CN101954124B - Tissue engineered skin with basilar membrane and construction method thereof - Google Patents

Abstract

The invention relates to the technical fields of tissue engineering and medical wound repair. At present, polylactic acid, polyglycolic acid, collagen, hyaluronic acid and other materials are used as active skin substitutes constructed by dermal scaffolds. On the one hand, it is difficult to extract the matrix material, the production process is complicated, and the cost is expensive, so it is difficult to be widely used in clinical practice. It does not have the basement membrane structure of the skin, so the healing skin is not resistant to pressure and wear, the epidermis is not firmly adhered, it is easy to fall off and break or form blisters, and it affects the development of normal epidermal structure and shape. However, allogeneic acellular dermis is obtained from cadaver skin, and its source is limited and expensive, which limits its clinical application. The purpose of the present invention is to provide a skin substitute with surface-modified amniotic membrane as the basement membrane and plasma as the matrix, which has wide sources of materials, low cost and simple preparation method, and can be kept intact in vivo transplantation as proved by animal experiments. The basement membrane and hemidesmosomes accelerate and promote the formation of epidermal structure and morphology.

Description

Construction method of tissue engineered skin containing basement membrane

technical field

The invention relates to the technical field of tissue engineering and medical wound repair, in particular to a tissue-engineered skin (i.e., an active skin substitute) in which the surface of amniotic membrane is modified and modified as a basement membrane, and the composite plasma is used as a matrix, which is cultured in vitro to construct a tissue-engineered skin containing a basement membrane. , especially suitable for skin defect repair of burns and trauma and scar shaping. the

Background technique

At present, biodegradable materials such as polylactic acid, polyglycolic acid, collagen, and hyaluronic acid are used to construct dermal scaffolds, and active skin substitutes are constructed on this basis, which have been initially applied to the repair of deep skin defect wounds and scar plastic surgery. However, on the one hand, it is difficult to extract the matrix material, the manufacturing process is complicated, and the cost of the skin substitute is difficult to be widely used in clinic. On the other hand, it does not have the basement membrane structure of the skin, which makes the healing skin not resistant to pressure and wear , the epidermis is not firmly adhered, it is easy to fall off and damage or form blisters, and it will affect the development of normal epidermal structure and shape. However, allogeneic acellular dermis is obtained from cadaver skin, and its source is limited and expensive, which limits its clinical application. the

Human amnion tissue is a translucent tissue containing a single layer of epithelial cells, without blood vessels, nerves, and lymphatic vessels, with a thickness of 0.02-0.5mm. Collagen composition (Zhao Min, Lu Jing, Zhang Qi, et al. Expression of HGF, bFGF, ColIV, LN in preserved amnion. Ophthalmology Research, 2006, 24(5): 514-517.). In addition, human amniotic membrane is a medical waste tissue with a wide range of sources and is convenient to obtain. As a biological dressing, it was used to cover burn wounds earlier, which can not only reduce wound inflammation, promote epithelialization, but also inhibit scar formation. At present, there are commercialized amniotic membrane products such as the freeze-dried bio-amniotic membrane of Jiangxi Ruiji Bioengineering Technology Co., Ltd. the

There is no literature report at home and abroad to use amniotic membrane as the basement membrane and composite plasma as the matrix to construct tissue-engineered skin containing basement membrane through in vitro culture. the

Contents of the invention

The purpose of the present invention is to provide a skin substitute with surface-modified amniotic membrane as the basement membrane, autologous/allogeneic plasma as the matrix, and a construction method thereof. The material of the invention has a wide range of sources, a simple preparation method, and low cost, and can maintain a complete basement membrane and hemidesmosome during in vivo transplantation, and accelerate and promote the formation of epidermal structure and morphology. the

The amniotic membrane modified by surface modification of the present invention is the basement membrane, and the blood plasma is the construction method of the tissue-engineered skin containing the basement membrane of the matrix, comprising the following steps:

A) Separation and cultivation of keratinocytes and fibroblasts (reference: Liu Dewu, Li Guohui, Zou Ping, Liu Deming. Epidermal cells, fibroblasts and acellular dermal matrix to construct tissue-engineered skin. Chinese Clinical Rehabilitation, 2004, 8(8) : 1439-1441.). the

B) surface modification modified amniotic membrane:

After removing the chorion, the amniotic membrane was repeatedly soaked and washed in normal saline, digested in 0.02% EDTA solution at 37°C for 2 hours, washed with ultrasonic vibration to completely remove residual cell fragments and chorion tissue, and placed in a solution containing 1000U/ml Qingda Mycin, 2.5μg/ml amphotericin B in normal saline for 20 to 50 minutes. the

C) Construct tissue engineered skin;

The amniotic membrane prepared in step B) is compounded with the plasma matrix containing fibroblasts, and then keratinocytes are seeded on the epidermal surface of the amniotic membrane at a density of 1-5×10 ⁵ /cm ² and cultured in vitro for 2-3 weeks.

The specific steps of the construction method of the present invention are as follows:

1. Isolation and culture of human keratinocytes and fibroblasts

The discarded foreskin tissue after circumcision or the excess skin tissue after plastic surgery were used to separate and culture keratinocytes and fibroblasts by enzyme digestion method and tissue block attachment method respectively. Prepare single-cell suspensions, inoculate in culture flasks at a density of 2-3×10 ⁵ cells/ml, place in a 37°C incubator, culture keratinocytes in serum-free medium, and culture fibroblasts in complete The DMEM medium is used for culture, and when the cells reach 70%-80% confluence, the cells are subcultured and expanded.

2. Surface modification modified amniotic membrane

Strip off the fresh amniotic membrane under aseptic conditions (taken from the placental tissue of caesarean section women who are negative for hepatitis virus antibody, syphilis antibody and HIV), bluntly separate the chorion, soak and wash repeatedly in normal saline, and dissolve in 0.02% EDTA solution Digest at 37°C for 2 hours, then put it into a high-frequency ultrasonic cleaner and wash it with ultrasonic oscillation for 2 hours to completely remove residual cell fragments and chorionic tissues, then soak and wash repeatedly in distilled water, and place amnicin and 2.5 μg/ml amphotericin B in physiological saline for 30 minutes to prepare surface-modified amniotic membranes. the

3. Construction of tissue engineered skin

Firstly, spread the prepared amniotic membrane on the culture plate with the basal surface upward, and wait for it to dry completely and adhere to the wall. Take patient or allogeneic whole blood and centrifuge to obtain plasma, remove cell debris through a microporous filter, add fibroblasts to the prepared plasma to prepare a concentration of 5×10 ⁵ cells/ml, mix well and add the above-mentioned amniotic membrane-covered culture dish. Then add 1M calcium chloride solution at a volume ratio of 1:40 to promote its polymerization, after it polymerizes into a gel, add 1×DMEM solution containing 20% fetal bovine serum, and in vitro culture for 3-5 days. Subsequently, keratinocytes were inoculated on the amnion at a density of 1-5×10 ⁵ cells/cm ² , and after the cells were fused, they were cultured at the air-liquid interface for 2-3 weeks, and the medium was changed every 2-3 days to form the following Active skin substitute with amniotic membrane as basement membrane and plasma gel as matrix.

The present invention also provides tissue engineered skin constructed according to the above method. the

The present invention also provides the application of the tissue-engineered skin constructed according to the above-mentioned method as a graft material for wound repair. the

The invention uses the amniotic membrane as the basement membrane and the plasma gel as the active skin substitute, and is mainly used for repairing full-thickness skin defect wounds and providing skin sources for patients with severe large-area deep burns. On the other hand, it can also be applied to chronic deep skin ulcers, skin avulsions and other skin defect wounds and scar plastic surgery. The active skin substitute prepared by the present invention is simple in method, convenient in obtaining materials, and low in cost. In addition, it is found in in vitro culture that the epidermis grows more rapidly and its structure is closer to normal skin than traditional tissue engineering skin without amniotic membrane. basement membrane, while hemidesmosomes are more mature and firm. Animal (nude mice) transplantation experiments showed that when the active skin substitute was implanted into full-thickness skin defect wounds, the survival rate reached 88%. The newborn skin can be seen with complete basement membrane, well-developed epidermis and hemidesmosomes. the

Therefore, in the present invention, amniotic membrane is surface-modified as basement membrane, combined with plasma gel containing fibroblasts as a matrix, keratinocytes are planted on the surface, and an active skin substitute containing basement membrane is constructed through in vitro culture. The preparation method of the invention is simple, the source of materials is wide, the cost is low, and the effects of infection, rejection and the like that may be caused by heterogeneous sources are eliminated at the same time. the

Detailed ways

Now, the present invention will be further described in conjunction with the embodiments, but the implementation of the present invention is not limited thereto. the

Embodiment 1. preparation contains the active skin substitute of basement membrane

1. Isolation and culture of human keratinocytes and fibroblasts

The discarded foreskin tissue after circumcision was used to separate and culture keratinocytes and fibroblasts by enzymatic digestion. Single cell suspensions were prepared respectively, inoculated in culture flasks at a cell density of 2-3×10 ⁵ cells/ml, placed in a 37°C incubator, and keratinocytes were cultured in serum-free medium (DK-SFM, Gibco, USA), the fibroblasts were cultured with complete DMEM medium (Gibco, USA), and passaged and expanded when the cells reached 70%-80% confluence.

2. Surface modification modified amniotic membrane

Strip off the fresh amniotic membrane under aseptic conditions (taken from the placental tissue of caesarean section women who are negative for hepatitis virus antibody, syphilis antibody and HIV), bluntly separate the chorion, soak and wash repeatedly in normal saline, and dissolve in 0.02% EDTA solution medium (Gibco, USA), digest at 37°C for 2 hours, then put it into a high-frequency (59KHz) ultrasonic cleaner (SK8200H, Kedao, Shanghai), and wash with ultrasonic oscillation for 2 hours to completely remove residual cell debris and chorionic tissue , and then soaked and washed repeatedly in distilled water, soaked in physiological saline containing 1000 U/ml gentamicin and 2.5 μg/ml amphotericin B for 30 minutes for later use. the

3. Construct tissue engineering skin:

Firstly, spread the prepared amniotic membrane on the culture plate with the basal surface upward, and wait for it to dry completely and adhere to the wall. Take the patient or allogeneic whole blood and place it in an anticoagulant tube containing sodium citrate anticoagulant, centrifuge at 600g for 5 minutes, collect the plasma, sterilize it through a filter with a pore size of 20um to remove cell debris, add fibroblasts to the prepared plasma to prepare the concentration After mixing thoroughly, the plasma containing fibroblasts was added to the culture dish covered with amnion at a ratio of 1: ⁶ volume (ml) to culture plate area (cm ² ). Then add 1M calcium chloride solution at a volume ratio of 1:40 to promote its polymerization, after it polymerizes into a gel, add 1×DMEM solution containing 20% fetal bovine serum, and in vitro culture for 3-5 days. Subsequently, keratinocytes were inoculated on the amnion at a density of 1-5×10 ⁵ cells/cm ² , and after the cells were fused, they were cultured at the air-liquid interface for 2-3 weeks, and the medium was changed every 2-3 days to form the following Active skin substitute with amniotic membrane as basement membrane and plasma gel as matrix.

Embodiment 2. Animal transplantation test of tissue engineered skin of the present invention

Male Balb/c-nu mice (nude mice, provided by Shanghai Xipuer-Bikay Experimental Animal Co., Ltd.), body weight 18 ± 2 grams, 16, were divided into two groups, respectively, the tissue engineered skin containing amniotic membrane of the present invention Group A and the traditional active skin substitute without amniotic membrane Group B: purely constructed with fibrin as a matrix (see A.L.Mazlyzama, B.S.Aminuddinb, et al.Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold.Burns , 2007, 33(3): 355-363). the

After the nude mice were anesthetized with ketamine intraperitoneally, the skin and hair on the back were shaved, and the full-thickness skin on the ventral side of the spine and the deep fascia up to the muscle layer were excised. Group A: The tissue-engineered skin cultured in vitro for 2-3 weeks described in Example 1 was transplanted on the wound surface. Group B: The traditional active skin substitute without amniotic membrane was transplanted on the wound. In order to prevent periwound skin shrinkage and epidermis crawling, which would affect the observation of transplantation results, an isolation ring was used to isolate the wound surface from the surrounding skin. The composite skin was covered with oil sand, and the dressing was changed regularly and the wound healing was observed. Two weeks after composite skin transplantation, the wounds were opened to observe the survival rate. The materials were collected and made into paraffin sections with a thickness of about 5 μm, and routine HE staining and transmission electron microscope observations were performed on the tissue sections. the

The results showed that there was no significant difference in the survival rate between the tissue engineered skin of the present invention and the traditional group. Histological HE staining observation shows that the epidermal structure of the tissue engineered skin of the present invention is well-developed 4 weeks after transplantation, and the shape is closer to normal skin. The transmission electron microscope observation shows that there is a perfect basement membrane, and at the same time, hemidesmosomes are more mature, while the control No continuous basement membrane formation was seen in the group, and hemidesmosome development was defective. In addition, in the active skin substitute prepared by the present invention, the formed stratified epidermal cells adhere more closely and firmly to the dermis substitute, and are not easy to detach. the

Claims (1)

1. a method for constructing tissue-engineered skin containing basement membrane, characterized in that the concrete steps of the method are as follows: A) Separation and cultivation of human keratinocytes and fibroblasts: Use the discarded foreskin tissue after circumcision, separate and culture keratinocytes and fibroblasts by enzymatic digestion, prepare single cell suspensions, and inoculate in culture flasks at a cell density of 2-3× ¹⁰⁵ cells/ml , placed in a 37°C incubator, keratinocytes were cultured in serum-free medium, fibroblasts were cultured in complete DMEM medium, and passaged and expanded when the cells reached 70%-80% confluence; B) surface modification modified amniotic membrane: The fresh amniotic membrane was peeled off under sterile conditions, the chorion was bluntly separated, after repeated immersion and washing in physiological saline, it was digested in 0.02% EDTA solution at 37°C for 2 hours, and then placed in a high-frequency ultrasonic cleaner for ultrasonic vibration washing for 2 hours, thoroughly remove the residual cell fragments and chorionic tissue, then soak and wash repeatedly in distilled water, and then soak in physiological saline containing 1000U/ml gentamicin and 2.5μg/ml amphotericin B for 30min for later use; C) Construct tissue engineered skin: Put the amniotic membrane prepared in step B) on the culture plate with the basal surface upward, wait until it is completely dry and adhere to the wall, take the patient or allogeneic whole blood and place it in an anticoagulant tube containing sodium citrate anticoagulant, centrifuge at 600g for 5min, collect Plasma, sterilized by a filter with a pore size of 20um to remove cell debris, add fibroblasts to the prepared plasma to prepare a concentration of 5×10 ⁵ cells/ml, mix well, and mix the volume ml with the culture plate area cm ² at a ratio of 1:6 Add the plasma containing fibroblasts to the culture dish covered with amniotic membrane; then add 1M calcium chloride solution at a volume ratio of 1:40, after it polymerizes into a gel, add 20% fetal bovine serum 1×DMEM solution, culture in vitro for 3-5 days; then inoculate keratinocytes on the amniotic membrane at a density of 1-5×10 ⁵ cells/cm ² , and conduct air-liquid interface culture for 2-3 days after the cells are fused Weekly, change the medium every 2-3 days.