CN109078219A - A kind of preparation method for the autologous collagen albumen tissue filler cooperating facial line engraving - Google Patents
A kind of preparation method for the autologous collagen albumen tissue filler cooperating facial line engraving Download PDFInfo
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Abstract
The present invention relates to a kind of preparation methods of autologous collagen albumen tissue filler for cooperating facial line engraving, belong to medical cosmetology technical field, comprising the following steps: step 1 obtains self at fibr tissue；Step 2, secondary culture；Step 3, collagen accumulation；The separation of step 4, fibroblast and collagen；Step 5, hyaluronic acid derivatives preparation；Step 6, the preparation of autologous collagen albumen tissue filler.The technical solution is filled into wrinkle bottom using autologous collagen albumen tissue filler, to improve the adhesion effect of facial line engraving link protein line and subcutaneous cell, reduce rejection, and hyaluronic acid derivatives and autologous collagen albumen also provide sufficient nutrition for subcutaneous cell growth, framework material in subcutaneous cell growth course as adherency, hyaluronic acid derivatives and autologous collagen albumen are also gradually degraded after cell growth shaping, and histocompatbility is good.
Description
Technical field
The present invention relates to a kind of preparation methods of autologous collagen albumen tissue filler for cooperating facial line engraving, belong to medicine
Beautifying technique field.
Background technique
Facial line engraving is a kind of novel medical cosmetology method, is also protein line face lift, is that spy is inserted into skin
Different albumen esthetic line, while skin and fascia layer are stimulated, so that stiff or sagging musculature is re-started arrangement, thus
Reproduce the newest Minimally Invasive Surgery method of beautiful face.Operative site is known as SMAS layers, between located subcutaneously fat and muscle, connection
The skin special layers of skin and muscle.The layer is since aging is sagging together with skin, and by the stimulation to these positions, promotion changes
Kind wrinkle.
Wrinkle refers to that skin is influenced by external environment, forms free free radical, and free radical destroys normal cell membrane tissue
Interior collagen, active material, oxidative cell and the small microgroove, the wrinkle that are formed.Wrinkle, which sums up, 4 originals
Cause: natural aging, centrifugal force effect, the irradiation of sunlight middle-ultraviolet lamp make skin that light aging and light injury and facial expression muscle occur
Excessive contraction.The wrinkle that wherein the excessive contraction of facial expression muscle generates claims dynamic property wrinkle, is mainly shown as on the outside of socket of the eye
Line and platysma line on crow's feet, forehead line and glabella line, Nasolabial furrow wrinkles and lip.Smoothing wrinkle has various methods, and part is outer
Appearance can only be improved with various anti-wrinkle creams, anti-wrinkle cream, suncream, it does not have biggish effect to the elimination of wrinkle.
Medical collagen implantation is effective, but because being non-Self substances, can only exist will be removed some months by body, infuse simultaneously
Certain allergic reaction is had after penetrating.
Collagen is the main component of extracellular matrix, accounts for about the 85% of collagenous fibres solids, accounts for egg in animal body
The 25%~30% of white matter total amount, it is widely present in the connective tissue (bone, cartilage, skin, tendon, tough etc.) of animal, to machine
Body and internal organs play support, protection, combination, and formed boundary every the effects of.Also there is the fine and close collagen arranged in human skin lower layer
Albumen exists, and the thickness of skin and changes with age at branch, and the skin of the elderly is easy to injury, and is not easy to be cured
It closes, this is likely due to caused by overlaying skin fibroblast missing, if these cells can be stimulated to grow, it is possible to restore
The elasticity of skin, equally can also hair follicle stimulating formed, reduce scar.Wrinkle is removed by way of autologous collagen protein injection,
Allergic reaction can be substantially reduced, but simple collagen injections are easy to be degraded by body, are unable to the solution wrinkle of essence
Line problem has the removal effect of wrinkle to improving for this purpose, improving to existing medical collagen injection fillers object
Significance.
Summary of the invention
To solve problems of the prior art, the embodiment of the invention provides a kind of fillings of autologous collagen albumen tissue
Gel products are made using hyaluronic acid in the preparation method of object, then form tissue filler with autologous collagen protein combination.Tool
Body technique scheme is as follows:
A kind of preparation method for the autologous collagen albumen tissue filler cooperating facial line engraving, comprising the following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, and be put into digestion 20min~60min in 37 DEG C of constant temperature oscillation boxes,
Be then allowed to stand after its layering after, draw upper layer fibroblast liquid, move into the DMEM culture medium containing fetal calf serum in, seal from
Heart separation, removes supernatant, and the DMEM culture medium containing fetal calf serum in right amount is added again, and fibroblast is made in piping and druming cell
Suspension is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation regularly replaces culture medium until at fiber
Cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, the secondary culture after fibroblast grows to 80%~90% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base is cultivated in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 3%~10%, and stirring to abundant dissolution is added crosslinking agent and stirs evenly, crosslinking agent is in the solution
Mass concentration be 2%~5%, 35 DEG C~50 DEG C of reaction temperature, reaction time 2h~10h is filtered to remove hydrochloric acid solution, adds
Enter D-Hanks buffer, cleans for 24 hours~48h, obtain hyaluronic acid derivatives；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, obtain autologous collagen albumen tissue and fill out
Fill object.
As an improvement of the above technical solution, in step 1, digestive juice is pancreatin and Type I collagen enzyme according to volume ratio 1:
1 mixes, and wherein the mass fraction of pancreatin is 0.25%, and the mass fraction of Type I collagen enzyme is 0.1%.
As an improvement of the above technical solution, in step 1, the speed of centrifuge separation is 800rpm~1800rpm, from
The heart time is 5min~15min.
As an improvement of the above technical solution, in step 2, enzyme solution is the pancreatin that mass fraction is 0.25%.
As an improvement of the above technical solution, in step 3, fibroblast incubation time in serum free medium
For 12h~60h.
As an improvement of the above technical solution, in step 5, crosslinking agent is ethylene glycol diglycidylether, diethylene glycol (DEG) two
Glycidol ether, polyethyleneglycol diglycidylether, polypropylene glycol diglycidyl ether, in 1,6- hexanediol diglycidyl ether
It is any.
As an improvement of the above technical solution, in step 6, collagen liquid, hyaluronic acid derivatives, ringer's solution and
The volume ratio of Glucose Liquid is 1:2~10:1~2:1~2.
As an improvement of the above technical solution, in step 6, Porcine HGF, the cell growth factor have been additionally added
Son is epidermal growth factor, fibroblast growth factor, keratinocyte growth factor, nerve growth factor, hepatic cell growth
One of the factor, transforminggrowthfactor-α, vascular endothelial growth factor are a variety of.
As an improvement of the above technical solution, it in step 6, is added in every 10ml autologous collagen albumen tissue filler
Growth factor-21 mg~5mg.
Above-mentioned technical proposal is filled into wrinkle bottom using autologous collagen albumen tissue filler, to improve facial line engraving
The adhesion effect of link protein line and subcutaneous cell reduces rejection, and hyaluronic acid derivatives and autologous collagen albumen
Also sufficient nutrition is provided for subcutaneous cell growth, the framework material in subcutaneous cell growth course as adherency is raw in cell
Hyaluronic acid derivatives and autologous collagen albumen are also gradually degraded after long molding, and histocompatbility is good.
Specific embodiment
The embodiment of the invention provides a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving,
Characterized by comprising the following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, and be put into digestion 20min~60min in 37 DEG C of constant temperature oscillation boxes,
Be then allowed to stand after its layering after, draw upper layer fibroblast liquid, move into the DMEM culture medium containing fetal calf serum in, seal from
Heart separation, the speed of centrifuge separation are 800rpm~1800rpm, and centrifugation time is 5min~15min, remove supernatant, again
The DMEM culture medium containing fetal calf serum in right amount is added, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle,
Under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；The step
Digestive juice is that pancreatin and Type I collagen enzyme are mixed according to volume ratio 1:1 in rapid, and wherein the mass fraction of pancreatin is 0.25%, I
The mass fraction of Collagenase Type is 0.1%；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80%~90% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 12h~60h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen, due to collagen egg
It is white for self fibroblast generation, therefore rejection can be effectively reduced as injection fillers object；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 3%~10%, and stirring to abundant dissolution is added crosslinking agent and stirs evenly, crosslinking agent is ethylene glycol
Diglycidyl ether, diethylene glycol (DEG) diglycidyl ether, polyethyleneglycol diglycidylether, polypropylene glycol diglycidyl ether, 1,
Any one of 6- hexanediol diglycidyl ether, the mass concentration of crosslinking agent in the solution are 2%~5%, reaction temperature 35
DEG C~50 DEG C, reaction time 2h~10h, it is filtered to remove hydrochloric acid solution, D-Hanks buffer is added, for 24 hours~48h is cleaned, obtains
Hyaluronic acid derivatives；The basic structure of hyaluronic acid is made of two dissacharide units D-Glucose aldehydic acid and N-acetyl-glucosamine
Large-scale polysaccharide, different from other mucopolysaccharides, sulfur-bearing, its hyalomitome molecule can not carry 500 times or more of moisture, be for it
Best moisturizing ingredient recognized by current, is widely applied in skin care products and cosmetics；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:2~10:1~2:1~2, obtains autologous collagen albumen tissue filler；
Porcine HGF is additionally added in the step, the Porcine HGF is epidermal growth factor, Desmocyte growth factor
Son, keratinocyte growth factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial cell are raw
One of long factor is a variety of, and growth factor-21 mg~5mg is added in every 10ml autologous collagen albumen tissue filler.
Ringer's solution is also referred to as compound sodium chloride injection, be because Ringer's solution in addition to containing sodium chloride composition, also containing sodium from
Son, potassium ion, calcium ion, magnesium ion, chloride ion and lactate ion.Ringer's solution is more complete than physiological saline ingredient, can replace
Physiological saline uses, and to adjust body fluid, electrolyte and acid-base balance, sodium lactate ringer's are then suitable for acid poisoning or have acid poisoning to incline
To dehydration case, so operating room is commonly used.
Traditional facial rhytidectomy is generally subcutaneously removed using operation, excess skin excision, then carries out suture promotion,
To achieve the effect that treatment, line engraving technology is to directly adopt the mode of implantation collagen line to lift skin.Line engraving
Although technology goes the principle of wrinkle class product to be also not quite similar by the way of implantation, with general injection fillers, generally injection class
Product is to be smoothed facial wrinkles by way of filling, and line engraving art is promoted art and formed using absorbable proteins
Line is implanted to and needs the position that is promoted, by the lifting of wire body, phenomena such as so as to improve wrinkle, relaxation, can more act on body its
His position, maintains that shape is tall and straight and skin is whole young.Facial line engraving needs to cooperate hyaluronic acid filler, is filled out using filler
Be charged to wrinkle bottom, and this programme then mixes hyaluronic acid derivatives with autologous collagen protein liquid, thus improve protein line and
The adhesion effect of subcutaneous cell reduces rejection.And hyaluronic acid derivatives and autologous collagen albumen are also that subcutaneous cell is raw
It is long that sufficient nutrition, the framework material in subcutaneous cell growth course as adherency, the hyalomitome after cell growth shaping are provided
Acid gel and autologous collagen albumen are also gradually degraded, and histocompatbility is good.
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, right below in conjunction with specific embodiment
Technical solution in the embodiment of the present invention is clearly and completely described.
Embodiment one
The autologous collagen albumen tissue filler for cooperating facial line engraving is prepared using following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, is put into 37 DEG C of constant temperature oscillation boxes and digests 20min, be then allowed to stand
After its layering, upper layer fibroblast liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation,
The speed of centrifuge separation is 800rpm, centrifugation time 15min, removes supernatant, is added again in right amount containing fetal calf serum
DMEM culture medium, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2
Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 12h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 4%, and stirring to abundant dissolution is added ethylene glycol diglycidylether and stirs evenly；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:2:1:1, obtains autologous collagen albumen tissue filler；In the step also
Addition has Porcine HGF, and Porcine HGF is epidermal growth factor, fibroblast growth factor, Keratiocyte growth
The factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mix,
Growth factor-21 mg is added in every 10ml autologous collagen albumen tissue filler.
Embodiment two
The autologous collagen albumen tissue filler for cooperating facial line engraving is prepared using following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, is put into 37 DEG C of constant temperature oscillation boxes and digests 30min, be then allowed to stand
After its layering, upper layer fibroblast liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation,
The speed of centrifuge separation is 1000rpm, centrifugation time 12min, removes supernatant, is added again in right amount containing fetal calf serum
DMEM culture medium, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2
Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 20h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 4%, and stirring to abundant dissolution is added ethylene glycol diglycidylether and stirs evenly；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:3:1:1, obtains autologous collagen albumen tissue filler；In the step also
Addition has Porcine HGF, and Porcine HGF is epidermal growth factor, fibroblast growth factor, Keratiocyte growth
The factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mix,
Porcine HGF 2mg is added in every 10ml autologous collagen albumen tissue filler.
Embodiment three
The autologous collagen albumen tissue filler for cooperating facial line engraving is prepared using following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, is put into 37 DEG C of constant temperature oscillation boxes and digests 30min, be then allowed to stand
After its layering, upper layer fibroblast liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation,
The speed of centrifuge separation is 1000rpm, centrifugation time 12min, removes supernatant, is added again in right amount containing fetal calf serum
DMEM culture medium, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2
Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 20h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 4%, and stirring to abundant dissolution is added ethylene glycol diglycidylether and stirs evenly；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:5:1:1, obtains autologous collagen albumen tissue filler；In the step also
Addition has Porcine HGF, and Porcine HGF is epidermal growth factor, fibroblast growth factor, Keratiocyte growth
The factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mix,
Porcine HGF 2mg is added in every 10ml autologous collagen albumen tissue filler.
Example IV
The autologous collagen albumen tissue filler for cooperating facial line engraving is prepared using following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, is put into 37 DEG C of constant temperature oscillation boxes and digests 30min, be then allowed to stand
After its layering, upper layer fibroblast liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation,
The speed of centrifuge separation is 1200rpm, centrifugation time 12min, removes supernatant, is added again in right amount containing fetal calf serum
DMEM culture medium, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2
Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 20h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 4%, and stirring to abundant dissolution is added diethylene glycol (DEG) diglycidyl ether and stirs evenly；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:10:1:1, obtains autologous collagen albumen tissue filler；In the step
It is additionally added Porcine HGF, Porcine HGF is epidermal growth factor, fibroblast growth factor, horn cell life
The long factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mixing and
At addition Porcine HGF 3mg in every 10ml autologous collagen albumen tissue filler.
Embodiment five
The autologous collagen albumen tissue filler for cooperating facial line engraving is prepared using following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, is put into 37 DEG C of constant temperature oscillation boxes and digests 30min, be then allowed to stand
After its layering, upper layer fibroblast liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation,
The speed of centrifuge separation is 1500rpm, centrifugation time 10min, removes supernatant, is added again in right amount containing fetal calf serum
DMEM culture medium, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2
Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 20h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 4%, and stirring to abundant dissolution is added diethylene glycol (DEG) diglycidyl ether and stirs evenly；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:5:2:2, obtains autologous collagen albumen tissue filler；In the step also
Addition has Porcine HGF, and Porcine HGF is epidermal growth factor, fibroblast growth factor, Keratiocyte growth
The factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mix,
Porcine HGF 4mg is added in every 10ml autologous collagen albumen tissue filler.
Embodiment six
The autologous collagen albumen tissue filler for cooperating facial line engraving is prepared using following steps:
Step 1 obtains and is removed remaining blood with D-Hanks buffer solution for cleaning at fibr tissue self and organized broken
Piece will be cut into small pieces at fibr tissue self, and digestive juice is added, is put into 37 DEG C of constant temperature oscillation boxes and digests 20min, be then allowed to stand
After its layering, upper layer fibroblast liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation,
The speed of centrifuge separation is 1800rpm, centrifugation time 5min, removes supernatant, is added again in right amount containing fetal calf serum
DMEM culture medium, piping and druming cell is made fibroblast suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2
Constant temperature incubation regularly replaces culture medium until fibroblast confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fibroblast
Secondary culture after to 80% fusion；
Fibroblast is put into free serum culture after fibroblastic growth three generations by step 3, collagen accumulation
Base culture 20h cultivates in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
The separation of step 4, fibroblast and collagen first takes out the serum free medium in step 3, so
50k ultrafiltration membrance filter is used afterwards, then with 0.22 μm of membrane filtration, obtains collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate exists
Mass concentration in solution is 4%, and stirring to abundant dissolution is added diethylene glycol (DEG) diglycidyl ether and stirs evenly；
Step 6, the preparation of autologous collagen albumen tissue filler, the collagen liquid that step 4 is obtained and step 5
It obtains hyaluronic acid derivatives to be uniformly mixed, adds ringer's solution and Glucose Liquid, collagen liquid, hyalomitome acid cure
The volume ratio of glue, ringer's solution and Glucose Liquid is 1:10:2:2, obtains autologous collagen albumen tissue filler；In the step
It is additionally added Porcine HGF, Porcine HGF is epidermal growth factor, fibroblast growth factor, horn cell life
The long factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mixing and
At addition Porcine HGF 5mg in every 10ml autologous collagen albumen tissue filler.
Above-mentioned autologous collagen albumen tissue filler has no toxic side effect, and has preferable biocompatibility and tissue compatible
Property, it can be used as the body tissue filler in medical cosmetology, wherein Porcine HGF can promote subcutaneously to regenerate and skin is created
Face healing, and skin epidermal cells can be promoted mature, increases skin elasticity, moisturizing, smoothes away wrinkles and prevents color spot,
With good beauty functions.
In the above-described embodiments, multiple groups part solution mixing in case of no particular description according to mass percent into
Row mixing.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of inventive embodiments, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (9)
1. a kind of preparation method for the autologous collagen albumen tissue filler for cooperating facial line engraving, which is characterized in that including following
Step:
Step 1, acquisition remove remaining blood and fragment of tissue with D-Hanks buffer solution for cleaning at fibr tissue self, will
It is cut into small pieces self at fibr tissue, digestive juice is added, be put into digestion 20min~60min in 37 DEG C of constant temperature oscillation boxes, it is then quiet
It sets after its layering, draws upper layer fibroblast liquid, move into the DMEM culture medium containing fetal calf serum, sealing centrifugation point
From the DMEM culture medium containing fetal calf serum in right amount is added in removal supernatant again, and it is outstanding that fibroblast is made in piping and druming cell
Liquid is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation regularly replaces culture medium until at fiber finer
Born of the same parents' confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fibroblast that step 1 is obtained are taken out from culture medium, and D-Hanks buffer is added
It cleans up, enzyme solution digestion process is then added, the secondary culture after fibroblast grows to 80%~90% fusion；
Fibroblast is put into serum free medium after fibroblastic growth three generations by step 3, collagen accumulation,
It is cultivated in 37 DEG C of serum free mediums, fibroblasts to secrete is made to go out collagen；
Serum free medium in step 3 is taken out first, is then used by the separation of step 4, fibroblast and collagen
50k ultrafiltration membrance filter, then with 0.22 μm of membrane filtration, obtain collagen liquid；
Step 5, hyaluronic acid derivatives preparation, Sodium Hyaluronate is dissolved in sodium hydroxide solution, Sodium Hyaluronate is in solution
In mass concentration be 3%~10%, stirring to abundant dissolution is added crosslinking agent and stirs evenly, the matter of crosslinking agent in the solution
Measuring concentration is 2%~5%, 35 DEG C~50 DEG C of reaction temperature, reaction time 2h~10h, is filtered to remove hydrochloric acid solution, D- is added
Hanks buffer cleans for 24 hours~48h, obtains hyaluronic acid derivatives；
Step 6, the preparation of autologous collagen albumen tissue filler obtain collagen liquid and step 5 that step 4 obtains
Hyaluronic acid derivatives are uniformly mixed, and add ringer's solution and Glucose Liquid, obtain autologous collagen albumen tissue filler.
2. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as described in claim 1,
It is characterized in that, in step 1, digestive juice is that pancreatin and Type I collagen enzyme are mixed according to volume ratio 1:1, wherein the matter of pancreatin
Measuring score is 0.25%, and the mass fraction of Type I collagen enzyme is 0.1%.
3. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as described in claim 1,
It is characterized in that, in step 1, the speed of centrifuge separation is 800rpm~1800rpm, and centrifugation time is 5min~15min.
4. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as described in claim 1,
It is characterized in that, in step 2, enzyme solution is the pancreatin that mass fraction is 0.25%.
5. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as described in claim 1,
It is characterized in that, in step 3, fibroblast incubation time in serum free medium is 12h~60h.
6. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as described in claim 1,
It is characterized in that, in step 5, crosslinking agent is ethylene glycol diglycidylether, diethylene glycol (DEG) diglycidyl ether, polyethylene glycol two
Any one of glycidol ether, polypropylene glycol diglycidyl ether, 1,6- hexanediol diglycidyl ether.
7. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as described in claim 1,
Be characterized in that, in step 6, collagen liquid, hyaluronic acid derivatives, ringer's solution and Glucose Liquid volume ratio be 1:2~
10:1~2:1~2.
8. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as claimed in claim 7,
It is characterized in that, in step 6, has been additionally added Porcine HGF, the Porcine HGF is epidermal growth factor, at fibre
Tie up Porcine HGF, keratinocyte growth factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, blood
One of endothelial cell growth factor is a variety of.
9. a kind of preparation method of autologous collagen albumen tissue filler for cooperating facial line engraving as claimed in claim 8,
It is characterized in that, in step 6, growth factor-21 mg~5mg is added in every 10ml autologous collagen albumen tissue filler.
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| CN201810930857.1A CN109078219A (en) | 2018-08-15 | 2018-08-15 | A kind of preparation method for the autologous collagen albumen tissue filler cooperating facial line engraving |
| CN202110556660.8A CN113135993A (en) | 2018-08-15 | 2018-08-15 | Autologous collagen tissue filler matched with facial line carving |
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| CN201810930857.1A CN109078219A (en) | 2018-08-15 | 2018-08-15 | A kind of preparation method for the autologous collagen albumen tissue filler cooperating facial line engraving |
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| CN202110556660.8A Withdrawn CN113135993A (en) | 2018-08-15 | 2018-08-15 | Autologous collagen tissue filler matched with facial line carving |
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| CN112980001A (en) * | 2021-03-16 | 2021-06-18 | 杭州基智生物科技有限公司 | Collagen composite hyaluronic acid gel, extracellular matrix bionic material and preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333349A (en) * | 2013-06-28 | 2013-10-02 | 陕西巨子生物技术有限公司 | Hyaluronic acid-collagen composite hydrogel for injection and preparation method thereof |
| CN107523533A (en) * | 2017-08-22 | 2017-12-29 | 四川驰鼎盛通生物科技有限公司 | The acquisition methods of autologous skin fibroblast, PRP and collagen liquid |
-
2018
- 2018-08-15 CN CN201810930857.1A patent/CN109078219A/en active Pending
- 2018-08-15 CN CN202110556660.8A patent/CN113135993A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333349A (en) * | 2013-06-28 | 2013-10-02 | 陕西巨子生物技术有限公司 | Hyaluronic acid-collagen composite hydrogel for injection and preparation method thereof |
| CN107523533A (en) * | 2017-08-22 | 2017-12-29 | 四川驰鼎盛通生物科技有限公司 | The acquisition methods of autologous skin fibroblast, PRP and collagen liquid |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980001A (en) * | 2021-03-16 | 2021-06-18 | 杭州基智生物科技有限公司 | Collagen composite hyaluronic acid gel, extracellular matrix bionic material and preparation method |
| CN112980001B (en) * | 2021-03-16 | 2024-03-19 | 杭州基智生物科技有限公司 | Collagen composite hyaluronic acid gel, extracellular matrix bionic material and preparation method |
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