CN109621012A - A kind of compound protein gel and preparation method thereof - Google Patents
A kind of compound protein gel and preparation method thereof Download PDFInfo
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- CN109621012A CN109621012A CN201910092866.2A CN201910092866A CN109621012A CN 109621012 A CN109621012 A CN 109621012A CN 201910092866 A CN201910092866 A CN 201910092866A CN 109621012 A CN109621012 A CN 109621012A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3616—Blood, e.g. platelet-rich plasma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
Abstract
The invention discloses a kind of compound protein gels and preparation method thereof, and preparation method includes the following steps: acquiring blood with the heparin tube with anti-coagulants；Above-mentioned blood is centrifuged 10-20min at room temperature with the centrifugal acceleration of 300-700g；Blood after centrifugation is transferred in Biohazard Safety Equipment, upper plasma layer is drawn, then places it in quick freeze in -80 DEG C of ultra low temperature freezer, cooling time 24-72h；Then the blood plasma after freezing is taken out, is -55--65 DEG C in temperature, pressure is to carry out frozen dried under 50-100Pa, and frozen dry blood plasma is made；Calcirm chloride injection is added with the volume ratio of 1:0.5-2 into frozen dry blood plasma, 5-15min is then reacted in 60-90 DEG C of water-bath, is taken out, is made.The protein gel can effectively solve to easily cause skin existing for sodium hyaluronate and autologous fat and ossify, collapse, the problem of body absorption rate height and filler stability difference.
Description
Technical field
The invention belongs to beauty product fields, and in particular to a kind of compound protein gel and preparation method thereof.
Background technique
It in recent years, is mainly following two applied to the filler of skin: hyaluronic acid and fat.
Hyaluronic acid (HA) is widely used to skin treating field as skin corium filler, and hyaluronic acid has enhancing
The effects of soft tissue elasticity, promotes the proliferation and differentiation of epidermal cell, high-biocompatibility.However, hyaluronic acid is after injection
Have and skin is caused to ossify, collapse, the high disadvantage of body absorption rate, while it does not have resisting age of skin yet and promotes regeneration
Ability.
Autologous fat filling is also the more technology of clinical application.What autologous fat generated after preliminary treatment in vitro
Vascular stroma tissue (SVF) contains a large amount of Porcine HGF, such as: vascular epidermal growth factor (VEGF), alkalinity are at fibre
Porcine HGF (bFGF), epidermal growth factor (EGF) and transforming growth factor (TGF-β) are tieed up, wherein the rush blood of VEGF
Pipeization effect can provide energy supply for stem cell and fibroblast proliferation.But autologous fat filling materials to body wound compared with
Greatly, experience sense is poor；Also, many clinical datas show that the absorptivity in autologous fat filling 3-6 months up to 50% or more, is filled out
It is poor to fill object structure stability.
Summary of the invention
For the above-mentioned problems in the prior art, the present invention provides a kind of compound protein gel and preparation method thereof,
The protein gel can effectively solve sodium hyaluronate or the autologous fat existing skin that easily causes in filling and ossify, collapse, in vivo
The problem of absorptivity height and filler stability difference.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
A kind of preparation method of compound protein gel, comprising the following steps:
(1) blood is acquired with the heparin tube with anti-coagulants；
(2) blood in step (1) is centrifuged 10~20min at room temperature with the centrifugal acceleration of 300~700g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it under the conditions of -80 DEG C, when freezing
Between be 24~72h；
(4) blood plasma in step (3) after freezing is taken out, is -55~-65 DEG C in temperature, pressure be under 50~100Pa into
Frozen dry blood plasma is made in row frozen dried；
(5) by calcirm chloride injection that frozen dry blood plasma made from step (4) and concentration are 20~50 μm of ol/L by 1:0.5~
2 volume ratio mixing, 5~15min is then reacted in 60~90 DEG C of water-bath, is taken out, is made.
Further, the blood in step (1) is Cord blood or autologous vein blood.
Further, the anti-coagulants in step (1) is 3.8% sodium citrate.
Further, centrifugal acceleration is 600~700g in step (2), and centrifugation time is 10~15min.
Further, the upper plasma in step (3) is placed under the conditions of -80 DEG C, cooling time 36h.
Further, the temperature of frozen dried is -58 DEG C in step (4), pressure 80Pa.
Further, the concentration of calcirm chloride injection is 30~40 μm of ol/L in step (5).
Further, frozen dry blood plasma is mixed with calcirm chloride injection according to the volume ratio of 1:0.5 in step (5).
Further, the bath temperature of step (5) is 75~78 DEG C, and the reaction time is 5~7min.
A kind of compound protein gel, is made by above-mentioned preparation method.
It is had the beneficial effect that caused by the present invention
1, the present invention use Cord blood to be used as the raw material for preparing protein gel, doing in Cord blood containing largely undifferentiated
Cell and the advantageous growth factor of cell proliferation and tissue repair, and the immune cell function in bleeding of the umbilicus is immature, NK and T
Cell is all in cubhood, and antigen presentation and immune function activity are low, and immunoglobulin is few, and the power of regeneration of stem cell is strong, fills out
Immunological rejection after filling is small, and compound protein gel is made after passing it through processing, makes using the protein gel as filler
With；The protein gel also has low immunogenicity, moulding effect and filling effect naturally, the phenomenon that without rigid collapsing, after filling
It holds time the advantages that long.
2, variation of the frozen dry blood plasma in present invention under the action of heat treatment and calcium ion on occurred conformation forms albumen
Matter gel, the protein gel are three-dimensional netted structure, and the tridimensional network is mainly by objects such as hydrophobic effect, hydrogen bond actions
Reason effect maintains, and the effect enhancing after heat treatment between protein, the hydrophobic group of protein interior is exposed, and promotes disulfide bond
Be further formed, the presence of a large amount of disulfide bond so that intermolecular cross-linking effect to reinforce, form stable reticular structure
Gel, the gel structure are not easy the absorption that is decomposed, this is gel-filled after skin, can be subcutaneous group as the bracket of skin
It knits to grow with cell and 3 D stereo growing environment is provided, wherein calcium ion can also activate platelet growth factor, promote cell point
Change regeneration etc., improve cytothesis ability, promote the microcirculation of reticular structure surrounding tissue, subcutaneous tissue and cell pass through one section
After the growth of time, the inside full of tridimensional network can reach raising filling effect, extend the purpose held time；
Due to providing energy to the cell growth at filling position after protein gel filling, cell is largely proliferated at the position, skin group
It knits and is regenerated, therefore the phenomenon for collapsing, being absorbed and ossifing is not present after filling, filling effect is good.
3, the present invention in using Freeze Drying Technique be made frozen dry blood plasma, frozen dry blood plasma sealing after can long term storage, freeze-drying
Blood plasma afterwards is convenient to use and transports, and is convenient for large-scale production and sale；Calcium chloride solvent dissolution when use through suitable concentration,
Protein structure inside frozen dry blood plasma restores rapidly, the basic free of losses such as protein therein and growth factor, and continuation is being closed
Heating water bath can be prepared by protein gel at a temperature of suitable, use for filling, and without being prepared in situ, improve procedure efficiency.
Specific embodiment
Embodiment 1
A kind of compound protein gel, preparation method, comprising the following steps:
(1) Cord blood, the volume of sodium citrate and Cord blood are acquired with the heparin tube with 3.8% sodium citrate anticoagulant
Than for 1:13；
(2) Cord blood in step (1) is centrifuged 10min at room temperature with the centrifugal acceleration of 300g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it in -80 DEG C of ultra low temperature freezer
Middle quick freeze, cooling time are for 24 hours；
(4) blood plasma after freezing in step (3) is taken out, is -55 DEG C in temperature, pressure is to carry out at freeze-drying under 50Pa
Frozen dry blood plasma is made in reason；
It (5) is that the calcirm chloride injection of 20 μm of ol/L presses the volume of 1:0.5 by frozen dry blood plasma made from step (4) and concentration
Than mixing, 5min is then reacted in 60 DEG C of water-bath, is taken out, and is made.
Embodiment 2
A kind of compound protein gel, preparation method, comprising the following steps:
(1) autologous vein blood, sodium citrate and autogenous vein are acquired with the heparin tube with 3.8% sodium citrate anticoagulant
The volume ratio of blood is 1:13；
(2) autologous vein blood in step (1) is centrifuged 20min at room temperature with the centrifugal acceleration of 700g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it in -80 DEG C of ultra low temperature freezer
Middle quick freeze, cooling time 72h；
(4) blood plasma after freezing in step (3) is taken out, is -65 DEG C in temperature, pressure is to carry out at freeze-drying under 100Pa
Frozen dry blood plasma is made in reason；
It (5) is that the calcirm chloride injection of 50 μm of ol/L presses the volume ratio of 1:2 by frozen dry blood plasma made from step (4) and concentration
Mixing, then reacts 15min in 90 DEG C of water-bath, takes out, and is made.
Embodiment 3
A kind of preparation method of compound protein gel, comprising the following steps:
(1) Cord blood, the volume of sodium citrate and Cord blood are acquired with the heparin tube with 3.8% sodium citrate anticoagulant
Than for 1:13；
(2) Cord blood in step (1) is centrifuged 15min at room temperature with the centrifugal acceleration of 500g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it in -80 DEG C of ultra low temperature freezer
Middle quick freeze, cooling time 48h；
(4) blood plasma after freezing in step (3) is taken out, is -65 DEG C in temperature, pressure is to carry out at freeze-drying under 70Pa
Frozen dry blood plasma is made in reason；
It (5) is that the calcirm chloride injection of 30 μm of ol/L presses the volume of 1:1.5 by frozen dry blood plasma made from step (4) and concentration
Than mixing, 10min is then reacted in 80 DEG C of water-bath, is taken out, and is made.
Embodiment 4
A kind of compound protein gel, preparation method, comprising the following steps:
(1) Cord blood, the volume of sodium citrate and Cord blood are acquired with the heparin tube with 3.8% sodium citrate anticoagulant
Than for 1:13；
(2) Cord blood in step (1) is centrifuged 12min at room temperature with the centrifugal acceleration of 600g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it in -80 DEG C of ultra low temperature freezer
Middle quick freeze, cooling time 36h；
(4) blood plasma after freezing in step (3) is taken out, is -58 DEG C in temperature, pressure is to carry out at freeze-drying under 80Pa
Frozen dry blood plasma is made in reason；
It (5) is that the calcirm chloride injection of 35 μm of ol/L presses the volume ratio of 1:1 by frozen dry blood plasma made from step (4) and concentration
Mixing, then reacts 6min in 76 DEG C of water-bath, takes out, and is made.
Comparative example 1
A kind of compound protein gel, preparation method, comprising the following steps:
(1) Cord blood, the volume of sodium citrate and Cord blood are acquired with the heparin tube with 3.8% sodium citrate anticoagulant
Than for 1:13；
(2) cord blood in step (1) is centrifuged 5min at room temperature with the centrifugal acceleration of 900g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it in -80 DEG C of ultra low temperature freezer
Middle quick freeze, cooling time 36h；
(4) blood plasma after freezing in step (3) is taken out, is -38 DEG C in temperature, pressure is to carry out at freeze-drying under 110Pa
Frozen dry blood plasma is made in reason；
It (5) is that the calcirm chloride injection of 35 μm of ol/L presses the volume ratio of 1:1 by frozen dry blood plasma made from step (4) and concentration
Mixing, then reacts 6min in 76 DEG C of water-bath, takes out, and is made.
Comparative example 2
A kind of compound protein gel, preparation method, comprising the following steps:
(1) blood, the volume ratio of sodium citrate and Cord blood are acquired with the heparin tube with 3.8% sodium citrate anticoagulant
For 1:13；
(2) blood in step (1) is centrifuged 12min at room temperature with the centrifugal acceleration of 600g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it in -80 DEG C of ultra low temperature freezer
Middle quick freeze, cooling time 36h；
(4) blood plasma after freezing in step (3) is taken out, is -58 DEG C in temperature, pressure is to carry out at freeze-drying under 80Pa
Frozen dry blood plasma is made in reason；
It (5) is that the calcirm chloride injection of 15 μm of ol/L presses the volume ratio of 1:3 by frozen dry blood plasma made from step (4) and concentration
Mixing, then reacts 17min in 55 DEG C of water-bath, takes out, and is made.
Test example
One, 90 female subjects are randomly selected, the age, average age was 40 years old, by its average mark between 30-50 years old
At 6 groups, every group of 15 people, according to blood group matching principle, respectively this 6 groups of subjects inject embodiment 1-4 and comparative example 1-2 and are made
Compound protein gel, injection mode is a point note formula, compound protein gel is injected in skin corium, the maximum injection at each position
Amount is 2ml, 7 days after injection, 15 days, 30 days, 90 days, observe and record subject after half a year, 1 year and 2 years and inject position
Form, concrete outcome are shown in Table 1.
It is learnt by table 1, protein gel made from 1-4 of the embodiment of the present invention and comparative example 1-2 is used equally for facial filling,
Its immunological rejection is small, disappears after 15 days, is filled using protein gel made from embodiment 1-4 and comparative example 1-2 latter
Fix time interior face overall effect it is good, no rigid phenomenon of collapsing, stability is preferable；Albumen made from embodiment 1-4
Gel starts the absorption that is gradually decomposed after generally after the implantation 1 year, after injection 2 years or so injection position saturation degree decline compared with
It is more, wherein the time longest that the protein gel in embodiment 4 maintains, filling effect are best；Albumen made from comparative example 1-2 is solidifying
Glue starts the absorbing phenomenon that is decomposed occur after injecting half a year, and face shows as three-dimensional sense decline, small collapsing, injection one occurs
Nian Hou, face is basic to restore to the original state, and saturation degree disappears, no three-dimensional sense.
Two, it after frozen dry blood plasma made from step (4) in embodiment 1-4 and comparative example 1-2 being sealed 1 year, takes out, after
Protein gel is made in the continuous operation according to step (5), injects the face of user according to above-mentioned method using the protein gel,
Its filling effect substantially be directly injected into consistent, hold time and be basically unchanged, it was demonstrated that, freeze according to the operation of step (4) is obtained
After dry plasma sealing storage 1 year, protein therein and growth factor etc. do not disappear or reduce, and Cord blood is handled
It is saved by the way of freeze-drying afterwards, facilitates and subsequent use and store.
Claims (10)
1. a kind of preparation method of compound protein gel, which comprises the following steps:
(1) blood is acquired with the heparin tube with anti-coagulants；
(2) blood in step (1) is centrifuged 10~20min at room temperature with the centrifugal acceleration of 300~700g；
(3) the upper plasma layer in the blood in aspiration step (2) after centrifugation, places it under the conditions of -80 DEG C, cooling time is
24~72h；
(4) blood plasma after freezing in step (3) is taken out, is -55~-65 DEG C in temperature, pressure is to be frozen under 50~100Pa
Frozen dry blood plasma is made in dry-cure；
(5) calcirm chloride injection that frozen dry blood plasma made from step (4) and concentration are 20~50 μm of ol/L is pressed into 1:0.5~2
Volume ratio mixing, then reacts 5~15min in 60~90 DEG C of water-bath, takes out, and is made.
2. the preparation method of compound protein gel as described in claim 1, which is characterized in that the blood in step (1) is navel
Band blood or autologous vein blood.
3. the preparation method of compound protein gel as described in claim 1, which is characterized in that the anti-coagulants in step (1) is
3.8% sodium citrate.
4. the preparation method of compound protein gel as described in claim 1, which is characterized in that centrifugal acceleration in step (2)
For 600~700g, centrifugation time is 10~15min.
5. the preparation method of compound protein gel as described in claim 1, which is characterized in that the upper plasma in step (3)
It is placed under the conditions of -80 DEG C, cooling time 36h.
6. the preparation method of compound protein gel as described in claim 1, which is characterized in that frozen dried in step (4)
Temperature is -58 DEG C, pressure 80Pa.
7. the preparation method of compound protein gel as described in claim 1, which is characterized in that calcium chloride is injected in step (5)
The concentration of liquid is 30~40 μm of ol/L.
8. the preparation method of compound protein gel as described in claim 1, which is characterized in that in step (5) frozen dry blood plasma with
Calcirm chloride injection is mixed according to the volume ratio of 1:0.5.
9. the preparation method of compound protein gel as described in claim 1, which is characterized in that the bath temperature of step (5) is
75~78 DEG C, the reaction time is 5~7min.
10. a kind of compound protein gel, which is characterized in that be made by any one of claim 1~9 preparation method.
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