNA content, DMMB for quantifying GAG content, hydroxyproline assay for quantifying total collagen content, and ELISA for quantifying amounts of specific collagens (I and II), and RT-PCR for analysis of mRNA expression of proteins associated with the extracellular matrix (e.g. collagen and aggrecan).
  • Constructs also may be evaluated using one or more of incremental tensile stress relaxation incremental compressive stress relaxation, and biphasic creep indentation testing to obtain moduli, strengths, and viscoelastic properties of the constructs.
  • Incremental compressive testing under stress relaxation conditions may be used to measure a construct' s compressive strength and stiffness.
  • Incremental tensile stress relaxation testing may be used to measure a construct's tensile strength and stiffness.
  • indentation testing under creep conditions may be used to measure a construct' s modulus, Poisson's ratio, and permeability.
  • the functionality index is an equally weighted analysis of ECM production and biomechanical properties that includes quantitative results corresponding to the constructs' salient compositional characteristics (i.e., amounts of collagen II and GAG) and biomechanical properties (compressive and tensile moduli and strengths).
  • ⁇ 4 ⁇ ⁇ ⁇ fa ⁇ G nat )lif ⁇ i fc C- na q t Jl )X ⁇ fc l ⁇ - a C t ) JV 2f ⁇ 1 ( C i-£ ⁇ , JV 2f[ 1 fc £ ⁇ ,J JjX 2 ⁇ fc &-'tT
  • G represents the GAG content per wet weight
  • C represents the collagen II content per wet weight
  • E ⁇ represents the tensile stiffness modulus
  • E c represents the compressive stiffness modulus
  • S ⁇ represents the tensile strength
  • S 0 represents the compressive strength.
  • Each term is weighted to give equal contribution to collagen, GAG, tension, and compression properties.
  • the subscripts nat and sac are used to denote native and self-assembled construct values, respectively.
  • the aggregate modulus is not used in Eq. 1, as it is expected to mirror the compressive modulus obtained from incremental compressive stress relaxation. Similarly, the amount of collagen I is not be used in Eq.
  • a hydrogel coated culture vessel or shaped hydrogel negative mold is seeded with chondrogenically induced ASID cells to produce new tissue, such as tissue of the knee meniscus, tendons, and ligaments.
  • the hydrogel coated culture vessel or shaped hydrogel negative mold is typically seeded with cells; the cells are allowed to self-assemble to form a tissue engineered construct.
  • applications of the tissue engineered construct include the replacement of tissues, such as cartilaginous tissue, the knee meniscus, joint linings, the temporomandibular joint disc, tendons, or ligaments of mammals.
  • the constructs may be treated with collagenase, chondroitinase ABC, and BAPN to aid in the integration of the constructs with native, healthy tissue surrounding the desired location of implantation.
  • a wound is naturally anti-adhesive, but debridement with chondroitinase ABC and/or collagenase removes anti-adhesive GAGs and enhances cell migration by removing dense collagen at the wound edge.
  • BAPN a lysyl oxidase inhibitor, may cause the accumulations of matrix crosslinkers and may, thus, strengthen the interface between the construct and native tissue at the desired location of implantation.
  • the tissue engineered constructs may be implanted into a subject and used to treat a subject in need of tissue replacement, hi certain embodiments, the constructs may be grown in graded sizes (e.g. small, medium, and large) so as to provide a resource for off-the-shelf tissue replacement. In certain embodiments, the constructs may be formed to be of custom shape and thickness. In other embodiments, the constructs may be devitalized prior to implantation into a subject.
  • Examples of constructs of the present disclosure were prepared using adult goat skins from 5 animals.
  • the skins were separated from underlying adipose tissue using sterile scissors, washed in sterile phosphate-buffered saline (PBS) and cut into small pieces (1x1 cm 2 ).
  • PBS sterile phosphate-buffered saline
  • the skin tissue was then digested with 0.5% dispase in 4 0 C overnight and then fixed onto a sterile plate, with the epidermis upward.
  • the epidermis was removed by scraping with a blade and the dermis was meticulously cleaned to remove all adipose tissue and blood coagulates in vessels.
  • the dermis was washed three times in sterile PBS, and minced into small pieces (2-3 mm 2 ), and digested in PBS solution containing 200U/ml collagenase type II (Worthington, Lakewood, NJ) at 37 0 C for 15 h under gentle shaking conditions. After incubation, the cell suspension was suspended in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum, 1% penicillin-streptomycin (Gibco/Invitrogen, Carlsbad, CA) and 1 % fungizone (Gibco/Invitrogen) and centrifuged at 1 ,200 rpm for 5 min at room temperature. The supernatant was aspirated away.
  • Dulbecco's modified Eagle's medium Gibco
  • penicillin-streptomycin Gibco/Invitrogen, Carlsbad, CA
  • fungizone Gibco/Invitrogen
  • Cells were resuspended in cell culture medium and seeded in flasks. Media changes were performed every 3-4 days. After cells reached confluency, cells were treated with 0.5% dispase for 15 minutes, and the floating cells were discarded. Then, after cultured for 3 days, cells were harvested as normal fibroblast and passaged using a solution containing 0.25% trypsin and 5 mM EDTA (Sigma). To obtain a homogeneous culture of ASID cells, harvested cells were seeded in a tissue culture treated flask and allowed to attach for 10 min, after which the floating cells were discarded. The remaining cells were washed 3 times with PBS and continued to be cultured in culture medium.
  • Negative control surface was pre-coated with water. Well surface were photographed using a Nikon CoolPix 990 digital camera mounted on a Nikon Eclipse TS-100 inverted microscope. As shown in FIGURE 2, the data illustrated that the aggrecan-coated surfaces formed micropatterned templates (parallel ridge/groove type structures) compared to the tissue culture treated control. Furthermore, the ridge width of these grooves increased with the increase of aggrecan concentration, while groove width decreased. The highest coating density resulted in grooves with ridge width/groove width of about 100-200/1-10 ⁇ m in aggrecan 10 ⁇ g/cm 2 groups.
  • Aggrecan is highly negatively-charged and functions to bind and organize water molecules and repel negatively charged molecules within the articular cartilage.
  • the aggrecan molecule is too large and immobile to redistribute itself; thus the addition of water causes aggrecan-rich matrix network to swell and expand, and results in substrate topography variation as well as surfaces charge variation in vivo. Based on these in vivo characteristics of aggrecan, it was hypothesized that aggrecan can be used as a specific ECM molecule to coat TCP surfaces for ASID cells to chondrogenically differentiate. After aggrecan coating, it was found that aggrecan molecules deposit on TCP surfaces and orient into special grooves.
  • FIGURE 2G, H and I TCP surfaces
  • FIGURE 4 A and B show the effect of different aggrecan concentration on the expression of collagen type I and II in ASID cells, further suggesting an optimal concentration of aggrecan of lO ⁇ g/cm .
  • Example 3 Chondrogenic Differentiation in Mono-layer culture.
  • FIGURE 5 shows aggrecan induced morphological changes in chondrocytes, ASID cells, and fibroblasts after 1 day in culture. Fibroblasts plated on tissue culture treated plastic alone attached to the surface, elongated, and spread to become spindle- shaped cells, maintaining a fibroblastic appearance. The majority of fibroblasts were shown to align strictly along the direction of the ridges/grooves formed by aggrecan. In sharp contrast, ASID cells grown on aggrecan-coated surfaces appeared to be small, round cells suspended in culture medium when first plated.
  • FIGURE 5E shows that different dimensions of the ridge/groove patterns only affected fibroblast distribution. All concentrations of aggrecan induced different degrees of directional migration of fibroblasts with the growing direction aligning the microgrooves. However, the wider microgrooves seemed to trap more fibroblast than the narrow ones.
  • chondrocytes respond sensitively to aggrecan-coated surfaces by organizing themselves into nodules (FIGURE 5A), suggesting a different interacting pathway against aggrecan-coated surface between chondrocytes and fibroblasts.
  • ASID cells employed an aggrecan- sensitive pathway significantly different from fibroblasts, but similar to that of chondrocytes by forming nodules with similar size and numbers on aggrecan-coated surfaces (FIGURE 5 A, C), suggesting similar cell-matrix interaction mechanisms may exist between ASID cells and chondrocytes when cultured on an aggrecan substrate.
  • Example 4 Detection of Cartilage Extracellular Matrix
  • FIGURE 6 shows the results of staining.
  • Example 5 Detection of Gene Expression by Semi Quantitative RT-PCR Analysis of Cell Grown on Tissue Culture Treated Polystyrene With or Without Aggrecan.
  • RNA was incubated with buffer, ImM dNTPs, 1 mM random hexamers, RNase inhibitor and 100 U Stratagene StrataScript RT enzyme (La Jolla, CA) at 42 0 C for 60 minute.
  • samples were either stored at -2O 0 C or used immediately for PCR amplification using the Rotor-gene 3000 real-time PCR machine (Corbett Research, Sydney, AU).
  • the real-time analysis used a 10 minute denaturing step, followed by 45 cycles of 30 seconds at 95 0 C, 30 seconds at 58 0 C, and 1 minute at 72 0 C, followed by a 2 minute extension. Fluorescence measurements were taken every cycle at 6O 0 C to provide a quantitative, real-time analysis of the genes analyzed. Primer sequences and concentrations are provided in Table 1 below.
  • Table 1 Primer sequences used for semi-quantitative real time PCR.
  • ASID cells and fibroblasts were grown on either aggrecan-coated tissue culture polystyrene or tissue culture treated polystyrene without aggrecan for 14 days. Steady-state levels of mRNA from each test group were collected for type II collagen and aggrecan measurement using quantitative real-time PCR.
  • the aggrecan-coated surfaces strongly reduced aggrecan expression of ASID cells from day 1 to day 7 compare to those of tissue culture treated control surface (FIGURE 7) However, at 14 days the effect of aggrecan on aggrecan gene expression faded away. In contrast, no obvious differences could be observed between fibroblast groups with or without aggrecan (data not shown). Aggrecan treatment can inhibit aggrecan gene expression in ASID cells.
  • aggrecan treatment can inhibit collagen type I expression in ASID cells (FIGURE 8 A).
  • FIGURE 10 and FIGURE 11 indicate the effect of aggrecan on aggrecan and collagen type I and II expression of ASID cells cultured on tissue culture treated and non-tissue culture treated polystyrene coated with or without aggrecan.
  • the results indicate that aggrecan- coated non-tissue culture surfaces are better for ASID expression of collagen I and collagen II.
  • the ratio of collagen I and collagen II indicate that non-tissue culture treated surfaces are better differentiated (FIGURE 10).
  • FIGURE 11 indicates that aggrecan expression was suppressed in the presence of aggrecan coating. As a result, further investigation using non- tissue culture treated surfaces was performed.
  • the results of the study of ASID cells and fibroblasts cultured on non-tissue culture treated plates with or without aggrecan can be seen in FIGURES 15-18.
  • FIGURE 15 Gene expression of collagen type I can be seen in FIGURE 15 across all groups over a 14 day period of culture.
  • Cartilage oligomeric protein gene expression can be seen in FIGURE 16.
  • FIGURE 17A and B show aggrecan abundance and gene expression over the 14 day culture period.
  • FIGURE 18 shows the collagen type II abundance in cell types over the 14 day culture period.
  • Chondrogenic medium comprises Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L-glucose and L- glutamine supplemented with 10 "7 M dexamethasone, 50 ⁇ g/ml ascorbic acid, 40 ⁇ g/ml proline, 100 ⁇ g/ml sodium pyruvate, and 50 mg/ml ITS+Premix.
  • DMEM Dulbecco's Modified Eagle Medium
  • L- glutamine supplemented with 10 "7 M dexamethasone, 50 ⁇ g/ml ascorbic acid, 40 ⁇ g/ml proline, 100 ⁇ g/ml sodium pyruvate, and 50 mg/ml ITS+Premix.
  • FIGURE 12, FIGURE 13, and FIGURE 14 indicate the results of this study.
  • Large quantities of Safranin-O stained positive nodules could be found in both aggrecan treated groups with normal medium and chondrogenic medium (FIGURE 12).
  • No nodule could be found in the groups grown on non aggrecan-coated surface with normal medium.
  • the data imply that chondrogenic medium combined with non-tissue culture treated surfaces enhance nodule formation of ASID cells at day 1.
  • No nodules could be found in fibroblast group with normal medium from day 1 to day 14.
  • no nodules could be found in ASID cells in normal medium after day 7, while large quantities of nodules could be found in chondrogenic medium groups. These nodules stain positive with Safranin-O for proteoglycans (FIGURE 13) and stain positive for type II collagen (FIGURE 14). Compared to previous experiments performed with tissue culture treated plates, aggrecan is required to get nodules on tissue culture treated surfaces, whereas with non-tissue culture treated surfaces, aggrecan is not needed but could obviously improve the formation of nodules. Non-tissue culture surfaces combined with chondrogenic medium could keep the nodules in culture for as long as 14 days.
  • Example 7 Immunofluorescence of Cell Samples
  • Cells for use in immunofluorescence experiments were grown directly on tissue culture treated plastic coverslips with and without aggrecan coating. After cultured for 36 hrs, they were rinsed with PBS, fixed in 4% paraformaldehyde, and permeabilized with a Triton- X solution. The cells were then blocked for 30 min in 1% BSA. For vinculin visualization, cells were incubated with monoclonal anti-vinculin IgG (1 :300; Sigma), followed by Alexa 488-conjugated goat anti-mouse IgG (1:200, Molecular Probes, Eugene, OR). F-actin was visualized by a 30 min exposure to rhodamine phalloidin (2 U/per coverslip; Molecular Probes, Eugene, OR).
  • coverslips were then mounted between a microscope slide and glass coverslip using ProLong Gold with DAPI (Molecular Probes, Eugene, OR). These samples were viewed with an Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany) and a CoolSNAP-HQ CCD camera (Photometries, Tuscon, AZ). Images were acquired and analyzed using Metamorph 4.15 (Universal Imaging Corp., Downingtown, PA). After 36 hrs in culture, differences in the organization of F-actin and vinculin of chondrocytes, ASID cells and fibroblasts grown on aggrecan-coated surfaces, as compared with cells grown on uncoated surfaces, were much more prominent.
  • chondrocytes and ASID cells Similar shape, size, and cytoskeletal effects were observed between chondrocytes and ASID cells (FIGURE 9A, C and a, c). Chondrocyte and ASID cells grown on aggrecan- coated surfaces showed an increase in the presence of actin stress fibers and vinculin- containing focal adhesion points than cells grown on the uncoated TCP surfaces, and occupied larger surface area on the substratum. It is important to note that chondrocytes and ASID cells are shown to perform similar f-actin and vinculin reorganization, which implied similar cell-ECM interaction and the consequent cellular events.
  • Example 8 Analysis of the Morphology of Constructs After culture on aggrecan coated non-tissue culture treated surfaces for 14 days, ASID cells, fibroblasts, and chondrocytes were transferred to hydrogel coated well surfaces and allowed to self-assemble.
  • 96-well plates were coated with 100 ⁇ l 2% agarose (w/v), and the plates were shaken vigorously to remove excess agarose.
  • the surface area at the bottom of the well in a 96-well plate is 0.2 cm 2 . Chilled plates were then rinsed with culture medium before the introduction of cells.
  • Chondrogenically induced ASID cells were then introduced into the hydrogel-coated wells at 4.8xlO 6 cells per well in 300 ⁇ l of culture medium (4.8 x 10 6 cells/0.2 cm 2 of hydrogel coated surface). The cells aggregated within 24 hrs, from which time 500 ⁇ l of the medium was changed every 2 days. After 2 weeks of culture, these cell aggregates were analyzed for extracellular matrix production. Fibroblasts and chondrocytes were used as control cells.
  • FIGURE 20 is an image of developing constructs formed from fibroblasts and ASID cells.
  • ASID cells self-assemble into cartilage-like constructs, outperforming fibroblast constructs; they also formed a much bigger construct than fibroblasts.
  • FIGURE 22 is an image of constructs formed by self-assembly of ASID cells and fibroblasts cultured on aggrecan- coated non-TCP surfaces for 14 days. The results indicate that ASID cells self-assemble better than the fibroblast group. Chondrocytes formed a much bigger construct than both ASID cells and fibroblasts (not shown). Both ASID and fibroblast constructs contracted, while no or light contraction was found in the chondrocyte group.
  • FIGURE 21 indicates the results of staining.
  • ASID cell constructs produce less collagen type I than the fibroblast constructs.
  • FIGURE 23 indicates the results of staining of ASID cell constructs, fibroblast constructs, and chondrocyte constructs. All cells were initially cultured on aggrecan-coated non-tissue culture treated surfaces for 14 days. Large quantities of proteoglycan and collagen type II were shown in chondrocyte and ASID groups, while less cartilage specific extracellular matrix were shown in fibroblast group. Slight collagen type I was shown in fibroblast group, while no or less collagen type II was found in this group. Moreover, as illustrated in FIGURE 19, oil red staining indicated differentiated ASID cells.
  • fibroblastic cells could be isolated from goat skin dermis considering their fast adhering characteristic to TCP surfaces(FIGURE 3), and these cells were demonstrated to have the potential of chondrogenic differentiation on aggrecan-coated surfaces by producing rich cartilage specific extracellular matrix (FIGURE 6) and expressing cartilage specific gene (FIGURE 7 and FIGURE 8).
  • the data presented herein also shows that ASID cells rearranged their cytoskeleton organization by aggrecan-coated surfaces stimuli as chondrocytes did under same experimental condition (FIGURE 8).
  • Full-thickness abdomen skin specimens were obtained from 5 goats, separated from underlying adipose tissue, and digested with 0.5% Dispase at 4°C overnight. The epidermis was then removed by scraping with a blade, and meticulously cleaned to remove all adipose tissue and blood coagulates in vessels. The dermis specimens were then washed, minced, and digested in phosphate buffered saline (PBS) containing 200 units/ml type II collagenase (Worthington, Lakewood, NJ) at 37°C for 15 hours with gentle rocking.
  • PBS phosphate buffered saline
  • the cell suspensions were diluted at a ratio of 1 :4 with expansion medium (Dulbecco's modified Eagle's medium [DMEM; Gibco, Grand Island, NY] supplemented with 10% fetal bovine serum [FBS; BioWhittaker, Walkersville, MD], 1% penicillin-streptomycin-amphotericin B [BioWhittaker], and 1% nonessential amino acids [Life Technologies, Gaithersburg, MD]) and centrifuged at 300g for 5 minutes. The cell pellets were resuspended in expansion medium and cultured in flasks. Cell yields were 5-12 million/ cm of skin. Medium was changed every 3-4 days.
  • expansion medium Dulbecco's modified Eagle's medium [DMEM; Gibco, Grand Island, NY] supplemented with 10% fetal bovine serum [FBS; BioWhittaker, Walkersville, MD], 1% penicillin-streptomycin-amphotericin B [BioWhittaker]
  • FBS fetal bo
  • ASID cells were chondroinduced by plating on aggrecan coated surfaces (ACS).
  • the concentration of aggrecan (Sigma) was 10 ⁇ g/cm per 24-well plate.
  • ASID cells, chondrocytes, and fibroblasts were seeded on ACS at a concentration of 2 x 10 5 cells/well in 0.3 ml of expansion medium.
  • Example 11 Chondroinduction Effects of Agggrecan on ASID Cells in Monolayer Culture. Triplicate samples from each cell group were collected at 24 hours, 1 week, and 2 weeks and assessed for chondrocyte specific matrix using the following analyses. For chondrocytic nodule formation, samples were collected and photographed using a CoolPix 990 digital camera (Nikon, Melville, NY) mounted on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany). For glycosaminoglycan (GAG) detection, Safranin O staining was performed after 10 minutes of formalin fixation. Cells were incubated with 1 % acetic acid, and Safranin O was applied for 2 minutes. Cells were then photographed after a water rinse.
  • chondrocytic nodule formation samples were collected and photographed using a CoolPix 990 digital camera (Nikon, Melville, NY) mounted on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany).
  • GAG glycosamino
  • Type II collagen was detected using immunohistochemistry. Briefly, formalin fixed cells were incubated with CII primary antibody (Chondrex, Redmond, WA) and detected using the Vectastain ABC kit (Vector, Burlingame, CA) according to the instructions provided. A quantitative sandwich enzyme linked immunosorbent assay (ELISA) for CII was also performed, using a monoclonal capture antibody (6009) and a polyclonal detection antibody (7006) (Chondrex).
  • Example 12 Quantification of Cartilage-Specific Matrix Gene Expression and Protein Production.
  • Semiquantitative reverse transcriptase-polymerase chain reaction (PCR) analyses were performed to measure the expression of type I collagen (CI), CII, cartilage oligomeric protein (COMP), and aggrecan.
  • RNA isolated using an RNAqueous kit (Ambion, Austin, TX) was reverse-transcribed using StrataScript RT enzyme and kit (Stratagene, La Jolla, CA) at 600 ng RNA per reaction.
  • PCR was performed using the Rotor-Gene 3000 real-time PCR system (Corbett Life Science, Sydney, New South Wales, Australia).
  • the real-time analysis consisted of 15 minutes at 95°C, followed by 55 cycles of 15 seconds at 95°C, and 30 seconds at 60 0 C. Primer and probe sequences and concentrations are shown in Table 1 above.
  • the day 0 control was obtained by isolating messenger RNA (mRNA) from fibroblasts prior to seeding onto ACS.
  • mRNA messenger RNA
  • ASID cells were shown to possess a greater chondroinduction potential compared with fibroblasts (FIGURE 25). Specifically, after seeding onto ACS, aggrecan gene expression in ASID cells was significantly higher (P ⁇ 0.05) than that in fibroblasts at 7 and 14 days (FIGURE 25B). Similarly, COMP expression by ASID cells was also significantly higher (P ⁇ 0.05) than that in fibroblasts (FIGURE 25C) at 7 and 14 days. By day 14, COMP expression in ASID cells was 5-fold higher than in fibroblasts.
  • chondrocytes, ASID cells, or F- ASID cells were plated on 24-well ACS at 2 x 10 5 cells/well. After 7 days, cells were harvested by scraping and were seeded to form self- assembled constructs, as previously described in Hu JC, Athanasiou KA. A self-assembling process in articular cartilage tissue engineering. Tissue Eng 2006;12:969-79. Briefly, a silicon-positive die consisting of cylindrical prongs (3 mm diameter x 10 mm long) was used to form a 2% agarose mold.
  • the mold was then separated from the silicon-positive die and saturated with defined medium containing 1% FBS.
  • defined medium containing 1% FBS For each construct, cells harvested from the 24 wells were combined and suspended in 50 ⁇ l of defined medium with 1% FBS and seeded into the agarose molds. Within 24 hours, the cells formed attached constructs, and these constructs were maintained in the agarose molds for 2 weeks. Medium was changed every 2 days. For the 3 D portion of this study, day 0 was defined as the day that cells were seeded into the agarose wells. After 2 weeks, constructs were collected to evaluate cartilage-specific matrix deposition, using Safranin O to determine GAG distribution and immunohistochemistry to detect CII, CI, chondroitin 4-sulfate, and chondroitin 6-sulfate.
  • Results are expressed as the mean ⁇ SD. Data were assessed by 3-factor analysis of variance. P values less than 0.05 were considered significant.
  • a modified rapid adherence process was developed to isolate ASID cells from goat dermis for chondroinduction. Instead of selecting all rapidly adhering cells from the dermis, the Dispase-sensitive subpopulations are first removed (since these populations also contain rapidly adhering cells). Rapidly adhering cells from the remaining sub populations are then isolated based on their adherence time. Cells that adhered to the plastic surface within 10 minutes were chosen because they produced the highest nodule numbers when seeded on ACS compared with cells from other time points (data not shown).
  • ASID cells were chondroinduced when seeded on ACS, and were phenotypically, morphologically, and functionally similar to chondrocytes.
  • In situ activity of ASID cells might be suppressed in the in vivo microenvironment through signaling from skin ECM and/or from mature fibroblasts.
  • the chondroinduction process may be initiated due to the presence of an enriched environment of ASID cells and/or exposure to aggrecan or other cartilage-specific ECM components.
  • Chondrocytes, ASID cells, and fibroblasts were seeded on ACS in this study. Fibroblasts showed a spindle-like morphology on ACS 24 hours after seeding.
  • chondrocytes responded sensitively to ACS by organizing into nodules, suggesting the presence of a different interacting pathway between chondrocytes and fibroblasts.
  • ASID cells used an aggrecan-sensitive pathway significantly different from that of fibroblasts.
  • ASID cells formed nodules similar in size and number to those in chondrocytes on ACS, suggesting that analogous early-stage cell-matrix interaction mechanisms may exist between ASID cells and chondrocytes when cultured on ACS.
  • ASID cells have a higher potential for chondroinduction compared with unpurified, heterogeneous fibroblast subpopulations.
  • nodules formed by ASID cells seeded on ACS were shown to stain positively for Safranin O and for CII.
  • both ASID and fibroblast cells seeded on uncoated surfaces showed negative staining for both GAG and CII under the same conditions, which is common for dermis- derived cells.
  • ASID cells exposed to ACS expressed cartilage marker genes more rapidly and more potently than did fibroblasts.
  • ACS appeared to inhibit the fibroblastic phenotype in ASID cells, as evidenced by significant inhibition of collagen type I gene expression at 1 day and 7 days.
  • chondrocytes, ASID cells, and fibroblasts were cultured on ACS for 36 hours. Chondrocytes and ASID cells were found to organize their F-actin on ACS in a similar pattern, which was significantly different from that of fibroblasts. Fewer stress fibers were found in ASID cells and chondrocytes than in fibroblasts. Furthermore, the distribution of vinculin in each group mirrored its F-actin distribution (FIGURE 26). The observed F-actin patterns of ASID cells and chondrocytes in this study were similar to those reported for chondrocytes in monolayer. This implies that the 2 cell types have similar cell-matrix interactions.
  • F-actin organization plays an important role in a large number of cellular events, including shape alteration, cell signaling, secretion, and ECM assembly. Any one or a combination of the above described events may thus be precipitated by the F-actin organization brought about by cell matrix interactions.
  • chondrocytes were found to respond to ECM components, including hyaluronic acid) and CI), by reorganizing their F-actin in vitro, resulting in the regulation of various chondrocyte behaviors such as cell shape determination, chondrogenesis initiation, chondrocytic phenotype maintenance, and chondrocyte hypertrophy.
  • any one or a combination of these events may have occurred as chondrocytes were seeded onto ACS.
  • specific cell-matrix interactions led to F-actin and vinculin reorganization. This reorganization may have resulted in the subsequent changes in various ASID cell events that ultimately led to chondrogenic phenotype formation of these cells in 2-D.
  • These specific cell matrix interactions may also lead to a temporal and spatial self-assembly process in 3-D.
  • the assembly of cells into functional multicellular organisms in 3 dimensions involves F-actins, the primary sites at which cells detect and adhere to their ECM.

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Abstract

La présente invention concerne des améliorations apportées à des procédés de génie histologique. L'invention concerne plus particulièrement, d'une part des procédés permettant d'induire une différenciation de cellules dérivées du derme pour servir de source de chondrocytes, et d'autre part des procédés associés convenant à la formation de constructions obtenues par génie histologique. L'invention concerne ainsi essentiellement un procédé permettant d'induire la différenciation de cellules en chondrocytes en prenant des cellules isolées de dermes et réagissant à l'aggrécan, et à ensemencer ces cellules sur une surface enduite d'aggrécan.
PCT/US2007/066085 2004-07-09 2007-04-05 Cellules dérivées du derme pour des applications de génie histologique Ceased WO2007136936A2 (fr)

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AU2007254048A AU2007254048A1 (en) 2006-04-05 2007-04-05 Dermis-derived cells for tissue engineering applications
EP07760205A EP2007875A4 (fr) 2006-04-05 2007-04-05 Cellules dérivées du derme pour des applications de génie histologique
CA002648648A CA2648648A1 (fr) 2006-04-05 2007-04-05 Cellules derivees du derme pour des applications de genie histologique
US12/246,367 US20090142307A1 (en) 2004-07-09 2008-10-06 Shape-Based Approach for Scaffoldless Tissue Engineering
US12/246,320 US8637065B2 (en) 2004-07-09 2008-10-06 Dermis-derived cells for tissue engineering applications
US12/246,306 US20090136559A1 (en) 2004-07-09 2008-10-06 Chondrocyte Differentiation from Human Embryonic Stem Cells and Their Use in Tissue Engineering
US13/029,325 US20110212894A1 (en) 2004-07-09 2011-02-17 Decellularization method for scaffoldless tissue engineered articular cartilage or native cartilage tissue

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EP2007312A2 (fr) 2008-12-31
EP2007881A2 (fr) 2008-12-31
WO2007115337A8 (fr) 2007-12-13
WO2007115336A3 (fr) 2008-05-22
AU2007234366A1 (en) 2007-10-11
WO2007136936A3 (fr) 2008-10-23
EP2007875A2 (fr) 2008-12-31
CA2648327A1 (fr) 2007-10-11
EP2007312A4 (fr) 2012-08-22
CA2648332A1 (fr) 2007-10-11
CA2648648A1 (fr) 2007-11-29
EP2007875A4 (fr) 2009-12-16
AU2007234365A1 (en) 2007-10-11
WO2007115336A2 (fr) 2007-10-11
AU2007254048A1 (en) 2007-11-29
EP2007881A4 (fr) 2009-07-08
WO2007115337A2 (fr) 2007-10-11
WO2007115337A3 (fr) 2008-07-03

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