Soft tissue filler composition for injection and method for preparing thereof

FIELD: medicine.

SUBSTANCE: there is offered a method for preparing a soft tissue filler composition for injection to relief or treat skin damages caused by mechanical or physiological reasons, including the stages as follows: 1) digestion of autologous dermal tissue recovered from autologous skin of a patient by processing with pancreatine/EDTA solution, and cell isolation; 2) cultivation and proliferation of the recovered dermal cells by serum-free cultivation in vitro in a medium containing a growth factor and activation factor for preparing autologous cellular culture material of dermal nature containing dermal fibroblastic stem cells, dermal fibroblastic "transitional" dividing cells, dermal fibroblasts and collagen; 3) centrifugation of autologous cellular culture material of dermal nature for separation of autologous cellular sediment of dermal nature; and 4) slurrying of autologous cellular culture material of dermal nature in glucose solution for injection or any solution for injection to prepare suspension for injection. There is offered a composition prepared by specified method which contains 1×107 to 8×107 cells/ml of autologous cells of dermal origins and 10 to 100 mg/ml of collagen as an effective ingredient.

EFFECT: invention provides therapeutic effect over a short period of time and maintains it for a long time.

13 cl, 7 ex, 8 dwg

 

The SCOPE of the INVENTION

The present invention relates to the composition of the filler for soft tissue injection, which is obtained by digestion of autologous dermal tissue, selected from the own patient's skin, the division into separate cells, culturein vitroand the proliferation of divided cells in serum-free medium. The present invention additionally relates to a method for obtaining the above-mentioned composition.

BACKGROUND of the INVENTION

Ordinary methods of filling used for healing skin defects are divided into two groups, i.e. dermal filling and subcutaneous filling. All components of the filler used in the above methods, as a rule, contain synthetic biological materials or heterologous proteins, instead of completely consist of autologous activators tissue of origin. Paraffin was first used as a material for filling in the 19th century. However, it has caused many side effects, and the effect of filling with it proved unsatisfactory. Was later developed method with the use of exogenous proteins as a material for filling. In particular, it is found that the means for filling using bovine collagen leads to rapid onset of therapeutic effect. However, PR the problem was when a single implantation, the collagen is gradually absorbed back into the skin after 3-6 months. Receipt and use of bovine collagen are described in U.S. patent No. 3949073, 4424208 and 4488911. Bovine collagen entered the market under the trade name of Zyderm I, II and III, in which the collagen is in the range from 35 mg/ml and 65 mg/ml, However, discovered that Sidearm does not give satisfactory results, since antibodies to bovine collagen are formed in 90% of patients after treatment with Ziderman (and have from 1 to 3% of them see severe allergies). To resolve these problems developed injectable collagen, cross-linked by glutaraldehyde, and it is sold under the trade name Siplast. This protein does not cause any allergic reactions, but its viscosity is too high to obtain good therapeutic effects (U.S. patent No. 4582640 and 4642117).

Furthermore, in U.S. patent No. 4969912 and 5332802 described method using autologous injectable human collagen to prevent immune reactions due to the use of bovine collagen, which is available under the trade name AUTOLOGIN. However, its therapeutic effect persists only for a few months. Fibril created on the basis of porcine collagen. In fact, to obtain Fibril combined three components i.e. pork gelatin, aminocaproic acid, and plasma from the subject to be treated. Fibril has desirable effects in some patients. However, most of the treated patients are prone to the formation of swelling at the injection site, and you cannot save its therapeutic effects in a long time.

BOTOX, which is approved by the FDA in April 2002 to conduct clinical trials, is a solution for injection to temporarily remove wrinkles. BOTOX has therapeutic effects in removing wrinkles by anestesiologia facial muscles and has several advantages, such as ease of use and immediate onset of therapeutic effects. However, numerous problems are that the expression of the patient's face becomes frozen after treatment, the duration of therapeutic effect is too small. Moreover, there is a risk of paralysis of the facial nerve.

Artecoll is a complex artificially synthesized particles and bovine collagen, which is successfully used in Canada, Mexico and Europe for several years. Artecoll has a prolonged therapeutic effects through the formation of autologous collagen by stimulating the local der the s. However, it also creates problems because the subject after treatment, you may feel the presence of particles, and in the treatment site may be swelling.

As mentioned above, these heterologous materials for filling cause side effects, such as the possibility of allergic reactions and rejection reactions, and the limited duration of therapeutic effects.

In U.S. patent No. 5591444, 5665372 and 5660850 belonging Isolagen Technologies, Inc., described is a method of treatment of skin defects and soft tissue using autologous fibroblasts of the skin. In particular, these patents describes a method of injection by means of: receiving autologous dermal tissue from a subject to treatment of a subject; culturing thein vitrothis tissue in a medium containing bovine serum; and premises of cultured autologous fibroblasts in ringer's solution. This injection does not cause any allergic reactions and reactions heterologous exclusion, but it is not free from security problems due to the use of serum of animal origin. Moreover, as the ringer's solution is not conducive to long-term maintenance of activity of active ingredient specified injection must be performed within a short period of time after receiving it, which causes inconvenience in practical terms the ski use.

In Beijing Yiling Bioengineering Co., Ltd, a method of treating wrinkles and scars with the use of injections, you get: selection autologous skin from being treated of the subject; its cultivation and proliferation of thein vitro; and suspendirovanie cultivated cells in glucose solution for injection (ringer's solution) or the solution, it is appropriate (Chinese patent No. 03155833.X).

Effective ingredient injection, described in the Chinese patent No. 03155833.X obtained by culturing autologous sample skin in partial addition of serum of animal origin. However, this method has several disadvantages. First, because the collagen content in the specified injection, which is responsible for the immediate onset of therapeutic effects, so small, the effects of the disappearance of wrinkles and scars come late from the point of view of effort, thus, taking from 4 to 6 months to obtain a satisfactory therapeutic effects. Moreover, subjected to treatment for a subject to worry about security in connection with the use of serum of animal origin as a component of culture medium.

Moreover, in international publication No. WO 2003/094837 described composition containing autologous, passaged of fibral the texts and muscle cells (not necessarily biodegradable non-cellular components of the matrix/biodegradable non-cellular fillers (such as collagen)). It further describes how to restore tissues that have undergone degeneration in a subject due to illness, disorder or defect. Further, in international publication No. WO 2004/048557 described a composition comprising: (i) autologous undifferentiated mesenchymal cells (UMC) and autologous fibroblasts; (ii) autologous UMC; or (iii) autologous fibroblasts and autologous keratinocytes as an effective ingredient. It also describes the method of production thereof and method of tissue repair using them. However, all the compositions described in these patents, a significant proportion of autologous muscle cells, autologous UMC or autologous keratinocytes, which are completely different from the composition of the filler for soft tissue containing dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells and dermal fibroblasts, described below.

Thus, the authors of the present invention have tried to overcome the problems of the prior art and has developed an autologous cell culture material from the dermis, containing dermal fibroblast stem cells, dermal fibroblast who's transitional dividing cells and dermal fibroblasts obtained in serum-free cultivationin vitrofrom autologous skin tissue together with a large number of collagen as an integrated filler. Moreover, it is confirmed that the thus obtained complex filler has therapeutic effects on removal and recovery of wrinkles and scars in a short period of time and stores these therapeutic effects for a long time.

DESCRIPTION

Technical problem

Accordingly, the principal objective of the present invention is to obtain a new composition of filler for soft tissue injection, where there is no problem from a security perspective because it does not use serum of animal origin. It is also directed to a composition which is very effective to remove wrinkles and scars by immediate onset and long-term maintenance of therapeutic effects, and which can be used effectively to improve the color and elasticity of the skin. The present invention also relates to a method for obtaining the above-mentioned composition.

Technical solution

According to one aspect of the present invention presents a method of obtaining a composition filler for soft tissue injection, which involves the following stages:

1) digestion autologinuser biopsies, selected from the own patient's skin, and separation into individual cells;

2) the cultivation and proliferation of selected dermal cells by serum-free cultivationin vitroto obtain autologous cell culture material from the dermis, containing dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells, dermal fibroblasts and collagen;

3) centrifugation of autologous cell culture material from the dermis for the Department of autologous cellular sediment dermal origin; and

4) suspension of autologous cellular sediment dermal origin in glucose solution for injection or arbitrary solution for injection to obtain a suspension for injection.

According to another aspect of the present invention includes a composition filler for soft tissue augmentation for injections received by the specified method.

Further, the present invention is described in more detail.

At the beginning of autologous dermal sampling used in stage 1), obtained by disinfection with an alcohol selected area of skin being treated patient, local anesthesia, cutting him epidermis and dermis in size from 1 to 30 mm2and then storing them in metachemistry for fabrics. The skin tissue, suitable for serum-free cultivation and proliferation ofin vitroautologous dermal cells at this stage may include any of the skin, epidermis and dermis derived from the back of the ear, eyebrows, lower part of the eyes and other areas. The thus obtained sample of autologous dermal tissue is subjected to the procedure of digestion of tissue and cell isolation. In particular, a sample of autologous dermal tissue can be placed in a Cup for cultivation and subjected to digestion of tissue treated with a solution of Pancreatin/EDTA. Further digestion of the tissue can be stopped by adding a solution of DMEM containing 10% FBS. Digested tissue in the reaction solution can be broken down and divided into individual cells by the use of careful re-suction using the suction pipette and centrifuged to remove supernatant, thereby obtaining cellular precipitate.

In stage 2) cells, obtained in stage 1), cultivatedin vitroin serum-free medium according to the conventional method in this field, such as the method of cultivation digested tissue or method of cultivation of a fragment of tissue.

Method of culturing cells according to the present invention differs from the methods of the prior art from the standpoint of the use of serum-free RL is turalei environment. In particular, the method of culturing cells according to the present invention is different: the use of serum-free culture medium with the addition of growth factors (e.g. epidermal growth factor and dermal growth factor) and activation factors (e.g., cortical hormone and pituitary extract the bull) instead of adding serum of animal origin, such as bovine serum, to the basic medium; culturing the cells in serum-free culture medium for 6 to 10 weeks; and the induction of cell proliferation and satisfactory secretion of collagen. Confirmed that the activity and morphology of cells cultured in serum-free culture medium containing growth factors and activation factors instead serum of animal origin according to the method of the present invention, are normal. In particular, the thus obtained culture solution contains a large amount of collagen, and autologous cell culture material from the dermis, containing dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells and dermal fibroblasts as an effective ingredient. In the case of receiving a filler using a culture solution of contents number is of the agents in the filler can vary from 10 to 100 mg/ml

The basal medium suitable for the present invention can be obtained using conventional media components, well known to ordinary skilled in the art, for example, "Experimental Cell Protocols" (2001, Science Press, Chief editor: D.L. Speycutter, Translation: Peitang Huang).

Serum-free culture medium, growth factors and activation factors used in the present invention, can include all products that are commercially available in this area. Growth factors that can be added to serum-free culture medium of the present invention, may include epidermal growth factor (EGF), dermal growth factor, basic fibroblast growth factor (bFGF), etc. These factors can be used alone or in the form of their mixtures. The concentration of growth factors in serum-free culture medium may preferably range from 0.1 to 5 ng/ml or more, preferably from 0.5 to 2 ng/ml. moreover, the activation of the factors that can be added to serum-free culture medium of the present invention, may include cortical hormone, pituitary extract the bull (BPE), insulin, hydrocortisone, etc. These factors can be used individually or in the form of their mixtures. The concentration of activation factors in serum-free culture medium may preferably be varied from 0.1 d is 1% or more, preferably from 0.2 to 0.5%.

Dermal cells isolated from autologous dermal tissue, inoculant in serum-free culture medium and cultured in an incubator at 37°C in 5% CO2within 6 to 10 weeks. During this time, it is preferable to replace the medium with fresh at intervals of 3 days.

At stage 3) solution with a culture of autologous cells from the dermis, obtained above, centrifuged at temperatures ranging from 4 to 32°C, at speeds ranging from 800 to 1200 rpm to remove supernatant, thereby obtaining only autologous cellular sediment dermal origin containing proliferating dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells, dermal fibroblasts and secretory their collagen.

Cultivated and proliferating dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells and dermal fibroblasts, and the secretory their collagen according to the present invention can be confirmed as follows.

Dermal fibroblast stem cells: they can be analyzed according to the method with antibodies to BrDU (5-bromosuccinimide), described in literature

Normally what I morphology of the dermal fibroblastic stem cells observed in the optical microscope at magnification 100× is a refined, elongated fusiform shape. They are painted in dark brown color according to the method with antibodies to BrdU (see figure 1).

Dermal fibroblasts transitional dividing cells: normal morphology of the dermal fibroblastic transient dividing cells observed in the optical microscope with magnification 100x is a subtle and small, elongated spindle-like shape, similar to the dermal fibroblast stem cells. They are painted in brown color according to the method with antibodies to BrdU (see figure 2).

Dermal fibroblasts: normal morphology of the dermal fibroblasts when observed through an optical microscope at magnification 100× is an elongated fusiform shape, with many processes (see figure 3).

Collagen: the concentration of collagen can be measured according to the method of measurement of collagen described in the literature (Ministry of Health P.R.China, Pharmacopoeia Committee, "Chinese Pharmacopoeia" (2ndEd.), Beijing Chemical Industry Press, 2000).

Stage 4) consists of obtaining the composition of the filler for soft tissue for injection suspendirovanie autologous cellular sediment dermal origin containing outlined above effective ingredients, glucose solution for injection at a concentration of 5% or the total solution for injection. When the thus obtained suspension can give is to include suitable additional ingredients. The composition of the filler for soft tissue according to the present invention as an effective ingredient contains cultured dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells and dermal fibroblasts isolated from native tissue of the patient together with secretively their collagen. The final concentration of effective cells in the injection preferably ranges from 1×107up to 8×107cells/ml, and the collagen content preferably ranges from 10 to 100 mg/ml, more preferably from 20 to 60 mg/ml

The composition of the filler for soft tissue according to the present invention can be obtained in the composition of the injectable products intended for human skin, which can be obtained according to the conventional method, well known to ordinary skilled in the art. For example, the composition of the present invention may be in the form of a solution, viscous solution, suspension or gel. This composition may further contain suitable excipients adapted for injection into the skin. Suitable excipients must be well-tolerated, stable and get in consistency, which provides an easy and pleasant to use. In this work, suitable examples of excipients include as non-limiting examples fo Paty buffer saline, bacteriostatic saline, propylene glycol, starch, sucrose and sorbitol.

Further, the composition of the filler for soft tissue according to the present invention may optionally contain an additional substance, such as an inert and pharmaceutically acceptable carrier or diluent, excipient, thickener, emulsifying agent, preservative, and their mixture. Suitable examples of the above-mentioned additional substances typically include substances that are commonly used in pharmaceutical preparations and preparations for skin care. More specifically, such examples are inert and pharmaceutically acceptable carrier or diluent include as non-limiting examples of the salt solution and purified water. Suitable thickeners include copolymers of acrylamide, carbomer, hydroxyethyl cellulose, hydroxypropylcellulose, polyacrylic acid, polymethacrylic acid and polyvinyl alcohol, but certainly not limited to.

Suitable emulsifying tools include Caprylic/capric triglyceride, ceteareth-7, cetyl alcohol, cetilistat, isostearate-11 and isostearate sodium, but are not limited to them. Preservatives give the compositions of the present invention is resistant to microbial exposure and toxicity towards microorganisms. Suitable examples include Speer is s, any parabens, diazolidinylurea, DMDMH-as Phenoxyethanol and adpropinquabant, but are not limited to them. Examples of the above-mentioned additional substances other than those listed, can also be used in embodiments of this invention, as will be taken into account as an ordinary specialist in the field.

The composition of the filler for soft tissue injection, obtained according to the method according to the present invention, contains large amounts of collagen, which operates by removing wrinkles and scars directly in the injection area. Moreover, other effective ingredients in the composition of the filler for soft tissue for injection of the present invention play an important role in the removal of wrinkles and scars at the same time functioning to improve the local environment in the skin through continuous production of collagen. It gives youth and beauty being treated subject.

Accordingly, the composition of the filler for soft tissue for injection of the present invention can be effectively used not only to cure all types of wrinkles and scars on the face and neck, stretch marks after pregnancy, wrinkles on the back surface of the hand, etc. but also to enhance the thickness, elasticity and skin texture.

As the composition of the filler for soft tissue injection by us is oedema invention contains autologous cells from the dermis as an effective ingredient, it does not cause any allergic reactions and reactions heterologous exclusion. Moreover, since the present invention does not use serum of animal origin in the course of obtaining the composition of the filler for soft tissue injection, there is no possibility of invoking infectious diseases or side effects. Accordingly, the composition of the filler for soft tissue for injection according to the present invention can directly call good cosmetics and therapeutic effects, to provide a natural expression of the face being treated subject after injection and save pronounced therapeutic effects for a long time.

The beneficial effects

The composition of the filler for soft tissue for injection according to the present invention has a prolonged therapeutic effects without causing any immune response and side effect due to the use of autologous dermal cells. Moreover, as it eliminates the risk of using the serum of animal origin by means of serum-free cultivationin vitroand directly causes therapeutic effects by producing significant amounts of collagen, the composition of the filler for soft tissue for injection of the present invention can effectivement to improve the color and elasticity of the skin, as well as smoothing and removal of wrinkles and scars.

Description of the drawings

The above and other objectives and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings, in which, respectively, represented by:

Figure 1 is a photograph (× 100)observed in the optical microscope, which presents dermal fibroblast stem cells, proliferating in serum-free cultivationin vitrofrom autologous dermal cells according to the method according to the present invention;

Figure 2 is a photograph (×100)observed in the optical microscope, which presents dermal fibroblasts transitional dividing cells, proliferating in serum-free cultivationin vitrofrom autologous dermal cells according to the method according to the present invention;

Figure 3 is a photograph (×100)observed in the optical microscope, which presents dermal fibroblasts, proliferating in serum-free cultivationin vitrofrom autologous dermal cells according to the method according to the present invention;

Figure 4 represents the result of the comparison of the morphology of autologous cell culture material dermal origin is placed (B), obtained according to the method of the present invention, with the morphology of cells derived from the adipose tissue (A), using phase-contrast microscope;

Figure 5 represents the result of immunofluorescence analysis that compare the expression profiles of nestin and fibronectin in autologous cell culture material from the dermis (A)obtained according to the method according to the present invention, the expression profiles of the cells derived from adipose tissue (B), where nestin detects a red spot at 594 nm, fibronectin detects a green spot at 488 nm, and cell nuclei detected as blue spots staining of DAPI;

6 represents the result of immunofluorescence analysis that compare the expression profiles of nestin and vimentin in autologous cell culture material from the dermis (A)obtained according to the method according to the present invention, the expression profiles of the cells derived from adipose tissue (B), where nestin detects a red spot at 594 nm, vimentin detected as green spots at 488 nm, and cell nuclei detected as blue spots staining of DAPI;

7 represents the result of immunofluorescence analysis that compare the expression profiles of nest is on and FSP1 in autologous cell culture material from the dermis (A), obtained according to the method according to the present invention, the expression profiles of the cells derived from adipose tissue (B), where nestin detects a red spot at 594 nm, FSP1 detects a green spot at 488 nm, and cell nuclei detected as blue spots when DAPI staining; and

Fig is the result of polymerase chain reaction with reverse transcription (RT-PCR), which analyze the expression profile of the marker proteins predecessor of nerve cells in autologous cell culture material of dermal origin, obtained according to the method of the present invention.

The best option

The present invention will now be described in more detail with reference to the following examples which are not intended to limit the scope of the present invention.

Example 1. A composition filler for soft tissue

1-1. The way to obtain

After the rear part of the ear disinfected with 70% ethanol, and its local surface area was anestesiologi 10% lidocaine and epinephrine (1:100000), it removed the tissue of the epidermis and dermis size 4 mm2and it was stored in the mother solution (mother solution for cells DMEM, Hyclone, USA).

The tissue sample was placed in a 35 mm Cup for cultivation, it was added 2 ml of 0.05% pancreati the a/EDTA, and then a Cup for cultivation was placed in CO2-incubator at 37°C for 10 minutes for the induction of digestion cells. The digestion reaction was stopped by adding 5 ml of DMEM with addition of 10% FBS.

Digested tissue was crushed to pieces and destroyed through careful suction using the suction pipette, to thereby divide it into separate cells. Separated dermal cells were centrifuged at 25°C, 1000 rpm for 5 minutes to remove the supernatant, thereby obtaining only the cellular precipitate.

Cell precipitate obtained above, was added 1 ml of medium for culturing dermal cells (Cascade, Cat. No. 106, with the addition of 1 ng/ml EGF as a growth factor and 0.2% BPE as the activation factor, USA), and the mixture was subjected to primary cultivation in CO2-incubator at 37°C with 5% CO2.

The culture medium was replaced by fresh with intervals of 2 to 3 days, and the primary cultured cells were subjected to secondary subculturing up until their confluences reaches a range from 50 to 70%. The resulting solution was cultured cells were digested with Pancreatin, centrifuged to remove supernatant, and optionally cultured under proliferative index 1:3.

The culture solution obtained in serum-free sub is oliviomani in vitrowithin 4 to 6 weeks, centrifuged to separate the cell sediment dermal origin with subsequent suspendirovanie in 5% glucose solution for injection, thereby obtaining 1 to 4 ml of the composition of the filler for soft tissue for injection containing autologous cells from the dermis at a concentration of 2×107up to 6×107cells/ml as an effective ingredient.

1-2. Tests for viral infection and immune response

To investigate the presence of viral infection in cell culture material of dermal origin, obtained by culturing autologous tissue sample within 4 to 6 weeks in the example, "1-1", 1 ml of cell culture material from the dermis subjected to testing for HIV and viral hepatitis infection according to the conventional Protocol KFDA, which confirmed that the culture of the cellular material of the present invention is free of viruses.

Next, confirmed that the cell culture material from the dermis does not cause any immune response, by subcutaneous experiment intended for skin subject to treatment of a patient, using 0.1 ml of cell culture material. In this work, such subcutaneous experiment conducted modi is zirovanii skin test for allergies to penicillin, which determined the sample, do not possess a significant area of symptom red swelling as negative after adding 0.05 ml of a suspension of penicillin on the epidermis of the inner part of the wrist and observations within 30 minutes. In addition, in the aseptic test according to Chinese Biological Product Regulation (2000 Ed.), General Principle of "Biological Product Aseptic Test Regulation", Article A/B, it is confirmed that autologous cell culture material from the dermis of the present invention meets the requirement of medical asepsis.

The composition of the filler for soft tissue for injection of the present invention, proven security, as described above, was a bright white suspension. In the test with antibodies to BrDU

share dermal fibroblastic stem cells in the composition of the filler was in the range of from 10 to 20%. Morphological examination of the cells showed that the share of dermal fibroblastic transient dividing cells and dermal fibroblasts are in the range of from 30 to 40% and from 50 to 70%, respectively. Next, measure the amount of collagen according to the method described in the literature (Ministry of Health P.R.China, Pharmacopoeia Committee, "Chinese Pharmacopoeia" (2ndEd.), Beijing Chemical Industry Press, 2000), the collagen content in whom is osili filler of the present invention was in the range of from 20 to 60 mg/ml

The composition of the filler for soft tissue for injection according to the present invention was stored and transported at 4°C using specific container, and kept in conditions of the environment where it is cool and dry, can be stored away from direct sunlight and in the absence of gas causes corrosion and high pressure.

Example 2. Therapeutic effect of filler for soft tissue removal of wrinkles

therapeutic effect of the composition of the filler for soft tissue injection obtained in example 1, wrinkle, clinically investigated as follows.

Volunteers for this clinical test became subject to treatment patients who are 60 years or less, and suffering from wrinkles on the face, such as forehead wrinkles, glabellar grooves (the space between the eyebrows and above the nose), nasolabial folds, marionette lines, etc. Among the volunteers were excluded patients who had autoimmune disease, a chronic skin disorder, disease that is transmitted by contact way, acute and chronic infectious disease, infection in the area of wrinkles, pregnancy, severe heart disease, liver and kidneys, sensitive physique, etc. Have finally selected 20 patients had blood tests and urine, did the m electrocardiogram, tested liver and kidney, were tested for the presence of HIV and HBs Ag, etc.

The wrinkled areas of the patient were sterilized with 70% alcohol followed by anesthesia of the dermis of the patient by injection of 1% lidocaine. 1.5 ml of the composition of the filler for soft tissue, obtained in example 1-1, was filled with 3 ml syringe equipped with a needle 4,5 length of 2.2 cm Needle was made injections at several points shot of the dermis, tilted on one side, and then the composition were injected with between the top layer and middle layer of the dermis. In this case the angle between the needle and the area of injection on the skin was kept in the range from 20 to 45°. During the treatment the skin is completely delayed up until she became pale, and left in this position to provide free space in the injection area. After processing, the area of the injection massaged bubble with ice for 2 hours. Total repeated three injections at the same dose injection once every two weeks, as described above. Then for injection were followed for 12 months.

The presence of pathological symptoms in the area of injection was examined by observation of local or systemic conditions of the patient, and patients took vitamin C twice a day (200 mg per dose, 400 mg/day) for 6 months. Patients indicated that it is necessary to prevent a direct impact on the region in which eccii sunlight and contact with irritating cosmetics within 3 days after treatment.

The effect on the cure of wrinkles was evaluated according to the following standards.

Fine wrinkles disappeared, and the skin has fully recovered.

Good: there was a decrease and a clear improvement of wrinkles.

Bad: there is no cure wrinkles.

In the observation of facial wrinkles after treatment therapeutic effects at time points 3, 6, 9 and 12 months after treatment was 68,3%, to 79.2%, 88.1 percent and 91,7%, respectively. therapeutic index for each area of wrinkles on the face amounted to 91.4% for wrinkles, 94,9% for fine wrinkles, 93% for glabellar grooves, 88,2% for lip groove and 88.9% for nasolabial folds and marionette lines, and the average time duration of therapeutic effects was 3.5 months. All treated patients had a significantly higher satisfaction in the statistical analysis compared with the control. Furthermore, no significant side effects except for temporary redness in the injection area.

Example 3. Therapeutic effect of filler for soft tissue to remove scars

The composition of the filler for soft tissue for injection was obtained according to the same method as described in example 1-1, except that the subculture of the cells were carried out over 8 weeks. The thus obtained composition of the filler contained autologin the e cells from the dermis in concentration, ranging from 3×107up to 7×107cells/ml as an effective ingredient.

Security composition filler for soft tissue, obtained above, was confirmed by the implementation of tests for viral infection and immune response, and aseptic test according to the same methods as described in example 1-2".

Further, the study of the distribution of cells for autologous dermal cells of origin in the composition of the filler for soft tissue, obtained by culturing for 8 weeks, the percentage of dermal fibroblastic stem cells was in the range of from 15 to 20%, the share of dermal fibroblastic transient dividing cells was in the range of from 20 to 50%, and the share of dermal fibroblasts were within the range of from 30 to 50%. In addition, the collagen content in the composition of the filler was in the range of from 30 to 80 mg/ml

therapeutic effect of the composition of the filler for soft tissue injection obtained in example 1 to remove scars clinically investigated as follows.

Twenty volunteers with scars caused by facial acne or wounds, were taken under the same conditions and were subjected to preliminary testing according to the same methods as described in example 2. Thus the selected subjects were subjected to treatment with the composition of the filler for soft the tissues in the area of scars once in two weeks. Total repeated three injections at the same dose for 6 weeks as described in example 2, and the area of the injection site was observed within 12 months after injection. This effect on healing scars were evaluated according to the following standards.

Fine: scars disappeared, and the skin has fully recovered.

Good: there was a decrease and a clear improvement of scars.

Bad: there is no cure scars.

In therapeutic effect on scar removal at time 3, 6, 9 and 12 months after treatment was 67.5%, of 92.5%92,5% 92,5%, respectively, and the average time duration of these therapeutic effects were 3.4 months. All treated patients had a significantly higher satisfaction in the statistical analysis compared with the control. Furthermore, no significant side effects, except for temporary redness in the injection area.

Example 4. Improving skin texture and beauty effects of filler for soft tissue

The composition of the filler for soft tissue for injection was obtained according to the same method as described in example 1-1, except that subcultivation cells was carried out for 8 weeks. The thus obtained composition of the filler contained autologous cells from the dermis in conc the tion, ranging from 1.5×107to 5.5×107cells/ml as an effective ingredient.

Security composition filler for soft tissue, obtained above, was confirmed by the implementation of tests for viral infection and immune response, and aseptic test according to the same methods as described in example 1-2".

Further, the study of the distribution of cells for autologous dermal cells of origin in the composition of the filler for soft tissue, obtained by culturing for 8 weeks, the percentage of dermal fibroblastic stem cells was in the range of from 5 to 20%, the share of dermal fibroblastic transient dividing cells was in the range of from 20 to 50%, and the share of dermal fibroblasts were within the range of from 30 to 70%. In addition, the collagen content in the composition of the filler was in the range of from 30 to 60 mg/ml

Improving skin texture and beauty effects of the composition of the filler for soft tissue injection obtained in example 1, clinically investigated as follows.

Twenty volunteers with rough and wrinkled skin texture and pale skin color, or those who wanted to increase the lips or the filter (the groove between the upper lip and nose), were taken under the same conditions and were subjected to preliminary testing according to the same methods as the op is Sano in example 2. Thus the selected subjects were subjected to treatment with the composition of the filler for soft tissue in the selected area once in two weeks. Total repeated three injections at the same dose for 6 weeks as described in example 2, and the injection sites were observed within 12 months after the injection.

The result confirmed that the treated skin of patients becomes more smooth and flat, thin wrinkles on her disappear. Moreover, the skin elasticity is restored, the skin becomes smooth by the time 3 months after treatment. Also improved the obvious image quality and increased lip, and a groove between the upper lip and the nose became more flat.

Comparative example 1

The composition of the filler for soft tissue augmentation for injections to remove wrinkles and scars were obtained according to the method described in Chinese patent application No. 03155833.X, and investigated its therapeutic effect by removing wrinkles and scars. In particular, the composition of the filler was obtained according to the same method as described in example 1-1, except that the cells were maintained in DMEM for culturing dermal cells (D5546, Sigma, USA) supplemented with fetal bovine serum for 5 weeks. The thus obtained composition of the filler contained cells at a concentration ranging from 1×107up to 2×107 cells/ml as an effective ingredient.

Security composition filler for soft tissue, obtained above, was confirmed by the implementation of tests for viral infection and immune response, and aseptic test according to the same methods as described in example 1-2".

The study of the distribution of cells for cells in the composition of the filler for soft tissue, obtained by culturing for 5 weeks, the share of dermal fibroblastic stem cells was in the range of from 5 to 15%, the share of dermal fibroblastic transient dividing cells was in the range of from 30 to 50%, and the share of dermal fibroblasts were within the range of from 40 to 60%. In addition, the collagen content in the composition of the filler was in the range of from 2 to 5 mg/ml

To study therapeutic effect to remove wrinkles and scars composition filler for soft tissue, obtained above, clinical test for twenty-eight volunteers according to the same method as described in example 2.

The result found that the composition of the filler has a pronounced therapeutic effects to remove wrinkles and scars only with time from 6 to 9 months after treatment. 6 months after the treatment of fine wrinkles at the corners of the eyes disappeared, frontal line became smaller or disappeared, and happened invites the texture and elasticity of the skin.

Next, to confirm that the cells comprise autologous cell culture material of dermal origin, obtained according to the method of the present invention are cells derived from the predecessor of nerve cells possessing characteristics that are specific to fibroblasts from the dermis, which distinguish them from the stem cells of mesenchymal origin, the following experiments were carried out using as a comparative control cells derived from adipose tissue.

Example 5. Morphological comparison of cells from the dermis with cells derived from adipose tissue

To obtain a cell culture material from the dermis according to the present invention, the tissue sample size from 3 to 4 mm2was collected from the back of the ear. This tissue sample was subjected to digestion of the tissue with a solution of Pancreatin/EDTA, which was stopped by addition of DMEM with addition of 10% FBS. Digested tissue was crushed into pieces and separated into single cells by pipetting several times, and centrifuged to remove supernatant, thereby obtaining only the cellular precipitate. Thus the selected cell sediment resuspendable in serum-free RL is turalei environment and cultured in 5% CO 2-incubator at 37°C. this serum-free culture medium was obtained by adding 1 g/ml hydrocortisone, 10 ng/ml hEGF, 3 ng/ml bFGF and 10 g/ml of heparin to the culture the main medium for fibroblasts (DMEM or 106; Cascade). The morphology of the cells was investigated in a phase-contrast microscope (Olympus IX 71) at time 3 weeks after cultivation and recorded using a digital camera.

Meanwhile, stem cells derived from adipose tissue was obtained following method, i.e. the solution with the aspiration of fat obtained after liposuction were subjected to lysis by treatment with 0.1% collagenase at 37°C for 45 minutes followed by centrifugation of the resulting solution to remove the supernatant, thereby obtaining only the cellular precipitate. Thus the selected cell sediment resuspendable in DMEM with addition of 10% FBS and cultured in 5% CO2-incubator at 37°C. the morphology of the cells was investigated in a phase-contrast microscope (Olympus IX 71) at time 3 weeks after cultivation and recorded using a digital camera.

As a result, as shown in figure 4, while the cells of the dermal origin according to the present invention had a fibroblast-like elongated shape, cells derived from adipose tissue, had a flattened form, open the in all directions. Such morphology of each cell coincided with the morphology, previously found in the literature (Patricia A. separation et al., Tisseu Engineering 7:211-228, 2001; and Patricia A. separation et al., Molecular Biology of the Cell 13:4279-4295, 2002).

Example 6. Immunological comparison of cells from the dermis with cells derived from adipose tissue

6-1. Immunofluorescent analysis using nestin and fibronectin as a marker

The cells of the dermal origin were cultured in serum-free culture medium for 2 weeks according to the same method as described in example 5. When the cells from the dermis of confluently 90% of the culture solution was added a solution of 0.25% trypsin/EDTA for detaching cells from culture plates with subsequent resuspending cells in serum-free culture medium. From the thus obtained cell suspension did a PAP smear on a glass slide and incubated in a CO2-incubator at 37°C for 12 to 16 hours. When cells proliferated to the appropriate concentration, the slide was washed in DPBS, dried and then used as a sample for subsequent immunofluorescence analysis.

Next, cells derived from adipose tissue were cultured in DMEM with addition of 10% FBS according to the same method as described in example 5. When achievement of the cells, originating from adipose tissue, confluently 90%, the culture solution was added a solution of 0.25% trypsin/EDTA for detaching cells from culture plates with subsequent resuspending cells in DMEM with addition of 10% FBS. From the thus obtained cell suspension did a PAP smear on a glass slide and incubated in a CO2-incubator at 37°C for 12 to 16 hours. When cells proliferated to the appropriate concentration, the slide was washed in DPBS, dried and then used as a sample for subsequent immunofluorescence analysis.

Each glass slide, obtained above, was placed in 3.7% paraformaldehyde in PBS as the solvent for 10 minutes with shaking for fixation cells were washed in 1% skim milk in PBS for 10 minutes. Each glass slide was then placed in 0.5% Triton X-100 in PBS for 10 minutes with shaking to enhance cell permeability, followed by washing with 1% skim milk in PBS for 10 minutes.

Nestin (anti-rabbit polyclonal antibody, CHEMICON) and fibronectin (mouse polyclonal antibodies to fibronectin, Santa cruz) was diluted with 1% skimmed milk in PBS 1:500, respectively. Each of the diluted solutions markers were added on a glass slide and left to interact for 1 hour with rocking. PEFC is this glass slide was washed three times with 1% skim milk in PBS for 10 minutes.

Alexa 594 (anti-rabbit IgG antibody, Molecular probe) and Alexa 488 (antimurine antibodies IgG, Molecular probe) was diluted with 1% skimmed milk in PBS 1:500, respectively. Each of the solutions diluted antibody was added to a glass slide and left to interact for 30 minutes with rocking.

DAPI (4',6-diamidin-2-phenylindolizine, SIGMA) was diluted with PBS to 1:20000. A glass slide was then placed in a solution of DAPI for staining cells were washed three times in DPBS for 10 minutes. The washed glass slide was examined in a fluorescent microscope (Olympus IX 71) and recorded using a digital camera.

As a result, as described in figure 5, nestin and fibronectin expressibility in the cells of the dermis according to the present invention. Then examined the cells expressing both of them. However, in cells derived from adipose tissue, were detected in the expression of fibronectin, but nestin not expressively. These results confirm that, as the cells of dermal origin, cultivated according to the method of the present invention, have the profile of expression of cellular markers, other than cells derived from adipose tissue, and Express nestin (a marker of progenitor cells nerve cells), they have immunological characteristics corresponding to the stem cells derived from the predecessor of nerve cells, and not relevant to stem cells of mesenchymal origin (Toma et al., Stem Cells 23:727-737, 2005; Fernandes et al., Nature 6:1082-1093, 2004; Toma et al., Nature 3:778-784, 2001).

6-2. Immunofluorescent analysis using nestin and vimentin as token

Samples of slides with cells from the dermis and the cells derived from adipose tissue for immunofluorescence analysis were obtained according to the same method as described in example 6-1," respectively, and were treated with paraformaldehyde for fixation of the cells, followed by treatment with Triton X-100 to improve cell permeability.

Nestin (anti-rabbit polyclonal antibody, CHEMICON) and vimentin (mouse monoclonal antibodies to vimentin, CHEMICON) was diluted with 1% skimmed milk in PBS 1:500, respectively. Each of the diluted solutions markers were added on a glass slide and left to interact for 1 hour with rocking. Then the slide was washed three times with 1% skim milk in PBS for 10 minutes.

Alexa 594 (anti-rabbit IgG antibody, Molecular probe) and Alexa 488 (antimurine antibodies IgG, Molecular probe) was diluted with 1% skimmed milk in PBS 1:500, respectively. Each of the solutions diluted antibody was added to a glass slide and left to interact for 30 minutes with rocking.

DAPI (4',6-diamido the-2-phenylindolizine, SIGMA) was diluted with PBS to 1:20000. A glass slide was then placed in a solution of DAPI for staining cells were washed three times in DPBS for 10 minutes. The washed glass slide was examined in a fluorescent microscope (Olympus IX 71) and recorded using a digital camera.

As a result, as illustrated in Fig.6, nestin and vimentin expressibility in cells from the dermis of the present invention. Then examined the cells expressing both of them. However, in cells derived from adipose tissue, were detected expression of vimentin, but nestin not expressively. These results are identical to the previous message, as described in the literature (Toma et al, Stem Cells 23:727-737, 2005; Fernandes et al., Nature 6:1082-1093, 2004).

6-3. Immunofluorescent analysis using nestin and surface protein of fibroblasts 1 (FSP1) as a marker

Samples of slides with cells from the dermis and the cells derived from adipose tissue for immunofluorescence analysis were obtained according to the same method as described in example 6-1," respectively, and were treated with paraformaldehyde for fixation of the cells, followed by treatment with Triton X-100 to improve cell permeability.

FSP1 (monoclonal antibodies against the surface protein in human fibroblasts, clone 1B10, SIGMA) were diluted with 1% skimmed m the beginning in PBS 1:500 was added to the slide. The slide was left to interact for 1 hour with rocking. Then the slide was washed three times with 1% skim milk in PBS for 10 minutes.

After washing each glass slide was placed in 3.7% paraformaldehyde in PBS for 10 minutes with shaking for fixation of the cells and washed with 1% skim milk in PBS for 10 minutes. A glass slide was placed in 0.5% Triton X-100 in PBS for 10 minutes to enhance cell permeability, followed by washing with 1% skim milk in PBS for 10 minutes.

Nestin (anti-rabbit polyclonal antibody, CHEMICON) was diluted with 1% skimmed milk in PBS 1:1000 was added to the slide. The slide was left to interact for 1 hour with shaking and then washed three times with 1% skim milk in PBS for 10 minutes.

Alexa 594 (anti-rabbit IgG antibody, Molecular probe) and Alexa 488 (antimurine antibodies IgG, Molecular probe) was diluted with 1% skimmed milk in PBS 1:500, respectively. Each of the solutions diluted antibody was added to a glass slide and left to interact for 30 minutes with rocking.

DAPI (4',6-diamidin-2-phenylindolizine, SIGMA) was diluted with PBS to 1:20000. A glass slide was then placed in a solution of DAPI for staining cells were washed three times in DPBS for 10 minutes. The washed glass slide researched what about fluorescent microscope (Olympus IX 71) and recorded using a digital camera.

As a result, as described in Fig. 7, nestin and FSP1 expressibility in cells from the dermis of the present invention. Then examined the cells expressing both of them. However, in cells derived from adipose tissue, were detected the expression of FSP1, but nestin not expressively.

These results confirmed that cell culture material of dermal origin, obtained by culturing in serum-free medium according to the present invention, has the signals for fibronectin and FSP-1, known as a marker protein of fibroblasts, as well as for nestin, known as a marker protein of stem cells derived from the predecessor's nerve cells, which obviously differ from stem cells of mesenchymal origin, such as cells derived from adipose tissue.

Example 7. RT-PCR to study the expression profile of the marker proteins predecessor of nerve cells in the dermal cells of origin

The cells of the dermal origin were cultured in serum-free culture medium for 2 weeks according to the same method as described in example 5. When the cells from the dermis of confluently 90% of the culture solution was added a solution of 0.25% trypsin/EDTA to release cells from cultural the th Cup with subsequent isolation of RNA using a kit for RNA extraction (Purelink Micro-to-midi Invitrogen).

The reaction solution was obtained by mixing the thus selected RNA, dNTP and 20-gauge primer oligo-dT and bringing the final volume to 10 μl. The reaction solution was incubated at 65°C for 5 minutes and then stored at 0°C for one minute. After it was added 10 μl of the mixture for the synthesis of cDNA (10× buffer RT, 25 mm MgCl2, 0.1 M DTT, not containing RNase, Superscript III RT), the resulting solution was subjected to reverse transcription at 50°C for 50 minutes and 85°C for 20 minutes. To the reaction solution was added 1 μl of RNase H and left at 37°C for 20 minutes, to thereby synthesize cDNA.

PCR was carried out to study the expression profiles of p75NTR, Pax3, Snail, Slug, nestin and vimentin, known as marker protein precursor neural cells, using the synthesized cDNA as the template. The PCR conditions were such as follows is an initial denaturation at 95°C for 2 minutes followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. While GAPDH was used as control, and a pair of forward and reverse primers used in PCR for amplification of each marker protein, was such, as follows:

SEQ ID NO:1 nestin-1 and SEQ ID NO:2 nestin-2 for amplification nestina

SEQ ID NO:3 vimentin-1 and SEQ ID NO:4 imenti is a-2 for amplification of vimentin

SEQ ID NO:5 p75NTR-1 and SEQ ID NO:6 p75NTR-2 for amplification p75NTR

SEQ ID NO:7 pax3-1 and SEQ ID NO:8 pax3-2 for amplification Pax3

SEQ ID NO:9 snail-1 and SEQ ID NO:10 snail-2 for amplifying the Snail

SEQ ID NO:11 slug 1 and SEQ ID NO:12 slug-2 for amplification Slug

SEQ ID NO:13 GAPDH-I and SEQ ID NO:14 GAPDH-2 for amplification of GAPDH

As a result, as illustrated in Fig, confirmed that p75NTR, Pax3, Snail, Slug, nestin and vimentin expressed on the mRNA level in cells from the dermis of the present invention. In particular, vimentin in a significant number expressively in cells from the dermis of the present invention. These results show the level of mRNA that cell culture material from the dermis of the present invention contains stem cells that originate from the predecessor of nerve cells.

Industrial applicability

As described above, the composition of the filler for soft tissue for injection according to the present invention has a prolonged therapeutic effects without causing any immune response and side effects due to the use of autologous dermal cells. Moreover, as it completely eliminates the risk of using serum of animal origin by means of serum-free cultivationin vitroand it has a direct therapeutic effect and by producing large amounts of collagen, the composition of the filler for soft tissue for injection of the present invention can be effectively used to improve the color and elasticity of the skin, and smoothing and removal of wrinkles and scars.

1. A method of obtaining a composition filler for soft tissue injection for the relief or treatment of skin damage due to mechanical or physiological reasons, which includes stages:
1) digestion of autologous dermal tissue, selected from the own patient's skin by treatment with a solution of Pancreatin/EDTA, and separation into individual cells;
2) the cultivation and proliferation of selected dermal cells by serum-free cultivation in vitro in a medium containing growth factor and activation factor for obtaining autologous cell culture material from the dermis, containing dermal fibroblast stem cells, dermal fibroblasts transitional dividing cells, dermal fibroblasts and collagen;
3) centrifugation of autologous cell culture material from the dermis for the Department of autologous cellular sediment dermal origin and
4) suspension of autologous cellular sediment dermal origin in glucose solution for injection or arbitrary solution for injections received the eat suspension for injection.

2. The method according to claim 1, in which autologous dermal tissue from stage 1) is subjected to defrost once after the dermal tissue selected from the own patient's skin, dried and stored.

3. The method according to claim 1, in which the composition of the filler for soft tissue contains from 1×107up to 8×107cells/ml of autologous cells from the dermis and from 10 to 100 mg/ml of collagen as an effective ingredient.

4. The method according to claim 1, in which the percentage of dermal fibroblastic stem cells, dermal fibroblastic transient dividing cells and dermal fibroblasts in the composition of the filler for soft tissue is 5-20:20-50:30-70.

5. The method according to claim 1, wherein the growth factor is one or more selected from the group consisting of epidermal growth factor, dermal growth factor, bFGF (basic fibroblast growth factor), and mixtures thereof.

6. The method according to claim 5, in which the content of the growth factor is from 0.1 to 1 ng/ml.

7. The method according to claim 1, in which the activation factor is one or more selected from the group consisting of cortical hormone, pituitary extract the bull (TIME), insulin, hydrocortisone, and mixtures thereof.

8. The method according to claim 7, in which the content of the activation factor is from 0.1 to 1%.

9. The method according to claim 1, in which passionato the second cultivation in vitro spend from 6 to 10 weeks.

10. The composition of the filler for soft tissue injection for the relief or treatment of skin damage due to mechanical or physiological reasons, which is obtained by the method according to claim 1, where the composition of the filler for soft tissue contains from 1×107up to 8×107cells/ml of autologous cells from the dermis and from 10 to 100 mg/ml of collagen as an effective ingredient.

11. The composition of the filler for soft tissue for injection of claim 10, which is intended for the relief or treatment of wrinkles or scars in the area containing the damage to the skin due to skin defects.

12. The composition according to claim 11, where the treatment of damage to skin because of skin defects approved for wrinkle reduction, scar removal or lip augmentation.

13. The composition according to item 12, where wrinkles include wrinkles on the face and neck, stretch marks after pregnancy and wrinkles on the back of the hand.



 

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3 ex

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7 cl, 4 tbl

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2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

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12 cl, 9 ex, 2 tbl

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2 ex, 2 dwg, 3 cl

FIELD: medicine.

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2 ex, 2 tbl, 52 cl

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1 tbl

FIELD: medicine.

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2 cl, 2 ex, 3 tbl

FIELD: medicine.

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32 cl, 1 ex, 3 dwg

FIELD: veterinary science.

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3 tbl

FIELD: medicine; oncology.

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32 cl, 3 tbl, 7 ex

FIELD: medicine.

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10 cl, 2 tbl, 5 dwg

FIELD: medicine; cellular technologies.

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1 ex, 1 dwg

FIELD: medicine.

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EFFECT: invention ensures high therapeutic effect with minimal expenditure of labour and finance.

3 tbl, 6 ex

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