Method for treating a patient using a cultured connective tissue construct

Field of the Invention

The This invention is in the field of tissue engineering, and in particular, it relates to the use of a cultured living connective tissue construct in surgical correction indications.

Background of the Invention

injury the disc are for the patient painful, debilitating and costly. The intervertebral disc is located between the articular surfaces of adjacent Vertebral body. The disc consists of concentric layers of colleagues fibers the fiber ring (annulus fibrosis) and the central gelatinous core (nucleus pulposus), which it surrounds. The nucleus consists of a viscous fluid connective tissue matrix and cells. The cells of the nucleus pulposus, physalide cells are in irregular aggregates on extracellular Matrix consisting of ground substance, distributed. On the outside the vortex are the fiber ring and the vortex by peripherally Longitudinal bands supported, the parallel to the spine extend.

The Disc works as a kind of hydraulic shock absorber, wherein the nucleus pulposus acts as the hydraulic fluid. With increasing age colleagues fibers of the fiber ring thinner and weaker. Of the Nucleus can break the ring back and the nerve endings press together, a condition which usually is known as a "slipped disc". The are in throat / neck injuries intricate slices between the fifth and sixth (C5-C6) vertebra and the sixth and seventh (C6-C7) Vortex, and injuries in the lower back are the fourth and fifth Lumbar (L4-5) or the fifth lumbar vertebra and the first sacral vertebra (L5-S1) most affected. In cases where the disk does not rupture, a nerve compression, by a causes bone spur from a degenerative disease Scheibener will. The bone spur protrudes into the intervertebral foramen and compressed the nerve ending, which occur on a continuous, periodic resulting chronic pain.

If a surgeon the whole or parts of a broken (ruptured) Disc from the back away, he enlarged the opening in the annulus through which the ruptured disc and loose Disc tissue can be removed in the gap. healed the opening formed Never what both the susceptibility opposite to another disc breaking through the existing opening as also against an inflammatory cascade elevated, resulting from the exposure of the disk inside relative to the epidural space. The inflammation and fibrosis occur in the intervertebral space, and often lead to changes in the surrounding tissues including the vertebrae, hyaline plates, the dura mater and the spinal cord.

Each Year about 200 000. discectomy performed. A biological repair of the ring hole or -defektes is essential to the standard care of patients with spinal disc disease to improve. The<patcit><text>WO 98/40111</text></patcit> describes a biomechanical implant which comprises at least two matrix components , wherein the first matrix component of collagen with a porous Macro structure suitable tensile forces or shear to resist exists, and the second matrix component is a hydrogenated is alginate gel which substantially the porous macrostructure of the first component fills and a swelling pressure exerts wherein the implant additionally has a population of cells, cartilage cells, Fibrochondrozyte, contains fibroblasts or osteoblasts or precursor thereof. The<patcit><text>WO 91/16867</text></patcit> describes a prosthetic spinal disc, which can be implanted in a human skeleton can, and which as a scaffold for a can serve regrowth of disc material. The<patcit><text>WO 99/04720</text></patcit> describes a A method of using a cell with a hydrogel suspension Treating a herniated disc by implanting a cell suspension in a patient, whereby a cell formed with a hydrogel is, which of the at at least one surface of the fiber ring and Disc is adhered, wherein said cells cartilage cells, fibroblasts or Osteoblasts. The present invention addresses these repair Fiber ring by using a cultured connective tissue construct, which, when applied surgically to the defect in the ring is, the opening closes and a barrier is formed, an inflammation and another Bandscheibenbersten to prevent.

Summary

The This invention relates to a method for repairing a injured intervertebral disc of a patient using a cultured connective tissue construct. The method includes modifying the opening in the fibrous ring of the Disc that has been generated by the bursting of the disc, and selective removal of the nucleus pulposus, and then a Transplanting the cultured connective tissue construct into the opening. The closure of the fiber ring is important in order to prevent re-rupturing of the disc, and to the inflammatory response and to alleviate the epidural fibrosis in the cavity that a too cause nerve damage can. The cultured tissue-Kon&lt;?page 3?&gt;struct is a sophisticated living tissue with connective tissue cells and an extracellular matrix, such as Collagen. Due to the fabric-like Features of the cultured tissue construct is bioremodellierbar, which means that it is able to join a Zellrepopulation, namely by the ingrowth of host cells, neovascularization and the reorganization and replacement of matrix components of implanted construct by host cells and enzymes.

DESCRIPTION OF THE FIGURES

<figref idrefs="S24">1</figref> shows an apparatus for forming a connective tissue Equivalentes.

Detailed description

The Invention is directed to a method of tissue replacement and -reparieren using a cultured connective tissue construct. the Connective tissue construct comprises collagen and living cells and Therefore bioremodellierbar, which means that if a patient with a need is transplanted to a tissue replacement and repair, it immediately functions as a replacement tissue, while at the same time begins with the patient's tissue at the Implantierstelle to integrate to form new tissue. In the most preferred embodiment The invention is directed to a method for repairing the Annulus of the intervertebral disc, after a Diskektomieoperation, wherein open the fiber ring was, with a cultured connective tissue construct.

the cultured connective tissue construct is used to the annular wall after removal of damaged Disc tissue, such as the nucleus pulposus and the annulus, close, for correcting spinal deformities after multiple discectomy in reconstructive spinal surgery or complete or partial removal of an injured disc due to a Injury such as a Ringriß, a ring damage, a ring breaking, a herniated disc, a herniated disc or Bandscheibenperforation. A closure of the Ring by this method seals the cavity to the inflammatory response reduce and mitigate postoperative epidural fibrosis, usually the removal of the tissue from the cavity follows. The closure of the Fiber ring also prevents a subsequent herniation in the remaining disc tissue, which in the intervertebral space remains, and slowed further disc degeneration.

the The method further comprises closing the fiber ring using a cultured connective tissue construct after implantation of a disc replacement. A Support the vortex with a disc replacement or a spacer to distance washers is occasionally performed, so that the disc can maintain its function, and a Nerve damage to prevent. The replacement is the intervertebral space through an opening provided in the fiber ring, either of the same, the for to access the damaged disc was used or an additional opening. Disc replacements are used to maintain the normal disc space height, a Colliding to avoid the posterior facet joints. A pressure on the spinal nerves can cause pain or numbness effect in the area of ​​their distribution. transmit Functional Spacer implants and attenuate Compression and torsion and stabilize the joint. Disc replacement materials, metal, Metal alloys, synthetic solids and synthetic fabrics include hydrogels and as long as it is biocompatible with the tissue of the patient.

Around the damaged remove disc is any technique in the prior the art of intervertebral disc removal usable, including, but not limited to discectomy, discectomy, laminectomy, or Laminotomy. dependent on the type and degree of disc injury can on the disc on any number of routes including anterior, posterior or be accessed posteriorlateral. The method for accessing the disc includes laparotomy-discectomy techniques. On Access by the fiber ring includes uniportale and multi portal approaches, which either one or more openings in the fiber ring for removal of the disc material and to implant and positioning an intervertebral spacer prosthesis are made.

After this the discectomy has been performed in the patient, the cultured Connective tissue construct in the in the fibrous ring at a surgical Operation generated opening introduced to seal the inner area of ​​the disc. Dependent on the size of the patient and the size of the hole can in the ring one or more parts may be used to plug the opening, these pieces through a built-up of the surrounding tissues to pressure their Body be kept. Optionally, and in addition is a refined Connective tissue construct on the opening and applied, and at this point along the tissue boundary of the opening the construct fixed. If the construct over the opening in the ring applied was the construct can by a seam or a surgical Adhesive, as in&lt;?page 4?&gt;example fibrin glue or other biologically compatible, used in surgical operations adhesives, are attached.

As soon as the method of implanting the construct has been completed, takes the construct immediately a function as a barrier between the Cavity on the inside of the nucleus pulposus and the exterior of the fiber rings.

at another preferred embodiment, the invention, the cultured connective tissue construct in a surgical repair of the central nervous system used. The cultured Connective tissue construct is used to seal the dura mater, which the brain and spinal cord covers. The layer construct is an appropriate form for closing Duradefekten after an injury or after spinal or cranial surgery, to a separation of the interior of the central nervous system from other body cells and tissues to maintain.

the cultured connective tissue construct is bioremodellierbar, meaning that it is capable of a Zellrepopulation by ingrowth host cells, neovascularization and reorganization and replacement of matrix components of the implanted join construct by host cells and enzymes. The graft prosthesis reserves its functional characteristics while the patient's cells the entire or substantially remodel the entire construct, to integrate it with the host tissue, and functioned as such it as an analog of the tissue it repairs or replaces.

the cultured connective tissue construct comprises connective tissue cells by an extracellular matrix, primary are made of collagen, bound. The aspect of the extracellular matrix of the Construct can respect the organization and composition vary, yet a connective tissue construct for use in the inventive method be. The connective tissue construct can a contracted collagen network be with fibroblasts as described by Bell in the <patcit><text>US Pat. No. 4,485,096</text></patcit> or by Kemp, et al. in the<patcit><text>US Pat. No. 5,536,656</text></patcit> described was where the contracted collagen network on an acellular collagen gel is arranged. However, this embodiment does not form part of the present invention, it provides merely provide background information represents, for which the understanding the invention is useful are. Another construct for the use in the process is a bioengineered tissue construct from cultured cells and endogenously produced extracellular matrix components without the requirement of exogenous matrix components or network support or Scaffolding elements, in the PCT publication No. <patcit><text>WO 00/29553</text></patcit> from Murphy, et al. has been described. Connective tissue constructs such as those the synthetic or bioresorbable tissue element with cultured comprise fibroblasts ends, enclose it with endogenously produced Matrix, as in the <patcit><text>US patents No. 5,580,781</text></patcit>. <patcit><text>5,443,950</text></patcit>. <patcit><text>5,266,480</text></patcit>. <patcit><text>5,032,508</text></patcit>. <patcit><text>4,963,489</text></patcit> Naughton, et al. described is, can also be used, and a fibrous collagen matrix with cultured cells therein as described by <patcit><text>US Patent Nos. 4,505,266</text></patcit> and <patcit><text>4,280,954</text></patcit> Yannas, et al. Xenogeneic materials, such as de-epidermalisierte and de-zellularisierte dermis and other flat layer fabrics were freed of antigenic determinants and cellular debris, as can a matrix component are used, the cells with nichtallogenen to re-populate the construct is cultured.

cell types for the Use in the invention are fibroblasts, or at the most most preferred embodiment, human fibroblasts. Human Fibroblastzellstämme can of a number are obtained from sources including but not limited to the foreskin of male newborns Dermis, a tendon that Lun ge, umbilical cords, cartilage, the faucet tube Corneal stroma, the mucosa and the intestines. A preferred fibroblast type is obtained from the dermis. The human cells may include, but are not limited to: fibroblasts, smooth muscle cells, Chondrocytes or other connective tissue cells of mesenchymal origin. Embryonic Progenitor cells, such as mesenchymal stem cells, can in the invention Manufacture of cultured construct used and can either be induced in vitro or in vivo in order to give the desired developing tissue. It is for the skilled artisan that the cultured connective tissue construct either by intentional addition or as a result of Culturing fibroblasts from primary sources contain other cells may, in the connective tissue, or other extracellular matrix components being found. It is preferred but not required that the origin of the used in the production of the tissue construct matrix-producing cell is derived from a tissue type, the or the application to the cultivation method of the invention is similar or pretending. For example, a construct with fibroblasts is cultured a living tissue construct obtain, or myoblasts for a skeletal muscle construct. In producing a tissue construct more than one cell type can be used will.

Although human cells in USAGE&lt;?page 5?&gt;development of the invention are preferred, are methods of the invention in which cells to be used is not limited to cells from human sources. Cells from other mammalian species include, but are not limited to coming from the horse, from Dog originating, porcine, bovine-derived, cats derived, derived from goats and sheep originating from sources can be used. Mouse cells, cells from rodent sources can also be used. Cell donors can vary in development and age. Cells from donor tissues of Embryos, neonates, or older individuals, including Adults are obtained.

Furthermore can genetically modified Cells transfected without outside action, chemically or virally are used in this invention. For such embodiments, the use more than one cell type, mixtures of normal used and genetically modified or transfected cells be, and mixtures of cells of two or more species or Tissue sources, or both can be used. Recombinant or genetically altered cells can be used in the production of the tissue construct to a to produce tissue construct that, as a drug delivery implant can be used in a patient, the increased level of natural Cell products or treatment with a therapeutic agent needed. The cells can recombinant cell products, growth factors, hormones, peptides or proteins for a continuous period or produce needed if this due to the states is signaled biologically, chemically or thermally in the culture. The cells can further genetically modified be proteins or different types of extracellular matrix components to express what either be "normal" but expressed at higher levels or are modified in some way so that a graft device extracellular comprising matrix and living cells, the therapeutically beneficial for a improved wound healing and simplified or directed neovascularization are. These methods are known in the art and are described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), herein incorporated by reference. All of the above Cell types in this invention for producing a cultured skin construct may be used, that the dependent Media containing synthesize cytokines.

While collagen the most preferred extracellular matrix composition for use is in the production of skin equivalents, the cytokines for produce and secrete conditioning the culture media, others can extracellular Matrix components are used. This extrazel-cellular matrix components can be used alone be used or can preferably includes the collagen, a natural dermal matrix to imitate. This extracellular Matrix components include: other collagens, both fibrous and non-fibrous Collagen from the collagen family such as collagen types II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, other matrix proteins, which may include but not limited to are elastin, proteoglycans such as Dicorin or biglycan, or glycoproteins such as tenascin recordable, Vitronectin, fibronectin, laminin, glycosaminoglycans and ThrombospondinI (GAG) such as hyaluronic acid (HA). The Dermal Matrix can respect the composition and the structure vary. Collagen sponges, biocompatible, bioremodelable, de-zellularisierte dermis or collagen gels. Instead of the skin cells to provide extracellular matrix components, can them on biodegradable fabric elements (such as Nylon or Polygalactin (PGA)) are cultured to provide a culture carrier, and cultured to produce an extracellular matrix, to the cells and their matrix surround the carrier. at the preferred embodiment, is the cultured connective tissue construct a contracted collagen gel contracted described by fibroblasts, such as those in the <patcit><text>US Pat. No. 4,485,096</text></patcit> from Bell. In an alternative embodiment, the contracted Collagen gel on an acellular Basic collagen layer can be arranged on a porous membrane, to fix the gel to the membrane and to an excessive radial To prevent contraction of the gel. A method for receiving acellular collagen layers are described in <patcit><text>US Patent No. 5,536,656</text></patcit> Kemp, et al. However, this embodiment forms no part of the present invention, but merely a background item of information which is useful for understanding the invention.

the inventive fabric can Equivalent be prepared using collagen and of the skin a tendon, including a rat tail tendon, calf skin collagen and Kalbextensorsehne, was obtained. Other collagen sources are suitable. A particularly preferred obtained from the common Kalbfingerextensorsehne Collagen composition and method for obtaining such collagen compositions are in the <patcit><text>US Pat. No. 5,106,949</text></patcit> Kemp described.

On Skin Equivalent is a directly on the lower surface given tissue culture dish, or directly on a porous member such as a relative a liquid permeable membrane, and &lt;?page 6?&gt;Although using methods according to the aforementioned Teachings of Kemp, et al. and as described here is. A casting mixture with collagen and fibroblasts is in the inner container <figref>20</figref> given and maintained under conditions which permit formation of the tissue-equivalents. The concentration of collagen, the number of cells and the volume the casting mixture can be controlled in order to control the diameter and thickness of the living-tissue-Equivalentes. The casting mixture has cells with a concentration of 1.25 × 10⁴ to 5 × 10⁴ Cells / ml and collagen at 0.5 to 2.0 mg / ml in a nutrient medium on. A preferred cell concentration is 2.5 x 10⁴ Cells / ml. The cultures are in a Incubator kept at sufficient environmental conditions of controlled Temperature, humidity and gas mixture to ensure the cell culture. Preferred Conditions are between 34 ° C and 38 ° C, in particular 37 ± 1 ° C, at a atmosphere between 5-10 ± 1% CO₂ and a relative humidity (Rh) between 80 and 90%. Once contracted collagen networks can have it for used surgical applications for treating a patient are in need of tissue repair or tissue replacement.

at an alternative embodiment, an acellular formed collagen gel prior to the formation of connective tissue construct on the growing medium be to provide the substrate an adhesive. In some make needed providing a substrate of adhesive, and in other cases it is not needed. If acellular Kollagengelkomponente used in the preparation of the cultured connective tissue construct is, it was found that the ratio of the volume of the casting mixture for the Tissue Equivalent to the Volumenen the casting mixture for the acellular hydrogenated Collagen gel has an effect on the cell viability and the cell differentiation has. Useful ratios, Volume to volume (v / v), of the casting mixture of tissue-Equivalentes to casting mixture of the collagen gel are 3: 1 to 1: 3. A preferred ratio wherein the cell concentration in the colleague network 2.5 x 10⁴ Cells / ml is 3: 1. The acellular hydrogenated collagen gel <figref>25</figref> is prepared from a composition colleagues, 0.5 to 2.0 mg / ml of collagen, preferably about 0.9 to 1.1 mg / ml, and culture medium contains. These colleagues composition is the inner container <figref>20</figref> added and kept conditions which allow the composition colleagues, settle and an acellular hydrogenated collagen gel of suitable dimensions, usually 1 to 5 mm thick to form, with a preferred thickness range between 2 mm and 3 mm. an acellular hydrogenated collagen <figref>25</figref> is preferably thick enough that a portion of acellular remaining when cells from the tissue-equivalent in an acellular hydrogenated Collagen migrate, and thin enough so that the tissue equivalent is not undesirably from the outer in the container <figref>10</figref> provided a source of nutrition Will get removed. However, this embodiment does not form part of the present invention, but merely background information is that for the understanding are the invention helpful.

at another alternative embodiment, the cultured tissue construct of cells is formed, under conditions for producing a layer of extracellular matrix have grown, which synthesized by the cultured fibroblast cells and is assembled with the cultured fibroblast cells included in the synthesized extracellular matrix layer are, wherein the extracellular Matrix by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the Cultivation conditions is produced. Methods for preparing these cultured connective tissue constructs are described in PCT Publication No. <patcit><text>WO 00/29553</text></patcit> from Murpy, et al. described.

As soon as obtain a sufficient number of cells after isolation and Scale-Up was, the cells are harvested and carried on a suitable culture surface and under suitable growth conditions cultured to a confluent to obtain cell layer. In another embodiment, the cells are on a porous recognized membrane that is submerged to a media contact from above below the culture through the pores and directly to enable. Preferably, the cells are either a base or a suspended growth medium and on the cell culture surface with a density between 1 x 10⁵ Cells / cm² to 6.6 × 10⁵/cm², Preferably between 3 x 10⁵ Cells / cm² to 6.6 × 10⁵/cm² and most preferably 6.6 × 10⁵ Cells / cm², Set (cells per square centimeter of surface area). The Cultures are cultured in a growth medium to culture to establish, and are grown to a confluency of between 80% and 100% cultured, whereby they are chemically induced at this time, by the medium is changed to a matrix production medium, high heading to the synthesis and secretion of extracellular matrix. At a alternative method, the cells are directly to production media attached to the need the change to avoid from the basic media to the production media but This is a method, the higher Approach densities required.

During the Culturing fibroblasts organize the secreted matrix molecules to a three-dimensional tissue-like form structure, but have no significant Kontrakti&lt;?page 7?&gt;ation forces on, to the formation of the cell-matrix construct to cause to contract and itself from the culture substrate lift. Media exchanges are changed every two to three days with a fresh made matrix production medium and with time takes the abgeson-made matrix in thickness and organization. The time is necessary to generate a cell-matrix construct is dependent on the ability the initial Approach density, the cell type, the age of the cell line and the ability the cell line to synthesize and secrete matrix. After complete Training have the constructs of the invention due to the generated and through the cells organized fibrous matrix or a base thickness Bulk-thickness; they are not usually confluent or overly confluent Cell cultures in which the cells can adhere to each other loosely. The fiber quality gives the constructs cohesive tissue-like Properties unlike conventional Cultures, since they, physical damage, such as tearing and columns or routine handling in a clinical setting resist. In the production of a cultured skin construct are the Cells an organized matrix around themselves on the cell culture surface, preferably at least 30 microns or more thick, more preferably between 60 to 120 micrometers thick, about the surface of the membrane. However, thicknesses have been achieved of more than 120 microns and these are for a use in testing or clinical applications suitable where such greater thicknesses needed will.

at a further alternative embodiment , the cultured connective tissue construct before application to a patient to be treated, the implant acceptance or cure, or both, to increase at the site of application in the patient. The constructs can be treated with other components by those with a solution brought to the components by, for example, immersion in contact will. components for treating a construct before application comprises extracellular matrix components such as hyaluronic acid, collagen, Proteoglycan, Orglycosaminoglycane or cytokines including growth factors such as: basic fibroblast growth factor (bFGF), epidermal Growth factor (EFG), keratinocyter growth factor (KGF), transforming growth factor Alpha (TFGα) Transforming growth factor beta (TGF), including transforming growth factor Beta 1 (TGFβ1) and transforming growth factor-beta 2 (TGFβ2), granulocyte colony stimulating Factor (FCSF), insulin-like Growth factor (IFG), Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF). It should be noted that the aforementioned terms in parentheses Abbreviations are generally known and in the art for the formal Nomenclature Before parties are used.

The following examples are provided for the execution of the to better explain the present invention and are not intended as the Scope of this invention be interpreted as limiting. The skilled artisan will appreciate that various modifications to the herein Methods described may be made without departing from the scope the present invention depart.

EXAMPLES

Reference Example 1: Preparation of Connective tissue construct with a contracted collagen network

Human Neonatal foreskin fibroblasts (derived from Organogenesis, Inc. Canton, MA) were incubated at 5 x 10⁵ Cells / 162 cm² tissue culture recognized in treated flasks and cultured in a growth medium. the Growth media consists of: Dublecco's Modified Eagle's medium (DMEM) (high glucose formulation, without L-glutamine, BioWhittaker, Walkersville, MD) supplemented with 10% newborn calf serum (NBCS) (HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine (BioWhittaker, Walkersville, MD). The cells are cultured in an incubator at 37 ± 1 ° C with a the atmosphere of 10 ± 1% CO₂ kept. The medium was every two to three days replaced with freshly prepared medium. After 8 days in culture, the cells were grown to confluence, the is the Cells had a packed monolayer along the bottom of the tissue culture flask formed, and the medium was aspirated from the culture flask. Around rinse the monolayer A sterile-filtered phosphate buffered physiological Saline when added to the soil of each culture flask and then out of the bottles aspirated. The cells were removed from the bottle by adding 5 ml trypsin-Versene-glutamine (BioWhittaker, Walkersville, MD) in each bottle and gently shaking to ensure full released coverage of the monolayer. The cultures were in the Incubator returned. As soon as the cells were released 5 ml of SBTI were (soybeans trypsin inhibitor) each bottle was added and mixed with the suspension to the stop action of trypsin Versene. The cell suspension was prepared from the bottles removed and evenly distributed on sterile conical centrifuge tubes. Cells were up by centrifugation at approximately 800 1000.times.g for 5 minutes collected.

A similar device the in <figref idrefs="S24">1</figref> shown was in carrying out the described below Works used. The cover is removed for servicing, is but otherwise the On&lt;?page 8?&gt;legally obtain sterility closed kept. Pertinent information regarding the apparatus are listed: The outer container <figref>10</figref> has a Diameter of 100 mm or more. The inner container 200 has a diameter of 75 mm. The permeable element 24 is from a polycarbonate membrane having a pore size of about 3 microns and a Thickness of 5 microns.

A "vorgeschmischte" solution 16.2 ml 10X Minium Essential Medium (MEM), 1.6 ml 200 mM L-glutamine, 0.2 ml of 50 mg / ml gentamycin, 18.0 ml of embryonic bovine serum, 5.0 ml 71.2 mg / ml sodium bicarbonate. The stock solutions were aseptically in the above order and combining at 4 ° C for 30 minutes in a sterile 50 ml tube stored. About 27.44 ml of a 2.2 mg / ml collagen solution (extracted by acid from the common Kalbfingerextensorsehne) in 0.05% v / v acetic acid were in a 50 ml tube be balanced and at 4 ° C for 30 minutes stored. Dulbecco's Minium Essential Medium (DMEM) complete (comprising 10% FBS, 4 mM L-glutamine, 50 .mu.m / ml Gentamycin) was added and 1 ml aliquots were applied to the membrane of the inner container <figref>20</figref> Pipette and gelation at room temperature was allowed.

The Connective tissue layer, a hydrogenated collagen gel with cells was cast with human dermal fibroblasts. A general description of Procedures and reagents may also be found in the <patcit><text>US Pat. No. 4,485,096</text></patcit> from Bell, the <patcit><text>US Pat. No. 5,536,656</text></patcit> from Kemp, et al. and the<patcit><text>US Patent No. 5,712,163</text></patcit> by Parenteau. The casting mixture for preparing each connective tissue construct includes about 12.5 ml of the premix described above, to which 27.44 ml a 2.2 mg / ml collagen solution in 0.05% v / v acetic acid, also described above, and 6.25 × 10⁵ Human skin fibroblasts are added. After the components to a total volume of approximately 40 ml were mixed, this was in the container <figref>20</figref> distributed and allowing the gel formation. The collagen gel containing the suspended fibroblasts in Dulbecco's minimum Essential Medium (DMEM) dipped completely into the outer container which <figref>20</figref> given was, and then at 36 ° C / 10% CO₂ for 4 to 8 days incubated to allow the cells to the Collagen contract to a contracted collagen network to form which the cultured tissue construct <figref>52</figref> represents.

Example 1: Preparation of a connective tissue construct from cultured cells and endogenously produced extracellular matrix components without the requirement of exogenous matrix components or network support or scaffolding elements

the prepared connective tissue construct of cultured cells and endogenously extracellular matrix components was prepared according to the teachings of Murphy, et al., described in PCT Publication <patcit><text>WO 00/29553</text></patcit> produced. Human neonatal foreskin fibroblasts were cultured to their number to increase, by using the procedure described in the Reference Example 1 Method, but in a chemically defined medium, that is, without Serum or organ extracts of animals. The cells were then resuspended to a concentration of 3 × 10⁶ Cells / ml and carried on treated membrane inserts 0.4 microns Pore ​​size, 24 mm Diameter in a magazine having 6 wells at a density of 3.0 × 10⁶ Cells / TW (6.6 × 10⁵ Cells / cm²). The cells in this example were completely cultured in a chemically defined medium.

the Medium contains: a base 3: 1 mixture of DMEM, Hams F-12 medium (Quality Biologics, Gaithersburg, MD) 4 mM Gluta MAX- (Gibco BRL, Grand Iceland, NY) and additives: 5 ng / ml human recombinant Epidermal growth factor (Upstate Biotechnology, Lake Placid, NY) 1 x 10^-4 M Ethanolamine (Fluka, Ronkonkoma, NY, Cat. No. 02400 ACS purity) 1 x 10^-4 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 ug / ml Transferrin (Sigma, St. Louis, MO), 20 ρM Triiodothyronine (Sigma, St. Louis, MO), 5 ug / ml insulin (Sigma, St. Louis, MO), 0.4 ug / ml Hydrochortison (Sigma, St. Louis, MO), 6.78 g / ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 50 ug / ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 ug / ml L-proline (Sigma, St. Louis, MO), 0.1 ug / ml Glycine (Sigma, St. Louis, MO).

rehearse for histological Analyses were taken at days 7, 14 and 21 and fixed in formalin, then in paraffin for a Hemotoxylin- and eosin staining for a Light microscope analysis embedded. One the collagen content of the construct measured biochemical analysis shows 170.88 ± 9.07 g / cm², Next Fibrillarkollagen endogenously produced were also decorin and glycosaminoglycan included in the cell-matrix constructs. The formed cultured skin constructs exhibit dermal fibroblasts and endogenously produced matrix. All have completely formed collagen fibers arranged in packed organization between the cells, on. Their give fiber qualities, thickness and cohesive integrity the construct considerable Strength, which allows this, peeling off to be removed from the culture membrane and handled, when it transferred to the construct being treated is, as a transplanted tissue or implant.

&lt;?page 9?&gt;

Example 2: Using a tissue construct to repair the fiber ring following a partial discectomy

Around the persistence of transplanted fibroblasts in the cultured to monitor connective tissue construct Constructs are marked with porcine fibroblasts with grünfluoriszierendem Protein (GFP) in a pig model implanted.

six young pigs of different sexes were up to 50 kg individually mounted at least two days before surgery, wherein they with standard fed pig feed were.

experimental animals were voranästhesiert with Telazol and atropine and intubated. you are set to an inhalable gas from isoflurane and oxygen and held in the anesthesia. You will be given also an antibiotic.

defects on the discs are produced by 5 × 10 mm large incisions made on the fiber ring be followed by a standard discectomy with uniform core removal in every room. A total of three discs per pig operates. Two points are with cultured tissue constructs treated with GFP-labeled fibroblasts and the remaining site serves as a control. To apply the connective tissue construct, is it first in three or four smaller portions separately and then inserted into the ring opening. Two Animals are euthanized each at weeks 2, 4 and 6 and the operation areas being deleted. The discs are before histological processing in formalin and then 70% Enthanol arranged. The discs are sectioned consecutively and fluorescence detection of GFP-labeled fibroblasts examined.

Example 3: Using a tissue construct with intervertebral disc spacer to maintain the intervertebral space

Around the persistence of transferred watching fibroblasts in the cultured connective tissue construct, Constructs are implanted with fibroblasts in a pig model.

test bulls be voranästäsiert with Telazol and Antropin and intubated. you will be set to an inhalable gas from isoflurane and oxygen and and kept under anesthesia. You will also be an antibiotic administered.

defects on the discs are produced by 5 × 10 mm large incisions made on the fiber ring be followed by a standard discectomy with uniform core removal in every room. A total of three discs per pig operates.

About the produced in the fiber ring opening the intervertebral space opens and the disc removed, namely limited to the forward rails and middle third section. The intervertebral disc spacer has a Dacron fabric, and a hydrogel is placed in the thoracic cavity, where it through the opening introduced into the fiber ring is. Proper positioning the implant is ensured using radiological Method, and then the spacer is fixed in place. the cultured connective tissue construct is then applied to the annular opening, by the construct first on the size of the annular hole opening is matched, and then using absorbable suture with which the opening surrounding fabric of space is sutured. While all three points with a The intervertebral spacers are provided, are two places with Cultured treated tissue constructs and the remaining point is as a control.

Two Animals are euthanized respectively after 2, 4 and 6 weeks, and the Surgical sites are removed. The discs are in formalin and then in 70% ethanol prior to histological processing arranged. The discs are sectioned serially and under fluorescent light examined for the presence of GFP labeled fibroblasts.

at all Samples remain the intervertebral disc spacer to its original Put. In control samples of the nucleus pulposus showed a significant Loss of proteoglycan and collagen and an increase of other Non-collagenous in the cavity. In test samples constituting the Connective tissue construct a complete scar over the in the fiber ring produced opening. The biochemical composition of the cavity has been changed, was However, closer on the composition of the negative control samples, indicating that the fibrosis of the cavity by the closure of the ring to the disk injury was significantly reduced.

Example 4: A study regarding the time profile with the use of tissue constructs in the correction of the fiber ring in pigs.

A Discectomy to remove a damaged and out crowded gelatinous core is a common clinical practice for mitigating pain and neurological Disorder. The operation produces a defect in the annulus, often with fibrous tissue fills, a situation which &lt;?page 10?&gt;after all leads to a collapse of the disc and fusion of the adjacent requires vertebral segments.

It porcine tissue constructs were prepared, namely using porcine dermal fibroblasts in accordance with the in Reference Example 1 procedure set forth for expressing green Floureszenzprotein (GFP) were transfected. Each construct was prepared from one derived from bovine Type I collagen with 20 to 30 million viable GFP-transfected porcine-derived dermal fibroblasts and was about 2 mm thick. It was the purpose of this study, the feasibility or appropriateness of connective tissue constructs to repair the fiber ring in a to examine from a pig-derived model and biocompatibility, the persistence and the remodeling of constructs to determine in this model.

six three to four-month-old pigs were used for the investigation. In each animal three consecutive discs were a Laminotomy exposed posterior. For each exposed plate a surgical ring defect was created. Several pieces of a Connective tissue construct, each having a diameter of about 2 to 5 mm, were divided into two Defects of each animal implanted. The other sheet effect was to Check left blank. The pigs were in pairs 2, euthanized 4 and 6 weeks after implantation. The who operated Discs containing spine was removed and fixed in 10% neutral buffered formalin. In Hemotoxillin- and Eosineingefärbten sections A Brightfield microscopy for A general observation carried out, and a fluorescence microscopy was to identify with a green fluorescent protein-labeled Cells in unstained Sections performed.

The Microscopy provided in several of the treated rings of the two groups of animals which were euthanized after 2 and 4 weeks, a clear evidence of residues of implanted tissue constructs. It was further identified a remodeling of the radicals of the Connective tissue construct by the host tissue. The implanted defects showed less inflammation and a more advanced healing than controls at all Time. The implanted defects reported the opening bridging cartilaginous tissue , whereas the control defects still a significant Volume fibrous tissue showed. The results of this feasibility study show that the implanted pig tissue construct with respect to the was host tissue biocompatibility, up to four weeks may consist and the restoration activities of the ring can be increased by six weeks.

Although the foregoing invention has for purposes of clarity and understanding, described in detail by way of illustration and examples was, it is for the expert course, that certain changes and modifications within the scope of the appended claims be performed can.