MXPA97009293A - Methods and uses of the connective tissue growth factor as an inducc agent - Google Patents

Abstract

The present invention relates to novel method and compositions related to the administration of connective tissue growth factor, alone or in combination with other growth factors, compositions or compounds, to induce the formation of connective tissue, including bone, cartilage and bone pi

Description

METHOD AND USES OF THE CONNECTIVE TISSUE GROWTH FACTOR AS AN INDUCTION AGENT Description The information described in this description was made in part with government support through grant No. GM 37223, awarded by the National Institutes of Health. The government of the United States may have some rights over the invention described in this description. 1. Relationship of Related Cases This application is related to, and is a continuation of, part of the application Serial Number 08 / 459,717, entitled "Connective Tissue Growth Factor", filed on June 2, 1995, which is a continuation in part of the application Serial Number 08 / 386,680, filed on February 10, 1995, which has the same title, which is one is a divisional of the application Serial Number 08 / 167,628, filed on December 14, 1993, now U.S. Patent No. 5,408,040, which is a continuation of the application Serial Number 07 / 752,427, filed on August 30, 1991, now abandoned. 2. Field of the Invention The present invention relates, in general terms, to the field of growth factors and specifically to the Factor of Growth of the Connective Tissue (CTGF) and to the methods of use thereof. 3. Background of the Invention A. The Role of Growth Factors in Bone and Cartilage Formation. Bone and cartilage formation. The formation of tissue and organs in all multicellular organisms that arise from a single fertilized egg requires the differentiation of specialized cell types from undifferentiated stem cells. As embryogenesis progresses, more specialized cell types and more complex structures are formed. Currently, however, there is very little concrete information available on the identification of specific factors or the mechanism of action of these factors on skeletal or cartilage formation in vertebrate animals, including humans. There are two common types of bone formation in the mammalian system: intramembranous ossification and endochondral ossification. The formation of skull bones is an example of intramembranous ossification. There, the mesenchymal cells of the neural crest interact with the extracellular matrix of the cranial epithelial cells and form bone. Hall, Amer. Sci. , 1988, 76174-181. Mesenchymal cells condense into small islands and differentiate into osteoblasts and capillaries. Osteoblasts secrete a specific type of extracellular matrix, (osteoid) that is capable of fixing calcium salts. Endochondral ossification is the process by which the long bones of the axial skeleton (arms and legs), and the vertebrae and ribs are formed. Hal supra. During this process bone formation occurs via an intermediate stage of cartilage tissue. In mammals, long bones are formed from mesenchymal cells in the embryonic buds of the limbs. These cells form chondrocytes, and secrete a cartilaginous matrix. Other surrounding mesenchymal cells form the perichondrium (finally, the periosteum). In some cases, the chondrocytes adjacent to the region where the chondrocytes are proliferating and form differentiated hypertrophic chondrocytes. Hypertrophic chondrocytes produce a different type of matrix, and alter their tissue orientation to form the physis. The structure of the physis is accommodated in multiple cellular columns composed of areas with cellular hypertrophy, proliferation, ossification and vascularization. Hall above; Gilbert "Trans-criptional regulation of gene expression", DEVELOPMENTAL BIOLOGY, 5a. edition. Sinaur Assoc., P 387-390 (1994). This results in a graduation of the transformation of the cells from chondrocytes to osteoblasts that form the mineralized bone. Endochondral ossification is an active, progressive process that occurs in mammals during growth from childhood to adult life. The differentiation of mesenchymal cells into chondrocytes, their proliferation and replacement by osteoblasts depends on the growth factors (including the TGB-β family), and on the mineralization of the matrix Tuan, 1984, J. Exp. Zool. (suppl.) 1: 1-13 (1984); Syfestad and Caplan, 1984, Devel. Biol. 104: 348-386. With respect to the connective tissue, it is felt that all the skeletal elements in mammals are derived from a single cell stalk that is capable of differentiation into specific cell types that make up muscles, cartilage, bones and tendons. These cells also appear to be able to differentiate into adipose tissue. The Relevant Technique Related to the Factors of Growth and 2nd Formation of Bone and Cartilage. Prior to the present invention, it was known, in general terms, that growth factors comprise a class of secreted polypeptides that stimulate cells to proliferate, differentiate and organize developing tissues. Typically, an activity of the development factor depends on its ability to set specific receptors, thereby stimulating a signaling event within the cell. Examples of some well-studied growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor of the beta family (TGF-β), transforming growth factor of the alpha family (TGF-a), epidermal growth factor (EGF), and fibroblast growth factors (FGF). Effect of TGF-β on the growth of chondrocytes, differentiation and cartilage formation. TGF-β play a role in chondrogenesis. As previously reported, transforming growth factors TGF-ßl and TGF-ß2 increase chondrogenesis in embryonic rat mesenchymal cells (Seyedin et al., 1987, J., Biol. Chem. 262: 1946-1947), and any isoform can induce the formation of chondroblasts from mesenchymal cells of murine muscles in culture, Seyedin, et al., 1986, J. Biol. Chem. 261: 5693-5695. The application of TGF-ß to prechondrial embryonic tissues of increase the differentiation of mesenchymal cells, the production of proteoglycans, and the replication of chondroblasts, Centrella et al., 1994, Endocrine Reviews 15: 27-38, Thorp and Ja owlew, 1994, Bone 15: 59-64. in if you found decreased levels in the growth plates of animals with three separate disorders where chondrocytes cease to differentiate. Id. In cultures of bovine articular cartilage organs, colágen or type II and the synthesis of proteoglycan were increased after administration of TGF-β. Morales and Roberts, 1988, J. Biol. Chem. 263: 12828-12831. In contrast, TGF-β showed a decrease in the expression of speci fi c collagens of type II and type X cartilage, the synthesis of chondrocyte proteoglycans, and the activity of alkaline phosphatase in cultured chondroid cells. Mundi, "The effects of TGB-ß on bone", Clinical Applications of TGF-ß. ", 1991, Wiley Chichester, Ciba Foundation Symposium 157: 137-151, the differentiation of chondrocytes from the growth plate of rabbits is inhibited by TGF-β, while mitogenesis in the growth plate increases. collaborators, 1988, Proc. Nati, Acad. Sci. USA 85: 9552- 9556. In addition, high concentrations of TGF-ßl or TGF-ß2 added to an osteoinductive model favors cartilage, instead of preferring bone formation, when smaller doses are used Mundy, supra This accumulation of apparently contradictory information has hampered efforts to define a function of TGF-β in chondrogenesis Morphogenic Bone Proteins and Bone Formation A family of proteins called bone morphogenetic proteins (BMPs) are capable of inducing ectopic bone formation in some mammalian species, with the exception of BMP-1, which encodes a metalloprotease, all of these p Roteins have structures that are related to TGF-β. However, it is not known which, if any of the BMP is responsible for the regulation of bone formation during normal embryogenesis. BMPs were first isolated from demineralized bone as factors that induce bone in extra skeletal ectopic sites. Originally three peptides were identified as BMP-1, BMP-2A, and BMP-3. Celeste, et al., 1990, Proc. Nati Acad. Sci. USA 87: 9843-9847; Kubler and Urist, 1980, Clin. Orthopedics and Reí. Res. 258: 279-294. The last two BMPs are members of the TGF-β superfamily. Subsequently, five more closely related members of the BMP group were identified and cloned. BMP-5, BMP-6, and BMP-7 are more like vgr / 60A, while BMP-2 and BMP-4 are more like Decapentaplegic. Both vga / 60A and Decapentaplégico are Drosophila genes that control the formation of the dorsal / ventral axis pattern. Hoffman, 1992, Mol. Repro and Dev. 32: 173-178. Hybridization in itself has localized transcription of BMP genes for areas of bone formation in the bud of the limbs at specific times during development, suggesting a physiological role. BMPs induce adventitious post-fetal mesenchymal cells to establish the connection of the formation of a fibroretic to chondro-osteoprogenetic pattern. Kubler and Urist, supra. Several lines of information suggest that BMPs can act synergistically with TGF-β to initiate the osteoinduction cascade in vivo. In murine subcutis, TGF-ßl increases ectopic bone production by most BMPs. BMP-6, (also known as VGR-1) is expressed in hypertrophic cartilage at the same time and in the same areas as TGF-β, and is associated with the expression of type X collagen. See, Celeste, et al. supra The addition of TGF-β to the bone explants that have been treated with either BMP-2 or BMP-3 results in increased osteoinductive activity and an increased ratio of cartilage to bone when compared to one or the other factor. alone. Bentz, et al., 1991 Matrix 11: 269-275. However, the synergistic effect of these proteins by TGF-β is not universal. It has been shown that TGF-β1 directly decreases BMP-2 expression in fetal rat calvary cultures. Harris, et al., 1994, "Bone and Mineral Res. 9: 855- 863. Since BMP-2 appears to be important in the differentiation of bone cells, it has been suggested that TGF-β1 may be acting as a contact for monitor the destinies of the chondro-osteoblastic precursors Other Factors Found to be Expressed in the Developing Tissue Cyr61 is a growth regulator that has been found to be expressed in embryonic and extraembryonic mouse tissues in development O'Brien and Lau, 1992, Cell Growth Differ 3: 645-654 Cyr61 is related but distinct from the connective tissue growth factor and prior to the present invention, the specific activity of Cyr61 was not known B. The Role of Growth Factors in Wound Healing: Platelet-derived Growth Factor and Wound Healing The PDGF is a molecule consisting of an A chain and a B chain. Eros and homodimers and all the combinations isolated to date are biologically active. With respect to factor activity, PDGF has been characterized as a cationic protein, stable to the heat found in the granules of the circulating platelets. The molecule has also been characterized as a mitogen and a chemotactic agent for connective tissue cells such as fibroblasts and smooth muscle cells. Due to the biological activity of PDGF and its release during wound healing, PDGF has been identified as a growth factor involved in wound healing, as well as in pathological conditions showing an overproduction of connective tissue, including arteriosclerosis and fibrotic diseases. It has been hypothesized that other growth factors other than PDGF may play a role in the normal development, growth and repair of human tissue. TGF-ß and Wound Healing. The formation of new tissue and regeneration requires the coordinated regulation of several genes that produce both regulatory and structural molecules that participate in the process of cell growth and tissue organization. As with bone induction, it seems that TGF-β plays as a central regulatory component of this process. TGF-β is released by the platelets, macrophages and neutrophils that are present in the initial phases of the repair process. TGF-β can act as a growth stimulating factor for mesenchymal cells and as a growth inhibitory factor for endothelial and epithelial cells. It has been suggested that the growth stimulating action of TGF-β appears to be mediated via an indirect mechanism involving the induction of other genes that include growth factors such as PDGF. Many members of the TGF-β superfamily have activities that suggest possible applications for the treatment of cell proliferative disorders, such as cancer. In particular, TGF-β has been shown to be a potent growth inhibitor for a variety of cell types (Massague, 1987, Cell 49: 437.), MIS has been shown to inhibit the growth of carcinoma tumors. human endometrium in hairless mice (Donahoe, et al., 1981, Ann. Surg. 194: 472), and inhibition has been shown to suppress the development of tumors in both the ovary and the testis (Matzuk, et al., 1992 , Nature, 360: 313). Many of the members of the TGF-β family are also important mediators of tissue repair. TGF-β has been shown to have remarkable effects on the formation of collagen and causes an attack on the angiogenic response in the neonate mouse (Roberts, et al., 1986, Proc. Na ti. Acad. Sci., USA 83 .: 4167). Morphogenic bone proteins (BMPs) can induce the growth of new bone are effective for the treatment of fractures and other skeletal defects (Glowacki, et al., 1981 Lancet, 3 .: 959; Ferguson et al., 1988, Clin. Orthoped. Relat. Res. , 227: 265; Johnson and collaborators 1988, Clin. Orthoped. Relat. Res. , 227: 257). C. Connective Tissue Growth Factor A growth factor previously unknown, related to PDGF, and called the Connective Tissue Growth Factor (CTGF), has been reported in a related patent. See United States Patent Number: 5,408,040. The connective tissue growth factor is a mitogenic peptide rich in cysteine that is selectively induced in fibroblasts after activation with TGF-β. Igarashi, et al., 1993, Mol. Biol. Cell 4: 637-645. The connective tissue growth factor is a member of a peptide family that includes products of the cef10 gene induced in serum (Simmons, et al., 1989, Proc. Nati, Acad. Sci. USA 86: 1178-1182), cyr61 ( O'Brien, et al., 1990, Mol. Cell, Biol. 10 .: 3569-3577), fispl2 / lG Ml (Ryseck et al., 1993, Cell Growth &Dif. 2: 225-233), and a chicken transforming gene, Nov (Joliot et al., 1992, Mol.Cell Biol. 12: 10-21 (1992).) The connective tissue growth factor also shares sequence homology with a drosophila, twisted gastrulation gene product. twg) (Mason et al., 1994, Genes &Develop, 8: 1489-1501, which determines cell fate during the formation of the dorsal / ventral pattern in the embryo.) As reported in that patent, the factor of connective tissue growth is the product of a different gene, as also reported in the United States patent number: 5,408,040 connective tissue growth factor possesses mitogenic activity. The final result of this mitogenic activity in vivo is the growth of the target tissue. The connective tissue growth factor also possesses chemotactic activity, which is the movement of cells induced chemically as a result of the interaction with particular molecules. Although the molecule is antigenically related to PDGF, there is little, if any, homology in the peptide sequence between connective tissue growth factor and PDGF. The Anti-PDGF antibody has a high affinity with the non-reduced forms of the PDGFF isomers and the connective tissue growth factor molecule and ten times less affinity with the reduced forms of these peptides lacking biological activity. A second protein, identified as "connective tissue growth factor 2", or "CTGF-2" has also been reported. See PCT application No. PCT / US94 / 07736 (International Publication No. WO 96/01896). According to the PCT Application, the connective tissue growth factor can also be used to increase repair of the connective tissue and support tissue. Although it is identified as a connective tissue growth factor, CTGF-2 is not closely related to the DTGF of the present invention. Specifically, the connective tissue growth factor family is comprised of three distinct protein groups, as compared to CTGF-2, which falls with the cyr61 group. PCT Application number PCT / US94 / 07736 in 4. Irrespective of the identification of several growth factors related to PDGF, including the connective tissue growth factor, prior to the present invention, those factors have not proven to be an agent of effective induction for the production of matrices, including the induction of bone and / or cartilage tissue. 4. Summary of the Invention The subject of the invention provides novel methods and compositions for the treatment of diseases, disorders or conditions where the production of matrix and / or connective tissue is desired, including the production of bone and / or cartilage. . The present invention is likewise directed to the treatment of diseases, disorders or conditions where the promotion of wound healing is desired. More specifically, the compositions of the present invention comprise the connective tissue growth factor and / or fragments and / or derivatives thereof (hereinafter collectively "connective tissue growth factor"), alone or in combination with other factors of increase. The connective tissue growth factor used in the present compositions can be obtained by isolation from natural sources, from synthetic manufacture, or by production by recombinant genetic engineering techniques. In one aspect of the invention, the methods of the present invention comprise administering an effective amount of connective tissue growth factor, alone or in combination with one or more compounds, to treat diseases, disorders or conditions where induction is desired. of bone or cartilage tissue. In a preferred embodiment of this method that additional compound is a growth factor. In another aspect of the invention, the methods of the present invention comprise administering an effective amount of connective tissue growth factor, alone or in combination with one or more compounds, again preferably one or more growth factors, to treat diseases , disorders or conditions where the promotion of wound healing is desired. In a preferred embodiment of the invention, the composition comprising the connective tissue growth factor is administered directly on or within the site in which the induction of the bone or cartilage is desired in order to induce the formation of that bone or cartilage. In another embodiment, the composition is formulated for targeted administration or alternatively, is designed for the release of novel compositions at the relevant site (eg, the wound in which cartilage formation is desired). In each case, the composition containing connective tissue growth factor is formulated appropriately for administration to a patient in need thereof. 5. Definitions As used herein, the term "connective tissue growth factor" will mean: (1) a protein encoded by the amino acid sequence set forth in Figure 1C, (2) a protein having factor connective tissue growth wherein that protein is encoded by the amino acid sequence of Figure 1C wherein one or more amino acids have been added, deleted, mutated, substituted or altered in some other way ("derivative"), and the sequence of nucleotides encoding that protein can hybridize to the nucleic acid sequence of Figure 1C under stringent conditions, or (3) a fragment of connective tissue growth factor or a derivative thereof. As used in this description, the term "induces" as used herein, shall mean to produce, to form, to cause to produce or to cause to form. As used in this description, the phrase "induction agent" will mean an agent, including proteins or other biological materials, that cause the production or formation of a specific end result (e.g., the production of connective tissue). As used herein, the term "polynucleotide" denotes DNA, cDNA and / or RNA encoding untranslated sequences that flank the structural gene encoding the connective tissue growth factor. For example, a polynucleotide of the invention includes 5 'regulatory nucleotide sequences and 3' untranslated sequences associated with the structural gene of connective tissue growth factor. A polynucleotide of the invention that includes the 5 'and 3' untranslated region is illustrated in Figure 1C. The 5 'regulatory region, which includes the promoter, is illustrated in Figure IB. A more detailed description of the polynucleotides contemplated by the present invention can be found in U.S. Patent No. 5,408,040. As used in this description, the phrase "stringent conditions", as used herein, refers to the hybridization conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl / 0.0015 M sodium citrate / 0.1 percent of SDS at 50 ° C; (2) employing during denaturation a denaturing agent such as formamide, eg, 50 percent (vol / vol) formamide with 0.1 percent bovine serum albumin / 0.1 percent Ficoll / 0.1 percent polyvinylpyrrolidone / 50 mM of sodium phosphate buffer at a pH of 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 ° C; or (3) use 50 percent formamide, 5 x SSC (0.75 M Nací, 0.075 M sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 grams / milliliter), 0.1 percent SDS, and 10 percent dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC and 0.1 percent SDS As used in this description, the phrase "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art which has been manipulated by insertion or incorporation of the genetic sequences of the connective tissue growth factor.As used herein, the phrase "therapeutically effective" means the amount of factor of growth of connective tissue that is effective in inducing the formation of bone or cartilage or wound healing 6. Brief Description of the Drawings The Figure shows the structural organization of the connective tissue growth factor gene. s are indicated by framed regions, with the solid areas in the gene corresponding to the open reading frame. Figure lb shows a coison of nucleotide sequences between the connective tissue growth factor promoter and the fisp-12 promoter. Identical nucleotides are marked with asterisks. The TATA box and other consensus sequences are indicated by shading. The site of the initiation of the transcript is indicated in the position number +1. Figure 1C shows the complete nucleotide and deduced amino acid sequence for the connective tissue growth factor structural gene and the 5 'and 3' untranslated sequences. Figure 2 shows the results of the experimental hybridization in itself related to the expression of the connective tissue growth factor transcripts in the long bone growth plate in neonatal mice. The in-situ hybridization experiments were performed using an antisense CTGF RNA probe as described below. The chondrocytes in the proliferation zone are strongly positive for the expression of the connective tissue growth factor gene, indicating that the connective tissue growth factor occurs in cartilage growth sites. Figure 3 shows expression of the connective tissue growth factor gene during embryogenesis where a transgenic mouse is constructed using a fusion gene constructed from a connective tissue growth factor promoter and the β-structural gene. -galactosidase. This gene, introduced into the germ line, expresses β-galactosidase in the expression sites of connective tissue growth factor and can be detected by histochemical elements expressing sections of the transgenic animal in development to the substrate X-gal that deposits a blue color at the sites of β-galactosidase activity. Panel A is a 12-day mouse embryo from a transgenic mouse. The blue dyeing is an area destined to become Meckel's cartilage, which is the first cartilage that is formed. Panel B is a photograph of the hind limb and leg showing the dyeing at the ends of the long bones and the paw in the metatarsal growth plates. Figure 4 provides evidence of the induction of cartilage and bone in the culture of mouse embryonic stem cell C3H10T1 / 2. C3H10T1 / 2 cells were cultured as described within the methods. The cells were treated with either nothing (Panel A), 5-azacytodyne (Panel B), connective tissue growth factor at 50 ng / milliliter. (Panel C) or 5-azacytidine followed by connective tissue growth factor (Panel D). Figure 5 sets forth the Northern blot analysis of the expression of the connective tissue growth factor gene in wound chambers implanted in bone regeneration sites. Figure 6 sets out the evidence related to the expression of connective tissue growth factor in human osteoblasts in response to TGF-β. Figures 7A-7D set forth the results of a chondrogenic assay.

Figure 7A provides the results of the chondrogenic assay for the control culture. Figure 7B provides the results of the chondrogenic assay for a culture in which 5 ng / milliliter of TGF-β1 was added. Figure 7C provides the results of the chondrogenic assay for a culture in which 5 ng / milliliter of TGF-ßl and 10 ng of cholera toxin were added. Figure 7D provides the results of the chondrogenic assay for a culture in which 5 ng / milliliter of TGF-ßl and 10 ng / milliliter of cholera toxin, and 5 ng / milli-liter of tissue growth factor were added. connective. Figure 8A is a Scatchard plot that reflects the attachment of connective tissue growth factor to NRK cells. Figure 8B is a Sctachard plot that reflects the attachment of connective tissue growth factor to rat chondroblasts. 7. Detailed Description of the Invention 7.1. Methods for producing connective tissue growth factor Nucleic Acid Sequences for Coding Factor Connective Tissue Growth: According to the invention, the nucleotide sequences encoding the connective tissue growth factor or its functional equivalents can be used to generate recombinant DNA molecules that direct the expression of the protein or a functional equivalent of it, in the appropriate host cells. Alternatively, nucleotide sequences that hybridize, within a strict position, to portions of the connective tissue growth factor sequence can also be used in nucleic acid hybridization assays, Southern and Northern blot analysis, and so on. . In yet another method, DNA molecules encoding connective tissue growth factor can be isolated by hybridization methods comprising selection of antibodies from expression libraries to detect shared structural features. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence can be isolated and used in the practice of the invention for the cloning and expression of connective tissue growth factor. Those DNA sequences include those that are capable of hybridizing to the human connective tissue growth factor sequence under stringent conditions. The altered DNA sequences that can be used according to the invention include deletions, additions or substitutions of different nucleotide residues that result in a sequence encoding the same or an equivalent gene product. This gene product may contain by itself eliminations additions or substitutions of amino acid residues within the sequence of connective tissue growth factor, which results in a silent change, thus producing a functionally equivalent protein. These amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophobia, and / or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; the positively charged amino acids include lysine and arginine; amino acids with uncharged polar front groups that have similar hydrophilic values include the following: leucine, isoleucine, valine; glycine, analin; Asparagine, glutamine, serine, threonine, phenylalanine, tyrosine. The DNA sequences of the invention can be engineered for the purpose of altering the sequence of the protein for a variety of purposes including, but not limited to, alterations that modify the processing and expression of the gene product. For example, mutations can be introduced using techniques that are well known in the art, for example, site-directed mutagenesis, for example, inserting new restriction sites. For example, in some expression systems such as yeast, the host cells can over-glycosylate the gene product. When using such expression systems it may be preferable to alter the coding sequence of the connective tissue growth factor to eliminate any N-linked glycosylation site. The sequence of connective tissue growth factor can be linked to a heterologous sequence to encode a fusion protein. For example, for selection of peptide libraries it may be useful to encode a chimeric connective tissue growth factor protein that expresses a heterologous epitope that is recognized by a commercially available antibody. A fusion protein can also be engineered to contain a localized dissociation site between the sequence of connective tissue growth factor and the heterologous protein sequence (eg, a sequence encoding a growth factor related to PGDF) , so that the connective tissue growth factor can be dissociated from the heterologous fraction. The coding sequence of the connective tissue growth factor can also be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers, et al., 1980, Nucleic Acids Res. Symp. Ser. 7: 215-233; Crea and Horn, 1980, Nucleic Acids Res. 9_: ~ (10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 1: 719; and Chow and Kempe, 1981, Nucleic Acids Res .9. (12): 2807-2817.

Alternatively, the protein itself could be produced using chemical methods to synthesize the amino acid sequence of the connective tissue growth factor in whole or in part. For example, the peptides can be synthesized by solid phase techniques, dissociated from the resin, and purified by preparative high performance liquid chromatography. See for example, Creighton, 1983, Proteins Structures And Molecu -lar Principies. WH. Freeman and Co., N.Y. pp. 50-60. The composition of the synthetic peptides can be confirmed by analysis or sequencing of amino acids. See, for example, for the Edman degradation procedure, see, Creighton, 1983, Proteins, Structures And Molecular Principles. WH. Freeman and Co. , N.Y. pp. 34-49. A more detailed description of the nucleic acid sequences comprising the present invention and methods for identifying those sequences can be found in U.S. Patent No. 5,408,040, which is incorporated herein by reference. Expression of the Factor of Growth of Connective Tissue. In order to express a biologically active connective tissue growth factor, the coding of the nucleotide sequence for the protein, or a functional equivalent as described above, above, was inserted into an appropriate expression vector, i.e. a vector containing the necessary elements for the transcription and translation of the inserted coding sequence. More specifically, methods that are well known to those skilled in the art can be used to construct expression vectors containing the connective tissue growth factor sequence and suitable transcription / translation control signals. These methods include DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausbel et al., 1989, Current Protocols in Molecular Biology, Grene Publishing Associates and Wiley Interscience, N.Y. A variety of host expression vector systems can be used to express the coding sequence of connective tissue growth factor. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, DNA of plasmid DNA expression vectors or cosmid DNA containing the coding sequence of connective tissue growth factor; yeasts, including Pichia pastoris and Hansenula polymorpha, transformed with recombinant expression vectors containing the coding sequence of connective tissue growth factor; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the sequence encoding connective tissue growth factor; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid vectors (eg, Ti plasmid) containing the connective tissue growth factor-encoding sequence; or animal cell systems infected with recombinant virus expression vectors (eg, adenovirus, vaccinia virus, human tumor cells (including HT-1080)) including cell lines engineered to contain multiple copies of growth factor DNA of connective tissue either stably amplified (CHO / dhfr) or unstable amplified in two-minute chromosomes (eg, murine cell lines). As used in this, the term "host expression vector systems" is understood and more generally, the term "host cells" includes any progeny of the host cell or host expression vector system. It is further understood that although all progeny may not be identical to the parent cell, since mutations may occur during replication, that progeny is included within the scope of the invention. The expression elements of this system vary in their strength and specificities. Depending on the host / vector system used, any of numerous suitable transcription or translation elements, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloning into bacterial systems, inducible promoters such as bacterio-phage pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.; When cloning into plant cell systems, promoters derived from the genome of plant cells (eg, heat shock promoters), the promoter for the small subunit of RUBISCO, the promoter for the binding protein a / b can be used. chlorophyll): or from plant viruses (for example, the CaMV RNA35S promoter, the TMV coat protein promoter); when cloning into mammalian cell systems, promoters derived from the genome of mammalian cells (eg, the adenovirus late promoter, the vaccinia virus 7.5 K promoter); When cell lines containing multiple copies of the SV40- CTGF DNA are generated, vectors based on BPV- and EBV- can be used with a suitable selectable marker. In bacterial systems, numerous expression vectors may conveniently be selected depending on the intended use for the expressed connective tissue growth factor. For example, a suitable vector for bacterial expression includes the vector based on T7 as described by Rosenberg et al., 1987, Gene 56: 125. As a further example, when large amounts of connective tissue growth factor are going to be produced to select the peptide libraries, vectors that direct the expression of high-level protein products that are easily purified may be convenient. Such vectors include, but are not limited to, the pUR278 expression vector of E. coli (Ruther et al., 1983, EMBO J. 2: 1791), in which the coding sequence of connective tissue growth factor can be bound within the vector in the frame with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke / Schuster, 1989, J. Biol. Chem. 264: 5503-5509); and similar. PGEX vectors can also be used to express foreign polypeptides such as GTGF with glutathione-S-transferase (GST). In general, these fusion proteins are soluble and can be easily purified from the lyzed cells by adsorption against glutathione-agarose beds followed by levigation in the presence of free glutathione. The pGEX vectors are designed to include thrombin or dissociation sites of the protease factor Xa so that the cloned polypeptide of interest can be released from the GST fraction. More generally, when the host is a prokaryote, the competent cells that are capable of collecting DNA can be prepared from the cells grown after exponential growth and subsequently treated by the CaCl 2 method, or alternatively MgCl 2 or RbCl, using well-known procedures in the technique. When the host cell is a eukaryote, several methods of DNA transfer can be used. These include transfection of DNA by calcium phosphate precipitates, conventional mechanical procedures, including microinjection, insertion of a plasmid embedded in liposomes, or the use of virus vectors. the eukaryotic cells can also be transformed together with the DNA sequences encoding the polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express proteins. See, Eukaryotic Viral Vectors, 1992, Cold Spring Harbor Laboratory, Gluzman, Ed.). Eukaryotic host cells include yeast, mammalian cells, insect cells and plant cells. In yeast, numerous vectors containing constitutive or inducible promoters can be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ausubel et al., Ed., Greene Publish. Assoc. & Wiley Interscience, Chapter 13; Grant et al., 1987, Methods in Enzimology, Wu & Grossman, Eds., Acad. Press, N.Y. , 153: 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash. , D.C., Ch. 3; Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Berger & Kimmel, Eds., Acad. Press, N.Y. , 152: 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Strathern et al., Eds., Cold Spring Harbor Press, Vols. I and II. For example, several shuttle vectors have been reported for the expression of foreign genes in yeast. Heinemann et al., 1989, Nature 340: 205; Rose, et al., 1987, Gene. 60: 237. In cases where plant expression vectors are used, the expression of the connective tissue growth factor coding sequence can be driven by any of numerous promoters. For example, viral promoters such as RNA 35S and RNA 19S, CaMV promoters (Brisson et al., 1984, Nature 310: 511-514), or the TMV coat protein promoter (Takamatsu et al., 1987, EMBO J. 6 .: 307-311); alternatively, plant promoters such as the small subunit of RTJBISCO (Coruzzi et al., 1984, EMBO J. 3: 1671-1680, Broglie et al., 1984, Science 224: 838-843) or shock promoters can be used. heat, for example, hspl7.5-E or soy hspl7.3-B (Gurley et al., 1986, Mol Cell. Biol. 6.559-564). These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electron-troevaporation, and so on. For the review of those techniques, see, for example, Weissbsch & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; Grierson & Corey, 1988, Plant Molecular Biology, 2a. edition, Blackie, London, Chapters 7-9. In an insect system, an alternative expression system can be used to express the connective tissue growth factor. In one of these systems, Baculovirus is used as a vector to express foreign genes. The virus then grows in the insect cells. The sequence encoding connective tissue growth factor can be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under the control of a Baculovi-rus promoter. These recombinant viruses are then used to infect cells in which the inserted gene is expressed. See, for example, Smith et al., 1983, J ". Virol. 4.6: 584; Smith, U.S. Patent No. 4,215,051.In mammalian host cells, numerous virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the sequence encoding connective tissue growth factor can be ligated to an adenovirus transcription / translation control complex, for example the late promoter and the tripartite forward sequence. chimeric can then be inserted into the adenovirus genome by in vitro or in vivo recombination.Insertion into a non-essential region of the viral genome (e.g., El or E3 region) will result in a recombinant virus that is viable and capable of expressing the connective tissue growth factor in infected hosts, see, for example, Logan &Shenk, 1984, Proc. Nati. Acad. Sci. (USA) 8: 1: 3655-3659. you can use the vaccinia 7.5K promoter. See, for example, Mackett et al., 1982, Proc. Nati Acad. Sci. 79: 7415-7419; Macket et al., 1984 J. Virol 4_9: 857-864; Panicali et al., 1982, Proc. Nati Acad. Sci. 79: 4927-4931. In another embodiment, the connective tissue growth factor sequence is expressed in human tumor cells, such as HT-1080, which have been stably transfected with calcium phosphate precipitation and a neomycin-resistant gene. In yet another embodiment, the expression vector pMSXND or a similar one is used for expression in a variety of mammalian cells, including COS, BHK 293 and CHO cells. Lee and Nathans, 1988, J. Biol. Chem. 263: 3521. Specific initiation signals may also be required for the efficient translation of the inserted connective tissue growth factor coding sequences. These signals include the ATG start codon and the adjacent sequences. In cases where the entire connective tissue growth factor gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, additional translation control signals may not be required. However, in cases where only a portion of the connective tissue growth factor coding sequence is inserted, the exogenous translation control signals, including the ATG start codon, must be provided. In addition, the initiation codon can be in the phase with the reading frame of the coding sequence of the connective tissue growth factor to ensure translation of the entire insertion. These exogenous translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Efficiency of expression can be increased by the inclusion of appropriate elements transcription enhancers, transcription terminators, and so on. See, for example, Bitter et al., 1987, Methods in Enzymol. 153 .: 516-544. In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the product of the gene in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, dissociation) of protein products may be important for the function of the protein. The different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells possessing the cellular machinery can be used for the proper processing of the primary transcription, glycosylation, and phosphorylation of the gene product. These mammalian host cells include, but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, HT-1080, and so on. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express connective tissue growth factor can be engineered. Instead of using expression vectors containing viral replication origins, the cells can be transformed with connective tissue growth factor DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, terminators). transcription, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, the engineered cells can be allowed to grow for 1-2 days in an enriched medium, and then put in contact with a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. Numerous screening systems can be used, including, but not limited to, herpes simplex virus kinase-thymidine (Wigler et al., 1977, Cell. 11: 223), hypoxanthine-guanine phospho-ribosyltransferase (Szybalska &; Szybalski, 1962, Proc. Nati Acad. Sci. (USA) 48.:2026), and adenine phosphoribosyl-transferase (Lowy, et al., 1980, Cell 22: 817) can be used genes in cells, tk, hgprt or aprt, respectively. Antimetabolite resistance can also be used as the basis for the selection of genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Nati, Acad. Sci. (USA) 77: 3567; O'Hare et al. collaborators, 1981, Proc. Nati, Acad. Sci. (USA) 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan &Berg, 1981. Proc. Nati, Acad. Sci. (USA) 7.8: 2072); neo, which confers resistance to aminoglycoside G-418 (Colberre-Garapin, et al., 1981, -T. Mol. Biol. 150: 1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147). Recently, additional selectable genes have been described, namely, trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to use histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Nati, Acad. Sci. (USA) 85:8047), and ODC (ornithine-decarboxylase) which confers resistance to ornithine decarboxylase inhibitor, 2- (difluoromethyl) -DL-ornithine, DFMO (McConloque, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory). The isolation and purification of the polypeptides expressed in host cells of the invention can be done by any conventional means, such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibody. 7.2 Identification of Transfectants or Transformants that Express the Factor of Growth of Connective Tissue The host cells that contain the coding sequence and that express the product of the biologically active gene can be identified by at least four general approaches: (a) DNA-hybridization DNA or DNA-RNA; (b) the presence or absence of functions of a "marker" gene; (c) estimating the level of transcription as measured by the expression of the mRNA transcripts of connective tissue growth factor in the host cell; and (d) detection of the product of the gene measured by an assay or by its biological activity. In the first approach form, the presence of the coding sequence of the connective tissue growth factor inserted into the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the coding sequence of the connective tissue growth factor, respectively, or portions or derivatives thereof. In the second approach form, the recombinant expression vector / host system can be identified and selected based on the presence or absence of certain "marker" gene functions (eg, resistance to antibiotics, resistance to methotrexate, phenotype of transformation, occlusion body formation in the baculovirus, etc.). For example, in a preferred embodiment, the sequence encoding the connective tissue growth factor is inserted into a sequence of the neomycin resistance marker gene of the vector, and the recombinants containing the coding sequence of the growth factor of the vector. Connective tissue can be identified by the absence of the marker gene function. Alternatively, the marker gene may be placed in a duo with the sequence of the connective tissue growth factor under the control of the same or different promoter used to control the expression of the connective tissue growth factor-coding sequence. The expression of the marker in response to induction or selection indicates the expression of the coding sequence of the connective tissue growth factor. In the third form of approach, the transcription activity for the coding region of the connective tissue growth factor can be estimated by hybridization assays. For example, RNA can be isolated and analyzed by Northern blotting using a probe homologous to the coding sequence of connective tissue growth factor or particular portions thereof. Alternatively, the total nucleic acids of the host cell can be extracted and tested for the hybridization of those probes. The fourth approach form involves the detection of the biologically active or immunologically reactive connective tissue growth factor gene product, including, but not limited to, the assays described in U.S. Patent No. 5,408,040. Indications of Treatment The methods, compounds and formulations of the present invention are each directed to the treatment of disorders, diseases or conditions related to the low production of connective tissue in bone, cartilage, or other organs such as skin and muscle alternatively, for disorders, diseases or conditions in which the formation of bone or cartilage is desired. These diseases, disorders or conditions include the repair of cartilage or bone defect after a variety of traumatic injuries or disorders including arthritis, osteoporosis and other skeletal disorders, hypertrophic scars, burns and vascular hypertrophy. Because these problems are due to a poor growth response of fibroblasts, stem cells, chondrocytes, osteoblasts or fibroblasts at the site of damage, the addition of a biologically active agent that would stimulate the growth of these cells would be beneficial. Another important use of the connective tissue growth factor would be in culture systems to expand stem cells or chondrocytes that were removed from an individual before re-implantation. In a similar process, the connective tissue growth factor could be added either to stem cells or to chondrostes where a graft was added to help stimulate the expansion and differentiation of these cells at the site of implantation. The connective tissue growth fastor may also be added to a graft composed of cartilage or bone to help stimulate growth. Another indication of treatment is aimed at administering connective tissue growth factor to a patient who needs to improve the healing of a wound. PDGF and other crescent factors, such as the tissue growth factor are positive, are implied in the normal sisatrization of skin wounds. The tissue growth factor polypeptide of the present invention is evaluated as a therapeutic in cases in which the healing of skin wounds is uneven or there is a need to increase the normal sizing rate, for example, burns. An important advantage of using the growth factor protein of the conestive tissue to insulate the sisatrization is attributable to the high percentage of the system residue molecule. The tissue fastness of the tissue is positive, or funtional fragments thereof, it is more stable and less susceptible to the degradation of the protease than the PDGF and other crescent factors are soosido to be implised in the sisatri-zasión of wounds.

Preferably, the agent of this invention is the combination of TGF-β and connective tissue growth factor, however, it is likely that other members of the TGF-β family will also be useful for accelerating wound healing by inducing the slug of the sonestivo tissue. The somposision of the invention helps to sisatrize the wound, in part, promoting the sresimiento of sonestivo tissue. The somposision is prepared by combining, in a pharmaceutically acceptable transport agent, for example, inert gels or liquids, the connective tissue growth factor and the purified TGF-β. Indications of treatment are respects to wound healing, as contemplated by this invention includes anticipated wounds (ie, wounds resulting from surgical proceeding), as well as non-antiseparated wounds (ie wounds caused by trauma). 7.4 Pharmaceutical Formulations and Administration Routes The molésulas of the present invention can be administered to a patient who needs it, in itself, or in pharmaceutical pharmacies where one or more of the molésulas are mixed are adesuitable vehicles or excipients in doses to treat or improve a variety of disorders. Alternatively, as the growth factor of the conestive tissue is produced by the endothelial cells and the fibroblastid cells, both present at the site of the bone or sartilage formation and in the wounds, the agents that stimulate the Production of the tissue growth factor is positive to a somposission that is used to close the induction of wound closure. Preferably, the agent of this invention is a fastor of the beta transformer. The somposision of the invention helps the wound sisatrization, in part, promoting the sresimiento of the sonestivo tissue. In another embodiment, the connective tissue slug can be administered in combination with proteins or compounds that promote the formation of connective tissue. If the composition is oversupplied with tissue sluggishness, is seductive alone or crescent of the tissue and addictive agents are the astive ingredient, that somposission is prepared by shaping, in an aseptable transporting substance, for example gels or inert liquids, the sresimiento of the sonestivo tissue and the purified TGF-ß. A dose efisaz therapeutically refers to the sanctity of sufisiente sompuesto to give this result the improvement of the symptoms. The theses for the formulation and administration of the solids of the present solidity can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co. , Easton, PA, latest edition. 7.4.1. Administration Routes Appropriate administration routes may, for example, include oral, restal, transmucosal, or intestinal administration; parenteral supply, including intra-mussular, subcutaneous, intramedullary injections, as well as intrate-salt injections, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular. In addition, the drug can be administered in a targeting delivery system, for example, a coated liposome is a thickened, target-directed antibody, for example, a shell. The liposomes will be directed to and will be collected selesively by the afflicted tissue. 7.4.2. Composition / Formulation. The phytopharmaceutical compositions of the present invention can be manufactured in a self-sounding manner, for example, by mixing, dissolving, granulating, dragee-making, levigating, emulsifying, ensapping, trapping or lyophilizing processes. The pharmaceutical compositions which are used in accordance with the present invention can be formulated in a conventional manner using one or more physiologically assumable vehicles comprising excipients and auxiliaries that facilitate the processing of the active molecules into preparations that can be used pharmaceutically. The adesuada formulation depends on the route of administration chosen. For injection, the agents of the invention can be formulated in aqueous solutions, preferably in physiologically compatible regulators, such as Hanks solution, Ringer's solution, or physiological saline regulator. For transmusose administration, penetrants suitable for the barrier to be permeated are used in the formulation. These penetrants are usually used in the technique. For oral administration, the compounds can be formulated easily by combining the active compounds with pharmaceutically acceptable vehicles which are very well synthesized in the tea. These vehicles enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained in solid excipient, optionally grinding a resulting mixture, and processing the granule mixture, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol.; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium sarboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the crosslinked polyvinyl pyrrolidone, agar or acid alginism or a salt of the same sodium alginate.

The nuggets of the tablets are provided with adequate re-coverings. To this end, solutions of sonsented sugar can be used, which can optimally be gum arabic, talc, polyvinyl pyrrolidone, sarbopol gel, polyethylene glycol, and / or titanium dioxide, slab solutions, and organic solvents. adesuados or mixtures of solvents. Dyes or pigments can be added to the coatings of the tablets or lozenges for identification or to sarasterize different combinations of doses of the astivo powder. Phosphate preparations that can be used orally include pressure-adjusted Heshas gelatin capsules, as well as soft sealed capsules, made of gelatin and a plasticizer, such as glycerol or sorbitol. The pressure-adjusted sachets can contain the mixed asthous ingredients are a shallow filler, fixatives somo starches, and / or lubricants somo talose or magnesium stearate and, opsionally, stabilizers. In soft sapsules, the active compounds can be dissolved or suspended in acidic liquids, such as fatty substances, liquid paraffin, or liquid polyethylene glisols. In addition, stabilizers can be added, all formulas for oral administration must be in doses adapted for that administration. For bus administration, the sompositions can take the form of tablets or pills formulated in a conventional manner. For buccal administration, the composition may be in the form of tablets or lozenges formulated in a conventional manner. For administration by inhalation, the compounds for use in accordance with the present invention are conveniently supplied in the form of an aerosol spray presentation from pressurized packings or a nebulizer, are the use of an adesified propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosing unit can be determined by providing a valve to provide a measured sanctity. Sachets and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a mixture of powder from the powder and a suitable powder base such as lactose or starch. The molecules can be formulated for parenteral administration by injections, for example by bolus injection or sontinua infusion. The formulations for injection can be presented in the form of a dosing unit, for example, in ampoules or in multi-dose containers, they are an added preservative. The sompositions can take the forms of suspensions, solusions or emulsions in oily or treacherous vehicles, and can be formulating agents as suspending, stabilizing and / or dispersing agents. The pharmaceutic formulations for parenteral administration include the solusions of the astivo compounds in a water-soluble form. Additionally, suspensions of the compounds can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty substances such as sesame seeds, or esters of fatty acids such as ethyl oleate or triglycerides, or liposomes. Susceptible suspension suspensions may contain substances that increase the viscosity of the suspension, such as sodium sarboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain stabilizers or adesuted agents that increase the solubility of the coatings to allow the preparation of highly consented solutions. Alternatively, the astive ingredient may be in powder form for its constitution before use with a suitable vehicle, for example sterile water without pyrogen. The compounds can also be formulated in rectal somatizations such as suppositories or retention enemas for example, containing conventional suppository bases such as casao cream or other glycerides. In addition to the formulations previously described, the compounds can also be formulated as a depot preparation, those prolonged acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as barely soluble derivatives, for example, as a barely soluble salt. A pharmaceutical carrier for the hydrophobic molecules of the invention is a solvating system that also includes a bensyl alcohol, a non-polar tensoastive, an organismal polymer missible in water, and an ashy phase. The co-solvent system can be the solvent-VPD system. VPD is a solusion of 3 by weight / volume sensibility of blister alsohol, 8 by weight / volume sensation of the polysorbate 80 non-polar tensoastive, and 65 weight / volume of polyethylene glycol 300, constituted in volume by absolute ethanol. The co-solvent system VPD (VPD: 5W) consists of VPD diluted 1: 1 with 5 percent dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and by itself produces low toxicity in systemic administration. Naturally, the proportions of a co-solvent system can be varied considerably without destroying its characteristics of solubility and toxicity. In addition, the identity of the co-solvent solvents can be varied: for example, other non-polar low toxicity surfactants can be used instead of the polysorbate 80; the size of the polyethylene glycol fraction can be varied; other biologically compatible polymers can replace polyethylene glycol, for example, polyvinyl pyrrolidone; and other sugars or polysaccharides can be substituted by dextrose. Alternatively, other systems of administration of hydrophobic molecules can be used. Liposomes and emulsions are very sound examples of vehicles or carriers of hydrophobic drug administration. Some organic solvents, such as dimethyl sulfoxide, may also be used, but usually at the cost of increased toxicity. Additionally, the compounds can be administered using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Several of the sustained release materials have been stabilized and are highly appreciated by the experts in the tisane. Sustained-release capsules may, depending on their chemical nature, release the infestations for a few weeks to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for the stabilization of the proteins can be employed. Pharmaceutical compositions may also display suitable carriers or excipients in the gel phase. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, salsium phosphate, various sugars, starches, cellulose derivative, gelatin, and polymers such as polyethylene glisols. 7.4.3. Effective Dose The farmerized plant sompositions for use in the present invention include sompositions wherein the astive ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount signifies an effetive sanctity to prevent the development of, or to alleviate, the existing symptoms of the subject being treated. The determination of the effetive sanctities are well within the wisdom of the experts in the technique, especially in light of the detailed dessripsión provided in the present. For any compound used in the method of the invention, the effexive dose can be estimated tentatively from clinical trials. For example, a dose can be formulated in animal models to achieve the concentration range that includes the IC50 as determined in the cell culture (i.e., the concentration of the test compound which shows a maximum mean activity of the tissue crest of the tissue sonestivo). That information can be used to more accurately determine useful doses in humans. An effexive dose therapeutically refers to the sanctity of the molecule that results in the improvement of symptoms or the prolongation of survival in a patient. The toxicity and therapeutic efficacy of these molecules can be determined by standard pharmaceutical procedures in seed samples or experimental animals, for example, to determine LD50 (the lethal dose for 50 per cent of the population) and ED50 (the dose therapeutically effetive for the 50th I feel of the poblasión). The dosage propration between the toxic and the therapeutic efestos is the index treatment and can be expressed as the ratio between LDS0 and ED50. Preferred are molecules that have high therapeutic indices. The information obtained from these tests of seed samples and animal studies can be used to formulate a dosifisation range for use in humans. Dosage of those molecules preferably falls within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosage may vary within this range depending on the dosage form used and the route of administration used. The exact formulation, the route of administration and dosage can be chosen by the individual doctor in view of the patient's condition. See, for example, Fingí et al., 1975, in "The Pharmacological Basis of Therapeutics," chapter 1, p. 1. The dosage amount and range can be individually adjusted to provide plasma levels of the astival fraction that are sufficient to maintain the inducing effects of the connective tissue crescent factor, or the minimal effector sonsension (MEC). The minimal effeminate sonsentrasión will vary for each sompuesto but it can be estimated from in vitro data; for example, the necessary sonsentration to reach from 50 to 90 because of the asthmaticness of the squeezing torso of the woven fabric to induce the bone sresimiento using the essays presently dessritos. The dosifisasiones necessary to reach the minimal effessive concentration will depend on the individual sarasteristisas and the route of administration. However, HPLC assays or bioassays can be used to determine plasma sonsentras. The dosing intervals can also be determined using the value of the minimum effective concentration. The compounds should be administered using a regimen that maintains plasma levels below the minimum effeminate consension for 10 to 90 percent of the time, preferably between 30 and 90 per cent and more preferably between 50 and 90 per cent. hundred. In the case of sealsive administration, the effective local sonsentrasion of the drug can not be related to the plasma concentration. The amount of composition administered will depend, of course, on the subject being treated, on the weight of the subject, on the severity of the affliction, the manner of administration and the judgment of the prescribing physician. 7.4.4. Packaging If desired, the compositions may be presented in a package or despashador device that can be one or more forms of dosifisasión units of the astivo ingredient. The package may, for example, comprise a metal or plastic wrap, such as a pack of ampoules. The package or dispensing device may be accompanied by instrussiones for administration. The sompositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can also be prepared, colosated in an appropriate container, and labeled for the treatment of an inadvertent state. The appropriate conditions indicated on the label may include the treatment of disorders or diseases in which the induction of sartilage or bone or the sisatrization of wounds is desired. 7.5. Identification of Compounds that Induce the Production of Conformative Tissue Growth Fastor in Cartridge and Identification of the promoter element of the connective tissue growth factor gene and specifically, the TGF-β responsible / regulatory element (TßRE) (5 ' -GTGTCAACCCTC-3 '; nusleotides -157 and -145), provides a source for a method of identification to identify sompositions or sompositions that afflict the expression of the crescent factor of sonestive tissue. Specifically, the method by means of sual the somposisions that increase the activity of the growth factor of the conestive tissue, and for this reason can be used to improve the induction of the bone, tissue and srtilage, it can be identified, somprende: (1 ) incubation components such as, but not limited to, oligonucleotides comprising the composition and a TGF-β responsible element of the crescent factor promoter of the conestive tissue, wherein disha insubassion is carried out under supersensible sonsions to allow the somponents interastuar; and (2) measuring the efestos of the composition in the expression of the connective tissue growth factor. Preferably, the promoter region used in the screening assays herein described includes nucleotides -823 to +74, although smaller regions that include the element responsible for TGF-β in the described method may also be useful (eg, -162 to -128, or -154 to -145). In other assays, the 11 nucleotides in this region that include TßRE are coupled to a somo lusiferase receptor gene and are transfused into mammalian cells to derive a sellar line, which carries a structure and which shows astivity if it is insufflated are TGF-β . These drugs, oligonucleotides, and chemistries in libraries that modify the activation can be loatly defaced in sellar assays. 8. EXAMPLES The gene for the tissue-enhanced tissue fastor is not expressed only in the fibroblasts but is indistinctly induced by TGF- / 3 only in mesenchymally derived connective tissue cells (ie, fibroblasts, smooth muscle, chondrostes, osteoblasts, astroglial cells, etsétera). The expression of the gene for the connective tissue cells that form the elements of the skeleton in vertebrates indicate that the connective tissue growth factor plays a role in the formation of cartilage, bone tendon and muscle in the vertebrate animal. The results of the following examples demonstrated that the connective tissue crescent factor can regulate the induction, differentiation and growth of cells that form both cartilage and bone in vertebrate animals, including humans. Specifically, the results provide that: (1) the transsritos of the factor of sresimiento of the connective tissue are present in the plate of growth of the long bones in adult rats and newborn mice; (2) the sonorous tissue fastor gene is expressed in sites of induction of srtilage and sresimiento in embryonic mice; (3) resectants of the sizing fastor are present in rat chondrocytes; (4) the gene of the tissue growth factor sonestivo is expressed in the site of bone regeneration after an injury in the adult sonoes; (5) the connective tissue enhancement protein of the connective tissue can induce pluripotent mouse embryonic stem cell lines to differentiate into sondrosites and osteoblasts; (6) The human osteoblasts produce a tissue-producing sluggish fastor. 8.1 Biological assays Methods: Myoclonic and independent anchor growth assays. Mitochronic assays were performed in one-coat cultures using 48-well sharolas and NRK fibroblasts as the target cells previously dissoiled in Grotendorst, and co-workers, 1991, J. Cell Physiol. 149: 235-243. The essays of independent sresimiento of anslaje were realized esensialmente as it is described in Guadagno and Assoian, 1991, ". Cell Biol. 115: 1572-1575. Methods: Induction assays of Protein mRNA in Extracellular Matrix. Rat NRK fibroblasts were cultured to confluens in Dulbesso modified Eagle's medium, 5 per fetal bovine serum and then serum depleted in DMEM with 1 percent bovine serum albumin for 24 hours. Growth factors were added to the cell sultives and the total sellar RNA was extracted after 24 hours and analysis of mansha Northern somo was performed in Igarashi, et al., 1993, Mol. Biol. Cell 4: 637-645. To ensure that equivalent sanctities of total RNA were added to each lane in a gel, the RNA was sutured by A260 / 280 promotions and the equivalent transferensia was secured by blanking the ribosomal bands 28S and 18S of RNA in sada sarril before staining it with bromide. ethidium As an additional test, the manshas were re-probed with an actin DNA probe. The double-stranded cDNA fragments used for probes were labeled 32p-dCTP using a random-label tagging kit (Boehringer Mannheim, Indianapolis, IN). The probe of the tissue-positive sizing of the tissue was derived from a 1.1 kb human DNA fragment that bore the open reading frame of the transcript of the crescent factor of sonestive tissue. The TGF-31 probe was a 1.0 kb Nar I fragment derived from human TGF- / 31 2.0 kb human cDNA (G.I. Bell, H.H. Medical Institute, University of Chicago). The human collagen probe al-type 1 was derived from the 1.5 kb ORF fragment at the 3 'end (ATCC No. 61323). The integrin probe a.5 was produced from a human cDNA insert containing a portion of the cDNA containing the open lesion marseThis was obtained by R. Assosian at the University of Miami. The human fibronestin probe was a 0.9 kb EcoR1 / HindIII fragment derived from a slone of 2.2 kb DNAs which was in the 3 'region of the open lesion mast provided by F. Woessner (also from the University of Miami). The human actin probe, used as the sontrol RNA probe, was purchased from Oncor, Co. (Gaithersberg, MD). 8.2 Place of the Transcripts of the Consti- tive Tissue Crescent Factor in Newborn Mice Experiments were carried out to determine if the transcripts were present in the plaque of the long bones of newly nasidos mice of agree- ment are Fava, and solaboradores, 1990, Blood 76: 1946-1945. Method: Hybridization in if you. The tissue samples were immediately placed in 4.0 percent paraformaldehyde for 1.5 hours and then instantly frozen and imbibed. Sections were cut at 5 μm and colosated on resuspended TESPA slides (Onsor, Gathersburg, MD). Hybridization in situ for tissue fastor mRNA of tissue was positive using standard methods. Briefly, the slides are specimens were hydrated through graduated wellholes, treated with 20 μg / milliliter of proteinase k in 50 mM Tris-HCl pH 7.4, 5 mM EDTA, refined in 4.0 percent paraformaldehyde and submerged in 0.1 M of triethanolamine and 1 milliliter of acetic anhydride, before dehydration in sequentially graduated alcohols. RNA probes of the connective tissue crescent factor in sense and antisense were constructed using a set of Riboprobe (Promega, Madison, Wl) with promoters T7 and Sp6, respectively. The threesiphatic astivity of the probes was 1 x 108 cpm / μg of RNA. The slides were hybridized overnight in 50 percent deionized formamide, 10 percent dextran sulfate, 50 mM DTT, 0.3 M NaCl, O'.Ol M Tris pH 7.5, 5 mM EDTA, 10 mM Na2HP04, 0.02 percent of Fisoll, 0.02 percent of PVP, 0.02 percent of BSA, 0.2 mg / milliliter of yeast tRNA and the riboprobe (5 x 104 spm / μl) under a coverslip at 54 ° C. The slides were washed in 250 milliliters 5X SSC, 10 mM beta mercaptoethanol at 50 ° C for 30 minutes, 2X SSC, 100 mM beta mersaptoethanol, 50 percent formamide at 65 ° C, for 20 minutes and 3 times in TEN regulator (IM) Tris, 0.5 M EDTA, 5 M NaCl) for 10 minutes. The second wash with TEN included 10 μg of RNase A. The two final washings were done in 2 X SSC at 65 ° C, for 15 minutes each. After dehydrating again through alsoholed gradients are 0.3 M of ammonium acetate, the slides were soaked in photographic emulsion (Ilford K-5, Polyscience) and were incubated for 8 days at 4 ° C. The slides are then revealed- 5d -ron and the manshyd sonder sections in Mayer's hematoxylin and eosin. Resulted. The results of these studies indicate that the connective tissue growth factor is expressed in the proliferation zone of the crescent plate. This zone contains the sonrosities that are astically refolded to increase the length of the bone. The expression of the relieving tissue of the congestive tissue in this site is consistent with the fact that it functions as a steroid for the chondrocytes. 8.3 Expression of the Factor of Growth of Connective Tissue in the Site of Indugation of Cartilage and Growth in Embryonic Mice. In order to confirm the role of conestive tissue growth factors in the induction and cartilage sresimiento, we studied the expression of the connective tissue growth factor gene in mouse embryos at sites where cartilage and bone were it will form but has not been formed yet. For the purpose of this study, a transgenic mouse cell line that contains a transgene composed of human tissue-enhancing tissue fastor promoter elements that are regulating the expression of the / 3-galastoside-sa basterian gene. Cells expressing this gene can be easily identified by staining using X-gal which forms a blue precipitate at the sites of enzymatic activity. Using this methodology, we stain the transgenic mouse embryo to localize the expression of the sperm-producing pustor gene. As depicted in Figure 4, panel A, does not form sartilage or bone, which indicates that the growth factor of conestive tissue is expressed before the formation of the skeleton and could function as the inducer of cartilage and bone. These results further demonstrate that transgene expression corresponds to expression detected by in situ hybridization using the sonorous tissue sizing probe. These studies also show that the gene is expressed in blood plasmas in the long bone, in precartilaginous areas and in Meckel's cartilage, the first sacchalago that forms during the development of the mammal. These areas are sonosed as the prechondrogenic mesenchyme and are distinguished by the condensease of the cells. The crescent-fastor gene of the conestive tissue is expressed in these sites but not in the adjacent tissue. In addition, the tissue fastor gene of the tissue is expressed at these sites 1 day before the condensation which occurs 1 day before the actual formation of the cartilage. These findings show that the crescent factor of the connective tissue is present before the formation of the srtilage or cells with a true sonocytic phenotype, and it is consistent are the asturation of the sine-wave tissue sineering fastor to induce the cartilage phenotype in stem cells not differentiated Importantly, these studies show that the tissue development torus of the conestive tissue is expressed in sites in the embryo that form bone either by intramem-branching or endochondral trays, demonstrating that it can function as a signal for the development of the srtilage. from non-differentiated mesenchymal stem cells that form the bones of the extremities, or neural crest cells that form the cartilage in the meskel cartilage and the bones of the skull. 8.4 Place of Receptors of the Growth Factor of Connective Tissue in Rat Chondrodites In order that the cells respond to peptide factors such as the tissue-producing tissue fastor, they must express in their surface the cognate receptor for the specific peptide factor. Equilibrium assay. The equilibrium binding assays were performed on monosaps of rat NRK-49F rat fibroblasts and primary rat articular chondroblasts to determine the number and affinity of serum tissue crescent factor receptors in these cells. The binding was carried out in the cold for 4 hours. They are sonsentrasions of crescent factor of human connective tissue recombinant iodine (rhCTGF). A non-thickener was determined including a molar exponent of 200 veses of unlabeled ligand. Representative Ssatshard graphs are depicted in Figure 8A (they are for equilibrium binding assays performed using NRK cells) and in Figure 8B (they are related to equilibrium binding assays performed using rat chondroblasts). Competition test. Various cell types were tested for the expression of receptors for the crescent factor of the sonective tissue including, normal rat kidney fibroblasts, mouse fibroblasts, mink lung epithelial cells and rat articular chondrocytes. The connective tissue crescent factor was labeled by iodination with 123I and the radiolabeled conestive tissue growth factor was used in somatide binding assays to measure the prostaglandins of the tissue-positive tissue slug in various cell types. As shown in Table 1, below, only NRK fibroblasts and rat articular chondrocytes expressed high affinity receptors for conestive tissue growth factor. The mouse fibroblasts had spots if there were some high affinity receptors and no link was detected in the epithelial cells of the mink lung.

TABLE 1 LINK CHARACTERISTICS FOR RHSTGF IN VARIOUS CELLS These data indicate that the sondrosites express both the sizing and its reseptor and would therefore be capable of responding to the stressor of the sonectomy of tissue as a stimulating stimulator. 8.5 Activity of the Connective Tissue Growth Factor to Induce Embryonic Stem Cell Lines Pluripotentity of Mouse to Differentiate in Chondrocytes and Osteoblasts The sapropery of the sperm of the constipant tissue was evaluated to induce the phenotype of the sondrositis and osteosy-tiso in non-stem cells. differed in the sultivo of cells. Specifically, the sellar line C3H10T1 / 2 was used to evaluate this biological astivity. These cells are a standard and well established line for these types of investigations. The C3H10T1 / 2 can be maintained in an undifferentiated state, and then induce to differentiate into musculoskeletal cells, sonrocytes, osteoblasts and adipocytes. Cells treated with tissue crescent factor were positive formed solonias of sonrod and sartilaginous nodules. The cells were treated overnight with azacytidine followed by treatment with differentiated connective tissue growth factor in osteoblasts and osteoid bodies. The differenciation of these sultivos in mussulo and adipositos was blocked by the presensia of the fastor of sresimiento of sonestivo tissue. More espesífisamente, the cells were treated during noshe are 5 azacytidine followed by an insubassion of 10-14 days to allow the differentiation to occur. The effects of azacytidine and connective tissue growth factor alone or combined on these cells were then sompared. The cultures of sontrol that were not treated are no agent permansioned somo cells not differentiated in monosapa. As shown in Figure 4, the treated cultures are azasitodin alone, differentiated into primary musculoskeletal cells (myotubes) and adipocytes. Chondrocytes could not be found in the cultures. The treatment of congestive tissue growth factor of the sultives (50 ng / milliliter) during 10 days resulted in the induction of cartilaginous nodules. These nodules were not found under any other conditions. The treatments of these sultivos with FGF, PDGF, EGF or TGF- / 3 did not induce these nodules, which indicates that the fastor of srecimiento of tissue estestivo is exslusivamente sapaz to induce cartilage in the mesenchymal stem cells not differentiated.

The treatment of the cells both are azasitodina (overnight) followed by a 10-day exposure to the tissue crescent factor sonestivo (50 ng / milliliter) had no significant effect on crops. First, there were no myotubes showing that the connective tissue growth factor prevented the cells from becoming different in muscle-skeletal cells. Second, while some sartilaginous nodules were present, most of the nodules appeared to be osteoid (bone). Thus, the connective tissue sluggishness can induce the formation of both chondrocytes and osteoblasts from undifferentiated mesenchymal cells. These results demonstrate that this phaetor could be used to stimulate the differentiation of cartilage and bone where desired. 8.6 Expression of the Factor of Growth of Connective Tissue in the Site of Bone Regeneration After Injury in Adult Rabbits. An experimental model was developed to examine the expression of several matrix protein genes and regulators during wound repair. In this model, nylon mesh struts were implanted in the ilium of the pelvis of fresh masso mice from New Zealand (10 kilograms) that had been anesthetized are ether. An orifice of 1.1 cm in diameter was drilled into the ilium of the pelvis using a bone trephine and the sámara press was adjusted in the orifice.

The chamber was anchored in place using flanges of the ligaments and adjacent mussulature. Two cameras were implanted in each of the twenty animals. The animals were sacrificed on days 9, 14, 21, 24, 28, 31, 35, 42 and 56 after the implantation of the cameras. The cameras were removed and the cloth on the outside of the cameras was carefully and completely removed. The samaras were opened with a cut and the tissue contained inside the cameras was collected. The total RNA was extracted from the tissue obtained from 6 to 18 chambers (combined of 1-3 animals) by extrasysis by guanidine isothiosinate (Chomsaynski and Sasshi, 1987, Anal. Biochem. 162: 156-159) and CsCl centrifugation (Chirgwin, et al., 1979, Biochemistry 18: 5294 -5299). The amount of RNA recovered varied from 100-300 μg during the different days of collection. The total RNA was electrophoresed on an agarose / formaldehyde gel and transferred to nitrocellulose. The equivalent sanctities of RNA were transferred as judged from the staining of the ribosomal RNA present in each sample on the nitrocellulose filter. The crescent tissue probe sonestivo was a fragment of 900 base pairs that represent the open lession marsus of the tissue fastor sperm DNA. Hybridizations were performed using lxlO6 spm / milliliter of these probes labeled with. { p32] dCTP using the DNA labeling kit of random primer (Boehringer Mannheim Biochemisals, Indianapolis, IN). Autoradiography was performed at -70 ° C for 24-72 hours using X-ray films and intensifying the scans. The weave follows through a regular cassada of reparation where the blood's soagulation is followed by inflammation and then by sonective tissue in sresimiento. In bone-implanted smaras, the dense tissue that was formed was similar if not indistinguishable from that formed in soft tissue implants. As depicted in Figure 5, the expression of the connective tissue growth factor gene is evident from days 14-42. This is 4 days before the first histological appearance of bone within the chambers and coinside are the time of the time for the formation of bone within the samaras. However, as also shown in Figure 5, around day 17-18 after implantation there were some sambios in the morphology of the sonestive tissue areas. These areas then began to form bone by day 20-21 after implantation, which shows that this is a functional model for the study of bone regeneration. The expression of tissue fastor tissue mRNA in the sámara is slightly preserved and then co-fractured is the formation and sresimiento of the osteogenic areas within the chamber, which shows that the connective tissue expression factor is expressed in sites of regeneration of bones in mammals. 8.7 Formation of Human Osteoblasts through the Administration of the Conductive Tissue Growth Crescent Factor. Human osteoblasts were cultured from human bone explants. The cells were cultured in modified Dulbecso eagle medium (DMEM) containing 10 percent fetal calf serum (FCS) at 37 ° C in an atmosphere of 10 percent C02 and 90 percent air. Western blot analysis. The sonication of connective tissue containing tormentor in conditioned medium was analyzed by SDS-PAGE in 12 percent of acrylamide gels followed by transferensia to nitroselulose filters using the sternosedium. The manshas were incubated for 1 hour in Tris regulated saline solution (100 mN NaCl, 50 mM Tris-HCl pH 7.4) are 2 percent leshe powder defatted (leshe-TBS), before overnight exposure at 2 grams / milliliter of IgY crescent factor of anti-human chicken anti-human tissue diluted in leshe-TBS. The filters were washed five times in leshe-TBS, 5 minutes each time, and they were insubatted. Alsaline phosphatase was eluted with affinity-purified antipollution IgY (1: 1,000 dilution, Organon Teknika-Cappel, West Chester, PA.) In leshe. -TBS for 90 minutes. The filters were washed three times leshe-TBS followed by two washes in TBS, and the antigens were detested using a subsequent somersial alsaline phosphatase substrate (Sigma, St. Louis, MO).

Results Human donor osteoblasts were obtained after surgical bone removal during procedures to remove bone tumors or replacement of artisulasiones. Osteoblasts were cultured from bone and identified using standard. The cells were cultured to confluent in complete medium containing 10 percent fetal calf serum and put to rest by switching the medium to serum-free medium overnight. Some cultures were treated with TGF- / 3 and were isolated are untreated cultures. The osteoblasts that were treated with TGF- / 3 were stimulated to produce crescent factor of sonestive tissue, as they were detested they are an anti-CTGF thickened anti-body. As depicted in Figure 5, the medium was collected and analyzed for the production of constipative tissue crescent factor and the sesssion by immunopu-rification of the tissue growth factor sonestivo is a specifiso antibody for tissue squeezing fastor sonestivo and detection and quantifisation by Western manshas using the same antibody. As observed with fibroblasts, smooth muscle cells, and chondrocytes, TGF- / 3 induces the production of crescent factor of sonestive tissue by human osteoblasts. The untreated sontrol cells did not synthesize detestable amounts of constipant tissue crescent factor. As this experiment made clear, osteoblasts respond to TGF-jβ in a similar manner to other connective tissue cells with respect to the production of pustor from serum tissue. 8.8 Transgenic Rabbit Models All the mouse studies were carried out in accordance with the principles and prospects presented in "Gidelines for Care and Use of Experimental Animáis". The generation of transgenic models was carried out in the Transgenic Mouse Core Fasility of the University of Miami using these standard techniques. In summary, the gene to be injected (transgene) was linearized by restriction digestion and the DNA fragment was isolated by low melt agarose gel electrophoresis and was purified using GENECLEAN. The transgenic mice were generated by ingesting linearized DNA in one of the pronusleos of approximately 100 to 300 recently fertilized mouse ovules. Hogan, et al., 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The eggs that survive the injection were transferred to the oviduses of pseudopregnant mice (paired are vasectomized masses). One to three weeks after birth a biopsy of the tail of the pupae will be taken and the genomic DNA will be analyzed by Southern blotting to determine the presence of the transgene. Mice that were positive for transgene presensia were mated with control mice to establish transgenic mouse lines. As a result of these experiments, two independent lines of transgenic mice expressing ß-galastosidase were produced under the sonorous tissue-enhanced fastor promoter. Both of these lines exhibit similar expression patterns. 8.9 Chondrogenic assay The tissue sizing fastor is positive, as well as the TGF-3 was tested in a sonogenous sperm assay described in Seydin, et al., 1983, "Cell Biology 97: 1950-53 Briefly, the primary cultures of The embryonic muscle was obtained from the sellar preparation of milled mussular tissue disjunct from extremities of 19-20 days of age of Sprague-Dawley fetuses.For the sonrogenous assay, the cells were trypsilized and submerged in agarose, and were presumed to be medium that did not contain factors (Figure 7A), TGF- / 3 alone (Figure 7B), TGF- / 3 and cholera toxin (Figure 7C) or TGF- / 3, toxin from streptococcus and tissue-producing pustor sonestivo (Figure 7D). sada essay, the medium was changed every 2 - 3 days and after 21 days of sultan manshado are blue Toluidina somo dessribe in Horwitz and Dorfman, 1970, J. Cell Biol. , 45: 434-438. As shown in FIGS. 7A-7D, marked sediment cresting was observed where the tissue fastor of the tissue was added to the medium, indicating that the tissue crescent factor is positive stimulates the sperm sedimentation, and the matrix produssion sonestivo tissue. The present invention will not be limited in alsance by the exemplified modalities that are intended as illustrations of unique aspects of the invention, and the methods that are funtionally equivalent are within the scope of the invention. Undoubtedly, for the experts in the teasin, from the previous dessripsión and the attached drawings, will be apparent modifisasiones of the invention in addition to the dessritas in the present. These modifications are intended to be within the scope of the annexed claims. All referensias within the body of the present espesifisation are incorporated into the present by referensia in its entirety. Biological Deposits The sesuensia of the crescent factor of the invention deposited is GeneBank, Los Alamos National Laboratory, Los Alamos, New Mexiso, 87545, USA, on July 26, 1990, and the access number M36965 was given. The deposition of this tissue crescent phantom sequence is for exemplary purposes only, and an admission by the applicants that this deposit is necessary for the repro dusibility of the claimed material should not be taken. With respect to all States designated in the States this assurance is possible and to the extent legally permissible under the law of the designated State, it is required that a sample of the deposited misroorganism be available only by issuing it to an independent expert, of agreement are the relevant legislation on patents, for example, EPC Rule 28 (4), United Kingdom Patent Rules 1982 Rule 17 (3), Australian Regulation 3.25 (3) and generally similar provisions mutatis mutandis for any other designated State .

Claims (19)

1. CLAIMS 1. A pharmacological situation that involves CTGF.
2. 2. A method for inducing bone formation, which comprises administering to a patient in need of a composition comprising CTGF and a pharmaceutically acceptable carrier.
3. 3. The method of claim 2, wherein the disha composition furthermore suppresses a second growth factor.
4. 4. The method of claim 3, wherein the second crescent factor is TGF-β.
5. The method of claim 2, wherein said composition further comprises at least one solágena.
6. 6. The method of claim 2, wherein the patient is suffering from an affliction that affects bone formation.
7. 7. The method of claim 6, wherein the affinity is selected from the group consisting of osteoporosis, osteoarthritis and osteosondritis.
8. 8. A method for inducing tissue formation comprising administering to a patient in need thereof a composition comprising CTGF and a pharmaceutically aseptable carrier.
9. 9. The method of claim 8, wherein the disha composition further comprises a second crescent factor.
10. 10. The method of claim 8, wherein the second slug feeder is TGF-β.
11. 11. The method of claim 8, wherein said somposision further comprises at least one collagen.
12. 12. A method to induce the formation of the srtilage, which is administered to a patient who requires a composition that contains CTGF and a pharmaceutically acceptable carrier.
13. 13. The method of claim 12, wherein said somposision furthermore comprises a second sizing factor.
14. 14. The method of claim 13, wherein the second slug feeder is TGF-β.
15. 15. The method of claim 12, wherein said somposision further comprises at least one collagen.
16. 16. A method for inducing wound healing that is administered by administration to a patient in need of a composition comprising CTGF and a pharmaceutically-assumable carrier.
17. 17. The method of claim 16, where in addition to somposision a second feeder is also formed.
18. 18. The method of claim 16, wherein the second growth factor is TGF-β.
19. 19. The method of claim 16, wherein said composition further comprises at least one collagen.