RU2396084C1 - Soft tissue filler composition for injection and method for preparing thereof - Google Patents

Abstract

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

Description

FIELD OF THE INVENTION

The present invention relates to a soft tissue filler composition for injection, which is prepared by digesting an autologous dermal tissue isolated from a patient’s own skin, separating into individual cells, culturing in vitro and proliferating the separated cells in a serum-free medium. The present invention further relates to a method for producing the above composition.

BACKGROUND OF THE INVENTION

Conventional philling methods used to heal skin defects are divided into two groups, i.e. dermal philling and subcutaneous filing. All components of the filler used in these methods, as a rule, contain biological synthetic materials or heterologous proteins, instead of being entirely composed of autologous activators of tissue origin. Paraffin was first used as a material for philling in the 19th century. However, it caused a lot of side effects, and the philling effect with its help was unsatisfactory. Subsequently, a method was developed using exogenous proteins as philling material. In particular, it was found that the philling agent using bovine collagen leads to a rapid onset of therapeutic effect. However, the problem was that with a single implantation, collagen was gradually absorbed back into the skin after 3-6 months. The preparation and use of bovine collagen is described in US Pat. 3949073, 4424208 and 4488911. Bovine collagen was marketed under the trade name Siderm I, II and III, in which the collagen content is in the range from 35 mg / ml to 65 mg / ml. However, it was found that Siderm does not give satisfactory results, since antibodies to bovine collagen are formed in 90% of patients after treatment with Siderm (and pronounced allergic reactions are observed in 1 to 3% of them). In order to solve these problems, injectable collagen, cross-linked with glutaraldehyde, has been developed and is sold under the trade name Ziplast. The specified protein does not cause any allergic reactions, but its viscosity is too high to obtain good therapeutic effects (US patent No. 4582640 and 4642117).

Moreover, in US Pat. 4969912 and 5332802 describe a method using autologous injectable human collagen to prevent an immune response due to the use of bovine collagen, which is available under the trade name AUTOLOGEN. However, its therapeutic effect lasts only for several months. Fibrel is based on porcine collagen. In fact, three components are combined to obtain Fibrell, i.e. pork gelatin, aminocaproic acid and plasma from the subject to be treated. Fibrel has desirable effects in some patients. However, most treated patients are prone to swelling at the injection site, and its therapeutic effects cannot be maintained for a long time.

BOTOX, which was approved by the FDA in April 2002 for clinical trials, is an injection for temporary removal of wrinkles. BOTOX has therapeutic effects in removing wrinkles by anesthetizing facial muscles and has several advantages, such as ease of use and immediate onset of therapeutic effects. However, numerous problems are that the patient's facial expression becomes frozen after treatment, the duration of the therapeutic effect is too short. Moreover, there is a risk of facial paralysis.

Artecall is a complex of artificially synthesized particles and bovine collagen, which has been successfully used in Canada, Mexico and Europe for several years. Artecall has prolonged therapeutic effects through the formation of autologous collagen through stimulation of the local dermis. However, it also creates problems in that the subject may experience the presence of particles after treatment, and swelling may occur in the treatment site.

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

U.S. Pat. 5591444, 5665372 and 5660850, owned by Isolagen Technologies, Inc., describe a method for treating skin and soft tissue defects using autologous skin fibrocytes. In particular, in these patents, an injection method is described by: obtaining autologous dermal tissue from a subject to be treated; in vitro cultivation of this tissue in a medium containing bovine serum; and placing cultured autologous fibrocytes in Ringer's solution. This injection does not cause any allergic reactions or heterologous rejection reactions, but it is not free from safety problems due to the use of animal serum. Moreover, since Ringer's solution does not contribute to the long-term maintenance of the active ingredient, this injection must be carried out within a short period of time after receiving it, which causes inconvenience in terms of practical use.

Beijing Yiling Bioengineering Co., Ltd has developed a method for treating wrinkles and scars using an injection that is obtained by: extracting autologous skin from a subject being treated; its cultivation and proliferation in vitro ; and suspending cultured cells in an injection glucose solution (Ringer's solution) or a solution corresponding thereto (Chinese Patent No. 03155833.X).

The effective injection ingredient described in Chinese Patent No. 03155833.X, obtained by culturing an autologous skin sample in an environment with partial addition of animal serum. However, this method has several disadvantages. Firstly, since the collagen content in this injection, which is responsible for the immediate onset of therapeutic effects, is so small, the effects of the disappearance of wrinkles and scars come late in terms of the effort spent, thus taking from 4 to 6 months to obtain satisfactory therapeutic effects . Moreover, the subject being treated may experience safety concerns regarding the use of animal serum as a component of the culture medium.

Moreover, in the international publication No. WO 2003/094837 describes a composition comprising autologous, passaged fibroblasts and muscle cells (optionally biodegradable components of the non-cellular matrix / biodegradable non-cellular fillers (e.g., collagen)). It further describes a method for repairing tissues that have undergone degeneration in a subject as a result of a disease, disorder or defect. Further, in the international publication No. WO 2004/048557 describes a composition comprising: (i) autologous undifferentiated mesenchymal cells (UMC) and autologous fibroblasts; (ii) autologous UMCs; or (iii) autologous fibroblasts and autologous keratinocytes as an effective ingredient. It also describes a method for their preparation and a method for tissue repair using them. However, all the compositions described in these patents substantially consist of autologous muscle cells, autologous UMCs, or autologous keratinocytes, which are completely different from a soft tissue filler composition containing dermal fibroblast stem cells, dermal fibroblast transition dividing cells, and dermal fibroblasts described below.

Thus, the authors of the present invention tried to overcome the problems of the prior art and developed autologous cell culture material of dermal origin containing dermal fibroblast stem cells, dermal fibroblast transition dividing cells and dermal fibroblasts obtained in serum-free cultivation in vitro from autologous skin tissue together with a large amount collagen as a complex filler. Moreover, it was confirmed that the complex filler thus obtained has therapeutic effects for removing and treating wrinkles and scars in a short period of time and retains these therapeutic effects for a long time.

DESCRIPTION

Technical problem

Accordingly, the main objective of the present invention is to provide a new soft tissue filler composition for injection where there is no safety problem due to the fact that it does not use animal serum. It also aims to provide a composition that is very effective for removing wrinkles and scars through the immediate onset and long-term maintenance of therapeutic effects, and which can be effectively used to improve skin color and elasticity. The present invention also relates to a method for producing the above composition.

Technical solution

According to one aspect of the present invention, there is provided a method for preparing a soft tissue filler composition for injection, which comprises the steps of:

1) digestion of an autologous skin biopsy isolated from the patient’s own skin and separation into individual cells;

2) culturing and proliferating the isolated dermal cells through serum-free in vitro cultivation to obtain autologous cell culture material of dermal origin containing dermal fibroblast stem cells, dermal fibroblast transition dividing cells, dermal fibroblasts and collagen;

3) centrifuging an autologous cell culture material of dermal origin to separate an autologous cell pellet of dermal origin; and

4) suspending an autologous cell pellet of dermal origin in a glucose solution for injection or an arbitrary solution for injection to obtain a suspension for injection.

According to another aspect of the present invention, there is provided a soft tissue filler composition for injection obtained by this method.

Further, the present invention is described in more detail.

At the beginning, an autologous skin biopsy, used in stage 1), is obtained by disinfecting with alcohol a selected area of the skin of the patient being treated, local anesthesia, cutting out the epidermis and dermis from 1 to 30 mm ² and then storing them in a mother tissue solution. Skin tissue suitable for serum-free cultivation and proliferation of in vitro autologous dermal cells at this stage may include any skin, epidermis and dermis obtained from the back of the ear, eyebrows, lower eyes and other areas. The autologous dermal tissue sample thus obtained is subjected to tissue digestion and cell isolation procedures. In particular, a sample of autologous dermal tissue can be placed in a culture dish and digested with a pancreatin / EDTA solution. Further, tissue digestion can be stopped by adding a DMEM solution containing 10% FBS. The digested tissue in the reaction solution can be destroyed and separated into individual cells by careful re-aspiration using a suction pipette and centrifugation to remove the supernatant, thereby obtaining a cell pellet.

In step 2), the cells obtained in step 1) are cultured in vitro in a serum-free medium according to a conventional method in the art, such as a method for culturing digested tissue or a method for culturing a tissue fragment.

The cell culture method of the present invention differs from the prior art methods in terms of using a serum-free culture medium. In particular, the cell culture method of the present invention is different: using a serum-free culture medium supplemented with growth factors (e.g., epidermal growth factor and dermal growth factor) and activation factors (e.g., cortical hormone and bovine pituitary gland extract) instead of adding animal serum such like bovine serum, to the main medium; culturing cells in a serum-free culture medium for 6 to 10 weeks; and inducing cell proliferation and satisfactory collagen secretion. It was confirmed that the activity and morphology of cells cultured in a serum-free culture medium containing growth factors and activation factors instead of animal serum according to the method of the present invention are normal. In particular, the culture solution thus obtained contains a large amount of collagen, as well as autologous cell culture material of dermal origin, containing dermal fibroblast stem cells, dermal fibroblast transition dividing cells and dermal fibroblasts as an effective ingredient. In the case of obtaining a filler using a culture solution, the collagen content in the filler can vary from 10 to 100 mg / ml.

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

The serum-free culture medium, growth factors, and activation factors used in the present invention may include all products commercially available in the art. Growth factors that can be added to the serum-free culture medium of the present invention may include epidermal growth factor (EGF), dermal growth factor, basic fibroblast growth factor (bFGF), and the like. These factors can be used individually or in the form of mixtures thereof. The concentration of growth factors in a serum-free culture medium may preferably vary from 0.1 to 5 ng / ml or more, preferably from 0.5 to 2 ng / ml. Moreover, activation factors that can be added to the serum-free culture medium of the present invention may include cortical hormone, bovine pituitary extract (BPE), insulin, hydrocortisone, and the like. These factors can be used individually or in the form of mixtures thereof. The concentration of activation factors in a serum-free culture medium may preferably vary from 0.1 to 1% or more, preferably from 0.2 to 0.5%.

Dermal cells isolated from autologous dermal tissue are inoculated into a serum-free culture medium and cultured in an incubator at 37 ° C in 5% CO ₂ for 6 to 10 weeks. During this time, it is preferable to replace the medium with fresh at intervals of 3 days.

In step 3), a solution of a culture of autologous cells of dermal origin obtained above is centrifuged at a temperature ranging from 4 to 32 ° C, at a speed ranging from 800 to 1200 rpm, to remove the supernatant, thus obtaining only autologous cell a sediment of dermal origin containing proliferating dermal fibroblast stem cells, dermal fibroblast transition dividing cells, dermal fibroblasts and the collagen secreted by them.

Cultured and proliferating dermal fibroblast stem cells, dermal fibroblast transition dividing cells and dermal fibroblasts, and the collagen secreted by them according to the present invention can be confirmed as follows.

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

The normal morphology of dermal fibroblast stem cells when observed under an optical microscope at a magnification of 100 × is a subtle, elongated fusiform shape. They are stained dark brown according to the method with antibodies with BrdU (see figure 1).

Dermal fibroblast transitional dividing cells: The normal morphology of dermal fibroblast transitional dividing cells when viewed with an optical microscope at a magnification of 100x is a thin and small, elongated fusiform shape similar to dermal fibroblast stem cells. They are browned according to the method with antibodies with BrdU (see figure 2).

Dermal fibroblasts: The normal morphology of dermal fibroblasts when observed under an optical microscope at a magnification of 100 × is an elongated fusiform shape with many processes (see FIG. 3).

Collagen: the concentration of collagen can be measured according to the method for measuring collagen described in the literature (Ministry of Health PRChina, Pharmacopoeia Committee, "Chinese Pharmacopoeia" (2 ^nd Ed.), Beijing Chemical Industry Press, 2000).

Stage 4) consists in preparing a soft tissue filler composition for injection by suspending an autologous cell pellet of dermal origin containing the above effective ingredients in a glucose solution for injection at a concentration of 5% or a total solution for injection. However, the suspension thus obtained may further include suitable additional ingredients. The soft tissue filler composition of the present invention contains cultured dermal fibroblast stem cells, dermal fibroblast transitional dividing cells and dermal fibroblasts isolated from the patient’s own tissue together with the collagen secreted by them as an effective ingredient. The final concentration of effective cells in the injection preferably ranges from 1 × 10 ⁷ to 8 × 10 ⁷ cells / ml, and the collagen content preferably ranges from 10 to 100 mg / ml, more preferably from 20 to 60 mg / ml.

The soft tissue filler composition of the present invention can be formulated as injectable products intended for human skin, which can be prepared according to a conventional method well known to those of ordinary skill in the art. For example, the composition of the present invention may be in the form of a solution, a viscous solution, suspension or gel. The composition may further comprise suitable excipients adapted for injection into the skin. Suitable excipients must be well tolerated, stable and obtained in a consistency that provides easy and pleasant use. In this work, suitable examples of the excipient include, but are not limited to, phosphate buffered saline, bacteriostatic saline, propylene glycol, starch, sucrose, and sorbitol.

Further, the soft tissue filler composition of the present invention may further comprise an additional substance, such as an inert and pharmaceutically acceptable carrier or diluent, excipient, thickener, emulsifying agent, preservative, and a mixture thereof. Suitable examples of the aforementioned additional substances, as a rule, include those substances that are commonly used in pharmaceuticals and skin care products. More specifically, such examples of an inert and pharmaceutically acceptable carrier or diluent include, but are not limited to, saline and purified water. Suitable thickeners include, but are not limited to, acrylamide copolymers, carbomer, hydroxyethyl cellulose, hydroxypropyl cellulose, polyacrylic acid, polymethacrylic acid and polyvinyl alcohol.

Suitable emulsifying agents include, but are not limited to, caprylic / capric triglyceride, ceteareth-7, cetyl alcohol, cetyl phosphate, isostearate-11, and sodium isostearate. Preservatives give the composition of the present invention resistance to microbial attack and toxicity to microorganisms. Suitable examples include, but are not limited to alcohols, any of parabens, diazolidinyl urea, DMDM hydantoin, phenoxyethanol, and iodopropyl butyl carbamate. Examples of the above additional substances, other than those listed, can also be used in embodiments of this invention, as will be appreciated by one of ordinary skill in the art.

The soft tissue filler composition for injection obtained according to the method of the present invention contains a large amount of collagen, which functions to remove wrinkles and scars directly in the injection area. Moreover, other effective ingredients in the soft tissue filler composition for injection of the present invention play an important role in removing wrinkles and scars while functioning to improve the local environment in the skin through continuous production of collagen. This gives youth and beauty to the subject being treated.

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

Since the soft tissue filler composition for injection of the present invention contains autologous cells of dermal origin as an effective ingredient, it does not cause any allergic reaction or heterologous rejection reaction. Moreover, since animal serum is not used in the present invention during the preparation of soft tissue filler composition for injection, there is no possibility of causing infectious diseases or side effects. Accordingly, the soft tissue filler composition for injection of the present invention can directly induce good cosmetic and therapeutic effects, provide a natural facial expression to the treated subject after the injection, and maintain pronounced therapeutic effects for a long time.

Beneficial effect

The soft tissue filler composition for injection of the present invention has prolonged therapeutic effects without causing any immune response or side effect due to the use of autologous dermal cells. Moreover, since it completely eliminates the risk of using animal serum through serum-free in vitro cultivation and directly induces therapeutic effects by producing a significant amount of collagen, the soft tissue filler composition for injection of the present invention can be effectively used to improve skin color and elasticity, as well as smoothing and removing wrinkles and scars.

Description of drawings

The above and other objects 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 are presented:

Figure 1 is a photograph (× 100) observed under an optical microscope, which shows dermal fibroblast stem cells proliferating upon serum-free cultivation in vitro from autologous dermal cells according to the method of the present invention;

Figure 2 is a photograph (× 100) observed under an optical microscope, which shows dermal fibroblast transition fissile cells proliferating upon serum-free cultivation in vitro from autologous dermal cells according to the method of the present invention;

Figure 3 is a photograph (× 100) observed under an optical microscope, which shows dermal fibroblasts proliferating upon serum-free cultivation in vitro from autologous dermal cells according to the method of the present invention;

4 is a result of comparing the morphology of an autologous cell culture material of dermal origin (B) obtained according to the method of the present invention with the morphology of cells derived from adipose tissue (A) using a phase contrast microscope;

5 is an immunofluorescence assay that compares the expression profiles of nestin and fibronectin in an autologous cell culture material of dermal origin (A) obtained according to the method of the present invention with expression profiles in cells derived from adipose tissue (B), where Nestin is detected as a red spot at 594 nm, fibronectin is detected as a green spot at 488 nm, and cell nuclei are detected as a blue spot when stained with DAPI;

6 is an immunofluorescence assay that compares the expression profiles of nestin and vimentin in an autologous cell culture material of dermal origin (A) obtained according to the method of the present invention with expression profiles in cells derived from adipose tissue (B), where Nestin is detected as a red spot at 594 nm, vimentin is detected as a green spot at 488 nm, and cell nuclei are detected as a blue spot when stained with DAPI;

7 is an immunofluorescence assay that compares the expression profiles of nestin and FSP1 in an autologous cell culture material of dermal origin (A) obtained according to the method of the present invention with expression profiles in cells derived from adipose tissue (B), where Nestin is detected as a red spot at 594 nm, FSP1 is detected as a green spot at 488 nm, and cell nuclei are detected as a blue spot when stained with DAPI; and

Fig. 8 is a result of reverse transcription polymerase chain reaction (RT-PCR) in which the expression profile of marker proteins of nerve cell precursor is analyzed in an autologous cell culture material of dermal origin obtained according to the method of the present invention.

The best way

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. Obtaining a soft tissue filler composition

1-1. Production method

After the rear part of the ear disinfected with 70% alcohol, and its local surface area was anesthetized 10% lidocaine and adrenalin (1: 100,000) of it removed sample epidermal tissue and dermis 4 mm ^2, and was stored in the mother liquor (mother DMEM cell solution, Hyclone, USA).

A tissue sample was placed in a 35 mm culture dish, 2 ml of a 0.05% pancreatin / EDTA solution was added thereto, and then the culture dish was placed in a CO ₂ incubator at 37 ° C. for 10 minutes to induce cell digestion. The digestion reaction was stopped by the addition of 5 ml of DMEM with the addition of 10% FBS.

The digested tissue sample was crushed into pieces and destroyed by careful suction using a suction pipette, so that it was divided into separate cells. The separated dermal cells were centrifuged at 25 ° C, 1000 rpm for 5 minutes to remove the supernatant, thus obtaining only a cell pellet.

To the cell pellet obtained above, 1 ml of dermal cell culture medium was added (Cascade, Cat. No. 106, with 1 ng / ml EGF as a growth factor and 0.2% BPE as an activation factor, USA), and the mixture was subjected to primary cultivation in a CO ₂ incubator at 37 ° C with 5% CO ₂ .

The culture medium was replaced with fresh at intervals of 2 to 3 days, and the primary cultured cells were subjected to secondary subculture until their confluency reached a range of 50 to 70%. The resulting solution with cultured cells was digested with pancreatin, centrifuged to remove the supernatant, and further cultured at a proliferative index of 1: 3.

The culture solution obtained by serum-free subcultivation in vitro for 4 to 6 weeks was centrifuged to separate the cell pellet of dermal origin, followed by suspension in a 5% glucose solution for injection, thereby obtaining from 1 to 4 ml of soft tissue filler composition for injection containing autologous cells of dermal origin in a concentration of from 2 × 10 ⁷ to 6 × 10 ⁷ cells / ml as an effective ingredient.

1-2. Tests for viral infection and immune response

To test for the presence of viral infection in a cell culture material of dermal origin obtained by culturing an autologous tissue sample for 4 to 6 weeks in Example 1-1, 1 ml of cell culture material of dermal origin was tested for HIV and viral hepatitis infection according to the generally accepted KFDA protocol, which confirmed that the cell culture material of the present invention is virus free.

Further, it was confirmed that the cell culture material of dermal origin does not elicit any immune response through a subcutaneous experiment designed for the skin of the patient being treated using 0.1 ml of cell culture material. In this work, such a subcutaneous experiment was carried out by modifying a skin test for penicillin allergy, in which a sample was determined that did not have a significant area of the symptom of red swelling, as negative after applying 0.05 ml of a suspension of penicillin to the epidermis of the inner part of the wrist and observing for 30 minutes. In addition, aseptic test according to the Chinese Biological Product Regulation (2000 Ed.), General Principle "Biological Product Aseptic Test Regulation", Article A / B, confirmed that the autologous cell culture material of dermal origin of the present invention meets the requirement of medical asepsis.

The soft tissue filler composition for injection of the present invention, confirmed to be safe as described above, was a light white suspension. As a result of the antibody test with BrDU

the proportion of dermal fibroblast stem cells in the filler composition was in the range from 10 to 20%. A morphological study of cells showed that the fractions of dermal fibroblast transitional dividing cells and dermal fibroblasts are in the range from 30 to 40% and from 50 to 70%, respectively. Further, as a result of measuring the amount of collagen according to the method described in the literature (Ministry of Health PRChina, Pharmacopoeia Committee, "Chinese Pharmacopoeia" (2 ^nd Ed.), Beijing Chemical Industry Press, 2000), the collagen content in the filler composition of the present invention was in the range of 20 to 60 mg / ml.

The soft tissue filler composition for injection of the present invention was stored and transported at 4 ° C. using a specific container, and stored in an environment where it is cool and dry, storage away from direct sunlight and in the absence of a corrosive gas and high pressure.

Example 2. The therapeutic effect of a soft tissue filler to remove wrinkles

The therapeutic effect of the soft tissue filler composition for injection obtained in Example 1 on wrinkle removal was clinically investigated as follows.

Volunteers for this clinical test are patients who are 60 years old or less and who suffer from facial wrinkles, such as frontal wrinkles, glabellar grooves (the space between the eyebrows and above the nose), nasolabial folds, puppet lines, etc. Among the volunteers, patients who had an autoimmune disease, chronic skin disorder, contact-transmitted disease, acute and chronic infectious disease, wrinkle infection, pregnancy, severe heart, liver and kidney diseases, sensitive physique, etc. were excluded. Blood and urine tests were taken from finally selected 20 patients, an electrocardiogram was made, liver and kidney functions were tested, HIV and HBs Ag were tested, etc.

The wrinkle area of the patient was sterilized with 70% alcohol, followed by anesthesia of the patient's dermis by injection of 1% lidocaine. 1.5 ml of the soft tissue filler composition obtained in Example 1-1 was filled with a 3 ml syringe equipped with a 4.5 cm 2.2 cm needle. The needle was injected at several points of the anesthetized dermis, tilted on one side, and then the composition was injected between the upper layer and the middle layer of the dermis. The angle between the needle of the syringe and the injection area on the skin was maintained in the range from 20 to 45 °. During processing, the skin was completely pulled back until it became pale, and left in this position to provide free space in the injection area. After treatment, the injection area was massaged with an ice bladder for 2 hours. Three injections were repeated in total at the same injection dose once every two weeks, as described above. Then, the injection area was monitored for 12 months.

The presence of pathological symptoms in the injection area was examined by monitoring the patient's local or systemic conditions, and patients took vitamin C twice daily (200 mg per dose, 400 mg / day) for 6 months. Patients were advised that it was necessary to prevent direct exposure to the injection area of sunlight and contact with irritating cosmetics for 3 days after treatment.

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

Fine: the wrinkles disappeared and the skin was completely restored.

Good: there was a reduction and clear healing of wrinkles.

Bad: there is no cure for wrinkles.

As a result of observing facial wrinkles after treatment, the therapeutic effects at time points 3, 6, 9, and 12 months after treatment were 68.3%, 79.2%, 88.1%, and 91.7%, respectively. The therapeutic index for each area of facial wrinkles was 91.4% for wrinkles, 94.9% for fine wrinkles, 93% for glabellar grooves, 88.2% for the labial groove and 88.9% for the nasolabial folds and puppet lines, and the average duration of therapeutic effects was 3.5 months. All treated patients had significantly higher satisfaction in the statistical analysis compared with the control. Moreover, there were no significant side effects except for temporary reddening of the skin at the injection site.

Example 3. The therapeutic effect of a soft tissue filler to remove scars

The soft tissue filler composition for injection was prepared according to the same method as described in Example 1-1, except that the cell subculture was maintained for 8 weeks. The filler composition thus obtained contained autologous cells of dermal origin in a concentration ranging from 3 × 10 ⁷ to 7 × 10 ⁷ cells / ml as an effective ingredient.

The safety of the soft tissue filler composition obtained above was confirmed by testing for viral infection and immune response, and an aseptic test according to the same methods as described in Example 1-2.

Further, as a result of studying the distribution of cells for autologous cells of dermal origin in the soft tissue filler composition obtained by culturing for 8 weeks, the proportion of dermal fibroblast stem cells was in the range from 15 to 20%, the proportion of dermal fibroblast transitional dividing cells ranged from 20 to 50%, and the proportion of dermal fibroblasts ranged from 30 to 50%. In addition, the collagen content in the filler composition was in the range of 30 to 80 mg / ml.

The therapeutic effect of the soft tissue filler composition for injection obtained in Example 1 on the removal of scars was clinically examined as follows.

Twenty volunteers with facial acne or wound scars were selected according to the same conditions and subjected to preliminary testing according to the same methods as described in Example 2. Thus, selected subjects were treated with a soft tissue filler composition in the scar area once every two weeks. Three injections were repeated in total at the same dose for 6 weeks, as described in Example 2, and the injection area was monitored for 12 months after the injection. Moreover, the effect of healing scars was evaluated according to the following standards.

Fine: the scars disappeared, and the skin was completely restored.

Good: there was a decrease and a clear healing of the scars.

Bad: there is no cure for scars.

As a result, the therapeutic effect of scar removal at time points 3, 6, 9 and 12 months after treatment was 67.5%, 92.5%, 92.5% and 92.5%, respectively, and the average duration of these therapeutic effects was 3.4 months. All treated patients had significantly higher satisfaction in the statistical analysis compared with the control. Moreover, there were no significant side effects, with the exception of temporary redness of the skin at the injection site.

Example 4. Improving the texture of the skin and cosmetic effects of the filler for soft tissues

The soft tissue filler composition for injection was prepared according to the same method as described in Example 1-1, except that the cells were subcultured for 8 weeks. The filler composition thus obtained contained autologous cells of dermal origin in a concentration ranging from 1.5 × 10 ⁷ to 5.5 × 10 ⁷ cells / ml as an effective ingredient.

The safety of the soft tissue filler composition obtained above was confirmed by testing for viral infection and immune response, and an aseptic test according to the same methods as described in Example 1-2.

Further, as a result of studying the distribution of cells for autologous cells of dermal origin in the soft tissue filler composition obtained by culturing for 8 weeks, the proportion of dermal fibroblast stem cells was in the range from 5 to 20%, the proportion of dermal fibroblast transitional dividing cells ranged from 20 to 50%, and the proportion of dermal fibroblasts ranged from 30 to 70%. In addition, the collagen content in the filler composition was in the range of 30 to 60 mg / ml.

The improvement in skin texture and cosmetic effects of the soft tissue filler composition for injection obtained in Example 1 were clinically investigated as follows.

Twenty volunteers with a rough and wrinkled skin texture and pale skin color, or those who wanted to enlarge their lips or filter (groove between the upper lip and nose), were selected according to the same conditions and subjected to preliminary testing according to the same methods as described in the example 2. Thus, the selected subjects were treated with a soft tissue filler composition in a selected area once every two weeks. Three injections were repeated in total at the same dose for 6 weeks, as described in Example 2, and the injection areas were observed for 12 months after the injection.

As a result, it was confirmed that the treated skin of patients becomes smoother and more even, fine wrinkles on it disappear. Moreover, the skin's elasticity is restored, the skin becomes smooth by the time 3 months after treatment. Quality also improved in an obvious way and lip enlargement occurred, and the groove between the upper lip and nose became flatter.

Comparative Example 1

A soft tissue filler composition for injection to remove wrinkles and scars was prepared according to the method described in Chinese Patent Application No. 03155833.X, and investigated its therapeutic effect in removing wrinkles and scars. In particular, the filler composition was prepared according to the same method as described in Example 1-1, except that the cells were cultured in DMEM dermal cell culture medium (D5546, Sigma, USA) supplemented with fetal bovine serum for 5 weeks. The thus obtained filler composition contained cells in a concentration ranging from 1 × 10 ⁷ to 2 × 10 ⁷ cells / ml as an effective ingredient.

The safety of the soft tissue filler composition obtained above was confirmed by testing for viral infection and immune response, and an aseptic test according to the same methods as described in Example 1-2.

As a result of studying the distribution of cells for cells in the soft tissue filler composition obtained by culturing for 5 weeks, the proportion of dermal fibroblast stem cells was in the range of 5 to 15%, the proportion of dermal fibroblast transition dividing cells was in the range of 30 to 50%, and the proportion of dermal fibroblasts ranged from 40 to 60%. In addition, the collagen content in the filler composition was in the range of 2 to 5 mg / ml.

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

As a result, it was found that the filler composition has pronounced therapeutic effects for removing wrinkles and scars only from the time from 6 to 9 months after treatment. 6 months after the treatment, fine wrinkles in the corners of the eyes disappeared, the frontal lines became smaller or disappeared, and the texture and elasticity of the skin improved.

Further, to confirm that the cells that are part of autologous cell culture material of dermal origin, obtained according to the method of the present invention, are cells derived from a precursor of nerve cells with characteristics specific for fibroblasts of dermal origin that distinguish them from stem cells of mesenchymal origin, the following experiments were carried out using cells originating from adipose tissue.

Example 5. Morphological comparison of cells of dermal origin with cells derived from adipose tissue

To obtain a cell culture material of dermal origin according to the present invention, a tissue sample of 3 to 4 mm ^{2 in} size was taken from the back of the ear. This tissue sample was digested with the addition of a pancreatin / EDTA solution, which was stopped by the addition of DMEM supplemented with 10% FBS. The digested tissue sample was crushed into pieces and separated into individual cells by pipetting several times, and centrifuged to remove the supernatant, thus obtaining only a cell pellet. Thus, the isolated cell pellet was resuspended in a serum-free culture medium and cultured in a 5% CO ₂ incubator at 37 ° C. In this case, a serum-free culture medium was prepared by adding 1 g / ml hydrocortisone, 10 ng / ml hEGF, 3 ng / ml bFGF and 10 g / ml heparin to the fibroblast culture stock medium (DMEM or 106; Cascade). Cell morphology was examined under a phase contrast microscope (Olympus IX 71) at time 3 weeks after cultivation and recorded using a digital camera system.

Meanwhile, stem cells originating from adipose tissue were obtained in the following manner, i.e. the suction fat solution obtained after liposuction was lysed by treatment with 0.1% collagenase at 37 ° C for 45 minutes, followed by centrifugation of the resulting solution to remove the supernatant, thus obtaining only a cell pellet. Thus, the isolated cell pellet was resuspended in DMEM supplemented with 10% FBS and cultured in a 5% CO ₂ incubator at 37 ° C. Cell morphology was examined under a phase contrast microscope (Olympus IX 71) at time 3 weeks after cultivation and recorded using a digital camera system.

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

Example 6. Immunological comparison of cells of dermal origin with cells derived from adipose tissue

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

Cells of 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 reached dermal origin confluency of 90%, a solution of 0.25% trypsin / EDTA was added to the cell solution to detach cells from the culture plate with subsequent resuspension of cells in a serum-free culture medium. From the thus obtained cell suspension, a smear was made on a glass slide and incubated in a CO ₂ incubator at 37 ° C for 12 to 16 hours. When the cells proliferated to the appropriate concentration, the glass slide was washed with DPBS, dried and then used as a sample for subsequent immunofluorescence analysis.

Next, the cells derived from adipose tissue were cultured in DMEM supplemented with 10% FBS according to the same method as described in Example 5. When the cells derived from adipose tissue reached 90% confluency, a solution of 0.25% was added to the culture solution trypsin / EDTA to detach cells from the culture plate, followed by cell resuspension in DMEM supplemented with 10% FBS. From the thus obtained cell suspension, a smear was made on a glass slide and incubated in a CO ₂ incubator at 37 ° C for 12 to 16 hours. When the cells proliferated to the appropriate concentration, the glass slide was washed with DPBS, dried and then used as a sample for subsequent immunofluorescence analysis.

Each slide obtained above was placed in 3.7% paraformaldehyde in PBS as solvent for 10 minutes with shaking to fix the cells and washed with 1% skim milk in PBS for 10 minutes. Each 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 antibodies, CHEMICON) and fibronectin (mouse polyclonal antibodies to fibronectin, Santa cruz) were diluted with 1% skim milk in PBS 1: 500, respectively. Each of the diluted marker solutions was added to a glass slide and allowed to interact for 1 hour with shaking. After that, the slide was washed three times with 1% skim milk in PBS for 10 minutes.

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

DAPI (4 ', 6-diamidin-2-phenylindole dihydrochloride, SIGMA) was diluted with PBS 1: 20,000. The slide was then placed in a DAPI cell staining solution and washed three times with DPBS for 10 minutes. The washed glass slide was examined under a fluorescence microscope (Olympus IX 71) and recorded using a digital camera system.

As a result, as described in FIG. 5, nestin and fibronectin were expressed in cells of dermal origin according to the present invention. Then examined cells expressing both of them. However, fibronectin expression was detected in cells derived from adipose tissue, but nestin was not expressed. These results confirm that since cells of dermal origin cultured according to the method of the present invention have an expression profile of cell markers other than cells derived from adipose tissue and express nestin (a marker of nerve cell progenitors), they have immunological characteristics corresponding to stem cells derived from a precursor of nerve cells and not corresponding to stem cells of mesenchymal origin (Toma et al., Stem Cells 23: 727-737, 2005; Fern andes et al., Nature 6: 1082-1093, 2004; and Toma et al., Nature 3: 778-784, 2001).

6-2. Immunofluorescence analysis using nestin and vimentin as a marker

Samples of slides with cells of dermal origin and 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 to fix the cells, followed by treatment with Triton X-100 to improve cellular permeability.

Nestin (anti-rabbit polyclonal antibodies, CHEMICON) and vimentin (mouse monoclonal antibodies to vimentin, CHEMICON) were diluted with 1% skim milk in PBS 1: 500, respectively. Each of the diluted marker solutions was added to a glass slide and allowed to interact for 1 hour with shaking. After that, the slide was washed three times with 1% skim milk in PBS for 10 minutes.

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

DAPI (4 ', 6-diamidin-2-phenylindole dihydrochloride, SIGMA) was diluted with PBS 1: 20,000. The slide was then placed in a DAPI cell staining solution and washed three times with DPBS for 10 minutes. The washed glass slide was examined under a fluorescence microscope (Olympus IX 71) and recorded using a digital camera system.

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

6-3. Immunofluorescence analysis using nestin and fibroblast surface protein 1 (FSP1) as a marker

Samples of slides with cells of dermal origin and 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 to fix the cells, followed by treatment with Triton X-100 to improve cellular permeability.

FSP1 (monoclonal antibodies to the surface protein of human fibroblasts, clone 1B10, SIGMA) was diluted with 1% skim milk in PBS 1: 500 and added to a glass slide. A glass slide was allowed to interact for 1 hour with swaying. After that, the slide was washed three times with 1% skim milk in PBS for 10 minutes.

After washing, each slide was placed in 3.7% paraformaldehyde in PBS for 10 minutes while shaking to fix the cells and washed with 1% skim milk in PBS for 10 minutes. A 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 antibodies, CHEMICON) was diluted with 1% skim milk in PBS 1: 1000 and added to a glass slide. The slide was allowed to interact for 1 hour while shaking and then washed three times with 1% skim milk in PBS for 10 minutes.

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

DAPI (4 ', 6-diamidin-2-phenylindole dihydrochloride, SIGMA) was diluted with PBS 1: 20,000. The slide was then placed in a DAPI cell staining solution and washed three times with DPBS for 10 minutes. The washed glass slide was examined under a fluorescence microscope (Olympus IX 71) and recorded using a digital camera system.

As a result, as described in FIG. 7, nestin and FSP1 were expressed in cells of dermal origin of the present invention. Then examined cells expressing both of them. However, FSP1 expression was detected in cells derived from adipose tissue, but nestin was not expressed.

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

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

Cells of 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 reached dermal origin confluency of 90%, a solution of 0.25% trypsin / EDTA was added to the cell to detach the cells from the culture plate with subsequent RNA isolation using an RNA isolation kit (Purelink Micro to midi, Invitrogen).

The reaction solution was prepared by mixing the thus isolated RNA, dNTP and 20-mer oligo-dT primer and adjusting the final volume to 10 μl. The reaction solution was incubated at 65 ° C for 5 minutes, followed by storage at 0 ° C for a minute. After 10 μl of the mixture for cDNA synthesis was added to it (10 × buffer for RT, 25 mM MgCl ₂ , 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, so that cDNA was synthesized.

PCR was performed to study the expression profiles of p75NTR, Pax3, Snail, Slug, nestin and vimentin, known as a marker protein of nerve cell precursor, using synthesized cDNA as a template. PCR conditions were as follows, i.e. 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. GAPDH was used as a control, and the pairs of forward and reverse primers used in PCR for amplification of each marker protein were as follows:

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

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

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 of Pax3

SEQ ID NO: 9 snail-1 and SEQ ID NO: 10 snail-2 for amplification of Snail

SEQ ID NO: 11 slug-1 and SEQ ID NO: 12 slug-2 for amplification of 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. 8, it was confirmed that p75NTR, Pax3, Snail, Slug, nestin and vimentin are expressed at the mRNA level in cells of dermal origin of the present invention. In particular, vimentin was expressed in significant quantities in cells of dermal origin of the present invention. These results indicate at the mRNA level that the cell culture material of dermal origin of the present invention contains stem cells derived from a precursor of nerve cells.

Industrial applicability

As described above, the soft tissue filler composition for injection of the present invention has prolonged therapeutic effects without causing any immune response and side effects due to the use of autologous dermal cells. Moreover, since it completely eliminates the risk of using animal serum through serum-free in vitro cultivation and has immediate therapeutic effects by producing large amounts of collagen, the soft tissue filler composition for injection of the present invention can be effectively applied to improve color and elasticity skin, as well as smoothing and removing wrinkles and scars.

Claims (13)

1. A method of obtaining a soft tissue filler composition for injection to alleviate or treat skin damage due to mechanical or physiological reasons, comprising the steps of:
1) the digestion of autologous dermal tissue isolated from the patient’s own skin by treatment with a pancreatin / EDTA solution and separation into individual cells;
2) cultivation and proliferation of isolated dermal cells by serum-free in vitro cultivation in a medium containing growth factor and activation factor to obtain autologous cell culture material of dermal origin containing dermal fibroblast stem cells, dermal fibroblast transition dividing cells, dermal fibroblasts and collagen;
3) centrifuging an autologous cell culture material of dermal origin to separate an autologous cell pellet of dermal origin and
4) suspending an autologous cell pellet of dermal origin in a glucose solution for injection or an arbitrary solution for injection to obtain a suspension for injection. 2. The method according to claim 1, wherein the autologous dermal tissue from step 1) is thawed once the dermal tissue isolated from the patient’s own skin is lyophilized and stored. 3. The method according to claim 1, in which the soft tissue filler composition contains from 1 × 10 ⁷ to 8 × 10 ⁷ cells / ml of autologous cells of dermal origin 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 fibroblast stem cells, dermal fibroblast transition dividing cells and dermal fibroblasts in the soft tissue filler composition is 5-20: 20-50: 30-70. 5. The method according to claim 1, in which the growth factor is one or more selected from the group consisting of epidermal growth factor, dermal growth factor, bFGF (main 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, bovine pituitary gland extract (BPE), 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 serum-free cultivation in vitro is carried out from 6 to 10 weeks. 10. The soft tissue filler composition for injection to alleviate or treat skin damage due to mechanical or physiological reasons, which is obtained by the method according to claim 1, where the soft tissue filler composition contains from 1 × 10 ⁷ to 8 × 10 ⁷ cells / ml autologous cells of dermal origin and from 10 to 100 mg / ml of collagen as an effective ingredient. 11. The soft tissue filler composition for injection of claim 10, which is intended to alleviate or treat wrinkles or scars in the area containing skin damage due to skin defects. 12. The composition according to claim 11, where the treatment of skin damage due to skin defects is intended to reduce wrinkles, remove scars or enlarge lips. 13. The composition according to item 12, where the wrinkles include wrinkles on the face and neck, striae after pregnancy and wrinkles on the back of the hand.

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