WO2021128763A1 - Preparation method for placenta tissue engineering de-immunized skin scaffold - Google Patents

Abstract

The present invention relates to the technical field of medical materials, and specifically, to a preparation method for a placenta tissue engineering de-immunized skin scaffold. The preparation method for a placenta tissue engineering de-immunized skin scaffold of the present invention comprises the following steps: 1) separating a placental membrane from a placenta in a sterile manner; 2) cleaning the separated placental membrane and then soaking same in an antibiotic solution for disinfection; 3) repeatedly washing and soaking the placenta with sterile distilled water; 4) performing gradient dehydration on the disinfected placental membrane by using ethanol-physiological saline solutions of different concentrations to remove protein and inactivate the immunogen; and 5) soaking the dehydrated placental membrane in ethanol, and then taking out and air drying the placental membrane to prepare a skin scaffold. The present invention can achieve the purpose of preparing medical artificial skin conveniently, quickly, efficiently and on a large scale, and the artificial skin is easy to store and convenient to transport.

Description

Preparation method of placental tissue engineering deimmunogenized skin scaffold

Cross reference of related applications

This application claims the priority of the Chinese patent application number 201911356292.1 filed on December 25, 2019, which is fully incorporated herein by reference.

Technical field

The invention relates to the technical field of medical materials, in particular to a method for preparing a placental tissue engineering de-immunogenized skin scaffold.

Background technique

The placenta (placenta) is an important organ for material exchange between the fetus and the mother. It is the combined organ of mother-child tissue formed by the combination of embryonic embryonic membrane and maternal endometrium during human pregnancy. The fetus develops in the uterus and depends on the placenta to obtain nourishment from the mother, while the two sides maintain considerable independence. The placenta also synthesizes a variety of hormones, enzymes and cytokines to maintain normal pregnancy. The placenta is still a traditional Chinese medicine blindly, and it is called the Ziheche, and it is also called the human fetal girth, cell garment, hematuria, and fetal membrane. Since ancient times, the placenta has been regarded by doctors as a good medicine, which can strengthen the immune system and is widely used, and it is more reliable in terms of safety.

Placental tissue discarded during the perinatal period is one of the important sources of regenerative medicine seed cells. It has a wide range of sources, is easy to obtain, and has no ethical and moral controversy. It is a research hotspot in recent years. As an immune-exempt biological graft, the placenta has biological properties such as anti-inflammatory, anti-adhesion, pain relief, and promotion of epithelialization. In recent years, with the further understanding of the placenta and the development of placental preservation and preparation technology, the placenta has once again become Research and application hotspots are widely used in burn treatment and other fields. It can be seen that human placenta is a tissue engineering material for wound repair and has a good application prospect.

At present, similar studies mostly use amniotic membrane as the matrix material of the skin scaffold, but the amniotic membrane is light and thin, and it is easily damaged during the stretching process. The present invention uses placenta to make skin stents, which are fundamentally different from the materials (amniotic membrane tissue components) used in the existing inventions and related processing techniques. Since ancient times, the medicine component of "Ziheche" is placenta instead of amniotic membrane. Modern research surface There are a variety of hormones and immune factors in the placenta, which contribute to the regeneration of local damaged tissues (amniotic skin stents do not have this function). In addition, the output of placental skin stents is much higher than that of amniotic skin stents (amniotic membrane is only a thin layer of connective tissue on the inner side of the placenta, which can only cover the surface of damaged tissues after being made into stents). The placental skin stents obtained by this process are extremely tough. Good, compared with the previously reported amniotic membrane stent, the stretch and extension performance is significantly improved. In actual treatment applications, it can be extended arbitrarily according to the size and shape of the burn wound, which is more convenient. Therefore, the present invention is an original invention in terms of stent material selection and process flow.

Summary of the invention

Aiming at the deficiencies of the prior art, the present invention discloses a preparation method of a human-derived placental tissue engineering de-immunogenized skin scaffold, which can realize the purpose of simple, rapid, efficient, large-scale preparation of medical artificial skin, and is easy to store , Easy to transport.

The specific technical scheme of the present invention is as follows:

The preparation method of the placental tissue engineering de-immunogenized skin scaffold of the present invention includes the following steps:

1) Take the placental tissue discarded after childbirth, wash the placenta with sterile distilled water, remove villi, blood vessels, amniotic membrane and other connective tissues to prepare sterile placental membrane;

2) Wash the separated placental membrane and soak it in a sterile antibiotic-physiological saline solution for antibacterial treatment;

3) Dehydrate the treated placenta membrane with different concentrations of ethanol-physiological saline solution in a gradient to remove protein and inactivate the immunogen;

4) Soak the dehydrated placental membrane in a sterile antibiotic-physiological saline solution, and then take it out to air dry to make a skin stent.

According to the preparation method of the present invention, wherein preferably, the antibiotic solution is a sterile physiological saline solution containing antibiotics.

According to the preparation method of the present invention, wherein preferably, the antibiotic in step 2) is one or more of gentamicin, penicillin and streptomycin. More preferably, it can be gentamicin to prevent penicillin allergy and streptomycin toxicity.

According to the preparation method of the present invention, preferably, step 2) uses physiological saline to wash the separated placental membrane, and after washing, it is soaked in an antibiotic solution for 5-24 hours.

According to the preparation method of the present invention, preferably, the ethanol concentration in the different ethanol-physiological saline solution in step 3) is 75%, 35%, and 15%, respectively.

According to the preparation method of the present invention, preferably, step 4) soak the dehydrated placental membrane in sterile normal saline for 1 to 3 days; air-dry in a sterile environment.

According to specific embodiments of the present invention, the preparation method of the placental tissue engineering de-immunogenized skin scaffold of the present invention may specifically include the following steps:

1. Aseptically separate the placental membrane from the whole placenta:

Select the placenta that has been separated from the human body, and exclude virus, bacteria and other pathogen infections by serum testing, such as HIV, syphilis, HBV and HCV infection, peel off the placenta from the inner layer of the placenta under aseptic conditions, and place it in sterile distilled water In the process, repeated washing to remove blood stains on the surface of the placenta, and blunt peeling of villi, blood vessels and other connective tissues from the latent space between the placenta and chorion with tweezers. Rinse repeatedly with sterile distilled water, and the aforementioned materials are all completed under aseptic conditions;

2. Wash with normal saline for 2 to 3 times, then soak in normal saline containing antibiotics (gentamicin, penicillin, streptomycin, etc.) for 5-24 hours for disinfection;

3. Take out the placental membrane, wash it with normal saline, and use different concentrations of ethanol-normal saline (75%, 35%, 15% high to low concentration ethanol) gradient dehydration for 1-3 days (removal of protein, inactivation of immunogen denaturation), and finally Immersion in sterile physiological saline containing antibiotics, this process can effectively improve the toughness of the placenta without destroying the structure of the placenta matrix. At the same time, it can effectively inactivate allogeneic cells, remove a large amount of heterologous proteins and unknown immunogens, and greatly reduce transplantation. Possibility of adverse immune reactions during the process.

4. Place the placental membrane soaked in sterile normal saline in a sterile ultra-clean workbench to air dry (the placental membrane is fixed to prevent retraction when air-dried);

5. Cut into placental membrane patches of different specifications according to clinical needs, make artificial skin stents, sterile vacuum packaging, and store them aseptically at room temperature in a cool and dry place for later use;

6. Before use, take out the placental skin stent patch and apply it to patients with skin defects.

According to the preparation method of the present invention, in step 1) the placental membrane is aseptically removed from the placenta. This process can be obtained by any method known in the art. The present invention is not particularly limited. For example, the following steps may be preferred:

a) Select the placenta separated from the mother's body, and after the virus, bacteria and pathogen infection are excluded by the serum test, the amniotic membrane, villi and vascular connective tissue are removed from the inner layer of the placenta under aseptic conditions, and the placental membrane tissue is placed in sterile water repeatedly Wash and remove blood stains on the surface; (serum testing mainly excludes pathogenic viruses and bacteria and other pathogen infections (such as HIV, syphilis, HBV, CMV and HCV)

b) The remaining villi, intermuscular membrane, blood vessels and other connective tissues in the washed placenta tissue are stripped clean to prepare placental membranes.

The placenta used in the present invention can be of human origin or other animal origin (for example: pig).

In the present invention, the stripped placental membrane needs to be repeatedly washed with sterile water, and the entire process of obtaining and separating the placental membrane from the placenta is completed under aseptic conditions.

The reagents used in the present invention, including water, need to be sterile, such as sterile physiological saline, distilled water and so on. The sterile water used can be sterile distilled water or sterile ultrapure water.

The present invention can cut the air-dried placental membrane into placental membrane patches of different specifications according to clinical needs to make artificial skin stents, which are sterile vacuum packaged, and stored in a cool and dry place at room temperature aseptically for later use; before use, remove the placental skin stent patches It can be used for patients with skin defects, as well as for patients with nonunion and bone defects.

The present invention also provides a placental tissue engineering de-immunogenized placental membrane scaffold, wherein the skin scaffold material is a placental membrane with immunogen removed.

Further preferably, the skin scaffold of the present invention can be prepared by any of the above-mentioned preparation methods.

The present invention uses devascularized placenta membrane as the artificial skin matrix material, and its toughness and thickness are significantly improved compared with other amniotic membrane-derived skin stents. The application range of the present invention will be broader, and the process of obtaining, storing and transporting will be more convenient.

After washing with normal saline, use different concentrations of ethanol-normal saline (75%, 35%, 15% high to low concentration ethanol) gradient dehydration for 1-3 days (removal of protein, inactivation of immunogen), and finally immersed in antibiotic-containing sterile physiological Salt water, this process can effectively improve the toughness of the placental membrane without destroying the matrix structure of the placental membrane. At the same time, it can effectively inactivate allogeneic cells, remove a large number of heterologous proteins and unknown immunogens, and greatly reduce the occurrence of poor immunity during the transplantation process. Possibility of reaction.

The preparation method of the present invention requires rinsing and disinfecting the placenta, continuous gradient dehydration with ethanol, air drying in a sterile table, cutting into placental membrane patches of different specifications according to clinical needs, and sterile vacuum packaging for later use. Prepared into placental membrane patch that is convenient for storage and transportation, and at the same time fully denatures foreign cells and immunogenic proteins in the placenta, without mechanical damage to the placenta itself, and can effectively improve the toughness of the placental membrane; it will be stored separately before use The placental membrane patch can be applied directly to the affected area to help the wound heal quickly. The method adopted by the invention can realize the purpose of preparing medical artificial skin stents quickly, efficiently and on a large scale. And the material we use is human placenta, which has a wide range of sources, and the human placenta is the waste of pregnant women after giving birth. The cost is low and there is no ethical problem. The present invention can obtain a large number of placenta-derived skin stent patches with the aid of a new technology and method. At present, the skin regeneration verification after burn has been carried out on pigs (large animal models), with significant effects and no adverse reactions.

Description of the drawings

Figure 1 is a diagram of the preparation of the placental membrane skin stent in Example 1. The left picture is a fresh placental stent without immunogen, and the right picture is a placental membrane patch without immunogen;

Figure 2 is a picture of the treatment of the pig skin scaffold in Example 2 (the pig models are all deep III degree scalds).

Detailed ways

The present invention will now be described in further detail with reference to the accompanying drawings.

Example 1

Prepare the skin scaffold:

1. Aseptically separate the placenta from the placenta:

Select the placenta that has been separated from the human body, and exclude virus, bacteria and other pathogen infections through serum testing, such as HIV, syphilis, HBV and HCV infection, peel off the placenta from the inner layer of the placenta under aseptic conditions, and place it in sterile distilled water In the process, repeated washing to remove blood stains on the surface of the placenta, and blunt peeling of villi, blood vessels and other connective tissues from the latent space between the placenta and chorion with tweezers. Rinse repeatedly with sterile distilled water and soak overnight until the placental membrane is white. The aforementioned materials are all completed under aseptic conditions;

2. Wash with normal saline for 2 to 3 times, then soak in normal saline containing antibiotics (gentamicin) for 5-24 hours for disinfection;

3. Take out the placental membrane, wash it with normal saline and use gradients of ethanol-normal saline (75%, 35%, 15% high to low concentration ethanol) to dehydrate for 1-3 days (remove protein and inactivate immunogen). This process Without destroying the structure of the placenta matrix, it can effectively improve the toughness of the placenta, at the same time it can effectively inactivate allogeneic cells, remove a large number of heterologous proteins and unknown immunogens, and greatly reduce the possibility of adverse immune reactions during the transplantation process.

4. After immersing the placental membrane dehydrated by ethanol in sterile normal saline, it is placed in a sterile ultra-clean workbench to air dry;

5. Cut into placental membrane patches of different specifications according to clinical needs, make artificial skin stents, sterile vacuum packaging, and store them in a cool and dry room at room temperature aseptically for later use;

6. Before use, take out the placental membrane skin stent patch, which can be used for patients with skin defects, as well as for patients with nonunion and bone defects.

The skin scaffold prepared in this example is shown in Figure 1, (left picture) fresh placental scaffold without immunogen, and (right picture) dehydrated immunogen placental scaffold.

Example 2

Preparation of the skin scaffold: the specific method is the same as in Example 1.

Grouping situation: group A control group without stent bandaging treatment group

Group B Experimental group: stent bandaging treatment group

Operation steps: heat the soldering iron for 5 minutes, and place the piglet directly on the back for 2 minutes. The perm will cause severe third-degree burns. After 3 days of scalding, surgery will be performed to remove the necrotic tissue, followed by bandaging treatment with or without stents, detailed modeling and treatment details as following:

(Group A control group) No stent group:

Diannan small-ear pig, number 152709, gender; boar, 115 days old, weight 10.6KG, 25% pentobarbital 10ml, intramuscular injection, 10 minutes;

a. Heat for 5 minutes with an electric soldering iron, and directly place the pig's back for 2 minutes

b. Electric perm is severely Ⅲ degree, and the burn wound area is 10*11cm (skin surface is burnt and pasty, blisters);

c. After 3 days, perform necrotic skin resection on a sterile operating table. The wound area is 10*11cm, and the deep intermuscular membrane of the wound is 1.0-2.0cm;

d. Sterile petroleum jelly oil gauze dressing;

e. The wound area expanded to 11*14cm after 5 days, infection, strange smell, local blisters, redness and ulceration, thick scabs, maggots crawling around, local use antibiotic saline to wash; intramuscular injection of amoxicillin 0.2g/day for anti-inflammatory and antibacterial treatment;

d. Infectious death within 6 days.

The experimental results are shown in Figure 2, where AD is the wound condition of the control group. The wound area on the day of the wound was 10*11cm (A), the surgical wound area was 10*11cm (B) after three days, and the wound area was expanded to 11*14cm after 5 days. Concomitant infection (C), infectious death within 6 days, maggots in the wound (D);

(Group B experimental group) Stent group

Southern Yunnan small-ear pig, number 000001, gender; boar, 115 days old, weight 11.5KG, 25% pentobarbital 11ml, intramuscular injection, 10 minutes,

a. Heat for 5 minutes with an electric soldering iron, and place the piglet directly on the back for 2 minutes;

b. Electric perm is severely Ⅲ degree, and the burn wound area is 10*11cm (skin surface is burnt and pasty, blisters);

c. After 3 days, perform necrotic skin resection on a sterile operating table. The wound area is 14*19cm, and the deep intermuscular membrane of the wound is 1.5-2.0cm.

d. Stick the prepared immunogen-free sterile skin stent on the wound surface during the operation;

e. Sterile petroleum jelly gauze dressing;

f. In the stent group, the wound area was 9*13cm for 5 days without infection;

g. Stent group 25 days, wound area 2*4cm

h. The wound healed in the stent group within 30 days, and new hairs could be observed.

The experimental results of the stent group are shown in Figure 2. Among them, EH is the wound condition of the stent group. The wound area on the day of the wound is 10*11cm (E), the surgical wound area is 14*19cm after 3 days, and the deep intermuscular membrane of the wound is 1.5-2.0cm( F). The wound area of the stent group was 5*13cm on 5 days. In the stent group, the wound area was 2*4cm (G) on 25 days, and the wound in the stent group was healed after 30 days, and the local hair growth started (H), indicating that hair follicles had formed.

The analysis of the above-mentioned experimental results shows that the skin stent used in the present invention has a good treatment effect for severe burns, and can effectively prevent wound infection and cause death during the treatment process (3 pigs in the control group, died within a week after the burn Due to infection, as shown in Figure 2, he died on the 6th day after the burn). However, in the skin stent treatment group of the present invention (n=3), no post-burn infection occurred (may be related to the skin stent soaked in antibiotics during the treatment of the present invention. At the same time, the skin stent can effectively block the burn wound from contact with the outside air. Cut off the wound infection induced by pathogenic microorganisms), the wound is completely healed in about 1 month, and signs of hair growth appear.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that modifications or equivalent replacements to the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and should be covered by the present invention. Within the scope of the claims.

Claims (9)

1. A preparation method of placental tissue engineering de-immunogenized skin scaffold includes the following steps:

   1) Take the placental tissue discarded after childbirth, wash the placenta with sterile distilled water, remove villi, blood vessels, amniotic membrane and other connective tissues to prepare sterile placental membrane;

   2) Wash the separated placental membrane and soak it in a sterile antibiotic-physiological saline solution for antibacterial treatment;

   3) Dehydrate the treated placenta membrane with different concentrations of ethanol-physiological saline solution in a gradient to remove protein and inactivate the immunogen;

   4) Soak the dehydrated placental membrane in a sterile antibiotic-physiological saline solution, and then take it out to air dry to make a skin stent.
2. The preparation method according to claim 1, wherein the antibiotic solution is a sterile physiological saline solution containing antibiotics.
3. The preparation method according to claim 1 or 2, wherein the antibiotic in step 2) is one or more of gentamicin, penicillin and streptomycin.
4. The preparation method according to claim 1, characterized in that, in step 2), the separated placental membrane is washed with normal saline, and after washing, it is soaked in an antibiotic solution for 5-24 hours.
5. The preparation method according to claim 1, wherein the ethanol concentration in the different ethanol-physiological saline solution in step 3) is 75%, 35%, and 15%, respectively.
6. The preparation method according to claim 1, characterized in that in step 4) the dehydrated placental membrane is soaked in sterile normal saline for 1 to 3 days; air-dried in a sterile environment.
7. The preparation method according to claim 1, wherein in step 1) the step of aseptically separating the placental membrane from the placenta is as follows:

   a) Select the placenta separated from the mother's body, and after the virus, bacteria and other pathogen infections are excluded by the serum test, the amniotic membrane, villi, blood vessels and other connective tissues are stripped from the inner layer of the placenta under aseptic conditions to obtain placental tissue, and the placental tissue is placed on Rinse and soak in sterile distilled water to remove blood stains on the surface of placental tissue;

   b) The remaining villi, intermuscular membrane, blood vessels and other connective tissues in the washed placenta tissue are stripped clean to prepare placental membranes.
8. A placental tissue engineering de-immunogenized skin scaffold is characterized in that the skin scaffold material is a placental membrane with immunogen removed.
9. The placental tissue engineering de-immunogenized skin scaffold according to claim 8, wherein the skin scaffold is prepared by the preparation method according to any one of claims 1-7.

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