CN114681674A

CN114681674A - Filling material for promoting blood vessel, collagen regeneration and adipose tissue repair, and preparation method and application thereof

Info

Publication number: CN114681674A
Application number: CN202210351631.2A
Authority: CN
Inventors: 刘宏伟; 丁煜勋; 林灿祥; 向科蓉; 卢金强; 陈芷菁; 陈若曦
Original assignee: First Affiliated Hospital of Jinan University
Current assignee: First Affiliated Hospital of Jinan University
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-07-01

Abstract

The invention provides a preparation method of a filling material for promoting blood vessel and collagen regeneration and adipose tissue repair, which is characterized in that adipose tissue is repeatedly frozen and thawed for a plurality of times; centrifuging the fully thawed adipose tissue; the adipose tissues are crushed evenly by violent stirring, and are centrifuged after standing and demulsification; treating flocculent solid obtained by centrifugation with TritonX-100 solution and sodium deoxycholate, and lyophilizing to remove water to obtain acellular matrix; mixing the acellular matrix, hyaluronic acid, sodium alginate and calcium chloride, grinding to obtain powder, and mixing with normal saline in proportion to obtain the acellular matrix/hyaluronic acid composite gel. The filling material prepared by the method can obviously improve the filling effect and has better functions of blood vessel regeneration, collagen regeneration, cell chemotaxis and the like.

Description

Filling material for promoting blood vessel, collagen regeneration and adipose tissue repair, and preparation method and application thereof

Technical Field

The invention relates to the field of medical cosmetic materials, in particular to a filling material for promoting blood vessel and collagen regeneration and adipose tissue repair, a preparation method and application thereof.

Background

The research and development of the human body filling material is one of the fields of the key development of the medical beauty treatment (hereinafter, medical beauty) industry in China. At present, medical and aesthetic filling materials mainly comprise fillers such as hyaluronic acid, polylactic acid, silica gel and the like. However, the filling material at the present stage has poor effect, which mainly comprises: the hyaluronic acid and polylactic acid are heterogeneous materials with single function, only play a role in filling, have no regeneration and repair function, need to be injected for many times after being degraded in vivo, and are expensive. Research has shown that adipose tissue regeneration and repair depends on the properties of the filling material such as promoting blood vessels, collagen regeneration and fat cell chemotaxis. The development of the filling material with the functions is a key link for promoting the further development of the medical and beauty industry.

Research shows that the acellular matrix is rich in a large number of growth chemotactic factors and has good angiogenesis and adipocyte chemotactic properties. Document CN108178868 discloses a method for preparing acellular matrix, but organic reagents such as isopropyl alcohol are used in the process, which easily destroy growth chemotactic factors and natural physical space structures in the acellular matrix, and influence the tissue regeneration performance of the acellular matrix. At present, acellular matrix preparation by a physical method is developed, but the obtained acellular matrix has overlarge particles and poor dispersibility, and is difficult to be clinically popularized as an injection filler. And the injected cell matrix can not reach a uniform dispersion system under the skin, thereby seriously influencing the filling plasticity effect. Therefore, it is necessary to develop an acellular matrix filling material which has good dispersion performance and can obviously promote the regeneration of tissue collagen, so as to be suitable for the development of the medical cosmetology industry.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide the filling material for promoting the regeneration of blood vessels and collagen and the repair of adipose tissues, and the preparation method and the application thereof.

The purpose of the invention is realized by the following technical scheme:

one of the purposes of the invention is to provide a preparation method of a filling material for promoting blood vessel, collagen regeneration and adipose tissue repair, which comprises the following steps:

repeatedly freezing and thawing adipose tissues for several times;

centrifuging the completely thawed adipose tissues to remove water and partial grease in the adipose tissues;

the adipose tissues are crushed evenly by violent stirring, and are centrifuged after standing and demulsification;

treating flocculent solid obtained by centrifugation with TritonX-100 solution and sodium deoxycholate, and lyophilizing to remove water to obtain acellular matrix;

mixing the acellular matrix, hyaluronic acid, sodium alginate and calcium chloride, grinding to obtain powder, and mixing with normal saline in proportion to obtain the acellular matrix/hyaluronic acid composite gel.

Further, the method comprises the following steps:

step 1) collecting and sub-packaging adipose tissues into a centrifuge tube, freezing for 20-30 minutes at-24 ℃, then unfreezing in a water bath at 37 ℃, and repeating the operation for 5-7 times;

step 2) centrifuging the completely thawed adipose tissues at the centrifugal rotation speed of 3000-4000 rpm for 10-15 minutes;

step 3) adding the adipose tissues centrifuged in the step 2) into a tissue pulverizer, pulverizing and dispersing the adipose tissues through violent stirring, extruding the grease out of the adipose tissues through stirring shearing force, wherein the stirring revolution is 3000rpm-4000rpm, and the stirring time is 5 minutes-10 minutes;

step 4) placing the adipose tissues uniformly stirred and crushed in the step 3) at 4 ℃, standing for 0.5-1 hour to remove emulsification, and then centrifuging at the rotating speed of 3000-4000 rpm for 5-10 minutes;

step 5) collecting the lower-layer flocculent solid obtained after centrifugation in the step 4), washing with physiological saline, adding 1% TritonX-100 solution, soaking for 5-10 minutes, centrifuging to remove the TritonX-100 solution, and washing with physiological saline;

step 6), adding 1% sodium deoxycholate, incubating for 5-10 minutes, centrifuging, collecting lower flocculent solids, and washing with normal saline;

step 7) putting the flocculent solid collected in the step 6) into a refrigerator at the temperature of-24 ℃ for freezing for 2-4 hours, and carrying out freeze-drying and dewatering by a freeze-drying machine to obtain a solid massive acellular matrix;

step 8) mixing 0.5g of the acellular matrix prepared in the step 7), 0.45g of hyaluronic acid, 0.05g of sodium alginate and 0.02g of calcium chloride, and grinding for 30-60 minutes by using a vibration ball mill at a grinding speed of 2000-3000 r/min, wherein the specification of grinding balls is 20 mm, and the sample size is less than 10 microns;

step 9) placing the powder obtained by grinding in the step 8) under an ultraviolet lamp for irradiating for 2-4 hours, sterilizing, and placing in a refrigerator at-24 ℃;

step 10) mixing the sterilized powder obtained in the step 9) with normal saline according to the ratio of 1: 5-1: 50, stirring uniformly, standing in a refrigerator at 4 ℃ for 24-36 hours, and removing bubbles in the gel to obtain the acellular matrix/hyaluronic acid composite gel.

Further, the adipose tissue in the step 1) is human adipose tissue or porcine adipose tissue.

Further, the hyaluronic acid in the step 8) is 180 ten thousand molecular weight.

Another object of the present invention is to provide a filling material for promoting blood vessel, collagen regeneration and adipose tissue repair, prepared according to the above method.

The invention also aims to provide the application of the filling material for promoting blood vessel, collagen regeneration and adipose tissue repair in soft tissue filling and medical cosmetic filling.

The invention has the outstanding effects that:

the preparation method of the filling material for promoting blood vessel and collagen regeneration and adipose tissue repair, disclosed by the invention, has the advantages that the acellular matrix is prepared by a physical method instead of a method using a chemical agent in the prior art, the growth chemotactic factor and the natural physical space structure in the acellular matrix are not damaged, the tissue regeneration performance of the acellular matrix is not influenced, the obtained acellular matrix has small particles, good dispersibility and good plastic filling effect, and can be clinically popularized as an injection filling agent. The filling material for promoting blood vessel and collagen regeneration and adipose tissue repair can replace the existing filling material, and simultaneously has the functions of regeneration and repair, reduces degradation in vivo and injection times, thereby reducing the treatment cost.

The following detailed description of the embodiments of the present invention is provided in connection with the examples to facilitate understanding and understanding of the technical solutions of the present invention.

Drawings

FIG. 1 is a graph showing the trend of the change in volume of hyaluronic acid gel, human adipose acellular matrix aqueous solution of comparative example 1 and human DAT/HA composite gel of example 1 after being injected subcutaneously into mice;

FIG. 2 is a graph showing the results of B-mode ultrasound detection in each group of hyaluronic acid gel, human adipose acellular matrix aqueous solution of comparative example 1 and human DAT/HA composite gel of example 1 after 2 months of injection;

FIG. 3 is the anatomical morphology of the human-derived adipose acellular matrix aqueous solution of comparative example 1 and the human-derived DAT/HA composite gel of example 1 after 2 months of injection;

FIG. 4 is a comparison graph of morphology of the human-derived adipose acellular matrix aqueous solution of comparative example 1 and the human-derived DAT/HA composite gel of example 1 before and after injection under an electron microscope;

FIG. 5 is a graph comparing HE staining, Masson staining and immunohistochemical staining of injections of each group 2 months after injection of the aqueous solution of the human adipose acellular matrix of comparative example 1 and the human DAT/HA complex gel of example 1;

fig. 6 is liver and kidney function index diagrams of various groups of mice after hyaluronic acid gel, human-derived adipose acellular matrix aqueous solution of comparative example 1 and human-derived DAT/HA composite gel of example 1 are injected.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in 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 of the embodiments.

Example 1: preparation of human source acellular matrix/hyaluronic acid composite gel

The preparation method of this example includes the following steps:

step 1) collecting and sub-packaging human adipose tissues into a 50ml centrifuge tube, freezing for 20 minutes at-24 ℃, then unfreezing in a 37 ℃ water bath condition, and repeating the operation for 5 times;

step 2) centrifuging the completely thawed adipose tissues at the centrifugal rotating speed of 3000rpm for 15 minutes;

step 3) adding the adipose tissues centrifuged in the step 2) into a tissue pulverizer, pulverizing and dispersing the adipose tissues through violent stirring, extruding the grease out of the adipose tissues through stirring shearing force, wherein the stirring revolution is 3000rpm, and the stirring time is 10 minutes;

step 4) placing the adipose tissues uniformly stirred and crushed in the step 3) at 4 ℃, standing for 0.5 hour to remove emulsification, and then centrifuging at the rotating speed of 3000rpm for 10 minutes;

step 5) collecting the lower-layer flocculent solid obtained after centrifugation in the step 4), washing the lower-layer flocculent solid with physiological saline for 3 times, adding 1% TritonX-100 solution to soak the flocculent solid for 5 minutes, centrifuging the flocculent solid to remove the TritonX-100 solution, and washing the flocculent solid with the physiological saline for 3 times;

step 6), adding 1% sodium deoxycholate, incubating for 5 minutes, centrifuging, collecting lower-layer flocculent solids, and washing for 3 times by using normal saline;

step 7) putting the flocculent solid collected in the step 6) into a refrigerator at the temperature of-24 ℃ for freezing for 2 hours, and carrying out freeze-drying and dewatering by a freeze-drying machine to obtain a solid massive acellular matrix;

step 8) mixing 0.5g of the acellular matrix prepared in the step 7), 0.45g of hyaluronic acid (180 ten thousand molecular weight), 0.05g of sodium alginate and 0.02g of calcium chloride, and grinding for 30 minutes by using a vibration ball mill at a grinding speed of 2000 revolutions per minute, wherein the specification of a grinding ball is 20 millimeters, and the sample size is less than 10 micrometers;

step 9) spreading the powder obtained by grinding in the step 8) in a culture dish, placing under an ultraviolet lamp for 2 hours, sterilizing, and placing in a refrigerator at-24 ℃;

step 10) mixing the sterilized powder obtained in the step 9) with normal saline according to the ratio of 1: 5, stirring uniformly, standing in a refrigerator at 4 ℃ for 24 hours, and removing air bubbles in the gel to obtain the acellular matrix/hyaluronic acid composite gel (DAT/HA composite gel). The bubble-free DAT/HA composite gel was packaged in 1 mL/vial and refrigerated at 4 ℃ for use.

Example 2: preparation of pig source acellular matrix/hyaluronic acid composite gel

The preparation method of this example includes the following steps:

step 1) collecting and sub-packaging pig-derived adipose tissues into 50ml centrifuge tubes, freezing for 30 minutes at-24 ℃, then unfreezing in a 37 ℃ water bath condition, and repeating the operation for 7 times;

step 2) centrifuging the completely thawed adipose tissues at the rotation speed of 4000rpm for 10 minutes;

step 3) adding the adipose tissues centrifuged in the step 2) into a tissue pulverizer, pulverizing and dispersing the adipose tissues through violent stirring, extruding the grease out of the adipose tissues through stirring shearing force, wherein the stirring revolution is 4000rpm, and the stirring time is 5 minutes;

step 4) placing the adipose tissues uniformly stirred and crushed in the step 3) at 4 ℃, standing for 1 hour to remove emulsification, and then centrifuging at the rotating speed of 4000rpm for 5 minutes;

step 5) collecting the lower-layer flocculent solid obtained after centrifugation in the step 4), washing the lower-layer flocculent solid with physiological saline for 3 times, adding 1% TritonX-100 solution to soak the flocculent solid for 10 minutes, centrifuging the flocculent solid to remove the TritonX-100 solution, and washing the flocculent solid with the physiological saline for 3 times;

step 6), adding 1% sodium deoxycholate, incubating for 10 minutes, centrifuging, collecting lower-layer flocculent solids, and washing for 3 times by using normal saline;

step 7) freezing the flocculent solid collected in the step 6) in a refrigerator at the temperature of-24 ℃ for 4 hours, and performing freeze-drying and dewatering by using a freeze dryer to obtain a solid massive acellular matrix;

step 8) mixing 0.5g of the acellular matrix prepared in the step 7), 0.45g of hyaluronic acid (180 ten thousand molecular weight), 0.05g of sodium alginate and 0.02g of calcium chloride, and grinding for 60 minutes by using a vibration ball mill at the grinding speed of 3000 revolutions per minute, wherein the specification of grinding balls is 20 millimeters, and the sample size is less than 10 micrometers;

step 9) spreading the powder obtained by grinding in the step 8) in a culture dish, placing under an ultraviolet lamp for 4 hours, sterilizing, and placing in a refrigerator at-24 ℃;

step 10) mixing the sterilized powder obtained in the step 9) with normal saline according to the ratio of 1: 50, stirring uniformly, standing in a refrigerator at 4 ℃ for 36 hours, and removing bubbles in the gel to obtain the acellular matrix/hyaluronic acid composite gel (DAT/HA composite gel). The bubble-free DAT/HA composite gel was packaged in 1 mL/vial and refrigerated at 4 ℃ for use.

Comparative example 1: preparation of human-derived adipose acellular matrix (DAT) solution

This example differs from example 1 in that hyaluronic acid (180 ppm molecular weight), sodium alginate and calcium chloride were not added in step 8), and the others were identical to example 1.

Comparative example 2: preparation of porcine-derived fat acellular matrix (DAT) solution

This example differs from example 2 in that hyaluronic acid (180 ppm molecular weight), sodium alginate and calcium chloride were not added in step 8), and the others were identical to example 2.

Examples of the experiments

The human DAT/HA composite gel filling effect evaluation experiment and the experimental results in the embodiment 1 of the invention are as follows:

1. tendency of volume change of filler:

the hyaluronic acid gel content of each group was 2.5%, and the fat acellular matrix content was 2.5%, as measured by measuring the volume change of the injection every 1 week by injecting 1ml of Hyaluronic Acid (HA) gel, human fat acellular matrix (DAT) aqueous solution (comparative example 1), and human DAT/HA complex gel (example 1) into the skin on the back of the mouse, respectively.

The experimental results are as follows:

fig. 1 shows that the volumes of the materials injected into the back skin are similar, hyaluronic acid is basically absorbed by the body after being injected into the skin for 3 weeks, the human fat acellular matrix aqueous solution (comparative example 1) and the human DAT/HA complex gel (example 1) have a temporary volume reduction due to water loss in the first 3 weeks after injection, and then gradually increase due to physiological processes such as chemotactic cell generation and collagen secretion. And the human DAT/HA composite gel (example 1) HAs a pore structure, so that the cell chemotactic growth is more facilitated, and the corresponding volume of the human DAT/HA composite gel is higher than that of the human fat acellular matrix aqueous solution (comparative example 1).

2. And (3) observing the morphology of the filler:

after 2 months of injection of the hyaluronic acid gel, the human adipose acellular matrix aqueous solution (comparative example 1) and the human DAT/HA composite gel (example 1), the morphology and size of each group of injection materials were observed by B-ultrasound.

The experimental results are as follows:

the results in fig. 2 show that no graft retention is seen at the site of hyaluronic acid gel injection under ultrasonography B, which indicates that hyaluronic acid gel is completely absorbed by the body; after 2 months of injecting the human-derived fat acellular matrix aqueous solution (comparative example 1), the center of the B-ultrasonic downward-moving plant presents uneven echo, the injected substance presents a block shape and forms an irregular low-echo-shadow liquid sac cavity, and the periphery of the graft is formed by a high-echo-shadow envelope; after 2 months of injection of the human DAT/HA complex gel (example 1), the B-ultrasound injection showed a uniform flat echogenic mass image, fused with subcutaneous tissue and dorsal musculature, with clear borders and no envelope around. The experimental result shows that the pure hyaluronic acid gel is easy to digest and absorb by organisms and does not have a long-term filling effect, the human-derived fat acellular matrix aqueous solution (comparative example 1) is not easy to digest and HAs long filling time, but the human-derived DAT/HA composite gel (example 1) can improve the dispersibility of the fat acellular matrix and prevent aggregation in the filling process through the hyaluronic acid gel, and the filling effect is optimal.

3. Evaluation of the effect of angiogenesis:

after 2 months of injection of hyaluronic acid gel, human-derived fat acellular matrix aqueous solution (comparative example 1) and human-derived DAT/HA composite gel (example 1), the injection is surgically excised, and the morphology, size and blood vessel growth status of each group of injection materials are observed.

The experimental results are as follows:

FIG. 3 shows that hyaluronic acid is completely absorbed by the body, and the human adipose acellular matrix aqueous solution (comparative example 1) is white spherical tissue, hard in texture, provided with a significant envelope, and has blood vessels crawling on the spherical surface, which can promote the regeneration of blood vessels. The human DAT/HA complex gel (example 1) presents a yellowish-brown opaque fat pad-like tissue, is soft, HAs no obvious envelope, and HAs microangiogenesis on the surface of the filler and grows into the tissue. The human DAT/HA composite gel (example 1) is proved to have good function of promoting angiogenesis and the best filling effect.

4. Evaluation of cell chemotactic Effect:

scanning electron microscope samples are prepared by the prepared human-derived adipose acellular matrix aqueous solution (comparative example 1) and human-derived DAT/HA composite gel (example 1) and filling after 2 months of injection, and the microscopic morphology and the cell abundance of the gel are observed by the scanning electron microscope.

The experimental results are as follows:

FIG. 4 shows that the aqueous solution of human adipose acellular matrix (comparative example 1) before injection has a microscopic morphology of filaments, and has partial aggregation and nonuniform dispersion. The human DAT/HA complex gel (example 1) was uniformly dispersed and no significant blocky aggregates were present. After injection, the human-derived fat acellular matrix aqueous solution (comparative example 1) had a small number of spherical objects (cells) on the filler, but the number was small, and the dispersion was not uniform, whereas the human-derived DAT/HA composite gel (example 1) had a large number of spherical objects (cells) on the filler, and the spherical objects (cells) were uniformly dispersed. Compared with the human adipose acellular matrix aqueous solution (comparative example 1), the human DAT/HA complex gel (example 1) is more beneficial to the growth of cells and HAs stronger cell chemotactic function.

5. Microscopic adipocyte chemotaxis and regeneration, collagen and angiogenesis effect evaluation:

after 2 months of injection of the human-derived adipose acellular matrix aqueous solution (comparative example 1) and the human-derived DAT/HA complex gel (example 1), the material microcontaining effect was observed by HE staining, Masson staining, and immunohistochemical staining, respectively.

The experimental results are as follows:

FIG. 5 shows that HE sections after injection of human adipose acellular matrix aqueous solution (comparative example 1) showed significant envelope formation, accumulation of irregular eosinophilic staining mass in the bursa, massive inflammatory cell infiltration and debris residue; after the human DAT/HA composite gel (example 1) is injected, no obvious envelope is formed, a small amount of inflammatory cells infiltrate into the center of the graft, obvious adipocyte generation and aggregation are realized, and eosinophilic staining extracellular matrix is densely arranged and relatively regular. Masson staining showed that human adipose acellular matrix in water (comparative example 1) had significant collagen fiber formation in the graft envelope capsule, and the density of arrangement of collagen fibers in the capsule was loose and not closely related. The human DAT/HA composite gel (example 1) injected is free from the formation of a capsule, the outer edge of the gel HAs obvious angiogenesis (arrow), collagen fibers are regularly and orderly arranged, collagen in the center of the graft is relatively less, and the collagen is infiltrated by more macrophages. Immunohistochemical staining showed that injection of human adipose acellular matrix aqueous solution (comparative example 1) had only a small amount of small angiogenesis (arrows) staining positive for a-SMA in the envelope, without any angiogenesis in the capsule. Injection of human DAT/HA complex gel (example 1) the graft exhibited diffuse small angiogenesis (arrows) and apparently vascular luminal formation in the center of the graft.

6. DAT/HA Complex gel safety assessment:

after hyaluronic acid gel, human adipose acellular matrix aqueous solution (comparative example 1) and human DAT/HA composite gel (example 1) were injected, blood of each group of mice was collected, lactate dehydrogenase LDH, glutamic pyruvic transaminase ALT and serum creatinine Scr which reflect the functions of the heart, liver and kidney of the body were detected, and the safety of the material was verified.

The experimental results are as follows:

the results in fig. 6 show that there is no obvious functional damage to heart, liver and kidney of mice injected with hyaluronic acid gel, human fat acellular matrix aqueous solution (comparative example 1) and human DAT/HA composite gel (example 1), and there is no statistical difference in functional indexes of heart, liver and kidney, such as LDH, ALT and Scr, of each group, which are within the range of heart, liver and kidney functional indexes of mice.

In conclusion, the DAT/HA composite gel is prepared by a physical method, shows excellent regeneration performance compared with common hyaluronic acid and fat acellular matrix in the aspects of volume after injection, anatomical morphology, under-lens observation, B-ultrasonic display, immunofluorescence, liver and kidney functions and the like, HAs small particles, better dispersibility and good plastic filling effect, and is a high-quality novel medical and beauty filling product.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A preparation method of a filling material for promoting blood vessel, collagen regeneration and adipose tissue repair is characterized by comprising the following steps:

repeatedly freezing and thawing the adipose tissue for several times;

centrifuging the fully thawed adipose tissue;

2. A method for preparing a filling material for promoting blood vessel, collagen regeneration and adipose tissue repair according to claim 1, comprising the steps of:

step 3) adding the adipose tissues centrifuged in the step 2) into a tissue pulverizer, wherein the stirring speed is 3000rpm-4000rpm, and the stirring time is 5 minutes-10 minutes;

step 5) collecting the lower-layer flocculent solid obtained after centrifugation in the step 4), washing with physiological saline, adding 1% Triton X-100 solution, soaking for 5-10 minutes, centrifuging to remove the Triton X-100 solution, and washing with physiological saline;

step 10) mixing the sterilized powder obtained in the step 9) with normal saline according to the ratio of 1: 5-1: 50, stirring uniformly, and standing in a refrigerator at 4 ℃ for 24-36 hours to obtain the acellular matrix/hyaluronic acid composite gel.

3. The method for preparing a filling material for promoting blood vessel, collagen regeneration and adipose tissue repair according to claim 1, wherein: the adipose tissue in the step 1) is human adipose tissue or porcine adipose tissue.

4. A method for preparing a filling material for promoting blood vessel, collagen regeneration and adipose tissue repair according to claim 2, wherein: the hyaluronic acid in step 8) was 180 ppm molecular weight.

5. A filling material for promoting blood vessel, collagen regeneration and adipose tissue repair prepared by the method according to any one of claims 1 to 4.

6. Use of the filling material for promoting blood vessel, collagen regeneration and adipose tissue repair according to claim 7 in soft tissue filling and medical cosmetic filling.