MX2008001710A - Use of adipose tissue-derived stromal cells in spinal fusion - Google Patents

Abstract

The present invention encompasses methods and compositions for treating a bone condition. The isolated adipose tissue-derived stromal cell of the invention and products related thereto have a plethora of uses, including but not limited to research, diagnostic, and therapeutic applications such as in spinal fusion procedures.

Description

USE OF STRIPTIC CELLS DERIVED FROM ADIPOSE TISSUE IN SPINAL FUSION BACKGROUND OF THE INVENTION There are generally two types of bone conditions: 1) non-metabolic bone conditions, such as bone fractures, bone / spinal cord deformation, osteosarcoma, myeloma, bone dysplasia and scoliosis, and 2) metabolic bone conditions, such as osteoporosis, osteomalacia, rickets, osteitis fibrosa, renal bone dystrophy and Paget's bone disease. Osteoporosis, a metabolic bone condition, is a systemic disease characterized by fragility and increased bone fracturability due to decreased bone mass and change in the fine structure of bone tissue, its main clinical symptoms including spinal kyphosis, and fractures of the dorsolumbar bones, vertebral centers , femoral necks, lower end of the radius, ribs, upper end of the humerus, and others. In bone tissue, bone formation and destruction occurs constantly due to bone resorption. After the deterioration of the balance between bone formation and bone destruction due to bone resorption, a quantitative reduction occurs in the bone. Traditionally, suppressors of bone resorption, such as estrogens, calcitonin and bisphosphonates, have been used primarily to treat osteoporosis. With respect to bone / spinal conditions, more than 75% of The American population suffers from back pain at some time in their life. Underlying medical conditions can contribute to back pain. These include scoliosis, spinal stenosis, degenerative disease of the intervertebral discs, infectious processes, tumors and trauma. The repair of large segment defects in diaphyseal bone is a significant problem faced today by orthopedic surgeons. Although such bone loss can occur as a result of acute injury, these massive defects commonly occur secondary to congenital malformations, benign and malignant tumors, bone infection and lack of union in fractures. The use of fresh autologous bone graft material has been considered as the standard of historical treatment, but it is associated with substantial morbidity including infection, malformation, pain and loss of function (Kahn et al., 1995, Clin. Orthop. Res. 313: 69-75). Complications resulting from graft harvesting, combined with limited supply, have inspired the development of alternative strategies for the repair of clinically significant bone defects. The primary procedure for this problem has been focused on the development of effective bone implant materials. Three general classes of bone implants have emerged from these research efforts, and these classes can be grouped as osteoconductive, osteoinductive or directly osteogenic. Allograft bone is probably the best known type of osteoconductive implant. Although widely used for many years, the risk of transmission of diseases, rejection by the host and lack of osteoinduction, compromises their convenience (Leads, 1988, JAMA 260: 2487-2488). Synthetic osteoconductive implants include titanium metal fibers and ceramics composed of hydroxyapatite and / or tricalcium phosphate. The favorably porous nature of these implants facilitates bone ingrowth, but their lack of osteoinductive potential limits their usefulness. A variety of osteoinductive compounds have also been studied, including the demineralized bone matrix, which is known to contain bone morphogenic proteins (BMPs). Since the original discovery of BMPs, others have characterized, cloned, expressed and implanted purified or recombinant BMPs in orthotopic sites for the repair of large bone defects (Gerhart et al., 1993, Clin Orthop., Res. 293: 317 -326; Stevenson et al., 1994, J. Bone Joint Surg. 76: 1676-1687; Wozney et al. , 1988 Science 242: 1528-1534). The success of this procedure depended on the presence of mesenchymal cells capable of responding to the inductive signal provided by BMP (Lañe et al., 1994, at the First International Conference on Homogenous Morphogenic Proteins). It is these mesenchymal progenitors that undergo osteogenic differentiation, and are ultimately responsible for the synthesis of new bone at the surgical site. An alternative to the osteoinductive procedure is the implantation of living cells that are directly osteogenic. Since it has been shown that the bone marrow contains a population of cells that have osteogenic potential, some have designed experimental therapies based on the implantation of fresh syngeneic or autologous marrow in sites that need repair of the skeleton (Grundel et al., 1991, Clin Orthop, Res. 266: 244-258, Werntz et al., 1996, J. Orthop Res 14: 85-93; Wolff et al., 1994, J. Orthop Res 12: 439-446). Although deep in principle, the viability of obtaining enough bone marrow with the required number of osteoprogenitor cells is limiting. The emerging field of regenerative medicine seeks to combine biomaterials, growth factors and cells as novel therapy to repair damaged tissues and organs. As this specialty grows, there is a demand for a reliable, safe and effective source of human adult stem cells that will serve in tissue engineering applications. For regulatory purposes, these cells must be defined by quantifiable purity measures. For practical purposes at the clinical level, these cells must be available as a "standard manufacturing" product, immediately available upon demand at the point of care. From a commercial point of view, the ability to use allogeneic adult stem cells, as opposed to adult autologous stem cells for transplantation, would have a significant positive impact on product development. Under these circumstances, a single batch of cells derived from a donor could be transplanted to multiple mammals, reducing the costs of quality control and quality assurance. Studies have shown the existence of stem cells adults in multiple tissue sites. Bone marrow-derived cells, known as mesenchymal stem cells (MSCs) or bone marrow stromal cells (BMSCs), have been extensively characterized (Castro-Malaspina et al., 980, Blood 56: 289-30125; Piersma et al. , 1985, Exp. Hematol 13: 237-243, Simmons et al., 1991, Blood 78: 55-62, Beresford et al., 1992, J. Cell. Sci. 102: 341-351, Liesveld et al., 1989, Blood 73: 1794-1800, Liesveld et al., Exp. Hematol 19: 63-70, Bennett et al., 1991, J. Cell. Sci. 99: 131-139). Recent studies have shown that MSCs derived from allogenic bone marrow can be transplanted (Bartholomew et al., 2002, Exp.Hematol.30: 42-8), and used to repair a critical size orthopedic defect in a canine model (Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A: 1927-35). However, MSCs represent approximately 1 in 10,000 to 100,000 nucleated bone marrow cells or approximately 200 cells per ml of bone marrow aspirate (Bruder er al., 2000, Principies of Tissue Engineering, 2nd edition, Academic Press). To obtain sufficient numbers of MSCs for tissue engineering applications, it is necessary to expand the MSCs derived from bone marrow through multiple in vitro passages. In contrast to the bone marrow, adipose tissue is easily accessible for surgical harvest and abundant in the average adult American. Recently, it has been shown that adipose tissue can serve as a source of stem cells (known as adult stem cells derived from adipose tissue or ADAS cells). These cells are able to show differentiation along multiple lineage pathways. In response to chemicals, hormones and / or specific cytokines, human and rodent ADAS cells express biochemical and histological characteristics consistent with neuronal cells and muscle, cartilage, bone and adipose tissue. In a recent study, murine ADAS cells accelerated the repair of a critical-sized skullcap defect (Cowan et al., 2004, Nat. Biotechnol.22: 560-7). Bone grafting is frequently used for the treatment of bone conditions. Of course, more than 1.4 million bone graft procedures are performed annually in the world. The success or failure of the bone graft depends on many factors including the vitality of the graft site, the processing of the graft and the immunological compatibility of the grafted tissue. In view of the prevalence of bone conditions, there is a need for novel bone sources for therapeutic, diagnostic and research purposes. The present invention satisfies this need.

BRIEF DESCRIPTION OF THE INVENTION The invention includes a method for improving bone fusion after a spinal fusion procedure in a mammal, which comprises administering an adult stromal cell derived from adipose tissue (ADAS) isolated in the mammalian spine, wherein the cell ADAS differs in vivo in a cell that expresses at least one characteristic of a bone cell. The invention also includes a method for performing one or more spinal fusions in a mammal, which comprises administering an ADAS cell into the mammalian spine to facilitate single or multiple level spinal fusion. Preferably, after administration of the ADAS cell into the mammalian spine, the ADAS cell differentiates in vivo in a cell that expresses at least one characteristic of a bone cell. In one aspect, the ADAS cell is cultured in vitro for a period without being induced to differentiate prior to administration of the cell to the mammal. In another aspect, the ADAS cell is allogenic with respect to the mammal. In another aspect, the ADAS cell induces bone formation by spinal fusion of intervertebral bodies. In another aspect, the ADAS cell induces bone formation by spinal fusion of intertransverse processes. In one aspect, the ADAS cell further comprises a biocompatible matrix. Preferably, the biocompatible matrix is selected from the group consisting of calcium alginate, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan sulfate and bone matrix gelatin.

In another aspect, the ADAS cell is genetically modified. In another aspect, the ADAS cell is administered in one or more interbody spaces in the mammalian spine. In another aspect, the spinal fusion is in a segment of the spine selected from the group consisting of cervical, thoracic, lumbar, lumbosacral, and sacral-iliac (SI) joints. In another aspect, the ADAS cell is administered in one or more interbody spaces by a method selected from the group consisting of a subsequent procedure, a posterolateral procedure, a prior procedure, an anterolateral procedure and a lateral procedure. In another aspect, the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, certain embodiments of the invention are described in the drawings. However, the invention is not limited to the precise arrangements and means of the embodiments described in the drawings. Figures 1A-1 D are an image describing a spinal fusion procedure. Figure 1A describes the intervertebral space in the lumbar spine. Figures 1 B and 1C show the introduction of a mechanical device and bone grafts that stabilize the space, respectively. Figure 1 D is an image that describes the spine. Figure 2 is an image that describes the potential of ADAS cells to differentiate along multiple lineage pathways. In response to specific cocktails of chemicals and growth factors, human ADAS cells can differentiate into chondrocytes, osteoblasts, adipocytes and glial and neuronal cell-like cells in vitro. Figures 3A-3D are an image describing the osteogenesis of human ADAS cells. Figure 4 is a diagram describing the expression of aldehyde phosphatase in ADAS cells during adipogenic and osteogenic differentiation. Figures 5A and 5B are an image demonstrating that ADAS cells form bone in vivo.

DETAILED DESCRIPTION OF THE INVENTION The present invention encompasses methods and compositions for treating bone disease. In a preferred embodiment, an adult adipose-derived stromal cell (ADAS) isolated from the invention is used to improve bone fusion after a spinal fusion procedure in a mammal.

Definitions As used herein, each of the following terms has the meaning associated therewith in this section. The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means an element or more than one element. The term "approximately" will be understood by those skilled in the art, and will go to some degree in the context in which it is used. The term "cell derived from adipose tissue" refers to a cell that originates from adipose tissue. The initial cell population isolated from adipose tissue is a population of heterogeneous cells that includes, but is not limited to, cells of the stromal vascular fraction (SVF). As used herein, the term "adipose-derived stromal cells", "adipose-derived adult stromal cells (ADAS)" or "adipose-derived stem cells (ASCs)" is used reciprocally, and refers to stromal cells that originate from adipose tissue that can serve as stem cell-type precursors for a variety of different cell types such as, but not limited to, adipocytes, osteocytes, chondrocytes, and glial / neuronal and muscle cell lineages. The term "adipose" refers to any fat tissue. Adipose tissue can be brown or white adipose tissue. Preferably, the adipose tissue is subcutaneous white adipose tissue. Said cells may comprise a primary cell culture or an immortalized cell line. Adipose tissue can be from any organism that has fat tissue. Preferably, the adipose tissue is mammalian, more preferably the adipose tissue is human. A convenient source of human adipose tissue is one that is derived from liposuction surgery. However, the source of adipose tissue or the adipose tissue isolation method is not critical to the invention. The term "allogeneic" refers to a graft derived from a different animal of the same species. As used herein, the term "autologous" is used to refer to any material derived from the same individual to which it will then be reintroduced into the individual. The term "xenogenic" refers to a graft derived from a mammal of a different species. As used herein, the term "biocompatible network" is used to refer to a substrate that can facilitate formation in three-dimensional structures that lead to tissue development. Thus, for example, cells can be cultured or seeded in said biocompatible network, such as one that includes extracellular matrix material, synthetic polymers, cytokines, growth factors, etc. The network can be molded into desired shapes to facilitate the development of fabric types. Also, at least at an early stage during the cultivation of cells, the medium and / or substrate is supplemented with factors (for example, growth factors, cytokines, extracellular matrix material, etc.) that facilitate the development of structures and appropriate tissue types. As used herein, the term "condition (or bone injury or disease)" refers to disorders or diseases of the bone that include, but are not limited to, acute, chronic, metabolic and non-metabolic conditions of the bone. The term covers conditions caused by disease, trauma or tissue failure to develop normally. Examples of bone conditions include, but are not limited to, a bone fracture, a bone / spinal cord deformation, osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis, osteomalacia, rickets, osteitis fibrosa, renal bone dystrophy and bone disease. Paget. The term "differentiation means" is used herein to refer to a cell culture medium comprising an additive or a lack of an additive such as a stem cell, adult stromal cell derived from adipose tissue or some other of said progenitor cells, which is not completely differentiated when it is incubated in the medium, and develops in a cell with all the characteristics of a differentiated cell, or some of them. The term "expandability" is used herein to refer to the ability of a cell to proliferate, for example, to expand in number, or in the case of a population of cells, to undergo duplications of the population. The term "graft" refers to a cell, tissue, organ or otherwise any compatible biological network for transplantation. By "growth factors", the following specific factors are understood to include, but are not limited to, growth hormone, erythropoietin, thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophage colony stimulation factor, cell factor mother / ligand c-kit, osteoprotegerin ligand, insulin, insulin-like growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor, ciliary neurotrophic factor, growth factor-derived platelets (PDGF) and bone morphogenetic protein at concentrations between picogram / ml to milligrams / ml. As used herein, the term "growth medium" is used to refer to a culture medium that promotes cell growth. A growth medium will generally contain animal serum. In some cases, the growth medium may not contain animal serum. An "isolated cell" refers to a cell that has been separated from other components and / or cells that naturally accompany the isolated cell in a tissue or mammal. As used herein, the term "multipotential" or "multipotential" is used to refer to the ability of a stem cell of the central nervous system to differentiate into more than one cell type.

As used herein, the term "modular" is used to refer to any change in the biological state, i.e., increase, decrease, and the like. As used herein, the term "non-immunogen" is used to refer to the discovery that ADAS cells do not induce proliferation of T cells in an MLR. However, the term non-immunogenic should not be limited to the proliferation of T cells in an MLR, but rather should also be applied to ADAS cells that do not induce T cell proliferation in vivo. The term "proliferation" is used herein to refer to the reproduction or multiplication of similar forms, especially of cells. That is, the term proliferation encompasses the production of a greater number of cells, and can be measured, inter alia, simply by counting the number of cells, measuring the incorporation of tritiated thymidine in the cell, and the like. The term "progression of or through the cell cycle" is used herein to refer to the process by which a cell prepares for mitosis and / or meiosis and / or enters them. Progression through the cell cycle includes progression through the G1 phase, S phase, G2 phase and M phase. The terms "precursor cell", "progenitor cell" and "stem cell" are used reciprocally in the technical and in the present, and refer to a pluripotent progenitor cell or non-committed lineage, which is potentially capable of displaying a limited number of mitotic divisions to renew itself, or to produce cells of progeny that will differentiate into the type of cell desired. Unlike pluripotent stem cells, it is generally considered that compromised lineage progenitor cells are unable to give rise to numerous cell types that phenotypically differ from one another. Rather, the progenitor cells give rise to a cell type or possibly two cell types of committed lineage. The term "stromal cell medium", as used herein, refers to a medium useful for the culture of ADAS cells. A non-limiting example of a stromal cell medium is a medium comprising DMEM / Ham's F12 medium, 10% fetal bovine serum, 00 U penicillin / 100 pg streptomycin / 0.25 pg fungizone. Typically, the stromal cell medium comprises a medium used as a base, serum and an antibiotic / antifungal. However, ADAS cells can be cultured with stromal cell media without an antibiotic / antifungal and supplemented with at least one growth factor. Preferably, the growth factor is human epidermal growth factor (hEGF). The preferred concentration of hEGF is about 1-50 ng / ml, more preferably the concentration is about 5 ng / ml. The preferred base medium is DMEM / F12 from Ham (1: 1). The preferred serum is fetal bovine serum (FBS), but other sera including horse serum or human serum can be used. Preferably, up to 20% FBS will be added to the previous medium to support the growth of stromal cells. Without However, a defined medium could be used if the growth factors, cytokines and hormones necessary in FBS for the growth of stromal cells are identified and provided at appropriate concentrations in the growth medium. It is further recognized that additional components can be added to the culture medium. Such components include, but are not limited to, antibiotics, antifungals, albumin, growth factors, amino acids, and other components known in the art for cell culture. Antibiotics that can be added in the medium include, but are not limited to, penicillin and streptomycin. The concentration of penicillin in the culture medium is from about 10 to about 200 units per ml. The concentration of streptomycin in the culture medium is from about 10 to about 200 pg / ml. However, in no way should be considered that the invention is limited to some means for the cultivation of stromal cells. Rather, any means capable of sustaining stromal cells in tissue culture can be used. The term "pharmaceutically acceptable carrier (or medium)", which may be used reciprocally with the biologically compatible medium or carrier, refers to reagents, cells, compounds, materials, compositions and / or dosage forms that are within the scope of be suitable for use in contact with the tissues of humans and animals, without excessive toxicity, irritation, allergic response, or other complication in proportion to a reasonable ratio of benefit / risk. A "suitable interbody space," as the term is used herein, means the space between adjacent vertebrae where a disc resides on a healthy spine, but which at least partially lacks disc material due to wear and tear in the disc. the spine, or has been prepared using techniques known in the art to surgically create a space in the disc space. As used herein, a "therapeutically effective amount" is the amount of ADAS cells sufficient to provide a beneficial effect to the subject to which the cells are administered. The term "treatment (or treatment)" refers to the improvement of the effects of, or delay in, healing or reversing the progress of, or delay or prevention of, the onset of a bone condition. As used herein, the term "endogenous" refers to any material of an organism, cell or system, or produced therein. The term "exogenous" refers to any material introduced from an organism, cell or system, or produced outside it. The term "coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA or mRNA, which serve as templates for the synthesis of other polymers and macromolecules in biological processes having a sequence defined nucleotides (ie, rRNA, tRNA and mRNA), or a sequence defined amino acids, and the biological properties that result from them. In this way, a gene encodes a protein if the transcription and translation of the messenger RNA that corresponds to that gene, produces the protein in a cell or other biological system. It may be referred to that both the coding strand, whose nucleotide sequence is identical to the messenger RNA sequence and is usually provided in sequence listings, such as the non-coding strand, used as the template for the transcription of a gene or cDNA, encode the protein or another product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other, and that code for the same amino acid sequence. The nucleotide sequences encoding proteins and RNA may include introns. An "isolated nucleic acid" refers to a segment or fragment of nucleic acid that has been separated from sequences that flank it in a state of natural occurrence, that is, a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment, ie, the sequences adjacent to the fragment in a genome in which it occurs naturally. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, a virus or replicator plasmid »autonomous, or in the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (ie, as a cDNA or a genomic or cDNA fragment produced by PCR or digestion with restriction enzymes), independent of other sequences. It also includes a recombinant RNA that is part of a hybrid gene that codes for additional polypeptide sequences. In the context of the present invention, the following abbreviations are used for commonly occurring nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine. The phrase "under the control of transcription" or "operably linked", as used herein, means that the promoter is in the correct location and orientation relative to the polynucleotides that control the initiation and expression of the polynucleotides by the RNA polymerase. As used herein, the term "promoter / regulator sequence" means a nucleic acid sequence that is required for the expression of a gene product operably linked to the promoter / regulator sequence. In some cases, this sequence may be the central promoter sequence and, in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are required for the expression of the gene product. The promoter / regulator sequence can be, for example, one that expresses the gene product in a tissue-specific form.

A "constitutive" promoter is a sequence of nucleotides which, when operably linked to a polynucleotide that encodes a gene product or specifies the same, causes the gene product to be produced in a cell under all physiological conditions of the cell, or most of them An "inducible" promoter is a sequence of nucleotides which, when operably linked to a polynucleotide that encodes a gene product or specifies the same, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present. present in the cell. A "tissue-specific" promoter is a nucleotide sequence which, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if the cell is a cell of the type of tissue that corresponds to the promoter. A "vector" is a composition of matter comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. In this manner, the term "vector" includes a virus or a plasmid of autonomous replication. It should also be considered that the term includes non-plasmid and non-viral compounds that facilitate the transfer of a nucleic acid in cells such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises cis-acting elements sufficient for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (i.e., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

Description The present invention relates to the discovery that adult stromal cells derived from adipose tissue (ADAS), can be differentiated into a variety of different cell types including, but not limited to, adipocytes, osteocytes, chondrocytes, and cell lineages glial / neuronal and muscle. In particular, the invention relates to the observation that ADAS cells can be differentiated along the osteogenic lineage in vivo. Based on the present disclosure, an ADAS cell can be used successfully in cell therapy and / or gene therapy for experimental / therapeutic purposes. For example, cells may be used in the treatment of bone diseases. Preferably, the cells are used to improve bone fusion after a spinal fusion procedure. Spinal fusion is a common orthopedic and neurosurgical procedure used to treat back pain in mammals suffering from degenerative disease of the intervertebral discs, spinal stenosis, scoliosis, spinal fracture, tumor, and the like.

Isolation and culture of ADAS cells ADAS cells useful in the methods of the present invention can be isolated by a variety of methods known to those skilled in the art. For example, such methods are described in U.S. Patent No. 6,153,432, which is incorporated herein by reference in its entirety. In a preferred method, ADAS cells are isolated from a mammalian subject, preferably a human subject. In humans, ADAS cells are typically isolated from liposuction material. If the cells of the invention will be transplanted into a human subject, it is preferred that the ADAS cells be isolated from that same subject to provide an autologous transplant. In another aspect of the invention, ADAS cells administered can be allogeneic with respect to the receptor. The allogeneic ADAS cells are isolated from a donor that is a different individual from the same species than the receiver. After isolation, the cells are cultured using the methods described herein to obtain an allogeneic product. The invention also encompasses ADAS cells that are xenogeneic with respect to the receptor. Without limiting the invention in any case, stromal cells can be isolated from adipose tissue using the methods described herein. In summary, human adipose tissue from subcutaneous deposits is removed by liposuction surgery. The adipose tissue is then transferred from the liposuction cup into a sterile 500 ml beaker, and allowed to settle for approximately 10 minutes. The precipitated blood is removed by suction. Approximately one volume of 1 25 ml (or less) of the tissue is transferred to a 250 ml centrifuge tube, and the tube is then filled with a Krebs-Ringer pH regulator. The tissue and the pH regulator are allowed to settle for approximately three minutes, or until a clear separation is achieved, and then the pH regulator is removed by aspiration. The fabric can be washed with Krebs-Ringer's pH regulator for another four to five times, or until the tissue becomes yellow-orange, and until the pH regulator becomes light tan. The stromal cells of adipose tissue can be dissociated using collagenase treatment. In summary, the pH regulator is removed from the tissue and replaced with approximately 2 mg of collagenase solution / ml of Krebs pH regulator (Worthington, ME), at a ratio of 1 ml of collagenase solution / ml of tissue. The tubes are incubated in a 37 ° C water bath with intermittent agitation for approximately 30 to 35 minutes. The stromal cells are isolated from other components of adipose tissue by centrifugation for 5 minutes at 500 X g at room temperature. The oil and the adipocyte layer are removed by aspiration. The remaining fraction can be resuspended in approximately 100 ml of pH regulated saline with phosphate (PBS) by vigorous swirl action, and can be divided into 50 ml tubes and centrifuged for five minutes at 500 Xg. The pH regulator is removed by aspiration, leaving the stromal cells. The stromal cells are then resuspended in stromal cell medium, and seeded at a suitable cell density and incubated at 37 ° C in C02 at 5% overnight. Once attached to the flask or tissue culture plate, the cultured stromal cells can be used immediately, or they can be kept in culture for a period or a number of passages before the use of the cells in accordance with the methods described herein. . However, in no way should be considered that the invention is limited to some method of isolation of stromal cells. Rather, any method for isolating ADAS cells should be encompassed by the present invention. Any medium capable of supporting fibroblasts in cell culture can be used to culture ADAS cells. Media formulations that support the growth of fibroblasts include, but are not limited to Eagle's essential minimum medium, ADC-1, LPM (free of bovine serum albumin), F10 (HAM), F12 (HAM), DCCMI, DCCM2, RPMI 1640, medium BGJ (with and without Fitton modification) -Jackson), Eagle basal medium (BME-with the addition of Earle's salt base), Dulbecco's modified Eagle's medium (DMEM-without serum), Yamane, IMEM-20, Eagle's medium with Glasgow modification (GMEM ), medium L-15 by Leibovitz, medium 5A by McCoy, medium M199 (M199E-based on Earle's salt), medium M199 (M199H-based by Hank's salt), minimum essential medium of Eagle (MEM-E- with Earle's salt base), Eagle's minimal essential medium (MEM-H-with Hank's salt base), and Eagle's minimal essential medium (MEM-NAA with non-essential amino acids), and the like. A preferred medium for the culture of ADAS cells is DMEM, more preferably DMEM / F 2 (1: 1). Other non-limiting examples of media useful in the methods of the invention may contain fetal bovine serum or another species at a concentration of at least 1% to about 30%, preferably at least about 5% to 15%, more preferably about 10%. Embryonic extract of chicken or other species may be present at a concentration of about 1% to 30%, preferably at least about 5% to 15%, more preferably about 10%. After isolation, the ADAS cells are incubated in stromal cell medium in a culture apparatus for a period or until the cells reach confluence before passage of the cells to another cultivation apparatus. The culture apparatus may be any culture apparatus commonly used in in vitro cell culture. Preferably, the level of confluence is greater than 70% before the passage of the cells to another culture apparatus. More preferably, the level of confluence is greater than 90%. A period can be any suitable time for cell culture in vitro. The stromal cell medium can be replaced during the culture of the ADAS cells at any time. Preferably, the stromal cell medium is replaced every 3 to 4 days. The ADAS cells are then harvested from the culture apparatus, after which the ADAS cells can be used immediately, or they can be cryopreserved to be stored for use at a later time. ADAS cells can be harvested by trypsinization, EDTA treatment, or any other method used to harvest cells from a culture apparatus. Several terms are used to describe cells in culture. Cell culture generally refers to cells taken from a living organism, and developed under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. The cells expand in culture when placed in a growth medium under conditions that facilitate the growth and / or division of the cells, resulting in a larger population of the cells. When cells expand in culture, the rate of proliferation of cells is typically measured by the amount of time it takes for cells to double their number, known from another way like the time of duplication. Each round of subculture is referred to as a passage. When the cells are subcultured, it means that they have suffered a passage. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times a passage has suffered. For example, a population of cultured cells that has undergone a passage ten times can be referred to as a P10 culture. The primary culture, that is, the first culture after isolation of the cells from the tissue, is designated PO. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), etc. Those skilled in the art will understand that there may be many duplications of the population during the period of passage through a passage; therefore, the number of duplications of the population of a crop is greater than the number of passages. The expansion of cells (ie, the number of population doublings) during the period between passage through a passage depends on many factors including, but not limited to, the density of sowing, the substrate, the medium and the time between passing through a passage.

Genetic modification The cells of the present invention can also be used to express a foreign protein or molecule for a therapeutic purpose, or in a method of tracking the assimilation of the cell and / or its differentiation in the receiver. In this manner, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into ADAS cells with concomitant expression of the exogenous DNA in ADAS cells. Methods for introducing and expressing DNA in a cell are well known to those skilled in the art, and include those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley &Sons, New York). The isolated nucleic acid can code for a molecule used to track the migration, assimilation and survival of ADAS cells once they are introduced into the recipient. Useful proteins for screening a cell include, but are not limited to, green fluorescent protein (GFP), any of the other fluorescent proteins (e.g., green, cyan, yellow, blue, and red fluorescent proteins enhanced; Clontech Palo Alto, CA) , or other marker proteins (e.g., LacZ, FLAG-tag, Myc, His6, and the like). The tracking of the migration, assimilation and / or differentiation of an ADAS cell of the present invention is not limited to the use of detectable molecules expressed by a vector or virus. The migration, assimilation and / or differentiation of a cell can also be evaluated using a series of probes that facilitate the localization of transplanted ADAS cells within a mammal. Tracing an ADAS cell transplant can also be achieved using antibodies or nucleic acid probes for markers specific cells detailed elsewhere in the present. The term "genetic modification", as used herein, refers to the stable or transient alteration of the genotype of an ADAS cell by the intentional introduction of exogenous DNA. The DNA can be synthetic, or derived naturally, and can contain genes, gene portions, or other useful DNA sequences. The term "genetic modification", as used herein, is not intended to include alterations of natural occurrence, such as those occurring through natural viral activity, natural genetic recombination, or the like. Exogenous DNA can be introduced into an ADAS cell, using viral vectors (retroviruses, modified herpes viruses, herpes viruses, adenoviruses, adeno-associated viruses, lentiviral vectors, and the like), or by direct transfection of DNA (lipofection, calcium phosphate transfection) , DEAE-dextran, electroporation, and the like). When the purpose of the genetic modification of the cell is the production of a biologically active substance, the substance will generally be one that is useful for the treatment of a particular disorder. For example, it may be desirable to genetically modify the cells, so that they secrete a certain growth factor product associated with bone formation. The cells of the present invention can be genetically modified having exogenous genetic material introduced into the cells, to produce a molecule such as a trophic factor, a growth factor, a cytokine, and the like, that is beneficial for the culture of the cells. In addition, by having the cells genetically modified to produce said molecule, the cell can provide an additional therapeutic effect to the mammal when it is transplanted into a mammal in need of it. For example, the genetically modified cell can secrete a molecule that is beneficial to neighboring cells in the mammal. As used herein, the term "growth factor product" refers to a protein, peptide, mitogen or other molecule that has a growth effect, proliferative, differentiation or trophic in a cell. For example, growth factor products useful in the treatment of bone disorders include, but are not limited to, FGF, TGF-β, insulin-like growth factor and bone morphogenetic protein. In accordance with the present invention, constructs of genes comprising nucleotide sequences encoding heterologous proteins are introduced into ADAS cells. That is, the cells are genetically altered to introduce a gene whose expression has a therapeutic effect in the mammal. In accordance with some aspects of the invention, the ADAS cells of the mammal to be treated or of another mammal, can be genetically altered to replace a defective gene, and / or to introduce a gene whose expression has a therapeutic effect on the mammal that is being treated. In all cases in which a gene construct is transfected into a cell, the heterologous gene is operably linked to regulatory sequences required to achieve gene expression in the cell. Said regulatory sequences typically include a promoter and a polyadenylation signal. Gene construction is preferably provided as an expression vector that includes the coding sequence for a heterologous protein operably linked to essential regulatory sequences, so that when the vector is transfected into the cell, the coding sequence will be expressed by the cell. The coding sequence is operably linked to the regulatory elements necessary for the expression of that sequence in the cells. The nucleotide sequence encoding the protein can be cDNA, genomic DNA, synthesized DNA or a hybrid thereof, or an RNA molecule such as messenger RNA. Gene construction includes the nucleotide sequence that codes for the beneficial protein operably linked to the regulatory elements, and can continue to be present in the cell as a functional cytoplasmic molecule, a functional episomal molecule, or it can be integrated into the chromosomal DNA of the cell. Exogenous genetic material can be introduced into the cells, where it remains as a separate genetic material in the form of a plasmid. Alternatively, linear DNA that can be integrated into the chromosome can be introduced into the cell. When DNA is introduced into the cell, reagents that promote the integration of DNA into chromosomes can be added. DNA sequences that are useful for promoting integration can also be included in the DNA molecule. Alternatively, RNA can be introduced into the cell. Regulatory elements for gene expression include: a promoter, a start codon, a stop codon and a polyadenylation signal. It is preferred that these elements be operable in the cells of the present invention. Furthermore, it is preferred that these elements be operably linked to the nucleotide sequence encoding the protein, so that the nucleotide sequence can be expressed in the cells, and in this way the protein can be produced. It is generally considered that the start codons and stop codons are part of a nucleotide sequence encoding the protein. However, it is preferred that these elements be functional in the cells. Also, the promoters and polyadenylation signals used must be functional within the cells of the present invention. Examples of promoters useful for practicing the present invention include, but are not limited to, promoters that are active in many cells, such as the cytomegalovirus promoter, the SV40 promoters and retroviral promoters. Other examples of promoters useful for practicing the present invention include, but are not limited to, tissue-specific promoters, ie, promoters that function in some tissues but not in others; likewise, promoters of genes normally expressed in cells, with or without specific or general intensifier sequences. In some embodiments, promoters are used that constitutively express genes in the cells, with or without enhancer sequences. They are provided intensifier sequences in said modalities when appropriate or desirable. The cells of the present invention can be transfected using well known techniques readily available to those skilled in the art. Exogenous genes can be introduced into the cells using standard methods, wherein the cell expresses the protein encoded by the gene. In some embodiments, cells are transfected by calcium phosphate precipitation transfection, DEAE-dextran transfection, electroporation, microinjection, liposome-mediated transfer, chemical-mediated transfer, ligand-mediated transfer or transfer by recombinant viral vectors. In some modalities, recombinant adenovirus vectors are used to introduce DNA with desired sequences in the cell. In some embodiments, recombinant retrovirus vectors are used to introduce DNA with desired sequences into the cells. In some embodiments, standard transfection techniques mediated by CaP04, DEAE-dextran or lipid vehicles are used to incorporate the desired DNA into dividing cells. Standard antibiotic resistance selection techniques can be used to identify and select transfected cells. In some embodiments, DNA is introduced directly into the cells by microinjection. Also, well-known particle bombardment or electroporation techniques can be used to introduce foreign DNA into the cells. A second gene is usually co-transfected or linked to the gene therapeutic. The second gene is often a selectable antibiotic resistance gene. Transfected cells can be selected by developing the cells in an antibiotic that will kill the cells that do not absorb the selectable gene. In most cases, where the two genes are unlinked and co-transfected, cells that survive antibiotic treatment have both genes in them, and express the same. It should be understood that the methods described herein can be carried out in many ways, and with various modifications and permutations thereof which are well known in the art. It can also be appreciated that in no way should any theory exposed for modes of action or interactions between cell types be construed as limiting this invention, but presented so that the methods of the invention can be more fully understood.

Therapeutic Use of ADAS Cells In addition to the fact that ADAS cells can be differentiated along different lineages of cells, the invention also relates to the discovery that ADAS cells lack immunogenic characteristics with respect to the induction of cell proliferation. T. This characteristic is an indication that there is a reduced likelihood of an immune rejection by the immune cells of the recipient. In addition, it has been shown that ADAS cells do not stimulate allogeneic PBMCs in a mixed reaction of lymphocytes. In some embodiments of the invention, it may not be necessary or desirable to immunosuppress a mammal prior to the initiation of cell / gene therapy with ADAS cells. Accordingly, transplantation with allogenic or even xenogeneic ADAS cells is included in the invention. The use of ADAS cells for the treatment of a disease, disorder or bone condition provides an additional advantage, because ADAS cells can be introduced into a recipient without the requirement of an immunosuppressant agent. It is believed that successful transplantation of a cell requires permanent grafting of the donor cell without inducing an immune response of rejection of the graft generated by the recipient. Typically, to prevent a graft rejection response, non-specific immunosuppressive agents are used, such as cyclosporin, methotrexate, steroids and FK506. These agents are administered on a daily basis, and if administration is interrupted, graft rejection usually results. However, an undesirable consequence in the use of non-specific immunosuppressive agents is that they work by suppressing all aspects of the immune response (general immune suppression), thereby greatly increasing a recipient's susceptibility to infection and other diseases. The present invention provides a method for treating a bone disease, disorder or condition, by introducing undifferentiated or differentiated ADAS cells in the recipient, without the requirement of agents immunosuppressants. There is therefore a reduced susceptibility by the recipient of the transplanted ADAS cell to incur infection and other diseases that include cancer-related conditions, which is associated with immunosuppression therapy. The present invention includes the administration of a cell ADAS allogenic or a xenogeneic ADAS cell, or otherwise an ADAS cell that is genetically different from the recipient, in a receptor, to provide a benefit to the recipient. The present invention provides a method of using ADAS cells to treat a bone disease, disorder or condition, without the requirement of the use of immunosuppressive agents when administering the cells to a recipient. In another embodiment, the ADAS cell used in the present invention can be isolated from adipose tissue of any mammalian species including, but not limited to, human, mouse, rat, simian, gibbon and bovine. Preferably, the ADAS cell is isolated from a human, a mouse or a rat. More preferably, the ADAS cell is isolated from a human. The ADAS cell can be administered to a mammal after a period of in vitro culture. The ADAS cell can be cultured in a way that induces the ADAS cell to differentiate in vitro. However, it is preferred that the ADAS cell be implanted in the receptor in an undifferentiated state, and that the implanted ADAS cell be differentiated to express at least one characteristic of a bone cell in vivo. The ADAS cells of this invention can be transplanted in a mammal using techniques known in the art such as, for example, those described in the U.S.A. Nos. 5,082,670 and 5,618,531, each incorporated herein by reference, or at any other suitable site in the body. Transplantation of the cells of the present invention can be achieved using techniques well known in the art, as well as those described herein or as they will be developed in the future. The present invention comprises a method for transplanting, grafting, infusing, or otherwise introducing the cells into a mammal, preferably a human. The number of ADAS cells administered to a mammal can be related to, for example, the performance of cells after processing of the adipose tissue. A portion of the total number of cells can be retained for later use, or they can be cryopreserved. In addition, the dose delivered depends on the route of delivery of the cells to the mammal. The dosage of ADAS cells varies within wide limits, and can be adjusted to the individual requirements in each particular case. The number of cells used depends on the weight and condition of the recipient, the number and / or frequency of administrations, and other variables known to those skilled in the art. This number can be adjusted by orders of magnitude to achieve the desired therapeutic effect. Between approximately 105 and approximately 1013 cells ADAS per 100 kg of body weight can be administered to the individual. In some embodiments, they are administered between about 1.5 x 10 6 and about 1.5 x 10 12 cells per 100 kg of body weight. In some embodiments, are administered between about 1 x 109 and about 5 x 1011 cells per 100 kg of body weight. In other embodiments, they are administered between about 4 x 109 and about 2 x 1011 cells per 100 kg of body weight. In other embodiments, between about 5 x 10 8 cells and about 1 x 10 10 cells per 100 kg of body weight are administered. The ADAS cells can be suspended in a suitable diluent, at a concentration of about 0.01 to about 5 x 106 cells / ml. Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the ADAS cells and the receptor, such as pH regulated saline or other suitable excipients. The composition for administration can be formulated, produced and stored according to standard methods that meet the appropriate sterility and stability. The cells can also be encapsulated and used to deliver biologically active molecules, according to known encapsulation technologies, including microencapsulation (see, for example, U.S. Patent Nos. 4,352,883, 4,353,888, and 5,084,350, incorporated herein by reference). or macroencapsulation (see, for example, U.S. Patent Nos. 5,284,761, 5,158,881, 4,976,859, and 4,968,733, and International Publication WO 92/19195 and WO 95/05452, all of which are incorporated herein by reference). For macroencapsulation, the number of cells in the devices can be made to vary; preferably, each device contains between 103-109 cells, more preferably about 105 to 107 cells. Several macroencapsulation devices can be implanted in the mammal. Methods for macroencapsulation and implantation of cells are well known in the art and are described, for example, in the U.S. patent. 6,498,018. The mode of administration of the cells of the invention to the mammal can vary, depending on several factors including the type of disease being treated, the age of the mammal, whether the cells are differentiated or not, whether the cells have heterologous DNA introduced. in them, and similar. The cells can be introduced at the desired site by direct injection, or by any other means used in the art for the introduction of compounds administered to a mammal suffering from a particular bone disease or disorder. ADAS cells can be administered in a host in a wide variety of ways. Management modes include, but are not limited to, intravascular, intracerebral, parenteral, intraperitoneal, intravenous, epidural, intraspinal, intrasternal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac or intramuscular. Preferably, the cells are used in spinal fusion procedures.

Composition The invention also provides a matrix for implantation in a mammal, wherein the matrix comprises an ADAS cell of the invention. The The matrix may also include, but is not limited to, an ADAS cell, an ADAS cell lysate, a conditioned medium of ADAS cells, and an extracellular matrix produced by an ADAS cell. The matrix may also contain, or be treated with, one or more bioactive factors including, but not limited to, an antiapoptotic agent (ie, erythropoietin, thrombopoietin, insulin-like growth factor I and insulin-like growth factor II, growth factor of hepatocytes, caspase inhibitors); an anti-inflammatory agent (ie, p38, MAPK inhibitors, TGF-beta inhibitors, statins, inhibitors of IL-6 and IL-1, and nonsteroidal anti-inflammatory drugs); an immunosuppressive / immunomodulatory agent; an mTOR inhibitor; an antiproliferative agent; a corticosteroid (ie, prednisolone, hydrocortisone); an antithrombogenic agent; and an antioxidant. The presence of a bioactive factor may contribute to the proliferation and / or differentiation of ADAS cells. The invention further provides in some aspects, methods for regenerating bone tissue in a mammal in need thereof, by administering a composition comprising an ADAS cell, a matrix, an ADAS cell lysate, an ADAS cell product of the invention (i.e. , molecules secreted by the ADAS cell), or any combination thereof in a mammal. As such, the invention encompasses a pharmaceutical composition, wherein the composition can be used in the treatment of a bone condition. For example, the bone condition includes, but is not limited a, a bone fracture, a bone / spinal cord deformation, osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis, osteomalacia, rickets, osteitis osa, renal bone dystrophy and Paget's bone disease. Preferably, the invention provides compositions and methods for improving bone fusion after a spinal fusion procedure. In a non-limiting embodiment, a formulation comprising the cells of the invention is prepared for administration directly to the site where the production of new bone tissue is desired. For example, the cells of the invention can be suspended in a hydrogel solution for injection. Alternatively, the hydrogel solution containing the cells may be allowed to harden, for example, in a mold, to form a matrix having cells dispersed therein prior to implantation, or once the matrix has been hardened, the cell formations can be cultured so that the cells are mitotically expanded before implantation. The hydrogel is an organic polymer (natural or synthetic) that is entangled by means of covalent, ionic or hydrogen bonds, which create a three-dimensional open network structure that traps water molecules to form a gel. Examples of materials that can be used to form a hydrogel, include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines and polyacrylates, which are ionically entangled, or block polymers such as polyethylene oxide-polypropylene glycol block copolymers, which are intertwined by the use of temperature or pH, respectively. In some embodiments, the polymers are at least partially soluble in aqueous solutions, such as water, salt solutions regulated in their pH, or aqueous alcoholic solutions, which have charged side groups, or a monovalent ionic salt thereof. Examples of polymers with acidic side groups which can be reacted with cations are poly (phosphazenes), poly (acrylic) acids, poly (methacrylic) acids, copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate) and sulfonated polymers, such as sulfonated polystyrene. Copolymers having acidic side groups formed by the reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used. Examples of acid groups are carboxylic acid groups, sulfonic acid groups, halogenated alcohol groups (preferably fluorinated), phenolic OH groups and acid OH groups. Examples of polymers with basic side groups that can be reacted with anions are poly (vinyl amines), poly (vinyl pyridine), poly (vinyl imidazole), and some polyphosphazenes substituted with one another. The quaternary or ammonium salt of the polymers can also be formed from nitrogens of the base structure or pendant amino groups. Examples of basic side groups are amino and imino groups. Other examples of polymers include, but are not limited to, poly-alpha-hydroxy esters, polydioxanone, propylene fumarate, poly-ethylene glycol, poly-ortho esters, polyanhydrides and polyurethanes, poly-L-lactic acid, poly glycolic acid and polylactic acid-polyglycolic acid copolymer.

ADAS cell transplantation using scaffolds The cells of the invention can be seeded on or in a three-dimensional scaffold, and can be implanted in vivo, where the seeded cells proliferate in the structure and form a replacement tissue in vivo in cooperation with mammalian cells. . In some aspects of the invention, the scaffold comprises extracellular matrix, cell lysate (e.g., soluble cell fractions), or combinations thereof, of ADAS cells. In some embodiments, the scaffold comprises an extracellular matrix protein secreted by the cells of the invention. Alternatively, the extracellular matrix is an exogenous material selected from the group consisting of calcium alginate, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan sulfate and gelatin of bone matrix. In some aspects, the matrix comprises natural or synthetic polymers. The invention includes biocompatible scaffolds, networks, self-assembly structures, and the like, whether biodegradable or not, liquid or solid. Such scaffolds are known in the techniques of cell-based therapy, surgical reconstruction, tissue engineering and wound healing. Preferably, the scaffolds are pretreated (for example, seeded, inoculated, put in contact) with the cells, matrix extracellular, conditioned medium, cell lysate, or combination thereof. In some aspects of the invention, the cells adhere to the scaffold. The seeded scaffold may be introduced into the body of a mammal in any manner known in the art including, but not limited to, implantation, injection, surgical fixation, transplantation with other tissue, injection, and the like. The scaffolding of the invention can be configured to the shape and / or size of a tissue or organ in vivo. For example, but not by way of limitation, the scaffold may be designed so that the structure of the scaffold supports the seeded cells without subsequent degradation; support the cells at the time of sowing until the tissue transplant is remodeled by the host tissue; and allow the seeded cells to unite, proliferate and develop into a tissue structure that has sufficient mechanical integrity to sustain itself. Scaffolds of the invention may be administered in combination with any of one or more growth factors, cells, drugs or other components described elsewhere herein, which stimulate tissue formation or otherwise enhance or improve the practice of the invention. . The ADAS cells that will be planted on the scaffolds can be genetically engineered to express growth factors or drugs. In another preferred embodiment, the cells of the invention are seeded on a scaffold, where the material exhibits specified physical properties of porosity and biomechanical resistance that mimics the characteristics of the true bone, promoting in this way the stability of the final structure and the access and egress of cellular metabolites and nutrients. That is, the material must provide structural support, and can form a scaffolding in which vascularization and migration of host cells can occur. In the preferred embodiment, the ADAS cells are first mixed with a carrier material before application to a scaffold. Suitable carriers include, but are not limited to, calcium alginate, agarose, types I, II, IV or other isoform of collagen, fibrin, polylactic acid / polyglycolic acid, hyaluronate derivatives, gelatin, laminin, fibronectin, starch, polysaccharides, saccharides, proteoglycans, synthetic polymers, calcium phosphate and ceramic materials (ie, hydroxyapatite, tricalcium phosphate). The external surfaces of the three-dimensional structure can be modified to improve the union or growth of cells and tissue differentiation, such as by plasma coating the structure, or the addition of one or more proteins (eg, collagen, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (eg, heparin sulfate, chondroitin 4-sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate), a cell matrix and / or other materials such as, but not limited to, gelatin, alginates, agar and agarose. In some embodiments, it is important to recreate in culture the cellular microenvironment present in vivo, such as the degree to which the cells of the invention develop prior to implantation in vivo. In addition, they can Growth factors, osteogenic inducing agents and angiogenic factors are added to the culture medium before, during, or subsequent to inoculation of the cells to trigger differentiation and tissue formation by ADAS cells after implantation in the mammal.

Therapeutic applications of ADAS cells The present invention encompasses methods for administering an ADAS cell to a mammal, including a human, to treat a disease wherein the introduction of ADAS cells provides therapeutic relief. The cells of the invention can be administered alone or as mixtures with other cells and / or a bioactive factor, as discussed elsewhere herein. A cell that can be administered in conjunction with ADAS cells of the invention includes, but is not limited to, other multipotent or pluripotent cells, an osteocyte, an osteoblast, an osteoclast, a bone-coating cell, a stem cell and a bone marrow cell. that is. The different cell types can be mixed with the ADAS cells immediately or shortly after administration to a mammal, or they can be co-cultured together for a period before their administration to a mammal. The person skilled in the art will readily understand that cells ADAS can be transplanted into a mammal, and after they receive signals and indications from the surrounding environment, the cells differentiate into mature cells in vivo by effect of the neighboring cell environment. Preferably, ADAS cells differentiate into a cell that exhibits at least one characteristic of a bone cell. Alternatively, ADAS cells can be differentiated in vitro into a desired cell type, and the differentiated cell can be administered to a mammal in need thereof. The invention also encompasses the grafting of ADAS cells in combination with other therapeutic methods to treat bone diseases. Preferably, the cells are useful for improving bone fusion after a spinal fusion procedure. ADAS cells can be co-grafted with other cells, both genetically modified cells and non-genetically modified cells that exert beneficial effects in the mammal. Therefore, the methods described herein may be combined with other therapeutic methods as would be understood by those skilled in the art, once provided with the teachings provided herein. ADAS cells can be administered with other drugs or beneficial biological molecules (growth factors, trophic factors). When ADAS cells are administered with other agents, they can be administered together in an individual pharmaceutical composition, or in separate pharmaceutical compositions, simultaneously or sequentially with the other agents (either before or after the administration of the other agents). Bioactive factors that can be co-administered include, but are not limited to, an anti-apoptotic agent (ie, erythropoietin, thrombopoietin, insulin-like growth factor I, and growth factor II). insulin type, hepatocyte growth factor, caspase inhibitors); an anti-inflammatory agent (ie, p38, MAPK inhibitors, TGF-beta inhibitors, statins, inhibitors of IL-6 and IL-1, and nonsteroidal anti-inflammatory drugs); an immunosuppressive / immunomodulatory agent; an mTOR inhibitor; an antiproliferative agent (i.e., azathioprine, mycophenolate mofetil); a corticosteroid (ie, prednisolone, hydrocortisone); an antithrombogenic agent; and an antioxidant. The invention encompasses the administration of ADAS cells to a mammal as undifferentiated cells, ie, as grown in growth medium. Alternatively, ADAS cells can be administered after exposure in culture to conditions that stimulate differentiation to a desired phenotype, e.g., an osteogenic phenotype. The cells of the invention can be surgically implanted, injected, supplied (for example, by means of a catheter or syringe), or otherwise administered directly or indirectly to the site in need of reconstruction or augmentation. The cells can be administered by means of a matrix (for example, a three-dimensional scaffold). The cells can be administered with conventional pharmaceutically acceptable carriers. Administration routes of the cells of the invention or components (eg, extracellular matrix, cell lysate, conditioned medium) thereof, include intramuscular, ophthalmic, parenteral (including intravenous), intraarterial, subcutaneous, oral and nasal. Particular routes of parenteral administration include, but are not limited to, intramuscular, subcutaneous, intraperitoneal, intracerebral, intraventricular, intracerebroventricular, intrathecal, intraclastemal, intraspinal and / or perispinal routes of administration. Preferably, the cells are used in spinal fusion procedures. The cells of the invention can be introduced alone or in a mixture with a composition useful in the reconstruction of wounds and bone defects. Such compositions include, but are not limited to, bone morphogenetic proteins, hydroxyapatite / tricalcium phosphate (HA / TCP) particles, gelatin, poll-L-lysine and collagen. For example, the cells of the invention can be combined with demineralized bone matrix (DBM) or other matrices that form the mixed osteogenic material (which forms bone as such) as well as osteoinductive. To improve the differentiation, survival or activity of the implanted cells, additional bioactive factors may be added as discussed elsewhere herein. For example, a bioactive factor may include, but is not limited to, a bone morphogenetic protein, vascular endothelial growth factor, fibroblast growth factors, and other cytokines that have osteoconductive and / or osteoinductive capacity. To improve the vascularization and survival of the transplanted bone tissue, angiogenic factors such as VEGF, PDGF or bFGF can be added, either alone or in combination with endothelial cells or their precursors. Alternatively, the ADAS cells that will be transplanted they can be genetically engineered to express such growth factors, antioxidants, anti-apoptotic agents, anti-inflammatory agents or angiogenic factors.

Pharmaceutical compositions Also encompassed within the scope of the invention, are ADAS cell products that include, but are not limited to, extracellular matrices secreted by the ADAS cells themselves, cell lysates (e.g., soluble cell fractions) of ADAS cells and conditioned medium of ADAS cells. As such, in terms of the administration of a composition comprising an ADAS cell, the invention includes a pharmaceutical composition comprising at least one of the following: an ADAS cell, an extracellular matrix produced therefrom, a cell lysate of the same, or a conditioned medium of ADAS cells. The pharmaceutical composition of the invention preferably includes a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is preferably used for the treatment of bone conditions as defined herein. The pharmaceutical compositions of the invention may comprise homogeneous or heterogeneous populations of ADAS cells, extracellular matrix or cell lysate thereof, or conditioned medium thereof, in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers for the cells of the invention, include suitable organic or inorganic carrier substances that do not detrimentally react with the cells of the invention or compositions or components thereof. To the extent that they are biocompatible, suitable pharmaceutically acceptable carriers include water, saline (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, and polyvinyl pyrrolidine. Said preparations can be sterilized, and if desired, can be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that influence the osmotic pressure, pH regulators and coloring agents. Pharmaceutical carriers suitable for use in the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (seventeenth edition, Mack Pub. Co., Easton, Pa.) And in WO 96/05309, each of which is incorporated herein by reference. As another example but not by way of limitation, the cells of the invention can be administered alone, in a pharmaceutically acceptable carrier, or they can be seeded on or in a matrix as described elsewhere herein, they can be used to reconstruct or replace tissue bone damaged or destroyed, to augment existing bone tissue, to introduce new or altered tissue, or to modify artificial prostheses. When cells are administered in semi-solid devices or In the case of solids, surgical implantation at a precise site in the body is typically a suitable means of administration. In the case where the cells are administered in the form of a liquid or fluid pharmaceutical composition, the cells can be administered to a more general site (ie, throughout a diffusely affected area), from which they migrate to a particular site ( that is, responding to chemical signals). Other embodiments encompass treatment methods, administering pharmaceutical compositions comprising cellular components of ADAS cells (e.g., cell lysates or components thereof) or products (e.g., extracellular matrix, trophic factors and other biological factors naturally produced by ADAS cells. or through genetic modification, ADAS cell culture conditioned medium). Again, these methods may further comprise administering other active agents as described elsewhere herein. ADAS cells can also be applied with additives to improve, control or otherwise direct, the desired therapeutic effect. Also, the cells can be applied with a biocompatible matrix that facilitates the tissue engineering in vivo sustaining and / or directing the fate of the implanted cells. Prior to administration of ADAS cells in a mammal, cells can be stably or transiently transfected or transduced with a nucleic acid of interest, using a plasmid, viral or alternative vector strategy. The cells can be administered after genetic manipulation, so that they express gene products that are intended to promote the therapeutic responses provided by the cells. The ADAS cells of the invention can be used to treat mammals that require repair or replacement of bone tissue resulting from disease or trauma or tissue failure to develop normally. The treatment may involve the use of the cells of the invention to produce new bone tissue. For example, undifferentiated or osteogenic cells of the invention induced by differentiation can be used to treat bone conditions including metabolic and non-metabolic bone diseases. Examples of a bone condition include, but are not limited to, a bone fracture, a bone / spinal cord deformation, osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis, osteomalacia, rickets, osteitis fibrosa, renal bone dystrophy and bone disease. of Paget.

Spinal fusion As discussed here, back pain continues to be a major public health problem, especially among the elderly. Severe and persistent back pain often causes weakness and disability. This pain is closely associated with abnormalities of the intervertebral discs of the spine. Based on the present disclosure, discs may be treated degenerated by restoring damaged tissues inside the disc. The ADAS cells of the invention can be used to stimulate bone development, and thus restore the intervertebral discs in various stages of degeneration. However, it is often necessary to remove at least a portion of the damaged and / or malfunctioning back component. For example, when a disc becomes fractured, a surgical discotomy procedure can be performed to remove the fractured disc, and fuse the two vertebrae together between the removed disc. Spinal fusion is a procedure by which two or more of the vertebrae that make up the spine are fused together with bone grafts and internal devices (such as rods) that heal in an individual solid bone. Spinal fusion can eliminate unnatural movement between the vertebrae and, in turn, can reduce the pressure on the nerve endings. In addition, spinal fusion can be used to treat, for example, injuries of spinal vertebrae caused by trauma; protrusion and degeneration of the cushion disc between the vertebrae (sometimes called a dislocated intervertebral disc or a herniated disc); abnormal curvatures (such as scoliosis or kyphosis); and weak or unstable spine caused by infections or tumors. The present invention encompasses compositions and methods for improving the success rates of spinal fusion procedures. Since it has been shown that the ADAS cells of the present Invention form bone in vivo, ADAS cells can be used in place of bone grafts conventionally used in spinal fusion surgeries. Specifically, ADAS cells can be used to stimulate bone formation between two adjacent vertebrae (within the vertebral body), as well as between adjacent transverse processes (within the spaces of the intertransverse process on either side of the spine). The ADAS cells of the present invention have numerous applications in the treatment of spinal disorders, including the promotion of proteoglycan-rich matrix production in the repair of intervertebral discs, the production of bone for the intervertebral body in spinal fusion. of the intertransverse process, and the production of bone for the healing of long bone fractures. The present invention is based on the discovery that ADAS cells can be used to facilitate fusion of bone in a spinal fusion procedure. When a disc becomes fractured, a surgical discotomy procedure may be performed that removes the fractured disc and fuses the two vertebrae between the removed disc. Details regarding typical implementations of methods for fusion of vertebrae are described in the U.S. Patents. Nos. 6,033,438 and 5,015,247, the contents of which are hereby incorporated by reference in their entirety. The degeneration of the intervertebral discs is commonly treated with a fusion of segments, whereby the elements of the anterior and posterior spine of the interbody space are fused together. Is It is important to consider the mechanical stresses imposed on the anterior and posterior elements when considering a fusion technique. The elements of the anterior movement segment (vertebral bodies and disc) have approximately 80% of the compression force at that determined level in the spine. The posterior 1/3 of the vertebral body and disc represents the central point for axial compression in the spine. These mechanics are critical to evaluate which type of fusion will have the best clinical result for a certain pathology. The invention provides a spinal fusion method in which ADAS cells are used as a source of a bone substitute. In one embodiment, the invention includes methods for performing one or more spinal fusions in a mammal, comprising introducing an effective amount of ADAS cells into one or more suitable interbody spaces in the mammal, by injecting the cells through a syringe. , catheter or cannula that facilitates spinal fusion at the individual or multiple levels. ADAS cells will be established under physiological conditions, that is, in vivo over time, where the cells differentiate and form bone. The presence of ADAS cells improves bone fusion in a spinal fusion procedure. In another embodiment, the invention includes methods for performing one or more spinal fusions in a mammal, comprising placing in the posterior portion of at least one suitable interbody space, a metal implant selected from pedunculated rods and screws or plates and pedunculated screws by joining them to the adjacent vertebrae; Inject an effective amount of ADAS cells into the anterior portion of the interbody space; and allowing the ADAS cells to differentiate into a cell that exhibits at least one characteristic of a bone cell, and thereby form bone in vivo. The invention includes methods for performing spinal fusions using an anterior, posterior, or posterolateral approach to the interbody space. The posterolateral procedure (unilateral or bilateral) reduces surgical morbidity over a previous procedure, but caution is required while working around the equine tail and nerve roots protruding into the spinal canal. The posterior access and the visualization of the interbody space is more limited than with the previous procedure, but many spinal surgeons are trained in how to treat those circumstances. As discussed elsewhere herein, ADAS cells may also comprise an amount of one or more active bioactive agents suitable for promoting bone growth, such as a growth factor, a bone morphogenetic protein, or a pharmaceutical carrier for same. One mechanism by which ADAS cells can provide a therapeutic or structural benefit is by incorporating them or their progeny into newly generated, existing or reconstructed tissues or tissue components. For example, ADAS cells and / or their progeny can incorporate in the newly generated bone, another structural or functional tissue, and thus cause a therapeutic or structural improvement, or contribute to it. Another mechanism by which ADAS cells can provide a therapeutic or structural benefit, is by expressing and / or secreting molecules, for example, growth factors, that promote the creation, retention, restoration and / or regeneration of the structure or function of a tissue or tissue component determined. ADAS cells can also be used in combination with other cells or devices such as synthetic or biological scaffolds, materials or devices that provide factors, drugs, chemicals or other agents that modify or ameliorate the relevant characteristics of cells as further described herein. . In accordance with the invention described herein, ADAS cells can be delivered to the mammal shortly after harvest of the adipose tissue of the mammal. For example, cells can be administered immediately after processing of the adipose tissue and obtaining an ADAS cell composition. Finally, the regulation of the supply will depend on the availability of the mammal and the processing time required to process the adipose tissue. In another embodiment, the regulation for delivery may be relatively longer, if the cells that will be reinfused into the mammal are subjected to modification, purification, stimulation or other additional manipulation, as discussed herein. The number of cells administered to a mammal can relate to, for example, the performance of cells after processing of adipose tissue. A portion of the total number of cells can be retained for later use, or can be cryopreserved. The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or description of the present invention as set forth herein.

EXAMPLES The invention is now described in relation to the following examples. These examples are provided for the purpose of illustration only, and the invention is not limited to these examples, but rather encompasses all variations that are apparent as a result of the teachings provided herein. The following experiments were performed to determine the function of ADAS cells on the result of a spinal fusion procedure. For example, the effect of syngenic or allogenic ADAS cells on spinal fusion procedures. The results of the present demonstrate that the ADAS cells are osteogenic, and contribute to the improvement in spinal fusion. Based on the present disclosure, ADAS cells can be used to treat mammals including, but not limited to, trauma victims, osteoporotic mammals lacking adequate numbers of osteogenic cells, and mammals with fractures without union.

EXAMPLE 1 Alternatives for autograft bone in spinal fusion surgery More than 75% of the population of North America suffers from back pain. In some cases, underlying medical conditions can contribute to back pain. These include scoliosis, spinal stenosis, degenerative disease of the intervertebral discs, infectious processes, tumors and trauma. For 1% of the population, back pain is so severe that they are forced to suffer disability during the course of their life; An additional 1% of the population suffers from disability due to back pain for a limited period. Most mammals with back pain are treated with conservative therapies; however, when modalities such as bed rest and medication fail, doctors often recommend spinal fusion surgery. The purpose of this operation is to form ectopic bone between two or more adjacent vertebrae, "fusing" them into a solid structure. The immobilization of the vertebral joint reduces the pressure on the nerve roots that leave the spinal cord, and the resulting painful sensation. Surgeons use a posterolateral procedure for the lumbar spine, introducing a bone graft or osteoinductive material with a mechanical support between the vertebral bodies that form ectopic bone (Figures 1A-1 D).

Unless it is desired to be limited by a particular theory, it is believed that the "ideal" graft material for a spinal fusion would provide the following properties: mechanical support (the material stabilizes the spinal / surgical site during the period of Recovery); osteoconductive (the material facilitates inward growth and the integration of adjacent bone on itself); osteoinductive (the material recruits and stimulates bone formation and growth from cells that may not naturally do so); and osteogenic (the material contains cells that by themselves are capable of forming new bone). Currently, the "gold standard" for spinal fusion reconstruction is autologous bone, usually harvested from the individual's iliac crest. Surgeons transplant the bone of the mammal itself to the site that needs it. However, this is far from being a perfect solution. In 30% of mammals, the donor site becomes infected, bruised, fractured or painful after surgery. Of course, when a mammal requires autograft bone for multiple spinal fusions, the iliac crest may not provide sufficient material. The underlying health of the mammal also influences the outcome of spinal fusion surgery. Mammals with osteoporosis or vascular insufficiency due to diabetes, smoking or age, exhibit reduced formation of new bone and lack of union at the site of spinal fusion (Whang, et al., 2003, Spine J. 3: 155- 65). Thus, there is a need for alternatives for autograft bone in spinal fusion surgery.

While there are alternative materials available, they all face a common limitation: none exhibits osteogenic capacity. The allograft bone of cadavers can be used, stored and used in the operating room, as needed. These materials can be preconfigured for specific use, or sprayed, allowing them to be applied as a paste to the surgical site; however, allografts can cause inflammation, can induce an immune response and have been an infectious source in a limited number of cases (Whang et al., 2003, Spine J. 3: 155-65). Because the allograft bone is sterilized, it stops containing viable native cells that form bone (osteoblasts, osteocytes), and lack osteogenic properties. In clinical trials, allograft bone is inferior to autograft bone in multiple-level spinal fusions (Whang et al., 2003, Spine J. 3: 155-65). Ceramic materials such as hydroxyapatite and tricalcium phosphate (HA / TCP) are osteoconductive, and promote new bone formation. However, they lack osteogenic and osteoinductive properties, limiting their usefulness. While osteoinductive growth factors such as bone morphogenetic proteins (BMPs) are commercially available (Sofamor / Medtronics Infuse ™), they require the presence of osteogenic cells within the spinal fusion site to promote new bone formation (Whang et al. al., 2003, Spine J. 3: 155-65). The development of an osteogenic cell has the potential to improve the result with any of these alternative spinal fusion materials.

Many animal species have served as models in preclinical spinal fusion tests. These include rat, rabbit, dog, sheep, goat and non-human primate (Khan et al., 2004, Biomaterials 25: 1475-85, Liebschner er al., 2004, Biomaterials 25: 1697-714, Sandhu er al., 2001 , Eur. Spine J. 10: S 122-31). Of these, the rat (Boden et al., 1998, Spine 23: 2486-92, Cui et al., 2001, Spine 26: 2305-10, Wang er al., 2003, J. Bone Joint Surg. Am. -A: 90511) and the rabbit (Khan et al., 2004, Biomaterials 25: 1475-85, Kruyt er al., 2004, Biomaterials 25: 1463-73) have been used for "proof of concept" studies due to the size and cost of the animal. In each species, surgeons can use a posterolateral procedure for the lumbar spine, similar to that used to treat other mammals. The rabbit is most commonly used for viability studies of spinal fusion (Khan et al., 2004, Biomaterials 25: 475-85), due in part to the size of the animal and the confirmed observation that the speed of spinal fusion in the rabbit is similar to that observed in a human. However, the rabbit has certain disadvantages compared to the rat model. Unlike rats, where well-characterized inbred strains are available, laboratory rabbits do not exhibit syngeneic or congenic haplotypes. In this way, it may not be possible to routinely transplant cells from one rabbit to another without the risk of rejection. The rat posterolateral spinal fusion model has been used successfully to demonstrate the osteoinductive effect of the bone morphogenetic protein 7 when it is presented on a collagen scaffold (Salamon er al., 2003, J. Spinal Disord. Tech. 16: 90-5). Several groups have successfully used the rat model to evaluate the osteogenic effect of bone marrow stromal cells in spinal fusion (Boden et al., 1998, Spine 23: 2486-92, Cui et al., 2001, Spine 26: 2305-10; Wang er a /., 2003, J. Bone Joint Surg. Am. 85-A: 905-1 1). They achieved statistically significant improvements in spinal fusion within 4 to 9 weeks after the implantation of bone marrow stromal cells, compared to scaffolding alone. Each study used groups of n = 4 to 8 animals. The following experiments are designed to evaluate the function of ADAS cells in spinal fusion procedures.

Isolation of ADAS cells Subcutaneous adipose tissue is harvested from male Fischer rats (8 to 10 weeks of age, n = 25, yielding approximately 3 grams of tissue per rat). ADAS cells are prepared according to published methodologies (Aust et al., 2004, Cytotherapy 6: 7-14, Halvorsen et al., 2001, Metabolism 50: 407-413, Sen et al., 2001, Journal of Cellular Biochemistry 81 : 312-319). Briefly, adipose tissue is shredded, washed and suspended in an equal volume of pH regulated saline with phosphate containing 1% bovine serum albumin and 0.1% type I collagenase (Worthington Biochemical, Lakewood NJ). After digestion for 60 minutes at 37 ° C with shaking (50 rpm), the suspension is centrifuged at 1200 rpm for 5 minutes at room temperature, and the stromal cells of the vascular fraction are transformed into pellets. The stromal vascular cells are seeded at a density of 0.1 grams of tissue digesta per cm2 in "stromal cell medium" (DMEM / Ham's F-12 medium supplemented with 10% fetal bovine serum (Hyclone, Logan UT), and 1% antibiotic / antifungal The cells are incubated for 3 to 6 days in a humidified CO2 incubator at 5% until they reach 75% confluence.This gives approximately 25-30 X 10 4 cells / cm2. ADAS cells are harvested by digestion with trypsin / EDTA, and passed through a passage at a stocking density of 5 X 10 3 cells / cm 2. Cells are expanded by up to 2 passages to obtain more than 60 million cells (table 1) Cells are evaluated in vitro for their osteogenic and adipogenic capacity using standard tests for an inductive period of 1 to 3 weeks as described in Halvorsen et al., 2001, Tissue Eng. 7: 729-41; Hicok et al. ., 2004, Tissue Engineering 10: 371-380). The cells can be cryopreserved in liquid nitrogen before use. To track the cells histologically, the cells are labeled during the initial passage with a retroviral vector possessing the LacZ gene expressing β-galactosidase to provide a traceable marker. The stable integration of retroviral vectors reduces the risk that the marker will be lost during the implantation time. This method has been commonly used to track implanted cells.

TABLE 1 Expansion and estimated yield of ADAS cells EXAMPLE 2 Osteogenesis of ADAS cells in vitro It has been shown that human ADAS cells exhibit an in vitro osteogenic phenotype when cultured in the presence of ascorbate, β-glycerophosphate, dexamethasone and 1.25 dihydroxyvitamin D3 (Halvorsen et al., 2001, Tissue Eng. 7: 729-41) . Under these conditions, ADAS cells mineralize their extracellular matrix as demonstrated by positive staining with alizarin red or von Kossa by calcium phosphate deposition (Figures 3A-3D). It was observed that the osteogenesis of human ADAS cells during a period of 10 days, was accompanied by an increase in alkaline phosphatase activity. At the end of the culture period, the osteogenic (mineralized) cells exhibited a 3-fold higher level of alkaline phosphatase than cells maintained under control conditions (Figure 4). Likewise, there was a time-dependent increase in secreted levels of osteocalcin protein under osteogenic conditions. ADAS cells expressed a number of gene markers consistent with an osteoblast phenotype, including osteocalcin, osteopontin, bone morphogenetic proteins (BMP) 2 and 4, and BMP receptors IA, IB, and II. It has also been shown that ADAS cells have the potential to differentiate along multiple lineage pathways. In response to specific cocktails of chemicals and growth factors, ADAS cells can differentiate into chondrocytes, osteoblasts, adipocytes, and glial and neuronal cell-like cells in vitro (Figure 2).

EXAMPLE 3 ADAS cells are osteogenic in vivo To extend the in vitro findings, human ADAS cells were transplanted into immunodeficient SCID mice. The ADAS cells were loaded in cubes of 3 cm3 of hydroxyapatite / tricalcium phosphate scaffold (HA / TCP), and implanted subcutaneously. After a period of 6 weeks, the implants were harvested, fixed, decalcified and stained with hematoxylin / eosin or with specific antibodies of human nuclear antigens (Figures 5A and 5B). Based on hematoxylin and eosin staining, it was observed that new bone was formed adjacent to the hydroxyapatite / tricalcium phosphate scaffold in the presence of human ADAS cells. Human cells were identified within the bone based on an immunofluorescence analysis with antigen-specific antibody humans. In the presence of the scaffold alone (no cells) new bone was not formed, and no human cells were detected. These studies demonstrate that ADAS cells are capable of exhibiting osteogenesis in vivo.

EXAMPLE 4 ADAS cells can be transplanted allogeneically The following experiments serve to provide proof of concept regarding the allogeneic transplantation of ADAS cells in the spinal fusion model. It has been shown that ADAS cells can not induce a proliferative response from allogeneic lymphocytes in a mixed lymphocyte reaction. While not wishing to be limited by any particular theory, it is believed that ADAS cells release a factor that inhibits the immune response of lymphocytes to allogeneic antigens. The presence of ADAS cells prolonged skin graft survival in the baboon model, and thus indicates that adult stem cells can be transplanted allogeneically for tissue engineering applications. By using a canine model, a critically sized segment defect can be created in the femoral diaphysis of dogs. Defects can be repaired with hydroxyapatite / tricalcium phosphate scaffolds alone or in combination with autologous or allogeneic ADAS cells; the allogenic cells are desorbed for the HLA-1 and HLA-2 antigens (Table 2). Transplant recipients do not receive any immunosuppressive therapy. HE sacrifices the animals 16 weeks later, and the degree of bone repair observed in the presence of the ADAS cells can be compared to the scaffold transplant alone (without ADAS cells). While not wishing to be limited by any particular theory, it is believed that there will be no observable significant difference between the repair obtained with autologous ADAS cells against allogeneic ADAS cells, nor will there be evidence of any immune response to allogeneic cells. These experiments serve to demonstrate the fact that allogeneic stem cell transplantation in a designed tissue construct is feasible, and in some cases does not require immunosuppressive therapy.

TABLE 2 Histomorphometric analysis of bone and ceramics in canine segment defects ** ** The percentage of ceramic was the percentage of the total area of the implant occupied by the ceramic, and the percent of bone was the percentage of the porous space occupied by bone. The values are given as the mean ± standard deviation.

In comparison with cell-free implants, the difference was significant (p <0.05) (Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A: 1927-35).

EXAMPLE 5 Syngenic ADAS cells in spinal fusion The following experiments serve to construe the hypothesis that ADAS cells are osteogenic in vivo and, in combination with a suitable biomaterial vehicle, can improve and accelerate spinal fusion in animal models. Table 3 summarizes the experimental design. The initial studies are carried out with syngenic ADAS cells (cells of the same rat strain), which mimic the conditions existing in a transplant of human autologous cells. By removing problems related to immune response and rejection, these experiments focus on the osteogenic capacity of ADAS cells for spinal fusion. These experiments using the rat as a spinal fusion model are compared with methods known in the art (Boden et al., 995, Spine 20: 412-20, Wang et al., 2003, J. Bone Joint Surg. 85-A: 905-1 1; Cui et al., 2001, Spine 26: 2305-10; Sandhu et al., 2001, Eur. Spine J. 10 Suppl. 2: S122-31; Wang et al., 2003, Spine J. 3: 155-65).

Surgical procedure and euthanasia An intertransverse spinal arthrodesis of individual level (L4-L5) was performed on 96 female Fischer rats, as described by Cui (Cui et al., 2001, Spine 26: 2305-10). The animals are anesthetized with ketamine (80 mg / kg) and xylazine (7 mg / kg), shaved, wrapped, and their skin disinfected with Betadine and 70% ethanol. A posterior longitudinal incision is made in the midline of L3 to L5. The periosteum is elevated along the lamina and the spinous processes towards the lateral aspect of the facets. The facets are removed using a rongeur, and the wound is irrigated with saline. The animals are randomized into groups of n = 32. Group A does not receive treatment. Group B receives the implantation of hydroxyapatite / tricalcium phosphate scaffold (40 mg) only in the fusion bed. Group C receives implantation of hydroxyapatite / tricalcium phosphate scaffold (40 mg) in combination with 2 X 10 6 ADAS cells derived from the subcutaneous adipose tissue of Fischer rats (syngeneic cells) in the fusion bed. After the placement of the implant, the deep fascia and the incisions of the skin are closed. Animals receiving buprenorphine hydrochloride (0.1 mg / kg) for postoperative analgesia are monitored for recovery of morbidity and function for up to 24 hours after the procedure. Groups of 16 animals from each group are slaughtered by asphyxia with C02 6 and 12 weeks after the surgical procedure. At that time, serum samples and the lumbar spine are collected for analysis.

Radiographic complementary treatment The animals are subjected to posteroanterior and lateral radiographs of the lumbosacral spine, after surgery and at 6-week intervals after surgery. Radiographic analysis is used to detect ectopic bone formation and callus formation in the lumbar spine at the surgical site. Microcomputing tomography (micro-CT) is performed on the samples dissected after sacrifice. The structure and volume of the new bone formation can be determined using methods known in the art (Mankani et al., 2004, Radiology 230: 369-76).

Manual palpation of the spinal fusion At the time of sacrifice of the animals, the lumbar spine is dissected at L3-L5 of the animals. The samples are palpated by extension and flexion in L3-4 and L4-5. Samples are classified for the presence or absence of some movement. Those samples with movement in any direction receive a score of "0", while those without movement in any dimension are considered as "merged" with a score of "1" (Cui et al., 2001, Spine 26: 2305-10 Grauer et al., 2004, Spine J. 4: 281 -6).

Biomechanical test of the spinal fusion Before the test, all the muscle is cleared, and the intervertebral disc in L4-L5 is divided, so that only the fusion mass is connecting the two vertebrae. Steel wire pins (3.2 mm) are placed in an anteroposterior direction in the vertebral bodies. Uniaxial tensile tests are carried out at a displacement speed of 0.5 cm / minute with the load applied through wire k. Offsets are measured using extensometers, and loads are measured using a load cell. Peak load to failure is measured from a computer generated load displacement graph. The stiffness is determined as the slope of the line between two points (at 50% and 75% load to the fault) in the load displacement curve. The adjacent segment in L3-4 is tested in a similar way.

Histological analysis Lumbar spine samples (n = 8 at each time point for each group) are fixed in formalin for 48 hours, decalcified in 0.25 M ethylenediaminetetraacetic acid in pH regulated saline with phosphate for 2 weeks at 4 ° C , and incubated for 16 hours in a solution of X-gal (1 mg / ml) at 37 ° C. The sample is included in paraffin, cross section (5 pm) and stained with hematoxylin and eosin. Ten sections of each sample are analyzed using Medivue software (Nikon) to quantify the average percentage (± standard deviation) of each implant occupied by ectopic bone. Without wishing it to be limited by any particular theory, it is believed that ADAS cells are successful for spinal fusion if the following results are achieved: 1) minimum fusion evidence is observed (melting score of 0 by manual manipulation in 90% of the animals, without radiographic evidence of ectopic bone, and less than 5% of the area of the surgical site occupied by matrix bone in 10 sections per sample based on histology and CT analysis) in group A (without treatment) at the 6 and 12 week time points; 2) detection of HA / TCP scaffold in the histological analysis of animals in groups B and C at the 6 and 12 week time points; 3) minimum fusion evidence (melting score of 0 for manual manipulation in 90% of the animals, without radiographic evidence of ectopic bone, and less than 5% of the area of the surgical site occupied by bone matrix in 10 sections per sample based on in histology and CT analysis) in group B (HA / TCP alone) at the 6 and 12 week time points; 4) detection of transplanted ADAS cells in groups C for 6 weeks after surgery based on the activity of the β-galactosidase enzyme or immunodetection in the histological analysis; and 5) superior spinal fusion in the presence of ADAS cells (groups C) (fusion score of "1" by manual manipulation in 90% of the animals, radiographic evidence of ectopic bone at the surgical site, and more than 30% of the area of the HA / TCP implant occupied by bone matrix in 10 sections per sample based on histology and CT analysis) with respect to the scaffold alone (group B) or missing lesion controls (group A) at the time points of 6 and 12 weeks The experiments presented in this example serve to record the utility of ADAS cells to accelerate and improve fusion lumbar spinal in a rat model.

TABLE 3 Sketch of the experimental design EXAMPLE 6 Allogenic ADAS cells in spinal fusion The following experiments serve to construe the hypothesis that ADAS cells can be allogeneically transplanted with a biomaterial scaffold to achieve superior spinal fusion compared to a biomaterial scaffold alone. Table 4 summarizes the experimental design. It has been shown that it is possible to transplant MSCs derived from bone marrow to repair bone defects without evidence of significant immune rejection (Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A: 1927-35). The experiments described herein demonstrate the utility of allogeneic ADAS cells in a lumbar spinal fusion model. Strains of inbred Fischer and ACI rats are selected for the following experiments, based on previous studies in the literature (Akahane et al., 1999, J. Bone Miner Res. 14: 561-8; Yoshikawa et al., 2000, J. Bone Miner Res. 15: 1147-57). These animals exhibit poor coupling of histocompatibility antigens and rejection of osteogenic tissue transplants, unless immunosuppressive therapy is given (Akahane et al., 1999, J. Bone Miner Res. 14: 561-8; Yoshikawa et al. , 2000, J. Bone Miner Res. 15: 1 147-57). Allogenic ADAS cells isolated from ACI rats are used for implantation in the lumbar spinal fusion of Fischer rats. The experiments herein can be carried out in parallel with the experiments related to the autologous syngeneic ADAS cells, thus allowing a comparative analysis. The absence or presence of an immune response to allogenic ADAS cells can be assessed based on mixed unidirectional lymphocyte reactions and flow cytometric analysis of serum samples obtained from the groups.

TABLE 4 Sketch of the experimental design Subcutaneous adipose tissue from male ACI rats is harvested (8 a weeks of age, n = 25, giving approximately 3 grams of tissue per rat), as discussed elsewhere herein. The number of cells obtained with each passage follows the estimates outlined in Table 1. The ADAS cells of the ACI rats are subjected to the same in vitro analyzes that were used for the ADAS cells of the Fischer rats. Complementary and surgical treatment procedures (ie, radiographic complementary treatment, manual palpation of the spinal fusion) are performed as described elsewhere herein. The HA / TCP implants can contain approximately 2 X 10 6 cells in a volume of 100 μ ?. For histological analysis, lumbar spine samples are fixed in formalin for 48 hours, decalcified in 0.25 M ethylenediaminetetraacetic acid in pH regulated saline solution with phosphate for 2 weeks at 4 ° C, and incubated for 16 hours in a solution of X -gal (1 mg / ml) at 37 ° C. The samples are included in paraffin, sectioned transversely (5 pm) and stained with hematoxylin and eosin. Ten sections of each sample are analyzed using Medivue software (Nikon) to quantify the average percentage (± standard deviation) of each implant occupied by ectopic bone. Sections are evaluated for the presence or absence of infiltrating lymphocytes. Without being desired to be limited by any particular theory, an antibody against the panhematopoietic antibody (anti-CD45) can be used for immunohistochemical staining of cells to identify any immune cells in or around the implants. The number of infiltrating lymphocytes can be determined in 10 sections per sample, and can be quantified using the Medivue software.

Immune response of serum The binding of serum antibodies to the ADAS cells of the ACI strain is evaluated by flow cytometry. ADAS cells of ACI rats are thawed rapidly from storage in liquid nitrogen, and put in culture for 5 days to facilitate maximum viability and expression of surface antigens. Cells are harvested by trypsinization, washed in a pH buffer (1x DPBS, 5% FBS, 0.5% BSA, 0.1% sodium azide), and resuspended at 5 x 10 6 cells / ml. 90 μ? of cells (5 x 10 5 cells) are distributed in aliquots in Eppendorf tubes of 2 ml. 10 μ? of undiluted rat serum, or serum diluted 1: 10 in pH buffer of staining, are added to each tube to give effective dilutions of serum that are 1: 10 and 1: 100. All tubes are incubated on ice for 30 minutes, washed with washing pH regulator (1X DPBS, 0.5% BSA and 0.1% sodium azide), and then resuspended in 100 μ? of staining pH regulator. Secondary antibody labeled with goat anti-rat FITC (IgG / lgM) is added to all tubes at a final dilution of 1: 100. Control tubes receive ADAS ACI cells only with secondary antibody (negative control), or with a positive control Fischer anti-ACI rat serum that is produced by repeated immunization of Fischer rats with ADAS ACI cells. The suspensions are incubated in the dark on ice for 15 minutes, and washed twice with washing pH regulator as discussed elsewhere herein. The cells are then fixed at 200 μ? of paraformaldehyde at 1%, and allowed to incubate on ice, in fixative for at least 15 minutes before acquisition. 20,000 events are acquired for the analysis of flow cytometry. The results are expressed as the percentage of ACI cells stained with the secondary antibody based on the increased mean fluorescence intensity relative to the secondary antibody alone. as a negative control.

Unidirectional Mixed Lymphocyte Reaction (MLR) This test is based on the following rationale. If T cells are primed in vivo for ACI alloantigens, they will respond to in vitro restimulation with faster kinetics. Activation of recipient rat T cells to ADAS cells of the allogeneic ACI strain can be assessed by the MLR test. MLR tests are performed on individual rats using cervical positive mesenteric LN cells grouped as responder cells. Eight animals are evaluated by a group of 3 groups: Without treatment (group A); scaffolding only (group B); scaffold + allogenic cells (group D) (table 5). The test is established by culturing the responder cells in medium, with irradiated syngenic Fischer spleen stimulator cells (5000R), or with irradiated allogeneic ACI spleen stimulator cells. The proliferation of T cells in response to medium or syngeneic spleen cells represents background responses; the syngeneic response is typically subtracted from the response to allogeneic cells to evaluate true proliferation. As positive and negative controls, tests are established with irradiated (positive) and syngeneic (negative) Fischer ACI lymphocytes; its expression of allogenic HLA 1 and 2 antigens against syngeneic ensures a vigorous proliferative response by responder, lymphocytes derived from Fischer, or absence of response. The MLR test is carried out in a 96-well plate using cavities tripled by treatment. Responding cells are seeded at 4 x 10 5 cells / well, and spleen cell stimulators are seeded at 1 x 10 5 cells / well. The medium used is Dulbecco's modified medium of Iscove + 10% FBS (Hyclone) supplemented with non-essential amino acids, sodium pyruvate, 2-mercaptoethanol and antibiotics / antifungals. Culture plates were prepared as a replica for harvest on days 3 and 7 of culture. The cultures are pulsed on days 2 or 6 with tritiated thymidine (1 pCi / cavity), and the cells are harvested approximately 16 hours later for scintillation counting. The results are reported as counts per minute (cpm) that reflect the degree of proliferation of T cells in the culture cavities.

TABLE 5 Mixed unidirectional lymphocyte reaction that you want to be limited by some theory In particular, it is believed that allogeneic ADAS cells are successful for spinal fusion if the following results are achieved: minimal fusion evidence is observed (melting score of 0 by manual manipulation in 90% of the animals, without radiographic evidence of ectopic bone , and less than 5% of the area of the surgical site occupied by bone matrix in 10 sections per sample based on histology and CT analysis) in group A (without treatment) at the 6 and 12 week time points; detection of HA / TCP scaffold in the histological analysis of all animals in groups B and D at the 6 and 12 week time points; minimum fusion evidence (melting score of 0 by manual manipulation in 90% of the animals, without radiographic evidence of ectopic bone, and less than 5% of the area of the surgical site occupied by bone matrix in 10 sections per sample based on histology and CT analysis) in group B (HA / TCP alone) at the 6 and 12 week time points; minimum fusion evidence (melting score of 0 by manual manipulation in 90% of the animals, without radiographic evidence of ectopic bone, and less than 5% of the area of the surgical site occupied by bone matrix in 10 sections per sample based on histology and CT analysis) in group B (HA / TCP alone) at the 6 and 12 week time points; detection of ADAS cells transplanted in groups D for up to 6 weeks after surgery based on the activity of the β-galactosidase enzyme in the histological analysis; superior spinal fusion in the presence of ADAS cells (D groups) (fusion score of "1" by manual manipulation in 90% of the animals, radiographic evidence of ectopic bone at the surgical site, and more than 30% of the area of the HA / TCP implant occupied by bone matrix in 10 sections per sample based on histology and CT analysis) with respect to the scaffold alone (group B) or injury controls missed (group A) at the 6 and 12 week time points; less than a 1.5-fold increase in the level of anti-ADAS antibodies in groups C and D (ADAS cell implants) with respect to groups A and B (without cell treatment); and without evidence of improved proliferation of responder cells stimulated by spleen cells derived from allogeneic cells compared to medium alone or spleen cells derived from syngeneic cells in the mixed unidirectional lymphocyte reaction when comparing groups A, B and D. Positive controls of the mixed reaction of lymphocytes will exhibit a proliferation response of at least 10,000 cpm.

EXAMPLE 7 Comparison and contrast of the relative efficacy of syngenic and allogenic ADAS cells in a spinal fusion model The description presented here provides data that allow the determination of whether allogenic ADAS cells (poorly adapted for HLA) and syngeneic cells (compatible with HLA), exhibit equal function in the achievement of a spinal fusion. The experimental design is summarized in Table 6. Without being desired to be limited by any particular theory, it is believed that both cell populations are equivalent, based on previous studies that achieved a successful repair of a critical size bone defect in dogs using allogeneic MSCs (Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A: 1927 -35). The comparison of syngenic and allogenic ADAS cells provides significant medical and commercial implications. The description presented herein provides for the use of allogeneic ADAS cells for tissue regeneration therapy.

TABLE 6 Comparison of spinal fusion with allogeneic versus syngeneic ADAS cells Unless desired to be limited by any particular theory, it is believed that allogeneic ADAS cells are comparable to syngenic ADAS cells in terms of success in spinal fusion, if all The parameters in Table 6 do not show a statistically significant difference between groups of allogenic and syngeneic ADAS cells (p> 0.05, preferably p> 0.30). The descriptions of each and any patent, patent application and publication cited herein are hereby incorporated by reference in their entirety. While this invention has been described in relation to specific embodiments, it is evident that other embodiments and variations of this invention may be conceived by those skilled in the art without departing from the real spirit and scope of the invention. It is intended that the appended claims be considered to include such modalities and equivalent variations.

Claims (25)

NOVELTY OF THE INVENTION CLAIMS

1 .- The use of an adipose-derived adult stromal cell (ADAS) isolated in the preparation of a drug useful for improving bone fusion after a spinal fusion procedure in a mammal, wherein the drug is adapted to be administrable in the spine of said mammal, and wherein said ADAS cells differentiate in vivo in a cell that expresses at least one characteristic of a bone cell.

2. The use as claimed in claim 1, wherein said ADAS cell is cultured in vitro for a period without being induced to differentiate prior to administration of said cell to the mammal.

3. The use as claimed in claim 1, wherein said ADAS cell is allogenic with respect to said mammal.

4. The use as claimed in claim 1, wherein the medicament is useful for inducing bone formation by spinal fusion of intervertebral bodies.

5. The use as claimed in claim 1, wherein the medicament is useful for inducing bone formation by spinal fusion of intertransverse processes.

6. The use as claimed in claim 1, wherein said ADAS cell further comprises a biocompatible matrix.

7. - The use as claimed in claim 1, wherein said biocompatible matrix is selected from the group consisting of calcium alginate, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin A, dermatan sulfate and bone matrix gelatin.

8. - The use as claimed in claim 1, wherein said ADAS cell is genetically modified.

9. The use as claimed in claim 1, wherein the medicament is adapted to be administrable in one or more interbody spaces in the mammalian spine.

10. - The use as claimed in claim 1, wherein the spinal fusion is in a segment of the spine selected from the group consisting of cervical, thoracic, lumbar, lumbosacral, and sacral-iliac (SI) joints.

11. The use as claimed in claim 1, wherein the medicament is adapted to be administrable in one or more interbody spaces by a procedure selected from the group consisting of a subsequent procedure, a posterolateral procedure, a prior procedure, a anterolateral procedure and a lateral procedure.

12. - The use as claimed in claim 1, wherein said mammal is a human.

13. The use of an adult adipose-derived stromal cell (ADAS) isolated in the preparation of a drug useful for performing one or more spinal fusions in a mammal, wherein the drug is adapted to be administrable in the mammalian spine to facilitate a spinal fusion of individual or multiple level.

14. The use as claimed in claim 13, wherein said ADAS cell differentiates in vivo in a cell that expresses at least one characteristic of a bone cell.

15. The use as claimed in claim 13, wherein said ADAS cell is cultured in vitro for a period without being induced to differentiate prior to administration of said cell to said mammal.

16. - The use as claimed in claim 13, wherein said ADAS cell is allogenic with respect to the mammal.

17. The use as claimed in claim 13, wherein the medicament is useful for inducing bone formation by spinal fusion of intervertebral bodies.

18. The use as claimed in claim 13, wherein the medicament is useful for inducing bone formation by spinal fusion of intertransverse processes.

19. The use as claimed in claim 13, wherein said ADAS cell further comprises a biocompatible matrix.

20. - The use as claimed in claim 13, wherein said biocompatible matrix is selected from the group consisting of calcium alginate, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan sulfate and bone matrix gelatin.

21. - The use as claimed in claim 13, wherein said ADAS cell is genetically modified.

22. The use as claimed in claim 13, wherein the medicament is adapted to be administrable in one or more interbody spaces in the spine of said mammal.

23. - The use as claimed in claim 13, wherein the spinal fusion is in a segment of the spine selected from the group consisting of cervical, thoracic, lumbar, lumbosacral, and sacral-iliac (SI) joints.

24. - The use as claimed in claim 13, wherein the medicament is adapted to be administrable in one or more interbody spaces by a procedure selected from the group consisting of a subsequent procedure, a posterolateral procedure, a prior procedure, a anterolateral procedure and a lateral procedure.

25. - The use as claimed in claim 13, wherein said mammal is a human.