Method for correction of age-specific and pathological human skin changes

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to dermatology, and can be used for treatment or rejuvenation of human skin. It is ensured by introduction of autogenous fibroblasts suspended in physiologic saline and hyaluronic acid in treatment or rejuvenation region. The skin is prepared with growth fibroblast medium with biologically active substances (BAS) - growth medium factors including fibroblast growth in the form of gel.

EFFECT: invention provides smoothing of wrinkles, higher skin elasticity.

2 ex, 2 tbl, 52 cl

 

The invention relates to medicine, namely to dermatology and cell therapy, rejuvenation and treatment of human skin.

The method comprises the introduction into the area of treatment or rejuvenation of tissues or organs of a suspension of autologous (same organism) fibroblasts in physiological solution with addition of hyaluronic acid.

In addition, it is proposed to stimulate proliferative and synthetic activity of the injected fibroblasts using products fibroblast growth contained in the environment, which is used for their growth. This serves to distinguish these factors from the environment and use them as components in cosmetic tool, which then can be used before cell therapy for skin preparation, and after therapy to increase the effect of the methodology and its prolongation.

Currently, cellular therapy is carried out as follows. The syringe is injected cells in place requiring correction and then expect results. When this is used as autologous and allogeneic cells (U.S. Patent 5660850 26.08.1997; Boss W.K. Jr, Usal H, Fodor P.B, Chemoff G. Autologous cultured fibroblasts: a protein repair system. Ann Plast Surg. 2000 May 44(55): 536-542).

A similar description of the methodology is provided in another patent (Patent No. 2281776 2.08.2006), which is based on the fact that to increase the efficiency of the cell is therapy are only fibroblasts with high efficiency cloning.

Despite the increasingly widespread method is still an open question about the effectiveness of autologous cells for cell therapy, especially in adults and older people (Kregel EA, Zgurski A.A., Terekhov S.M. the Use of autologous fibroblasts in cosmetology. Aesthetic medicine. 2004. V.3. No. 4. S-342). The problem is that not always take root cells and they are not always effectively correct wrinkles or other defects.

The proposed method can significantly improve cell survival and increase the efficiency of cell therapy.

Hyaluronic acid acts as a dermal filler matrix, creating a vital volume of injected autologous cells, increases the retention of the cells prior to their introduction into the area of treatment and better survival rate of the cells after their introduction. Input solutions products is carried out with a syringe in the field of therapy. The concentration of drugs and their actions selected individually in accordance with the specific conditions and depending on the patient's age, physical condition, specific organ and tissue.

Previously, experiments were carried out on animals. For example, investigated the effects of skin fibroblasts in conjunction with the preparation of stabilized hyaluronic acid (Restylane) the devil is musnah mice (E.S. Yoon et al. Advantages of the presence of living dermal fibroblasts within restylane for soft tissue augmentation // Ann Plast Surg. 2003 Dec; 51(6). P. 587-592). The authors evaluated the possibility of increasing the validity of Restylane in combination with dermal fibroblasts. After subcutaneous injection to mice mixture 5x105fibroblasts suspended in 200 µl of phosphate-saline buffer (FSB) in combination with 200 ál of Restylane, weight of nodules formed was slightly decreased during the first 2 weeks and then did not change up to 16 weeks. The control nodules formed after injection of Restylane, diluted FSB, decreased during the observation period, 2 times. Immunochemical staining showed the presence of human collagen bundles of the experimental group compared with the control group. The obtained results indicate that Restylane, mixed with cultured fibroblasts of human skin, can be successfully injected as a living graft with the possibility of long-term effect.

We have conducted studies in which skin fibroblasts people was injected into the problem areas of the skin of the patient in a physiological solution and also together with hyaluronic acid. The ratio was as follows. Take 0.5 ml of suspensie fibroblasts with 1 million cells in physiological solution there was added 0.5 ml of hyaluronate acid. Received 0.5 million cells/ml. The original solution of hyaluronic acid which you was in the range of 0,02-0,1%, so that the final concentration in the suspension was 0.01 to 0.05%. The suspension was stirred and held around the syringe problem areas.

The results were as follows. Papules injection of fibroblasts with hyaluronic acid-kept 2 times longer than without it. This means that the injected fibroblasts have more opportunities to survive and perform their inherent functions - production of collagen and elastin, which naturally leads to wrinkles and actually seems to reduce their depth, softening the skin, improving its elasticity and firmness. Visually it is observed to reduce the visibility of wrinkles, smoothing wrinkles, evening out skin surface. The speed reduction tucks is increased at least 1.5 times that talks about real skin rejuvenation is also equivalent value.

The obtained results indicate that hyaluronic acid mixed with cultured fibroblasts of human skin, can be successfully injected as a living graft with the possibility of long-term effect.

The second proposal is to stimulate proliferative and synthetic activity of the injected fibroblasts. For this purpose it is proposed to use the products fibroblast growth contained in the environment, which is used for their growth.

Usually in cosmetic products is used substances of vegetable origin or synthesized chemically, also used hyaluronic acid (Patent RF № 94040904 from 1994.08.19).

During culturing in growth medium skin fibroblasts synthesize and secrete into the environment a large number of biologically active substances, among which are the various growth factors (EGF, FGF, TGF, KGF), components of the extracellular matrix (GAG, hyaluronic acid, chondroitin sulfate) and enzymes (EGF - like growth factor epidermis, regulates the migration and proliferation of epidermal skin cells. FGF - fibroblast growth factor stimulates the growth of all types of cells and production of extracellular matrix, TGF - trasformarsi growth factor, is involved in the formation of new blood vessels; KGF - keratinocyte growth factor (Krikheli E.A., Zgurski A.A., Terekhov S.M. the Use of autologous fibroblasts in cosmetology. Aesthetic medicine. 2004. V.3. No. 4. S-342). Synthesized also collagen and elastin components of the extracellular matrix.

The authors concluded that this environment can be used for the preparation of cosmetic products, which can be applied both before and after the application of cell therapy. This cosmetic product can be called individual (personal), because it uses the factors of the original owned by the recipient.

The presence of skin growth factors and other listed substances from the body's own cells Bud is t to contribute to the improvement of its condition to ensure the survival of existing and introduced from outside, resulting in cell therapy, cells.

On the basis of these factors serves to produce individual cosmetics under the General title: medical individual cosmetic product (MIX).

Its composition may be as follows. Lyophilized growth factors and protein ingredients of the growth medium, which increased autologous fibroblasts of the patient, glycerin, as a filler, hyaluronic acid to create a matrix in the dermis, the aromatic components to give an attractive smell.

Following the recipe.

1. Cells are grown 14-28 days on a medium containing serum, and serum-free environment.

The compounding gel, option 1:

Growth serum-free medium with BAS49%
Hyaluronic acid (sodium hyaluronate)0,5-1,5%;
Glycerin (as a base)a 49.5-48.5 per cent;
Flavor0-0,5%

The compounding gel, option 2:

Growth medium with zaworotko is, containing BAS49%
Hyaluronic acid (sodium hyaluronate)0,5-1,5%;
Glycerin (as a base)a 49.5-48.5 per cent;
Flavor0-0,5%

2. Biologically active substances produced by cells in the environment are concentrated using techniques "Minhan", "Pillicon" -"Millipore" USA or are subjected to lyophilization. The content of biologically active substances (BAS), including vitamins and amino acids growth medium, growth factors, and others, is of the order of 10 mg/ml in the growth medium.

The compounding gel, option 3:

BAS (concentrate or freeze-dried)0.5 to 3%
Glycerin (as a base)70%;
Collagen of cattle1,5%;
Hyaluronic acid (sodium hyaluronate)1,5%;
Flavor0-0,5%
Purified waterthe required quantity to 100%.

The term storage is of 50-60 days at +4°C.

The rate of application of 10 ampoules of 2 ml for 20 days through the day.

After 5-7 days the second course of 10 vials in a day - the minimum rate.

Courses can be applied both before and after cell therapy. Cosmetics applied as follows. Opened 1 ampoule and its volume is applied in the evening to problem areas. After that the product is not washed away until the next morning. After a day on the following night, use the second ampoule. In the following days similar uses 3-I and then the other of the ampoule 10. After cell therapy after 10 days also used other 10 ampoules. After 5-7 days the second course of 10 vials every other day.

Thus, you have the following options for the use of the individual cell cosmetics:

1) 1st year to cellular therapy;

2) 2nd year after cell therapy;

3) 2-year and 2nd year after cell therapy.

Of course, there remains the possibility of using cellular technology without the use of MIX.

MIX is a cosmetic product for external use.

Due to these factors and after their application on the skin, intercellular substance will gradually be updated and, hence, to rejuvenate.

It also creates favorable conditions for engraftment of autologous fibroblasts and their better performance in the future.

Trials were conducted at dobrovo izah. The results of the tests described below and in table 1 and confirm the industrial applicability of the method.

Table 1
The results of therapy with autologous fibroblasts without hyaluronic acid and together with the last
OptionsParameter estimation to therapyTherapy:
fibroblastsfibroblasts + hyaluronic acid
VolunteersBOK, 52 gDSC, 54 g
Wrinkles1063
Turgor356
Skin color975
Sub. score357
VolunteersSEASSC
Wrinkles864
Turgor567
Skin color765
Sub. score
Volunteers KVADAM
Wrinkles954
Turgor345
Skin color754
Sub. score
VolunteersVBASIZES
Wrinkles863
Turgor2 34
Skin color653
Sub. score
VolunteersFTMSSC
Wrinkles764
Turgor677
Skin color765
Sub. scorethe
VolunteersGRPMAP
Wrinkles863
Turgor456
Skin color643
Sub. score
Note. Wrinkles, points 10-1 (depth). Turgor, points 1-10. The color of the skin in the area of the wrinkles, the score 10-0 (dark - light). Subjective assessment of 1-10 points.

Volunteer B. O.K. 53,, female [Control]. When viewed from the skin of the face detected: decreased skin turgor in the cheeks, chin, deep wrinkles in the area of nosegun the x folds, on the nose, forehead, around the eyes; dehydration and change the contour of the face.

Diagnosis: age-related changes of the skin.

Correction of problem areas of the skin was carried out declared by way of using autologous cells and hyaluronic acid.

Purpose: transplantation of autologous fibroblasts in the problem areas of the skin together with hyaluronic acid.

To do this, the volunteer took a piece of skin from the inner surface of the forearm. Biopsy (4 mm diameter) after washing (2 times) in a solution of nutrient medium, DMEM with antibiotics penicillin (50 µg/ml) and streptomycin (50 u/ml) was placed in a Petri dish, and crushed with a scalpel in 0.1 ml of 0.25% solution of collagenase type II. After grinding the pieces of cloth covered the cover glass and the Cup was filled with 2 ml of the same solution of collagenase. The Petri dish was placed in CO2-incubator. After 1.5 h, the Cup was removed, the collagenase solution was poured off, washed with medium, DMEM with 20% fetal calf serum (ETS) and the newly poured growth environment. After 1 day, the medium was changed for fresh. After 5 days the skin pieces were washed away, with pieces of skin were placed in a centrifuge tube, centrifuged 3 min at 1000 rpm, adosados poured, the precipitate was placed in a new Petri dish was closed cover glass, poured the environment DMEM with 20% ETS and put in C02-incubator. Per adicheskii after 3 days the medium was changed for fresh. Within 15 days after reaching monolayer pieces of skin with the medium were placed in a centrifuge tube and perform similar actions. Then in a Petri dish 2 times washed the cells with EDTA, and then filled with 1 ml of trypsin solution. The Petri dish was placed in CO2-incubator. After 15 min when cells were ukreplenii from the growth surface in a bowl filled with 2 ml of growth medium, the cells are suspended and transferred into a culture flask (75 cm2and added to 10 ml of growth medium with 20% ETS, the vial was placed in CO2-incubator. In 7 days after reaching the monolayer of cells was removed from the surface of the growth in the same way and moved the cage into the vial (150 cm2. During the month of cells were perseval in a similar way to achieve monolayer in 10 vials (150 cm2. Next, cells 7 vials were removed in a similar manner, the suspension was centrifuged 2 times in saline and prepared suspension in physiological solution with a concentration of 2 million cells/ml and poured into vials, 1,5 ml After that produced the input of the suspension in the region of wrinkles insulin syringe. Before therapy, the skin surface was treated with a solution of 70% ethanol or 0.5% chlorhexidine, then did the local anesthesia 5% cream EMLA. Enter the suspension produced in the surface or medium from the second dermis 0.05 ml with an interval of 5-6 or 8 to 10 mm depending on the kind of wrinkles. Did 2 courses with an interval in 2 weeks

The surface appearance of the skin was recorded in photographs and visual observations and subjective evaluation of the patient before and after therapy.

Volunteer DSC 55,, female [Experience]. Had similar signs of skin aging with volunteer B.

Volunteer performed the same actions as the volunteer B. the Difference was that he was administered a suspension of autologous fibroblasts together with hyaluronic acid.

For this likewise were preparing a suspension of autologous fibroblasts in saline solution at a concentration of 2 million cells/ml and poured into vials, 1,5 ml After that, immediately before the procedure to cell suspension 0.5 ml) was added 0.5 ml of hyaluronic acid (sodium hyaluronate) and produced the input of the suspension in the region of wrinkles insulin syringe. Also did 2 courses with an interval in 2 weeks

The surface appearance of the skin was recorded in photographs and visual observations and subjective evaluation of the patient before and after therapy.

The results of therapy without hyaluronic acid (Volunteer B. O. K.). 4-5 months after transplantation of fibroblasts was observed smoothing of wrinkles and large creases in the forehead; smoothing wrinkles on the nose and in the lateral infraorbital region of the eyes; decreased the severity and depth of the nasolabial folds; significantly improved skin turgor, uchilsa contour and complexion.

The results of therapy with hyaluronic acid (Volunteer DSC). 4-5 months after transplantation of fibroblasts the effect is the same as the volunteer B, however, the degree of wrinkles is higher by 2-3 points on 10-point scale, the degree of patient's satisfaction above.

Similar results were obtained for another 10 patients, 5 of whom therapy without hyaluronic acid, and 5 with hyaluronic acid. Picked up a pair of volunteers so that their parameters were approximately equal.

Also, tests were carried out on volunteers individual cosmetics MIX.

The results are described below and summarized in table 2.

The volunteers carried out the same steps described above, i.e. in the same way received culture of autologous cells and also prepared cell suspension. The difference was that part of the volunteers were introduced only a suspension of fibroblasts, and the other was conducted, except of course of cell therapy, courses own cosmetics before and after application of cell therapy.

For this likewise were preparing a suspension of autologous fibroblasts in saline solution at a concentration of 2 million cells/ml and poured into vials, 1,5 ml After that produced the input of the suspension in the region of wrinkles insulin syringe 2 courses with an interval in 2 weeks.

The surface appearance of the skin was recorded in photographs, is also on visual observations and subjective evaluation of the patient before and after therapy.

Part of the volunteers did a skin treatment accessorizes MIX to cell therapy and after. This was prepared individual cosmetics, as described above.

Cosmetics MIX was used according to the following scheme:

1) 1st year to cellular therapy (10 vials every other day);

2) 2nd year after cell therapy (2 courses of 10 vials every other day).

Table 2
The results of therapy with autologous fibroblasts without individual cosmetics MIX, and together with the last
PatientThe estimated ParametersThe status parameter Estimation to therapyTherapy:
fibroblastsfibroblasts + MIX
VolunteersBOK, 52 gDSC, 54 g
Wrinkles1063
Turgor 356
Skin color975
Sub. score
VolunteersSEASSC
Wrinkles864
Turgor567
Skin color765
Sub. score
VolunteersKVA DAM
Wrinkles954
Turgor345
Skin color754
Sub. score

After applying the MIX observed the following effects compared to control.

Volunteer A.S., male, 54, skin alive, has been the absorption of seals formed after cell therapy, smoothed skin, fine wrinkles become less noticeable.

Volunteer F.A., female, 40 years: the skin has become softer, firmer, fresher, wrinkles smoothed out.

Volunteers FO, female, 45 years, LE, female, 53,, O.E., male, 43, was encountered similar changes.

In all cases there were no negative effects of the MIX, both objective and subektivniy.

Thus, the technique has proven its effectiveness and lack of allergies the x etc. negative reactions.

1. The way of rejuvenation and treatment of human skin, including the use of autologous fibroblasts with the addition of hyaluronic acid, wherein the autologous fibroblasts injected into the treatment or rejuvenation in physiological solution, while the skin is pre-treated with growth medium of fibroblasts with biologically active substances (BAS) - factors in the growth environment, including including the fibroblast growth factors.

2. The method according to claim 1, characterized in that prepare 0.5 ml of a suspension containing 1 million fibroblasts in physiological solution with the addition of 0.5 ml of a solution of hyaluronic acid at a concentration of 0,02-0,1%, the suspension is stirred and 0.1-0.2 ml injected by syringe into the area rejuvenation or treatment.

3. The method according to claim 1, characterized in that use growth environment in the composition of the gel containing fibroblasts within 14-28 days in serum-free medium, containing BAS 49%, hyaluronic acid or sodium hyaluronate 0.5 to 1.5%, glycerol is 49.5 48.5%, and flavoring 0-0,5%.

4. The method according to claim 1, characterized in that use growth environment in the composition of the gel containing fibroblasts within 14-28 days with serum content of BAS 49%, hyaluronic acid or sodium hyaluronate 0.5 to 1.5%, glycerol is 49.5 48.5%, and flavoring 0-0,5%.

5. The method according to claim 1, otlichuy is the, what factors growth medium before use concentrated using known methods or subjected to lyophilization.

6. The method according to 5, characterized in that the factors of growth medium is added to the gel in 0.5-3%, the gel also contains glycerin 70%, collagen cattle of 1.5%, hyaluronic acid and 1.5%, purified water required amount to 100%.



 

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8 cl, 31 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: method relates to medicine, namely to oncology, and can be used for treating patients with locally spread or generalised lung cancer. Essence of invention lies in the following: sample of bone marrow from posterior spine of iliac crest in amount 150-180 ml is taken, incubated at 37°C during 24 hours with 1 mln ronkoleikin IU (IL-2), after that autohemochemical therapy (AHCT) is performed together with automieloimmunotherapy (AMIT), medications are introduced intravenously with drip-feed, two courses of treatment are performed with 3 week interval. In case of small cell cancer the following medications are introduced: cysplatin - 80 mg/m2 on the 1-st day, vepesid - 120 mg/m2 on the 1-st, 2-nd, 3-rd day, ronkoleikin - 1 mln IU on the 2-nd, 3-rd, 4-th, 5-th day. In case of non-small cell cancer the following medications are introduced: cysplatin - 80 mg/m2 on the 1-st day, vepesid - 120 mg/m2 on the 1-st, 3-rd, 5-th day, ronkoleikin - 1 mln IU on the 2-nd, 4-th, 6-th, 7-th day.

EFFECT: method allows enhancing anti-tumor activity of immune system and stabilising neoplastic process.

3 cl, 2 ex

FIELD: food industry.

SUBSTANCE: invention relates to primary processing of medical-and-technical raw materials, namely to preservation of reindeer unossified horns (antlers). Reindeer unossified antlers preservation method includes freezing, thermal treatment using infrared radiation spectrum till normal moisture content is reached. Unossified antlers, frozen at -20-24°C are sawn into plates 0.5-0.7 mm thick, put on reticulate cassettes (without defrosting) and thermally treated with active ventilation at wave length 1.6-2.4 mcm, 6 hours and at 45°C. After that they are left for cooling during 12 hours, plates are turned over and thermal treatment process is repeated till production with moisture content 12-19% is obtained.

EFFECT: invention provides for production of high-quality raw material with high content of biologically active substances, reduction of technological operations amount and preservation time.

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