CN108478864B - Composite fiber stent - Google Patents
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- CN108478864B CN108478864B CN201810206824.2A CN201810206824A CN108478864B CN 108478864 B CN108478864 B CN 108478864B CN 201810206824 A CN201810206824 A CN 201810206824A CN 108478864 B CN108478864 B CN 108478864B
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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
- A61L27/56—Porous materials, e.g. foams or sponges
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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
- A61L27/20—Polysaccharides
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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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/225—Fibrin; Fibrinogen
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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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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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
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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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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Abstract
The invention provides a composite fiber scaffold extracted from whole blood, wherein the preparation process comprises the steps of mixing the whole blood with HPMC physiological saline solution before coagulation; and centrifuging the mixed solution to obtain an upper layer transparent gel-like substance, namely the composite fiber scaffold. Compared with the traditional fibrin scaffold, the composite fibrous scaffold has higher viscosity and stable properties. HPMC of different concentrations and viscosities can modify the rheological properties of the fibrous scaffold. The composite fiber scaffold is rich in various growth factors, and the growth factors cannot be secreted due to the tight combination with the fiber mesh and are released when being stimulated (such as wound healing).
Description
The application is a divisional application with application number 2017106649584, named composite fiber scaffold and a preparation method thereof, and the application date is 8 months and 7 days in 2017.
Technical Field
The invention relates to the field of medical stents, in particular to a composite fiber stent.
Background
Tissue regeneration repair is based on stem cells, growth factors and scaffolds. Wherein the scaffold is a three-dimensional matrix which can provide an environment for the stem cells to act under the induction of growth factors. A good stent needs to meet the following requirements: 1 mimics the natural extracellular matrix; 2, facilitating cell attachment, migration, proliferation and differentiation; 3 promoting angiogenesis; 4, good biodegradation rate; 5 appropriate physical properties to support the construction of regenerative tissue; the aperture of 6 is about 15-20um, which is convenient for the growth of new blood vessels.
Existing scaffolds are classified into bioscaffolds and synthetic scaffolds according to their origin, wherein bioscaffolds (such as platelet rich plasma, PRP, platelet rich fibrin, PRF, collagen, chitosan, hyaluronic acid, etc.) are superior to synthetic scaffolds due to their good biocompatibility. Among them, PRP and PRF are fibrin containing concentrated platelets extracted from blood, and have been widely used in the field of regeneration in recent years. The PRP preparation process is that anticoagulated whole blood is centrifuged in two steps, and then calcium ions and thrombin are added to prepare the PRP; the PRF preparation process is to directly prepare whole blood after one-time centrifugation. Since the PRP and the PRF have fiber network structures and contain various growth factors, the PRP and the PRF provide good mediums for the proliferation and differentiation of stem cells and the promotion of tissue regeneration, and are good biological scaffolds at present.
However, PRP and PRF also have drawbacks: they do not have stable viscoelasticity and cannot be shaped according to the regeneration requirements of biological tissues. And the pore diameter of PRP is small (about 1-5 μm), while the pore diameter of PRF is slightly larger (about 15-20 μm), the pore diameter of PRF is reduced to less than 10 μm after the PRF is deformed by external force, so the pore diameter of PRP or PRF does not completely meet the requirement of stem cell scaffold.
In view of the above, it is necessary to change the conventional preparation method of extracting fibrin from whole blood to overcome the above-mentioned drawbacks and obtain a new ideal stem cell scaffold.
Reference documents:
1 lie Ruizhi, Zhang Xiu Ming, Yi Nei, Jian politan and hydroxypropyl methyl cellulose in vitro free radical degradation research, chemistry and biological engineering 2013, vol.30No. 6.
2.Dohan,D.M.,J.Choukroun,A.Diss,S.L.Dohan,et al.,Platelet-rich fibrin(PRF):a second-generationplatelet concentrate.Part II:platelet-relatedbiologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2006.101(3):p.e45-50.
Disclosure of Invention
The invention provides a composite fiber scaffold, which solves the problems that in the prior art, the shape is unstable, the pore diameter is small, and the composite fiber scaffold can not be shaped according to the regeneration requirement.
In the invention, a high molecular material hydroxypropyl methylcellulose (HPMC) and fibrin are added in the process of polymerizing fibrinogen to form a fibrin mesh structure to form a novel fiber scaffold.
The technical scheme of the invention is realized as follows:
a preparation method of a composite fiber scaffold comprises the following steps,
mixing the physiological saline solution of HPMC and whole blood to obtain a mixed solution;
and centrifuging the mixed solution before coagulation of the mixed solution to obtain an upper layer transparent gel sample substance, namely the composite fiber scaffold.
As a preferred solution, said HPMC is replaced by collagen, hyaluronic acid, cellulose, chitin or a synthetic product of hyaluronic acid and cellulose.
Wherein the chemical structure of the hyaluronic acid is as follows:
the chemical structural general formula of the cellulose is as follows:
the chemical mechanism general formula of chitin is:
as a preferable technical scheme, the concentration of the HPMC physiological saline solution is 6-10%.
As a preferable technical scheme, the concentration of the HPMC in the mixed solution is 3-5%.
As a preferred technical scheme, the centrifugation time is 8-15 minutes.
As a preferred technical scheme, the preparation process of the HPMC physiological saline solution comprises the following steps: the HPMC powder is dissolved by using 1/3 final volume of physiological saline with 70-80 degrees, and then is poured into 2/3 normal temperature physiological saline to be stirred and dissolved.
A composite fiber scaffold, which is the transparent gel-like substance prepared above.
Advantageous effects
(1) The composite fiber scaffold of the present invention has higher viscosity than PRP and PRF. This is because different concentrations and viscosities of HPMC can tailor the rheological properties of the present fibrous scaffold.
(2) Compared with PRP and PRF, the composite fiber scaffold has no obvious fibrous structure, is a homogeneous gel substance with viscosity, has stable properties, and accords with various clinical applications such as wound surface smearing, skin grafting and the like.
(3) The composite fiber scaffold is rich in various growth factors, and the growth factors are tightly combined with the fiber mesh to be stored and released when being stimulated (such as wound healing). The concentration measurement results of ELISA also confirm that the external secretion amount of the growth factor of the invention is less than PRP (p <0.05), and the growth factor has no obvious difference with PRF.
(3) Compared with PRP and PRF, the fiber pore diameter of the composite fiber scaffold is larger (see electron microscope photos 1-3 of attached drawings). The pore diameter of PRP is about 1-5um, the pore diameter of PRF is about 5-10um, the pore diameter of the invention is about 15-20um, which is more beneficial to the growth of new blood vessels. The reason is that: HPMC is a polymeric material whose presence affects the polymerization of fibrin monomers during the formation of fibrin fibers. On the other hand, hydroxyl in HPMC and carboxyl in fibrinogen form hydrogen bond, which changes the secondary structure of fibrin.
(5) The HPMC polymer material used in the preparation method of the invention has small content, and the HPMC is used as the implant material without rejection reaction and can be degraded in vivo [1 ].
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive exercise.
FIG. 1 is an electron micrograph of the present invention.
FIG. 2 is a PRP electron micrograph.
FIG. 3 is a PRF electron micrograph.
FIG. 4 is a diagram after culturing adipose-derived stem cells on a medium;
a is live/dead staining after 5 percent HPMC and adipose-derived stem cells are co-cultured for 7 days;
and B is the adipose-derived stem cells cultured in a common culture medium for 7 days.
FIG. 5 shows the result of ELISA detection of in vitro secretion of cytokines by PRF and PRP according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
The reagents used in the following examples are all commercially available.
Example 1
A preparation method of a composite fiber scaffold comprises the following steps,
(1) preparing HPMC normal saline solution;
dissolving HPMC powder with 1/3 final volume of 70-80 deg.C physiological saline, adding 2/3 normal temperature physiological saline, stirring, and dissolving to obtain 6-10% HPMC physiological saline solution.
The HPMC is selected from E50 (medical type).
(2) 5-20mL of whole blood from an animal or patient was collected using a vacuum blood collection tube.
(3) Putting the HPMC physiological saline solution prepared in the step (1) and the whole blood collected in the step (2) in a centrifugal tube 1: 1 to obtain a mixed solution, and diluting the HPMC to 3-5% in final concentration.
Wherein the mixing is completed before coagulation within about 1 minute.
(4) And (3) centrifuging (300-500g) for 8-15 minutes before the coagulation of the mixed solution in the step (3) to obtain an upper layer transparent gel-like substance, wherein the lower layer is a red blood cell layer, and the volume of the upper layer and the lower layer is about 1: 1.
(5) collecting the upper gel sample substance with disposable plastic dropper to obtain the composite fiber bracket.
It is more viscous and shape stable than conventional platelet-concentrated fibrin products PRP and PRF. HPMC is a common thickening agent in clinic, and the rheological property of the fiber scaffold can be adjusted by adjusting the concentration of HPMC and selecting HPMC with different viscosities.
The composite fiber scaffold is rich in various growth factors. The literature states that PRP secretes significantly higher concentrations of growth factors than serum, whereas PRF secretes significantly lower concentrations of growth factors than serum; the reason is that the fiber forming process of the PRF is close to physiological condition, the structure is uniform and ordered, and the growth factors are well locked in the fiber net structure; whereas the fibers of PRP are formed suddenly, the structure is rigid and disordered, and growth factors cannot bind to the fiber network and are secreted out [2 ]. The composite fiber scaffold of the present invention is formed in a process similar to PRF, and the growth factors are combined with the fiber mesh, cannot be secreted outside, and are released when being stimulated (such as wound healing). This was also confirmed by the concentration measurement results of ELISA.
Compared with the pure PRP and PRF, the fiber pore diameter of the composite fiber scaffold is larger, about 15um, and is more favorable for the growth of new blood vessels (see attached figures 1-3). The reason is that: HPMC is a polymeric material whose presence affects the polymerization of fibrin monomers during the formation of fibrin fibers. On the other hand, hydroxyl in HPMC and carboxyl in fibrinogen form hydrogen bond, which changes the secondary structure of fibrin.
Conventional PRP and PRF are composed of solid phase fiber and liquid phase serum, and if the serum and the fiber web are separated by external force such as squeezing or stirring, the fiber web is rapidly collapsed and cannot be applied to actual clinical practice. The invention has no obvious fibrous structure, is a homogeneous gel substance with viscosity, and accords with various clinical applications such as wound surface smearing, skin grafting and the like.
(II) in vitro experiment:
HPMC at 5% concentration was co-cultured with adipose stem cells for 7 days.
Green fluorescence in live/dead staining is the cytoplasm of live cells and red is the nucleus of dead cells. See FIG. 4 for live/dead staining after 7 days of co-culture of 5% HPMC with adipose-derived stem cells, which indicates that 5% HPMC has no toxic effect on cells. Therefore, the scaffold can be used as a stem cell scaffold for regenerative medicine.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
1. A composite fiber scaffold is characterized in that the composite fiber scaffold is a transparent gel-like substance;
the preparation method comprises the following steps:
mixing the physiological saline solution of HPMC and whole blood to obtain a mixed solution;
and centrifuging the mixed solution before coagulation of the mixed solution to obtain a transparent gel-like substance, namely the composite fiber scaffold.
2. A composite fibre scaffold according to claim 1, wherein said HPMC is selected as model E50.
3. The composite fiber scaffold according to claim 1, wherein the HPMC in saline is formulated as follows: the HPMC powder is dissolved by physiological saline with the temperature of 1/3 of the final volume being 70-80 ℃, and then is poured into 2/3 normal temperature physiological saline to be stirred and dissolved.
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WO1999020288A1 (en) * | 1997-10-17 | 1999-04-29 | Harvest Technologies Corporation | Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma |
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