Composition for stimulating the growth of osteoblasts containing frpt and vitamin d

 

(57) Abstract:

The invention relates to medicine, namely to the chemical-pharmaceutical industry and relates to a composition for stimulating the growth of osteoblasts containing FRPT and vitamin D. the invention includes a composition comprising a growth factor derived from platelets, and vitamin D in sufficient quantity to stimulate the growth of osteoblasts and stimulate bone growth in a patient. The technical result is to increase the proliferation of osteoblasts and thereby intensify the processes of bone regeneration. 4 C. and 16 h. p. F.-ly, 7 ill., table 2.

Reconstruction of bone is a dynamic process through which the fabric weight and structure of the skeleton. This process is characterized by the balance between resorption and bone growth with the participation of two cell types, the players are assumed to be the main role.

These cells are osteoclasts and osteoblasts. Osteoblasts synthesize and lay new bone tissue in the cavities formed by osteoclasts. The activity of osteoblasts and osteoclasts is regulated by many factors both systemic and local, including growth factors.

In vitro studies have shown that PPT is a mitogenic factor for osteoblasts (Abdennagy and other Cell Bid. Internal. Rep. 16 (3):235-247, 1992). Mitogenic activity and chemotactic properties associated with FRPT, were shown when adding a growth factor to the normal osteoblastogenesis cells (Tuskamota and others, Biochem. Biophys. Res. Comm., 175 (3): 745-747, 1991) and primary cultures of osteoblasts (Centerella and others , Endocrinol. 125 (1):13-19, 1989).

Recent studies have shown that osteoblasts produce the AA isoform FRPT (Zhang and others, Am. J. Physiol. 261: S. 348-354, 1991). The exact mechanism by which FRPT affects the growth of osteoblasts, which are still not well understood, but there is agreement about the fact that this growth factor plays a key role in regulating both normal reconstruction of the skeleton, and the healing of fractures.

Therapeutic use FRPT includes, for example, treatment of injuries that need to heal proliferation of osteoblasts, such as fractures. Stimulation of proliferation of mesenchymal cells and the synthesis intramembranous bones indicate how aspects of sage the new Orleans, Louisiana).

Vitamin D is traditionally considered necessary to prevent rickets, a disease of inadequate bone mineralization. The need for this is connected with the role of vitamin D to facilitate calcium absorption in the gastrointestinal tract, and significance levels of calcium in the serum for homeostasis of bone tissue.

Recent observations suggest that osteoblasts have receptors for the metabolite of vitamin D 1, 25-dihydroxycholecalciferol that specifies them as the main target of this hormone (Suda and others, J. Cell. Biochem., 49:53-58,1992).

I believe that vitamin D plays an important role in the activation of the osteoblast resorption, mediated by osteoclasts (Watrous and others, Sem. in Arthritis anl Rheum., 19 (1): 45-65, 1989). Vitamin D was used in the culture of osteoblasts in vitro (Kurihara and others, Endocrinol. 118 (3): 940-947, 1986), that was accompanied by an increase in the number of alkaline phosphatase as a marker of differentiation of cells into osteoblasts.

However, in bone cells stimulation of alkaline phosphatase are associated with reduced cell proliferation (Huffer, Lab. Investig. 59(4):418-442, 1988). In cultures of bone cells of the cranial vault adding vitamin D increases calcium release into the culture medium and associated with the blasts, requires induction by vitamin D (Yoon and others, Biochem. 27:8521-8526, 1988). The exact role of vitamin D in bone homeostasis and how it affects osteoblasts and osteoclasts, needs to be clarified.

Due to the important role of osteoblasts in wound healing and regeneration of bone, the ability to enhance the proliferation of these cells is highly desirable. The present invention creates the opportunity, and also has other advantages, which become apparent through the following detailed description and the accompanying drawings.

The present invention focuses on ways to stimulate growth of osteoblasts by applying a composition that includes a growth factor derived from platelets (FRPT), and vitamin D. In one of the embodiments of the invention, the composition contains almost no chain FRPT. In a related embodiment, the invention composition includes a recombinant FRPT CENTURIES. In another embodiment of the invention, cells are grown in vitro.

Another feature of the present invention is to provide methods of stimulating bone growth in a patient by introducing this patient an effective amount of com is etenia vitamin D is 9,10-SECO-cholesta-5,7,10[19] -triene-3-ol or 1, 25-dihydroxycholecalciferol.

Another feature of the present invention is to provide methods of stimulating the growth of osteoblasts by culturing these cells in the presence of an effective amount of a composition that includes FRPT and vitamin D; however, the above composition contains almost no A-chain FRPT.

These and other features of the present invention become apparent through the following detailed description and the accompanying drawings.

Fig. 1 shows that vitamin D increases the stimulation FRPT-HV absorption primary osteoblasts pigs ³H-thymidine.

Fig. 2 shows that a twofold increase of mitogenesis requires the simultaneous presence FRPT and vitamin D.

Fig. 3 shows that Energeticheskiy effect FRPT and vitamin D varies depending on the concentration of vitamin D.

Fig. 4 shows that when using basic FGF and vitamin D Energeticheskiy effect is not observed.

Fig. 5 shows that vitamin D affects FRPT-induced mitogenic fibroblasts Swiss ZTZ.

Fig. 6 shows that FRPT causes many times more powerful and the ASS="ptx2">

Fig. 7 shows that FRPT and vitamin D stimulates bone resorption in case of separate addition in culture of cells of the cranial vault, but while adding to the culture of cells of the cranial vault, showing a decrease in bone resorption.

The present invention is based in part on the discovery of the fact that FRPT and vitamin D demonstrate Energeticheskiy effect on the growth of osteoblasts. In addition, it was found that PPT can inhibit bone resorption induced by vitamin D.

As noted above, osteoblasts play a Central role in bone formation and maintenance of bone homeostasis in General. The methods of the present invention are useful for promoting the growth of osteoblasts in cell cultures in vitro, and (iv vivo (for example, when the Union of fractures), and thus, healing.

In the context of the present invention assumes that FRPT includes AA, BB and AB isoforms FRPT, separately or in combinations, as well as their biologically active analogs. In addition, it is assumed that the BB isoform FRPT includes viral homolog (gene product of the v-sis).

FRPT can be derived from both natural and recombinant sources. JV is built entirely in this document as a reference. PPT can also be obtained from bacteria (see Tackney and other WO 90/04035).

How the selection FRPT from natural sources described Raines and Ross (J. Biol. Chem. 257:5154-5160, 1982), Hart and others (Biochemistry 29:166-172, 1990) and in U.S. patent N 4479896.

In some of the aforementioned issued patent States that it is possible directly to obtain the biologically active material using secretory path of eukaryotic cells expressing recombinant FRPT. Expression and secretion of the corresponding gene product of eukaryotic cells provides proper handling and Assembly, resulting in molecules of native and biologically active structure.

When using an appropriate promoter transcription and secretion signal sequences, as a rule, any eukaryotic cell can Express and secrete FRPT in biologically active form for use according to the present invention. On the other hand, polypeptide chain PPT can be expressed prokaryotic cells, isolated and cultured in vitro for the production of biologically active molecules.

For expression FRPT yeast cells posledn promoter and secretory signal sequence. Promoters that can be used for yeast cells include the promoter of Alba factor (MF 1) and yeast promoter triosephosphate yeast (TFI) (U.S. patent N 4559311).

Promoters can also be obtained from other yeast genes, for example, alcohol dehydrogenase 1 (ADH) or alcohol dehydrogenase 2 (ADG). Can be used with appropriate promoters for other species of eukaryotes, is well known to specialists in this field.

Secretion of gene products FRPT can be performed through the use of pre-Pro secretion signal sequence of the alpha factor for yeast pheromone mating (Kurjan and Herskowitz, Cell 30:933, 1982; Julius and others , Cell 36:309, 1984; and Brake and others, Proc. Natl. Acad. Sci. USA 81:4642, 1984) or use of the leader of the BAR1 gene of yeast and sequences of the third domain (see U.S. patent N 5037743), although you can use other signals secretion.

To ensure the effective termination of transcription and polyadenylation of mRNA can add the termination sequence of the yeast cells, such as the terminator triosephosphate (Alber and Kawasaki, J. Molec. Appl. Genet. 1: 419, 1982).

Methods of crosslinking of DNA fragments adequately described (Sambrook and other , Molecular Cloning: A Lab structures units in the expression of them is inserted into the appropriate expression vector.

It is preferable to use the expression vector, which is stably maintained in the cell host to obtain more biologically active substances per unit of culture. In this sense convenient sectors of yeast expression plasmids are pCPOT (ATCC 39685 and pMPOT2 (ATCC 67788), which comprise the gene of Schizosaccharomyces/pombe, encodes the enzyme of glycolysis triosephosphate (POT1 gene).

The inclusion of the POT1 gene ensures stable preservation of plasmids in the cell host with a deletion of the gene TFI, thanks to its ability complementary to compensate for the deletion of the gene in the cell-master, reveals how the U.S. patent N 4931373, incorporated herein by reference.

After the preparation of the design DNA comprising a selective marker POT1 and unit of expression, including, for example, the promoter TFI, leader sequence BAR1 and sequence of the third domain, the corresponding DNA sequence encoding FRPT and terminator TFI, it is transferred into a yeast cell host with a deletion of the gene TPI. Methods of transformation of yeast cells are well known and extensively described in the literature.

Transformed yeast cells may be selected by growing on STN use standartniy Wednesday, such as JEPD (20 grams of glucose, 20 grams Bacto-peptone, 10 g of yeast extract per liter).

Selected transformants containing the appropriate expressing constructs, grown to stationary base on the standard complex environments, cells are removed by centrifugation or filtration, and the environment focus. Because FRPT is a hydrophobic protein with a high positive charge (Rainer and Ross, ibid: Antoniades, Proc, Natl. Acad. Sci. USA 78:7314, 1981; Deuel and others, J. Biol. Chem. 256:8896, 1981), recombinant FRPT has the same parameters, that can be used to highlight ion-exchange chromatography. For example, recombinant FRPT-CENTURIES in the broth for cultivation of yeast is separated from the cells and fractionary by cation-exchange chromatography.

FRPT, desorbed from the column is acidified and again fractionary by chromatography with reversed phase in serial mode. Facing a product containing PPT, acidified, passed through a strong cation exchange column and elute step gradient of NaCl. The eluate is collected, and FCT-CENTURY precipitated with (NH₄)₂SO₄.

The resulting material absoluut by gel filtration and separated according to 4HCO₃at pH 8-10. The eluate is collected, and FCT-CENTURY precipitated by addition of (NH₄)₂SO₄. The precipitate is dissolved in acetic acid and fractionary by gel filtration. The eluate absoluut and lyophilizers.

Expression of biologically active proteins of eukaryotic cells (other than yeast) can be made by the specialist through the use of corresponding signals in the expression/regulation.

The transcription promoters capable of driving expression of the sequences FCT, chosen for their ability to provide effective and/or regulated expression in a specific type of eukaryotic cells. The signal sequence capable of directing gene product on the way, secretion by cell, chosen by their function in the cell host. A selection of other useful regulatory signals, such as signals the end of transcription, polyadenylation signals, and enhancers of transcription, is obvious to the expert.

It is shown that recombinant FRPT has practical the same biological activity as native FRPT. The main biological activity FCT, in particular, the induction of chemotaxis and mitogenesis in feeling the Asti physiological role of this protein, including its role in tissue regeneration.

In preferred embodiments, the implementation of the present invention FCT contains almost no chain A. Because homodimeric isoforms FRPT (AA and BB) are homologous, but not identical, and the monomers have a molecular weight of 12.5 to 14.3 kDa (chain A) and 13-14 kDa (chain B), the purity of the product is confirmed by obtaining a single large band in polyacrylamide gel.

Compositions with FRPT that are used in some embodiments to implement the present invention, preferably are pure, i.e., in General, do not contain contaminants that could interfere with their therapeutic application. Particularly preferred preparations that do not contain toxic, antigenic, causing inflammation, pyrogenic or other harmful substances more than 90%, preferably more than 99% purity.

Mentioned herein vitamin D refers to biologically active forms of the compounds, and to their predecessors, which may iv vivo to turn into biologically active forms. Therefore, the vitamin D refers to, among others, vitamin D₂vitamin D₃and their active metabolites.

Vitamin D

You can also use other metabolisable forms and analogues of these compounds, including 1-hydroxy vitamin D₃, 25-hydroxy vitamin D₃, 24,25-hydroxy vitamin D₃, 1,25-dihydroxy vitamin D₃, 25-hydroxy vitamin D₃, 1,25-dihydroxy vitamin D₂, 24,25-dihydroxy vitamin D₂and other known compounds.

The preferred compound is vitamin D₃(9,10-SECO-cholesta-5,7,10[19] -triene-3-ol), and the most preferred is the biologically active form of this compound, 1,25-dihydroxycholecalciferol, commercially available compounds.

In one embodiment, the implementation of the present invention is intended to stimulate the growth of osteoblasts in vitro. Often osteoblasts isolated from primary cultures, i.e. cultures obtained directly from tissue containing a heterogeneous population of cell types.

Primary cultures of bone tissue may contain osteoclasts, fibroblasts, cells that are the precursors of osteoblasts and endothelial cells. Primary cultures can be obtained in several different ways. For example, to obtain primary cultures you can use the code codeistvia collagenase, collect and cultivate (Aubin and others , J. of Cell Biol, 92: 452-461, 1982). Alternative methods use svezhesobrannye pieces of bone, which affect the collagenase and which are then washed, cultured, allowing the cells to migrate out of the bone pieces, and use environment containing a small amount of Ca²⁺that is selective for the growth of osteoblasts after exposure to collagenase (Robey and others, Calif. Tiss. 35: 453-460, 1985).

The identification of osteoblasts in primary culture initially is phenotypic. Phenotypic markers of osteoblasts include the expression of alkaline phosphatase (Manduca etc., J. Bone Min. Res. 8: 281, 1993), synthesis of collagen type 1 (Kurihara and others, Endocrinol. 118 (3): 940-947, 1986), the production of osteocalcin (Yoon and others, ibid) and sensitivity to parathyroid hormone (Aubin and others, ibid.).

Osteoblasts are usually cultivated at a temperature of 37^oC in an atmosphere containing 5% CO₂in a medium comprising a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors normally found in the serum of the fetus cows. Experts know a lot of suitable culture media.

The present invention can also be used to steam is know osteogenic sarcoma person (ATCC N HTB 85); U-2 OS, primary osteogenic sarcoma of the person (ATCC N HTB 964); HOS TE 85), osteogenic sarcoma person (ATCC N CRZ 1543); MG-63, human osteosarcoma (ATCC N CRL 1427) and UMR 106, the rat osteosarcoma (ATCC N CRZ 1661).

In another embodiment of the present invention, the composition comprising FRPT and vitamin D, is used as a drug to enhance osteoblast-mediated bone formation. The methods of the present invention can be applied to accelerate the regeneration of bone defects and damage, such as closed, open and non-consolidation of fractures; to accelerate bone healing in plastic surgery; stimulation of bone ingrowth in racemethionine prosthetic joints and dental implants; for treatment of diseases and defects of the periodontium; to increase bone formation during osteogenesis by traction for the treatment of other lesions in the skeleton, which can be treated by stimulation of osteoblast activity, such as osteoporosis and arthritis.

The composition of the present invention can be entered locally or systemically. Local administration can be by injection into the site of the lesion or defect or inserting or attaching solid media into the injury site, reportergene places may be facilitated by applying compositions of the dosed release of the medicinal agent, such, for example, described in patent application U.S. N 07/871246 under consideration (corresponding WIPO publication WO 93/20859), which is fully incorporated herein by reference.

In brief, made of biodegradable polyester films, such as films of polylactic acid, polyglycolic acid, politican or copolymer film of polylactic acid/polyglycolic acid containing PPT, and cover their plates, pins, screws, etc. to attach to bone or to be placed in the bone.

Such compositions provide a gradual release of FCT in the area of the target. The preferred film of 50:50 PLA/ISC. These films can optionally include a carrier, such as albumin, detergent, polyoxyethylenesorbitan or glutamic acid.

In the case of the inclusion in the composition of the albumin ratio FRPT and albumin should be supported, as a rule, at the level of between 0.125 and 2.5 µg/mg In principle, as a carrier you can use any substance that facilitates the degradation of the polymer, forming pores in the film or reduces the adsorption of growth factors (growth factors) film.

Albumin is particularly preferred n is Rutana monooleate, polyoxyethylenesorbitan monolaurate, polyoxyethylenesorbitan monopalmitate, polyoxyethylenesorbitan the monostearate and polyoxyethylenesorbitan trioleate.

Films of this type are particularly suitable for coating prosthetic devices and surgical implants. These films, for example, can wrap the outer surface of the surgical screws, pins, plates, rods, etc., Implantable devices of this type are widely used in orthopedic surgery.

These tapes can also be used for coating materials, sealing bone, such as hydroxyapatite blocks, swabs from demineralized bone substance, collagen matrix, etc., Usually film or devices described herein, is applied to the bone at the site of fracture. This is usually implantation into the bone or attachment to the surface using standard surgical technique.

In addition to the copolymers, growth factors and media referred to above, the biodegradable film may include other active or inert components. Of particular interest are agents that stimulate tissue growth or infiltration. Particularly preferred agents that stimulate bone growth, such as mortgages and others, J. Biomed. Mat. Res. 23: 571-589, 1989).

In order to load the film FRPT and media use powders or liquid solutions. For example, freeze-dried FRPT and albumin can be uniformly dispersed on one surface of the film, after which the film roll. Different proteins can be applied in aqueous solution (for example, in physiological solution with phosphate buffer or 0.1 M acetic acid), which is left to dry.

Biodegradable materials containing PPT, can form various implants, according to techniques known in the art. It is possible to make rods, plates, blocks, bolts, etc. to attach or premises in the bone at the site of fracture or other defect.

Formulations of biodegradable materials are such that the number FRPT ranged from 0,0375 to 1.25 μg per mg of the copolymer.

Alternative methods for local delivery PPT and/or vitamin D include osmotic Minnesota ALZET (Alza Corp. Palo Alto, CA); materials with the gradual release matrix, such as described by Wang and others (WO 90/11366); dextranase granules with an electric charge, described Bao and others , (WO 92/03125); delivery systems based on collagen, as described Ksaz and systems on the basis of alginate as described Edelman and others (Biomaterials 12: 619-626, 1991).

Other known methods of local delivery with a gradual release of the active agent into the bone include metal prosthesis covered with a porous film that can be impregnated, and solid plastic rods incorporating drugs.

The delivery compositions of the present invention for resorptive actions can be facilitated by conjugation FRPT, vitamin D, or both together, with the target molecule. "Target molecule" refers to a molecule that binds to a tissue of interest.

For example, molecules, natselilsya on bone include tetracyclines; calcein; biophosphonate; poliasparaginovaya acid; polyglutamine acid; amino-photoshare; peptides associated with the phase of mineralization of bone, such as osteonectin, bone sialoprotein, osteopontin; antibodies to bone tissue; proteins containing domains, joining the minerals of bone, etc., See, for example, descriptions Bentz and others, (EP 0512844) and Murakami and others (EP 0341961).

The composition used according to the present invention may be in the form of pharmaceutically acceptable salts, especially the organic acid, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, etc.

Extension acid salt of the basic amino acid residues obtained by treatment of the peptide corresponding acid or inorganic substance by methods well known in the art; or the desired salt can be obtained by lyophilization of the appropriate acid. These salts can then be used for the preparation of injectable, local and aqueous solutions for local or systemic injections of the composition of the present invention.

Materials and methods for the production of drugs for injection and topical preparations can be found in Remington's Pharmaceutical Sciences, 17th ed., 1985, incorporated herein entirely by reference.

According to the present invention, "effective amount" of a composition refers to an amount that provides a statistically significant effect. When used to stimulate the growth of osteoblasts in vitro usually Gelateria cells, growing in the presence of PPT and in the absence of vitamin D. For therapeutic purposes "effective amount" is an amount of a composition containing FRPT and vitamin D, which is required to obtain a clinically meaningful increase the speed of healing of a fracture, reverse the development of bone loss in osteoporosis, stimulation and/or increase bone formation in non-consolidation of fractures and osteogenesis by extension, improvement and/or growth of bone on the inside of prosthetic devices, and repair of dental defects.

Such amounts will be partially depend on the specific condition of the person being treated and other factors evident to the specialist. For example, for osteoporosis increase bone formation is expressed in a statistically significant difference in bone mass between the control group and groups exposed to the treatment.

One can observe, for example, 10-20% or more increase bone mass. Other dimensions clinically significant increase healing may include, for example, tests on the gap and tension, tensile and torsion, bending up to 4 points, and other biomechanical tests well known in the art. General guidelines for sheeni.

The preferred dose of vitamin D for systemic treatment of a 70-kg patient is from about 1 ng to 1 mg, preferably from about 10 ng to 500 μg, and most preferably from about 20 ng to 1 μg. The preferred dose of vitamin D for local use it in combination with FRPT is from about 1 ng to 1 mg, preferably from about 5 ng to 500 ng, and most preferably from about 10 ng to 100 ng.

The preferred dose FCT for systemic treatment of a 70-kg patient is from about 1 PG to 10 mg, preferably from about 100 PG to 1 mg, and most preferably from about 10 ng to 100 μg.

The preferred dose FCT for local use in combination with vitamin D is from about 1 ng to 10 mg, preferably, from about 1 μg to 1 mg, and most preferably from about 10 μg to 500 μg. In General, compositions with a gradual release of the active agent should be prepared in such a way as to provide a dose in the region of the upper limit of the indicated doses. Doses should be adjusted to the speed of release. Liquid compositions usually contain from 1 to 1000 μg/ml FER from 1 ng/ml to 100 ng/ml, preferably from 5 ng/ml to 40 ng/ml and most preferably from 6 ng/ml to 20 ng/ml

It was found that the best Energeticheskiy effect FRPT and vitamin D is obtained by combining FCT and vitamin D in the ratio of 6:0.1 to 6: 1000 (FRPT: vitamin D), preferably from 6:1 to 6:500, more preferably from 6:10 to 6:100, most preferably about 6:40.

The compositions described above are entered into for periods of time varying from one day to 6 months or more, depending on the condition being treated. In General, the dose is administered with a frequency of 5 times a day to once a month, preferably once a day to once a month, until the actual completion of healing. Effective treatment will depend on such factors as the age and General condition of the patient, the disease, about which treatment is carried out, and a way of introduction. The schema definition of treatment is the responsibility of the specialist.

The present invention is illustrated by the following non-limiting examples.

Example 1. Was allocated a primary culture of osteoblasts pigs. The pigs (weighing approximately 30 pounds) of the femur was removed areas atoi temperature in phosphate-buffered saline (FSB) to remove blood.

The bone pieces were placed in a solution containing 1 mg/ml collagenase type 11 Clostridium histolyticum (Sigma Chemical Co., St. Louis, MO) in the environment of Dulbecco (DMEM) (Fred Hutchinson Cancer Research Center, Seattle WA), before use, sterilized by filtration. Slices were incubated in the medium with collagenase for 2 hours at 37^oC and Strahovanie. After incubation with collagenase medium was removed, and the bone pieces were placed in the FSB and washed up until in a wash medium had no cells.

Slices were placed in a minimal supportive environments Needle (GIBCO-BRL, Gaithersbutg, MD) containing 10% fetal serum cows (SEC) (Hyclone Logan, UT), 1 mm sodium pyruvate, and to 0.29 mg/ml L-glutamine, at low density and incubated at temperature 37^oC in an atmosphere containing 5% CO₂.

The medium was changed every 4-5 days. Migration of osteoblasts from bone pieces were observed within 7-10 days. Cells were used for studies immediately after the formation of the monolayer, and then destroyed.

Cells were tested for expression of alkaline phosphatase to confirm osteoplastische phenotype. Histochemical staining was carried out with a set for histochemical staining of AR 86R (Sigma, St. Louis, MO) according to the manufacturer's instructions. Razreshenie density of the cells corresponded to the increase in the number of cells alkaline phosphatase+.

Example 2. Primary osteoblasts pigs (prepared as described above) were tested against the relative mitogenic activity in the presence FRPT-BB, PPT-BB and z, 25-dihydroxycholecalciferol and 1, 25-dihydroxycholecalciferol separately. Mitogenic activity of merals of incorporation³H-thymidine by the method of Rainer and Ross (Meth. Enzymology 109: 749-773, 1985).

In short, inactive primary osteoblasts pigs was obtained by seeding cells at a density of 310⁴cells/ml in minimal supportive environments Needle (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal serum cows (SEC), in 96-well plates, and then gave them to grow for 3-4 days.

The medium was removed and to each well was added 180 mg DFC^*(table 1), containing 0.1% SEC. To half of the total number of holes was added 1, 25-dihydroxycholecalciferol (Biomol. Research Labs, Plymouth Meeting, PA) to a concentration of 10 nm. Cells were incubated for 3 days at a temperature of 37^oC in an atmosphere containing 5% CO₂.

As 1, 25-dihydroxycholecalciferol was dissolved in ethanol, the remaining wells were used as control containing an equivalent amount of ethanol, as in the wells, where it was added as a solvent of 1, 25-dihydroxycholecalciferol. Seth/ml.

Negative controls were set without adding FRPT-HV and +/-vitamin D. the plates were incubated for 20 hours at a temperature of 37^oC, then the medium was removed. To each well was added a hundred microlitres DFC^*containing 0.1% SEC and cu/ml³H-thymidine, and the plates were incubated for further 3 hours at a temperature of 37^oC.

The medium was aspirated and each well was added 150 l l trypsin. The plates were incubated at temperature 37^oC as long as the cells were not detached (at least 10 minutes). Internationally unrecognized breakaway cells collected on the filter power harvester LKB Wallac 1295-001 (LKB Wallec, Pharmacia, Gaithersbirg, MD).

The filters were dried by heating in a microwave oven for 10 minutes and counted on a scintillation counter LKB Betaplate 1250 (LKB Wallac) according to the manufacturer's instructions.

Table 1

250 ml of modified according to the method of Dulbecco eagle medium (DMEM)

250 ml of medium ham F12

to 0.29 mg/ml L-glutamine

1 mm sodium pyruvate

25 mm HEPES (Sigma, St. Louis, MO)

10 μg/ml fetuin^**(Aldrich, Milwaukee, W1)

50 µg/ml insulin (GIB CO-BRL)

3 ng/ml selenium (Aldrick, Milwaukee, W1)

20 μg/ml transferrin (JRH, Lenexa, KS)

The results presented in Fig. 1, show that FRPT-BB stimulates the absorption Tim is 6-10 ng/ml FRPT together with 1, 25-dihydroxycholecalciferol has doubled the maximum uptake of thymidine. In the presence of only one of 1, 25-dihydroxycholecalciferol stimulation of growth was observed.

Example 3. To determine whether the simultaneous presence of 1, 25-dihydroxycholecalciferol and FCT to get sinergeticheskogo impact on growth, undertook a study of mitogenome as described in example 2, with the following modifications: a) the final concentration of 1, 25-dehydrocholesterol was 100 nm; (b) preparing an additional number of wells no 1, 25-dihydroxycholecalciferol in the presence FRPT by removing the medium used for pre-treatment of cells 1, 25-dihydroxycholecalciferol, and add fresh medium containing only the relevant breeding FRPT.

The results, shown in Fig. 2, indicate that FRPT and 1, 25 - dihydroxycholecalciferol must be present simultaneously to obtain two-fold increase of mitogenesis.

Example 4. To determine whether the effect of 1, 25-dihydroxycholecalciferol and FRPT-induced mitogenic primary osteoblasts pig dose-dependent, took ili ranged from 0.01 nm to 100 nm and (b) FRPT was present in only one con centration, namely to 6.2 ng/ml

The results presented in Fig. 3, indicate that Energeticheskiy effect PPT and 1, 25-dihydroxycholecalciferol changed depending on the concentration of 1, 25-dihydroxycholecalciferol.

Example 5. To determine whether the effects observed with vitamin D-specific FRPT, vitamin D was tested in combination with basic fibroblast growth factor (FGF).

Recombinant basic FGF man (BPL-GIBCO) was tested at concentrations of 0.4 ng/ml to 100 ng/ml in the test for mitogenome as described in example 2. The results presented in Fig. 4, shows no effect when using basic FGF and 1, 25-dihydroxycholecalciferol.

Example 6. Undertook a study to determine whether fibroblasts, potentially contaminating the type of cells in primary cultures derived from bone, to call Energeticheskiy effect observed using PPT and 1, 25-dihydroxycholecalciferol.

The research was carried out as described in example 2, using a murine fibroblast cell line Swiss ZTZ (ATCC N CCL92), except that the fibroblasts were placed in DMEM containing 1 is 1, 25-dihydroxycholecalciferol had no effect on FRPT-induced mitogenic fibroblasts.

Example 7. To confirm that Energeticheskiy the effect of 1, 25-dihydroxycholecalciferol and FRPT observed in osteoblasts, used the stable cell line of mouse osteoblasts, MSSTS (Suda and others, L. Cell Biol. 96:191-198, 1983).

The study was performed mainly as described in example 2, except that the cells were placed in a medium-MEM (GIBCO-BRL) containing 10% of the SEC, with a density of 310⁴cells / ml In contrast to fibroblasts Swiss ZTZ and primary osteoblasts pigs, 1, 25-dihydroxycholecalciferol inhibited cell growth MSSTS.

Thus, data were analyzed from the point of view of the multiplicity of induction of thymidine uptake. The multiplicity of induction is defined as the ratio of the number of pulses per minute ³H-thymidine incorporated in the presence of FRPT, to that in the absence of FCT. The results, shown in Fig. 6, indicate that FRPT induces a large multiplicity of induction in the presence of 1, 25-dihydroxycholecalciferol than in the absence of this vitamin.

Example 8. To check the response of osteoblasts in primary cultures of pig bones ). Investigated the absorption of³H-thymidine and the expression of a marker of osteoblasts, alkaline phosphatase, primary bone cells pigs.

Primary bone cells pigs were sown on plates and processed as described above, except that: (a) the cells were placed on glass slides with cameras Lab-tek N 177445 (ALL World Scientific, Seattle, WA); (b) used only one concentration of 10 nm 1, 25-dihydroxycholecalciferol and (C) used only one concentration of 100 ng/ml PPT CENTURIES.

After incorporation³H-thymidine slides three times washed FSB and stained for alkaline phosphatase as previously described. After staining, the slides were dried in air and covered NTB3 emulsion (Kodak, Rochester NY). The emulsion was dried in air and glass was kept for one week at a temperature of 4^oC.

Then the glass showed 5 minutes in developer D-19 (Kodak) at room temperature, washed in water and fixed in rapid fixer (Kodak) for 5 minutes. Then made contractarian glasses methylene blue (Sigma) by dilution of the initial solution 1:100 in water and applied to cells for one minute.

Cells expressing alkaline phosphatase (apase+), identified by pink crebra around nuclei. In the absence of FRPT-BB only 2-4% of the cells incorporated³H-thymidine, and the amount of addition of 1, 25-dihydroxycholecalciferol had no effect.

Adding FRPT-BB increased the percentage of cells incorporeality³H-thymidine, approximately ten times. As alkaline phosphatase+ and alkaline phosphatase-cells were sensitive to PPT CENTURIES. I calculated the percentage of alkaline phosphatase+cells that incorporated ³H-thymidine in response to exposure FRPT, in the presence and absence of 1, 25-dihydroxycholecalciferol. The same calculations were also performed for alkaline phosphatase-cells.

The results are summarized in table 2. The addition of 1, 25-dihydroxycholecalciferol increased the number of alkaline phosphatase+cells that incorporated³H-thymidine, almost twice, while cells alkaline phosphatase - showed a more moderate increase. These results suggest that osteoblasts in culture, the results of the expression of alkaline phosphatase, demonstrate Energeticheskiy response to 1, 25-dihydroxycholecalciferol and FRPT CENTURIES.

Alkaline phosphatase-cells, demonstrating Energeticheskiy response to 1, 25-dihydroxycholecalciferol and FRPT-BB may be or may not be the osteoblasts, because the osteoblasts do not Express alkaline phosphatase on any stage of their differentsirovki in vitro experiment to cells of the cranial vault, while FRPT in the same experiment demonstrates a moderate effect on bone resorption. Because bone resorption is a contraindication to stimulate bone formation, a study was conducted to assess the interaction FRPT and vitamin D in bone resorption.

In 4-day-old mice CD-1 (Charles River Laboratories, San Diego, CA) took the cranial vault, which included the parietal part of the sagittal suture. The bones were placed in a 6-hole Petri dishes (American Science Products, McGraw Park, IL) with 1 ml of growth medium (DMEM, to 0.29 mg/ml L-glutamine, 1 mm sodium pyruvate and 15% inactivated by heating with horse serum (IPLA) and incubated at temperature 37^oC in atmosphere containing 5% CO₂within 24 hours when lightly shaken.

After incubation the medium was removed from the wells and replaced it in 1.5 ml growth medium containing either 200 ng/ml PPT-BB or 10^-8M 1, 25-dihydroxycholecalciferol, or FRPT and vitamin D. In each group had five bones, and groups were incubated with shaking for 72 hours at a temperature of 37^oC in atmosphere containing 5% CO₂.

After incubation the medium was removed from the wells and determined the levels of Ca²⁺using analyzer total calcium NOVA-7 (Nova Biomedical, Waltham, MA is the ed after 24-hour incubation, to make sure that the bones are not destroyed during the collection process. Shattered bones release into the environment of large amounts of calcium and therefore in the final analysis they are not used.

The results, shown in Fig. 7, indicate that as PPT and 1, 25-dihydroxycholecalciferol added to the bones separately stimulated bone resorption. However, while adding levels of release of Ca²⁺due ratably bones observed with vitamin D, decreased, indicating an inhibiting effect FRPT.

Example 10. To test the ability FRPT and vitamin D Energetichesky to increase bone ingrowth into porous implants of hydroxyapatite produced cylindrical implants with a length of 25 mm and a nominal diameter of 4 mm (Interpore, Iwine, CA) from a hydroxyapatite of mateiral with pore size 190 - 230 microns.

These cylinders contain clippings of 10 mm diameter and 3 mm Implants were loaded FRPT by immersion in a solution FRPT, resulting in a received dose of 0.5 - 50 ng of the implant. Vitamin D was administered locally using Micronase ALZET (Alza Corp.), resulting in a received dose of vitamin D 10 - 100 ng of the implant. The dose ratio FRPT and vitamin D was 1 : 6.

Intramedullary implants were inserted using the distal access (Anderson and others , J Orthop Res. 10 : 588 - 595, 1992). Made of 2.5 cm lateral parapatellar incision, getting access to korennomu joint along the lateral edge of the tendon of the patella. The patella was displaced medially with outstretched limbs.

The edge of the 4.0 mm drill bit was placed between the femoral mewelcome directly proximally. A hole was drilled through the articular cartilage and metamizol bone for fixation intramedullary position. The implant was inserted and pushed proximally to the end position. For closing the distal inlet of the used surgical bone wax. The wound was closed in layers 4 - 0 absorbable suture material and brackets in stainless steel.

The research produced histological and biomechanical. Produced cross-sections of the femur length 3.5 mm, containing implants, so that it was possible to study the following is egusa to the endosteal cortex srednespetsialnoe part of the thigh.

For each plot, the sample was cut into two parts, one of which was used for histological analysis and the other for biomechanical research. Each experimental plot was compared with the control plot, loaded only substance carrier of the opposite thigh.

Histological samples were fixed in formalin phosphate buffer and obezvozhivani increasing concentrations of ethanol: 95% ethanol for 25 hours, then three changes of 100% ethanol for 24 hours each. After the last treatment, 100% ethanol, the samples were prosvetlili two changes of xylene for 24 hours each.

Did the shading fabric plastic. Samples were prepared for cross sections in plastic rods and embedded in methacrylate at room temperature in a vacuum desiccator under nitrogen atmosphere, as described by Bain and others (Stain Technol. 65 (4): 159, 1990).

From each sample were prepared slices with a thickness of 150 m using a low-speed diamond disk knife (Struwrs Accutom-2, Torrance, CA). Thick slices then cut manually on a more subtle, approximately 30 μm between two glass coated abrasive film 1200. Histological slices were placed glass with Measurement of bone formation were determined using morphometric program for bone Bioquant (Biometrics, Inc., Nashville, TN), which is coupled through a camera luceda with light/epifluorescent microscope (Olympus BH-2 (Scientific Instruments, Inc., Redmond, WA).

Total bone ingrowth into the implant was measured under fluorescent microscope at 40-fold magnification. Settings mineral opposition and bone formation was determined by fluorochromes marks at 100-fold increase in vivo. For calculating the speed of mineral opposition average distance between marks on the inner surface of the implant was divided by the time interval between labels.

The overall rate of bone formation was determined by the stroke of the contours of the area of newly formed bone (i.e., bone, limited fluorochrome label) and dividing the total area of newly formed bone on the time interval between labels.

Ingrowth of bone were evaluated by mechanical means, using a test push. The samples were placed in an Instron testing device and to the center of the implant has applied a constant force of 0.5 mm/sec. The force required to eject the implant were determined using biomechanical testing techniques, well known in the art, as described Knowler and others (Biomaterials, 13 (8): 491 - 496, 1992).

Example 11. Determined the ability FRPT-BB and vitamins 5-week-old mice, using FRPT, vitamin D or a combination FRPT and vitamin D. For experiments on thighs dose FRPT ranged from 2 to 200 ng per day. Doses of vitamin D ranged from 20 ng to 2 μg per day.

These compounds were injected with the periosteum of the middle part of the front surface of the left femur newborn rats (2 to 3 days of age) for ten consecutive days. The opposite thigh served as a control substance carrier. The bones were marked for histomorphometry by intraperitoneally injection tetracycline (10 mg/kg) on day 5 and intraperitoneally injection calcein (10 mg/kg) on 17 and 22 days.

In each experimental group were used for eight rats. On the 24th day of the animals were killed, removed both the femur and processed for histomorphometry nedeklarirovanny bones.

The study of bone formation in the vault of the skull was performed by injection FRPT, vitamin D, or a combination FRPT and vitamin D subcutaneously in the periosteal tissue covering the sagittal suture. The injection was done once a day for 10 days. Dose FRPT ranged from 2 to 200 ng per day. The dose of vitamin D ranged from 20 ng to 2 μg per day.

Newly formed bone was marked for measurement by intrapersonal on the 28th day collected the arches of the skull and processed for histological evaluation.

Although the present invention has been described with some detail through illustrations and examples for purposes of clarity of understanding, it is obvious that certain changes and modifications may be made within the scope of the attached claims.

1. Way to stimulate the growth of osteoblasts, characterized in that it includes the announcement of these cells a composition comprising a growth factor derived from platelets (FCT), in combination with vitamin D in sufficient quantity to stimulate the growth of osteoblasts.

2. The method according to p. 1, characterized in that the composition contains almost no chain AND FCT.

3. The method according to p. 1, characterized in that the composition comprises recombinant FRPT CENTURIES.

4. The method according to p. 1, characterized in that the osteoblasts grown in vitro.

5. The method according to p. 4, characterized in that the concentration FRPT in the specified composition is in the range from 1 to 100 ng/ml

6. The method according to p. 4, characterized in that the concentration FRPT in the specified composition is in the range of 5 to 40 ng/ml

7. The method according to p. 1, characterized in that colocalize fact, the ratio FRPT : vitamin D in the specified composition is in the range of 6:10 - 6:100.

9. Method of stimulating bone growth in a patient, characterized in that the specified patient is administered an effective amount of a composition comprising FRPT and vitamin D.

10. The method according to p. 9, wherein the vitamin D is 9,10-SECO-cholesta-5,7,10(19)-triene-3-ol or 1,25-dihydroxycholecalciferol.

11. The method according to p. 9, characterized in that the composition is delivered locally to a wound or defect of the bone.

12. The method according to p. 9, characterized in that the ratio FRPT : vitamin D in the specified composition is in the range 6:1 - 6:500.

13. The method according to p. 9, characterized in that the ratio FRPT : vitamin D in the specified composition is in the range of 6:10 - 6:100.

14. Way to stimulate the growth of osteoblasts, characterized in that exercise culturing these cells in the presence of an effective amount of a composition comprising FRPT and vitamin D, with the specified composition contains almost no A-chain FRPT.

15. Pharmaceutical composition for stimulating the growth of osteoblasts, characterized in that it comprises a growth factor derived from platelets FRPT, and vitamin D the vitamin D is 9,10-SECO-cholesta-5,7,10(19)-triene-3-ol or 1,25-dihydroxycholecalciferol.

17. The pharmaceutical composition according to p. 16, characterized in that the concentration FRPT in these compositions is in the range of 10 to 500 μg/ml.

18. The pharmaceutical composition according to p. 16, characterized in that the ratio FRPT : vitamin D is in the range 6:1 - 6:500.

19. The pharmaceutical composition according to p. 16, characterized in that the ratio FRPT : vitamin D is in the range of 6:10 - 6:100.

20. The pharmaceutical composition according to p. 15, characterized in that the composition contains almost no A-chain FRPT.

21. The pharmaceutical composition according to p. 15, characterized in that it is useful for stimulating the growth of bone.