CN114404660A - Cell culture method based on three-dimensional scaffold - Google Patents
Cell culture method based on three-dimensional scaffold Download PDFInfo
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- CN114404660A CN114404660A CN202210073743.6A CN202210073743A CN114404660A CN 114404660 A CN114404660 A CN 114404660A CN 202210073743 A CN202210073743 A CN 202210073743A CN 114404660 A CN114404660 A CN 114404660A
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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
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
- A61L27/58—Materials at least partially resorbable by the body
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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
- A61L27/60—Materials for use in artificial skin
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Abstract
The invention discloses a cell culture method based on a three-dimensional scaffold, which comprises the following components in percentage by weight: 28.5-37.4% of Si, 21-28% of Si-OEt, 1.2-4.6% of Si-OH and 25.6-39.4% of Si-O; the invention can be used as a cell proliferation and capillary growth scaffold to promote the growth, proliferation, differentiation and the like of target cells and form certain tissues so as to promote wound healing by providing the three-dimensional structure scaffold for promoting the growth of skin tissues, and the three-dimensional structure scaffold can provide a space for full growth of defective internal cell tissues, is gradually absorbed and replaced by the tissues in the wound healing process and can be used for repairing the wounds of the lower limb venous ulcer skin tissues.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a cell culture method based on a three-dimensional support.
Background
The biological scaffold can effectively promote self-renewal, proliferation, directional differentiation and the like of stem cells by simulating the physicochemical characteristics of a natural microenvironment of a human body, is widely applied to the field of regenerative medicine in recent years, generally needs to be used in the nursing of venous ulcer of lower limbs, and has the following basic principle that a tissue-guided regenerative membrane technology is a tissue engineering application technology which is more successful in clinical application at present: the tissue defect area is isolated from surrounding tissues by using a membrane technology, so that invasion of surrounding fiber cells/tissues is blocked, and a certain favorable environment is created for growth of tissues in the defect. The preparation method has the unique characteristics of simple preparation, strong tissue applicability, simple and convenient clinical operation and the like. At present, the membrane has extensive and intensive research on the aspects of bone science, dentistry, nerve and blood vessel regeneration. Currently, collagen membrane materials (such as BioGide) and hyaluronic acid (such as HYAFF 11) are used clinically.
However, the traditional stent can only be applied to surface repair of defective tissues, cannot form a three-dimensional structure, and can prevent the transfer or communication of macromolecular nutrients and cytokines to influence tissue regeneration when regenerated tissues fully grow into the stent, and meanwhile, the traditional stent has more local degradation products, unsmooth excretion and influence tissue generation.
Disclosure of Invention
The invention aims to provide a cell culture method based on a three-dimensional scaffold, which aims to solve the problems that the traditional scaffold can only be applied to surface repair of a defective tissue, cannot form a three-dimensional structure, and can block the transfer or communication of macromolecular nutrients and cytokines to influence tissue regeneration when a regenerated tissue fully grows into the scaffold, and meanwhile, local degradation products are more, the excretion is not smooth, and the tissue generation is influenced.
In order to achieve the purpose, the invention provides the following technical scheme: a cell culture method based on a three-dimensional scaffold comprises the following three-dimensional scaffold, wherein the three-dimensional scaffold comprises the following components in percentage by weight: 91.85-98.05% of polymer and 1.95-8.15% of liquid;
wherein the polymer comprises the following components in percentage by weight: 28.5-37.4% of Si, 21-28% of Si-OEt, 1.2-4.6% of Si-OH and 25.6-39.4% of Si-O.
Preferably, the liquid comprises the following components in percentage by weight: 1.3-4.1% of water, 0.38-3.26% of ethanol and 0.04-0.19% of nitric acid.
Preferably, the three-dimensional scaffold is a three-dimensional structural scaffold.
Preferably, the three-dimensional scaffold is a net structure.
A cell culture method based on a three-dimensional scaffold comprises the following steps:
s1, putting the polymer, water, ethanol and nitric acid into a stirring tank together for hydrolysis condensation reaction, wherein the reaction time is 15-29 h;
s2, putting the raw materials reacted in the step S1 into an evaporation kettle, reacting for 4-8 hours, introducing compressed air, and releasing water and ethanol in the evaporation process;
s3, curing the raw materials in the step S2, controlling the temperature to be 2-6 ℃, and controlling the viscosity to be 40-70 Pa.s;
s4, spinning and cutting the spun fiber obtained in the step S3, and introducing compressed air, wherein the mass is controlled to be 380-480 mg;
s5, finally, packaging the fiber dressing in the step S4, and performing radiation sterilization on the fiber dressing to obtain the three-dimensional scaffold.
Preferably, in the step S1, the temperature is 35-41 ℃, and the stirring speed is 100-140 r/min.
Preferably, in the step S2, the temperature is 52-79 ℃, and the stirring speed is 110-150 r/min.
Preferably, in the step S2, the raw material viscosity is 0.6 to 1.9Pa · S.
Preferably, in the step S4, the spun fiber compression ratio is 16 to 26%.
Preferably, in the step S4, the density of the spun fiber is 73-123 mg/cm3。
Compared with the prior art, the invention has the beneficial effects that:
the invention can be used as a cell proliferation and capillary growth scaffold to promote the growth, proliferation, differentiation and the like of target cells and form certain tissues so as to promote wound healing by providing the three-dimensional structure scaffold for promoting the growth of skin tissues, and the three-dimensional structure scaffold can provide a space for full growth of defective internal cell tissues, is gradually absorbed and replaced by the tissues in the wound healing process and can be used for repairing the wounds of the lower limb venous ulcer skin tissues.
Drawings
FIG. 1 is a flow chart of the preparation method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
referring to fig. 1, the present invention provides a technical solution: a cell culture method based on a three-dimensional scaffold comprises the following three-dimensional scaffold, wherein the three-dimensional scaffold comprises the following components in percentage by weight: si32.6%, Si-OEt 21.4%, Si-OH 3%, Si-O35.6%, water 4.1%, ethanol 3.26% and nitric acid 0.04%.
Wherein the three-dimensional scaffold is a three-dimensional structure scaffold.
Wherein, the three-dimensional support is a net structure.
A cell culture method based on a three-dimensional scaffold comprises the following steps:
s1, putting the polymer, water, ethanol and nitric acid into a stirring tank together for hydrolysis condensation reaction, wherein the reaction time is 18h, the temperature is 37 ℃, and the stirring speed is 120 r/min;
s2, putting the raw materials reacted in the step S1 into an evaporation kettle, reacting for 6 hours at 65 ℃, stirring at the rotating speed of 130 r/min, introducing compressed air, releasing water and ethanol in the evaporation process, wherein the viscosity of the raw materials is 1.2Pa S;
s3, curing the raw material in the step S2, controlling the temperature at 4 ℃ and the viscosity at 60Pa S;
s4, spinning and cutting the spinning fiber in the step S3, and introducing compressed air, wherein the mass is controlled to be 420mg, the compression ratio of the spinning fiber is 21%, and the density is 103mg/cm3；
S5, finally, packaging the fiber dressing in the step S4, and performing radiation sterilization on the fiber dressing to obtain the three-dimensional scaffold.
Example 2:
referring to fig. 1, the present invention provides a technical solution: a cell culture method based on a three-dimensional scaffold comprises the following three-dimensional scaffold, wherein the three-dimensional scaffold comprises the following components in percentage by weight: si 34.5%, Si-OEt 25%, Si-OH 2.5%, Si-O33.3%, water 2.7%, ethanol 1.45% and nitric acid 0.1%.
A cell culture method based on a three-dimensional scaffold comprises the following steps:
s1, putting the polymer, water, ethanol and nitric acid into a stirring tank together for hydrolysis condensation reaction, wherein the reaction time is 18h, the temperature is 37 ℃, and the stirring speed is 120 r/min;
s2, putting the raw materials reacted in the step S1 into an evaporation kettle, reacting for 6 hours at 65 ℃, stirring at the rotating speed of 130 r/min, introducing compressed air, releasing water and ethanol in the evaporation process, wherein the viscosity of the raw materials is 1.2Pa S;
s3, curing the raw material in the step S2, controlling the temperature at 4 ℃ and the viscosity at 60Pa S;
s4, spinning and cutting the spinning fiber in the step S3, and introducing compressed air, wherein the mass is controlled to be 420mg, the compression ratio of the spinning fiber is 21%, and the density is 103mg/cm3；
S5, finally, packaging the fiber dressing in the step S4, and performing radiation sterilization on the fiber dressing to obtain the three-dimensional scaffold.
Example 3:
referring to fig. 1, the present invention provides a technical solution: a cell culture method based on a three-dimensional scaffold comprises the following three-dimensional scaffold, wherein the three-dimensional scaffold comprises the following components in percentage by weight: si32.75%, Si-OEt24.4%, Si-OH 3%, Si-O32.6%, water 4.1%, ethanol 3.26% and nitric acid 0.19%.
A cell culture method based on a three-dimensional scaffold comprises the following steps:
s1, putting the polymer, water, ethanol and nitric acid into a stirring tank together for hydrolysis condensation reaction, wherein the reaction time is 18h, the temperature is 37 ℃, and the stirring speed is 120 r/min;
s2, putting the raw materials reacted in the step S1 into an evaporation kettle, reacting for 6 hours at 65 ℃, stirring at the rotating speed of 130 r/min, introducing compressed air, releasing water and ethanol in the evaporation process, wherein the viscosity of the raw materials is 1.2Pa S;
s3, curing the raw material in the step S2, controlling the temperature at 4 ℃ and the viscosity at 60Pa S;
s4, spinning and cutting the spinning fiber in the step S3, and introducing compressed air, wherein the mass is controlled to be 420mg, the compression ratio of the spinning fiber is 21%, and the density is 103mg/cm3；
S5, finally, packaging the fiber dressing in the step S4, and performing radiation sterilization on the fiber dressing to obtain the three-dimensional scaffold.
As described in the above embodiments, the present invention provides a three-dimensional scaffold for promoting skin tissue growth, which can be used as a scaffold for cell proliferation and capillary growth, promote the growth, proliferation, differentiation, etc. of target cells, and form a certain tissue, thereby promoting wound healing, and the three-dimensional scaffold can provide a space into which cell tissue inside a defect can grow sufficiently, and can be gradually absorbed and replaced by the tissue during wound healing, thereby being used for wound repair of lower limb venous ulcer skin tissue.
Products and degradation products thereof:
the product is an amorphous polyester of orthosilicic acid, which is completely different from crystalline silica in form, composition, structure, chemical behavior and degradation products. After the product is degraded, the degradation product containing silicon is ortho silicic acid [ SiO (OH)2]4, and the difference exists between the product and silica sol. Chemically, silica sol particles differ from the present product in the presence of ethoxy groups, the ethoxy group content of the silica sol particles being very low, and the content of orthosilicic acid formed upon contact with water being very low or not formed at all. The product has relatively high ethoxy content (up to 30%). These ethoxy groups avoid condensation reactions of the fibers, leading to the formation of hardly/undegradable-Si-O-Si-chains. Thus, the silicon product or its degradation products are not to be confused with crystalline silica, silica gel or silica sol (amorphous). Orthosilicic acid is inert in neutral pH environments, but can condense in saturated solution to form silicic acid dimers and small oligomers. Condensation of orthosilicic acid reduces its solubility and thus bioavailability, however, this condensation does not occur in the physiological environment in vivo, since saturation conditions are never reached.
The other degradation product, ethanol, is released from the fibers in water or physiological fluids due to hydrolysis of the ethoxy groups. The maximum release of ethanol was theoretically calculated in an aqueous environment with a piece of dressing (specification 430 ± 50 mg): the ethanol content in the product comprises free ethanol (the technical requirement is not more than wt.6%) and ethanol after ethoxy (the technical requirement is wt.20 +/-10%) hydrolysis, so the maximum release amount of the ethanol is as follows: 480mg × (30+ 6)% ≈ 173mg ═ 0.173 g. In the human body, the released alcohol can be rapidly diffused into the blood of the human body, while the blood of normal adults contains a small amount of alcohol, and the content of the alcohol in the blood of the normal human body is about 0.003%. We analyze the worst case scenario, if a piece of dressing instantaneously releases all of the ethanol, the percentage of ethanol concentration calculated from the 5L blood volume (adult normal blood volume) of the human body is: 0.173g/0.8g/ml/5000ml ≈ 0.004% (0.8g/ml is ethanol density), which is close to the normal ethanol content in human blood. On the other hand, in practical clinical application, the product using process and the wound environment need to be kept in a wet state, but one dressing can be infiltrated by at least about 50ml of physiological saline, and the release amount of the dressing after instant degradation of ethanol is 0.173g, and the maximum release volume of the converted ethanol is as follows, regardless of the accumulation of tissue fluid: 0.173g/0.8g/ml ≈ 0.2ml, the local volume concentration of ethanol is about 0.2ml/50ml ≈ 0.4%, which is much lower than the ethanol content that produces disinfection effect (70% to 75% of ethanol is used for disinfection according to the data).
The product has the using points: the product can be cut at will until the size of the wound is suitable; debridement before use: so as to ensure that the wound surface has no necrotic tissue and the wound edge contains living tissue; the wet working environment: applying the three-dimensional scaffold-based cell culture method to a wound in a dry state, and then wetting the wound with sterile physiological saline; use in combination with a secondary dressing: the secondary dressing is used for fixing and maintaining a wound moist environment; and (3) dressing replacement: the fiber is blended into the granulation tissue for replacement, or the fiber is replaced when the product is peeled off and falls off due to other external factors.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A cell culture method based on a three-dimensional scaffold comprises the three-dimensional scaffold, and is characterized in that the three-dimensional scaffold comprises the following components in percentage by weight: 91.85-98.05% of polymer and 1.95-8.15% of liquid;
wherein the polymer comprises the following components in percentage by weight: 28.5-37.4% of Si, 21-28% of Si-OEt, 1.2-4.6% of Si-OH and 25.6-39.4% of Si-O.
2. The three-dimensional scaffold-based cell culture method of claim 1, wherein: the liquid comprises the following components in percentage by weight: 1.3-4.1% of water, 0.38-3.26% of ethanol and 0.04-0.19% of nitric acid.
3. The three-dimensional scaffold-based cell culture method of claim 1, wherein: the three-dimensional support is a three-dimensional structure support.
4. The three-dimensional scaffold-based cell culture method of claim 1, wherein: the three-dimensional support is of a net structure.
5. A cell culture method based on a three-dimensional scaffold is characterized in that: the method comprises the following steps:
s1, putting the polymer, water, ethanol and nitric acid into a stirring tank together for hydrolysis condensation reaction, wherein the reaction time is 15-29 h;
s2, putting the raw materials reacted in the step S1 into an evaporation kettle, reacting for 4-8 hours, introducing compressed air, and releasing water and ethanol in the evaporation process;
s3, curing the raw materials in the step S2, controlling the temperature to be 2-6 ℃, and controlling the viscosity to be 40-70 Pa.s;
s4, spinning and cutting the spun fiber obtained in the step S3, and introducing compressed air, wherein the mass is controlled to be 380-480 mg;
s5, finally, packaging the fiber dressing in the step S4, and performing radiation sterilization on the fiber dressing to obtain the three-dimensional scaffold.
6. The three-dimensional scaffold-based cell culture method of claim 5, wherein: in the step S1, the temperature is 35-41 ℃, and the stirring speed is 100-140 r/min.
7. The three-dimensional scaffold-based cell culture method of claim 5, wherein: in the step S2, the temperature is 52-79 ℃, and the stirring speed is 110-150 r/min.
8. The three-dimensional scaffold-based cell culture method of claim 5, wherein: in the step S2, the viscosity of the raw material is 0.6 to 1.9 pas.
9. The three-dimensional scaffold-based cell culture method of claim 5, wherein: in the step S4, the spinning fiber compression ratio is 16-26%.
10. The three-dimensional scaffold-based cell culture method of claim 5, wherein: in the step S4, the density of the spinning fiber is 73-123 mg/cm3。
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