CN101775366A - Preparation method of tissue engineering skin containing hair follicles - Google Patents
Preparation method of tissue engineering skin containing hair follicles Download PDFInfo
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- CN101775366A CN101775366A CN201010107206A CN201010107206A CN101775366A CN 101775366 A CN101775366 A CN 101775366A CN 201010107206 A CN201010107206 A CN 201010107206A CN 201010107206 A CN201010107206 A CN 201010107206A CN 101775366 A CN101775366 A CN 101775366A
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Abstract
The invention relates to a preparation method of tissue engineering skin containing hair follicles. In the invention, tissue engineering hair follicles are evenly inoculated in a tissue engineering dermal layer formed by compounding a bracket material and cells, then the tissue engineering dermal layer is covered by epidermal cells, and the tissue engineering skin containing the hair follicles is formed after culture; the invention overcomes the defects of little number of the hair follicles in the skin, no uniformity and uncontrollable quantity of the hair follicles caused by mixed injection formerly, and the prepared tissue engineering skin contains an epidermis, dermis and hair follicle type tissue structure, wherein the number of the hair follicles approaches to the natural number, the hair follicles are evenly distributed in artificial pores and develop continuously after in-vitro culture and transplantation, a multi-layer concentric circle structure is formed, an inner layer is hair papilla cells wrapt by micro-capsule membranes, and an outer layer is epithelium class cells; and the skin cultured in vitro or transplanted in vivo finally forms functional skin containing a hair follicle accessory structure, and the regeneration of the hair follicles after skin transplantation can be realized.
Description
Technical field
The invention belongs to the tissue engineering technique field of biomaterial, be specifically related to a kind of preparation method who contains the organization engineering skin of hair follicle.
Background technology
Organizational project is the research focus of life science, and relevant research relates to all respects.Its content is that the functioning cell of vitro culture is planted in corresponding timbering material, forms tissue or the organ with certain function after cultivating.Along with developing rapidly of tissue engineering technique, organization engineering skin has begun to be extensive use of clinically.Present organization engineering skin mainly is that dermis of skin cell and epidermic cell are planted in the dermal scaffold material, the organization engineering skin of Gou Jianing has corium similar to normal skin and epidermis two-layer structure thus, but do not have the accessory organ of skins such as hair follicle, sebiferous gland, on weave construction, still have difference with normal skin tissue.
Skin is the organ of human body maximum, has functions such as protection body, regulate body temperature, drainage refuse.But, make the impaired or forfeiture of skin function, thereby cause internal organs to lose protection, even influence life owing to reasons such as inflammation, ulcer, wound, burn, tumor post-operation and congenital malformation cause the damaged or unusual of skin and mucous membrane.Employing can cause the new wound of skin donor site from body flap and auto-skin grafting, often can't satisfy the needs that big area is transplanted because the skin donor site source is limited.This crisis has been alleviated in the application of organization engineering skin, but does not contain accessory organ's organization engineering skin, can not satisfy patient demand on function.
Hair follicle is the important accessory organ of skin, and important effect is arranged in the physiological process of skin.The disappearance of head hair follicle and can cause alopecia areata or whole alopecia unusually influences the quality of life that has also influenced the patient time attractive in appearance; The hair follicle of body part has unusually then had a strong impact on the metabolism and the thermal exchange of health, causes the patient to live in the homothermic environment.Therefore, the regeneration of hair follicle becomes the key that addresses the above problem.
In developmental biology research, hair follicle is the interactional typical organ of epithelium and mesenchyme, early stage in fetal development, the downward projection of epidermic cell forms a mao nail, and hair is followed closely and extended, expands the blastema that forms sebiferous gland downwards, expands and locates to be the blastema of hair muscle, further form pin feather, sebiferous gland, hair muscle are shaped gradually, form hair papilla, inner root sheath at last, and the hairiness form becomes in the hair follicle.Sophisticated hair follicle is divided into hair shaft, epithelial root sheath, dermal sheath and hair papilla four parts.Epithelial root sheath is close to hair shaft, be made up of the multilayer superficial cell, can be divided into internal root sheath and external root sheath from inside to outside, internal root sheath links to each other with the epidermis that caves in sebiferous gland opening level, be similar to the transparent layer and the cutinized layer of epidermis, the cell of external root sheath is similar to the cell of basal layer of epidermis and spinous layer; Dermal sheath and dense connective tissue on every side thereof are to be transformed by corium, are equivalent to the skin corium of skin; Hair papilla cell also is the cell of corium source property, has important effect for the developmental biology of hair follicle, can induce hair follicle to generate in vivo and in vitro.Evidence suggests that hair follicle has important effect qualitatively in the formation of minimizing cicatrix of skin, raising skin wound healing speed and wound healing; And in the hair follicle outer root sheath cell at hair follicle knuckle position, have the hair follicle stem cells of supporting that the follicular epithelium cell upgrades, and not only in periodically growing, hair follicle plays an important role, and also be simultaneously the cell source of epidermis self; In addition, dermal sheath cell and hair papilla cell can transform mutually under certain condition, play the effect of keeping hair follicle growth, and demonstrate the characteristics of some stem cells, behind skin wound, can be converted into skin flbroblast and participate in or participate in directly the reconstruction of dermis of skin tissue, promotion skin healing.
Microcapsulary is that the various materials of dispersive (comprising solid, liquid, gas and biological substance) are encapsulated in the film that one deck is made up of macromolecular material, and forming diameter is the technology of the spherical microcapsule of 50～1000 μ m.Biologically active substances such as proteolytic enzyme and hormone are encapsulated in the spherical microcapsule that form in the selective permeation film, are called bio-microcapsule.The research report, microcapsulary is combined with the histocyte transplanting, cell is encapsulated in the film, is characterized in that the selective permeation film can avoid the attack of immunity system to the capsule inner cell, and biologically active substance and meta-bolites can freely be come in and gone out in micromolecular nutritive substance and the capsule.
At present, the research of tissue engineering skin containing hair follicles has become focus.The condition that the early stage hair follicle of simulation embryo forms with epithelium class cell and mesenchymal cytomixis, can realize the regeneration of hair follicle through culturing in vivo; Mostly be to adopt to have the hair papilla cell of inducing the hair follicle regeneration effect and mix back injection dermal scaffold with hair follicle outer root sheath cell or hair follicle stem cells and cultivate, in the hope of the formation tissue engineering skin containing hair follicles; No matter the wherein formation of hair follicle still be formed hair follicle structure from quantity, all undesirable.Early stage hair follicle is that epithelial cell depression and gathering hold agglomerating mesenchymal cell after grow formation gradually, contact unordered with mesenchymal cell and mix the epithelium class cell that injects, make iuntercellular need identification back self rearrangement mutually, hair follicle structure of Xing Chenging and size are uneven thus.Hair follicle in the skin should be to be evenly distributed and enormous amount, and the part forms or the hair follicle of a small amount of formation is only arranged is to play corresponding physiological action.
Summary of the invention
At the existing problem of prior art, the purpose of this invention is to provide a kind of preparation method of tissue engineering skin containing hair follicles, make prepared organization engineering skin contain a large amount of hair follicle structure, can realize the hair follicle regeneration after the dermatoplasty.
The preparation method of tissue engineering skin containing hair follicles proposed by the invention, it is characterized in that, be that the organizational project hair follicle evenly is inoculated in the tissue engineered dermal equivalent layer that is compounded to form by timbering material and cell,, after cultivating, promptly form the organization engineering skin that contains hair follicle again coated with epidermic cell; Described organizational project hair follicle is with the hair papilla cell in mesenchyme source or can be wrapped in that to form particle diameter in the macromolecular material be the micro-capsule of 200～1000 μ m to the stem cell of hair papilla cell differentiation, and adopting the rotating and culturing method that the hair follicle outer root sheath cell of epithelial origin or the epithelium class cell with stem cell function are wrapped in micro-capsule again, peripheral to form particle diameter be the organizational project hair follicle of 250～1100 μ m; Described timbering material is the same with described macromolecular material, be the acceptable Biodegradable material of body, can select any or several combinations of extracellular matrix, collagen, hyaluronic acid, chondroitin sulfate, gelatin, Fibrin Glue, chitosan, alginates, protein-polysaccharide, polylysine, poly(lactic acid), polyglycolic acid.
Described various types of cells is from body or allosome derived cell; Wherein, the hair papilla cell in the micro-capsule or can select any of embryonic stem cell, IPS cell, mesenchymal stem cells MSCs, hemopoietic stem cell or dermal sheath stem cell to the stem cell of hair papilla cell differentiation; Outer field hair follicle outer root sheath cell of micro-capsule or the epithelium class cell with stem cell function can be selected any of epidermal stem cells, hair follicle stem cells or hair follicle knuckle portion stem cell.
Preparation method's of the present invention concrete steps comprise:
Step 1, preparation nutrient solution
Nutrient solution A: in containing the commercial DMEM nutrient solution 500ml that V/V is 15% foetal calf serum, be added with Urogastron EGF 2～10 μ g, fibroblast growth factor bFGF 5～50ng, Niu Chuiti extract B PE 3～15g;
Nutrient solution B: in the commercial Williams E of serum-free nutrient solution 500ml, be added with EGF 2～10 μ g, bFGF 5～50ng, BPE 3～15g, insulin-like growth factor I GF-15～50ng;
Nutrient solution C: in containing the commercial DMEM nutrient solution 500ml that V/V is 10% foetal calf serum, be added with Regular Insulin 2～50ng, hydrocortisone 10～500 μ g, VITAMIN B4 10～17mg, Vc 5～30mg, EGF 2～10 μ g, bFGF 5～50ng, BPE 3～15g;
Nutrient solution D: the calcium chloride that in nutrient solution C, contains 0.1～1mM concentration;
Nutrient solution E: the calcium chloride that in nutrient solution C, contains 0.2～2mM concentration;
Nutrient solution F: the calcium chloride that in nutrient solution C, contains 0.3～3mM concentration;
Step 2, obtain cell
But hair papilla cell obtain and cultivate reference literature [Cheng Bo etc. improvement hair papilla cell culture methods " Chinese journal of dermatology " 2,001 34 (5) 387～388] or other prior arts, it is good and have a hair papilla cell of compendency growth characteristics to obtain growth conditions; The stem cell of different sources is through the inducing culture of hair papilla cell culture supernatant, have the hair papilla cell feature after, can be used as the micro-capsule inner cell of organizational project hair follicle.But the hair follicle outer root sheath cell obtain and cultivate reference literature [cultivation of people's hair follicle outer root sheath cells such as Tang Jianbing " China burn magazine " 2,003 19 (1) 47～48] or other prior arts, obtain the good hair follicle outer root sheath cell of growth conditions; The stem cell of other epithelial origins can obtain with reference to prior art, need not to induce promptly the okioplast as the organizational project hair follicle.Obtaining and cultivating of inoblast and epidermic cell can be with reference to [press of " organizational engineering philosophy and technique " banket chief editor The Fourth Military Medical University 239～240] or other prior arts acquisitions.
Step 3, preparation organizational project hair follicle
But the preparation reference literature of celliferous micro-capsule [intensity, permeability and the Study on biocompatibility " Third Military Medical University's journal " 2,009 31 (17) 1653～1656 of ACA such as Liu Rui, APA micro-capsule] or other prior arts, preparation macromolecular material micro-capsule contains hair papilla cell or can be to the stem cell of hair papilla cell direction differentiation in the micro-capsule; The micro-capsule that will contain cell mixes with the hair follicle outer root sheath cell that obtains or the stem cell of other epithelial origins, adopted rotating and culturing 1～3 day with nutrient solution A, treat that cell is attached at micro-capsule, with the centrifugal supernatant discarded of 300rpm, change nutrient solution B and cultivated 2～4 days, obtain the organizational project hair follicle; The preparation of step 4, tissue engineering skin containing hair follicles
Under 4 ℃ of conditions, with the acetum of 0.5M timbering material is mixed with the solution of 6～20mg/ml, uviolizing under the ice bath adds the foetal calf serum of 10% volume ratio and the commercial substratum of DMEM that final concentration is 80～150mg/ml more respectively, transfer pH7.2～7.4, make gelating soln; Nylon membrane is soaked into gelating soln, place culture dish to solidify to form support membrane; The inoblast of vitro culture with 10
5～10
6The density of individual/ml is mixed in gel, and it is dropped to solidified support membrane surface, cultivates for 37 ℃ to solidify back formation skin corium, adds nutrient solution C and cultivates 2～4 days, changes liquid every day; Inhale after skin corium is cultivated and finished and abandon nutrient solution, evenly punch on the surface with syringe needle, pitch of holes is 2～5mm; The organizational project hair follicle suspension of cultivating is pressed 10～100/cm
2Density drip on the skin corium surface, make suspension evenly infiltrate in the hole; Press 10 on the skin corium surface then
5～10
6Individual/cm
2Density inoculation epidermic cell, add nutrient solution D and cultivate after 1 day, move to and carried out liquid under cultivation on the support 2～4 days; Change nutrient solution E and carry out solution-air face cultivation 1～2 day; Changing nutrient solution F cultivated 1～2 day; Change liquid every day between incubation period.
The present invention adopts microcapsule technology with hair papilla cell or can wrap up with macromolecular material to the stem cell of hair papilla cell differentiation, utilize the rotating and culturing method with the hair follicle outer root sheath cell or can be inoculated in the micro-capsule periphery again to the stem cell of hair follicle outer root sheath cell differentiation, make hair papilla cell be in hair follicle inside, wrapped up by dermal sheath cell on every side, just like natural micro-capsule, give the more perfect developmental condition of hair follicle, after cultivating, form the organizational project hair follicle of enormous amount; It evenly is inoculated in the skin corium of organization engineering skin, promptly forms tissue engineering skin containing hair follicles through cultivating maturation coated with epidermic cell.
The present invention has overcome little, inhomogeneous, the uncontrollable shortcoming of hair follicle amount of hair follicle quantity in the skin that hybrid injection caused in the past, prepared organization engineering skin contains epidermis, corium and hair follicle sample weave construction, wherein hair follicle quantity is near natural quantity and being uniformly distributed in the artificial pore, hair follicle is educated in vitro culture and through transplanting follow-up supervention, form the multiwalled concentric structure, internal layer is the hair papilla cell that microcapsule membrane wrapped up, and skin is an epithelium class cell; The final functional skin that contains the hair follicle accessory structure that forms of the skin of transplanting in vitro culture or body can be realized the hair follicle regeneration after the dermatoplasty.
Embodiment
Embodiment below in conjunction with the example in detail technical solution of the present invention.
Step 1, preparation nutrient solution
Nutrient solution A: in containing the commercial DMEM nutrient solution 500ml that V/V is 15% foetal calf serum, be added with Urogastron EGF 6 μ g, fibroblast growth factor bFGF 10ng, Niu Chuiti extract B PE 25 μ g/ml;
Nutrient solution B: in the commercial Williams E of serum-free nutrient solution 500ml, be added with EGF 6 μ g, bFGF 10ng, BPE 25 μ g/ml, insulin-like growth factor I GF-110ng;
Nutrient solution C: in containing the commercial DMEM nutrient solution 500ml that V/V is 10% foetal calf serum, be added with Regular Insulin 10ng, hydrocortisone 100 μ g, VITAMIN B4 15mg, Vc 10mg, EGF 6 μ g, bFGF 10ng, BPE 25 μ g/ml;
Nutrient solution D: the calcium chloride that in nutrient solution C, contains 0.8mM concentration;
Nutrient solution E: the calcium chloride that in nutrient solution C, contains 1.9mM concentration;
Nutrient solution F: the calcium chloride that in nutrient solution C, contains 2.6mM concentration;
Step 2, obtain cell
1), the obtaining and cultivating of hair papilla cell: clip head part skin, insert and contain in two anti-PBS liquid flushing 3 times, microscopically separates hair follicle; Place centrifuge tube to add 2ml collagenase solution (625U/ml) isolating hair follicle, digestion is 1 hour in 37 ℃ of incubators, and hair papilla is loose free from hair matrix, and piping and druming makes it to dissociate out gently; Shift the hair follicle dermal papilla tissue to culturing bottle with inspection embryo pin, add the DMEM nutrient solution (it is two anti-to be added with 100U/ml) that contains 4% serum, at 5%CO
2Cultivate in 37 ℃ of incubators, obtain the good hair papilla cell of growth conditions, as the micro-capsule inner cell of organizational project hair follicle;
2), the obtaining and cultivating of hair follicle outer root sheath cell: hair follicle is placed centrifuge tube, adding 4 ℃ of digestion of dispase enzyme spends the night, under the Stereo microscope hair shaft extruded from hair follicle together with the root sheath epithelial cell and put into 24 orifice plates, the cover glass lid is pressed on the hair shaft tissue, add serum-free K-SFM (GBICO) nutrient solution and cultivate, obtain the good hair follicle outer root sheath cell of growth conditions;
Obtaining and cultivating of inoblast and epidermic cell can be with reference to [press of " organizational engineering philosophy and technique " banket chief editor The Fourth Military Medical University 239～240] or other prior arts acquisitions.
Step 3, preparation organizational project hair follicle
Hair papilla cell is digested to unicellular with 0.25% trypsin solution, is suspended in the sodium alginate soln of 20g/L concentration, cell concn is 5 * 10
8Individual/L, splash in the calcium chloride solution of 11g/L through the high-voltage electric field generator for microcapsules, form little glueballs, physiological saline cleans 3 times; Act on 10min in 0.3% chitosan solution, physiological saline cleans 3 times; Act on 4min in the 2g/L sodium alginate soln, physiological saline cleans 3 times; In the 0.05mol/L liquor sodii citratis, act on 6min again, clean 3 times with physiological saline; Obtain sodium alginate-chitose-sodium alginate microcapsule, contain hair papilla cell in the micro-capsule.To contain cell microcapsule and mix, and add nutrient solution A and put into agravic rotating generator cultivation 2 days, treat that outer root sheath cell is attached at micro-capsule, and, change nutrient solution B and cultivated 3 days, obtain the organizational project hair follicle with the centrifugal supernatant discarded of 300rpm with the hair follicle outer root sheath cell that obtains.
The preparation of step 4, tissue engineering skin containing hair follicles
Under 4 ℃ of conditions, with the acetum of 0.5M extracellular matrix, collagen and hyaluronic acid are mixed with the solution of 8mg/ml, uviolizing under the ice bath adds the foetal calf serum of 10% volume ratio and the commercial substratum of DMEM that final concentration is 100mg/ml more respectively, transfer pH7.3, make gelating soln; Nylon membrane is soaked into gelating soln, place culture dish to solidify to form support membrane; The inoblast of vitro culture with 10
5The concentration of individual/ml is mixed in the gel, and it is dropped to solidified support membrane surface, cultivates for 37 ℃ to solidify back formation skin corium, adds nutrient solution C and cultivates 3 days, changes liquid every day; Inhale after skin corium is cultivated and finished and abandon nutrient solution, evenly punch on the surface with the 0.7mm injection needles, pitch of holes is 2mm; The organizational project hair follicle suspension of cultivating is pressed 25/cm
2Density drip on the skin corium surface, make suspension evenly infiltrate in the hole; Press 10 on the skin corium surface then
5The density inoculation epidermic cell of individual/ml adds nutrient solution D and cultivates after 1 day, moves to and carries out on the support cultivating 2 days under the liquid; Change nutrient solution E and carry out solution-air face cultivation 2 days; Changing nutrient solution F cultivated 2 days; Change liquid every day between incubation period, and the tissue engineering skin containing hair follicles preparation is finished.
Claims (2)
1. the preparation method of a tissue engineering skin containing hair follicles, it is characterized in that, be that the organizational project hair follicle evenly is inoculated in the tissue engineered dermal equivalent layer that is compounded to form by timbering material and cell,, after cultivating, promptly form the organization engineering skin that contains hair follicle again coated with epidermic cell; Described organizational project hair follicle is with the hair papilla cell in mesenchyme source or can be wrapped in that to form particle diameter in the macromolecular material be the micro-capsule of 200～1000 μ m to the stem cell of hair papilla cell differentiation, adopt the rotating and culturing method with the hair follicle outer root sheath cell or the epithelium class cell with stem cell function is wrapped in the micro-capsule periphery again, forming particle diameter is the organizational project hair follicle of 250～1100 μ m; Described timbering material is the same with described macromolecular material, is the acceptable Biodegradable material of body.
2. preparation method according to claim 1 is characterized in that concrete steps comprise:
Step 1, preparation nutrient solution
Nutrient solution A: in containing the commercial DMEM nutrient solution 500ml that V/V is 15% foetal calf serum, be added with Urogastron EGF 2～10 μ g, fibroblast growth factor bFGF 5～50ng, Niu Chuiti extract B PE 3～15g;
Nutrient solution B: in the commercial Williams E of serum-free nutrient solution 500ml, be added with EGF 2～10 μ g, bFGF 5～50ng, BPE 3～15g, insulin-like growth factor I GF-15～50ng;
Nutrient solution C: in containing the commercial DMEM nutrient solution 500ml that V/V is 10% foetal calf serum, be added with Regular Insulin 2～50ng, hydrocortisone 10～500 μ g, VITAMIN B4 10～17mg, Vc 5～30mg, EGF 2～10 μ g, bFGF 5～50ng, BPE 3～15g;
Nutrient solution D: the calcium chloride that in nutrient solution C, contains 0.1～1mM concentration;
Nutrient solution E: the calcium chloride that in nutrient solution C, contains 0.2～2mM concentration;
Nutrient solution F: the calcium chloride that in nutrient solution C, contains 0.3～3mM concentration;
Step 2, obtain cell
Obtain the good hair papilla cell of growth conditions, need be to the stem cell of different sources through the inducing culture of hair papilla cell culture supernatant; Obtain the good hair follicle outer root sheath cell of growth conditions, the stem cell of other epithelial origins be need not to induce;
Step 3, preparation organizational project hair follicle
The micro-capsule that will contain cell mixes with the hair follicle outer root sheath cell that obtains or the stem cell of other epithelial origins, adopted rotating and culturing 1～3 day with nutrient solution A, treat that cell is attached at micro-capsule, with the centrifugal supernatant discarded of 300rpm, change nutrient solution B and cultivated 2～4 days, obtain the organizational project hair follicle;
The preparation of step 4, tissue engineering skin containing hair follicles
Under 4 ℃ of conditions, with the acetum of 0.5M timbering material is mixed with the solution of 6～20mg/ml, uviolizing under the ice bath adds the foetal calf serum of 10% volume ratio and the commercial substratum of DMEM that final concentration is 80～150mg/ml more respectively, transfer pH7.2～7.4, make gelating soln; Nylon membrane is soaked into gelating soln, place culture dish to solidify to form support membrane; The inoblast of vitro culture with 10
5～10
6The density of individual/ml is mixed in gel, and it is dropped to solidified support membrane surface, cultivates for 37 ℃ to solidify back formation skin corium, adds nutrient solution C and cultivates 2～4 days, changes liquid every day; Inhale after skin corium is cultivated and finished and abandon nutrient solution, evenly punch on the surface with syringe needle, pitch of holes is 2～5mm; The organizational project hair follicle suspension of cultivating is pressed 10～100/cm
2Density drip on the skin corium surface, make suspension evenly infiltrate in the hole; Press 10 on the skin corium surface then
5～10
6Individual/cm
2Density inoculation epidermic cell, add nutrient solution D and cultivate after 1 day, move to and carried out liquid under cultivation on the support 2～4 days; Change nutrient solution E and carry out solution-air face cultivation 1～2 day; Changing nutrient solution F cultivated 1～2 day; Change liquid every day between incubation period.
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| CN201010107206A CN101775366A (en) | 2010-02-05 | 2010-02-05 | Preparation method of tissue engineering skin containing hair follicles |
| PCT/CN2010/071123 WO2011094964A1 (en) | 2010-02-05 | 2010-03-18 | Method for preparing tissue engineering skin containing hair follicle |
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| CN201010107206A CN101775366A (en) | 2010-02-05 | 2010-02-05 | Preparation method of tissue engineering skin containing hair follicles |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102172337A (en) * | 2011-02-18 | 2011-09-07 | 中国人民解放军第四军医大学 | Tissue engineering skin with sebaceous gland-like structure and preparation method thereof |
| CN102335459A (en) * | 2010-07-21 | 2012-02-01 | 中国科学院动物研究所 | Hair follicle reconstruction method based on hair follicle stem cells and fibroblast |
| CN102552323A (en) * | 2012-01-17 | 2012-07-11 | 浙江大学 | Medicine for accelerating skin repair and regeneration, preparation method thereof and application thereof |
| CN103320383A (en) * | 2013-07-10 | 2013-09-25 | 杭州市萧山区中医院 | Culture medium for culturing rat hair follicle stem cells |
| CN104548209A (en) * | 2015-02-03 | 2015-04-29 | 苏州磐升生物技术有限公司 | Tissue-engineered epidermis and preparation method thereof |
| CN104667352A (en) * | 2015-02-03 | 2015-06-03 | 济南磐升生物技术有限公司 | Preparation method of tissue engineering epidermis having hypodermal cells |
| CN104726396A (en) * | 2015-04-17 | 2015-06-24 | 陕西博溪生物科技有限公司 | Method for building full-thickness skin models |
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| CN108472410A (en) * | 2015-11-17 | 2018-08-31 | 柏林工业大学 | Method for producing skin equivalent and its purposes transplanted for testing in vitro and in vivo |
| CN109321513A (en) * | 2018-09-30 | 2019-02-12 | 洛阳师范学院 | A kind of organization engineering skin construction method with physiological function |
| 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 |
| CN113544259A (en) * | 2020-01-31 | 2021-10-22 | 瑞帝安有限公司 | Method for differentiating human adipose-derived mesenchymal stem cells into hair papilla cells |
| CN114085804A (en) * | 2021-11-09 | 2022-02-25 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | Skin organoid formed by in vitro induction and differentiation of human embryonic stem cells and high-efficiency culture method and application thereof |
| CN114958733A (en) * | 2021-07-29 | 2022-08-30 | 北京博百悦生物技术有限公司 | Preparation method of autologous hair follicle mesenchymal stem cells |
| CN115537373A (en) * | 2022-07-06 | 2022-12-30 | 西南医科大学 | Renewable artificial hair follicle and preparation method and application thereof |
| CN115627256A (en) * | 2022-12-02 | 2023-01-20 | 上海尚瑞生物医药科技有限公司 | Multi-layer tissue engineering skin composed of hair follicle cells and preparation method and application thereof |
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| CN110167609A (en) * | 2016-11-10 | 2019-08-23 | 奥加诺沃公司 | Hair follicle of biometric print and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102335459A (en) * | 2010-07-21 | 2012-02-01 | 中国科学院动物研究所 | Hair follicle reconstruction method based on hair follicle stem cells and fibroblast |
| CN102172337A (en) * | 2011-02-18 | 2011-09-07 | 中国人民解放军第四军医大学 | Tissue engineering skin with sebaceous gland-like structure and preparation method thereof |
| CN102172337B (en) * | 2011-02-18 | 2013-10-23 | 中国人民解放军第四军医大学 | Tissue engineering skin with sebaceous gland-like structure and preparation method thereof |
| CN102552323B (en) * | 2012-01-17 | 2013-12-04 | 浙江大学 | Medicine for accelerating skin repair and regeneration, preparation method thereof and application thereof |
| CN102552323A (en) * | 2012-01-17 | 2012-07-11 | 浙江大学 | Medicine for accelerating skin repair and regeneration, preparation method thereof and application thereof |
| CN103320383B (en) * | 2013-07-10 | 2016-04-06 | 杭州市萧山区中医院 | A kind of substratum cultivating rat hair follicle stem cell |
| CN103320383A (en) * | 2013-07-10 | 2013-09-25 | 杭州市萧山区中医院 | Culture medium for culturing rat hair follicle stem cells |
| CN104548209A (en) * | 2015-02-03 | 2015-04-29 | 苏州磐升生物技术有限公司 | Tissue-engineered epidermis and preparation method thereof |
| CN104667352A (en) * | 2015-02-03 | 2015-06-03 | 济南磐升生物技术有限公司 | Preparation method of tissue engineering epidermis having hypodermal cells |
| CN104667352B (en) * | 2015-02-03 | 2016-11-09 | 济南磐升生物技术有限公司 | A kind of preparation method of the tissue engineering epidermis with hypodermal cell |
| CN104548209B (en) * | 2015-02-03 | 2017-01-25 | 苏州磐升生物技术有限公司 | Tissue-engineered epidermis and preparation method thereof |
| CN104726396A (en) * | 2015-04-17 | 2015-06-24 | 陕西博溪生物科技有限公司 | Method for building full-thickness skin models |
| CN108472410A (en) * | 2015-11-17 | 2018-08-31 | 柏林工业大学 | Method for producing skin equivalent and its purposes transplanted for testing in vitro and in vivo |
| CN107952117A (en) * | 2016-10-14 | 2018-04-24 | 三鼎生物科技股份有限公司 | Method for 3D printing artificial skin |
| CN107952117B (en) * | 2016-10-14 | 2021-01-22 | 三鼎生物科技股份有限公司 | Method for 3D printing artificial skin |
| CN107802891A (en) * | 2017-11-09 | 2018-03-16 | 清华大学深圳研究生院 | Organization engineering skin and preparation method thereof |
| CN108324993B (en) * | 2018-01-15 | 2020-11-03 | 朱剑虹 | Stem cell complex for inducing hair regeneration, preparation method and application thereof |
| CN108324993A (en) * | 2018-01-15 | 2018-07-27 | 上海神因生物科技有限公司 | A kind of stem cell complex, preparation method and the application of induction hair regeneration |
| CN109321513A (en) * | 2018-09-30 | 2019-02-12 | 洛阳师范学院 | A kind of organization engineering skin construction method with physiological function |
| 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 |
| CN113544259A (en) * | 2020-01-31 | 2021-10-22 | 瑞帝安有限公司 | Method for differentiating human adipose-derived mesenchymal stem cells into hair papilla cells |
| CN114958733A (en) * | 2021-07-29 | 2022-08-30 | 北京博百悦生物技术有限公司 | Preparation method of autologous hair follicle mesenchymal stem cells |
| CN114085804A (en) * | 2021-11-09 | 2022-02-25 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | Skin organoid formed by in vitro induction and differentiation of human embryonic stem cells and high-efficiency culture method and application thereof |
| CN114085804B (en) * | 2021-11-09 | 2023-12-26 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | Human embryonic stem cell in-vitro induced differentiation formed skin organoid and efficient culture method and application thereof |
| CN115537373A (en) * | 2022-07-06 | 2022-12-30 | 西南医科大学 | Renewable artificial hair follicle and preparation method and application thereof |
| CN115627256A (en) * | 2022-12-02 | 2023-01-20 | 上海尚瑞生物医药科技有限公司 | Multi-layer tissue engineering skin composed of hair follicle cells and preparation method and application thereof |
| WO2024113917A1 (en) * | 2022-12-02 | 2024-06-06 | 上海尚瑞生物医药科技有限公司 | Multi-layer tissue engineered skin composed of hair follicle cells, preparation method therefor, and use thereof |
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