CN107090429B

CN107090429B - Treatment method of small-particle active adipose tissues

Info

Publication number: CN107090429B
Application number: CN201710425122.9A
Authority: CN
Inventors: 程辰; 何际洲; 李青峰; 谢芸
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2017-06-08
Filing date: 2017-06-08
Publication date: 2020-09-01
Anticipated expiration: 2037-06-08
Also published as: CN107090429A

Abstract

The invention provides a method for treating small-particle active adipose tissues, which comprises the following steps: taking large-particle adipose tissues; placing the large-particle adipose tissues under a probe of a low-frequency ultrasonic instrument for acting for 1-1.5 minutes; the working condition of the low-frequency ultrasonic instrument is that the power is 80-95W, and the pulse is in an intermittent working mode; filtering the fat tissue after the ultrasonic treatment, and removing fibrous tissue to obtain the small-particle active fat tissue. The invention uses ultrasonic equipment and adopts a pulse discontinuous working mode, and the activity of original tissues is still kept when the adipose tissues are treated. The method is simple, has high repeatability and is suitable for large-scale popularization and application. The prepared fat particles have finer particle size and more uniform tissues, can pass through an injection needle with the aperture of 0.6mm, and the cell size of the fat tissue is about 1/3-1/2 of the fat cell obtained by the traditional method, thereby greatly meeting the clinical requirements.

Description

Treatment method of small-particle active adipose tissues

Technical Field

The invention relates to the technical field of adipose tissue treatment, in particular to a method for treating small-particle active adipose tissues.

Background

Soft tissue defects are common diseases causing human morphology and dysfunction, but the reconstruction of soft tissue defects is always a difficult problem to solve due to the poor safety and effectiveness of artificially synthesized materials and the like. With the progress of medical science in recent years, people find that autologous adipose tissues are the best materials for soft tissue repair, have the advantages of sufficient sources, no biological rejection and the like, but the adipose tissue particles obtained by the traditional adipose extraction technology are large, and a 2-3mm injection needle is generally adopted for fat transplantation injection operation after the adipose tissue particles are extracted from the autologous tissues, so that the autologous adipose tissues are generally only filled in the tissue gaps such as subcutaneous tissues, adipose tissues and muscle gaps with loose tissues; in addition, the fat tissue particles extracted by the traditional method are thick, cannot pass through an injection needle with the diameter within 1mm, and is difficult to transplant and inject at a position with compact tissues such as the skin, but in clinical practical operation, the skin is often filled by fine injection with the diameter of 0.6-0.8mm, so that the problem of fine soft tissue defect is corrected. Therefore, the conventional fat transplantation technology cannot be widely applied in clinic.

The previous method of obtaining fine particles of fat requires the use of a custom-made suction tube 1mm in diameter to aspirate the fat, which is time consuming, inefficient to obtain, and significantly compromised in viability due to the extrusion of the adipose tissue within the fine needle cannula. Therefore, there is still a technical difficulty in obtaining fine fat particles efficiently.

Disclosure of Invention

The invention provides a method for treating small-particle active adipose tissues, which solves the technical problem of activity reduction in the process of preparing small-particle fat in the prior art and improves the obtaining efficiency.

The technical scheme of the invention is realized as follows:

a method of treating small particle, living adipose tissue, comprising:

taking large-particle adipose tissues;

placing the large-particle adipose tissues under a probe of a low-frequency ultrasonic instrument for acting for 1-1.5 minutes; the working condition of the low-frequency ultrasonic instrument is that the power is 80-95W, and the pulse is in an intermittent working mode;

filtering the fat tissue after the ultrasonic treatment, and removing fibrous tissue to obtain the small-particle active fat tissue.

As a preferable technical scheme, in the pulse intermittent working mode, the action time is 1.5-3 seconds. More preferably 2 seconds.

In a preferable technical scheme, in the pulse intermittent working mode, the pause time is 2.5-5 seconds. More preferably 3 seconds.

As a preferable technical scheme, the filter screen used for filtering is a metal filter screen, and the aperture is 1 mm.

As a preferable technical scheme, the frequency of the low-frequency ultrasonic instrument is 20 KHz.

As a preferred technical scheme, the small-particle active adipose tissues prepared by the method can pass through a 0.6 mm-diameter injection needle.

The prepared small-particle active adipose tissues are applied to fine injection.

Advantageous effects

(1) The invention uses ultrasonic equipment and adopts a pulse intermittent working mode, and the introduced interval time can avoid the damage of the heat effect generated during ultrasonic treatment on the tissues, thereby still retaining the activity of the original tissues when the adipose tissues are treated. Viable cell proliferation after tissue digestion and in vitro culture is seen in figure 1. Since the activity of the original tissue is preserved, the thin needle intradermal injection is realized while the survival rate of fat after transplantation is not affected.

(2) The method is simple, has high repeatability and is suitable for large-scale popularization and application.

(3) The fat particles prepared by the method have smaller particle size and more homogeneous tissues, and can pass through an injection needle with the aperture of 0.6mm, and the cell size of the fat tissue is about 1/3-1/2 of that of fat cells obtained by the traditional method, thereby greatly meeting the clinical requirements.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive exercise.

FIG. 1 is a diagram of a living cell culture obtained by digesting and centrifuging the small-grained adipose tissue of example 1.

FIG. 2 is a diagram of a culture of living cells obtained by digesting and centrifuging large granular adipose tissues according to example 1.

FIG. 3 is a staining chart of a small particle-size reactive adipose tissue section of example 1.

FIG. 4 is a staining diagram of a large-grained adipose tissue section obtained by a conventional method.

FIG. 5 is a graph showing the results of adipogenic staining of the cultured adipocytes of example 1; wherein A: adipogenic staining results of traditional adipose cultured cells; b: results of adipogenic staining of small particle live adipose cultured cells.

FIG. 6 shows the results of osteoblast staining of the cultured adipocytes of example 1; wherein A: osteogenic staining results of conventional adipose cultured cells; b: osteogenic staining results of small granular activated adipose cultured cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, 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.

The ultrasound apparatus used in the following examples is a relatively conventional general apparatus (commercially available) in medical and biological laboratories, which is simple and easy to carry and is inexpensive. The instrument is simple and convenient to operate and high in repeatability.

Example 1

A method of treating small particle, living adipose tissue, comprising:

(1) collecting 15ml of large-particle adipose tissues obtained by a traditional fat suction method;

(2) placing under a probe of a 20KHz ultrasonic instrument, acting for 1 minute, wherein the power is 95W, the working mode is a pulse discontinuous mode, and the acting time is 2s and the pause interval is 3 s;

(3) removing fibrous tissues in the adipose tissues of the fat treated by the ultrasonic instrument through a metal filter screen, wherein the aperture of the metal filter screen is 1 mm; the obtained small-particle active adipose tissue is more homogeneous and can pass through an injection needle with the aperture of 0.6 mm.

Validation of the activity of cells within the tissue:

the specific experimental method comprises the following steps: collecting 5ml of small-particle active adipose tissues, adding 5ml of 1% collagenase solution, placing the small-particle active adipose tissues in a constant temperature shaking table at 37 ℃ for 60min, placing the small-particle active adipose tissues in a centrifuge for centrifugation for 5min at 800g, collecting cell precipitates, and inoculating the cell precipitates to a culture dish for in-vitro cell culture after heavy suspension by using a cell culture solution. Cell growth was seen after incubation, see figure 1.

Control experimental method: collecting 5ml of adipose tissue obtained by a traditional method, adding 5ml of 1% collagenase solution, placing the mixture into a constant temperature shaking table at 37 ℃ for 60min, placing the mixture into a centrifuge for centrifugation for 5min at 800g, collecting cell precipitates, and inoculating the cell precipitates into a culture dish for in vitro cell culture after resuspension by using cell culture solution. Cell growth was seen after incubation, see figure 2.

Referring to FIG. 1: the small-grained adipose tissue obtained in this example was subjected to digestion and centrifugation to obtain a culture of viable cells, and it was confirmed that viable cells having proliferative capacity were present in the small-grained adipose tissue.

As can be seen from fig. 1 and 2, the small particle activated adipose tissue of the present embodiment does not destroy the activity of cells in the tissue by the ultrasonic treatment method, compared to the large particle fat extracted by the conventional method. Since the activity of the original tissue is preserved, the thin needle intradermal injection is realized while the survival rate of fat after transplantation is not affected.

In addition:

the small particle active adipose tissues obtained in the embodiment and cells obtained by culturing the adipose tissues obtained by the traditional method are subjected to cell induction adipogenesis experiments and osteogenesis experiments, and oil red staining is carried out after adipogenesis induction; alizarin red staining is carried out after osteogenic induction, and the staining result is observed under a microscope. See fig. 5-6 for experimental microscopic examination. From these figures it can be seen that: the cultured cells of fine-grained homogeneous fat obtained by the method have no obvious difference in the fat forming and bone forming abilities from the cultured cells of adipose tissue obtained by the traditional method.

And (3) obtaining time comparison:

the conventional method has a time of 3-5 minutes for sucking 15ml of large granular fat from the subcutaneous part of a patient through a fat suction tube having a diameter of 2-3mm, and a time of 2-3 minutes for obtaining 15ml of fine granular fat by subjecting the large granular fat to ultrasonic treatment for a total time of 5-8 minutes. Fine granular fat can also be obtained through a custom made 1mm diameter fat suction tube, but 15-20 minutes are required to suction 15ml of fine granular fat from the patient subcutaneously.

Example 2

A method of treating small particle, living adipose tissue, comprising:

(1) collecting 25ml of large-particle adipose tissues obtained by a traditional fat suction method;

(2) placing under 20KHz ultrasonic instrument probe, acting for 1.5 min, with power of 80W, working mode being pulse discontinuous mode, acting for 2s, and pausing for 5 s;

(3) removing fibrous tissues in the adipose tissues of the fat treated by the ultrasonic instrument through a metal filter screen, wherein the aperture of the metal filter screen is 1 mm. (ii) a The obtained small-particle active adipose tissue is more homogeneous and can pass through an injection needle with the aperture of 0.6 mm. The cell size of the adipose tissue is about 1/3-1/2 of the adipose cells obtained by the traditional method, and the clinical requirement is greatly met.

In other embodiments, the power of 80-95W, the action time of 1.5-3 seconds, and the pause time of 2.5-5 seconds may be selected by one skilled in the art in the pulse interruption mode of operation as desired. These data combinations all meet the requirements of the present invention. The obtained tissue is more homogeneous and can pass through the injection needle with the aperture of 0.6 mm. The cell size of adipose tissue is approximately 1/3-1/2 of adipocytes obtained by conventional methods.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for treating small particle reactive adipose tissue, comprising:

taking large-particle adipose tissues;

placing the large-particle adipose tissues under a probe of a low-frequency ultrasonic instrument for acting for 1-1.5 minutes; the working condition of the low-frequency ultrasonic instrument is that the power is 80-95W, and the pulse is in an intermittent working mode; in the pulse intermittent working mode, the action time is 1.5-3 seconds; the pause time is 2.5-5 seconds;

2. The method as claimed in claim 1, wherein the pulse interruption mode is performed for 2 seconds.

3. The method as claimed in claim 1, wherein the pause time in the pulse interruption mode is 3 seconds.

4. The method for treating small particle activated adipose tissue as claimed in claim 1, wherein the filtering mesh is a metal mesh with a pore size of 1 mm.

5. The method for treating small particle activated adipose tissue as claimed in claim 1, wherein the frequency of the low frequency ultrasound instrument is 20 KHz.

6. The method of claim 1, wherein the small-sized particles of the adipose tissue are prepared to pass through a 0.6mm diameter needle.

7. The method of claim 1, wherein the small-grained adipose tissue is applied to a fine injection.