CN116251236A - A kind of biological breast patch and preparation method thereof - Google Patents

Abstract

The invention discloses a biological breast patch and a preparation method thereof. The preparation method of the biological breast patch comprises the following steps: 1) Collecting dermis of mammal, cleaning with water, and filtering; 2) Carrying out virus inactivation treatment on the skin tissue treated in the step 1); 3) Degreasing the skin tissue treated in the step 2); 4) Performing decellularization treatment on the skin tissue treated in the step 3) to obtain a decellularized dermis matrix; 5) Immersing the acellular dermal matrix obtained in the step 4) in an aqueous solution of sodium hyaluronate, and taking out; 6) And 5) freeze-drying the acellular dermal matrix treated in the step 5) to obtain the acellular dermal matrix. The biological breast patch adopts the acellular dermal matrix material, can guide the host autologous cells to grow in, provides a template for reconstructing damaged tissues by cells, can be rapidly vascularized, and gradually degrades along with the construction of the new tissues of the host until the new tissues of the host are completely replaced by the host tissues.

Description

A kind of biological breast patch and preparation method thereof

technical field

The invention belongs to the technical field of tissue engineering, and in particular relates to a biological breast patch and a preparation method thereof.

Background technique

Breast reconstruction for breast cancer patients with indications has been widely recognized at home and abroad. Implant breast reconstruction has become the most commonly used breast reconstruction method due to its advantages of short operation time, fast postoperative recovery, no donor site damage, and safety and reliability. .

The first condition for breast reconstruction with implants is to cover as much tissue as possible on the surface of the implanted prosthesis, but mastectomy will cause a large amount of muscle loss, and the remaining autologous tissue often cannot achieve the effect of effectively wrapping and supporting the prosthesis. Complications such as sagging or even exposure of the prosthesis will occur. To address the lack of prosthetic coverage, some biomaterial products (such as acellular dermal matrix, bovine pericardium, etc.) or synthetic materials (such as titanium-coated polypropylene mesh, etc.) are increasingly used in breast implants Reconstructive surgery, in which the breast is reconstructed with or without muscle tissue to cover the implant. Among the currently marketed patch products for breast reconstruction in China, a cross-linking agent is introduced in the preparation process of the patch made of pericardium, which hinders its degradation in the body while changing the natural structure of the material; The synthetic patch will exist in the patient's body for a long time because it is not degradable itself, which will increase the psychological burden of the patient.

Invention patent (CN100372511C) discloses a decellularized dermal matrix and its preparation method. It introduces the peeling of animal skin, cleaning, virus inactivation, immunogen removal, fat removal, molding, packaging and sterilization to obtain different sizes and specifications. medical products. As a natural medical material for tissue transplantation, the acellular dermal matrix can guide autologous cells to grow in and provide a template for cells to rebuild damaged tissues. At the same time, the acellular dermal matrix rapidly vascularizes and builds with host new tissue And gradually degrade until it is completely replaced by host tissue. However, there are few reports on acellular dermal matrix materials used in breast reconstruction in China.

Contents of the invention

The purpose of the present invention is to provide a biological breast patch and its preparation method. The biological breast patch can guide the growth of the host's own cells, provide a template for the cells to rebuild damaged tissues, and can rapidly vascularize, and with The host's new tissue is constructed and gradually degraded until it is completely replaced by the host tissue.

A kind of preparation method of biotype breast patch provided by the invention comprises the following steps:

1) Take the dermis of the mammal, wash it with water and filter it dry;

2) performing virus inactivation treatment on the skin tissue treated in step 1);

3) Degreasing the skin tissue treated in step 2);

4) performing decellularization on the skin tissue treated in step 3) to obtain decellularized dermal matrix;

5) Soak the acellular dermal matrix obtained in step 4) in an aqueous solution of sodium hyaluronate, and take it out;

6) The acellular dermal matrix treated in step 5) is freeze-dried to obtain the biological breast patch.

In a specific embodiment of the present invention, the mammal is a cow; the dermis is obtained by peeling off the surface skin and subcutaneous tissue.

In the above method, the virus inactivation treatment described in step 2) includes: soaking the skin tissue with an aqueous solution of hydrogen peroxide, and cleaning after soaking;

The mass concentration of the aqueous solution of hydrogen peroxide may be 0.06-0.1 g/ml, specifically 0.06 g/ml;

The soaking time may be 1 to 1.5 hours, specifically 1.5 hours;

The cleaning can be ultrasonic cleaning.

In the above method, the degreasing treatment described in step 3) includes: soaking and treating the skin tissue with a mixed solution composed of chloroform and ethanol, and cleaning after soaking;

In the mixed solution, the volume ratio of chloroform and ethanol may be 1:(1-5), specifically 1:3;

The soaking time may be 30 to 60 minutes, specifically 45 minutes;

The cleaning can be ultrasonic cleaning.

In the above method, the decellularization treatment in step 4) includes: soaking the skin tissue in an aqueous solution of an alkaline reagent, and cleaning after soaking;

The alkaline reagent can be NaOH or KOH;

The concentration of the alkaline reagent in the aqueous solution of the alkaline reagent may be 0.5-5.5 mol/L, specifically 3 mol/L;

The soaking time may be 30 to 60 minutes, specifically 30 minutes;

The cleaning can be ultrasonic cleaning;

The number of times of the decellularization treatment may be 3 to 5 times, specifically 3 times.

In the above method, the concentration of the aqueous solution of sodium hyaluronate in step 5) can be 0.01 to 0.1 g/ml, and the hydration time of the sample after compounding sodium hyaluronate is significantly shortened. The concentration of the aqueous solution is preferably 0.03-0.1g/ml, more preferably 0.03-0.05g/ml or 0.05-0.1g/ml, further preferably 0.03g/ml, 0.05g/ml, 0.1g/ml;

The immersion in step 5) includes the first immersion and the second immersion after the first immersion is completed and the acellular dermal matrix is turned over, and the time of the first immersion and the second immersion can be 10 to 20min (such as 10min).

In the above-mentioned method, freeze-drying described in step 6) may comprise the following steps:

a) Spread the sample flat on the freeze-drying tray and place it on the partition in the freeze-drying machine;

b) Product pre-freezing: Turn on the freeze dryer, first cool the box down to -50°C, and start at least 1 hour since the cooling;

c) Vacuuming: After pre-freezing, turn on the vacuum pump, and the vacuum degree of the box will drop and remain below 20Pa;

d) The first drying: the temperature of the box rises to -30°C, and enters the temperature maintenance stage after the end of the temperature rise. The total time from the start of the temperature rise is 3 hours;

e) The second drying: the temperature of the box continues to rise to -15°C, and enters the temperature maintenance stage after the end of the temperature rise. The total time from the start of the temperature rise is 1h;

f) The third drying: the temperature of the box continues to rise to 0°C, and enters the temperature maintenance stage after the end of the temperature rise. The total time from the start of the temperature rise is 1h;

g) The fourth drying: the temperature of the box continues to rise to 25°C, and enters the temperature maintenance stage after the temperature rise is completed, and the total time since the start of the temperature rise is at least 1h;

h) After the freeze-drying is completed, the sample is taken out.

Further, after step 6), the method may further include: sub-cutting the freeze-dried acellular dermal matrix; and,

Perforating the cut out acellular dermal matrix.

Still further, the method further includes the steps of packaging, sterilizing and analyzing in sequence after the punching process;

The sterilization is ethylene oxide sterilization or radiation sterilization.

Preferably, the conditions for ethylene oxide sterilization are as follows: the temperature is 37°C-47°C (42±5°C), the humidity is 30%-80%, the concentration of ethylene oxide is 500 mg/L, and the sterilization time is 3 to 5 hours (such as 4 hours);

Preferably, the desorption is carried out in a desorption oven at a temperature of 37°C-47°C (ie 42°C±5°C), specifically 42°C, for 4-6 days, specifically 6 days.

The biological breast patch prepared by any of the methods described above is also within the protection scope of the present invention.

In the above biological breast patch, the biological breast patch can be rectangular or crescent-shaped;

The diameter of the through holes in the biological breast patch is 0.5-2.5 mm, and the distance between two adjacent through holes can be 1-2 cm; the through holes can be specifically round holes;

The diameter of the through holes in the biological breast patch can be 0.5-2.5 mm (such as 2 mm), and the distance between two adjacent through holes can be 1-2 cm (such as 2 cm);

The biological breast patch is crescent-shaped, and the through holes are distributed in the middle of the biological breast patch.

The breast patch of the present invention is an acellular dermal matrix material and has the following advantages:

(1) The breast patch provided by the present invention has been effectively processed for virus inactivation and antigen removal, eliminated the risk of spreading zoonotic diseases, has excellent biocompatibility, and has little rejection in the implanted body;

(2) The breast patch provided by the present invention does not use a fixative in the process of processing, and introduces sodium hyaluronate. The compound sodium hyaluronate increases the hydration speed and effect of the material on the one hand, and can also promote Proliferation and differentiation of host cells in the acellular dermal matrix, which is conducive to tissue regeneration;

(3) The breast patch provided by the present invention is processed by vacuum freeze-drying technology, which retains the natural loose porous structure of the acellular dermal matrix to the greatest extent, and can effectively guide cell growth and tissue regeneration;

(4) The surface of the breast patch provided by the present invention is perforated, and the "crescent-shaped" specification is designed in combination with clinical needs, which can avoid the occurrence of complications such as seroma caused by surgical effusion, and greatly reduce clinical symptoms. Doctor's pruning process;

(5) The breast patch provided by the present invention is sterilized by ethylene oxide, which greatly reduces the damage of radiation sterilization to the collagen fiber bundle of the material, increases the mechanical strength of the patch, and prolongs the degradation time, and is more suitable for breast implants reconstructive surgery.

Description of drawings

Fig. 1 is a schematic diagram of perforating a breast patch in Example 1 of the present invention, and the shapes of the patches are rectangle (A) and crescent (B).

Fig. 2 is the physical photograph of the wet state (A) and dry state (B) of the breast patch in Example 1 of the present invention.

Fig. 3 is a scanning electron microscope image of the smooth surface (A), the rough surface (B) and the section (C) of the breast patch in Example 1 of the present invention.

Fig. 4 is an HE staining image of the breast patch in Example 1 of the present invention.

Fig. 5 is a graph of HE staining results of the breast patch implanted in the leg muscles of New Zealand white rabbits in Example 1 of the present invention for 12 weeks.

Fig. 6 is a comparison chart of test data of tensile strength and tearing force of breast patches prepared in Example 1 and Example 2 of the present invention respectively.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials and reagents used can be obtained from commercial sources.

Embodiment 1, prepare biotype breast patch through ethylene oxide sterilization

1. Raw material processing: thaw the cowhide, peel off the surface skin and subcutaneous tissue, only keep the dermis, wash the skin with purified water and drain the water;

2. Virus inactivation: Soak the skin tissue in 0.06g/ml hydrogen peroxide solution for 1.5h, and clean it in an ultrasonic cleaner after soaking;

3. Degreasing treatment: soak the skin tissue in a mixed solution containing chloroform:ethanol at a ratio of 1:3 (v/v) for 45 minutes, and clean it in an ultrasonic cleaner after soaking;

4. Decellularization treatment: soak the skin tissue in 3mol/L NaOH solution for 30 minutes, and then wash it in an ultrasonic cleaner after soaking. Repeat this step 3 times to obtain the decellularized dermal matrix;

5. Compounding of active substances: soak the acellular dermal matrix in 0.03g/ml sodium hyaluronate solution for 10 minutes, turn over the acellular dermal matrix after soaking, and continue soaking for 10 minutes to make it compound on the acellular dermal matrix;

6. Freeze-drying: spread the acellular dermal matrix obtained in step (5) on a stainless steel freeze-drying tray, then place it in a vacuum freeze dryer, and perform freeze-drying according to a preset procedure to obtain a freeze-dried acellular dermal matrix Material;

The freeze-drying procedure is:

a) Spread the sample flat on the freeze-drying tray and place it on the partition in the freeze-drying machine;

b) Product pre-freezing: Turn on the freeze dryer, first cool the box down to -50°C, and start at least 1 hour since the cooling;

c) Vacuuming: After pre-freezing, turn on the vacuum pump, and the vacuum degree of the box will drop and remain below 20Pa;

d) The first drying: the temperature of the box rises to -30°C, and enters the temperature maintenance stage after the end of the temperature rise. The total time from the start of the temperature rise is 3 hours;

e) The second drying: the temperature of the box continues to rise to -15°C, and enters the temperature maintenance stage after the end of the temperature rise. The total time from the start of the temperature rise is 1h;

f) The third drying: the temperature of the box continues to rise to 0°C, and enters the temperature maintenance stage after the end of the temperature rise. The total time from the start of the temperature rise is 1h;

g) The fourth drying: the temperature of the box continues to rise to 25°C, and enters the temperature maintenance stage after the temperature rise is completed, and the total time since the start of the temperature rise is at least 1h;

h) After the freeze-drying is completed, the sample is taken out.

7. Splitting and cutting: the freeze-dried acellular dermal matrix material is cut according to the size and specifications required by the product. According to the clinical application requirements of breast reconstruction, the present invention specially designs a crescent-shaped product, as shown in FIG. 1 . During the use of the crescent-shaped product, the upper short arc is sutured with the patient's pectoralis major, and the lower long arc is sutured with the patient's chest wall, which greatly reduces the clinician's operating time and reduces the risk of infection caused by trimming the patch;

8. Hole punching and packaging: use a special puncher to punch holes in the divided breast patch with the parameters of a round hole diameter of 2 mm and a spacing of 2 cm. The punching effect is shown in Figure 1 (A) and Figure 1 (B). Show. For crescent-shaped products, the through holes are mainly distributed in the middle where fluid is easy to accumulate, and the upper and lower ends of the patch that need to be sutured are not perforated to ensure that the mechanical properties of the patch are not affected. The amount of operations performed by Chinese doctors. After the punching is completed, the breast patch is packaged; it is packaged with blister & Tyvek lid material, which requires aseptic transfer and operation.

9. Sterilization analysis: The material is sterilized and analyzed by ethylene oxide. The sterilization conditions are: temperature 42±5°C, humidity 30%-80%, ethylene oxide concentration 500mg/L, sterilization time is 4 hours; analysis process: In a dedicated analysis oven, the temperature is controlled at 42°C for 6 days to obtain breast patches.

Embodiment 2, prepare biotype breast patch through irradiation sterilization

1. Raw material processing: the same as 1 in Example 1.

2. Virus inactivation: same as 2 of embodiment 1.

3. Degreasing treatment: same as 3 of embodiment 1.

4. Decellularization treatment: the same as 4 in Example 1.

5. Compounding of active substances: the same as 5 in Example 1.

6. Freeze-drying: the same as 6 in Example 1.

7. Sub-cutting and cutting: the same as 7 of embodiment 1.

8, punching, packaging: the same as 8 of embodiment 1.

9. Sterilization: The product is sterilized with e-book radiation after punching and packaging, and the radiation dose is 15KGy.

Embodiment 3, preparation of biological breast patch compounded by different concentrations of active substances

1. Raw material processing: the same as 1 in Example 1.

2. Virus inactivation: same as 2 of embodiment 1.

3. Degreasing treatment: same as 3 of embodiment 1.

4. Decellularization treatment: the same as 4 in Example 1.

5. Combination of active substances: soak the acellular dermal matrix in 0, 0.01, 0.03, 0.05 and 0.1g/ml sodium hyaluronate solution for 10 minutes, turn over the acellular dermal matrix after soaking, and continue soaking for 10 minutes, so that It is complexed to the acellular dermal matrix;

6. Freeze-drying: the same as 6 in Example 1.

7. Sub-cutting and cutting: the same as 7 of embodiment 1.

8, punching, packaging: the same as 8 of embodiment 1.

9. Sterilization Analysis: Same as 9 in Example 1.

Embodiment 4, the detection of the biotype breast patch of different sterilization methods, the biotype breast patch compounded by different concentrations of active substances

1. Detection of biological breast patch sterilized by ethylene oxide

The schematic diagram of before and after hydration of the breast patch in Example 1 is shown in FIG. 2 . The breast patch processed by vacuum freeze-drying technology removes most of the free water in the material, greatly reduces the risk of bacterial contamination, and is more conducive to long-term storage.

The microstructure of the breast patch in embodiment 1 is shown in the scanning electron microscope figure of accompanying drawing 3, as can be seen from Fig. 3, the breast patch prepared by the present invention has smooth surface (Fig. 3A), rough surface (Fig. 3B) and section (Fig. 3C ), the arrangement of collagen fiber bundles on the smooth surface is relatively more orderly and compact, while the arrangement of collagen fiber bundles on the rough surface is relatively sparse.

The cell removal effect of the breast patch in embodiment 1 is shown in accompanying drawing 4, as can be seen from Fig. 4, the breast patch prepared by the present invention completely removes all kinds of cells in the skin tissue, greatly reduces the immunogenicity of the material, and ensures Implantation of the breast patch into the patient does not elicit an immune response from the host.

In Example 1, the breast patch was implanted into the leg muscles of New Zealand white rabbits, and samples were taken after 12 weeks of implantation, and stained with hematoxylin-eosin (HE). The results are shown in Figure 5. It can be seen from Figure 5 that the material is relatively complete, the collagen fiber structure of the material under the microscope is intact, fibroblasts and fibroblasts can be seen between the collagen fibers, and the degree of inflammatory reaction is extremely low. The above phenomenon shows that the breast patch prepared by the present invention can be maintained in the body for a long time and play a role of long-term mechanical support. In addition, it can effectively induce surrounding host cells and tissues to grow into it, and realize the integration of materials and tissues.

2. Testing the mechanical properties of biological breast patches with different sterilization methods

The breast patches prepared by different sterilization methods in Example 1 and Example 2 were tested for mechanical properties such as tensile strength and tearing force. According to the method stipulated in GB/T 1040.3-2006 "Determination of Tensile Properties of Plastics Part 3: Test Conditions for Films and Sheets", the sample is cut into a dumbbell shape with a width of about 10mm and a length of about 30mm, purified water For hydration, use a universal testing machine to test, set the speed of the testing machine to 25mm/min, and test the tensile strength respectively. Set the speed of the testing machine to 100mm/min, and test the tearing force respectively. The test results are shown in Table 1 and Figure 6.

Table 1 Comparison of mechanical properties of breast patch with different sterilization methods

Sterilization method Tensile strength/MPa Tear force/N Irradiation sterilization 25.09±6.25 16.50±2.39 Ethylene oxide sterilization 24.92±7.59 28.07±3.56

It can be seen from Table 1 and Figure 6 that the tensile strengths of breast patches sterilized by ethylene oxide and radiation sterilized were 24.92±7.59 and 25.09±6.25 respectively, with no significant difference; The tear strength of the breast patch can reach 28.07±3.56, while the tear strength of the irradiated breast patch is only 16.50±2.39. The reason for this result may be that irradiation sterilization will break the hydrogen bonds that maintain the orderliness of the collagen structure between some collagen molecules, destroying the orderliness of the collagen structure, and the tearing force that is most affected by this is significantly reduced, while epoxy Ethane sterilization does not have any effect on collagen fibers, so the tear force can maintain a high level. Therefore, the present invention selects ethylene oxide sterilization as the final sterilization method of the breast patch. In summary, the breast patch prepared by the present invention has good mechanical strength, and can suspend and bear the weight of the prosthesis and surrounding tissues after being implanted in the human body, ensuring the aesthetic feeling of the reconstructed breast patch.

3. Detection of hydration time of biological breast patches compounded with different concentrations of active substances

Put the bio-type breast patches compounded with different concentrations of active substances prepared in Example 3 in purified water, start timing from the moment of immersion until the product is completely hydrated, and compare the breast patches soaked in different concentrations of sodium hyaluronate The results of the differences in hydration time are shown in Table 2.

Table 2. Effect of sodium hyaluronate concentration on hydration time

It can be seen from Table 2 that when sodium hyaluronate is not compounded, the hydration time of the sample is 5.5s, and the hydration time of the sample is significantly shortened after compounding sodium hyaluronate, but as the concentration increases to 0.05g/ml or even 0.1g /ml, the hydration time was not significantly shortened. Combined with the hydration effect and cost analysis, the parameter for determining the concentration of the breast patch compound sodium hyaluronate is set at 0.03g/ml.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application.

Claims (10)

1. A method for preparing a biological breast patch, comprising the following steps:

1) Collecting dermis of mammal, cleaning with water, and filtering;

2) Carrying out virus inactivation treatment on the skin tissue treated in the step 1);

3) Degreasing the skin tissue treated in the step 2);

4) Performing decellularization treatment on the skin tissue treated in the step 3) to obtain a decellularized dermis matrix;

5) Immersing the acellular dermal matrix obtained in the step 4) in an aqueous solution of sodium hyaluronate, and taking out;

6) And 5) freeze-drying the acellular dermal matrix treated in the step 5) to obtain the biological breast patch.

2. The method according to claim 1, characterized in that: the virus inactivation treatment in step 2) comprises: soaking the skin tissue with an aqueous solution of hydrogen peroxide, and cleaning after the soaking is finished;

the mass concentration of the aqueous solution of the hydrogen peroxide is 0.06-0.1 g/ml;

the soaking time is 1 to 1.5 hours;

the cleaning is ultrasonic cleaning.

3. The method according to claim 1 or 2, characterized in that: the degreasing treatment in step 3) includes: soaking the skin tissue with a mixed solution of chloroform and ethanol, and cleaning after the soaking is finished;

in the mixed solution, the volume ratio of chloroform to ethanol is 1: (1-5);

the soaking time is 30-60 min;

the cleaning is ultrasonic cleaning.

4. A method according to any one of claims 1-3, characterized in that: the decellularization treatment in step 4) includes: soaking the skin tissue in an aqueous solution of an alkaline reagent, and cleaning after the soaking is finished;

the alkaline reagent is NaOH or KOH;

the concentration of the alkaline reagent in the aqueous solution of the alkaline reagent is 0.5-5.5 mol/L;

the soaking time is 30-60 min;

the cleaning is ultrasonic cleaning;

the times of the decellularization treatment are 3-5 times.

5. The method according to any one of claims 1-4, wherein: the concentration of the aqueous solution of sodium hyaluronate in the step 5) is 0.01-0.1 g/ml;

the soaking in the step 5) comprises a first soaking and a second soaking after the first soaking finishes turning over the acellular dermal matrix, wherein the time of the first soaking and the time of the second soaking are respectively 10-20 min.

6. The method according to any one of claims 1-5, wherein: the method further comprises the following steps of 6): cutting the acellular dermal matrix after freeze drying; and, a step of, in the first embodiment,

and (3) punching the acellular dermal matrix after the shearing and cutting.

7. The method according to claim 6, wherein: the method further comprises the steps of packaging, sterilizing and analyzing sequentially after the punching treatment;

the sterilization is ethylene oxide sterilization or irradiation sterilization.

8. The method according to claim 7, wherein: the conditions for sterilizing the ethylene oxide are as follows: the temperature is 37-47 ℃, the humidity is 30-80%, the concentration of ethylene oxide is 500mg/L, and the sterilization time is 3-5 hours;

the analysis is carried out in an analysis oven at 37-47 ℃ for 4-6 days.

9. A biologic breast patch prepared by the method of any one of claims 1-8.

10. The biologic breast patch of claim 9, wherein: the biological breast patch is rectangular or crescent;

the diameter of the through holes in the biological breast patch is 0.5-2.5 mm, and the distance between two adjacent through holes is 1-2 cm;

the biological breast patch is crescent-shaped, and the through holes are distributed at the middle position of the biological breast patch.

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