ES2414285T3 - Composition and procedures for the production of biological tissues and tissue constructions - Google Patents

Abstract

A method for preparing an implantable tissue construct, said method comprising: (a) providing an implantable multilayer structure comprising: (i) an integration layer; and (ii) a cell support layer comprising a sponge matrix, the cell support layer being operably linked to the first integration layer; and (b) introducing a solution comprising suspended chondrocytes into said multilayer structure to create a seeded device; and (c) culturing said seeded device under conditions comprising cyclic hydrostatic pressure with a dormant phase at atmospheric pressure for one to sixty days.

Description

Composition and procedures for the production of biological tissues and tissue constructions

Field of the Invention

The present invention relates to methods for preparing tissues and tissue constructions. In particular, the invention lists methods that incorporate cells into biological matrices.

Background

Vertebrate tissues and organs often fail to regain normal form and function when they are damaged and often fail (due to metabolic and / or mechanical trauma) to integrate properly with a host when they are transplanted. In an attempt to reconstruct tissues and organs, surgeons have implanted living tissues or non-living materials instead of damaged or resected native anatomical structures. These traditional techniques have limitations. For example, although autografts are histocompatible, loss of donor site tissue often creates anatomical and / or physiological pathologies in the same host. On the other hand, transplanted allografts and xenografts often cause a pathological immune response that approximates rejection of these grafts by the host.

Non-living transplant materials have a marginal utility in the repair and transplantation of native tissues, tissue constructions and organs. Specifically, transplanted non-living materials are especially subject to extrusion, infection and scar tissue formation.

The use of biotechnology modified tissue as an alternative to transplanted native tissues and non-living materials has also been described. For example, U.S. Patent No. 5,902,741 to Purchio et al. teaches a procedure for culturing cartilaginous tissue in vitro using a framework inoculated with a nutrient medium that carries cartilaginous cells or precursors. Similarly, U.S. Patent No. 5,928,945 to Selektar et al. teaches a process for producing neo-cartilage obtained by biological engineering by applying flow shear flow stress to chondrocytes. In another example, US Patent No. 6,080,194 to Pachence et al. It teaches a mold for producing articular cartilage comprising a matrix of dense collagen located on the surface of the cartilage defect to prevent cellular migration and internal vascular growth from the sub-condral plate.

SMITH R L et al .: “In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure” JOURNAL OF ORTHOPAEDIC RESEARCH, JOHN WILEY & SONS, INC, NEEDHAM, MA, vol. 14, No. 1, January 1, 1996 (), pages 53-60, ISSN: 0736-0266, discloses a procedure comprising: providing a solution comprising chondrocytes; b- sowing the chondrocytes in a device to obtain a high density monolayer in a sown device; c- treating said seeded device under conditions comprising subjecting said chondrocyte seeded device to culture with a cyclic hydrostatic pressure of 10 MPa at 1 Hz for 4 hours. Cyclic hydrostatic pressure simulates physiological hydrostatic pressure and activates chondrocytes.

However, these compositions and procedures (in the prior art) only approximate a fraction of the anatomical and physiological repertoire of the native tissue they are trying to replace. More specifically, these defects in the prior art can, in part, be attributed to the inability to uniformly sow, cultivate and differentiate cells across the entire cross section of a three-dimensional cell support layer. What is needed, therefore, are transplantable tissue and tissue constructs that can support cells in their normal metabolic state and can be anatomically and physiologically integrated with a host without causing a pathogenesis.

Summary of the invention

The present invention relates to processes for the preparation of tissues and tissue constructions. In particular, the invention relates to the construction of a multi-layered biological structure (ie system framework) comprising living cells incorporated in a cell support matrix. The process of the invention is defined in claim 1. The present invention contemplates a composition comprising a first integration layer; a second cellular support layer (comprising a sponge matrix) operatively attached to said first integration layer in which said support layer is seeded with cells (suspended in a gel), and a third protective layer operatively linked to said first and second layers.

It is not intended that the process of the present invention be limited to providing a composition comprising a multilayer system having a specific number of layers. In one embodiment, said multilayer system comprises two layers. In a preferred embodiment, said multilayer system comprises three layers. In another embodiment, said multi-layer system comprises more than three layers.

It is not intended that the gel element of said cell support layer be limited to a single composition. In one embodiment, said gel element comprises gelatin. In another embodiment, said gel element comprises

collagen In a preferred embodiment, said gel element comprises Type I collagen.

It is not intended that the methods of the present invention be limited to cells collected from any particular species. In one embodiment, said cells are mammalian cells. In a preferred embodiment, said cells are from a human being. The cells used in the methods of the present invention are chondrocytes collected from connective tissue. In a preferred embodiment, said connective tissue is cartilage.

In one embodiment, the present invention describes methods for preparing tissue or tissue constructs using a multilayer system that includes a cell support matrix seeded with live cells directly isolated and / or expanded from viable tissue. In a preferred embodiment, the present invention contemplates a method that provides a cell support matrix, a gel and cells; mixing said cells with said gel to form a cell suspension; and contacting said cell suspension with said cell support matrix to create a cell seeded matrix.

In another embodiment, the present invention further comprises applying heat to said matrix seeded with cells under conditions in which said gel is cured to a substantially solid form, thereby forming a matrix seeded with heat cured cells.

The present invention further comprises culturing said matrix seeded with heat cured cells under conditions comprising cyclic hydrostatic pressure, said cell support matrix being able to consist essentially of porous type I collagen and said chondrocyte cells being.

In one embodiment, the present invention describes a method, comprising:

a) provide, i) a solution comprising suspended chondrocytes; ii) a sponge matrix; and iii) a means of treatment; b) introducing said sponge matrix into said solution under conditions such that said solution enters said sponge by capillary action and said chondrocytes come into contact with said matrix to create a seeded device; and c) treating said seeded device with said treatment medium under conditions such that at least a portion of said solution solidifies within said device.

In an embodiment of the present invention, said treatment medium comprises a culture chamber. In another embodiment, the present invention contemplates a treatment of said seeded device, which comprises heating in the absence of carbon dioxide.

In one embodiment of the present invention, the sponge matrix is configured to transplant into a joint of a human being.

In one embodiment of the present invention, the chondrocyte suspension solution comprises neutralized aqueous type I collagen.

In another embodiment, the present invention further comprises, after step (c) above, culturing said chondrocytes under conditions such that said chondrocytes develop gene products associated with the extracellular matrix. In one embodiment, said culture is performed under constant or cyclic hydrostatic pressure with said cyclic hydrostatic pressure being in a range of about 0.01-10.0 MPa at 0.5 Hz. In another embodiment, said cyclic hydrostatic pressure is in a range. 0.5-5.0 MPa at 0.5 Hz. In a preferred embodiment, said cyclic hydrostatic pressure is approximately 3.0 MPa pressure at 0.5 Hz. In one embodiment the duration of said cyclic pressure is 1 - 30 days. In another embodiment, the duration of said cyclic pressure is 5-8 days. In a preferred embodiment, the duration of said cyclic pressure is approximately 6 days.

In one embodiment the present invention contemplates a resting phase (after exposure to cyclic pressure). In one embodiment, the duration of said resting phase is in the range of 1-60 days. In one embodiment, the duration of said resting phase is in the range of 4-21 days. In a preferred embodiment, said resting phase is approximately 12 days.

In another embodiment the present invention describes a method, comprising:

a) providing i) a solution comprising chondrocytes suspended in aqueous collagen; ii) a cell support matrix; and iii) a means of treatment; b) introducing said matrix into said solution under conditions such that said solution enters said sponge by capillary action and said chondrocytes come into contact with said matrix to create a seeded device; and c) reacting said seeded device with said treatment medium under conditions such that at least a part of said solution solidifies within said device.

In an embodiment of the present invention, said chondrocyte suspension solution is at least partially gelled.

In one embodiment of the present invention, the treatment medium comprises a culture chamber. In another embodiment the treatment comprises heating in the absence of carbon dioxide.

In one embodiment of the present invention, the sponge matrix comprises type I collagen configured to be transplanted into a joint of a human being.

In another embodiment, the present invention further comprises, after step (c), culturing said chondrocytes under conditions such that said chondrocytes develop gene products associated with the extracellular matrix.

In one embodiment of the present invention, said culture is performed under cyclic hydrostatic pressure, said cyclic hydrostatic pressure being in a range of about 0.01-10.0 MPa at 0.5 Hz. In another embodiment, said cyclic hydrostatic pressure is in a range of 0.5-5.0 MPa at 0.5 Hz. In a preferred embodiment, said cyclic hydrostatic pressure is approximately 3.0 MPa pressure at 0.5 Hz. In one embodiment the duration of said cyclic pressure It's 1 -30 days. In another embodiment, the duration of said cyclic pressure is 5-8 days. In a

In a preferred embodiment, the duration of said cyclic pressure is approximately 6 days.

The present invention contemplates a resting phase (after exposure to cyclic pressure), the duration of said resting phase being in the range of 1-60 days. In another embodiment, the duration of said resting phase is in the range of 4-21 days. In a preferred embodiment, said resting phase is approximately 12 days.

It is not intended that the present invention be limited to a situation in which said chondrocytes are suspended.

15 in a sol-gel solution. It is not intended that the present invention be limited to any specific degree of gelation for said sol-gel solution containing chondrocytes.

It is not intended that the present invention be limited to a situation in which the complete chondrocyte suspension solution solidifies. That is, it is sufficient for a significant part of the solution to solidify, thereby substantially immobilizing in one embodiment the cells suspended within a matrix.

20 seeded.

In one embodiment, the present invention contemplates a system, comprising: i) a cell support layer configured to allow the growth of cells in three dimensions, said cell support layer having an integration side and a functional side, and in the that said cell support is seeded with at least one cell type to create seeded cells; ii) an integration layer that is in contact with said integration side of

Said cellular support layer, said integration layer being configured to be inserted into the tissue; and iii) a protective layer that is in contact with said functional side of said cellular support layer, said protective layer being configured to protect said seeded cells from the effects of blood transported agents.

In an embodiment of the present invention, said blood transport agents are factors of

30 coagulation, cytokines and / or cytotoxic cells. In an embodiment of the present invention, said seeded cells are in contact with a nutrient fluid. In another embodiment of the present invention, said integration layer is configured to be inserted into tissue after surgery.

The present invention can be used in a method for inserting a system comprising: i) a cell support layer configured to allow the growth of cells in three dimensions, said layer having

A cellular support an integration side and a functional side, and wherein said cellular support is seeded with at least one cell type to create seeded cells; ii) an integration layer that is in contact with said integration side of said cellular support layer, said integration layer being configured to be inserted into tissue; and iii) a protective layer that is in contact with said functional side of said cellular support layer, said protective layer being configured to protect said seeded cells from the effects of agents

40 carried by the blood and tissues of a patient. The insertion of the system in the patient is after surgery in said patient.

Description of the drawings

Figure 1 projects data on the effect of cyclic hydrostatic pressure, compared to static atmospheric pressure, on glycosaminoglycan (GAG) production by chondrocytes grown within the matrix

45 of TESS. Specifically, this figure shows that there is a 300% increase (p <0.001) in the production of GAG in TESS matrices in the test group (3.0 MPa of CHP at 0.5 Hz) with respect to the matrices of TESS in the control group (atmospheric pressure only). Figure 2A is a photomicrograph showing the production of Safranin-O staining of the extracellular matrix by chondrocytes, grown under static atmospheric pressure (control group) within the matrix

50 of TESS. In this figure the honeycomb sponge element of the cell support matrix (for example the TESS matrix) is marked with "HC" and an area of the extracellular matrix (made by cultured chondrocytes) is marked with a "*" . Figure 2B is a photomicrograph showing the preparation of Safranin-O staining of the extracellular matrix by chondrocytes, cultured under cyclic hydrostatic pressure (test group) within the matrix

TESS 55. In this figure the honeycomb sponge element of the cellular support matrix (for example the TESS matrix) is marked with "HC" and an extracellular matrix area (made by cultured chondrocytes) is marked with a "*". Figure 3A is another photomicrograph showing the staining with matrix Safranin-O

extracellular by chondrocytes, grown under static atmospheric pressure (control group), within the TESS matrix. In this figure the honeycomb sponge element of the cellular support matrix (for example the TESS matrix) is marked with "HC" and an extracellular matrix area (made by cultured chondrocytes) is marked with a "*". Figure 3B is another photomicrograph showing the production of Safranin-O staining of the extracellular matrix by chondrocytes, cultured under cyclic hydrostatic pressure (test group), within the TESS matrix. In this figure the honeycomb sponge element of the cellular support matrix (for example the TESS matrix) is marked with "HC" and an extracellular matrix area (made by cultured chondrocytes) is marked with a "*". Figure 4 illustrates data on the effect of cyclic hydrostatic pressure, compared to static atmospheric pressure, on the production of glycosaminoglycans (GAG) by chondrocytes cultured with the TESS matrix. Specifically, this figure shows that there is a 132% increase (p <0.006) in the production of GAG in TESS matrices in the test group (3.0 MPa of CHP at 0.5 HZ) compared to TESS matrices in the control group (atmospheric pressure only).

Definitions

As used herein, the term "tissue" refers to a collection of similar cells and the intercellular substances that surround them. Those skilled in the art will appreciate that there are four basic tissue types in the body, 1) epithelium; 2) connective tissue including blood, bone and cartilage; 3) muscle; and 4) nerve.

As used herein, the term "neo-tissue" refers to a composition, which comprises cells, that approximates the shape and function of a native tissue in situ.

As used herein, the term "neo-organ" refers to a synthetic composition, which comprises cells, that approximates the form and function of a native organ in situ.

As used herein, the term "neo-cartilage" refers to a plurality of chondrocytes in a transitional stage of histogenesis. This transitional state of histogenesis is characterized by the elaboration of gene products associated with the extracellular matrix (ie GAG) and a morphology comprising partial encapsulation in lagoons. In one embodiment, this neo-cartilage is manufactured by Histogenics Corporation (Easthampton, MA) under the trademark NEOCART.

As used herein, the term "tissue construction" refers to a population of substantially avascular mixed cells that (in a preferred embodiment) is propagated in a tissue culture system including, but not limited to, the TESS tissue processor. .

The term "neo-tissue construction" refers to tissue constructs formed de novo from cells or tissue by the methods of the present invention.

As used herein, the term "native environment" refers to the in situ biological environment that supports a native tissue or tissue construction.

As used herein, the term "biocompatible" refers to compositions (eg cells, tissues, matrices, etc.) that do not substantially disrupt the normal biological functions of other compositions with which they come into contact. For example, the present invention contemplates a composition comprising a multilayer system that has a physical and chemical structure that is biocompatible with the cells and tissue produced by said system (eg neo-tissues), and that is also biocompatible with cells or tissues in which said system is supplied (and its cells or tissue). In selected embodiments, the present invention also contemplates biocompatible materials that are both biodegradable and non-biodegradable.

As used herein, the term "matrix" refers to a composition comprising a plurality of pores that divide the free space into partially closed interstices in which said interstices are in fluid communication. One type of matrix is a "sponge matrix" comprising a matrix that captures fluid by capillary action. In selected embodiments, the present invention contemplates a composition comprising a cell support layer, wherein said cell support layer comprises a collagen sponge matrix.

As used herein, the term "TESS matrix" refers to a commercial embodiment (a patented composition manufactured by Koken Company LTD., Tokyo, Japan) of the cell support layer described in the present invention.

As used herein, the term "pore size" refers to the average cross-sectional dimension of the interstices in a matrix.

As used herein, the term "functional surface" refers to the surface directed towards a region of a cell, tissue or organ that performs at least one function. For example, in a system for

produce cartilage, the term "functional surface" refers to the surface directed towards the synovium when the system is implanted in a host. Similarly, in a system for producing oral mucosa, the term "functional surface" refers to the surface directed towards the inside of the mouth once the system has been implanted in a host.

As used herein, the term "donor" refers to a human or non-human being from which cells, tissues, blood or an organ for transplantation are taken.

As used herein, a "host" refers to an individual (either human or non-human) in which cells, tissue, blood or organs are transplanted. For example, the present invention contemplates procedures in which cells (eg chondrocytes) are taken from a donor for in vitro multiplication and transplantation into a host (if the donor and the host are the same individual or are different individuals).

As used herein, the word "chondrocyte" refers to a specific type of cartilaginous cell. In vivo, these cells are characterized by defined morphologies and physiologies (observed in situ) compared to morphologies and physiologies in vitro (observed in tissue culture) for these same cells. For example, chondrocytes collected from adult mammalian trochlea substantially do not divide and do not produce substantially any extracellular matrix (ie GAG). In contrast, substantially quiescent chondrocytes isolated from adult mammalian trochlea are activated in vitro (for example when placed in tissue culture) so that a majority of these cultured cells divide and produce, in vitro, gene products associated with the matrix extracellular (ie GAG).

As used herein, the term "suspended chondrocytes" refers to any number of chondrocytes in a fluid. In a preferred embodiment said fluid comprises aqueous type I collagen.

As used herein, the term "supply tissue" refers to the procedure of introducing, inserting, injecting or transplanting cells or tissue from a donor to a host. For example, the present invention contemplates methods in which tissue that has grown in the multilayer system of the present invention (i.e. neo-tissue) is supplied in a host at, for example, the site of an injury (i.e. target lesion).

As used herein, the term "cyclic hydrostatic pressure" (and the abbreviation "CHP") refers to the application of repeated periods (for example two or more) of applied hydrostatic pressure (within a defined load range ) that preferably creates a sine waveform of measured pressure. In a preferred embodiment said CHP is 3.0 MPa at 0.5 Hz.

As used herein, the term "constant pressure" refers to the application of a substantially non-fluctuating pressure load (for example more or less 20% or less, or more preferably 5%

or less) or non-cyclic pressure for a period of time.

As used herein, the term "load" or "load range" refers to a period of applied CHP loading followed by a return to ambient pressure (for example in which no external pressure is applied).

As used herein, the term "resting phase" refers to a variable period of time in which the cells are maintained in culture at approximately atmospheric pressure after exposure to a culture under cyclic hydrostatic pressure.

As used herein, the term "de novo formation" refers to the production of cells (for example, chondrocytes, fibroblasts, fibrochondrocytes, tenocytes and osteoblasts) or tissues (for example cartilaginous connective tissue, fibrocartilage, tendon and bone ) within a support structure (for example multi-layer system, frame or collagen matrix).

As used herein, the term "Type I collagen" is characterized by two alpha 1 (I) chains and an alpha 2 (I) chain (heterotrimeric collagen). The alpha 1 (I) chains are approximately 300 nm in length. Type I collagen is found predominantly in bone, skin (in sheet-like structures) and tendon (in rope-like structures). Type I collagen is further typified by its reaction with the protein core of another connective tissue component known as proteoglycan.

As used herein, the term "type II collagen" is characterized by three identical alpha 1 (II) chains (homotrimeric collagen) present on chromosome 12. Type II collagen in cartilage is presented as thin fibrils in a loose mesh, which interacts strongly with other components (especially proteoglycans). Type II collagen is covalently crosslinked with type IX collagen and other FACIT collagen, which aids in the formation of type II collagen. This molecule is also able to bind and interact with integrin and decorin molecules.

As used herein, "neutralized aqueous type I collagen" refers to type I collagen without charge.

As used herein, "MMP" refers to "matrix metalloproteinase." MMP is associated with degeneration of cartilage in a diseased joint. MMP can be further distinguished as MMP-1, MMP-2, MMP-9, etc.

As used herein, the term "MPa" means MegaPascal. An MPa is equal to 145 psi.

As used herein, the term "mandrel" refers to a tapered, cylindrical shaft, spindle or rod that acts as a core around which the material can be cast by casting, forged, twisted or otherwise modeled.

As used herein, the term "epithelial tissue" refers to the cell layer (usually avascular) that encompasses the free, cutaneous, mucous and serous surfaces, including the glands and other structures derived therefrom. The epithelial tissue has squamous, cubic and / or columnar cells after histological examination. In addition, epithelial tissue can be described as simple, stratified or pseudostratified.

As used herein, the term "connective tissue" refers to tissue that protects and supports the body and its organs, and tissues that bind organs together. Examples of such tissues include (but not limited to) mesenchyme, mucous connective tissue, areolar (loose), adipose, reticular, elastic, collagenous, bone, blood or cartilaginous (for example, hyaline, fibrous and elastic cartilage).

As used herein, the term "muscle tissue" refers to tissue that is characterized by the ability to contract after stimulation and can be categorized as skeletal, cardiac and smooth.

As used herein, the term "nerve tissue" refers to tissue (comprising nerve fibers, dendrites and support tissues) that initiates and transmits nerve impulses.

As used herein, the terms "membrane tissue" and "membranous tissue" refers to a combination of epithelial tissue and connective tissue that forms a membrane. Examples of such membranes include (but not limited to) mucous, serous, cutaneous and synovial membranes.

As used herein, the term "sol-gel solution" refers to a colloidal suspension that can change from a liquid (sol) to a more solid material (gel). In a preferred embodiment, the "sun" is an aqueous collagen suspension that changes, by heat treatment, to a gel.

As used herein, the term "gel" or "gelled" refers to the state of matter between liquid and solid. As such, a "gel" has some of the properties of a liquid (that is, the shape is elastic and deformable) and some of the properties of a solid (that is, the shape is discrete enough to maintain three dimensions on a surface two-dimensional).

As used herein, the term "partially gelled" refers to the state of matter between liquid and solid so that a substance demonstrates more of the properties of a liquid (ie, the shape is elastic and deformable) and less than the properties of a solid (that is, the shape is discrete enough to maintain three dimensions on a two-dimensional surface).

As used herein, the term "solidifies" or "solidified" refers to any process whereby a substance becomes solid, compact or hard.

As used herein, the term "partially solidified" or "partially solidified" refers to any process whereby a substance is made to some degree more compact or hard solid compared to the substance on which it is acted.

As used herein, the words "cure" or "cure" refers to the application of a chemical or physical process to further solidify a gel.

As used herein, the term "treatment medium" refers to any device that can regulate the temperature, atmospheric pressure and / or gas saturation.

As used herein, the term "seeded device" refers to a matrix that contains any number of cells. In a preferred embodiment, said seeded device comprises chondrocytes disposed within a collagen sponge matrix.

As used herein, the term "make gene products" refers to the production of peptides and / or proteins by a cell. In a preferred embodiment, the chondrocytes produce proteins associated with the extracellular matrix of cartilage (including but not limited to GAG).

Detailed description of the invention

The present invention relates to processes for the preparation of neo-tissues and tissue constructions. In particular, the invention relates to the construction of a multi-layer biological structure (for example, any three-dimensional shape) that includes at least one matrix layer seeded with living cells.

I. General design of a system for tissue production and tissue construction

The present invention provides a process for the preparation of a composition comprising a matrix containing cells, originally collected from an organ or tissue in situ, which have subsequently been expanded (by in vitro cell culture) before seeding into said matrix. In one embodiment, this composition, which comprises a seeded matrix, is placed (as a cassette) in a tissue culture apparatus (in an example a tissue processor) so that the cells arranged within said system can expand in number and / or differentiate. It is not intended that the present invention be limited to any specific type of cell. These cells are chondrocytes that multiply and differentiate when grown under conditions that include cyclic hydrostatic pressure.

The present invention is not limited to a specific configuration. In one embodiment, however, the present invention prepares a system comprising two layers.

The cell support layer provides a platform that contains the cells seeded in the system and provides support for said cells as they: a) divide and differentiate (in a tissue culture apparatus) into a neo-tissue and b) when said system is implanted in a host.

The integration layer protects the neo-tissue as it is divided, ex vivo, into a tissue culture apparatus. Once said neo-tissue is implanted in a host, the integration layer is oriented to face a tissue surface native to said host, thereby exposing said neo-tissue to the medium, including humoral factors (e.g., hormones and trophic factors), at the transplant site.

In some embodiments, the system also comprises a protective layer that substantially encapsulates the cell support layer and the integration layer. The multi-layer design of the systems can be manufactured in a variety of ways. In one embodiment, the system may be a sheet. In another embodiment, the system can be formed to approximate the three-dimensional structure of a tissue or organ as seen in vivo including, but not limited to tubes, ellipsoids and claviforms.

Neo-tissues (or "products") propagated in accordance with the procedures listed in the present invention substantially replicate native tissues. Specifically, the systems and procedures of the present invention produce tissues that substantially reproduce the anatomy and physiology of native tissues. In one embodiment, the neo-tissues prepared in the present invention are propagated from cells originally collected from the intended host (for example, they are autologous). In some embodiments the cell support and integration layers of the present invention are biodegradable. In these embodiments, the non-cellular components of the system are biodegraded so that, after implantation in a host, only the neo-tissue is integrated into the host.

As described above, the multilayer system comprises at least two layers, a cell support layer and an integration layer. In selected embodiments, the system also incorporates a protective layer. Each of these layers is described in more detail later. However, the present invention also contemplates the incorporation of complementary layers in the system. These complementary layers may contain a variety of trophic factors including, but not limited to, epidermal growth factor (EGF), transforming growth factor P (e.g., TGF β-1, TGF β-2, etc.), growth factor platelet-derived (e.g. PDGF-AA, PDGF-AB and PDGF-BB), fibroblast growth factors (FGF), insulin-like growth factors (IGF), tumor necrosis factors (TNF), stimulatory factors colonies (CSF), nerve growth factors (NGF), connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), pro-angiogenic or anti-angiogenic factors, and others therapeutic agents including, but not limited to, cytokines, interleukins (e.g., IL-1, IL-2) or other cofactors such as heparin or calmodulin, antibiotics, antineoplastic and antibacterial agents, to further stimulate or control the remodeling tissue ion or to combat sepsis.

In other embodiments, such complementary layers may provide ontogenic or morphogenic factors such as bone morphogenic proteins (BMP 1-8); gene products from the Hedgehog family (hh); gene products of the Hox family; gene products of the Pax family; Notch gene products; osteoprotegerin; and osteoprotegerin binding proteins.

It is important to note that although the general design of the system of the present invention enables a system comprising more than one layer, the present invention also contemplates embodiments comprising only the cellular support layer.

II. The multilayer system components

1. The cell support layer (and "TESS matrix")

The cellular support layer of the present invention (in a specific commercial embodiment called "TESS matrix") comprises a matrix that facilitates three-dimensional propagation of tissue and tissue constructs. The function of the cell support layer is, in part, to provide a growth platform for chondrocytes. Desirable characteristics of the cell support layer include (but not limited to) biocompatibility, hydrophilicity, and neutral charge. In one embodiment, the cellular support layer comprises a matrix (or honeycomb reticular structure) made substantially of type I collagen. In a preferred embodiment said type I collagen matrix in honeycomb is called a "TESS matrix."

It is not intended that this cell support layer be limited to any specific material or formulation. For example, the matrix described in Japanese Patent No. 6,022,744 is suitable for use as a cell support layer as described in the present invention. This matrix is prepared by gelling a solution of acidic aqueous collagen with ammonia gas to form a collagen gel and then lyophilizing said gel to substantially evaporate all of the water, thereby forming a substantially porous dry matrix.

In one example, 300 grams of a 1% aqueous atelocollagen solution (maintained at pH 3.0) is poured into a 10 x 20 cm tray. This tray is then placed in a 5 liter container. An open 50 ml container containing 30 ml of a 3% aqueous ammonia solution is placed next to the tray, in the 5 liter chamber, containing 300 grams of said 1% aqueous solution of atelocollagen. The 5 liter container (containing the open trays of atelocollagen and ammonia) is then sealed and allowed to stand at room temperature for 12 hours. During this period the ammonia gas released from the open container of aqueous ammonia, and confined within the sealed 5 liter vessel, is reacted with the aqueous atelocollagen, thereby gelling said aqueous solution of atelocollagen.

This collagen gel is then washed with water overnight and subsequently lyophilized to produce a sponge-like matrix. This lyophilized matrix is then cut into squares, sterilized and stored under a sterile envelope.

The present invention contemplates matrices prepared in accordance with other protocols. In one embodiment, the matrix prepared according to the protocol set forth in US Patent No. 5,206,028 to Li will be used. In another embodiment, the collagen matrices prepared in accordance with the protocols set forth in US Patent No. 5,656,492 to Glowacki et al.

The pore size of these collagen matrices can be adjusted by varying the pH of the dispersion, the concentration of collagen and the lyophilization cycle (for example, freezing time, temperature range and cycle time). See, U.S. Patent No. 4,522,753 to Yannas et al. Although it is not intended that the present invention be limited to any specific mechanism, adjustment to pore size may be advantageous in promoting the uniform distribution of cells with different morphologies.

For example, chondrocytes cultured in a type I collagen matrix show increased infiltration in said matrix when the average pore size of said matrix was greater than 100 micrometers. See, U.S. Patent No. 6,080,194 to Pachence et al. Although it is not intended that the support layer of the present invention be limited to a specific pore size, in an embodiment of the present invention said pore size is in a range of about 50-500 micrometers. In another embodiment, the pore size is in a range of 100-300 micrometers. In another embodiment, the pore size is about 200 micrometers.

It is not intended that the present invention be limited to a cellular support layer comprising type I collagen. In other embodiments, the cellular support layers of the system are composed of other materials, including (but not limited to): other collagens such as type II and type IV collagen; collagen gels contracted by cells containing other components of the extracellular matrix such as proteoglycans, glycosaminoglycans, glycoproteins (eg, fibronectin, laminin, etc.); elastin and / or bioactive peptide growth factors or cytokines; other biopolymers such as fibrin; synthetic biodegradable fibers composed of such polymers such as polylactic or polyglycolic acids, polycaprolactones or polyamino acids, or their copolymers; and construction structures comprising collagen and polylactic acid.

2. Gel component

In a preferred embodiment of the process of the present invention, the cell support matrix of the present invention acts as a sponge that captures by capillarity (and evenly distributes along the interstices of the matrix) cells suspended in a solution of sol- gel in which said sol-gel solution is in its sol state. It is not intended that the present invention be limited to a specific type of sol-gel. However, preferred features of this sol-gel include, but are not limited to, a viscosity (such as a sun) that promotes easy mixing of cells under conditions that require only nominal shear forces to mix said cells with said gel. In a preferred embodiment this sol-gel is substantially composed of type I collagen and is commercially available (for example, VITROGEN produced by Cohesion Corporation of

Palo Alto, CA.). VITROGEN is a bovine dermal collagen solubilized in purified pepsin (i.e. 99.9% pure) dissolved in 0.012 N HCl and maintained as a sterile solution.

Another preferred feature of this sol-gel is the ability to cure (or change) to a solid form, said solid form characterized by a substantial inability to pour or mix freely. It is not intended that the present invention be limited to any specific mechanism for curing said sol-gel to a solid form (eg, gel). In one embodiment, said curing comprises the application of heat. In a preferred embodiment, the sol gel is cured by application at a temperature of approximately 37 ° C. In other embodiments said sol-gel can be cured to a substantially solid form by exposure to chemical compounds or ultraviolet radiation.

In another embodiment, the present invention contemplates the use of polypeptide vehicle gels.

3. The integration layer

The process of the present invention also contemplates a support matrix, seeded with cells, further comprising an integration layer. Integration facilitates the incorporation (in situ) of said support matrix, seeded with cells, into a lesion or defect in the body. In one embodiment, the integration layer is contacted with an organ or tissue in situ. That is, the integration layer forms an interface between the support matrix introduced (surgically) seeded with cells and tissue or organ in situ native to a receptor.

Depending on the particular type of cells seeded in the support matrix and the tissue in which said cells will be incorporated, the integration layer may provide different functions. In one embodiment, the protective layer protects the seeded matrix with implanted cells from endocrine or paracrine signals produced by the host's native tissue. In another embodiment the integration layer can be configured to facilitate the transmission of endocrine or paracrine signals from the host's native tissue to the matrix seeded with implanted cells.

It is not intended that the process of the present invention be limited to any specific mechanism of action for the integration layer. In one embodiment, the integration layer prevents infiltration of blood vessels and unwanted cells of the underlying bone tissue in a matrix seeded with chondrocytes implanted in a mobile joint of a host.

In preparing the integration layer of the present invention, a dense collagen barrier can be prepared in accordance with the procedure presented in US Patent No. 5,206,028. A porous collagen matrix, which is approximately 4 mm to 10 mm thick, is hydrated using a chamber with controlled humidity (with a relative humidity of 80% at 25 ° C) for 60 minutes. The collagen material is compressed between two sheets of Teflon at a thickness of less than 0.2 mm. The compressed material is then crosslinked in a solution of 0.5% formaldehyde, 1% sodium bicarbonate at pH 8 for 60 minutes. The crosslinked membrane is then rinsed thoroughly with water, and lyophilized for approximately 48 hours. The dense collagen barrier has an internal construction of densely packed fibers that intersect in a multilayer structure.

In another embodiment, the integration layer is prepared from dispersions or solutions based on collagen that are air dried in the form of sheets. Drying is carried out at temperatures ranging from about 4 to 40 ° C for a period of time from about 7 to 48 hours.

In another embodiment, the integration layer is comprised of a copolymer of polyethylene glycol and collagen that slowly absorbs the liquid and biodegrades slowly. The adhesive nature of the polyethylene glycol and collagen copolymer can allow the system to be more effectively placed in, and adhere to, a lesion or defect.

It is not intended that the integration layer listed in the present invention be limited to a copolymer of polyethylene glycol and collagen. For example, in other embodiments, the system integration layer or layers may be comprised of collagen, fibrin, agarose, alginate, chitin, polylactic acid, polyglycolic acid, polylactic and polyglycolic acid copolymers, and collagen and fibrin copolymers. In addition, the integration layer may also include materials to promote, stimulate or inhibit the growth and development of specific tissues and cells.

Examples of such materials include (but not limited to): demineralized bone powder, growth and development factors such as bone morphogenetic proteins; epidermal growth factors; enzymes that stimulate the alteration of the extracellular matrix, such as chondroitinase ABC (US Patent No. 5,916,557); and pro-angiogenic factors such as vascular endothelial cell growth factor (VEGF).

In embodiments of the present invention designed to promote vascularization, the integration layer of the present invention may contain anti-thrombogenic factor such as heparin, or highly negatively charged artificial materials such as styrene-ethylene or butylene-ethylene copolymers that produce a antithrombogenic effect.

In another embodiment of the present invention designed to promote bone tissue formation (eg, in which osteoblasts are planted in the support matrix) the integration layer may contain osteogenic factors and / or demineralized bone powder to stimulate tissue. subchondral in situ.

4. The protective layer

Preferred methods of the present invention also contemplate a support matrix, seeded with the cells, further comprising a protective layer. The protective layer encapsulates the outer surface of a support matrix seeded with cells. The protective layer isolates a support matrix seeded with mechanical and physiological trauma cells which, in some applications, is associated with the implantation of a matrix seeded with cells in a host; simultaneously allowing nutrients to move to said matrix seeded with cells. In one embodiment, the protective layer is applied to the matrix seeded with cells before and / or after said matrix is removed from a tissue processor.

In a preferred embodiment, the protective layer comprises a patented solution, designated CT3, which is manufactured by Cohesion Technologies of Palto Alto, CA that is applied on the surface of a chondrocyte-plated matrix that has been removed after a given cultivation period. in a tissue processor. In one embodiment, a surgeon applies this CT3 in aerosol to subchondral bone in situ. By placing the cell support layer, a surgeon places the polymeric material in the lesion and, without waiting for it to completely polymerize, places the neo-cartilage in position. In this way, the layer directed towards the subchondral bone seals the subchondral bone and keeps the cell support layer (eg, neo-cartilage) in place. The outer protective layer comprising said polymer is then applied in a form that rapidly polymerizes, and is applied in such a way as to keep it below the mobile surface of the joint to prevent friction travel.

In one embodiment, once the system has been supplied or implanted in the target defect or lesion, the protective layer protects the new tissue from forces or processes directed towards it from the functional surface, thereby facilitating the integration of the cellular support with the native tissue surrounding the lesion. For example, when used to protect implanted neo-cartilage, this protective layer can replace other types of protective surgical interventions making it unnecessary to remove a thin layer of the patient's periosteum from a canalicular bone, and its subsequent securing in place. on the system to contain the neo-cartilage.

In a system for the production of neo-cartilage, it is further desirable that the protective layer be positively charged or neutrally charged to chemically contain the natural matrix produced by the neo-cartilage as it develops and produces negatively charged components such as chondroitin sulfate and other glycosaminoglycans.

The protective layer of the system can take different forms. The use of one form against the others depends on the nature of the lesion and the site of the lesion in which the neo-tissue must be delivered, returned or implanted. In one embodiment the present invention contemplates an injury or part of an injury, in situ, with said protective layer to create a liner and subsequently contact a support matrix seeded with cells with said liner.

It is not intended that the present invention be limited to a multilayer system comprising a protective layer composed of any specific material. For example, in one embodiment, the protective layer comprises an adhesive sealant that is applied by the doctor or surgeon after placing the integration layer and cell support layer in the lesion, and is therefore not attached to the system in the time of initial planting of cells. To form an effective seal, it is desirable that the adhesive sealant be sprayed or applied with an instrument such as a large gauge syringe so that the adhesive sealant can be easily handled by the practitioner before polymerization (or fixation) of the copolymer.

It is further desirable that this material (ie sealant) be able to withstand 0.01-15.00 MPa of tension. The material used for this application should be a biologically reliable polymer that will eventually biodegrade in the body. An example of a preferred material is a copolymer (or construction) of polyethylene glycol and collagen. In fact, for embodiments of the present invention in which chondrocytes are planted in a collagen matrix, collagen based sealants are preferred. Some of the advantages of using said copolymer (or construction) in the protective layer include that said copolymer is effective, safe, strong and durable and that it can also be used by the physician as a surgical sealant after delivery of the multi-layer system or matrix (and corresponding cells or neo-tissue) in the target lesion.

In another embodiment, the outer / protective layer comprises a protective layer of a sealant (ie, material) sprayed on the surface of the multilayer system before cultivating said system, and then reinforced with more of the same material once it is locates in the injury.

In another embodiment, any of the fast gelling biocompatible polymer compositions described in US Patent Application Serial No. 20010055615 (corresponding to Serial No. 09 / 293,708) by Wallace (published December 27, 2001) can be used It can be used as the protective layer in the present invention.

In yet another embodiment, the protective layer comprises a rigid, but flexible material, which can be applied by fitting it to a site of injury, effectively sealing the lesion and additionally fulfilling the protective function. Said embodiment may be referred to as a protective coating layer. Examples of materials suitable for use in this embodiment include, but are not limited to, polylactate-polyglycolic acid polylactate or copolymer forms, and a non-woven condensed N-glucosamine structure of varying degrees of acetylation. The present invention contemplates the use of such materials either used individually or in combination. In addition, the present invention contemplates additional embodiments in which the adhesive sealant (referred to above) can also be used together with said protective layer of calage as an extra sealant around the periphery of the inserted system.

III. Tissue culture devices and tissue processors

1. Conventional incubators

The methods of the present invention employ a variety of tissue culture devices. Conventional tissue culture incubators and incubation environments are used to expand (or multiply) cells collected from tissues or organs. In a preferred embodiment, this cell expansion is associated with the removal of dead cells and cellular debris (eg, residual native extracellular matrix). That is, conventional tissue culture techniques can be used to select and multiply a desired population or populations of cells. These expanded cells can then be incorporated into the three-dimensional cell support matrices listed in the present invention. In a preferred embodiment, for example, chondrocytes collected from articular cartilage are expanded in culture. These expanded chondrocytes are then seeded in the solid support matrix to form a seeded matrix. This seeded matrix can then be placed in a tissue processor.

2. Tissue processors

A tissue processor has the ability to regulate / modify tissue culture conditions including the application of constant or cyclic hydrostatic pressure, the recirculation of the medium, and the regulation of oxygen concentration. In a preferred embodiment said tissue processor is the tissue engineering support system (TESS) described in published US Patent Application 20010021529 (corresponding to US Patent Application Serial No. 09 / 796,422) assigned to Takagi Industrial Co. Ltd.

The TESS processor provides an apparatus for culturing cells or tissue comprising: a culture unit having a culture chamber containing cells or tissue and culture medium; a means of applying pressure to apply pressure to the cells or tissue in the culture chamber; and a culture medium contribution medium for the contribution of continuous or intermittent supply of culture medium to the culture unit. (See Figure 1 and the corresponding text of US Patent Application Serial No. 09 / 796,422). The TESS processor may also comprise a control means (for example depressurization valve) for controlling the pressure application medium or culture medium supply medium. Generally, the pressure applied to the cells or tissue is established depending on the type of cells or tissue to be cultured. The TESS processor further comprises a medium capable of absorbing gases such as nitrogen, carbon dioxide and oxygen. In addition, the TESS processor further comprises a hermetically sealed space that includes a heating medium and a humidifying medium.

3. Applications of tissue processors

Certain cells are extremely difficult to grow in three-dimensional (3-D) culture so that they can form a neo-tissue (eg, neo-cartilage). The TESS processor not only facilitates the growth and culture of these difficult-to-grow cells but also allows a mixed cell culture to be grown together. These mixed cell populations can be cultured in the TESS under conditions such that they self-organize in a tissue construction that approaches the histological organization of an organ in situ. Although it is not intended that the present invention be limited to any specific tissue culture mechanism or protocol, the TESS perfuses the medium under pressure, thereby facilitating the supply of oxygen and nutrients into 3-D matrices seeded with cells.

Neo-tissues can be composed of healthy or cancerous human cells. Cancer cells could be used as a model system to explore antineoplasia and allow the researcher to develop, in vitro, the optimal pharmacological regimen before clinical trials.

For example, a 3-D neo-tissue comprised of secretory cells derived from epithelium and stromal cells derived from mesoderm could approximate the morphology of prostate tissue in situ. Said "prostatic type" tissue could allow a researcher to explore, as a prostate tumor repression drug, the many vitamin D analogues.

In addition, these 3-D cultures could be comprised of cancer cells (from a metastatic tumor in an organ) and "normal" cells that are the target for metastasis of said cancer cells, thus facilitating the study of interactions metastatic Such systems would be especially important in cases where cancer mortality does not result from a primary cancer of secretory cells.

epithelial (for example, in prostate or breast cancer), but of bone transformation by interaction with migratory cells of the primary cancer site. Such 3-D culture would be valuable for evaluating the combinatorial pharmacological regimens indicated for prostate metastases (eg, chemotherapeutic agents / androgen repressors such as carboplatin).

IV. Overview of neo-tissue formation

The present invention provides methods for the formation of neo-tissue using the components of the multilayer system described above. More specifically, these components are combined and reacted under conditions such that a viable three-dimensional neo-tissue is produced. The combination and reaction conditions for these components can be varied according to the type of neo-tissue produced and the intended application for this same neo-tissue. In a preferred embodiment, however, neocartilage is produced according to the following scheme.

1. Isolation of chondrocytes from tissue source

Chondrocytes were enzymatically isolated from cartilage collected under sterile conditions from the hind limbs of pigs 6 months of age. The femur separated from the tibia and exposed the head of the trochlea. Strips of cartilage were removed from the trochlea using a surgical sheet. The cartilage was chopped, digested in a 0.15% type 1 collagenase solution in DMEM: 1: 1 F-12 (DMEM / F-12) nutrient mixture with 1% penicillin-streptomycin (P / S ) and stirred gently for 18 hours at 37 ° C. Chondrocytes were collected and rinsed twice with PBS by centrifugation at 3000 rpm for 5 minutes. The chondrocytes were resuspended in DMEM / F-12 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% P / S.

2. Planting of cells in the TESS matrix

Isolated chondrocytes were incubated for a period of five days at 37 ° C in a conventional incubator. The cells were then collected by trypsinization. A cell suspension of 150,000 cells was seeded in 18 μl of VITROGEN solution per matrix (said matrix having an approximate volume of 19 μl) with nine matrices per group. It is not intended that the present invention be limited to a specific cell support matrix size or shape or number of cells. In fact, in one embodiment of the present invention, the seeded matrix can be scaled up to a larger volume (in which approximately 1 µl of the cell suspension described above is seeded in 1 µl of matrix). The matrices of the control group were incubated in an incubator at 37 ° C and the test group was incubated in the TESS. (See, Example 1 and Table 1).

In another experimental example, the isolated chondrocytes were incubated for a period of five days at 37 ° C in a conventional incubator. The cells were then collected by trypsinization. A cell suspension of

300,000 cells in 18 μl of VITROGEN solution per matrix (said matrix having an approximate volume of 19 μl) with eight matrices per group. It is not intended that the present invention be limited to cell numbers or size

or specific cell support matrix form. In fact, in one embodiment of the present invention, the seeded matrix can be scaled up to a larger volume (in which approximately 1 µl of the cell suspension described above is seeded in 1 µl of matrix). The matrices of the control group were incubated in an incubator at 37 ° C and the test group was incubated in the TESS. (See, Example 2 and Table 2).

3. Biochemistry (DMB test)

At the end of the culture, six matrices of each group were used in the biochemistry test. Briefly, the matrices were transferred to microcentrifuge tubes and digested in 300 μl of papain (125 μg / ml in 0.1 M sodium phosphate, 5 mM disodium EDTA and 5 mM L-cysteine-HCl) for 18 hours at 60 ° C. GAG production in the matrices was measured using a modified dimethylene blue (DMB) microassay with shark chondroitin sulfate as a control (reference: Connective Tissue Research, 1982, vol. 9, pp. 247-248)

4. Histology

The remaining matrices of each group were fixed in 4% paraformaldehyde. The matrices were processed and included in paraffin. Sections of 10 μm were cut in a microtome and stained with safranine-O (Saf O).

V. Preferred protocols for neo-tissue growth

The present invention contemplates protocols for the growth (i.e., in vivo, ex vivo and in vitro) of neo-tissues, using the multilayer system component described in the present application. In preferred embodiments, the TESS processor is used to deliver constant or cyclic hydrostatic pressure to the matrix seeded with cells. In this preferred embodiment, these cell-seeded matrices are kept at rest (in an example in a conventional incubator or TESS processor maintained at 37 ° C at atmospheric pressure) after exposure to constant or cyclic hydrostatic pressure.

1. Production of neo-cartilage

In a preferred embodiment, autologous pig chondrocytes are seeded in the cell support matrix and incubated under cyclic hydrostatic pressure at 37 ° C and 5% CO2. In this preferred embodiment, said cyclic hydrostatic pressure is about 3.0 MPa of pressure at 0.5 Hz. In this preferred embodiment the duration of said cyclic pressure is about 6 days followed by a 12-day resting phase in a incubator maintained at 37 ° C at atmospheric pressure. At the end of this resting phase, the matrices were collected for biochemical and histological analysis as described in Example 1 and Example 2.

2. Alternative protocol

In another embodiment, the present invention contemplates an algorithm for the growth (i.e., in vivo, ex vivo and in vitro) of cells, using the components of the present invention, which involves the application of hydrostatic pressure to cartilage isolated in situ ( or chondrocytes adhered to a matrix) and subjected to the regime comprising the cyclic application of hydrostatic pressure for approximately 1-8 hours followed by approximately 16-23 hours of recovery period.

3. Reduced oxygen concentration

In an alternative embodiment the cell support matrix seeded with chondrocytes described above can be grown at reduced O2 concentration (i.e. less than 20% saturation) during neocartilage formation in the TESS processor. Although it is not intended that the present invention be limited to any specific mechanism, the reduced oxygen concentration of cartilage, observed in vivo, may be due to its (normal) lack of vascularization resulting in a lower partial pressure of oxygen (compared to adjacent tissues). In one embodiment, neo-tissues are grown at oxygen concentration between about 0% and about 19% saturation. In another embodiment, said oxygen concentration is about 2% saturation.

SAW. Medical applications

The methods of the present invention prepare tissue constructs that are suitable for use in a variety of medical applications. In one embodiment, the heat-cured cell seeded matrix prepared in the present invention can be implanted in vivo to repair damaged tissue or cells. In another embodiment, the heat-cured cell seeded matrix prepared by the present invention can replace

or increase existing tissue or join together biological tissues or structures as described below.

1. Tissue repair applications

It is not intended that the use of the methods of the present invention to prepare tissue be limited to repair, replacement, reconstruction or augmentation of any specific tissue (or cellular) type. For example, in one embodiment, damaged cartilaginous tissue is repaired, replaced, rebuilt or augmented. In another embodiment, tissue defects caused by tumor, trauma, infection or congenital malformation (involving various tissue or cell types) are repaired, replaced, reconstructed or increased.

A. Cartilage repair

Although the present invention is useful in procedures for the repair of full-thickness cartilaginous defects, it can also be directed to the repair of partial thickness defects. In addition, the methods of the present invention can be combined with techniques for forming molds of defective areas so that custom-formed neo-tissues can be produced, which are adapted and adjusted dimensionally for the defect of a particular patient. In addition, the systems and methods of the present invention can be used to create complete cartilaginous structures such as menisci, or they can be used to create materials for partial or total replacement of an articular surface. In other embodiments, cartilaginous neo-tissues (ie, neo-cartilage) may be available in accordance with the present invention to combine with alloplastic materials to form artificial joints. In one embodiment, the cartilaginous neo-tissues (ie, neo-cartilage) described in the present invention will repair and / or replace tendons and ligaments. In a specific embodiment said ligament is the anterior cruciate ligament (ACL). In another embodiment, the cartilaginous neo-tissues (ie, neocartilage) described in the present invention will repair and / or replace vertebral discs.

The methods of the present invention can be adaptable to neo-tissue construct formation by combining, for example, formed cartilage and formed bone to be used for replacement of a complete joint surface including subchondral bone, or will be used for replacement of a small covered bone. of cartilage as it can be found on the wrist. Either used to form cartilaginous neo-tissue, or to form neo-tissue constructs, these compositions can be molded into implantable structures for total joint replacement and for replacement of half joint in small joints such as those in the hand

or the temporomandibular joint.

In another embodiment, these procedures can be used to create a full-thickness, implantable cartilaginous neo-tissue to place in a joint that supports a large weight such as the knee. Arthroscopic procedures can be used profitably in the collection of native chondrocytes to incorporate into a neotechid formed by these compositions and procedures.

A first arthroscopic procedure will be used to assess the extent of the potential cartilaginous defect and to collect native chondrocytes for culture and subsequent delivery to the cell support matrix prepared by the present invention. Based on a mold formed at the time of the first arthroscopic procedure, or based on measurements made during the procedure, a dimensional model of the anticipated defect will be formed and the neo-tissue will be formed to fit this model in size and shape.

A second arthroscopic procedure will be carried out to debride a damaged area of the native cartilage in situ and replace the damaged area with the neo-cartilage prepared in accordance with the present invention.

Other adaptations of conventional surgical techniques will be employed at other anatomical sites to collect native cartilage cells, to debride damaged cartilaginous tissue, to create a defect within the host's articular surface (for example including a complete excision of a joint surface or a partial excision of a joint surface), to implant neo-tissue in the host defect and to conserve the neo-tissue at that site.

B. Repair of tissue defects

Defects caused by tumor, trauma, infection or congenital malformation are well known in medical and surgical techniques. Reconstruction of such defects may require replacement of full thickness tissue, or may require replacement of tissue constructions. For example, in one embodiment, it may be necessary to reconstruct an excision taken from the lower eyelid (for example, in the case of a basal cell carcinoma) by an extensive flap procedure that "employs" adjacent tissues or upper eyelid tissues. Alternatively, graft procedures can be used using the other eyelids or the nasal septum as donor sites (despite the fact that there may be donor site problems, and that some local tissue rearrangement will be required in the reconstructed area).

The present invention can be adapted to repair these defects. For example, a neotisular construction can be placed that provides both neo-cartilage and a covering layer (conjunctive or epidermal) in said defect to simplify the reconstruction task. As an alternative, a neotech that comprises the three layers of eyelid tissue could omit the need for any substantial local tissue rearrangement. A neo-tissue comprising neo-cartilage would obviate the need to pick up a cartilage graft from another site with the consequent risks to the donor site.

In another embodiment, a nasal septal deformation defect will be repaired using neo-cartilage. The repair of persistent fistulas after cleft palate procedures may also be susceptible to treatment with the neo-cartilage described in the present invention. These fistulas caused by cleft palate repairs may require reconstruction using a tissue construction with two outer mucous layers that cover an internal bone layer.

In another embodiment, the neo-tissues prepared in the present invention will also adapt to difficult reconstructive problems. Using the methods of the present invention, oral mucosa could be produced in significant amounts to fill or coat intraoral defects with specialized tissue having functional characteristics appropriate for intraoral localization. Reconstruction of other specialized areas such as nail bed and articular cartilage for large and small joints can also be performed using the compositions and methods of the present invention. The present invention may also provide reconstructive solutions for damaged temporomandibular joint or may be applied to repair degenerative diseases such as rheumatoid arthritis.

C. Ligament and tendon

The methods of the present invention will be adapted to the production of tendon and ligaments in vitro. In one embodiment, this will be promoted by stretching ligament and tendon progenitor cells, over a framework, ex vivo during exogenous ligament or tendon development. The ligaments contemplated by the present invention will be derived from ligament progenitor cells, mesenchymal stem cells, or fibroblasts especially fibroblasts derived from the sheath cells surrounding the ligament and tendon, or fibroblasts or tenocytes derived from the ligament or tendon themselves. Autologous ligaments and tendons can be used to replace damaged ligaments and tendons in the patient's own body without fear of immune response or other adverse responses.

The stretched frame, referred to above, can be considered another layer of the multilayer composition described in the present invention. This framework will allow progenitor cells to stretch ex vivo (for example in a modified TESS processor) to apply a physical stimulus that causes them to produce proteins and matrix components that are consistent with true neo-ligaments or neo-tendons.

In one embodiment, the stretched frame will consist of a fibrous material that can withstand tensile stresses that are within the range of tensile stresses to which the cartilage and ligament are normally subjected over the course of life. More specifically, the fibrous material can withstand a range of tensile stresses that are necessary to induce said cells to start producing normal gene products in the correct relationship that differentiates ligament and tendon from other tissues or organs, and allows these products genetics take orientation and fitness, so that a ligament can be developed

or healthy and morphologically reliable tendon in said framework. In addition, the fibrous material should have a degree of elasticity that can accommodate the amount of progenitor cell stretching required to induce said cells to form neoligament or neotendon.

The ligament and tendon developed on said framework will acquire a tensile strength, elasticity and structure to support bone and muscle in vivo, which is equivalent to the tensile strength and ligament and tendon structure developed within the body during the normal course of Growth and differentiation. The framework is also designed so that once the neoligament or neotendon is sufficiently functional, the framework reintroduces the ligament or tendon into the lesion and supports it until it is integrated into the body. In one embodiment the frame will degrade after implantation.

In one embodiment, the cells located in the framework will be selected from cells in a stage in their development life so that (when they are located in the framework and are subjected to the physical and chemical conditions of a three-dimensional tissue culture apparatus designed to induce differentiation of cartilage and ligament) these cells produce substantially all gene products made by morphologically reliable ligaments or tendons.

In one embodiment, the framework is selected from a variety of materials, including but not limited to, polyesters (including polyalkinoates), polyglycolate, polylactates, biodegradable suture materials. Other materials may also be suitable as long as these materials can be safely degraded within the body, and as long as the degradation products do not interfere with the normal metabolic function of the cartilage and ligament or the surrounding tissue.

It is necessary that the physical structure of the frame be consistent with its intended function (for example the uniform application of tensile force at the cellular (micrometric) level). These tensions are very mild compared to the tensions to which the mature cartilage or ligament is subjected. It is also important that the fibrous structure resists rupture at the required tensile forces. In one embodiment, fibers of 1-10 micrometers in diameter will be entwined in a variety of interlacing patterns including, but not limited to, a solid interlacing, a hollow center interlacing (such as Chinese puzzle interlacing) so that the application of tensile force at the ends of the frame produces a uniform stretch of the surface of the frame. In order to produce a ligament with its own bone anchor points, it would be possible to incorporate such substances as demineralized bone powder into the polar ends of the framework, which would be induced under the correct physical stimulus that would form bone to incorporate into the existing bone when it is placed in situ in the body.

D. Other applications

The methods of the present invention can also be used as a model to elucidate the mechanism of certain diseases. For example, in one embodiment, the multilayer systems of the present invention can also be used as models for the study of physiological or pathological conditions in vitro. For example, in one embodiment of the invention the system can be used as a model for the blood brain barrier. In another embodiment, a three-dimensional mucosal epithelium culture (produced using the compositions and methods of the present invention) can be used as a model system for studying herpes virus or papillomavirus infection. Such a model system could be used to test the efficacy of antiviral drugs.

Alternatively, the methods of the present invention can be used to study the pharmacokinetics of various drugs and growth factors. For example, the neo-tissues prepared in the present invention can also be used for in vitro exploration of cytotoxic compounds, allergens, growth factors and regulators, pharmaceutical compounds. For this purpose, the neotejido can be maintained in vitro and exposed to the test compound. For example, the activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture. This can easily be evaluated by known vital staining techniques.

The effect of growth factors or regulators can be assessed by analyzing the cellular content of a neotech, for example, by total cell counts and / or differential cell counts. This can be achieved using conventional cytological and / or histological techniques including the use of immunocytochemical techniques that employ antibodies that define type-specific cell antigens. The effect of various drugs on normal cells cultured in the three-dimensional system can be evaluated. For example, drugs that increase the formation of red blood cells can be tested in three-dimensional bone marrow cultures. Drugs that affect cholesterol metabolism, for example by reducing cholesterol production, could be tested in the three-dimensional liver system. Three-dimensional cultures of tumor cells can be used as model systems to test, for example, the efficacy of antitumor agents.

The methods of the present invention can also be used in the diagnosis and treatment of malignant tumors and diseases. For example, a biopsy of any tissue (for example, bone marrow, skin, liver, etc.) can be taken from a patient suspected of having a malignant tumor. If the biopsy cells are cultured in the system of the invention, the malignant cells can be expanded by cloning during the proliferation of said culture, thereby increasing the chances of detecting a malignant tumor and, therefore, increasing the accuracy of the diagnosis and punctuality of treatment. Said application of the present invention may be especially useful in diseases such as AIDS in which the population of infected cells is impoverished in vivo. In addition, in that case, the patient's culture could be used in vitro to explore compounds (for example cytotoxic or pharmaceutical compounds) to identify those that are most effective (i.e. those that destroy malignant or diseased cells, but avoid normal cells) . These agents could then be used to treat the patient therapeutically.

VII. Alternative realizations

Although certain embodiments of the multilayer system for producing neotextiles or neotextile constructions comprise a three layer construction, the present invention also contemplates multilayer systems comprising more than three layers.

For example, by producing a neotextile or neotextile construction, the layers of the multilayer system may be arranged to include: a lower layer of tissue attachment; a second layer that supports a biological matrix, in which a first type of tissue cells resides; a third integration layer deployed on the second layer to integrate the second layer with an additional layer of biological matrix, in which a second type of tissue cell resides; and a protective top layer disposed on the additional layer of biological matrix. Such an arrangement could provide a tissue construction that includes a layer of cartilage protected by a layer of mucosa. Preferably, the integration layer would be formed of a material that would enhance the normal union of the cartilage layer and the mucous layer so that their unitary behavior under physiological conditions would replicate the behavior of the native tissue.

More complex structures are also contemplated with multiple cell support matrices seeded with cells of at least two tissue types, with integration layers located between each matrix. For example, a tissue construction of the nasal septum (according to these principles) could be made with an external mucous layer, an internal cartilaginous layer and a second external mucous layer.

In another embodiment, a neo-woven construction can be produced in accordance with the present invention that includes the three layers of the nasal septum.

In other embodiments, the neotides prepared by the present invention can be molded in a variety of ways. For example, tubular structures such as blood vessels, channels or other specialized ducts (for example, ureter, esophagus, etc.) can be formed by sculpting a neotejido around a mandrel or other tube-shaped structure.

In other embodiments the methods of the present invention could be used to create luminal, tubular or cylindrical structures such as, for example, a multilayer blood vessel.

In another embodiment, the methods of the present invention can be used to produce a replica of a tendon or ligament (i.e. neotendon or neoligament). The multilayer system of the present invention can be formed within an external guide tube to produce a structure that functionally replicates a tendon. To mold a neoligament, a cell support matrix seeded can be inserted into an external guide tube that would act as a modeling mold for the neoligament. In another embodiment, a mandrel that slowly degrades using a material such as lactic polyglycolic acid (PGLA) can be employed.

In yet another embodiment, the methods of the present invention can be used to form a tissue replica of the oral mucosa (i.e. neomucosa). The system for forming oral mucosa may include a matrix to support the growth and differentiation of keratinocytes, and may also include a protective layer directed towards the functional surface of the oral mucosa (ie towards the oral cavity). Said protective layer would be constructed to protect the underlying cellular support layer from the chemical and enzymatic impact of contact with a saliva, as well as physical forces inherent in the intraoral anatomical location.

The system for forming oral mucosa may also include an integration layer adapted to facilitate the union of the cells formed in the cell support layer with the underlying bone. The integration layer may, for example, include osteogenic factors or demineralized bone powder to stimulate and induce the development of bone ligament and periosteum. As another example, this layer may comprise cadaveric dermis included in (or stratified with) osteogenic factors or demineralized bone powder.

In another embodiment, the methods of the present invention can be used to form a replica of skin tissue (eg neoepidermis or neodermis). For example, a cell support layer can be provided that allows the growth and differentiation of keratinocytes into stratified, keratinized epithelium. Attached to said cell support layer there may be an integration layer that also contains fibroblasts,

growth and appropriate nutrient materials capable of producing a tissue replica of the dermis (ie neodermis). The integration layer can be further adapted to join the replica of skin tissue with its underlying tissue bed.

A skin tissue can have numerous medical uses. For example, said neotexture can be used in acute or reconstructive burn surgery, in the reconstruction of excisions, or in the treatment of chronic wounds or pressure sores.

In a further embodiment, the multilayer system of the present invention is molded into an in vitro tissue construct to explore cytotoxic and pharmaceutical compounds, and to evaluate biological responses to cytokines and growth / regulatory factors. For example, a tissue construction that approximates the shape and function of a prostate gland of a human or non-human animal (i.e. a neoprostate or neogland as characterized by a compound of stromal and glandular cells may comprise a system, which it includes a protective layer and multiple layers of cellular support.In said embodiment, the protective layer can act to contain the neo-organ within a desired volume and help preserve its shape and integrity.This system may optionally have an integration layer. of integration can anchor the system, for example, in the in vitro culture substrate used (for example a culture plate, flask, slides, etc.) Each cell support layer can provide a main cell type present in (and collected de) the native prostate gland Examples of such cell types include, but are not limited to, secretory epithelium of the prostate gland, mu Smooth stroma muscle and fibrous connective tissue. To form a neo-organ with dimensions that approximate those of the native prostate gland, a multi-layer system can be molded to include a protective layer, an optional integration layer, and a plurality of cell support layers. Said plurality of cell support layers may comprise, for example, several sets of three cell support layers stacked together and deployed within a single system. The combination of cell types can approximate the structure and function of the prostate gland in situ.

Experimental part

The following examples serve to illustrate preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

Example 1

This example provides a case in which tissues were formed in vitro in an embodiment of the present invention. Briefly, articular pig chondrocytes (sAC) were isolated enzymatically from cartilage with type I collagenase. The cells were suspended in collagen (VITROGEN) as described above and introduced by capillary into the honeycomb sponge element of the cell support matrix (for example the TESS matrix). The cells were incubated on their three-dimensional support (i.e., the TESS matrix) at 37 ° C, 5% CO2 and 20% O2. After 24 hours, some of these cell matrices (i.e. cell support layers comprising the test group) were transferred to the TESS ™ processor and incubated at 3.0 MPa of cyclic hydrostatic pressure as described above for 6 days. followed by a 12-day rest phase.

The control group comprised of TESS matrices seeded with chondrocytes was incubated for 18 days at 37 ° C, 5% CO2 and 20% O2 and at atmospheric pressure.

At the end of the culture period, the matrices were collected for biochemical and histological analysis as seen in Table 1. For biochemical analysis, the production of glycosaminoglycans (GAG) was measured using a modified dimethylmethylene blue (DMB) microassay. Figure 1, described in the "Description of the Drawings" of the present specification, represents, in graphical form, the data set forth in Table 1. For histological analysis, the formation of neotejido was measured by staining with Safranin-O. The histology of the control group is illustrated in Fig. 2A. The histology of the test group is projected in Fig. 2B.

Table 1

Group (n = 8 matrices per group)

Pressure Conditions Total days in cultivation GAG production (μg / ml) (mean +/- DT) (n = 6 matrices)

In TESS processor (Cyclic Pressure 3 MPa, at 0.5 Hz)

In incubator (Atmospheric Pressure)

Control

- 18 days 18 4.71 ± 0.71

Test

6 days 12 days 18 14.11 ± 1.10

NOTE: 1) All cultures were incubated at 37 ° C, 5% CO2 and 20% O2. 2) In TESS culture, the flow rate of the medium was 0.05 ml / min. 3) Two matrices of each group were collected for histological analysis.

Example 2

This example provides another case in which tissues were formed in vitro in another embodiment of the present invention. Articular articular pig chondrocytes (sAC) of cartilage with type I collagenase were isolated enzymatically. The cells were suspended in collagen (VITROGEN) as described above (eg.

5 300,000 cells in 18 μl of VITROGEN) and were introduced by capillarity in the honeycomb sponge element of the cell support matrix (ie the TESS matrix), each matrix having a volume of approximately 19 μl.

The cells in their cell support matrix were incubated at 37 ° C, 5% CO2 and 20% O2. After 24 hours, some of these cell matrices (the test group) were transferred to the TESS ™ processor and incubated at 3.0 MPa of cyclic hydrostatic pressure as described above for 6 days followed by incubation for 12 days.

10 at atmospheric pressure at 37 ºC, CO2 5% and O2 20%.

The remaining cell matrices comprising the control group were incubated at atmospheric pressure for 18 days at 37 ° C, 5% CO2 and 20% O2.

At the end of the culture period, the matrices were collected for biochemical and histological analysis as seen in Table 2. For biochemical analysis, the production of glycosaminoglycans (GAG) was measured using a microassay.

15 of modified dimethylmethylene blue (DMB). Figure 4, described in the "Description of the Drawings" of the present specification, represents, in graphical form, the data set forth in Table 2. For histological analysis, the formation of neotexture was measured by staining with Safranin-O. The histology of the control group is illustrated in Figure 3A. The histology of the test group is projected in Figure Fig. 3B.

Table 2

Group (n = 8 matrices per group)

Pressure Conditions Total days in cultivation GAG production (μg / ml) (mean +/- DT) (n = 6 matrices)

In TESS (Cyclic Pressure 3 MPa, at 0.5 Hz)

In incubator (Atmospheric Pressure)

Control

- 18 days 18 19.24 ± 2.14

Test

6 days 12 days 18 25.44 ± 1.37

NOTE: 1) All cultures were incubated at 37 ° C, 5% CO2 and 20% O2. 2) In TESS culture, the flow rate of the medium was 0.05 ml / min. 3) Two matrices of each group were collected for histological analysis.

Claims (12)

1. A procedure for preparing an implantable tissue construction, said procedure comprising:

(to)

 provide an implantable multilayer structure comprising:

(i)

 an integration layer; Y

(ii)

 a cell support layer comprising a sponge matrix, the cell support layer being operatively attached to the first integration layer; Y

(b)

 introducing a solution comprising chondrocytes suspended in said multilayer structure to create a seeded device; Y

(C)

 culturing said seeded device under conditions comprising a cyclic hydrostatic pressure with a resting phase at atmospheric pressure for one to sixty days.

2. The method of claim 1, wherein the implantable multilayer structure further comprises: (iii) a protective layer that insulates an outer surface of the cell support layer, the protective layer being operatively attached to the cell support layer and the integration layer.

3.

 The method of claim 1, wherein said chondrocytes are suspended in an aqueous Type 1 collagen solution.

Four.

 The method of claim 3, wherein said solution is a sol-gel.

5.

 The method of claim 4, wherein said sol gel is at least partially gelled.

6.

 The method of claim 5, wherein said culture results in the activation of said chondrocytes to make gene products associated with an extracellular matrix.

7.

 The method of claim 1, wherein the resting phase is four to twenty one days.

8.

 The method of claim 1, wherein said culture is performed under the cyclic hydrostatic pressure of 3 MPa at 0.5 Hz for six days followed by twelve days of the resting phase.

9.

 The method of claim 1, wherein said sponge matrix of said cell support layer is composed of a Type 1 collagen in biodegradable honeycomb.

10.

 The method of claim 1, wherein the integration layer comprises a copolymer of polyethylene glycol and collagen, collagen, fibrin, agarose, alginate, chitin, polylactic acid, polyglycolic acid, a polylactic acid copolymer, a polyglycolic acid copolymer, a collagen copolymer or a fibrin copolymer.

eleven.

 The method of claim 9, wherein said Type 1 collagen in biodegradable honeycomb is prepared from Type 1 collagen by gelling an acidic aqueous solution of Type 1 collagen with ammonia gas to form a collagen gel and lyophilizing said gel to give a dry porous matrix.

12.

 The method of claim 1, wherein said seeded matrix is grown in reduced oxygen concentration of less than 20% saturation.