WO2011094964A1

WO2011094964A1 - Method for preparing tissue engineering skin containing hair follicle

Info

Publication number: WO2011094964A1
Application number: PCT/CN2010/071123
Authority: WO
Inventors: 金岩; 凡孝菊; 张勇杰; 王爱军
Original assignee: 中国人民解放军第四军医大学; 陕西瑞盛生物科技有限公司
Priority date: 2010-02-05
Filing date: 2010-03-18
Publication date: 2011-08-11
Also published as: CN101775366A

Abstract

A method for preparing tissue engineering skin containing hair follicles includes implanting tissue engineering hair follicle evenly in the dermal tissue formed by combining scaffold material and cell, coating with epidermal cell, then culturing, and forming the tissue engineering skin containing hair follicles. The quantity of hair follicles in the tissue engineering skin containing hair follicles is nearly as the natural quantity, and the hair follicles are distributed in artificial pores evenly. The skin can overcome defects of the skin forming by injection with its hair follicles being of small quantity, uneven distribution, and uncontrolled quantity.

Description

Preparation method of hair follicle tissue engineering skin

Technical field

The invention belongs to the technical field of tissue engineering of biological materials, and particularly relates to a method for preparing tissue engineering skin containing hair follicles.

Background technique

Tissue engineering is a research hotspot in the field of life sciences, and related research involves various aspects. The content is that the functional cells cultured in vitro are planted in the corresponding scaffold materials, and after being cultured, a tissue or an organ having a certain function is formed. With the rapid development of tissue engineering technology, tissue engineered skin has begun to be widely used clinically. The current tissue engineering skin mainly implants skin dermal cells and epidermal cells on skin scaffold materials, and the tissue engineering skin thus constructed has a dermis and epidermis two-layer structure similar to normal skin, but no accessory organs of skin such as hair follicles and sebaceous glands. There is still a difference in tissue structure from normal skin tissue.

The skin is the largest organ of the human body, and has functions of protecting the body, regulating body temperature, and discharging waste. However, due to inflammation, ulcers, trauma, burns, postoperative tumors, and congenital malformations, skin and mucous membrane defects or abnormalities may cause damage or loss of skin function, resulting in loss of protection of internal organs and even life. The use of autologous flaps and autologous skin grafts can cause new trauma to the donor site, often due to the limited source of donor sites and the inability to meet large-area transplants. The application of tissue-engineered skin alleviates this crisis, but tissue-engineered skin without accessory organs is functionally unable to meet patient needs.

Hair follicles are important accessory organs of the skin and play an important role in the physiological processes of the skin. Deletion and abnormality of the hair follicles of the head can lead to alopecia areata or total baldness, which affects the aesthetics and affects the quality of life of the patient. The abnormal hair follicles in the body part seriously affect the metabolism and heat exchange of the body, so that the patient can only be at a constant temperature. Living in the environment. Therefore, the regeneration of hair follicles is the key to solving the above problems.

In developmental biology research, hair follicles are typical organs of epithelial and mesenchymal interaction. In the early stage of embryonic development, epidermal cells protrude downward to form hairpins, and the nails extend downward and expand to form the embryonic base of sebaceous glands. The embryonic base of the pilose muscle further forms a hair cone, and the sebaceous gland and the pilose muscle gradually form, and finally the hair nipple and the inner hair root sheath are formed, and hair follicles are formed in the hair follicle. Mature hair follicles are divided into four parts: hair shaft, epithelial root sheath, dermal sheath and dermal papilla. The epithelial root sheath is closely attached to the hair shaft and consists of multiple layers of keratinized epithelial cells. It can be divided into an inner root sheath and an outer root sheath from the inside to the outside. The inner root sheath is at the level of the sebaceous gland opening and the invaginated epidermis. Even, similar to the transparent layer and the keratinized layer of the epidermis, the cells of the outer root sheath are similar to the basal layer of the epidermis and the cells of the spinous layer; the dermal sheath and the surrounding dense connective tissue are transformed from the dermis, which is equivalent to the dermis of the skin. The dermal papilla cells are also dermis-derived cells, which have an important role in the developmental biology of hair follicles, and can induce hair follicle production in vitro and in vivo. There is evidence that hair follicles play an important role in reducing skin scar formation, improving skin wound healing speed and wound healing quality; and in hair follicle outer root sheath cells of hair follicles, there are hair follicle stem cells supporting hair follicle epithelial cell renewal. It not only plays an important role in the periodic growth of hair follicles, but also is the source of cells for self-renewal of the epidermis; in addition, dermal sheath cells and dermal papilla cells can transform each other under certain conditions to maintain the growth of hair follicles and show some The characteristics of stem cells can be transformed into skin fibroblasts after skin trauma or directly involved in the reconstruction of skin dermal tissue, promoting skin healing.

The microcapsule technology is a technique in which a dispersed variety of substances (including solid, liquid, gas, and biological substances) is encapsulated in a film composed of a polymer material to form spherical microcapsules having a diameter of 50 to 1000 μm. A spherical microcapsule formed by encapsulating a biologically active substance such as a protease and a hormone in a selectively permeable membrane is called a biological microcapsule. Research reports that microcapsule technology is combined with tissue cell transplantation to encapsulate cells in the membrane. It is characterized by selective permeable membranes to prevent the immune system from attacking intracapsular cells, while small molecules of nutrients and cysts Biologically active substances and metabolites are freely accessible.

At present, research on tissue engineering skin containing hair follicles has become a hot spot. Simulating the conditions of early hair follicle formation in embryos, mixing epithelial cells with mesenchymal cells, and regenerating hair follicles by in vivo culture; mostly using dermal papilla cells with hair follicle regeneration and hair follicle outer root sheath cells or hair follicles The stem cells are mixed and injected into the skin scaffold to form a tissue containing hair follicle tissue; wherein the formation of hair follicles, whether from the number or the structure of the hair follicle formed, is not ideal. Early hair follicles are formed by the epithelial cells sag and aggregated into a group of mesenchymal cells, and the mixed injected epithelial cells are in contact with the mesenchymal cells, so that the cells need to recognize each other and self-organize rearrangement. The hair follicle structure and size thus formed are not uniform. The hair follicles in the skin should be distributed and hooked, and the number of hair follicles formed locally or only a small amount can not play a corresponding physiological role.

Summary of the invention

In view of the problems existing in the prior art, an object of the present invention is to provide a method for preparing a tissue containing hair follicle tissue, which comprises a large amount of hair follicle structure, which can realize hair follicle regeneration after skin transplantation. The preparation method of the hair follicle tissue-engineered skin proposed by the invention is characterized in that the tissue engineering hair follicle is uniformly inoculated into the tissue engineering dermis layer formed by the composite material of the scaffold and the cells, and then covered with epidermal cells, after being cultured, Forming a tissue-engineered skin containing hair follicles; the tissue-engineered hair follicles are obtained by encapsulating mesenchymal-derived dermal papilla cells or stem cells which can differentiate into dermal papilla cells into a polymer material to form micro-particles having a particle size of 200-1000 μm The sac is then subjected to a rotary culture method to coat epithelial-derived outer root sheath cells or epithelial cells having stem cell function on the periphery of the microcapsule to form a tissue-engineered hair follicle having a particle diameter of 250 to 1100 μm; Like the polymer material, it is an organism-acceptable biodegradable material, and can be selected from extracellular matrix, collagen, hyaluronic acid, chondroitin sulfate, gelatin, fibrin glue, chitosan, alginate, proteoglycan. Any one or a combination of polylysine, polylactic acid, polyglycolic acid.

The various types of cells are autologous or allogeneic cells; wherein, the dermal papilla cells in the microcapsules or the stem cells which can differentiate into the dermal papilla cells can select embryonic stem cells, IPS cells, bone marrow mesenchymal stem cells, and hematopoietic stem cells. Or any of the hair follicle dermal sheath stem cells; the outer root sheath cells of the outer layer of the microcapsule or the epithelial cells having the stem cell function, and any one of the epidermal stem cells, the hair follicle stem cells, or the hair follicle bulge stem cells may be selected.

Specific steps of the preparation method of the present invention include:

Step one, preparing the culture solution

Culture medium A: Epidermal growth factor EGF 2~10 g, fibroblast growth factor bFGF 5~50ng, bovine pituitary extract BPE 3 in 500ml of commercial DMEM containing V/V 15% fetal bovine serum ~15g;

Culture medium B: In serum-free commercial Williams E medium 500ml, EGF 2~10 w g, bFGF 5~50ng, BPE 3~15g, insulin-like growth factor IGF-1 5~50ng;

Culture medium C: In 500 ml of commercial DMEM containing V/V 10% fetal bovine serum, 2 to 50 ng of insulin, 10 to 500 ug of hydrocortisone, 10 to 17 mg of adenine, and 5 to 30 mg of Vc were added. EGF

2~10 g, bFGF 5~50ng, BPE 3~15g;

The culture solution D: calcium chloride having a concentration of 0.1 to 1 m M in the culture solution C;

The culture solution E: calcium chloride having a concentration of 0.2 to 2 m M in the culture solution C;

The culture solution F: calcium chloride having a concentration of 0.3 to 3 m M in the culture solution C;

Step 2, get the cell The acquisition and culture of dermal papilla cells can be referred to the literature [Cheng Bo et al.

Chinese Journal of Dermatology 2001 34 (5) 387~388] or other prior art, obtaining dermal papilla cells with good growth and agglutinative growth characteristics; stem cells of different origins are cultured from dermal papilla cells Induced culture, with the characteristics of dermal papilla cells, can be used as microcapsule cells in tissue engineering hair follicles. The acquisition and culture of outer root sheath cells of hair follicles can be obtained by referring to the literature [Tang Jianbing et al., Culture of Hair Follicles of Hair Follicles, Chinese Journal of Burns, 2003 19 (1) 47~48] or other prior art techniques to obtain hair follicles with good growth conditions. Outer root sheath cells; other epithelial-derived stem cells can be obtained by reference to the prior art and do not require induction as the outer layer of tissue engineered hair follicles. The acquisition and culture of fibroblasts and epidermal cells can be obtained by reference to [Organizational Engineering and Technology, Jin Yan, ed., Fourth Military Medical University Press 239~240] or other prior art.

Step 3, preparing tissue engineering hair follicles

Preparation of cell-containing microcapsules can be referred to the literature [Liu Rui et al. ACA, APA microcapsules strength, permeability and biocompatibility study "Journal of the Third Military Medical University" 2009 31 (17) 1653~1656] or other In the prior art, a microcapsule of a polymer material is prepared, and the microcapsule contains dermal papilla cells or stem cells which can differentiate into dermal papilla cells; the microcapsule containing cells is mixed with the obtained outer root sheath cells of hair follicles or other epithelial-derived stem cells. Using culture medium A, spin culture for 1 to 3 days, until the cells are attached to the microcapsules, centrifuge at 300 rpm to discard the supernatant, and change culture medium B for 2 to 4 days to obtain tissue engineering hair follicles; Step 4, containing hair follicle tissue Preparation of engineered skin

4°C, the scaffold material was prepared into a solution of 6~20mg/ml with 0.5M acetic acid solution, irradiated with ultraviolet light under ice bath, and then added 10% by volume of fetal bovine serum and the final concentration was 80~150mg. /ml of DMEM commercial medium, adjusted pH 7. 2~7. 4, into a gel solution; the nylon membrane is saturated with the gel solution, placed in a petri dish to form a support film; the cultured fibroblasts in 10 ⁵ to 10 ⁶ / ml density mixed in the gel, which is added dropwise to the surface of the cured support film, cultured at 37 ° C to form a dermis layer, added to the culture solution C for 2 to 4 days, daily change; After the dermal layer is cultured, the culture solution is aspirated, and the needle is punched on the surface with a hole spacing of 2 to 5πππ _; the cultured tissue engineering hair follicle suspension is added to the surface of the dermis at a density of 10 to 100 pieces/cm ² . The suspension is evenly infiltrated into the well; then the epidermal cells are inoculated at a density of 10 ^{5 to} 10 ⁶ /cm ² on the surface of the dermis, and cultured for 1 day in the culture medium D, and then transferred to the scaffold for subcultivation for 2 to 4 days. Replace the culture solution E for gas-liquid level culture for 1~2 days; replace the culture solution F Incubate for 1 to 2 days; change the solution every day during the culture. The invention adopts the microcapsule technology to coat the dermal papilla cells or the stem cells which can differentiate into the dermal papilla cells with a polymer material, and then inoculates the outer root sheath cells of the hair follicle or the stem cells which can differentiate into the outer root sheath cells of the hair follicle by spin culture. On the periphery of the capsule, the dermal papilla cells are placed inside the hair follicle, surrounded by dermal sheath cells, like natural microcapsules, giving hair follicles more perfect development conditions, and forming a large number of tissue engineering hair follicles after culture; uniformly inoculation into tissue engineering In the dermis layer of the skin, the epidermal cells are covered with epidermal cells to form hair follicle tissue-engineered skin.

The invention overcomes the shortcomings of small hair follicles, uneven hooks and uncontrollable hair follicles in the skin caused by the mixed injection in the past, and the prepared tissue engineering skin contains epidermis, dermis and hair follicle-like tissue structure, wherein the number of hair follicles is close to the natural quantity and Evenly distributed in artificial pores, hair follicles continue to develop in vitro and after transplantation, forming a multi-layered concentric structure, the inner layer is the dermal papilla cells surrounded by microcapsules, and the outer layer is epithelial cells; Or the skin grafted in the body eventually forms a functional skin containing a hair follicle accessory structure, which can achieve hair follicle regeneration after skin transplantation.

detailed description

Specific embodiments of the technical solution of the present invention will be described in detail below with reference to examples.

Step one, preparing the culture solution

Culture medium A: Epidermal growth factor EGF 6 μg, fibroblast growth factor bFGF 10 ng, bovine pituitary extract BPE 25 g/ml in 500 ml of commercial DMEM containing V/V 15% fetal bovine serum. ;

Culture medium B: EGF 6 g, bFGF 10 ng, BPE 25 g/ml, insulin-like growth factor IGF-1 10 ng were added to 500 ml of serum-free commercial Williams E medium;

Culture medium C: 10 ng of insulin, 100 gg of hydrocortisone, 15 mg of adenine, 10 mg of VCl, 10 mg of EGF, 6 μg of EGF, bFGF 10 ng in 500 ml of commercial DMEM containing V/V 10% fetal calf serum. , BPE 25 g / ml ;

The culture solution D: a calcium chloride having a concentration of 0.8 mM in the culture solution C;

Culture medium E: calcium chloride having a concentration of 1. 9 mM in the culture solution C;

Culture medium F: calcium chloride having a concentration of 2. 6 mM in the culture solution C;

Step two, get the cell

1), acquisition and culture of dermal papilla cells: Cut the human head skin, rinse it with PBS solution containing double antibody for 3 times, separate the hair follicle under the microscope; place the separated hair follicle in a centrifuge tube and add 2 ml collagenase solution (625U/ml), digested in the incubator at 37 °C for 1 hour, the dermal papilla was loosely released from the hair matrix, and gently patted to release it; the embryonic needle was used to transfer the dermal papilla tissue of the hair follicle into the culture flask, and the inclusion was included. 4% serum DMEM medium (with lOOU/ml double antibody) was cultured in a 5% C0 ₂ 37 °C incubator to obtain well-grown dermal papilla cells as microencapsulated cells of tissue engineering hair follicles;

2) Acquisition and culture of outer root sheath cells of hair follicles: Place the hair follicles in a centrifuge tube, digest the enzyme with dispase at 4 ° C overnight, and extrude the hair shaft and root sheath epithelial cells from the hair follicle into a 24-well under a stereo microscope. In the plate, the cover slip is pressed onto the hair shaft tissue, and cultured in a serum-free K-SFM (GBICO) culture medium to obtain a hair follicle outer root sheath cell with good growth state;

The acquisition and culture of fibroblasts and epidermal cells can be obtained by referring to [Organizational Engineering Principles and Techniques, Jin Yan, ed., Fourth Military Medical University Press, 239~240] or other prior art.

Step 3, preparing tissue engineering hair follicles

The dermal papilla cells were digested into single cells with 0.25% trypsin solution, suspended in a sodium alginate solution at a concentration of 20 g/L, and the cell concentration was 5×10 ⁸ /L. Drip into 11 g / L of calcium chloride solution to form a micro-gel ball, washed 3 times with normal saline; 10 min in chitosan solution for 10 min, 3 times with normal saline; 2 g / L seaweed The sodium solution was used for 4 min, and the physiological saline was washed 3 times; then it was applied to 0. 05 mol/L sodium citrate solution for 6 min, and washed with physiological saline for 3 times; sodium alginate-chitosan-alginic acid was obtained. Sodium microcapsules, which contain dermal papilla cells. The cell-containing microcapsules were mixed with the obtained outer root sheath cells of the hair follicles, and the culture liquid A was added to the gravity-free rotary generator for 2 days, and the outer root sheath cells were attached to the microcapsules, and the supernatant was discarded by centrifugation at 300 rpm, and replaced. Culture medium B was cultured for 3 days to obtain tissue engineering hair follicles.

Step 4: Preparation of skin tissue containing hair follicles

The extracellular matrix, collagen and hyaluronic acid were formulated into a solution of 8 mg/ml with an acetic acid solution of 0.5 M at 4 ° C, irradiated with ultraviolet light under ice bath, and then added with 10% by volume of fetal calf serum and DMEM commercial medium with a final concentration of 100 mg/ml, adjusted to pH 7.3, to prepare a gel solution; the nylon membrane was immersed in the gel solution, and placed in a Petri dish to form a support film; the fibroblasts cultured in vitro were 10 The concentration of ⁵ / ml was mixed in the gel, and it was added dropwise to the surface of the cured support film. After culturing and solidifying at 37 ° C, the dermis layer was formed, and the culture medium was added for C C. for 3 days, and the liquid was changed every day; After the culture solution was aspirated, the hole was evenly perforated with a 0.7 mm injection needle at a distance of 2 mm _; the cultured tissue engineering hair follicle suspension was added to the surface of the dermis at a density of 25/cm ² to make the suspension uniform. Penetrate into the hole; then in the true The surface of the cortex was inoculated with epidermal cells at a density of 10 ⁵ /ml, and cultured for 1 day in culture medium, and then transferred to a scaffold for subculture for 2 days; the culture medium E was replaced for gas-liquid surface culture for 2 days; The culture was carried out for 2 days; the liquid was changed every day during the culture, and the hair follicle tissue engineering skin preparation was completed.

Claims

Claim

A method for preparing a hair follicle tissue-engineered skin, characterized in that a tissue engineering hair follicle is uniformly inoculated into a tissue engineering dermis formed by a composite material of a scaffold and a cell, and then covered with epidermal cells, which are formed after being cultured. Tissue-engineered skin containing hair follicles; the tissue-engineered hair follicles are obtained by encapsulating mesenchymal-derived dermal papilla cells or stem cells which can differentiate into dermal papilla cells into a polymer material to form microcapsules having a particle diameter of 200 to 1000 μm. Then, the outer root sheath cells of the hair follicle or the epithelial cells having the stem cell function are wrapped around the periphery of the microcapsule by a rotary culture method to form a tissue engineering hair follicle having a particle diameter of 250 to 1100 μm; the scaffold material and the Like polymer materials, it is a biodegradable material that is acceptable to the body.

2. The preparation method according to claim 1, wherein the specific step comprises: Step 1: preparing a culture solution

Culture medium: In 500 ml of commercial DMEM containing V/V 15% fetal calf serum, epidermal growth factor EGF 2~10 g, fibroblast growth factor bFGF 5~50ng, bovine pituitary extract BPE 3 ~15g;

Culture medium C: In 500 ml of commercial DMEM medium containing V/V 10% fetal bovine serum, 2 to 50 ng of insulin, 10 to 500 ug of hydrocortisone, 10 to 17 mg of adenine, and 5 to 30 mg of Vc were added. EGF 2~10 g, bFGF 5~50ng, BPE 3~15g;

Step two, get the cell

The dermal papilla cells with good growth state are obtained, and the stem cells of different sources are required to be cultured by the culture supernatant of the dermal papilla cells; the outer root sheath cells of the hair follicles with good growth state are obtained, and the stem cells of other epithelial sources are not required to be induced;

Step 3, preparing tissue engineering hair follicles

The microcapsules containing the cells are mixed with the obtained outer root sheath cells of hair follicles or other epithelial-derived stem cells, and cultured in the culture medium A for 1 to 3 days, and the cells are attached to the microcapsules, and centrifuged at 300 rpm. The supernatant is cultured for 2 to 4 days, and the tissue engineering hair follicle is obtained;

Step 4: Preparation of skin tissue containing hair follicles

4°C, the scaffold material was prepared into a solution of 6~20mg/ml with 0.5M acetic acid solution, irradiated with ultraviolet light under ice bath, and then added 10% by volume of fetal bovine serum and the final concentration was 80~150mg. /ml of DMEM commercial medium, adjusted pH 7. 2~7. 4, into a gel solution; the nylon membrane is saturated with the gel solution, placed in a petri dish to form a support film; the cultured fibroblasts in 10 ^{5 to} 10 ⁶ / ml density mixed in the gel, which was added dropwise to the surface of the cured support film, cultured and solidified at 37 ° C to form a dermis layer, added to the culture solution C for 2 to 4 days, and changed every day; After the dermal layer is cultured, the culture solution is aspirated, and the needle is uniformly perforated on the surface with a hole spacing of 2 to 5 mm; the cultured tissue engineering hair follicle suspension is added to the surface of the dermis at a density of 10 to 100 pieces/cm ² . The suspension is evenly infiltrated into the well; then the epidermal cells are inoculated at a density of 10 ^{5 to} 10 ⁶ /cm ² on the surface of the dermis, and cultured for 1 day in the culture medium D, and then transferred to the scaffold for subcultivation for 2 to 4 days; Change culture medium E for gas-liquid surface culture for 1~2 days; replace culture medium F culture 1~ 2 days; change the liquid every day during the culture.