CN114886922A - Medicine containing fat micro-segment and application thereof in treatment of chronic wound surface difficult to heal - Google Patents
Medicine containing fat micro-segment and application thereof in treatment of chronic wound surface difficult to heal Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a medicament containing fat micro-segment and application thereof in treatment of chronic wound surface difficult to heal, wherein the fat micro-segment is prepared by the following steps: (1) extracting adipose tissue; (2) removing impurities; (3) emulsifying fat tissue: cutting adipose tissue into small pieces by using tissue scissors, adding PBS buffer solution containing soybean lecithin, stirring and emulsifying; (4) centrifugal separation: the adipose tissue was observed to be divided into 3 layers by centrifugation, and the middle layer was taken as the fat micro-segment. The invention effectively improves the effect of the medicament containing fat micro-fragments on treating chronic difficult-to-heal wounds by a specific preparation process, particularly by emulsifying adipose tissues by using a specific emulsifier.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a medicine containing fat micro-fragments and application thereof in treatment of chronic wound surfaces which are difficult to heal.
Background
Adipose tissue is an important place for storing substances in our human body, and not only stores huge energy, but also contains rich multifunctional and multipotent important elements. The fat micro-segment structure (NICHE) mainly comprises a three-dimensional biological scaffold and a cluster cell, wherein the three-dimensional biological scaffold is composed of collagen, a connective tissue network and a micro-vascular network, the cluster cell comprises Pericytes (Perichytes), fat cells, preadipocytes, Mesenchymal Stem Cells (MSC), endothelial progenitor cells, Hematopoietic Stem Cells (HSC), lymphocytes, macrophages and the like, and the cluster cell, the cytokine and exosome are embedded into the three-dimensional biological scaffold, so that the microenvironment for cell growth can be reduced to the maximum extent.
Sunmanning et al (one example of the autologous adipose tissue function micro-fragment transplantation for treating the diabetic foot ulcer), Chinese journal of diabetes 9.7(2017): 2), obtains good effect by using the autologous adipose tissue function micro-fragment transplantation for treating one example of diabetic foot patients, and provides a new idea and method for clinical treatment of the diabetic foot.
In addition, Libaijian autologous fat micro-segment is applied to knee osteoarthritis patients for treatment, and the curative effect of Libaijian autologous fat micro-segment is researched and analyzed. The method selects 60 patients as the study objects, the patients with knee osteoarthritis are treated in the hospital from 2015 8 months to 2017 8 months, and are divided into two groups according to a random digital table method, wherein 30 patients in a control group are treated by sodium hyaluronate injection, and the other 30 patients in an observation group are treated by utilizing Libaijian autologous fat micro-segments, and the pain degree and the knee joint function condition of the two groups of patients before and after treatment are contrastively analyzed. As a result: the HSS score (89.16 +/-6.35) and VAS score (1.03 +/-0.26) scores of the observation group are remarkably better than those (71.59 +/-9.84) and (3.12 +/-0.52) scores of the control group after treatment, and the difference has statistical significance (P0.05). The conclusion shows that the knee osteoarthritis patients have obvious treatment effect by applying Libaijian autologous fat micro-segments.
Although drugs containing fat micro-fragments have been widely used in the medical field, treatment of chronic and difficult-to-heal wounds still faces a great challenge. The invention aims to improve the application of the medicament containing fat micro-fragments in treating chronic difficult-to-heal wound surfaces.
Disclosure of Invention
Based on the reasons, the invention provides a medicament containing fat micro-fragments and application thereof in treating chronic difficult-to-heal wound surfaces. Specifically, in order to achieve the purpose of the present invention, the following technical solutions are proposed:
one aspect of the present invention relates to a medicament comprising fat micro-fragments, said fat micro-fragments being prepared by the steps of:
(1) extracting adipose tissue;
(2) removing impurities;
(3) emulsifying fat tissue: cutting adipose tissue into small pieces by using tissue scissors, adding PBS buffer solution containing soybean lecithin, stirring and emulsifying;
(4) centrifugal separation: the adipose tissue was observed to be divided into 3 layers by centrifugation, and the middle layer was taken as the fat micro-segment.
In a preferred embodiment of the present invention, the step (2) refers to removing macroscopic blood vessels and fibrous parts in the fat.
In another preferred embodiment of the present invention, the step (3) means: cutting adipose tissue to about 1mm by tissue scissors 3 Adding 1-3 times of PBS buffer solution with pH of 7.0-7.6, wherein the PBS buffer solution contains 1-3 wt% of soybean lecithinAnd stirring at 500-3000rpm for 5-30 min for emulsification.
In another preferred embodiment of the present invention, the step (3) means: centrifugation is carried out for 1-5 minutes at a rotation speed of 1000-3000 g/min.
In a preferred embodiment of the present invention, the adipose tissue is autologous adipose tissue. By using autologous adipose tissue, it helps to avoid immune reactions.
In a preferred embodiment of the present invention, the medicament further comprises a dressing including, but not limited to, sterile gauze or sterile non-woven fabric.
The invention also relates to the application of the medicine in preparing the medicine for treating the wound surface.
In a preferred embodiment of the present invention, the wound is a chronic refractory wound, including but not limited to pressure sores, venous leg ulcers, diabetic ulcers, unexplained wounds.
Advantageous effects
The invention effectively improves the effect of the medicament containing fat micro-fragments on treating chronic difficult-to-heal wounds by a specific preparation process, particularly by emulsifying adipose tissues by using a specific emulsifier.
Drawings
FIG. 1: electron micrograph of fat micro-fragments prepared in example 1.
FIG. 2: schematic diagram of the results of the antibacterial proliferation assay.
Detailed Description
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1:
(1) extracting autologous adipose tissues: 100-150 ml of autologous adipose tissues from the abdomen and buttocks were extracted and rapidly placed in 0.01M PBS buffer at pH7.2 containing gentamicin for preservation.
(2) Removing impurities: in a GMP manufacturing plant, a portion of autologous adipose tissue was placed into a 50 ml centrifuge tube and the macroscopic vascular and fibrous portions of the fat were removed.
(3) Emulsifying autologous adipose tissues: cutting adipose tissue to about 1mm by tissue scissors 3 Adding 2-fold mass of PBS buffer solution with pH7.2 and containing 2 wt% of soybean lecithin, and stirring at 1000rpm for 10 minutes for emulsification.
(4) Centrifugal separation: after centrifugation at 2000 g/min for 3 min, the adipose tissue was observed to be divided into 3 layers, and the middle layer was taken as the final product fat micro-segment, the electron micrograph of which is shown in FIG. 1.
Example 2:
the same as in example 1 except that 1 wt% of soybean lecithin was added to the PBS buffer solution of step (3), and the mixture was stirred at 1000rpm for 20 minutes to emulsify.
Comparative example 1:
the preparation method was the same as in example 1 except that no soybean lecithin was added to the PBS buffer solution, and the same amount by weight of Tween 80 was added.
Example 3:
antibacterial proliferation assay: the fat micro-fragments obtained in examples 1-2 and comparative example 1 were put into a 96-well plate, 1mg of the fat micro-fragment sample was added to each well, and then 200. mu.L of the bacterial suspension (2X 10) 7 CFU/mL) were uniformly placed in a plate, incubated overnight at 37 ℃, after which the bacteria were dispersed in PBS solution using sonication and the number of bacteria was counted using a plating method. .
As can be seen from FIG. 2, the fat micro-segment drugs of the present invention significantly reduced the proliferation potency of bacteria, both for gram-negative Pseudomonas aeruginosa and for gram-positive Staphylococcus aureus, while the anti-bacterial proliferation potency of the fat micro-segment drugs of examples 1 and 2 was significantly further improved.
Example 4:
clinical treatment was performed using the dressing prepared in example 1, as illustrated below:
1. case selection
All cases were selected from 100 patients in emergency department and hospitalization at my hospital from 1 month in 2018 to 12 months in 2018, and were randomly divided into 50 cases for each of the treatment group and the control group. And (3) inclusion standard: the wounds which can not be healed for more than 3 months comprise 48 pressure sores (25 cases in a treatment group and 23 cases in a control group), 18 lower limb venous ulcers (9 cases in the treatment group and 9 cases in the control group), 25 diabetic ulcers (11 cases in the treatment group and 14 cases in the control group) and 9 unexplained wounds (5 cases in the treatment group and 4 cases in the control group). The difference between the two groups has no significance (P > 0.05) by respectively testing the age, the sex and the disease condition with left chi square, and the two groups have comparability.
2. The treatment method comprises the following steps:
2.1 treatment group: the patient was coated with the fat micro-segment prepared in example 1, bandaged with sterile vaseline gauze and other dressings, washed and replaced once every 1-2 days, and the efficacy was evaluated after 8 weeks.
2.2 control group: after the wound surface is cleaned by a furacilin solution with the ratio of 1:5000, the wound surface is wrapped by sterile vaseline gauze and other dressings, the wound surface is cleaned and replaced once in 1-2 days, and the curative effect evaluation is carried out after 8 weeks.
3. Criteria for therapeutic effect
The grading standard of curative effect is formulated by referring to the Chinese medicine industry standard of the people's republic of China, namely ' the standard of curative effect of diagnosis of surgical diseases in traditional Chinese medicine ' and ' the guide of clinical research on new Chinese medicines ' of the Ministry of health, published by the State administration of traditional Chinese medicine.
And (3) healing: the wound surface is completely healed, clinical symptoms disappear, and no relapse occurs after 2 months of follow-up visit.
The effect is shown: the area of the wound surface is reduced by more than 70 percent, and clinical symptoms basically disappear.
Improvement: the area of the wound surface is reduced by more than 30 percent, and clinical symptoms are improved.
And (4) invalidation: the area of the wound surface is reduced by less than 30 percent or does not change or expand, and the clinical symptoms are not improved.
4. The treatment effect is as follows: the therapeutic effect of the two groups of patients after treatment is shown in Table 1
Table 1: comparison of therapeutic effects of two groups of patients after treatment
| Group of | N | Recovery method | Show effect | Improvement of life | Invalidation | Total effective rate |
| Treatment group | 50 | 3 | 16 | 15 | 16 | 68％ |
| Control group | 50 | 0 | 5 | 12 | 33 | 34％ |
Note: comparison with control group P < 0.05
Compared with the two groups of curative effects, the cure rate and the total effective rate of the treatment group are obviously superior to those of the control group, and the treatment group has comparability, P < 0.05.
The above description is of the preferred embodiment of the present invention, but it is not intended to limit the present invention. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (9)
1. A medicament comprising fatty micro-fragments, said fatty micro-fragments being prepared by:
(1) extracting adipose tissue;
(2) removing impurities;
(3) emulsifying fat tissue: cutting adipose tissue into small pieces by using tissue scissors, adding PBS buffer solution containing soybean lecithin, stirring and emulsifying;
(4) centrifugal separation: the adipose tissue was observed to be divided into 3 layers by centrifugation, and the middle layer was taken as the fat micro-segment.
2. The medicament according to claim 1, wherein the step (2) is to remove macroscopic blood vessels and fiber parts in fat.
3. The medicament according to claim 1, wherein the step (3) is: cutting adipose tissue to about 1mm by tissue scissors 3 Adding 1-3 times of PBS buffer solution with pH value of 7.0-7.6 by mass, wherein the PBS buffer solution contains 1-3 wt% of soybean lecithin, and stirring for 5-30 minutes at 3000rpm for emulsification.
4. The medicament according to claim 1, wherein the step (3) is: centrifugation is carried out for 1-5 minutes at a rotation speed of 1000-3000 g/min.
5. The medicament of claim 1, wherein the adipose tissue is autologous adipose tissue.
6. The medicament of claim 1, further comprising a dressing selected from sterile gauze or sterile non-woven fabric.
7. Use of a medicament according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of wounds.
8. The use according to claim 7, wherein the wound is a chronic refractory wound.
9. Use according to claim 8, wherein the chronic refractory wounds are selected from pressure sores, venous leg ulcers, diabetic ulcers or wounds of unknown cause.
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