CN115537373A - Renewable artificial hair follicle and preparation method and application thereof - Google Patents
Renewable artificial hair follicle and preparation method and application thereof Download PDFInfo
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- CN115537373A CN115537373A CN202210796892.5A CN202210796892A CN115537373A CN 115537373 A CN115537373 A CN 115537373A CN 202210796892 A CN202210796892 A CN 202210796892A CN 115537373 A CN115537373 A CN 115537373A
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- skin
- hair follicle
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- hair
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Abstract
The invention discloses a preparation method and application of a renewable artificial hair follicle, which comprises the steps of culturing hair follicles in vitro, preparing biological 3D printing ink and a gel block, planting the hair follicles and sebaceous gland hanging drops on the biological 3D printing gel block, performing induction culture by using an induction culture medium to form a required skin accessory, printing the skin accessory and an ink box layer by layer to form a head skin, a dermis layer and an epidermis layer according to a set skin shape, and culturing the printed artificial head skin, wherein the hair follicles break dormancy and realize regeneration by virtue of a microenvironment provided by the artificial head skin. The hair loss area repairing device can repair the hair loss area in a targeted manner, the requirement of a user on nursing and planting the scalp in the future is low, the pain of the patient is low in the treatment process, the safety is high, the survival rate of the hair follicle in the later period is high, and the hair regeneration capacity is higher. Is not limited by the number of hair follicles on the scalp, and is suitable for patients with various types of alopecia.
Description
Technical Field
The invention relates to the field of biotechnology, and on the one hand, the invention discloses a preparation method of a renewable artificial hair follicle, and on the other hand, the invention also discloses the renewable artificial hair follicle prepared by the preparation method and application thereof.
Background
Hair loss caused by hair loss becomes an increasingly serious sub-health problem which troubles modern men (including some women), and although hair loss is not a serious physiological health problem, the hair loss seriously affects the psychological state of an individual, so that the quality of life of the individual is seriously reduced. According to incomplete statistics, more than 2 hundred million people in China currently have alopecia in different degrees, and the population of the same class is more than 10 hundred million worldwide. Pathological abnormal hair loss, such as male baldness, is that under the action of certain factors, hair follicles are in a resting period for a long time and cannot enter a growth cycle, at present, clinically, few medicines for pathological hair loss are provided, and the medicines only have effects on a small proportion of patients, such as minoxidil and finasteride, and the two medicines are two anti-hair-loss medicines which are widely applied clinically, are not only required to be taken for a long time and have side effects of different degrees, but also have weak anti-hair-loss effect.
Hair transplantation is currently the most effective and widely used technique, and includes FUT and FUE. Elliptical scalp cutting harvest was the preferred method of obtaining hair transplant hair follicle colonies since the mid-90 s of the 20 th century. Follicular Unit Extraction (FUE) has become an increasingly popular method of obtaining donor hair over the past 20 years. FUEs use manual or robotic devices to harvest individual follicular units from a donor area, and the main advantage of FUEs over oval donor harvesting is that no donor hair is harvested and then the closed linear scar is sutured or sutured. This is a great advantage, especially for those with short hairs, and no noticeable scars will be visible. Although FUE has the advantage of a wireless scar, other problems and potential long-term side effects remain. As with the full-thickness skin incision, harvesting each follicular unit results in scarring of the punch to a diameter of 1 mm. These scars can have an effect on the pigmentation and development of the remaining hair follicles. Furthermore, whether the FUE is performed by manual, motorized, or robotic perforation, as with the harvesting of follicles by oval scalp cutting of the donor, there is a risk of significant depletion of hair from the donor area, i.e., the donor area will have less detail.
In summary, in addition to drug treatment, hair follicle transplantation has become an emerging solution to the problem of hair loss in recent years. Aiming at the defects, a preparation method and application of an artificial hair follicle with high safety, low treatment limit and high hair follicle survival rate are urgently needed in the industry at present.
Disclosure of Invention
The present invention is directed to solving the technical problems in the related art at least to some extent. Therefore, the invention improves the traditional method: the design and application of 3D biological printing technology and tissue engineering dermatology are combined, artificial hair follicles containing epidermal stem cells, bone marrow mesenchymal stem cells, hair papilla cells and the like are implanted into a 3D biological printing microenvironment to realize artificial scalp micro units capable of regenerating hair follicles, the micro units are implanted between an epidermal layer and a dermal layer of a hair loss area of human scalp to realize high fusion with the skin micro units per se, the artificial scalp micro units are used for perfecting and forming a new healthy scalp environment, the aim of synchronously regenerating skin true epidermis and various skin accessories is realized physiologically, and the effect like real hair roots is visually conveyed. The skin micro-unit model prepared by the method realizes synchronous regeneration of the skin cuticle and various skin appendages, is not limited to printing hair follicles any more, but aims to help patients to construct a healthy scalp environment and enable the hairs to independently realize the regeneration function on human scalps.
The invention is realized by the following technical means:
a method for preparing regenerative artificial hair follicle, comprising:
(1) Culturing artificial hair follicles in vitro:
(1) separating and culturing human epidermal stem cells:
carefully separating the digested skin from the epidermis and dermis with an ophthalmic insert, immersing the epidermis in 0.25% trypsin, and digesting at 37 deg.C for 30min to obtain cell suspension. The digestion was stopped by adding DMEM containing 10% FBS, centrifuged at 15cm radius and 1000r/min for 5min, and the supernatant was discarded.
Placing the centrifuged cell suspension in a 60mm culture dish, adding 2-5 mL of culture medium (containing DMEM,10ng/mL EGF, 5ug/mL insulin, 100ug/mL gentamicin), placing at 37 deg.C, 5% 2 And culturing in an incubator with saturated humidity, changing the culture solution after 24 hours, and changing the culture solution 1 time every 2 days. Culturing until day 19, connecting the epidermal stem cells into sheets, and subculturing when 80% confluence is reached.
And (3) completely sucking the residual culture solution in the culture dish by using a suction pipe, adding 1-2 mL of 0.25% trypsin (based on the fact that the digestive juice can cover the bottom of the whole culture dish), standing for 2min, sucking out the trypsin solution, and adding DMEM culture solution to stop digestion. Centrifuging at a centrifugation radius of 15cm and 1000r/min for 5min, discarding the supernatant, and performing subculture according to a ratio of 1.
(2) And (3) separating and culturing hair papilla cells:
adding glutamine with final concentration of 2mmol/L, hepes (hydroxyethyl piperazine ethanesulfonic acid) 2mmol/L, hydrocortisone 10ug/L, transferrin 10mg/L, sodium selenite 10ug/L, and N-acetyl-L-D-arginine to Williams E serum-free medium 5 Preparing a hair follicle culture medium by using U/L penicillin and 100mg/L streptomycin, and adding 10mg/L bovine insulin before use;
the scalp was rinsed 3 times with PBS, scrubbed sterilized with 75% ethanol gauze, transferred to another sterile petri dish, and rinsed again with PBS. The surgical blade cuts the separated scalp to individual follicular units in succession. Visually selecting hair follicle with plump papilla, smooth and mellow appearance and black color, placing under a microscope to select complete follicular unit with complete papilla and no separation of inner and outer root sheaths, placing in 24-well plate (1 root/hole), adding prepared Williams E medium (0.5 mL/hole) 2 The culture was carried out in an incubator at 37 ℃ with 1 change of culture medium every 3 days.
(3) And (3) isolated culture of bone marrow mesenchymal stem cells:
taking bone marrow by puncturing bone marrow under aseptic condition, gently blowing and beating with PBS and suspending the bone marrow, centrifuging at 1500r/min for 10min, removing supernatant and fat layer, adding PBS and blowing and beating into single cell suspension. Slowly adding Percoll separating medium with relative density of 1.073 along the tube wall, and centrifuging at 1500r/min for 20min to obtain milky cloudy mononuclear cell layer;
washing the mononuclear cells with PBS for 3 times, centrifuging to collect the cells, suspending the cells in DMEM/F12 culture solution containing 100mL/L fetal calf serum for culture, changing the culture solution every other day, and discarding the non-adherent cells. Purifying and amplifying after 80-90% of adherent cells are fused;
pouring out the old culture solution, adding pancreatin, placing in an incubator for 2-3 min, observing the increase of intercellular gaps or the floating of a small amount of cells under an inverted microscope, and adding a culture medium containing serum to stop the pancreatin;
and (3) gently blowing and beating the cells to completely suspend, transferring the cells into a centrifuge tube, centrifuging the centrifuge tube at 800r/min for 7min, removing supernatant, collecting cell sediments, adding culture solution to suspend the cells, inoculating the cells into 3 culture bottles again according to the passage of 1.
(4) Separating and culturing human adipose-derived stem cells:
the neonate umbilical cord was placed in a sterile stainless steel box and the blood was flushed with PBS. Clamping one end of an umbilical cord by using hemostatic forceps, sucking PBS by using a 10mL coarse suction pipe to wash residual blood cells in the cavity of the umbilical vein for 5 times, sucking 1.25% collagenase by using the 10mL coarse suction pipe to fill the umbilical vein until the umbilical cord is full, and clamping the other port of the umbilical cord by using the hemostatic forceps;
the clamped umbilical cord is placed into a stainless steel box, the box cover is covered, the umbilical cord is moved to an incubator at 37 ℃ to be digested for 20min and is moved to a super clean workbench, hemostatic forceps at two ends are loosened, and a 10mL coarse suction tube is used for sucking PBS to wash umbilical vein for 5 times.
(5) Mixing the seed cells, and culturing in sterile environment to obtain in vitro artificial hair follicle.
(2) Preparing biological 3D printing ink and rubber blocks:
various nutrient substances required for promoting the growth of hair follicles are collected and uniformly mixed with the printing material to prepare biological 3D printing ink and printing rubber blocks.
Further, the printing material includes: one or more of lactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, poly (dl-lactide-co-poly (trimethylene carbonate)), nano beta-tricalcium phosphate, nano hydroxyapatite, nano calcium phosphate, gelatin, sodium alginate hydrogel or I type collagen hydrogel.
(3) In vitro culture of the required skin appendages:
and (3) planting the hair follicle and the sebaceous gland hanging drop on the biological 3D printing rubber block, then performing induction culture by using an induction culture medium, finally adding epidermal stem cells, and performing gas-liquid planar culture to form the required skin attachment foundation.
Further, the induction medium includes sweat gland induction medium, hair follicle induction medium and sebaceous gland induction medium.
Further, after the induction culture time is 5-10d, adding epidermal stem cells, and carrying out gas-liquid plane culture to form the required skin accessory foundation.
(4) Constructing the composite tissue engineering head skin:
and according to the set skin shape, printing the head skin layer by using the prepared skin attachment and the prepared ink box together. And after the dermis layer is printed, paving a layer of sheep acellular dermis matrix support, and continuously printing the epidermis layer part. Culturing the artificial head skin constructed by printing according to a conventional tissue engineering skin culture method to obtain the regenerative artificial hair follicle.
The invention also discloses a reproducible artificial hair follicle prepared by the preparation method.
The invention also discloses an application of the renewable artificial hair follicle to the surface of autologous skin.
The invention has the beneficial effects that:
(1) The performance is excellent: the 3D printing technology has great achievements in the medical field, the microenvironment in which the hair follicles can grow is printed by the 3D printing technology, the hair loss area can be repaired purposefully, similar printed skins have certain achievements at present, and the requirement on nursing and planting the scalp of a user in the future is low.
(2) The efficiency is high: 3D printing technique is high-efficient convenient, implants 3D artificial scalp micro unit between skin corium layer and epidermis through appointed biological instrument in alopecia area, can accomplish the implantation of 3D artificial scalp micro unit in the short time, and 3D prints the scalp micro unit and more lets people no longer endure the misery of hair transplantation in-process extraction hair follicle.
(3) The universality is high: simple hair transplantation transplants the hair follicle of the back of the head to the forehead area, and the hair just becomes position quantity and does not become more, and if the hair follicle is damaged, the hair transplantation effect will be discounted greatly. And through the combination of 3D biological printing technology and tissue engineering dermatology, a microenvironment containing renewable hair follicles is artificially cultured and implanted between the dermis layer and the epidermis layer without the limitation of the number of hair follicles on the scalp, so that the hair loss treatment device is suitable for various types of alopecia patients.
(4) The safety is high: the artificial hair follicle is obtained by taking autologous bone marrow mesenchymal stem cells, vascular endothelial cells, hair papilla cells, adipose-derived stem cells and the like as seed cells and culturing the seed cells by a tissue engineering technology. After the hair follicle is implanted into the self body, no obvious rejection reaction exists, and the high fusion with the self hair follicle can be realized.
(5) The survival rate is high: the 3D bioprinting microenvironment realizes synchronous regeneration of various skin appendages, enables the hair to independently realize the regeneration function on the scalp of a person again, and combines the manual work and the scalp of the person, so that the hair can survive for a long time after being transplanted, and the regeneration capability of the hair is stronger.
Detailed Description
The following detailed description of the embodiments of the present invention is intended to be illustrative, and not to be construed as limiting the invention.
Example 1
A method for preparing regenerative artificial hair follicle, comprising:
(1) Culturing artificial hair follicles in vitro:
(1) separating and culturing human epidermal stem cells:
carefully separating the digested skin from the epidermis and dermis with an ophthalmic insert, immersing the epidermis in 0.25% trypsin, and digesting at 37 deg.C for 30min to obtain cell suspension. Adding DMEM containing 10% FBS to stop digestion, centrifuging at a radius of 15cm, centrifuging at 1000r/min for 5min, and discarding the supernatant.
Placing the centrifuged cell suspension in a 60mm culture dish, adding 2-5 mL of culture medium (containing DMEM,10ng/mL EGF, 5ug/mL insulin, 100ug/mL gentamicin), placing at 37 deg.C and 5% C0 2 And culturing in an incubator with saturated humidity, changing the culture solution after 24h, and changing the culture solution for 1 time every 2 days. Culturing until day 19, connecting the epidermal stem cells into sheets, and subculturing when 80% confluence is reached.
And (3) completely sucking the rest culture solution in the culture dish by using a suction pipe, adding 1-2 mL of 0.25% trypsin (based on the fact that the digestive juice can cover the bottom of the culture dish), standing for 2min, sucking out the trypsin solution, and adding a DMEM culture solution to stop digestion. The cells were centrifuged at a centrifugation radius of 15cm at 1000r/min for 5min, and the supernatant was discarded and subcultured at a ratio of 1.
(2) And (3) isolated culture of hair papilla cells:
adding glutamine with final concentration of 2mmol/L, hepes (hydroxyethyl piperazine ethanesulfonic acid) 2mmol/L, hydrocortisone 10ug/L, transferrin 10mg/L, sodium selenite 10ug/L, and N-acetyl-L-D-arginine to Williams E serum-free medium 5 Preparing a hair follicle culture medium by using U/L penicillin and 100mg/L streptomycin, and adding 10mg/L bovine insulin before use;
the scalp was rinsed 3 times with PBS, scrubbed sterilized with 75% ethanol gauze, transferred to another sterile petri dish, and rinsed again with PBS. The surgical blade cuts the separated scalp to individual follicular units in succession. Visually selecting hair follicles with plump hair papillae, smooth and mellow appearance and black color, placing under a mirror to select complete follicular units with complete hair papillae and no separation of inner and outer root sheaths, placing in a 24-pore plate (1 root/hole), adding prepared Williams E culture medium, each hole is 0.5mL, and the content of C0 is 0.5% 2 The culture was carried out in an incubator at 37 ℃ with 1 change of culture medium every 3 days.
(3) And (3) isolated culture of bone marrow mesenchymal stem cells:
taking bone marrow by puncturing bone marrow under aseptic condition, gently blowing and beating with PBS and suspending the bone marrow, centrifuging at 1500r/min for 10min, removing supernatant and fat layer, adding PBS and blowing and beating into single cell suspension. Slowly adding Percoll separating medium with relative density of 1.073 along the tube wall, and centrifuging at 1500r/min for 20min to obtain milky cloudy mononuclear cell layer;
washing the mononuclear cells with PBS for 3 times, centrifuging to collect the cells, suspending the cells in DMEM/F12 culture solution containing 100mL/L fetal calf serum for culture, changing the solution every other day, and discarding the cells which are not attached to the wall. And purifying and amplifying after 80-90% of adherent cells are fused.
Pouring out the old culture solution, adding pancreatin, placing in an incubator for 2-3 min, observing the increase of intercellular gaps or the floating of a small amount of cells under an inverted microscope, and adding a culture medium containing serum to stop the pancreatin action.
Gently blowing and beating to completely suspend the cells, transferring the cells into a centrifuge tube, centrifuging the centrifuge tube at 800r/min for 7min, removing supernatant, collecting cell sediments, adding culture solution to suspend the cells, inoculating the cells into 3 culture bottles again according to the passage of 1;
(4) separating and culturing human adipose-derived stem cells:
the neonate umbilical cord was placed in a sterile stainless steel box and the blood was flushed with PBS. Clamping one end of an umbilical cord by using a hemostatic forceps, sucking PBS by using a 10mL coarse suction pipe to wash residual blood cells in the cavity of the umbilical vein for 5 times, sucking 1.25% collagenase by using the 10mL coarse suction pipe to fill the umbilical vein until the umbilical cord is full, and clamping the other port of the umbilical cord by using the hemostatic forceps.
Placing the clamped umbilical cord into a stainless steel box, covering the box cover, transferring to an incubator at 37 ℃ for digestion for 20min, transferring to a super clean workbench, loosening the hemostatic clamps at two ends, and sucking PBS by using a 10mL coarse suction pipe to wash umbilical vein for 5 times.
(5) Mixing the seed cells, and culturing in sterile environment to obtain in vitro artificial hair follicle.
(2) Preparing biological 3D printing ink and rubber blocks:
various nutrient substances required for promoting the growth of hair follicles are collected and uniformly mixed with a printing material to prepare biological 3D printing ink and printing rubber blocks.
Further, the printing material includes: lactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, racemic polylactic acid-poly-dispersed methylene carbonate copolymer, nano beta-tricalcium phosphate, nano hydroxyapatite, nano calcium phosphate, gelatin and sodium alginate hydrogel.
(3) In vitro culture of the required skin appendages:
and (3) planting the hair follicle and the sebaceous gland hanging drop on the biological 3D printing rubber block, then performing induction culture by using an induction culture medium, finally adding epidermal stem cells, and performing gas-liquid planar culture to form the required skin attachment foundation.
Further, the induction medium includes sweat gland induction medium, hair follicle induction medium and sebaceous gland induction medium.
Further, after the induction culture time is 8d, epidermal stem cells are added to carry out gas-liquid plane culture to form the required skin appendage foundation.
(4) Constructing the composite tissue engineering head skin:
and according to the set skin shape, printing the head skin layer by using the prepared skin attachment and the prepared ink box together. And after the dermis layer is printed, laying a layer of the sheep acellular dermal matrix support, and continuously printing the epidermis layer part. Culturing the artificial head skin constructed by printing according to a conventional tissue engineering skin culture method to obtain the regenerative artificial hair follicle.
Example 2
A method for preparing regenerative artificial hair follicle, comprising:
(1) In vitro artificial hair follicle culture:
(1) separating and culturing human epidermal stem cells:
carefully separating the digested skin from the epidermis and dermis with an ophthalmic insert, immersing the epidermis in 0.25% trypsin, and digesting at 37 deg.C for 30min to obtain cell suspension. The digestion was stopped by adding DMEM containing 10% FBS, centrifuged at 15cm radius and 1000r/min for 5min, and the supernatant was discarded.
Placing the centrifuged cell suspension in a 60mm culture dish, and adding 2-5 mL of culture solution (containingDMEM,10ng/mL EGF, 5ug/mL insulin, 100ug/mL gentamycin) at 37 ℃ 5% 2 And culturing in an incubator with saturated humidity, changing the culture solution after 24 hours, and changing the culture solution 1 time every 2 days. Culturing until day 19, connecting the epidermal stem cells into sheets, and subculturing when 80% of the epidermal stem cells are combined.
And (3) completely sucking the residual culture solution in the culture dish by using a suction pipe, adding 1-2 mL of 0.25% trypsin (based on the fact that the digestive juice can cover the bottom of the whole culture dish), standing for 2min, sucking out the trypsin solution, and adding DMEM culture solution to stop digestion. The cells were centrifuged at a centrifugation radius of 15cm at 1000r/min for 5min, and the supernatant was discarded and subcultured at a ratio of 1.
(2) And (3) isolated culture of hair papilla cells:
adding glutamine with final concentration of 2mmol/L, hepes (hydroxyethyl piperazine ethanesulfonic acid) 2mmol/L, hydrocortisone 10ug/L, transferrin 10mg/L, sodium selenite 10ug/L, and N-acetyl-L-D-arginine to Williams E serum-free medium 5 Preparing a hair follicle culture medium by using U/L penicillin and 100mg/L streptomycin, and adding 10mg/L bovine insulin before use;
the scalp was rinsed 3 times with PBS, scrubbed sterilized with 75% ethanol gauze, transferred to another sterile petri dish, and rinsed again with PBS. The surgical blade cuts the separated scalp to individual follicular units in succession. Visually selecting hair follicles with plump hair papillae, smooth and mellow appearance and black color, placing under a mirror to select complete follicular units with complete hair papillae and no separation of inner and outer root sheaths, placing in a 24-pore plate (1 root/hole), adding prepared Williams E culture medium, each hole is 0.5mL, and the content of C0 is 0.5% 2 The culture was carried out in an incubator at 37 ℃ with 1 change of culture medium every 3 days.
(3) And (3) separating and culturing the bone marrow mesenchymal stem cells:
taking bone marrow by puncturing bone marrow under aseptic condition, gently blowing and beating with PBS and suspending the bone marrow, centrifuging at 1500r/min for 10min, removing supernatant and fat layer, adding PBS and blowing and beating into single cell suspension. Slowly adding Percoll separating medium with relative density of 1.073 along the tube wall, and centrifuging at 1500r/min for 20min to obtain milky cloudy mononuclear cell layer;
washing the mononuclear cells with PBS for 3 times, centrifuging to collect the cells, suspending the cells in DMEM/F12 culture solution containing 100mL/L fetal calf serum for culture, changing the culture solution every other day, and discarding the non-adherent cells. And purifying and amplifying after 80-90% of adherent cells are fused.
Pouring out the old culture solution, adding pancreatin, placing in an incubator for 2-3 min, observing the increase of intercellular gaps or the floating of a small amount of cells under an inverted microscope, and adding a culture medium containing serum to stop the pancreatin action.
And (3) gently blowing and beating the cells to completely suspend, transferring the cells into a centrifuge tube, centrifuging the centrifuge tube at 800r/min for 7min, removing supernatant, collecting cell sediments, adding culture solution to suspend the cells, inoculating the cells into 3 culture bottles again according to the passage of 1.
(4) Separating and culturing human adipose-derived stem cells:
the neonate umbilical cord was placed in a sterile stainless steel box and the blood was flushed with PBS. Clamping one end of an umbilical cord by using a hemostatic forceps, sucking PBS by using a 10mL coarse suction pipe to flush residual blood cells in the cavity of the umbilical cord vein for 5 times, sucking 1.25% collagenase by using the 10mL coarse suction pipe to fill the umbilical cord vein until the umbilical cord is full, and clamping the other port of the umbilical cord by using the hemostatic forceps.
Placing the clamped umbilical cord into a stainless steel box, covering the box cover, transferring to an incubator at 37 ℃ for digestion for 20min, transferring to a super clean workbench, loosening the hemostatic clamps at two ends, and sucking PBS by using a 10mL coarse suction pipe to wash umbilical vein for 5 times.
(5) Mixing the seed cells, and culturing the mixture in a sterile environment to obtain the in-vitro artificial hair follicle.
(2) Preparing biological 3D printing ink and rubber blocks:
various nutrient substances required for promoting the growth of hair follicles are collected and uniformly mixed with the printing material to prepare biological 3D printing ink and printing rubber blocks.
Further, the printing material includes: lactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, racemic polylactic acid-poly-dispersed methylene carbonate copolymer, nano beta-tricalcium phosphate, nano hydroxyapatite, nano calcium phosphate, gelatin and type I collagen hydrogel.
(3) In vitro culture of the required skin appendages:
and (3) planting the hair follicles and the sebaceous glands on the biological 3D printing glue blocks, then performing induction culture by using an induction culture medium, finally adding epidermal stem cells, and performing gas-liquid planar culture to form the required skin appendage foundation.
Further, the induction medium includes sweat gland induction medium, hair follicle induction medium and sebaceous gland induction medium.
Further, after the induction culture time is 9d, epidermal stem cells are added to carry out gas-liquid plane culture to form the required skin appendage foundation.
(4) Constructing the head skin of the composite tissue engineering:
and according to the set skin shape, the prepared skin attachment and the prepared ink box are used together to print the head skin layer by layer. And after the dermis layer is printed, paving a layer of sheep acellular dermis matrix support, and continuously printing the epidermis layer part. Culturing the artificial head skin constructed by printing according to a conventional tissue engineering skin culture method to obtain the regenerative artificial hair follicle.
Example 3
A method for preparing regenerative artificial hair follicle, comprising:
(1) Culturing artificial hair follicles in vitro:
(1) separating and culturing human epidermal stem cells:
carefully separating the digested skin from the epidermis and dermis with an ophthalmic insert, immersing the epidermis in 0.25% trypsin, and digesting at 37 deg.C for 30min to obtain cell suspension. Adding DMEM containing 10% FBS to stop digestion, centrifuging at a radius of 15cm, centrifuging at 1000r/min for 5min, and discarding the supernatant.
Placing the centrifuged cell suspension in a 60mm culture dish, adding 2-5 mL of culture medium (containing DMEM,10ng/mL EGF, 5ug/mL insulin, 100ug/mL gentamicin), placing at 37 deg.C, 5% 2 And culturing in an incubator with saturated humidity, changing the culture solution after 24 hours, and changing the culture solution 1 time every 2 days. Culturing until day 19, connecting the epidermal stem cells into sheets, and subculturing when 80% confluence is reached.
And (3) completely sucking the rest culture solution in the culture dish by using a suction pipe, adding 1-2 mL of 0.25% trypsin (based on the fact that the digestive juice can cover the bottom of the culture dish), standing for 2min, sucking out the trypsin solution, and adding a DMEM culture solution to stop digestion. Centrifuging at a centrifugation radius of 15cm and 1000r/min for 5min, discarding the supernatant, and performing subculture according to a ratio of 1.
(2) And (3) isolated culture of hair papilla cells:
adding glutamine with final concentration of 2mmol/L, hepes (hydroxyethyl piperazine ethanesulfonic acid) 2mmol/L, hydrocortisone 10ug/L, transferrin 10mg/L, sodium selenite 10ug/L, and N-acetyl-L-D-arginine to Williams E serum-free medium 5 Preparing a hair follicle culture medium by using U/L penicillin and 100mg/L streptomycin, and adding 10mg/L bovine insulin before use;
the scalp was rinsed 3 times with PBS, scrubbed sterilized with 75% ethanol gauze, transferred to another sterile petri dish, and rinsed again with PBS. The surgical blade cuts the separated scalp to individual follicular units in succession. Visually selecting hair follicles with plump hair papillae, smooth and mellow appearance and black color, placing under a mirror to select complete follicular units with complete hair papillae and no separation of inner and outer root sheaths, placing in a 24-pore plate (1 root/hole), adding prepared Williams E culture medium, each hole is 0.5mL, and the content of C0 is 0.5% 2 The culture was carried out in an incubator at 37 ℃ with 1 change of culture medium every 3 days.
(3) And (3) separating and culturing the bone marrow mesenchymal stem cells:
taking bone marrow by puncturing bone marrow under aseptic condition, gently blowing and beating with PBS and suspending the bone marrow, centrifuging at 1500r/min for 10min, removing supernatant and fat layer, adding PBS and blowing and beating into single cell suspension. Slowly adding Percoll separating medium with relative density of 1.073 along the tube wall, and centrifuging at 1500r/min for 20min to obtain milky cloudy mononuclear cell layer;
washing the mononuclear cells with PBS for 3 times, centrifuging to collect the cells, suspending the cells in DMEM/F12 culture solution containing 100mL/L fetal calf serum for culture, changing the solution every other day, and discarding the cells which are not attached to the wall. And purifying and amplifying after 80-90% of adherent cells are fused.
Pouring out the old culture solution, adding pancreatin, placing in an incubator for 2-3 min, observing the increase of intercellular gaps or the floating of a small amount of cells under an inverted microscope, and adding a culture medium containing serum to stop the pancreatin action.
Gently blowing and beating to completely suspend the cells, transferring the cells into a centrifuge tube, centrifuging the centrifuge tube at 800r/min for 7min, removing supernatant, collecting cell sediments, adding culture solution to suspend the cells, inoculating the cells into 3 culture bottles again according to 1;
(4) separating and culturing human adipose-derived stem cells:
the neonate umbilical cord was placed in a sterile stainless steel box and the blood was flushed with PBS. Clamping one end of an umbilical cord by using a hemostatic forceps, sucking PBS by using a 10mL coarse suction pipe to flush residual blood cells in the cavity of the umbilical cord vein for 5 times, sucking 1.25% collagenase by using the 10mL coarse suction pipe to fill the umbilical cord vein until the umbilical cord is full, and clamping the other port of the umbilical cord by using the hemostatic forceps.
The clamped umbilical cord is placed into a stainless steel box, the box cover is covered, the umbilical cord is moved to an incubator at 37 ℃ to be digested for 20min and is moved to a super clean workbench, hemostatic forceps at two ends are loosened, and a 10mL coarse suction tube is used for sucking PBS to wash umbilical vein for 5 times.
(5) Mixing the seed cells, and culturing in sterile environment to obtain in vitro artificial hair follicle.
(2) Preparing biological 3D printing ink and rubber blocks:
various nutrient substances required for promoting the growth of hair follicles are collected and uniformly mixed with a printing material to prepare biological 3D printing ink and printing rubber blocks.
Further, the printing material includes: lactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, racemic polylactic acid-polydimethylene carbonate copolymer, nano beta-tricalcium phosphate, nano hydroxyapatite, nano calcium phosphate, gelatin, sodium alginate hydrogel and I type collagen hydrogel.
(3) In vitro culture of the required skin appendages:
and (3) planting the hair follicle and the sebaceous gland hanging drop on the biological 3D printing rubber block, then performing induction culture by using an induction culture medium, finally adding epidermal stem cells, and performing gas-liquid planar culture to form the required skin attachment foundation.
Further, the induction medium includes sweat gland induction medium, hair follicle induction medium and sebaceous gland induction medium.
Further, after the induction culture time is 10 days, epidermal stem cells are added to carry out gas-liquid plane culture to form the required skin appendage foundation.
(4) Constructing the head skin of the composite tissue engineering:
and according to the set skin shape, printing the head skin layer by using the prepared skin attachment and the prepared ink box together. And after the dermis layer is printed, paving a layer of sheep acellular dermis matrix support, and continuously printing the epidermis layer part. Culturing the artificial head skin constructed by printing according to a conventional tissue engineering skin culture method to obtain the regenerative artificial hair follicle.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (7)
1. A method for preparing regenerative artificial hair follicle, comprising:
(1) Culturing artificial hair follicles in vitro;
(2) Preparing biological 3D printing ink and rubber blocks: collecting various nutrient substances required for promoting hair follicle growth, and uniformly mixing the nutrient substances with a printing material to prepare biological 3D printing ink and printing glue blocks for later use;
(3) In vitro culture of the required skin appendages: planting the hair follicles and sebaceous gland hanging drops on the biological 3D printing glue block, performing induction culture by using an induction culture medium, and finally adding epidermal stem cells to perform gas-liquid planar culture to form a required skin attachment;
(4) Constructing the composite tissue engineering head skin: according to the set skin shape, printing the skin of the head layer by the skin accessory and the ink box; after the dermis layer is printed, laying a layer of sheep acellular dermis matrix support, and continuously printing the epidermis layer part; culturing the artificial head skin printed and constructed according to a conventional tissue engineering skin culture method to obtain the regenerative artificial hair follicle.
2. The production method according to claim 1, wherein:
the culture of the in vitro artificial hair follicle in the step (1) comprises the following steps:
carrying out isolated culture on human epidermal stem cells, hair papilla cells, bone marrow mesenchymal stem cells and human adipose stem cells, mixing all seed cells, and culturing the mixture into the in-vitro artificial hair follicle in a sterile environment.
3. The production method according to claim 1, wherein:
the printing material of step (2) comprises: one or more of lactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, racemic polylactic acid-poly-dispersed methylene carbonate copolymer, nano beta-tricalcium phosphate, nano hydroxyapatite, nano calcium phosphate, gelatin, sodium alginate hydrogel and type I collagen hydrogel.
4. The production method according to claim 1, wherein:
the induction culture medium in the step (3) comprises a sweat gland induction culture medium, a hair follicle induction culture medium and a sebaceous gland induction culture medium.
5. The production method according to claim 1, wherein:
and (4) the induction culture time in the step (3) is 5-10 days.
6. A regenerative artificial hair follicle produced by the production method according to any one of claims 1 to 5.
7. Use of the artificial hair follicle according to claim 6, inoculated onto an autologous skin surface.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775366A (en) * | 2010-02-05 | 2010-07-14 | 中国人民解放军第四军医大学 | Preparation method of tissue engineering skin containing hair follicles |
| CN204521701U (en) * | 2015-03-30 | 2015-08-05 | 朱家源 | By the organization engineering skin that 3D prints |
| CN108525021A (en) * | 2018-04-17 | 2018-09-14 | 山西医科大学 | Contain blood vessel and hair follicle structure organization engineering skin and preparation method thereof based on 3D printing |
| CN111265723A (en) * | 2020-01-21 | 2020-06-12 | 南京师范大学 | 3D printed skin and preparation method thereof |
| CN111330082A (en) * | 2020-01-16 | 2020-06-26 | 中国人民解放军总医院 | Preparation method for constructing biological 3D printing skin micro-unit model containing skin accessory |
-
2022
- 2022-07-06 CN CN202210796892.5A patent/CN115537373A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775366A (en) * | 2010-02-05 | 2010-07-14 | 中国人民解放军第四军医大学 | Preparation method of tissue engineering skin containing hair follicles |
| CN204521701U (en) * | 2015-03-30 | 2015-08-05 | 朱家源 | By the organization engineering skin that 3D prints |
| CN108525021A (en) * | 2018-04-17 | 2018-09-14 | 山西医科大学 | Contain blood vessel and hair follicle structure organization engineering skin and preparation method thereof based on 3D printing |
| CN111330082A (en) * | 2020-01-16 | 2020-06-26 | 中国人民解放军总医院 | Preparation method for constructing biological 3D printing skin micro-unit model containing skin accessory |
| CN111265723A (en) * | 2020-01-21 | 2020-06-12 | 南京师范大学 | 3D printed skin and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张菊芳: "毛发整形美容学", 28 February 2013, 浙江科学技术出版社, pages: 332 * |
| 蔡景龙: "瘢痕整形美容外科学", 31 March 2015, 浙江科学技术出版社, pages: 514 * |
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